Median IC50 values determined by HINT were similar for both subtypes of seasonal viruses, A(H1N1)pdm09 and A(H3N2), across three seasons. Human seasonal viruses with PB2 substitutions S324C, S324R, or N510K displayed a 27–317-fold reduced pimodivir susceptibility by HINT. In addition, pimodivir was effective at inhibiting replication of a diverse group of animal-origin viruses that have pandemic potential, including avian viruses of A(H5N6) and A(H7N9) subtypes. A rare PB2 substitution H357N was identified in an A(H4N2) subtype poultry virus that displayed >100-fold reduced pimodivir susceptibility. Our findings demonstrate a broad inhibitory activity of pimodivir and expand the existing knowledge of amino acid substitutions that can reduce susceptibility to this investigational antiviral.Pimodivir exerts an antiviral effect on the early stages of influenza A virus replication by inhibiting the cap-binding function of polymerase basic protein 2 (PB2). In this study, we used a combination of sequence analysis and phenotypic methods to evaluate pimodivir susceptibility of influenza A viruses collected from humans and other hosts. Screening PB2 sequences for substitutions previously associated with reduced pimodivir susceptibility revealed a very low frequency among seasonal viruses circulating in the U.S. during 2015–2020  and among non-seasonal viruses collected in various countries during the same period . Pimodivir potently inhibited virus replication in two assays, a single-cycle HINT and a multi-cycle FRA, with IC Two subtypes of seasonal influenza A viruses, A(H1N1) and A(H3N2), have been causing recurrent epidemics of variable severity for several decades. Moreover, a vast natural reservoir for influenza A viruses of diverse subtypes exists in wild birds. Avian viruses are known to cause outbreaks with varying severity in humans and can also trigger a pandemic. Zoonotic infections with avian viruses of A (H5Nx), A(H7N9), A(H9N2) have been reported in recent years and pose a serious public health threat . The firVaccination is the principal tool to mitigate the burden of influenza. However, vaccine effectiveness can be reduced due to antigenic drift, or vaccines may not be available at the early stages of a pandemic. In such circumstances, antivirals can play an important role in controlling the impact of influenza, especially in populations that are at high-risk of developing complications.Three classes of antiviral drugs – M2 blockers, neuraminidase (NA) inhibitors, and polymerase acidic (PA) inhibitor – are approved in numerous countries to control influenza A infections. However, M2 blockers are not recommended for use due to widespread resistance in contemporary seasonal influenza viruses. Although the M2 blocker amantadine has been used since 1970s, widespread resistance has occurred only after 2003 . The inc7 GTP) cap structures of host mRNA and inhibits early stages of viral transcription (7 GTP guanine base (Pimodivir (VX-787) is an orally administered inhibitor of the polymerase basic protein 2 (PB2). It prevents binding of PB2 to the 7-methyl GTP  analysis, which is supplemented with phenotypic testing . In this2.2.1.Influenza viruses were submitted to the World Health Organization (WHO) Collaborating Center for Surveillance, Epidemiology and Control of Influenza at CDC by laboratories participating in the WHO Global Influenza Surveillance and Response System (GISRS). Viruses were propagated in Madin-Darby canine kidney (MDCK) cells , MDCK-SIAT1 cells, or fertilized chicken eggs, depending on subtype and origin. All the procedures involving swine and low pathogenicity avian influenza viruses collected in the U.S. were conducted at biosafety level 2 enhanced containment, while other non-seasonal viruses were handled at biosafety level 3 enhanced containment.2.2.Illumina MiSeq platform was utilized for codon-complete genome sequencing of influenza viruses and sequence analysis was performed as described . Sequenc2.3.2 for 18–24 h. For HINT assay, single-cell suspension was added to wells of a 96-well microplate containing a mixture of pimodivir and virus (~1000 infectious units) and microplates were incubated at 37 °C and 5% CO2 for 16–18 h; a single-cycle replication was achieved by omitting TPCK-trypsin from virus growth media.Pimodivir was purchased from a commercial source (MedChemExpress), dissolved in DMSO as a 10 mM stock, aliquoted and stored at −80 °C. Viruses were tested by either focus reduction assay (FRA) or high-content imaging neutralization assay (HINT) as previously described . Both as50) values were calculated by curve-fitting analysis.Visualization of virus infected cells in both assays was done by immunostaining with mouse anti-viral nucleoprotein (NP) antibody , followed by incubation with goat anti-mouse IgG antibody conjugated to Alexa Fluor-555 (ThermoFisher Scientific), and Hoechst 33,258 dye (AnaSpec Inc.) was used to stain cellular DNA. For FRA, the number of foci formed by virus-infected cells was counted under a fluorescence microscope; while for HINT, infected cell population was detected and quantified using either CellInsight CX5 (ThermoFisher Scientific) or Celigo (Nexcelom Bioscience) image cytometers. Pimodivir 50% inhibitory concentration (IC50 fold increase compared with the sequence matched control (wild-type) viruses from the same subtype. The arbitrary criteria define influenza virus inhibition as normal (≤3-fold increase) or reduced (>3-fold increase).Currently, there is no established criteria available for the definition of pimodivir resistance or reduced susceptibility. Therefore, we used the arbitrary criteria based on IC3.3.1.In this study, NGS-generated PB2 sequences of 11,934 influenza A viruses were analyzed, which included 10,555 viruses collected by the U.S. virological surveillance during five recent seasons  and 1379 viruses collected for vaccine effectiveness studies during 2015–2017 seasons. Most (~61%) of the sequences belonged to the A(H3N2) subtype. Interrogation of PB2 sequences at nine residues of interest  revealed3.2.50 value. In the multi-cycle replication-based FRA assay, S324C, N510K, and S324R conferred a 20-, 283-, and 688-fold reduction in pimodivir susceptibility, respectively   .50 values within and across multiple tests . As this mutation was shown to slow virus replication in MDCK-SIAT1 cells  viruses was included in each test; their genomes were nearly identical, except substitution I38M in PA protein . Both cole tests . HoweverT1 cells , it migh50 values were similar, 4.46 nM vs 5.22 nM, respectively. It is worth noting that A(H1N1)pdm09 viruses displayed a somewhat broader IC50 range, 0.85–12.31 nM (~15-fold difference between the minimum and maximum), compared to A(H3N2) viruses (~5-fold range). Nevertheless, as shown in the scatter plots, subtype-specific IC50 values remained consistent between seasons pdm09 (n = 110) and A (H3N2) (n = 96) subtypes collected during 2016–2019 were tested using HINT. The median subtype-specific IC seasons .3.3.We next interrogated 8971 PB2 sequences of non-seasonal viruses collected from human and non-human hosts between October 1, 2014–April 20, 2020 . A total of 18 viruses carrying PB2 substitutions of concern were detected, indicating a low  frequency of naturally occurring resistance to pimodivir. These substitutions were as follows: F325L (n = 1), S337P (n = 1), K376R (n = 3), T378S (n = 3), M431L (n = 3), and N510K (n = 7) . They we50 values ranged 1.12–27.77 nM (~25-fold range) with a median of 8.03 nM v had the highest. Notably, viruses from the same zoonotic outbreak showed similar IC50 values. For example, pimodivir IC50 range was much narrower, 5.40–10.10 nM, for five A(H1N2)v viruses collected during the 2018 zoonotic outbreak in California and Michigan. In addition, FRA was used to test a subset of viruses, including three A(H1N1)v collected in the U. S., Europe, and China (50 0.57 nM); similar to HINT results, the range by FRA was also broad (~35-fold range). The significant genetic heterogeneity of the viruses in the panel may be the underlining reason for the broad ranges of IC50 observed in both assays.Next, a panel of 23 ‘variant’ viruses of A(H1N1)v (n = 3), A(H1N2)v (n = 8), and A(H3N2)v (n = 12) subtypes was assembled for phenotypic testing using HINT. It comprised of viruses collected during outbreaks that took place in the U.S. during 2008–2018. All ‘variant’ viruses tested were resistant to M2 inhibitors due to the presence of V27T or S31N substitutions in M2 protein. The panel was complemented by classic swine influenza A(H1N1) viruses (n = 3). HINT pimodivir IC 8.03 nM ; A/swinend China . All tes50 values (~46-fold range) , A(H5N6), and A(H9N2) . As highd range) .Pimodivir effectively inhibited replication of a broad variety of swine and avian-origin influenza A viruses in cell culture.3.4.50, 644.17 nM. Notably, it’s NA inhibitor-sensitive counterpart (wild-type ty/MN/80-NA-R292) showed similarly high elevated average pimodivir IC50, 468.43 nM (data not shown).Based on published data  and the 3.5.50 values were low, average 3.48 nM, we concluded that H357N alone conferred the observed pimodivir resistance of ty/MN/80 virus.To elucidate the molecular basis for the observed pimodivir resistance of NA inhibitor-sensitive and -resistant ty/MN/80 viruses, their PB2 sequences were analyzed. None of the previously reported PB2 substitutions  were fou4.NCT03376321). The drug development program for pimodivir has been discontinued by the commercial developer . Nonetheless, RNA-dependent RNA-polymerase remains an attractive target for antiviral development due to its critical roles in virus replication and the high degree of sequence conservation v viruses is higher (2.10–27.77 nM) than other variant subtypes. However, not equal number of viruses were tested for each subtype. Moreover, for A(H1N2)v, out of eight viruses tested, five viruses were collected from same zoonotic outbreak. While in case of A(H3N2)v, we tested 12 viruses collected during different outbreaks in the U.S. from 2013 to 2017. Interestingly, two A(H3N2)v viruses (A/Ohio/27/2016 and A/Ohio/28/2016) collected from same outbreak, showed very similar pimodivir IC50 values (within ~2-fold range). As pimodivir impedes virus replication at an early stage, efficiency of virus internalization into cells may affect the IC50 values. Therefore, it is tempting to speculate that this range may reflect the difference in replicative fitness or internalization efficiency of these variant viruses in MDCK-SIAT1 cells, a cell line optimized for propagation of human viruses, which would require further experiments and would be focus of our future investigation. Consistent with this notion, seasonal A(H1N1)pdm09 viruses, which originated from swine, also displayed a broader range of IC50 values compared to A(H3N2) viruses. Moreover, it is noteworthy to mention that the PB2 gene segment of A (H1N1)pdm09 virus was derived from avian-origin viruses through reassortment (Zoonotic infections with swine- and avian-origin viruses are concerning due to their pandemic potential. Swine-origin ‘variant’ viruses have caused multi-state outbreaks during the last two decades  and are sortment .in vitro replication capacity compared to wild-type virus (in vitro replicative fitness and pathogenicity in mice (While HINT has its advantages, FRA is better suited for testing highly pathogenic avian viruses and showed pimodivir to be highly potent. Interestingly, sequence analysis revealed that substitutions associated with pimodivir resistance were more likely to be found in viruses isolated from wild birds and poultry rather than seasonal viruses. In addition, characterization of viral replicative fitness due to PB2 amino acid substitutions associated with reduced drug susceptibility would be beneficial. However, reports on replicative fitness of pimodivir-resistant viruses remain sparse. In the phase 2a clinical trial of pimodivir, variant virus containing M431I was reported to display 12.5-fold reduced pe virus . One limpe virus . Our stupe virus . Zhu and in mice . EngineeIn summary, this study demonstrates the high potency of pimodivir as an inhibitor of influenza A virus replication in cell culture, including viruses with pandemic potential.Supp


Dysregulation of m6A modifiers promotes oncogenesis through enzymatic functions that disrupt the balance between the deposition and removal of m6A modification on critical transcripts. However, the roles of mRNA m6A in multiple myeloma (MM) are poorly understood. The present study showed that RNA demethylase ALKBH5 was overexpressed in MM and associated with a poor prognosis in MM patients. Knocking down ALKBH5 induced apoptosis and inhibited the growth of MM cells in vitro. Xenograft models and gene set enrichment analysis with patient transcriptome datasets also supported the oncogenic role of ALKBH5 in MM. Mechanistic studies showed that ALKBH5 exerted tumorigenic effects in myeloma in an m6A-dependent manner, and TNF receptor-associated factor 1 (TRAF1) was a critical target of ALKBH5. Specifically, ALKBH5 regulated TRAF1 expression via decreasing m6A abundance in the 3'-untranslated region (3'-UTR) of TRAF1 transcripts and enhancing TRAF1 mRNA stability. As a result, ALKBH5 promoted MM cell growth and survival through TRAF1-mediated activation of NF-κB and MAPK signaling pathways. Collectively, our data demonstrated that ALKBH5 played a critical role in MM tumorigenesis and suggested that ALKBH5 could be a novel therapeutic target in MM.N Multiple myeloma (MM) is a hematological malignancy characterized by uncontrolled expansion of monoclonal plasma cells in the bone marrow (BM) . The mal6-methyladenosine (m6A) is a prevalent and abundant chemical modification in messenger RNA (mRNA) in eukaryotes  and and6A moors Fig.  30]. Mo. Mo6A moors Fig. . The Canors Fig. . In addi6A modifiers in MM. According to the Arkansas myeloma cohort (GEO accession number GSE4581) [6A demethylases (ALKBH5 and FTO) was correlated with poor patient survival  to clarify their functions in MM  could promote tumorigenesis in MM. Therefore, we compared the expression pattern of FTO with that of ALKBH5 in MM. We also found that FTO was not significantly overexpressed in MM cells based on another MM atlas [Next, we investigated the prognostic value of these five canonical mGSE4581) , the eleal Figs.  and S1E.al Figs. . We alsoal Figs.  and S1F. MM Fig. . Similar MM Fig. ; S1B–F, CLs Fig. . These pMM atlas  and our Both gain- and loss-of-function studies were performed to investigate the role of ALKBH5 in MM. We conducted shRNA-mediated KD experiments (shA5#1 and shA5#2) to decrease ALKBH5 expression in HMCLs Fig. . ConsequPrkdc Il2rGamma (NCG) mice to evaluate the role of ALKBH5 in MM cell growth in vivo . As a result, shALKBH5-ON (Dox) group displayed significantly alleviated tumor burden, as revealed by the concentration of immunoglobulin kappa (Igκ) light chain in serum  ALKBH5 or ALKBH5-H204 A mutant  . Consistent with previous reports [6A-seq data showed that m6A peaks were mainly distributed in the coding region (CDS) and 3'-untranslated region (3'-UTR) of transcripts in MM cells, displaying the consensus motif DRACH  was conducted to compare the gene expression profile of MM cells with or without ALKBH5 KD to explore the potential mechanisms underlying ALKBH5 function in MM. A total of 2896 and 4570 genes were differentially expressed in cell lines RPMI-8226 and CAG, respectively Fig. . SeveralACH Fig. . Given tACH Fig.  and S4E.aks Fig. .Fig. 5Id6A-seq data identified 10 downregulated and 1 upregulated potential targets of ALKBH5 in MM -associated factor 1 (TRAF1) is an adapter molecule known for its role in TNFR-induced cell survival . In normTRAF1 following ALKBH5 KD in MM cells , followed by TRAF1-specific qPCR to substantiate the change in TRAF1 m6A methylation levels following ALKBH5 KD. Consistent with the current m6A-seq results  in TRAF1 3'-UTR resulted in resistance to the effect of ALKBH5 KD  , 51. Replls Fig. .Fig. 8ALWe analyzed ALKBH5 expression in several MM cohorts containing different genetic subgroups, most of which were defined via immunoglobulin heavy-chain locus  translocations and cyclin D overexpression (TC classification) , 52, to 6A represents a new layer of the regulatory mechanism controlling gene expression [6A modulators are dysregulated in various cancers [6A eraser ALKBH5 was highly expressed in MM, and increased ALKBH5 expression was correlated with a poor prognosis in MM patients. Using various HMCLs and xenograft models, we demonstrated that ALKBH5 and its demethylation activity were required for myeloma cell growth and survival. RNA-seq, m6A-seq, and ALKBH5-RIP assay identified TRAF1 as a direct target of ALKBH5. We showed that ALKBH5 positively regulated TRAF1 mRNA stability in an m6A-dependent manner. Further functional studies validated that TRAF1 was a pivotal contributor to the oncogenic effect of ALKBH5 in MM. Of course, other potential targets of ALKBH5 may also contribute to the overall function of ALBKH5 in MM, which needs to be investigated systematically. Additionally, we examined the mechanisms underlying the MM-promoting effects of the ALKBH5-TRAF1 axis. Activation of NF-κB and MAPK signaling pathways plays a critical role in tumorigenesis in MM [RNA mpression . Accumul cancers , 20. In is in MM . We demo6A writers and erasers  and their impacts on MM proliferation. Several other reported m6A regulators were not covered in this study. However, we found YTHDF2 was a crucial reader protein that mediates the effect of ALKBH5 on TRAF1 transcripts. YTHDF2 was dysregulated in various human malignancies and played an oncogenic or tumor-suppressive role by facilitating the degradation of m6A-modified transcripts [6A modification was also found to be involved in the self-renewal of cancer stem cells, tumor metastasis, and drug response/resistance [6A modification in MM.The current study also provides insights into the expression profile of the other canonical mnscripts –66. The sistance , 21, whiRecent studies have linked ALKBH5 overexpression to epigenetic changes in some cancers , 67, 68.6A demethylation in MM pathogenesis. ALKBH5 promoted MM cell survival and proliferation via posttranscriptional regulation of TRAF1 expression, which activated NF-κB and MAPK signaling pathways. Given the critical role of ALKBH5 in MM tumorigenesis, targeting ALKBH5 is a promising therapeutic strategy for treating MM patients.In summary, the current study uncovered the importance of ALKBH5-mediated mRNA mAll samples from newly diagnosed MM patients were collected from BM aspirations after obtaining informed consent. CD138 + MM cells were isolated using CD138 beads . PBMCs from healthy donors were purified using Ficoll . All experiments using primary human samples were conducted in accordance with the ethical regulations and were approved by the ethics committees of the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.5 selected cells were injected via the tail vein into 4- to 5-week-old NCG mice . In vivo MM growth was monitored via chemiluminescence imaging of mice following intraperitoneal injection with luciferin  every 5 days. The mice were randomly assigned to the experimental groups. All animal experiments were approved by and conducted in accordance with the guidelines of the Animal Experimental Ethical Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University.CAG, RPMI-8226, and U266 cells were transduced with indicated lentiviruses and selected using 2 μg/mL puromycin for 2 days. 5 × 10t-test. Statistical analyses were performed using GraphPad Prism 9.0.The quantification data were presented as mean ± standard deviation (SD) of three independent experiments. The log-rank (Mantel-Cox) test was performed for Kaplan–Meier survival analysis. All the other data were analyzed using the two-tailed Student’s Additional methods are provided in supplemental Methods.Supplemental materials


Childhood cancer patients receiving treatment containing alkylating agents are at risk of infertility, yet inter-individual variability in treatment-related ovarian damage is observed. Alkylating agents are metabolized by cytochrome P450 (CYP450) enzymes and polymorphisms in these CYP450 enzymes may explain this variability in ovarian damage. This study on genetic variation in CYP450 enzymes of chemotherapy-induced gonadotoxicity, using anti-Müllerian hormone (AMH) levels as a proxy for ovarian reserve, in female childhood cancer survivors (CCSs) may identify patients at risk of infertility. This unique global collaboration of two large CCS studies shows the significant gonadotoxic effect of enzyme CYP3A4*3 and significant protective effect of CYP2B6*2 on gonadal function in CCSs receiving alkylating agents. Genetic variation in CYP3A4 and CYP2B6 have previously been associated with gonadotoxicity after cancer treatment. These findings could guide risk prediction models determining patients at risk of chemotherapy-induced gonadal impairment.n = 743; age (years): median 25.8, interquartile range (IQR) 22.1–30.6). Using two additive genetic models in linear and logistic regression, nine genetic variants in three CYP450 enzymes were analyzed in relation to cyclophosphamide equivalent dose (CED) score and their impact on AMH levels. The main model evaluated the effect of the variant on AMH and the interaction model evaluated the modifying effect of the variant on the impact of CED score on log-transformed AMH levels. Results were validated, and meta-analysis performed, using the USA-based St. Jude Lifetime Cohort : median 31.3, IQR 26.6–37.4). Results: CYP3A4*3 was significantly associated with AMH levels in the discovery and replication cohort. Meta-analysis revealed a significant main deleterious effect (Beta (95% CI): −0.706 (−1.11–−0.298), p-value = 7 × 10−4) of CYP3A4*3 (rs4986910) on log-transformed AMH levels. CYP2B6*2 (rs8192709) showed a significant protective interaction effect (Beta (95% CI): 0.527 (0.126–0.928), p-value = 0.01) on log-transformed AMH levels in CCSs receiving more than 8000 mg/m2 CED. Conclusions: Female CCSs CYP3A4*3 carriers had significantly lower AMH levels, and CYP2B6*2 may have a protective effect on AMH levels. Identification of risk-contributing variants may improve individualized counselling regarding the treatment-related risk of infertility and fertility preservation options. Background: Female childhood cancer survivors (CCSs) carry a risk of therapy-related gonadal dysfunction. Alkylating agents (AA) are well-established risk factors, yet inter-individual variability in ovarian function is observed. Polymorphisms in CYP450 enzymes may explain this variability in AA-induced ovarian damage. We aimed to evaluate associations between previously identified genetic polymorphisms in CYP450 enzymes and AA-related ovarian function among adult CCSs. Methods: Anti-Müllerian hormone (AMH) levels served as a proxy for ovarian function in a discovery cohort of adult female CCSs, from the pan-European PanCareLIFE cohort ( Over the past decades, major improvements in the treatment of childhood cancer have resulted in five-year survival rates that exceed 80% . FactorsCyclophosphamide and ifosfamide are among the most commonly used alkylating agents in childhood cancers, effective against a wide range of cancers, including neuroblastoma, osteosarcoma, soft tissue sarcoma, leukemia, and lymphoma. Cyclophosphamide is a prodrug, in which the activation to 4-hydroxycyclophosphamide (4-OH-CPA) is catalyzed by the hepatic cytochrome P450 (CYP) isozymes including CYP2A6, 2B6, 3A4, 3A5, 2C9, 2C18, and 2C19 . The higCYP2B6 and CYP2C19 on the pharmacokinetics of cyclophosphamide in Chinese patients treated for systemic lupus erythematosus. Their investigation identified star-alleles CYP2C19*2 and CYP2B6*1G that significantly influenced the 4-OH-CPA concentration; moreover, the combination of these two SNPs was significantly associated with short-term outcomes and side effects [CYP3A4, CYP2B6, and CYP3A5. Patients with the CYP3A4*1B variant who were aged <45 years when receiving treatment showed a significantly longer time to ovarian failure than patients homozygote for CYP3A4*1A [CYP2C19*1/*1 genotype had an increased risk of cyclophosphamide-related toxicity compared with carriers of the CYP2C19*2 allele [Single nucleotide polymorphisms (SNPs) in CYP genes are associated with cyclophosphamide metabolism and toxic effects on ovarian function including impaired fertility and premature ovarian insufficiency (POI) in adult premenopausal women, but this has not been studied in exposed children with cancer ,19. Shu  effects . A study2 allele . Additio2 allele ,22,23,242 allele ,11,25.Thorough evaluation of possible genetic determinants mandates large cohorts and independent replication cohorts. Here, we aimed to evaluate the associations between polymorphisms in candidate genes of CYP enzymes and treatment-related gonadal impairment that have been previously identified in adult patients receiving cyclophosphamide ,19, in tEligible participants were females diagnosed with cancer before the age of 25 years and treated with chemotherapy. They had survived at least five years after diagnosis, were ≥18 years of age at evaluation and provided a blood sample to quantify anti-Müllerian hormone (AMH) levels and for extraction of DNA. Exclusion criteria included: history of bilateral ovarian radiotherapy , central nervous system (CNS) irradiation, total body irradiation (TBI), or stem cell transplantation. Further details of the study protocol have been published previously .This international retrospective study is part of PanCareLIFE, a pan-European research project including 28 institutions from 13 countries addressing ototoxicity, fertility, and quality of life . DemograFemale CCSs enrolled in the SJLIFE study served as the replication cohort. SJLIFE is a retrospectively-constructed cohort with prospective follow-up of patients diagnosed and treated at St. Jude Children’s Research Hospital  between 1962–2012 and includes detailed treatment data, clinical assessment, patient-reported outcomes, whole-genome germline sequencing, and collection of biospecimens for research . SJLIFE The outcome of this study was serum level of AMH reflecting ovarian function. AMH levels of both the discovery and replication cohort were determined in the same endocrine laboratory  using an ultra-sensitive Elecsys AMH assay , as previously described . Intra-aCYP2C19*2 (rs4244285), CYP2C19*17 (rs12248560), CYP3A4*1B (rs2740574), CYP3A4*3 (rs4986910), CYP3A4*22 (rs35599367), CYP2B6*2 (rs8192709), CYP2B6*6 (rs2279343), CYP2B6*9 (rs3745274), and CYP2B6*1G (rs4802101). Details of genotype data and the quality control protocol are provided in the Details about DNA processing are described in the 2; ≥4000–8000 mg/m2; ≥8000 mg/m2) [For each survivor, the administered cumulative dose of alkylating agents was quantified using the Cyclophosphamide Equivalent Dose (CED)-score . To eval0 mg/m2) , as prev0 mg/m2) .2) and 10 genetic principal components to account for ancestry. Additionally, we conducted a logistic regression analysis, adjusted for CED score  and principal components. For the logistic regression analysis, cases and controls for gonadal impairment were defined as low versus high AMH levels per age category , respectively. We defined cases and controls based on tertiles (the lower versus the highest tertile) and on a standard deviation (SD) based threshold (<−1.5 SD or <−2.0 SD versus above the threshold)  . In the The modifying effect of SNPs on the impact of CED score on gonadal impairment was also investigated. The association between the selected SNPs and reduced ovarian function is based on two models. The main effect model (1) was adjusted for age at time of serum sampling, CED score, and genetic principal components. We fit an interaction model (2) that, aside from the terms included in the main effect model, additionally included an interaction term (SNP*CED category) for the genetic variant and CED score categories to evaluate the modifying effect of the variant on the impact of CED score on the log-transformed AMH levels.Results of linear and logistic regression analyses are presented as regression coefficients (beta) with standard errors (SE) and odds ratios (OR) with a 95% confidence interval (95% CI), respectively. Sensitivity analyses performed to assess the robustness of our findings are provided in the p-values < 0.05) association with log-transformed AMH levels or reduced ovarian function in either model or an interaction effect with CED were selected for replication of both models. Statistical analyses were conducted using Statistical Package for Social Sciences (SPSS) version 24.0.0.1 and R version 3.5.1.SNPs that showed a statistically significant  were considered to be statistically significant. The SNPs within one CYP are not independent, as the presence or absence of one or multiple SNPs in the CYP leads to changed activity/transcription and therefore a possible effect on the gonadotoxicity of chemotherapy. We evaluated findings from the PanCareLIFE cohort in the SJLIFE replication cohort using identical models. In addition, we combined the data from the discovery and replication cohort and performed a meta-analysis using R version 3.5.1, package “rmeta” . DetailsFinally, for all SNPs with a significant main effect, we calculated the cumulative betas for every genotype per CED category to allow interpretation of the findings for clinical applicability. The calculation reflects the expected difference in AMH for each genotype and every CED category compared to the AMH level of survivors with a wild-type genotype treated without alkylating agents. To this end, we back-transformed the betas of the logAMH.2, 15.9% received ≥ 4000–8000 mg/m2, and 23.7% received ≥ 8000 mg/m2. All SNPs were in Hardy–Weinberg equilibrium (significance level < 1 × 10−7). CYP3A4*3 (rs4986910) on log-transformed AMH levels . Eight patients were homozygous for this allele and, therefore, while the effect is statistically significant, it only affects few patients. Moreover, a significant interaction effect was seen with ≥ 4000–8000 mg/m2 and ≥ 8000 mg/m2 CED score, based on three patients (2 CED. CYP2C19*17 (rs12248560) showed a negative interaction effect with the presence of an alternative allele T  and CYP2B6*2 (rs8192709)  showed a positive interaction effect. The logistic regression based on tertiles did not show any significant effects   . CYP3A4*3 remained significantly associated with decreased log AMH levels in the main model ; p-value 7 × 10−4)  . In the H levels . While tED score . HoweverCYP3A4 gene leading to the CYP3A4*3 variant (rs4986910) [CYP3A4. Similarly, the effect of the presence of the CYP3A4*1B allele is controversial. In our study, CYP3A4*1B had no significant effect on AMH. The presence of the alternative allele has been previously associated with increased ovarian toxicity in breast cancer survivors receiving cyclophosphamide as part of the treatment regimen [CYP3A4 expression [CYP3A4*1B allele with the CYP3A5*1 expressor allele has previously been suggested based on increased total CYP3A activity (rather than increased CYP3A4 activity) [2 CED are expected to have half (0.585-fold) the level of AMH compared to women receiving 0 mg/m2 CED  . An addi4986910) , results4986910) . The CYP4986910) . This is4986910) . Neverth regimen  and in vpression ,38. In cpression ,39,40,41ctivity) ,44,45,46g/m2 CED . Howeverg/m2 CED .CYP2B6*2 allele (rs8192709) has not previously been associated with gonadotoxicity. Yet, rs8192709 is in high linkage disequilibrium (LD) (EUR: D’ 0.9729) with the poor metabolizer rs4802101 (CYP2B6*1G) in both Caucasian and Asian populations (D’ = 0.894) [CYP2B6*2 allele in interaction with CED scores of ≥8000 mg/m2. The presence of rs4802101 (CYP2B6*1G) was not significantly associated with AMH levels in our cohort . The intronic variant CYP2C19*17 has an effect on promotor activity and is correlated with increased CYP2C19 activity [We also analyzed the effect of two other alleles on ovarian function. A protective effect of the presence of the = 0.894) . This maactivity ,48,49,50CYP2B6 and CYP2C19 and to a lesser extent by CYP3A4 to their active metabolites. CYP3A4 is also involved in the formation of dechloromethyl-CPA  in the discovery cohort showed no significant effects on AMH levels. Yet, when using a standard deviation-based threshold of AMH to classify cases and controls for logistic regression, a significant main effect of CYP3A4*3 was seen, yielding similar results to the linear regression . This maNPY2R) associated with premature menopause. The prevalence of premature menopause was increased in homozygous carriers of this haplotype after exposure to ovarian radiotherapy [BRSK1 gene on the association between alkylating agents and AMH levels [BRSK1), rs365132 (UIMC1) and rs16991615 (MCM8), relevant for age at natural menopause or premature ovarian insufficiency (POI) in the general population [One genome-wide association study (GWAS) in CCSs using data from the SJLIFE and the Childhood Cancer Survivor Study identified a haplotype formed by 4 of 13 SNPs, located upstream of the neuropeptide receptor 2 gene  in a healthy population ,68,69,70CYP3A4*3 is based on a small number of participants, eight participants in the discovery and 12 in the replication cohort with the alternative allele. Due to the small numbers, no conclusions could be drawn from the interaction model of CYP3A4*3 and thus the main model was used to create This study presents results from the largest cohort of female CCSs with available AMH levels and DNA samples and replicates findings in a large representative childhood cancer cohort. In addition, this is the first study to evaluate the effect of SNPs in CYP450 enzymes on gonadotoxicity of exposure to alkylating agents in CCSs. Moreover, all AMH samples including those of the replication cohort were processed at the same laboratory ensuring consistent and comparable results. As we have categorized survivors on CED score instead of tumor type in our analyses, we can generalize the findings of this study towards patients treated in the future, even though some survivors received therapeutic strategies that are no longer in use. A limitation of our study is that the discovery cohort and replication cohort were not completely comparable regarding the distribution of CED categories. However, this variability in the cohort also makes our results more generalizable. Moreover, the main effect of Our findings may enhance clinical practice by identifying patients at high risk of ovarian impairment who more than others may benefit from referral and counseling about fertility preservation options. Recently, the International Guideline Harmonisation Group (IGHG) published recommendations advising that all patients should be informed about their potential risk of gonadal damage ,75,76. TCYP2B6*2 may have a protective effect on AMH levels in patients receiving CED scores of 8000mg/m2 or more. Identifying pharmacogenetic risk factors for alkylating agent-related gonadotoxicity such as the presence of a CYP3A4*3 allele may improve risk prediction models for reduced ovarian function and consequent infertility in female CCSs. This may lead to a better identification of patients at high risk to whom fertility preservation options may be offered. Upfront fertility preservation programs, including ovarian tissue cryopreservation, would benefit from optimized prediction models to identify paediatric cancer patients at the highest risk for gonadotoxicity for whom the balance of benefits of fertility preservation—including ethical considerations—likely outweighs harms [In conclusion, adult female CCSs carrying the CYP3A4*3 allele appear to have a five-fold lower ovarian function after treatment for childhood cancer, and hs harms .


Clinical validation further showed that the expression of ST6GAL1 was significantly increased in patients infected with high-risk HPV subtypes and showed a positive association with E6 in cervical scraping samples. Interfering ST6GAL1 expression markedly blocked the oncogenic effects of E6 on colony formulation, proliferation, and metastasis. Importantly, ST6GAL1 overexpression enhanced tumorigenic activities of both E6-positive and E6-negative cells. Mechanistical investigations revealed that E6 depended on activating YAP1 to stimulate ST6GAL1 expression, as verteporfin (inhibitor of YAP1) significantly suppressed the E6-induced ST6GAL1 upregulation. E6/ST6GAL1 triggered the activation of downstream cGMP/PKG signaling pathway and ODQ (inhibitor of GMP production) simultaneously suppressed the oncogenic activities of both E6 and ST6GAL1 in cervical cancer cells. Taken together, these findings indicate that ST6GAL1 is an important mediator for oncogenic E6 protein to activate the downstream cGMP/PKG signaling pathway, which represents a novel molecular mechanism and potential therapeutic targets for cervical cancer.Alterations in glycosylation regulate fundamental molecular and cellular processes of cancer, serving as important biomarkers and therapeutic targets. However, the potential association and regulatory mechanisms of E6 oncoprotein on glycosylation of cervical cancer cells are still unclear. Here, we evaluated the glycomic changes  Cervical cancer remains the fourth most frequently diagnosed cancer and the fourth leading cause of cancer-related deaths among women worldwide . The majvice versa for HPV18 E6. Furthermore, HPV16 E6 also promotes NHERF1 degradation to enhance the metastasis of cervical cancer cells, whereas NHERF1 is not targeted by HPV18 E6  integrated into the host cell DNA . The E6 HPV18 E6 . Recentl cancers . Thus, aGlycosylation alterations frequently occurred in tumor cells and participate in numerous fundamental biological processes, including cellular signaling, proliferation, adhesion, extracellular matrix interactions, proximal and distal communication, inflammation, and immune surveillance . For exa− C33A cells via using Lectin microarray, and ST6GAL1 is an important mediator for E6 to perform oncogenic activities. In details, E6 depends on activating YAP1 to stimulate ST6GAL1 expression and subsequently results in the activation of downstream cGMP/PKG pathway. Importantly, knockdown ST6GAL1 or blocking cGMP/PKG pathway with specific inhibitor ODQ suppressed the oncogenic activities of both E6-positive and E6-negative cervical cancer cells. Therefore, our findings provide novel insights into the molecular mechanisms involved in HPV16 E6-induced oncogenesis and suggest that targeting ST6GAL1 or cGMP–PKG signaling might be potential therapeutic strategies for cervical cancer.Recently, increased sialylation, GlcNAcylation, and reduced fucosylation have been found to be upregulated in carcinomas of the cervix –28 and p−) cells, were purchased from the cell bank of the Chinese Academy of Sciences  as we previously described (2 at 37°C.The human embryonic kidney (HEK293T) cells, C33A . The instructions for use of these cervical sampling brush were as follows: expose the cervix, wipe off the secretions with a cotton swab, carefully insert the brush head into the endocervical canal until the outer bristles touch the outer cervical neck, rotate clockwise five to eight times, and finally insert the brush head into the preservation solution bottle. Genomic DNA was first extracted from the collected samples using a DNA extraction kit  according to the manufacturer’s instructions. According to the clinical detection results of HPV typing as we presented in our previous study , 10 sampHPV16 E6 stable-expressing C33A cells were constructed according to our previous study . In brieN- and O-linked glycans and analyzed as previously described  with different binding preferences covering escribed , 34. WhoShort-hairpin RNA targeting human ST6GAL1 (pSilencer 1.0-U6-shST6GAL1) and negative control shRNA (pSilencer 1.0-U6-shNC) were designed and chemically synthesized by the TSINGKE Biological Technology . According to the targeting sequences, oligonucleotides coding for each short-hairpin RNA (shRNA) were designed and listed as follows: shRNA ST6GAL1, forward 5′-GGGAGTTACTATGATTCCTTTTTCAA GCTTAAAGGAATCATAGTAACTCCCTTTTT-3′; reverse, 5′-AATTAAAAAGGG AGTTACTATGATTCCTTTAAGCTTGAAAAAGGAATCATAGTAACTCCCGGCC -3′); shRNA-negative control (NC) . Messenger RNA (mRNA) of ST6GAL1 (GenBank accession no. NM_173216.2) was synthesized and inserted into the pcDNA3.1 vector ; empty vector transfection was transfected as the control. C33A or C33A-E6 cells were grown to a density about 70–80% in 6-cm dishes and then transfected with indicated plasmids by Lipofectamine 2000 Transfection Kit  following the manufacturers’ instruction. Forty-eight hours posttransfection, the selective overexpression or silencing of ST6GAL1 was detected by Western blot.p < 0.05. ST6GAL1-associated mRNAs were analyzed using R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) as we previously described  pathway enrichment analysis. MEV software was used for hierarchical cluster analysis of mRNA expression according to the protocol provided by OEbiotech.Total RNA was extracted from C33A and C33A-E6 cells with Trizol reagent . Then, the RNA was quantified using a NanoDrop ND-2000 (Thermo Scientific), and its integrity was assessed using the Agilent Bioanalyzer 2100 . Next, the mRNA molecules were purified from the total RNA using oligo (dT) magnetic beads and fragmented into small pieces and then subjected to transcriptome analysis . The E6-dysregulated mRNAs were identified by the combination of the absolute value of the |log2 [fold change of reads per kilo base per million mapped reads (RPKM)]| ≥ 1 and escribed ; the cut−ΔΔCt method. The gene-specific primer sequences are listed in Total RNA extracted from indicated cervical cancer cells with Trizol reagent . Subsequently, complementary DNA was generated from 1 μg total RNA using Prime Script RT Master Mix . Real-Time PCR (qRT-PCR) was conducted using 2× SYBR green PCR master mix  according to the manufacturer’s protocol to detect mRNA levels of indicated genes. The ABI 7500  was used to perform the amplification reaction. Each experiment was performed in triplicate. Gene expression values were normalized relative to expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by the 2S397 , VASP , p-VASPSer239 , Smad2 , p-Smad2S467 , E-cadherin , sGC , PKG1 , PKG2 , PDE5A , and GAPDH  at 4°C overnight. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized by chemiluminescence (Pierce ECL Western Blotting Substrate).Cells were lysed on ice with radioimmunoprecipitation assay (RIPA) buffer  containing a protease inhibitor cocktail. The concentration of protein was detected by a BCA Protein Assay Kit (Beyotime). Protein samples (30 µg) were separated by 8–12% (v/v) sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane  for subsequent experiments. The membranes were blocked by 5% defatted milk for 2 h at room temperature and then incubated with primary antibodies against HPV16E6 , ST6GAL1 , B3GNT2 , FUT2 , B4GALT1 , Bcl-2 , Bax , p53 , YAP1 , p-YAP13) were seeded into 96-well plates and incubated with 5% CO2 at 37°C, after incubation for 24, 48, 72, and 96 h, 10 μl Cell Counting Kit-8 (CCK-8) reagent  was added into each well and incubated for another 4 h. Cell viability was assessed by detecting the absorbance of the dye solution at 450 nm in a microplate reader . All samples were prepared in triplicate and normalized to a blank control.Cells (1.5 × 103) were plated in six-well plates for 24 h and then received indicated transfection or drug treatment. Cells were then cultured in fresh medium for another week. Colonies were fixed with methanol and stained with crystal violet  for 30 min. Photographs were acquired, and colonies containing more than 50 cells were counted. All the experiments were performed in triplicate.Cells  according to the manufacturer’s protocol at 4°C in the dark for 30 min. The fluorescence of the stained cells was examined by flow cytometry  and analyzed by FlowJo 10.5.4 software.5) were seeded in 24-well plates for growth to 80–90% confluence. Then, the monolayers were wounded by scratching the surface as uniformly as possible with a pipette tip. The remaining cells were washed thrice in phosphate-buffered saline (PBS) to remove cellular debris and incubated at 37°C with serum-free medium. Migrating cells at the wound front were photographed under an Olympus IX73 microscope after 24 h. The area of the wound was measured with ImageJ software .A wound-healing assay was used to evaluate the migration of C33A and C33A-E6 cells. Briefly, cells (1 × 104) were resuspended in serum-free medium and placed into the upper chamber. Complete medium in the lower chamber was considered as a chemical attractant. After incubating for 24 h, the invaded cells attached to the lower surface were fixed with 4% formaldehyde for 15 min and then stained with 0.05% crystal violet for 30 min. The invasion cells were photographed under an Olympus IX73 microscope. Experiments were performed in three independent times and six random fields were scanned in each time.Transwell assay was used to investigate cells invasion in C33A and C33A-E6 cells as we described previously , 32. Brit-test or one-way ANOVA followed by Newman–Keuls post-hoc test was performed for data analysis. A p < 0.05 was considered as statistically significant .The data were presented as mean ± standard deviation of at least three independent experiments and analyzed by GraphPad Prism 8.0 Software . Unpaired − C33A cervical cancer cells (C33A-E6) and detected the glycan repertoire of cellular glycoproteins via using 95-lectins microarray. In general, C33A-E6 cells showed higher levels of α-2,6 sialic acids (bound by lectin SNA-I), N-acetylgalactosamine , α-1,3- and α-1,6-linked mannose , β-galactosidase (GAL9), and lower levels of lactose/galactose , and α-1,2 fucose structures (UEA-I) than the corresponding control C33A cells  or a non-targeting control (shNTC)  and Smad was significantly increased by ST6GAL1 overexpression and nockdown , 36. Thu+ Siha and HPV18+ Hela cells , α-1,3- and α-1,6-linked mannose (MAN2A1), and N-acetylgalactosamine , and decreased levels of lactose/galactose (B4GALT1) and α-1,2 fucose structures .HPV16 E6 oncoprotein plays multifunctional roles on post-translational modifications (PTMs), including ubiquitination, acetylation, and phosphorylation, and thereby contributes to the viral life cycle and to the induction of malignancy –40. Servp110 and therefore confers chemoresistance of pancreatic ductal adenocarcinomas  expression  can be found in The studies involving human participants were reviewed and approved by Institutional Review Board of the Wuhan Children’s Hospital . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.DT, YH, YX, and TX: study conception and design. JW, GL, ML, QC, CYao, and HC: data acquisition. CYuan and NS: analysis and data interpretation. JW and GL: drafting of the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (81760540 and 82060539), Natural Science Foundation of Wuhan Municipal Health Commission (grant no. WX18Q27), Natural Science Foundation of Hubei Municipal Health Commission (grant no. WJ2021M016), The Top Medical Young Talents of Hubei Province (2016), and The Open Research Fund Program of the State Key Laboratory of Virology of China (grant no. 2015KF010).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The central role of eukaryotic translation initiation factor 4E (eIF4E) in controlling mRNA translation has been clearly assessed in the last decades. eIF4E function is essential for numerous physiological processes, such as protein synthesis, cellular growth and differentiation; dysregulation of its activity has been linked to ageing, cancer onset and progression and neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). The interaction between eIF4E and the eukaryotic initiation factor 4G (eIF4G) is crucial for the assembly of the translational machinery, the initial step of mRNA translation. A well-characterized group of proteins, named 4E-binding proteins (4E-BPs), inhibits the eIF4E–eIF4G interaction by competing for the same binding site on the eIF4E surface. 4E-BPs and eIF4G share a single canonical motif for the interaction with a conserved hydrophobic patch of eIF4E. However, a second non-canonical and not conserved binding motif was recently detected for eIF4G and several 4E-BPs. Here, we review the structural features of the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs, focusing on the implications of the recent structural and biochemical evidence for the development of new therapeutic strategies. The design of novel eIF4E-targeting molecules that inhibit translation might provide new avenues for the treatment of several conditions. Translation initiation, the rate-limiting step in protein synthesis, is finely regulated by several mechanisms . In the eIF4F is an heterotrimeric complex composed by eIF4E, the DEAD-box helicase eIF4A and eIF4G , 6 and i4E-BPs are a family of inhibitory proteins that sequester eIF4E through the interaction with the same binding site recognized by eIF4G, therefore, preventing the assembly of the eIF4F complex –10. 4E-BIn the last decades, the three-dimensional (3D) structure of eIF4E alone or in complex with its molecular partners from different organisms has been widely characterized using experimental and theoretical approaches , 25–35.Briefly, three main regions can be identified in the 3D structure of eIF4E Fig. a: a ventThis review provides an overview of the roles of eIF4E and 4E-BPs in physiological and pathological conditions. We focus on the structural features of the interactions between eIF4E and different 4E-BPs and discuss new therapeutic strategies targeting eIF4E.The main function of eIF4E is to mediate ribosome recruitment on the mRNA to start protein synthesis. Nevertheless, other roles for this protein have been described. eIF4E strongly enhances the helicase activity of eIF4A, independently of its cap-binding function . The nuceif4e transcription via binding to the E-boxes located in the eIF4E promoter ; , another member of the 4E-BP family, has been extensively characterized, showing a specific role in the central nervous system . CYFIP1 sability . Lack ofsability . CYFIP1 sability , 82, thesability . Furthersability . Synaptisability , 82. A csability . In partdiseases , 86. CYFdiseases –89.D. melanogaster CUP protein . Unlike 4E-BP1, these three proteins form an additional main-chain contact between the nitrogen atom of Arg 375 [CUP] (Lys 43 in 4E-Tand Arg 81 in Thor) and the carbonyl oxygens of His 111 [Dm 4E] [Sc 4E-BPs in complex with Sc eIF4E, confirming the conserved binding mode between the non-canonical site and the lateral surface, despite the poor sequence conservation [Sc p20 and Sc eIF4E  residues Leu 633, Ile 636, Val 639 and Val 640 interact with the hydrophobic residues Phe 47, Ile 63, Leu 75 and Ile 79of Hs eIF4E, while in Dm eIF4G (PDBID: 4UEC) the residues involved in the hydrophobic interaction are Val 641, Ile 646, Leu 647 that engage Tyr 80, Ile 96, Leu 108 and Ile 112, respectively, of Dm eIF4E. The linker region engages the surface of eIF4E through the interaction with two conserved eIF4E residues: Hs Asn 77 (or Dm Asn 110) and Hs His 78 (or Dm His 111) [eIF4G interacts with eIF4E in the same way as the other 4E-BPs. Structurally, the eIF4G surface deputed to interact with eIF4E is composed by the same three regions found in 4E-BPs . The linker region, that is located immediately after the C:4E-BM, forms an elbow loop that orients the NC:4E-BM towards the lateral surface of eIF4E and engages this area with hydrophobic interactions. Specifically, the G PDBID: T46 resid. [Hs eIF4G non-canonical binding site (572Tyr-Asp-Arg-Glu-Phe-Leu-Leu78) that, unlike the fungal Chaetomium thermophilum homologous protein, does not have a defined secondary structure  and Hs eIF4G fragment peptides with eIF4E, establishing that eIF4G non-canonical motif has a lower binding affinity than the non-canonical binding motif of 4E-BP2 for the cap-binding protein [Saccharomyces cerevisiae eIF4E–eIF4G complex (PDBID: 1RF8) [Sc eIF4G also binds Sc eIF4E through the conserved bipartite mechanism, although the crystal structure of the complex is not available yet [Sc eIF4G–eIF4E demonstrating that the complex likely adopts a conformation very similar to Ct eIF4E–eIF4G [Chaetomium thermophilum) eIF4G (PDBID: 6FC0) shows the conserved bipartite binding mechanism described for metazoan 4E-BPs but with some striking differences. In addition to the NC:4E-BM α-helix binding to the lateral surface of eIF4E, in the N-terminal segment two additional α-helices wrap around the N-terminus region of eIF4E forming a bracelet-like structure which significantly contributes to enlarge the Ct eIF4E–eIF4G interface, as observed only for Sc eIF4E–eIF4G complex [In 2011 Umenaga et al.  have dis protein . This diD: 1RF8)  reveals D: 1RF8) . CHAPS mable yet . They ha4E–eIF4G . The thr complex . In conc complex , and whaOne of the main features of cancer onset and progression is the malfunctioning of the translation machinery, resulting in an increase of protein synthesis due to eIF4E dysregulation. eIF4E has thus been recognized as an attractive pharmacological target in particular for anticancer therapy. To date, several inhibitors targeting eIF4E have been developed. A subgroup of these inhibitors acts directly repressing the translation of eIF4E mRNA; others by blocking the interaction between eIF4E and its partners. Furthermore, indirect strategies implying the use of inhibitors that act on phosphorylation pathways have been also developed , 15. BecStreptomyces hygroscopicus that is able to exert an allosteric inhibitory effect on mTORC1. Rapamycin has shown antineoplastic effects towards several cancer cell lines and mouse models [eIF4E is also indirectly targeted by inhibitors of the mTOR pathway, which interrupt the upstream signals for the phosphorylation of 4E-BPs, consequently preventing the dissociation between eIF4E and 4E-BPs. The best characterized compound is rapamycin (or sirolimus), a macrolide produced by e models . Howevere models , 156–161e models –166, whie models . Other se models , 15. Thee models .Structural properties play a key role in the regulation of cap-dependent translation; targeting eIF4E/eIF4G interaction is thus another promising approach. High-throughput screening assays identified small-molecule inhibitors , such as. described the development of second-generation hydrocarbon stapled-peptides in complex with eIF4E, showing increased binding kinetics and improved scaffold in terms of their degree of ordered helical structure [Peptides and peptidomimetics are an emerging class of molecules able to modulate and inhibit a wide range of protein–protein interactions (PPIs) and cell membrane morphology. Their stability, specificity and low toxicity make peptidomimetics more suitable for therapeutic application than small compounds –174. An tructure , 35, 177Hence, peptides and peptidomimetics represent a new and attractive class of biomolecules, which will most likely play a central role in pharmacological applications for the development novel therapeutics.The eukaryotic translation initiation factor eIF4E plays a critical role in promoting the first step of the translation process. It is clearly established that dysregulation in the activity of eIF4E can be the primary cause of different types of diseases, such as cancer and neurodevelopmental disorders. Thus, it represents an attractive biological target for the development of drugs aimed to interfere with the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs. The plethora of information accumulated in the last years on the structural features of these interactions, and their detailed analysis, is cardinal for the advancement of new therapeutic strategies. This structural information can be used in a near future to design novel and more specific eIF4E-targeting molecules with inhibitory potential, thus having an impact on the mRNA translation process.


Swine influenza virus (SIV) is an important zoonosis pathogen. The 2009 pandemic of H1N1 influenza A virus (2009/H1N1) highlighted the importance of the role of pigs as intermediate hosts. Liaoning province, located in northeastern China, has become one of the largest pig-farming areas since 2016. However, the epidemiology and evolutionary properties of SIVs in Liaoning are largely unknown. We performed systematic epidemiological and genetic dynamics surveillance of SIVs in Liaoning province during 2020. In total, 33,195 pig nasal swabs were collected, with an SIV detection rate of 2%. Our analysis revealed that multiple subtypes of SIVs are co-circulating in the pig population in Liaoning, including H1N1, H1N2 and H3N2 SIVs. Furthermore, 24 H1N1 SIVs were confirmed to belong to the EA H1N1 lineage and divided into two genotypes. The two genotypes were both triple reassortant, and the predominant one with polymerase, nucleoprotein (NP), and matrix protein (M) genes originating from 2009/H1N1; hemagglutinin (HA) and neuraminidase (NA) genes originating from EA H1N1; and the nonstructural protein (NS) gene originating from triple reassortant H1N2 (TR H1N2) was detected in Liaoning for the first time. According to our evolutionary analysis, the EA H1N1 virus in Liaoning will undergo further genome variation. Swine have been considered as an intermediate host of influenza virus, as they contain both avian-like receptors  and human-like receptors  in the up respiratory track epithelia cells, meaning that both avian and mammalian influenza viruses can replicate in swine ,2. DiffeChina is one of the largest pork-consuming countries, with nearly 500 million pigs slaughtered annually. For environmental reasons, numerous large pig-farming companies in southern China have changed their expansion strategies by moving their pig farms to northeastern China since 2016. As a result, tens of large pig farms have been built in Liaoning province in recent years. As the pig population increases rapidly, the epidemiological situation of SIVs in Liaoning is becoming more and more complicated . In thisDuring SIV active surveillance in 2020, 33,195 pig nasal swabs were collected from apparently healthy pigs from 30 different pig farms in 11 different cities of Liaoning. All the pig farms utilized all-in and all-out breeding methods. According to the biosecurity requirements, pig nasal swabs were collected from the finishing pig herds of each pig farm just before transferring them to slaughterhouses. In total, 66 finishing pig herds were sampled. The pig farms types, sizes, and sampling details can be found in PCR based on influenza virus matrix (M) gene was conducted for the selection of positive samples . Viral RPig sera were collected from herds of pigs of different ages, including suckling pigs, nursery pigs, gilts, sows, and boars, by pig farm resident veterinarians as part of routine surveillance, and the remaining sera were given to us for an SIV HI test. More pig farms and sampling details can be found in The viral genome was sequenced by Sanger sequencing. The nucleotide sequences of our SIVs isolates were processed using the Lasergene sequence analysis software. The reference sequences for phylogenetic tree construction were retrieved from the 3 main influenza virus databases: the NCBI influenza virus database, the GISAID influenza database, and the Influenza Research database. The MEGA X  softwarehttp://www.datamonkey.org/), accessed 22 January 2022 [p-value were set at 0.05.The selection pressure and positive selection sites of Liaoning EA H1N1 SIVs were estimated by calculating synonymous (dS) and non-synonymous (dN) substitution rates at every codon using the Datamonkey web server , polymerase basic protein 1 (PB1), polymerase acid protein (PA), nucleoprotein (NP), matrix protein (M), and nonstructural protein (NS) genes of the 24 viruses, at 94.17–100%, 94.90–99.96%, 93.90–100%, 93.30–100%, 91.85–100%, and 94.51–100%, respectively, at the nucleotide level. The NP and M genes formed two groups. The PB2, PB1, PA, and NS genes formed one group. The PB2, PB1, PA, and NP genes originated from the 2009/H1N1 lineage. Most SIV M genes originated from the 2009/H1N1 lineage except for one SIV , which originated from the EA H1N1 lineage. The NS group originated from the TR H1N2 lineage as shown in According to our genetic analysis results, the predominant genotype of EA H1N1 SIVs in Liaoning was a triple reassortant, which was recombinated with the EA H1N1, 2009/H1N1, and TR H1N2 lineages. Only one virus, DL 1007, belonged to a different genotype due to its M gene from the original EA H1N1 lineage. We named the two genotype groups G1 and G2, respectively.All the EA H1N1 viruses possessed a single basic amino acid in the HA cleavage site, PSIQSR/G, which was characteristic of low-pathogenic avian influenza virus. Mutations in the HA protein that contributed to the increased binding to human-type receptors, transmission, and replication, such as 190D (H3 numbering), 225E, and 226Q, were detected in the EA H1N1 SIVs ,28,29. AThe amino acids at positions 251, 271, 588, 431, 590, and 591 of the PB2 protein suggested that these viruses could adapt to mammalian hosts ,31,32,33Other amino acid mutations, which could increase mammalian adaptation, pathogenicity, and anti-influenza drugs resistance, were also found in the NP, M2, and NS1 genes as shown in Given that the EA H1N1 SIV infection is common in the Liaoning pig population, we further analyzed its genomic evolution properties. There are only 33 Liaoning EA H1 SIVs sequences available online (including 29 EA H1N1 and 4 EA H1N2 SIVs) for now. The EA H1N1 SIV was first detected in Liaoning in 2006 and became prevalent gradually. Since 2014, reassortant EA H1N1 or H1N2 SIVs have been detected in Liaoning . From 20The selection pressure of the HA and NA genes from P1 (11 HA and NA genes), P2 (22 HA genes and 18 NA genes except 4 N2 NA), and P3 (24 HA and NA genes) were analyzed. As shown in There were three positive selected sites, 60, 219, and 361, in the HA gene across the P3 time period. HA 219 was located at the 220 loop; the mutation might affect the receptor binding property of the virus . PositivIn 2020, we performed extensive epidemiology surveillance of SIVs in Liaoning. Pig nasal swabs and sera were collected from 11 main pig-farming cities. Our results indicated that three subtypes of SIVs, namely H1N1, H1N2, and H3N2, are circulating in the pig population of Liaoning and that EA H1N1 SIV is the majority lineage. EA H1N1 SIVs in Liaoning evolved into two genotypes, G1 and G2, through recombination with the 2009/H1N1 and TR H1N2 virus. Serological surveillance indicated that the EA H1N1 virus exhibits different infection rates in different age groups of pigs. We found that these SIVs contain adaptation molecular characteristics to mammalian hosts and will acquire adaptive evolutionary mutation in the future.The EA H1N1 SIVs began to circulate in China in 2001. A complex and extensive reassortment event occurred among EA H1N1, TR H1N2, and 2009/H1N1 SIV after the 2009 human influenza pandemic ,46,47. IThe SIV vaccine is rarely used in Liaoning pig farms, so serological studies will reflect the SIV infection rate. None of the sampled pig farms in our study applied the influenza vaccine either. There is no specific requirement concerning pig age for serological studies. Usually, we cannot obtain enough information about the infection rate among pigs of different ages. In our study, we collected sera from herds of pigs of various ages and found that the EA H1N1 SIV infection rate is related to the pig production stages. Suckling and nursery pigs had relatively lower EA H1N1 SIV infection rates, which might be due to their isolated breeding environment. Except for the unknown age of the herd, gilts had the highest SIV infection rate, which might be due to the breeding process of gilts. Generally, after the nursery stage, pigs that were healthy and met the breeding criteria were picked out from different herds and developed into gilt herds. The multiple sources of gilt herds might have contributed to their high EA H1N1 SIV seroprevalence. Sows and boars may have more SIV infection opportunities because they live longer than other herds.Previous studies have shown that EA H1N1 SIVs are a great threat to human health: EA H1N1 can transmit efficiently among ferrets, and serological evidence has indicated that the EA H1N1 SIVs infection rates could reach 10.4% and 4.4% among swine workers and the general population ,28,48. ILiaoning has a complex SIV ecosystem. Two genotypes of EA H1N1 SIVs are circulating in Liaoning, and more extensive mutations might be generated in EA H1N1 SIVs in Liaoning. The SIV epidemiology of Liaoning might be underestimated; thus, routine and extensive surveillance studies should be performed in Liaoning.


Supplemental Digital Content is available in the text. The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt–Oram syndrome  and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients.Tbx5G125R) and performed electrophysiologic analyses , transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling .We analyzed ECGs of an extended family pedigree of Holt–Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome  that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties.We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt–Oram syndrome patients. Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers. A pathogenic variant in the fifth exon of TBX5 ([T-box transcription factor 5] p.G125R) found in a Dutch, atypical Holt–Oram syndrome family with early onset atrial fibrillation was modeled in mice.This is the first human pathogenic variant based on a patient family in this key cardiac transcription factor explored in vivo.We identify widespread electrophysiologic, transcriptional, and epigenetic changes  in the atria of Tbx5-p.G125R heterozygous mice distinct from the changes in atria of Tbx5 insufficiency models.The characterization of the Tbx5 p.G125R mouse model indicates that a patient-specific pathogenic variant in TBX5 induces changes in regulatory element activity, altered balance in the regulatory network of atrial cardiomyocytes, and clinically relevant changes in cardiomyocyte function.These findings shed light on the target and off-target genes of Tbx5-p.G125R because of its altered properties, which are at the basis of distinct electrophysiologic changes observed in a subset of Holt–Oram syndrome patients with atrial fibrillation.This work may provide insight into the epigenetic changes and transcriptional underpinning of arrhythmia in the general population caused by small increases in TBX5-expression caused by common variants predisposing to atrial fibrillation.TBX5 (T-box transcription factor 5) have been linked to Holt–Oram syndrome (HOS), which is typically characterized by varying degrees of limb and cardiac malformations and conduction defects (OMIM [Online Mendelian Inheritance in Man] #142900).3 Tbx5 is dose-dependently required for heart development and specification and function of the conduction system, and determines the working myocardial phenotype of the atrium.8 Tbx5 has been put forward as key regulator of ion-handling protein-encoding gene expression and rhythm control in vivo.13 Genome-wide association studies uncovered several variants near TBX5 associated with atrial fibrillation (AF). Moreover, transcriptome-wide analysis of human heart tissue identified an association of increased TBX5 expression with AF risk.14Pathogenic variants in T-box transcription factor (TF) TBX5 causing a glycine to arginine substitution  in the T-box domain was found in a Dutch, atypical HOS family.15 In this family, affected members show HOS symptoms such as mild skeletal abnormalities and septal defects, as well as atypical early onset paroxysmal AF. The function of missense variants in this key TF and the impact on gene regulation in the context of heart function in vivo are poorly understood. Therefore, we engineered the p.G125R variant in the highly conserved T-box of mouse Tbx5, and used this model to gain insight into the molecular mechanisms underlying structural and electrophysiological changes predisposing to AF in TBX5 missense variant carriers.A pathogenic variant in the fifth exon of Supplemental Material.Detailed Methods are provided in the Whole-tissue RNA-sequencing , cardiomyocyte (CM) nucleus H3K27ac (histone H3 lysine 27 acetylation) CUT&RUN (cleavage under targets and release under nuclease), CM nucleus assay for transposase-accessible chromatin using sequencing (ATAC-seq), and single-nuclei RNA-seq are available on Gene Expression Omnibus (GSE167082). All other data and supporting materials have been provided with the published article.The study was conducted according to the guidelines of the Declaration of Helsinki. Written informed consent was obtained from the families presented in the paper.Animal care and experiments conform to the Directive 2010/63/EU of the European Parliament. All animal work was approved by the Animal Experimental Committee of the Amsterdam University Medical Centers , was performed in compliance with the Dutch government guidelines, and was approved by the Central Committee Animal Experiments.Supplemental Material which also describes the specific statistical tests used to analyze large data sets. Results of statistical analysis are given in the text or the figure legends.A detailed description of the statistics can be found in the 15 Here, we reanalyzed in detail the available ECG traces (as obtained by 24-h Holter or 12-lead ECG) of the patients previously analyzed.15 We found a range of additional abnormalities—including irregular RR interval, right bundle branch block, atrioventricular (AV) junctional escape, AV junctional rhythm, sinus arrest, left ventricular noncompaction, atrial extras, and sick sinus syndrome—that were not checked for previously . This shows that these patients suffer from other types of supraventricular arrhythmia that indicate dysfunctionality of the atrial muscle, including the sinoatrial and AV nodes.Heterozygous TBX5-p.G125R carriers show atypical HOS, including cardiac septal defects, mild skeletal abnormalities, and early onset AF . Interbreeding of Tbx5G125R/+ animals showed that from embryonic day (E) 12.5 to E16.5, homozygous Tbx5G125R/G125R fetuses were necrotic or dead (Table S2), revealing that homozygosity of the mutation is incompatible with life. Tbx5G125R/G125R fetuses showed ventricular and atrial septal defects (E14.5–E16.5), as well as enlarged right atria at E16.5 , which were also present in patients heterozygous for this pathogenic variant . We did not observe differences in CM proliferation rates in embryonic (E12.5) right atria or left ventricles between different genotypes , nor differences in the heart weight–tibia length ratio between control (n=6) and Tbx5G125R/+ mice (n=10) at 20 days after birth (Table S3).To investigate how the p.G125R pathogenic variant in TBX5 affects heart function, we generated a mouse model with this point mutation in Tbx5G125R/+ mice  and Wenckebach cycle lengths compared with controls . Hearts isolated from Tbx5G125R/+ mice also showed longer and more variable RR intervals, as well as shorter PR intervals, than controls . In addition, these experiments show that the atrial ectopic beats occurred during or after the QRS complex, and occurred in all cases in response to a premature ventricular beat  and Tbx5G125R/+ mice were susceptible to atrial arrhythmias, we performed programmed atrial stimulation in vivo using a transesophageal catheter . Similarly, recorded action potentials in isolated atrial CMs  and at all action potential repolarization phases .To assess whether r Figure A. We wers Figure D reveales Figure E. ActionTbx5G125R/+ nuclei were obtained . Only the CM cluster showed some degree of segregation between genotypes . CMs showed 59 genes that were significantly differentially expressed; endothelial cells showed 17 genes and fibroblasts showed 16 . Analyses of the Gene Ontology terms for the differentially expressed genes in CMs showed mostly terms involving muscle contraction regulation and ion transport (Table S7). The fraction of CMs and fibroblasts differed between genotypes . Picrosirius red–staining sections of 8-month-old control (n=4) and Tbx5G125R/+ (n=5) atria revealed a trend toward a smaller fibrotic area in the atria of Tbx5G125R/+ mice compared with controls; however, it was not significant . Together, these findings suggest that the Tbx5G125R/+ atria do not show elevated fibrosis even though fibrosis has been associated with AF.To gain insight into the cell types affected by Tbx5-p.G125R, we performed single-nucleus RNA-seq on right atrial tissue. Profiles from 3475 wild-type nuclei and 2933 Tbx5G125R/+ (n=5) animals . The point mutation may affect mRNA levels of Tbx5G125R . Using RNA-seq reads and Varscan,16 we quantified the number of Tbx5 transcripts tags containing the wild-type allele (G) and the number containing the mutant allele (C). In control animals, the wild-type allele had a frequency of 100%. In Tbx5G125R/+ animals, the variant allele had a frequency of 54% , indicating both the wild-type and Tbx5G125R/+ transcripts are present at equal levels in Tbx5G125R/+ animals.To determine at greater depth the transcriptional responses to heterozygosity of Tbx5-p.G125R, RNA-seq was performed on whole right atria of adult female control (n=5) and Padj < 0.05; Table S8; Figure Tbx5G125R/+ atria, 610 were upregulated. While the cardiac function of many of the most significantly differentially expressed genes (Table S8) has not been defined, Lgals3  and Padi2 (peptidyl arginine deiminase-2) have been implicated in cardiac dysfunction.18 We observed differential expression in 33 of 300 analyzed gene-encoding proteins involved in ion handling/electrophysiology, among which were several intracellular Ca2+-modulating and gap junction genes , as well as K+ channel genes such as Kcnj15 (potassium inwardly rectifying channel subfamily J member 15) and Kcnn2 . Reduced expression of Scn5a (sodium voltage-gated channel α subunit 5), Ryr2 (ryanodine receptor 2), and Atp2a2 (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2) was linked to conduction slowing and altered intracellular Ca2+ concentrations in Tbx5 haploinsufficient mice.11 However, these and other genes that were previously found to respond to Tbx5 insufficiency  were not responsive to Tbx5-p.G125R. Gene Ontology term analysis yielded general terms for genes downregulated in the Tbx5G125R/+, but pointed to actin filament and muscle process, as well as ion transport, for upregulated genes . Transcriptional profiles of left atria of mutants and controls were in accordance with those of the right atria (Table S8).We found 1316 genes differentially expressed  in whole-tissue RNA-seq of right and left atria, of which only Postn (activated fibroblast marker) was decreased in expression in right atria of Tbx5G125R/+ animals . These data further support the absence of fibrosis and activated fibroblasts in Tbx5G125R/+ atria. Furthermore, as the right atrial samples all contained sinoatrial node tissue, we explored the expression of sinoatrial node–specific genes23 in the whole-tissue RNA-seq data. Of 30 markers tested, 14 showed differential expression. Notably, transcripts for key TFs Tbx3, Isl1 (insulin gene enhancer protein ISL-1), and Shox2 (short stature homeobox 2), and key ion channels Hcn4 (hyperpolarization activated cyclic nucleotide gated potassium channel 4), Hcn1 (hyperpolarization activated cyclic nucleotide gated potassium channel 1), and Cacna2d2  were not differentially expressed between genotypes. However, Cacna1d, , Cacna2d3, calcium voltage-gated channel auxiliary subunit α2 δ3), Cacna1g , Cacna1h , and Ryr3 (ryanodine receptor 3), as well bone morphogenetic protein (BMP)–signaling components , showed significant differential abundance in the data sets .We explored fibrotic tissue markers  using ATAC-seq  and reduced accessibility  compared with sites of unchanging accessibility . TBX5 was reported to interact and cooperatively regulate cardiac target gene expression with KLF13 (Krüppel-like factor 13).26 These data suggest that Tbx5-p.G125R may have reduced interaction with KLF/SP factors, leading to reduced accessibility of sites normally co-occupied by Tbx5 and KLF/SP factors.To gain insight into the changes in epigenetic state and regulatory element deployment in response to Tbx5-p.G125R, we assessed the chromatin accessibility profile of control and q Figure A. There Tbx5G125R/+ (n=6) pooled left and right atria . We detected 127 286 regions enriched for H3K27ac association and 1653 sites with significantly differential H3K27ac regions , of which 1335 regions have increased signal in Tbx5G125R/+ and 318 regions have reduced signal . Of the 127 286 H3K27ac regions, 18% (22 911/127 286) overlapped with Tbx5 binding sites in the fetal mouse heart24 (Table S16), whereas in the regions with increased H3K27ac association, 40%  overlapped Tbx5 binding sites . For example, accessibility and H3K27ac signal of Tbx5-bound regulatory elements in Padi2 and Wdr3 (WD repeat-containing protein 3) are strongly increased in Tbx5G125R/+ atria . Interestingly, the H3K27ac regions that are reduced in the Tbx5G125R/+ atria all overlap Tbx5 binding sites . These data are in agreement with the chromatin accessibility analysis, and together indicate that Tbx5-p.G125R induces altered epigenetic states of sites (including putative enhancers) normally occupied by Tbx5 and—to a lesser extent—of sites not occupied by Tbx5.To further examine the impact of Tbx5-p.G125R on genome-wide regulatory element activity, we examined the genomic localization of H3K27ac association by CUT&RUN in isolated PCM1+  CM nuclei of 8-week-old control (n=5) and 12 By crossing our RNA-seq data with the ATAC-seq and CUT&RUN data sets , we detected 128 enhancer-derived transcripts  that were differentially expressed between control and Tbx5G125R/+ animals in the right atria , of which 42 were more abundant in the control and 86 more abundant in the Tbx5G125R/+ animals . Out of the 128 thus identified elements, 71 showed Tbx5 occupation (Table S17).24 The remaining 57 ncRNA-associated elements often showed Tbx5 occupation of accessible/H3K27ac-marked elements at a few-kbp distance, but these were considered to not colocalize. An example of a differentially expressed ncRNA with a transcriptional start site overlapping differential accessibility and H3K27ac is shown in Figure Bmp6 (bone morphogenetic protein 6). Of these differentially active regulatory elements, we identified 166 candidate target genes that were differently expressed between genotypes (Table S17). Highly interesting candidates include Tbx20 (T-box transcription factor 20), Sema3a (semaphorin 3A), Kcna4 (potassium voltage-gated channel subfamily A member 4), Slc8a1, and Tgfb2 (transforming growth factor β2), which are likely to impact atrial function in Tbx5G125R/+ animals 31 Differentially transcribed ncRNAs, in combination with differential accessibility and differential target gene expression, are shown for the Tgfb2, Sema3a, and Kcnj15 loci in Figure S8. Interestingly, previously identified prominent Tbx5-dependent ncRNAs (close to Ryr2 and to Atp2a2 [Serca2a (sarcoplasmic/endoplasmic reticulum CA2+ ATPase 2a])12 and their putative target genes  were not affected in Tbx5G125R/+ atria .Previously, Tbx5-dependent enhancers and noncoding RNA (ncRNA) were identified by analyzing Tbx5-dependent enhancer transcription.15 Here, we show that these patients suffer from other types of supraventricular arrhythmias that indicate dysfunctionality of the atrial muscle, including the sinoatrial node and AV node, that were not identified previously (Table S1). Typical HOS is usually associated with loss-of-function variants leading to haploinsufficiency; the atypical HOS phenotype of TBX5-p.G125R carriers suggest the pathogenic missense variant causes TBX5 to gain specific function(s). In the mouse model with this variant, the quantity of Tbx5 (wild-type) and Tbx5-p.G125R mRNA is equal in the adult Tbx5G125R/+ mouse atria, indicating that loss of expression  causing haploinsufficiency does not occur. We did identify broadly changed expression profiles and epigenetic states induced by heterozygosity for Tbx5-p.G125R. However, important genes and enhancers previously found to respond to Tbx5 insufficiency 12 did not respond to Tbx5-p.G125R. This indicates Tbx5-p.G125R has gained functions invoking molecular mechanisms different from those invoked by reduced Tbx5 dose. Intriguingly, expression quantitative trait loci analysis revealed that AF risk variant carriers in the human population express significantly more, not less, TBX5 in the heart.14 It will be interesting to investigate whether the Tbx5-p.G125R TF and increased TBX5 dose in risk variant carriers share transcriptional responses underlying AF propensity.Previous characterization of a Dutch family with the pathogenic TBX5-p.G125R missense variant revealed atypical HOS, with mild skeletal abnormalities and lower penetrance of congenital heart defects but a high prevalence of AF.33 The right atrial RNA-seq data sets indicated differential expression in 14 of 30 selected sinoatrial node markers. While these findings require validation, they suggest that the sinoatrial node may be transcriptionally affected in Tbx5G125R/+ mice. The differentially expressed genes included genes encoding calcium channels and BMP-signaling components important for sinoatrial node development and function,22 which may contribute to dysfunction of the sinoatrial node in Tbx5G125R/+ mice. Moreover, the balance between Tbx5 and the transcriptional repressor Tbx3 was found to determine the function of conduction system components.35 The altered DNA binding properties of Tbx5-p.G125R could affect both the regulation of important regulators in the nodes, and change the competitive balance with Tbx3. Further studies are required to explore the impact and underlying mechanisms of Tbx5-p.G125R on the sinoatrial and AV nodes.The patients displayed AV junctional escape beats, atrial extras, sinus bradycardia, and sick sinus syndrome in addition to AF. These arrhythmias were reproduced in the Tbx5-p.G125R mouse model, indicating Tbx5-p.G125R function and impact on cardiac electrophysiology in vivo are conserved between human and mice. Tbx5 is critical for the development and gene regulation in the sinoatrial and the AV nodes.Tbx5-haploinsufficient and -deficient mouse models implicate reduced expression of Ryr2 and Atp2a2 in AF propensity,36 these genes are not deregulated in atria of heterozygous Tbx5-p.G125R mice. However, we observed changes in expression of many other ion-handling protein-encoding genes, and in particular, genes associated with atrial function. Notably, the transcriptional profiling and differential regulatory element usage analysis identified Tbx5-p.G125R–mediated increased atrial expression of Tbx20, Sema3a, Kcna4, and Pitx2c , and decreased atrial expression of Tgfb2. Each of these genes has been causally linked to heart function and rhythm control, further indicating that multiple complex mechanisms underlie the arrhythmia phenotype. Tbx20 is an essential cardiac TF, controlling CM function, ion channel gene expression, and atrial development.28 Because T-box TF function is highly dose-sensitive, increased Tbx20 expression in Tbx5+/G125R atria is likely to broadly affect cardiac gene expression and function. Sema3a, produced by CMs, controls cardiac sympathetic innervation and is associated with arrhythmia when disrupted or overexpressed in mice.29 Moreover, Sema3a was identified as a naturally occurring Kv4.3 (potassium voltage-gated channel) inhibitor,37 suggesting its induction could affect Kv4.3. An induction of Kv1.4 (encoded by Kcna4) has been observed in pathophysiologic conditions in the human heart . An increase of Kv1.4 (partly replacing Kv4.3) in atria was suggested to prolong ADP and to alter its dynamics, causing AF predisposition.31Transcriptional profiling of the atria showed that heterozygosity of Tbx5-p.G125R leads to extensive dysregulation of gene expression in the atria, indicating multiple and complex mechanisms underlie the arrhythmia phenotype. While studies of 40 In the formed left atrium, Pitx2c was shown to interact with Tbx5 to regulate ion channel gene expression and electrophysiology.10 Ectopic induction of Pitx2c in the right atrium could potentially affect sinoatrial node development and function, consistent with the observation that both human and mouse TBX5-pG125R carriers display sinoatrial node dysfunction. Furthermore, the atrial TF network around Tbx5 is likely affected by ectopic presence of Pitx2c in the right atrium, leading to altered ion channel gene expression.10 The decreased expression of Tgfb2 in the atria of heterozygous Tbx5-p.G125R mice may lead to reduced TGF-β signaling and diminished fibrosis,30 as is observed in the atria of these mice.Pitx2c is specifically expressed in the left atrium, where it imposes left identity on the atrium, initiates pulmonary vein myocardium formation, and suppresses sinoatrial node development during embryogenesis.Tbx5G125R/+ CMs show a prolongation of the APD. While APD is often shortened in AF patients, mainly in the setting of sustained arrhythmias,42 APD prolongation is associated with increased risk of developing AF in patients.45 In atria of Tbx5G125R/+ mice, APD was prolonged as well, which is consistent with the patient clinical picture as well as with the increase in atrial arrhythmias we observed. The decreased Ca2+ amplitude and intracellular Ca2+ concentrations may be a result of longer Ca2+ currents because of reduced Ca2+-induced Ca2+ current inactivation, as well as decreased activation of Ca2+-activated K+ channels, and both mechanisms may contribute to the prolonged ADP that was observed. Moreover, deregulation of genes encoding channel subunits for various voltage-gated potassium currents ], Kcna4 [Kv1.4]; see Figure 2+-modulating proteins such as Cacna1c and Cacna1g could be at the basis of the decreased of Ca2+ transient amplitude observed in the mutant mice. Another explanation could involve Mrvi1 (murine retrovirus integration site 1 homolog), which uniquely responds to Tbx5-p.G125R and is decreased in the Tbx5G125R/+ atria. Mrvi1 is associated with Pln and plays a key role in cardiac contractility by modulating nitric oxide–induced inhibition of Ca2+ signaling.47Patch clamp measurements of isolated atrial 24 suggesting TBX5-p.G125R may display increased interaction with genuine Tbx5-occupied sites in vivo, in keeping with its increased DNA-binding affinity in vitro.15 However, sites normally not occupied by Tbx5 also showed increased accessibility, and the sites displaying decreased accessibility were enriched for Tbx5-binding and T-box motifs. Together, these observations suggest Tbx5-p.G125R has acquired properties that both quantitatively and qualitatively influence chromatin interaction. Additional analyses  are required to define these altered interaction properties of Tbx5-p.G125R. The changed chromatin interaction may involve a shift in affinity for binding partner TFs or cofactors, as was previously also reported for missense mutations in GATA4 (GATA-binding protein 4) and NKX2-5.49 The sites of reduced accessibility in heterozygous mutants were highly enriched for motifs of Sp/KLF, MEF2, and T-box families of TFs, pointing to candidate TFs that normally interact with Tbx5, and to a lesser extent, Tbx5-p.G125R. Mef2c and Klf13 are reported functional interaction partners of Tbx5 in the regulation of cardiac development and gene regulation.26 The Sp4 TF is expressed in the heart, including the atria and AV node, and Sp4-deficient mice display a high incidence of sinus bradycardia and AV block.50 Future studies could explore whether the Tbx5-p.G125R interactions with Mef2c—as well as the Sp and Klf TF family members expressed in the atria—are affected.A relatively large number of sites with increased accessibility and with increased H3K27ac association were observed in heterozygous Tbx5-p.G125R atrial CMs. These sites were enriched for Tbx5 binding in the normal heart,In conclusion, Tbx5-p.G125R displays altered properties that induce changes in the epigenetic state, such as chromatin accessibility and H3K27ac, that underlie deregulated expression of both Tbx5 target genes and off-target genes, which is at the basis of distinct electrophysiologic changes that recapitulate clinical observations in patients heterozygous for TBX5-p.G125R.We thank Berend de Jonge, Aho Ilgun, and Corrie de Gier-de Vries for their contributions. We thank Jan Ruijter for assisting with statistical analysis and presentations.This work was supported by CardioVasculair Onderzoek Nederland , Leducq Foundation (14CVD01 to V.M.C.), and Dutch CardioVascular Alliance (OUTREACH to V.M.C.).None.Supplemental MethodsFigures S1–S9Table S1–S18References 51–72