Single nucleus sequencing reveals evidence of inter-nucleus recombination in arbuscular mycorrhizal fungi. Published 5, December 2018Recently a reader contacted us about potential inconsistencies between the data in Supplementary File 6 and Figure 2. Figure 2 is directly built from data in Supplementary File 6. After checking the data and the scripts used to generate Figure 2, we found that an earlier version of Figure 2 was mistakenly uploaded.The earlier version Figure 2 was based on an analysis of the isolate SL1 that allowed the maximum number of Blast hits for regions surrounding SNPs to be of 1, instead of 2 as written in the method section of our paper. The results of this parameter change produced minor differences that do not alter the overall patterns of nuclear diversity originally observed. In all cases, the figure shows that isolates A4 and A5 harbour a structured nuclear genome organization, while in the isolate SL1 nuclear genetic diversity appears to be more diverse.As a result, Figures 2 (SL1 portion), Supplementary File 10 (SL1 portion), Supplementary File 11 (SL1 portion), Supplemental File 5 (SL1 portion) and Supplementary File 4 (SL1 portion) have been corrected.Corrected Figure 2:Original Figure 2:Corrected Supplementary file 10:Original Supplementary file 10:Corrected Supplementary file 11:Original Supplementary file 11:Supplementary file 4 and Supplementary file 5 have also been corrected.In Supplementary 4 we also identified a correction of number of SNPs in A4 after stringent filtering, from 2363 to 2362. The increase in SNP counts for SL1 with the updated parameter after stringent filter changes the reduction of SNP from 94.20% to 93.81%. The corresponding passage in the article has now been corrected:Original text: “This conservative approach has likely removed some true positives from our analysis - for example this filtering method resulted in an average 94.2%”The corrected text now reads as follows:“This conservative approach has likely removed some true positives from our analysis - for example this filtering method resulted in an average 93.8%”Our conclusions are unaffected by these changes, and we apologize for this oversight.The article has been corrected accordingly.


Atherosclerosis is a chronic inflammatory disorder of large- and medium-sized arteries and remains one of the most common causes of age-related death worldwide. It is well established that effector T cell responses promote both atherosclerotic plaque growth and instability . In contin vitro [FOXP3 exon 7, whereas, a reduced portion of FOXP3Δ2 is associated with an unstable plaque phenotype [FOXP3 appears to represent a layer of regulation that can reveal functional differences of Treg cell populations in different patient categories.Treg cells are a potential mediator of future intervention strategies for atherosclerosis. These cells depend on the lineage-defining transcription factor FOXP3 for their development and function. Human FOXP3 isoforms are generated through the exclusion of exon 2 and/or exon 7. In contrast, mice do not produce any isoforms of FOXP3. Full-length FOXP3 (FOXP3fl) and FOXP3 lacking exon 2 (FOXP3Δ2) isoforms confer a suppressive ability to Treg cells in vitro –6, whereFOXP3 exon 7 [The two dominant FOXP3 isoforms, FOXP3fl and FOXP3Δ2, are coexpressed in Treg cells and normally makes up 95% of the total amount of FOXP3 protein. The expression pattern does however vary. For instance, we have found that IL-1β could promote splicing of 3 exon 7 . Another3 exon 7 . However3 exon 7 . As we hin vivo function of FOXP3 isoforms in humans.What will the future bring to FOXP3 isoforms and their involvement in chronic inflammatory disorders? First, we hope that the studies involving FOXP3 isoforms will be expanded upon as it remains essential to validate the current findings in other patient cohorts. This should go hand in hand with developing better tools for identification of specific FOXP3 isoforms. Another interesting line of research will be to better define the molecular mechanisms of action of distinct FOXP3 isoforms including their interactomes and their differential ability to control gene expression. Finally, we have together with collaborators, identified patients with atypical forms of the immunodysregulation polyendocrinopathy enteropathy X-linked syndrome that lack expression of specific FOXP3 isoforms. We believe that these patients will give us a better understanding of the


Plasmodium falciparum is predominant. However, their usefulness has been threatened by the emergence of gene deletion on P. falciparum histidine rich protein 2 (pfhrp2) and P. falciparum histidine rich protein 3 (pfhrp3). Use of standard and recommended methods is key for accurate investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion.Malaria rapid diagnostic tests based on histidine-rich protein-2 have played a vital role in improving malaria case management and surveillance particularly in Africa, where pfhrp2 and pfhrp3 gene deletion in Africa. An online search was done using PubMed and MEDLINE Google Scholar for all articles published in English on pfhrp2/3 gene deletion in Africa. Relevant articles that met the inclusion criteria were summarized and assessed based on the protocol recommended by the World Health Organization for confirmation and reporting of pfhrp2/3 gene deletion.A systematic review was conducted to assess the status, methods and approaches that have been used for investigation, confirmation and reporting of fhrp2 and pfhrp3 gene deletion studies were conducted and reported. The level of pfhrp2/3 gene deletion across selected studies in Africa ranged from the highest 62% to the lowest 0.4%. There was wide variation in methods and approaches including study designs, size and sampling and whether both pfhrp2 and pfhrp3 double deletions or pfhrp2 single deletion were investigated, with a wide variation in laboratory methods.The search identified a total of 18 articles out of which 14 (77.7%) fulfilled the criteria for inclusion and were retained for review. The articles were distributed across 12 countries where the ppfhrp2/3 gene-deleted P. falciparum parasites in Africa. The approaches and methods used for investigation, confirmation and reporting of pfhrp2/3 deleted parasites have varied between studies and across countries. Countries that are considering plans to investigate, confirm and report pfhrp2/3 deletion should use recommended standard and harmonized methods to prevent unnecessary recommendations for costly switch of RDTs in Africa.Based on the review, there is evidence of the presence of  Plasmodium falciparum is the most prevalent malaria species in the WHO African region, accounting for 99.7% of estimated malaria cases in 2017 [The World Health Organization (WHO) estimated that there were 219 million cases of malaria and 435,000 malaria deaths and nearly half of the world’s population was at risk of malaria infection in 2017 [P. falciparum, over 90% of RDTs used for the diagnosis of malaria in sub-Saharan Africa are HRP2-based [Plasmodium falciparum specific RDTs specifically recognize HRP2 antigen that encodes for the pfhrp2 gene and whose antibodies cross-react with histidine-rich protein 3 (pfhrp3) antibodies due to high degree of similarity in amino acid sequence [pfhrp2 and pfhrp3 genes. Plasmodium falciparum parasites lacking the pfhrp2/3 gene do not express HRP2 protein antigen threatening the usefulness of HRP2 RDTs in malaria diagnosis [P. falciparum parasites with pfhrp2 and pfhrp3 gene deletions were reported in the Amazon basin in 2010 by Gamboa et al. [pfhrp2/3 gene deletions outside the Amazon region in Africa and India [P. falciparum with missing pfhrp2/3 genes pose a public health threat as a large number of malaria infected patients will go undetected by the HRP2 RDTs and, therefore, remain untreated leading to increased risk of malaria morbidity and mortality, and continued malaria transmission [Efforts to reduce the burden of malaria in Africa have mostly included the use of long-lasting insecticide-treated nets (LLINs), indoor residual spraying (IRS) with insecticides, intermittent preventive therapy (IPT), diagnosis and treatment. Case management which involves testing and treatment with artemisinin-based combination therapy (ACT) is a major intervention for malaria control , 2. The P2-based , 2. Plassequence . Howeveriagnosis , 6. The a et al. . Howeversmission , 6.pfhrp2-deleted parasites meets or exceeds the lower 90% confidence interval for 5% prevalence, or a plan for change over a longer time frame if deletions are present but < 5% [pfhrp2 and pfhrp3 gene deletions [pfhrp2/3 gene deletion, countries, such as Eritrea have introduced non-HRP2 alternative RDTs that are able to detect gene-deleted parasites [P. falciparum parasite confirmation in Africa and the limited options available of WHO approved non-HRP malaria RDTs [pfhrp2 RDTs are based on quality data generated from well conducted studies using recommended methods to avoid unnecessary costly switch of RDTs [pfhrp2/3 gene deletion studies in Africa have varied. There have been variations in; (1) the size of the studies, (2) source of participants used , (3) clinical classifications of the participants including symptomatic versus asymptomatic individuals, and (4) investigation of pfhrp2 deletion alone versus pfhrp2 and pfhrp3 double deletions and flanking genes and (5) the laboratory methods.The WHO recommends a policy switch to more effective alternative non-HRP2 RDTs, when the prevalence of but < 5% . In Afrieletions –18. Due arasites . Howeverarasites . The thrria RDTs , 7. It i of RDTs . Howeverpfhrp2 and pfhrp3 gene deletion [pfhrp2 gene deletion and the methods and approaches being used for its estimation, confirmation and reporting in Africa.Due to this variability in study designs, methodologies and reporting, the WHO Global Malaria Programme published a standard protocol on the recommended approaches and methods required for investigation, confirmation and reporting of deletion . This repfhrp2 gene deletion in P. falciparum parasites in Africa since 2010 when the first deleted parasites were identified in clinical samples in the Amazon region, (2) assess the methodologies and approaches being used for pfhrp2/3 gene deletion estimation, confirmation and reporting in Africa.The review aimed to (1) assess the status of pfhrp2/3 gene deletion in Africa between January 2010 and June 2019. Literature search was done using PubMed and MEDLINE google Scholar for all articles published in English about pfhrp2/3 gene deletion in Africa. The following were used as search words; ‘Malaria’, ‘Plasmodium falciparum’, ‘pfhrp2’, ‘pfhrp3’ ‘Gene deletion’, ‘Malaria Rapid diagnostic tests’, ‘Africa’. All searches were restricted to paper titles and abstracts.A systematic search of literature was conducted electronically for published studies on pfhrp2/3 gene deletion in clinical samples in Peru until June 2019 [pfhrp2 deletion, (3) conducted in Africa, and (4) published during the selected review period. In order to expand on the scope, the papers referenced or cited in the selected papers were also reviewed for additional evidence. The WHO recommended protocol for investigation, confirmation and reporting of pfhrp2/3 gene deletion  fulfilled the criteria for inclusion and were retained for review (Fig. The review considered published articles on P. falciparum pfhrp2 gene deletion based on the WHO recommended methods for confirmation and reporting of pfhrp2 and pfhrp3 gene deletions is shown in Table pfhrp2/3 gene deletion across Africa is shown in Fig. The summary of findings from the reviewed articles on pfhrp2/3 gene deletions in Africa were assessed and reviewed where studies were conducted and reported between 2010 and June 2019. There was wide variation in methods and approaches used across studies (Table pfhrp2 gene deletions (Table pfhrp2 and pfhrp3 double deletions or pfhrp2 single deletion alone with wide variation in laboratory methods.The status, methods and approaches that have been used for confirmation and reporting of pfhrp2 and pfhrp3 gene deletion in Africa where P. falciparum is the predominant parasite and where huge volumes of HRP2 based RDTs are used for malaria diagnosis [pfhrp2/3 gene deletion across malaria endemic countries in Africa range from the highest 62% in Eritrea to the lowest 1.45% and 0.4% in Mozambique and Angola, respectively [pfhrp2/3gene deletion in these studies could be due to selection pressure caused by exclusive use of HRP2 RDTs over time as suggested in previous studies [pfhrp2/3 negative parasites that remained undetected and continued to increase [pfhrp2/3-negative parasite populations leading to their increase in frequency [pfhrp2/3-negative parasites once they emerge [pfhrp2/3 deleted parasites reported by studies in Western Kenya and Southern Mozambique may be due to the absence of these ideal conditions for pfhrp2 selective pressure, such as are high transmission settings and use of malaria microscopy as the major diagnostic tool [pfhrp2/3 gene deletions above the 5% recommended WHO cut-off, Eritrea has introduced non-HRP2 RDTs to detect pfhrp2 and pfhrp3 deleted parasites [A total of 14 research articles satisfied our criteria for inclusion in the review Table . These aiagnosis –18. Baseectively , 12, 20.ectively . Gene deectively . The obs studies . In Eritincrease . This serequency . Low maly emerge . The exttic tool . Due to arasites .pfhrp2 and pfhrp3 gene deletion across all studies. The major differences and limitations in methods and approaches across the studies are highlighted below.There was wide variation in the approaches and methods used for investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion [pfhrp2 and pfhrp3 deletion studies [pfhrp2/3 gene deletion studies is because parasite density is generally higher in the former compared to the low-density infections in the latter and hence provide better quality samples for confirmation of gene deletions by molecular tests [pfhrp2/3 gene deletion investigation including blood donors in Mali [pfhrp2 gene deletions as 6.4%, 4.8% and 1.45%, respectively [pfhrp2 gene deletions up to 62% and 23%, respectively [pfhrp2/3 gene deletion studies. The Mali study however gives contrary findings and reports a significant association between asymptomatic population and pfhrp2/3 gene deletion [pfhrp2/3 gene deletion studies across malaria endemic countries in Africa as the use of appropriate study participants is fundamental for the gene deletion study outcomes.All the reviewed studies but one used a cross-sectional design as recommended by the WHO protocol for confirmation and reporting deletion –18. The  studies . The reaar tests , 7. Howe in Mali . The DRCectively , 16, 21.ectively . The WHOdeletion . These vpfhrp2/3 deletion recommends the recruitment of a minimum of 370 or 318 symptomatic individuals with suspected P. falciparum infection to estimate a prevalence of 3.2% and 8.0%, respectively per sampling region or province [pfhrp2 gene deletion of > 80% at one of the study hospitals [P. falciparum DNA samples [P. falciparum population [pfhrp2/3 gene deletion across countries.The WHO protocol for investigation of ospitals . The Zam samples . Based opulation , 24. Nonpfhrp2 and pfhrp3 double deletion versus pfhrp2 single deletion alone: The WHO protocol for pfhrp2/3 studies recommends estimation and reporting of both pfhrp2 and pfhrp3 in P. falciparum gene deletion studies [P. falciparum-based RDTs are designed to specifically recognize HRP2 antigen, however pfhrp2 and pfhrp3 are homologous genes whose antigens may cross-react due to high degree of similarity in their amino acid sequence [pfhrp2 and pfhrp3 gene deletions warranting investigation of both genes. Evidence from the Eritrean study showed that every sample that was pfhrp2 deleted was also pfhrp3 deleted suggesting a possible association [pfhrp2 and pfhrp3 double deletion while others reported pfhrp2 deletion alone. Gene deletions studies in Ghana, Mali and Rwanda investigated and reported single pfhrp2 deletion alone [pfhrp2 and pfhrp3 double deletions [pfhrp2 gene deletion alone is a possible underestimation as some of the samples may test positive with HRP2 RDT even when parasites are pfhrp2 deleted due to cross-reactivity with HRP3 antibodies. These observations and variations in methods call for harmonization and standardization of investigative and reporting approaches for pfhrp2/3 gene deletions.Reporting of  studies , 7. Thissequence , 25, 26.ociation . Howeveron alone , 9, 17, eletions –16, 18. pfhrp2/3 gene deletion in P. falciparum parasites have been previously published [pfhrp2/3 deletion should be negative by HRP2 based RDT and positive with expert microscopy or Pf-pLDH RDT [P. falciparum mono-infection and exclude other non-P. falciparum species. Samples that are PCR confirmed as P. falciparum are amplified in the exon1 and exon 2 regions of the pfhrp2/3 gene to detect the presence or absence of the gene [pfhrp2 or pfhrp3 in the exon region are considered pfhrp2 and pfhrp3 deleted after ascertaining the quality of parasite DNA by amplification of MSP1 and MSP1 single copy genes [pfhrp2/3 gene deletion study as opposed to the use of dried blood spots as preferred samples [pfhrp2/3 gene deletion outcome is poorly understood. Four of the reviewed articles that reported pfhrp2 deletion in four countries missed an essential procedural requirement of demonstrating the quality of parasite DNA by PCR amplification of MSP1 and MSP2 single copy genes of P. falciparum [P. falciparum MSP1 and MSP1 single copy genes as an essential confirmation of P. falciparum DNA quality in suspected pfhrp2/3 deleted samples is a major methodological flaw that creates uncertainty on the validity and correctness of the reported deletion estimates [pfhrp2/3 in the subtelomeric region is optional and not essential requirement for confirmation and reporting of P. falciparum parasite gene deletion [pfhrp2 and pfhrp3 gene regions and can extend in the neighbouring flanking genes [The recommended laboratory-based testing methods required for confirmation of suspected ublished , 25–35. pLDH RDT , 33, 35.py genes , 6, 7. H samples . The efflciparum , 15, 17.lciparum . Detailelciparum . Failureng genes . Under tpfhrp2/3 surveys that aims to achieve representativeness of a country’s malaria parasites population across all epidemiological settings [pfhrp2 and pfhrp3 gene deletion in parasites collected across the various states and provinces [P. falciparum parasites population [The WHO recommends the design of settings . The improvinces , 31. Howpulation , 16–18. P. falciparum infections as measured by microscopy, others used PCR confirmed or number of RDT-/microscopy + discordant samples that is a smaller denominator [pfhrp2/3 gene deletion estimates with possible overestimation or underestimation. The WHO standard protocol recommends the use of total P. falciparum infections measured by microscopy the suitable denominator to avoid overestimation of gene deletion estimates [P. falciparum species and false positives that are misclassified by poor quality microscopy. This potentially inflates the denominator leading to under estimation of deletions [Across all the reviewed studies, there were differences in the denominators used in the final computation of gene deletion estimates. While others used total ominator –18. The stimates . Howevereletions , 12, 13.pfhrp2/3 gene deletion across studies in malaria endemic countries in Africa. The direct implication of the use of non-Standardised and non-harmonized methods for confirmation and reporting of parasite gene deletion is the risk of unnecessary recommendations for a costly switch from HRP2 based RDTs to non-P. falciparum RDTs. Non-HRP2 RDTs are more expensive, less sensitive with poor field thermal stability [P. falciparum is predominant and where large volumes of HRP2 based RDTs are used for malaria diagnosis [P. falciparum RDTs in the context of gene deletions.Our review found a wide variation in methodologies and approaches for investigation of tability . Unnecesiagnosis , 2. HoweP. falciparum, the search identified only 13 published articles on pfhrp2/3 gene deletion in Africa. This observation could explain the limited data available on the occurrence and status of pfhrp2/3 gene deletion in malaria endemic countries in Africa. The WHO recommends initiation of surveys and surveillance systems to allow early detection and containment of pfhrp2/3 gene-deleted parasites in countries at risk of this threat [pfhrp2/3 gene deletion, the WHO recommends the need for periodic monitoring to assess if levels are increasing or exceeded the 5% prevalence cut-off required for change of diagnostic policies [Despite the high burden and dominance of s threat . Specifis threat . Howevers threat , 37. Evepfhrp2 and pfhrp3 gene-deleted parasites in all malaria epidemiological setting including low and high transmission zones [pfhrp2/3 gene-deleted parasites to spread and cause disease [pfhrp2/3 deleted parasites are drug sensitive compared to gene harbouring parasites and whether current treatment is effective for pfhrp2/3 deleted parasites.Previous studies have demonstrated the possible occurrence and survival of on zones , 15, 31. disease , 9, 11. pfhrp2/3 gene-deleted parasites in P. falciparum infected samples [pfhrp2/3 could still occur due to low quality parasite DNA particularly in low parasitaemia samples. Missed deletions could occur if an infection with a deleted parasite occurs subsequent to an infection with a wild type parasite, since circulating HRP2 can persist for up to a month [Studies have shown the failure of HRP2 based and the ability of non-HRP2 RDTs to detect  samples , 11, 12. samples , 38, 39. samples . However a month . In highpfhrp2/3 gene-deleted P. falciparum parasites in Africa. The approaches and methods used for investigation, confirmation and reporting of pfhrp2/3 deleted parasites have varied between studies and across countries. The available evidence on the occurrence of pfhrp2/3 deletion comes from a limited number of countries leaving it largely unknown and unreported in many malaria endemic countries in Africa. Countries that are considering plans to confirm and report pfhrp2/3 deletion should use recommended standard and harmonized methods to prevent unnecessary recommendations for costly switch of RDTs in Africa.Based on the review, there is evidence of the presence of


HvCKX2.1 is the regulatory target for the most abundant heterochromatic small RNAs in drought-stressed barley caryopses. We investigated the diversity of HvCKX2.1 in 228 barley landraces and 216 wild accessions and identified 14 haplotypes, five of these with ten or more members, coding for four different protein variants. The third largest haplotype was abundant in wild accessions (51 members), but absent from the landrace collection. Protein structure predictions indicated that the amino acid substitution specific to haplotype 3 could result in a change in the functional properties of the HvCKX2.1 protein. Haplotypes 1–3 have overlapping geographical distributions in the wild population, but the average rainfall amounts at the collection sites for haplotype 3 plants are significantly higher during November to February compared to the equivalent data for plants of haplotypes 1 and 2. We argue that the likelihood that haplotype 3 plants were excluded from landraces by sampling bias that occurred when the first wild barley plants were taken into cultivation is low, and that it is reasonable to suggest that plants with haplotype 3 are absent from the crop because these plants were less suited to the artificial conditions associated with cultivation. Although the cytokinin signalling pathway influences many aspects of plant development, the identified role of HvCKX2.1 in the drought response raises the possibility that the particular aspect of cultivation that mitigated against haplotype 3 relates in some way to water utilization. Our results therefore highlight the possibility that water utilization properties should be looked on as a possible component of the suite of physiological adaptations accompanying the domestication and subsequent evolution of cultivated barley.The cytokinin dehydrogenase gene  Triticum monococcum L.), emmer wheat (T. dicoccum (Schrank) Schübl.) and barley (Hordeum vulgare L.)–occurs during the 8th millennium bc  250 matrix); periods indicate positions occupied by amino acids with weakly similar properties (<0.5 and >0 in the Gonnet PAM 250 matrix). The barley sequence is the HvCKX2.1 consensus sequence as determined in this study . The other sequences are taken from Genbank: maize, (TIFF)Click here for additional data file.S3 FigThe ellipses indicate the regions within which 95% of the data points for each haplotype are expected to fall. Black dots and ellipses, haplotype 1; cyan, haplotype 2; orange, haplotype 3.(TIFF)Click here for additional data file.S4 FigtSNE was run with perplexity = 30, iterations = 2000 and theta = 0.5. Black dots and ellipses, haplotype 1; cyan, haplotype 2; orange, haplotype 3.(TIFF)Click here for additional data file.


Pyruvate Kinase M2 (PKM2) mediates metabolic reshuffling and is ubiquitously upregulated in several cancer types. The non-metabolic function of PKM2 as key nuclear kinase and modulator of gene expression is instrumental in cancer progression and tumorigenesis. Here, we attempt to discern the non-canonical function of PKM2 as an epigenetic modulator and the underlying implication of this activity. Using 5’-FAM labelled reconstituted mononucleosome we have shown that PKM2 interacts with the complex through Histone H3 and possibly obstruct the access to DNA binding factors. Subsequently, the interaction negatively impacts the ATP dependent remodeling activity of Chromodomain Helicase DNA binding protein-7 (Chd7). Chd7 remodeling activity is required to ameliorate DNA damage and is crucial to genome stability. Our study shows that PKM2 blocks the Chd7 mediated sliding of nucleosome. It can be conjectured that stalling Chd7 may lead to impaired DNA damage and increased genomic instability. We propose a mechanism in which PKM2 negatively regulate nucleosome repositioning in chromatin and may exacerbate cancer by altering the nucleosome architecture. This research is imperative to our understanding of how altered cancer metabolism can potentially modulate the gene expression and sustain incessant proliferation by tweaking the chromatin topography. Metabolism and gene expression are stringently regulated physiological process that are concurrently modulated by a wide array of enzymes and regulatory protein. The concerted control mechanism exerted by the milieu of governing factors lead to dynamic regulation of the cellular machinery and contribute to incessant cell proliferation and tumorigenesis . Metabol−1 , 150 mM NaCl, 3 mM EDTA, and 0.05% NP-40 surfactant. The association kinetics for PKM2 was monitored for 400 s, followed by dissociation kinetics for 300 s. Different concentrations of Histone H3 (100 nM- 1000 nM) diluted in running buffer were injected across the sensor surface. SPR signals and resultant sensogram for PKM2-Histone H3 interactions were analyzed. Resultant sensogram was analyzed by subtracting change in the signal from activated/blocked control panel from that obtained as a consequence of binding of histone H3 to PKM2. At last the surface was regenerated with 75 mM NaOH. All the data were recorded at room temperature . Autolab SPR Kinetic Evaluation software was used for data analysis.2, 5% sucrose, 0.1mg/ml of BSA, and 1 mM DTT.500 nM of PKM2 was incubated with 100 nM of FAM-labeled nucleosome on ice overnight. Buffer composition was as 20mM HEPES buffer, pH 7.6, 50mM KCl, 5mM MgClVarying concentration of ExoIII enzyme was added to the 10ul of reaction mixture. After incubation at 23 °C reaction was terminated with 2X stop buffer containing 20mM EDTA and 2% SDS and addition of ~20μg of glycogen. DNA was extracted using phenol–chloroform–isoamyl alcohol (25:24:1). The samples were subjected to denaturing electrophoresis in a 7% polyacrylamide—7 M urea gel in 1X TBE buffer. The gel was scanned by using Typhoon Phosphorimager FLA9000 .Nucleosome sliding experiment was performed using an electrophoretic mobility shift assay (EMSA)-based method as described previously –47 with GCG⌃C has been inserted at dyad position 73 after each 208 bp and is usually obscured in nucleosome. Remodeling results in exposed restriction site and subsequent digestion of the DNA. To summarize 5nM of the pJ201 (34 × 601) chromatinized plasmid was incubated with 50 nM of Chd7 remodeler and 2.5mM of ATP with or without PKM2 at 23°C for 120 mins. Buffer used was 20mM HEPES, pH 7.6, 50mM KCl, 5 mM MgCl2, 1 mM EDTA, 1mM DTT, 0.1 mg/ml of BSA. Post incubation restriction digestion was performed using 1 U of HhaI enzyme for a time period of 30 min. The reaction was allowed for the defined time and stopped using Glycogen stop buffer . Samples were loaded on to 1.4% agarose gels. Electrophoresis was carried at 120V for 2hrs and gel was stained with ethidium bromide [To study the effect of PKM2 on nucleosome accessibility at chromatin level restriction digestion of pre-assembled nucleosome array of plasmid was carried out using HhaI enzyme. HhaI digestion site  bromide .kd value of very stable interactions is difficult and at the same time might be misleading under mild or physiological conditions. The complex decay in such cases is extremely slow and kd value should be only a qualitative measure and not quantitative. We can conclude from the aforementioned binding parameters that immobilized PKM2 interacts with Histone H3 and there is a gradual increase in the response unit with increase in Histone H3 concentration. A kinetic equilibrium is obtained represented by plateau in the association phase of the plot. Affinity Constant (KD) value for the binding kinetics is 6.39E-06M (Affinity error 3.20E-07) and calculated from the equilibrium equation. The obtained value is mean of the experiment repeated in triplicate. However the rate of complex decay is remarkably low as we do not observe any significant dissociation of the PKM2-Histone H3 complex suggesting that some other factor might be needed to break the association between the two biomolecules. This dissociation phase kinetics is interesting and needs to be further investigated. Fluorescence spectroscopy experiment using FITC labelled PKM2 and Histone H3 further validate the interaction between the two proteins. Interestingly we observe a decrease in the fluorescence intensity of labelled PKM2 when titrated with increasing concentration of Histone H3. With subsequent increase in the Histone H3 concentration in the PKM2 microenvironment the fluorescence is gradually quenched and a steady depreciation in the FITC fluorescence intensity is recorded which finally becomes stabilized and no further change in the intensity corresponding to higher concentrations of Histone H3 is observed 2 tetramer and two units H2A–H2B dimers onto the Widom 601 nucleosome positioning sequence DNA is critical for the nucleosome assembly. Microscale reconstitution by employing Salt gradient dialysis with 1:1.5 DNA-to-octamer ratio as previously determined and subsequent titrations by varying the molar ratio PKM2 envince that nucleosome reconstitution can be possibly altered by PKM2 interaction. As can be inferred from native PAGE electrophoresis at a 1:2.4 PKM2-to-octamer ratio PKM2 interacts with the nucleosome and forms a complex of higher molecular weight , Lane 2 observed . The DisExonuclease III possess the phosphomonoesterase activity and catalyzes the 3' exonucleolytic digestion of the duplex DNA. The activity is hindered by the DNA-histone interaction and at regulated low levels of ExoIII, digestion of the nucleosome monomers yield a compact core of 145 bp which defines the boundary of the nucleosome. On extensive ExoIII digestion the nucleosome core is further chopped to yield discrete bands that are integer multiples of 10 bases signifying sequential disruption of the DNA–histone contacts at every turn of the helix with the advance of ExoIII . We usedAs evident from the restriction digestion assay PKM2 interacts with nucleosome and reduces the accessibility to the complex. We were interested in exploring the possible outcomes of this finding and how it might contribute to altered gene expression. Chd7 is a member of the SNF2-protein superfamily and is reported to play a role in DDR (DNA Damage Repair). To cite as per the reports any aberrant expression or somatic mutations of Chd7 often in many cases may lead to cancer. Chd7 exhibits an intrinsic ATP-dependent nucleosome remodeling activity by sliding the histone octamer from one end of the DNA fragment to the centered position . This suSince DNA exist as chromatin in the nucleus and DNA-directed processes have majorly evolved with the chromatin  we furthTo dissect the impact of PKM2 on nucleosome positioning in a chromatin array and investigate if it could hinder Chd7 driven remodeling we used 34-mer nucleosome array for our study. The pJ201 (34 × 601) construct has restriction site for HhaI at the dyad position. The 34-mer reconstituted chromatin when digested by HhaI do not show any digested fingerprint , lane1 sHere, we report that PKM2 potentially acts as a modulator of chromatin remodeling activity by directly interacting with chromatin and thus contribute to oncogenesis. We highlight a rather unaddressed role of PKM2 that may be pivotal in exacerbating genomic integrity and trigger cell proliferation. The metabolic functions of PKM2 as key catalytic enzyme of glycolytic cycle and regulator of the energy flux in a cell is well established. However, the non-metabolic functions of PKM2 and its various aspects need to be discerned. The oligomeric switch as a consequence of oncogenic signal mediated post-translational modification subsequently facilitate nuclear translocation. Using fluorescence labelled NCP (nucleosome core particle) and 34-mer chromatin array we have demonstrated direct interaction of PKM2 with the nucleosome complex. We examined the possible interaction of PKM2 through nucleosome reconstitution. Gel mobility shift assay result of the reconstituted complex validate the potential binding of PKM2 to nucleosome. It can be inferred that PKM2 may have a role to play in nucleosome assembly and thereby dictate a likely control over gene expression. However the implications of this interaction is immense and needs to be further explored. Intuitively the nature of this interaction was further verified by establishing an evident association of PKM2 with Histone H3. Employing native PAGE gel electrophoresis we confirmed that PKM2 binds with Histone H3 and there is a possible interaction with Histone H4 (as seen in the native PAGE) which was although not as promising as Histone H3. The possibility of interaction with Histone H4 is a subject of study and further interpretation.Our finding was consistent as confirmed by SPR and fluorescence spectrometry experiments. This study was further extended to the validation of 34-mer chromatin array emphasizing the veracity of this interaction to the chromatin level. The results further prove the possibility of wider involvement of PKM2 in the chromatin structural organization and the downstream implication it may exert on gene regulation.Nucleosome restrict the accessibility of transcription factors and impede transcription and other chromatin transactions. This can be attributed to the 14 contact points between DNA and the histone octamer. However, protein complexes like chromatin remodelers can dynamically reposition and slide nucleosome to alter gene expression suggesting multi-layered regulation To dissect the impact of PKM2 on nucleosome accessibility we performed Exonuclease III digestion of PKM2-Nucleosome complex. Constrained digestion of nucleosome at low concentration of enzyme yield a nucleosome core with 145 bp wrapped around it which is further cleaved with the increase in enzyme units. However, in case of PKM2 bound nucleosome it is remarkable to observe from the lack of cleaved bands on the denaturing gel that digestion cannot proceed and the enzyme activity is significantly compromised. Results suggest that PKM2 probably impose a barrier to the remodeling enzymes, insulate nucleosome and obstruct access.As previously noted, in presence of nucleosome, transcription machinery face a roadblock and the highly coordinated process of gene regulation is impaired. Several chromatin remodelers relieve this transcriptional blockade by facilitating nucleosome sliding. CHD family of remodelers harness the energy generated from ATP hydrolysis to alter the chromatin structure. The interplay between the repair machinery and the remodeler is crucial to gain an access to DNA double-strand breaks (DSB) and prevent genomic instability . Chd7 isNucleosome mapping reactions with HhaI using 34-mer pre-assembled nucleosome array imply that PKM2 impair remodeling in chromatin without altering the structure. In contrast to chromatin digestion in the presence of the ΔChd7 which was on account of the exposed HhaI site at the dyad position we fail to observe any digestion in presence of PKM2. This was evidently on account of inhibition of nucleosome sliding.To summarize, we have shown that PKM2 can directly interact with the nucleosome through Histone H3 and obscure the chromatin from undergoing epigenetic modifications catalyzed by remodeling complexes. The basis of this prediction was evident from the suspended remodeling activity of Chd7 in the presence of PKM2 which can possibly lead to dysfunctional DDR machinery and genomic instability . As per In a cancer cell metabolic overhaul often commensurate with dysfunctional metabolic enzymes that synergistically impact the epigenetic blueprint and promote altered gene expression. In this research article we report a so far unaddressed role of PKM2 as a modulator of chromatin remodeling activity. Our study show that PKM2 can directly interact with nucleosome through Histone H3 and inhibit Chd7 mediated remodeling. At present non-metabolic functions of PKM2 is an intensely researched domain and its role in cancer progression and tumorigenesis is widely being acknowledged. Besides acting as a key nuclear kinase it is known to regulate the expression of several oncogenes. Here, we attempt to answer how PKM2 can directly alter the expression of genes by hindering nucleosome positioning and inhibiting remodeling. Exonuclease III digestion of Pyruvate Kinase M2- NCP complex suggest that PKM2 obscure nucleosome and create a roadblock for the Chd7 remodeler and thereby impede nucleosome sliding. Chd7 is implicated in a number of developmental disorder and is reported to play a role in cancer as well. As an important factor for DNA Damage Repair machinery any impairment in its functions can have detrimental implications. It can be inferred that PKM2 by negatively modulating Chd7 catalyzed chromatin remodeling may probably exacerbate genomic instability and in turn contribute to cancer. Through this study we aim to highlight the impact of PKM2 on chromatin architecture and its impact on the epigenetic landscape of the cell.S1 Fig(A) Interaction of Chd7 with PKM2 with increasing molar concentration of PKM2. (B) Interaction of PKM2 with Chd7 with increasing molar concentration of Chd7.(PDF)Click here for additional data file.


GST genes have been reported to be associated with increased susceptibility to cancer development and anticancer drug resistance. In this study, we investigated the association between genetic variants in GSTM1 and GSTP1 and patients’ clinicopathological parameters. The prognostic values of such associations were evaluated among bladder cancer patients.The glutathione S-transferases (GSTs) are a superfamily of phase II detoxifying enzymes that inactivates a wide variety of potential carcinogens through glutathione conjugation. Polymorphic changes in the GSTM1 and GSTP1 in bladder cancer patients was assessed using polymerase chain reaction followed by DNA sequencing. Overall survival was estimated using the Kaplan-Meier method and multiple logistic regression and correlation analysis were performed.Genotyping of GSTM1 null genotype was significantly associated with poor overall survival compared with the wild-type GSTM1 genotype. There was a trend towards better overall survival in patients with wild-type GSTP1 allele (AA) compared with GSTP1 (AG/GG) genotype. Interestingly, Kaplan-meier survival curve for GSTM1 null patients adjusted for sub-cohort with amplified HER2 gene showed poor survival compared with the GSTM1 null/ non-amplified HER2 gene. Also the same population when adjusted with HER2 protein expression, data showed poor survival for patients harboring GSTM1 null/high HER2 protein expression compared with low protein expression.The GSTM1 null genotype on bladder cancer patients’ outcome. Further investigations are required to delineate the underlying mechanisms of combined GSTM−/− and HER2 status in bladder cancer.This study focuses on the impact of  Bladder cancer is the 9th most common cancer and a leading cause of cancer-related death worldwide. It has been estimated that around 550,000 new bladder cancer cases and 199,922 deaths occurred in the year 2018 worldwide and these numbers are expected to double in the upcoming years . TherefoIt is well documented that occupational exposure to chemical carcinogens including aromatic amines and polycyclic aromatic hydrocarbons is associated with the risk of bladder cancer development , 4. KellProtecting against carcinogen-induced and chemotherapy-induced oxidative stress involves a series of event characterized by the activation of phase-II cellular detoxifying enzymes; Glutathione S-transferases (GSTs) or N-acetyltransferases (NATs) . GSTs enGSTM1 gene is located on chromosome 1p13.3 and the most common polymorphic variant of GSTM1 gene is the homozygous deletion (GSTM1 null genotype) characterized by abolished enzyme activity [GSTM1 and the risk of cancer, but the association remains controversial among different populations. Previous epidemiological studies showed an association between the homozygous deletion of GSTM1 and increased risk of lung, colorectal and head and neck cancers [GSTM1 null and the risk of several types of cancers [activity . Many st cancers –17. Howe cancers –21.GSTP1 polymorphism at codon 105 is an A to G substitution resulting in an amino acid switch from isoleucine to valine  and lowering the catalytic activity of GSTP1enzyme [GSTP1 Val105 genotype was shown to be associated with a variety of tumors, such as ovarian, breast, colon, lymphoma, and pancreas [GSTP1 variants modulate the risk of urinary bladder cancer has also been investigated [GSTP1 gene polymorphisms and the risk of bladder cancer: while a number of studies identified an obvious association between GSTP1 polymorphisms Ile105Val and bladder carcinoma risk [GSTP1 Ile105Val polymorphism and bladder cancer [GSTP1 is encoded by a single gene located on chromosome 11 . The comP1enzyme . The decpancreas . The hypstigated , 25. Howoma risk –28, other cancer , 30.ERBB2 amplification and signalling [HER2 may be involved in cancer susceptibility and clinical management of cancer patients. In the present study, we aim to investigate the prognostic value of GSTM1 and GSTP1 genetic polymorphisms in patients with bladder cancer and evaluate their association with patients’ clinicopathological parameters. We also attempted to evaluate the clinical significance of HER2 status in cases confirmed to have GSTM1/ GSTP1 variants with bladder cancer prognosis.HER2 is a trans-membrane glycoprotein receptor tyrosine kinase of the epidermal growth factor receptor family EGFR/ErbB. It plays an important role in the development and progression of many tumor types including breast, gastric and bladder cancers . Recent gnalling . HER2 isgnalling , 33, 34.Formalin-fixed paraffin-embedded (FFPE) tissue samples were obtained from histologically confirmed bladder cancer patients who underwent bladder resection between 2005 and 2012 at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia. The study group consists of 93 patients; only specimens containing more than 80% cellular composition were used in the analysis. All patients have not been subjected to any chemotherapy or radiotherapy prior to sample collection. Clinical and pathological data including age, gender, tumor grade, tumor stage, lymph node, vascular invasion, metastasis, and survival were gathered from patients’ medical records and summarized in Table Genomic DNA was extracted from FFPE tissue samples using QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s instructions. Purified DNA was eluted in 50 μl elution buffer and stored at − 80 °C until use. Purity and concentration of eluted DNA was analyzed using a spectrophotometer system .GSTM1 (present/null) and GSTP1 Ile105Val polymorphisms was performed as described previously [GSTM1 and GSTP1 oligonucleotide primers were purchased from MWG-Biotech  to amplify the GSTM1 fragments, , GSTP1  PCR was performed on a Thermal Cycler 480 apparatus . Thermo cycler parameters included: an initial denaturation at 94 °C/ 15 min; followed by 35 cycles of denaturation at 94 °C/ 1 min, annealing at 57 °C /1 min, and extension at 74 °C/ 1 min; and a final extension at 72 °C/10 min. Confirmation of PCR products were examined by 2% agarose gel electrophoresis and visualized using a Syngene UV transilluminator.Genotyping for the detection of eviously . GenotypGSTP1 PCR products, sequencing kit  was used according to the manufacturer’s instructions using Genetic analyzer 3500 . The resulting sequence data was analyzed using Applied Biosystems sequence analysis software (v 5.4). GSTP1 genotypes were determined as wild type Ile/Ile (AA), heterozygous type Ile/Val (AG) or homozygous variant type Val/Val (GG) as shown in Fig. GSTM1, the PCR products were separated on a 2% agarose gel and determined as null/ present genotypes.To sequence the amplified HER2 immunostaining was undertaken earlier . The expGST polymorphisms with aggressiveness of bladder cancer. Odds Ratios (OR) and their 95% Confidence Intervals (95% CI) were used to calculate the results. The wild type of all genotypes was used as the reference group. Interactions between GSTM1 and GSTP1 polymorphisms and aggressiveness bladder cancer phenotypes were analyzed using Spearman correlation analysis. In all tests, the values p ≤ 0.05 were considered as statistically significant.Statistical data analysis was performed using SPSS . Appropriate, Chi-square test and Fisher’s exact test were used to establish any significant differences in polymorphism incidences between bladder cancer cases. Multivariate Cox regression model were used to evaluate the prognostic significance of GSTs genes, HER2 and other clinicopathological factors. Cumulative survival probabilities were estimated using the Kaplan-Meier method, with log-rank comparison test. Multiple logistic regression analysis was performed to assess the association between In the current study, 93 patients with urinary bladder carcinoma were genotyped for two polymorphisms in two important genes of the glutathione-s-transferase family involved in xenobiotic metabolism. The distribution of the clinicopathological characteristics of the bladder cancer patients is presented in Table GSTM1 and GSTP1 to the susceptibility of bladder cancer . Our data revealed that a total of 44 bladder cancer patients out of 93 (47.31%) had a GSTM1-deleted genotype (−/−). GSTM1 specific bands showing on agarose gel electrophoresis was seen in 45 out of 93 patients (48.38%). No further investigations were carried out to discriminate between heterozygous deletion (+/−) and wild-type (+/+) GSTM1 variants hence both heterozygous deletion and wild-type variants are considered GSTM1 present , whereas the lower percentage was with GSTM1 null and the GSTP1 wild allele 14 (15.05%) shown in Fig. GSTs different groups.A higher frequency within our cohort was found between those carrying GSTM1 null genotype was associated with poor overall survival in comparison to GSTM1 present genotype, log rank p = 0.038 . GSTP1 AG carriers had the worst overall survival compared to GSTP1 AA or GG genotypes carriers . When merging GSTM1 survival and GSTP1 polymorphisms .Kaplan-Meier curve showed that HER2) gene amplification, after breast and gastric cancers, and also demonstrates frequent overexpression of HER2 protein [GSTM1 and GSTP1 polymorphisms in respect to HER2 status of the same cohort. HER2 protein expression and gene amplification data [GST genotypes and HER2 status, bright field double in situ hybridization (BDISH) and immunohistochemistry (IHC) data were used to analyze HER2 gene amplification and protein expression within the GSTP1/ GSTM1 analyzed cohort. Our data indicated no association between HER2 protein level and both GSTP1 (p = 0.07) and GSTM1 (p = 0.75) polymorphic status . Such a relationship was not established for amplified HER2 gene and GSTM1 null/present variants  showed a significant impact on patients’ overall survival. Figure GSTM1 null and amplified HER2 gene , though this was not the case with non-amplified HER2 patients  compared to GSTM1 null/ low HER2 protein expression counterpart  are members of a large gene family of cytosolic phase II xenobiotic metabolizing enzymes involved in catalyzing and detoxifying a variety of carcinogens including reactive electrophilic compounds [GST of carcinogen-detoxifying gene family as well as in NAT2 confer increased risk of bladder cancer [increased expression of GST family members, especially GSTP1 and GSTM1, was reported in several human solid tumors and is believed to confer resistance to various platinum-base chemotherapy drugs and metformin through regulation of many genes and molecular pathways [Globally, bladder cancer is a leading cause of mortality , 38. It ompounds . Membersompounds , 40. It r cancer . Moreovepathways , 42. MecGSTP1 and GSTM1 variants in a cohort of 93 bladder cancer patient from Saudi Arabia. We also evaluated the association between GSTP1 and GSTM1 gene polymorphisms with a set of clinical and pathological parameters as well as the prognostic value of both genes polymorphisms in bladder cancer patients.In our investigation we examined the frequency of GSTM1 and GSTP1 gene variants was represented in Table GSTM1 present and null is equally distributed in our cohort 48.38 and 47.31% respectively. This data is in agreement with previous report on the frequency of the GSTM1 null genotype in the Caucasian population [GSTM1 null genotype was 59.1% in patients with muscle invasive bladder cancer (MIBC) [GSTM1 null genotype varies significantly among populations from different ethnic groups [GSTP1 gene polymorphism when we considered patients holding at least one copy of the dominant allele, data indicated that the frequency of AA and AG genotypes were found to be significantly high in our study group with a combined ratio of 77.4% for both genotypes compared to the GG genotype (6.45%). The reported frequency of GSTP1 AA/AG genotypes is around 67% of the Iranian patients [GSTP1 AA/AG variants was observed in in the Caucasian population with bladder cancer [The frequency and distribution of pulation . In an ir (MIBC) . Nonethec groups . As for patients  and Indipatients . HoweverGSTP1 and GSTM1 genes and patients’ outcome. Our results indicated a significant association between the null GSTM1 genotype and poor overall survival among bladder cancer patients. The association between GSTs and poor survival was previously highlighted in many cancer types including bladder cancer [GSTP1 genotypes, our data show trend for better survival for patients with the wild allele homozygote AA in comparison to heterozygote AG and variant allele homozygote GG genotypes or to GG/AG combined though data are not significant. When GSTP1 GG/AG and GSTM1 null genotypes were present together, poor overall survival increased in comparison to GSTP1 alone.We next sought to evaluate the association between polymorphism of the r cancer –50. As fGSTs leads to reduced detoxification potential which may result in increased DNA adduct levels in the target tissues and eventual mutations in the driver genes leading carcinogenesis. Therefore, the association of GSTP1/ GSTM1 variants with highly malignant disease and poor prognosis in cancer patients was suggested [The accumulating data suggested that genetic polymorphism of uggested .GSTM1 were at high risk of developing bladder cancer [GSTM1 null and other cancers such as breast [GSTM1 null distribution in bladder cancer patients than in healthy individuals [GSTM1 in our cohort did not show any significant difference in comparison to the wild-type allele which may indicate that the null genotype is not the only factor in determining the increased risk and aggressiveness of bladder cancer but is certainly one of many combined genetic factors that contribute to the pathogenesis of the disease. To-Figueras et al. suggested a relation between GSTM1 null genotype and p53 mutation in increasing the risk of lung cancer susceptibility among smokers [GSTP1 Ile105Val polymorphism were characterized by frequent mutations in the tumor suppressor gene p53 and high-grade/ high stage tumors in bladder cancer [p53 mutation (data not published) which may suggest the involvement of p53 mutation in association with GSTP1 in the risk of bladder cancer development and drug resistance. This suggestion is valid knowing that GSTP1 gene contains a functional canonical p53 binding motif and the capacity of p53 to transcriptionally activate the human GSTP1 gene [Previous studies on patients from different ethnic origins revealed that individuals with the null r cancer , 51–54. s breast , lung [5s breast  and colos breast . Anwar e smokers . In an er cancer . In an iTP1 gene .GSTP1/GSTM1 variants and Human Epidermal growth factor Receptor 2 (HER2) gene/ protein status in bladder cancer patients. Our data indicated that patients with high HER2 protein expression/ gene amplification and null GSTM1 genotype had significant poor survival compared to patients with low HER2 expression and null GSTM1 genotype, suggesting that combining HER2 status with GSTM1 genotype may have a prognostic value for bladder cancer patients. The exact mechanism of the influence of GSTM1 and HER2 on bladder cancer is yet to be elucidated. Together, our data showed that GSTM1 gene deletion either alone or in combination with HER2 may serve as markers for bladder cancer prognosis.In the same context and for the first time we investigated the relationship between different GSTP1 Ile105Val genotype, GSTM1 genotype alone or in combination with HER2 status and patients’ clinicopathological features. This is consistent with previous published reports [GSTP1 Val/Val genotype and GSTM1/GSTT1 double null genotypes.We observed no association between the  reports , 58, and reports  who founGSTM1 null genotype is significantly associated with poor overall survival in urinary bladder cancer patients. Furthermore, combined GSTM1 deletion and amplified HER2 gene might be considered as the worse prognostic genotype combination in bladder cancer. To the best of our knowledge, this is the first study to investigate the association between GSTs genes polymorphisms and HER2 status in Saudi bladder cancer patients. One of the limitations of the current investigation is scarcity of the sample size and clinical data used for correlation analysis. Therefore, further analyses using larger sample size are needed to investigate the functional significance of combined GSTM1 deletion and HER2 on bladder cancer prognosis. Furthermore, larger epidemiological studies are needed to assess the relationship between these genes and response to therapies (chemotherapy and anti-HER2 therapy) which may support their use as potential predictive biomarkers for bladder cancer treatment.The present study revealed that Additional file 1: Figure S1. Histograms showed the frequency of expression patterns of HER2 protein receptors in 93 of bladder cancer by IHC.Additional file 2: Figure S2. Kaplan-Meier survival curves demonstrating the overall survival of GSTP1 adjusted with HER2 status. (A)GSTP1 genotypes with HER2 gene amplification. (B)GSTP1 genotypes with HER2 gene Non-amplification. (C)GSTP1 genotypes with HER2 Protein expression. (D)GSTP1 genotypes with No HER2 Protein expression.


Salmonella enterica serotype I 4,[5],12:i:- has been increasingly isolated from swine. However, its pathogenic potential is not well characterized. Analysis of swine cases confirmed a strong positive association between isolation of I 4,[5],12:i:- and lesions of enteric salmonellosis and suggested a similar pathogenic potential as that for Salmonella Typhimurium. Salmonella enterica serotype I 4,[5],12:i:- has emerged as a major public health threat in Europe  is a National Animal Health Laboratory Network accredited laboratory that receives >75,000 case submissions annually, of which most are derived from swine production systems located throughout the United States. Histopathologic analysis is performed by veterinary diagnostic pathologists on ≈10,000 cases from swine per year. The ISU-VDL laboratory information management system provided the initial dataset for this analysis.Salmonella I 4,[5],12:i:- from samples submitted from pigs was associated with microscopic lesions consistent with enteric salmonellosis, we compared cases from which Salmonella I 4,[5],12:i:- was isolated with cases from which neither Salmonella I 4,[5],12:i:- nor Salmonella Typhimurium were isolated; these samples were collected during July 2016–December 2017. We also reviewed cases from which Salmonella Typhimurium or 1 of 3 Salmonella serogroup B serovars  were isolated to determine the potential comparative pathogenicity of Salmonella I 4,[5],12:i:-. All of these cases met the following qualifying criteria: animals were 3–13 weeks of age, a Salmonella serovar as outlined above was isolated by direct culture performed on enteric tissues, and histopathologic analysis was performed on the large intestine. To determine the association between the presence of Salmonella I 4,[5],12:i:- and lesions consistent with enteric salmonellosis, we also reviewed 40 additional cases that met the previously stated inclusion criteria but from which neither Salmonella I 4,[5],12:i:- nor Salmonella Typhimurium were isolated; we randomly selected these cases from an Excel  data file by using the RAND function.To determine whether isolation of Salmonella were routinely processed and reported (https://www.sas.com) to perform all analyses. We used the Pearson χ2 test and odds ratios to determine the association between isolation of Salmonella I 4,[5],12:i:- and pathologic diagnosis of enteric salmonellosis. We considered a p value <0.05 significant.Enteric samples submitted for isolation of reported . MicroscSalmonella I 4,[5],12:i:- from 138 porcine cases that met all of the qualifying criteria. We isolated Salmonella Typhimurium from 18 cases, and other potentially lesser pathogenic Salmonella serogroup B serovars, including Salmonella Derby, Agona, and Heidelberg, from 35 cases.We isolated Salmonella I 4,[5],12:i:-   of 40 cases  . We conf<0.0001)  for 100 isolated  Table 1.40 cases  Table 2.Salmonella was isolated from 6 of the 40 cases, 3 of which had lesions consistent with enteric salmonellosis. We confirmed compatible histologic lesions of salmonellosis in 17 (94%) of 18 cases in which Salmonella Typhimurium was isolated and 11 (31%) of 35 cases in which another serogroup B Salmonella was isolated. Other agents of enteric disease that were concurrently detected in some cases included rotaviruses, coronaviruses, coccidians, and hemolytic Escherichia coli. However, none of these agents caused lesions consistent with those used to define salmonellosis in this report  was isolated than for cases from which Salmonella Typhimurium (94%) was isolated. Based on these data, we believe that Salmonella I 4,[5],12:i:- is a likely cause of enteric salmonellosis that has a similar or perhaps slightly lower pathogenic potential in swine than Salmonella Typhimurium. Pathogenicity studies are needed to further characterize the pathogenic potential and fitness of Salmonella I 4,[5],12:i:- compared with that of Salmonella Typhimurium in swine.Although increased isolation of Salmonella I 4,[5],12:i:- has evolutionary advantages that have resulted in its predominance as one of the most common Salmonella serotypes responsible for swine enteric salmonellosis. Accordingly, it is essential to determine the putative attributes that facilitate its rapid spread and ecologic success. Specifically, antimicrobial drug resistance genes and genes that encode resistance to heavy metal micronutrients should be evaluated, given their current and common use in US swine production.We suspect that Salmonella enterica I 4,[5],12:i:- associated with lesions typical of swine enteric salmonellosis.Additional information on


Xenopus embryos causes a loss of neural crest progenitors, a phenotype that is rescued by expression of Lrp6 or β-catenin. Dkk2 overexpression expands the neural crest territory in a pattern reminiscent of Wnt8, Lrp6 and β-catenin gain-of-function phenotypes. Mechanistically, we show that Dkk2 mediates its neural crest-inducing activity through Lrp6 and β-catenin, however unlike Wnt8, in a GSK3β independent manner. These findings suggest that Wnt8 and Dkk2 converge on β-catenin using distinct transduction pathways both independently required to activate Wnt/β-catenin signaling and induce neural crest cells.Neural crest progenitors are specified through the modulation of several signaling pathways, among which the activation of Wnt/β-catenin signaling by Wnt8 is especially critical. Glycoproteins of the Dickkopf (Dkk) family are important modulators of Wnt signaling acting primarily as Wnt antagonists. Here we report that Dkk2 is required for neural crest specification functioning as a positive regulator of Wnt/β-catenin signaling. Dkk2 depletion in  The neural crest is a population of cells unique to the vertebrate embryo. They are induced at the neural plate border during gastrulation, and around the time of neural tube closure, leave the neuroepithelium to produce a diverse array of cell types, contributing to multiple tissues, including the heart, the peripheral nervous system, and the craniofacial skeleton.Neural crest cells are generated through a sequence of events orchestrated by the modulation of several signaling pathways and the activation of a complex network of transcription factors . A largeXenopus embryos and promote head formation (Xenopus embryos (The Dickkopf (Dkk) family of secreted glycoproteins acts primarily as negative modulators of Wnt signaling, this is especially true for Dkk1 and Dkk4 . Dkks inormation . Dkk1 in embryos . Similar embryos . During  embryos . The rol embryos , howeverHere we show that Dkk2 knockdown prevents neural crest formation in vivo and in neuralized animal cap explants injected with Wnt8. Furthermore, Dkk2 gain-of-function increases the neural crest progenitor pool, reminiscent of Wnt8, Lrp6 and β-catenin gain-of-function phenotypes. We demonstrate that Dkk2 mediates its neural crest-inducing activity by activation Wnt/β-catenin signaling in a GSK3β independent manner. We propose that during neural crest formation, Lrp6 mediates two independent signaling events triggered by Wnt8 and Dkk2 converging on β-catenin to specify the neural crest.dkk2 mRNA . A Dkk2MO was designed to specifically interfere with translation of kk2 mRNA . The spees of MO . Unilate embryos . Concomited side . To confjunction , resultijunction . The phexpansion . snai2 ehenotype . Later iype  was significantly repressed in Dkk2-depleted (Dkk2SMO-injected) animal cap explants, while co-injection of a control MO (CoMO) had no effect on snai2 induction  by BMP inhibition or mesoderm (bra) by fibroblast growth factor 8b (FGF8b) in these explants , consistent with a potential role in neural crest induction. This expression increases over time to reach a maximum around stage 20 and then progressively declines  results in axis duplication, while Wnt activation post-MBT expands neural crest progenitors . For thefficient . This redkk2 mRNA (500 pg) or plasmid DNA (25 pg) in one cell at the 2 cell stage. Dkk2 overexpression in both cases resulted in a lateral expansion of snai2 expression domain in the vast majority of the embryos . This phenotype is very reminiscent of Wnt8, Lrp6 and β-catenin gain-of-function phenotypes , Dr. Tatiana Piotrowski  and Dr. Sergei Sokol , respectively. A flagged version of Xenopus Dkk2 was generated by adding a Flag tag (DYKDDDDK) at the C-terminal using the following primers F: GAATTCGCCACCGAGATGAACATGGTTTTGCTGGGGA; R: ctcgagTTACTTGTCGTCGTCGTCCTTGTAGTCTATTTTCTGGCAAATG. The PCR product was cloned into pCS2+ (pCS2+ Dkk2 Flag). Control (CoMO), Dkk2 , Lrp6 , 200 pg (wnt8) or 50 pg  was injected per embryo. MOs, mRNAs and plasmid DNA were injected in one blastomere at the 2 cell stage (NF stage 2) and embryos were analyzed by in situ hybridization at the neurula stage (NF stage 14–17). To identify the injected side, 500 pg of β-galactosidase mRNA was coinjected as a lineage tracer. Only embryos with co-localized expression of the lineage tracer with the cell type marker were considered for analysis. Control and injected embryos were treated in the dark with 10 μM of GSK3 inhibitor  at the gastrula stage (NF stage 11/11.5), and collected at NF stage 14–17. For the axis duplication assay, embryos were injected in the equatorial region in both ventral blastomeres at 4 cell stage (NF stage 3) and analyzed at NF stage 32. For animal cap explant experiments, both blastomeres at the 2 cell stage (NF stage 2) were injected in the animal pole region. Explants were dissected at the blastula stage (NF stage 9) and cultured for 8 hr in NAM 0.5X. In rescue experiments, the injections of mRNAs/plasmid DNA and MOs were performed sequentially. For whole embryo injections and animal cap explant assays each experiment was performed on at least three independent batches of embryos.8 25 pg; , noggin n 10 pg; , dkk2  to visualize the lineage tracer  on the injected side and processed for in situ hybridization. Antisense digoxygenin-labeled probes  were synthesized using template cDNA encoding snai1 , snai2 , containing HaltTM Protease Inhibitor Cocktail . After two consecutive centrifugations to eliminate lipids, the lysate was concentrated on an Amicon Ultra Centrifugal Filter , 5 μl of the concentrated lysate was resolved on a 10% NuPAGE Bis-Tris gel and transferred onto a PVDF membrane using the iBlot system (Invitrogen). Blots were subsequently incubated overnight with one of the following primary antibodies: monoclonal anti-Flag M2 antibody  and anti α-tubulin antibody . The blots were then washed and incubated with anti-mouse IgG coupled to horseradish peroxidase . Peroxidase activity was detected with the Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and imaged on a ChemiDoc MP Biorad gel documentation system . Membranes were stripped using Restore Western Blot Stripping Buffer (ThermoFisher Scientific) according to the manufacturer recommendations.Embryos were injected at the 4 cell stage with 10 ng of Power SYBR Green PCR Master Mix  on a QuantStudio 3 Real-Time PCR System  with the following primer sets: bra , sox2 , snai2 , luciferase  and odc . The PCR conditions were as follows: denaturation 95°C (15 s), annealing and extension at 60°C (1 min) for 40 cycles.Total RNAs were extracted from embryos or animal cap explants using the RNeasy micro RNA isolation kit . The RNA samples were digested with RNase-free DNase I before RT-PCR. The amount of RNA isolated was quantified by measuring the optical density using a Nanodrop spectrophotometer . Approximately 250 ng of total RNAs from animal caps was reverse transcribed using the Superscript VILO cDNA Synthesis Kit  and 2 μl of 1:1000 dilution of the synthesized cDNA was amplified using  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior and Reviewing Editor.Thank you for submitting your article "Dkk2 promotes neural crest specification by activating Wnt/β-catenin signaling in a GSK3β independent manner" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This is an interesting paper that addresses the role of Dkk2 in neural crest formation, showing that it is required for the expression of both Snai2 and Sox10. Loss of Dkk2 can be rescued by β-catenin and Lrp6, as well as Wnt8. Over-expression of Dkk2 leads to an expansion of Snai2. While a GSK3β inhibitor expands Snai2 expression anteriorly, the prospective neural crest territory remains sensitive to Dkk2 MO, suggesting it functions in a GSK3β independent manner.Essential revisions:1) It is critical to provide better expression profile of Dkk2 and it is suggested to move the images to main figures. The published expression pattern for Dkk2 does not show expression in the neural fold or neural crest. Furthermore the RNA seq data available on Xenbase shows a very low expression of Dkk2 during neural crest induction. It is even more striking when the expression data is compared to the Wnt partner namely Wnt8 and Lrp6. It is only in the Discussion that the authors refer to expression data presented in the supplemental material.2) The expression data suggest that Dkk2 is an extremely potent activator of the pathway that despite its minute expression it is essential for proper Wnt signaling. This is not entirely consistent with the fact that loss of Dkk2 can be rescued by overexpression of Wnt8, Lrp6 or β-catenin since all of these mRNA are expressed at 10 to 100 folds excess when compared to Dkk2. Does the Dkk2 protein possess some particular property such as extreme stability or extreme binding affinity to the co-receptors? Following a tagged Dkk2 protein in the embryo (by blot and IF) could provide some clues. Dose response in the embryo or animal cap could be used to determine the effectiveness of Dkk2 compare to Dkk1. What is the minimal dose at which Dkk2 expression can expend the neural crest?3) While the results strongly suggest that Dkk2 potentiates WNT signaling towards neural crest formation, the mechanism of action is unresolved. The authors should assess whether Dkk2 triggers expression of known direct targets of WNT/β-cat pathway, or non-canonical WNT pathways. They should also test mesoderm markers to rule out possible secondary effects.4) The authors should test a broader repertoire of neural crest markers (in addition to Sox10 and Snai2). In addition, analysis of early stages (st. 13) is required to test effects on early NC specification markers.snai2 and sox10 expression is not conclusive evidence for loss of neural crest cells. The authors should examine non-neural ectoderm markers. They should also let embryos grow to later stages, as loss of neural crest should result in complete unilateral absence of craniofacial cartilage and cranial peripheral nervous system. If so, is this rescued by β-catenin and Lrp6 injections in Dkk2-depleted embryos?5) In the absence of lineage tracing, the loss of 6) While the loss of function phenotype is compelling, the in vitro translation does not allow one to test the effective amount to inject in an embryo. Blocking the translation of a tagged Dkk2 mRNA would help define that dose. In addition, rescuing Dkk2 MO with an insensitive mRNA would confirm the specificity of the phenotype. The splice morpholino need to be more carefully quantified. Using realtime qPCR what is the percentage of properly spliced mRNA with various doses of the MO?Other suggested revisions:1) The authors should references previous studies that provide molecular insights into how Dkk2 activates Wnt/β-cat. For example, Li et al. (2002) suggested that it is the C2 domain of Dkk2 that is critical in its WNT activating functions, and further provide evidence suggesting that Dkk2 WNT inductive role operates through DVL and GSK3 independent mechanisms. Mao and Niehrs (2003) suggested that Krm2 is responsible for the WNT-antagonistic effect of Dkk2, and that in its absence, Dkk2 operates as a WNT activator.2) It would be nice to examine biochemical interactions between Dkk2 function with Lrp5/6, Ror, Krm, during neural crest development.3) The authors present a model where Dkk2 and Wnt8 appear to function in parallel pathways that converge on β-catenin. What's not clear is why eliminating Dkk2 prevents Wnt8 from its normal activation of β-catenin and neural crest cell formation and also why vice versa, Dkk2 can't activate β-catenin and specify neural crest cells in the absence of Wnt8. Do Dkk2 and Wnt8 somehow impact each other's function?4) Better quantification would be appropriate.5) On BIO experiments, it is puzzling that the Dkk1+BIO does not display expected anterior expansion of Snai2 on the control side, unlike the Dkk2+Bio…?6) This statement in the Discussion is debatable:"While overexpression studies have previously shown that Dkk2 can function as an activator of Wnt/β-catenin signaling when co-expressed with Fzd8 or Lrp6 , this is the first study that provides evidence for its positive role in a Wnt/β-catenin regulated developmental process."Xenopus arguing for such a relationship and maintenance of pluripotency in neural crest cells.7) It should be noted that the expanded domain of Sox2 does not correlate with neural crest cell generation. This contrasts with recent studies in 8) Given the proposed role of Dkk2 one would expect that it could rescue the overexpression of GSK3 to complement the BIO- inhibitor data. Has this been tested? Essential revisions:1) It is critical to provide better expression profile of Dkk2 and it is suggested to move the images to main figures. The published expression pattern for Dkk2 does not show expression in the neural fold or neural crest. Furthermore the RNA seq data available on Xenbase shows a very low expression of Dkk2 during neural crest induction. It is even more striking when the expression data is compared to the Wnt partner namely Wnt8 and Lrp6. It is only in the Discussion that the authors refer to expression data presented in the supplemental material.dkk2 expression data as a main figure of the manuscript . Dkk2 is broadly expressed during embryogenesis and is not distinctly enriched in the neural crest territory. It is likely that the regionalized expression of other components of the pathway, such as Fzd receptors and Wnt ligands, which are expressed at higher levels, provides the spatiotemporal cues to achieve a localized response. The expression pattern of dkk2 is presented and discussed in the Results section of the manuscript .Although the Xenbase RNA-seq data indicates that Wnt8 and Lrp6 are expressed at higher levels than Dkk2 it is difficult to correlate this information to the amount of protein present at the time of neural crest induction. As suggested we have moved the 2) The expression data suggest that Dkk2 is an extremely potent activator of the pathway that despite its minute expression it is essential for proper Wnt signaling. This is not entirely consistent with the fact that loss of Dkk2 can be rescued by overexpression of Wnt8, Lrp6 or β-catenin since all of these mRNA are expressed at 10 to 100 folds excess when compared to Dkk2. Does the Dkk2 protein possess some particular property such as extreme stability or extreme binding affinity to the co-receptors? Following a tagged Dkk2 protein in the embryo (by blot and IF) could provide some clues. Dose response in the embryo or animal cap could be used to determine the effectiveness of Dkk2 compare to Dkk1. What is the minimal dose at which Dkk2 expression can expend the neural crest?Regarding the mRNA expression levels of Dkk2, Wnt8 and Lrp6 see response to point #1. As suggested we have performed Western blot analyses to evaluate the accumulation overtime of a Flag-tagged version of HuDkk2 (see 3) While the results strongly suggest that Dkk2 potentiates WNT signaling towards neural crest formation, the mechanism of action is unresolved. The authors should assess whether Dkk2 triggers expression of known direct targets of WNT/β-cat pathway, or non-canonical WNT pathways. They should also test mesoderm markers to rule out possible secondary effects.snai2, dkk2 overexpression did not significantly upregulate other known direct targets of Wnt/β-catenin signaling such as pax3, znf703 or kremen2. This result suggests that dkk2 alone is a poor activator of Wnt/β-catenin signaling, consistent with the view that it is acting in concert with a Wnt ligand to activate the pathway. The expression of the mesoderm marker myoD was unaffected in dkk2-depleted embryos ruling out any possible secondary effects .We have performed the suggested experiment and found that with the exception of 4) The authors should test a broader repertoire of neural crest markers (in addition to Sox10 and Snai2). In addition, analysis of early stages (st. 13) is required to test effects on early NC specification markers.pax3, sox8, snai1, twist1 and sox9 . We found that early neural crest markers  such as pax3, sox8 and snai1 were only mildly affected – their expression levels were unchanged but their expression domain appeared to be shifted laterally – while other markers  such as twist1, and sox9 to a lesser extent, were downregulated, consistent with the phenotype observed for sox10 and snai2. This result suggests that dkk2 may not participate in neural plate border specification but rather in the neural crest progenitors formation and/or maintenance.We have performed additional experiments to increase the repertoire of neural crest genes analyzed, which now include 5) In the absence of lineage tracing, the loss of snai2 and sox10 expression is not conclusive evidence for loss of neural crest cells. The authors should examine non-neural ectoderm markers. They should also let embryos grow to later stages, as loss of neural crest should result in complete unilateral absence of craniofacial cartilage and cranial peripheral nervous system. If so, is this rescued by β-catenin and Lrp6 injections in Dkk2-depleted embryos?sox2 expression domain .We have analyzed the expression of keratin, a gene expressed in the non-neural ectoderm, and show that its overall expression is reduced/shifted laterally consistent with the expansion of We also show that neural crest-derived craniofacial cartilages in addition to pigment cells are affected upon Dkk2 depletion , pointing to a loss of neural crest lineages in these embryos.6) While the loss of function phenotype is compelling, the in vitro translation does not allow one to test the effective amount to inject in an embryo. Blocking the translation of a tagged Dkk2 mRNA would help define that dose. In addition, rescuing Dkk2 MO with an insensitive mRNA would confirm the specificity of the phenotype. The splice morpholino need to be more carefully quantified. Using realtime qPCR what is the percentage of properly spliced mRNA with various doses of the MO?snai2 and sox10 expression, associated with an expansion of sox2 expression domain. This phenotype was observed in both whole embryos and animal cap explants. We also used a control MO, which did not affect the expression of these genes. Importantly, this phenotype was efficiently rescued by expression of Lrp6 and β-catenin, two components of Wnt/β-catenin signaling pathway. We believe that this combination of approaches makes a compelling case for a specific requirement of dkk2 in neural crest formation. It is difficult to estimate the efficacy of a translation blocking MO without a good antibody, and we have not been able to identify one that could detect endogenous dkk2 in Xenopus. As suggested, we have performed qRT-PCR to evaluate the proportion of spliced versus unspliced transcripts generated upon injection of increasing doses of MO. These data are presented in Figure 1—figure supplement 1.In this study, we used two MOs interfering with Dkk2 mRNA translation and splicing, respectively. Both gave a similar phenotype resulting in the inhibition of Other suggested revisions:1) The authors should references previous studies that provide molecular insights into how Dkk2 activates Wnt/β-cat. For example, Li et al. (2002) suggested that it is the C2 domain of Dkk2 that is critical in its WNT activating functions, and further provide evidence suggesting that Dkk2 WNT inductive role operates through DVL and GSK3 independent mechanisms. Mao and Niehrs (2003) suggested that Krm2 is responsible for the WNT-antagonistic effect of Dkk2, and that in its absence, Dkk2 operates as a WNT activator.Both studies are now referenced in the text .2) It would be nice to examine biochemical interactions between Dkk2 function with Lrp5/6, Ror, Krm, during neural crest development.We agree with the reviewer, and this is something we are planning to explore in order to elucidate the precise mechanism of Dkk2-mediated signaling during neural crest specification, however we feel that this is beyond the scope of the current study.3) The authors present a model where Dkk2 and Wnt8 appear to function in parallel pathways that converge on β-catenin. What's not clear is why eliminating Dkk2 prevents Wnt8 from its normal activation of β-catenin and neural crest cell formation and also why vice versa, Dkk2 can't activate β-catenin and specify neural crest cells in the absence of Wnt8. Do Dkk2 and Wnt8 somehow impact each other's function?Our model predicts that signaling by Dkk2 and Wnt8 are both independently required to induce neural crest cells, this is specifically supported by the BIO experiment. Further work will address the mechanisms underlying this dual requirement during neural crest formation.4) Better quantification would be appropriate.For all injection experiments we provide a rigorous quantification of the phenotypes, which are summarized in the form of graphs for all figures, supplemented with numerical data provided as “source data” files.5) On BIO experiments, it is puzzling that the Dkk1+BIO does not display expected anterior expansion of Snai2 on the control side, unlike the Dkk2+Bio?We have included a more representative image for Dkk1+BIO .6) This statement in the Discussion is debatable:"While overexpression studies have previously shown that Dkk2 can function as an activator of Wnt/β-catenin signaling when co-expressed with Fzd8 or Lrp6 , this is the first study that provides evidence for its positive role in a Wnt/β-catenin regulated developmental process."We have revised this statement .7) It should be noted that the expanded domain of Sox2 does not correlate with neural crest cell generation. This contrasts with recent studies in Xenopus arguing for such a relationship and maintenance of pluripotency in neural crest cells.sox2 expression does not behave in a way that is compatible with the model of pluripotency retention in the neural crest lineage – a model that is still up for debate. At the neurula stage, we consider sox2 as a marker for neural progenitors and not as much an indicator of pluripotency.We agree with the reviewer, 8) Given the proposed role of Dkk2 one would expect that it could rescue the overexpression of GSK3 to complement the BIO- inhibitor data. Has this been tested?dkk2 and wnt8 are both independently necessary to induce neural crest we did not expect that overexpressing dkk2 would be sufficient to restore neural crest formation in embryos injected with GSK3. We have performed the suggested experiment and found that Dkk2 expression was indeed unable to rescue neural crest formation in embryo overexpressing GSK3 (see Because our model predicts that signaling by GSK3 see .


COPD) is a multifactorial and heterogeneous disease that creates public health challenges worldwide. The underlying molecular mechanisms of COPD are not entirely clear. In this study, we aimed to identify the critical genes and potential molecular mechanisms of COPD by bioinformatic analysis. The gene expression profiles of lung tissues of COPD cases and healthy control subjects were obtained from the Gene Expression Omnibus. Differentially expressed genes were analyzed by integration with annotations from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, followed by construction of a protein‐protein interaction network and weighted gene coexpression analysis. We identified 139 differentially expressed genes associated with the progression of COPD, among which 14 Hub genes were identified and found to be enriched in certain categories, including immune and inflammatory response, response to lipopolysaccharide and receptor for advanced glycation end products binding; in addition, these Hub genes are involved in multiple signaling pathways, particularly hematopoietic cell lineage and cytokine‐cytokine receptor interaction. The 14 Hub genes were positively or negatively associated with COPD by wgcna analysis. The genes CX3CR1,PTGS2,FPR1,FPR2, S100A12,EGR1,CD163, S100A8 and S100A9 were identified to mediate inflammation and injury of the lung, and play critical roles in the pathogenesis of COPD. These findings improve our understanding of the underlying molecular mechanisms of COPD.Chronic obstructive pulmonary disease ( ARG1arginase 1BPbiological processCCcellular componentCOPDchronic obstructive pulmonary diseaseCScigarette smokeCX3CR1C‐X3‐C motif chemokine receptor 1DALYdisability‐adjusted life yearDEGdifferentially expressed geneEGR1early growth response 1FCfold changeFGGfibrinogen gamma chainFPR1formyl peptide receptor 1FPR2formyl peptide receptor 2GOGene OntologyGSgene significanceKEGGKyoto Encyclopedia of Genes and GenomesMFmolecular functionMMmodule membershipORM1orosomucoid 1PPBPproplatelet basic proteinPPIprotein‐protein interactionPTGS2prostaglandin‐endoperoxide synthase 2S100A12S100 calcium binding protein A12S100A8S100 calcium binding protein A8S100A9S100 calcium binding protein A9VCAM1vascular cell adhesion molecule 1wgcnaweighted gene coexpression network analysisYLLsyears of life lostChronic obstructive pulmonary disease (COPD), characterized by long‐term poorly reversible airway limitation and persistent respiratory symptoms, is a common and preventable disease As a large‐scale and efficient technique for acquiring genomic data, microarray‐based gene expression profiles have been widely used to seek new insights for biomarkers in many human diseases GSE27597 and GSE106986). DEGs were identified by r software  and subsequently analyzed using bioinformatic methods including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments and construction of protein‐protein interaction (PPI) and weighted gene coexpression analysis (wgcna) networks. We screened the DEGs for potential association with the development and progression of COPD. Our work may further the understanding of the potential molecular mechanisms of COPD.We selected two Gene Expression Omnibus microarray datasets on COPD . GSE27597 comprises the expression profile from 64 lung tissue samples from COPD cases and 16 samples from healthy donors GSE106986 includes molecular profiling of 19 lung tissue samples, containing 14 samples from COPD cases and 5 from smokers GSE27597 and GSE106986 were conducted on the Affymetrix Human Exon 1.0 ST GeneChip  and Agilent‐026652 Whole Human Genome Microarray 4x44K v2 , respectively.#AuthorQuery RepTwo gene expression datasets, r software and annotation packages. The two datasets were merged into one array dataset and then batch normalized using r packages (sva and limma 3.40.6). The DEGs between COPD cases and control subjects were identified using the limma package in r 3.60. A P‐value <0.05 after being adjusted by false discovery rate and |log2FC > 1, where FC represents fold change, were applied together as the cutoff for DEGs screening.The probe set IDs of two datasets were converted into gene symbols using the david online tool (https://david.ncifcrf.gov/) http://kobas.cbi.pku.edu.cn/) GO enrichment of the DEGs on the biological process (BP), molecular function (MF) and cellular component (CC) categories was performed using a https://string-db.org/) is frequently used for identifying the protein interactions cytoscape is an open source bioinformatic software platform used for integrating gene expression profiles and visualizing molecular interaction networks. cytoscape plugin cytoHubba provides multiple topological analysis methods on Hub genes, regulatory networks and drug targets for experimental biologists cytoscape version 3.6.1  with cytoHubba plugin according to the rank of connection degree (number) for each gene, which is represented by the different degrees of color (from red to yellow): the role of the gene is greater in the PPI network with the darker color of the gene STRING database (wgcna may be used for screening modules (clusters) of highly correlated genes and for calculating module membership (MM) measures, in which the module eigengene or an intramodular Hub gene is used to summarize such modules, and eigengene network methodology is used for relating modules to one another and to external sample traits wgcna package was used to identify coexpression modules for the merged and standardized array datasets (GSE27597 and GSE106986). In brief, first, a weighted adjacency matrix containing pairwise connection strengths was constructed based on the selected soft threshold power (β = 11) on the matrix of pairwise correlation coefficients. Then, the connectivity measure per gene was calculated by summing the connection strengths with other genes; modules were defined as branches of a hierarchical clustering tree by using a dissimilarity measure, and each module was assigned a color. Subsequently, module preservation r function was used to assess the module structure preservation. Finally, the module eigengene was used for summarizing the gene expression profiles of each module, and each module eigengene was regressed on case trait (COPD) and smoking status by using the linear model in the limma r package.GSE27597 and GSE106986 by the sva and limma packages, 139 DEGs were identified using the limma package  , proplatelet basic protein (PPBP), prostaglandin‐endoperoxide synthase 2 (PTGS2), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), vascular cell adhesion molecule 1 (VCAM1), S100 calcium binding protein A12 (S100A12), arginase 1 (ARG1), early growth response 1 (EGR1), CD163, fibrinogen gamma chain (FGG), orosomucoid 1 (ORM1), S100 calcium binding protein A8 (S100A8) and S100 calcium binding protein A9 (S100A9).The 139 DEGs were applied for construction of PPI networks using STRING. After removing the discrete and partially connected nodes, the PPI network data of DEGs were imported into the cytoHubba of wgcna network was first constructed using lung tissue expression data from cohorts GSE27597 and GSE106986, independent of COPD status and smoking status (ever/current smoking versus nonsmoking). A total of 2942 DEGs were selected (a corrected P < 0.05) and subsequently used to identify modules of coexpressed genes using a hierarchical clustering procedure. The corresponding branches of the resulting dynamic clustering tree and module are shown as colored bands underneath the cluster tree. We then merged the highly similar dynamic clustering modules into the merged dynamic modules (cut height = 0.25)  versus each MM. We found that the 14 Hub genes were also either positively or negatively associated with COPD , Red  and Grey  genes most significantly correlated with GS for COPD were also the important MM elements , an important mediator of inflammation, was shown to be involved in inflammation response and associated with COPD pathogenesis PTGS2 may protect smokers against the development of COPD FPR1 and FPR2 were reported to be involved in recruitment and activation of inflammatory cells induced by CS, and play important roles in lung inflammation, injury and the pathogenesis of COPD S100A8, S100A9, and S100A12 might induce neutrophil and monocyte chemotaxis, adhesion to fibrinogen and diapedesis, and neutrophil migration to inflammatory sites S100A8, S100A9 and S100A12 may be regulated by RAGE, which was shown to contribute to CS‐induced airway inflammation in COPD S100A8, S100A9 and S100A12 is important in the development of COPD.EGR1 is an autophagy regulator gene that plays important roles in cellular homeostasis, airway remodeling and control of inflammatory immune response; it is also a significant risk factor for susceptibility to COPD EGR1 may be induced by CS and involved in proinflammatory mechanisms that are likely associated with the development of COPD −/− mice were observed to resist CS‐induced autophagy, apoptosis and emphysema EGR1 in CS‐induced inflammatory immune response and COPD, and effective inhibition of EGR1 may attenuate airway remodeling and inflammation associated with the pathology of COPD.CD163, a carefully regulated component of the innate immune response to infection and a macrophage receptor for bacteria, was shown to play important roles in functional pulmonary defense elements and the inflammatory immune response of the respiratory system CD163 on lung alveolar macrophages may be implicated in the pathogenesis of COPD and poor lung function ARG1 was shown to contribute to asthma pathogenesis by inhibiting nitric oxide production, modulating fibrosis, regulating arginine metabolism and inhibiting T cell proliferation, and it involves the initiation of adaptive T helper 2 cell‐mediated allergic lung inflammation by regulating group 2 innate lymphoid cells ARG1 ablation in the lung may enhance peripheral lung function but have little effect on airway inflammation ARG1 in COPD needs to be studied in the future.ORM1 appears to function in regulating the activity of the immune system during the acute‐phase reaction and has been identified as an acute exacerbation of COPD‐specific immunomodulatory mediator PPBP serves as a potent neutrophil chemoattractant and activator, and its elevated expression in the bronchial mucosa might be involved in the pathogenesis of COPD VCAM1 was shown to express in endothelial cells of atopic asthma cases, but not COPD cases FGG was found to be involved in blast lung injury resistance via promoting tissue‐protective adenosine signaling CX3CR1, PTGS2, FPR1, FPR2, S100A12, EGR1, CD163, S100A8 and S100A9, potentially mediated inflammation and injury of the lung, and play critical roles in the pathogenesis of COPD. The roles of five Hub genes, including PPBP, ARG1, FGG, VCAM1 and ORM1, identified to be associated with COPD in this study need to be confirmed in the future. These findings could improve our understanding of the underlying molecular mechanisms of COPD and provide us with insights for drug target discovery for the disease.In conclusion, we have identified 139 candidate DEGs associated with the progression of COPD. The results from bioinformatic analysis are in agreement with those from previous cell and animal models and human studies. Our results showed that nine Hub genes, The authors declare no conflict of interest.XK, FY and QW conceived and designed the project. XH and YL acquired the data. XG and ZZ analyzed and interpreted the data. XH and YL wrote the paper.