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+{"text": "Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease. Know your enemy. Description of the \u2018virus gene chip\u2019 that helped to classify the SARS virus as a novel coronavirus Over the past two decades, technological advances in molecular biology have fuelled progress in the discovery of new pathogens associated with human diseases. The identification of novel viruses such as hepatitis C virus , sin nomWe have previously described a prototype DNA microarray designed for highly parallel viral detection with the potential to detect novel members of known viral families . This miDuring the initial phase of research into the etiology of SARS, an unknown virus was cultured in Vero cells from a patient suffering from SARS . Total nNSP11 gene . A pair of PCR primers was designed to amplify the intervening sequences between the two conserved regions, and a fragment that possessed 89% identity over 37 amino acids to MHV, a murine coronavirus, was obtained  , we wereA key feature of this approach is that direct recovery of hybridized material from the microarray provides a rapid route for obtaining sequences of novel viruses. By contrast, conventional strategies for subsequent sequence identification would require time-consuming steps such as library screening or additional rounds of PCR primer design and synthesis. In the case of SARS, we were able to ascertain within 24 h that a novel coronavirus was present in the unknown sample, and partial genome sequences of this virus were obtained over the next few days without the need for specific primer design. To our knowledge, this is the first demonstration of the feasibility and utility of directly recovering nucleic acid sequences from a hybridized DNA microarray. In light of the continuous threat of emerging infectious diseases, this overall approach will greatly facilitate the rapid identification and characterization of novel viruses.Total nucleic acid was purified using the automated NucliSens extraction system . Following the manufacturer's instructions, 100 \u03bcl of each specimen was added to tubes containing 900 \u03bcl of prewarmed NucliSens lysis buffer and incubated at 37\u00b0C for 30 min with intermittent mixing. Fifty microliters of silica suspension provided in the extraction kit was added to each tube and mixed. The mixtures were then transferred to a nucleic acid extraction cartridge and loaded onto the extractor workstation for processing. Approximately 50 \u03bcl of total nucleic acid eluate was recovered.For the culture supernatants, 450 ng of nucleic acid was used as input for the amplification protocol. In parallel, 50 ng of HeLa cell RNA was used as a positive amplification control and water was used for a negative control. Samples were amplified using a random-primer protocol as described by infection-Cy5mock) > 1500 intensity units are listed in DNA microarrays were printed and hybridized essentially as described by PCR primers were designed by aligning the hybridizing oligonucleotides (Oligo IDs 15081544_766 and 12175745_728) to the IBV genome  and selecting stretches of near-identity. Primer-B-amplified material was used as the template for 35 cycles of thermocycling using the following program: 94\u00b0C for 30 s, 56\u00b0C for 30 s, and 72\u00b0C for 60 s.Amplified viral sequences hybridized to individual microarray spots were recovered by scraping a 100 \u03bcm area of the microarray using a tungsten wire probe  mounted on a micromanipulator while visualized by fluorescence microscopy (Nikon TE300). Recovered material was PCR amplified using primer-B, cloned into pCR2.1TOPO (Invitrogen), and sequenced. A detailed protocol is available as Primer-B-amplified nucleic acid (see above) was cloned in pCR2.1TOPO, plated on 2xYT/kan plates, and grown overnight at 37\u00b0C. White colonies were picked into 384-well plates containing 2xYT/kan plus 8% glycerol and incubated overnight at 37\u00b0C. DNA was purified by magnetic bead isolation. DNA sequencing involved adding 3 \u03bcl of water to each bead pellet, followed by 3 \u03bcl of Big Dye terminator (v3.1) sequencing cocktail, and incubation for 35 cycles of 95\u00b0C for 5 s, 50\u00b0C for 5 s, and 60\u00b0C for 2 min. Reaction products were ethanol precipitated, resuspended in 25 \u03bcl of water, and loaded onto the ABI 3730xl sequencer. The resulting sequence reads were trimmed to remove primer sequences from the RT-PCR step and then assembled by Phrap . Resulting contigs were screened by blast to remove any contigs with high human or monkey sequence similarity. The remaining contigs were edited to high quality, making any obvious joins. (Sequences are available as Data S1(91.5 KB DOC)Click here for additional data file.Protocol S1(28 KB DOC)Click here for additional data file.Protocol S2(39.5 KB DOC)Click here for additional data file.Table S1(97 KB DOC)Click here for additional data file.Table S2(2.2 MB XLS)Click here for additional data file.The Gene Expression Omnibus accession number for the array sequence is GSE546."}
+{"text": "Coccolithoviridae is a recently discovered family of viruses that infect the marine coccolithophorid Emiliania huxleyi. Following on from the sequencing of the type strain EhV-86, we have sequenced a second strain, EhV-163.The We have sequenced approximately 80% of the EhV-163 genome, equating to more than 200 full length CDSs. Conserved and variable CDSs and a gene replacement have been identified in the EhV-86 and EhV-163 genomes.The sequencing of EhV-163 has provided a wealth of information which will aid the re-annotating of the EhV-86 genome and identified a gene insertion in EhV-163. Coccolithoviridae strain EhV-86, a giant dsDNA algal virus from the family Phycodnaviridae that infects the marine coccolithophorid Emiliania huxleyi [Phycodnaviridae [Phycodnaviridae and the presence of six RNA polymerase subunits (unique among the Phycodnaviridae) we suggested this genus would eventually be renamed as a subfamily of the Phycodnaviridae termed Coccolithovirinae.We recently determined the whole genome sequence of the  huxleyi . Core geaviridae . Due to Emiliania huxleyi bloom in the English Channel [Emiliania huxleyi, and their possible placement within a putative subfamily, we have undertaken to sequence a second coccolithovirus genome, EhV-163.Strain EhV-86 was originally isolated, along with many others, in 1999 from an  Channel ,4. In co Channel . Both vi Channel . In ordeThe sequencing of EhV-86 was hindered by the highly repetitive nature of the genome (three different types of repeat family were identified ), which Poxviridae, Iridoviridae, Asfariviridae, Phycodnaviridae and Mimiviridae families, it is likely that for ehv167, at least, the high degree of conservation is due to a high selection pressure [Phycodnaviridae (PBCV-1 and ESV-1).Of the 202 CDSs that had complete sequence, 20 were identical at DNA level and a further 17 were identical at the amino acid level . These 3pressure ,6. This No sequence was obtained for 88 of the 472 CDSs predicted to be encoded in the EhV-86 genome. The similar size of the EhV-163 genome in comparison with that of EhV-86 and the high levels of similarity in other regions suggests that the majority of these CDSs are likely to be present. Indeed, a hybridisation of EhV-163 genomic DNA to the EhV-86 based coccolithovirus microarray has revealed that of the 425 EhV-86 CDSs probed for, only 28 appear to be absent in EhV-163 (unpublished data). However, one notable gene deletion in EhV-163 is a putative phosphate permease found at approximately 115 kb on the EhV-86 genome  after only two rounds for the EhV-163 version of ehv142. PSI-BLAST searches using the corresponding EhV-86 CDS reveal no matches for KELCH-like proteins, suggesting ehv142 may play a different role in each virus strain. Both EhV-86 and EhV-163 are capable of infecting many of the same strains (with varying virulence) [E. huxleyi that are susceptible to infection by only one or other of the viruses (unpublished data). Intriguingly, KELCH-like proteins have been identified in poxviruses and are found to be highly variable [There appears to be a high degree of variation in ehv142 between the two strains. The CDS has approximately 86.9 % identity at the nucleotide level (183 of the 1398 nucleotides are different) and 79.1% identity at the amino acid level (97 of the 465 amino acids are different) . Growth was monitored by cell counts in a Reichert haemocytometer under a light microscope. Four days post-inoculation, the decimated cultures were subjected to a filtration, concentration and purification regime [Six 1L cultures of exponentially growing n regime .DNA was extracted from CsCl-purified EhV-163 by initially treating the sample with proteinase K (5 mg/ml) in a lysis buffer containing 20 mM EDTA, pH 8.0 and 0.5% SDS (w/v) at 65\u00b0C for 1 h. 0.1 \u00d7 volume aliquots of phenol were added to the samples, after which the DNA was extracted with an equal volume of chloroform:isoamyl alcohol (24:1). The DNA was precipitated with the addition of 0.5 \u00d7 volume 7.5 M ammonium acetate, pH 7.5 and 2.5 \u00d7 volume absolute ethanol. Virus DNA was stored in molecular grade water (Sigma) prior to genome sequencing. under EnvBase accession number egcat:00010. When a PCR product was obtained, it was sequenced directly using both primers and the resulting sequence added to the contig library. The depth of sequence coverage varied across the genome due to the random nature of the initial sequencing strategy. Depth of coverage varied from just one sequence read for some regions to up to18 for others, with an average coverage of approximately 3. In areas of low coverage, sequence reads containing ambiguous results were removed from the analysis. 267 contigs were generated, covering approximately 80% of the EhV-163 genome. These contigs have been submitted to Genbank under the accession numbers DQ127552-DQ127818. This data is also available from , EnvBase accession number egcat:00010.Genomic DNA was sheared by sonication, ligated into pCR-Blunt (Invitrogen) and sequenced using M13 forward and reverse primers. After 2700 reads, the sequence was assembled into contigs and analysed using SeqMan (DNAstar). Following alignment to the backbone of EhV-86, 229 primer pairs were designed, specific to the EhV-163 gDNA sequence, to attempt to amplify the missing gaps. The sequence, annealing temperature and genomic location (in relation to EhV-86) of the primers designed can be found in the NERC environmental genomic data catalogue at . Artemis Comparison Tool (ACT)  was used to compare the EhV-163 contigs against the EhV-86 genome.The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences by comparing nucleotide or protein sequences to sequence databases and calculating the statistical significance of matches. Protein-protein BLAST (BLAST-P) and Position-specific iterated BLAST (PSI-BLAST) were performed on CDSs of interest online at The author(s) declare that they have no competing interests.MJA helped coordinate the study, carried out the molecular genetic studies, sequence alignment and drafted the manuscript. DSCH prepared the EhV-163 DNA for the construction of the shotgun library, helped coordinate the study and draft the manuscript. AD and DSCH constructed the EhV-163 clone library. AD screened the library. AD and KJC performed the sequencing and participated in the sequence alignment. WHW conceived, designed and coordinated the study and helped to draft the manuscript. All authors read and approved the final manuscript.Click here for file"}
+{"text": "RHD genotype data on at least one parent and at least one child diagnosed with schizophrenia or related disorder. Meta-analysis inclusion criteria were (1) well-defined sample of schizophrenia patients with majority born before 1970, (2) Rhesus D incompatibility phenotype or genotype data available on mother and offspring, and by offspring sex. Two of ten studies, plus the current genetic study sample, fulfilled these criteria, for a total of 358 affected males and 226 affected females. The genetic study found that schizophrenia risk for incompatible males was significantly greater than for compatible offspring (p = 0.03), while risk for incompatible and compatible females was not significantly different (p = .32). Relative risks for incompatible males and females were not significantly different from each other. Meta-analysis using a larger number of affected males and females supports their difference. Taken together, these results provide further support that risk of schizophrenia due to Rhesus D incompatibility is limited to incompatible males, although a weak female incompatibility effect cannot be excluded. Sex differences during fetal neurodevelopment should be investigated to fully elucidate the etiology of schizophrenia.Rhesus D incompatibility increases risk for schizophrenia, with some evidence that risk is limited to male offspring. The purpose of this study is to determine whether risk for schizophrenia due to Rhesus D incompatibility differs by offspring sex using a nuclear family-based candidate gene approach and a meta-analysis approach. The genetic study is based on a sample of 277 nuclear families with  Schizophrenia is a debilitating disorder with a mixture of characteristic signs and symptoms, both positive and negative, affecting ~1% of the population . The posSex differences in schizophrenia have been noted since Kraepelin found that dementia praecox occurred primarily in young men , and morAlthough the explanation for sex-specific differences is not yet known, some researchers suggest that male\u2013female differences in schizophrenia may be explained through a neurodevelopmental perspective, where more males than females are likely to experience a form of the disease due to a neurodevelopmental anomaly resulting from early environmental influences . The neuAlthough the prenatal environment offers one plausible source for male\u2013female differences in schizophrenia, there have been few detailed sex-specific studies and the results of such studies as yet are inconclusive. Some studies have documented an increased frequency of obstetric complications in pregnancies of males with schizophrenia , others RHD locus [MIM 111680] . Genotype data were available on both parents in 132 families, while they were available only for the mother in 107 families, and only for the father in 38 families. 58 affected offspring were determined to be either unambiguously Rhesus D incompatible with their mothers  because genotype information was available on mother and offspring, or potentially incompatible with their mothers  in the case of heterozygous offspring with Rhesus D positive fathers, missing maternal genotypes, and sibling genotypes that are consistent with a Rhesus D negative mother. In 21 independent families the unambiguously or potentially incompatible affected off-spring were male, in 6 families the incompatible offspring were female, and in 10 families both male and female incompatible offspring were present. Birth years for the offspring ranged from 1923\u20131976, with only 12 offspring born in or after 1970, the era when prophylaxis against maternal isoimmunization became widely used in Finland . HoweverRHD genotypes  (p=0.6). Among the 409 genotyped parents, 52 were dd, 189 were Dd, and 168 were DD; the frequency of the D and d alleles were 64.2% and 35.8%, respectively. These data are comparable to the frequency of Rhesus D negative (dd) individuals in Finland (~12%) (Finnish Red Cross), and the frequency of the d allele in the Finnish population (~34%) (A standard procedure was used to extract DNA from EDTA blood  and a PCand d/d) . PCR wasand d/d)  resultinn (~34%) .RHD gene is treated as diallelic, with D denoting all polymorphisms that lead to protein expression and d alleles denoting all polymorphisms that do not. An individual who inherits two d alleles (dd) is Rhesus D negative while those who inherit at least one D allele (Dd or DD) are Rhesus D positive. Rhesus D incompatibility occurs when the mother is dd and the child is Dd. To test for association of Rhesus D incompatibility and schizophrenia, we used a conditional log-linear model  and ln; however we present the non-logged values throughout this paper.cel from . The parAs previously , the modp-values are reported because prior evidence suggests that the Rhesus D incompatibility effect is deleterious =3.21, p=0.036 (one-sided), leading us to reject the null hypothesis of \u03bcmale=1 in favor of the alternative hypothesis that there is a positive male incompatibility effect. The model that constrains the female effect to one but allowsfor a male effect  =0.220, p=0.319 (one-sided), consistent with the hypothesis that there is no female incompatibility effect. To test the sex independent model originally proposed =0.620, p=0.431 [two-sided]) and thus fails to provide additional support for the hypothesis that the incompatibility effect is exclusively for male offspring.In order to test the hypothesis that lue of 1 . This coproposed , we thenQ statistic for male offspring data=2.07, p=0.356; Q statistic for female offspring data=0.485, p=0.785) indicating that pooling their results is justified . However, when we compared a model with these estimates of the male and female incompatibility effects to a model that constrained the sex-specific effects to be equal, we found that the two models were not statistically different, thus failing to provide additional support for the hypothesis that the incompatibility effect is exclusively for male offspring.\u03bcfemale = 1.07, 90% CI 0.72\u20131.58; \u03bcmale = 1.64, 90% CI 1.25\u20132.17], it also does not fall within the 90% CI for the male incompatibility effect, and it is contained within a 90% CI that includes the null value. Furthermore, the 90% CI for the male incompatibility effect does not cover the null value of one and the effect estimate for males does not fall within the 90% CI for the female effect.By combining three datasets in the meta-analysis, we increased the sample size for affected males to 358 and affected females to 226 (a 12\u201318% increase from the Finnish sample). The results of our meta-analysis yielded the strongest evidence thus far that the incompatibility effect is limited to male offspring. The point estimate of the pooled female incompatibility effect is not only smaller than that for males [The results of our candidate gene analysis and meta-analysis suggest that the effect of Rhesus D incompatibility on the development of schizophrenia is limited to male offspring. However, because the sample size for females was ~35% less than that of the males, our study may have lacked sufficient statistical power to detect a small female effect. On the other hand, the estimated pooled relative risk for females in the meta-analysis is 1.07, which is very small in magnitude, leaving in question its substantive meaningevenif found to be statistically significant in a larger sample. A null or very small female effect relative to males is an important finding that allows for hypothesis generation and testing to determine why schizophrenia effects of Rhesus D incompatibility are so much greater for male offspring compared to female offspring.immunized by male fetuses than female fetuses , we are unable to provide this additional piece of evidence. Nevertheless, our sex-dependent finding is particularly interesting in light of recent work demonstrating that HLA-B matching as a schizophrenia risk factor is limited to females . Not onl"}
+{"text": "Patients with colorectal cancer undergoing surgical treatment require planning and preparation of interventions for prevention of postoperative complications, especially considering the complexity and commitment of its clinical and psychosocial condition.To identify and synthesize the factors that influence the occurrence of postoperative complications and establish the implications of these scientific evidence for nursing care.It is an integrative review of literature where descriptors were used, wound, colorectal cancer and complications with search in the databases Medline, CINAHL and Lilacs, resulting in a sample of 10 scientific articles.Results indicated that the preoperative bowel preparation, staging and tumor location, surgical technique and care of the wound in the postoperative period as the factors influencing the occurrence of postoperative complications.Thus, measures of prevention and infection control can be implemented that are related to the rigor of the completion of the enema, ensure the conduct, preparation and guidance of patients for diagnostic tests and specialized  , fitness and education on pre-operative surgery and its consequences, evaluation of patient outcome and the surgical wound and hospital discharge planning with primary care for the patient and family, encouraging the physiological recovery and wound healing. These nursing interventions could help in decreasing the rates of postoperative complications and mortality of this clientele, as well as improve the quality of perioperative nursing care.J. Silva Other review, H. Sonobe: None declared, D. Andrade: None declared, A. Giordani: None declared, C. Naka Shimura: None declared, E. Watanabe: None declared."}
+{"text": "These proteins inhibit viral infectivity, binding to the HCV envelope glycoproteins E1 and E2 and block viral entry into human hepatocytes. In addition, we demonstrate that the most potent of these agents, the protein griffithsin, is readily bioavailable after subcutaneous injection and shows significant in vivo efficacy in reducing HCV viral titers in a mouse model system with engrafted human hepatocytes. These results indicate that HCV viral entry inhibitors can be an effective component of anti-HCV therapy and that these proteins should be studied further for their therapeutic potential.Hepatitis C virus (HCV) infection is a significant public health problem with over 170,000,000 chronic carriers and infection rates increasing worldwide. Chronic HCV infection is one of the leading causes of hepatocellular carcinoma which was estimated to result in \u223c10,000 deaths in the United States in the year 2011. Current treatment options for HCV infection are limited to PEG-ylated interferon alpha (IFN-\u03b1), the nucleoside ribavirin and the recently approved HCV protease inhibitors telaprevir and boceprevir. Although showing significantly improved efficacy over the previous therapies, treatment with protease inhibitors has been shown to result in the rapid emergence of drug-resistant virus. Here we report the activity of two proteins, originally isolated from natural product extracts, which demonstrate low or sub-nanomolar  These include the NS3/4A protease, the NS5b polymerase, the multifunctional NS5a protein, and the surface glycoproteins E1 and E2. Effort is also being put into the development of immunomodulators that affect toll-like receptors or inhibit cyclophilin The HCV envelope glycoproteins E1 and E2 are responsible for viral attachment and entry in vivo activity Viral entry of HCV offers several specific cellular targets in addition to the viral envelope glycoprotein E2. Antibodies to all of these targets have inhibited HCV entry to some extent Here we report the activity of the proteins griffithsin (GRFT) and scytovirin (SVN) against HCV. GRFT and SVN were originally isolated from red 50) values of >200 nM (genotype 1b) and \u223c1000 nM (genotype 2a) \u200a=\u200a34 \u00b5M and a selectivity index (SI) of 84,000  . In conttype 2a) . SVN wasf 84,000 . The sig50\u200a=\u200a0.30 nM), TH (1b) intermediate (EC50\u200a=\u200a2.2 nM) and J6(2a) the least sensitive (EC50 of 14.1 nM). The pattern of sensitivity was similar for SVN although SVN had less potent overall activity  the most sensitive   & S2. WePrior to initiating animal efficacy studies, we determined GRFT's bioavailability following sub-cutaneous injection. GRFT (20 mg/kg) was injected into Alb-uPA/SCID mice (n\u200a=\u200a5) for 10 days (Q24) and blood samples were taken on days \u22124, 3, 11, 18 and 25. Quantification of the GRFT present in the mouse plasma was determined using capture ELISA. GRFT was bioavailable following subcutaneous injection, with plasma levels reaching a high of 3.3 \u00b5g/mL (\u223c260 nM) on day 11 and slowly reducing to 0.59 \u00b5g/mL (\u223c47 nM) on day 18 .Monitoring of health parameters confirmed that GRFT treatment was well tolerated by the animals and resulted in only mild and transient alterations in body weights and morbidity scores in a minority of animals.Following bioavailability and tolerability studies in the Alb-uPA/SCID chimeric mice, we initiated efficacy studies in these same mice after successful engraftment of human hepatocytes. Study animals were split into two groups (n\u200a=\u200a6 each) and were dosed (Q24) with either 20 mg/kg/day of GRFT or with saline control. Drug treatment began on day 1 and continued through day 10. Animals were challenged with genotype 1a HCV 8 hr after the first GRFT treatment on Day 1. GRFT treatment was well tolerated and all animals completed the study. Assay of blood samples drawn on days \u22124 (control), 3, 11, 18 and 25 confirmed that serum hAAT levels remained high and relatively constant for the study animals throughout the study. HCV titers for both GRFT-treated and control animals rose from their pre-infection values of 0 on Day 0 to similar values at day 3 of the study. Thereafter, however, control animal HCV titers continued to rise while GRFT-treated animals showed no increase in titers at day 11 and a reduction in viral titers at day 18  . In compin vivo study showed efficacy for GRFT in the transgenic mouse model system, the results were not conclusive. To further elaborate on the initial in vivo findings, a second study was conducted with several changes. In this study, GRFT was administered on day \u22121, day 0 and day 1 to allow blood levels to reach therapeutic levels prior to viral challenge, and GRFT (20 mg/kg/day) was administered for a total of 18 days. In addition, GRFT efficacy was compared to IFN- \u03b1-treatment  in a second study arm while in a third arm animals were treated with both agents to determine if there were any additive effects. As before, all three arms were compared to saline-treated control animals. Four hours after drug treatment on day 1 animals in all groups received \u223c2\u00d7105 infectious units HCV/dose by i.p. injection. Serum samples were taken on day-4 (control), day 3, 10, 17, 24 and 31 and measured for HCV RNA and hAAT.Though the initial In this second efficacy study the prolonged dosing of GRFT for an additional 8 days appeared to have a deleterious effect on the overall health of test animals with two of seven GRFT\u2013treated mice euthanized after the completion of treatment on day 17 due to excessive weight loss and poor health index scores in addition to one found dead on day one. One animal in the combined GRFT/IFN-\u03b1 group died on day 11 and two additional mice were euthanized on day 17 in the combination study group. None of the animals in either the saline control or IFN-\u03b1-treated groups had to be sacrificed prior to the end of the study. hAAT levels remained relatively constant for all animals in the study except one mouse in the saline control group. N-linked glycosylation sites on E1 and 9\u201311 on E2 50 values of >200 nM (genotype 1b) and \u223c1000 nM (genotype 2a) (50 concentration of 0.4 nM (50\u200a=\u200a17 nM) and CV-N (EC50\u200a=\u200a7.6 nM) and showing 30-fold better potency than that recently reported for GRFT in vitro selectivity index exhibited by GRFT  and SVN .The need for additional therapeutic agents to treat HCV infections has led to increased attempts to identify both drug targets and candidate drugs that bind to these targets. The envelope proteins of HCV, E1 and E2, are critical for viral infectivity and have not yet been successfully exploited for HCV therapy. Both E1 and E2 are heavily glycosylated with 4\u20135 conserved type 2a) . The factype 2a) . GRFT inf 0.4 nM  comparin50's ranging from 0.3\u201314.1 nM while SVN inhibited in the range from 3.2\u201396 nM , J6 (genotype 2a) and TH (genotype 1b). GRFT and SVN inhibited all of the HCV pseudoparticles from infecting cells while having no effect on controls . This co.2\u201396 nM . The bro.2\u201396 nM ; S1 & S2in vitro selectivity index. Bioavailability studies  and genotype 2a (JFH-1) replicon cells were grown and treated as previously reported Human immunodeficiency virus-1 (HIV-1)-based pseudoparticles containing HCV E1E2 or vesicular stomatitis virus (VSV) glycoprotein G envelope proteins were prepared as described Whole cell lysates of Huh7.5 cells infected with HCV and uninfected were used: 20 \u00b5g protein per lane was used. Samples were resolved in duplicate on two 4\u201320% Bis-Tris pre-cast gradient SDS-PAGE gels  and blotted onto polyvinylidine difluoride membranes. Both membranes were blocked with 5% BSA overnight. Membrane 1 was used for detection of HCV E1 and E2 proteins while membrane 2 was used for GRFT and SVN detection. Membrane 1 was incubated overnight (4\u00b0C) with mouse anti-E1 HCV antigen monoclonal antibody , before being washed three times with PBST and further incubated with goat anti-mouse IgG (H+L), peroxidase conjugated secondary antibody  at a 1\u22361000 dilution for 1 hr. After washing with PBST, signals were detected on Hyblot CL autoradiography film  using the SignalFire ECL reagent system  according to the manufacturer's protocol. Membrane 1 was then stripped to remove all bound antibodies and blocked with 5% blocking buffer for 3 hrs before incubation with anti-gp70(E2) HCV monoclonal antibody  overnight (4\u00b0C). Washing, incubation with secondary antibody and detection were done as previously outlined. For detection of where GRFT binds, membrane 2 was incubated overnight (4\u00b0C) with 1 \u00b5g/ml GRFT before being washed three times and further incubated with rabbit anti-GRFT polyclonal antibody (1\u2236200 dilution) overnight (4\u00b0C). Membrane 2 was washed as before and incubated with goat anti-rabbit IgG (H+L), peroxidase conjugated secondary antibody  at a 1\u22361000 dilution for 1 hr. Washing and detection was done as before. For detection of SVN binding, membrane 2 was stripped and blocked as previously outlined and incubated with 1 \u00b5g/ml SVN overnight (4\u00b0C) before being washed three times and further incubated with rabbit anti-SVN polyclonal antibody (1\u22365000 dilution) overnight (4\u00b0C). Washing, incubation with secondary antibody and detection were done as previously outlined.100 \u00b5l of a 1 \u00b5g/ml solution of recombinant HCV E2  in sterile filtered PBS was added to each well of a protein-binding 96-well plate . ELISA was then performed with both GRFT and SVN as previously reported either in the presence of increasing concentrations of mannose (0\u2013100 mM) To test the effect of pretreatment with GRFT or SVN on E2 binding to the cell surface protein CD81, 100 ng/well of recombinant HCV 70 kDa E2 envelope protein  was bound to high-binding 96-well Greiner plates  at 4\u00b0C overnight. The wells were emptied and then blocked with 200 \u00b5l/well of 3% BSA  overnight before being washed three times with 200 \u00b5l/well PBST. Serial dilutions of GRFT or SVN were added (100 \u00b5l/well) to the test wells while 100 \u00b5l/well PBST was added to the control wells; incubation done at room temperature for 1 hr 30 min. Wells were washed as before and serial dilutions of human recombinant CD81 (25\u2013127) protein  were added to both test and control wells; incubation done for 1 hr 30 min. Wells were washed as before and 100 \u00b5l/well monoclonal mouse anti-CD81 (25\u2013127) antibody  was added and incubated for 1 hr30 min at room temperature, followed by three washes with PBST. Goat anti-mouse IgG (H+L), peroxidase conjugated secondary antibody  was added to each well (100 \u00b5l/well of a 1\u22361000 dilution) and further incubated at room temperature for 1 hr, followed by three washes with PBST. The TMB 2-component Microwell peroxidase substrate kit  was used for peroxidase detection: 100 \u00b5l/well added, incubation for 30 min (GRFT test plate) or 100 min (SVN test plate) before reaction was stopped with 100 \u00b5l/well 0.5M HCl. Absorbance read at 450 nm.via the central tail artery from all animals, allowed to clot and serum removed. Dosing began on Day 1 and continued daily up to and including Day 10. Blood samples were collected on Days 3, 11, 18 and 25. Serum samples were stored at \u221280\u00b0C prior to assay. GRFT levels in mouse serum were quantified by ELISA on gp120 coated plates as previously reported Ten albumin-urokinase plasminogen activator/severe combined immunodeficient (Alb-uPA/SCID) mice with low engraftment of human liver cells (human \u03b11-antitrypsin (hAAT) values less than 20 at 6 weeks) were used. Individual body weights were recorded prior to treatment and daily during the study. Animals were evaluated daily for morbidity and mortality. Study animals were allocated into 2 groups of 5 animals. GRFT or saline solutions were administered daily by subcutaneous injection. Dosing volume was 2.0 mL/kg for both groups. Blood was collected Twelve Alb-uPA/SCID mice with high engraftment of human liver cells  were allocated to this study. Animals were evaluated as before except that study animals were allocated into 2 groups of 6 animals.5 IU). Blood samples were collected on Days 3, 11, 18 and 25 and the sera separated. Serum samples were stored at \u221280\u00b0C until used for measurement of hAAT and HCV titers. The minimum detectable limit of the virus load assay is 200IU.Treatment began on Day 1 and continued daily up to and including Day 10. Animals were challenged with HCV 8 hours after the first drug dose on Day 1 by intra-peritoneal injection of 100 \u00b5l of a standard patient serum laden with genotype 1a HCV  received saline control, Group 2 animals (n\u200a=\u200a6) received INF-\u03b1 (1350 IU/g), Group 3 animals (n\u200a=\u200a6) received GRFT (20 mg/kg) and Group 4 animals (n\u200a=\u200a7) received a combination of GRFT and INF-\u03b1.5 IU) and then dosed with INF-\u03b1 (for the Group 2 INF-\u03b1 group and the Group 4 combination group). Dosing with GRFT, INF-\u03b1 or the combination of the two continued daily up to Day 16. Blood samples were collected on Days 3, 10, 17, 24 and 31. Serum samples were prepared and stored at \u221280\u00b0C until assayed for hAAT and HCV titers. The minimum detectable limit of the virus load assay is 200IU.Baseline blood draws were obtained on Day \u22124 and daily GRFT administered on Day \u22121 and Day 0. On Day 1 of the study, animals were treated with GRFT, challenged 4 hours later by the intra-peritoneal injection of 100 ul of the HCV genotype 1a inoculum ; lanes 2\u20139: whole cell lysates from HCV infected Huh7.5 cells (20 \u00b5g protein/lane); lanes 10: whole cell lysate from uninfected Huh7.5 cells (20 \u00b5g protein/lane). After overnight Incubation with GRFT (1 \u00b5g/ml), bound GRFT was detected with rabbit anti-GRFT polyclonal antibodies at a 1\u2236200 dilution.(TIFF)Click here for additional data file.Figure S2Western blot analysis of SVN interaction with HCV infected and uninfected Huh7.5 cells. Lane 1: molecular weight marker ; lanes 2\u20139: whole cell lysates from HCV infected Huh7.5 cells (20 \u00b5g protein/lane); lane 10: whole cell lysate from uninfected Huh7.5 cells (20 \u00b5g protein/lane):. After overnight Incubation with SVN (1 \u00b5g/ml), bound SVN was detected with rabbit anti-SVN polyclonal antibodies at a 1\u22365000 dilution.(TIFF)Click here for additional data file.Figure S3ELISA study on the effect of GRFT (A) or SVN (B) on HCV E2 binding to CD81. Plate-bound E2 protein at 100 ng/well, was pre-treated with (\u25aa) or without (\u2022) GRFT or SVN (100 ng/well) for 1 hr30 minutes, before serial dilutions of CD81 were added. Mouse anti-CD81 monoclonal antibodies were used to detect the bound CD81 as indicated by absorbance readings. Points are averages of triplicate samples .(TIFF)Click here for additional data file."}
+{"text": "Setaria italica) is an important food and fodder grain crop that is grown for human consumption. Production of this species is affected by several plant diseases, such as rust. The cultivar Shilixiang has been identified as resistant to the foxtail millet rust pathogen, Uromyces setariae-italicae. In order to identify signaling pathways and genes related to the plant\u2019s defense mechanisms against rust, the Shilixiang cultivar was used to construct a digital gene expression (DGE) library during the interaction of foxtail millet with U. setariae-italicae. In this study, we determined the most abundant differentially expressed signaling pathways of up-regulated genes in foxtail millet and identified significantly up-regulated genes. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to analyze the expression of nine selected genes, and the patterns observed agreed well with DGE analysis. Expression levels of the genes were also compared between a resistant cultivar Shilixiang and a susceptible cultivar Yugu-1, and the result indicated that expression level of Shilixiang is higher than that of Yugu-1. This study reveals the relatively comprehensive mechanisms of rust-responsive transcription in foxtail millet.Foxtail millet ( R) proteins that recognize and inhibit specific pathogen effectors, a process called effector-triggered immunity (ETI) [During the protracted course of plant-pathogen co-evolution, plants have evolved a series of complex mechanisms to protect themselves effectively against attack by pathogens . Plants ty (ETI) . Plants ty (ETI) . SAR canty (ETI) . The indty (ETI) .Uromyces setariae-italicae. This rust is one of the most important diseases of foxtail millet worldwide. The disease can be epidemic in the short term, and in severe cases can cause losses of ~10\u201330% of yields. Based on our screening appraisal, the foxtail millet cultivar Shilixiang has the best rust resistance among 16,800 millet germplasm resources collected from eight countries. Disease resistance is coherent in the seedling and adult stages, making it an ideal subject for research to reveal the mechanism of resistance to rust [U. setariae-italicae have been limited mainly to basic biology [Studies about differential gene expression during interactions with rust fungi have been reported in a number of cereals, including wheat  and pear biology ; the patU. setariae-italicae infection, using the DGE system on a whole-genome scale. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was also used to validate the expression patterns of some important genes.Compared to expression microarray technology, digital gene expression (DGE) profiling offers many advantages, including an unbiased view of the transcriptome, greater precision, simpler preprocessing, and consistent results compared to qPCR . Thus, iU. setariae-italicae  and incubated for 48 h at 28\u00b0C with 95% relative humidity, and then maintained in a growth chamber at 28\u00b0C under a photoperiod of 14h light and 10 h dark with a light intensity of 6000 lux. Leaf samples were collected at 0, 24, and 48 h after inoculation. One leaf was collected per pot, so that, a total of ten leaves were collected at each inoculation time. The collection was repeated three times at each time point. Samples were immediately quick-frozen in liquid nitrogen and then stored at -80\u00b0C until total RNA was isolated. These leaf samples were used as starting material for the DGE and qRT-PCR analysis.The foxtail millet cultivar Shilixiang and Yugu-1, respectively, have been identified as resistant and susceptible to attack by italicae . The ShiTotal RNA was isolated from the leaf samples taken at 0, 24, and 48 h post-inoculation. RNA was extracted using an RNAprep Pure Plant Kit  according to the manufacturer\u2019s instructions. The quality and concentration of total RNA was checked using a Nanodrop ND 1000 spectrophotometer , and 6 \u03bcg of total RNA from the Shilixiang cultivar was used for Illumina sequencing.et al, tag libraries were constructed for the three different inoculation time samples  [Samples for establishing DGE libraries were prepared using an Illumina Gene Expression Sample Prep Kit. As described by Tang nd 48 h) . After 1Raw image data obtained by sequencing was transformed by base calling into sequence data, which is called raw data or raw reads. Raw sequences have 3' adaptor fragments, some low-quality sequences, and several types of impurities. Raw sequences were transformed into Clean Tags by removing 3' adaptor sequences, empty reads (reads with only 3' adaptor sequences but no tags), low quality tags (those with unknown sequences 'N'), tags that were too long or too short, and those with a copy number of one (probably due to sequencing error). Clean tags were 21 nt long.http://www.phytozome.net) was used as a reference to align the DEGs. According to the ascending order of physical position, DEGs were located individually on the foxtail millet chromosomes. Homology searches of DEGs were performed using the BLAST program against the non-redundant (nr) protein database in GenBank. GO enrichment analysis was conducted to look for significantly enriched GO terms in DEGs. Pathway enrichment analysis was used to identify significantly enriched metabolic pathways or signal transduction pathways in DEGs, compared to the whole genome background.For gene expression annotation, we used a virtual library containing all the possible CATG+17 base length sequences of the reference gene sequences. All clean tags were mapped to the reference sequences, and only 1 bp mismatch was considered. Clean tags mapped to reference sequences from multiple genes were filtered, and any remaining clean tags were designated as unambiguous clean tags. The number of unambiguous clean tags for each gene was calculated and then normalized to TPM (number of transcripts per million clean tags). A rigorous algorithm was used to identify differentially expressed genes (DEGs) between every pair of samples. Foxtail millet genome sequence available at Phytozome . The products of first strand cDNA synthesis were ready to be used directly in qPCR. PCR primers were designed using Primer Premier 5.0 software and synthesized by Sangon Biotech Co., Ltd. . The 18S rRNA gene  was used as an internal control to normalize the amount of gene-specific qRT-PCR products; primers used in the reactions are listed in T method .U. setariae-italicae could facilitate the identification of signaling pathways and genes related to millet defense against rust.Solexa/Illumina DGE analysis was performed to obtain a global view of the foxtail millet transcriptome after plants were inoculated with rust. Three Shilixiang millet DGE libraries were sequenced: at 0 h after inoculation with rust (SRX659703), at 24 h after inoculation (SRX659704), and at 48 h after inoculation (SRX659705)\u2014this generated approximately three million raw tags in each library. After removing the low quality tags, the total number of clean tags for each of the three libraries ranged from 3.2 to 3.3 million and the number of tag entities with unique nucleotide sequences ranged from 80,668 to 105,050 . A foxtaTo specifically identify genes related to rust inoculation in foxtail millet, the DEGs between two samples were identified. Comparison of DEGs in Shilixiang at 24 h after inoculation with rust versus Shilixiang inoculated with rust 0 h showed that 4542 genes differed significantly  between the two samples. Among them, 3442 genes were up-regulated and 1100 genes were down-regulated. Between Shilixiang at 48 h after inoculation with rust and Shilixiang inoculated with rust 0 h, a total of 5112 DEGs were detected, including both up-regulated (3941) and down-regulated genes (1171). We identified 968 genes that were expressed differently at 48 h post-inoculation compared to after only 24 h. Of those, 518 were up-regulated and 450 were down-regulated. A Venn diagram illustrating all of the DEGs is shown in Gene ontology (GO) analysis of differentially expressed genes provided comprehensive evaluation of transcriptional variation in molecular functions, biological processes and cellular components. The numbers and assortment of the allocated GO categories provide a good indication of the diversity of the genes. A total of 3415, 3828 and 723 DEGs were categorized into the three main GO categories of three comparison groups . And GO Pathway enrichment analysis was used to identify significantly enriched metabolic pathways or signal transduction pathways in DEGs by comparing them with the whole genome background. Among all the genes with KEGG pathway annotation, 1121 differentially expressed genes were identified in the libraries between 0 and 24 h, 1258 differentially expressed genes between 0 and 48 h, and 222 differentially expressed genes between 24 and 48 h after inoculation. Among the top-10 most abundant differentially expressed signaling pathways of up-regulated millet genes, from every comparison group, the cluster for Ribosome represented the largest group . In ordeThrough pathway enrichment analysis in DGE, defense-related genes were discovered in foxtail millet. Some of the significantly up-regulated genes were described . They inU. setariae-italicae. Peroxidase is an oxidative enzyme and it may be involved in defense reactions of foxtail millet against the rust pathogen at later stages of attack. Thus it might be possible that cooperation of PR proteins improves the ability of foxtail millet to fight against U. setariae-italicae infection. Expression of PR genes indicates the activation of SAR, and PR1 proteins have been used as markers for SAR [PR proteins have been reported to be induced by pathogen infection, and they are known to have antifungal activity and enzymatic or inhibitory activity that improves the defensive abilities of plants , 25. In  for SAR . Hence, Rboh was phosphorylated by CDPK, which was activated by elicitor-induced calcium influx. Co-infiltration of CDPK with Rboh led to the rapid production of ROS, which was accompanied by HR and resulted in resistance to the fungus , calcium-dependent protein kinases (CDPKs), and mitogen-activated protein kinases (MAPKs)\u2014that were up-regulated in the millet-rust plant-pathogen interactions. CDPK has been reported to trigger the production of ROS in response to pathogen infection in potato and tobacco , 28. In e fungus . Cai et n attack . In our duced HR , 31. WRKitalicae .SGTlb (one of two highly homologous Arabidopsis SGTl genes) disabled early plant defenses that are conferred by multiple R genes [Disease resistance (R) genes in plants encode R proteins, which act as primary receptors of pathogen effector proteins or play indirect roles in this process . In foxt R genes . Therefo R genes . UltimatPhaeoisariopsis personata, PAL transcript levels increase in peanut plants [U. setariae-italicae  catalyzes the first committed step of the core pathway, is a sign of plant responses to incompatible pathogens . PAL is t plants . Cytochrt plants . In thisitalicae .To validate the results of the DGE, nine genes were selected for confirmation using qRT-PCR . They inAdditionally, in order to determine whether the genes contribute to resistance to millet rust, expression levels of the genes were compared between the resistant cultivar Shilixiang and the susceptible cultivar Yugu-1. Results indicated that the genes can be induced in both cultivars, but with differences in their levels of gene expression: the expression level was higher in the resistant cultivar .Induced expression of WRKY70, PAL and PER was continuous in both cultivars, but the expression level was higher in the resistant cultivar. Induced expression of SGT and MKK1/2 were continuous in Shilixiang, but in Yugu-1 they were down-regulated or up-regulated slightly. RPM1/RPS2, HSP90, GST, GLU were induced in Shilixiang and gene expression level reached peak 24 h post-inoculation, while the expression of these genes showed almost no change or fluctuation within a narrow range in Yugu-1.S1 Fig(DOC)Click here for additional data file.S2 Fig(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file."}
+{"text": "The MObile Technology for Improved Family Planning (MOTIF) trial assessed a mobile phone-based intervention comprising voice messages and counsellor support to increase post-abortion contraception at four Marie Stopes International clinics in Cambodia. The aim of this process evaluation was to assess women\u2019s views and experiences of receiving the MOTIF intervention, gain insights into the mechanism of action of the intervention and seek recommendations for improvements.We conducted a qualitative study comprising15 semi-structured interviews with women who had received the intervention and undertook a simple thematic analysis.We identified themes relating to communication via mobile phone, supporting contraception use, broader post-abortion care, interaction with family and friends and suggestions for improvement. The majority of women were positive about the mobile phone-based intervention to support contraception use and reported it to be a convenient way to ask questions or get advice without going to a health centre, although a few women found the voice messages intrusive.The intervention supported contraception use by provision of information, encouragement, reminders to return to clinic, reassurance and advice for problems and had a positive effect on contraceptive uptake and continuation. Women reported a sense of being cared for and received support for additional physical and emotional issues. Most women thought that the duration of the intervention and frequency of messages were acceptable.The majority of women were positive about the mobile phone-based intervention which provided support for contraception use as well as additional physical and emotional issues. The study provides some insights into how the intervention might have worked and considers how the intervention could be improved. Our MObile Technology for Improved Family Planning (MOTIF) trial assessed a mobile phone-based intervention comprising six interactive voice messages +/\u2212 counsellor support to increase contraception use amongst women seeking abortion services at four Marie Stopes International clinics in Cambodia. The aim of this study was to explore women\u2019s views and experiences of receiving the MOTIF intervention, gain further insights into how the intervention worked, and seek recommendations for improvements.We conducted 15 interviews with women who had received the intervention. We recorded and analysed the interview transcripts to identify the main themes. We found that the majority of women were positive about the mobile phone-based intervention to support contraception use and reported it to be a convenient way to ask questions or get advice in confidence without going to a health centre, although a few women found the voice messages intrusive. Women reported that the intervention, in particular phone counselling, supported contraception use by provision of information, encouragement, reminders to return to clinic, reassurance and advice for problems. Women reported a sense of being cared for and received support for additional physical and emotional issues. Most women thought that the duration of the intervention and frequency of messages were acceptable.In conclusion, the majority of women were positive about the mobile phone-based intervention which provided support for contraception use as well as additional physical and emotional issues. The study provides some insights into how the interventions might have works and considers how the intervention could be improved.In recent years several randomised controlled trials have assessed interventions delivered by mobile phone  to improve contraception use . InterveThe study reports women\u2019s views and experiences of receiving voice messages and counsellor support for post-abortion contraception in the MOTIF trial. The MOTIF trial protocol, results and description of the intervention development are reported elsewhere , 10, 11.Amongst 249 women that received the intervention, around half actively requested to speak to a counsellor (pressed \u20181\u2019) and over 90% spoke to a counsellor at some stage. Women who spoke to the counsellor having requested to (by pressing \u20181\u2019) were more likely to be using effective contraception at 4\u00a0months compared to women who didn\u2019t request or speak to the counsellor . Increased parity, a history of >2 previous induced abortions, lower socio-economic status, and medical abortion were associated with requesting to speak to a counsellor . This stThis qualitative study involved 15 semi-structured interviews with women within a few weeks of receiving the intervention. Participants for interview were selected purposively by the counsellor (author LS) delivering the intervention to include women from urban and rural areas and those who did or did not appear to respond to the intervention; both users and non-users of contraception. The counsellor telephoned participants to ask if they were willing to participate in the interview study.The topic guide was developed to explore women\u2019s experience of the intervention, aiming to identify active components of the intervention, and seek recommendations for improvements Table . QuestioInterviews were conducted between 30 October and 23 November 2013. Author CS conducted six interviews  with author UV interpreting, author RW conducted three interviews (at the clinic) with UV interpreting, and UV conducted six interviews . Participants were provided with an information sheet to read, or it was read to them, and provided signed or thumb-printed consent, or recorded verbal consent for the phone interviews. Interviews were recorded and transcribed by Cambodian research assistants  to English. NVivo 11 software was used to store and code all transcripts , spend money to clinic directly, and we discuss with her and if we don\u2019t discuss with her and we have to come to PET, we spend money first for travel fee and second for PET fee\u201d (interview 6)Several women reported that receiving messages or speaking to the counsellor was a convenient way to obtain support for health issues or remind them about contraception and saved the time and costs of clinic attendance.\u201cThe message just tell me to click one or two for answer\u2026 at that time I still work but if I\u2019m busy, it\u2019s ok but sometimes when I\u2019m very busy and like stress and sometimes it\u2019s annoying\u201d (interview 2)Conversely, a few women reported missing calls if they were away from their phone. Two women reported that messages could be intrusive if received at an inconvenient time e.g. when busy at work.\u201cShe explained even about drug, IUD/implant, injection, oral pill, and IUD, and condom. She explained all. She told me a lot\u201d (interview 5)\u201cThe message ended and a short while, she would call me back\u2026 I asked her that, for example, we forgot to take the pill\u2026 What should I do if [I] forgot taking till 3 days?\u201d (interview 11)Many women reported receiving information and increasing their knowledge on a range of contraceptive methods. Several women reported that the counsellor would ask if they were using a method, and the phone call provided an opportunity for women to ask questions about contraception.\u201cAfter I got information from counsellors I then went to clinic to insert IUD\u2026I learnt how to insert IUD and taking pills. Inserting of IUD is much more easier. Talking pill has to be on time and take it daily but for IUD we do not have to do like this. We can have sex whenever! Counselling service made me feel confident because I thought that medical science is better than our thought. Some people said that IUD can move around, but when I came to ask counsellor, she said that IUD did not have legs to move around\u2026. I also told people I know to insert IUD as well\u201d (interview 10)\u201cShe explained me to have contraception here, use implant at this clinic, there was discount\u201d (interview 6)Some women reported that they decided to adopt a long-acting method of contraception after speaking to the counsellor. A few women described being given information on discounts or where to access services.\u201cShe said that the side effects of IUD lasted 3\u00a0months, I remember that. And after these 3\u00a0months, our body can tolerate with it, it will be alright\u2026and now I am alright\u201d (interview 3)Several women reported that the counsellor provided advice if they were experiencing side-effects from contraception; either advising the women to attend the clinic for examination or providing reassurance which led to the women continuing to use the method.\u201cI was confident because she said that when we took the pill, we had to take regularly. If we didn\u2019t take the pill regularly, it was possible to be pregnant\u2026 so I changed my habit according to her\u201d (interview 1)Three women received a specific pill reminder message at one-month, but only one woman recalled receiving this. However, several women using oral contraceptive reported that the counsellor had emphasized the importance of taking the pill regularly and the risk of consequent pregnancy, resulting in a change in behaviour.\u201cFor that voice message that I received, I think that it\u2019s good. It\u2019s good and like makes me feel warm like there\u2019s someone take care about us\u201d (interview 8)\u201cWhen I received that voice message, I felt that\u2026 she encouraged me and she loved our health so that she wanted to know about our health if we were healthy\u201d (interview 12)An important aspect of the intervention was the sense of care and emotional support provided as well as support for specific problems. Many women reported feeling happy because somebody was taking care of them, or asked about their health or more generally about what they were doing. Several women reported that the counsellor provided encouragement and that they gained confidence and felt less afraid.\u201cStruggle in my life, because sometimes I want to commit suicide, but counsellor not allow me to do this, they encourage me to be strong\u201d (interview 15)One woman reported receiving support for suicidal feelings.\u201cAfter I used medicine I asked her my problem \u201cwhy around my abdominal still painful? Why I cannot do something? Even just walking, I cannot walk\u201d. Counselor said that bleeding it just side effect of medicine, but if you has much bleeding you need to go back to clinic. I followed up myself and I found that there was just small bleeding, so I decided not coming to clinic\u201d (interview 9)The counsellor provided support for post-abortion health concerns such as abdominal pain, vaginal bleeding or discharge. In some cases the counsellor would provide reassurance or otherwise suggest the women attend the clinic for a check up.\u201cIf I make comparison, I see a lot of changing. Before I seldom to hear information, by the time I have started to use services here, I can consult at anytime and I know more about illness problem that often happen to women and its prevention\u201d (interview 9)A few women reported gaining general information about illnesses and health promotion.\u201cThe reason why I believe her because she encouraged me that she wouldn\u2019t tell other about my secret and what she talked with me she kept in secret\u2026I believe that they can help a lot of other women, sister, because woman is shy, and sometimes she can\u2019t talk to other\u2026 it just sometimes we felt embarrassed. There\u2019re some matters that we don\u2019t want other to know\u201d (interview 12)In most cases women listened to the voice messages on their own. Some women reported that they were able to discuss topics in confidence that they couldn\u2019t discuss elsewhere or with others, for example, at a hospital or with family members.\u201cSometimes there was my mother; I didn\u2019t want to talk because I was shy and I didn\u2019t want her to know. But if I stayed with my family, my couple, there was no problem. But sometimes\u2026he didn\u2019t stay with me every time, but we didn\u2019t want like questioning words like I was sick like this or like that I didn\u2019t want to talk in front of my mother, and my younger siblings. But she called me when I was alone or with my family, I would talk and there was no problem\u201d (interview 8)A few women reported that they would hang up, or arrange to speak to the counsellor at a different time if messages were received or the counsellor called in the presence of others.\u201cNo one listens to my voice messages because my phone stays with me all the time. Even my husband, he also does not hack and listen alone. When new message coming, my husband told me\u2026 Moreover my husband always asks me questions when I finished listen to messages because he concerns much about my health condition\u201d (interview 9)If few women reported that family members or friends might ask questions after listening to a voice message in their presence, but husbands\u2019 were generally supportive.\u201cI never let anyone listen to it but I brought my younger sister who wanted to use IUD like me. I always told her that I felt well\u201d (interview 3)In other cases, women reported deliberately sharing intervention content. One woman reported using the speakerphone so that others could listen to the message. There were a few instances where women reported having subsequent conversations with friends or family members to recommend Marie Stopes services or contraception methods.There were a few instances where someone else listened to the message or spoke to the counsellor because the phone had been shared with another family member but there were no reported instances of harm as a consequence.\u201cI do not have any request because it is good enough already. I rarely to see services like this in others organization\u201d (interview 9)All of those interviewed thought that the service should be offered to women in the future. One participant suggested focusing on less educated women.\u201cI want direct phone call\u2026 I am not interested in voice message at all because it takes long time, but if you call me, I once pick up the phone call\u201d (interview 1)\u201cText message. Sometimes when I go bathroom when comeback, I can see message rather than missed call.\u201d (interview 15)Most women were positive about voice messages, although a few women expressed a preference for direct phone calls or text message. Although most women couldn\u2019t remember how frequently they received messages, most reported that a frequency of two times a month was enough.\u201cnot really necessary for me because I do not have any problem now\u201d (interview 14). However, a few women reported that they would have liked the voice messages to continue beyond 3\u00a0months in case they experienced problems in the future.\u201cThere was a message said that this message was the last message. To me, when the message said that it was the last message, I felt regret and I didn\u2019t want\u201d (interview 3)\u201cSometimes I don\u2019t know in the future I will meet what problem, sister, so I am difficult to call her. When she sends voice message to me, she has my number and that number is easy for me to call to ask her too\u201d (interview 12)Most women thought that the intervention duration of three-months was sufficient and that the messages were no longer required e.g. \u201cWe go to small pharmacies, we have to spend the money too; therefore, I don\u2019t mind about paying this amount. This is for our health too\u201d (interview 3)\u201cI am interested in general health short voice messages if we send you weekly? For example, messages tell you how to prevent from diseases, how prevent from pregnancy, STI and so on\u2026I want these services still providing for long period\u201d (interview 14)Most women reported that they would be happy to pay a small fee (e.g. $1-2USD) for such a service, but a few thought it should remain free of charge. A few women suggested that the messages support other health topics.The majority of women were positive about the mobile phone-based intervention to support contraception use and reported it to be convenient in a number of ways.Most women liked being able to respond to the voice message to request to speak to a counsellor or not. However a few women found the voice messages intrusive. Phone counselling was a convenient way to ask questions or get advice without going to a health centre. Women reported that the intervention, in particular phone counselling, supported contraception use by provision of information, encouragement, reminders to return to clinic, reassurance and advice for problems and had a positive effect on contraceptive uptake and continuation. Women reported a sense of being cared for and received support for additional physical and emotional issues. Counselling allowed women to discuss issues with the counsellor in confidence, although in some cases the intervention content was shared with others, either deliberately or unintentionally. Most women thought that the duration of the intervention and frequency of messages were acceptable.This study provides insights to the intervention gained by in-depth interviews with women. A strength of our methodology is that most of the interviews were conducted by female researchers, which is considered more appropriate for reproductive health research in order to minimise \u2018social distance\u2019 between researchers and subjects .Our study also has some limitations. It is possible that women would have been more likely to agree to be interviewed if they had had a positive experience of the intervention. We did not document if any women refused to participate. Most of the women interviewed were using a contraceptive method and hence we were unable to assess differences in accounts between contraception users and nonusers. As in the trial, most of the women interviewed were married, and single women and entertainment workers were under-represented . The intAnother potential limitation is that it wasn\u2019t always clear if the woman was referring to voice messages or counselling when analysing the interviews. Hence it was not always possible to attribute women\u2019s reports to the automated or counsellor-delivered components of the intervention. As a consequence of these potential biases, it is not possible to conclude that all relevant themes were identified.To our knowledge, this is the first in-depth interview study reporting participants\u2019 perceptions of a mobile phone-based intervention to support contraception use alongside a randomised controlled trial, although participants were asked questions regarding their satisfaction in two RCTs of oral contraceptive adherence interventions in the USA , 3. Our Communication via mobile phone was a convenient way to discuss contraception or health issues, saving money and time in comparison to going to a health centre, as previously reported . Our preOur finding that messages could be inconvenient and intrusive for some women highlights the limitation of real-time voice messages as a delivery mechanism, contrasting with other studies of interventions delivered by mobile phone where participants could check messages at their convenience , 20.It is unclear if the voice messages improved contraceptive use. Participants in other trials reported that daily educational text messages helped them remember to take oral contraceptive or HIV medication , 3, 16. Interview findings suggest that the intervention included components identified as best practices for contraception counselling including developing close personal relationships, building trust, and adequate counselling regarding side-effects , which mThe intervention provided additional benefits that were broader than the trial definition of success (i.e. contraception use). Our findings resonate with other qualitative studies of mHealth interventions that reported a feeling that someone cares , 19. TheOur finding that women received support for management of physical and emotional health issues is consistent with studies elsewhere; post-abortion mobile phone follow up was demonstrated to reduce women\u2019s anxiety and stress in South Africa , and be Although there were no reports of any adverse advents as a result of others listening to messages, our interview participants were mostly married and more likely to have disclosed having had an abortion to others. Women concerned about others listening to messages may have elected not to receive the intervention. Phone sharing is common in Cambodia and possible unintended consequences should be considered when developing future intervention content .Other evaluations of text message interventions for contraception and sexual health have found that participants would re-read messages which might lead to conversations with family or friends , 28. AltThis study provides some insight into the mechanism by which the intervention resulted in behaviour change. The voice messages appeared to act as a conduit for additional support rather than directly influencing behaviour change, but may have promoted engagement in the intervention, as previously reported .intrapersonal determinants of contraceptive use, in particular health concerns, as well as capability, motivation and opportunity to use contraception, as per our conceptual framework [The majority of support for contraception uptake and continuation and other post-abortion issues appeared to be provided by the counsellor calls rather than the voice message. The counselling addressed ramework \u201332. The ramework .provide instruction, and prompt practice) and three to the counselling  but the long-term effects require further evaluation. Future interventions for post-abortion contraception could consider including messages to support comprehensive post-abortion care more broadly and should anticipate that women may have a range of issues and be prepared to manage these safely. The intervention could be adapted for use on smart phones and could utilise password protected voice or text messages that are retained on the participants phone. This might improve the response to messages and facilitate sharing of intervention content with others. We recommend that mobile phone-based interventions for on-going support for PAFP should be integrated with counselling at the time of seeking services. Operational research could examine clients\u2019 actual willingness to pay for such interventions, and the effect of varying the duration of the intervention. Further adequately powered trials of mobile phone-based interventions to support contraception use are needed.The majority of women were positive about the mobile phone-based intervention which provided support for contraception use as well as additional physical and emotional issues. The study provides some insights into the possible mechanism of action and considers how the intervention could be improved."}
+{"text": "The products have been characterized by powder X-ray diffraction (XRD), optical microscopy, and scanning electronic microscopy (SEM). The results show the as-grown product is pure tubular aragonite crystalline whiskers with a diameter of 5\u201310 \u03bcm and a length of 100\u2013200 \u03bcm, respectively. The concentration of Mg2+ plays an important role in determining the quality and purity of the products. Furthermore, the method can be extended to fabricate CaSO4 fibers. The high quality of the product and the mild conditions used mean that the present route has good prospects for the growth of inorganic crystalline whiskers.In this paper, we have developed a facile MgCl The influencing factors such as concentration, time, and Mg2+ additive have been investigated. Microscopy images show that the length of whisker is up to several hundred micrometers. Moreover, the method can be extended to prepare CaSO4 fibers. The high quality of whiskers, environmental benignity and low-temperature used mean the present method is promising in industrial applications.Herein, we have developed a convenient hydrothermal route with the assistance of magnesium chloride . No calcite phase can be detected in the pattern, indicating that pure aragonite CaCO3 has been produced in the present process. The optical image in The phase and morphology of the products have been determined by XRD and optical microscopy and SEM. 3 is essential to achieve pure aragonite crystals. In our system, the presence of appropriate magnesium ions inhibit the nucleation of calcite and thereby reducing carbonate activity below that required for calcite nucleation [It is known that high precipitation rate facilitates the formation of calcite phase because it hass greater entropy than aragonite . Adjusticleation ,19. The 2+ ions, in which metastable aragonite is precipitated first [2+ played an important role in producing pure aragonite fibers in the carbonation process. Therefore, the purity of the aragonite can be controlled by changing the Mg2+ concentration. If excess Mg2+ is introduced, according to reaction (1) and (3), CO2 releases from reaction 1 will be fast and nucleation favors the formation of calcite. When little Mg2+ is used, the ability to control the nucleation rate of CaCO3 is weak and thus results in the occurrence of calcite. Here, we investigated the influence of the ratio of Ca2+/Mg2+ on the formation of aragonite tubular whiskers. It is found that the proper ratio of Ca2+/Mg2+ is from 63 to 8. For example, if Ca2+ concentration is kept at 0.36 mol\u2219L\u22121, MgCl2 can be introduced in a range of 0.1~0.2 mol\u2219L\u22121 and the resulting products are mainly composed of aragonite whiskers  also leads to the increase of calcite in the products 2CO, and MgCl2 are varied in an appropriate ratio, 2CO and 0.07~0.61 mol\u2219L\u22121 for MgCl2, respectively), the shape of the obtained aragonite crystals is well retained. It is notable that the yields of the whiskers are reduced as the feedstock concentration decreases. Moreover, high concentrations of feedstocks produce aragonite whiskers in high yields 2SO4, and MgCl2 as starting materials. The SEM image and XRD pattern , urea (NH2)2CO, 10.9 g) and magnesium chloride  were dissolved in 42 mL of distilled water to form a homogenous solution. Then the solution was transferred to a Teflon-lined autoclave with the capacity of 50 mL and maintained at 155 \u00b0C for 6\u20138 h. After the reaction was complete, CaCO3 whiskers filled the autoclave. The whiskers were washed with distilled water several times and collected for characterization.In a standard procedure, anhydrous calcium chloride (CaClK\u03b1 radiation (\u03bb = 0.1541874 nm). The morphology observation was performed on a polarizing microscope  and Hitachi S-4800 field-emission scanning electron microscope .The X-ray powder diffraction (XRD) pattern was carried out on a Philips X\u2019Pert PRO SUPER X-ray diffractometer equipped with graphite monochromatized Cu 2 has been developed to grow well-defined tubular aragonite whiskers on a large scale. Mg2+ plays an important role in determining the purity and quality of the product. Meanwhile, the present route can be extended to prepare CaSO4 fibers, indicating that the method may be generalized to grow functional oxide materials. Of course, further studies are still necessary to provide insights into unraveling the crystal growth mechanism.In summary, a facile hydrothermal route with the assistance of MgCl"}
+{"text": "There are over 6 billion vaccine doses administered each year, most containing aluminium-based adjuvants, yet we still do not have a complete understanding of their mechanisms of action. Recent evidence has identified host DNA and downstream sensing as playing a significant role in aluminium adjuvant  activity. However, the cellular source of this DNA, how it is sensed by the immune system and the consequences of this for vaccination remains unclear. Here we show that the very early injection site reaction is characterised by inflammatory chemokine production and neutrophil recruitment. Intravital imaging demonstrates that the Alum injection site is a focus of neutrophil swarms and extracellular DNA strands. These strands were confirmed as neutrophil extracellular traps due to their sensitivity to DNAse and absence in mice deficient in peptidylarginine deiminase 4. Further studies in PAD4\u2212/\u2212 mice confirmed a significant role for neutrophil extracellular trap formation in the adjuvant activity of Alum. By revealing neutrophils recruited to the site of Alum injection as a source of the DNA that is detected by the immune system this study provides the missing link between Alum injection and the activation of DNA sensors that enhance adjuvant activity, elucidating a key mechanism of action for this important vaccine component. Researchers have identified the mechanism of action in which aluminium hydroxide  boosts the efficacy of co-administered vaccines. In this study, James Brewer and colleagues from the University of Glasgow, UK, studied the effects of alum co-administration in a mouse model, finding that the compound induces immune cells, called neutrophils, to swarm around the site of immunization and produce neutrophil extracellular traps (NETs). NETs, webs of DNA and cell contents formed under certain conditions of neutrophil cell death, immobilize pathogens and promote their elimination. This study builds on previous research and demonstrates that alum artificially stimulates this pathway to boost the adaptive immune response to vaccine antigens, increasing their immunogenicity. The authors also suggest further study into neutrophil components as potential therapeutic agents. Alum is known to induce a Th2 immune response, characterised by the production of interleukin (IL)-4 and murine IgG1 antibodies.2 However, despite several theories being proposed, the mechanism of action of Alum remains unclear. Glenny originally suggested that Alum functioned through the formation of a depot at the site of immunisation, resulting in the slow release of antigen and/or sustained tissue inflammation.3 However, our recent work clearly demonstrates that removal of the Alum injection site, as soon as 2\u2009h after immunisation, had no impact on the resulting adaptive immune response.4 While the ability of Alum to trigger innate immune responses via the NLRP3 inflammasome and the subsequent production of proinflammatory cytokines has been highlighted,7 the role of the inflammasome in mediating Alum function is controversial, with others showing that key components of the inflammasome, such as Nlrp3 and caspase 1, are dispensable for adjuvant activity.9 More recently, interest has grown in the role of endogenous danger signals, such as host DNA, in Alum adjuvant function. It has been demonstrated that host DNA is accessible to enzyme (DNase) degradation following Alum immunisation and plays a role in driving antigen-specific T-cell response and B-cell response via DNA sensors such as IRF3.10 Similarly, sensing of host DNA by STING1 was shown to drive enhanced antigen presentation via Dendritic Cells \ufeff(DCs) and prolonged T cell\u2013DC interactions following Alum immunisation.11 Overall, these studies suggest that release of host DNA plays a pivotal role in the adjuvant function of Alum. However, the cellular source of this host DNA and the mechanism of DNA release remain unclear. Here we analyse the very early responses to Alum at the injection site and demonstrate that neutrophils are the principal cell recruited within 2\u2009h of injection. Intravital imaging revealed neutrophil swarming and cell death focussed around Alum, and the presence of DNA strands within the tissue. These strands were subsequently confirmed as neutrophil extracellular traps (NETs) via their sensitivity to DNase treatment. NETs were also absent in PAD4-deficient mice, which also displayed markedly reduced immune responses following Alum injection. These studies demonstrate that the mechanism of neutrophil death plays an important role in the adjuvant activity of Alum, and explain how the cellular reaction at the injection site drives the liberation of host DNA, which subsequently impacts on adjuvant activity.Following its discovery in 1926,4 Clearly, for any inflammatory response to play a role in adjuvant activity, it would have to occur within that narrow time frame. Analysis of Ly6G+CD11b+ neutrophils at the site of immunisation 2\u2009h after ovalbumin (OVA)/Alum injection demonstrated an increased frequency  analysis 2\u2009h after OVA/Alum injection of PAD4ko mice failed to identify DNA strands  lower OVA-specific, OTII T cells as a proportion of the T-cell population  compared with WT control mice  and B cell (MD4) responses following adoptive transfer to PAD4ko and wild-type (WT) mice. At the peak of the antigen-specific T-cell response, 5 days after immunisation,ion Fig.\u00a0. Analysiion Fig.\u00a0, howeverice Fig.\u00a0. Subsequged Fig.\u00a0, f. OVA-ged Fig.\u00a0, h. CollOur studies demonstrate that Alum induces a rapid recruitment, swarming and PAD4-dependent netosis following injection and furthermore that this response is responsible for a significant portion of the adjuvant effect induced by Alum.4 Recruitment of neutrophils to the site of Alum injection was particularly rapid compared with LPS, observable within minutes of injection and reaching a peak within 2\u2009h, consistent with previous reports in models of sterile inflammation.16 Following injection of antigen alone we observed a small increase in neutrophil recruitment in tissue by both FACS and intravital MPLSM; however, the presence of NETs was associated with a significant increase in neutrophil recruitment, suggesting NETs may act to amplify neutrophil recruitment by Alum, as previously observed in models of sterile tissue injury.16These findings are consistent with our previous studies demonstrating that key events in Alum adjuvant function occurred within the first 2\u2009h following immunisation.17 our data and those of previous studies19 support a role for NETs in shaping the adaptive immune response. NETs have been described in response to a number of infectious20 and sterile challenges22in vivo; however, this is the first description of Alum inducing extracellular trap formation by neutrophils. Previous studies reported that Alum was able to induce fibrin extracellular traps at the site of immunisation,23 which were composed of fibrin, extracellular chromatin, citrullinated histones and were dependent on fibrinogen for formation. In contrast to our findings, Munks et al. suggested that these extracellular structures were independent of neutrophils and were not required for the adjuvant activity of Alum.23 This discrepancy with our current findings likely arises from the characterisation and location of these structures. Munks et al. focussed on the fibrin component, showing that neutrophil-depleted mice formed fibrin nodules without assessing the nucleic acid composition. Furthermore the intraperitoneal route of immunisation exposes a number of specialised resident cell populations that are not found in subcutaneous tissue,25 in particular the large peritoneal macrophages that disappear rapidly after inflammatory challenge.24 In the skin, neutrophils are the principal cell population recruited to the skin in response to Alum, and we now show for the first time these cells swarm towards Alum injection sites. PAD4 expression is broadly restricted to neutrophils and peritoneal macrophages within the murine immune system,26 and the absence of NETS observed in PAD4-deficient mice indicates that neutrophils are the most likely source of NETS forming in the skin in response to Alum.Although first described as having an important role in innate immunity against bacteria,10 However, the cellular source and sensing of this DNA remained unclear. We now show that the very early inflammatory response to Alum drives neutrophil recruitment and generates free DNA via PAD4-dependent netosis. This response enhances antigen-specific T expansion and B-cell differentiation leading to an increased, class-switched antibody response. In the context of vaccination, these studies demonstrate that the later inflammation evident at the Alum injection site is superfluous for adjuvant activity and furthermore suggest that components released during the neutrophil netosis response may have utility as vaccine adjuvants.A role for DNA released at the Alum injection site in adjuvant function has previously been demonstrated as treatment with DNAse following Alum immunisation reduced antigen-specific CD4 T-cell activation and IgG and IgE antibody responses.hi cells.12 PAD4-floxed mice were a kind gift from Dr Kerri Mowen.13 To generate PAD4-KO mice and control littermate mice, PAD4-floxed mice were crossed with the ella cre deleter strain (B6.FVB-Tgn(Ella-Cre)C5379Lmgd . Male and female PAD4-KO mice and control littermate mice were used at 7\u201316 weeks of age. OT-II TCR Tg mice27 and MD4 BcR Tg mice28 on C57BL/6 backgrounds were used as a source of OVA-specific T cells and HEL-specific B cells, respectively. All mice were either injected subcutaneously (s/c) in the ear pinna with 10\u2009\u03bcl or in the scruff with 100\u2009\u03bcl of antigen/adjuvant suspensions. Mice were immunised with either 100\u2009\u03bcg of chromatographically purified chicken OVA , or OVA-HEL conjugate prepared using glutaraldehyde to couple the chicken OVA and HEL  as published.15 Antigens were prepared in a 1% Alum suspension  or in 1IU ISCOMATRIX  or 1\u2009\u03bcg LPS . To identify the site of injection, mice were also injected Alexa-647-labelled OVA prepared using an Alexa-647 protein labelling kit (Thermo Fisher Scientific) according to the manufacturer\u2019s instructions. All buffers and media were purchased endotoxin-free and absence of appreciable endotoxin contamination of proteins was confirmed by absence of bone marrow-derived dendritic cell activation at a dose of 500\u2009\u00b5g. Animals were allocated to experimental groups by a blinded observer, and were maintained under standard animal house conditions at the University of Glasgow and procedures were performed according to UK Home Office regulations.Six to eight-week-old female BALB/c mice were purchased from Harlan . LysM-eGFP mice were a kind gift from Professor Sussan Nourshargh. These mice have the gene for eGFP knocked into the Lysozyme (Lys) M locus resulting in mice with fluorescent myelomonocytic cells, with neutrophils comprising the highest percentage of eGFPlater (Qiagen). RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer\u2019s instructions and was homogenised using a gentleMACs Dissociator . RNA was converted to cDNA using a reverse transcription kit from Primer Design . One\u2009\u03bcg of cDNA was then loaded onto customised Taqman low-density array (TLDA) plates  and probed for the presence of cytokine and chemokine genes. Samples were subsequently analysed on an Applied Biosystems 7900HT Fast Real-Time PCR System, using the SDS2.4 software. Data were analysed on the RQ Manager 1.2.1 software. Data are shown as fold change over the appropriate untreated control tissue. For analysis of the injection site (ear) n\u2009=\u20093 for each treatment group; for analysis of the dLNs n\u2009=\u20095 for the OVA-treated group, n\u2009=\u20097 for the OVA/ALUM-treated group and n\u2009=\u20093 for the untreated, the PBS and the LPS-treated groups. Where a target transcript was undetectable an arbitrary value of 0.001 was assigned. The data shown are representative of two independent experiments.Ear tissue and superficial cervical dLNs were excised and stored in RNA29 we estimated three mice would provide 80% power to detect significant differences (a\u2009<\u20090.05) in numbers of neutrophils recruited to tissue in response to adjuvant vs. PBS control. For the analysis of T-cell transfer and B-cell transfer into PAD4KO and WT mice on day 5 the group size was n\u2009=\u20093 and on day 7 the group size was n\u2009=\u20094 for WT mice and n\u2009=\u20093 for PAD4KO mice. The data shown are representative of two independent experiments. Using previous data,30 we estimated three mice per time point will provide 80% power to detect significant differences (a\u2009<\u20090.05) in numbers of Plasma B cells induced by antigen and adjuvant vs. antigen alone.Cell suspensions were prepared from ear tissue by digestion in 2\u2009mg/ml Collagenase IV and 2\u2009mg/ml Hyaluronidase (both from Sigma Aldrich) and DNaseI  at 37\u2009\u00b0C for 30\u2009min, followed by homogenisation using a gentleMACs Dissociator (Miltenyi Biotech). The draining superficial cervical lymph node was gently teased apart using 26G needles and digested in 2.68\u2009mg/ml collagenase D (Roche) for 25\u2009min at 37\u2009\u00b0C. The enzymatic reaction was stopped with a final concentration of 10\u2009mM EDTA. A single cell suspension was then obtained by passing the lymph node suspension through a 70\u2009\u03bcm cell strainer. Single cell suspensions were then stained with combinations of the following antibodies CD11c\u2013PerCP-Cy5.5 , CD11b\u2013PE-Cy7 , F4/80\u2013APC  CD45-eFluor\u00ae450 , Ly6-G\u2013PE , MHCII\u2013FITC , anti-B220-eFluor\u00ae450 , anti-GL-7-FITC , anti-FAS-PE-Cy7 , anti-CD45.1-APC , anti-CD4-PE-Cy5 , anti-Vb5.1/5.2-PE  and biotinylated anti-IgMa . Streptavidin-APC-eFluor\u00ae780  was used to detect the biotinylated antibody. The viability of the cells was confirmed using a fixable viability stain conjugated to eFluor\u00ae450 (eBioscience). All antibodies and isotype controls were added to samples at a dilution of 1:200. Samples were read using a MACSQuant flow cytometer  and analysed using Flowjo software . For analysis of innate cell recruitment to the injection site group sizes of six mice were used. The data shown are representative of three independent experiments. For analysis of innate cell recruitment to the dLN a group size of four was used for OVA treatment; five for OVA/ALUM treatment and three for LPS treatment. Using data from previous studies1 and IgG2a/IgG2c titres were determined in serum samples collected 14 days after immunisation as previously described.2 For the analysis of antibody responses to OVA/ALUM the group sizes were n\u2009=\u20093 while for the analysis of antibody responses to OVA/IMX the group sizes were n\u2009=\u20094 with n\u2009=\u20092 for PBS-treated controls. The data shown are representative of three independent experiments.OVA-specific IgGin vivo imaging, animals were anaesthetised with fentanyl/fluanisone  and midazolam injectable anaesthesia  given intraperitoneally. Core body temperature was continuously monitored and maintained by a thermostatically controlled heat mat. The ear of interest was mounted on a purpose-built, heated stand with the use of veterinary-grade glue , enabling maintenance of tissue at 37\u2009\u00b0C throughout the experiment.31 Intravenous injection of non-targeted quantum dots  allowed highlighting of blood vessels. Cells were visualised in the ear pinna using the cell-permeable DNA dye Hoechst 33342  and extracellular DNA visualised with SYTOX orange . The dyes were administered subcutaneously along with the antigen/adjuvant preparations. Mice were also treated subcutaneously in the ear pinna with 5\u2009mg of DNase along with antigen/adjuvant preparations to block NET formation at the injection site.17 Videos were acquired for 15\u201330\u2009min at an X\u2013Y pixel resolution 512\u2009\u00d7\u2009512 with 2.5-\u03bcm increments in Z. 3D tracking was performed with both Imaris 7 (Bitplane) and Volocity 5.5 (Perkin Elmer) after correction for tissue drift.Multiphoton imaging was performed with a Zeiss LSM7 MP system equipped with both a 10\u00d7/0.3 numerical aperture (NA) air and a 20\u00d7/1.0 NA water immersion objective lens (Zeiss) and a tunable titanium/sapphire solid state two-photon excitation source . For Ears were carefully split into dorsal and ventral halves and fixed in 2% paraformaldehyde prepared in PBS (PFA/PBS) (eBiosciences) for 30\u2009min on ice. Fixed ears where then permeabilised in 0.2% Triton X-100 (Sigma)/PBS on ice for 15\u2009min, and blocked overnight at 2\u20138\u2009\u00b0C with 10\u2009\u00b5g/ml anti-CD16/CD32 , 5% Normal mouse serum, 0.2% Triton X-100 in PBS. FITC conjugated anti-MPO  and purified rabbit anti-citrulinated Histone H3  were added to give a final concentration of each at 2\u2009\u00b5g/ml and incubated at 2\u20138\u2009\u00b0C overnight. Tissues were washed in three changes of 0.2% triton X-100/PBS at 2\u20138\u2009\u00b0C for 1\u2009h. The Alexafluor 647-conjugated secondary antibody  was added at 2\u2009\u00b5g/ml in 2% NMS/0.2% Triton X-100/PBS overnight at 2\u20138\u2009\u00b0C. Tissues were washed in three changes of 0.2% Triton X-100/PBS at 2\u20138\u2009\u00b0C for 1\u2009h. Sytox Orange was added at a 1/10,000 dilution in 0.2% Triton X-100/PBS and incubated at 2\u20138\u2009\u00b0C for 1\u2009h. Tissues were then fixed in 1% PFA/PBS and imaged on the multiphoton microscope.P\u2009\u2264\u20090.05 was considered significant.For flow cytometry and enzyme linked immunosorbent assay data, intergroup significance was determined by either a one-way ANOVA or a two-way ANOVA with a Tukey post test. For TLDA data intergroup significance was determined using the non-parametric Kruskal\u2013Wallis test with a Dunns post test. Statistical analysis was performed using GraphPad Prism 6 . A value of Supplementary Figure 1Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5"}
+{"text": "Introduction: Milk fat globule membrane (MFGM) is a protein- and phospholipid-rich membrane that surrounds the lipid droplet in milk. We have previously reported that a diet composed of a combination of prebiotics, bovine MFGM (bMFGM), and lactoferrin (bLf) supported brain development in young pigs. Due to the growing interest of its potential benefits in neurodevelopment, the present study focused on the effects of dietary bMFGM alone using the pig as a translational model.Methods: Male pigs were provided ad libitum access to milk replacer with added whey protein-lipid concentrate (source of bMFGM) at 0 (CONT), 2.5 (MFGM-2.5), or 5 (MFGM-5.0) g/L from postnatal day (PND) 2 to 31. Blood was collected from pigs at PND 15 and 31, and pigs underwent behavioral testing using the novel object recognition task starting at PND 25. At PND 31, magnetic resonance imaging was conducted and animals were subsequently euthanized for tissue collection.Results: No group differences in body weight gain or milk intake were observed. At PND 31, few group differences were detected in absolute and relative brain volumes, brain water diffusivity outcomes, or behavioral parameters using the novel object recognition task. Serum lipoprotein was higher in pigs receiving diets with added dietary bMFGM compared with the CONT group. Serum cholesterol and high-density lipoprotein significantly higher  in the MFGM-2.5 compared with the CONT group. However, cholesterol concentrations within the brain prefrontal cortex and hippocampus did not differ among dietary groups.Conclusion: In this pig model, dietary supplementation with bMFGM was well-tolerated and supported growth and dietary intake similar to the control formula. Added dietary bMFGM was associated with increased serum lipoprotein, but no group differences in early brain cholesterol concentrations, macrostructure, microstructure, or recognition memory pigs at 31 days of age. Further examination of longitudinal brain development and myelination in the pig, particularly at later ages/maturation, is warranted. Proper early-life nutrition is critical to the growing infant due to direct influence on brain development and effects on growth and cognition later in life. It is well-known that human milk is the gold-standard source of nutrition for understanding the necessary macronutrients and bioactive compounds needed for a developing child . HoweverMFGM is a triple-layer membrane that surrounds the lipid droplet in the milk of multiple mammalian species \u201312. It fTherefore, the objective of this study was to assess the influence of added dietary bMFGM on structural brain development, cognitive behaviors, and cholesterol and lipoprotein profiles using the translational pig model. The young pig was used due to its similarities in gastrointestinal system and resemblances in gross neuroanatomy when compared with human infants \u201331. WhenClostridium perfringens antitoxin C and D  upon initial arrival to the Piglet Nutrition and Cognition Laboratory on PND 2. Contrary to standard agricultural procedures, pigs on this study were not provided an iron dextran shot as all diets contained adequate concentrations of iron. Additionally, pigs did not receive standard agricultural processing, meaning needle teeth, tails, and testicles were not removed. Pigs were individually housed in custom rearing units, as previously described  male pigs were obtained from a commercial swine farm  on postnatal day (PND) 2 and transferred to the University of Illinois Piglet Nutrition and Cognition Laboratory where they were artificially reared until PND 30 or 31. The trial was completed in three cohorts with a total of 18 pigs per diet group selected from 16 litters to control for initial body weight and genetics. All pigs were provided with a single dose of prophylactic antibiotic  administered at 5.0 mg/kg body weight on day of birth and two doses of n = 18 per group) were provided one of three experimental milk replacer diets with added whey protein-lipid concentrate  at 0 (CONT), 2.5 (MFGM-2.5), or 5 (MFGM-5.0) g/L (ad libitum from 1000 until 0600 the next day (20 h continuous feeding period each day). Individual body weights were recorded daily and health checks recorded twice daily to track the well-being of each pig. Leftover milk from the previous feeding period was subtracted from allotted volume to quantify milk disappearance over the daily feeding period for each pig, which is referred heretofore as milk intake.All milk replacer formulas were produced by Mead Johnson Nutrition  using a proprietary blend of nutrients formulated to meet the nutritional needs of growing pigs . Individ5.0) g/L . These c5.0) g/L , 36. All5.0) g/L , pigs wen = 8; MFGM-2.5, n = 8; MFGM-5.0, n = 8). Blood was collected from the jugular vein into evacuated serum tubes , then left at room temperature for at least 45 min to allow for proper clotting. Samples were then centrifuged at 4\u00b0C and 1,250 \u00d7 g for 20 min , aliquoted, and stored at \u221280\u00b0C. Serum samples  were analyzed by Dr. Frankie Stentz at the University of Tennessee for analyses of serum cholesterol, triglyceride, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) using validated methods.Blood serum samples were collected on PND 15 as well as at study conclusion on PND 30/31 from 24 pigs  were euthanized via exsanguination after CO2 asphyxiation. The hippocampus and prefrontal cortex were collected exclusively from the right hemisphere of each pig. Samples were snap-frozen in liquid nitrogen and stored at \u221280\u00b0C. Brain cholesterol was measured in samples (n = 36) using a cholesterol colorimetric test . The colorimetric test was conducted according to the manufacturer's instructions and all measures were assessed in duplicate. Samples with results above the upper limit of the test's detection range were diluted and re-analyzed.On PND 30 or 31, all pigs (n = 36) were transported to the Biomedical Imaging Center at the Beckman Institute for Advanced Science and Technology  and anesthetized upon arrival via an intramuscular injection of a telazol:ketamine:xylazine solution  at a dose of 0.03 mL/kg of body weight. Once fully sedated, pigs were placed in the MRI scanner and maintained on 2% isoflurane/98% oxygen for the duration of the 60 min scan. Imaging was performed using a Siemens MAGNETOM Prisma 3T scanner with a custom 8-channel head coil specific to the pig. Oxygen saturation levels and heart rate were monitored every 5 min throughout the entirety of the scan. Detailed neuroimaging sequences and post-imaging analysis of volumetric assessment and diffusion tensor imaging have been previously described , which has been described in detail previously , 39. Tespost-hoc Tukey adjustment using the MIXED procedure of SAS 9.3 . All statistical models included replicate as a random effect, and fixed main effects of diet and PND, as well as their interaction. The level of significance was set at P < 0.05 while trends were defined as 0.05 < P < 0.10. Data collected at a single time-point  were analyzed as a one-way ANOVA whereas data collected from the sample animal on more than one occasion  were analyzed using a repeated-measures ANOVA.All data were analyzed by an analysis of variance (ANOVA) with a P < 0.01) as evident in P \u2265 0.62; Main effects of diet and PND were observed for daily body weights and milk intake  and HDL . No differences in serum lipoprotein fractions were detected in comparisons of CONT and MFGM-5.0 or MFGM-2.5 and MFGM-5.0. No group differences in the ratio of LDL-to-HDL were detected. A main effect of PND for triglycerides was observed, concentrations of triglycerides concentrations were lower at PND 30 compared to PND 15 .No interactive effects were observed, so only main effects of diet and PND are reported (P \u2265 0.21). Moreover, there were no dietary differences observed in relative concentrations of free cholesterol in the prefrontal cortex or hippocampus  across dietary groups.No significant differences between dietary treatments were observed in absolute volumes of whole brain, gray matter, or white matter . AnalysiP = 0.044) at 0.69 \u00d7 10\u22123 mm2/s in pigs provided MFGM-2.5 compared with the mean diffusivity of 0.66 \u00d7 10\u22123 mm2/s in pigs provided CONT  in Indonesia, infants received formula with added complex milk lipids  that increased the ganglioside content of the diet from ~ 2\u20138 weeks to 24 weeks of age. Infants fed formula containing complex milk lipids demonstrated improved hand and eye coordination IQ, performance IQ, and general IQ on the Griffiths Mental Development Scale . In a DBMFGM has also been shown to improve cognitive development and performance in preclinical rodent models. Rodents fed MFGM exhibited improved performance on spatial and passive avoidance tasks , increasDiscrepancies between the behavioral outcomes in this study compared to other studies may be due variations in MFGM composition. Before milks are commercially available, raw milk must undergo various mechanical and thermal processes so that it is safe for human consumption. Thus, MFGM may have an altered structure between varied dairy products , 75. AddWe examined the growth and development of pigs provided supplemental dietary MFGM early in life. The addition of bMFGM to the diet did not appear to negatively impact body weight gain or milk intake, yet increases in serum lipoprotein fractions displayed similar patterns as those found in breastfed infants as well as infants who received added dietary bMFGM. Implications of such findings suggest that infants receiving added bMFGM in formula may exhibit developmental patterns more similar to breastfed infants than to infants given formula without added bMFGM.To our knowledge, this has been the first study to assess the influence of dietary complex bMFGM on neurodevelopment using neuroimaging outcomes in the pig. Our previous studies have shown that polydextrose, galactooligosaccharides, and bLf in formula in addition to added bMFGM supported neurodevelopment. It is possible that synergistic efficacy among those bioactives influenced the physiological response related to brain development in young pigs. In the present study, direct influence associated with added dietary bMFGM on brain macrostructure and microstructure and recognition memory appeared limited in early life. Longitudinal examination of brain development and myelination in the pig, particularly at later ages, is warranted.The datasets generated for this study will not be made publicly available. The data are protected as they were generated as a result of a commercially-funded project.All animals and experimental procedures were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and approved by the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee.JF, SF, MC, GG, BB, and RD contributed to the design of the study. JF, SF, and RD interpreted the study and prepared the manuscript. All authors were involved in data acquisition, analysis, interpretation and read and approved the final version of this manuscript.The authors declare that this study received funding from Mead Johnson Nutrition. The funder had the following involvement with the study: contribution to study design, reviewing final manuscript before submission."}
+{"text": "The structures were elucidated by spectroscopic analyses. In the present research, compounds 1, 3, 4, 5a/5b and 6a/6b, also showed in vitro cytotoxicity against six cancer cell lines . Among them, compound 1 exhibited significant cytotoxicity against these cancer cell lines, with IC50 of 0.24-6.57 \u03bcM. Furthermore, HPLC profiles were developed for qualitative and quantitative analysis of these active constituents in different parts of this plant, including mature and immature seeds, leaves, stems and roots. The results revealed that compounds 3 and 4 have the highest concentrations, which are found in the roots part of the plant.Two new alkaloids, 9-methoxy-18,19-dehydrocamptothecin ( Camptotheca acuminata   was approved as a medicine for treating several cancers in Japan, France, and United States with camptothecin originating from Taiwanese N. foetida.The development of camptothecin-containing plants as a cash crop is becoming an important issue in southeastern Asia. , Taiwan . In 1995 foetida . MeanwhiN. foetida is an important resource for anticancer drugs, this plant has been reinvestigated. A number of camptothecinoids, other alkaloids and phytochemicals have been reported from this plant + ion at m/z 377.1135, corresponding to the molecular formula C21H17N2O5. The IR spectrum indicated the presence of hydroxyl (3406 cm-1), lactone carbonyl and lactam carbonyl (1745 cm-1 and 1652 cm-1) and aromatic functional (1500 cm-1) groups. By comparing to mass data of 9-methoxy-camptothecin (4), the molecular formula reduces two protons of 1. According to its 1H- and 13C-NMR spectra, new compound 1 was similar to 9-methoxy-camptothecin (4). Compound 1 was found to be a 18,19-dehydro-analog of 4 with the following changes in the NMR chemical shifts: a typing ABC spin coupling pattern attributed to a vinyl group of 1  instead of an ethyl group of 4 + ion at m/z 485.1925, corresponding to the molecular formula C25H29N2O8. The IR spectrum indicated the presence of hydroxyl (3397 cm-1), lactam carbonyl (1670 cm-1) and aromatic functional (1596 cm-1) groups. In the 1H-NMR spectrum of compound 2, the resonances and multiplicities largely matched those of the A, B, and D rings in camptothecin (3), however, in 1H- and 13C-NMR spectra, the overlapping signals and two sets of signals for part of molecules could be distinguished, revealing 2 to be a racemic mixture. The absence of geminal protons (H-7) at \u03b4 5.43, the absence of lactone carbonyl (C-21) at \u03b4 172.5, and the presence of a singlet at \u03b4 2.25  and a triplet at d 5.27  suggested that 2 lacked the E ring of camptothecin. As another difference in the 1H-NMR spectra, compound 2 showed a pair of downfield-shifted singlets at \u03b4 6.97/7.00 (1H for 2a and 2b in a ratio of 1:1) in place of the proton singlet at \u03b4 5.28  found in 3. In addition, in the 13C-NMR spectrum, C-5 was found at \u03b4 84.3/84.4 in 2, rather than 50.6 as in 3. Based on this data, a hydroxyl group was attached on C-5. The NMR data for 5-hydroxyl substitution of camptothecinoids could be assigned on the basis of data of the known synthetic product, 5-hydroxycamptothecin +, .5-Hydroxy-mappicine-20-O-\u03b2-glucopyranoside (2a/2b). White amorphous powder (2.07 mg);  -39.2\u00b0 ; UV  nm (log \u03b5): 223 (3.96), 255 (3.82), 357 (3.63); IR (neat) \u03bdmax 3397, 2929, 1670, 1596, 1457, 1340, 1195, 1113 cm-1; 1H- and 13C-NMR (CD3OD) see m/z 485.1925 [M+H]+, .Dried leaves, stems, roots, mature seeds, and immature seeds were ground, 10 g were weighed and extracted with methanol at 24-25\u00b0C for 5 days, to mimic the large scale extraction conditions. All extracts were evaporated under reduced pressure to give five residues . Each dry extract (5.0 mg) was dissolved in DMSO/MeCN (0.6 mL), filtered on a pre-column and injected to HPLC (each injection was 30 \u00b5L).N. foetida and their structures were determined by spectroscopic methods. These reference compounds were used for qualitative analysis. Standard stock solutions containing 1 mg/mL of compounds 3, 4 were prepared with 1 mg compounds 3, 4 in 1.0 mL DMSO for quantitative analysis. Standard sample solutions were injected (injection volume: 10 \u00b5L) directly into the HPLC system.Camptothecinoids were isolated from immature seeds of 2O  at a flow rate of 1 mL/min. The solvents were filtered through a 0.45 \u03bcm filter prior to being used. Total HPLC running time for the assay was 22 minutes.HPLC analyses were performed on a Shimadzu model LC-10AT HPLC (Japan) equipped with a two solvent delivery system, a SIL-10A automatic sample injector and a model SPD-M10A diode array detector. The detector was at 365 nm. Data acquisition and quantification were performed using Shimadzu Class-VP software (version: 6.12SP5). Chromatography was carried out on a Varian Polaris 5 C18-A (250\u00d74.6mm i.d.) column. Isocratic elution was performed with water and HPLC-grade acetonitrile/H3 and 4, in the linear range of 0.015 to 0.5 mg/mL, were prepared in DMSO, respectively. Six replicates (n=6) of each concentration were subjected to HPLC.In the standard HPLC chromatogram, six different concentrations of compounds 50 is the concentration of agent that reduced cell growth by 50% under the experimental conditions.Fractions and isolates were tested against lung (A549), breast (MEA-MB-231 and MCF7), and liver (HepG2 and Hep3B) cancer cell lines using established colorimetric MTT assay protocols . Doxorub"}
+{"text": "During our investigation of antibiotic resistance mechanisms in a sample of Irish thermophilic Campylobacter broiler isolates, it was determined that 100% of tetracycline-resistant isolates (n\u2009=\u2009119) harboured tet(O). Accessory tetracycline-resistance mechanisms were considered as tetracycline minimum inhibitory concentrations ranged from 4 to \u2265\u200964\u00a0mg/L. Primers previously reported for the detection of tet(A) in Campylobacter failed to produce an amplicon using a positive control strain (Escherichia coli K12 SK1592 containing the pBR322 plasmid) and a selection of Campylobacter isolates. Accordingly, we designed new tet(A)-targeting primers on SnapGene2.3.2 that successfully generated a 407\u00a0bp product from the positive control strain only. Further in silico analysis using BLASTn and SnapGene2.3.2 revealed that previously reported Campylobacter tet(A) sequences deposited on GenBank shared 100% homology with Campylobacter tet(O). We postulate that this gave rise to the erroneous report of a high tet(A) prevalence among a pool of Kenyan broiler Campylobacter isolates that were tested using primers designed based on these apparent tet(A) sequences. In conclusion, further work would be required to determine whether the homology between tet(A) potentially present in Campylobacter and known tet(A) genes would be sufficient to allow amplification using the primers designed in our study. Finally, the existence of tet(A) in thermophilic Campylobacter spp. remains to be demonstrated.The true prevalence of  It is aCampylobacter spp. is usually mediated by a ribosomal protection protein Tet(O), which confers resistance by preventing tetracycline ribosomal binding, thus abolishing the inhibitory effect of the antibiotic by preventing bacterial protein synthesis via association of aminoacyl tRNA with the bacterial ribosome [Tetracycline-containing therapeutics are the most commonly administered antimicrobial in poultry production and animal husbandry in Ireland, used for the treatment of enteric, respiratory and dermal infections , 4. Tetrribosome , 5.Campylobacter spp. isolates, we were especially interested in tetracycline resistance genes, as resistance to tetracycline was most prevalent (34%) by phenotypic sensitivity testing (Unpublished 2019). Tetracycline-resistant isolates were preliminarily screened for the presence of tet(O), using the method described by Aminov et al. [tet(O) gene. However, accessory tetracycline-resistance mechanisms were considered as minimum inhibitory concentrations ranged from 4 to \u2265\u200964\u00a0mg/L. Moreover, the mobilisation of tetracycline-resistant determinants is associated with the presence of tet genes on plasmids [tet(A) gene, which codes for an efflux protein and has been reported to co-exist with tet(O) in Campylobacter, was considered as part of the investigation [tet(A) among thermophilic Campylobacter spp.In our study, during the investigation of antibiotic resistance mechanisms among a sample of 350 Irish broiler v et al.  and it wtet(A) primers described by Abdi-Hachesoo et al. [Escherichia coli K12 SK1592 containing the pBR322 plasmid (DSM 3879). In addition, a selection of tetracycline-resistant thermophilic Camplyobacter spp. isolates also failed to generate an amplicon. New tet(A)-targeting primers were thus designed on SnapGene2.3.2, based on homologous regions of the tet(A) gene from the pBR322 plasmid (GenBank J01749.1) and from the Pseudomonas putida strain Fars110 (GenBank JN937120.1) (Table\u00a0tet(A) positive control. A 407\u00a0bp product was successfully amplified using the new primers (Tet(A)-Camp-F and Tet(A)-Camp-R) with the E. coli K12 positive control strain but none of the tetracycline-resistant thermophilic Camplyobacter spp. isolates generated a product.We tested the o et al.  (Table\u00a01tet(O) and tet(A) products were resolved by electrophoresis in a 2% and a 1.5% agarose gel, respectively. All primers used are listed in Table\u00a0With reference to the methods used in our study, DNA was extracted using PureLink\u2122 Genomic DNA Mini Kit . PCR mixtures (50\u00a0\u00b5L) contained 2.5U Amplitaq\u2122 DNA polymerase , 1\u00d7 buffer I , 2.5\u00a0mM magnesium chloride, 0.2\u00a0mM of each dNTP , 200\u00a0\u00b5M forward and reverse primer (Table\u00a0tet(A) primers listed by Abdi-Hachesoo et al. [E. coli (DSM 3879 positive control strain), under less stringent conditions, prompted further investigation within our study. In the Abdi-Hachesoo et al. [tet(A) primer (Tet(A)-F and Tet(A)-R) reference is not listed in their bibliography, although these primers were previously reported by Maynard et al. [tet(A) among Canadian swine E. coli isolates  sequences, Shiraz3 and Shiraz4  deposited in GenBank in the Abdi-Hachesoo et al. [Campylobacteraceae (taxid 72294) sequences using BLASTn. Multiple tet(O) sequences were returned with 100% identity, including C. jejuni 81-176 (GenBank NG_048260.1). Furthermore, our alignment studies using SnapGene2.3.2 demonstrated absolute homology between tet(O) (GenBank M18896.2) and Shiraz3 and 4 tet(A) sequences . We propose that the true identity of the Shiraz 3 and 4 sequences are Campylobacter tet(O).We scanned the tet(A) among thermophilic Campylobacter spp. isolated from extensively reared Kenyan broilers was published by Nguyen and coworkers [tet(A) primers included the same primers as those used by Abdi-Hachesoo et al. [tet(A) primers (designated tet-A-1 and tet-A-2) [tet(O). To confirm this, we performed an in silico PCR using SnapGene2.3.2 with the tet-A-1 and tet-A-2 primers [Campylobacter tet(O) sequences (GenBank M18896.2 and NG_048260.1). A 486\u00a0bp product was predicted, which correlates to the amplicon length reported by Nguyen et al. [tet(A) among this subset (n\u2009=\u200953) of thermophilic Campylobacter isolates is erroneous. Our opinion also explains why clusters of tet(A) harbouring Campylobacter spp. isolates are not described in any database, to our knowledge.Furthermore, in 2016, a second study reporting a high prevalence of tet(A) potentially present in Campylobacter and known tet(A) genes would be sufficient to allow amplification using the primers designed in our study. The investigation of alternative Campylobacter-associated tetracycline resistance mechanisms is certainly worthwhile, but the presence of tet(A) in Campylobacter spp. is an open question.In conclusion, further study would be required to determine whether the homology between"}
+{"text": "To understand how DPSCs sense and respond to the mechanics of their microenvironments, it is essential to determine how these cells convert mechanical and physical stimuli into function, including lineage specification. This review therefore covers some aspects of DPSC mechanoresponsivity with an emphasis on the factors that influence their behavior. An in-depth understanding of the physical environment that influence DPSC fate is necessary to improve the outcome of their therapeutic application for tissue regeneration.Dental pulp is known to be an accessible and important source of multipotent mesenchymal progenitor cells termed dental pulp stem cells (DPSCs). DPSCs can differentiate into odontoblast-like cells and maintain pulp homeostasis by the formation of new dentin which protects the underlying pulp. DPSCs similar to other mesenchymal stem cells (MSCs) reside in a niche, a complex microenvironment consisting of an extracellular matrix, other local cell types and biochemical stimuli that influence the decision between stem cell (SC) self-renewal and differentiation. In addition to biochemical factors, mechanical factors are increasingly recognized as key regulators in DPSC behavior and function. Thus, microenvironments can significantly influence the role and differentiation of DPSCs through a combination of factors which are biochemical, biomechanical and biophysical in nature. Under  Dental pulp tissue contains an accessible source of multipotent mesenchymal progenitor cells, known as dental pulp stromal/stem cells (DPSCs), which participate in dentin and pulp regeneration . In the A key study by The teeth are essential for mastication and are subjected to various mechanical stresses due to jaw movement and occlusal forces which are transmitted to the dental pulp tissue and can subsequently influence DPSC fate . MoreoveThe signals involved in regulating SC fate are not only ECM proteins, adjacent differentiated cells, secreted and cell surface molecules but also mechanical signals. Notably it has been reported that in response to degradation, disruption and mechanical erosion DPSCs can differentiate into odontoblast-like cells to form dentin. Indeed several studies have now demonstrated that mechanical stresses transmitted to the pulp tissue can affect the behavior of DPSCs . TherefoDental pulp stem cells not only play a crucial role in dentinogenesis but also provide a promising source of cells for use in regenerative medicine . DPSCs cin vivo, many studies have investigated the effects of mechanical forces using in vitro models , promotes their osteoclastogenic differentiation which occurs during physiologic root resorption of deciduous teeth . NotablyThe overall aim of this review therefore is to report the effects of mechanical stimuli on the biological behavior of DPSCs as well as describing the associated intracellular signaling and odonto/osteogenic differentiation in DPSCs. The data discussed in this review indicates that appropriate mechanical stresses are important biological stimuli that can effectively promote proliferation and differentiation of DPSCs. The understanding of how these cells respond to mechanical stimuli (mechanosensitivity) is important for bone and tooth tissue engineering applications using DPSCs alone or in conjunction with biomaterials and other bioactive molecules such as growth factors and cytokines.Dental pulp stem cells are mechanosensitive cells that can recognize mechanical changes and transform this information into cellular responses . For exain vitro , equiaxial static tensile strain and micro- and nano-scale surface topographies have also been shown to induce osteogenic differentiation of DPSCs , prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) (of DPSCs . For exaof DPSCs  are able (COX-2) , 2011.As has been observed for osteocytes, the rapid stimulation of NO production by PFF in DPSCs appears to be related to the activity of the enzyme endothelial nitric oxide synthase (eNOS) but not to the activity of inducible NOS (iNOS) . ImportaAn interesting study by in vivo on DPSCs could be used to promote bone formation and limit bone resorption -6 and -1\u03b2, tumour necrosis factor (TNF)-\u03b1 as well as the expression of the antioxidant genes heme oxygenase-1 (HO-1) and superoxide dismutases (SOD). The same authors also showed that mechanical stimulation using cyclic tensile strain induced expression of the odontoblastic markers DSPP, DMP-1, OPN, and BSP. Moreover, they demonstrated that the odontoblastic differentiation of DPSCs was mediated by the NF-E2-related transcription factor 2 (Nrf2)/HO-1 pathway (The biological response of DPSCs to mechanical stimuli occurs not only during orthodontic tooth movement but also during normal mastication. For example, DPSCs can produce growth factors, angiogenic changes and a mild inflammatory-type reaction in response to orthodontic forces , 2010. F pathway . These d pathway . In supp pathway .Dental pulp tissue is subjected to mechanical stress during normal masticatory process and also during pathological trauma or orthodontic tooth movement .Moreover, it is also well known that occlusive force plays a role in physiological root resorption of deciduous teeth, in which dental pulp cells from these mechanically stressed teeth secrete cytokines that are important for odontoclast activation and subsequent root resorption .Therefore, DPSCs located near the roots of teeth are subjected to higher levels of oral mechanical stress by jaw movement and occlusal forces. Moreover, orthodontic forces transfer horizontal stretch to DPSCs, and also during tooth eruption dental pulp tissue is stretched vertically .Furthermore, during physiological mechanical loading of teeth, the dentin is subjected to fluid flow which can activate the nocireceptors and mechanoreceptors on odontoblast processes present in the dentin tubules that regulate the maintenance of tooth integrity .Importantly, To the best of our knowledge, until now only the study by Regenerative medicine is an important application of DPSCs and this necessitates a thorough knowledge of mechanobiology. However, the studies analyzed in this review demonstrate that the response of DSPCs to mechanical stimuli differs according to the type and source of the mechanical forced applied Table . ImportaThe understanding of the mechanical stimuli that regulate DPSC behavior will not only improve our knowledge relating to mechanisms involved in their differentiation but could also provide valuable insights for optimizing DPSC-based therapies. In fact, the particular mechanoresponsiveness of DPSCs to mechanical stimuli may be of utility for future bone and teeth tissue engineering applications.in vivo environment on DPSCs to promote the regeneration of dental and bone tissues.Although physical and mechanical factors are known to play a key role in regulating DPSC fate, further studies are required to elucidate the detailed molecular mechanisms and signaling pathways involved in the mechanosensitive response of DPSCs to various types of forces.Ultimately it may be possible to use mechanical forces that mimic the dynamics of the FP, MM, BC, RS, BS, PC, and MT conceived and wrote the manuscript. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "A new strategy is introduced combining the use of Multi-Criteria Optimization-based Trade-Off Exploration (TO) and RapidPlan\u2122 (RP) for the selection of optimisation parameters that improve the trade-off between sparing of organs at risk (OAR) and target coverage for head and neck radiotherapy planning. Using both approaches simultaneously; three different workflows were proposed for the optimisation process of volumetric-modulated arc therapy (VMAT) plans. The generated plans were compared to the clinical plans and the plans that resulted using RP and TO individually.+ respectively. The TO management was standardised to maximise the sparing of the parotid glands without compromising a clinically acceptable PTV coverage. Resulting plans were inter-compared based on dose-volume parameters for OAR and PTVs, target homogeneity, conformity, and plans complexity and deliverability.Twenty clinical VMAT plans previously administered were selected. Five additional plans were created for each patient: a clinical plan further optimised with TO (Clin+TO); two plans generated by in-house built RP models, RP_1 with the model built with VMAT clinical plans and RP_TO with the model built with VMAT plans optimised by TO. Finally, these last two plans were additionally optimised with TO for the creation of the plans RP_1\u2009+\u2009TO and RP_TO+ plans, which reported a mean parotid dose average of 15.0\u2009\u00b1\u20094.6\u2009Gy vs 22.9\u2009\u00b1\u20095.5\u2009Gy (left) and 17.1\u2009\u00b1\u20095.0\u2009Gy vs 24.8\u2009\u00b1\u20095.8\u2009Gy (right). However, at the same time, RP_TO+ showed a slight dose reduction for the 99% volume of the nodal PTV and an increase for the 95%  but remained within clinical acceptance. Plans optimised with RP and TO combined, showed an increase in complexity but were proven to be deliverable.The plans optimised using TO in combination with RP showed significantly improved OAR sparing while maintaining comparable target dose coverage to the clinical plans. The largest OAR sparing compared to the clinical plans was achieved by the RP_TO+ plans, acceptance of a slight deterioration in nodal PTV allowed the largest reduction in OAR dose to be achieved.The use of TO combined with RP during the optimisation of VMAT plans enhanced plan quality the most. For the RP_TO The challenge in radiotherapy planning is to manage the compromise between tumour irradiation and sparing of healthy tissue. The planning objectives involved can be inherently contradictory, which restricts an optimal result for all objectives at the same time. This situation is reflected in the irradiation of head and neck cancer (HNC) where there are many organs at risk (OAR) and their close proximity to the target volume makes it difficult to achieve clinically acceptable target coverage without radiation-induced toxicity and decreased of quality of life \u20133.For the treatment of HNC, volumetric-modulated arc therapy (VMAT) has shown good dose conformity and sparing of OAR with a decrease in treatment delivery time compared to other techniques \u20137. For tEclipse treatment planning system (TPS) Varian Medical Systems, Palo Alto, CA, version 15.5 introduces the tools for a MCO approach based on trade-offs exploration (TO). A copy of an initial plan optimisation, called the balanced plan, is used together with the chosen N optimisation objectives as the base for the subsequent generation of the 3\u2009N\u2009+\u20091 alternative plans for objective. The user can then explore the trade-offs along the possible solutions and select the plan that best fulfils the treatment goals. The initial plan influences the subsequent Pareto frontier approximation, therefore, trade-offs exploration around a starting promising plan is desirable , 17.Recently, knowledge-based radiotherapy treatment planning has been incorporated into clinical practice. RapidPlan\u2122 (RP) (Eclipse v15.5) operates with a knowledge-based treatment planning algorithm that is able to generate dose volume histogram (DVH) estimates for a new patient geometry by analysis of a database of previous plans. The DVH estimates are then translated into optimisation objectives parameters for intensity modulated radiotherapy (IMRT) or VMAT plans. The resulting plans have shown to reduce planner interaction while improving the quality and consistency , 19. HowIn this study the key question addressed was how to maximise the performance of RP and TO for the radiotherapy treatment of HNC. We suggested a combination of both approaches, RP and TO used simultaneously during the optimisation process of a plan. Three different workflows were proposed for the VMAT planning of HNC patients and an inter-comparison of the resulting plans was performed. At the same time, these were compared with the plans optimised using RP and TO on their own and the clinical plans for reference.Clinical HNC plans previously administered were available from a database with tumours originating in various sites and stages, i.e. tongue, glottis, supraglottis, lip, oral cavity, nasal cavity, tonsillar region, larynx, pharynx , neck and parotids. Plans for the region of optical structures were not included. The clinical plans were generated with the in-house optimisation template (based on the plan constraints shown on Table\u00a0Two different sets of plans were used in this work. Set_1 consisted of a collection of 54 plans that were selected randomly from the database for the creation of the RP models described in the methods section part III. These plans had a mean PTV1 volume of 325.0\u2009cc (range 68.6\u2013786.6\u2009cc) and mean PTVLR volume of 182.0\u2009cc (range 0\u2013375.7\u2009cc).Set_2 included 20 different patients; these had a mean PTV1 volume of 291.8\u2009cc (range 75.5\u20131069.4\u2009cc) and a mean PTVLR volume of 274.2\u2009cc (range 124.7\u2013389.0\u2009cc). Set_2 was selected from cases with no full parotid involvement , with mean volume overlap of parotids and PTVs of 19.9%, (range 0\u201340.8%). Set 2 was used for the creation, comparison, and analysis of plans using TO and/or RP.Upper or lower objectives: dose-volume points that define the maximum or minimum dose that a structure may receive.Mean objective: mean dose level that should not be exceeded for a structure.Line objective: it is located under the lower bound of the DVH estimate range. If the OAR overlaps with the target, the highest upper objective of the target is the defining level in that region.The inclusion of MCO in radiotherapy planning aims to allow the exploration of the trade-offs of the treatment objectives in an efficient way to then select a plan that best fulfils the prescribed clinical goals. To commence, it is required to have a starting plan (that will be the centre of the approximation of the Pareto surface). The optimisation objectives used in inverse radiotherapy planning are based on dose statistics to regions of interest, for instance:A starting plan will be used together with a selection of the optimisation objectives for trade-off exploration as the base for a generation of alternative plans.max), PRV spinal cord combined (upper points D1 and Dmax), and larynx line dose only when the tumour did not originate in it. Upper and lower point objectives were selected for PTV1 and lower point objectives for PTVLR [The optimisation objectives selected for the trade-off exploration of this study were: right and left parotids line dose or mean dose , PRV brainstem upper point  was trained with the 54 standard clinical VMAT plans. The second model (RP_TO) used the same VMAT plans but this time further optimisation was carried out using TO (following the process in Section II) prior to adding them to the model. Set_1 consisted of plans previously used for the head and neck RP model that is currently in use at our clinic, therefore, these plans are clinically acceptable and the model was processed for elimination of outliers. Both models were generated by training the two PTV targets and 12 OAR structures . The optimisation objectives selected are shown in Table\u00a0, RP_1 and RP_TO. The resulting plans were then used as the initial plans for a further optimisation using TO for the creation of the last two types of plans for each patient, RP_1\u2009+\u2009TO and RP_ TO+ (following the same trade-offs exploration procedure described on section II). In this way, the trained models suggested the optimisation objectives parameters for an initial plan that would be the centre of the later approximation of the Pareto surface. Figure\u00a0The two models were used to generate a plan for each patient of Set 2 without manual interventionFor the clinical plans and the 5 different plans generated for each patient, PTVs and OAR doses were evaluated following the dose-volume constraints specified. Additionally, the homogeneity of the PTVs was assessed by calculating the Homogeneity Index according to Eq. 1 .1\\documeAlso, the dose delivered to normal tissue was assessed by Eq. Where Vx% is the volume receiving at least x% of the dose and VPTVs is the sum of both target volumes PTV1 and PTVLR. This was calculated for V100%, V80%, V50%, and V10%.To investigate plan deliverability, RadCalc version 6.3 was used to quantify several complexity parameters: the total number of monitor units (MU), the modulation factor (MF) and the average leaf pair opening (ALPO) , 31.For the plans that resulted in lowest MF, plan specific quality assurance (QA) measurements were performed at the treatment room for further evaluation of the delivered dose. A Varian TrueBeam Slim linear accelerator was used and the phantom Octavius 729 detector array together with the Octavius 4D rotating unit. A comparison between the delivered and the planned dose was performed with a 3D gamma evaluation  using the verification software VeriSoft 6.1 (6.1.0.46) and an acceptance of 95% points passing the criteria of 3\u2009mm for the distance to agreement (DTA) and a dose difference tolerance level of 3% , 33. ForSignificant differences between the 6 final sets of plans were assessed for all the investigated parameters by the Wilcoxon signed-rank test at a level of 0.05.All plans resulting from implementing TO and/or RP were deemed clinically acceptable according to the criteria of PTV coverage and OAR doses Table . While m99, D95) and the homogeneity index remained comparable across techniques as well. For PTVLR, the dose homogeneity was shown to be slightly affected and D95 increased significantly but a D99 significant reduction resulted only for RP_TO+.For the plans created, it is shown in Table + plans. For OAR, RP_1\u2009+\u2009TO and RP_TO reported no significant difference between them. Furthermore, the combined use of RP and TO showed improvements in CI when comparing with the solely use of TO (Clin+TO plans) however whereas RP_TO plans are comparable with the clinical plans, for RP_1\u2009+\u2009TO and RP_TO+ the results showed an increase. The dose distribution of a representative case is illustrated in Fig.\u00a0Figure\u00a0+) were selected for plan specific QA. Table\u00a0Table\u00a0+). The dosimetric impact was assessed on the resulting plans for a set of 20 patients. In addition, for each patient, two more plans were created using TO and RP on its own during the optimisation stage . For the five optimisation strategies and the clinical plans, a dosimetric comparison across the resulting plans was carried out. Additionally, investigation of complexity and deliverability of the plans was performed.In this study, the combined use of RP and TO was proposed focusing on HNC radiotherapy VMAT planning. Three workflows were followed using RP together with TO during the optimisation process: the use of an in- house RP model built with TO VMAT plans (RP_TO); the use of an in-house RP model, built with clinical VMAT plans, to obtain the starting optimisation objectives parameters and then further TO (RP_1\u2009+\u2009TO); and the use of RP_TO to generate the initial objectives parameters and further TO (RP_TO+) this led to the largest dose reduction for all OAR. This reveals that for a new patient, the knowledge-based model that was trained with the plans that were already optimised by TO helped to obtain optimisation parameters that are closer to the desired trade-offs, providing the best starting point for TO. The results are in agreement with Wang J et al. [Although the use of RP and TO separately presents advantages over the clinical plans , 19, it J et al.  who show+ plans achieved the lowest average OAR doses for all the dose-volume parameters investigated. Most notably, a significant reduction in mean dose of 7.9\u2009Gy and 7.7\u2009Gy for the left and right parotids respectively was accomplished with respect to the clinical plans. Despite the dose improvement for the OAR, the CI results showed that the dose to normal tissue was slightly compromised. Further work with NTO and the inclusion of a structure, considering normal tissue not being recognised as OAR, as part of the trade-offs exploration objectives is of our interest to investigate improvement [+ but this plans provided an OAR dose improvement respect to clinical plans while maintaining comparable CI and HI to them, being this influenced by the upper PTVLR objectives used in the RP model. The results from both workflows point to the capacity of TO to decide on the trade-offs pursued for this cohort of patients and agree with ongoing HNC trials that have shown decreased toxicity to the surrounding organs as a result of dose de-escalation to nodal PTV. The implementation of RP and TO for the treatment of this cohort of patients could lead to an improved patient quality of life [RP_ TOrovement . On the  of life , 35.+) resulted in a consistent reduced number of plans failing to meet the OAR constraints. The reason three plan parameters failed with RP_TO+ as opposed to two with Clin+TO is that the additional failing plan had the largest PTV1 in the set (1069.0\u2009cc) and this lay outside the range of the plans that constitute the RP models used; therefore, following our trade-offs management, PTV1 D99 and PTVLR D99 were marginally below the constraint (94.9 and 74.7% respectively). This patient was re-planned with different trade-offs exploration management and the constraints of the PTVs were met at the expense of a 2.4Gy increase in the left parotid mean dose. This example points to the fact that the success of a RP model is dependent upon the quality and robustness of the model itself [The results displayed in Table l itself . SimilarThe time required to create these plans is equally of relevance. The estimated time in our clinics to plan a HNC case, including optimisation and dose calculation until the final plan is produced, ranges between 50 and 300\u2009min depending on the complexity and the experience of the planner. The use of RP and MCO in clinical practice has proven to reduce planning time , 36. Fol+. Although RP_TO+ showed better relationship between OAR sparing and PTV coverage, it can be observed that the homogeneity of the targets is slightly better when using RP_TO which has proved to have relation with an increase in complexity [Kyroudi, et al.  reportedmplexity . Despite+ workflows in clinical practice and analysis of the physicians\u2019 preference on the resulting trade-offs. The use of the proposed optimisation approaches for other treatment sites is of interest and the opportunity to use RP and TO in combination to escalate the dose delivered to the tumour while maintaining or reducing OAR doses.Future work will focus on the implementation of the RP_TO and RP_TOThe use of RP combined with TO improved OAR sparing on VMAT HNC radiotherapy plans while maintaining clinically acceptable target coverage. The simultaneous use of both approaches reflects in the resulting plans the individual benefit that they provide. The best OAR sparing was obtained when planning starting by generating a plan with a RP model built with plans optimised with TO and then further individualised TO optimisation. For this workflow, the trade-off between a minor deterioration of the nodal PTV, while maintaining clinically acceptable PTVs coverage, allowed the most significant reduction in OAR doses. RP_TO plans, created with the RP model optimised with TO plans, also provided an OAR dose improvement respect to clinical plans while maintaining comparable conformity and target homogeneity. Plans optimised with RP and TO were proven to be deliverable despite an increase in plan modulation."}
+{"text": "Following the publication of this article , concernThere are similarities in dot plot patterns between the right panel of Fig 3A and the right panel of Fig 3C. Although the plots are not exact duplicates, the similarities observed are not expected from plots derived from distinct datasets.The original data underlying Fig 3 are no longer available and thus it is not possible to resolve the concerns pertaining to the reliability of the integrity of the published figure.The Commission on Research Integrity at Oslo University Hospital and Institute of Clinical Medicine, University of Oslo has completed an investigation and concluded that there was evidence of scientific misconduct.PLOS ONE Editors retract this article.In light of the outstanding concerns with panels in Fig 3 that question the integrity of these data and the outcome of an internal institutional investigation, the PW, QG, ZS, EM, LM, and GG did not agree with the retraction. SS, MW, NACW, and GK did not respond to the retraction decision."}
+{"text": "Scientific Reports 10.1038/srep18210, published online 15 December 2015Retraction of: 1.The Editors have retracted this Article because significant portions of the text and equations were taken from Roland Molontay\u2019s BSc thesis without attributionThe following parts of the paper are copied verbatim or are adapted from those appearing in the thesis: the definition of dB and preceding and subsequent sentences; Definition 3.2; Proof of Lemma 3.4; Lemma 3.5; Proof of Lemma 3.5; Lemma 3.6; Proof of Lemma 3.6; Equation 6; Lemma 3.7; Proof of Lemma 3.7; Lemma 3.8; Equation 8; Proof of Theorem 3.3.The authors do not agree to the retraction of the Article."}
+{"text": "Moreover, the aggregation of A\u03b2 is increased by oxidative stress, and the neurotoxicity induced by the oligomers and fibrils is in part mediated by free radicals. Interestingly, it has been reported that oxidative stress can also induce BACE1 transcription and expression. BACE1 is the key enzyme in the cleavage of the amyloid precursor protein to produce A\u03b2, and the expression of this enzyme has been previously shown to be enhanced in the brains of Alzheimer's patients. Here, we have found that BACE1 expression is increased in the hippocampi from AD patients at both the early (Braak stage II) and late (Braak stage VI) stages of the disease as studied by immunohistochemistry and western blot. To address the role of A\u03b2 and oxidative stress in the regulation of BACE1 expression, we have analyzed the effect of subtoxic concentrations of A\u03b2 oligomers (0.25\u2009\u03bcM) and H2O2 (10\u2009mM) on a human neuroblastoma cell line. Firstly, our results show that A\u03b2 oligomers and H2O2 induce an increase of BACE1 mRNA as we studied by qPCR. Regarding BACE1 translation, it is dependent on the phosphorylation of the eukaryotic initiation factor 2\u03b1 (eIF2\u03b1), since BACE1 mRNA bears a 5\u2032UTR that avoids its translation under basal conditions. BACE1 5\u2032UTR contains four upstream initiating codons (uAUGs), and its translation is activated when eIF2\u03b1 is phosphorylated. Consistently, we have obtained that A\u03b2 oligomers and H2O2 increase the levels of BACE1 and p-eIF2\u03b1 assayed by western blot and confocal microscopy. Our results suggest that A\u03b2 oligomers increase BACE1 translation by phosphorylating eIF2\u03b1 in a process that involves oxidative stress and conforms a pathophysiological loop, where the A\u03b2 once aggregated favors its own production continuously by the increase in BACE1 expression as observed in AD patients.Alzheimer's disease (AD) is tightly linked to oxidative stress since amyloid beta-peptide (A AD symptoms consist in dramatic memory deficits and an irreversible cognitive decline. They start with neuronal death in the hippocampus and the brain structure for learning and memory, and later, the neuronal loss progresses to other cortical areas. The histopathological characteristics of AD patients are extracellular senile plaques and intracellular neurofibrillary tangles. The senile plaques are mainly composed of amyloid  protein , 2.\u03b2 is a peptide having from 36 to 43 amino acids [\u03b21-40 is the most abundant. A\u03b2 is produced by the consecutive enzymatic action of the beta- and gamma-secretase activities on an integral type I transmembrane glycoprotein termed amyloid precursor protein (APP) [\u03b21-42 production increases with aging [\u03b2 species production is due to the high aggregant properties of the A\u03b21-42 [Ath aging , in patith aging . The pate A\u03b21-42 , 9, whic\u03b2 [\u03b2 aggregates produce H2O2 [\u03b2 production pathway.The oligomeric forms are the responsible for the most neurotoxic effects associated to the A\u03b2 \u201312. A\u03b2 auce H2O2  inducinguce H2O2 , 15. Moruce H2O2 , 17. BACBACE1 gene promoter [BACE1 mRNA bears a particular 5\u2032UTR that contains four upstream initiation codons (uAUGs) and possesses a rich GC content. These sequence motifs confer a particular secondary structure to that region of the mRNA that impairs ribosomes to reach the main AUG in order to start BACE1 translation [BACE1 mRNA translation is only activated when the eukaryotic initiation factor 2\u03b1 (eIF2\u03b1) is phosphorylated at serine 51 [\u03b1 phosphorylation is to block the translation of the most of the proteins under stressful conditions and to induce only the translation of a group of special proteins that bear 3 or more uAUGs in the 5\u2032UTR [\u03b1 kinases: heme-regulated eukaryotic initiation (HRI), general control nonderepressible 2 kinase (GCN2), double-stranded RNA-activated protein kinase (PKR), and double-stranded RNA-activated protein kinase-like (PERK), which phosphorylate eIF2\u03b1 at serine 51 blocking translation initiation [BACE1 expression is physiologically repressed at the transcriptional and translational level. There are different pathways, mostly related to different types of stress, that induce the nuclear translocation of transcription factors, such as cJun/cFos, to bind to promoter , 19. Hownslation \u201323. In berine 51 \u201326. The he 5\u2032UTR . These sitiation \u201330.\u03bcm) were treated with 4% H2O2 and incubated o.n. at 4\u00b0C with 1\u2009:\u2009100 rabbit anti-BACE1 Antibody . The secondary Ab was 1\u2009:\u2009500 donkey anti-rabbit peroxidase-conjugated Ab, which was incubated for 1\u2009h at room temperature (RT). A Peroxidase Substrate Kit DAB (Vector) was used. Slides were stained with hematoxylin and fixed. The images were taken by a Leica DMR microscope.Human hippocampal samples were supplied by the Neurological Tissue Bank of the Biobank-Hospital Cl\u00ednic-IDIBAPS, Barcelona, Spain. The procedure was carried out according to the rules of the Helsinki Declaration and to the Ethics Committee of the Institut Municipal d'Investigacions M\u00e8diques-Universitat Pompeu Fabra (EC-IMIM-UPF). Hippocampal samples were obtained from 3 nondemented controls , 2 AD patients at Braak stage II , and 4 AD patients at Braak stage VI . Sections , 50\u2009mM Tris-HCl, 1\u2009mM dithiothreitol, 1\u2009mM sodium orthovanadate, and protease inhibitor cocktail (Roche), pH\u20098. Membranes were blocked for 1\u2009h at RT with Tween 20-Tris buffer solution  plus 5% skimmed milk. Then, membranes were incubated overnight (o.n.) at 4\u00b0C with 1\u2009:\u20094,000 mouse anti-actin Ab (Sigma) or 1\u2009:\u20091,000 rabbit anti-BACE1 Ab. 1\u2009:\u20092,000 secondary Abs were horseradish peroxidase-conjugated donkey anti-mouse and anti-rabbit , which were incubated for 1\u2009h at RT. Bands were visualized with Super Signal (Pierce) and analyzed with the Quantity One system in a BioRad Universal Hood II.Human brain tissue sections, obtained as indicated in the subsection 2.1., were lysed with 50\u20092.Human neuroblastoma cells (SH-SY5Y cells) were grown with Ham's F12 GlutaMax  supplemented with 15% fetal bovine serum  and 1% penicillin/streptomycin (Gibco). Cells were incubated at 37\u00b0C in a humidified atmosphere of 5% CO\u03b21-42 (Anaspec) went to solution with 250\u2009\u03bcL of ultrapure MilliQ water. Therefore, we adjusted pH to \u226510.5 with NaOH (1\u2009M) to keep away from the A\u03b2 isoelectric point. The solution was sonicated for 1\u2009min in 250\u2009\u03bcL of phosphate buffer . To favor the proper oligomer formation, the A\u03b2 aliquots were dissolved in F12 medium at 0.4\u2009mg/mL and incubated for 24 at 4\u00b0C.1\u2009mg of lyophilized human A4 cells/well. After 12\u2009h, the growth medium was removed. Cells were treated with increasing concentrations of A\u03b21-42 oligomers or H2O2 (Sigma) in F12 medium. Treatments were carried out along 24\u2009h at 37\u00b0C. Then, cell survival was assayed by 3--2,5-diphenyltetrazolium bromide (MTT) reduction method. It consists of the addition of 10% (regarding cell medium volume) of MTT stock solution at 5\u2009mg/mL. Cells were incubated with the MTT for 2\u2009h. Medium was discarded and 100\u2009\u03bcL of DMSO were placed per well. MTT absorbance was measured in an absorbance plate reader (BioRad) at A540\u2009nm and A650\u2009nm (as reference). Control cells treated with phosphate buffer saline (PBS) were the 100%. Bridge field images were obtained with a Leica DM IL microscope.SH-SY5Y cells were seeded in a 96-well plate at a density of 2.5 \u00d7 104 cells/well. After 12\u2009h, the growth medium was removed and cells were treated for 24\u2009h with subtoxic concentrations of A\u03b21-42 oligomers (0.25\u2009\u03bcM) or H2O2 (10\u2009\u03bcM) and the toxic ones for A\u03b21-42 oligomers (15\u2009\u03bcM) and H2O2 (100\u2009\u03bcM) in F12 medium. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 at RT. Coverslips were incubated o.n. at 4\u00b0C with 1\u2009:\u2009100 rabbit anti-cleaved Caspase-3 Ab . Cells were washed thrice and incubated with 1\u2009:\u20092,000 Alexa Fluor 555 goat anti-rabbit Ab (Life Technologies) and Hoechst 1\u2009:\u200910,000 (Thermo Scientific) for 1\u2009h at RT. Coverslips were mounted with Fluoromount (Southern Biotech). Digital images were taken with a Leica TCS SP confocal microscope and analyzed with the Leica confocal software.Cells were seeded on coverslips in 24-well plates at a density of 3 \u00d7 105 cells/dish. After 12\u2009h, the growth medium was removed and cells were treated for 24\u2009h with subtoxic concentrations of 0.25\u2009\u03bcM A\u03b21-42 oligomers or 10\u2009\u03bcM H2O2 in F12 medium. The mRNA was extracted by using the NucleoSpin RNA extraction kit (Macherey Nagel) and quantified with NanoDrop ND-1000 (Thermo Fisher Scientific). BACE1 and HPRT cDNAs were obtained with the SuperScript III Reverse Transcriptase (Invitrogen). Finally, a quantitative PCR was performed by using the fluorophore Sybr Green (Thermo Fisher Scientific). Samples were quantified with QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific).Cells were seeded on 60\u2009mm Petri dishes at a density of 6 \u00d7 105 cells/well. After 12\u2009h, the growth medium was removed and cells were treated for 24\u2009h with subtoxic concentrations of 0.25\u2009\u03bcM A\u03b21-42 oligomers or 10\u2009\u03bcM H2O2 in F12 medium. To study phosphorylated proteins, the cells were lysed on ice with lysis solution as indicated in subsection 2.2. Extracts were homogenised using vortex for 30\u2009min at 4\u00b0C; afterwards, samples were centrifuged at 10,000 for 5\u2009min to obtain the supernatant. A Bio-Rad kit was used to calculate protein concentrations. Aliquots of 20\u2009\u03bcL (for phosphorylated proteins) or 80\u2009\u03bcg (for the other proteins) were loaded into a 10% SDS-PAGE gels. Afterwards, proteins were transferred onto 0.2\u2009\u03bcm pore nitrocellulose membranes. Membranes were blocked for 1\u2009h at RT with TTBS plus 5% bovine serum albumin (BSA) for phosphorylated proteins or 5% skimmed milk for the other proteins. Then, membranes were incubated o.n. at 4\u00b0C with 1\u2009:\u20091,000 rabbit anti-BACE1 Ab (Invitrogen), 1\u2009:\u2009500 rabbit anti-p-eIF2\u03b1 (Ser51) Ab (Invitrogen), 1\u2009:\u2009500 mouse anti-eIF2\u03b1 Ab (Abcam), and 1\u2009:\u20095,000 mouse anti-tubulin Ab (Sigma). 1\u2009:\u20092,000 secondary Abs were horseradish peroxidase-conjugated donkey anti-mouse and anti-rabbit  for 1\u2009h at RT. Bands were visualized with Super Signal (Pierce) and analyzed with the Quantity One system in a BioRad Universal Hood II.Cells were seeded on 6-well plates at a density of 3 \u00d7 104 cells/well. After 12\u2009h, the growth medium was removed, and cells were treated for 24\u2009h with subtoxic concentrations of 0.25\u2009\u03bcM A\u03b21-42 oligomers or 10\u2009\u03bcM H2O2 in F12 medium plus 15% FBS. Then, cells were fixed with 4% paraformaldehyde (PFA). Cells were permeabilized with 0.1% Triton X-100. Coverslips were incubated o.n. at 4\u00b0C with 1\u2009:\u2009100 mouse anti-eIF2\u03b1 Ab, rabbit anti-p-eIF2\u03b1 Ab, or rabbit anti-BACE1 Ab. After primary Abs, cells were incubated with 1\u2009:\u20092,000 Alexa Fluor 555 goat anti-rabbit Ab or 1\u2009:\u20092,000 Alexa Fluor 647 goat anti-mouse Ab (Life Technologies) for 1\u2009h at RT. Coverslips were mounted with Fluoromount. Digital images were taken with a Leica TCS SP confocal microscope and analyzed with Leica confocal software. Immunofluorescence was quantified by ImageJ program.Cells were seeded on coverslips in 24-well plates at a density of 3 \u00d7 10t-test using the GraphPad software.Data are expressed as mean \u00b1 SEM of n experiments as indicated in the corresponding figures. Statistical analyses were performed by one-way ANOVA using Tukey's posttest or Student's \u03b2 production that will induce the onset and progression of the disease. Consistently, A\u03b21-42 oligomers have been reported to be present in the hippocampi from AD patients since the early stages of the disease [\u03b2 is widespread in the brain and dementia is severe). We found in both stages an increased expression of BACE1 . The maintained BACE1 expression even in the late stage of the disease suggest that oxidative stress, which has been reported to be significantly increased in AD [\u03b21-42 oligomers, which generate free radicals, as we address in the present work.There are previous works that reported increased expression of BACE1 in AD patients \u201333 relat disease . Here, wof BACE1 , linkingern blot  when theed in AD , would b\u03b21-42 for 24\u2009h (\u03b21-42 oligomers were significantly neurotoxic at 5\u2009\u03bcM (p < 0.005), 10\u2009\u03bcM (p < 0.001), and 15\u2009\u03bcM (p < 0.001). The production of H2O2 by the A\u03b21-42 oligomers is a continuous process, while the A\u03b21-42 is present, along 24\u2009h in our experiments. Therefore, we have also studied the effect of the treatment with H2O2 on cell viability (2O2 is cytotoxic at 100\u2009\u03bcM (p < 0.001).Human neuroblastoma cells were treated with increased concentration of oligomeric Afor 24\u2009h  in orderiability . We have\u03bcM A\u03b21-42 oligomers and 100\u2009\u03bcM H2O2 showed a low reduction of MTT and high caspase activation as expected.The cell cytotoxicity was also studied by images obtained after the reduction of the MTT to blue formazan by bright field microscopy Figures  and with\u03bcM A\u03b21-42 oligomers and 10\u2009\u03bcM H2O2. These concentrations were producing neither cytotoxicity when assayed by MTT reduction . The treatment with 10\u2009\u03bcM H2O2 for 24\u2009h produce the same effect on BACE1 mRNA transcription (p < 0.05). These results suggest that both A\u03b21-42 oligomers and H2O2 share common mechanisms at the transcriptional level.Human neuroblastoma cells were treated with 0.25\u2009\u03bcM A\u03b21-42 oligomers increased BACE1 expression after 24\u2009h as it was analyzed by western blot (p < 0.05 by western blot and p < 0.001 by immunofluorescence). Similar results were obtained when cells were treated with 10\u2009\u03bcM H2O2 for 24\u2009h (p < 0.05 by western blot and p < 0.001 by immunofluorescence). Interestingly, the phosphorylation of eIF2\u03b1 was increased with both 0.25\u2009\u03bcM A\u03b21-42 oligomers and 10\u2009\u03bcM H2O2  supporting that BACE1 expression is due to an increase in its translation. These results also correlated with the increased fluorescence showed by p-eIF2\u03b1 after the treatment with 0.25\u2009\u03bcM A\u03b21-42 oligomers and 10\u2009\u03bcM H2O2 (Figures Attending to the effects on BACE1 translation, we have obtained that 0.25\u2009 Figures .\u03b2, which increases by oxidative stress [2O2 [\u03b2 aggregates has been reported to be mediated by oxidative stress [\u03b2 aggregation is considered the key factor of AD, according to the amyloid cascade hypothesis, we have studied the effect of A\u03b2 oligomers and oxidative stress in BACE1 expression and the mechanisms that control its pathophysiological expression.There are many evidences that relate oxidative stress with the ethiopathogenesis of AD, a devastating neurodegenerative disease whose major risk factor is aging. This tight relationship starts with the aggregation of Ae stress . Once thess [2O2  and hydress [2O2 , 37. In e stress , 38, 39.e stress , 17. We \u03b21-42 oligomers, mimicking the environment of neurons in AD patients, increase BACE1 transcription and expression in vitro. The relevance of this finding is supported by the increased expression of BACE1 found in AD hippocampi. This would suggest the existence of a loop of amyloid production that will activate BACE1 to release more amyloid contributing to accelerate the dysregulation of BACE1 and the characteristic amyloidosis of AD. This effect should be based in the capability of A\u03b21-42 oligomers to produce oxidative stress since similar results has been obtained when we used hydrogen peroxide.BACE1 is an enzyme that accomplishes some physiological functions as dendritic spine growth in the hippocampus where contributes to memory formation . In fact\u03b1 carried out by the enzyme HRI [\u03b1, can be activated by oxidative stress [\u03b21-42 oligomers yield to the phosphorylation of the eIF2\u03b1 in vitro. The dysregulation of the different eIF2\u03b1 kinases by oxidative stress would explain these results.BACE1 expression is controlled physiologically by the phosphorylation of eIF2zyme HRI . HRI is zyme HRI , 43 and e stress \u201348. In o\u03b2 oligomers increase BACE1 transcription and translation. Our data support that the mechanism involved in the increased BACE1 expression is the dysregulation of the phosphorylation of the eIF2\u03b1 that would generate the amyloid burden in AD.Summarizing, our results suggest that oxidative stress induced by A"}
+{"text": "Drosophila TLX homologue, Tailless, initiate tumourigenesis by reverting intermediate neural progenitors to a stem cell state. Strikingly, we could block tumour formation completely by re-expressing Asense (homologue of human ASCL1), which we show is a direct target of Tailless. Our results predict that expression of TLX and ASCL1 should be mutually exclusive in glioblastoma, which was verified in single-cell RNA-seq of human glioblastoma samples. Counteracting high TLX is a potential therapeutic strategy for suppressing tumours originating from intermediate progenitor cells.Understanding the sequence of events leading to cancer relies in large part upon identifying the tumour cell of origin. Glioblastoma is the most malignant brain cancer but the early stages of disease progression remain elusive. Neural lineages have been implicated as cells of origin, as have glia. Interestingly, high levels of the neural stem cell regulator TLX correlate with poor patient prognosis. Here we show that high levels of the  The underlying mechanisms of glioblastoma initiation and growth have proved challenging to elucidate. This is due, in part, to the extensive molecular heterogeneity of glioblastoma, both between patients and within individual tumours. As such, there are many potential routes to tumourigenesis and the cell fate changes that contribute to glioblastoma initiation and progression remain to be fully elucidated. Cell fates can be altered in many different ways during tumourigenesis, depending upon the combination of genetic mutations present and the tumour cell of origin.Mouse models have revealed many of the different cell types that can give rise to glioblastoma. In the central nervous system (CNS), tumours have been induced experimentally from differentiated glial cells, glial precursors and neural stem/progenitor cells . A recenDrosophila CNS has proved extremely valuable for understanding the fundamental principles of cancer  have been observed in glioblastoma and been shown to correlate with poor patient prognosis . TLX is Drosophila, different NSC lineages exhibit distinct vulnerabilities to tumour-inducing mutations , is required to direct the identity of Type II NSCs during development. We found that high levels of Tll are sufficient to initiate tumours from differentiating Type II NSC lineages by directing a cell fate change from INP to NSC. To identify downstream effectors of Tll action, we mapped the genome-wide targets of Tll and identified the proneural gene asense as a direct target of Tll repression, both during development and in tumourigenesis. Strikingly, we were able to rescue Tll tumours completely, and restore normal neurogenesis, by re-expressing asense. Our results demonstrate a reciprocal relationship between Tll and Asense expression and we hypothesized that this relationship might hold true in glioblastoma. We found that expression of TLX and ASCL1 (human counterparts of Tll and Asense) are also mutually exclusive in glioblastoma, suggesting a potentially conserved route to tumourigenesis.Here we show that the tll mRNA in Type II NSCs but not in their progeny (INPs) (Drosophila Type II lineages (To understand the role Tailless (Tll) plays in the development of Type II NSC lineages, we first assessed its expression pattern. We found that Tll was expressed in Type II NSCs throughout larval development . We detey (INPs) , while Ty (INPs) . Tll shay (INPs) : their Dy (INPs)  and recry (INPs) . TLX is y (INPs) . In the y (INPs) , which ilineages .wor-GAL4 using two independent RNAi constructs that target different regions of the tll coding\u00a0sequence   clones  . We also) clones . We foun) clones , demonstlineages . This sulineages . Furtherl brains .tll RNAi  with pntP1-GAL4  in combitP1-GAL4  and foll II NSCs , suggest+ Pros+. In addition, the Ets transcription factor PointedP1 (PntP1) is expressed in Type II NSCs and immature INPs but not in Type I lineages (earmuff (erm), which is expressed from INPs onwards  to their daughter cells (GMCs). In GMCs, Ase is expressed and Pros, a pro-differentiation transcription factor, translocates to the nucleus. In contrast, Type II NSCs express Dpn but not Ase or Pros. Type II NSCs give rise to INPs, which express Dpn, Ase and cortical Pros. INPs then generate GMCs that are Aselineages . Type II onwards  but not tll knockdown, no INPs  could be found in Type II lineages and instead GMCs (Ase+ Pros+) were positioned adjacent to the NSCs   might convert them to a Type II fate . To determine if tumour initiation occurred via conversion of Type I to Type II NSCs, we assessed the expression of Ase in tumours in the ventral nerve cord, which normally contains only Type I NSCs. Remarkably, Tll-induced tumours consisted almost entirely of NSCs that were negative for Ase, indicating a Type II-like NSC fate  was associated with a reduction in GMCs and neurons, as assessed by expression of Pros  and were then transformed into Type II NSCs (erm-GAL4 negative). We conclude that Tll expression is sufficient to induce a cell fate change from INP to NSC and our results implicate INP reversion to NSCs as the mechanism of tumour initiation.To determine the cell of origin of Tll-induced tumours, we used G-TRACE   to follol brains . Expresspression . This woerm-GAL4 expression only   . We profD (TaDa)  and idenDa)  (As ectopic expression of Tll results in repression of (n\u00a0=\u00a010) . Strikin(n\u00a0=\u00a010) . However(n\u00a0=\u00a010)  nor does(n\u00a0=\u00a010) . Therefo(n\u00a0=\u00a010) .Drosophila, Tll represses Ase both during development and in tumourigenesis. In other words, high levels of Tll correspond to low levels of Ase.We showed that, in In human glioblastoma, high TLX expression is correlated with poor patient prognosis . IntriguTo determine if TLX and ASCL1 expression are mutually exclusive in glioblastoma, we analysed a previously published single-cell RNA sequencing (scRNA seq) data set that profiled glioblastoma samples from 28 patients, including both adult and pediatric tumours . Our anaDrosophila CNS , Ay-GAL4, UAS-lacZ(nls) (BL4410), btd-GAL4 (MZ1407) (14-94-GAL4 (OK371VGlut-GAL4) (BL26160). tub-GAL80ts (BL7018) was used to restrict GAL4 activity to larval stages as indicated.The following GAL4 lines were used: Ay-GAL4, UAS-btd-GAL4 ,\u00a0erm-GALbtd-GAL4  (R9D11-Gbtd-GAL4  (BL39575(MZ1407) , pntP114-94-GAL4 , wor-GAL-94-GAL4 , OK371-Gase (FLP (BL4539 and BL4540), UAS-lacZ (LT3-NDam (LT3-NDam-tll (this study), UAS-mCD8-GFP (BL5130 and BL5137), UAS-mCD8-mCherry (BL27391), UAS-myr-mRFP (BL7118\u00a0and\u00a0BL7119), UAS-tll (tll-miRNA[s] (tll-shRNA (VDRC 330031), UAS-TLX (this study), G-TRACE (BL28280 and BL28281)1118. w was used as a reference stock.The following UAS-transgenes were used: UAS-ase , UAS-FLPUAS-lacZ , UAS-LT3LT3-NDam  and UAS- UAS-tll   (erm-mCD8-GFP (R9D11-mCD8-GFP) (erm-lacZ (R9D11-lacZ) . Pnt-GFP (BL42680) is a protein fusion under the control of a 90.7 kb insert containing the pnt coding sequence and surrounding regulatory sequences. This construct can rescue the lethality of pnt amorphic heteroallelic combinations (The following reporter lines were used: dTomato) , erm-mCDCD8-GFP) , erm-lac11-lacZ)  and Tll-11-lacZ)  ; FRT82B, tub-GAL80 were crossed to male flies carrying w; erm-lacZ; FRT82B or w; erm-lacZ; FRT82B, l49tll/TM6B. l49tll is strong tll point mutation that is homozygous embryonic lethal . Samples were blocked with 10% normal goat serum before overnight incubation with the following antisera: rabbit anti-Ase 1:2,000  a gift , rat anttll coding sequence and labeled with Quasar 570. Third instar larval brains were fixed in 4% formaldehyde/PBS for 45 min at room temperature and then permeabilized in 70% ethanol/PBS for 6 hr at 4 \u00b0C. Brains were washed with Wash Buffer  for 5 min before being incubated with probes (125 nM) in hybridisation buffer  overnight at 45 \u00b0C. Brains were washed with Wash Buffer, stained with DAPI and mounted in Vectashield (Vector Laboratories) for imaging.A set of 38 Stellaris FISH probes was designed against the Fluorescent images were acquired using a Leica SP8 confocal microscope. Images were analysed using Fiji , which wwww.graphpad.com) was used for statistical analysis. No data were excluded.GraphPad Prism version 7.00 for Mac OS X (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) and UniProt alignment  tools.Sequence alignment performed using EMBOSS Needle (TLX (AGATGAATTCATGAGCAAGCCAGCCGG-3\u2019 and rev: 5\u2019-ATGACTCGAGTTAGATATCACTGGATTTGTACATATCTGAAAGCAGTC-3\u2019. The amplified product was cloned (using restriction enzymes EcoR1 and XhoI) into pUAST-attB and then integrated into attP40 by standard methods.The coding sequence of human TLX  was ampltll was amplified from an embryonic cDNA library using the primers fwd: 5\u2019-cagaaactcatctctgaagaggatctgcgagatctaATGCAGTCGTCGGAGG-3\u2019 and rev: 5\u2019 acagaagtaaggttccttcacaaagatcctctagaTCAGATCTTGCGCTGACT 3\u2019. The amplified product was cloned via Gibson assembly into pUASTattB-LT3-NDam  and the Gene Expression Omnibus (GEO: GSE131928).Single cell sequencing analysis was performed using Seurat version 3. Data was obtained from  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This paper demonstrates very elegantly that the transcription factor Tailless that is expressed in type II neuroblasts is required to repress Asense (the ortholog of ASCL1), and that its loss leads to the switch of identity from type II to type I neuroblasts. Interestingly, the paper shows that tumorgenesis resulting from overexpression of Tll is due to the reversion of intermediate neural progenitors to dividing neuroblasts, and that this is due to the Ase down-regulation. But the most exciting point of this paper is the demonstration that these brain tumors can be avoided by restoring Ase expression, which might be an important tool to eventually address why human glioblastoma have a poor prognosis when ASCL1 is also affected.Decision letter after peer review:asense/ASCL1\" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Utpal Banerjee as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Yan Song (Reviewer #1).Thank you for submitting your article \"Tailless/TLX reverts intermediate neural progenitors to stem cells driving tumourigenesis via repression of The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.In short, the three reviewers are very positive about the paper and its relevance to human cancer.eLife. However, all three reviewers are conscious of the mechanistic insights that your paper provides, and, more importantly, the connection to human brain tumors and the different prognosis depending on the levels of ASCL1/Ase.One of the reviewers mentioned that the fact that overexpression of Tll causes brain tumors was known and so was its role in Type II/Type I neuroblasts, in particular with a recent paper from the Thor lab in However, before proceeding with publication, the reviewers would like you to add two points, which you should be able to achieve in the coming two months:\u2013 One is to present the DamID data in more detail, in particular genes other than Ase that might play a role in the process.\u2013 The reviewers would also like you to explore the link between Tll and the Notch pathway since some of the phenotypes are quite similar. It should be fairly simple in your hands to look at interactions between these pathways.Reviewer #1Drosophila larval brain neural stem cell lineages as model system to tackle these important questions. The authors showed that fly TLX homolog, Tailless (Tll), is specifically expressed in type II NSCs. Downregulation of Tll led to upregulation of Asense  and a NSC identity switch from type II to type I. The authors further showed that high levels of Tll induced tumorigenesis by reverting intermediate neural progenitors (INPs) to a NSC state and identified INPs as the cellular origin of Tll-induced tumors. More importantly, the authors identified Ase as a key direct target of Tll. Ase is downregulated in high Tll-induced brain tumors and restoring Ase expression prevented Tll-induced tumorigenesis and reinstated normal neurogenesis. Finally, the authors suggested that such mutually exclusive relationship between Tll and Ase holds true in human glioblastoma samples via analysis of single-cell RNA-seq.The orphan nuclear receptor TLX is an important neural stem cell regulator in both normal and tumorigenic conditions. TLX is expressed in neural stem cells (NSCs) during mouse embryonic development and in adulthood and is required for neurogenesis. High levels of TLX have been detected in glioblastoma and correlate with poor patient prognosis. However, the cellular origin and the molecular mechanisms underlying high TLX-induced brain tumor formation remain unclear. In the study, the authors used Overall, this is a very interesting study of potential therapeutic values. Most experiments are well-controlled and well performed. However, before recommending publication the following points need be addressed:1) Through DamID-seq, the authors identified Ase as the major target of Tll in NSC lineages. Indeed, high Tll-induce tumor could be completely blocked by Ase co-expression. However, since Ase inactivation itself does not lead to tumor formation , downregulation of Ase seems to be a permissive but not sufficient step in Tll-induced tumorigenesis. Therefore, other self-renewal or differentiation gene(s) is very likely to be important target gene of Tll in NSC lineages. Can the authors re-examine their DamID-seq results and find out such important target gene(s)?2) Figure 4 showed that Tll can induced tumor from type I NSCs. However, downregulation of Ase alone might lead to a type I to type II NSC identity switch, but not tumorigenesis . What is the cellular origin and molecular mechanisms underlying Tll-induced tumorigenesis in type I NSC lineages?3) Whether Ase, in turn, inhibits Tll expression in INPs or type I NSCs?Reviewer #2tll in the developing Drosophila CNS, in Type II neuroblasts (NBs). They identify ase as a key target of tll, and find that simultaneous overexpression of ase can suppress the tll overexpression effects. They furthermore use DamID to identify ase as a putative direct target of Tll. These are very interesting findings. However, three major points temper my enthusiasm for this study.They identify a role for Major points:tll in Drosophila NB biology is not novel. tll was previously shown to be important for brain NB generation  and MBNB proliferation . Even more importantly, tll was recently show to be expressed in Type II NBs in the embryo and necessary for their generation/development, and tll could act with erm to convert Type I NBs in the embryonic nerve cord to Type II NBs, as well as act in the developing wing disc to generate ectopic Type II NBs . Against this backdrop, the current study does take major strides in our understanding of Type II NB biology.1) The role of tll and these other regulators is not addressed (at least 24 publications). In particular, the connection between Notch pathway and tll is certainly worthy of more scrutiny, as Notch signalling, similar to tll, acts in Type II to repress Ase and Erm expression, hence preventing NBs from prematurely becoming INPs. Moreover, there is growing connection between tll/Tlx and Notch: tll mutants show loss of expression of the proneural gene l'sc , which is negatively regulated by Notch; tll and Notch signalling intersect in the developing Drosophila embryonic optic placodes ; the C. elegans tll orthologue nhr-67 regulates both lin-12 (Notch) and lag-2 (Delta) during uterus development ; mouse tll orthologue Nr2E1 (aka Tlx) negatively regulates the canonical Notch target gene Hes1 . Hence, a key issue to address would be the intersection between tll and Notch, and given that NHRs are able to act both as transcriptional repressors and activators, two simple models emerge: (A) Tll acts with NotchICD-Su(H)-Mam on E(spl), and the obligate E(spl) repressors then act to directly repress ase, (B) alternatively, Tll acts combinatorially with E(spl) to directly repress ase. A straightforward way to test these two models would be to analyse expression of E(spl)-reporters in Tll LOF and GOF. In addition, it would be important to analyse Tll/tll expression in Notch pathway GOF and LOF.2) In the same vein, there is a large body of work on the NB->INP->GMC->Neuron transition in Type II NBs. This has identified key \"gating\" roles for Notch-Dpn-E(spl), as well as for Brm, Brat, Klu, PntP1, Btd, Erm, Mediator-Complex, Barc and Trx. However, the possible connection between pnt, erm, E(spl), btd, klu, pros, dpn? Importantly, the evidence for Tll binding to the ase gene, but possibly also to dpn and E(spl), has bearing on the model for tll action. In addition, the DamID results are not described in great detail.3) DamID: Does Tll-Dam bind to other genes in the NB-INP-GMC-neuron pathway e.g., Reviewer #3Drosophila Tailless. It covers both the role of Tll in normal neurogenesis and the tumourigenic effect of Tll overexpression.The Hakes and Brand manuscript contains detailed insightful information on the function of tll is required to maintain the Type II state: strikingly, upon tll loss, Type II neural stem cells are transformed into Type I, hence significantly compromising neurogenesis due to the lack of intermediate neural progenitors. They also show tll levels are critical: TLL over expression in the larval brain causes tumours derived from both Type II and Type I lineages. In the former, tumours develop as a consequence of intermediate progenitors reverting to a neural stem cell-like state. In the later, ectopic Tll results in the loss of Ase and in some cases even in the generation of intermediate neural progenitors, both suggestive of a certain degree of transdetermination from Type I towards Type II neural stem cells.The authors show that The manuscript goes on to demonstrate that Asense is a direct target of Tll repression during normal development as well as in the tumours that develop upon Tll upregulation to the extent that forcing Ase expression suppresses Tll-induced tumoursDrosophila Tll, has been linked to the development of malignant astrocytomas. Interestingly, the authors find that TLX and ASCL1 (human Ase) are mutually exclusive in cells from human glioblastomas, which indeed is closely reminiscent of the results reported in the manuscript regarding fly neurogenesis and brain tumour development.Overexpression of the human homologue TLX, which shares a remarkable level of protein sequence identity, cofactors, and function with The reported studies take advantage of state-of-the-art techniques to perform sophisticated cell type-specific experimental manipulations to create the mutant conditions, and to trace the cell lineages of interest. The results are very well documented, presented, and discussed. Altogether, this is a high quality manuscript that I am happy to recommend for publication. Reviewer #1[\u2026]Overall, this is a very interesting study of potential therapeutic values. Most experiments are well-controlled and well performed. However, before recommending publication the following points need be addressed:1) Through DamID-seq, the authors identified Ase as the major target of Tll in NSC lineages. Indeed, high Tll-induce tumor could be completely blocked by Ase coexpression. However, since Ase inactivation itself does not lead to tumor formation , downregulation of Ase seems to be a permissive but not sufficient step in Tll-induced tumorigenesis. Therefore, other self-renewal or differentiation gene(s) is very likely to be important target gene of Tll in NSC lineages. Can the authors reexamine their DamID-seq results and find out such important target gene(s)?ase, Tll has multiple targets that could contribute to the Tll phenotypes we observe .We have included the full list of Tll target genes as an excel file . In addition to 2) Figure 4 showed that Tll can induced tumor from type I NSCs. However, downregulation of Ase alone might lead to a type I to type II NSC identity switch, but not tumorigenesis . What is the cellular origin and molecular mechanisms underlying Tll-induced tumorigenesis in type I NSC lineages?We showed that Tll must be downregulated in Type II lineages to allow differentiation to proceed. When expressed in Type I NSCs, Tll induces a cell fate change from Type I to Type II NSC and Tll is maintained at high levels in these transformed lineages. Once a Type I NSC has been transformed into a Type II NSC, differentiation is blocked due to continued high levels of Tll, which results in tumourigenesis. Tumourigenesis likely occurs through the inactivation of Ase in addition to other Tll target genes (see above).3) Whether Ase, in turn, inhibits Tll expression in INPs or type I NSCs?We show that ectopic expression of Ase in Type II NSCs does not repress Tll nor does expressing Ase in Tll-induced tumours. We have added these data as Figure 6\u2014figure supplement 2. Therefore, Ase does not act directly on Tll but through other downstream targets.Reviewer #2Drosophila CNS, in Type II neuroblasts (NBs). They identify ase as a key target of tll, and find that simultaneous overexpression of ase can suppress the tll overexpression effects. They furthermore use DamID to identify ase as a putative direct target of Tll. These are very interesting findings. However, three major points temper my enthusiasm for this study.They identify a role for tll in the developing Major points:Drosophila NB biology is not novel. tll was previously shown to be important for brain NB generation  and MBNB proliferation . Even more importantly, tll was recently show to be expressed in Type II NBs in the embryo and necessary for their generation/development, and tll could act with erm to convert Type I NBs in the embryonic nerve cord to Type II NBs, as well as act in the developing wing disc to generate ectopic Type II NBs . Against this backdrop, the current study does take major strides in our understanding of Type II NB biology.1) The role of tll in tll null mutants are not viable and the effect of tll loss of function on Type II NSCs specifically has never been addressed. We have identified a previously unknown role for Tll in larval Type II NSCs. Our data show that Tll is necessary and sufficient to promote Type II NSC fate. In the absence of Tll, Type II NSCs switch to a Type I identity, whereas ectopic expression of Tll alone is sufficient to induce Type II NSC fate in Type I NSCs.tll mutant embryos fail to generate many different types of NSC (not just Type II NSCs) due to lack of l\u2019sc expression, which precedes NSC delamination. This would explain why Curt et al., 2019 found that tll mutant embryos lack Type II NSCs. Curt et al. promoted Type II NSC identity in the embryonic brain by co-expression of Tll and Erm, not Tll alone. Later in development, Tll is indeed required for the proliferation of larval mushroom body NSCs and GMCs , demonstrating that Tll has diverse roles in brain development.It was shown some years ago  that We have added these points to our Discussion.2) In the same vein, there is a large body of work on the NB->INP->GMC->Neuron transition in Type II NBs. This has identified key \"gating\" roles for Notch-Dpn-E(spl), as well as for Brm, Brat, Klu, PntP1, Btd, Erm, Mediator-Complex, Barc and Trx. However, the possible connection between tll and these other regulators is not addressed (at least 24 publications). [\u2026] Hence, a key issue to address would be the intersection between tll and Notch, and given that NHRs are able to act both as transcriptional repressors and activators, two simple models emerge: (A) Tll acts with NotchICD-Su(H)-Mam on E(spl), and the obligate E(spl) repressors then act to directly repress ase,ase locus. Therefore, the simplest explanation is that Tll acts directly on ase. Furthermore, we found that expression of the Notch signalling reporter E(spl)-m\u03b3-GFP  remains unchanged in Tll LOF Type II NSCs (see We show that Tll binds directly to the NSCs see . These d(B) alternatively, Tll acts combinatorially with E(spl) to directly repress ase. A straightforward way to test these two models would be to analyse expression of E(spl)-reporters in Tll LOF and GOF.+ cells in Tll GOF lineages. Indeed, we found that ectopic Type II NSCs in Tll GOF lineages express the Notch signalling reporter E(spl)-m\u03b3-GFP  , we would expect to see an increase in E(spl)-m\u03b3-GFP005) see .In addition, it would be important to analyse Tll/tll expression in Notch pathway GOF and LOF.+ cells in N GOF Type II lineages would be consistent with previous literature and the data we have presented in this study.It is known that N GOF in Type II lineages results in ectopic Type II NSCs  and we have shown that normal Type II NSCs express Tll. Therefore, an increase in Tlli.e. they are not Type I NSCs . In contrast, erm expression is absent entirely from Tll LOF Type II lineages. Indeed, all Type II NSC and lineage features are absent in Tll LOF Type II NSCs . We have added the effect of Tll LOF on PntP1 expression as Figure 2\u2014figure supplement 2C-D\u2019.There are some important differences between the Tll and N LOF phenotypes. We have shown that Type II NSCs become Type I NSCs without Tll, whereas Type II NSCs appear to undergo premature differentiation without N signalling . Although it was reported previously that Type II NSCs switch to Type I NSCs without N signalling, Erm is precociously expressed in the NSC and maintained in the lineage, ase) and that N signalling acts within Type II lineages to ensure timely differentiation of INPs (by repressing erm in Type II NSCs). However, as the reviewer states, there are many genes that regulate cell fate changes in Type II lineages. How Tll interacts with each of these genes will be an interesting topic for future study.We propose that Tll and N signalling act in parallel in Type II NSCs: Tll acts upstream to establish Type II NSC identity (including repression of 3) DamID: Does Tll-Dam bind to other genes in the NB-INP-GMC-neuron pathway e.g., pnt, erm, E(spl), btd, klu, pros, dpn? Importantly, the evidence for Tll binding to the ase gene, but possibly also to dpn and E(spl), has bearing on the model for tll action. In addition, the DamID results are not described in great detail.ase, Tll as a transcription factor has multiple targets, including pnt, erm, E(spl), btd, klu, pros and dpn, that could contribute to the Tll phenotypes we observe.We have included the full list of Tll target genes as an excel file . In addition to"}
+{"text": "Controlling of the CO I.1.A volume control breath delivers a fixed volume specified by an operator. per minute automatically increases or decreases to ensure the delivery of the requested mandatory or minimum minute volume. The concept of MMV appears attractive in that it provides the ability to increase or decrease the number of volume control breaths in a given time span. Thus, if the patient is apneic, an appropriate number of volume control breaths are delivered, and as the patient resumes spontaneous ventilation, the number of volume control breaths decreases gradually. This support scheme provides flexibility for a patient to automatically take up the work load of breathing and for the machine to gradually wind down at the same time.Modern lung ventilators are equipped with a variety of support techniques that provide the clinician various ways of artificially ventilating patients. Mandatory minute ventilation (MMV) is one such ventilation technique that interact with the patient in such a way that the delivery of a preset minimum minute volume  is guaranteed. The number of volume control breaths2 tension, the MMV setting may be decreased or increased respectively by the clinician to correct the disturbed arterial CO2 tension. Regulating the MMV setting to maintain an acceptable arterial CO2 tension could be performed automatically. This paper deals with such an automatic ventilation scheme that maintains the arterial CO2 tension less than a specified level determined by a clinician.For a patient ventilated in MMV, the clinician selects the level of minimum minute ventilation and this setting remains unaltered until a change is requested by the clinician. In the event of an increase or decrease in arterial COII.2 content of the exhaled air, Bohr formulated the following relationship 2 tension, and 2 tension in exhaled air. A typical trace of exhaled CO2 tension against exhaled volume  consists of three phases 2-free gas from the airways, phase 2 a transition phase characterized by a S-shaped up-swing in the tracing representing the washout of airway with alveolar gas, and phase3 comprising CO2-bearing gas from the alveoli , the patient descends down line AB and reaches the desired CO2 level in one step. The one-step-ahead control law can be written as2 tension, 2 tension, and 2 tension. The above control strategy is derived using 2 production changes , the patient instead of descending from point A to point B moves from point A to point C. In this situation, the target value is not reached in one step due to the disturbance in the CO2 production; the controller again attempts to push the patient from point C to the new desired point D by calculating a new alveolar ventilation of 5.4 L. It is clear that desired performance of the controller can be guaranteed even under sudden changes in CO2 production, provided it remains stable after the change. However, if the CO2 production is unstable and oscillates about a mean CO2 production value of 2 tension oscillates about the desired CO2 tension with an amplitude of 2 production oscillates between 150 to 250 mL/min for a constant effective alveolar minute ventilation of 4.3 L represents an oscillatory movement of a point between 2 tension will oscillate between 30 to 50 mmHg as shown in the horizontal oscillations. If the magnitude and direction of CO2 production oscillations are2 tension by oscillating the alveolar minute ventilation with an amplitude of 2 production oscillations are in phase with the effective alveolar minute ventilation oscillations. The vertical oscillations in 2 tension oscillations. If the CO2 production oscillations and the 2 tension will oscillate with amplitude2 tension oscillations when there is no CO2 control action present.A patient with an unacceptable blood CO2 production depends on body metabolism, it cannot be predicted, though it can be continuously measured. Controlling CO2 tension when oscillatory disturbances are present in CO2 production may intensify the existing oscillations. When consistent oscillations are present in the CO2 production, the best performance the controller can achieve is by regulating the CO2 tension using a running mean CO2 production value instead of regulating it using the single CO2 production value at the time of sampling.Since the disturbance in CO2 level, it is necessary that the assumption the CO2 production is relatively constant be valid and regulation of effective alveolar minute ventilation be possible. 2 production over a period of 30 min. Here the average CO2 production varies within a band of 10 mL and from 2 tension to oscillate with an amplitude 2 tension oscillations have amplitudes ranging from 1.1 mmHg to 0.5 mmHg. Since the target 2 production is, therefore, not a performance limiting factor of the control system.From the above discussion it is clear that to control the arterial COThe minute ventilation for a mechanically ventilated patient depends on the patient's own respiratory drive and the number of mandatory breaths the operator specifies. The mandatory breaths contribute to a certain mandatory minute ventilation and the patient's respiratory drive contributes to a certain spontaneous minute volume. Both the spontaneous and mandatory minute volumes contain respiratory dead space volume, and subtraction of the two gives the effective minute volume carried by spontaneous and mandatory breaths. The controlling of 2 level below a certain maximum level  serves as the input to the VMMV algorithm. The periodic MMV selection is based on 2 tension and minute ventilation, both averaged over 5\u2013min intervals, the MMV selector outputs a new MMV setting every 5 min. The following is a clinical trial of a patient ventilated in the new VMMV mode.To maintain arterial CO2 tension of 41 mmHg. Based on the arterial CO2 tension in this minute, a blood gas efficiency of 0.646 was calculated  to ensure that arterial CO2 level stays below the set limit. The ventilation mode while providing all features of the MMV mode can ensure that arterial CO2 remains below a set limit."}
+{"text": "A part of localized prostate cancer (PC) was an incidental finding in patients who received transurethral resection of the prostate (TURP) for urinary symptoms. The present study examined whether changes in prostate-specific antigen (PSA) levels after TURP possess a predictive value for localized PC. Our data revealed that patients at intermediate risk who are associated with tumor involvement \u22645% in TURP specimens, PSA_TURP \u2264 4 ng/mL, and \u226568% PSA reduction following TURP might be suitable for conservation management instead of immediate local therapy. Moreover, for patients with no pre-TURP PSA, Gleason score (GS) < 7, and low PSA_TURP could potentially be utilized to select which patients could be considered for conservative management after TURP. The findings suggest the pathologic finding of TURP and changes in PSA could be used as adjuvant markers to guide a risk-adaptive strategy for patients with localized PC.Regarding localized prostate cancer (PC), questions remain regarding which patients are appropriate candidates for conservative management. Some localized PC was an incidental finding in patients who received transurethral resection of the prostate (TURP) for urinary symptoms. It is known that TURP usually affects the level of prostate-specific antigen (PSA). In the present study, we examined whether changes in PSA levels after TURP possess a predictive value for localized PC. We retrospectively reviewed the clinical data of 846 early-stage PC patients who underwent TURP for urinary symptoms upon diagnosis at our hospital. Of 846 patients, 687 had tumor involvement in TURP specimens, and 362 had post-TURP PSA assessment. Our data revealed that, in addition to low GS and PSA levels at diagnosis, \u22645% tumor involvement in TURP specimens, greater PSA reduction (\u226568%) following TURP, and post-TURP PSA \u2264 4 were significantly associated with better progression-free survival (PFS). Survival analysis revealed that the addition of prostate-directed local therapy significantly improved PFS in intermediate- and high-risk groups, but not in the low-risk group. Moreover, in the intermediate-risk group, local therapy improved PFS only for patients who were associated with post-TURP PSA > 4 ng/mL or <68% PSA reduction following TURP. We also found that local therapy had no obvious improvement in PFS for those with post-TURP \u2264 4 ng/mL regardless of pre-TURP PSA. In conclusion, conservative management is considered for patients at low or intermediate risk who have greater PSA reduction following TURP and low post-TURP PSA. Therefore, the levels of PSA following TURP might be helpful for risk stratification and the selection of patients for conservative management. Screening with prostate-specific antigen (PSA) testing has led to the identification of more patients with localized prostate cancer (PC) . For theIn addition to the Gleason score (GS), PSA is used for determining tumor involvement and prognosis ,10. The p < 0.001) and five-year PFS , higher rates of disease failure , and poor PFS compared to those with \u226568% PSA reduction (p < 0.001). Furthermore, as shown in p < 0.001), but did not significantly affect patients with greater PSA reduction (p = 0.122). For high-risk patients, the survival benefit brought about by the addition of local therapy was independent of the PSA reduction level. Based on our data, for patients with early-stage PC at intermediate risk, the level of PSA reduction following TURP could be utilized to assist with patient selection for local therapy.In total, data regarding PSA_Dx and rechecked PSA levels after TURP (PSA_TURP) were obtained from 362 patients. Among these patients, 334 had lower PSA_TURP than PSA_Dx, with a median 66% reduction in PSA level following TURP. To assess whether the change in PSA following TURP had an impact on the prognosis, the level of PSA reduction was redefined as a binary variable by finding the value from a receiver-operating characteristic (ROC) curve that maximized the percentage correctly classified for biochemical failure rate. Accordingly, we divided the patients into two groups with 68% PSA reduction following TURP (67% sensitivity and 62% specificity). In a univariate analysis, patients with <68% PSA reduction had higher rates of biochemical failure rate  and low (PSA_TURP \u2264 4 ng/mL). The clinical characteristics of both groups are shown in Traditionally, clinical and pathologic staging systems were used alone to categorize outcomes, but now cancer is often evaluated using risk-stratification systems. The appropriate treatment policy for localized PC requires further investigation. Local treatment of PC is associated with a reduced quality of life, so these risks must be carefully balanced against any benefit in terms of survival ,17. The The PSA level can increase due to many causes . The PSAThe limitations of our study are inherent to investigations based on hospital registries. We were unable to ascertain the reasons for the choice of local therapy or conservative management. Furthermore, we could not adjust for potential unmeasured selection biases regarding performance status, access to healthcare, or other patient-related factors. Accordingly, the study was designed to assess changes in PSA after TURP and how they affect selection of patients for conservative treatment, but not to investigate whether local therapy is better than conservative management for patients with early stage prostate cancer.Our data revealed that local therapy improved PFS for patients with PSA_TURP > 4 ng/mL, but led to no obvious improvement in PFS for patients with PSA_TURP \u2264 4 ng/mL regardless of pre-TURP PSA \u2264 10 or >10 ng/mL Based on our data, PSA_TURP might help rule out local treatment for patients with no pre-TURP PSA.Data were obtained from our hospital\u2019s system, a high-quality cancer registry that provides information regarding each patient\u2019s demographics, disease stage, tumor histology, and primary treatment details. This retrospective study was approved by the institutional review board of Chang Gung Memorial Hospital (No. 201800593B0), and a waiver of informed consent was obtained. This study adhered to strict confidentiality guidelines, in accordance with regulations regarding personal electronic data protection. From 2001 to 2017, there were 20,037 patients newly diagnosed with PC at our hospital. We excluded patients with other cancer diagnoses before PC diagnosis and those who had clinical stage T3\u2013T4, with lymph node involvement, or with distant metastasis at diagnosis, and those who received chemotherapy or hormone therapy prior to reaching biochemical failure. We included 846 patients who were diagnosed with clinical stages T1\u2013T2N0M0 and underwent TURP prior to definite treatment for urinary symptoms; they then underwent follow-up at our hospital. The timing of these enrolled patients receiving TURP was simultaneous with prostate biopsy (85%), and within two months (15%). As shown in We used the Kaplan\u2012Meier method for survival curves, and the log-rank test to determine differences in survival curves between groups. Cox proportional hazards models were used for hazard ratios with 95% confidence intervals (CIs) after adjustment for clinical characteristics. All analyses were conducted using SAS statistical software, version 9.2 , and SPSS version 17.2 for Windows .The findings indicate the predictive value of the pathologic finding of TURP and changes in PSA, and may be helpful for treating men with localized PC. Based on our data, we suggest that conservation management might be suitable for patients at low or intermediate risk associated with tumor involvement \u22645% in TURP specimens, PSA_TURP \u2264 4 ng/mL, and \u226568% PSA reduction following TURP. Moreover, for patients with no pre-TURP PSA, GS < 7 and low PSA_TURP could potentially be utilized to select which patients could be considered for conservative management after TURP. Therefore, tumor involvement in TURP specimens and changes in PSA and PSA_TURP could be used as adjuvant markers to guide the implementation of a risk-adaptive strategy for selected PC patients."}
+{"text": "In this review, the underlying mechanisms of health benefits and the risk of habitual behaviours such as internet use and media multitasking were explored, considering their associations with the reward/motivation system. The review highlights that several routines that are beneficial when undertaken normally may evolve into excessive behaviour and have a negative impact, as represented by \u201cthe inverted U-curve model\u201d. This is especially critical in the current era, where technology like the internet has become mainstream despite the enormous addictive risk. The understanding of underlying mechanisms of behavioural addiction and optimal level of habitual behaviours for mental health benefits are deepened by shedding light on some findings of neuroimaging studies to have hints to facilitate better management and prevention strategies of addictive problems. With the evolution of the world, and the inevitable use of some technologies that carry the risk of addiction, more effective strategies for preventing and managing addiction are in more demand than before, and the insights of this study are also valuable foundations for future research. Dopamine (DA) is a critical neurotransmitter for maintaining and promoting favourable human mentality, including healthy mood and motivation, through its function within the reward/motivation system. On the other hand, the relationship between addictions, such as cocaine addiction, food addiction, and the problematic use of the Internet (PUI), one of the recent types of behavioural addiction, and the deficits of the reward system have been widely demonstrated by neuropsychological studies, including neuroimaging studies \u20133.Excessive behaviours, which characterise behavioural addiction, as well as substance addictions, are thought to be underpinned by a dysregulation of the DA reward/motivation system, which is typically based on the reward deficiency syndrome (RDS) hypothesis , 5. The It needs to be ascertained if environmental factors influence the development of the deficiency in the neurotransmission of the reward-related circuitry. Several positron emission tomography (PET) studies have shown that postsynaptic DA receptor change/downregulation occurs as a consequence of high-level DA release induced by conditions such as sleep deprivation, nicotine abstinence, and PUI \u20138. It alIn this review article, we shed light on the health benefits/negative aspects of daily behaviours, including up-to-date life habits, Internet use (IU) and media multitasking, physical exercises, gambling, and related psychological resilience, and their potential neural correlates in the reward/motivation system.The reward system is a well-characterised network of regions across the limbic, prefrontal, striatal , and midbrain regions . Neuroim2 receptor availability)  , 16 and ability) . Amongstability) \u201320, as wability) . These Mability) , 22, 23.per se  study revealed that online social networking service size, typically represented by the number of online friends, was positively associated with regional brain volumes in healthy individuals , indicatper se \u201328. TherAn fMRI study by Fujiwara et al.  was condhttp://www.fil.ion.ucl.ac.uk/spm). Twenty-two spherical clusters (10-mm diameters) were specified and peak coordinates based on a previous motivation-related fMRI study (p < 0.05). Mediation analysis was performed to investigate whether autistic traits (indexed by the autism spectrum quotient (AQ) mediated the association between the degree of IU and FC values of the network. A nonparametric bootstrap method (2000 bootstrap samples) was used to test the mediation path .For the MRI dataset analysis, a region of interest (ROI)-to-ROI FC analysis was applied to the resting-state fMRI scans using the CONN-fMRI Functional Connectivity toolbox (ver.17e) with the statistical parametric mapping software package SPM12  may be implicated in media multitasking because it may include several forms of IoT usage, such as different kinds of social media, online gaming, shopping, and pornography at the same time. Another unique example of a comorbid problem of multitasking is obesity , 35; it Concerns have been raised about the impact of media multitasking on attention and two opposing hypotheses have been provided: the scattered attention hypothesis and the trained attention hypothesis . On the Several studies have suggested that a higher cognitive load for switching between tasks is needed for heavy multitaskers , 38, whiIn an fMRI study by Kobayashi et al. , the assThe mean MMI (1.68) was lower than the scores reported by previous studies, indicating that the extent of multitasking was low to intermediate but pathological among the participants of the study  and (2) the latter 180 s of the task [\u201cOdd 2\u201d]. As for the relationship between task performance and resilience, no correlation was found between the reaction time and the CD-RISC scores. In addition, task performance did not differ between the low (CD-RISC score \u2264 58) and high (CD-RISC score \u2265 59) resilience groups.Miyagi et al.  attempteNeuroimaging data were analysed using the CONN software. The preprocessed fMRI time series in the ROIs of DMN were extracted for four-time windows (180 s each): (1) the former half of the resting state [\u201cRest1\u201d], (2) the latter half of the resting state [\u201cRest 2\u201d], (3) [\u201cOdd 1\u201d], and (4) [\u201cOdd 2\u201d]. Significant differences in FCs between [switching] \u201cRest 2\u201d and \u201cOdd 1\u201d and \u201cOdd 1\u201d and \u201cOdd 2\u201d [sustaining] are shown in The increased DMN FCs in the study may be explained as follows: (1) In general, the regions consisting of the DMN are deactivated by performing any cognitive tasks , 65. How2 receptor availability in the striatum, which supports the RDS hypothesis. However, in PET studies of GD patients, D2 receptor availability in the striatum did not differ from that in the healthy group , accumulating evidence of biological similarities has led to the classification of gambling disorder (GD) in the same category as substance use disorders in the DSM-5 . Howeverhy group . Taken tMRI studies can provide explanations for this problem. There is a widely used task called the monetary incentive delay task (MIDT) . MIDT isIn a meta-analysis of MIDT, both GD patients and SUD patients showed reduced striatal activation during reward anticipation . This reIn addiction literature, the striatum has attracted attention related to DA, but the insula may be important as well. The insula has gained attention in the field of addiction because studies have shown that damage to the insula abolishes nicotine addiction . In an MExercise is widely known to have health benefits for the body and the mind. However, it may potentially damage our health if has excessive intensity, frequency, and duration. Therefore, excessive exercise could be regarded as a \u201cbehavioural addiction\u201d from a psychiatric point of view , 85. ExcShibata et al.  investigIPED use was significantly higher for weightlifting (61.1%) and ECPT (60.3%) but lower for walking (24.5%) than for other disciplines. EAI and AAI were positively associated with IPED use among individuals with habitual exercise. As indicated in Considering the association between EAI and IPED use for these disciplines, a higher EAI was associated with greater IPED use, indicating that excessive exercise leads to the risk of \u201ccross-addiction\u201d with substance use, which may lead to excessive enhancement like doping. Given the increased online accessibility (12.2% of those who use IPEDs purchased them online), the risk of \u201ctriplet\u201d cross-addiction, including excessive Internet use and excessive exercise and IPED abuse, may be considered, together with the commonality among these issues in terms of the need for regulation. In this study, professional athletes were excluded from participation to investigate the relationship between exercise habits and IPED use in non-professionals. In future studies, both professional and non-professional athletes should be investigated and compared, considering the differences in exercise levels such as intensity, frequency, and skills. In particular, the personal need of individuals for exercise in their lives  would vary; hence, terms such as \u201cexcessive\u201d and \u201caddictive\u201d could be cautiously defined by considering the entire body of evidence non-professional and professional athletes. Future studies should consider adequate DA neurotransmission (regulation of the reward system as a consequence) for establishing optimised strategies for dealing with these habits in future studies. Interestingly, the self-compassion scale score was significantly higher, and the EAI and AAI scores were relatively lower for cycling than for other disciplines. In this context, high self-compassion may have contributed to the lower EAI and AAI scores for cycling. These health benefits may not be directly interpreted as cycling-specific, and \u201cmind-body\u201d integrated training programs for different types of sports, in general , may regulate DA neurotransmission, leading to the improvement in coping skills or reducing the risk of addiction as a consequence.The possibility of \u201coptimal, hyper-normal\u201d level behaviours and future directions are discussed below: The previous reports introduced in this current review are from cross-sectional comparisons, and several interpretations of the causality of habitual behaviours and mental health would be possible. (1) Considering the low to intermediate media multitasking and IU as a part of the background of the multitasking, these may be potential mental/cognitive health benefits, as long as the level is not excessive. (2) A homeostatic attention-related network connectivity is assumed to be a possible interpretation of fewer changes in network FCs in individuals with higher resilience and low to intermediate levels of media multitasking. (3) Habitual Budo training, which closely overlaps with Zen meditation, can help foster the motivation that can partially influence its potential benefits related to the cultivation of self-compassion as \u201cZen in action\u201d. (4) Habitual physical exercise has health benefits; however, the risk of related addictive problems should be considered in the same light as other habitual behaviours, and its potential to lead to cross-addiction with substance misuse should not be overlooked.Regardless of differences in interpretation, there are several limitations to each study cited in this article. First, the studies in which the positive benefits of each behaviour were discussed  were conducted using a cross-sectional design. Therefore, caution should be exercised when discussing the causality between life habits (behaviours) and the changes in cognition, including those related to the reward system. Future studies with a longitudinal design would be needed to address this point. Second, the sample sizes were relatively small, particularly in the study of the benefits of Budo on motivation . SimilarHabitual behaviours introduced in the current review cannot be avoided in our social lives in this era. Gambling is not always necessary in daily life; however, it is still a form of entertainment for many people when engagement is not excessive . It is important to monitor and estimate the level of each behaviour and other related psychological backgrounds to better understand the optimal frequency, intensity, and duration of engagements for each behaviour. Direct comparisons of various levels of multitasking revealed that intermediate multitasking requires the most attention processing, suggesting an inverted U-shape association between multitasking tendency and attention function . In thisKT, MS, KK, TMi, NO, TMu, TU, and HF wrote, edited, and supervised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by Grant-in-Aid for Scientific Research (B) , Grant-in-Aid for Transformative Research Areas (A) , Grant-in-Aid by the Smoking Research Foundation, Grant-in-Aid for Scientific Research (A) , The Strategic International Brain Science Research Promotion Program (Brain/ MINDS Beyond) (21dm0307102h0003) from Japan Agency for Medical Research and Development (AMED).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The aim of the review was to present the state of knowledge about the respiratory pathology in former premature neonates  other than chronic lung disease, in order to provide reasons for a respiratory follow-up program for this category of patients. After a search of the current evidence, we found that premature infants are prone to long-term respiratory consequences due to several reasons: development of the lung outside of the uterus, leading to dysmaturation of the structures, pulmonary pathology due to immaturity, infectious agents or mechanical ventilation and deficient control of breathing. The medium- to long-term respiratory consequences of being born before term are represented by an increased risk of respiratory infections  during the first years of life, a risk of recurrent wheezing and asthma and a decrease in pulmonary volumes and airway flows. Late preterm infants have risks of pulmonary long-term consequences similar to other former premature infants. Due to all the above risks, premature neonates should be followed in an organized fashion, being examined at regular time intervals from discharge from the maternity hospital until adulthood\u2014this could lead to an early detection of the risks and preventive therapies in order to improve their prognosis and assure a normal and productive life. The difficulties related to establishing such programs are represented by the insufficient standardization of the data gathering forms, clinical examinations and lung function tests, but it is our belief that if more premature infants are followed, the experience will allow standards to be established in these fields and the methods of data gathering and evaluation to be unified.  Due to improvements in prenatal care  and neon-Support for the child\u2014early identification of concerns and early intervention.-Support for the families\u2014monitoring normal/abnormal growth and development and helping in the decisions about the future of the child. -Support for the healthcare system\u2014resource allocation and planning of health care services.Considering this, there has been an increased interest in the follow-up of these categories of neonates, with the following objectives :-SupportThe follow-up programs have focused mainly of the neurological outcomes of the NICU graduates, especially premature infants ,5, althoA respiratory follow-up program for NICU graduates seems more than appropriate, for several reasons .First, as in the case of the development of the central nervous system , the devThe second issue regarding the long-term risks for the respiratory system of a premature infant is represented by the specific pulmonary pathology of the neonate related either to immaturity, specific to the premature neonate: respiratory distress syndrome , transieA third reason for the long-term risks related to the respiratory system are the issues related to control of breathing  . This to-Based on the current knowledge, the respiratory consequences of prematurity can be classified in:-Chronic lung disease of the premature infants (bronchopulmonary dysplasia);-Risks of respiratory tract infections;-Risk of wheezing and asthma;-Modification of the pulmonary function tests.The last three topics\u2014except BPD\u2014will be covered in the present review. Bronchopulmonary dysplasia represents, indeed, the main pulmonary pathology of the extremely premature neonates ,30. TherFormer premature neonates are at risk for respiratory tract infections during the first years of life. In a study published in 2003, Doyle et al. found that ELBW (extremely low birthweight neonates\u2014neonates with birthweight < 1000 g ) have siThe duration of mechanical ventilation and the birthweight seem to be predictive of the occurrence of respiratory infections in former premature neonates. In a study published in 2018, MacBean and co-workers identified three clusters related to the risk of low respiratory tract infections\u201448% of the neonates ventilated less than 5 days had a lower respiratory tract infection during the first year, compared with 65% of those ventilated more than 5 days with birthweights more than 882 g and 100% of those ventilated more than 5 days with a birthweight less than 882 g . The patAs mentioned above, two pathogens account for most of the respiratory infections in the former premature neonates: respiratory syncytial virus (RSV) and human rhinovirus (HRV). Even if RSV has been studied in more detail, rhinoviruses make up an important part of the pathology of preterm neonates. In a cohort study performed in Argentina, 65% of the VLBW (very low birthweight neonates\u2014neonates with birthweight < 1500 g ) infantsPrematurity represents a risk factor for both the infection with RSV and the most severe form of it\u2014bronchiolitis. Thus, a premature infant has a seven-fold risk factor for acute viral bronchiolitis during the first RSV season after their birth . There aA large amount of the medical literature addresses the topic of bronchial hyperresponsiveness and wheezing disorders directly linked to premature birth. Furthermore a number of reviews have given more statistical power to this association.A systematic review published in January 2014  found a Such findings support the idea that the relationship between asthma-like conditions and prematurity is a complex, multifactorial one and one should be aware of as many of these aspects as possible in order to be able to find the best medical approach in a former premature child with recurrent wheezing.Another review, published in 2018 , has a mHowever there is an increasing amount of evidence regarding prematurity and prematurity-related exposers and the risk of developing atopy and other inflammatory disorders.In a perspective-type article, a group of immunologists drew attention to several factors altering the inflammatory responses in premature babies .In consequence, the maturational process of the immune system of the fetus\u2014a highly complex process\u2014is altered in the case of a premature birth. Soon after birth, the premature neonate establishes a large number of class-switched B cells, but their IG G and IG A production maintains fetus-like characteristics such as small amounts of antibody production and low affinity to antigens, as seen in response to vaccination. Furthermore, there is a lack of maternally-transmitted passive immunity through antibodies crossing the placenta in the third trimester with an amount of maternal antibodies in VLBW babies of just 10\u201320% of the amount found in a term neonate. As well as reducing the immune protection of the baby, there seems to be a more intricate relationship with idiotypic control of B and T cell receptors, thus contributing to the adaptative immune responses ,58. Age-A number of extrinsic factors have been associated with prematurity and/or asthma development risk.Stress has long been suspected to be both a cause for premature labor and a risk factor for developing abnormal immunologic responses in the offspring, thus leading to atopy and asthma. A large cohort study of a potent stressful factor, an electricity system failure for 45 days during an ice storm in Quebec in 1998, found larger proportions of premature birth and immunological dysregulation in the offspring of women pregnant at that time .Another mechanism for developing immunologic dysregulation might be premature, stimulation of the chemosensory system in a baby born prematurely. This hypothesis is sustained by the scientifically proven fact that chemosensory system stimulation is an important mechanism of bacterial recognition .A number of clinical trials yielded interesting results, finding statistically significant associations between atopy and/or wheezing on the one hand and, on the other hand, prematurity-associated conditions such as maternal diabetes, mechanical ventilation and antibiotic use rather than with prematurity per se ,63,64.A number of indices related to respiratory distress in the neonatal period have been associated with asthma in medical trials. Thus, increased oxygen exposure in the first days of life, a higher number of hypoxic episodes and a lower oxygen saturation level have been linked to a higher risk of asthma in a cohort study of premature babies < 28 weeks gestation . Other sOverall, we can conclude there is a definite association between prematurity and asthma-like features. Although some additional factors  seem to influence this relationship; when evaluating a premature baby, one must also consider not just their gestational age or birthweight but also all the additional perinatal factors that will definitely influence their future morbidity in the short as well as the long term.Evaluating lung function is an interesting topic for researchers as well as for clinicians dealing with former premature babies presenting with respiratory morbidity later in life. However, measuring lung function can be challenging, especially due to the lack of proper standardized techniques and the lack of cooperation from children, especially those who are very young or with neuropsychiatric disorders. Efforts are being conducted by a united task force of both the American Respiratory Society (ARS) and European Respiratory Society (ERS) toward proper standardized techniques of evaluating pulmonary function in children.For children older than 5 years, pulmonary function can be investigated using spirometry\u2014a procedure last updated in a paper published in 2019 in regard to proper investigation technique, investigation conditions and events that may alter a respiratory curve, thus diminishing measurement accuracy .For the pulmonary function of infants, the matter is more complicated. Recommendations regarding standardizing tidal breath analysis were issued by the ARS/ERS Task Force in 2000. The need for eliminating all sources of leak, minimizing dead space equipment and using a device with proper linearity and frequency response was emphasized. The authors admit there is still a challenge to proper identify the beginning and end of inspiration and expiration and establishing appropriate reference standards .In 2005, more recommendations were issued regarding the raised volume rapid thoracoabdominal compression (RVRTC) maneuver. In spite of considerable efforts made to develop and standardize this technique, and the fact that it provides capabilities of measuring an extended range of pulmonary volumes, its use has been limited to a few specialized laboratories throughout the world .Whole body plethysmography is another method used in research settings for measuring lung volume, airway resistance and functional residual capacity . AlthougThe multiple breath washout test has been rediscovered lately as a valuable tool for evaluating functional residual capacity and peripheric airway disfunction . There aThe method has its undisputable use for measuring lung function in children with cystic fibrosis. In measuring lung function for children with chronic lung disease, wheezing and asthma, the method has not yet proven its efficacy or fails to provide consistent result .In spite of all these difficulties, there is a growing amount of data on pulmonary function modifications in a former premature baby.Thus, in an article published in 2009, follow-up data in a randomized trial population of former premature babies showed an alteration in respiratory function at 11\u201314 years that manifested through lower volumes on spirometry more frequently in the male population . AnotherLung function using spirometry was also assessed in a cohort of school-age children who were born at 22\u201326 weeks gestation. Researchers found lower forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) in former premature babies compared to their normal controls. Asthma manifestations were also found in a greater proportion in the formerly premature baby group .A regional prospective cohort study conducted in Norway compared tidal respiratory function of babies born before 28 weeks gestation with or without a bronchopulmonary dysplasia (BPD) diagnosis to that of term babies using electromagnetic inductance plethysmography. Researchers aimed to study a normal breathing pattern with no interference from using a mask or performing a forced ventilation. Lung volumes of premature born babies measured at term equivalent age were significantly lower when compared to those of a control group of term born infants .A cohort study of 164 former premature babies, children that were born preterm (before 37 weeks of gestation) and are examined or evaluated after 40 weeks corrected age, published in 2021, found a worsening of respiratory function over time as measured by spirometry at 3\u20136 years of age, 7\u201311 years and 12\u201320 years. Mechanical ventilation, postnatal steroids and a maternal history of atopy and asthma were associated with a more adverse outcome .A management technique for improving respiratory function in preterm babies (GA < 32 weeks) was proposed by an article, which has been just approved for publishing, that found that feeding premature babies protein rich formula after discharge will improve lung function and diminish airway inflammation as measured through exhaled NO measured at 6 years of age In conclusion, prematurity represents a disturbing factor for the developing lung. Tests that are standardized and have been used for a long time such as spirometry and exhaled NO should be considered as soon as the child is old enough to cooperate. The others are worth being considered as possible diagnostic tools as they cross the line from research to clinical practice devices. Future technological development will for sure enable us to have more reliable clinical diagnostic devices.Late preterm infants are defined as infants born between 34 weeks and 36 6/7 weeks gestation . The stu-The late preterm neonates are born during the end of the saccular phase and the beginning of the alveolar phase of lung development . Two pro-There is an immune deficit at the level of the T lymphocytes, a deficient pulmonary cytotoxic lymphocyte response, leading to the attempt to clean the infection by the macrophages and neutrophils . The infThese infants are also at risk for medium- and long-term health problems, both neurologic and respiratory . The risSeveral studies have assessed the long-term respiratory problems of late preterm infants. In a study published in 2013 on a Dutch cohort, there was noticed a double number of hospitalizations in moderate-preterm children compared to term neonates . The difRegarding the risk of RSV infection, the rates of hospital admissions for RSV infections is similar between infants born at 33\u201335 weeks gestational age and infants born at less than 32 weeks . A revieA Canadian study  effectivInfants with scores higher than 49 (moderate and high risk) were eligible for prophylaxis. As can be seen, the risk factors are represented in the order of importance by birth during the peak of RSV season, siblings in daycare, crowding SGA and male status of the patient, and more than 1 smoker in the house. Using this score, 19% out of this population of premature infants was selected to receive prophylaxis. The score is based on a previous study performed by a research group in Spain .As can be seen, this category of former premature infants, forgotten in almost all follow-up programs, has important risk factors and important pathologies during infancy and childhood, both comparable to the early (less than 32 weeks) former premature infants. All late preterm neonates should be examined at discharge from the point of view of the respiratory risks. The patients that are included in the neurologic follow-up program will also be followed from the respiratory point of view . BecauseThe previous sections showed that the former premature infants are at risk for respiratory pathology due to factors related to prematurity. They present with a greater risk of respiratory infections, and especially of severe forms of bronchiolitis, than children born at term. They also present with lower than normal respiratory volumes and flows, and developmental lung and airways abnormalities, and this leads to an increased risk of airways disorders (recurrent wheezing and asthma). The sequelae are present from early infancy throughout childhood and until adult life.-Support for the child\u2014identifies from the maternity hospital a population at risk that could be followed and allows establishing preventive measures for populations at risk\u2014RSV prophylaxis for selected groups of patients, avoidance of triggers for patients at risk of asthma, etc. Periodic respiratory examination and pulmonary function tests could allow early identification of the children with a risk of wheezing and asthma and early treatments. According to the AAP policy statement in 2014  RSV prophylaxis for palivizumab is indicated for two categories of preterm neonates, during the first year of life:-Premature infants born before 29 weeks 0 days of gestation-Preterm infants with chronic lung disease defined as birth <32 weeks 0 days, and requirement for >21% oxygen for at least 28 days after birth .Based on these arguments, a respiratory follow-up program for former premature infants is perfectly justified. It fulfils all the roles of a follow-up program (as stated in one of the above sections):As another indication, the 33\u201335 weeks neonates selected to be at risk according to the tool quoted previously regarding the late preterm infants (see the previous section regarding late preterm infants).-Support for the families\u2014the program allows a partnership with the families. The respiratory risks could be explained to the family at discharge from the hospital (a flyer could be developed as in the cases for the neurologic follow-up). The family could be helped to avoid behaviours at risk both for the family  and for the patient  and encouraged to maintain a good hygiene with avoidance of infections and allergens. Moreover, knowing the respiratory status of the child would be of help in decisions about the practice of certain activities and sports-Support for the healthcare system: a good follow-up program could offer data as complete as possible regarding the respiratory outcome of former premature infants, allowing the stakeholders to manage the levels of care and services available. The data could also help as a feedback for the units, in order to try to correct risk factors that lead to unfavorable outcomes, if possibleEven if the medical literature states the need for the inclusion of a respiratory follow-up component in the follow-up program for premature neonates , there iThe structure of such a respiratory follow-up program could be linked to a regular neurologic and neurodevelopmental program ,5. The fAs in the case of the neurologic follow-up, the respiratory follow-up program should not only have a diagnostic component, but also a component of early intervention. There should be established links with pediatric emergency departments, pediatric infectious disease specialists and pulmonary function laboratories and clinics specialized in the monitoring and treatment of asthma. Moreover, information could be provided to the general practitioners in order to improve the care of their patients. The former premature infants present, due to lung immaturity at birth, extrauterine development of the respiratory system, pulmonary conditions in the neonatal period and deficient control of breathing, and a risk of medium- and long-term pulmonary problems. This consist of respiratory tract infections  during the first year of life, airway diseases such as recurrent wheezing and asthma during childhood and abnormalities of the lung function that could place them at risk for complications later in life. This is why a respiratory follow-up program is needed for every NICU as is the case for the neurodevelopmental follow-up program. Establishing such a program could lead to the early detection of risks and preventive therapies put in place in order to improve their prognosis and ensure a normal and productive life."}
+{"text": "Conclusion: Our study suggests an association between the immunological trait and cardiac dysfunction in severe COVID-19 patients.Background: We aimed to explore immune parameters in COVID-19 patients admitted to the intensive care unit (ICU) to identify distinctive features in patients with cardiac injury. Methods: A total of 30 COVID-19 patients >18 years admitted to the ICU were studied on days D1, D3 and D7 after admission. Cardiac function was assessed using speckle-tracking echocardiography (STE). Peripheral blood immunophenotyping, cardiac  and inflammatory biomarkers were simultaneously evaluated. Results: Cardiac dysfunction (DYS) was detected by STE in 73% of patients: 40% left ventricle (LV) systolic dysfunction, 60% LV diastolic dysfunction, 37% right ventricle systolic dysfunction. High-sensitivity cardiac troponin (hs-cTn) was detectable in 43.3% of the patients with a median value of 13.00 ng/L. There were no significant differences between DYS and nDYS patients regarding mortality, organ dysfunction, cardiac (including hs-cTn) or inflammatory biomarkers. Patients with DYS showed persistently lower lymphocyte counts (median 896 [661\u20131837] cells/\u00b5L vs. 2141 [924\u20133306] cells/\u00b5L,  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Although the predominant manifestation of COVID-19 is acute respiratory distress syndrome (ARDS), infection with SARS-CoV-2 is also associated with multiple cardiac manifestations . ImportaNevertheless, there are conflicting reports on the myocardial histology of patients with COVID-19 who had evidence of myocardial injury. A recent preliminary report on postmortem heart analysis demonstrated \u201cgross cardiac enlargement\u201d in 24 out of 25 cases, with evidence of left ventricular hypertrophy and moderate to marked atherosclerotic narrowing of the coronary arteries. Moreover, 60% of patients had evidence of a patchy epicardial mononuclear infiltrate with a predominance of CD4 T cells compared to CD8 T cells. Small vessel thrombi were observed in three cases, and one had hemophagocytosis within an area of epicardial inflammation [Few studies have addressed the evolution of the immunologic profile in COVID-19 patients. Laing et al.  accompliOur research focused on the immunologic profile evolution in COVID-19 patients with cardiac dysfunction (and not only injury detected by biomarkers), considering the scarcity of data on the underlying immune mechanisms related to cardiovascular disease in this context. Furthermore, we sought to determine if a specific immune activation in these patients could translate an underlying myocarditis-like mechanism driving the cardiac dysfunction. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options, offer insights into COVID-19 pathogenesis and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.This prospective observational study involves a comprehensive clinical and cellular characterization of COVID-19 patients over 18 years old, positive for SARS-CoV2 RT-PCR test, and admitted to the ICU. All patients were enrolled consecutively between June 2020 and September 2020 at Hospital Garcia de Orta-Almada . Exclusion criteria comprised type 1 acute myocardial infarction and pulmonary embolism as presenting diagnosis and inappropriate acoustic window for STE.Previous studies have shown the prognostic value of performing serial assessments of biomarkers at early disease onset . AccordiComprehensive STE was performed by an intensive care and echocardiography specialist at each time point. Besides evaluating invasive arterial pressure and central venous pressure, peripheral blood samples were collected to assess cardiac and inflammatory biomarkers, along with an immunophenotyping characterization by multiparametric flow cytometry. The criteria to choose these particular timepoints is related to the physiopathology and natural history of other cardiac dysfunctions induced by infection, such as septic-induced myocardial dysfunction (previously known as septic cardiomyopathy) ,11.Additionally, data on other markers were collected from the hospital patient file, including C-reactive protein (CRP), procalcitonin (PCT), ferritin, erythrocyte sedimentation rate (ESR), D-dimers, fibrinogen and complete blood count with platelets.All clinical data and medication were analyzed, namely demographic data: gender, age, previous history of hypertension, diabetes mellitus, chronic obstructive pulmonary disease (COPD), body mass index (BMI), and current medication with remdesivir or corticosteroid. Clinical outcomes such as mechanical ventilation, P/F ratio , shock, acute kidney injury, ICU and hospital length of stay, and mortality were also recorded. Besides cardiovascular risk factors, baseline cardiac function by echocardiography was searched for in the previous history through family physician files.All echocardiographic measures were obtained with simultaneous EKG display, and the three different sets of measures were obtained with the patient in the same position. All Doppler measurements were averaged from three measurements in sinus rhythm and ten measurements in atrial fibrillation. In addition to the qualitative examination of chambers and valves, the following measures were obtained via the standard parasternal and apical views: pulmonary artery systolic pressure; left atrium (LA) area and volume; left ventricle (LV) end-diastolic and end-systolic volumes (EDV and ESV) using biplane modified Simpson\u2019s rule, from which the ejection fraction (EF) was calculated; right ventricle (RV) end-diastolic area and fractional area change (FAC); tricuspid annular plane systolic excursion (TAPSE); mitral annular plane systolic excursion (MAPSE) and left ventricle outflow tract (LVOT) diameter, LVOT VTI measured on pulsed-wave Doppler from which stroke volume (SV) was calculated, and then cardiac output (CO) and cardiac index (CI) derived. Mitral flow measurements included: E maximal velocity, E deceleration time and A maximal velocity. Tissue Doppler measurements were: RV S\u2019 wave, LV septal e\u2019, lateral e\u2019 and systolic myocardial velocity during ejection (Sa).To determine LV, RV and LA strain, four-chamber views were acquired after optimizing the sector size, gain, depth, compress and time-gain compensation. The frame rate was maximized (76 \u00b1 24 fps) by decreasing the depth and sector width. The region of interest was manually traced at ED along the endocardial border. The software then automatically tracked the endocardial contours throughout the cardiac cycle. Manual adjustments were made to the contours as needed to optimize tracking. Images were analyzed offline using dedicated software  software). The peak strain value was derived from the maximal inflection point on the composite LA strain curve and graded based on recently published cutoffs .The cardiac dysfunction was classified according to the European Society of Cardiology (ESC) and American Society of Echocardiography (ASE) guidelines for chamber quantification classification . LV systVenous ultrasound was performed to assess inferior vena cava (IVC) variability ((Max-Min)/Max) measured in M-mode, and portal vein pulsatility index (PVPi) was measured by pulsed-wave Doppler in the portal vein ((Max-Min)/Max), according to the previous classification of venous congestion by ultrasound .Peripheral blood samples were collected into tubes without an anticoagulant agent and were centrifuged after coagulum retraction. Serum samples were then separated, immediately tested for cardiac biomarkers, or further aliquoted and stored at \u221220 \u00b0C for inflammatory biomarkers analysis.As for cardiac markers, high sensitive troponin T (reference range < 13 ng/L) and NT-proBNP (reference range < 125 pg/mL) were assessed by immunoassay with electrochemiluminescence technology (or \u201cECLIA\u201d) in Cobas (Roche) analyzers.\u00aeELISA kits  were used for IL-1\u03b2 and IL-6, presenting sensitivities of 1 pg/mL (assay range 3.9\u2013250 pg/mL) and 0.7 pg/mL (assay range: 3.1\u2013300 pg/mL), respectively. Low, medium and high control samples were run parallel at each series to assure curve verification. For ADM measurement, the RayBio\u00ae Human/Mouse/Rat Adrenomedullin Enzyme Immunoassay Kit was used . The assay has a sensitivity of 0.9 ng/mL (assay range 0.1\u20131.000 ng/mL). The SigmaPlot software version 14.5 (Systat Software Inc. (SSI), San Jose, CA, USA) was used to perform a four-parameter logistic regression curve and determine final ADM concentrations. Samples were run in duplicates (accepted whenever the coefficient of variation was below 15%), and the mean values were used for each sample.To quantify serum levels of IL-1\u03b2, IL-6 and adrenomedullin (ADM), commercial immunoassays were used according to the procedures defined by the respective manufacturers. The QuantikineFor immunophenotyping characterization, peripheral blood samples collected into EDTA tubes were analyzed within 24 h after collection. In brief, cells were incubated with a prevalidated panel of monoclonal antibodies for 15 min, lysed, washed, and acquired in an 8-color BD FACS Canto II Flow Cytometer. The panel of monoclonal antibodies included CD3, CD4, CD8, CD16, CD19, CD20, CD27, CD38, CD45, CD45RA, HLA-DR, IgD, IgM and TCR gamma delta, assuring the identification of several leukocyte populations, and particularly, distinct subsets of T cells  and B cells . A single-platform strategy with BD Trucount tubes was also used to assure absolute cell counts for each subset addressed.BD FACS Diva software was used for acquisition purposes, and Infinicyt 2.0 software  was used for data analysis. All gating strategies are presented in . Categorical variables were presented as percentages and analyzed with Fisher\u2019s exact test or Qui Square test as applicable. Continuous variables were expressed as mean and standard deviations (SDs) or median and interquartile range (IQR), depending on the normality of distributions. Group comparisons were performed using the unpaired t-test with Welch\u2019s correction or the nonparametric Mann\u2013Whitney U-test. Three or more paired groups were compared with Friedman\u2019s test, followed by the Dunn multiple comparison test. The nonparametric Spearman correlation coefficient assessed correlation studies. Statistical significance was considered for This was a prospective observational study on current practice, and all collected data were the standard monitoring and intervention data used in critical care. All monitoring and intervention attitudes have been extensively described in the literature, and patients were assessed and treated routinely. Data were anonymously collected, and no information allowing patient identification was collected. Patient anonymity was maintained both during data processing and publishing. A patient number was used in all echocardiograms recorded for later review by an expert. According to local legislation, consent from the patient or next of kin to record data and draw blood was obtained before study enrollment. The Ethics Committees approved the study of Hospital Garcia de Orta and NOVA Medical School (CE no. 44-2020-CEFCM).Between June and September of 2020, 40 patients met the inclusion criteria, from which 10 were excluded . The baseline characteristics of all patients also separated in patients with (DYS) and without (nDYS) cardiac dysfunction at ICU admission are shown in 2, and half of the patients were under ACEi or ARA treatment. The family physicians\u2019 previous history files showed that none of the patients had previous cardiac dysfunction detected by echocardiography, except three that had no registries. Overall, 22 patients (73.3%) had some cardiac dysfunction detected by STE, either left ventricle (LV) systolic dysfunction (40%), LV diastolic dysfunction (63.3%) or right ventricle (RV) systolic dysfunction (36.7%). Hypotension, acute kidney injury or mechanical ventilation occurred in 23.3% of patients. Five patients (16.7%) died during the study. Troponin was detectable in 43.3% of the patients with a median value of 13.00 ng/L, while NT-proBNP had a median value of 275 pg/mL. In addition, there were no significant differences between DYS and nDYS patients regarding mortality, organ dysfunction and cardiac or inflammatory biomarkers .Of the final 30 patients included, 63% were male with a mean age of 60.7 \u00b1 14.8 years. Hypertension was the most common risk factor (73.3%), followed by obesity (40%) with a mean BMI of 31 kg/mFrom the STE evaluation, 73.3% of patients demonstrated a cardiac dysfunction at ICU admission, and 80% of patients experienced a cardiac dysfunction within the first seven days of ICU stay  Supplem.p = 0.008). In addition, the DYS group presented lower median values of both LA RV  and LA CT  compared to the nDYS group, while no differences were observed regarding classic measurements, including E/A, e\u2019, E/e\u2019 and sPAP. Venous congestion evaluation (IVC% and PVP) did not differ significantly between groups with organ dysfunction such as AKI, P/F or mechanical ventilation. Nevertheless, PVP showed a stronger correlation with the congestion biomarker NT-proBNP than IVC% , considering patients in both groups and all time points. Inter- and intraobserver variability was calculated for LVEF, LV GLS, RV FAC, RV GLS, TAPSE, E/A, e\u2019, E/e\u2019, sPAP and LA volume. LA CT and LA RV, ranged from 0.8\u20131.8% \u00b1 0.9\u20131.1% for interobserver and 0.4\u20131.1% \u00b1 0.3\u20130.9% for intraobserver variability. Specifically for STE measurements: for the interobserver variability (median (IQR 25\u201375)): LV GLS 0.8 (0.5\u20131.1), RV GLS 0.7 (0.5\u20131.1), LA RV 1.3 (0.9\u20131.9), LA CT 1.1 (0.8\u20131.7); for the intra-observer variability: LV GLS 0.6 (0.5\u20130.8), RV GLS 0.7 (0.5\u20130.8), LA RV 1.3 (0.5\u20130.9), LA CT 0.9 (0.4\u20130.9).Speckle-tracking measures were similar to the 2D measures for both LV and RV systolic function, mainly LV GLS vs. LVEF, MAPSE, LV S\u2019 and RV GLS vs. RV FAC, TAPSE, RV S\u2019. Significantly different results for LV diastolic function were seen. Namely, assessment of LA strain markedly improved LV diastolic dysfunction detection compared to the classic criteria , there was a mild T lymphocytopenia . Nevertheless, T cell subsets were within the normal reference ranges.To understand the variations in the clinical and immune profiles of patients with COVID-19 during their ICU stay according to their cardiac manifestations, we divided patients into two groups, depending on whether they showed any cardiac dysfunction at admission to the ICU. These groups were compared throughout the three time points. However, as some patients were discharged from the ICU or eventually died during this period, the number of patients assessed was not constant at all time points.+) total T cell and CD4 T cell counts were similar between both groups at D1 and D3. However, nDYS patients showed an increase in the levels of both activated subsets at D7, compared to DYS patients . In addition, the percentages of activated HLA-DR+ CD8 T cells also decreased at D3 in DYS patients (p = 0.037), increasing afterwards at D7 (p = 0.013), while nDYS showed similar values in all time points.Considering the longitudinal assessment of immune data, both groups showed similar leucocyte and lymphocyte counts at admission  as shown in Regarding CD4 and CD8 T cell differentiation patterns, at admission, Tp = 0.007) and D7 evaluations (p = 0.046) regarding CD4 TEM cell percentages. Interestingly, the rates of CD8 TEM cells decreased over time in both groups, though without statistical significance (p = 0.058).Similar differences between groups were observed in D3 (p = 0.009). Although the IL-6 levels were not significantly different between groups, neither at baseline nor along the time, left atrial strain function parameters, such as LA RV and LA CT, correlated negatively with IL-6 levels at admission , as displayed in Considering other inflammatory biomarkers, DYS patients had lower platelets at D3, with slower recovery in platelet counts and CRP levels, and CRP decreased more at D7 in nDYS patients  or inverted (for LA CT) toward a positive correlation with IL-6 levels. Nevertheless, of the five nonsurvivors, three had the highest values of IL-6 (>300 pg/mL in two patients and another with 191 pg/mL), all at D7. Only one survivor had a similar peak of IL-6 (>300 pg/mL) at D3, decreasing afterwards at D7.Cardiac function parameters did not parallel the evolution in the other immuno-inflammatory markers. During the three time points, ventricular and atrial, systolic and diastolic function measurements did not change significantly in both groups , and patTo our knowledge, this is the first study to describe an association between the dynamic immunological and inflammatory profiles over time and cardiac dysfunction by STE, and not solely a cardiac injury defined by increased troponin levels in severe COVID-19 patients.CM) cells. Nevertheless, inflammatory markers were similar at admission in patients with and without cardiac dysfunction, and both groups had lymphopenia. In fact, lymphopenia has been described in COVID-19 patients with a different clinical spectrum of the disease [In the overall population, the clinical, imaging and routine laboratory findings of enrolled patients were similar to previously published data ,8,16. Pa disease , althoug disease , and our disease  the vira disease ,22. Ther disease . NeverthCM and TEM cells at admission and during the ICU stay. The striking loss of T cells, particularly of na\u00efve CD4 T cells, has been already characterized in COVID-19 patients [CM and TEM subsets [EM cells in G1 or S-G2/M cell cycle phases [Patients from our cohort with cardiac dysfunction showed a distinct T cell differentiation pattern, with a higher frequency of CD4 and CD8 Tpatients ,25, thou subsets , with 10e phases . Furthere phases , with pre phases . Additioe phases . In conte phases . In briee phases .Interestingly, although our patients had increased serum levels of IL-6, according to previous studies ,8,16,29,p = 0.007). More broadly, the immune features of COVID-19 remain largely unsettled. However, caution is needed when drawing inferences about the underlying processes that such markers might reflect as well as their potential causal roles in disease.A previous systematic review defied the role of IL-6 in COVID-19 . AlthougThis is the first study providing the incidence of cardiac dysfunction with multimodal description  in association with an extensive sequential immunological analysis (in different time points) in critically ill COVID-19 patients. Most previous studies have defined cardiac injury only by troponin elevation ,38, but Nonetheless, in our cohort of critically ill COVID-19 patients, 73,3% of patients demonstrated a cardiac dysfunction at ICU admission, and 80% of patients experienced cardiac dysfunction within the first seven days of ICU stay. The most frequent abnormalities were LV diastolic dysfunction, followed by LV and RV systolic dysfunction. Our data regarding cardiac dysfunction prevalence in COVID-19 is in line with recent evidence suggesting that 70% of patients with COVID-19 harboured a cardiac injury within the first ICU admission, identified by multimodal cardiac assessment . This inAlthough LA strain is advocated to be less load-dependent , it is sOn the other hand, venous congestion evaluation (IVC% and PVP) did not differ significantly between groups, nor was it correlated with acute kidney injury, an association we previously demonstrated in a small series . This coWe should highlight that remdesivir was given in only one-third of our patients, and no difference was found between groups. On the other hand, all patients had been under corticoterapy at some time point. Therefore, these are factors with weak discriminatory power to conclude from any association with their administration.Within our limitations, it is also worth mentioning the small sample size that could have underpowered our study for more clinically significant outcomes such as mortality, the need for mechanical ventilation, shock, ICU and hospital length of stay.Moreover, from a clinical point of view, the high rate of cardiovascular risk factors could also point out that pre-existing cardiac conditions of the patients studied may have determined subclinical systo-diastolic dysfunction independent from their ongoing SARS-CoV-2 infection at the time of the study. All the patients in the DYS group, except three who had no registries, had their clinical files checked to confirm the absence of cardiac dysfunction in previous echocardiograms,. Nevertheless, the repeated measures along the time course may have partially overcome this issue for the other results. Despite the extensive STE study, uncommon in other studies, we did not report the EKG abnormalities, which could enrich our population\u2019s spectrum of cardiac disease. Moreover, GLS measurements have been shown to be software-dependent . TherefoRegarding the immunological analysis, we should highlight different sample numbers between the three time points, mostly due to patients\u2019 discharge or death, and some practical limitations in assuring immunophenotypic assays in all samples. However, this was a longitudinal assessment in which each patient was evaluated continuously in subsequent time points, thus ensuring its control over time. Furthermore, only two trained operators processed and analyzed all samples, thus reducing a potential interference in reproducibility generally observed in a multioperator approach. Still, despite the extensive flow cytometric analysis being performed, functional assessments would have brought extra input on the characterization of the analyzed cells and their role in the disease. Finally, IL-1\u03b2 levels were inexpressive, eventually due to the low sensitivity of the kit used. Nevertheless, we aim to further explore other biomarkers, such as the IL-6 receptor, which may bring additional insights into the immune background of COVID-19 patients, particularly those with associated cardiac injury.Our study suggests a characteristic immunological profile in severe COVID-19 patients with cardiac dysfunction  and highlights the importance of early and repeated assessment of LV diastolic function.Moreover, in this group of patients, a more expressive effector memory T cell compartment at baseline, enriched in activated CD8 T cells, may suggest a persistent inflammatory state with its possible migration to peripheral tissues (such as the heart). Further studies are required to define immune-mediated tissue injury and identify specific host-directed therapy targets for the treatment of this disease.This study is a pioneer on the extensive longitudinal cardio-immunologic assessment of severe COVID-19 patients, and the associations found can further impact the COVID-19 pneumonia monitorization."}
+{"text": "Research from multiple perspectives to investigate adults\u2019 use of wearable activity-tracking devices is limited. We offer a multiperspective model and provide empirical evidence of what leads to frequent usage of wearable health technologies from a large, nationally representative survey sample.This study aims to explore factors affecting the use of wearable activity-tracking devices among health consumers from the perspectives of individual health beliefs  and information-seeking behaviors.Our Integrated Model of Wearable Activity Tracker (IMWAT) use and proposed hypotheses were validated and tested with data collected from a telephone survey with a national quota sample. The data were analyzed using a variety of statistical techniques, including structural equation analysis.The sample comprised 2006 participants. Our results showed that the perceived benefits of physical activity, perceived susceptibility, and self-efficacy toward obesity were significant predictors of information-seeking behaviors, which, in turn, mediated their effects on the use of wearable activity trackers. Perceptions of obesity severity directly promoted wearable device usage.This study provided a new and powerful theoretical model that combined the health beliefs and information-seeking behaviors behind the use of wearable activity trackers in the adult population. The findings provide meaningful implications for developers and designers of wearable health technology products and will assist health informatics practitioners and obesity prevention communicators. Obesity is regarded as an ongoing international health problem. Numerous studies have explored behavioral determinants of obesity such as an individual\u2019s psychological beliefs, unhealthy dietary habits, stress levels, and inadequate physical activity . PhysicaScientists introduced the concept of wearable health technology by suggesting that this type of technology can provide a meaningful solution to obesity issues ,7. WearaMany wearable health devices have been developed to detect and promote physical movement. Such wearable technology delivers accurate physical activity data and changes in dietary intake compared to the conventional method of collecting health information ,11. PrevThus, this study aims to fill this void; we begin by reviewing the prevailing consensus regarding psychological factors to predict obesity prevention behaviors from the Health Belief Model (HBM) ,17 and cTo develop a theoretical model of health consumers\u2019 wearable health device use, this paper adopts the HBM as a theoretical framework to understand the factors that trigger usage. The HBM was one of the first and best-known social cognition models to explain health-related behaviors . This moThe HBM explains certain beliefs in regard to threats to oneself , together with belief in the effectiveness of a proposed behavior, and predicts the likelihood of engaging in that behavior . In doinApplying this model to the obesity context, perceived susceptibility refers to the degree to which individuals perceive themselves to be susceptible to being obese; perceived severity refers to perceptions on risks or diseases among those who are overweight; perceived barriers equate to strong barriers that prevent individuals from obtaining obesity treatment or practicing intervention behaviors; and perceived benefits are one\u2019s understanding of the tangible benefits of health behavior change such as regular exercise to prevent obesity ,17,20. Rr=0.21), perceived susceptibility (r=0.15), perceived benefits (r=0.13), and perceived severity (r=0.08) were found to be significant factors across previous literature . Se. Se39]. Health information seeking was measured with the 5-point scale ranging from 1=\u201dnot at all\u201d to 5=\u201dvery frequently\u201d  41]. Us. Us41]. A total of 2006 participants were recruited to participate in the study. z test.The chi-square goodness-of-fit statistic is an index of model adequacy for which a nonsignificant value indicates a good fit of the model to the data. Our work used 4 additional fit indexes to decide how well the specified model explains the data: the comparative fit index (CFI), the root mean square error of approximation (RMSEA), the standardized root mean square residual (SRMR), and the Tucker\u2013Lewis index (TLI). CFI compares the difference between the chi-square value and degrees of freedom (df) of the null (independent) model to the difference between the chi-square value and df of the hypothesized model. Then, this difference is divided by the difference between the chi-square value and df of the null model. CFI is not sensitive to sample size and the recommended cut-off value is \u22650.90 . RMSEA pThe structural model (path model) is a special case of structural equation modeling (SEM). In the structural model, each measurement variable connects to each construct, that is, there exists a one-to-one mapping between constructs and measurement variables, and measurement errors become 0. The initially proposed model 1 is represented in lavaan package in R is an open-source program that is extremely powerful and flexible for SEM. The factor analytic models and the path model for model 1 were as follows:We assumed that all exogenous latent variables were correlated, and all data were analyzed using R, version 3.6.1 . The x1: the measurement variable of SUS;x2: the measurement variable of SEV;x3: the measurement variable of BEN;x4: the measurement variable of SE;x5: the measurement variable of IS;x6: the measurement variable of UOW;\u03be1: the exogenous variable (SUS);\u03be2: the exogenous variable (SEV);\u03be3: the exogenous variable (BEN);\u03be4: the exogenous variable (SE);\u03b71: the endogenous variable (IS);\u03b72: the endogenous variable (UOW).The factor analytic models for the exogenous and endogenous variables were as follows:The path model of structural coefficients was as follows:\u03b221 is the coefficient of \u03b71 on \u03b72,  is the coefficient of \u03be1 on \u03b71,  is the coefficient of \u03be2 on \u03b71,  is the coefficient of \u03be3 on \u03b71,  is the coefficient of \u03be4 on \u03b71, and  is the vector of equation errors to predict \u03b71 and \u03b72. Since there is a one-to-one mapping between constructs and measurement variables, the measurement errors are zero.where P value of the chi-square test was .07, which is greater than .05, indicating that this structural model fit the data well. The CFI value was 0.991, the RMSEA value was 0.024, the SRMR value was 0.017, and the TLI value was 0.967. z value), and P values for each path in model 1. The coefficients (\u03b2s and ) were estimated by maximum likelihood estimation. Susceptibility, benefits, and self-efficacy significantly predicted information seeking, and information seeking was significantly reflected in wearable activity tracker use . However, perceived severity did not give rise to information seeking . This may indicate that this model may not be adequate and needs improvement. Therefore, we considered an alternative model.Our model fit statistics included the following: chi-square, CFI, RMSEA, SRMR, and TLI. For the initial model, the SEM analysis indicated that the Our second model posits that susceptibility, benefits, and self-efficacy perceptions together contributed to information seeking, while perceived severity and information seeking together contributed to wearable activity tracker use . The onlThe factor analytic models for exogenous and endogenous variables were the same as model 1, but the path model of the structural coefficients was different from the model 1. The path model of structural coefficients was as follows:\u03b221 is the coefficient of \u03b71 on \u03b72,  is the coefficient of \u03be1 on \u03b71,  is the coefficient of \u03be3 on \u03b71,  is the coefficient of \u03be4 on \u03b71, and  is the coefficient of \u03be2 on \u03b72. Note that the only difference between model 1 and model 2 is the matrices in the middle term.where P values were also very close to the P values of the maximum likelihood estimates. In the initial model, ADF indicated that SEV (perceived severity) was not significant, whereas all paths were significant in the second model  estimator. Even though it is well known that the maximum likelihood estimator is relatively robust to violations of normality assumptions, and a large sample reduces the problem of multivariate nonnormality, it is worth checking our models with ADF. Consistent with maximum likelihood estimation, ADF estimates (not shown) had very similar values to the maximum likelihood estimates, and their Our multiple analyses generated a meaningful model of health care technology use that provides several contributions to theory and practice.First, it supports the application of the HBM in the use of wearable activity trackers, which was not present in most existing works. The key factors were identified within two paths: perceived benefits, perceived susceptibility, and self-efficacy influence health information\u2013seeking behaviors, while perceived severity is directly related to adults\u2019 use of wearable fitness trackers. It is meaningful to see how those factors are influencing the actual application of wearable activity-tracking devices with a varying range in significance and directional relationships.According to standardized estimates, we may identify a relatively stronger predictor of health information\u2013seeking behaviors: self-efficacy. This finding was consistent with a previous study  that four=0.14) was the second strongest predictor, followed by perceived barriers (r=0.22). In short, the above findings address two concerns: (1) additional research is needed to explore the predictive power of severity perceptions on other health-related contexts, as suggested by our data, and (2) future research should avoid the continued use of the direct and universal effects version of the HBM, as illustrated by our results.Interestingly, obesity severity perception was not related to online health information seeking, but rather directly related to adults\u2019 use of wearable fitness trackers. One possible explanation for this could be the nature of the predictor. Perceived severity has strong direct influences on behavioral outcomes, as supported by Lee and Kim . CarpentAnother thing to note is that our integrated model was confirmed by a large national cohort comprising over 2000 people, and the context was tailored to use of a specific technology\u2014wearable activity trackers. Previously, the TAM has been the only theoretical framework that dominantly explained the actual use of new technology. Our study contributes to the discipline by providing a new theoretical model combining the health beliefs and information-seeking behaviors for wearable activity tracker use. This could lead to additional contributions, such as gaining evidence-based knowledge on the precedents of wearable fitness trackers usage to promote physical activities and improve the outcomes of obesity interventions, and further evaluating its long-term effects. Using the proposed IMWAT, the mechanism underlying wearable health technology use can be explained.This study has limitations that should be addressed in future studies. First, we noticed that our endogenous variable, wearable activity tracker use, was skewed. This may have hindered the process of normal SEM; hence, we ran the same analysis to assess model fit and whether the paths were significant using the ADF estimator, which does not require any assumption in the data distribution. Consequently, the analysis with ADF generated the same result as our model tested with maximum likelihood, which does not make this limitation a major issue.Considering that our sample was skewed to those who were obese , a stratification analysis by individuals\u2019 weight status still needs to be explored in future research. To promote the wider use of wearable activity trackers, future studies also need to examine what triggers adults to adopt wearable health technologies and motivates them to continue using these devices.Finally, future studies could apply more variables from other major theories of health behaviors, such as the Theory of Planned Behavior. For instance, perceived behavioral control could be a meaningful addition to the current model. This variable is closely related to self-efficacy in the HBM, since they both reflect people\u2019s confidence in performing health behaviors .This study provides a theory-driven mathematical model of how different interactions between individual beliefs and multifactors influence wearable fitness tracker use among both obese and healthy adults. Our model holds several managerial implications for health informatics and health care practitioners utilizing wearable activity trackers for public obesity intervention programs . ChanginAs Feng and colleagues  pointed All in all, the state-level sample of over 2000 people produced nationally applicable results, ensuring the generalization of this study to a wider population and supporting the practical use of the results. The variables employed in this model will assist wearable health technology product developers and designers."}
+{"text": "We initiated a phase 3 clinical study (NCT04629703) of fostamatinib for the treatment of COVID-19.Key pathologies in severe COVID-19 include immune cell activation, inflammatory cytokine release, and neutrophil extracellular trap release (NETosis), which are mediated by spleen tyrosine kinase (SYK) . Fostamatinib, an oral SYK inhibitor approved for chronic immune thrombocytopenia, has shown activity in vitro using plasma from patients with severe COVID-19, by abrogating the hyperimmune response triggered by anti-spike IgG;\u200aA double-blind, randomized, placebo-controlled, adaptive design, multi-center, Phase 3 study (NCT04629703) is underway to evaluate the safety and efficacy of fostamatinib in 308 adult patients with COVID-19 . Hospitalized patients without respiratory failure  were included. Patients with ARDS or using extracorporeal membrane oxygenation (ECMO) were excluded. Patients will receive fostamatinib 150 mg BID or placebo for 14 days; both arms receive SOC. The primary outcome will be progression to severe/critical disease  within 29 days of the first dose of study drug. Fostamatinib is investigational for COVID-19.Blinded update of trial in progress as of 28 April 2021. 12 patients have been randomized in North and South America. The clinical status score at Baseline was 5  in all 12 patients. Five patients had 8 adverse events (AE) . One AE (PE) was serious and is resolving. No deaths have been reported. At least two patients have been discharged  with continued dosing at home.Fostamatinib has the potential to provide a treatment option for the hyperimmune complications of COVID-19.Ziad Mallat, MD, PhD, Rigel Pharmaceuticals, Inc. (Consultant) Sandra Tong, MD, Rigel Pharmaceuticals, Inc."}
+{"text": "Body image disturbance (BID) is a core feature of eating disorders, for which there are few objective markers. We examined the feasibility of a novel digital tool, \u201cSomatomap\u201d, to index BID related to anorexia nervosa (AN) severity. Fifty-five AN inpatients and 55 healthy comparisons (HC) outlined their body concerns on a 2-Dimensional avatar. Next, they indicated sizes/shapes of body parts for their current and ideal body using sliders on a 3-Dimensional avatar. Physical measurements of corresponding body parts, in cm, were collected for reference. We evaluated regional differences in BID using proportional z-scores to generate statistical body maps, and multivariate analysis of covariance to assess perceptual discrepancies for current body, ideal body, and body dissatisfaction. The AN group demonstrated greater regional perceptual inaccuracy for their current body than HC, greater discrepancies between their current and ideal body, and higher body dissatisfaction than HCs. AN body concerns localized disproportionately to the chest and lower abdomen. The number of body concerns and perceptual inaccuracy for individual body parts was strongly associated with Eating Disorder Examination Questionnaire  scores across both groups. Somatomap demonstrated feasibility to capture multidimensional aspects of BID. Several implicit measures were significantly associated with illness severity, suggesting potential utility for identifying objective BID markers. BID is associated with a higher risk of relapse4, which occurs in nearly half of all patients5, suggesting that a greater insight into the perceptual mechanisms underlying this process may be key for more effective and durable treatments. BID is a multifaceted construct that can be loosely divided into perceptual9 and attitudinal12 components. As there is a subjective element of this construct, it is difficult to assess the more objective sensory and affective components of how an individual perceives their own body, and few assessment tools exist to capture this.Body image disturbance (BID), defined as disruption of how one\u2019s body size is experienced, is a core diagnostic feature for anorexia nervosa (AN)10, commonly require patients to select from a predetermined menu of word indicators to describe their mentalized representation of their body. Perceptual inaccuracy  has been suggested to be a core characteristic of AN for decades15, and has been demonstrated empirically in multiple studies6. These perceptual distortions lead to both distress and subsequent eating disorder behaviors. For instance, an individual with AN may view their overall body or certain parts of their body as extremely large, when it is actually very thin. These perceptual inaccuracies and distortions are often related to negative emotions. Eating disorder behaviors may partly reflect attempts to correct misperceptions of body size and shape, and to temporarily reduce negative emotions, which are ultimately ineffective. Therefore, assessing the degree of these perceptual distortions as an indicator of eating disorder illness severity may inform treatment targets. Yet, it is still unclear whether and to what degree these abnormalities are due to a primary dysfunction of sensory encoding or another perceptual misrepresentation mechanism. Moreover, BID in AN has been linked to disrupted activity and connectivity in visual processing and parietal association networks20, raising the possibility that these brain areas play a disproportionate role in developing and/or maintaining over-estimations of one\u2019s body size and shape.Clinical measures of BID 24 are one commonly used method to overcome reliance on language-based inferences. Some figure scales have strong psychometric properties, provide quick assessments of overall body dissatisfaction24, and measure distinct dimensions of body dissatisfaction independently 22. Yet, they do not measure other details such as the types of body concerns , lack a focus on specific body parts , and they do not specifically measure the associated emotional impact of body concerns. Visual computer-based body-shaping tools have been developed, but they also typically only assess overall body size perception because they use multiple body parts that scale together, or else use preset virtual avatars29. This precludes identification of which aspects of the body are key to generating the disturbance, making it impossible to pinpoint the precise body areas that are misperceived as problematic, distorted, or obese, and to link these misperceptions with negative affect and eating disorder behaviors. Moreover, most tools also obscure the head/neck, preventing the identification of BID related to these features. The available studies assessing distinct body part distortions have found that individuals with AN overestimate individual body parts more than the whole body size30, suggesting a need for visual tools with this capability.Visual silhouette assessments31 for development details). These results raised the possibility that Somatomap might have utility for investigation of BID in clinical populations, such as in individuals with eating disorders.We recently created a multi-platform digital tool called \u201cSomatomap\u201d, which utilizes a visual representation of specific body concern areas on 2-dimensional (2D) and 3-dimensional (3D) avatars to facilitate BID assessment related to specific regions across the entire body. In a proof-of-concept study with female professional fashion models and nonmodels, this tool identified patterns of body image concerns localized to discrete body areas  to a clinical sample we made several refinements to the original tool, including increasing the number of 3D body part measurements to 23 independently manipulatable body areas and assessing additional body image measures . This allowed us to estimate a measure of \u201cbody dissatisfaction\u201d, defined by subtracting the ideal from the currently perceived body rating. We also evaluated the relationship between Somatomap BID measures and traditional estimates of illness severity . We predicted that inpatients with AN  would exhibit greater BID on the visual measures in Somatomap as evidenced by a significantly higher proportion of body image concern areas (Somatomap 2D), and significantly lower perceptual accuracy for the size/shape of individual body parts (Somatomap 3D). We also predicted that the degree of BID on Somatomap 2D and 3D would be associated with eating disorder symptom severity as measured by the EDE-Q. Finally, we employed a usability scale with the prediction that both groups would report acceptable levels of usability for each measure.The current study examined the feasibility of measuring BID characteristics in individuals with AN using Somatomap. In transitioning Somatomap for use from a nonclinical  Participants first used a mouse to outline an area of body concern directly on a 2D human avatar presented on a computer screen. They subsequently selected the specific concerns  and the emotions they related to each area  from a menu of visual icons. In addition to the 39 specific types of body concerns and the 26 specific emotions icon word-pairs they could select from, a free text-entry option was available for participants to indicate any other specific body concern areas or any other specific emotions related to their body concerns not listed in the menu.Participants also rated the level of defectiveness, emotion intensity, and perceived distress surrounding their body concern using visual-analogue scales . This process was repeated for each individual body concern. There was no limit on the number of body concerns that they could outline and rate, and no time limit.33. Participants used sliders to adjust the shape of each 3D avatar body area independently, in order to show how they perceived their current body, and then again to show how they would like their ideal body to look. Adjustable body areas included: neck girth, neck length, shoulder width, bust girth, chest girth, biceps girth, upper arms length, forearms girth, lower arms length, wrists girth, hands girth, hands length, torso length, waist, stomach form, hips, thighs girth, thighs length, calves girth, calves length, ankles, feet width, and feet length.After personalizing the avatar to match their own hair and skin color, the 3D avatar was displayed on screen. The size/shape of the 23 adjustable body parts were randomly permuted for the initial avatar to prevent potential priming effects associated with viewing anatomically proportioned bodies31 (adapted from the PhenX toolbox34), with the inclusion of 16 additional body areas for a total of 23 body areas. Areas with multiple parts  were measured individually and then averaged. We used the InBody bioimpedance scale  and a stadiometer to calculate each participant\u2019s actual body mass index (BMI).The protocol for obtaining the physical body measurements of participants was applied from our prior Somatomap study35, which includes four subscales: Shape, Weight, Eating, and Restraint concern. A summed average of the four subscale ratings yields an EDE-Q Global score; all scores range across a scale from 0-none to 6-severe. Scores\u2009\u2265\u20094.0 have often been considered to reflect clinically severe levels of eating disorder psychopathology37.To estimate the degree of eating disorder symptom severity, each participant completed the Eating Disorder Examination Questionnaire (EDE-Q 6.0)38. Chorus is a visual development platform supporting the creation of web-based digital health applications that can be accessed on various devices including mobile, tablet and desktop.This study was approved by the Western Institutional Review Board, and all methods were carried out in accordance with relevant guidelines and regulations. Prior to the experiment, each participant provided written informed consent, and informed consent was obtained from a parent and/or legal guardian for the participants under 18\u00a0years of age . All participants completed demographic questions, BID assessments , and self-report scales at LIBR on a computer. User experience surveys were collected after completing the 2D and 3D measures, and physical measurements were completed last. The data for the Somatomap app and survey was collected on the Chorus app platform31. To evaluate between-group differences in the body maps we used custom Matlab software  to calculate the test statistic for each pixel using the z-formula for proportion, and employed cluster correction (6 pixel threshold) and smoothing following our previously described procedures40. Across both groups, a linear regression model (lm) conducted in R (version 3.6.2) examined the relationship between the number of individual body concerns traced and eating disorder symptomatology .To create group-level proportional maps of body concerns, all pixelwise body concern tracings were merged for each person (overlapping pixels were set to 1) and the ensuing binary maps were overlapped as in our prior Somatomap study procedure31. Similar to our previous study, we used multivariate analysis of covariance (MANCOVA) to determine if there were group differences in each of these measurements. Covariates included BMI, height, and weight. If the MANCOVA results were significant, a follow-up analysis using analysis of covariance (ANCOVA) with the Benjamini\u2013Hochberg multiple comparison correction was used to determine which variables showed statistically significant differences between ANs and HCs. Multiple linear regression models were used to examine relationships between body dissatisfaction body part measures and body discrepancy body part measures (separately) with measures of psychopathology severity (EDE-Q), across both groups. Multiple linear regression models were used to examine whether intersecting measures on 2D and 3D together might explain more variance of symptom severity than when applied with 2D concerns and 3D body dissatisfaction and body discrepancy separately.The 3D assessment procedure yielded six distinct scores across each of the 23 body parts: (1) actual body, (2) current perceived body, (3) ideal perceived body, (4) current body discrepancy (2\u2009\u2212\u20091), (5) ideal body discrepancy (3\u2009\u2212\u20091), and (6) body dissatisfaction (3\u2009\u2212\u20092). Both current and ideal perceived body measurement scores were converted into centimeters from arbitrary units using piecewise linear interpolation as previously describedUsability surveys were collected after completing both measures to assess the ease and enjoyability of use, the degree to which the 2D tool effectively captured participants\u2019 BID concerns and associated emotions, and the degree of identification with the original and final 3D avatar.P\u2009<\u20090.001), which was driven by differences in body weight (P\u2009<\u20090.001) but not height (P\u2009=\u20090.70). No significant differences for overall race/ethnicity , or education levels  between the groups were identified.Fifty-five AN and 55 HC females completed testing (see Table P\u2009<\u20090.001)  and from 1 to 7 per individual for HCs , which significantly differed between groups . Across both groups, a linear regression revealed a significant relationship between the Global EDE-Q score and the number of body concern areas outlined  to complete Somatomap 2D .Proportional body maps showed that AN participants perceived body concerns across a broader area compared with HCs Fig.\u00a0A. The stM\u2009=\u200915.65; SD\u2009=\u200915.74) and from 0 to 20 per individual for HCs , which was significantly different between groups . The number of affective labels selected  for all concerns ranged from 0 to 93 per individual for ANs  and from 1 to 22 per individual for HCs ; which was also significantly different between groups  ranged from 1 to 73 per individual for ANs (Ps\u2009<\u20090.001) with regards to the level of defectiveness ; HC ), emotion intensity ; HC ), and degree of distress caused by body concerns ; HC ).Emotion ratings associated with body concerns were significantly higher for ANs relative to HCs \u2009=\u200933.73, Wilks \u039b\u2009=\u20090.097, P\u2009<\u20090.001; Table F\u2009=\u20091.73, Wilks \u039b\u2009=\u20090.675, P\u2009=\u20090.037; Table F\u2009=\u20093.40, Wilks \u039b\u2009=\u20090.515, P\u2009<\u20090.001; Table There were multiple statistically significant group differences in the measurements of the actual body, current perceived body, and ideal perceived body. A complete listing of these results is provided in the Supplement. Here, we summarize the statistically significant differences observed. The AN group had a significantly smaller measured bust girth, chest, biceps, waist, \u201cstomach form\u201d , thighs, and calves, and had a shorter upper arm length compared with HCs \u2009=\u200911.89, Wilks \u039b\u2009=\u20090.232, P\u2009<\u20090.001; Fig.\u00a0There were multiple statistically significant differences in the current body discrepancy, ideal body discrepancy, and body dissatisfaction measurements. A complete listing of these results is provided in the Supplement. In summary, the AN group had significantly higher discrepancy scores (overestimated current body sizes) for neck girth, biceps, stomach form, hips, thighs, calves, ankles, feet length, and overall body compared to HCs \u2009=\u20096.37, Wilks \u039b\u2009=\u20090.362, P\u2009<\u20090.001; Fig.\u00a0Relative to HCs, the AN group had significant differences in ideal body discrepancy scores such that they desired narrower shoulders, a thinner chest, a larger bust, a thinner waist, narrower feet, longer upper arms, torsos, and hands, compared with their actual body part sizes and shapes. Both groups desired a thinner stomach form, but the AN group desired it to be significantly thinner relative to their actual body \u2009=\u20092.59, Wilks \u039b\u2009=\u20090.582, P\u2009<\u20090.001; Fig.\u00a0M\u2009=\u20096.9; SD\u2009=\u20092.2; HC M\u2009=\u20096.4; SD\u2009=\u20092.0).The AN group showed significantly greater body dissatisfaction scores for neck girth, chest, biceps, forearms, waist, hips, thighs, calves, ankles, feet width, and overall body  relative to the HC group \u2009=\u20093.71, P\u2009<\u20090.001) \u2009=\u20097.57, P\u2009<\u20090.001) \u2009=\u20095.03, P\u2009<\u20090.001).To explore the degree to which aggregating the number of body concerns on 2D and all of the body dissatisfaction parts on 3D, were related to eating disorder severity, we conducted a multiple linear regression using the EDE-Q Global scores as the dependent variable, with the number of 2D body concerns plus all of the body dissatisfaction parts on 3D as independent variables, across groups. Together, the number of 2D concerns with all of the 3D body dissatisfaction body parts explained 59% of the variance of overall ED psychopathology \u2009=\u200923.4, P\u2009<\u20090.001).Chest and waist were body areas identified by both the 2D and 3D approaches as being significantly related to BID in AN relative to HCs. We conducted a multiple linear regression between the number of 2D body concerns and 3D chest and waist body dissatisfaction and the EDE-Q Global scores, which explained 40% of the variance of overall ED psychopathology \u2009=\u20099.73, P\u2009<\u20090.001). All pre-checks for all regression models performed showed minimal concern evidence for of multicollinearity (variance inflation factor\u2009<\u20095).Finally, to examine the degree to which aggregating relevant 2D and 3D measures explained symptom severity, we conducted a multiple linear regression using the EDE-Q Global as the dependent variable, with the number of 2D body concerns plus all of the current body discrepancy parts on 3D as independent variables, across groups. Together, the number of 2D concerns with all of the 3D current body discrepancy body parts explained a high percentage of the variance of overall ED psychopathology , and enjoyment , and how closely the final 3D avatar reflected their body  than the AN group. Participants \u201cliked least\u201d that Somatomap: \u201cwas lacking muscle tone\u201d, \u201cstarted randomized\u201d, and \u201cliked best\u201d that it was: \u201ceasy to use\u201d, \u201cenabled me to express myself\u201d, and \u201cvery realistic\u201d.Participants rated the 2D body map and 3D avatar as easy to use, as very reflective of their body concerns (2D), and that the final 3D avatar was mostly reflective of their actual perceived body Tables , S.9. HCThe current study tested the feasibility of a novel digital tool for assessing multidimensional BID characteristics, including objective measurements of perceptual disturbance for body part size estimation and subjective experiences of dissatisfaction, in acutely ill individuals with AN relative to HCs. We also tested the relationships between these measures and illness severity estimates. We found, as hypothesized, that there were significant BID differences between groups in terms of perceptual overestimations, thin-ideal body dissatisfaction, and body concerns , and in terms of the affective valence associated with body concerns. Finally, Somatomap 2D and 3D difference measures were associated with symptom severity measures across groups when examined individually and when aggregrated, with aggregated measures explaining the greatest symptom variance.Analysis of the Somatomap 2D data revealed that this tool detected a greater degree of BID concerns in AN than HCs for the chest and abdomen, with greater levels of perceived distress, intensity, and defectiveness related to AN body concerns. Moreover, across both groups, the number of outlined body concerns was positively associated with eating disorder symptom severity on the EDE-Q when controlling for age and BMI. This result raises the intriguing notion that a single perceptual rating about body concerns (involving a few mouse clicks and minutes) could provide information about an individual\u2019s eating disorder psychopathology severity. Such an implicit approach could potentially augment or replace more time-consuming screening methods which explicitly signal the search for a disorder, although additional study is needed to verify this possibility. At a minimum, this approach provides a clear indication of the visual perceptual body mapping and associated emotional characteristics related to BID in AN, at the individual and group levels.41, which might decrease their ability to accurately perceive their own body.Analysis of the Somatomap 3D data identified that, overall, the AN group (relative to HCs) showed a general over-estimation of current body girth and a preference for a thinner body than what they currently perceived. This was apparent from the current perceived body, current body discrepancy, ideal perceived body discrepancy, and body dissatisfaction scores, which were associated with symptom severity. These findings support the value of assessments of individual body parts to better understand perceptual and attitudinal aspects of BID. They raise the possibility of impaired cognitive and/or visual mechanisms operating in individuals with AN42, which suggests that mismatches between actual and ideal internal self-representations produce negative affect and poor health outcomes. These results also support a recent meta-analysis which found that more negative and less positive affect ranges for self-discrepancy were significantly related to psychopathology43. We might speculate that the AN inpatients\u2019 overestimations of their body size compared to HCs in our current study indicates an inability or difficulty to calibrate their body perception in relation to the general population. Another possibility is that they may \u2018re-calibrate\u2019 their body perception towards other underweight individuals present in their current treatment setting. The development and/or maintenance of body size and shape over-estimations in AN may be related to previously demonstrated brain activation and connectivity abnormalities in visual systems. For example, BID in AN may also be due to a disturbance in brain connectivity and abnormal functioning in body processing networks45. This might involve reduced connectivity between the left fusiform body area and extrastriate body area46, reduced neural activity in cortical visual systems and hyperconnectivity in dorsal visual and parietal networks20, which may be associated with body size misperception46 in AN. The connectivity of these networks could be further investigated in future studies utilizing Somatomap. For a discussion of the relevance of these findings to theories of body perception in HCs, see the Our observation that the AN group overestimated body sizes and reported increased negative emotion compared with HCs supports the self-discrepancy theory25. The chest is another focus area given that it is a region containing adipose tissue in females. Both regions show a lower percentage of body fat in the underweight stage followed by an increased ratio of truncal/extremity fat during weight restoration26, which may help to explain the common overlap across measures. Our exploratory aggregated regression analyses revealed that these measures explained a substantial degree of symptom severity on the EDE-Q. Specifically, while the chest and waist body dissatisfaction (3D) together with the number of 2D body concerns explained 40% of the variance of EDE-Q, and body dissatisfaction parts (3D) together with the number of 2D body concerns explained 59% of the EDE-Q variance, it was the 3D current discrepancy body parts (together with the number of 2D body concerns) that explained the most variance of symptom severity: 73% of the EDE-Q Global score variance. Although exploratory, these results suggest that combining convergent measures focusing on the visual perceptual characteristics or body dissatisfaction of discrete body parts, and total number of body parts of concern, might be used to make meaningful inferences about the degree of symptom severity across the spectrum of eating psychopathology.Two truncal areas of AN body concern were identified across Somatomap 2D and 3D: the abdomen and chest. Excessive fullness and/or bloating of the stomach and intestines is a frequent complaint for individuals with AN, which are often identified in clinical settings as motivators of restrained eatingObjectively distinguishing the perceptual and affective components of BID may be critical to better understanding AN, in terms of screening, prevention, diagnostic assessment, prognostic prediction, intervention targeting, and relapse prevention monitoring. Clinicians currently measure BID by relying upon subjective, language-based questionnaires or visual analogue scales which assess body dissatisfaction and body distortion constructs in a global manner. The present study tested the feasibility of a different approach, one that included quantification of visual perceptual distortions and body dissatisfaction for discrete body parts. This revealed: (1) both Somatomap 2D and 3D detected visual perceptual and affective aspects of BID in AN, explained unique variance in eating disorder symptom severity and greater variance when combined across groups, (2) both were tolerable, with no one quitting the measurement during test/retest, (3) usability was generally good, although the significantly reduced usability rating in AN (compared with HCs) means there is room for participant-suggested improvements, and (4) Use of a multi-platform digital tool means it can be remotely deployed such as in post-acute settings for monitoring and detecting illness trajectory. In addition to providing more specific information about distinct combinations of BID and illness severity, this tool may equip researchers to visually, statistically, and remotely pinpoint BID parameters that are uniquely related to ED severity.This study has several limitations. First, AN participation in this study was limited to females from a single inpatient treatment facility, and therefore, testing with a broader range of facilities, care settings, and demographic characteristics  would be important to demonstrate generalizability. Second, although enjoyability of the tool was rated by AN participants above average, this experience could be further improved (particularly in the AN patient group) by incorporating AN participants\u2019 usability assessment feedback to include other dimensions of the body . Longitudinal and reliability studies will be necessary to test the tool\u2019s ability to detect changes in illness severity , and comparing this assessment with other measures of BID to highlight the incremental validity will be essential. An additional approach could evaluate how changes in BID relate to (or predict) longitudinal clinical outcomes. A strength of the current approach is that it effectively captures a broad range of body dissatisfaction self-concepts in relation individual body parts, and illustrates their association with illness severity. The digital multi-platform nature of this tool could have broad applications including remote deployment, in multiple clinical settings.The current study demonstrates the initial feasibility of Somatomap for assessing multidimensional aspects of BID related to ED severity in AN. The novel visual perceptual mapping approach entailed by this tool can capture implicit responses and estimate perceptual disturbances at the level of individual body parts, yielding objective markers of BID. This positions Somatomap as a potentially useful tool both for research and clinical applications.Supplementary Information."}
+{"text": "Body weight dissatisfaction (BWD) and visual body perception are specific aspects that can influence the own body image, and that can concur with the development or the maintenance of specific psychopathological dimensions of different psychiatric disorders. The sexual orientation is a fundamental but understudied aspect in this field, and, for this reason, the purpose of this study is to improve knowledge about the relationships among BWD, visual body size-perception, and sexual orientation.A total of 1033 individuals participated in an online survey. Physical comparison, depression, and self-esteem was evaluated, as well as sexual orientation and the presence of an eating disorder. A Figure Rating Scale was used to assess different valences of body weight, and mediation analyses were performed to investigated specific relationships between psychological aspects.p\u2009<\u20090.001); instead, higher body misperception was present in gay men (p\u2009=\u20090.001). Physical appearance comparison mediated the effect of sexual orientation in both BWD and perceptual distortion. No difference emerged between women with a history of eating disorders and without, as regards the value of body weight attributed to attractiveness, health, and presence on social media.Bisexual women and gay men reported significantly higher BWD than other groups (This study contributes to understanding the relationship between sexual orientations and body image representation and evaluation. Physical appearance comparisons should be considered as critical psychological factors that can improve and affect well-being. The impact on subjects with high levels of eating concerns is also discussed.Level III: case\u2013control analytic study. The way people perceive their bodies is an essential aspect of daily intrapersonal and interpersonal interactions; indeed, a negative body image is associated with eating disorders (ED), adverse sexual experiences, low self-esteem, depression, and anxiety \u20133. Body The presence of a discrepancy between the body\u2019s perception and the ideal body that people struggle to achieve can be defined as body image disturbance . Body imThere is a broad consensus that heterosexual women experience normative pressure to achieve specific ideal body sizes . HoweverThe evaluation of one\u2019s body image includes the concerns about body weight and shape, the perception thereof, and the ideal body appearance, linked to a specific mood, self-esteem, and cultural environment , 34. DifHowever, in the literature, the psychometric instruments used have not been sufficiently sensitive to differences between subgroups with different socio-cultural pressures about body evaluation, and this could explain the mixed results obtained in the literature , 25. BodOur goal is to study different facets of body weight and body image perception in a significant sample of cisgender women and men with different sexual orientations. Recent evidence from the literature has shown psychological differences between cisgender and transgender people regarding body image and body experiences , 41. MorThe data were collected with an anonymous online survey (SurveyMonkey.com) completed by Italian speakers, 18\u00a0years old or older. The participants were recruited via online invitations through social media  and LGBTQ\u2009+\u2009group mailing lists from the area of the Veneto Region , through those responsible for managing personal data, without the involvement of researchers. The invitation consisted of a request to complete voluntarily and spontaneously in questionnaires on body image and body experiences to indicate that the questionnaires would be used for research purposes. The online survey was devised in such a way as to prevent multiple responses from the same IP addresses, but the IP addresses were hidden from investigators. The data were collected between February 2019 and October 2019. Each participant provided informed consent before receiving the survey. The research was in accordance with the Declaration of Helsinki, and an ethical evaluation of the study was obtained by the local research ethics committee. The anonymous nature of the data acquired does not require the application of European legislation about privacy. The duration of the survey was about 30\u00a0min.As suggested by the international literature, the gender of each participant was determined by asking them whether they identify as cisgender, transgender, or queer with forced choices about the gender they had at birth and about their current gender . Sexual \u03b1\u2009=\u20090.815).The Rosenberg Self-Esteem Scale (RSES) is a widely-used tool that measures positive and negative feelings about oneself . It is a\u03b1\u2009=\u20090.752).The Physical Appearance Comparison Scale (PACS)  was used\u03b1\u2009=\u20090.788).The Patient Health Questionnaire-9 (PHQ-9) is a screening tool for depression . It is a\u03b1\u2009=\u20090.898).The Eating Attitudes Test (EAT-26) is a well-established self-report questionnaire measuring eating symptoms and concerns characteristic of individuals with and without ED . It incl2 with an increase of 2\u00a0kg/m2 and a center of 25\u00a0kg/m2. The participants were asked to choose which figure best represented their body size and represented an ideal body; these questions were asked to identify possible biased representations and body weight dissatisfaction. Body weight dissatisfaction was evaluated by subtracting the selected ideal figure score from the estimated current figure score. Perceptual distortion was assessed by subtracting the current figure score  from the estimated current figure score ). Looking at men, the effect of sexual orientation on body weight dissatisfaction was significantly mediated only via eating concerns . Looking at women with an ED, no mediation effects were found.The first hypothesis tested with mediation analysis was that psychological constructs could mediate the relationship between body weight dissatisfaction and sexual orientation. Looking at women without an ED, the effect of sexual orientation on body weight dissatisfaction was mediated only via the physical appearance comparison . No significant mediation effect was found in the ED subgroup. See Fig.\u00a0The second hypothesis was about the distorted perception of the body. Women without ED as well as men, showed that the effect of sexual orientation on perceptual body distortion was mediated physical comparison  and sexual orientations have an influence on body weight dissatisfaction and body perception/representation in the cisgender population. This study found significant relationships between the sexual orientations of cisgender individuals and body weight dissatisfaction and body size perception. Bisexual women and gay men seem to be the most vulnerable to body weight dissatisfaction, which is a significant risk factor for psychiatric disorders with severe impacts on patients\u2019 lives. However, the data collected should be increase with more studies with integrated methodology  that allowed a deeper inside into the effect of body weight dissatisfaction or body perception. For this reason, sexual orientation should be systematically taken into consideration in the assessment and treatment of EDs or other body image disturbances .The relationship between sexual orientation and body weight dissatisfaction and body representation is a neglected topic, especially in women and sexual minorities. It has a role in the development and in the maintenance of body image disturbance, but there is a lack of research about the presence of any moderator constructs that could contribute to its modification.A large sample of cisgender women and men was included in the study, showing how that bisexual women and gay men are exposed to higher body weight dissatisfaction and distorted body shapes perception. Attractiveness analysis highlighted the relevance of internalized normative pressure for thinner female bodies in all subgroups, even though bisexual women and men prefer healthy weight bodies. Physical appearance comparison is a crucial psychological element in the assessment of body comparison and evaluation, and this should be taken into consideration in mental health prevention programs."}
+{"text": "Applications of advanced image and histological techniques revealed the presence of cortical alveoli oocytes (CAO), which prevailed as the most advanced oocyte phase for 4\u20135\u00a0months. This new finding of an extended and early appearance of CAOs in this gadoid was supported by that vitellogenesis first started to appear 3\u2009months later. The subsequent oocyte growth trajectories indicated that larger individuals [total length (TL)\u00a0=\u00a070\u2009cm] typically spawn in the order of 3\u2009weeks earlier than the smaller ones (TL\u00a0=\u00a040\u2009cm). The spawning season appeared stretched over about 3\u2009months. The majority of skipping females arrested oocyte growth at the CAO phase followed by atretic reabsorption. Compared to those individuals maturing for the spawning season, \u2018skippers\u2019 generally exhibited lower body condition, characterized also by relatively lower liver sizes at the time of the main spawning season. This study demonstrated well\u2010developed skipping dynamics, but also that the CAO period, i.e., when skipping takes place, may be exceedingly long in this commercially valuable gadoid and that its reproductive cycle in many ways deviates from that of the data\u2010rich, sympatric NEA cod (Gadus morhua).The present study tracked oocyte development over 9\u00a0months and noted incidences of \u2018skipping\u2019,  The largest stock of this species\u2014north\u2010east Arctic (NEA) hereafter referred to as M. aeglefinus (NEA stock)\u2014resides in the Barents Sea  does not follow this pattern  if not accounted for in assessment models  are excluded from annual SSB estimates, uncovering under what conditions insufficient energy reserves or associated trade\u2010offs  which continue oocyte development will migrate southwards to spawning grounds (~68 to 72\u00b0N) along the shelf break from the Lofoten archipelago and northwards , or advanced previtellogenic oocytes (PVO) . We took advantage of modern image analysis techniques in combination with histology to pin\u2010point the time course of reproductive events in field\u2010caught M. aeglefinus (NEA stock) through the initiation of the reproductive cycle until the onset of spawning to (i) examine oocyte development and timing of reproductive commitment, (ii) investigate the dominant mode and fundamental mechanisms determining skipping, and, finally, (iii) discuss and compare our findings to related studies on the sympatric Gadus morhua (L.) (NEA stock), a group\u2010synchronous and determinate batch spawner like M. aeglefinus  were collected in the Barents Sea at 181 sampling locations during autumn, winter and spring , ovary weight  and liver weight . The currently applied equations for Fulton's condition factor (K) and somatic hepatosomatic index (HSIS) were given as K\u00a0=\u00a0100\u2009\u00d7\u2009W/TL3 and HSIS\u00a0=\u00a0100\u2009\u00d7\u2009LW/(W\u2009\u2212\u2009OW) ; the 95% confidence interval of the exponent parameter ranged between 2.98 and 3.05. Ages were obtained from otoliths , whole weight (https://www.axlab.dk/) with 20\u2009ml of 4% formaldehyde.Just after completion of fish handling, an ovarian subsample (~2\u00a0g) was excised from the mid\u2010section of the right lobe  were subjected to digital image analysis according to the auto\u2010diametric method  with the plugin ObjectJ (https://sils.fnwi.uva.nl/bcb/objectj/). The mean size of the 20 largest oocytes, hereafter labelled as oocyte leading cohort diameter , reflected the female maturity stage  were analysed histologically to detail microscopic structures\u2014oocyte development phases as well as post\u2010ovulatory and atretic follicles .Based on detailed examinations of histological slides, immature, skipping, maturing or developing  as well as measuring the extent of multicollinearity . In this exploratory analysis, the minimum value of LC was set at 450\u2009\u03bcm to ensure that all females included were de facto maturing (see below), whereas the maximum LC value was set at 800\u2009\u03bcm assuming imminent spawning  maturity categories. Finally, a two\u2010way ANOVA was used to examine if the continuous response variables K and HSIS varied between maturity categories at different sampling months, including an interaction term (maturity category\u2009\u00d7\u2009month).Prior to performing any test on changes in LC over time, the date variable was converted into the number of days, where the earliest sampling day, August 14, 2020, was coded as day 1, and subsequent sampling days were given a number past this day, K and HSIS. Finally, Tukey's HSD post hoc test was used to examine differences detected by ANOVA. All analyses were performed in R  and rejection of the null hypothesis was always set at P\u2009<\u20090.05.Model assumptions were assessed by residual\u2010fit plots and tested for normality and homogeneity of variance using Shapiro\u2013Wilk and studentized Breusch\u2013Pagan tests. When parametric violations were detected, data were arcsine transformed in the case of ratios represented by 33.1LC of maturing individuals continued to increase over the entire study period Figure\u00a0. This oo3.2F4,88\u00a0=\u00a025.4, r2\u00a0=\u00a00.515, P\u2009<\u20090.001)  when combined with day (P\u2009<\u20090.001) and TL (P\u00a0=\u00a00.04). Hence, the resulting equation with LC as response variable became:Substantial individual variability in LC existed at a given sampling date for fish with LC \u2265450\u2009\u03bcm, reflecting an extensive spawning season  indicated individual variations in the start of spawning of at least 3\u00a0months: on March 15 the LC in one female was 477\u2009\u03bcm, while it was 785\u2009\u03bcm in another, i.e. a difference of 308\u2009\u03bcm, corresponding to 100\u2009days if the LC growth rate is 3.07\u2009\u03bcm/day.with the separate 3.3i.e., presence of CAO, were observed in August: four out of 13 females had at that time reached ECAO  of the studied immature females did not show any sign of oocyte development, remaining in PVO 4A Figure\u00a0, with LC Figures\u00a0 and 6. B Figures\u00a0 and 6. A Figures\u00a0 at LC 46 Figures\u00a0  and larger   compared to immature  and skipping  females.Within the material examined by histology, significant differences in age and TL existed among maturity categories Figure\u00a0, where m3.6S, appeared dependent on maturity category, where skipping females showed significantly lower values in March than spawning females , but the main effect, i.e., maturity category\u2009+\u2009month, was evidently in place . Consequently, overall, HSIS was a more sensitive parameter in these respects than K; maturity category\u2009+\u2009month explained 38.9% of the variability in HSIS but only 14.4% in K.Monthly changes in female energy storage, represented in the first instance by HSIs Figure\u00a0. In Apri4M. aeglefinus displayed several special features as a gadoid. Our histological analysis revealed an extended cortical alveoli phase, relative to the better\u2010studied G. morhua: in M. aeglefinus (NEA stock) CAO appeared as early as August  is likely start releasing eggs in the order of 1\u2009month later than G. morhua (NEA stock)    the CAO phase might persist throughout the whole reproductive cycle but under an exceptional reproductive style of two vitellogenic cohorts ultimately splitting apart supporting successive annual spawning  females proceed directly from late PVO to CAO  resembles that of a recent study of the North Sea saithe \u00a0 individuals was evident in that some females, at a given day, showed up to 300\u2009\u03bcm larger LC sizes compared to others. The reason for the observed and extrapolated variability in spawning time was partly related to size\u2010specific effects   during the months from February to March, with the remaining trivial proportions of skipping females classified as either reabsorbing\u2010CAO or \u2010VO. Consequently, the resting\u2010PVO mode should be expected to specifically refer to the end (closure) of the reabsorbing\u2010CAO mode, i.e., following completed CAO atresia, indicated presently by the gradual accumulation of resting skippers and the associated declining proportion of reabsorbing skippers. This way of thinking markedly differs from the conventional one, where resting skippers are said to not advance beyond PVO phases (see Introduction). We therefore claim that an individual M. aeglefinus becoming a skipper enters the CAO phase, as default, but becomes physiologically arrested at the following energetically demanding vitellogenin sequestration. Both steps are part of the gonadotrophin\u2010dependent oocyte growth, indicating that the animal is sexually mature . Compared to G. morhua (NEA stock), M. aeglefinus (NEA stock) not only show approximately 40% higher fecundity, selecting TL\u00a0=\u00a060\u2009cm as the overlapping reference point  , although a nonsignificant trend was noticed. Nonetheless, skipping females generally exhibited lower values of HSIS and K compared to maturing females. High variability in K and particularly HSIS was common, indicating that other factors than maturity status or temporal variability may have affected these parameters. Alternatively, reproductive decisions were potentially being made much earlier, following a critical post\u2010spawning feeding period   will do in the future under ongoing climate change. A recent climate impact study suggests that this stock will develop positively under the intermediate RCP 4.5 climate scenario until 2050, in stark contrast to M. aeglefinus (North Sea), due to increased gross primary and secondary production and less sea ice abundance in the Barents Sea . These results have implications for future reproductive studies of M. aeglefinus by in\u2010depth characterization of continuous oocyte growth of maturing individuals and terminated oocyte development of skippers. It would be beneficial if M. aeglefinus (NEA stock) ovary samples in future studies were collected earlier in the reproductive cycle  to assess the nutritional status of females at a time when the physiological decision to spawn might be predetermined.To conclude, the present study has highlighted important new processes related to the reproductive cycle of This contribution is a revision of an earlier Master of Science thesis in Fisheries Biology and Management, University of Bergen, Norway, defended by F.T in June 2021. E.J., O.S.K. and F.T. conceived the study. O.S.K designed the laboratory analysis. F.T. and M.A. processed samples in the laboratory. F.T., T.C.S.S. and K.C. analysed the data. F.T., O.S.K and E.J. wrote the manuscript. A.F provided supervision. E.J. and O.S.K acquired funding. All authors commented on manuscript drafts and approved the final version of the manuscript."}
+{"text": "The ZnO nanopowder, before it was doped into CsPbBr3 solution, was first put into a furnace to anneal at different temperatures, and formed the pa-ZnO. The properties of pa-ZnO were different from ZnO. The optimized doping conditions were 2 mg of pa-ZnO nanopowder and pre-annealing at 300 \u00b0C. Under these conditions, the highest sensitivity  of the ZnO-doped CsPbBr3 perovskite acetone sensor was 1726. In addition, for the limit test, 100 ppm was the limit of detection of the ZnO-doped CsPbBr3 perovskite acetone sensor and the sensitivity was 101.In this work, acetone gas sensors were fabricated using pre-annealing metal oxide zinc oxide (pa-ZnO)-doped perovskite cesium lead bromide (CsPbBr There are more than 3500 chemical components in the breath exhaled by the human body, most of which are volatile organic compounds (VOCs). Among the breath VOCs, acetone is a by-product of lipid metabolism that is closely related to blood glucose levels. Acetone in exhaled breath can thus monitor the metabolic state of the human body. Various techniques have been employed to measure the acetone of very low concentrations, such as gas chromatography\u2013mass spectrometry, proton transfer reaction mass spectrometry, vacuum-free ion mobility spectrometry, laser absorption spectroscopy, and colorimetric sensors ,2. HowevThe boiling point and autoignition temperature of acetone are 56 \u00b0C and 465 \u00b0C, respectively. Metal oxide semiconductors are commonly used as sensor materials for sensing chemical gases, but they need to have good sensitivity in high-temperature environments, while perovskite can have a good gas sensing effect in room-temperature environments ,8. Halid3 solution. In total, 0.1835 g of PbBr2 powder , 0.1064 g of CsBr powder , and 1 mL of DMSO solvent  were put into beaker and stirred with 500 ppm at a temperature of 70 \u00b0C for 30 min. Then, pa-ZnO nanopowders with various weights  were added into the beaker and stirred with 500 ppm at temperature of 70 \u00b0C for 1 day. Next, the third step was the formation of pa-ZnO-doped CsPbBr3 perovskite film for the acetone sensor. A total of 60 mL of pa-ZnO-doped CsPbBr3 solution was spin-coated on a glass substrate with an ITO  pattern at 5000 ppm for 30 s. Additionally, the pa-ZnO-doped CsPbBr3 perovskite film was baked on a hot plate at 100 \u00b0C for 10 min to complete the electro-resistance-type pa-ZnO-doped CsPbBr3 perovskite acetone sensor, as shown in In the device preparation, the ZnO nanopowder with a size of 30 nm  was first put into a furnace to anneal at different temperatures, and formed the pa-ZnO. The second step was to prepare the pa-ZnO-doped CsPbBr3 perovskite films. Additionally, a Keithley 2400 source meter was employed to measure the I-V characteristics and to estimate the sensitivity of the electro-resistance-type pa-ZnO-doped CsPbBr3 perovskite acetone sensors. In characteristic X-ray diffraction (XRD), a scanning electron microscope (SEM), photoluminescence (PL), UV/VIS spectrometers, and X-ray photoelectron spectroscopy (XPS) were used to measure and analyze the characteristics of pa-ZnO-doped CsPbBr3 perovskite films on glass substrates. The coverages of all samples are around 83%, as estimated by Image J software. The aggregation domain sizes of the pa-ZnO-doped CsPbBr3 perovskite films, with 2 and 5 mg of pa-ZnO nanopowder doping, exhibited larger than that of the samples with 1 and 10 mg of pa-ZnO nanopowder doping, as show in 3 perovskite films with 2 mg of the pa-ZnO nanopowder dopants had the largest aggregation domain size.3 perovskite films, with various amounts of pa-ZnO nanopowder dopants with treatments of different annealing temperatures of 200\u2013500 \u00b0C in UV-vis range. In total, two absorption peaks at 372 and 519 nm were observed. They are corresponding to the absorption of pa-ZnO and CsPbBr3. The peak position of the absorption did not shift with the doping amount of the pa-ZnO dopant and annealing temperature. This means that the nature of the materials of the pa-ZnO-doped CsPbBr3 perovskite films did not change. 3 perovskite films. As shown in 3 that are observed. Their positions are at approximately 14.97\u00b0, 21.24\u00b0, 25.23\u00b0, and 30.47\u00b0, and are corresponding to the (100), (110), (111), and (200) phases of the cubic lattice structure, respectively [3 perovskite film at an annealing temperature of 300 \u00b0C. This means that 300 \u00b0C is the optimized annealing condition. An XRD pattern was employed to understand the quality and composition variation of the pa-ZnO-doped CsPbBrectively ,23,24. A3 added with 2 mg of pa-ZnO annealed at 300 \u00b0C. As shown in 3 film) to 76.35 eV. As shown in 5/2 increased from 149.87 eV of the original sample to 150.97 eV, and the binding energy of Pb-4f7/2 increased from 144.97 eV of the original sample to 146.11 eV. As shown in 5/2 increased from 731.68 eV of the original sample to 732.56 eV, and the binding energy of Cs-3d3/2 increased again from 745.65 eV of the original sample to 746.61 eV. As shown in 3/2 increased from 1029.22 eV of the original sample to 1030.78 eV. The binding energy of Zn-2p1/2 was increased from 1052.36 eV to 1054.07 eV to compare with the original sample. Therefore, it can be found that when 2 mg of pa-ZnO is added, for any different atoms, the binding energy is increased in comparison to the original sample, which is the highest in all samples. The phenomenon of this binding energy displacement may be that when the atoms are oxidized, the bond energy will be enhanced, and the oxidation will increase the binding energy of the inner layer electrons. The more electrons that are lost during oxidation, the greater this increase, and the peak position of the O-1s is related to the oxygen vacancies of the crystal lattice. It can be seen that, compared with the original sample of XPS spectra, the intensity after annealing has a downward trend, and it is known that the annealing heating causes oxygen vacancies [acancies ,26.3 perovskite acetone sensors with the treatment of annealing at various temperatures and pa-ZnO doping amounts. The sensitivity, S, can be used to estimate the performance of the gas sensor [s sensor ,27: (1)S3 perovskite acetone sensors with the treatment of annealing at various temperatures and pa-ZnO doping amounts at 4.9 V, are summarized in 3 perovskite acetone sensors with the treatment of annealing at 300 \u00b0C and a doping amount of 2 mg shows the highest sensitivity, owing to the best conductivity, caused by a lot of oxygen vacancies. When the annealing temperature increases to 400 and 500 \u00b0C, the sensitivity decreases because of the description of oxygen vacancies, owing to the oxidation effect.The sensitivity of the pa-ZnO-doped CsPbBr3 perovskite is exposed to gas molecules to be measured, adsorbed oxygen on the pa-ZnO-doped CsPbBr3 perovskite film reacts with the acetone gas, thereby releasing electrons, as shown in 3 perovskite film. Therefore, we measured the current-voltage (I\u2013V) characteristics to evaluate the performance of the pa-ZnO-doped CsPbBr3 perovskite acetone sensors. 3 perovskite acetone sensors with the treatment of annealing at 300 \u00b0C and a pa-ZnO doping amount of 2 mg, under the ambient of various concentrations of acetone. Under the treatment of the champion condition: an annealing temperature of 300 \u00b0C and a pa-ZnO doping amount of 2 mg, the current of the pa-ZnO-doped CsPbBr3 perovskite acetone sensor decreases as the concentration of the acetone decreases. When the concentration of the acetone decreases to 0.01% (100 ppm), the I-V curve of the pa-ZnO-doped CsPbBr3 perovskite acetone sensor is a near-background I-V curve. Therefore, the 100 ppm is the limit of detection for the pa-ZnO-doped CsPbBr3 perovskite acetone sensor, and the sensitivity is 101. To compare the results of other works [3 perovskite acetone sensor in this work have a competitive edge.When the thin film of the pa-ZnO-doped CsPbBrer works , the per3 perovskite acetone sensors have been investigated. We have studied the morphology, absorption spectra, and XRD pattern of pa-ZnO-doped CsPbBr3 perovskite films for pre-annealing temperatures and doping amounts, respectively. The optimized pre-annealing temperature and doping amount are 300 \u00b0C and 2 mg of ZnO, respectively. According to the XPS spectra, the binding energy shifts with the amount of pa-ZnO nanopowder added. The effect this binding energy displacement may be that the atoms will be oxidized, the bond energy will be enhanced, and the oxidation will increase the binding energy of the inner layer electrons. Under the treatment of the champion condition, the highest sensitivity of the pa-ZnO-doped CsPbBr3 perovskite acetone sensor is 1726. In addition, 100 ppm is the limit of detection of the pa-ZnO-doped CsPbBr3 perovskite acetone sensor, and the sensitivity is 101. This work is not suitable for medical applications (1 ppm-level) [In this work, pa-ZnO-doped CsPbBrm-level) ,29,30,31"}
+{"text": "Objectives: The aim of the present study was to evaluate the risk of late mortality and major adverse cardiovascular and cerebral events after coronary artery bypass grafting (CABG) in patients with prior percutaneous coronary intervention (PCI). Methods: A total of 2948 patients undergoing isolated CABGs were included in a prospective multicenter registry. Outcomes were adjusted for multiple covariates in logistic regression, Cox proportional hazards analysis and competing risk analysis. Results: In all, 2619 patients fulfilled the inclusion criteria of this analysis. Of them, 2199 (79.1%) had no history of PCI and 420 (20.9%) had a prior PCI. An adjusted analysis showed that a single prior PCI and multiple prior PCIs did not increase the risk of 30-day and 5-year mortality. Patients with multiple prior PCIs had a significantly higher risk of 5-year myocardial infarction  and repeat revascularization . Similarly, 30-day and 5-year mortality were not significantly increased in patients with prior PCI treatment of single or multiple vessels. Patients with multiple vessels treated with PCI had a significantly higher risk of 5-year myocardial infarction , repeat revascularization  and stroke  at 5-year. The risk for repeat revascularization was also increased with a prior single vessel PCI, but not for other outcomes. Conclusions: Among patients undergoing CABGs, multiple prior PCIs seem to increase the risk of late myocardial infarction and the need for repeat revascularization, but not the risk of mortality.  Percutaneous coronary intervention (PCI) is the prevailing invasive treatment for coronary artery disease. In subsets of patients, PCI is associated with an increased risk of repeat revascularization compared to coronary artery bypass grafting (CABG) ,2. It isThe European Coronary Artery Bypass Grafting (E-CABG) registry was a prospective, multicenter study that included 7352 patients who underwent an isolated CABG at 16 European centers of cardiac surgery, which provided information for the evaluation of early outcomes after surgery from January 2015 to May 2017. The project is registered on ClinicalTrials.gov (Identifier: NCT02319083). Data on preoperative, operative and early postoperative variables and outcomes were prospectively collected. Eight centers  agreed to collect retrospective data on the late events of these patients. For the present study, only patients with complete data on the timing and number of PCIs and treated vessels, as well as data on the SYNTAX score, were included in this analysis. Patients who underwent a PCI \u2264 30 days from a CABG were excluded from this study because the indication for surgical revascularization might have been related to an acute complication of the PCI and/or severe myocardial ischemia not successfully treated by PCI. Data on the date of death, myocardial infarction, repeat coronary revascularization and stroke were collected retrospectively from electronic national registries as well as by contacting regional hospitals, patients and their relatives. The Ethical Committee of the sponsor institution granted the permission to perform this study .The Institutional Review Board or Ethical Committee of each participating center has further approved this study.A previous study  suggesteThe primary outcomes were 5-year all-cause mortality, myocardial infarction, stroke and repeat coronary revascularization. Secondary outcomes were 30-day mortality as well as a composite of major cardiac and cerebrovascular events (MACCE), i.e., all-cause mortality, myocardial infarction, stroke and repeat coronary revascularization. For the present study, myocardial infarction was defined as any myocardial infarction occurring 30 days after the index CABG procedure because the diagnosis of perioperative myocardial infarction might have differed between centers. Repeat revascularization and stroke were considered when occurring at any time after surgery.p-value < 0.20 for between-groups differences. Analyses on the impact of the number of prior PCIs as well as the impact of the number of vessels treated by prior PCIs were adjusted for the following covariates: age, dialysis, diabetes, atrial fibrillation, prior cardiac surgery, critical preoperative state, off-pump surgery, bilateral internal mammary artery grafting, radial artery grafting, number of diseased vessels, Synergy between Percutaneous Coronary Intervention with Taxus and Cardiac Sur-gery (SYNTAX) score and EuroSCORE II. The number of distal anastomoses was not included in the regression models because this might have reflected the number of vessels previously treated with PCIs. Indeed, revascularization can be considered complete in cases when previously stented vessels were free of signs of restenosis and therefore were not surgically revascularized. Cardiopulmonary bypass time and aortic cross-clamping time were not included in the regression models because a significant proportion of patients underwent revascularization with the off-pump technique. All statistical tests were two-sided, and p < 0.05 was set for the statistical significance. Statistical analyses were performed using SPSS v. 25  and Stata v. 15.1  statistical softwares.The Kruskal\u2013Wallis test and the linear-by-linear association test were used for univariate analysis of the baseline and operative data of the study cohorts. The Kaplan\u2013Meier method and the Cox proportional hazard method were employed to evaluate the impact of the number of prior PCIs and the number of the main coronary vessels treated by a PCI on mortality and any MACCE. Since myocardial infarction, stroke and repeat coronary revascularization may be hindered by a patient\u2019s death, a competing risk analysis using the Fine\u2013Gray regression model with all-cause death as a competing event was performed to estimate the unadjusted and adjusted subdistribution hazard ratios (SHRs) for the incidence of these events. Multivariable models assessing the outcomes in patients with an increasing number of PCIs and an increasing number of treated main coronary arteries by PCIs were adjusted risk factors with a Patient selection for this study is shown in Out of 2948 patients who were operated on at the participating institutions from January 2015 to May 2017 and who were included in the E-CABG registry, 2619 patients fulfilled the inclusion criteria of this analysis. Of them, 2199 (79.1%) had no history of PCI and 420 (20.9%) had a prior PCI. A single PCI procedure was previously performed in 272 patients and \u22652 prior PCIs in 148 patients. A PCI procedure was previously performed on a single vessel in 271 patients and on \u22652 vessels in 149 patients. The mean follow-up time of this series was 4.6 \u00b1 1.3 years. The mean time for myocardial infarction was 2.6 \u00b1 1.5 years, for repeat revascularization 1.7 \u00b1 1.7 years and for stroke 1.8 \u00b1 1.8 years.Patients\u2019 characteristics in these study groups are summarized in Patients with a single prior PCI or multiple prior PCIs did not have an increased risk of 30-day mortality when adjusted for multiple covariates . SimilarPatients\u2019 characteristics are summarized in Patients with a single vessel treated with a PCI and those with multiple vessels treated with PCIs did not have a significantly increased risk of 30-day mortality when adjusted for multiple covariates . SimilarThe present multicenter study demonstrated that having multiple prior PCIs may increase the risk of myocardial infarction and repeat revascularization after CABG at a 5-year follow-up. The novelty of this analysis resides in the information provided on major adverse cardiovascular events at 5-year follow-up in a rather large series of all-comers populations. In fact, there is some evidence from several studies that a prior PCI might not affect late survival after a CABG , but, toTaken together, the present findings, along with the results by Hakamada et al. , suggestThis study was not planned to address the causes of the increased risk of MACCE associated with a prior PCI. However, we speculate that multiple PCIs, as well as a PCI on multiple vessels, may prevent surgical revascularization on optimal sites of diseased coronary arteries, and coronary stents may not allow the development of collateral circulation. Furthermore, a failing stent may contribute to myocardial infarction and the need for repeat revascularization. Indeed, the present study showed that patients with prior PCIs, particularly those with multiple prior PCIs, had lower SYNTAX scores. This finding suggests that the extent of coronary artery disease per se should not be the main determinant of poor outcomes in these patients. By contrast, restenosis or even thrombosis of a previously implanted stent may contribute to some of such late coronary events. Cardiac surgeons planning CABGs often face questions about the durability of coronary stent, particularly in case of non-hemodynamically significant stent restenosis. In such cases, surgical revascularization might be considered incomplete. In fact, the prothrombotic state during the perioperative period, as well as the progression of coronary artery disease later, may further contribute to coronary stent failure and jeopardize the outcome of a CABG.This study demonstrated a higher risk of early and late stroke in patients with \u22652 vessels treated with a prior PCI, and the same trend was also observed in patients with \u22652 prior PCIs. We speculate that the non-significantly increased baseline prevalence of stroke or transient ischemic attack, atrial fibrillation and left ventricular ejection fraction might have contributed to an overall increased risk of stroke of peripheral vascular and/or cardiogenic origin. When these three risk factors were included in the regression models, patients with \u22652 vessels treated with a prior PCI still had a significantly higher risk of developing stroke after a CABG . Still, a history of stroke or transient ischemic attack  and atrial fibrillation , but not a depressed left ventricular ejection fraction, were independent predictors of stroke. However, we do not have data on the nature of stroke and its underlying causes.A few limitations might have affected the validity of the present findings. First, despite the prospective nature of the E-CABG registry, information on late events was retrospectively collected using different methods to gather this data. Second, we do not have information regarding the adverse events related to coronary stent or graft failure. Third, the fate of the previously implanted stents and atherosclerosis of native arteries might have been affected by the preoperative withdrawal of antiplatelet drugs and anticoagulants. Similarly, antithrombotic therapy might have had an impact on thrombotic events, early and late, after surgery . HoweverThe characteristics of the participating centers were somewhat different in terms of patient volume, referral pathway, and revascularization strategy. Such inter-institutional differences make this study based on an all-comers population and strengthen the generalizability of these findings.In conclusion, the results of the present study suggest that for patients undergoing a CABG, prior multiple PCIs may significantly increase the risk of late myocardial infarction and the need for repeat revascularization. Prior PCIs did not affect either early or late mortality after a CABG. These findings should be considered in the decision-making process, particularly when planning PCIs for complex coronary artery disease or when repeat revascularization after a prior PCI is indicated."}
+{"text": "Erosion caused by the repeated impact of particles on the surface of a substance is a common wear method resulting in the gradual and continual loss of affected objects. It is a crucial problem in several modern industries because the surfaces of various products and materials are frequently subjected to destructively erosive situations. Polymers and their hybrid materials are suitable, in powdered form, for use as coatings in several different applications. This review paper aims to provide extensive information on the erosion behaviors of thermoset and thermoplastic neat resin and their hybrid material composites. Specific attention is paid to the influence of the properties of selected materials and to impingement parameters such as the incident angle of the erodent, the impact velocity of the erodent, the nature of the erodent, and the erosion mechanism. The review further extends the information available about the erosion techniques and numerical simulation methods used for wear studies of surfaces. An investigation was carried out to allow researchers to explore the available selection of materials and methods in terms of the conditions and parameters necessary to meet current and future needs and challenges, in technologically advanced industries, relating to the protection of surfaces. During the review, which was conducted on the findings in the literature of the past fifty years, it was noted that the thermoplastic nature of composites is a key component in determining their anti-wear properties; moreover, composites with lower glass transition, higher ductility, and greater crystallinity provide better protection against erosion in advanced surface applications. Halting activities;Shortening productive service life;Decreasing performance;Requiring material replacement, including repair and maintenance costs;Decreasing productivity and efficiency;Reducing revenue;Impacting safety ;Health impacts .Erosion continues to evolve as an important research area; it has attracted the interest of researchers for over 125 years because the reliability of surface integrity is of interest to most industries. Erosion is a major issue since it results in several problems, such as failure/collapse, the degradation of surfaces, severe accidents, and vulnerabilities in many industrial systems and processes. Surface degradation by erosion is a slow but nevertheless continuous and unpreventable process in numerous industries, such as the oil and gas industry; erosion also affects aircraft, steam engines, and the rotor blades of power plant drive turbines, including wet-steam turbines, as well as other turbine plants that operate on wet steam . ErosionDevelopments in production methods and advancements in materials and coatings that offer exceptional performance and distinct properties represent one of the most crucial imperatives in modern industrial techniques ,5. DurinThis study aims to explore the techniques utilized for wear testing, the factors leading to surface erosion, and the wear behavior of neat polymers utilized in the past fifty years; to correlate modifications in erosive properties, the available data for hybrid glass and carbon-fiber-reinforced composites are compiled from the literature for the past twenty-five years. The study further proposes to identify gaps in the literature to aid researchers in directing their future work, with the goal of effectively utilizing optimal materials in industries where surfaces are exposed to wear-prone environments.Contact methods that involve the testing surface material in common contact with another material, including or not including supplementary media.Methods utilizing a flow of coarse erodents or loose solid coarse particles in a liquid or gas.Several methods of evaluating the wear resistance of materials are available ,11. SubjAir jet erosion testers;Slurry jet erosion testers;Water droplet erosion testers;Dry erosion testing with loose particles of the erodent;Wear testing using solid particle accelerators;The Taber method;Silica blasting;Shot blasting.In the first class of techniques, erosion is determined after a specified length of time sliding the surface to be tested against the surface of a reference material. In the second group of procedures, loose abrasive particles are dispersed onto the analyzed surface at a specified angle using compressed air or water as a medium. Considering both of the above cases, the most extensively utilized methods are:Erosion by fluid or particles is a vigorous phenomenon that includes gradual harm to a surface caused by an impinging material because of repeated collisions and interactions. Based on the physicomechanical properties of the substrate surface or the substrate coating, the wear of the surface also differs according to the flow of the abrasive material ,13. NumeThere have also been numerous studies carried out to explore the prompting contributors to solid particle erosion, comprising impact velocity ,15, impiMathematical theoretical models using artificial neutral network approaches and linear regression have also been successfully applied using factorial design. It has been suggested that the extent of the exposure to erosive conditions, the inclination angle, the velocity of erodent, the radial distance, the path of the sample\u2019s rotation in the medium, and the distance tracked all have an influence on the wear resistance of materials ,22.Overall, an ideal erosion-resistant material should have an elevated surface area/volume ratio, high hardness, high adhesive strength, small filler size, and a good dispersion of filler; these characteristics result in better mechanical properties, namely scratch resistance and high ductility without strength loss, as presented in For specific mechanical and physical applications, the wear resistance of the base material must be identified. The vital parameters in the wear process and their role in various classes of composites and polymers have been highlighted by Barkoula and Karger-Kocsis . The rolBased on a study by Zhang et al. , it was 5\u2013106 s\u22121; therefore, predicting stress conditions is difficult and correlating mechanical properties with erosion rates is also very complicated. Similarly, it is also tough to foresee their comparative impacts because there is a series of certain mechanisms that encourage the process of erosion. This is concluded based on a literature survey that found all these results in physical effects such as filler breakage/damage, fragmentation, pull-out, denaturing, debonding, etc. In addition, the reliance of the erosion rate of polymer composites on experimental factors is dependent on the wear process [To study erosion resistance and relate it to several mechanical properties of neat materials and their hybrid composites, researchers have carried out effective tests . Deforma process .The properties of a material play a major role in the erosion mechanism and the erosion rate in wear environments. Hutchings et al.  proposed\u22123 and 10\u22124. The mechanism and nature of erosion can be predicted to some extent using the wear coefficient/erosion efficiency (\u03b7). It takes into consideration that a dislodgment of surface material from the crater without any damage results in micro plowing . In contrast, if the surface erodes by ideal micro-cutting, then \u03b7 = 1.0 or 100%. It has been observed that a very low value of \u03b7 (\u03b7 \u2264 100%) results in surface loss by platelet formation or cutting in the form of a lip. Such material results in surface losses or fractures by continuous impacts and is said to be ductile material. Brittle materials usually erode by spalling and the interlinking of lateral or radial cracks, and the erosion of their surface results in material removal in the form of large chunks. In such cases, quite a high value of \u03b7 value is observed, i.e., a value greater than 100% (\u03b7 \u2265 100%).The erosion efficiency varies as a function of hardness; as the material becomes harder, the removed segment of the crater volume is increased. For the wear of neat resins, the value of k typically ranges between 10The velocity of impinging particles is used to calculate the erosion rate. The impact velocity (v) can be correlated to the erosion rate (E) using the expression:At various impact angles, the magnitudes of K and n can be obtained using this power equation by the least-square fitting of data points. The velocity exponents are found to range from 1.4 to 3.0 for several polymers at different impact angles. Mostly, greater values of n are associated with sharper impact angles for polymers. Pool et al.  concludeErosion by solid particles results in the thinning of components, surface crumpling, abrading, roughening, scratching, deprivation, and a drop in the practical lifespan of the affected components. Hence, wear resulting from particle impact has been deemed a critical challenge; it is responsible for many breakdowns, collapses, and failures in engineering applications. A robust struggle is ongoing to develop coatings that impede the activity of metal erosion, which is estimated to cost U.S. industries more than two hundred billion dollars yearly ,33. A suTo overcome problems with metallic coatings, the second-generation erosion-resistant coatings were introduced: multi-layered structures constituting metal/ceramic materials. These materials resulted in new drawbacks, sometimes in the form of cracks. Consequently, coatings comprising polymers (polymeric coatings) were introduced, owing to characteristics inherent to the polymers ,38.Several theories relating to tribology and polymer tribology have been developed ,39,40. MThe literature includes research whose authors discovered that plastic polymer-based materials displayed characteristics such as elevated deformation, plasticity, malleability, and great flexibility. It was assumed that they had the ability to absorb and release impact energy very productively in a cyclic way during the deformation and restoration process. Therefore, the employment of polymer-based materials was attempted, and they effectively exhibited better wear performance in erosive situations than ceramic-based materials or metal. Hence, the routinely used metal and metal/ceramic-based anti-erosive protective composites were gradually replaced by polymer materials. Polymeric materials can be handled easily and have better processability, low cost, good erosion resistance, and a greater ability to absorb impacts. Detailed information about the types of polymers and their composites is summarized and presented in Harsha et al.  conducteZhang et al.  used finIbrahim et al.  presenteThe above techniques described in the literature predict the roles of different parameters and have been found useful and relevant to wear applications.It has been shown in the literature that polymers and their associated composites are extensively utilized materials in erosive wear situations . Since tAs discussed earlier, the particle erosion behavior of polymers has been explored by many researchers. The specific materials studied include polystyrene , polyproThe available data for neat thermoset and thermoplastic polymers are presented in For the prediction of the erosion of polymers, the thermoplastic or thermoset nature of the resin is the deciding factor. Generally, thermosetting polymers demonstrate brittle behavior, while thermoplastics exhibit ductile behavior. Erosion efficiency values for thermoplastics using a heating rate of 5 \u00b0C/min for 2 h under an inert atmosphere of 3% are less than carbonization temperature at 400 \u00b0C and around 100% for thermosetting polymers . A literBesides simple polymers, fiber-reinforced polymers are also well regarded because these composites deliver additional advantages, including their outstanding mechanical properties, freedom in fabrication, strength, lightweight character, and tailorable anisotropy for specific applications . The fibA serious concern also exists in the case of polymer composites: in some works, relatively low erosion efficiencies have been observed compared to metallic substrates when fibers were used as reinforcement ,80. ReseThe cost of the manufacturing process is usually reduced by manufacturing polymer composites with a blend of the finest tribological, mechanical, and thermal characteristics. In the last few decades, hybrid composite materials have been productively deployed in erosion wear environments.2), can increase fracture toughness. The use of polymer nanocomposites has attracted considerable attention as a method of enhancing polymer properties and extending their utility by using molecular or nanoscale reinforcements rather than conventional particulate-filled composites [In the field of nanocomposites, there has been diverse research on using epoxy and polyurethane resin-based nanocomposites to synthesize coating materials because of their advantageous mechanical properties, such as excellent adhesion strength, high modulus, and low creep; however, the resulting materials sometimes prove brittle in different combinations ,83. Diffmposites ,85. Compmposites ,87,88.Coating technology is now actively using nanomaterials in different industrial sectors owing to their exceptional capabilities. The advantages include surface toughness, hardness, adhesive strength, temperature resistance, erosion resistance, corrosion resistance , the impPatnaik et al.  reportedKorostelyov et al.  exploredPool et al.  highlighChellaganesh et al.  studied The erodent volume, air jet velocity, and angle of impingement were evaluated as process parameters. In addition, an analysis of the surface morphology was conducted using an electron microscope. Wear rate was calculated based on mass change relative to time. It was demonstrated that filler improved the wear properties of the composite material compared to resin.Maclean et al.  studied 2/epoxy nanocomposites. The study discovered that adding nano-silica had a considerable effect on the curing reaction and cavitation erosion, as well as on the wear, mechanical, and thermal properties. Overall, a plasticizing effect was noted with the addition of nano-silica. It was concluded that a decrement in cumulative mass loss and an improvement in cavitation erosion, in terms of collective erosion and erosion rate, were observed for the nanocomposites due to the increment in root plastic deformation and shape fractures in certain regions.Abenojar et al.  evaluateDong et al.  worked oSuresh et al.  studied 2). The erosion rates (ERs) of the PEI composites were calculated at various angles of impingement (from 15\u00b0 to 90\u00b0) and impact velocities (from 30 to 88 m/s). The relationships of the erosion rate with mechanical properties such as tensile strength (T), impact strength (I), ultimate elongation to fracture (e), hardness (H), and shear strength (S) were discussed, and it was proved that these properties influenced the erosion rate of PEI composites. It was demonstrated that neat polyetherimide and its hybrid composites reinforced with carbon fibers displayed semi-ductile behavior, and the highest erosion rate was observed at a 60\u00b0 impingement angle. However, the PEI composite using glass fiber as its filler reached its maximum erosion rate at a 60\u00b0 impingement angle under impact velocities of 88 and 30 m/s. These findings demonstrate that fiber content, experimental parameters, and mechanical properties contributed to controlling the erosion rate of the composites. SEM investigation of the eroded surfaces demonstrated that the basis of erosion consisted of the phenomena of microcracking, sand particle embedment, fiber pull-out, chip formation, fiber cracking, and the removal of fibers. It was also concluded that, for the application of PEI in erosive wear environments, it is always desirable to use a lower glass fiber content when reinforcing a neat PEI polymer.Harsha et al.  investigFor aerospace and engineering applications, the tribological and mechanical benefits of hybrid thermoplastic composites are growing steadily. This is due to their favorable characteristics, including low density, stiffness, and high strength, especially as compared to the common metallic/monolithic metal alloys. The wear resistance of a surface material is an important protection against erosion. Nanotechnology using appropriate filler is the key to designing nanocomposites for high-performance applications. The effects of different types of filler are reported in the literature. Some of the most frequently used fillers are carbon-based because they influence mechanical parameters and lubrication while also improving the erosive performance of the matrix. The tribological and structural usage of glass and carbon fibers has been explored in engineering and aerospace applications; the available information related to erosion is given in For the most part, nano- and micro-fillers are combined with a matrix to modify and adjust the mechanical parameters, wear challenges, thermal characteristics, and curing procedure of polymers. Nanoparticles can affect the curing process by increasing the initial curing mechanism ; sometimes, in high quantities, they hamper the crosslinking of resin, and curing is inhibited.Pyrogenic silica is employed in coatings, paints, and adhesives due to its rheology. In particular, nano-silica has a pronounced effect on mechanical characteristics, wear parameters, and cavitation erosion properties, as well as on thermal properties and the curing reaction. Natural fibers have gained enormous attention as reinforcing materials for polymer-based composites. Natural fibers, such as kenaf, coconut, applewood, rice husk, and sisal banana, have been studied, and the effects of using short fiber fillers to reinforce polymers have been explored. Nano- and micro-fillers, such as cork nanoparticles, carbon black, and jute, have been explored for use in epoxy resins to assess their ability to modify the mechanical properties, thermal characteristics, curing reaction, and wear resistance of epoxy resins .Similarly, the addition of CNTs resulted in a considerable enhancement of the modulus properties, tensile strength, fracture resistance, and toughness of the matrix used. Hybrid composites modified with graphite have been reported to yield anti-wear and self-lubrication results compared to neat resin ,108,109.Chen et al.  exploredHarsha et al.  reportedArjula et al.  conducteFouad et al.  investig2. The composite had lower values for hardness and strength, but its ductility increased. It was concluded that wear was affected by the addition of the nanoparticles. It was suggested that it would be advantageous to use a lower percentage of silica (3%) because the Young\u2019s modulus decreases when a larger content of silica is added, particularly for bulk applications. It was recommended to avoid using silica epoxy nanocomposites for applications that require greater wear resistance.Abenojar et al.  exploredSuresh et al.  evaluateCao et al.  studied Kumar et al.  utilizedVery few comprehensive investigations have used polymers and their hybrid composites for erosion-resistant applications. The data given in Epoxy is a versatile coating with certain disadvantages, including brittleness and rigidity, that can be efficiently reduced by using hybrid polymers, i.e., its modification with polyurethane for surface protection.2O3 has the ability to benefit from hydrogen bonding with polymer chains; because of its low cost, it can be considered a promising nanoparticle for increasing wear resistivity.Nano\u2013AlCarbon-based materials, such as MWCNT, graphene, and graphite, can have covalent bonding within their matrix; thus, they can improve the modulus and tensile strength of the polymer matrix. However, graphite can exhibit Van der Walls bonding among its layers, so it may reduce the shear stress of the composite. Comparing graphene with MWCNT, we observe its better dispersion in the matrix in comparison with MWCNT.Replacing carbon fiber with aligned CNT films as the reinforcement material can lead to a change in erosive wear behavior from brittle to ductile. CNTs can absorb more energy upon fracture or bending, and it is possible to further functionalize CNTs to increase their erosive wear.The erosive wear of composites with glass fibers as a filler is greater than that of carbon fiber composites. The change in erosive rate is due to fiber/matrix interfacial bonding and the different properties of the fibers.Due to its minimal cost, nano-silica is recognized as the most widely used NP for manufacturing erosive endurance composites. It can develop effective bonding with the matrix because of its self-lubricating characteristics.2 can alter mechanical characteristics and erosive properties by effectively transmitting the cracks and developing a strong bridge between the filler and the polymer resin during the erosion process.Polymer hybrids containing nano\u2013TiOZnO nanoparticles have the major advantage of favorable mechanical properties in polymer hybrid materials; with a phase disruption mechanism, its surface functionalization can develop strong covalent bonds between polymer chains.The addition of nano-clay to a polymer matrix results in a phase disruption mechanism that can enhance the mechanical characteristics of the polymer composite to some extent by utilizing its Van der Wall interactions.For wear applications, the filler plays a key role in optimizing the polymer; important factors include its composition, the polymer\u2013filler interaction, and the homogeneous distribution of filler in the matrix. The erosion of the matrix surface varies depending on the nature of the erodent and the impingement conditions.At perpendicular angles, erosion was found to be lower; at inclined ranges, it varied due to the slicing of fiber material.Higher hardness values of composites do not indicate a higher wear resistance value. Lower Tg values suggest that the nanocomposites plasticize more effectively with the neat resin and convey better erosion resistance. The observed plasticization is in harmony with the greater strength and augmented ductility.Polymers and their composites have been utilized as structural and tribo-materials for erosion protection purposes in numerous industries owing to their advantageous characteristics, such as high strength, greater hardness, enhanced toughness, high modulus, and, in some cases, recyclability. The most beneficial features of polymers are their flexibility in design/shape and the ease of manufacturing them into intricate parts by extrusion or injection molding techniques. In the context of many fundamental applications, erosion is a major concern because it causes the failure of parts and components. The present work is a review of thermoplastic and thermoset polymeric composites that have been explored in the previous few years for their erosion/wear-related qualities. The polymer composites of EP, PU, PEI, PES, PEEK, PEK, PS, and BMI have been discussed, along with the particulars of solid particle fillers, such as clay alumina, titanium oxide, zinc oxide, silica, and carbon-based fillers. Based on the findings relating to the impacts of the mentioned fillers on the characteristics of composite materials with respect to solid particle erosive wear, the key conclusions of this work can be listed as follows:"}
+{"text": "Solid particle erosion inevitably occurs if a gas\u2013solid or liquid\u2013solid mixture is in contact with a surface, e.g., in pneumatic conveyors. Nowadays, an erosive failure of the component after the usage of a long period has been gaining the interest of the researchers. In this research work, carbon fibre-reinforced polymer (CFRP) composites are prepared by varying the tow sizes of fibres, such as 5k, 10k, and 15k. The prepared composites are subjected to erosion studies by varying the process parameters, such as the impact angle  and velocity . The Taguchi orthogonal array design has been employed for the experimental plan and the erosion rate and surface roughness are observed for each run. The changes in the responses are reported for varying process parameters. The higher erodent velocity of 129m/s leads to higher erosion rates and forms poor surface quality. The minimum impact angle of 30 degrees provides higher erosion rates and higher surface roughness than the other impingement angles. Finally, the eroded surface of each sample is examined through microscopic and 3D profilometer images and the erosion mechanism is analysed at different conditions. The eroded particles supplied at lower speeds do not penetrate the composite surface. However, it is well-known that the lower the collision force, the harder the traces on the surface, yet no sign of fibre breaking or pull-out is observed. The passage of erodent particles on the composite caused surface waviness (flow trace), which prevents the surface from degrading. Materials are the key factors in engineering, since the desired materials are required to meet the needs of the environment that they are meant for use in. Engineering materials are generally grouped into two parts, i.e., metals and non-metals. However, there are many other engineering materials, such as ceramics, polymers, semiconductors, composites, biomaterials, and advanced materials . PolymerReplacing the metals with polymer composites is the prime aim of most researchers since the polymer composites are lightweight materials. Various carbon fibre-reinforced polymer (CFRP) composites can be substituted for metals, with excellent specific stiffness, strength, and good corrosion resistance ,4. As a Solid particle erosion (SPE) is a repetitive process that removes the material from a surface, due to solid particle impacts. It happens wherever solid particles, e.g., sands, metallic particles, and foods, are carried out in a conveying system by liquids or gases. Several industries, such as energy producers, pharmaceuticals, and chemical engineering, have employed conveying systems. SPE may damage the conveyors, and hence, they require regular maintenance. Sometimes, substantial harm is caused to the system due to total failure of parts. Therefore, by estimating the erosion in the conveying systems, one can optimise the operating conditions to attain better designs and parts, for a longer lifetime, and maintenance and production costs can be reduced . The polOne of the many types of degradation classified as wear is the erosion of materials caused by the surface impact of hard particles. It is a complicated phenomenon that frequently involves mechanical, chemical, and material parameters. This complexity appears to defy simplification on the part of both the experimentalist seeking to carefully separate variables and the theorist attempting to accurately model the wear system. However, significant progress has been made in recent years in both gaining a basic understanding of the significant parameters of wear and applying a materials methodology to mitigate wear problems . On the It is observed that the maximum erosion is revealed while testing the polymer composites at higher impingement angles. When the erodent flows in the same direction as the fibre orientation in the matrix, the erosion rate becomes less, and vice versa. Moreover, a low erosion rate is noted for the effective adhesion between the fibre and the matrix . The bonSuihkonen et al. have produced glass fibre-reinforced vinyl ester-based composites and attempted to determine their erosive wear behaviour using the slurry erosion wear test rig. The experimental results have exhibited that the tested rubbers have better wear resistance than the fabricated composite materials . Zhang eFrom the literature review, it is clear from the existing experimental research that the importance of conducting an erosion study on various fibre-reinforced polymer composite is inevitable, as it has been suggested for various engineering applications. Most of the researchers have interest in finding the significant relationship existing between various erosion process variables and erosion resistance of metals and alloys only. However, limited research works have focused on finding the erosion performance of carbon fibre-reinforced polyester matrix composites. Still, the suitability of utilising carbon fibre-reinforced polymer composites for engineering applications needs to be ensured. The changes in the erosion performance of polymer composites for varying process inputs are also required to meet the needs of the industries. Hence, the need exists to focus on the performance of such CFRPs. Moreover, the surface quality of the component is also of interest to the researchers as it is important while fabricating the component used in such erosive environments. The primary aim of the present work is to provide a systematic experimental evaluation of the influence of the tow size (k) of the carbon fibre matrix on mechanical and erosion behaviours of the composite. In addition, this work examines the mechanism of erosion prevailing on the surface of the composite at varying conditions through an optical profilometer.The materials employed in this investigation were PAN-based carbon fibre as reinforcement, supplied by Dowaksa Company, Turkey, and the polyester resin as a matrix for hand-layup composite material fabrication.The fibres were never twisted, and the fabric was a 600 gsm twill-woven fibre fabric with a 45 percent fibre weight fraction, and four layers of lamina were piled at a 0.135 mm thickness. The mechanical properties were measured using an Instron (Series-3382) according to ASTM standards D3039-08 and D790-10, using a 100 mm gauge length and a 200 \u00d7 20 \u00d7 3 mm specimen sample. The impact strength of an unnotched specimen of 63 \u00d7 13 \u00d7 3 mm was tested using a 3.56 kg impact hammer on a Charpy impact tester in accordance with ASTM standard D256-10. Hardness has been evaluated in three distinct locations using a Rockwell hardness testing machine with a maximum load of 60 kg and a specimen dimension of 15 \u00d7 15 mm. In each test, the averages of three samples were analysed and reported. Mechanical properties of the composite laminate were tested in accordance with ASTM standards using the samples of the specified dimensions. Tensile and flexural strengths were also tested.The ASTM G76-13 solid particle erosion test has been carried out by utilising an air jet erosion tester . The air jet erosion tester is made up of the following components: an air compressor, a feeder unit, a mixing chamber, a control unit, an erosion unit, and a dust collector. The erodent was alumina, irregular in shape, with an average size of 50 micron, and it was stored in the hopper as well as transported to the mixing chamber by the conveyor belt unit. The erodent was mixed with dry, compressed, high-speed air of 0.2\u20135 bar in the mixing chamber. After mixing, the air\u2013erodent combination was pushed via the erosion unit\u2019s tungsten carbide nozzle  and the specimen was struck at high velocity. The sample was made to be 25 \u00d7 25 \u00d7 5 mm in size for the air jet erosion experiment. The experiment has been carried out with a continuous erosion time of 10 min and a standoff distance of 10mm. Each specimen was cleaned and weighted using a precision balance with 0.1 mg accuracy before and after the erosion test. Each experiment was performed three times, and the average weight loss was measured, then the erosion rate of each sample was computed using the weight loss. The erosion rate is the amount of material lost from the composite per unit time by the impact of the erodent released, and it may be computed using Equation (1) :Erosion The sample holder of varying impingement angles was employed to change the impact angle for the experimental work. By adjusting the air pressure, the erodent velocity was determined using the double-disc method. In terms of mechanical properties and for comparison, samples of carbon fibre-reinforced composites with varied tow sizes  were employed for erosion testing.When a significant number of elements are involved, statistical tools have been proven to play a vital role in assessing the product or process characteristics. Erosion is a frequent phenomenon in which the combined reaction of many control components influences the responses. The applications of various statistical approaches to investigate the influence of parameters are demonstrated in the existing literature. For the current experimental task, the Taguchi experimental design has been utilised to plan the experimental runs and to examine the importance of control variables in the output response. For the experiment, a typical L9 orthogonal array was used. Thus, by utilising Taguchi\u2019s factorial experimental design , the numThe mean response of the experimental data was computed by translating the output data (erosion rate) to a signal-to-noise ratio (S/N). As the erosion rate should be as low as feasible, the erosion rate\u2019s S/N ratio was judged to be \u201cthe smaller the better\u201d. The ratio value for the \u201cthe smaller the better\u201d criterion is stated as Equation (2) , which in represents the number of observations, and y represents the observed data. The equation has been used to calculate the optimal state of the erosion rate and surface roughness, with the erosion control parameters set to the minimum. Finally, an ANOVA test was run to examine the significant effect of specific parameters on the erosion rate.In the preceding equation, The fabricated composites were subjected to testing and the determined properties are shown in The samples were tested using the solid particle erosion test rig by varying the parameters based on the standard orthogonal array system. The weight loss of the sample for each experimental run was measured and the erosion rate was calculated by using Equation (1). The surface roughness tester has been used to measure the surface roughness, and average roughness values are presented in The erosion testing parameters and the material qualities had an impact on the solid particle erosion test. The solid particle erosion test on polymer composites heavily influenced the erosion test parameters, such as the impingement angle, erodent velocity, and erodent flow rate. The proper selection of these variables decides the performance, such as lower erosion rate and surface roughness. The composite erosion rate increased as the erodent velocity increased. When the hard abrasive particles strike the material surface at high speed, their kinetic energy is transferred into the impinging area and deforms the material plastically as well as separates the reinforcement from the matrix. However, the surface damage occurs on the composite surface as a result of the creation of micro-cuts, and craters are caused by the repetitive attack of hard erodent particles at a greater erodent velocity. Similarly, increasing the tow size increased the erosion rate of the composite.An analysis of variance (ANOVA) test has been carried out to understand the significance of each process parameter and their contribution to the output responses. a value measured on the composite material tended to increase when the tow size increased from 5k to 10k. Again, a decrement was noticed for the change in tow size from 10k to 15k. This was caused by the effective package of fibre in the matrix, and it led to protecting the impact impinged by the solid particle. A decrement was observed while increasing the impact angle from 30 to 90 degrees.The impact of hard particles on the surface in the perpendicular direction broke the fibre and formed the waviness . When thThe changes in the surface roughness were observed for varying erodent velocities, and they showed an increasing trend. Further, continuously increasing roughness was observed while increasing the velocity from 72 to 129 m/s. The fast-moving solid particle with kinetic energy was utilised for the removal of material from the composite. Here, the increased erodent velocity facilitated higher energy, which caused impacts on the surface of the material in larger quantities. On the other hand, it led to a higher number of small craters on the surface of the element. The continuous impact caused by the fast-moving solid particle led to the formation and clubbing of small craters, and wider, deeper craters on the surfaces. Subsequently, the surface roughness increased. Higher surface roughness was observed at an impact angle of 30 degrees and erodent velocity of 129 m/s.a was observed for all the experimental runs. As the roughness on the eroded component is required to be minimum, \u201cthe smaller the better\u201d, the objective function was also used for calculating the S/N ratio for the surface roughness.The eroded surface texture was measured using a profilometer. Ra, followed by erodent velocity and tow size factors. Moreover, F-values of all the parameters were also higher as per the F-test. It indicates the consequence of corresponding factors affecting the particular responses, and they were highly correlated.From a = 27.032 \u00b5m, Rz = 46.588 \u00b5m, Rt = 143.930 \u00b5m, and Rq = 35.721 \u00b5m. The variations in the surface profile and 3D image exhibit the surface topography of the sample after the erosion test. The microscopic image reveals the deformation region caused by the erodent particle\u2019s sliding movement.The surface topography has been examined using an optical microscope  and a 3D surface profilometer (Nano System NV-2000). The eroded surface of the composite was examined under varying conditions after the experimental work. The microscopic view depicts the scanning direction of the surface profile. The surface profile shows the eroded zone thoroughly with width and depth. The microscopic view, 3D profilometer image, and the surface profile observed on the eroded surface for the condition of a tow size value of 10k, angle at 90 degrees, and velocity of 129 m/s are shown in a = 27.032 \u00b5m, Rz = 46.588 \u00b5m, Rt= 143.930 \u00b5m, and Rq = 35.721 \u00b5m. The variations in the surface profile and 3D image exhibit the surface topography of the sample after the erosion test. The microscopic image reveals the deformation region caused by the erodent particle\u2019s sliding movement.The surface topography has been examined using an optical microscope  and a 3D surface profilometer (Nano System NV-2000). The eroded surface of the composite was examined under varying conditions after the experimental work. The microscopic view depicts the scanning direction of the surface profile. The surface profile shows the eroded zone thoroughly with width and depth. The microscopic view, 3D profilometer image, and the surface profile observed on the eroded surface for the condition of a tow size value of 10k, angle at 90 degrees, and velocity of 129 m/s are shown in The parametric influences affecting the response characteristics are understood from the experimental results and the interaction plot. Besides, the surface-level changes in the composite material caused by the erosive action of the sliding particle also need to be addressed. The physical surface of the component is also important in various applications, as it firmly develops contacts with its counterpart. Since the erosive action affects the surface texture of the component during the erosion test, the examination of surface topography of the sample is also equally important for understanding the parametric influence of process parameters.Bonding is easily broken at higher speeds, causing bigger materials to be removed from the work piece. The high impact energy is directly transferred to the composite, resulting in a faster erosion rate. The surface contact area of the eroded particles has a direct impact on the erosion at lower angles. At higher velocity, the impingement of erodent particles is also witnessed on the composite surface. The greater the angle, the greater the resistance to the erosion. This is because the erodent particles on the composite surface provide less impact stress. The microscopic and 3D profilometer images for varying tow sizes of 5k, 10k, and 15k are depicted in The eroded particles supplied at lower speeds did not penetrate the composite surface. The lower the collision force, the harder the traces on the surface, yet there was no sign of fibre breaking or pull-out. The microscopic view shows the fibre\u2019s strong interfacial connection with the matrix. The passage of erodent particles on the composite caused surface waviness (flow trace), which prevented the surface from degrading.The strength and the hardness of the composite increased to the maximum value for the increment of the tow scale from 5k to 10k, and then it tended to decrease.When the effect of erodent velocity was observed, it was higher, and the erosion rate and the surface roughness of the composite were also higher.The minimum erosion rate and a good surface finish were noticed at a higher impingement angle of 90 degrees, whereas the minimum impact angle of 30 degrees led to higher erosion rates and a poor surface finish.The erosion rate and the surface roughness of the composite were maximum for the 10k tow size fibre-reinforced polymer composite, whereas the 15k tow size fibre-reinforced polymer composite possessed an improved surface finish with a comparatively lower erosion rate.The eroded particles supplied at lower speeds did not penetrate the composite surface. The lower the collision force, the harder the traces on the surface, yet there was no sign of fibre breaking or pull-out.The passage of erodent particles on the composite caused surface waviness (flow trace), which prevented the surface from degrading.In the future, the same study can be extended to determine the erosion performance for varying percentages of fibre reinforcements and other fibre-reinforced polymer composites.A carbon fibre-reinforced polymer composite with varying tow sizes has been fabricated and the erosion behaviour has been determined. The observations are summarised as follows:"}
+{"text": "To the EditorA key step in biomedical innovation is problem identification and needs assessment. Clinicians can play an essential role in biomedical innovation because they are at the forefront of patient care and encounter clinical unmet needs on a regular basis . HoweverThe Magic Wand Initiative was founded at the Massachusetts General Hospital  in 2013 to empower clinicians to engage in problem-driven research and innovation. The demonstrated success of the single-institution program  led us thttps://www.advancing-derm.org) provided initial funding for administrative support of the program. No costs are incurred by residency programs whose trainees participate in the program.The VMW program is a 10-month course designed to educate dermatologists to engage in problem-driven innovation by identifying and defining an unmet clinical need in dermatology. The program is advertised by e-mail communication with residency program directors (through the listserv of the Association of Professors in Dermatology) and by announcement at the annual Next Generation of Innovators Luncheon organized by Advancing Innovation in Dermatology. Course meetings are through monthly video conferences, and the curriculum is composed of didactics, interactive group brainstorming, and pitch sessions. In a pitch session, participants present and discuss their chosen clinical problem in a 5-minute presentation. In the presentation, the problem is thoroughly characterized in terms of its clinical importance, its societal and economic impact, and all stakeholders impacted by it. A specification sheet containing the necessary and desirable characteristics of an ideal solution is outlined. The presentation is intended to deeply define the unmet need and motivate listeners, such as research faculty, engineers, and other stakeholders, to join the clinician-led team to work on solutions to the problem. Videoconferences take place on a monthly basis between July and April and usually last for 1\u20122 hours (6\u20138 PM Eastern Time). Before enrolling in the course, each participant who is a resident or fellow is required to provide a letter of support from their program director affirming that they will be granted time to attend the videoconferences. Attendance is monitored at each session, and participants are allowed to miss a maximum of 2 of 10 sessions. Although missing more than two sessions results in removal from the course, to date, this remedy has not been necessary for any participants. The course leadership and invited faculty speakers receive no remuneration for their participation. Advancing Innovation in Dermatology  by independently developing novel solutions and intellectual property, it will be up to the team members to address the ownership of intellectual property with each other and in accordance with their institutional policies.Each dermatologist enrolled in the course is asked to identify 2\u20123 unmet needs from their clinical practice that they are motivated to characterize. These are then presented in an initial brainstorming session attended by their colleagues and key opinion leaders in the field of dermatology, who assist in vetting and defining the scope of the problems. Participants then choose which problem\u2014their PWS\u2014to further characterize during the course. For PWS pitch sessions, key opinion leaders with specific expertise in the chosen PWS are invited to participate.Didactic sessions address practical issues to consider when formulating a PWS and its possible solution, such as stakeholders, market research, reimbursement, regulatory issues, and intellectual property concepts. Industry, regulatory, and intellectual property lawyers are also invited to speak about these important steps in the innovation pathway. Depending on the session topic, 2\u20124 invited faculty are present for each session. The course culminates in the creation of a white paper dedicated to the characterization of each participant\u2019s PWS and an outline of necessary and desirable characteristics of a solution. A sample course schedule and topic list are depicted in The VMW program has graduated three classes from 2017 to 2020. There were 7 participating dermatologists in the first class (2017\u20132018), 9 in the second (2018\u20122019), and 17 in the third (2019\u20122020). In the third year, the program was modified to enable participants to work in teams; a larger number of participants could therefore be accommodated. Participants were characterized by their demographics, by the PWS they identified, and by their subsequent involvement in dermatologic innovation. In addition, the 17 members of the 2019\u20132020 VMW class were surveyed before starting and after the completion of the course regarding their interest in and perceived ability to participate in problem-driven innovation.VMW has graduated 33 dermatologists, of whom 28 were residents, 1 was a clinical fellow, 3 were assistant professors, and 2 were associate professors. A total of 30 (91%) participants were affiliated solely with academic institutions, whereas 1 (3%) was affiliated solely with a private practice. One (3%) participant had affiliations with an academic institution and with a private practice over the course of their VMW participation, and another (3%) had academic and military affiliations. The course has attracted participants from 26 institutions . AlthougFour of the five faculty participants were female. We do not know the reason for the skew toward female participation at the faculty level. Although it could reflect greater appeal to women than to men, it may simply be an artifact of a small sample size or due to the female director (LG) and female cochair (KCL) actively encouraging faculty members to apply. Representation at the leadership level is known to be important in attracting underrepresented members. More investigation and a larger sample size are needed to understand the success of our program in attracting women to innovationA total of 29 volunteer faculty have delivered didactic lectures on specific topics or served as key opinion leaders to brainstorm with course participants. The faculty include academic dermatologists , dermatologists in private practice with experience in innovation, and industry representatives with subject matter expertise in specific areas such as intellectual property law and regulatory affairs.All the 17 participants from the 2019\u20122020 class responded to the presurvey, and 11 responded to the postsurvey. Overall, respondents indicated a greater understanding of and ability to engage in key aspects of the process of innovation after completion of the course. For example, 4 of 17 (24%) respondents to the precourse survey agreed or strongly agreed with the statement, \u201cI can identify potential stakeholders/team members to collaborate with when solving problems.\u201d In the post-course survey, 11 of 11 (100%) agreed or strongly agreed with the statement. In the precourse survey, 2 of 17 (12%) respondents agreed or strongly agreed with the statement, \u201cI can manage a team through the innovation process of turning an idea into a product.\u201d In the postcourse survey, 10 of 11 (91%) of respondents agreed or strongly agreed. After their introduction to problem-driven innovation through the VMW, 19 (58%) program graduates have become involved in other innovation-based initiatives in dermatology. Three have proceeded to lead other initiatives focusing on problem-driven innovation: one cofounded HealthAI @ Stanford, another cofounded Hacking Dermatology, and a third developed an externship program that pairs early-career physicians with established innovative companies. Hacking Dermatology is an Advancing Innovation in Dermatology program in which teams of participants propose innovative solutions to unsolved problems in Dermatology; winning teams receive seed funding to begin to develop their technologies. Four graduates of the VMW have participated in this externship program. Four graduates have served as analysts on due-diligence teams for an early-stage accelerator fund.http://www.hackingdermatology.org). At Hacking Dermatology 2019, VMW participants were represented on three winning teams during the initial round. In the subsequent final pitch competition, teams led by VMW graduates won first, second, and (tied for) third places . Nine graduates have cofounded companies. At the beginning of the COVID-19 pandemic, one graduate of our program founded a nonprofit to address the problem of the limited availability of personal protective equipment. The graduate formed a team that developed a mask offering significantly greater protection than conventional surgical masks and which through innovative use of discarded surgical instrument sterilization wrap also has the benefit of supporting environmental sustainability . AnotherIn conclusion, the VMW is a pilot program that has shown early success with an impact in teaching problem-driven innovation to dermatologists and engaging them in the process of innovation. The Magic Wand program, which emphasizes the importance of deeply characterizing a clinical problem before countenancing solutions, is applicable to participants at any career level. In the future, we plan to publicize the program more broadly to attract participants at all career stages. Given the success of the program with dermatologists in the United States, our program has already been expanded to Europe. In 2021, we launched the European VMW program to empower, educate, and engage European dermatologists in problem-based innovation . As thesProblem identification and characterization by clinicians is an essential first step in problem-driven innovation. After identification and characterization of a problem, a collaborative research team can be formed \u2014or joined\u2014to seek a solution. The VMW shows promise as an educational venue to encourage problem-driven innovation in dermatology and serves as an opportunity to seed collaboration between clinical and research faculty.magicwandinitiative.org. For further information, please contact info@magicwandinitiative.org. Hacking Dermatology is an initiative of Advancing Innovation in Dermatology. Further information can be found hackingdermatology.org.Further information about the Magic Wand Initiative can be found at No datasets were generated or analyzed during this study, except for the survey response data, which are presented in the manuscript in their entirety.http://orcid.org/0000-0001-8998-0030Yakir S. Levin: http://orcid.org/0000-0003-2107-8985Kachiu C. Lee: http://orcid.org/0000-0002-3389-5096Adam B. Raff: http://orcid.org/0000-0001-7868-5669Jamie J. Breslin: http://orcid.org/0000-0002-2193-8917William Ju: http://orcid.org/0000-0002-9266-0887Lilit Garibyan: The trademark for Magic Wand Initiative is owned by the Massachusetts General Hospital , which is the current employer of YSL and LG. The authors state no other conflict of interest. Some initial funding for this program was provided by Advancing Innovation in Dermatology, which is a nonprofit organization where JJB and WJ currently work."}
+{"text": "In this study, the performance of a support vector machine (SVM)-based classifier will be tested against pathological ground truths and its performance determined in cognitively healthy older adults.End-of-life studies have validated the binary visual reads of 18F-Flutemetamol end-of-life dataset to determine the regions with the highest feature weights in a data-driven manner and to compare between two different pathological ground truths: based on neuritic amyloid plaque density or on amyloid phases, respectively. We also trained and tested classifiers based on the 10% voxels with the highest amplitudes of feature weights for each of the two neuropathological ground truths. Next, we tested the classifiers\u2019 diagnostic performance in the asymptomatic Alzheimer\u2019s disease (AD) phase, a phase of interest for future drug development, in an independent dataset of cognitively intact older adults, the Flemish Prevent AD Cohort-KU Leuven (F-PACK). A regression analysis was conducted between the Centiloid (CL) value in a composite volume of interest (VOI), as index for amyloid load, and the distance to the hyperplane for each of the two classifiers, based on the two pathological ground truths. A receiver operating characteristic analysis was also performed to determine the CL threshold that optimally discriminates between neuritic amyloid plaque positivity versus negativity, or amyloid phase positivity versus negativity, within F-PACK.We applied SVM with a linear kernel to an CL\u2009=\u200948\u201351 for the different classifiers (area under the curve (AUC)\u2009=\u200999.9%), except for the classifier trained with amyloid phases and based on the 10% voxels with highest feature weights. There the cut-off was CL\u2009=\u200926 (AUC\u2009=\u200999.5%), which closely matched the CL threshold for discriminating phases 0\u20132 from 3\u20135 based on the end-of-life dataset and the neuropathological ground truth.The classifiers yielded adequate specificity and sensitivity within the end-of-life dataset . The regions with the highest feature weights corresponded to precuneus, caudate, anteromedial prefrontal, and also posterior inferior temporal and inferior parietal cortex. In the cognitively normal cohort, the correlation coefficient between CL and distance to the hyperplane was\u2009\u22120.66 for the classifier trained with neuritic amyloid plaque density, and\u2009\u22120.88 for the classifier trained with amyloid phases. This difference was significant. The optimal CL cut-off for discriminating positive versus negative scans was Among a set of neuropathologically validated classifiers trained with end-of-life cases, transfer to a cognitively normal population works best for a classifier trained with amyloid phases and using only voxels with the highest amplitudes of feature weights.The online version contains supplementary material available at 10.1007/s00259-022-05808-7. As an example, in the extended dataset from the pivotal 18F-Flutemetamol end-of-life study [18F-Flutemetamol PET by majority read for increased neuritic plaque density was 91% and specificity was 90% [18F-Florbetapir [18F-Florbetaben [Key evidence for the validity of amyloid PET tracers comes from end-of-life studies \u20133. In thfe study  based onfe study , sensiti was 90% , 5. Comp18F-Flutemetamol phase 2 data and compared to visual reads [In these pivotal clinicopathological studies, the visual read was based on a set of prior ad hoc rules for discriminating positive vs negative scans. In a previous study, a support vector machine (SVM) with a linear kernel was trained with the al reads . The cla18F-Flutemetamol end-of-life dataset (n\u2009=\u200968), all phase 0\u20132 cases were read as negative and 89% of phases 4\u20135 as positive, whereas 33% of the phase 3 cases were read as positive [SVM can also be trained with a different ground truth to see whether its diagnostic performance against different neuropathological dimensions outperforms that of the a priori chosen measure of modified neuritic amyloid plaque density. Such an alternative neuropathological classification scheme is based on amyloid phases determined from A\u03b2 immunochemistry , 8. Accopositive  and alsopositive , 12.Once the classifier has been trained and tested using post-mortem verification, and has proven to be accurate, it can readily be applied to different datasets without the need for laborious and time-intensive visual reads. This may be particularly useful for the detection of Alzheimer\u2019s disease (AD) in the asymptomatic phase, since levels in the early disease stage may be equivocal and more difficult to read visually. Amyloid imaging has been instrumental in defining the asymptomatic phase of AD . WhereasSD 8.9, range 60\u201396\u00a0years) from the phase 3 study [SD 205.6, range 0\u2013846\u00a0days).A total of 101 cases   [F-PACK is a prospective longitudinal community-recruited cohort of 180 cognitively intact older adults  Fig.\u00a0, who all18F-Flutemetamol PET scans were acquired on PET/CT scanners from 90 to 100\u00a0min post injection in 94 cases and from 90 to 110\u00a0min in 7 cases, with an injected dose of 185\u2013370\u00a0MBq  [18F-Flutemetamol PET images from an independent in-house dataset [18F-Flutemetamol Standardized Uptake Value Ratio (SUVR) image was created for each subject using the cerebellar grey matter (GM) as reference region obtained by intersecting the Automated Anatomical Labelling (AAL) atlas areas 91\u2013108 [In the GE067-026 trial, static  dataset . These 6 dataset . An 18F-For preprocessing of the PET scans from the F-PACK cohort, we made use of the individual\u2019s structural MRIs. This differs from the PET-only procedure of the end-of-life data.18F-Flutemetamol PET imaging on a 16-slice Biograph PET/CT scanner . The tracer was injected as a bolus in an antecubital vein . Further details of the standard image acquisition procedure can be found in the 18F-Flutemetamol PET was done using SPM12 running on MATLAB R2014b as described in detail elsewhere [All F-PACK participants underwent lsewhere , 20. In For reasons of comparability, the PET scans of the F-PACK cohort were also processed using the PET-only procedure as described above for the end-of-life data. For this a sumPET was created of the 90\u2013110\u00a0min frames.18F-Flutemetamol SUVR values were calculated for a composite region, which consisted of five bilateral cortical regions (SUVRcomp): frontal, parietal, anterior cingulate, posterior cingulate, and lateral temporal  Standard of Truth, A\u03b2 phase , Consort18F-Flutemetamol [In the phase 3 trial of emetamol , the groemetamol  and Ikonemetamol  and summAccording to the CERAD neuritic plaque density Standard of Truth, the end-of-life dataset was composed of 29 negative and 72 positive cases Table . These wAssessment of amyloid phases is described in previously published protocols , 8 and spROC [http://expasy.org/tools/pROC/), was applied to the CL values of the end-of-life dataset to determine the best CL threshold for neuritic plaque density  and, separately, for A\u03b2 phase (phase 0\u20132 vs 3\u20135). Area under the curve (AUC), and specificity and sensitivity at maximized Youden\u2019s index were used as performance measures. The best CL threshold for both neuropathology scores was 28.9, with a specificity of 86.2% and a sensitivity of 73.6% for neuritic plaque density (AUC\u2009=\u200982.2%) and a specificity of 90.9% and a sensitivity of 69.6% for amyloid phases (AUC\u2009=\u200983.6%).A receiver operating characteristic (ROC) analysis, using the R package pROC   using the function fitcsvm with a linear kernel and default settings. An SVM with a linear kernel is defined by a hyperplane which subdivides the two classes. The distance to the hyperplane can be considered a quantitative measure of the strength of evidence that a case belongs to one or the other class. All standard statistical analyses were conducted with R statistical software version 4.0.3 . A leave-one-out SVM was performed to discriminate the SUVR images of the end-of-life dataset in a binary manner based on normal versus abnormal neuritic plaque density as Standard of Truth (CERAD cut-off\u2009>\u20091.5), with accuracy, specificity, and sensitivity as outcome measures. To avoid bias, the number of cases in the positive class was equated to the number of normal cases (the negative class), which were the least abundant, resulting in 29 cases per class  for comparison of two overlapping correlations based on dependent groups was used. The performance of all classifiers was also evaluated relative to the visual reads of the F-PACK cohort. We also evaluated which CL threshold optimally separates the cases considered pathological by the classifier from those considered normal. ROC analysis, using the R package pROC [http://expasy.org/tools/pROC/), was applied to the CL values to determine which CL threshold best discriminates between classifier positive versus negative cases, for each of the classifiers. AUC, and specificity and sensitivity at maximized Youden\u2019s index will be used as performance measures.Next, we evaluated how performance of the classifier related to the CL scale in asymptomatic older adults who participated in the F-PACK study. This was then compared to the CL threshold derived directly from the end-of-life dataset and to that determined in other studies . This alge cocor  (http://age pROC  (http://cortex) and caudate nucleus (SUVRcaudatus) with the pons as reference region. The Thal et al. (2018) staging scheme consists of four PET A\u03b2 phase estimates: PET A\u03b2 phase estimate 0 (without visible 18F-Flutemetamol retention), PET A\u03b2 phase estimate 1 , PET A\u03b2 phase estimate 2 , and PET A\u03b2 phase estimate 3 (SUVRcortex\u2009\u2265\u20090.6 and/or SUVRcaudatus\u2009\u2265\u20091.0) [As a secondary analysis, we evaluated the performance of two recently proposed PET amyloid staging schemes against the performance of the classifier. The PET amyloid staging scheme from Hanseeuw et al. (2018) is based on visual reads and consists of three stages: PET amyloid stage 0 , PET amyloid stage 1 , and PET amyloid stage 2  . The PETs\u2009\u2265\u20091.0) . The PETU test was used as post hoc analysis to determine which PET amyloid phases differed significantly in distance to the hyperplane. P-values were adjusted using the Bonferroni multiple testing correction method.Using the end-of-life dataset, we examined how the distance to the hyperplane relates to these two PET amyloid staging schemes , 12. KruIn addition, we examined how accurately the classifiers trained based on neuritic plaque density could classify according to amyloid phase and vice versa for the classifiers trained with amyloid phase.SD 3.6). The mean specificity was 90.2% . The mean specificity was 98.4% . The mean sensitivity was 84.0% . Among the 77 abnormal cases (phases 3\u20135), seven out of 14 phase 3 cases, three out of 24 phase 4 cases and one phase 5 case were classified as normal . Among the phases 3, 4, and 5, the distance also differed significantly .Welch\u2019s one-way ANOVA with amyloid phase as between-subjects factor was used to test if the distance to the hyperplane differed between amyloid phases. Post hoc comparison revealed that there was no difference in the distance to the hyperplane between phases 0, 1, and 2. The distance to the hyperplane differed significantly from phases 0, 1, and 2 versus each of the other phases  and for classifier P\u2009<\u20092.2\u2009\u00d7\u200910\u201316). For classifier P\u2009=\u20090.024). For classifier P\u2009<\u20092.2\u2009\u00d7\u200910\u201316). These results are comparable to the results of the regression where the SUVR images were processed using the MRI-assisted procedure.A regression analysis was also performed with the distance from the hyperplane as predictor and CL value as outcome variable in all 180 AUC of 99.8%. When classifier AUC of 100%. When classifier AUC of 99.9%. On the other hand, when classifier AUC of 99.5%. In other words, for classifier We also determined for each classifier the optimal CL threshold for separating cases classified as positive versus negative in F-PACK. When classifier As a secondary analysis, we evaluated the performance of two recently proposed PET amyloid staging schemes against the performance of the classifier. One of these schemes is based on visual reads , the othAs described before , the Than\u2009=\u200912) that were placed in Hanseeuw phase 0 were false-negative. The classifier put 5 of these phase 3\u20135 cases in the pathological class, hence demonstrating superior performance.The Hanseeuw et al. (2018) amyloid PET classification, which is also based on amyloid deposition in cortical and striatal regions , misclasPcorrected\u2009\u2264\u20090.017; Hanseeuw: Pcorrected\u2009\u2264\u20090.0035).Kruskal\u2013Wallis rank sum test with amyloid PET stage as between-subjects factor was used to test if the distance to the hyperplane differed between amyloid PET phases. Post hoc comparison revealed a significant difference in the distance to the hyperplane per amyloid PET phase for the two amyloid PET classification schemes  as the median specificity reported by Ikonomovic et al. (2016)  for the A classifier trained with amyloid phases 0\u20132 versus phases 3\u20135 in the end-of-life dataset performed in line with what one would expect based on the visual read studies . InteresThe sensitivity of the Centiloid (CL) method based on an ROC analysis of the end-of-life dataset was 73.6% for the neuritic amyloid plaque density and 69.6% for Thal amyloid phase. The classifier had a sensitivity of 83.7% and 84%, respectively. The sensitivity of the majority read based on the visual reads in the pivotal phase 3 study was 86%, with a confidence interval ranging from 73 to 95%, and the median of the sensitivity of the 5 readers was 88%  . The sen18F-florbetapir amyloid PET scans in cognitively normal controls, mainly from the Alzheimer\u2019s Disease Neuroimaging Initiative [Recently, amyloid PET classification schemes have been proposed based on a combination of cortical and striatal amyloid levels or reads , 12. Theitiative . It has Visual reads are based on a set of explicit ad hoc rules, hence the interest of a data-driven definition of the anatomical distribution of the most discriminative regions. The pattern obtained for the amyloid phase-based classifier confirmed the regions that are also considered critical for visual read classification: precuneus and posterior cingulate, head of the caudate, rostral anterior cingulate, and ventromedial prefrontal cortex. These are in line with a previous SVM paper with visual reads as comparison  and confMore or less the same regions as for the amyloid phase-based classifier also had high feature weights for the neuritic plaque density-based classifier but the clusters for the latter classifier were more scattered and less confined. For the amyloid phase-based classifier, the visual appearance of the distribution of the highest feature weights corresponded better to regions commonly attended to for visual reads, than those of the neuritic plaque density-based classifier.18F-Flutemetamol dataset obtained in 180 cognitively intact older adults. A classifier may be particularly useful in the asymptomatic stage of the disease, when a substantial portion of participants is situated at an intermediary level. From the SVM classifier, we derived the distance to the hyperplane for each SUVR image. This is a quantitative measure of the strength of evidence that a case belongs to one or the other class. It may be compared to the \u201clevel of confidence\u201d of a visual read but is strictly objective. We used this measure of evidence strength to gain further insight in the link between the image classification and the continuous neuropathological measures underlying the binarized classification. When we extracted the distance from the hyperplane as a measure of classification likelihood, the distance from the amyloid phase-based classifier correlated more closely with the CL scale than when the same approach was taken for the neuritic plaque density-based classifier (P\u2009<\u20093.1\u2009\u00d7\u200910\u201315). A closer match with amyloid phases compared to neuritic plaque density may be due to several reasons: amyloid phase takes into account both diffuse and neuritic plaques, and 18F-Flutemetamol has affinity for both types [We applied the classifier to an independent th types .CL\u2009=\u200926) closely corresponded to that obtained when determining a CL threshold directly from the end-of-life data (CL\u2009=\u200928.9). Second, classification based on classifier CL\u2009=\u200926) was also very close to that reported by La Joie et al. (2019) (CL\u2009=\u200923.5) [Two other findings indicated that the transfer to a cognitively normal population worked best for classifier  density . Hence,  density . The sel\u2009=\u200923.5) .Some of the classifiers performed poorly on the F-PACK. One cannot simply assume good transfer of a classifier trained on end-of-life data for application in a cohort of a very different nature, who are cognitively normal. Among the four classifiers, classifier Five amyloid phases exist, which we dichotomized into amyloid phases 0\u20132 and 3\u20135. This was motivated by the relatively low number of cases in each of the phases 0, 1, and 2, and by the observation that amyloid PET is unable to detect increases in retention in phases 0\u20132 , 12. HowAutomated classification using a neuropathologically validated SVM classifier with a linear kernel has value in the detection of an increased amyloid phase or neuritic plaque density in cognitively intact older adults. This study establishes the distance to the hyperplane as an informative and integrative measure with a strong relationship to measures of neuritic amyloid plaque density and, in particular, to amyloid phases.Supplementary file1 (DOCX 45 KB)Below is the link to the electronic supplementary material."}
+{"text": "We investigated the role of m6A\u2010modified circRERE in osteoarthritis (OA) and its mechanism.NIRF2BPL were screened by microarrays. The role of m6A\u2010modification in circRERE was examined by methylated RNA precipitation and morpholino oligo (MOs) treatment. The axis of circRERE/miR\u2010195\u20105p/IRF2BPL/\u03b2\u2010catenin was determined using flow cytometry, western blotting and immunofluorescence in human chondrocytes (HCs) and corroborated using a mouse model of destabilization of medial meniscus (DMM) with intra\u2010articular (IA) injection of adeno\u2010associated viruses (AAV).CircRERE and CircRERE was decreased in OA cartilage and chondrocytes compared with control. CircRERE downregulation was likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2\u2010HRSP12\u2010RNase P/MRP in OA chondrocytes. MOs transfection targeting HRSP12 binding motifs in circRERE partially reversed decreased circRERE expression and increased apoptosis in HCs treated with IL\u20101\u03b2 for 6\u00a0h. CircRERE exerted chondroprotective effects by targeting miR\u2010195\u20105p/IRF2BPL, thus regulating the ubiquitination and degradation of \u03b2\u2010catenin. CircRere (mouse homologue) overexpression by IA\u2010injection of AAV\u2010circRere into mice attenuated the severity of DMM\u2010induced OA, whereas AAV\u2010miR\u2010195a\u20105p or AAV\u2010sh\u2010Irf2bpl reduced the protective effects. The detrimental effects of AAV\u2010sh\u2010Irf2bpl on DMM\u2010induced OA were substantially counteracted by ICG\u2010001, an inhibitor of \u03b2\u2010catenin.Our study is a proof\u2010of\u2010concept demonstration for targeting m6A\u2010modified circRERE and its target miR\u2010195\u20105p/IRF2BPL/\u03b2\u2010catenin as potential therapeutic strategies for OA treatment. Liu et al. demonstrate that circRERE expression is downregulated in human osteoarthritis (OA) cartilage and chondrocytes, and circRERE downregulation is likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2\u2010HRSP12\u2010RNase P/MRP. Mechanistically, circRERE downregulation leads to aberrant \u03b2\u2010catenin ubiquitin and degradation by targeting miR\u2010195\u20105p/IRF2BPL during OA pathogenesis. They are much more stable than linear mRNAs due to their covalently closed loop configuration without a free 3' or 5' end.6\u2010methyladenosine (m6A) is a prevalent reversible RNA methylation associated with mRNAs and ncRNAs with the RRACH  consensus sequence preferentially occurring around stop codons, within long exons and at 3' untranslated regions (3' UTRs).NInterferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) is an E3 ubiquitin protein ligase which has been shown to drive ubiquitination and degradation of \u03b2\u2010catenin, thus probably inhibiting Wnt/\u03b2\u2010catenin signalling. IRF2BPL belongs to the IRF2BP family, which also includes IRF2BP1, IRF2BP2.Here, using circRERE as an example, we highlighted the role of m6A\u2010modified circRNA in OA. We found that circRERE was down\u2010regulated in human OA cartilage and chondrocytes. In vitro and in vivo experiments revealed the chondro\u2010protective roles of circRERE in OA. What's more, circRERE downregulation in human OA chondrocytes is likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2\u2010HRSP12\u2010RNase P/MRP.2Experimental procedures are described in the supplementary materials and methods.33.1n\u00a0=\u00a04) and amputees without OA (n\u00a0=\u00a04) (GEO accession: GSE178724). The heat map showed 298 differentially expressed circRNAs by at least 2.0\u2010fold (p\u2009<\u20090.05)  were identified between humans and mice using the circbank database.n\u00a0=\u00a020)    Figure\u00a0. Figure\u00a0) Figure\u00a0A, B. QRT) Figure\u00a0, and the) Figure\u00a0, but difd Figure\u00a0B. Thus, d Figure\u00a0. CircRERt Figure\u00a0. FISH oft Figure\u00a0. Downregt Figure\u00a0. Further) Figure\u00a0. The mou) Figure\u00a0C. Decrea) Figure\u00a0. Circula) Figure\u00a0D\u2010F. Thes3.2RERE expression was unaffected by circRERE downregulation or overexpression , but also influences the corresponding biological function and processes by affecting the targeted gene expression.To investigate the effects of m6A modification on circRERE expression or function, we first examined whether circRERE contains m6A methylation, m6A\u2010specific immunoprecipitation (MeRIP) assays and subsequent qRT\u2010PCR analysis using divergent primers showed that circRERE was enriched in m6A antibody\u2010precipitated complexes Figure\u00a0. RNA pulIF (m6A)\u2010FISH (circRERE) staining and qRT\u2010PCR assays suggested that decreased circRERE expression in response to IL\u20101\u03b2 stimulation was partly abrogated by the downregulation of YTHDF2 in HCs Figure\u00a0. Given t3.4The cytoplasmic localization of circRERE indicated its potential molecular mechanism in regulating OA may be microRNA (miRNA) sponging, peptide encoding, or circRNA\u2010protein interactions.n\u00a0=\u00a020 for human, n\u00a0=\u00a010 for mice)  Figure\u00a0. Differe) Figure\u00a0.The functions of miR\u2010195\u20105p in vitro were investigated using FCM, EdU, and IF assays. Loss\u2010of\u2010function experiments suggested that downregulation of miR\u2010195\u20105p in HCs rescued IL\u20101\u03b2\u2010induced apoptosis and aberrant expression of anabolic and catabolic molecules Figure\u00a0, but didConsidering that circRERE is conserved between humans and mice, and nucleotides upstream and downstream of the miR\u2010195\u20105p target sequences in circRERE and circRere were also highly conserved Figure\u00a0D,E. We fMarked increases in the examined manifestations of DMM\u2010induced OA were observed in the DMM\u2009+\u2009AAV\u2010miR\u2010NC and DMM\u2009+\u2009AAV\u2010circRere\u2009+\u2009AAV\u2010miR\u2010195a\u20105p groups compared with the Sham+AAV\u2010miR\u2010NC and DMM\u2009+\u2009AAV\u2010circRere\u2009+\u2009AAV\u2010miR\u2010NC groups, including cartilage erosion, SBP thickness and pain, but not synovitis and osteophyte maturation Figure\u00a0, indicat3.5p\u2009<\u20090.05, Figure\u00a0IRF2BPL) was modulated by circRERE silencing  was performed using the same cartilage samples as the aforementioned circRNA microarray . For deprivation experiments, AAV\u2010circRere and AAV\u2010sh\u2010Irf2bpl were co\u2010IA injected into the affected knees (commencing 1\u00a0week after DMM surgery). Infected efficiency was determined by fluorescence examination of knee sections and qRT\u2010PCR of cartilage Figure\u00a0A.Marked increases in the examined manifestations of DMM\u2010induced OA were observed in the DMM\u2009+\u2009AAV\u2010shRNA\u2010NC and DMM\u2009+\u2009AAV\u2010circRere\u2009+\u2009AAV\u2010sh\u2010Irf2bpl groups compared with the Sham\u2009+\u2009AAV\u2010shRNA\u2010NC and DMM\u2009+\u2009AAV\u2010circRere+AAV\u2010shRNA\u2010NC groups, including cartilage erosion, SBP thickness and pain, but not synovitis and osteophyte maturation Figure\u00a0B, indicaFor the rescue experiments, AAV\u2010sh\u2010Irf2bpl (commencing 1\u00a0week after DMM surgery) and ICG\u2010001  were co\u2010IA injected into the affected knees. Infected efficiency was determined by fluorescence examination of knee sections and qRT\u2010PCR of cartilage Figure\u00a0C,D. Sign4In this study, circRERE was significantly downregulated in OA cartilage. Gain\u2010and loss\u2010of\u2010function experiments in vitro indicated that circRERE affected apoptosis, proliferation, and anabolic and catabolic biomarker synthesis in HCs. Furthermore, circRere overexpression in mouse knee joints reduced the severity of DMM\u2010induced OA by targeting the axis of miR\u2010195a\u20105p/Irf2bpl/\u03b2\u2010catenin.Importantly, we showed that the percentage of m6A\u2010modified circRERE was increased in OA chondrocytes, and the increased m6A level on circRERE may enhance its endoribonucleolytic cleavage by YTHDF2\u2010HRSP12\u2010RNase P/MRP. Considering that YTHDF2\u2010bound m6A\u2010modified circRNAs with HRSP\u201012 binding sites (GGUUC) are preferentially degraded by RNase P/MRP, MOs\u2010circRERE targeting GGUUC motifs in circRERE were transfected into HCs followed by IL\u20101\u03b2 treatment. Decreased circRERE expression in HCs stimulated with IL\u20101\u03b2 for 6\u2009h was partly reversed by MOs\u2010circRERE transfection, but not at 12 or 36\u2009h, which may corroborate the preferential degradation of circRNAs in an HRSP12\u2010dependent manner. Functionally, the increased apoptosis of HCs upon 6\u2009h IL\u20101\u03b2 stimulation was also partially rescued by MOs\u2010circRERE treatment.Although the origin of increased m6A\u2010methylated circRERE in OA chondrocytes is unclear, it is most likely to be connected with METLL3 activity. To validate the effects of METTL3 on m6A modification of circRERE, METTL3 was silenced by siRNA in human OA chondrocytes. Unsurprisingly, METTL3 downregulation significantly decreased the m6A level on circRERE, but had no impact on the expression level of circRERE Figure\u00a0A\u2010C, indiIn order to find associated pathways, we also performed KEGG analysis of differentially expressed mRNAs between the OA and control human cartilage Figure\u00a0R,S. ThusRIP assays showed that AGO2 was strongly bound to circRERE. Subsequent experiments in HCs showed that circRERE acted as an ceRNA to regulate target mRNA IRF2BPL and subsequent downstream target \u03b2\u2010catenin via sponging miR\u2010195\u20105p. Mouse circRere expresses a homology of human circRERE (94% similarity), and the binding sequences for miR\u2010195\u20105p in two circRNAs are conserved Figure\u00a0D. FurtheGiven that mass spectrometry analysis following RNA pull\u2010down in HCs to screen circRERE\u2010interacting proteins was not conducted, other proteins may interact with circRERE. Due to the existence of predicted ORFs and m6A modification in circRERE, the possibility of circRERE to encode proteins could not be excluded. Therefore, further exploration is needed to discover other possible mechanisms by which circRERE may affect the pathogenesis of OA. Another limitation of this work is that only surgically induced OA model (DMM) was examined, we did not examine other subsets of OA, such as natural age\u2010related OA. Follow\u2010up work is needed to test the function of circRERE in other subsets of OA. Furthermore, this study may be improved by employing transgenic mice with a chondrocyte\u2010specific promoter for circRere, rather than using AAVs to regulate circRere expression.In conclusion, circRERE downregulation in OA chondrocytes was likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2\u2010HRSP12\u2010RNase P/MRP, and circRERE downregulation led to aberrant \u03b2\u2010catenin ubiquitination and degradation by targeting miR\u2010195\u20105p/IRF2BPL during OA pathogenesis. Thus, concomitant targeting of m6A modification of circRERE and its downstream target miR\u2010195\u20105p/IRF2BPL/\u03b2\u2010catenin might provide a synergistic effect in OA clinical treatment.JL conceived this study and supervised the project. YXL conducted most of the experiments and acquired data with the help from YHY, YCL, BW, XYH, LX and WTZ. YXL and YHY contributed to data interpretation and analysis. JL wrote the manuscript. JL, YXL and YHY reviewed the manuscript. All authors approved the final manuscript.The authors declare no competing interests.APPENDIX S1 Supporting InformationClick here for additional data file.FIGURE S1 (A) For 13 circRNAs, three primers of each were designed to amplify them in cDNA and gDNA of human chondrocytes by RT\u2010PCR and agarose gel electrophoresis analysis. (B) Relative expressions of circZFHX4, circTBCK, circTENM3, circRERE, circARHGAP5 and RERE in human control and OA\u2010Damaged cartilage were detected by qRT\u2010PCR and agarose gel electrophoresis analysis (n=20). *p<0.05, **p<0.01 by Mann\u2010Whitney U test. (C) Pairwise alignment of the human circRERE and mouse circRere sequences. (D) Relative expressions of circRere and Rere in total RNA of MCs treated with or without RNase R (n=3). ***p<0.001 by two\u2010tailed unpaired t test. (E) Relative expressions of circRere and Rere in MCs treated with Actinomycin D (n=3). ***p<0.001 by two\u2010way ANOVA with Tukey's post hoc test. (F) FISH of circRere in MCs. Scale bar, 20\u00b5m. Data are presented as mean \u00b1 SEM.Figure S2 (A) \u03b2\u2010galactosidase staining and quantitative analysis for the \u03b2\u2010gal\u2010positive HCs infected with Ad\u2010circRERE or Ad\u2010vector and treated with doxorubicin . n=3. Scale bar, 200\u00b5m. NS, no significance. ***p<0.001 by One\u2010way ANOVA with Tukey's post hoc test. (B) Overview of DMM surgery, IA\u2010injection with AAV\u2010vector or AAV\u2010circRere, pain assays and sampling. (C) Relative circRere expression in mouse cartilage from Sham+AAV\u2010vector, DMM+AAV\u2010vector and DMM+AAV\u2010circRere groups. n=4. **p<0.01 by Brown\u2010Forsythe and Welch ANOVA test followed by Dunnett's T3 multiple comparison test. Data are presented as mean \u00b1 SEM.Figure S3 (A) Efficiency of METTL3 downregulation in human OA chondrocytes (isolated from damaged cartilage of OA patients) by si\u2010METTL3 (n=3). **p<0.01 by two\u2010tailed unpaired t test. (B) The percentage of m6A\u2010modified circRERE upon METTL3 downregulation in human OA chondrocytes (isolated from damaged cartilage of OA patients) (n=3). ***p<0.05 by two\u2010way ANOVA with Tukey's post hoc test. (C) Relative expression of circRERE upon METTL3 downregulation in human OA chondrocytes (isolated from damaged cartilage of OA patients) (n=3). NS by two\u2010tailed unpaired t test. Data are presented as mean \u00b1 SEM.Figure S4 (A) Number of RBP sites between RBPs and circRERE. (B) FMRP, IGF2BP3, EIF4A3, and DGCR8 RIP assays and subsequent qRT\u2010PCR analysis were performed to detect CO\u2010RIPed\u2010circRERE expression in HCs infected with Ad\u2010vector or Ad\u2010circRERE (n=6). *p<0.05, *p<0.01 by two\u2010tailed unpaired t test or Welch's t test. (C) Schematic to show the overlapping of the target miRNAs of circRERE, as predicted by miRanda, TargetScan and Arraystar's proprietary program. (D) The potentially conserved binding sites between two circRNAs  and miR\u2010195\u20105p, predicted by miRanda and TargetScan. The potential complementary residues are shown in blue. (E) Pairwise sequence alignment of hsa_circRERE and mmu_circRere, and the sequences highlighted in red are targeted by miR\u2010195\u20105p. (F) Schematic of luciferase reporter vectors containing wild\u2010type (WT) or mutant (Mut) putative miR\u2010195\u20105p binding sites of circRERE. (G) Schematic of luciferase reporter vectors containing wild\u2010type (WT) or mutant (Mut) putative miR\u2010195a\u20105p binding sites of mmu_circRere. (H) Relative luciferase activity of WT or MUT circRere luciferase reporter vector co\u2010transfected with miR\u2010195\u20105p mimics or negative control (n=4). ***p<0.001by two\u2010tailed unpaired t test. (I) Quantification of Figure 4H (n=3). *p<0.05 by two\u2010way ANOVA with Tukey's post hoc test.  HCs were transfected with miR\u2010195\u20105p inhibitor and stimulated with IL\u20101\u03b2 (36h). Then proliferation was determined by EdU assays (n=3). One\u2010way ANOVA with Tukey's post hoc test. (L) Quantification of Figure 4L (n=3). *p<0.05 by two\u2010way ANOVA with Tukey's post hoc test. (M) Relative expressions of circRere and miR\u2010195a\u20105p in knee cartilage from Sham+AAV\u2010miR\u2010NC, DMM+AAV\u2010miR\u2010NC, DMM+AAV\u2010circRere+AAV\u2010miR\u2010NC and DMM+AAV\u2010circRere+AAV\u2010miR\u2010195a\u20105p groups (n=4). **p<0.01 by Brown\u2010Forsythe and Welch ANOVA test followed by Dunnett's T3 multiple comparison test. NS, no significance. Data are presented as mean \u00b1 SEM.Figure S5 (A) Volcano plot illustrating the statistical significance of differentially expressed mRNAs between human OA and control cartilage. (B) Scatter plot of differentially expressed mRNAs. (C\u2010E) Gene Ontology (GO) analysis of upregulated genes in OA cartilage. (F\u2010H) GO analysis of downregulated genes in OA cartilage. (I) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of upregulated genes in OA cartilage. (J) KEGG analysis of downregulated genes in OA cartilage. (K) Schematic to show the overlapping of the target mRNAs of miR\u2010195\u20105p, as predicted by PITA, miRanda, Pictar, TargetScan and downregulated mRNAs in OA cartilage identified by mRNA microarray. (L) QRT\u2010PCR for relative expression of predicted target mRNAs of miRNA\u2010195\u20105p in HCs infected with Ad\u2010sh\u2010con or Ad\u2010sh\u2010circRERE. n=6. ***p<0.001 by two\u2010tailed unpaired t test. (M) Putative miR\u2010195\u20105p.Figure S6 (A) Relative expressions of circRere and Irf2bpl in knee cartilage from Sham+AAV\u2010shRNA\u2010NC, DMM+AAV\u2010shRNA\u2010NC, DMM+AAV\u2010circRere+AAV\u2010shRNA\u2010NC and DMM+AAV\u2010circRere+AAV\u2010sh\u2010Irf2bpl groups (n=4). **p<0.01, ***p<0.001 by one\u2010way ANOVA with Tukey's post hoc test. (B) Scoring of osteophyte maturity in four groups. one\u2010way ANOVA with Tukey's post hoc test. (C) To investigate the infected efficiency of AAVs, representative knee cartilage fluorescence (GFP) images in knee sections from DMM+AAV\u2010sh\u2010Irf2bpl+vehicle and DMM+AAV\u2010sh\u2010Irf2bpl+ICG\u2010001 groups were obtained by a confocal microscope. Scale bar, 200\u00b5m. (D) Relative expression of Irf2bpl in knee cartilage from Sham+vehicle, DMM+vehicle, DMM+AAV\u2010sh\u2010Irf2bpl+vehicle and DMM+AAV\u2010sh\u2010Irf2bpl+ICG\u2010001 groups (n=4). *p<0.05 by one\u2010way ANOVA with Tukey's post hoc test. (E) Scoring of osteophyte maturity in four groups (n=6). *p<0.05 by one\u2010way ANOVA with Tukey's post hoc test. NS, no significance. Data are presented as mean \u00b1 SEM.Click here for additional data file."}
+{"text": "Exploring the effect of bile salts on the properties of emulsion carriers containing hydrophobic bioactive compounds is particularly critical to understanding the stability and bioavailability of these hydrophobic bioactive compounds in the digestive process. In this study, the effects of bile salts on the stability and digestive characteristics of the ursolic acid (UA) self-stabilized water-in-oil (W/O) emulsion were investigated via static and dynamic (with or without enzyme) in vitro simulated digestive systems. The results showed that under the static system, the basic conditions had less interference, while the bile salts had a significant effect on the appearance and microstructure of the emulsion. The primary mechanism of emulsion instability is hydrophobic binding and depletion flocculation. Under the dynamic condition, it was found that the low concentrations of bile salts can promote the release amount and the rate of free fatty acids via displacement, while high concentrations of bile salts inhibit the decomposition of lipid, which may be related to the secondary coverage formed at the interface by the bile salts. These findings provide a theoretical basis for understanding the digestive behavior of the UA emulsion and its interaction with bile salts, which are conducive to developing and designing new emulsions to improve the bioaccessibility of UA. Pentacyclic triterpenoids are widely found in various plants. They are one of the active ingredients of many medicinal materials, which have rich biological activities, mainly reflected in liver protection and the regulation of glucose and lipid metabolism disorders . In receAn emulsion is a thermodynamically unstable dispersed system formed by dispersing one liquid in the form of tiny droplets  into another immiscible liquid . The emu2+ and short-chain fatty acids, to promote lipolysis [Only limited lipohydrolysis occurs in the oral and gastric phases of the emulsion . When paipolysis . HoweverPrevious studies have found that typical pentacyclic triterpenoids such as ursolic acid and oleanolic acid have self-assembly characteristics, can form stable W/O emulsions, and their bioaccessibility is significantly improved compared with direct oral administration ,23. It sUrsolic Acid  in the form of a white powder was purchased from Shaanxi Jin Kang Tai Biological Technology Co., Ltd. . UA was used without further purification. Rapeseed oil was purchased from a local store. Bile salt, pepsin (\u2265250 U/mg), pancreatic enzyme , and lipase  were obtained from Sigma Chemical Company. Phosphate buffered saline  was obtained from Gibco Life Technologies . All other reagents were of analytical grade.UA particles  were dispersed in rapeseed oil, and the UA oil dispersion was prepared via continuous stirring in a constant temperature water/oil bath at 80 \u00b0C for 20 min. The 30 wt% aqueous phase was homogenized with the UA oil dispersion in a T18 digital high-speed disperser Ultra-Turrax  for 2 min at 13,600 rpm to produce the W/O emulsion, which was sealed and stored under refrigeration immediately after preparation.2 solution (containing 110 mg of CaCl2) and 7.6 mL of a 0.1 mol/L NaOH solution were added. Different concentrations of bile salts were selected according to the experiment, pre-dissolved with PBS, stirred, and added to the system, and the pH was adjusted to the desired value (6.5 or 7.0). The prepared SIF should be preheated at 95 \u00b0C for 5 min to exclude any possible enzyme activity in the bile salts. After the SIF is cooled, 1 g of the UA emulsion is added and the resulting mixture is incubated for 2 h at 37 \u00b0C and 600 rpm.Synthesizing the methods of Qian , Sarkar The optical microstructures of the original emulsion, emulsion\u2013water, emulsion\u2013enteric salt, and emulsion\u2013SIF mixture were observed using a BX53M polarized light microscope . A drop of the upper emulsion was taken on a slide, covered with a coverslip, and compacted. Then, the structures were observed with 10\u00d7, 20\u00d7, and 50\u00d7 objectives and photographed and recorded, respectively.) was estimated using Nano Measure 1.2 software and 100 droplets were selected for counting. D was defined as:A plain microscopic image of the emulsion was obtained via a 50\u00d7 lens of a polarized light microscope, and the emulsion (~2 \u03bcL) was deposited on a slide and covered with a coverslip. The average surface diameter of the emulsion  before being analyzed via laser Doppler velocimetry and the phase light scattering (M3-PALS) technique, to measure the \u03b6-potential of the sample. Each individual \u03b6-potential data point was calculated from the mean and standard deviation of at least ten readings on the same sample.2, 975 \u03bcL of distilled water, and a final volume of 10 mL, and then incubated for 2 min at 37 \u00b0C.According to Ojeda-Serna  et al., SGF was prepared by mixing 10 mL of the oral phase with 10 mL of the NaCl solution (2 g/L) and adding 1.0 M of HCl to adjust the pH to 2.0 . Porcine2 solution (containing 110 mg of CaCl2) was added and adjusted to pH 7.0. Finally, the pancreatin (2.4 mg/mL) and lipase (3.6 mg/mL) powders were added to the mixture and the timing started. The resulting mixture was incubated at 37 \u00b0C at 600 rpm for 2 h. During the digestion period, 0.1 M of the NaOH standard solution was added dropwise to maintain the pH at 7.0, and the consumption volume of NaOH at different digestion times was recorded. During simulated intestinal digestion, the lipids are broken down into two molecules of free fatty acids and one molecule of monoglycerides. Using the pH\u2013stat method, the amount of total free fatty acids released in the simulated digestion was calculated according to the formula.According to the method of Qian et al. , with slNaOHC is the concentration of the NaOH standard solution (mol/L), NaOHV is the volume (mL) of the NaOH standard solution consumed at different digestion times (t), oilm is the mass of rapeseed oil contained in the digested emulsion (g), and oilM is the average molecular mass (g/mol) of rapeseed oil, which is 930 g/mol [In the formula, 30 g/mol .4,3) is measured via a laser particle size analyzer MASTERSIZER 3000 , and the measurement method of \u03b6-potential is the same as method 2.7. To avoid the effect of multiple scattering, all samples were diluted with deionized water at the same pH.The average particle size and \u03b6-potential of the samples were measured after the digestion system with different bile salt concentrations went through the various stages of simulated GIT. The average particle size (dA is the peak area of UA (mAUS), and m is the UA concentration (\u03bcg/mL). The correlation coefficient (R2) is 0.99743.HPLC determinations were performed using an Agilent 1100 LC system equipped with a quaternary solvent delivery system, autosampler, and diode array detector (DAD). The detection wavelength is 210 nm. The separation was performed on a YMC-Pack ODS-A (250 \u00d7 4.60 mm) column, and the oven and autosampler were kept at 30 \u00b0C. The mobile phase was HPLC grade methanol, the flow rate was 1 mL/min, the injection volume was 20 \u03bcL, and each sample was analyzed at least three times. A series of concentrations  of the ursolic acid standard was prepared. Taking the concentration of ursolic acid as the abscissa and the peak area as the ordinate, the standard curve of ursolic acid was drawn. The linear regression equation is:The UA content in the aqueous phase (micelles) of the digested samples was determined according to the method of Martin et al. , with slAll experiments were repeated three times, and the results were expressed as mean \u00b1 standard deviation. We used Excel 2016 , PowerPoint 2016 , and Origin 2022  for graph analysis.The basic conditions of the intestinal digestion model are intestinal salts, pH, bile salts, and pancreatic enzymes . Before 2, while the type and amount of salt ions affect the stability of the emulsion. It can be seen that the mechanisms of pH and ionic strength on emulsion stability are interrelated, and these two factors themselves also affect each other.2+, Na+, and other salt ions will cover and wrap around the droplet surface and even shield the droplets from mutual electrostatic interactions [The upper emulsion of the digested solution was made into microscope slides after resting. Comparing the pH 6.0 intestinal salt group and the pH 6.0 pure water group, some larger droplets appeared at the interface ; consideractions ,30,31, iCentrifugal stability can be used to simulate the state change in the samples after a long time of natural standing. It is a fast method to evaluate emulsion stability. The comparison of the images in the second and third rows shows that after centrifugation, part of the emulsion droplets increased significantly , but no Particle size and \u03b6-potential results of the emulsion in pure water (pH 6.0) and the emulsion in the intestinal salt solution  after simulated digestion are shown in +, Ca2+, and other cations added. pH is one of the important factors affecting the \u03b6-potential, and the \u03b6-potential decreases with the increase in pH. Comparing the pH 6.0 intestinal salt group with the pH 7.5 intestinal salt group, there was no significant decrease in potential. It is speculated that more UA particles are released in the enteric salt system at pH 6.0. In general, pH and enteric salt both have a certain degree of influence on the stability of the emulsion, but the effect is not large.\u03b6-potential reflects the electrostatic interaction between colloidal particles and can be used to determine the stability of the system. Generally speaking, the stability of the emulsion is proportional to the absolute value of the \u03b6-potential. It is known that the potential magnitude of the pure UA solid in the water phase is negative (\u221228.20 \u00b1 0.78 mV), so the negative potential of the emulsion\u2013pure water system is probably related to the part of UA released in the emulsion. Comparing it with the enteric salt group at pH 6.0, the absolute value of the uniform potential of the system decreases after the addition of salt ions, which may be related to the electrostatic repulsion between NaThe analysis of the test results in the first part shows that the pH and intestinal salts have little effect on the stability of the emulsion, so we further investigated the effect of different bile salt concentrations on the characteristics of the emulsion by keeping the pH and ionic strength constant without the addition of pancreatic enzymes. Since the ingestion of the emulsion is in the fed state and the intestinal pH fluctuates between 6~7.5, the pH was chosen to be fixed at 7.0 for this part of the experiment . In addiThe appearance and microstructure of the emulsion after the static simulated digestion conditions (without enzymes) at different bile salt concentrations is shown in As shown in 2+. When the bile salt concentration was 25 mg/mL, the droplet size became smaller and massive crystals appeared at the interface as UA crystals. So, it can be clearly seen from When the bile salt concentration was 15 mg/mL and 20 mg/mL, the droplet size in the field of view slowly became smaller. At this point, the emulsion morphology changed to a white flocculent form, and the original size droplets were broken down into smaller droplets. More precipitates in the background may be crystallites formed by the bile salts and CaAfter the above static in vitro simulated digestion test (without enzymes), we have confirmed that bile salts have a certain effect on the properties of the emulsion. In the following experiments, 2.4 mg/mL pancreatin and 3.6 mg/mL lipase will be added to more realistically simulate in vitro oral\u2013gastric\u2013gut digestion. Different bile salt concentrations of 0.0, 1.0, 2.0, 5.0, 10.0, and 15.0 mg/mL were also selected to further investigate the effect of bile salts on the digestion of the emulsion and UA bioaccessibility by observing the appearance of the digests, measuring the particle size and \u03b6-potential of the samples, and calculating the UA concentration in the aqueous phase.After the UA emulsion was digested in the oral, gastric, and intestinal phases and was left to stand for one day, the appearance was photographed. The results are shown in The FFA release rate of the emulsion in the external simulated digestion system with different bile salt concentrations is shown in The UA concentrations in the aqueous phase of the digestive juice under different bile salt concentrations are shown in This study explored the effects of bile salts on UA\u2019s emulsion stability and digestion characteristics. The results show that basic factors such as pH and intestinal salt had little effect on the emulsion itself. In the static simulation of small intestine digestion, bile salts can significantly affect the stability of the emulsion. With the increase in bile salt concentration, the emulsion morphology changes, the stability decreases, and the particle size also drops, which could be caused by the emulsification of bile salt and the hydrophobic interaction with UA. In dynamic oral\u2013gastric\u2013gut in vitro simulated digestion, the addition of bile salts can significantly affect the digestibility of emulsions by changing the degree of hydrolysis and the rate of the FFA release. Low concentrations of bile salts can promote emulsion digestion via displacement, while high concentrations of bile salts inhibit emulsion digestion due to the secondary coverage. Understanding the stability and digestion of the UA emulsion in the human body provides a theoretical basis for expanding the practical application of the UA emulsion and also gives information for improving the bioavailability of hydrophobic bioactive compounds."}
+{"text": "The risk factors for breast cancer have been defined in several studies but there is deficient data for specific subtypes. We report here the pathological characteristics of a breast cancer cohort and risk factors for patients with triple-negative disease. In this case\u2013control study, a prospective breast cancer cohort was evaluated for demographic, reproductive, obesity-related and other risk factors using a validated questionnaire. Tumors were characterized for routine pathological characteristics and immunohistochemical markers of basal-like breast cancer. Patients with triple-negative breast cancer (TNBC) constituted cases and those with non-TNBC were controls. Odds ratios (OR) were calculated for each risk factor and independent associations were tested in an unconditional logistic regression analysis. Between 2011 and 2014, 1146 patients were recruited, of whom 912  with sufficient pathology material were analysed. Reproductive factors of parity, breastfeeding, age-at-menarche, age at first full-term pregnancy and oral contraceptive use were not significantly associated with TNBC. Higher body mass index  was not significantly associated while lesser waist circumference  and lower waist-to-hip ratio were significantly associated , with TNBC. History of tobacco use was not significantly associated while lower socio-economic status was borderline associated with TNBC . No factor was significant after adjustment for covariates. Central obesity seems to be preferentially associated with non-TNBC, and lower socio-economic status with TNBC in India, while most other conventional risk factors of breast cancer show no significant association with TNBC versus non-TNBC. Epidemiological studies have shown an association between breast cancer and smoking, alcohol consumption, a high-fat diet, reproductive factors, and socioeconomic status, which may explain its more frequent occurrence among women with a Western lifestyle. However, the risk factors for specific pathological and molecular subtypes of breast cancer have not been accurately defined. Therefore, the differential effect of risk factors on breast cancer subtypes, if any, remains unclear. A few studies have evaluated the risk factors for estrogen receptor negative breast cancer and suggested that higher parity and younger age at first child-birth may be associated with higher risk of developing this type3.Breast cancer is the most common cancer among women in urban India, with its incidence recently surpassing that of cervical cancer5. Accordingly, the fraction of patients with triple-negative breast cancer has been reported to be higher (25\u201330%) in patients from India and other developing countries7. The differences in hormone receptor positivity between Indian and Caucasian patients could be a real ethnic variation or it could be a result of lower average age at diagnosis.The proportion of estrogen receptor (ER)-positive breast cancer in Indian women appears to be lower (about 45\u201360%) than that in their European and American counterpartsHence, we undertook this study to identify the risk factors for triple negative breast cancer using a case\u2013control design in patients with carefully characterised pathological breast cancer subtypes. We also report the detailed pathological characterisation of breast tumours from the same cohort of patients.This was a prospective case\u2013control study to elucidate the risk factors associated with the three major subtypes of breast cancer wherein patients with triple-negative breast cancer were considered to be cases while those with estrogen and/or progesterone receptor positive and HER2 negative and estrogen and progesterone receptor any status but HER2 positive or amplified, respectively, was considered as a common control group.Enrolment in the study was determined at the first presentation of patients to either institution. Patients eligible for the study were women between 18 and 70\u00a0years of age diagnosed with invasive breast cancer who had to be treatment na\u00efve except for surgery for the primary tumour. Those who had received neoadjuvant or adjuvant systemic therapy of any type were excluded, as also those with treatment for metastatic disease. If patients presented before surgery, core biopsy tissue was required for immunohistochemistry and molecular studies. If operated, the availability of paraffin-embedded blocks of the surgical specimen was essential for further studies. Patients should have been willing to provide informed consent for inclusion into the study including consent for blood samples for EBV and HPV testing.The study was designed by faculty members of the breast cancer groups of both institutions and approved by the Institutional Ethics Committee of Tata Memorial Centre and the Ethics Committee of Jehangir Clinical Development Center. All participants provided written informed consent before study participation. All research procedures were performed in accordance with the Declaration of Helsinki and local regulations.8.A detailed questionnaire that had previously been validated was administered to all participants at the time of study inclusion. Data on age, menopausal status, residential address , and contact details were collected. Information was also obtained on the following potential risk factors for breast cancer: socioeconomic status, tobacco use, alcohol consumption, diet (predominantly vegetarian vs non-vegetarian), number of pregnancies up to or beyond the stage of viability (28\u00a0weeks), age at first childbirth, age at menarche/menopause, history of breastfeeding, family history of breast or ovarian cancers, and number of members living in the same household for the preceding life period. The data for residence included any place of residence lived in for a minimum period of 1\u00a0year. The detailed definition of these risk factors and the methodology of their collection followed the methods described in previous epidemiologic studies and are described in the study protocol and questionnaire Specifically, some risk factors were further dichotomised using acceptable cut-offs as described in previous epidemiologic literatureThe following clinical and pathological parameters were recorded: weight, height clinical and (if available) pathological tumour size, clinical and (if available) pathological node status, grade, presence of lymphovascular invasion, immunohistochemistry (IHC)-based estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status, and Ki-67 index. Additionally, all triple-negative tumours were analysed for expression of core basal markers of cytokeratins (CK) 5/6, CK14, CK17, and epidermal growth factor receptor (EGFR) using standard immunohistochemical technique. Fluorescence in situ hybridisation (FISH) was performed if the HER2 score was 2+\u2009on IHC. All pathological evaluations were performed under the supervision of a single experienced breast pathologist at the Tata Memorial Centre, Mumbai. The standard recommendations for staging and diagnosis were followed in the patient work-up before therapy initiation.The association of various risk factors with the subtypes was calculated using odds ratios (ORs) for exposure to each risk factor for triple-negative breast cancer (TNBC) cases vs non-TNBC controls. The individual OR was obtained using an unconditional logistic regression model. We estimated a sample size of 1000 to have 80% power to detect a minimum OR of 1.65 assuming 1\u00a0\u2212\u00a0\u03ac\u2009=\u200995% and the prevalence of exposure to be 15%. The study was well-powered to detect an OR of 2.0 for exposure even with a very low prevalence among controls . In a sample of 1000, the estimated proportions of patients with triple-negative, hormone receptor-positive and HER2 negative, and HER2-positive breast cancer were assumed to be 20\u201330%, 50%, and 20\u201330%, respectively.Statistical analysis was performed using STATA version 15.0. All the variables of interest were cross-tabulated with the case\u2013control status of the patients. OR and the corresponding 95% confidence intervals (CIs) for each risk factor under consideration were estimated using unconditional logistic regression models. Odds ratios were tabulated without any adjustment and after adjustment for covariates.This was a case\u2013control study performed among newly diagnosed, previously untreated patients with invasive breast cancer of any stage who presented to two urban hospitals in Mumbai and Pune between July 2011 and December 2014 (n\u2009=\u20091267). Tissue samples for 355 patients were inadequate because the samples had been processed outside the two institutions and were either insufficient in quantity or had poor quality. Tissue samples for the remaining 912 patients were suitable for immunohistochemistry (IHC), and these patients were included in the study , 112 (23.2%) patients had T3 or T4 disease at diagnosis, 577 (63.2%) had pathologically confirmed axillary lymph node positive disease, 494 patients had ER-positive disease (54.0%) and 418 were ER-negative (46.0%). Of the hormone receptor-positive cases, 308 (78.5%) patients were both ER and PR positive and HER2 negative, 77 (19.6%) had ER positive and PR negative disease, and 7 (1.7%) had ER negative and PR positive tumors. The number of patients with HER2-positive disease (IHC 3+\u2009or FISH amplified) was 254 (27.9%), of whom 109 (42.9%) were ER-positive and 145 (57%) were ER-negative. TNBC was present in 266 (29.1%) patients, of whom 106 (39.8%) expressed both CK5/6 and CK14, 122 (45.8%) expressed CK5/6 and CK17, while 200 (75.2%) expressed three or more core basal markers i.e. CK5/6, EGFR, CK14 and/or CK17.Of the 912 patients whose blocks were analysed by IHC, 905 completed the questionnaire satisfactorily. This included 651 patients with non-TNBC and 254 with TNBC. Table Our results in a breast cancer patient cohort from two tertiary care cancer centres in urban India suggest that triple negative breast cancer constitutes a higher proportion of cases compared with that reported from developed countries and that TNBC phenotype is not significantly differentially associated with reproductive or body size related risk factors, compared with non-TNBC phenotype. This is one of the few studies that has prospectively analysed the association of breast cancer receptor-based subtypes with risk factors in a case-case analysis.It is worth noting that our study was designed to evaluate the association of risk factors using TNBC patients as cases and non-TNBC patients as controls, which was meant to bring out differential predispositions, if any, to these subtypes of breast cancer in the Indian population. This also means that our results cannot be directly compared with other studies that tested the associations between patients with specific breast cancer subtypes using women without breast cancer as controls. Importantly, our results imply that risk factor modification strategies do not need to be specifically tailored for breast cancer subtypes and that a broad strategy is likely to be effective in modifying the population-level predisposition to all types of breast cancer, with the possible exception of parity, as discussed below.9, although it is traditionally considered a protective factor for breast cancer. Our results suggest that parity is not significantly differentially associated with triple negative breast cancer compared with non-TNBC although the OR was 1.35. Given the limited power of finding risk factor associations in a case-case analysis, high parity being associated with TNBC remains a possibility, based on our results. There was no significant differential association of other reproductive risk factors like age at first full term pregnancy, age at menarche and breast feeding with TNBC, suggesting that these factors are likely to be similarly operative in predisposition to all types of breast cancer.One important previous study has suggested that high parity could be a risk factor for triple negative breast cancer10 found an association of waist-hip ratio with TNBC among both premenopausal and post-menopausal women. It is likely that central obesity as measured by waist-to-hip ratio is a more accurate descriptor of the underlying metabolic predisposition to breast cancer compared with BMI because it considers not only the total body fat composition but also its distribution11. Other studies have variably found an association of TNBC with various measures of body weight17. Since our study used non-TNBC patients as control, the association of waist circumference and waist-to-hip ratio in our results suggests that central obesity is preferentially associated with non-TNBC, especially hormone receptor-positive disease, which constituted a high proportion of our non-TNBC controls in our study.We did not find BMI to be differentially associated with TNBC compared with non-TNBC while a lower waist-to-hip ratio was borderline significantly associated with TNBC compared with non-TNBC. Some studies have suggested that a higher waist-to-hip ratio is associated with the risk of hormone receptor-positive breast cancer. In our study, we have reported that a lower waist-to-hip ratio was associated with the risk of TNBC. This is because ours was a case\u2013control study wherein TNBC patients were cases and non-TNBC patients were controls. Therefore, all risk factor associations are preferential associations with TNBC compared with the non-TNBC subtype. An association of lower waist-to-hip ratio with TNBC in our study is consistent with literature reports of higher waist-to-hip ratio being associated with estrogen receptor-positive breast cancer. However, this would still be consistent with an overall association of a higher waist-to-hip ratio with the risk of TNBC when healthy individuals are used as controls, albeit to a lesser extent than estrogen receptor-positive disease. The Carolina studyWe also collected data on tobacco chewing, a somewhat unique form of tobacco used in India. It was not significantly associated with breast cancer subtypes. Interestingly, lower socioeconomic status was borderline associated with TNBC, the reasons for which are unclear but could reflect the impact of other factors. However, since this association was not statistically significant in univariable and multivariable analyses, it could result from chance.6 in India. We also found that a high proportion of TNBC tumors express the immunohistochemical markers, i.e. CK5/6, CK14, CK17 or EGFR of basal-like cancers. A previous report in a subset of patients from this study reported a high prevalence of Epstein\u2013Barr Virus (EBV) in the tumor cells of TNBC tumors18.Our study confirms previous reports that triple-negative breast cancer phenotype constitutes a higher proportion of patientsOur study has several strengths. It was a prospective study with a large sample size that included patients who presented to two large tertiary care hospitals. The tissue samples were processed by a central laboratory, and all the tumours were subtyped by a single experienced pathologist at a tertiary cancer centre.BRCA1 and BRCA2 pathogenic variants. This information might be useful in evaluating the interaction between germline predisposition and risk factors.Nevertheless, our study also had a few limitations. Because women with non-TNBC cancers were used as controls, the odds ratios in our study indicate the association of each risk factor with these phenotypes. The absolute risk association of each factor with TNBC can only be analysed in a study that includes healthy persons as the control population. Moreover, we did not perform germline sequencing for variants that predispose to breast cancer, like In conclusion, the results of our case\u2013control analysis of the association of risk factors with breast cancer phenotypes suggests that lower waist-to-hip ratio, lower socio-economic status and possibly high parity could be differentially associated with triple-negative breast cancer compared with non-TNBC cancers, although these associations were not statistically significant. Most other reproductive and non-reproductive risk factors showed no significant association with breast cancer phenotypes. Broad risk factor modification strategies are likely to be useful as population-level interventions."}
+{"text": "Many musicians have suffered the consequences of drug addiction. What about cannabis use?To describe the epidemiological characteristics of cannabis users among musiciensTo study the prevalence of anxiety and depression disorders among these consumersA descriptive and retrospective study of the epidemiological characteristics and prevalence of depression and anxiety in a population of 37 musicians who consume cannabis. This sample was selected among 202 musicians having participated in an anonymous questionnaireThe prevalence of cannabis use among musicians in our study is about 18.31%. 76% of them are professionnals with sex ratio of 6.25. The mean age of these musicians is 27 years old. They started using cannabis at a mean age of 21 years old. The history of school failure was found in 1/3 of all cases, with a younger age at the onset of cannabis use (18 years old vs 22 years old in absence of school failure). 72.4% of cannabis users are single, 27.5% are in a relationship, 66.7% of the 37 musicians are Tabaco smokers, 55.6% are alchoolics, and 19,4% are using other drugs. 16.7% of these musicians are followed for depressive disorder, anxiety or bipolar disorder. The mean duration of cannabis use is 7 years, often in group of people. The first contact with cannabis occurs after the start of learning music in 44.4% of cases (a mean of 12 years after).The average consumption is about 4 times per week, mostly outside the musical activity in 3/4 of the cases. 53.6% believe that cannabis can cause a decline in their health. 10 musicians increased cannabis use and 8 of them believe that it can improve their performance and creativity. On the other hand, only 9 musicians wish to wean the use of cannabis. 19/29 musicians (65.5%) have an anxiety (A) and/or depression (D), that is proven to be moderate to severe respectivelly in 2/3 and half of cases, The mean of the A score and D score of the HAD scale is 10 and 9, respectivelly.The reasons of cannabis addiction are various: fleeing reality, seeking the anxiolytic or sedatif effects and improving performance.None Declared"}
+{"text": "High-resolution single-photon imaging remains a big challenge due to the complex hardware manufacturing craft and noise disturbances. Here, we introduce deep learning into SPAD, enabling super-resolution single-photon imaging with enhancement of bit depth and imaging quality. We first studied the complex photon flow model of SPAD electronics to accurately characterize multiple physical noise sources, and collected a real SPAD image dataset  to calibrate noise model parameters. With this physical noise model, we synthesized a large-scale realistic single-photon image dataset  for subsequent network training. To tackle the severe super-resolution challenge of SPAD inputs with low bit depth, low resolution, and heavy noise, we further built a deep transformer network with a content-adaptive self-attention mechanism and gated fusion modules, which can dig global contextual features to remove multi-source noise and extract full-frequency details. We applied the technique in a series of experiments including microfluidic inspection, Fourier ptychography, and high-speed imaging. The experiments validate the technique\u2019s state-of-the-art super-resolution SPAD imaging performance. High-resolution single-photon imaging is challenging due to complex hardware and noise disturbances. Here, the authors realise simultaneous single-photon denoising and super-resolution enhancement by physics-informed deep learning, with a physical multi-source noise model, two single-photon image datasets, and a deep transformer network. Such a single-photon imaging sensor has been widely applied in various fields such as fluorescence lifetime imaging5, fluorescence fluctuation spectroscopy6, time-off-light imaging9, quantum communication and computing11, and so on13. Compared with EMCCD and sCMOS cameras that also maintain high detection sensitivity, SPAD arrays acquire photon-level light signals at a low-noise level, and perform direct photon-digital conversion that can effectively eliminate readout noise and enhance readout speed14.Single-photon avalanche diode (SPAD) array has received wide attention due to its excellent single-photon sensitivity15, recent advance has exhibited higher fill factors approaching 100%16. Nevertheless, compared with the well-established manufacture craft of CMOS using which Kitamura et al. have reported a 33 megapixel standard CMOS imaging sensor (7680\u2009\u00d7\u20094320 pixels) in 201217, the array size of SPAD in academic literature was only 400\u2009\u00d7\u2009400 in 201918 and 1024\u2009\u00d7\u20091000 in 202015. Moreover, the commercial SPAD products available in the market maintain only thousand pixels19, and cost more than twenty thousand euros. In this regard, the average pixel cost of SPAD is 8 orders of magnitude higher than that of a commercial CMOS. To improve SPAD imaging resolution, Sun et al. reported an optically coded super-resolution technique20 with a high-SNR input with 5.2\u2009ms integration time, which is several orders higher than practical single-photon imaging applications with sub-nanosecond exposure time9. The underlying limitation originates from the employed single-source Poisson noise model22, which deviates from complex real SPAD noise containing a variety of different-model sources such as crosstalk, dark count rate, and so on1 .In the era of deep learning, another obstacle preventing super-resolution SPAD imaging is the lack of datasets containing image pairs of low-resolution single-photon acquisitions and high-resolution ground truth. Although one can accumulate multiple subframes to generate noise-free images, its pixel resolution is still low and far from meeting the Nyquist sampling requirement. One way to acquire high-resolution images is placing another CMOS or CCD camera to take images of the same target, which however introduces additional laboursome registration workload31 and VOC201232 datasets) to digitally synthesize a large-scale realistic single-photon image dataset containing 2.6 million image pairs.In this work, to tackle the great challenge of high-fidelity super-resolution SPAD imaging with low bit depth, low resolution and heavy noise in photon-limited scenarios, we first established a real-world physical noise model of SPAD arrays. As shown in Fig.\u00a034 has recently attracted increasing attention and produced an impressive performance on multiple vision tasks36. As presented below, the reported network mainly consists of three modules, including the shallow feature extraction module, the deep feature fusion module and the image reconstruction module. The framework can gradually extract multi-level frequency features of input images and perform adaptive weighted fusion to reconstruct high-quality images. The gated fusion transformer network was trained using the above large-scale single-photon image dataset and tested on various SPAD images. We built four experiment setups to acquire various macroscopic and microscopic images, two of which acquired target images for dataset collection and direct enhancement validation, and another two were applied for microfluidic inspection and Fourier ptychograpic imaging. A series of experiments validate the technique\u2019s state-of-the-art super-resolution single-photon imaging performance.Driven by the single-photon image dataset, we designed a gated fusion transformer network for single-photon super-resolution enhancement. The transformer frameworkWe first built an optical setup to acquire SPAD images of various targets, as shown in Fig.\u00a0mentclass2pt{minimmentclass2pt{minimTo validate the fidelity of the reported multi-source noise model, we used the synthesized images of different noise models to train different neural networks under the same transformer framework (without super resolution yet), and compared their enhanced results with the reference ground-truth (GT) image composed of 60,000 SPAD subframes. As shown in Fig.\u00a0Despite the above collected single-photon image dataset, further super-resolution enhancement cannot be achieved due to the lack of high-fidelity and high-resolution ground truth pairs. To tackle such a big challenge, we further synthesized a large-scale single-photon image dataset using the public copyright-free Pascal voc2007 and voc2012 images (24 bits). Specifically, the high-resolution images were cropped to 512\u2009\u00d7\u2009512 pixels and downsampled to different scales of 2, 4, 8, and 16 in each dimension. Then, to simulate acquired SPAD images, measurement noise of different illumination and bit depth conditions were added to the 32\u2009\u00d7\u200932 images using the above calibrated noise parameters. In this way, there produce 17250 image pairs of low-resolution SPAD images  and corresponding high-resolution ground truth. The dataset structure is demonstrated in Fig.\u00a0We trained the reported gated fusion transformer neural network (detailed in the Methods section) on the synthesized image dataset under different bit depths, illumination conditions, and super-resolution scales, and tested the network and state-of-the-art enhancing techniques on the test dataset including 505 synthetic noise images. Each test image is composed of different subframes ranging from 16 to 1024 (corresponding to the bit depth ranging from 4 to 10), where each pixel is 1 or 0 indicating whether a photon is detected. The competing methods are classified into two categories, including the popular optimization-based denoising techniques (BM3D and VST) together with subsequent bicubic interpolation (\u201cBM3D+bicubic\u201d and \u201cVST+bicubic\u201d), and the state-of-the-art neural networks . These networks were also trained on our synthesized dataset to converge for a fair comparison.We list a quantitative comparison of peak signal-to-noise ratio (PSNR) and structural similarity metric (SSIM) in Table\u00a0Further, we fixed the illumination flux to be 40\u2009mW, and the super-resolution scale to be 4, and studied these method\u2019s enhancement performance under different bit depths ranging from extremely-low 16 subframes to 1024 frames. The quantitative comparison results are presented in Table\u00a0We applied the trained neural network on real-acquired data for experiment validation. The acquired images were input into the enhancing network with the super-resolution scale being 4 (output 128\u2009\u00d7\u2009256 pixels). The targets include a plaster model, a cat toy and different printed shapes. The enhanced results are presented in Fig.\u00a0To further validate the enhancing ability on microscopic imaging, we built the second microscopy setup by mounting the SPAD array camera (MPD-SPC3) to a commercial microscope (Olympus BX53). The hardware integration time is still set as 37. Inspection of microfluidic chips is important to monitor fluid flow. We built a microfluidic inspection setup using SPAD, as shown in Fig.\u00a0Microfluidic technique manipulates and processes small amounts of fluids using mini channels of micrometer widthThe micro-droplet formation process was recorded using the SPAD camera, as presented in the upper row in Fig.\u00a0Using the microfluidic inspection setup, we can clearly study fluid flow and micro-droplet change process from frame sequence. As shown in the four frames in Fig.\u00a039. It works by acquiring multiple images under different angles of illumination, and inputting these images into phase retrieval algorithms for simultaneous amplitude and phase reconstruction. In our recent work40, we have successfully implemented FPM with SPAD array, realizing coherent imaging with higher resolution and larger dynamic range with single-photon inputs. The experiment setup is presented in Fig.\u00a0Fourier ptychographic microscopy (FPM) is a synthetic coherent imaging technique, providing high-throughput amplitude and quantitative phase images40 to stitch the acquired SPAD images of different illumination angles together, and the FPM reconstruction results are shown in the middle column. We can see that although FPM effectively enhances imaging resolution, there still exists obvious noise and aberration in the reconstruction that is introduced by heavy SPAD noise. Then, we applied the reported technique on the FPM reconstruction, and the enhanced results are presented in the last columns. We can see that in the improved results, the background is smoothed, while the super-resolution details are further enhanced. The experiment further validates the superior enhancing ability of the reported technique on different imaging modalities.The exemplar acquired images of an onion cell sample, a blood cell sample (simulated data), a USAF resolution test target and a plant sample  are presented in the left column of Fig.\u00a039. In this regard, it is our future work to investigate deep into these factors. One possible solution is to incorporate the reported enhancement technique into the iterations of FPM optimization, which may help reduce error accumulations and improve robustness.We also noticed that the enhancement of our technique on experiment data Fig.\u00a0 is not aSPAD imaging holds unique advantages in capturing ultra-fast scenes under limited illumination conditions. Our SPAD arrays can achieve a minimum integration time of 20\u2009ns to capture 1-bit images. We demonstrate the effectiveness of SPAD imaging for high-speed, microsecond timing scenarios in Fig.\u00a0The first demonstration involves recording a rapidly rotating fan. In this experiment, we captured images of the spinning fan using an experimental setup shown in Fig.\u00a041. We provided a high-speed single-photon imaging demonstration video in Supplementary Movie\u00a0The second demonstration features an electrostatic ball, as presented in Fig.\u00a0mentclass2pt{minimIn this work, we introduced deep learning into single-photon detection, and presented a enhancing technique for large-scale single-photon imaging. By tackling the big challenge of single-photon enhancement with extremely low resolution, low bit depth, complex heavy noise and lack of image datasets, the reported technique realizes simultaneous single-photon denoising and super-resolution enhancement , and the design of a deep transformer neural network with a content-adaptive self-attention mechanism for single-photon enhancement. The reported noise model studied the entire photon flow process of single-photon detection, and considered multiple noise sources that degrade single-photon imaging quality. The noise sources include shot noise from photon incidence, fixed-pattern noise from SPAD array\u2019s photon response, dark count rate, afterpulsing and crosstalk noise from blind electron avalanche, and deadtime noise from the quenching circuit. The first single-photon image dataset of 3600 single-photon images was collected with different illumination flux and bit depth, to calibrate the statistical parameters of the multi-source physical noise model. Employing the calibrated noise model, the second single-photon image dataset was synthesized, containing 1.1 million images over 17250 scenes, providing image pairs of low-resolution single-photon ideas and corresponding high-resolution ground truth ranging from thousand to mega pixels. This strategy saves great efforts to collect and register paired data, and makes it possible to study latent signal features of single-photon data. The dataset was applied to train the transformer network for single-photon image enhancement, providing a smooth background and fine details benefiting from its self-attention and gated fusion mechanism.42. This would save laboursome data collection and parameter calibration efforts for different experiments. Furthermore, big model training has gotten a big development for computer vision tasks43. It may be an effective method to generate synthetic datasets based on the reported physics model to train a big model for SPAD imaging.In the workflow, the multi-source noise model parameters were calibrated employing a set of collected images acquired using a specific SPAD camera. Although the reported noise model is generalized and applicable to various single-photon detection schemes, the noise parameters of different SPAD arrays may deviate from each other, even for the same version of cameras. In this regard, the automatic calibration of different SPAD arrays is worthy of further study. Besides, we consider that the transfer learning technique can be adapted to other single-photon detection hardware and settings45 and quantum key distribution46. In such schemes, we can integrate the reported enhancing technique with the sensing framework to achieve global optimum48. The technique may work as an enhancing solver in the alternating optimization process to improve resolution and attenuate noise and aberrations47, preventing error accumulation for global optimization.Besides direct enhanced imaging, single-photon detection has also been applied in multiple computational sensing modalities for broader applications, such as non-line-of-sight imaging49.The photon detection efficiency of the SPAD array is varied at different wavelengths. This property provides two hints for us to further develop the reported technique. First, we can further consider this wavelength-dependent noise in our multi-source physical noise model, which can compensate for the degradation introduced by varied photon efficiency. Second, we can employ various photon efficiency to retrieve spectral information of incident light, opening research avenues on single-photon multispectral imaging that would benefit a set of single-photon applications23 and fluorescence lifetime imaging in microscopy. Prior to the availability of the reported method, addressing issues with commercially available 64\u2009\u00d7\u200932 resolution SPAD for LiDAR systems was difficult, as they struggled to perform accurate and fast detection due to limited resolution. Using the reported approach, we can assist LiDAR systems in obtaining higher resolution and more precise scene information for enhanced detection. Furthermore, data collection in complex environments, such as rainy, foggy, or hazardous conditions, poses even greater challenges. Tackling these scenarios is vital for safety-related tasks. In this context, the reported technique presents an alternative solution for LiDAR systems confronted with these situations. In our future work, it is worth studying time gating in our enhancement framework to improve depth-selective resolution. Besides, considering time stamp into the multi-source physical noise model may further help improve noise robustness and enhancing ability.Besides single-photon detection sensitivity, another highlighted ability of SPAD is its picosecond-scale time gating, indicating the time stamp of photon arrivals. In our present work, we did not consider time gating in different applications such as 3D LiDAR imaging capabilities for autonomous vehiclesAs shown in Fig.\u00a0In the incident process that photons enter the photosensitive surface of SPAD arrays, photon arrival is a stochastic process due to the quantum properties of light, known as the shot noise 50, with the expectation to be calibrated.SPAD arrays absorb incident photons and generate electric charges through photoelectric conversion. In this process, each SPAD pixel responds with a certain probability, and an avalanche occurs under a certain probability to form a saturation current for a new photon count. The probability that the above process occurs is termed photon detection efficiency (PDE), which is defined as the ratio between the number of incoming photons and the number of output current pulses. Since PDE is mainly related to manufacture craft and hardware settings, it is approximated to be a fixed probability distribution, meaning that the fixed-pattern noise  of dark field without illumination. In such a case, we considered shot noise To calibrate the above multiple noise parameters, we first extract the noise maps of afterpulsing noise As stated in the technical manual provided by the SPAD manufacture, the typical probability of afterpulsing events is 1\u20132 orders of magnitude higher than that of crosstalk events. Therefore, we prioritize afterpulsing events during the calibration process. When both afterpulsing and crosstalk events are observed at a single pixel, we classify it as an afterpulsing event rather than a crosstalk event. By doing so, we can effectively ignore the influence of crosstalk events while ensuring that afterpulsing events receive the necessary attention. This strategy helps alleviate the problem of redundant calibration and enables us to obtain accurate parameter values.Using the sub-noise maps, the fixed probability of afterpulsing noise is calculated asFollowed the calibration of afterpulsing noise and crosstalk noise, the dark count rate noise is approximated as30, whose shift-window mechanism can effectively reduce network parameters by more than one order of magnitude compared to the conventional transformer networks34. On this basis, our network further introduces dense connections among different Swin-Transformer layers (STL), enabling long-distance dependency modeling and full-frequency information retrieval. As shown in Fig.\u00a0We designed a gated-fusion transformer network for single-photon enhancement. The network is inspired by the Swin-Transformer structureGiven a low-quality image This module is composed of convolution, batch normalization and activation layers  to extract different levels of medium-frequency and high-frequency features 54, as validated in Fig.\u00a0The last layer of the deep feature fusion module is the Gated Fusion layer, which fuses the outputs of different DCSTB operations with adaptively different weights. The process can be described asWe retrieve high-quality single-photon images by aggregating shallow features and multi-level deep fusion features. The operation is described as55 that lacks the densely connected gated fusion structure (as shown in Fig.\u00a0The shallow features We designed a hybrid loss function consisting of We randomly selected 505 image pairs from the synthetic large-scale single-photon image dataset as the testing set, and the remaining 16620 image pairs were used for network training. All the images were cropped to 32\u2009\u00d7\u200932 pixels when input into the network. We implemented the network on Ubuntu20 operating system using the Pytorch framework, and trained 1000 epochs until convergence using Adam optimization on NVIDIA RTX3090 with the Batch size set to 24. We set the initial learning rate as 0.0003, which was decreased by 10% for every 100 epochs. The default weight decay was set to 0.00005, and the Adam parameters of The minimum data generated in this study have been deposited in the Figshare database under accession code 10.6084/m9.figshare.23966922.v1. The complete data are available under restricted access for the funded project requirements, access can be obtained by the corresponding author within one month of the reasonable request to the corresponding author.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1"}
+{"text": "HAS-BREAD score was 4  in the hemodialysis patients and 3  in the non-hemodialysis patients (p\u2009<\u20090.001). All procedures were successful, except for a non-hemodialysis patient with a larger left atrial appendage. There were no major complications during index hospitalization and 45-day observational period, except for a hemodialysis patient with suspected bleeding and a non-hemodialysis patient who died due to cardiac amyloidosis. LAAC seems to be feasible in hemodialysis patients with high risks of thromboembolic events and bleedings.In the countries like Japan where anticoagulation is not recommended in hemodialysis patients, the feasibility of percutaneous left atrial appendage closure (LAAC) in hemodialysis patients with non-valvular atrial fibrillation (NVAF) accompanying high risks of thromboembolic stroke and bleeding remains unknown. Peri-procedural and 45-day clinical outcomes following LAAC using WATCHMAN system, which were performed in our institute between Jun 2020 and April 2022 according to the Japanese Circulation Society guidelines, were retrospectively compared between those with and without hemodialysis. 118 patients  consisting of 25 hemodialysis patients and 93 non-hemodialysis patients were included. CHADS score was 3  in the hemodialysis patients and 3  in the non-hemodialysis patients ( Non-valvular atrial fibrillation (NVAF) is the most common cardiac arrhythmia. The prevalence of NVAF has increased with the aging of the world-wide population, affecting approximately 10% in Western countries . One of The strategy for those with end-stage renal disease is controversial. The incidence of NVAF and its impact on increasing the risk of stroke are higher in patients with end-stage renal disease compared with general population . NeverthPercutaneous left atrial appendage closure (LAAC) is an established therapy for preventing thromboembolic stroke in patients with NVAF, who are not good candidates for long-term anticoagulation therapy Fig.\u00a0 9]. How. How9]. NVAF was defined as AF without moderate or severe mitral stenosis or mechanical prosthetic heart valve. Consecutive patients with NVAF who received LAAC using WATCHMAN system  and informed consents were obtained from all participants before inclusion.2 score and CHADS2-VASc score. On the contrary, they should also be at high risk of bleeding and satisfy either of them: HAS-BLED score equal to or above 3 points; multiple histories of trauma due to falling; cerebral amyloid angiopathy; requirement of multiple antiplatelets; histories of major bleeding with BARC type 3\u20135. All patients received transesophageal echocardiography for the anatomical assessments. The final indication was determined by the institutional heart-valve team conference.The indication of LAAC was according to the Japanese Circulation Society guidelines. Patients should be those with NVAF who were at high risk of systemic embolisms and were highly recommended to receive anticoagulation therapy according to CHADSThe indication of LAAC for the hemodialysis patients was similar to the non-hemodialysis patients. All hemodialysis patients satisfied \u201cA\u201d in the HAS-BLEAD score .The WATCHMAN2.5 and FLX are self-expanding, nitinol-framed structures ranging in diameter from 21 to 33\u00a0mm (WATCHMAN2.5) and from 20 to 35\u00a0mm (WATCHMAN FLX), respectively, to accommodate varying LAA anatomy and size. These devices are fixed by anchor at the LAA ostium to avoid embolization and to prevent blood flow in the LAA. LAAC was performed under general anesthesia according to the standard procedure by the board-certified interventionists using angiography, transesophageal echocardiography, and double curve sheath via a trans-septal puncture approach.Following the procedures, anticoagulation therapy using warfarin or DOAC as well as antiplatelets were continued for 45\u00a0days to allow time for device endothelialization: (1) single antiplatelet and DOAC/warfarin with the therapeutic international normalized ratio between 2.0 and 2.6 for those with non-hemodialysis; single antiplatelet and warfarin with a therapeutic international normalized ratio between 1.5 and 2.0 for hemodialytic patients .Continuous variables were expressed as mean and standard deviation and compared between the two groups  using unpaired t-test. Categorical variables were expressed as numbers and percentages and compared between the two groups using Fischer\u2019s exact test. A value of 2 score was 3  points, CHADS2-VASc score was 5  points, and HAS-BLEAD score was 3  points.118 NVAF patients were included. Median age was 79  years old and 81 (69%) were men . CHADS2 score and CHADS2-VASc score were not significantly different between the two groups, whereas the hemodialysis group had a higher HAS-BLEAD score (p\u2009<\u20090.001). Eighty patients received transesophageal echocardiography, which demonstrated no significant differences in the LAA parameters between the two groups . The incidences of multiple device use and partial recapture were not significantly different between the two groups (p\u2009>\u20090.05 for both).The procedure success rate was 99%: the procedure was unsuccessful in a patient in the non-hemodialysis group due to the inappropriately larger size of LAA Table . The prop\u2009=\u20090.76). All patients could be discharged alive.During the index hospitalization, there were no major complications including access site events, acute kidney injury, pericardial effusion, device embolization, infectious endocarditis, and stroke Table . One hemp\u2009<\u20090.05; Table At baseline, more hemodialysis patients received antiplatelets than the non-hemodialysis group p\u2009<\u20090.0; Table 5As for the antiplatelets, most of the patients (79%) received single antiplatelets at the index discharge according to the institutional protocol, irrespective of the dependence on hemodialysis Fig.\u00a0. No pati94 patients were followed until day 45. The proportion of prescriptions remained almost unchanged from the index discharge. In the hemodialysis patients, 20 (95%) received warfarin. In the non-hemodialysis patients, 5 (7%) received warfarin and others (68 [93%]) received DOAC.p\u2009=\u20090.63). No patients had right-to-left jet.94 patients completed a 45-day follow-up Table . Most ofDuring the 45-day observational period, no patients had major complications including pericardial effusion and device-related thrombus. There were no deceased patients, except for a patient with cardiac amyloidosis in the non-hemodialysis group who died suddenly without any obvious reasons 5\u00a0days following the index discharge.In this prospective study, we reported for the first time the short-term feasibility of LAAC in hemodialysis patients with high risks of thromboembolic events and bleedings.LAAC is an established alternative to anticoagulation therapy to prevent stroke events in patients with NVAF and high risk of bleedings . LAAC haIn non-hemodialysis patients, the existence of NVAF is a major trigger of cardiac stroke. The hemodialysis patients have multiple high-risk origins of thromboembolic strokes due to systemic atherosclerosis, whereas cardiac stroke is reported to be a major etiology in Japan .2 score, which is an established score to consider the risk of stroke in the general cohort, to the hemodialysis cohort remains unknown, given that the original study did not include them [The applicability of CHADSude them . Howeverude them .Anticoagulation therapy is an established strategy to prevent stroke in the general cohort with NVAF and high risk of stroke. In hemodialysis patients, all types of DOAC are contraindicated. The Japanese society for dialysis therapy guidelines states that warfarin therapy is, in principle, contraindicated . PreventGiven all together, strategies other than anticoagulation would be required for those with hemodialysis to prevent stroke.One of the strategies to answer the above request is LAAC. Gotzmann and colleagues retrospectively analyzed 128 candidates of LAAC. Of them, mortality, bleeding incidence, and thromboembolic events incidence were not statistically different between 33 hemodialysis patients and others . In anotThis is the first study that demonstrated the short-term feasibility of LAAC in Japanese hemodialysis patients. According to the recommended standard regimen for post-LAAC 45\u00a0days consisting of the combination of DOAC and low-dose aspirin , we admiThe incidences of peri-procedural complications were not significantly different between those with and without hemodialysis. There was no device-related thrombosis during the 45-day follow-up period among all cohort. Only a patient with hemodialysis required termination of warfarin during a 45-day follow-up period. The patient had a slight progression of anemia due to asymptomatic interstitial bleeding, which was ameliorated without blood transinfusion.LAAC might be a feasible strategy also in hemodialysis patients. Short-term warfarin therapy following LAAC would be safe in this cohort. Further studies are warranted to validate the long-term feasibility and implication of LAAC in this cohort.This study was conducted in a single center using a small sample size. Statistical non-significance does not guarantee similarity. There are several differences in background and procedure-related parameters, including the incidence of major bleeding history and HAS-BLEAD score as well as procedure time between the two groups. More patients with hemodialysis received LAAC due to high HAS-BLEAD score rather than a history of major bleeding. Initial recommendations to receive LAAC were dominantly performed by cardiologists. Thus, there might be selection bias. We cannot ignore the impact of two different devices (2.5 and FLX) and the learning curve of the operators. Of note, hemodialysis patients trended to receive WATCHMAN 2.5 rather than FLX. Post-procedural medication regimens were different between the two groups. Such a difference might have affected clinical outcomes. We observed just 45\u00a0days following LAAC and a longer observational study is the next concern.LAAC seems to be feasible in hemodialysis patients with high risks of thromboembolic events and bleedings."
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