This study aimed to reveal the mechanism by which long noncoding RNAs (lncRNAs) modulate hepatocellular carcinoma (HCC) by regulating mRNA via the N6-methyladenosine (m6A) enzyme. The expression and clinical data of 365 HCC patients and 50 healthy control samples were downloaded from the the Cancer Genome Atlas (TCGA) database. Differentially expressed lncRNAs (DElncRNAs) and differentially expressed mRNAs (DEmRNAs) screened using limma packages from the R. m6A2Target database were used to predict the relationship between m6A enzyme-lncRNA and m6A enzyme-mRNA. The mRNAs in the lncRNA-m6A enzyme-mRNA network were subjected to enrichment analysis. Cox regression analysis was used to screen for RNAs significantly related to HCC prognosis. The expression of differentially expressed RNAs (DERs) was verified using the TCGA dataset and GSE55092. Eighty-five DElncRNAs and 747 DEmRNAs were identified. The mRNAs in the lncRNA-m6A enzyme-mRNA network were primarily involved in mitotic cell division, the p53 signaling pathway, and the cell cycle. Three lncRNAs and 14 mRNAs were significantly associated with HCC prognosis. Furthermore, the expression of 12 DERs differed significantly between patients in the early and advanced stages. LINC00665 was predicted to regulate 11 mRNAs by modulating IGF2BP1, IGF2BP2, and YTHDF1. Thus, this study provides new insights into the roles of lncRNA and m6A enzymes in HCC. HCC usuN6-methyladenosine (m6A) accounts for approximately 50% of ribonucleotides in mammals . M6A metLong noncoding RNAs (lncRNAs) are functionally diverse species of noncoding RNA. Studies have demonstrated that lncRNA expression may be closely related to cancer development ,12. SeveBased on The Cancer Genome Atlas (TCGA), RNA-seq expression dataset, and m6A enzyme-targeting gene database, this study aimed to reveal how lncRNAs play a role in HCC by regulating m6A regulators to further influence mRNA .22.1https://gdc-portal.nci.nih.gov/) on April 28, 2021. A total of 423 samples were included, comprising 365 tumor samples with clinical information and 50 healthy control samples. The detected lncRNAs and mRNAs were annotated according to the annotation information of the detection platform .HCC gene expression data were downloaded from the TACG database  of R3.6.1  with a f2.3p <0.05 and |PCC| >0.5. Furthermore, the lncRNAs-m6A enzyme-mRNAs comprehensive network was constructed by combining the DElncRNAs-m6A enzyme and DEmRNAs-m6A enzyme network. All the networks constructed in this study were visualized using Cytoscape  software (http://www.cytoscape.org/).First, the m6A2Target database  was usedFinally, significant DEmRNAs in the comprehensive network underwent gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using DAVID  ,19. Term2.4Univariate and multivariate Cox regression analyses in the survival package (version 2.41-1)  of R3.6.2.5p value less than 0.05 was selected as the threshold for screening independent prognostic clinical factors.Independent prognostic clinical factors were screened using univariate and multivariate Cox regression analyses in the survival package (version 2.41-1) of R3.6.1. A log-rank t-test in R3.6.1.Based on the screened independent prognostic clinical factor information, the samples were divided into different clinical factor feature groups, and the variations in the expression levels of the DERs screened in the previous step were analyzed using the between groups 2.6The expression values of RNAs with significantly different expression levels in different independent prognostic clinical factors screened in the previous step were extracted from the TCGA dataset to analyze the expression differences of these RNAs between tumor samples and normal samples. GSE55092 was subsequently downloaded from the National Center for Biotechnology Information Search database Gene Expression Omnibus database to analyze the expression of DERs. A total of 49 HCC tumor samples and 91 control samples were included in the GSE55092.33.1After annotation, 1,241 lncRNAs and 14,164 mRNAs were identified. A total of 85 DElncRNAs and 747 DEmRNAs were screened between the tumor and control groups . Thereaf3.2m6A enzyme-related lncRNAs were intersected with DElncRNAs, following which 53 DElncRNAs related to m6A enzyme and 12 m6A enzyme enzymes were identified . This neIn conjunction with the results of the above analysis, a DElncRNAs-m6A enzyme-DEmRNAs network was constructed . Moreove3.3p < 0.05; From the DElncRNAs and DEmRNAs included in the comprehensive network – along with clinical prognosis information of TCGA samples – univariate Cox regression analysis was used to screen the DERs significantly associated with survival prognosis. A total of 83 DERs significantly associated with prognosis were screened, including 7 lncRNAs and 76 mRNAs (Table S1). Multivariate Cox regression analysis was subsequently performed on these 83 DERs for independent prognostic-related DERs. Finally, 17 significantly independent prognosis-related DERs were obtained . Patient3.4Univariate and multivariate Cox regression analyses revealed that the pathologic stage was an independent prognostic clinical factor . Consequp < 0.05) – including LINC00665, chromobox 2 (CBX2), cyclin A2 (CCNA2), cyclin B1 (CCNB1), centromere protein F (CENPF), FLVCR heme transporter 1 (FLVCR1), Fraser extracellular matrix complex subunit 1 (FRAS1), kinesin family member 20A (KIF20A), recQ like helicase 4 (RECQL4), solute carrier family 7 member 6 (SLC7A6), SPC24 component of NDC80 kinetochore complex (SPC24), and ZW10 interacting kinetochore protein  were screened and observed to be expressed significantly differently in various groups. LINC00665 plays a role in cancer development. An earlier study has reported that LINC00665 may be involved in HCC cell cycle regulation, and the high expression of LINC00665 in HCC patients is significantly associated with poor prognosis . This fiutophagy . HoweverCCNA2 and CCNB1 [CCNA2 and CCNB1 via the m6A enzyme. The protein encoded by CCNA2 is a cell cycle regulator [CCNA2 has been proven to be a target of miRNAs that regulate the proliferation of HCC cells [CENPF was identified as a potential prognostic biomarker and target for HCC [RECQL4 [SPC24 [KIF20A [ZWINT was associated with great prognosis in patients with HCC after surgery [Based on our hypothesis, LINC00665 could regulate 11 mRNAs by modulating the m6A enzymes IGF2BP1, IGF2BP2, and YTHDF1. In lung adenocarcinoma tissues, the expression of LINC00665 is closely related to the aggressive clinicopathological characteristics of patients and can be used as an independent predictor of relapse-free survival . CIP2A-Bnd CCNB1 . Furtheregulator . CCNA2 hCC cells . MoreoveCC cells ,32. In tCC cells . High exCC cells . CENPF w for HCC . It is r [RECQL4 36, SPC24 [SPC24 , and KIF [KIF20A  could pr surgery .http://m6a2target.canceromics.org) is the most comprehensive database of target genes corresponding to the three types of m6A enzymes  so far. In this database, the validated targets module contains experimentally verified relationship pairs between m6A enzymes and target genes, whereas the Potential Targets module stores the relationship pairs between m6A enzymes and target genes obtained through high-throughput sequencing data analysis. Several studies have confirmed that there was a direct interaction between the m6A enzyme and mRNA using eCLIP-seq [in vivo and in vitro experiments in the future.m6A2Target database (CLIP-seq ,41. Howe


Patients with stenosis at C2/C3 (n = 2) and C3/C4 (n = 6) presented a similar pattern, not reaching significance. Gender was a significant predictor of higher spinal cord dynamics among men with stenosis at C5/C6 (p = 0.048) and C6/C7 (p = 0.033). Age and severity of stenosis did not relate to spinal cord motion. Thus, the data demonstrates focally increased spinal cord motion depending on the specific level of stenosis. Gender-related effects lead to dynamic alterations among men with stenosis at C5/C6 and C6/C7. The missing relation of motion to severity of stenosis underlines a possible additive diagnostic value of spinal cord motion analysis in DCM.In degenerative cervical myelopathy (DCM), focally increased spinal cord motion has been observed for C5/C6, but whether stenoses at other cervical segments lead to similar pathodynamics and how severity of stenosis, age, and gender affect them is still unclear. We report a prospective matched-pair controlled trial on 65 DCM patients. A high-resolution 3D T2 sampling perfection with application-optimized contrasts using different flip angle evolution (SPACE) and a phase-contrast magnetic resonance imaging (MRI) sequence were performed and automatically segmented. Anatomical and spinal cord motion data were assessed per segment from C2/C3 to C7/T1. Spinal cord motion was focally increased at a level of stenosis among patients with stenosis at C4/C5 ( The anatomical degenerations of the cervical spine, which may lead to the syndrome of degenerative cervical myelopathy (DCM) are well established  ,2,3,4. WRecent findings based on phase-contrast MRI (PC-MRI) have demonstrated significantly increased craniocaudal spinal cord motion among patients with degenerative cervical myelopathy (DCM) at the most commonly affected segment C5/C6 ,10,11,12n = 12) ) [(C2/3 − C6/C7) ÷ ptp-amplitudeC7/T1]). The segments C2/C3 and C7/T1 are both suitable as references, as both have been reported to represent similar dynamics in healthy controls )  and corrcontrols ,12. A crcontrols ,12, refe2), spinal canal CSA (mm2), and the adapted maximum canal compromise  ÷ ]) reflecting the severity of the individual’s spinal stenosis unrelated to body size [The following anatomical parameters were automatically computed within the post-processing pipeline per segment: spinal cord CSA  has beep was required to be <0.05 to assume significance.Statistical analysis was conducted by SPSS Statistics® . Data is given as mean and standard deviation (SD). Quantitative data of patients and controls were compared segment by segment. Comparison of two groups, unrelated values, was conducted upon data distribution analysis (Shapiro-Wilk): normally distributed data was compared via t-test, non-normally distributed data via Mann–Whitney U-test. Comparison of multiple related variables was calculated via bonferroni-adjusted analysis of variance (ANOVA) with repeated measurements upon validation of distribution and sphericity; outliers were not excluded. Comparison of multiple unrelated variables was performed via Kruskal–Wallis Test. Prediction models were rated by multiple linear regressions upon validation of standard premises. Outliers were excluded if identified by two methods . Age did not differ between any other group of patients. Comparison of gender between groups showed no significant difference (p = 0.18–0.84), with exception of patients with stenosis at C3/C4 (100% men). Duration of the heartbeat was comparable between all groups of patients (p = 0.85) and between patients and controls (p = 0.25–0.78). The comparison of the mJOA score between groups of patients was not significant .Age, mJOA score, number of patients receiving decompressive surgery, aSCOR, and aMCC per group are listed in p = 0.026, C3/C4 p = 0.022, C4/C5, C5/C6, C6/C7 p < 0.001, respectively). The aMCC among patients with stenosis at C4/C5 (2.97 ± 0.2) was significantly higher compared to patients with stenosis at C3/C4  and at C5/C6 . The aMCC between other groups did not differ.As expected, the aMCC at the stenotic level was significantly higher per group compared to controls , but within patients with stenosis at C2/C3 (p = 0.16).aSCOR was expectedly significantly higher among patients (p > 0.5)  .p = 0.007; group C5/C6: 13.44 ± 6.4 mm/s vs. 7.89 ± 3.3 mm/s, p < 0.001; group C6/C7: 17.69 ± 7.5 mm/s vs. 7.31 ± 3.7 mm/s, p = 0.001). Similarly, the cranial and caudal adjacent segments showed significantly, or borderline significantly increased spinal cord dynamics .Spinal cord motion at level of stenosis was significantly higher among patients with stenosis at C4/C5, C5/C6, and C6/C7  at stenosis were significantly increased among patients with stenosis at C4/C5, C5/C6, and C6/C7 .p = 0.011), highlighting a primarily caudal compression of the spinal cord tissue  or  were calculated within all suitable groups of patients with stenosis at C3/C4, C4/C5, C5/C6, and C6/C7. Gender as a predictor could not be analyzed among patients with stenosis at C3/C4 (100% men).2 = 0.933, p = 0.042), gender being the only significant predictor (p = 0.033).aMCC, aSCOR, age, and mJOA score did not reach significance within any prediction models. One model  significantly predicted the C2-pAI at stenosis among patients with stenosis at C6/C7 , and of higher spinal cord strain among men in group C6/C7 .Gender was a significant predictor of higher spinal cord motion at stenosis among men in group C5/C6  showed significantly lower values. At the level of stenosis, spinal cord motion did not differ between genders. There was no significant difference of age, HB, mJOA score, spinal canal CSA, aSCOR, or aMCC between men and women per group with stenosis at C4/C5, C5/C6, or C6/C7, nor between men or women with stenosis at C5/C6 and men with stenosis at C6/C7.In contrast, men with stenosis at C6/C7 revealed atypically decreased spinal cord motion at segments cranial to the stenosis. Comparison to matched controls , the total displacement—a parameter comprising information of the entire velocity curve—remained similar between genders. As the mean duration of the heartbeat was not different between men and women, a lower peak but with similar total displacement indicated a flattened peak and a prolonged sinusoidal spinal cord motion curve over one heartbeat among women. This finding is complementary to the recently described spinal cord motion curve pattern among DCM patients by Hupp et al. In contrast to controls with a short, singular spinal cord oscillation within the heart cycle, DCM patients showed an ongoing spinal cord motion during the entire heart cycle . Among wAs an unexpected, possibly gender-related effect, a uniquely altered spinal cord motion pattern was observed among men with stenosis at C6/C7: Spinal cord motion was significantly slower at segments cranial to the stenosis followed by a vast acceleration at stenosis. Sufficient explanation for this observation cannot be concluded based on the currently assessed data , that did not differ between genders or groups. As the assessed parameters on spinal canal anatomy and age did not significantly differ between genders, possible compensatory mechanisms as elasticity of meninges leading toward differences of volume–compensation within the subarachnoid CSF-space may play a role. The possibility of gender effects within DCM is underlined by significantly worse functional outcome of men undergoing decompressive surgery, which has been recently reported .n = 19) and multisegmental (n = 36) stenoses based on validated analysis procedures was recently published by Hupp et al. [The only other trial on spinal cord motion across all cervical segments in 55 DCM patients with mono-  and sub-cohorts. This is mostly due to the known contribution of most to least affected segments in DCM, but also to the exclusion of multisegmental relevant stenoses. At the current state of basic research and the general aim of an understanding of intrathecal dynamics, we chose to limit the possible confounding effect of multisegmental stenosis.Effects on (yet) non-symptomatic spinal stenosis were not a topic of this study and should be investigated in further longitudinal trials. Differences in spinal canal degeneration, e.g., rather soft disc herniation vs. solid ossification of ligaments, and their possible influence on spinal cord dynamics were not systematically addressed. The study does not include a full analysis of physiological relations , nor associations to clinical function due to primary aims on pathophysiological ground research. Due to small group size, we cannot sufficiently analyze the association of clinical impairment to spinal cord motion based on the current data. The mJOA score reflects the range of patients included within the presented data, but it does not comprise details on mild spinal cord affection. First, more refined and reliable clinical and electrophysiological assessments are needed to assess the many aspects of DCM (e.g., reliable light-touch, pin-prick testing as part of the International Standards for Neurological Classification of Spinal Cord Injury  evoked pThe presented data presents focally increased spinal cord motion depending on the affected cervical stenotic segment among DCM patients. There is proof of an overall disarranged dynamic behavior resulting in mechanical strains on spinal cord tissue across the cervical spine. Men and women show significantly different spinal cord motion patterns depending on the affected cervical segment, thus indicating gender-related differences in DCM.


NR3C1 transcription and/or translation is strongly associated with GC resistance, but the underlying molecular mechanism and the clinical value of NR3C1 alterations with GC resistance in ALL treatment remain unclear. This study applied panel sequencing to 333 newly diagnosed and 18 relapsed ALL samples to characterize the link between NR3C1 and ALL further. We identified NR3C1 mutations in three patients with newly diagnosed ALL (0.9%) and two patients with relapsed ALL (11.1%). Functional analyses revealed that four of these five NR3C1 mutations  were loss-of-function (LoF) mutations. A drug sensitivity test further showed that LoF NR3C1 mutations influence GC resistance. Saturated mutagenesis of hotspot R477 demonstrated the importance of this residue for NR3C1 function. The dominant-negative effect of p. R477C and p. R477S and the non-dominant negative effect of p. R477H and p. Y478C suggests multiple mechanisms underlying GC resistance. Thus, primary or acquired genomic lesions in NR3C1 may play a critical role in GC resistance and contribute to ALL treatment failure and/or relapse.Treatment outcomes in children with acute lymphoblastic leukemia (ALL) have been improved substantially, with a cure rate exceeding 80% using conventional therapy. However, the outcome for patients with relapsed/refractory ALL remains unsatisfactory, despite the fact that these patients generally receive more intense therapy. Glucocorticoid (GC) resistance is a leading cause of treatment failure and relapse in ALL. Abnormal  Acute lymphoblastic leukemia (ALL), a highly aggressive hematologic malignancy, is the most common cancer in children. After a 60 years' endeavor, ALL is now considered a highly curable cancer in children with contemporary therapeutic regimens . Howeverin vitro is associated with unfavorable outcomes  are the most effective and frequently used agents for childhood ALL. They are essential drugs not only for ALL but also for other lymphoid malignancies . Variousoutcomes .IKZF1) (BTG1) (TBL1XR1) (CREBBP) (NR3C1). Among these, NR3C1 mutations are the most frequent genetic events contributing to GC resistance , functions as a transcription activator by binding to glucocorticoid response elements that are mainly found in the promoters of glucocorticoid-responsive genes. Thus, the disruption of GR expression and function influences the therapeutic effect of GCs in patients with ALL. Abnormalities in NR3C1, such as decreased expression and loss of protein function, contribute to the differential expression of alternatively spliced and translated GR isoforms and are the key to GC resistance  related to ALL relapse and GC resistance (NR3C1 mutations in ALL relapse may drive the generation of novel therapies.The advent of next-generation sequencing (NGS) has contributed to the identification of genetic factors related to GC resistance in ALL, including mutations involved in IKAROS family zinc finger 1 (IKZF1) , BTG Ant) (BTG1) , TBL1X RTBL1XR1) , CREB bi(CREBBP) , and nucsistance . NR3C1, sistance . Li et asistance . HoweverNR3C1 mutations in primary and relapsed ALL samples from patients enrolled in the CCCG-2015-ALL study in our hospital. The results may drive the improvement of clinical outcomes and help produce a generation of novel therapies for ALL.In this study, we screened and functionally examined N = 333) and relapsed (N = 18) B-ALL were enrolled. This study was approved by the Ethics Committees of Guangzhou Women and Children’s Medical Center  and was conducted in accordance with the ethical guidelines of the Declaration of Helsinki. According to institutional guidelines, informed consent was obtained from the parents or guardians.Patients with newly diagnosed (DUX4-IGH rearrangement), and REH cells (ETV6-RUNX1 rearrangements) were purchased from the American Type Culture Collection . 293T and Nalm6 cells were maintained in Dulbecco’s Modified Eagle Medium and RPMI1640  supplemented with 10% fetal bovine serum . The LookOut® Mycoplasma PCR Detection Kit was used to  to confirm the mycoplasma-free status of tested cell line in this study.293T cells, Nalm6 cells  with the primers listed in The full-length NR3C1 was ectopically overexpressed in Nalm6 cells (DUX4-IGH rearrangement) and REH cells (ETV6-RUNX1) by lentiviral transduction. Briefly, lentiviral particles were spun with 12 ng/ml polybrene for 2 hat 1,200 g, 4°C. Fourty-eight hours later, GFP+ cells were sorted using FACSAria™ Cell Sorter (BD) and the transduction was confirmed by western blot  or DEX  for 72 h. Cell viability was measured by the 3--2,5-diphenyltetrazolium bromide (MTT) assay using a Multiscan MS spectrophotometer . Experiments were performed in triplicate and repeated at least three times.Full-length NR3C1 cDNA was amplified and cloned into the cL20c-IRES-GFP lentiviral vector, and mutations were generated using Q5 Site-Directed Mutagenesis Kit  with primers listed in ern blot . All staGAPDH was used as an internal control. Primers are listed in Total RNA was extracted using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s protocol, and 1 μg of RNA was reverse transcribed into cDNA using the Invitrogen Second Strand cDNA Synthesis Kit with random hexamers . qRT-PCR was performed using the ABI Prism 7900HT Detection System  with FastStart SYBR Green Master Mix (Roche). https://www.R-project.org). All tests were two-sided Student’s t-tests. A p value less then 0.05 was considered statistically significant.Data are presented as mean ± SD. All statistical analyses were performed using GraphPad Prism  and R  were identified among 333 patients with newly diagnosed ALL. In the relapsed cohort, 2 of 18 patients (11.1%) had NR3C1 mutations (p.R477H and p. Y478C)  domain, DNA-binding domain (DBD), and ligand-binding domain (LBD).A total of 466 children with ALL were enrolled from March 2015 to June 2020 in the CCCG-ALL-2015 study of Guangzhou Women and Children’s Medical Center, including 18 patients with relapsed ALL. Among these 466 patients, 333 diagnostic DNA samples were available. The baseline characteristics of the ALL cohort are summarized in reatment . To syst project  and cBio project , as summNR3C1 mutations in GCs response, we cloned wild-type NR3C1 and its mutant forms, and evaluated the effects of mutations on DEX-induced NR3C1 transcriptional activity. First, we applied the glucocorticoid response element (GRE)-reporter assay to examine the effects of NR3C1 mutations on DEX-induced GRE activity. As shown in NR3C1 mutants by lentiviral transfection and tested the effect of mutations on the drug response to GCs. In accordance with the results for transcriptional activity, prednisolone resistance was identified in Nalm6 cells with NR3C1 mutants (50) for the NR3C1-Mutant  were 80.3, 84.1, and 19.8 µM, respectively, while the IC50 value of prednisone for wild-type Nalm6 cells was only 15.8 µM  and evaluated gene expression levels before and after prednisolone treatment. As shown in BCL2 and MCL1 were significantly up-regulated in Nalm6 cells with dominant-negative mutations  but not in cells with non-dominant-negative mutations (p.R477H and p. Y478C). However, the pro-apoptotic genes BAX, BAK, BOK, BIM, BID, and PUMA were down-regulated in the dominant-negative group, suggesting increased anti-apoptosis and decreased pro-apoptosis mediate NR3C1 mutation-induced GC resistance  were non-dominantly negatively down-regulated. Therefore, mutations in the DBD of NR3C1 can affect GC resistance via two mechanisms: dominant-negative regulation and non-dominant-negative regulation. Further investigation using RNA-seq and ChIP-seq technique is warranted to decipher the precise regulatory mechanisms and confirm our current findings.A previous study has shown that GC resistance can be caused by the intrinsic inhibition of apoptosis . The selNR3C1 mutations have functional effects; however, the R477 amino acid residue is critical for normal function. NR3C1 mutations are more frequent in ALL cases with relapse than in newly diagnosed cases. In the future, deep sequencing to trace these acquired genetic mutations, permitting the early detection and treatment of relapse, may be warranted as another means to improve clinical outcomes.In addition to somatic IKZF1, BTG1, TBL1XR1, and CREBBP lesions, NR3C1 also contributes to GC resistance and may contribute to relapse in children with ALL. Not all ALL-related


Endometrial cancer (EC) is characterized by high estrogen levels unopposed by progesterone. Treatment with progestins is standard for early EC, but the response to progestins is dependent on progesterone receptor (PGR) expression. Here, we show that the expression of PGR in endometrial epithelial cells is dependent on ARID1A, a DNA-binding subunit of the SWI/SNF chromatin-remodeling complex that is commonly mutated in EC. In endometrial epithelial cells with estrogen receptor overexpression, we find that ARID1A promotes estrogen signaling and regulates common gene expression programs. Normally, endometrial epithelial cells expressing estrogen receptors respond to estrogen by upregulating the PGR. However, when ARID1A expression is lost, upregulation of PGR expression is significantly reduced. This phenomenon can also occur following the loss of the SWI/SNF subunit BRG1, suggesting a role for ARID1A- and BRG1-containing complexes in PGR regulation. We find that PGR is regulated by a bivalent promoter, which harbors both H3K4me3 and H3K27me3 histone tail modifications. H3K27me3 is deposited by EZH2, and inhibition of EZH2 in the context of ARID1A loss results in restoration of estrogen-induced PGR expression. Our results suggest a role for ARID1A deficiency in the loss of PGR in late-stage EC and a therapeutic utility for EZH2 inhibitors in this disease. Endometrial cancer (EC) is the most commonly diagnosed gynecologic malignancy, with an estimated 66,570 new cases diagnosed in 2021 . EC is dThe functions of the endometrium are highly regulated through steroid hormone signaling. During the menstrual cycle, estrogen, through estrogen receptor α (ESR1), promotes proliferation of the endometrium, while progesterone inhibits estrogen-induced proliferation and promotes secretory differentiation through progesterone receptor (PGR) . ESR1 anIn several endometrial pathologies, including EC and endometriosis, the balance between estrogen signaling and progesterone signaling becomes perturbed, either through increased reliance on estrogen or diminished responsiveness to progesterone ,10. In tPGR. We further show that a subset of estrogen and ARID1A co-regulated gene promoters contain histone post-translational modifications associated with bivalent chromatin. Bivalent chromatin-containing genes are repressed by the polycomb repressive complex 2 (PRC2) yet remain poised for transcriptional activation [PGR gene repression. Our results suggest that when ARID1A is mutated, estrogen-dependent transcription of the critical estrogen target gene, PGR, does not occur because of PRC2 activity at the PGR locus, resulting in progesterone resistance and increased EC cancer risk.Genetic factors are associated with increased EC risk, including somatic mutations in ARID1A, a DNA-binding subunit of the SWI/SNF chromatin-remodeling complex. ARID1A is mutated in up to 16% of CAH and 40% of EC, and ARID1A mutations are more frequent in women diagnosed with EC at a younger age ,19,20,21tivation . Drug inESR1 plasmid from the Precision LentiORF Collection  and grown under Blasticidin conditions. Stable expression was confirmed by western blot.12Z immortalized human endometrial epithelial cells  were mai12Z-ESR1 cells were seeded at a density of 40,000 cells/mL DMEM/F12 media without phenol red supplemented with 10% FBS and 1% L-glutamine. The following day, the media was refreshed, and cells were transfected with 50 pmol/mL of siRNA  using the RNAiMax  lipofectamine reagent according to the manufacturer’s instructions at a ratio of 1:1 volume:volume in OptiMEM . After 24 h, the media was replaced with DMEM/F12 media without phenol red supplemented with 10% charcoal-stripped FBS 1% L-glutamine. The following day, the media was replaced with DMEM/F12 media without phenol red supplemented with 0.5% charcoal-stripped FBS, 1% L-glutamine containing either 10 nM β-Estradiol  or 0.1% ethanol vehicle.Protein lysates were quantified using the Micro BCA Protein Assay Kit (ThermoFisher) and a FlexSystem3 plate reader. Protein lysates were run on a 4–15% gradient SDS-PAGE gel  and transferred to the PVDF membrane using the TransBlot Turbo system (BioRad). Primary antibodies were used at the following dilutions: 1:1000 ARID1A (D2A8U) ; 1:1000 β-Actin ; 1:1000 PGR ; 1:1000 ESR1 , 1:100 BRG1 ; 1:1000 ARID1B . Horseradish peroxidase (HRP) conjugated secondary antibodies  were used at a dilution of 1:2000. Clarity Western ECL Substrate (BioRad) was used for protein band visualization, and western blot exposures were captured using the ChemiDoc XRS+ imaging system (BioRad). Uncropped western blots are shown in RNA samples were collected 72h-post siRNA transfection using the Quick-RNA Miniprep Kit . cDNA was synthesized from RNA, and qRT-PCR was performed using PowerUp SYBR Green Master Mix  and the Applied Biosystems  ViiA7 real-time PCR system. ® dsDNA System , and Kapa Illumina Library Quantification qPCR assays . Individually indexed libraries were pooled and 75 bp, single-end sequencing was performed on a NextSeq500 sequencer using a 75-cycle high output kit (v2)  and each library was sequenced to an average raw depth of 35 M reads. Base-calling was done by Illumina NextSeq Control Software (NCS) v2.0 and the output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.Libraries were prepared by the Van Andel Genomics Core from 500 ng of total RNA using the KAPA mRNA HyperPrep kit (v4.17) . RNA was sheared to 300–400 bp. Prior to PCR amplification, cDNA fragments were ligated to Bioo Scientific NEXTflex dual indexing adapters . Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip , QuantiFluorcutadapt [Trim Galore!  followed by quality control analysis via FastQC [STAR [DESeq2 [DESeq2 from the top 500 genes by variance across samples. Relative gene expression heatmaps were produced using relative regularized logarithm (rlog) [DESeq2 normalized counts table.Raw 75 bp reads were trimmed with cutadapt  and Trima FastQC . Trimmeda FastQC  v28 annoQC [STAR  aligner  [DESeq2 ,35 count [DESeq2  for downm (rlog)  counts bhttps://egg2.wustl.edu/roadmap/web_portal/ accessed on 28 December 2021), and samples were binarized by presence or absence of any H3K27me3 peak calls within 3 kb of the PGR gene TSS. Among the 56 samples with matched RNA-seq data, PGR gene expression  was then compared between samples with vs. without promoter H3K27me3. NIH Roadmap Epigenomics data were used to explore the relationship between H3K27me3 and PGR expression in human tissues . Broad HIGV [t-tests.Wild-type 12Z cell ARID1A ChIP-seq data and siARID1A vs. control 12Z cell total RNA-seq data were re-analyzed from GSE121198 as previously described . Genes eIGV  visualizIGV  was usedIGV . ComplexIGV  was usedIGV  functionIGV  was usedIGV ,52 was uIGV  was usedIGV  was usedEndometrial cancer cell line data for 40 cell lines were collected from the Cancer Cell Line Encyclopedia (CCLE)  using th12Z cells were seeded at a density of 4000 cells per well in a 96-well plate. After 24 h, cells were treated with EPZ-6438 at concentrations from 100 nM to 100 μM, or 0.1% DMSO vehicle. After 48 h, cells were incubated with 2 μg/mL calcein-AM for 1 h, and fluorescence was measured using a SpectraMax i3x .ESR1 open reading frame cDNA to develop a cell line with stable ESR1 expression. Stable expression of ESR1 was developed under selection with Blasticidin. Protein expression of ESR1 was confirmed by western blot  were transiently transfected with ARID1A siRNAs (siARID1A) or non-targeting RNAs [PGR), not augmented by ARID1A loss (C3), or augmented by ARID1A loss (CCNA1)  D. As witget gene ,60,61, sAmong the 1699 genes significantly differentially expressed upon E2 treatment of 12Z-ESR1 cells (FDR < 0.0001), 939 were further affected by the loss of ARID1A A. Most oSERPINE1, a gene regulated by ARID1A through repression of hyperacetylation of the SERPINE1 super-enhancer [SERPINE1 expression is upregulated following ARID1A loss but repressed following E2 treatment  [PGR (We next considered the regulation of s (PRCs) ,68,69. Ps (PRCs) ,74,75,76s (PRCs) ,77,78,79s (PRCs) . EZH2 acs (PRCs) . The H3Ks (PRCs) ,83. Duris (PRCs) ,85. Chros (PRCs) ,85. We ps (PRCs) . Among pCs) [PGR G,H. ThesPGR is regulated by a bivalent promoter in 12Z cells, we utilized the NIH Epigenomics Roadmap data to investigate if bivalent chromatin may be a mechanism regulating PGR expression across human tissues. Among 56 tissue samples that had both H3K27me3 ChIP data and matched transcript data, we found that those samples with H3K27me3 at the PGR promoter had significantly lower PGR expression than those without promoter H3K27me3 (PGR expression can be repressed by H3K27me3. As H3K27me3 is deposited by EZH2, we next tested whether PGR induction following E2 stimulation was being repressed by EZH2 enzymatic activity following ARID1A loss. To do so, we utilized a small molecule inhibitor of EZH2, EPZ-6438, which inhibits methyltransferase activity in a dose-dependent manner [PGR mRNA in ARID1A knockdown cells treated with E2 (PGR in the context of ARID1A loss in the endometrial epithelium, leading to progesterone resistance and an “unopposed estrogen” phenotype in endometrial cancer (Following our observation that H3K27me3 A, furthet manner ,87. EPZ- with E2 C–E. Thes with E2 A,B, whil with E2 A. These l cancer .PGR. Loss of either ARID1A or BRG1 SWI/SNF subunits is frequently observed in diseases in which PGR expression is lost, including breast cancer, in which ARID1A is mutated in 5% of cases [Here, we described roles for ARID1A and EZH2 in the regulation of PGR upon E2 stimulation. ARID1A co-regulates a large subset of genes regulated by estrogen, which may explain why ARID1A mutations are common in estrogen-dependent gynecologic diseases and cancers, wherein ARID1A mutations are observed in the epithelial compartment. Estrogen is a master regulator of the endometrial epithelium, and we found that both ARID1A and estrogen co-regulate epithelial identity genes, with ARID1A playing non-conventional roles in repression. ARID1A loss leads to EMT and invasion in the endometrial epithelium, and the results presented here suggest a role for wild-type ARID1A and estrogen signaling in the co-operative maintenance of epithelial identity. At other genes, ARID1A plays traditional activating roles following estrogen stimulation, which includes counteracting the repressive effects of PRC2 complexes at critical genes like of cases  and ARIDof cases . PositivThe exact role of PGR in the epithelial component of the endometrium remains unclear. PGR is expressed in both the epithelial and stromal compartments of the endometrium. However, in vivo mouse studies and in vitro human cell line studies have demonstrated a role for stroma PGR, rather than epithelial PGR, in reducing epithelial proliferation following estrogen stimulation or apoptosis following ovariectomy ,92,93,94PGR promoter is rich in CpG islands [PGR expression through the removal of activating H3K4me3 marks [ESR1 expression in breast cancer cells [ESR1 by EZH2, although EZH2 expression is negatively correlated with ESR1 expression in patient tumor tissues (Previous studies have shown that PGR expression is regulated through epigenetic mechanisms. The  islands . DNA met islands ,98, a ph islands . Howevere3 marks ,101. PGRe3 marks . EZH2 haer cells . In the  tissues A,B. EndoPgr-Cre system [In EC, EZH2 expression is increased in tumor samples compared to normal tissue, and high EZH2 expression correlates with decreased progression-free survival or overall survival ,108,109.e system  resultede system . RNA-seqe system , althouge system . AdditioESR1 in high-grade serous OC cells [In the past decade, targeting of EZH2 in SWI/SNF-deficient cancers has been the focus of many studies. The synthetic lethal relationship was first identified in SMARCB1 deficient rhabdoid tumors and lymphomas, in which tumor phenotypes could be rescued by inhibition of EZH2 ,86. EPZ-OC cells . HistoneOC cells . In the


G93A mice in order to obtain a synergistic effect. The results showed that the combinatorial gene therapy increased preservation of MNs and of innervated neuromuscular junctions and reduced glial reactivity in the spinal cord of the treated SOD1G93A mice. Moreover, NRG1 isoforms overexpression improved motor function of hindlimb muscles and delayed the onset of clinical disease. However, this combinatory gene therapy did not produce a synergic effect compared with single therapies, suggesting an overlap between NRG1-I and NRG1-III activated pathways and their beneficial effects.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the neuromuscular system for which currently there is no effective therapy. Motoneuron (MN) degeneration involves several complex mechanisms, including surrounding glial cells and skeletal muscle contributions. Neuregulin 1 (NRG1) is a trophic factor present particularly in MNs and neuromuscular junctions. Our previous studies revealed that gene therapy overexpressing the isoform I (NRG1-I) in skeletal muscles as well as overexpressing the isoform III (NRG1-III) directly in the central nervous system are both effective in preserving MNs in the spinal cord of ALS mice, opening novel therapeutic approaches. In this study, we combined administration of both viral vectors overexpressing NRG1-I in skeletal muscles and NRG1-III in spinal cord of the SOD1 Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of upper and lower motoneurons (MNs) leading to progressive paresis, muscle atrophy and death, often due to respiratory failure –3. In moG93A)  , 21. TheG93A) , 21, 22.G93A) .Regrettably there are no effective treatments for ALS, and the current standard of care is limited to symptomatic and palliative therapies . Only twInterestingly, the ALS field has entered a thrilling era of therapeutic development as shown by the potential of gene therapies in the recent years , 25, 26.G93A mice, with downregulation of NRG1 type III and upregulation of NRG1 type I in the spinal cord  yield neurotrophic factors that play essential roles in promoting MN survival, supporting axonal, and neuromuscular development and maintenance –35. NRG1nal cord , 40. In 93A mice , in deve93A mice  and in m93A mice . Thus, o93A mice . Therefo*G93A)1Gur mice expressing the mutation G93A in the human SOD1 gene were obtained from the Jackson Laboratory . Non-transgenic wild-type (WT) littermates were used as controls. Mice were maintained by crossing transgenic males with F1 hybrid females originally obtained from Janvier Laboratories (France). Transgenic mice were identified by analyzing tissue samples from the tail. Animals were maintained under standard conditions and handled in accordance with the guidelines of the European Union Council (Directive 2010/63/EU) and Spanish regulations on the use of laboratory animals. The Ethics Committee of the Universitat Autònoma de Barcelona approved the corresponding experimental procedures.Transgenic B6SJL-Tg, SOD Mock mice , SOD NRG1-I + NRG1-III FL mice . Then, mice were distributed in two subgroups at 16 weeks, one for analysis of mRNA expression: WT , SOD Mock , SOD NRG1-I + NRG1-III , and another for histological studies: WT , SOD Mock , SOD NRG1-I + NRG1-III .In this study we included three groups of B6xSJL female mice, one of WT mice and two of SOD1G93A mice can be found in the A time-chart showing the AAV administration and the follow-up made during progression of the disease in the SOD1http://www.viralvector.eu). AAV8 vector coding mock or NRG1-I, and AAVrh10 virus coding mock or NRG1 type III were produced as in previous studies , the spine was surgically exposed and 10 μl of viral vector (1 × 1011 vg of AAVrh10-CMV-Nrg1-III vector or mock vector) were delivered between lumbar vertebrae L3 and L4 into the cerebrospinal fluid (CSF) with a 30G needle attached to a Hamilton syringe, as previously described  and 1 μg per sample was reverse transcribed using the iScript™ Reverse transcriptase and Reverse Transcription Supermix (Bio-Rad). The reverse transcription cycle conditions were 25°C for 5 min, 42°C for 20 min and 95°C for 1 min. We analyzed mRNA expression of Nrg1-I and Nrg1-III, by means of specific primer sets: Nrg1-I Fwd TGGGAACGAGCTGAACCGCA; Nrg1-I Rev TCCAGAGTCAGCCAGGGATG; Nrg1-III Fwd TTCCCTTCTCCAGCTCGGACC; Nrg1-III Rev GTCCCAGTCGTGGATGTAGATG. Mouse 36B4 was used to normalize the expression levels . The thermal cycling conditions started with 5 min polymerase activation at 95°C, followed by 45 cycles of 15s at 95°C, 30s at 60°C, 30s at 72°C and 5s at 65°C to 95°C (increasing 0.5°C every 5s). Melting curves were analyzed by monitoring the SYBR Green decrease after extension. The Pfaffl method was used for quantification relative to controls using the 36B4 gene for mRNA or Cyclophilin for DNA .5 haploid genomes , followed by phenol/chloroform extraction. Real time primers for the housekeeping gene cyclophilin B, Desmin-NRG1-I or CAG-Nrg1-III constructs, with the forward primer anneals with the promoter sequence and the reverse primer with each Nrg1 isoform, are the following: mCycloB Fwd TCAACCTCTCCTCTCCTGCC; mCyclo Rev: GGTTTCTCCACTTCGATCTTGC; Desmin GGGTTTGGGGTTCTGAATGTG; Nrg1-I Rev CGGTCCTTCTTCTTGCCCTT; CMV AGAACCCACTGCTTACTGGC; Nrg1-III CGGTCCTTCTTCTTGCCCTT. Viral genome copies/cell were calculated using a standard curve generated from known amounts of a plasmid DNA containing a CMV-Nrg1-III or Des-Nrg1-I sequences or a 500 bp cyclophilin PCR product (CyclophilinB-Fwd5617: CATGCCTATGGTCCTAGCTT and CyclophilinB-Rv6141) purified by Geneclean (Q-Biogene) in 10 ng per ml of salmon's sperm DNA (Sigma) and assuming that 1 mg of mouse genomic DNA contains 3x10 genomes .Other subgroups of mice were perfused with 4% paraformaldehyde in PBS also at 16 weeks of age. Lumbar spinal cord and the GM muscle were obtained. Spinal cords were post-fixed and cryopreserved in 30% sucrose in PBS. GM muscles were directly cryopreserved in 30% sucrose in PBS.For spinal MN counting, transverse sections were cut at 20 μm thickness with a cryotome  and serially collected. Slides containing L4-L5 spinal cord sections separated 100 μm were stained with cresyl violet. Motoneurons were identified in the lateral side of the ventral horn and counted if fulfilling the criteria of having diameter larger than 20 μm, polygonal shape and prominent nucleolus.For astrocyte and microglia labeling, other sections of L4-L5 segments were immune-labeled with primary antibodies anti-ionized calcium binding adapter molecule 1 , and anti-glial fibrillary acidic protein , respectively , 43. ImaFor NMJ labeling, longitudinal sections of the GM muscle were cut at 60 μm thickness and collected in sequential series in Olmos solution. Sections were processed for immunolabeling with primary antibodies anti-synaptophysin  and anti-neurofilament 200  as previously reported (TM software (Bio-Rad). At least 3 animals per group and treatment and 3 different blots were quantified for each protein analyzed.Snap-frozen lumbar spinal cord samples were sonicated and homogenized in RIPA lysis buffer plus a mixture of protease inhibitors (Millipore). Total protein concentration was determined by BCA Protein Assay Kit (ThermoScientific). Protein separation was performed on 10% SDS-polyacrylamide electrophoresis gel (VWR Life Science), transferred to PVDF membranes  and immunoblotted. The following primary antibodies were used: rabbit anti-phospho Erk1/2 , total Erk , rabbit anti-phospho Akt , total Akt , and rabbit anti-GAPDH . The secondary antibodies were anti-rabbit HRP-conjugated secondary antibody  and Westar Eta C Ultra 2.0 ECL substrate (CYANAGEN). Image density was analyzed with Image Labpost-hoc test when necessary. Results of MUNEs and MEPs were analyzed with t-Student test. For clinical disease onset the Log-rank (Mantel-Cox) test was applied. Results of histological and molecular biology analyses were compared using t-Student or ANOVA with Tukey's post-hoc test. A difference was considered significant if p ≤ 0.05.To avoid bias, experiments were performed by researchers blinded regarding the treatment received by each group, and mice were randomly distributed in groups. Sample size was selected according to previous observations in our lab. Data are shown as mean ± SEM. Results of motor nerve conduction and locomotion tests were analyzed with repeated measurements ANOVA, followed by Bonferroni G93A mice. Vector injections were performed at the same intervention, previous to disease onset (at 7 weeks of age). mRNA analysis at the advanced-stage of the disease (16 weeks) showed that NRG1-I expression was increased in the GM muscle (38.77 ± 11.18) of treated in comparison with the control SOD1G93A mice (0.76 ± 0.41) (G93A control mice (0.68 ± 0.05)  , and NRG ± 0.05) . In agre93A mice . As exper vector .G93A compared to mock SOD1G93A control mice  compared to the control SOD1G93A mice (6.9 ± 0.5), and higher number of innervated NMJs (74.7 ± 3.7) than in SOD1G93A mock mice (43.0 ± 4.7)  whereas the untreated mice showed marked glial reactivity in the ventral horn   . The comctively) .G93A untreated mice  would activate protective pathways in the MNs, whereas overexpression of NRG1-I at the skeletal muscles (by means of systemic injection of AAV8-desmin-NRG1-I) would maintain the NMJ and promote re-innervation of the already denervated muscle  funds from the Instituto de Salud Carlos III of Spain.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Here, we report the coding-complete genome sequence of a clinical sample of influenza virus obtained from a pig at a livestock farm in Karaganda, Central Kazakhstan, during a pig study in 2020. Isolate A/Swine/Karaganda/04/2020 (H1N1) belongs to clade 1A.3.2.2 lineage 1A, which includes the 2009 H1N1 pandemic strains. Alphainfluenzavirus in the family Orthomyxoviridae and cause one of the most important respiratory diseases in pigs as well as humans (–Influenza A viruses (IAV) belong to the genus s humans –3. An im humans –.de novo using SPAdes v3.15.2. The resulting contigs were used to conduct a BLASTn search for reference sequences in the GenBank database. The search was restricted to only Influenza A virus sequences . Mapping against the 8 segments of the reference strains was performed using BWA software . The IAV segments were amplified by reverse transcriptase PCR (RT-PCR) using the protocol proposed by Zhou et al. , the reasoftware .The genome assembly of A/Swine/Karaganda/04/2020 (H1N1) was 13,611 bp long . An influenza virus sequence annotation tool  was usedUsing the Swine H1 Clade Classification Tool from the Influenza Research Database (IRD) , the KazThe obtained genome sequences will be used to study virus circulation and genetic variation for inducing the reversal of drug sensitivity . Swine sMZ363969.1 to MZ363976.1. The raw sequence reads can be accessed through the Sequence Read Archive (SRA) database under accession number SRR15011445 and BioProject accession number PRJNA742842.The data for the 8 segments of strain A/Swine/Karaganda/04/2020 (H1N1) are available at NCBI under GenBank accession numbers


Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016–2018. Most E30 cases affected persons 0–4 years of age (29%) and 25–34 years of age (27%). Sequences were divided into 6 genetic clades (G1–G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed  Echovirus 30 (E30) is a common cause of viral meningitis outbreaks and upsurges reported worldwide (Enterovirus B (EV-B) species within the Picornaviridae family of human enteroviruses and is divided into 2 genogroups (GG), I and II , and to members of the European Non-Polio Enterovirus Network . We requested pseudonymized data from 2016–2018 with sample identifier, sampling date, specimen type, and sequence in FASTA be sent to ECDC secure file transfer protocol server by January 7, 2019. We also collected optional data, such as patient age, clinical presentation, whether they were hospitalized, and infection outcome. We excluded submissions without virus sequence data  and 145 sequences from region 2 (nt positions 2916–3428). Of these, 1,329 sequences were collected during 2016–2018 and 78 during 2010–2015. We used the 2010–2015 sequences for phylogenetic reconstruction but excluded these from further data analysis  spin column at room temperature for next-generation sequencing (NGS) or to conduct 3Dpol sequencing of the 549 nucleotides, as previously described  region, we randomly selected records from each clade to ensure fair distribution of sequence data. We asked participants to send either extracted RNA in a QIAGEN  automated extraction kit or QIAGEN filters and eluted in 50 µL of elution buffer  was used according to the manufacturer’s instructions. Runs were performed on the Nextseq (Ilumina). Raw data were processed by using Jovian  (Complementary DNA (cDNA) and double stranded DNA (dsDNA) were generated and purified . For tag/Jovian) . We conducted VP1 phylogenetic reconstruction with the 1,407 study sequences and 324 sequences extracted from Genbank. We selected region 1 for clade analysis because it is more commonly used for enterovirus typing  Figure 1http://www.virus-evolution.org). We generated maximum-likelihood and neighbor-joining trees for VP1 and 3Dpol regions by using MEGA version 7 (https://www.megasoftware.net) with the optimal model  and 100 bootstraps  and 70 complete VP1 sequences extracted from GenBank  Figure 1https://nextstrain.org/community/enterovirus-phylo/echo30-2019/vp1).We aligned sequences by using MAFFT (−3 substitutions/site/year during the time-resolved tree reconstruction. We provided the resulting Nextstrain tanglegram build for viewing (https://nextstrain.org/community/enterovirus-phylo/echo30-2019/3D:community/enterovirus-phylo/echo30-2019/vp1) and the codes for both VP1 and 3Dpol analyses (https://github.com/enterovirus-phylo/echo30-2019).We used a dataset of 110 sequences to conduct 3Dpol analysis, including 48 complete genome sequences and 12 3Dpol sequences generated in this study and 50 sequences extracted from GenBank  Figure 1We deposited VP1 and complete genome sequences in GenBank under accession nos. KC309427–37, KY986976–7033, MK251835–6, MK372854–80, MK507733–7, MK814991–6288, and MK895104–9 and 3Dpol sequences under accession nos. MN395293–303. We deposited NGS fastq reads in European Nucleotide Archive database under accession nos. SAM17101211–58.During 2016–2018, a total of 1,329 E30 records representing 1,292 cases that fulfilled the study criteria were submitted from 22 countries  Figure 1<3 months of age also were heavily affected   cases and ranged from 0 to 73 years with a mean age of 18.7 years. Children <5 years of age  and adults 26–45 years of age  were most affected . Infants, 20.6%) .https://nextstrain.org/community/enterovirus-phylo/echo30-2019/vp1).Clinical information was available for 734 (56.8%) E30 cases, of which 380 cases had unknown symptomology. For most  cases, the recorded signs and symptoms suggested meningitis. Symptoms of acute flaccid paralysis were reported in 1 case, encephalitis in 3 cases, and meningoencephalitis in 8 cases. Fever, either as sole symptom or in combination with other signs and symptoms, was recorded in only 52 (7.1%) cases. Unfortunately, not all records were filled in completely, and clinical data were absent for some samples. Other signs and symptoms mentioned were gastrointestinal symptoms in 6 cases, respiratory symptoms in 6, rash in 2, other neurologic symptoms in 4, or other unspecified in 53 cases; 8 cases had no symptoms. We created an interactive representation of age and clinical features of sequences from E30 cases, which we made available on Nextstrain  of cases, only 5 of which had no hospitalization. The low fraction of hospitalization reported limited further analysis. No deaths were reported.Among the 1,329 curated VP1 study sequences, 1,019 (76.7%) could be subdivided into 5 distinct clades, G1–G5, that showed >5% sequence divergence from one another , panel A>2 distinct viruses dominating the upsurge in 2018    Figure 2Most E30 G1 viruses were detected among infants <3 months of age  and in young adults 26–45 years of age  . G2 100.https://nextstrain.org/community/enterovirus-phylo/echo30-2019/vp1).Most E30 VP1 sequences within clades G1, G2, G4, and G6 displayed specific amino acid substitutions. G6 sequences predominantly displayed amino acid changes at position 56 (Y-F), position 84 within the BC loop (V-A), position 87 within the BC loop (E-D), and position 145 (V/I) compared with G1, G2, and G4. Most G1 and G6 sequences had a valine at positions 54 and 120 compared with the G2 and G4 sequences, which had an isoleucine. At position 122, most G4 sequences contained a leucine, whereas G1, G2, and G6 sequences contained a phenylalanine. Interactive data are available on Nextstrain (https://nextstrain.org/community/enterovirus-phylo/echo30-2019/3D:community/enterovirus-phylo/echo30-2019/vp1)  .We found that 3Dpol sequences of G1–G6 formed 8 recombination groups, which were separated by other published E30 variants and by other species B types . We noteA large upsurge of E30 infections was reported in several countries in Europe during 2018 (>2 years. In total, 6 clades were identified during the study period and circulated in a pattern of rapid turnover of newly emerging genetic lineages and RFs and their relatively rapid disappearance over time, a pattern that is typical for other enteroviruses (The data clearly demonstrate that analysis based on phylogenetic clade assignment shows differential dominance of many different clades. The upsurge in 2018 was caused by appearance of several different clades or genogroups of E30 viruses; G1 in GGII (oviruses , 2. ThisThe cocirculation of different E30 clades during the 2018 upsurge and in previous years argues against the idea that the periodic emergence of E30 occurs through the evolution of more pathogenic or transmissible forms of the virus. The cocirculation of several different groups fits better with changes in population susceptibility from birth cohort effects and a breach of a critical immunity level that controls E30 spread within the population (As shown in the original description of the upsurge (Using Nextstrain, we visualized the various categories of demographic and clinical data, clades, and RFs. Unfortunately, G5 could not be inferred due to possible recombination events within the fragment. Complete reconstruction of E30 temporal events with geographic spread was hampered by the inevitably uneven sampling and testing in different years by the different contributing countries.This study underpins the strength of the ENPEN consortium, which brings together virologists, public health experts, infectious disease doctors, and scientists across Europe to enable rapid detection and early warning through standardized surveillance. Previous studies using Nextstrain with 2 EV-D68 datasets have shown the value of combining demographic and phylogenetic analysis, both as retrospective (Differences in surveillance systems, case definitions, and sample selection between institutes and countries make standardized data collection difficult, particularly for denominator data. The differences in data collection proved to be a limitation in our study, and the extent of the circulation of the different strains remains unknown. The emergence and disappearance of viruses from different clades across the years suggests that some form of predictive modeling might be undertaken if data were standardized and provided in real-time through networks such as ENPEN. The mechanisms underlying the complex cyclic pattern of E30 and other enteroviruses and the effects of changing population immunity, antigenic changes, virus diversification, pathogenicity, and recombination need further exploration. The emergence of different enterovirus types, and their associated periodicities and population penetrance, might be driven by multiple mechanisms (Additional information on molecular epidemiology and evolutionary trajectory of recent echovirus 30 emergence, Europe.


Arabidopsis thaliana contains six FLS genes, among which FLS1 encodes a functional enzyme. Previous work has demonstrated that the R2R3-MYB subgroup 7 transcription factors MYB11, MYB12, and MYB111 redundantly regulate flavonol biosynthesis. However, flavonol accumulation in pollen grains was unaffected in the myb11myb12myb111 triple mutant. Here we show that MYB21 and its homologs MYB24 and MYB57, which belong to subgroup 19, promote flavonol biosynthesis through regulation of FLS1 gene expression. We used a combination of genetic and metabolite analysis to identify the role of MYB21 in regulating flavonol biosynthesis through direct binding to the GARE cis-element in the FLS1 promoter. Treatment with kaempferol or overexpression of FLS1 rescued stamen defects in the myb21 mutant. We also observed that excess reactive oxygen species (ROS) accumulated in the myb21 stamen, and that treatment with the ROS inhibitor diphenyleneiodonium chloride partly rescued the reduced fertility of the myb21 mutant. Furthermore, drought increased ROS abundance and impaired fertility in myb21, myb21myb24myb57, and chs, but not in the wild type or myb11myb12myb111, suggesting that pollen-specific flavonol accumulation contributes to drought-induced male fertility by ROS scavenging in Arabidopsis. Commonly found flavonols in plants are synthesized from dihydroflavonols by flavonol synthase (FLS). The genome of  cis-element of the FLS1 promoter, which is essential for the reduced-fertility phenotype of the myb21 mutant.MYB21 modulates the accumulation of flavonols through directly binding to the GARE  Flavonoids are synthesized via the phenylpropanoid pathway and include flavonols, anthocyanins, and proanthocyanidins .Arabidopsis thaliana, there are six FLS genes, referred to as FLS1 to FLS6. Among their products, FLS1 is functional and shows strong enzymatic activity   or when overexpressing FLS1, the siliques of the myb21 mutant contained more seeds. Consistent with the accumulation of excess reactive oxygen species (ROS) in the myb21 mutant, treatment with the ROS inhibitor diphenyleneiodonium chloride (DPI) could partly rescue the sterility phenotype of myb21. Although the mechanism requires further investigation, we further hypothesized that a MYB21-mediated flavonoid metabolism in pollen grains was essential for stamen development and male fertility, and this is probably because flavonols can prevent ROS from reaching damaging levels.Here, we report that MYB21 and its homologs promote the expression of Arabidopsis thaliana ecotype Columbia-0 (Col-0) were grown in a growth room at 22 °C with a 16 h light/8 h dark cycle unless otherwise indicated. The Arabidopsis Genome Initiative identifiers of the three MYBs under study are as follows: MYB21 (At3g27810), MYB24 (At5g40350), and MYB57 (At3g01530). The myb21 (SALK_042711), myb24 (SALK_017221), myb57 (SALK_065776), and myb21myb24myb57 mutants were kind gifts from D. X. Xie. The seeds of chs/tt4-11 (SALK_020583) and myb11myb12my111 mutant were a gift from J. R. Huang.Plants of MYB21-FLAG (three copies of FLAG in tandem) in Col-0 and the myb21 mutant were generated by cloning MYB21 cDNA into the JW819 vector under the control of the 35S promoter. The overexpression of MYB21-FLAG was able to complement the phenotype of the myb21 mutant, including its shortened stamen and reduced fertility  were amplified by PCR and ligated into the pBI121 vector. β-Glucuronidase (GUS) histochemical analysis was performed as described previously  myb21, the FLS1 cDNA fragment was inserted into pCAMBIA1300 under the control of the 35S promoter. The overexpression of FLS1 in the myb21 mutant was able to rescue the infertility phenotype of myb21 in 16 of 21 individual plants. These vectors were introduced into Arabidopsis plants by the Agrobacterium tumefaciens-mediated floral dip method (Transgenic plants overexpressing ertility . To prodProAlcA:MYB21 plants were grown in soil until flowering (5 weeks old). The plants were then sprayed with 1% ethanol or water (mock control). One hour later, plant materials were harvested and total RNA was extracted.For ethanol treatment, the For the kaempferol and DPI treatments, 10 mM kaempferol (Sigma-Aldrich) and DPI (Sigma-Aldrich) in DMSO solution were used. Before floral stage 13 (flower in full bloom), the plants were sprayed with 2 μM kaempferol and 20 μM DPI five times at intervals of 2 days. The same concentration of DMSO was used as a mock treatment. The phenotypes of inflorescences and siliques were observed and analyzed.β-TUBULIN2 (At5g62690). The primers used in this study are listed in For total RNA extraction, plant materials were homogenized in liquid nitrogen and mixed with Trizol reagent (Invitrogen) following the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of total RNA using a reverse transcription system (TaKaRa). The fragments of interest were amplified by real-time PCR (qRT–PCR) using sequence-specific primers . qRT–PCRFLS1 was cloned into the pGreenII 0800-LUC vector. The 35S::REN gene (Renilla luciferase) in the vector was used as an internal control. Equal concentrations and volumes of transformed Agrobacterium strains were mixed and co-infiltrated into Nicotiana benthamiana leaves by using a needleless syringe. After culturing for 3 days, the infiltrated leaves were harvested and the fluorescent values of firefly luciferase (LUC) and Renilla luciferase (REN) were analyzed using a Dual-Luciferase Reporter Assay System (Promega). The ratio of LUC to REN represented the activity of the FLS1 promoter with or without the effect of the transcription factors MYB21, MYB24, and MYB57, as well as MYB12 and MYB99.Transient expression experiments and dual-luciferase assays were carried out as described previously  was then co-transformed with bait vectors into yeast strain Y187 and plated on SD/-Leu/-Trp medium. Colonies were then transferred to SD/-Leu/-Trp/-His deficient medium with 25 mM 3-amino-1,2,4-triazole for 3 days.The PCR products of Pro35S:MYB21-FLAG and wild-type plants were harvested for chromatin immunoprecipitation (ChIP)–qRT assays. The ChIP experiments were performed using an EpiQuikTM Plant ChIP Kit . According to the manufacturer’s instructions, the chromatin from the plant cells is extracted and sheared; the length of sheared DNA fragments is 200–500 bp. One-third of the crude chromatin extracts was saved for use as an input control, and the other two-thirds were treated with anti-FLAG antibody or (as a negative control) normal mouse IgG, separately. DNA was released from the antibody-captured protein–DNA complex and purified. Eluted DNA was further analyzed by qRT–PCR, with the Actin8 promoter as a reference, as described (Pro35S:MYB21-FLAG with that for the wild-type plants (Approximately 1 g of seedlings of 10-day-old MYB21 was cloned into the GST fusion vector (pGEX-4T-1) and then transformed into BL21 (DE3) Escherichia coli. The recombinant protein GST-MYB21 was purified using a Glutathione Sepharose 4 Fast Flow kit  according to the manufacturer’s protocol. The DNA fragments containing GARE motifs in the FLS1 promoter were amplified using biotin-labeled primers  glass plates . A system of ethyl acetate/formic acid/acetic acid/water  was chosen as the mobile phase for the chromatography (–1 at 30 °C. Compounds were separated by gradient elution with solvent A (0.2% acetic acid in water) and solvent B (0.2% acetic acid in acetonitrile) with the following elution profile: 0 min 98% A, 2% B; 1 min 98% A, 2% B; 2 min 85% A, 15% B; 15 min 78% A, 22% B; 17.5 min 50% A, 50% B; 19 min 100% B; 21.5 min 100% B; 22 min 98% A, 2% B.The profiling of the flavonols was implemented by an UPLC system (Agilent 1290) coupled to a Q-TOF mass spectrometer (Agilent 6545). The methods used were as described in previous studies  ion source. Full-scan mass spectra from 100 to 1000 m/z values of flavonoid glycosides [M + H]+ with 10 V cone voltage and patterns of MS2 fragmentation under 20 V cone voltage 2, 0.16 mM boric acid, 1 mM KNO3) containing 5 μM 5-(and 6)-chroromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) and 0.005% (v/v) Triton X-100. The PVS containing CM-H2DCFDA was then replaced with fresh PVS, and the stained pollen grains and stamen were placed on a microscope slide. Fluorescence was visualized on an inverted fluorescence microscope (ZEISS Axio Vert.A1) after excitation with a 488 nm laser. The quantification of viable pollen grains and stamens was performed using Fiji (Anthers were detached from 10 flowers during late flower stage 12 to early stage 13 (just beginning to open) and placed in a 2 ml conical tube. The pollen was then released from the anthers by vortexing (t-test when comparing two variables and ANOVA with Fisher’s least significant difference (LSD) test when comparing three or more conditions. Values of P<0.05 were considered statistically significant. All analyses were performed using ORIGIN 8 software.Differences were analyzed using Student’s MYB21 and its homologs (MYB24 and MYB57) participate in flavonol biosynthesis, we quantified the flavonol content of inflorescences in the myb single mutants myb21, myb24, and myb57 and in the triple mutant myb21myb24myb57. Two flavonoid-deficient mutants, myb11myb12myb111  and chs , were used as positive controls. Analysis by HPTLC with subsequent DPBA staining showed that the inflorescences of the three myb single mutants accumulated less flavonol than the wild type, and the reduction in myb21myb24myb57 was more drastic , CHI (AT3G55120), F3H (AT3G51240), F3′H (AT5G07990), FLS1, FLS3 (AT5G63590), UGT78D1 (AT1G30530), UGT78D2 (AT5G17050), and UGT79B6 (AT5G54010) were lower in the myb21, myb24, and myb57 mutants, and down-regulated to a greater extent in the myb21myb24myb57 triple mutant , under the control of the CaMV 35S promoter as effectors . A clear (nearly 2-fold) increase in the abundance of FLS1 transcripts was observed 1 h after the application of 1% ethanol to flowers of the 5-week-old ProAlcA:MYB21 plants  revealed that the FLS1 promoter (–837 bp) contains two GARE cis-elements  showed rescue of the defects in stamen development of myb21  fluorescence intensity (a measure of ROS content) of pollen grains was higher in the myb21 mutant than in the wild type (myb21 mutant when FLS1 was overexpressed (i.e. in the transgenic plants FLS1OE1myb21 and FLS1OE2myb21). To further verify the effect of ROS accumulation on stamen development in myb mutants, we treated myb21 and myb21myb24myb57 flowers with 20 μM DPI (a ROS inhibitor) before floral stage 13  regulate the expression of FLS1 does not follow those of the subgroup 7 MYBs , F5H (AT4G36220), and CCoAoMT (AT4G34050), which is responsible for lignin monolignol biosynthesis  overexpressing MYB21 exhibited different induction effects on FLS1 and F5H  mutant, and neither was the level of MYB21 transcripts in myb11myb12myb111  with no seed formation was characterized, and results indicated that flavonols were essential for male fertility in rice , myb21, FLS1OE1myb21, and FLS1OE2myb21.Fig. S5. Gene expression of myb11myb12myb111 and chs plants show normal stamen development.Fig. S6. Fig. S7. MYB21 and MYB11/MYB12/MYB111 probably mediate the biosynthesis of phenylpropanoid metabolites in their own distinct way.Table S1. Oligonucleotide primer sequences.Table S2. Quantitative UPLC/Q-TOF MS data of different flavonol derivatives.Table S3. Flavonol profiles in methanol–water extracts.erab156_suppl_Supplementary_Figures_S1-S7_Table_S1-S3Click here for additional data file.


This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient  Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated mutagenesis has been widely used to disrupt genes in mice, rats, zebrafish, fruit flies, and nematodes . Howeveri-GONAD) of Cas9 and guide RNAs (gRNAs) into mouse zygotes 1J inversion identified from C3H/HeJJcl. Appropriately marked inversions were used as balancer chromosomes to maintain mutations in the corresponding chromosomal region , and C3H/HeJJcl mice  were used in this study. The animals were maintained at a constant temperature (22  ± 2°C) and humidity (50 ± 10%), with a 12-hours light/12-hours dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee of Chubu University  and were conducted in accordance with institutional guidelines.https://ddbj.nig.ac.jp/DRASearch/, Accession: ERR008069 and ERR008070). Sequence read mapping was performed using BWA-mem software implemented in the MASER pipeline (In(6)1J breakpoints were identified as reported previously (Raw sequencing reads of C3H/HeJ were previously sequenced by In(6)1J, homozygous In(6)1J (C3H/HeJJcl background) females were mated with C57BL/6NCrSlc males, and the F1 heterozygotes were further backcrossed for six generations to obtain C57BL/6NCrSlc mice. The single-nucleotide polymorphism (SNP) genotype of each region was determined by PCR-restriction fragment length polymorphism (RFLP) analysis. We identified four SNPs (described in dbSNP) with mismatch PCR-RFLP based on the Mouse Genomes Project at Wellcome Sanger Institute (https://www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1505). PCR primers were used to amplify a genomic sequence containing a restriction site in C3H/HeJ mice but not in C57BL/6N mice. Following PCR amplification, the PCR products were digested for 4 hours at 37°C with 5 units of restriction enzyme and then analyzed by 1.2% agarose gel electrophoresis. The PCR primers used to validate recombination suppression are listed in Supplementary Table S1.To examine whether recombination was suppressed in https://phenome.jax.org/snp/retrievals, and cleavage efficiencies were retrieved from CHOPCHOP (http://chopchop.cbu.uib.no/) (Supplementary Table S2). CRISPR RNP consists of Alt-R S.p. Cas9 Nuclease 3NLS  and a custom guide RNA (crRNA): tracrRNA duplex, which includes the crRNA and a universal structural RNA (tracrRNA) (Integrated DNA Technologies). crRNA and tracrRNA were heated to 95°C for 10 minutes and slowly cooled to 25°C. This crRNA: tracrRNA duplex and the Alt-R S.p. Cas9 Nuclease 3NLS were incubated at 25°C for 10 minutes to form the RNP complex.Allele-specific CRISPR guide RNAs were designed using an SNP data retrieval utility, such as i-GONAD experiments, as previously described , and 1% sodium dodecyl sulfate], and DNA was examined by PCR amplification. PCR products were cloned into the pTAC-1 vector , and the sequences of individual clones were determined by Sanger sequencing . The PCR primers used for genotyping are listed in Supplementary Table S1.Reverse transcriptase (RT)-PCR was performed using total RNA. Total RNA was isolated from ear tissue using ISOSPIN Cell & Tissue RNA . Template cDNA was obtained using ReverTra Ace qPCR RT Master Mix . The RT-PCR products were directly analyzed by Sanger sequencing . The primers used for RT-PCR are listed in Supplementary Table S1.In(6)1J was selected to examine whether it could balance a recessive lethal mutation. A homozygous B6.C3H-em1CuIn(6)1J Mitf male was mated with a heterozygous em1CuTprkb female. Following inbreeding of F1 em1CuTprkb/B6.C3H-em1CuIn(6)1J Mitf mice, their offspring were phenotypically distinguishable. A schematic diagram of the assay and the expected results is depicted in Tprkb deletion. The first reaction utilizes the external primers to specifically amplify only the Tprkb-deleted product. In the second reaction, the internal primers are used to detect the Tprkb-exon 10 fragment, which was engineered to be deleted by the genome editing. Crude DNA derived from each embryo was prepared as a PCR template according to published methods 1JTprkb mice, the chi-square test was performed using Excel version 16.36 . The threshold for statistical significance was P < 0.05.For Mendelian genotype ratios of progeny obtained from sibling mating between em1CuIn(6)1J Mitf mice used in this study are available at the RIKEN BioResource Research Center and Center for Animal Resources. The C3H/HeJ genome assembly is available for download from the DRA search of DDBJ (https://ddbj.nig.ac.jp/DRASearch/). Supplementary material is available at figshare: https://doi.org/10.25387/g3.14677524.The B6.C3H-In(6)1J on chromosome 6 (In(6)1J breakpoints in C3H/HeJ, we retrieved available C3H/HeJ WGS data from the DDBJ DRA  and analyzed them. The In(6)1J breakpoints were predicted from alignment data using the BreakDancer tool version 1.4.5 (Gm38889 (In(6)1J encompassed approximately 40% of the chromosome from 63 to 120.8 Mb, which was notably larger than the inversion predicted in previous studies (In(6)1J (In(6)1J is expected to have occurred in the C3H/HeJ strain after the early 1970s (Previous studies showed that the inbred mouse strain C3H/HeJ carries an inversion on 1.4.5 . In the (Gm38889 . In(6)1J(In(6)1J ; these mIn(6)1J congenic strain was constructed by six generations of selective backcrossing into the C3H/HeJJcl to C57BL/6N background. Previous studies demonstrated that recombination between the wild-type and chromosomal balancer lines does not occur within these inversion events (In(6)1J suppresses crossing over in the inversion interval, we determined the recombination frequencies on chromosome 6. We analyzed four PCR-RFLPs that lie external (dbSNP no. rs387767483 and dbSNP no. rs242839954) and internal (dbSNP no. rs244130831 and dbSNP no. rs238042460) to inversion In(6)1J. Among all 16 meioses examined, the external region was recombined with the C57BL/6N strain 1J usage, we induced mutagenesis in the gene for which loss of function was expected to cause a visible phenotype. Mitf is a recessive gene within the In(6)1J region. Mitf mutations show a reduction or lack of pigmentation in the coat, eye, and inner ear of the mouse (i-GONAD method to generate a line of B6.C3H-In(6)1J mice lacking Mitf (In(6)1J female and electroporated the oviduct in vivo 1J Mitf strain was generated, which eliminated the mosaicism. Heterozygotes for B6.C3H-em1CuIn(6)1J Mitf had normal coat pigmentation. In contrast, homozygous individuals had white spots throughout the body and less-pigmented eyes of normal size 1J inversion chromosome acted as a coat color marker and enabled the inversion to be easily tracked.To facilitate B6.C3H-ing Mitf . This me in vivo . Four fo in vivo . To detemal size . The RT-deletion . Sequenc 266–274 . The Miti-GONAD-mediated mutagenesis in the presence of a non-targeted B6.C3H-In(6)1J in heterozygotes. We attempted to disrupt a potentially essential gene, Tprkb (encoding the Tp53rk binding protein), which was expected to result in lethal phenotypes based on a previous study  alleles (em1Cu-In(6)1J Mitf males, and seven B6 females that mated with B6 males were used as controls. Control B6/B6 strains had only three pups born through cesarean section, suggesting that most embryos died owing to the deletion of both Tprkb gene alleles. One of the three pups had a deletion mutation, but we could not obtain a surviving founder F0 (em1CuIn(6)1J Mitf hybrid strains, we obtained six F0 pups via cesarean section and found that two had large deletions in the target locus, resulting in one viable F0 mouse (In(6)1J females that mated with B6 males successfully obtained a viable F0 mouse with Tprkb deletion 1J could balance a recessive lethal mutation within the inversion interval, as described in em1CuIn(6)1J Mitf male was mated with a heterozygous female carrying a em1CuTprkb mutation, and the F1 trans-heterozygotes were further intercrossed. As mentioned previously, B6.C3H-em1CuIn(6)1J Mitf contains a coat color marker, which allows animal carriers to be easily identified. Following inbreeding of F1 mice, the balanced strain em1CuTprkb/B6.C3H-em1CuIn(6)1J Mitf segregated into two phenotypes: black progeny inherited the heterozygous em1CuTprkb mutation, whereas white spot progeny were genotyped as wild-type (In(6)1J . As homozygous em1CuTprkb mutants were not observed in live-born progeny, we collected embryos at embryonic days 14.5 (E14.5) and E7.5 and analyzed them. However, no em1CuTprkb homozygous embryo was found 1J makes it easier for researchers to maintain lethal mutations and more efficient experiments in which lethal mutants can be analyzed.We examined whether B6.C3H-ild-type . We alsoas found . These nhan E7.5 . PreviouIn(6)1J strains generated in this study will be deposited as frozen sperm at the RIKEN BioResource Research Center and Center for Animal Resources. The B6.C3H-i-GONAD method in B6.C3H-In(6)1J mice allowed us to efficiently generate recessive lethal strains without ex vivo handling of embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination did not occur within this inversion interval. Such heritable lethal mutations are common in many human inherited disorders 1J will be a useful tool for disrupting and analyzing disease-causing essential genes. In addition, B6.C3H-In(6)1J are applicable in N-ethyl-N-nitrosourea mutagenesis screens , as the balancers shown in In this study, the allele-specific


S100A1 expression is deregulated in a variety of human malignancies, but its role in normal and malignant endometrial cells is unclear.We used endometrial carcinoma (Em Ca) cell lines to evaluate the physical and functional interaction of S100A1 with p53 and its negative regulator, mouse double minute 2 (MDM2). We also evaluated the expression of S100A1, p53, and MDM2 in clinical samples consisting of 89 normal endometrial and 189 Em Ca tissues.p21waf1 and BAX. S100A1 overexpression enhanced cellular migration, but also sensitized cells to the antiproliferative and proapoptotic effects of Adriamycin, a genotoxic agent; these phenotypes were abrogated when S100A1 was knocked down using shRNA. In clinical samples from normal endometrium, S100A1 expression was significantly higher in endometrial glandular cells of the middle/late secretory and menstrual stages when compared to cells in the proliferative phases; high S100A1 was also positively correlated with expression of MDM2 and p21waf1 and apoptotic status, and inversely correlated with Ki-67 scores. However, such correlations were absent in Em Ca tissues.S100A1 interacted with MDM2 but not p53 in Em Ca cell lines. Treatment of cells stably overexpressing S100A1 with Nutlin-3A, an inhibitor of the p53/MDM2 interaction, increased expression of p53-target genes including The interaction between S100A1 and MDM2 may modulate proliferation, susceptibility to apoptosis, and migration through alterations in p53 signaling in normal- but not malignant-endometrial cells.The online version contains supplementary material available at 10.1186/s12885-022-09249-1. Endometrial carcinoma (Em Ca) is the most prevalent malignancy of the female genital tract in developing countries , 2. In JThe S100 protein family is comprises multigene calcium-binding proteins of the EF-hand type and has more than 25 members, each encoded by separate gene clusters on chromosome 1q21. This region is prone to chromosomal rearrangements and is frequently rearranged in cancers , 7. AlthS100 expression is deregulated in several human malignancies including carcinomas of the head, neck, lung, and breast –10. HoweBased on our previous work , 14, we BamH1-EcoRI sites of RNAi-Ready pSIREN-RetroQ vector , according to the manufacturer’s instructions. The primer sequences for the PCR reaction used in this study are listed in Table A FLAG-tagged S100A1 expression plasmid generated by polymerase chain reaction (PCR) was cloned into a p3xFLAG-CMV14 vector . S100A1-specific short hairpin RNA (shRNA) oligonucleotides were designed as described previously . Cytoplasmic and nuclear fractions were prepared using ProteoExtract Subcellular Proteome Extraction kit . Proteins were resolved by sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membranes, and probed with primary antibodies coupled to the enhanced chemiluminescence (ECL) detection system .2. Cell lysates were cleared and incubated with anti-FLAG M2, anti-p53 or anti-MDM2 antibodies, followed by incubation with Protein G-Sepharose (Amersham Pharmacia Biotechnology). Western blot assay was subsequently performed with anti-FLAG M2, anti-p53, and anti-MDM2 antibodies.For immunoprecipitation, cells overexpressing FLAG-tagged S100A1 were lysed with immunoprecipitated (IP) buffer  in the presence of 1 mM CaClCells were fixed using 70% alcohol and stained with propidium iodide (Sigma) for cell cycle analysis. Cells were analyzed by flow cytometry using BD FACS Calibur  and CellQuest Pro software version 3.3 (BD Biosciences).Apoptotic cells were identified in hematoxylin-eosin (HE)-stained sections according to the criteria of Kerr et al. . Five fiAfter transfection of FLAG-tagged S100A1, the cells were incubated with anti-FLAG M2 antibody. FITC- or rhodamine-labeled anti-mouse or rabbit IgG secondaries  were used as described previously .Cells were seeded into 24-well tissue culture plates and grown to reach almost total confluence. After a cell monolayer formed, a scratch wound was made with a sterile 200-μL tip. The area of the wound was analyzed using ImageJ software version 1.41 . Cell migration was calculated based on the number of pixels occupied by the wound closure compared to control scratches.Cell migration was determined using 24-well transwell chambers with 8-μm pore size . The lower chamber was filled with medium containing 10% serum. Cells were suspended in serum-free medium and transferred into the upper chamber. After 48 h, the number of HE-stained cells on the bottom surface of the polycarbonate membranes was counted using a light microscope.Histological findings were reviewed in hysterectomy specimens of endometrioid-type Em Cas from the case records of Kitasato University Hospital during the period from 2007 to 2020, according to the criteria of the 2014 World Health Organization classification . Each caEighty-nine biopsy specimens of normal endometrial tissues including 32 in the proliferative phase, 41 in the secretory phase (18 early and 23 middle and late), and 16 in the menstrual phase were also investigated. All tissues were routinely fixed in 10% formalin and processed for embedding in paraffin. Approval for this study was given by the Ethics Committee of Kitasato University School of Medicine (B19-144).IHC was performed using a combination of microwave oven heating and polymer immunocomplex  methods as described previously , 16.waf1, and Ki-67 in normal endometrial tissues was performed based on the percentage of immunopositive cells and the immunointensity; the values of these two parameters were multiplied together as described previously [waf1, and Ki-67 was counted in at least 1000 cells from five randomly selected fields and the labeling indices (LIs) were then calculated as a percentage. In addition, the number of cleaved PARP1-positive cells in five randomly selected fields was used to calculate the mean number of apoptotic figures per HPF.For evaluation of IHC findings, scoring of cytoplasmic and/or nuclear immunoreactivity for S100A1, p53, MDM2, p21eviously , 16. In To examine any association between S100A1 immunoreactivity and several clinicopathological factors, the score was divided into two categories (high and low) with a mean score of 4.5 used as cutoff.TP53 gene were amplified by PCR, and the products were subjected to direct sequencing PCR as described previously [MDM2 gene status was also extracted form cBioPortal .In Hec6, Hec251, and Ishikawa cells, exons 5 to 9 of the eviously . Cancer http://www.cbioportal.org/). The data were subcategorized into ‘high’ and ‘low’ groups  based on the median Z score (= 0) for mRNA expression levels in each category, and then examined for any correlation with overall survival (OS) or progression-free survival (PFS).Uterine Corpus Endometrial Carcinoma TCGA PanCancer data describing mRNA expression data (RNA Seq V2 PSEM) for the S100 family in 520 cases were extracted from cBioportal for Cancer Genomics  in the Hec251 cell line, whereas the mutations were not evident in the Hec6 and Ishikawa cell lines.Three Em Ca cell lines, Hec6, Hec251, and Ishikawa cells, had mutations in the DNA binding domains of the Some S100 family members are known to modulate the p53/MDM2 axis . To examwaf1 and/or BAX in S100A1 overexpressing cells as compared to control cells  and MDM2 genes (B). n, number of cases; Wt, wild-type; Mu, mutant type.Additional file 3: Figure S3. Relationship between expression of S100 family members and prognosis derived from TCGA Em Ca data analysis. Overall survival (OS) and progression free survival (PFS) between low and high mRNA expression categories of 18 S100 family members.Additional file 4: Figure S4.TP53 gene status in Hec6, Ishikawa, and Hec251 cell lines. *, stop codon.Additional file 5: Figure S5. Western blot analysis of the indicated proteins in total lysates from eight Em Ca cell lines (A), stable H6- and Ish-S100A1 cells (B), and H251-shS100A1 cells (C), as well as control cells (Con).Additional file 6: Figure S6. Interactions between S100A1, MDM2, and p53 in stable Ish-S100A1 cells. (A) Western blot analysis (WB) with anti-MDM2 (upper panel), anti-p53 (middle panel), and anti-FLAG M2 antibodies (lower panel) after immunoprecipitation (IP) with the indicated antibodies using stable Ish-S100A1 cell lysates. Input represents 5% of the total cell extract. Normal rabbit IgG was used as a negative control. In the middle panel (p53), the band indicated by an arrow in the S100A1 lane is non-specific, since the molecular weight is slightly higher compared to that of endogenous p53. (B) Western blot analysis of the indicated proteins in stable Ish-S100A1 cells in response to Nutlin-3A treatment for the times shown. The p53 band is indicated by arrows.Additional file 7: Figure S7. Changes in proliferation and apoptosis following overexpression of S100A1 in Ishikawa cells. (A) Upper left: two independent clones of stable Ish-S100A1 cells and control cells were seeded at low density. The cell numbers are presented as mean ± SDs. P0, P3, P6, and P9 are 0, 3, 6, and 9 days after cell passage, respectively. Upper right: FACS analyses of stable Ish-S100A1 and control cells at 3 days after seeding (P3). Lower: western blot analysis of the indicated proteins in stable Ish-S100A1 cells and controls following re-stimulation of serum-starved (24 h) cells with 10% serum for the indicated times. The p53 band is indicated by arrows. (B) Upper: stable Ish-S100A1 and control cells were treated with 1 μg/mL Adriamycin (ADR) for the times shown. Daggers indicate the sub-G1 fraction. Lower: the percentages of cells undergoing apoptosis (sub-G1 fractions) were calculated following flow cytometry. C, control (C) Left: after 1 μg/mL ADR treatment, stable Ish-S100A1 and control cells undergoing apoptosis are indicated by arrows. Original magnification, x400. Right: the numbers of apoptotic cells are shown as mean ± SDs. Con, control (D) Western blot analysis of the indicated proteins in total lysates from stable Ish-S100A1 and control cells treated with 1 μg/mL ADR for the times shown. The p53 band is indicated by arrows.Additional file 8: Figure S8. Changes in cell migration following overexpression of S100A1 in Ishikawa cells. Left: a scratch was made in the middle of a layer of confluent Ish-S100A1 cells or control cells, and phase contrast images were taken 24 h later. The red lines indicate the borderlines between confluent cell layers and wound area. Right: the wound areas were calculated using Image J software version 1.41, with the area at 0 h post-wounding set as 1. The experiments were performed in triplicate. Data are expressed as mean ± SDs. Con, controlAdditional file 9: Table S1. Relationship between mRNA, copy number variations, and mutations in S100 family members.Additional file 10: Table S2. Relationship between S100A1 score and several clinicopathological factors in endometrial carcinomas.