C-reactive protein (CRP), is casually determined by DNMT3A and TET2-tuned promoter methylation status. CRP features a CpG-poor promoter with its CpG motifs located in binding sites of STAT3, C/EBP-β and NF-κB. These motifs are highly methylated at the resting state, but undergo STAT3- and NF-κB-dependent demethylation upon cytokine stimulation, leading to markedly enhanced recruitment of C/EBP-β that boosts CRP expression. Withdrawal of cytokines, by contrast, results in a rapid recovery of promoter methylation and termination of CRP induction. Further analysis suggests that reversible methylation also regulates the expression of highly inducible genes carrying CpG-poor promoters with APRs as representatives. Therefore, these CpG-poor promoters may evolve CpG-containing TF binding sites to harness dynamic methylation for prompt and reversible responses.Acute phase reactants (APRs) are secretory proteins exhibiting large expression changes in response to proinflammatory cytokines. Here we show that the expression pattern of a major human APR, that is  CRP induction have been thoroughly examined by reporter assays and truncation analysis. A region of ~220 bp in the proximal promoter of CRP that contains  binding sites for STAT3, NF-κB and C/EBP-β is identified to be sufficient to mediate CRP induction by IL-6 and IL-1β (Acute phase reactants (APRs) are liver-produced plasma proteins constituting an integral part of innate defense . They arnd IL-1β .CRP expression. Rather, the major −286C allele constitutes a CpG motif, at which DNA methylation frequently occurs; whereas the minor alleles of −286A/T disrupt the CpG motif and are associated with enhanced CRP expression. Beside the −286CpG, there are only four additional CpGs within the proximal promoter of CRP. Importantly, two of those CpGs are located at the binding sites of STAT3 and NF-κB/C/EBP-β  associated with plasma levels of CRP is located at 286 bp upstream the transcription start site . This SN/C/EBP-β . Given t/C/EBP-β  and cont/C/EBP-β , it is nausality . In the CRP expression, we first compared methylation levels of CRP promoter in different human tissues. CRP is expressed predominantly, if not solely, by the liver  of DNMT3A enhanced  and ten-eleven translocations (TETs), respectively . A causaenhanced , whereas3B cells . Knockou  and serum amyloid P component   . By contn of CRP . We thusCRP promoter  of CpGs termed CpG islands (CGIs) that are resistant to DNA methylation . The CpGCRP expression have been identified, how their actions are coordinated remains unclear. This study demonstrates a previously unrecognized, epigenetic mechanism wherein methylation status of CRP promoter responds to and further modifies the effects of distinct TFs. At the induced state, the pioneered binding of STAT3 to CRP promoter in hepatocytes drives a minor wave of induction, and further licenses the subsequent recruitment of NF-κB p50. The two TFs probably work together to tip the balance of TET2 and DNMT3A at CRP promoter, leading to its demethylation. As significant cell proliferation was not noted, the methylated cytosine might be eventually removed by base excision repair mechanism ; rabbit CRP ; human SAA ; human SAP ). Subsequent cloning and sequencing were performed as described  according to the manufacturer’s instructions. Samples were then amplified with SureStart Taq DNA Polymerase . The primer sequences used were: human escribed . The exp2) to Hep3B cells (G28888.Hep_3B2.1.7.3) in Cancer Cell Line Encyclopedia project is 0.92 (p<10−24). Total RNA was extracted with RNAiso Plus reagent . cDNA was synthesized from 1 μg total RNA using reverse transcriptase M-MLV , Oligo d(T)15 Primers , dNTP mixture , and recombinant ribonuclease inhibitor . Gene expression was determined with quantitative PCR (q-PCR) using SYBR Premix Ex Taq II  in a CFX96 Real-Time PCR Detection System . The gene expression levels were normalized to that of ACTB. The primer sequences used were: human CRP ; human SAA ; human SAP ; human ACTB .Human hepatocellular carcinoma cell line Hep3B were obtained from cell bank of Chinese Academy of Sciences . Hep3B cells were cultured in MEM media  containing 10% FBS . Cells were tested for mycoplasma and have been authenticated by Cel-ID  using RNGene expression profiles of Hep3B cells with or without IL-6 and IL-1β treatment for 24 hr were determined with RNA sequencing services provided by GENEWIZ . Transcriptome and methylome of C57BL/6 mouse liver tissues at the resting or turpentine-induced state were determined with RNA sequencing and whole genome bisulfite sequencing services (WGBS) provided by GENEWIZ. All RNA-seq and WGBS data have been deposited in GEO under accession code GSE146797.Where appropriate, Hep3B cells were treated with 10 ng/ml rhIL-6 , 1 ng/ml rhIL-1β , 5-Azacytidine  , RG108  , BAY11-7082  , S31-201   or Stattic  .DNMT1 ; DNMT3A ; DNMT3B ; TET1 ; TET2 ; TET3 . For overexpression, coding sequence of target genes were cloned into pcDNA3.1 vector . Vectors were transfected into Hep3B cells with ViaFect Transfection Reagent . 48 hr later, gene expression was determined with q-PCR.For knockdown, targeting sequences were synthesized (GENEWIZ), and cloned into pcDNA6.2-GW/EmGFP-miR vector . The targeting sequences were: DNMT3A ; TET2 . Gene expression was determined with q-PCR. In some experiments, single clones of DNMT3A knockout cells were further selected with puromycin .For Cas9-mediated knockout , targetiCRP promoter were: #1 5’-GGGGACTGTTGTGGGGTGGG-3’; #2 5’-GAAGCTCTGACACCTGCCCC-3’. The catalytic domains of DNMT3A and TET2 were cloned into pcDNA-dCas9-p300 Core vector . sgRNA vector and DNMT3A or TET2 vector were co-transfected into Hep3B cells. 48 hr later, gene expression was determined with q-PCR.For dCas9-mediated targeting , targetiCas9 . The targeting sequences for C/EBP-β were: #1 5’-GGGCGCCTGGGGGCCGCCAA-3’; #2 5’-GGCGGCGGCGGCGGCGGGGG-3’.To prepare Hep3B cells stably expressing Cas9 , pspax2,CRP (−533 ~ +103 bp) was cloned into PGL4.10 (luc2) vector . Hep3B cells were transfected with 1.5 μg of PGL4.10 CRP reporter vector and 0.075 μg of phRL-TK  using X-tremeGENE 9 DNA Transfection Reagent . After 48 hr of transfection, luciferase activities were measured using Dual-Luciferase Reporter Assay System  on a Synergy HTX Multi-Mode Microplate Reader . Activities of firefly luciferase were normalized with that of co-transfected Renilla luciferase.The promoter fragment of CRP promoter were cross-linked with 1.42% formaldehyde at room temperature for 15 min. The reaction was stopped by addition of glycine to a final concentration of 125 mM. Cells were then sonicated in IP buffer  at 4°C for 10 min, followed by addition of anti-STAT3 , anti-p50  or anti-C/EBP-β  for 15 min in ice/water bath with sonification. Protein-DNA complexes were isolated with nProtein A Sepharose 4 Fast Flow , and eluted with 10% Chelex 100 . The eluates were treated with proteinase K  and subjected to DNA purification, and the crosslinking was reversed by heating at 55°C for 30 min and boiling for 10 min. DNA was then purified and analyzed with q-PCR. The primer sequences for human CRP used were: genomic binding ; vector binding .ChIP experiments were performed as described . BrieflyCRP promoter through vector PCR using appropriately methylated primers before transfection.In some experiments, vectors were either entirely methylated with CpG methyltransferase  or site-specifically methylated at −53 or −108 CpG sites of Data were presented as mean ± SEM. Statistical analysis was performed by the two-tailed Student’s t-test, one-way ANOVA with Tukey post hoc or K-S tests as appropriate. Values of p<0.05 were considered significant. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:CRP) belongs to the family of acute phase reactant genes, which play an essential role in inflammatory responses to infection or tissue injury in the liver. CPR needs to be quickly activated after stimulation by cytokines, and its activation is reversible upon resolution of inflammation. Zhang and colleagues report here that the dynamic expression of CPR is causally regulated by DNA methylation, and that this type of regulation may apply to other inducible genes that are endowed with CpG-poor promoters and have evolved CpG-containing binding sites for transcription factors specialized in rapid and reversible responses.The C reactive protein  the details of CRP regulation in sequential phases by STAT3, NFkB and DNA demethylation/5OHT, and subsequent CEBP binding and activity. The evidence is supported by a satisfactory range of complementary experiments, from functional screens to modulation of DNA methylation by chemical inhibitors and knock-out or knock-down of methylating (DNMT3A) or demethylating (TET2) enzymes, with validations by targeted epigenomic editing.The interesting novelty and appeal of the study comes from 1) the biological importance of However, the reviewers share the common concern that the manuscript is too concise in its current state to be fully instructive and convincing. More details are clearly required:– In the Introduction, to provide proper background.– In the Results section to explain the experimental design, methods and results and provide proper validation.– In the Discussion, to replace the results in the current state of knowledge and clarify the model.I agree that here is a need for further analyses of existing data and for inclusion of important missing controls to fully support interpretations and conclusions.Please address the following comments in a revised version of your manuscript:Essential revisions:1) Provide information (in main text and figure legends) about i) how the CRISPR KO was performed: which exons were targeted? Was the analysis performed on single clones or cell bulk? And ii) how was DNA methylation analyzed in Figure 1: bisulfite sequencing? Available GEO datasets (and please cite the source if it is the case)?TET2 and DNMT3A KO or KD by Western blot and RT-qPCR, respectively (as a supplementary figure).2) Provide validation of CRP promoter is directly changed in the experiments of functional modulation of DNA methylation . This is absolutely crucial for supporting conclusions. Along the same line, to further prove that the effect is not indirect, the expression of STAT3, p50 and C/EBP should be assessed (Western blot or RT-qPCR) in DNMT3A KO, TET2 KO and upon treatment with DNA methylation inhibitors.3) Provide evidence that DNA methylation of the CRP gene in various human tissues (available from public databases) to see how many CpG sites of the CRP promoter are specifically hypomethylated in liver.4) Throughout the manuscript, figures illustrating methylation profiles are needed. In Figure 1F, please show raw bisulfite sequencing data with individual clones rather than fold differences between the two alleles-an unusual and incomplete way to show the data. This will benefit interpretation of differences between the 286A and 286C alleles  on the A allele?). Also, please show WGBS tracks of the CRP demethylated upon inflammation in the liver (genome browser views)? How do changes in expression and methylation correlate? How many DMRs and do they preferentially locate to CpG poor promoters? These are obvious questions that need to be addressed, possibly in supplementary figures.5) At the end of the manuscript, the authors investigate if demethylation of CpG-poor promoters is a general feature for the activation of APR genes, in cells and liver. This is important and potentially interesting but again, the authors need to provide much more details on what was done and to perform a more thorough analysis of their WGBS and RNA-seq data. Otherwise, Figure 7 is a too vague genomic characterization of CpG frequency and expression associations. How many replicates of RNA-seq and WGBS? Do replicates correlate? How many genes upregulated and what are the genes upregulated? Is CRP, it would be beneficial if the authors could look by RT-qPCR at expression of several of these genes in the +/- 5aza conditions  to see if they go up, as they would predict. They could also include some gene sets based on PRG, CpG low status, etc… as well as known rapid response genes  that seem to be regulated in a very different manner than APR genes. These may be similar to the authors "cytokine" genes.6) To further support their assumption that APR genes may behave in general as TET2 KO cells  instead of DNMT3A KO cells, to determine which TF has reduced binding when demethylation is compromised.7) The sequential model proposed by the authors is confusing. First, according to Figure 4 and Figure 5B-D, all three TFs  are potentially sensitive to DNA methylation, which contradicts the model. Second, to test which TF requires demethylation for binding, the kinetic experiments described in Figure 5B-D should be performed in TET1 to demethylate specific promoters upon neuronal activation . Second, in relationship with inflammation and APR gene regulation, the authors should discuss the work of Stephen Smale  -and potentially others- about conventional categorization of CpG-rich promoters in inflammatory gene induction: nucleosome poor promoters, accessible, independent of remodeling, one step induction. This provides an interesting contrast with the sequential kinetics of CRP, regulated in part by changes in DNA methylation and temporal waves of TF binding, as demonstrated here by the authors.8) There is a lack of thorough introduction and/or discussion of existing evidence from the literature to place the authors' study in the current knowledge context. First, it is known that dynamic DNA methylation changes usually affect CpG-poor promoters, in part due to lack of protective binding of methyl DNA binding proteins, while CpG islands (CGI) promoters, on the contrary, are predominantly non-methylated and stable to ensure expression of housekeeping genes and primary response genes . Apart from the studies cited in the Discussion of this manuscript, acute changes in DNA methylation have been described in other contexts such as in neurons, upon activation . As in the present study, but at a genome wide scale, the authors described dynamic reversible methylation of low density CpG promoters in brain-specific genes, and correlated them with gene expression. Moreover, CpG methylation is known to affect transcription factor binding , and specifically STAT3 binding . And very recently, in neurons as well, TF were described to recruit Recommended changes:CRP hypomethylated in liver cells in unstimulated conditions? Is it because a basal level of expression is needed? This could be discussed.1) Why is CRP expression at the resting stage. However, they did not assess as to whether CRP expression is modified by these inhibitors after induction (it should not), while this would be an important point for their demonstration. Please include this analysis after Figure 2A.2) In Figure 2A, the authors show that DNA methylation inhibitors enhance 3) To assess whether DNA demethylation precedes or follows gene activation, please add a 24h time point for DNA methylation analysis , if possible. Essential revisions:1) Provide information (in main text and figure legends) about i) how the CRISPR KO was performed: which exons were targeted? Was the analysis performed on single clones or cell bulk? And ii) how was DNA methylation analyzed in Figure 1: bisulfite sequencing? Available GEO datasets (and please cite the source if it is the case)?TET2 and exon 14 or 2 of DNMT3A were targeted. The analysis was performed on cell bulk in Figure 3 and 5, and was performed on single clones in Figure 6.In CRISPR KO experiments, exon 3 or 7 of CRP promoter in different human tissues were obtained from available GEO datasets generated by whole-genome bisulfite sequencing: Liver 01-GSM916049, Liver 02-GSM1716965, Adipose-GSM1120331, Adrenal-GSM1120325, Aorta-GSM1120329, Esophagus-GSM983649, Gaster-GSM1120333, Lung-GSM983647, Ovary-GSM1120323, Muscle-GSM1010986, Atrium-GSM1120335, Colon-GSM983645, Spleen-GSM983652, Thymus-GSM1120322. In other figures of Figure 1, DNA methylation levels of CRP promoter were determined with bisulfite cloning sequencing.In Figure 1C, DNA methylation levels of These descriptions have been included in the revised manuscript.2) Provide validation of TET2 and DNMT3A KO or KD by Western blot and RT-qPCR, respectively (as a supplementary figure).TET2/DNMT3A KD/KO showed that the levels of TET2 or DNMT3A were effectively reduced .Immunoblotting and qPCR validation of 3) Provide evidence that DNA methylation of the CRP promoter is directly changed in the experiments of functional modulation of DNA methylation . This is absolutely crucial for supporting conclusions. Along the same line, to further prove that the effect is not indirect, the expression of STAT3, p50 and C/EBP should be assessed (Western blot or RT-qPCR) in DNMT3A KO, TET2 KO and upon treatment with DNA methylation inhibitors.CRP promoter in Hep3B cells following treatment with DNA methylation inhibitor 5-aza, or DNMT3A/TET2 KD/KO. The results indicated that 5-aza and DNMT3A KD/KO reduced, whereas TET2 KD/KO augmented DNA methylation of CRP promoter .We have performed additional experiments as suggested by the editor and reviewers. We have assessed the changes in DNA methylation of DNMT3A/TET2 KD/KO . 5-aza treatment reduced the expression of STAT3, but did not affect that of p50 and C/EBP-β. DNMT3A or TET2 KD showed little effect on the expression of the three TFs. DNMT3A or TET2 KO tended to reduce their expression. Because the observed effects on CRP expression by 5-aza or DNMT3A/TET2 KD/KO are consistent or complementary, the above results suggest that these manipulations regulate CRP expression by directly modulating promoter methylation.We have assessed the expression of STAT3, p50 and C/EBP-β in Hep3B cells following treatment with 5-aza, or CRP via dCas9-mediated targeting of the catalytic domains of DNMT3A or TET2. Enforced methylation of CRP promoter by DNMT3A-dCas9 reduced the expression of CRP in Hep3B cells, but showed little effect on that of serum amyloid A  and serum amyloid P component  . By contrast, enforced demethylation of CRP promoter by TET2-dCas9 only selectively enhanced the expression of CRP . We thus conclude that DNMT3A and TET2-tuned methylation status of CRP promoter constitutes a key part of the regulatory mechanism that causally determines the expression.Further support to this suggestion is provided by specifically modulating promoter methylation of 4) Throughout the manuscript, figures illustrating methylation profiles are needed. In Figure 1F, please show raw bisulfite sequencing data with individual clones rather than fold differences between the two alleles-an unusual and incomplete way to show the data. This will benefit interpretation of differences between the 286A and 286C alleles  on the A allele?). Also, please show WGBS tracks of the CRP gene in various human tissues (available from public databases) to see how many CpG sites of the CRP promoter are specifically hypomethylated in liver.CRP gene are shown in Figure 1C. -53, -108, -286 and -306CpG of CRP are all most demethylated in human livers with only -255CpG in Liver 01 being more methylated than in Adrenal.Figure 1 has been carefully revised according to the comments of the editor and reviewers. Raw bisulfite sequencing data with individual clones are shown in Figure 1F. The results indicate that most maintained CpGme were on -286C allele. The WGBS tracks of 5) At the end of the manuscript, the authors investigate if demethylation of CpG-poor promoters is a general feature for the activation of APR genes, in cells and liver. This is important and potentially interesting but again, the authors need to provide much more details on what was done and to perform a more thorough analysis of their WGBS and RNA-seq data. Otherwise, Figure 7 is a too vague genomic characterization of CpG frequency and expression associations. How many replicates of RNA-seq and WGBS? Do replicates correlate? How many genes upregulated and what are the genes upregulated? Is CRP demethylated upon inflammation in the liver (genome browser views)? How do changes in expression and methylation correlate? How many DMRs and do they preferentially locate to CpG poor promoters? These are obvious questions that need to be addressed, possibly in supplementary figures.We have performed additional experiments and analysis as suggested by the editor and reviewers.2) between the replicates are 0.991 for resting Hep3B cells, 0.998 for induced Hep3B cells, 0.884 for resting mouse livers and 0.803 for induced mouse livers . For WGBS, the correlations (R2) between the replicates are 0.828 for resting mouse livers and 0.810 for induced mouse livers .Two biological replicates were examined for both RNA-seq and WGBS. For RNA-seq, the correlations (RThe expression of 2705 and 3371 genes were changed by over 2-fold (up- and down-regulated) in induced Hep3B cells and in induced mouse livers, respectively. In Hep3B cells, the “regulation of inflammatory response”, “acute inflammatory response” and “regulation of immune effector process” are the top 3 activated biological processes. In induced mouse livers, the “acute inflammatory response”, “positive regulation of response to external stimulus” and “cell chemotaxis” are the top 3 activated biological processes .CRP, mouse CRP is not an acute phase protein [1-3] , and its promoter did not undergo demethylation in induced livers (In contrast to human d livers . Althougp < 10-15). Therefore, differential methylation prefers to occur in CpG-poor regions. However, there are only 85 promoters located in these DMRs. Such an under-representation is consistent with previous findings made in mouse neurons activated by electroconvulsive stimulation 4.Overall, 1262 differentially methylated regions  were identified in induced versus resting mouse livers. The CpG ratios of these DMRs  are significantly lower than that of non-DMRs  To further support their assumption that APR genes may behave in general as CRP, it would be beneficial if the authors could look by RT-qPCR at expression of several of these genes in the +/- 5aza conditions  to see if they go up, as they would predict. They could also include some gene sets based on PRG, CpG low status, etc… as well as known rapid response genes  that seem to be regulated in a very different manner than APR genes. These may be similar to the authors "cytokine" genes.We have performed additional experiments as suggested by the editor and reviewers. The effects of 5-aza treatment on the expression of 2 additional APRs with CpG-poor promoters, 3 class A PRGs with CpG-rich promoters, 4 class B PRGs with CpG-poor promoters, and 2 class B SRG with CpG-poor promoters in Hep3B cells were determined with q-PCR . 5-aza t7) The sequential model proposed by the authors is confusing. First, according to Figure 4 and Figure 5B-D, all three TFs  are potentially sensitive to DNA methylation, which contradicts the model. Second, to test which TF requires demethylation for binding, the kinetic experiments described in Figure 5B-D should be performed in TET2 KO cells  instead of DNMT3A KO cells, to determine which TF has reduced binding when demethylation is compromised.TET2 KO almost abrogated the recruitment of p50 and C/EBP-β to CRP promoter, but the recruitment of STAT3 was still apparent . This indicates that STAT3 can nevertheless be recruited to CRP promoter even when demethylation is compromised, consistent with the observations that vector binding of STAT3 was least sensitive to methylation . This also indicates that STAT3 can act largely independent of p50 and C/EBP-β to drive CRP expression, albeit markedly impaired . Indeed, the sole activation of STAT3 in wildtype cells drove CRP expression to a level comparable to the minor peak, whereas the sole activation of NF-κB was completely ineffective . NF-κB inhibition (with intact STAT3) at the induced state, however, resulted in an even stronger methylation of CRP promoter  and a reversal of allelic imbalance . Therefore, promoter demethylation requires p50 that acts downstream of STAT3.We have performed additional experiments as suggested by the editor and reviewers. DNMT3A KO not only tripled CRP expression during the entire course of induction, but eliminated the time lag between the two waves . The augmented amplitude can be explained by enhanced recruitment of three TFs, while the altered dynamics may correspond to the shifted timing of p50 recruitment . As such, p50 selectively recruited during the time lag could be responsible for promoter demethylation to prime the second wave. Accordingly, C/EBP-β appears to be the major effector that responds to promoter demethylation: its overexpression did not demethylate CRP promoter , but when combined with blockage of DNA methylation, it drove the resting expression of CRP to a level approaching to that induced by IL-6 and IL-1β . C/EBP-β KO, however, lowered the induced expression of CRP by ~70% . These results together are consistent with the proposed sequential model.Interestingly, enforced DNA demethylation by We have carefully revised the manuscript to elaborate the sequential model.8) There is a lack of thorough introduction and/or discussion of existing evidence from the literature to place the authors' study in the current knowledge context. First, it is known that dynamic DNA methylation changes usually affect CpG-poor promoters, in part due to lack of protective binding of methyl DNA binding proteins, while CpG islands (CGI) promoters, on the contrary, are predominantly non-methylated and stable to ensure expression of housekeeping genes and primary response genes . Apart from the studies cited in the Discussion of this manuscript, acute changes in DNA methylation have been described in other contexts such as in neurons, upon activation . As in the present study, but at a genome wide scale, the authors described dynamic reversible methylation of low density CpG promoters in brain-specific genes, and correlated them with gene expression. Moreover, CpG methylation is known to affect transcription factor binding , and specifically STAT3 binding . And very recently, in neurons as well, TF were described to recruit TET1 to demethylate specific promoters upon neuronal activation . Second, in relationship with inflammation and APR gene regulation, the authors should discuss the work of Stephen Smale  -and potentially others- about conventional categorization of CpG-rich promoters in inflammatory gene induction: nucleosome poor promoters, accessible, independent of remodeling, one step induction. This provides an interesting contrast with the sequential kinetics of CRP, regulated in part by changes in DNA methylation and temporal waves of TF binding, as demonstrated here by the authors.The manuscript has been carefully revised according to the comments of the editor and reviewers.4]. Though promoters were underrepresented in these regions, their methylation changes were nevertheless modestly anticorrelated with gene expression [4]. Such CpG content-dependent changes in DNA methylation, however, were not observed in another study examining neurons activated by contextual learning [6]. Moreover, DNA methylation changes in human dendritic cells following infection rarely occurred at promoters , and were claimed to be a consequence of gene expression [7]. Those findings thus argue that the regulation of reversible methylation on inducible expression of genes with CpG-poor promoters may be context-dependent, and that rigorously controlled case study should be integrated into genome-wide investigation to conclude on causality.Acute changes in DNA methylation have also been analyzed at a genome-wide scale in previous studies. One study examined mouse neurons activated by electroconvulsive stimulation, and found that methylation changes were preferentially occurred in CpG-poor regions [5]. Despite their distinct requirements for SWI/SNF complexes [5], preassembled Pol II [9] and new protein synthesis , both groups of genes exhibit a single-wave kinetics of induction . This is in contrast with CRP and probably other major APRs, which show a two-wave kinetics of induction with the second wave licensed by promoter demethylation. However, nucleosome remodeling may also act downstream in the second wave, as C/EBP-β has recently been shown to promote CRP expression by recruiting BRG1 via MKL1 [11]. Conversely, promoter methylation may also contribute to inducible expression of nucleosome remodeling-dependent genes in macrophages by directly regulating TF recruitment [12-14].Interestingly, genes showing inducible expression in macrophages activated by endotoxin have been classified into two groups: nucleosome remodeling-independent genes with CpG-rich promoters, and nucleosome remodeling-dependent genes with CpG-poor promoters , TETs and DNMTs can nevertheless mediate active demethylation and de novomethylation, respectively. This indicates that the methylation status of part of the genome may depend on the balance of the two types of enzymes [15-19]. Cellular signaling able to tip the balance of TETs and DNMTs could thus represent a regulatory mechanism that finely and reversibly tunes the expression of certain genes in response to environmental cues. In case of CRP, DNMT3A and TET2 appear to be involved in the regulation. Though the mechanism of TF-induced local demethylation is not fully understood [16], TFs including NF-κB and EGR1 have been reported to evoke DNA demethylation in neurons [20] for example by recruiting TET1 [21]. Future study is warranted to elucidate how TFs regulate the balance of TETs and DNMTs and to discover scenarios where regulation by reversible DNA methylation plays a prominent role.Despite that most CpGs in mammalian promoters not associated with CGIs are usually methylated . CRP has been proposed to act as a soluble pattern recognition receptor in host defense . Therefore, basal levels of circulating CRP may constitute an integral part of immune surveillance that contributes to the silent clearance of invading pathogens and apoptotic cells in homeostasis. These discussions have been included in the revised manuscript.As pointed by the editor and reviewers, hepatocytes secrete 2) In Figure 2A, the authors show that DNA methylation inhibitors enhance CRP expression at the resting stage. However, they did not assess as to whether CRP expression is modified by these inhibitors after induction (it should not), while this would be an important point for their demonstration. Please include this analysis after Figure 2A.CRP expression at the resting but not at the induced state  wherein CRP promoter underwent active demethylation .We have performed additional experiments as suggested by the editor and reviewers. Treating Hep3B cells with 5-aza significantly enhanced 3) To assess whether DNA demethylation precedes or follows gene activation, please add a 24h time point for DNA methylation analysis , if possible.TET/DNMT KO or KD . Before its completion, however, our lab had to be closed due to the SARS-CoV-2 epidemic. Therefore, we could not arrange this experiment in a reasonable time frame. Nevertheless, the results shown in Figure 5 suggest that the demethylation of CRP promoter might follow the minor peak driven by STAT3, but precede the major peak driven by C/EBP-β.This experiment was originally arranged to be performed following those assessing methylation changes induced by 5-aza, References:1) Baltz ML, de Beer FC, Feinstein A, Munn EA, Milstein CP, Fletcher TC, March JF, Taylor J, Bruton C, Clamp JR, Davies AJ, Pepys MB. Phylogenetic aspects of c-reactive protein and related proteins. Ann N Y Acad Sci. 1982;389:49-752) Pepys MB, Hirschfield GM. C-reactive protein: A critical update. 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DRR, DUM1, linc-MD1, linc-YY1, LncMyod, Neat1, Myoparr, Malat1, and SRA) and three genomic imprinting-related lncRNAs  in mouse skeletal muscle. The expression levels of these lncRNAs were examined by quantitative RT-PCR in six skeletal muscle atrophy models  and two skeletal muscle hypertrophy models . Cluster analyses of these lncRNA expression levels were successfully used to categorize the muscle atrophy models into two sub-groups. In addition, the expression of Gtl2, IG-DMR, and DUM1 was altered along with changes in the skeletal muscle size. The overview of the expression levels of lncRNAs in multiple muscle atrophy and hypertrophy models provides a novel insight into the role of lncRNAs in determining the skeletal muscle mass.Skeletal muscle is a highly plastic organ that is necessary for homeostasis and health of the human body. The size of skeletal muscle changes in response to intrinsic and extrinsic stimuli. Although protein-coding RNAs including myostatin, NF-κβ, and insulin-like growth factor-1 (IGF-1), have pivotal roles in determining the skeletal muscle mass, the role of long non-coding RNAs (lncRNAs) in the regulation of skeletal muscle mass remains to be elucidated. Here, we performed expression profiling of nine skeletal muscle differentiation-related lncRNAs ( Skeletal muscle is an organ that plays important roles in motion, postural maintenance, and metabolic adaptation. The adult skeletal muscle exhibits high plasticity and its mass can be altered by intrinsic and extrinsic stimuli. The skeletal muscle mass is decreased not only in diseases, such as cancer cachexia, neuromuscular disorders, chronic kidney disease, heart failure, and chronic obstructive pulmonary disease, but also as a consequence of aging, immobilization, and malnutrition ,5,6,7,8.SRA, is required for the terminal differentiation of myoblasts by enhancing the transcriptional activity of myoblast determination protein 1 (MyoD) [DRR enhancer RNA, also known as MUNC, is expressed from the distal regulatory element of the Myod1 gene, which codes for MyoD, and promotes myogenic differentiation [LncMyoD is in the immediate vicinity of the Myod1 gene and is required for terminal muscle differentiation through the translational regulation of genes involved in proliferation [linc-MD1, DUM1, linc-YY1, Malat1, and Neat1, also regulate myogenesis [Recent advances in techniques for nucleic acid detection have revealed the presence of numerous long non-coding RNAs (lncRNAs), which are defined as RNAs longer than 200 bases in length . Althoug1 (MyoD) , one of ntiation ,26. LncMferation . Other logenesis ,31,32,33Myoparr, from the promoter region of the myogenin gene, which codes for a transcriptional factor that is indispensable for skeletal muscle differentiation [Myoparr interacts with a transcriptional co-activator, DEAD (Asp-Glu-Ala-Asp) box polypeptide 17 (Ddx17), and is essential both for the specification of myoblasts into the differentiation lineage and for the myoblast cell cycle withdrawal. Intriguingly, Myoparr is also involved in the induction of muscle atrophy caused by surgical denervation in adult skeletal muscle in mice [myostatin deficiency increased the expression levels of genomic imprinting-related lncRNAs, Gtl2  and IG-DMR [Gtl2 promotes bovine myoblast differentiation by acting as an miR-135 sponge [H19, another genomic imprinting-related lncRNA, also regulates myogenic differentiation [H19 show muscle hypertrophy and hyperplasia [DRR, DUM1, linc-MD1, linc-YY1, LncMyod, Neat1, Myoparr, Malat1, and SRA), whose roles in skeletal muscle differentiation were well established [Gtl2, H19, and IG-DMR) in six muscle atrophy and two muscle hypertrophy models in mice. By using the expression data sets of these lncRNAs, we could classify the muscle atrophy conditions into two sub-groups. Our comprehensive expression data of 12 lncRNAs provide an important insight into the roles of these lncRNAs in the regulation of skeletal muscle mass.We previously identified an lncRNA, ntiation . Myoparr in mice ,35. Thesd IG-DMR . Gtl2 pr5 sponge . H19, anntiation ,39,40, aerplasia . Thus, iablished ,31,32,33Myoparr, linc-MD1, LncMyod, DRR, DUM1, linc-YY1, Malat1, Neat1, and SRA) in six muscle atrophy models in mice. The first three models, including denervation, casting, and tail suspension, are the disuse atrophy group. Sciatic nerve transection significantly decreased the tibialis anterior (TA) muscle weight by 18.9% compared to that in the control side that was sham operated after 7 days of surgical denervation  in six muscle atrophy models. Intriguingly, we found increased H19 expression after 7 days of surgical denervation  in the skeletal muscle hypertrophy condition. Because of the difficulty in inducing muscle hypertrophy in mice by resistance training, we used both genetic hypertrophy and mechanical overload (MOV)-induced chronic hypertrophy models for the analysis. As a genetic hypertrophy model, we used myostatin knockout mice [myostatin gene in mice resulted in a significant increase of 81.5% in the TA muscle mass compared to that in the wild-type mice  in the muscle hypertrophy condition. The expression level of H19 was significantly increased by the MOV treatment but not by the myostatin deficiency  in skeletal muscle differentiation have been reported [linc-MD1 and LncMyod; and (2) the expression levels of Gtl2, IG-DMR, and DUM1 are highly correlated with skeletal muscle mass. Collectively, these findings suggest that the lncRNAs involved in muscle differentiation and genomic imprinting might also be involved in the regulation of skeletal muscle mass.Recent findings indicate that denervation, immobilization, cancer cachexia, chronic kidney disease, fasting, and aging affect the expression of  muscles ,49,50,51ignaling ,52, whicignaling ,54. Othechanisms ,56,57. Treported ,31,32,33linc-MD1 and LncMyod; the expression levels of linc-MD1 and LncMyod were largely increased in the disuse models, including the denervation, casting, and tail suspension models, but not in the systemically wasting models, including the dexamethasone-administration, cancer cachexia, and fasting models. During muscle differentiation, the expression of linc-MD1 and LncMyod is controlled by the MyoD protein [Myod1 expression [DUM1, linc-YY1, Myoparr, and H19, whose expression levels are also positively controlled by the MyoD protein during muscle differentiation [linc-MD1 and LncMyod in the disuse-mediated muscle atrophy conditions. Denervation and MOV treatments also increased Myoparr expression; however, it remains unclear how these treatments increased the expression level of Myoparr. Therefore, future studies investigating factors whose expression changes in common in denervation and MOV models may reveal the molecular mechanism regulating Myoparr expression in adult skeletal muscle. Intriguingly, linc-MD1 increases the myocyte-specific enhancer factor 2C (MEF2C) and mastermind-like protein 1 (MAML1) expression by sponging miR-133 and miR-135 during skeletal muscle differentiation [linc-MD1 expression would work as a compensatory mechanism, attenuating the muscle atrophy in the disuse conditions by activating MEF2C. By contrast, despite the fact that IGF2-mRNA-binding protein 2 (IMP2) is required to maintain the skeletal muscle mass [LncMyoD blocks the function of IMP2 in muscle differentiation [LncMyoD expression might contribute to the cause of muscle atrophy in the disuse situations. The expression levels of linc-MD1 and LncMyod were also increased by MOV treatment. Therefore, in that case, linc-MD1 might contribute to inducing muscle hypertrophy and LncMyod might work as a compensatory mechanism to attenuate muscle hypertrophy, as opposed to the disuse situations. Both linc-MD1 and LncMyoD are not expressed in intact mature muscle [linc-MD1 and LncMyoD in multiple muscle atrophy models would be useful to reveal the roles of these lncRNAs in adult skeletal muscle.Our cluster analysis of comprehensive expression profiling of lncRNAs classified the six muscle atrophy models into two sub-groups, namely the disuse-mediated atrophy and systemically wasting atrophy sub-groups. This classification was mainly dependent on the expression of  protein ,28. Althpression ,59, consntiation ,30,34,60ntiation . MAML1 pntiation . Therefocle mass , LncMyoDntiation , suggeste muscle ,28. The Gtl2 and IG-DMR, are located in the Dlk1-Dio3 imprinting locus at chromosome 12qF1 in mice. A paternally inherited DNA mutation of this locus, called the callipyge mutation, results in a 30%–40% increase in the hindlimb muscle mass in sheep through activation of the Dlk1 or Rtl1 gene [Dlk1 expression and counteracts muscle hypertrophy [Gtl2 is also maternally expressed from this locus and promotes muscle differentiation by sponging miR-135 in bovine myoblasts [Gtl2 [IG-DMR is a maternal lncRNA and works like an enhancer RNA that controls gene expression on the maternal chromosome at the Dlk1-Dio3 locus in embryonic stem cells [Gtl2 and IG-DMR are involved in the regulation of adult skeletal muscle mass. Intriguingly, our data showed that the expression levels of Gtl2 and IG-DMR were increased in two muscle hypertrophy models and were decreased in multiple muscle atrophy models. We and others previously found that the expression levels of lncRNAs at this locus are gradually decreased during muscle growth and aging in mice and pigs [DUM1 expression, which was correlated with the skeletal muscle mass in this study, also gradually decreased during muscle growth and aging in mice [Genomic imprinting-related lncRNAs, tl1 gene . On the ertrophy ,66. Gtl2yoblasts . Skeletats [Gtl2 . IG-DMR em cells . Howeverand pigs ,69,70,71 in mice . Therefomyostatin deficiency cause a slow-to-fast fiber-type shift [myostatin deficiency [n numbers in each group would result in an increased false-positive change in lncRNA expression. Thus, it cannot be ruled out that the expression levels of lncRNA in our models might be influenced by the changes in these muscle properties and microenvironment.Besides muscle mass, all of the models used in this study affect several muscle properties. For example, the disuse atrophy  and the pe shift ,73, wherpe shift ,74. Morepe shift ,76. Hindpe shift ,78. Immope shift . Satellificiency . Expressficiency ,82,83. Ificiency ,86,87,88ficiency . The proficiency . It is aThe C57BL/6J and CD2F1 mice were purchased from the Japan SLC and Charles River Laboratories Japan, respectively. Mice were housed in cages with a constant temperature (24 °C) and a 12-h light:12-h dark cycle and were provided with water and food ad libitum, except during the fasting experiments. Animal experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee of Fujita Health University, Japan . Mechanical overload experiments were approved by the Animal Care and Use Committee of the National Cerebral and Cardiovascular Center in Japan  and were conducted under institutional and national guidelines. All animal experiments were performed in accordance with the ethical standards laid down in the 1964 declaration of Helsinki and its later amendments.l-glutamine , and PS  in an atmosphere of 5% CO2 at 37 °C.Cachexia-inducible colon-26 adenocarcinoma (C26) clone 20, described previously , was a kFor induction of muscle atrophy by denervation, a 3-mm segment of the sciatic nerve was excised from the hind leg of each 8-week-old male C57BL/6J mouse under anesthesia, as described previously . ContralLeft hindlimb of 8-week-old male C57BL/6J mice was immobilized in plaster casts under anesthesia, as described previously, for the induction of muscle atrophy by casting . The rigMuscle atrophy was induced by tail suspension as follows: 8-week-old male C57BL/6J mice were suspended for 3 days by their tails, as described previously, such that the hindlimbs could not touch the floor, wall, and the lid of the cage . Three dMuscle atrophy was also induced by dexamethasone as follows: 7-week-old male C57BL/6J mice were intraperitoneally administrated 25 mg/kg/day water-soluble dexamethasone  for 7 days under anesthesia, as described previously ,94. Mice6 cells resuspended in 200 µL of PBS were subcutaneously injected into the dorsum of 6-week-old male CD2F1 mice under anesthesia, as described previously, to induce muscle atrophy by cachexia [Trypsinized C26 cells were washed with PBS and 1 × 10cachexia . Equal vFor the induction of muscle atrophy by fasting, 8-week-old male C57BL/6J mice were allowed free access to water but were prohibited from eating solid food for 2 days, as described previously . ControlMyostatin knockout mice, described previously [eviously , were a eviously . The TA For muscle hypertrophy model induction using MOV, the soleus and gastrocnemius muscles were surgically removed from 10-week-old male C57BL/6J mice under anesthesia, as described previously . Sham-opRpl26 expression and shown as relative expression compared to the control samples. The raw data of qRT-PCR are shown in Myoparr (NR_160520), linc-MD1 (NR_131249), DUM1 (NR_028300), linc-YY1 (AK081464), Malat1 (NR_002847), Neat1 (NR_131212), SRA (NM_025291), H19 (NR_130973), and Gtl2 (NR_027652). The full-length sequences of DRR (MUNC) and LncMyoD were described in [IG-DMR lncRNA has not identified yet, we used the previously described primer-pairs [IG-DMR lncRNA. The primers used are listed in Total RNA was extracted from the TA or plantaris muscles and purified using the miRNeasy Mini Kit  with DNase I (QIAGEN) treatment, according to the manufacturer’s protocol. One microgram total RNA was subjected to the reverse transcription reaction using SuperScript III reverse transcriptase (Thermo Fisher Scientific) or Protoscript II reverse transcriptase  with random primers. The quantitative real-time PCR (qRT-PCR) was conducted using SYBR Premix Ex Taq . Data were normalized to Gtl2 NR_0652. The er-pairs  to detect-tests. A value of p < 0.05 was considered statistically significant. Data are presented as box-and-whisker plots and statistical significance is reported in the figure legends as * p < 0.05, ** p < 0.01, *** p < 0.001.Statistical analyses were performed using unpaired two-tailed Student’s https://bioconductor.org/packages/release/bioc/html/genefilter.html). The z-score was used for the cluster analysis of gene expression changes under muscle atrophy. Correlation analysis was performed by R software using Pearson correlation coefficient. A value of R > 0.7 was considered a strong correlation.The value of the log2 fold change in the expression levels of lncRNAs was converted to a z-score using R software with the Genefilter package (The expression profiling of 12 lncRNAs, which are related to skeletal muscle differentiation and genomic imprinting, in multiple muscle atrophy and hypertrophy conditions performed in this study provides a novel insight about lncRNAs in determining the skeletal muscle mass. However, further studies including expression analysis of lncRNAs using more suitable models or animal experiments using conditional transgenic or knockout mice of these lncRNAs are needed to mechanistically relate the changes in skeletal muscle mass to lncRNA expression.


KCND3 encodes the voltage-gated potassium ion channel subfamily D member 3, a six trans-membrane protein (Kv4.3), involved in the transient outward K+ current. KCND3 defect causes both cardiological and neurological syndromes. From a neurological perspective, Kv4.3 defect has been associated to SCA type 19/22, a complex neurological disorder encompassing a wide spectrum of clinical features beside ataxia. To better define the phenotypic spectrum and course of KCND3-related neurological disorder, we review the clinical presentation and evolution in 68 reported cases. We delineated two main clinical phenotypes according to the age of onset. Neurodevelopmental disorder with epilepsy and/or movement disorders with ataxia later in the disease course characterized the early onset forms, while a prominent ataxic syndrome with possible cognitive decline, movement disorders, and peripheral neuropathy were observed in the late onset forms. Furthermore, we described a 37-year-old patient with a de novo KCND3 variant [c.901T>C (p.Ser301Pro)], previously reported in dbSNP as rs79821338, and a clinical phenotype paradigmatic of the early onset forms with neurodevelopmental disorder, epilepsy, parkinsonism-dystonia, and ataxia in adulthood, further expanding the clinical spectrum of this condition.  KCND3, is a six trans-membrane segmented (S1–S6) ion channel, involved in the transient outward K+ current. Segments S1 to S4 form the voltage-sensing domain, while S5 and S6 segments with the pore loop constitute the ion selective pore (H5) [Voltage-gated potassium ion channels (Kvs), considered highly sophisticated voltage-gated ion channels from both a functional and a structural standpoint, play a fundamental role in the generation and propagation of the action potential in all living beings . Kvs areKv4.3 is highly expressed in the central nervous system, particularly in cerebellar Purkinje cells, deep nuclei, granule cells, and interneurons . Pre- and postmigrating Purkinje cells show different levels of Kv4.3 expression, suggesting a role in their migration and cerebellar development . KCND3 is the causative gene of SCA 19/22, an autosomal dominant cerebellar ataxia mapped to chromosome 1p21-q23 [1p21-q23 ,8.KCND3 deficiency to include epilepsy, intellectual disability/cognitive impairment, movement disorders, pyramidal signs, and peripheral neuropathy [KCND3 loss-of-function mutations have been associated with the neurological phenotype, while KCND3 gain-of-function has been associated with the cardiological syndrome, with only few exceptions [This condition was first identified through linkage analysis in two independent large families ,10 and nuropathy ,18,19,20uropathy . Althougceptions .KCND3 gene deficiency, we reviewed the literature, focusing on clinical presentation and evolution according to age of onset and highlighting less frequent and underrecognized clinical features of this condition. Finally, we report a new case presenting in childhood with neurodevelopmental disorder and focal epilepsy who later developed a complex movement disorder and, into adulthood, ataxic features and cerebellar atrophy.In order to better delineate the neurological phenotype associated with This 37-year-old man was born from a non-consanguineous healthy couple. Delivery was at term with no complications. Early development was normal, with first word pronounced at 12 months of age and walking at 15-months. He was first evaluated at three years of age for speech difficulties and clumsiness with frequent falls. Rehabilitative treatment improved clumsiness and falls. At the age of 5 years, neuropsychological tests revealed a mild intellectual disability (IQ = 67) on the Weschler Intelligence Scale for Children III (WISC-III). On neurological examination, he presented minimal upper limbs postural tremor, brisk lower limbs reflexes and oral and verbal dyspraxia. During the same period, he suffered from both focal and generalized motor seizures. EEG recording detected frontocentral epileptic abnormalities with slow generalized background activity. Carbamazepine treatment successfully controlled epilepsy. At that time, brain MRI was normal. At the age of 7, a further cognitive evaluation showed a moderate intellectual disability (IQ = 41 on WISC-III) and behavioral dyscontrol became a major issue. KCND3 variant  as rs79821338 but no clinical description was available. The variant was located in the S4 functional domain and mutated aminoacid was predicted to alter physical and chemical properties and interactions between the transmembrane protein domain and the lipid membrane, disturbing the S4 domain function.At the age of 14 years, he started to complain about recurrent migraine episodes. In the same period, carbamazepine was withdrawn due to a long-standing seizure freedom. The next year he suffered from episodes of left-sided facial numbness, dizziness, sweating, limb and head tremors with migraine. He also progressively exhibited cervical and upper limbs dystonia, more severe during evening hours. A levodopa trial was ineffective. Clinical status remained relatively stable over the next years, and repeated brain MRI failed to detect any alteration. By his twenties he showed a more pronounced cervical and oromandibular dystonia, a subcontinuous “no-no” head tremor, bradykinesia, and generalized rigidity. A slightly wide-based gait became evident. A brain MRI at the age of 23 years revealed isolated vermis atrophy. An extensive work-up, including plasma aminoacids, acylcarnitine on dried blood spot, urinary organic acids, copper metabolism study, cerebrospinal fluid neurotransmitters and pterins, alfafetoprotein, and lysosomal enzyme activities, was normal. Two next generation sequencing panels for genetic epilepsy and genetic movement disorders did not reveal any responsible genetic alteration. Finally clinical exome sequencing (CES) analysis disclosed a de novo c.901T>C (p.Ser301Pro)  variant , predictNeurological examination at the age of 37 revealed bradykinesia, rigidity, a mixed parkinsonian-ataxic gait, cervical and oromandibular dystonia with dystonic head tremor, dystonic posturing of the upper limbs (left > right), and cerebellar speech. He also exhibited saccadic ocular pursuit, saccadic dysmetria, and an ocular fixation instability  and dysarthria was detected in 22 patients ,9,14,16.While ataxia was the most frequent presenting sign in late-onset patients, a variable association of ataxia, neurodevelopmental disorder , and epilepsy characterized the presentation in the early-onset forms.The clinical course in the early-onset cohort was characterized by neurodevelopmental disorders (15/15), ataxia (14/15), and oculomotor disorders (10/15) as main symptoms ,17,18,19Epilepsy was reported exclusively in early-onset patients and occurred in nearly 50% of patients (7 out of 15 early-onset patients) ,16,19. EA pure cerebellar syndrome characterized a relevant number of the late-onset patients 22/53) [3 5,6,8,,14,16. PFixed and progressive ataxia, which was reported in nearly all patients 64/68), included a variable association of gait disturbance, limbs dysmetria, dysdiadochokinesia, cerebellar dysarthria, and swallowing disturbances. The course of ataxia in late-onset patients was usually slowly progressive, with patients bedridden or wheelchaired after 30–50 years of the disease 4/68, inc. In the KCND3 deficiency (39/68 patients). Saccadic smooth pursuit and gaze-evoked-nystagmus (GEN), usually considered expressions of vestibulocerebellar dysfunction [A proper oculomotor performance is strictly dependent on an intact cerebellum ; thus, ifunction ,28, werefunction , describfunction . Slow eyfunction ,8,18. CACNA1A and KCNA1  accounting for the majority of cases [KCND3 mutations have been reported with EA as the main disease feature [EA is a clinically heterogeneous disorder characterized by recurrent spells of ataxia lasting minutes to hours  which haof cases ,29. To d feature ,20. The  feature . Interic feature .A further patient reported by Lee and colleagues presented at the age of 30 years with intermittent diplopia and later suffered from progressive gait ataxia and intermittent cerebellar dysarthria . KCND3 mutations. Cognitive impairment was reported in four out of the nine members of the first family described [Cognitive impairment and neurodevelopmental disorders such as developmental delay (DD), ID and learning disabilities are variably present in patients with escribed . In the escribed ,16,18. Fescribed ,16,18. Nescribed ,9,16. Deescribed ,16. KCND3 deficient patients, occurring in 9 out of 53 late-onset patients and in 3 out of 15 early-onset patients [Movement disorders (MD) were present in 23 out of 68 patients. Myoclonus occurred in five cases, involving limbs, trunk, and abdomen ,16,17. Mpatients . Epilepsy was reported in 7 out of the 15 patients with disease onset during infancy or childhood ,16,19, oEEG abnormalities included: theta waves in the frontal or in both the frontal and the parietal area ,16; spikPyramidal signs including hyperreflexia, ankle clonus, spasticity, and extensor plantar reflex were observed in a minority of patients in both early and late onset group 16/68)  8,9,12,,20. . 20. Peripheral neuropathy was observed only in the late-onset group (five patients) ,9,18. BoCerebellar atrophy was the main finding on brain MRI in 30/35 patients ,17,18,20Neuroradiological findings of 35 literature patients that underwent brain MRI are summarized in KCND3 pathogenetic variants associated with a neurological phenotype have been reported. Among recurrent variants, p.Phe227del was found in four unrelated families [To date, 15 different families , while pfamilies ,14,18. Variants reported as pathogenic affect residues located from the first Kv4.3 extracellular loop p.Lys214Arg)  to the sArg  to t+ current, and increased protein degradation [Electrophysiological and immunohistochemical studies revealed a loss of function pathogenic mechanism leading to aberrant Kv4.3 functioning through different pathways, such as impaired protein trafficking, reduced outward Kradation ,8,13,18.KCND3 gain of function variants previously associated with sudden unexplained death and cardiological syndrome  [Two l392Ile) ,19, werel392Ile)  and in aKCND3 gene deficiency, associated to SCA19/22 [SCA19/22 ,8, is a The pathophysiology underlying this wide spectrum of manifestations is not fully understood.Based on our literature review, we propose two main clinical phenotypes, according to the age at onset.Patients with early onset forms present with neurodevelopmental disorder or epilepsy preceding the onset of cerebellar signs. Movement disorders can emerge later in the disease course in half of them. Late-onset patients show a pure slowly progressive cerebellar ataxia with possible cognitive decline, peripheral neuropathy, parkinsonism, and other movement disorders.PUM1 deficiency and a further Kvs disease due to [KCNC3 gene mutation (SCA type 13) [Such a different age-dependent clinical trajectory was observed in few other genetic neurological disorders presenting with ataxia, including e due to  KCNC3 getype 13) . Inherited neurological channelopathies are known to be causative of a great number of paroxysmal and non-paroxysmal neurological disorders ,33.KCND3 variants presenting with EA associated to interictal ocular motility abnormalities or pyramidal signs [KCND3 patients, EA may anticipate the development of a progressive ataxia as seen in other channelopathies [Our literature review identified two patients with different al signs ,20. Due opathies . KCND3 defects.Our patient showed paroxysmal neurological symptoms in a disease stage when fixed ataxic signs were not yet obvious. Further studies are needed in order to clarify the prevalence, phenomenology, and pathogenic mechanisms of EA and other paroxysmal neurological disorders in KCND3 patients are heterogenous and their prevalence is relatively high. A single post-mortem study revealed normal basal ganglia, except for a pallor of substantia nigra [Movement disorders in ia nigra , and PETia nigra . There iia nigra . The ceria nigra ,37,38. Tia nigra ,40,41,42ia nigra  includinCo-occurrence of dystonia and ataxia, named dystonia-ataxia syndrome, can be caused by more than 100 different genes . Our patThe existence of a neural integrator for the control of head movements, analogous of that in the ocular motor system leading to centripetal drift of the eyes and consequent gaze-evoked nystagmus was proposed by Klier and Colleagues in 2002 . RecentlKCND3 patients are consistent with CCAS. A role for the cerebellum in cognitive and affective functions was first pointed out by the description in adult patients of the cerebellar cognitive affective syndrome (CCAS) . CCAS isKCND3 early-onset patients remarks this concept. Later on, the importance of the cerebellum in neurodevelopmental trajectories was studied in patients with congenital or early acquired cerebellar disorders ,54. The Cognitive decline and ID are non-motor symptoms described in several types of SCAs ,56. A reKCNC3 defect) [KCND3 defect (early-onset presentation). ID was associated to SCA 13 (due to  defect) ,56 and, The association between SCAs and epilepsy is best known in SCA type 10 and 13 ,60; howeEpilepsy in SCA type 10 develops some years after the onset of ataxia . In SCA Brain MRI abnormalities include isolated vermis atrophy, isolated cerebellar hemispheric atrophy, or global cerebellar atrophy and possibly white matter lesions and cerebral atrophy. Interestingly, a number of early-onset patients had a normal brain MRI, with four of them showing ataxia at time of examination ,13,16. IKCND3 variant. Written informed consent was obtained in accordance with institutional review board regulations and protocols to disclose clinical information, neuroimaging, and video recordings. We reported clinical information, genetic data, neuroimaging, and video recordings of a new patient with a novel CES was performed using the Illumina Nextera Trusight One capture kit on the Illumina Miseq Dx platform  with a percentage of aligned sequences: 99.7%; mean region coverage depth: 95.5×; regions covered at least 20×: 91.3%; percentage of bases with Q ≥ 30: 95.9%; data referring to the index patient.http://compbio.charite.de/phenomizer/, last access date: 11 June 2020), and Decipher browser . Variants were filtered for: consequence , metrix (filter PASS), and population frequency . Variants analysis disclosed a never reported missense variant in KCDN3 gene. The sequencing product was aligned to the reference genome (GRCh37-hg19). The variants call, filtering, and prioritization were performed using an in-house pipeline and different tools, including Variant Interpreter (Illumina), Phenomizer browser  was conducted to identify all English-language papers reporting “KCND3 deficiency”, “SCA19”, “SCA22”, and “SCA19/22” were used as search terms ,18,19,20Patients were classified according to the age of onset in two main subgroups: infantile-childhood onset group , including 15 patients, and a juvenile-adult onset group , including 53 patients. Given the partial overlap between childhood and adolescence in terms of age range  and the Detailed clinical, radiological, and genetic data are summarized in KCND3 mutation with neurodevelopmental disorder and prominent dystonia, with a clinical course paradigmatic of the early onset forms. Although the pathophysiological underpinnings were not elucidated, based on our literature review, presentation patterns and disease course differ between early and late onset forms, thus delineating two well differentiated phenotypic trajectories according to the age of onset.In summary, we reported a new patient with


During the beginning of the coronavirus disease 2019 (COVID-19) pandemic, nursing homes were identified as congregate settings at high risk for outbreaks of COVID-19  were included in the analysis. Overall, 13,185 (86%) nursing homes had ≥50 beds, 10,750 (70.1%) were for-profit, and 14,349 (93.5%) had dual Medicare and Medicaid certification . Most nuDuring May 25–November 22, nursing homes reported 572,135 cases to NHSN, 296,762 (51.8%) of which occurred among residents and 275,373 (48.2%) among staff members. Among residents, cases per 1,000 resident-weeks increased during June and July, reaching 11.5 cases per 1,000 resident-weeks (week of July 26), and decreased during August–September . In November, rates increased again, reaching 23.2 cases per 1,000 resident-weeks (week of November 22) . Among shttps://stacks.cdc.gov/view/cdc/99807). During the July peak, rates among residents and staff members in HHS Regions 4, 6, and 9 ranged from 14 to 24 cases per 1,000 resident-weeks. In HHS Regions 5, 7, and 8, rates ranged from 2.5 to 15 cases per 1,000 resident-weeks during August–September and increased again in November, ranging from 32 to 44 cases per 1,000 resident-weeks during the week of November 22.Nursing homes in HHS Regions 1 and 2 reported peak incidences of >10.0 cases per 1,000 resident-weeks among residents and staff members during May or June before rates subsequently declined to <6.0 cases per 1,000 resident-weeks during June–October (Supplementary Figure, During May, population-level COVID-19 rates across the HHS regions ranged from 17 to 67 cases per 100,000 population, and during the July peak, increased to >178 cases per 100,000 in HHS Regions 4, 6, and 9. Rates declined in all HHS regions during August–September and began increasing again in October, with rates in HHS Region 5, 7, and 8 exceeding 615 cases per 100,000 during the week of November 22. For each HHS region, trends in nursing home incidence among residents and staff members were similar to population trends in the surrounding community.There has been a substantial incidence of COVID-19 among nursing home residents and staff since May 2020. Rates of COVID-19 among residents and staff members in nursing homes fluctuated during weeks ending May 31–November 22, with regional and temporal variability; however, trends resembled those in the surrounding communities. These data suggest that increases in community rates might be associated with increases in nursing home COVID-19 incidence and that nursing home mitigation strategies need to include a comprehensive plan to monitor local SARS-CoV-2 transmission and minimize high-risk exposures within facilities. Increased COVID-19 incidence in communities with nursing homes increases the risk for introduction of SARS-CoV-2 by staff members. In Minnesota, ≥34% of high-risk exposures among health care staff members involved nonpatient contacts, including household and social contacts, indicating potential lapses in adherence to mask use and social distancing recommendations during social interactions (The findings in this report are subject to at least four limitations. First, nursing homes reported aggregate weekly data to NHSN, preventing patient-level analysis. Second, reported data were not validated, and trends among nursing homes excluded because of missing data might have differed. Third, the sources of introduction and direction of transmission between residents and staff members could not be determined. Finally, these results might not be generalizable to residents and staff members of other long-term care facilities, such as those for the developmentally disabled and assisted living facilities because this analysis was restricted to nursing homes reporting COVID-19 data weekly, as required by CMS.Nursing homes are high-risk, congregate settings that require a comprehensive infection prevention and control strategy to reduce SARS-CoV-2 entry into the facility and mitigate transmission to prevent severe outcomes. CDC’s nursing home guidance provides tiered recommendations for different phases of a COVID-19 response and should be implemented in addition to CMS regulatory requirements (In the United States, COVID-19 among older adults living in nursing homes is associated with higher rates of severe illness and death.Rates of COVID-19 among nursing home residents and staff members increased during June and July 2020, and again in November. Trends in reported COVID-19 cases among nursing home residents and staff members were similar to trends in incidence of COVID-19 in surrounding communities.Increases in community rates might be associated with increases in nursing home COVID-19 incidence, and nursing home mitigation strategies need to include a comprehensive plan to monitor local SARS-CoV-2 transmission and minimize high-risk exposures within facilities.


Extra virgin olive oil (EVOO) is a major component of the Mediterranean diet and is appreciated worldwide because of its nutritional benefits in metabolic diseases, including type 2 diabetes (T2D). EVOO contains significant amounts of secondary metabolites, such as phenolic compounds (PCs), that may positively influence the metabolic status. In this study, we investigated for the first time the effects of several PCs on beta-cell function and survival. To this aim, INS-1E cells were exposed to 10 μM of the main EVOO PCs for up to 24 h. Under these conditions, survival, insulin biosynthesis, glucose-stimulated insulin secretion (GSIS), and intracellular signaling activation (protein kinase B (AKT) and cAMP response element-binding protein (CREB)) were evaluated. Hydroxytyrosol, tyrosol, and apigenin augmented beta-cell proliferation and insulin biosynthesis, and apigenin and luteolin enhanced the GSIS. Conversely, vanillic acid and vanillin were pro-apoptotic for beta-cells, even if they increased the GSIS. In addition, oleuropein, p-coumaric, ferulic and sinapic acids significantly worsened the GSIS. Finally, a mixture of hydroxytyrosol, tyrosol, and apigenin promoted the GSIS in human pancreatic islets. Apigenin was the most effective compound and was also able to activate beneficial intracellular signaling. In conclusion, this study shows that hydroxytyrosol, tyrosol, and apigenin foster beta-cells’ health, suggesting that EVOO or supplements enriched with these compounds may improve insulin secretion and promote glycemic control in T2D patients. Diabetes mellitus describes a group of metabolic disorders characterized by chronically elevated glycemia. It represents one of the fastest-growing health challenges of the 21st century, with the number of adults living with diabetes having more than tripled over the past 20 years [In its two main forms, diabetes is caused by immune-mediated beta-cell destruction (type 1 diabetes (T1D)) or by the loss of physiological beta-cell functional mass, often concomitant to reduced insulin sensitivity in peripheral insulin-dependent tissues (type 2 diabetes (T2D)).The loss of beta-cell functional mass is a necessary and early condition in the development of T2D [According to recommendations for the management of hyperglycemia in T2D from the American Diabetes Association and the European Association for the Study of Diabetes , lifestyExtra virgin olive oil (EVOO) is a major component of the Mediterranean diet (MedDiet) and is appreciated worldwide because of its nutritional benefits in metabolic diseases, including T2D . In the Traditionally, the high content of monounsaturated fatty acids (MUFAs), particularly oleic acid , was considered to be responsible for the beneficial effects of EVOO . Indeed,Although increasing data support the beneficial role of MedDiet and its components, especially PCs of EVOO, in T2D, the exact mechanisms responsible for these effects are not yet fully understood. Accordingly, in this study we investigated, for the first time, simultaneously but individually, the effects of the main EVOO PCs on beta-cell function and survival.To evaluate the impact of PCs on beta-cell survival, INS-1E cells were treated for 24 h with 10 μM of the most important PCs identified in EVOO. To simplify the presentation of data, each compound was assigned an alphanumeric code .Ki67 . Surprisingly, caffeic acid (C3), vanillic acid (C4), and vanillin (C5) also increased apoptosis levels .Hydroxytyrosol (C1), tyrosol (C2), vanillic acid (C4), and apigenin (C12) significantly increased Ins1) mRNA levels by approximatively 40–50% (p < 0.004 vs. DMSO) without inducing changes in insulin2 (Ins2) mRNA levels. Furthermore, hydroxytyrosol (C1), tyrosol (C2), p-coumaric acid (C6), ferulic acid (C7), sinapic acid (C8), (+)-pinoresinol (C10), oleuropein (C11), and apigenin (C12) significantly augmented insulin content levels to varying extents, ranging from 1.3- to 2.4-fold (p < 0.004 vs. DMSO). In addition, 24 h exposure to PCs did not induce any change in insulin release in the culture medium (Hydroxytyrosol (C1), tyrosol (C2), and apigenin (C12) also increased insulin1 . In contrast, p-coumaric acid (C6), ferulic acid (C7), sinapic acid (C8), and oleuropein (C11) reduced the ability of beta-cells to secrete insulin in response to stimulatory concentrations of glucose , INS-1E cells were treated for 1 h with 10 μM of each PC, cultured with 3 mM  of glucose Krebs–Ringer bicarbonate HEPES buffer (KRBH) for 1 h and then cultured with 25 mM (stimulatory) of glucose KRBH for another hour. Interestingly, vanillic acid (C4), vanillin (C5), luteolin (C9), and apigenin (C12) enhanced the GSIS by ~1.9-, 1.4-, 1.3-, and 1.3-fold, respectively .Furthermore, we evaluated the ability of a mixture of three PCs , 10 μM tyrosol (C2), and 10 μM apigenin C12)) to promote the GSIS in human pancreatic islets. The components of the mixture were chosen for different reasons: while apigenin was the only PC to enhance all observed biological effects in INS-1E cells without influencing apoptosis, hydroxytyrosol and tyrosol, in addition to inducing beta-cell proliferation and insulin biosynthesis, are the only two PCs in EVOO to have already been included in a health claim by the European Food Safety Authority (EFSA) for their ability to protect blood lipids from oxidative stress [) to promAs mentioned above, apigenin was the only phenolic compound to enhance all observed biological effects without influencing apoptosis .p < 0.017 vs. no apigenin).We, therefore, investigated whether apigenin is able to promote the phosphorylation/activation of protein kinase B (AKT) and cAMP response element-binding protein (CREB), which are key in the regulation of beta-cell mass and function ,14. Apigp-coumaric acid, ferulic acid, sinapic acid, (+)-pinoresinol, and oleuropein), and the GSIS . Surprisingly, several PCs also exerted detrimental effects. This was the case for caffeic acid, vanillic acid, and vanillin, which induced beta-cell apoptosis  reduced fasting plasma glucose, hemoglobin A1c (HbA1c) levels, and body mass index (BMI) in overweight T2D patients compared to the consumption of refined olive oil with no PCs . The exa2O2-induced cytotoxicity [Until now, only a few studies have examined the effects of EVOO PCs on beta-cell function and survival. Tyrosol was found to inhibit endoplasmic reticulum (ER)-induced beta-cell apoptosis , and caftoxicity ,23,24,25toxicity ,26,27,28Importantly, in our study, we used commercial standard PCs instead of PCs extracted directly from EVOO. This choice was driven by the need to reveal the specific effect of each individual PC on beta-cell function. In fact, while the ability of EVOO to improve metabolic control in T2D patients is already known ,5,6, thep-coumaric acid, ferulic acid, sinapic acid, and oleuropein). Indeed, we have shown that the same compound can exert both positive and negative effects. For example, vanillic acid and vanillin enhance the GSIS but increase apoptosis levels; similarly, p-coumaric acid, ferulic acid, sinapic acid, and oleuropein increase insulin content but reduce the GSIS. This could be due to the clonal heterogeneity of pancreatic beta-cells. In fact, it has recently emerged that different subpopulations of beta-cells may be differently targeted by insults or treatments, yielding potentially different responses in terms of function, proliferation, and survival [Relevant to this concept, it was recently demonstrated that the consumption of a diet rich in EVOO improves metabolic control and beta-cell survival and function in high-fat-diet-induced diabetes in mice; however, no additional beneficial effects were observed from EVOO containing higher levels of PCs . Accordisurvival .Most studies agree that the beneficial effects of PCs are due to their antioxidant action. Here, we have also shown that apigenin, the only PC able to enhance all observed biological effects without influencing apoptosis, is able to phosphorylate and activate AKT and CREB , which aOur study has some limitations. First, PCs undergo changes through intestinal and hepatic metabolism, in which they are hydrolyzed and later conjugated into their glucuronidated, methylated, or sulphated forms in order to be absorbed and become biologically active . In our In summary, this study shows that hydroxytyrosol, tyrosol, and apigenin, both alone and in combination, may preserve both function and survival of beta-cells, suggesting that EVOO or supplements enriched with these compounds may improve insulin secretion and promote glycemic control in T2D patients.2. For experiments, cells were seeded in 6-well dishes up to 80% confluence.Rat insulin-secreting INS-1E cells  were grown in RPMI-1640 medium with 11.1 mM glucose supplemented with 10% fetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 1% nonessential amino acids . Cultures were kept in a monolayer at 37 °C in a humidified incubator gassed with 5% COHuman pancreatic islets were isolated by collagenase digestion from pancreatic biopsies of non-obese, non-diabetic patients undergoing duodenocefalopancreasectomy (dpc) for Vater’s ampulla tumors. Biopsies were excised and processed with the approval of the regional ethics committee, and informed consent was obtained from each patient. Baseline characteristics of islets donors are reported in p-coumaric acid, ferulic acid, sinapic acid, luteolin, (+)-pinoresinol, oleuropein, and apigenin were all purchased from Sigma-Aldrich . Stock solutions of PCs were prepared in DMSO and stored at −20 °C. Cells were treated with 10 μM PCs, or a mixture thereof, for various times, as indicated. Under control conditions, cells were treated with DMSO only.Standards of hydroxytyrosol, tyrosol, caffeic acid, vanillic acid, vanillin, 2, 1 mM CaCl2, 10% glycerol, 10 mM NaPP, 10 mM NaF, and 4 mM EDTA) supplemented with protease and phosphatase inhibitors . Protein concentration was determined using the Bradford assay . Equal amounts of protein (40 μg) were separated by SDS-PAGE and blotting on polyvinylidene fluoride (PVDF) membranes was performed using a Trans-Blot Turbo Transfer System . Membrane blocking and incubation with primary antibodies were performed with 5% skim milk in Tris-buffered saline with Tween, and then membranes were incubated with primary antibodies overnight at 4 °C, followed by washing with tris-buffered saline (TBS) and incubating with a horseradish-peroxidase-conjugated secondary antibody for 1 h at room temperature. Clarity Western ECL Substrate  was used for visualization of proteins with a Model 3000 VersadDoc Imaging System . The signal intensity of each protein band was then measured using Quantity One Software  and normalized to the corresponding total protein band. Anti-phosphorylated (p)AKT antibody , anti-Akt antibody (4691), anti-pCREB antibody , and anti-CREB antibody (9197) were purchased from Cell Signaling Technology.For protein analysis, cells were lysed in lysis buffer  according to the manufacturer’ instructions.Apoptosis was measured using the Cell Death Detection ELISAGusb (glucuronidase beta) as an internal control.Total RNA was isolated using the RNeasy Mini Kit , with genomic DNA contamination eliminated via DNase digestion . Total RNA (500 ng) was used as a template for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit . Primers were designed using Primer Express 3.0 . Cell lysates were then cleared by centrifugation and frozen at −70 °C for subsequent determination of insulin concentrations (dilution factor 1:1000).2, 1.25 mM MgCl2, 5 mMK H2PO4, 25 mM NaHCO3, 10 mM HEPES, and 0.5% BSA (pH 7.4)) in the presence of PCs and finally cultured for 1 h in 3 mM glucose KRBH buffer and for another hour in 25 mM glucose KRBH buffer. Supernatants were collected and frozen at −70 °C for subsequent determination of insulin concentrations (dilution factor 1:10).For GSIS assessment, INS-1E cells and human pancreatic islets were treated for 1 h with 10 μM PCs or a mixture of 10 μM C1, 10 μM C2, and 10 μM C12. Cells and islets were then cultured for 1 h in 3 mM glucose Krebs–Ringer bicarbonate HEPES buffer .p-value < 0.05 was considered statistically significant) followed by Bonferroni-corrected paired t-test.Data are expressed as means ± standard deviation (SD). At least three independent experiments were performed. Statistical significance was determined by one-way ANOVA (


Salmonella enterica subsp. enterica serotype Enteritidis (SE) is recognized as a major cause of human salmonellosis worldwide, and most human salmonellosis is due to the consumption of contaminated poultry meats and poultry byproducts. Whole-genome sequencing  typing and core genome multilocus sequence typing (cgMLST). All isolates carried aminoglycoside resistance genes, aac(6’)-Iaa, and 56 isolates carried multiple antimicrobial resistance genes. The most frequent virulence gene profile, pef-fim-sop-inv.-org-sip-spa-sif-fli-flg-hil-ssa-sse-prg-pag-spv, was found in 90 isolates. The SNP analysis provided a higher resolution than the cgMLST analysis, but the cgMLST analysis was highly congruent with the SNP analysis. The phylogenetic results suggested the presence of resident SE within the facility of processing plants, environments of slaughterhouses, and the integrated broiler supply chain, and the phylogenetically related isolates were found in retail meats. In addition, the SE isolates from different origins showed close genetic relationships indicating that these strains may have originated from a common source. This study could be valuable reference data for future traceback investigations in South Korea. Salmonella enterica (S. enterica) is one of the most prevalent foodborne pathogens in the world ) . For theGiven the significance and repeated outbreaks of SE in South Korea together with the limited availability of WGS data, we sequenced the whole genome of 96 SE isolates isolated from poultry sources in South Korea during 2010–2017, including retail chicken meats, farms, slaughterhouses, an integrated broiler supply chain, and chicken transporting trucks. The presence of antimicrobial resistance genes, chromosomal mutation, and virulence genes in the SE isolates was investigated using WGS data. Then we conducted a phylogenetic analysis using SNP and cgMLST analysis to investigate the genetic relatedness of SE isolates sequenced in this study.n = 27, 28.1%), followed by pattern 10  and pattern 5 . Among 96 isolates, 56 isolates (58.3%) carried multiple antimicrobial resistance genes -Iaa, and four other different genes including aph(6)-Id , aph(3")-Ib , aac(3)-Iid , and aph(3’)-Ia  were also detected.All 96 isolates carried aminoglycoside resistance genes, sul2  and sul1 , and only one trimethoprim resistance gene, dfrA1, was detected in two (2.1%) isolates.Two sulfonamide resistance genes were found in 55 (57.3%) isolates including blaTEM-1B  and blaCTX-M-15 .Two beta-lactams resistance genes were detected in 56 (58.3%) isolates including tet(A), was detected in 48 (50%) isolates.Only one tetracycline resistance gene, catA2, was found in five (5.2%) isolates.One phenicol resistance gene, gyrA gene, with all being resistant to nalidixic acid and ciprofloxacin ; aspartate/asparagine, D87N in 50 isolates (52.1%). None of the isolates had point mutation n the gyrB, parC and parE genes (data not shown).A total of 75 (78.1%) out of 96 isolates presented point mutation in the floxacin . The nonpef-fim-sop-inv.-org-sip-spa-sif-fli-flg-hil-ssa-sse-prg-pag-spv)  followed by profile 2 (fim-sop-inv.-org-sip-spa-sif-fli-flg-hil-ssa-sse-prg-pag)  and profile 3 (pef-fim-sop-inv.-org-sip-spa-sif-fli-flg-hil-ssa-sse-prg-pag) . All isolates carried fim, sop, inv, org, sip, spa, sif, fli, flg, hil, ssa, sse, prg, and pag, and none of the strains carried sef, sfm, spr, stn, and bss  and E (n = 1) and the slaughterhouse A (n = 1) . Two isolates from the slaughterhouse G and the truck clustered in the group II . Group III included five isolates from the retail chicken meats of brand D (n = 2), slaughterhouse D (n = 1) and E (n = 1), and the hatchery 2 of the integrated broiler supply chain (n = 1) . In group IV, five of six isolates from the retail chicken meats of brand B were clustered together with the isolates from the retail chicken meats of brand D (n = 2) and the slaughterhouse C (n = 5), D (n = 4), and F (n = 1) . In group V, three isolates from the retail chicken meats of brand A clustered together . Group VI contained four isolates from the trucks (n = 3) and a chicken farm (n = 1) , and eight SE isolates from the retail chicken meats of brand A clustered together in the group VII . Group VIII included five isolates from the retail meat chicken meats of brand B (n = 1) and D (n = 3) and the slaughterhouse G (n = 1) . In group IX, seven isolates from chicken farms were clustered together with the isolates from the duck farm (n = 1), the slaughterhouse G (n = 1), and the truck (n = 1) . Group X included 27 isolates from the integrated broiler supply chain (n = 25), duck farm (n = 1) and chicken farm (n = 1) . Among the 26 isolates from the integrated broiler supply chain, 25 isolates were clustered together into group X.In SNP phylogeny , group IThe results of cgMLST analysis showed that the clustering of the isolates resembled that of the SNP analysis, such as the SNP group I, III, VIII, IX and X . HoweverSalmonella isolates from different origins, such as humans, food, animals or environmental samples in previous studies [WGS analysis has been used to investigate genetic characteristics, and phylogenetic relationships among  studies ,14,17,25Salmonella spp. [Salmonella spp. from poultry sources, in that there was high resistance to streptomycin, nalidixic acid, sulfisoxazole, and cephalothin, found in previous studies from South Korea [Salmonella spp. in the Korean poultry production food supply chain is concerning due to the potential transmission to humans. High resistance to antimicrobials and multidrug resistance of Salmonella spp. from poultry sources were also reported in other countries [Salmonella spp. from raw chicken meat in Malaysia [et al.’s study [Salmonella isolates recovered from commercial farms were simultaneously resistant to ampicillin, amoxicillin–clavulanic acid, ceftiofur, cefoxitin, and ceftriaxone [Several previous studies reported that phenotypic resistance and genotypic resistance correlated highly in lla spp. ,17,26,27lla spp. ,15,17,27th Korea . The finountries ,30. For Malaysia . In Zhou’s study , among 1triaxone .Salmonella isolates recovered from the animal, food, and/or environmental sources contained the same virulence genes as carried by human clinical isolates, and these findings highlight the pathogenic potential of Salmonella causing disease in humans via contaminated food.A total of 90 SE isolates (93.8%) from different poultry sources showed the same virulence gene profile involved in adhesion, invasion, cell survival, and virulence plasmid genes. However, neither enterotoxin-producing genes nor biofilm regulator genes were detected in the strains tested. In previous studies ,17,34, SMost SE isolates from the same origin phylogenetically clustered together even though sampling dates and sampling locations of the samples were different in this study. For example, the SE isolates from the retail chicken meats of brand A collected in April, July and August in 2011 clustered together in the group VII, and the SE isolates from the evisceration rooms, chilling rooms, and carcasses in slaughterhouse C all clustered together in the group IV . Togetheenterica [SE isolates from the different stages in the integrated broiler supply chain, such as grandparent stocks, parent stocks, hatcheries, and farms clustered together in the group X . Interesenterica . Taken tThe SE isolates from different origins showed close genetic relationships, and epidemiological or traceback evidence was provided to establish the connection between these SE isolates in the identical groups. In group IX, two SE isolates (KR 97 and KR 101) collected in the same year, 2012, from the slaughterhouse G and the truck which transported culled chickens to the slaughterhouse G were grouped together . TherefoConsistent with previous studies ,19, bothThe 96 SE isolates sequenced for this study were selected from SE isolates collected from poultry sources in South Korea between 2010 and 2017. The 83 isolates were isolated by Konkuk University in Seoul, South Korea, during 2010–2017, and 13 isolates were kindly provided by the Korean National Animal and Plant Quarantine Agency (APQA) in Gimcheon-si, South Korea .Salmonella isolation, 200 mL of buffered peptone water (BPW)  was added to the swab samples in sterile plastic jars with caps and incubated at 37 °C for 18–24 h. For the carcass rinse samples, rinsed BPW was transferred to a sterile plastic jar with a cap and incubated at 37 °C for 18–24 h for pre-enrichment. For selective enrichment, 100 µL of enriched BPW broth culture was transferred to 9.9 mL of Rappaport–Vassiliadis broth (Difco Laboratories) and incubated at 42 °C for 24–48 h. Following incubation, the Rappaport–Vassiliadis broth enrichment cultures were streaked onto xylose lysine deoxycholate agar (Difco Laboratories) and brilliant green agar (Difco Laboratories), and the plates were incubated overnight at 37 °C. Two presumptive Salmonella colonies from each sample were transferred to MacConkey agar (Difco Laboratories). Colonies with a positive result were confirmed by Salmonella-specific PCR, as previously described [For escribed . Serotypn = 19), two hatcheries (n = 2), two parent stocks (n = 2), and two broiler farms (n = 3).Those isolates included 28 isolates from retail chicken meats of five different brands (A to E), 17 isolates from six chicken slaughterhouses (A to G), four isolates from two duck slaughterhouses, 37 isolates from hatcheries and chicken farms, two isolates from duck farms, and eight isolates from chicken transporting trucks. Among 96 SE isolates, 26 SE isolates were from the integrated broiler supply chain A; two grandparent stocks . The hatchery and farm isolates were from environmental samples , carcasses, livers, feces, cloaca swabs, eggshells, and fluff.Sample DNA concentrations were determined using a Qubit BR dsDNA assay kit . Concentrations were diluted to 0.2 ng/µL, and libraries were prepared following the Illumina Nextera XT DNA library prep kit  reference guide with the following exceptions. Forty microliters of PCR product transferred to a new MIDI plate, 20 µL of AMPure XP beads  were added to each well and incubated at room temperature for 5 min without shaking. After 80% ethanol washes, beads were allowed to air dry for 12 min. Then beads were resuspended in 53 µL of RSB and incubated at room temperature for 2 min without shaking.The concentration of sample libraries was determined using the Qubit dsDNA HS assay kit , and libraries were diluted to a 2 nM concentration and combined in equal volumes to form the pooled library. The pooled library was denatured to obtain a 10 pM final library following the Illumina Denature and Dilute Libraries Guide-Protocol A. Six hundred µl of the denatured 10 pM library was loaded into the MiSeq reagent cartridge.Trimmomatic  was usedSalmonella serotype from raw sequencing reads.The trimmed reads were assembled using SPAdes  with thegyrA, gyrB, parC, and parE genes were determined using assembled genomes and ResFinder 3.2  with settings of a threshold of 90%, and a minimum length of 60% [The presence of acquired antimicrobial resistance genes as well as chromosomal mutations in the h of 60% .https://patricbrc.org/app/Annotation) [sef, pef, sfm and fim), invasion effectors , host cell survival effectors , virulence plasmid gene (spv), enterotoxin-producing gene (stn) and biofilm regulator (bss) [All genomes were annotated using the RASTtk algorithm  using thotation) . Proteinor (bss) .https://itol.embl.de/).High-quality SNPs were identified using CFSAN SNP Pipeline v2.0.2 using default quality filters . Specifihttps://cge.cbs.dtu.dk/services/cgMLSTFinder/) using the scheme developed by EnteroBase Salmonella database (https://enterobase.warwick.aC.uk) that considers a total of 3002 loci. The minimum spanning tree based on allelic profiles was created using GrapeTree version 1.5.0 [For cgMLST, raw sequence data files of the isolates were uploaded to cgMLST Finder 1.1 of the Center for Genomic Epidemiology (on 1.5.0 .Salmonella in the poultry industry, from farm to retail chicken meat, in conjunction with epidemiology and traceback findings. In addition, this study could be valuable reference data for future traceback investigations in the poultry industry in South Korea.In conclusion, this study demonstrated the application of WGS to predict antimicrobial resistance, detect virulence genes, and assess the phylogenetic relationship among SE isolates from different poultry sources in South Korea. In addition, both SNP and cgMLST analyses were highly congruent. Our results demonstrated that WGS is a powerful tool for investigating the epidemiology of