SLC22A12 (RHUC type 1) or SLC2A9 (RHUC type 2) genes, which encode urate transporters URAT1 and GLUT9, respectively. In this study, our goal was to identify mutations associated with twenty-one new cases with RHUC through direct sequencing of SLC22A12 and SLC2A9 coding exons. Additionally, we carried out an SNPs-haplotype analysis to determine whether the rare SLC2A9 variant c.374C>T; p.(T125M), which is recurrent in Spanish families with RHUC type 2, had a common-linked haplotype. Six intragenic informative SNPs were analyzed using PCR amplification from genomic DNA and direct sequencing. Our results showed that ten patients carried the SLC22A12 mutation c.1400C>T; p.(T467M), ten presented the SLC2A9 mutation c.374C>T, and one carried a new SLC2A9 heterozygous mutation, c.593G>A; p.(R198H). Patients carrying the SLC2A9 mutation c.374C>T share a common-linked haplotype, confirming that it emerged due to a founder effect.Renal hypouricemia (RHUC) is a rare inherited disorder characterized by impaired urate reabsorption in the proximal tubule resulting in low urate serum levels and increased urate excretion. Some patients may present severe complications such as exercise-induced acute renal failure and nephrolithiasis. RHUC is caused by inactivating mutations in the  SLC22A12 gene  encoding urate transporter 1 (URAT1) . T. TSLC2A9s mother D. Additihttps://varsome.com/) the new SLC2A9 variant, c.593G>A; p.(R198H), was considered as likely pathogenic using the criteria established by the American College of Medical Genetics and Genomics  [According to Varsome (Available online: and PP5) . It showSLC2A9 is frequent among Spanish patients with RHUC type 2  of T-G-T-C, . This haplotype was present between RHUC patients at a 0.694 frequency. The haplotype G-G-C-C-G-T-C was the most frequent among controls (0.429). Thus, we concluded that mutation c.374C>T; p.(T125M) occurred due to a founder effect.Our results showed that alleles carrying the c.374C>T mutation shared a common SNP haplotype . The disRHUC type 1 and RHUC type 2 are rare inherited disorders caused by defective UA reabsorption in the renal proximal tubule due to mutations in the urate transporter URAT1 or GLUT9, respectively ,14. The SLC22A12) pathogenic variants, mainly c.1400C>T; p.(T467M) and also c.1245_1253del; p(L415_G417del). The other four families were Caucasian and had shown the GLUT9 (SLC2A9) mutation, c.374C>T; p.(T125M), which had only been previously identified in an RHUC patient from Israel whose parents were both of Sephardi-Jewish origin living in Israel for many generations [Xenopus oocytes have revealed that mutation p.(T125M) significantly reduces the ability of GLUT9 to transport UA, and that this is due to a drastic decrease in the expression of the mutant protein [Our previous studies of RHUC with 13 Spanish families have identified mutations in these two genes ,33. Nine protein .SLC2A9 mutation [https://gnomad.broadinstitute.org/, accessed on 23 March 2023). On the other hand, based on the present findings and our previous results [SLC2A9 mutations.The cohort analyzed in the present study included 21 patients from 18 new families. The genetic analysis showed that eight of these families, which are of Roma origin, had RHUC type 1, while the other ten had RHUC type 2. Of the 31 RHUC Spanish families studied so far, 13  had the c.374C>T; p.T125M) mutation in GLUT9. Therefore, we confirmed that this mutation is recurrent in Spanish families with RHUC type 2. This finding suggested the hypothesis of a founder effect. To evaluate this hypothesis, a set of six SNPs surrounding the position of variant c.374C>T; p.(T125M) were genotyped. The results showed that the Spanish patients and the patient of Sephardi-Jewish origin share a common c.374C>T-linked haplotype, which was not identified in the controls, indicating that this variant originated from a single mutational event. None of the RHUC type 2 patients who have been studied in other parts of the world carry this 5M mutatimutation ,48, whicmutation . By the mutation . Therefo results , c.374C>Our results also confirmed that the heterozygous loss-of-function GLUT9 mutation p.T125M) can cause RHUC type 2 . This haM can cauXenopus expression system leading to a decreased UA transport activity [Patient 33, presenting the compound heterozygous mutations p.T125M) and p.L75R), showed serum UA levels (0.4 mg/mL) and UA excretions values (45%) similar to those patients with the p.(T125M) mutation in both alleles. This is consistent with previous findings reported for other compound heterozygous mutations 5R, showe. SimilarM and p.Lactivity . This muSLC2A9 in patient P38 (family 29) identified a very rare heterozygous missense mutation variant, c.593G>A; p.(R198H), which was inherited from his mother  abolishes urate transport activity [SLC2A9 variant p.(R198H) is pathogenic and causes renal hypouricemia type 2 by decreasing urate reabsorption in the proximal tubule. Nevertheless, functional studies using the Xenopus oocyte expression system and electrophysiology will be required to characterize the consequences of this novel SLC2A9 mutation.Sequence analysis of s mother . This vas mother B,C. It hactivity ,39. Thisactivity . All theSLC2A9 is the main cause of RHUC type 2 among Spanish patients and confirm that the SLC22A12 variant c.1400C>T; p.(T467M) is the most frequent in patients of Roma origin. These data considerably facilitate the genetic diagnosis of RHUC in Spain. Furthermore, we have expanded the mutation spectrum of RHUC type 2 with the finding of a novel SLC2A9 mutation. Finally, the haplotype analysis results confirmed that p.(T125M) is a founder mutation.In conclusion, our results indicate that the pathogenic variant c.374C>T; p.(T125M) of A total of 21 patients from different hospitals in Spain, 18 in pediatric age and 3 adults, belonging to 18 unrelated families diagnosed with idiopathic RHUC were investigated . All patSLC22A12 and SLC2A9 was performed using PCR amplification of the coding exons and direct DNA sequencing as previously described [SLC22A12 (NCBI: NG_008110.1) and SLC2A9 (NCBI: NG_011540.1) sequence and confirmed by sequencing additional independent amplification products.The mutational analysis of https://gnomad.broadinstitute.org/, accessed on 23 March 2023) [http://www.1000genomes.org/, accessed on 26 May 2022) [https://www.ncbi.nlm.nih.gov/clinvar/, accessed on 26 May 2022) [http://www.hgmd.cf.ac.uk/ac/index.php, accessed on 7 March 2023) [SLC2A9 variant identified.Several databases of genetic variants including the Genome Aggregation Database (gnomAD. Available online: ch 2023) , 1000 Geay 2022) , ClinVaray 2022) , and Humch 2023)  were inqSLC2A9 variant was evaluated using Varsome, an annotation tool and search engine for human genomic variants which estimates the impact of mutations on protein structure and function  [The pathogenicity of the novel st 2022) . With thst 2022) . The varhttps://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 10 October 2022) [Pan troglodytes GLUT9, XP_016806818.1; Mus musculus GLUT9, NP_001095884.1; Xenopus laevis GLUT9, NP_001121314.1; human GLUT7, NP_997303.2; human GLUT2, NP_000331.1; and human GLUT1, NP_006507.2.The multiple sequence alignment of proteins was performed using Clustal Omega (1.2.4) (available online: er 2022) . Proteinhttps://swissmodel.expasy.org/, accessed 16 November 2022) to generate the three-dimensional structure (3D) of wild-type GLUT9 using GLUT1 protein as a template. The web service HOPE  was used to analyze the structural effects of the novel missense mutation in GLUT9.We used the protein structure homology-modeling server SWISS-MODEL  with the criterion of R2 threshold 0.8. The SNPs were analyzed using PCR amplification followed by DNA sequencing. Primer3 software was used to design the specific oligonucleotide primers for all fragments to be amplified that included the selected SNPs , PHASE v2.1.1 , and SNPSTATS . The Hardy–Weinberg Equilibrium was verified for all the SNPs.In order to test whether the patients had a common c.374C>T; p.T125M)-linked haplotype, an SNPs-haplotype analysis was performed. In this analysis, we also included a DNA sample from the Israeli RHUC patient of Sephardi-Jewish origin in whom the mutation was originally identified (kindly provided by Drs. Dganit Dinour and Liat Ganon) [M-linked ted SNPs . The amp


Epithelial ovarian cancer is the most lethal of gynecological cancers. The therapeutic efficacy of chimeric antigen receptor (CAR) T cell directed against single antigens is limited by the heterogeneous target antigen expression in epithelial ovarian tumors. To overcome this limitation, we describe an engineered cell with both dual targeting and orthogonal cytotoxic modalities directed against two tumor antigens that are highly expressed on ovarian cancer cells: cell surface Muc16 and intracellular WT1. Muc16-specific CAR-T cells (4H11) were engineered to secrete a bispecific T cell engager (BiTE) constructed from a TCR mimic antibody (ESK1) reactive with the WT1-derived epitope RMFPNAPYL (RMF) presented by HLA-A2 molecules. The secreted ESK1 BiTE recruited and redirected other T cells to WT1 on the tumor cells. We show that ESK1 BiTE-secreting 4H11 CAR-T cells exhibited enhanced anticancer activity against cancer cells with low Muc16 expression, compared to 4H11 CAR-T cells alone, both in vitro and in mouse tumor models. Dual orthogonal cytotoxic modalities with different specificities targeting both surface and intracellular tumor-associated antigens present a promising strategy to overcome resistance to CAR-T cell therapy in epithelial ovarian cancer and other cancers. Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy with five-year survival below 30% , 2. Despin vivo, the immunosuppressive tumor microenvironment, limited CAR T-cell trafficking to the tumor, and long-term exhaustion of CAR T-cell functions all have proven to be key obstacles for the success of CAR T-cell therapy (Targeted immunotherapies using adoptive cell transfer of engineered autologous chimeric antigen receptor (CAR) T cells have shown remarkable clinical results in hematologic malignancies -8. In co therapy . In addi therapy , 2, 10.  therapy . A CAR-T therapy . Muc16  is correlated with a better prognosis and improved survival in patients with EOC 15,16).,16.15,16Here we propose to overcome antigenic heterogeneity of EOC and to enhance local antitumor activity in EOC, by engineering the well-studied CAR T cells specific for Muc16 (4H11) to also secrete the ESK1-BiTE against WT1 RMF/HLA-A2 complex, thereby generating dual specificities, as well as orthogonal modalities of killing. We demonstrated that this approach could mediate potent and specific antitumor activity against EOC cells, thereby potentially mitigating the effects of antigen heterogeneity and low expression of Muc16 in EOC.2. Cells were checked regularly for mycoplasma. SKOV-3 cell line was transduced with HLA-A*02:01 molecule as described by Latouche  from HLA-typed healthy donors and patients were obtained by Ficoll density centrifugation. The cell lines used in this study were obtained from ATCC . The cell lines include: Ovarian cancer cell line SKVO-3, AML cell lines AML-14 and HL-60, T leukemia cell line Jurkat. All tumor cells were HLA typed. Primary ovarian cancer cells were obtained from ascitic fluid, blood and tissue samples from patients with ovarian cancer, following Institutional Review Board (IRB) approval. patients with ovarian cancer. The cell lines were cultured in RPMI 1640 supplemented with 5% FCS, penicillin, streptomycin, 2 mmol/L glutamine, and 2-mercaptoethanol at 37 C/5% COLatouche , using Heviously . ESK1.BiMuc16 expression on ovarian cancer cell line SKOV-3 was measured by staining the tumor cells with 4H11 mAb conjugated to APC. SKOV-3/HLA-A2 + cell lines were stained with ESK1 mouse IgG1 conjugated to APC, which recognizes WT1-drived RMF epitope in the context of HLA-A0201. For binding by BiTEs, human T cells or cancer cells were incubated with different concentrations of ESK1-BiTE or control BiTE for 30 min on ice, washed, and incubated with secondary mAbs against His-Tag. In some cases, direct staining was performed by using ESK1-BiTE or its control conjugated to APC. HLA-A*02 expression was determined by direct staining of the cells with mAb clone BB7. WT1 intracellular staining was performed by staining the cells with mAb to WT1 (CAN-R9) and its IgG isotype both conjugated to Alexa488 using intracellular fixation and permeabilization buffer set . WT1RMF/HLA-A2 expression in Primary ovarian cancer cells were measured by staining the cells with anti-human CD45 mAb vs mouse ESK1. Flow cytometry data were collected on a LSR Fortessa (BD) and analyzed with FlowJo V10.Retroviral vectors were generated using the SFG backbone as previously shown , 27. We Human T cells were purified from healthy donor under an Institutional Review Board-exempt protocol. In brief, bulk human T cells were activated on day 0 using CD3/CD28 Dynabeads (Miltenyi) and cultured in RPMI 1640 medium supplemented with 10% FBS and 100 IU recombinant human IL-2. Lentiviral transduction of cells was performed on day 2 and day 3 by centrifugation of activated T cells in media containing virus from retroviral producers Galv-9 cells at 2000xg at room temperature for two hours on RetroNectin-coated plates . Transduction efficiency was determined by flow cytometry using a Alexa 647-labeled anti-idiotype antibody against the Muc16-targeted CAR (mAb clone 22G6), generated at MSKCC Antibody and Bioresource Core Facility).Secretion of the ESK1 BiTE was confirmed by analyzing conditioned media of Galv9 producer cells by Western blot as previously shown . Ni-NTA Supernatants of CAR T cells transduced with the CAR/BiTE construct was collected on day 5 and 7, pooled, concentrated 30–40 folds by centrifugation using Amicon Centrifugal Filter Units  at 4000xg for 20 minutes in room temperature. Supernatant fluids were stored at 4°C, and the secretion of ESK1-BiTE was measured by staining SKOV3 and Jurkat cells, and flow cytometric analysis. Recombinant ESK1-BiTE was used in parallel to determine the approximate concentrations of the secreted BiTE. Functional assay of ESK1-BiTE was measured by cytolytic activity of the supernatants by using isolated PBMC’s from the same donor as effector cells. In brief, supernatant fluids in serial dilution were incubated with target cells and isolated PBMC’s at E:T ratio of 10:1, and the cytotoxicity was measured by luciferase-based assay.51Cr-release assay. For measuring cytotoxicity of CAR T cells, T cells were incubated with luciferase-expressing tumor targets at various E: T ratios for 24 hours. Next day, additional Mock-T cells from the same donor were added for another 24 hours. Remaining luciferase activity was subsequently measured with a Tecan infinite M1000 Pro Microplate Reader. Percentage specific lysis was calculated by the following equation: Percentage = [ – total RLU)/(target cells only RLU)] × 100.For ESK1-BiTE-mediated cytotoxicity, PBMCs from healthy donors were incubated with targets at a E: T ratio of 20 or 30:1, in the presence or absence of ESK1 or control BiTEs at various concentration for 5 hr. The cytotoxicity was measured by standard Prkdc SCID IL2rgtm1Wjl/SzJ mice, known as NSG, were purchased from the Jackson Laboratory, and were used for xenograft animal tumor model. SKOV3 ovarian cancer cells transduced with HLA-A2 and GFP/luciferase were used. For ESK1 BiTE therapy, 2.5 million Muc16 low SKOV3/A2+/GFP + cells were ip injected into mice. On day3, after confirming the tumor engraftment by bioluminescence imaging (BLI), PBMCs (10 million/mouse) from a healthy donor were injected ip into mice.Seven to 8-week-old male NOD.Cg-A few hours later, ESK or control BiTE was ip injected into mice (5ug/mouse) and repeated on a daily basis. The tumor growth was monitored by BLI after 4 and 9 injections. For 4H11 CART cell therapy, the same Muc16 low SKOV3/A2 + ovarian cancer cells (2 million/mouse) were injected into the peritoneal cavity (ip) of mice and the tumor engraftment was confirmed on day 4 by bioluminescence imaging and mice were randomized into treatment groups. Two million of CAR T cells were ip. injected into mice, followed by 2 million Mock-T cell injection the next day. Tumor growth was monitored by firefly luciferase imaging once a week, for all the animal models. The P test used for the survival curve is Mantel-Cox.Expression of the WT1 protein and the WT1 RMF epitope in ovarian cancer cells SKOV3/HLA-A2 + was shown by intracellular staining and by binding of ESK1 to the RMF/HLA-A2 complex  and B. CESK1 has potential off target reactivity, possibly limiting its systemic use in humans , 30. TheWe tested if the ESK1-BiTE is secreted by the CAR T cells after transduction. Supernatant fluid from HEK-derived retroviral producer cells  demonstrated successful secretion of ESK1 BiTE by western blot at a predicted molecular weight of approximately 55kDa . Importahigh SKOV3 cells, whether A2 + or A2 negative. Consequent to the high Muc16 expression, ESK1-BiTE secretion by these cells did not have additional cytotoxic effects to either of these cell lines .To test if the ESK1-BiTE-secreting 4H11 CAR T cells were able to engage other bystander T cells leading to a greater cytolytic activity than 4H11 CAR T cells alone, we determined the expression of the appropriate antigens (Muc16 and WT1 RMF/HLA-A2 complex) in wild type SKOV3 ovarian cancer cells with “low” Muc 16 expression  and thosll lines . In contll lines . HoweverTo further investigate if the secreted ESK1 BiTE from 4H11 CART cells was responsible for mediating the T cell cytotoxicity against target cells, the supernatant fluids of 4H11 CAR T or 4H11-ESK1 CAR T cells (concentrated about 16-fold) were incubated with activated T cells (mock-T cells from the same experiments) as effectors along with SKOV3/HLA-A2 positive or negative target cells overnight. Serial diluted supernatant from ESK1-4H11 CART cells lysed the SKOV3/A2 +target cells, but not SKOV3/A2 negative target cells . The potlow tumor cells by engaging bystander effector T cells. Therefore, the anti-tumor activity of ESK1-BiTE-secreting 4H11 CAR-T cells was tested in vivo using the Muc16 low/HLA-A2 + SKOV3 cell line, in a relevant intraperitoneal  model. After confirming the tumor engraftment on day 4 post tumor injection, mice were randomized to different groups. Mock T cells, 4H11 CAR T cells or ESK1 BiTE-secreting 4H11 T cells were injected i.p. into mice. The following day (day 5), mock-T cells from the same donors and the same experiments were injected ip into mice of all groups, as bystander effector T cells, in order to test if ESK1 BiTE was able to redirect these T cells to kill the tumor cells. The tumor growth was monitored by bioluminescence imaging. The enhanced anti-tumor effects of ESK1 BiTE-secreting 4H11 CART cells as compared to the regular 4H11 CAR T cells became evident starting on day 19 and these mice continued to improve. On day 26 post tumor engraftment, there was significant reduction of tumor growth compared to 4H11 CAR T cells and other control groups , although expression is heterogeneous , 32. Bechomogeneously expressed throughout EOC have been identified. In this study, we chose to create a dual targeting CAR T cell strategy that not only is directed to 2 different antigens, thereby reducing possible escape due to heterogeneity, but also acts to kill by use of two different modalities, that also increases efficacy and reduces possible escape. 4H11 CAR T cells were engineered to secret the ESK1-BiTE, derived from a TCR mimic mAb directed against WT1 epitope presented by HLA-A2, allowing a simultaneous targeting of two antigens that are highly expressed on EOC. Because WT1 is an oncogenic protein, loss of this antigen is also less likely. In addition, the ESK1-BiTE may promote epitope spreading to further extend the killing to a third modality(in vitro and in vivo. While 4H11 CART cells were able to eradicate the SKOV3/HLA-A2 +tumor cells, ESK1-secreting CAR T cells significantly enhanced their anti-tumor activity in Muc16 low tumor cells and prolonged the survival of tumor bearing mice.The lack of significant efficacy in a trial of Muc16 CART cells armored with IL12 might be due to the heterogenous or low expression of Muc16 on tumor cells , 13. How modality. We demoWe selected WT1 RMF presented by HLA-A2 as a model antigen for Muc16 CART cells, because it is a well-validated intracellular tumor antigen found in a wide array of human cancers , 35. TarIn summary, this study has demonstrated that the targeted delivery by 4H11 CAR T cells, directed to the Muc16 antigen, of the ESK1 BiTE, which reacts to a second, intracellular, tumor antigen, WT1, and which kills by an orthogonal mechanism, could overcome the problem of low Muc16 expression on ovarian cancer cells and therefore is a possible strategy to improve clinical outcome of CAR T cell therapy.


We sequenced the draft genome of thraustochytrid strain 12B. This strain shows a high production of polyunsaturated fatty acids, such as docosahexaenoic acid. The draft genome sequence of approximately 65 Mbp will provide insights into metabolic engineering to improve the production of polyunsaturated fatty acids in the microorganism. A marine protist, thraustochytrid strain 12B (strain 12B) belonging to the family Thraustochytriaceae, was isolated from the mangrove area of Okinawa, Japan , which cd-glucose  at 30°C for 2 days. Genomic DNA was prepared using the DNAs-ici!-F reagent . The genomic DNA was quantified using the Qubit double-stranded DNA assay . For whole-genome sequencing, a 400-bp fragment library was prepared from 1 µg of the genomic DNA using the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific). The library was used to prepare a templated-beads sample by amplification and enrichment using the Ion PGM Hi-Q OT2 Kit (Thermo Fisher Scientific) on the Ion OneTouch 2 system (Thermo Fisher Scientific). The templated-beads sample was manually loaded onto the Ion 318 Chip v2 BC (Thermo Fisher Scientific) and sequenced on the Ion PGM system (Thermo Fisher Scientific) with the Torrent Suite software (ver. 5.0.3) by using the Ion PGM Hi-Q Sequencing Kit (Thermo Fisher Scientific).Strain 12B was obtained from the Biological Resource Center, National Institute of Technology and Evaluation (NBRC), Japan, as strain number NITE P-68, and was cultivated in BY+790 medium consisting of 50% artificial seawater , 1 g/L Bacto peptone , 1 g/L Bacto yeast extract (BD Bioscience), and 0.5 g/L de novo assembly using the MIRA software ver. 4.0.5  were obtained using the Torrent Suite software. The reads were used for


EH1Mut hearts and analysis of putative direct targets revealed misexpression of the retinoic acid pathway and cardiac progenitor genes. Further, we show that altered cardiac progenitor development and function contribute to the severe cardiac defects in our model. Our studies indicate that TLE-mediated repression is a primary mechanism by which Tbx20 controls gene expression.T-box transcription factor 20 (Tbx20) plays a multifaceted role in cardiac morphogenesis and controls a broad gene regulatory network. However, the mechanism by which Tbx20 activates and represses target genes in a tissue-specific and temporal manner remains unclear. Studies show that Tbx20 directly interacts with the Transducin-like Enhancer of Split (TLE) family of proteins to mediate transcriptional repression. However, a function for the Tbx20-TLE transcriptional repression complex during heart development has yet to be established. We created a mouse model with a two amino acid substitution in the Tbx20 EH1 domain, thereby disrupting the Tbx20-TLE interaction. Disruption of this interaction impaired crucial morphogenic events, including cardiac looping and chamber formation. Transcriptional profiling of Tbx20 Summary: The Tbx20-TLE repression complex is essential for mammalian heart development and regulates cardiac progenitor cell development. Embryonic heart development requires intricate regulation of transcription factor (TF) networks (TRNs) that coordinate cardiac cell specification, maturation and progression of crucial morphological events . The essXenopus and mice results in similar phenotypes in which cardiac looping is impaired and hearts fail to undergo chamber formation  and second heart field (SHF) cardiac progenitors, endocardial cells and cardiomyocytes . Expressormation .In addition, cardiac lineage-specific knockouts reveal that Tbx20 is required for the proliferation and maturation of both the myocardium and endocardial-derived primordial valves (cushions) . ClinicaIn vitro studies show that Tbx20 interacts with a network of cardiac TFs, including Tbx5, Nkx2.5, Gata4 and Casz1, to synergistically regulate cardiac gene expression  protein. TLE/Gro family members perform essential functions in diverse developmental processes through their interaction with a myriad of transcription factor families   proteins, a family of transcriptional corepressors . The TLEand Fox) . TLE/Groand Fox) . Tbx20 wogenesis . This stEH1Mut), thereby disrupting the Tbx20-TLE interaction. Using this model, we demonstrate that the Tbx20-TLE interaction is essential for embryonic heart morphogenesis, as mutant mice displayed impaired cardiac looping and chamber formation, resulting in embryonic lethality. Our transcriptional profiling of Tbx20EH1Mut hearts and analysis of Tbx20 direct targets revealed misexpression of the retinoic acid (RA) pathway and cardiac progenitor genes, which implied that the Tbx20-TLE interaction inhibits cardiac progenitor programs in the developing heart. In addition, our studies suggest that altered cardiac progenitor specification and impaired cardiomyocyte differentiation significantly contribute to Tbx20EH1Mut cardiac defects. Collectively our studies define a function for the Tbx20-TLE interaction in the developing heart and suggest that a TLE-mediated repression program is a primary mechanism by which Tbx20 regulates heart development.To investigate the function of the Tbx20-TLE complex in cardiogenesis, we generated a novel mouse model in which a two amino acid substitution was introduced into the Tbx20 EH1 domain  were viable, fertile and phenotypically indistinguishable from control littermates. In contrast, we failed to recover homozygous (Tbx20EH1Mut) mice postnatally  was sufficient to specifically disrupt the Tbx20-TLE interaction and impair the recruitment of chromatin remodeling proteins . To detegermline . Mice hetnatally . Immunoh embryos . In addi embryos  (Stennar embryos .EH1Mut embryos at embryonic day (E)9.5 revealed that mutant embryos displayed pericardial edema and hemorrhaging , left and right ventricles, and atria  and a concomitant decrease in the mitotic index (pHH3+/TPM1+)  . In addihenotype . Taken tEH1Mut hearts at E9.5. This analysis identified 2218 differentially expressed genes , of which 1363 genes were significantly upregulated and 855 genes were significantly downregulated in Tbx20EH1Mut hearts compared with controls . In addition, the ‘cyclins and cell cycle regulation’ pathway included several genes involved in cell cycle progression and proliferation . Downregulation of these pathways corroborated our findings that cardiomyocyte number and mitotic index were decreased in Tbx20EH1Mut hearts.TLE proteins function as transcriptional corepressors; therefore, we hypothesized that the Tbx20-TLE interaction represses inappropriate gene expression in the developing heart . To testcontrols . We perfcontrols . PathwayEH1Mut hearts. Surprisingly, analysis of upregulated genes identified several pathways associated with RA signaling, including ‘Retinoate Biosynthesis I’, ‘Retinoate Biosynthesis II’ and ‘RAR Activation’. Genes within these biological categories included the RA-synthesizing enzyme Aldh1a2, several RA binding proteins, Rbp1, Rbp2, Rbp7, and retinol dehydrogenase Rdh12. We confirmed that Aldh1a2 expression is significantly increased in Tbx20EH1Mut hearts compared with controls using fluorescence in situ hybridization (RNA-FISH)  . These fNA-FISH) .P-value≤0.05 and log2 fold change≥±0.585) with published Tbx20 chromatin immunoprecipitation with high throughput sequencing (ChIP-seq) data generated from embryonic hearts . In addition, we found genes associated with cardiomyocyte energy metabolism and contractility  , RA responsive genes . Based on our findings, we propose that the Tbx20-TLE complex suppresses aberrant RA signaling during embryonic heart development.To determine which subset of our differentially expressed genes are putative direct targets of Tbx20, we intersected our RNA-seq data (adjusted c hearts . We iden Atp2a2) . It is hby Tbx20 . IPA anaby Tbx20 . This suEH1Mut hearts was unexpected because these genes are primarily expressed in the SHF cardiac progenitors located outside the primary heart tube at E9.5. In combination with impaired looping and chamber formation defects in Tbx20EH1Mut hearts, we suspected that this finding indicated arrested cardiomyocyte development.Misexpression of RA pathway genes in Tbx20EH1Mut heart tube , previously identified by single-cell RNA-seq . We founart tube . These opulation   of OFT-enriched genes were specifically upregulated in Tbx20EH1Mut hearts  were primarily located in the proximal OFT  as we observed higher expression in the pSHF compared with the aSHF  give rise to the right ventricle and portions of the OFT. The posterior SHF (pSHF) progenitors give rise primarily to the atria and a subset of cardiac vessels . Analysi domains  . We conducted a quantitative spatial analysis of the cardiac crescent (CC) at E7.75. In control embryos, FHF progenitors, identified by expression of Hcn4, reside within the CC, whereas Isl1+ SHF progenitors lie medial to the CC (Hcn4 and Isl1 double-positive cells (Hcn4+Isl1+) in control embryos  of Isl1+ SHF progenitors was reduced in Tbx20EH1Mut embryos  and the pSHF (Tbx5), although overall expression of these both genes appeared to be decreased in the mutants  cells were primarily detected in the SHF, OFT and right ventricle (RV)  . In Tbx2 the OFT . These dt hearts . Consist embryos . Taken tTbx20 is essential for heart development, and its disease relevance is well established . Howeverin vivo.Tbx20 functions as both a transcriptional repressor and an activator, and studies indicate that its interactions with various protein networks dictate the transcriptional activities of Tbx20. Tbx20 directly interacts with crucial cardiac transcription factors, including Isl1, Gata4, Nkx2.5 and Casz1 . HoweverEH1Mut). We found that Tbx20EH1Mut mice had severely impaired heart formation and were arrested in development at E9.5. These findings demonstrated that the Tbx20-TLE repression complex is crucial for embryonic heart development.More recently, Tbx20 has been shown to interact with TLE proteins, a family of transcriptional corepressors . FurtherEH1Mut mouse phenotypically parallel the Tbx20 null and global loss-of-function mutants  are downregulated in the Tbx20EH1Mut heart. Therefore, Tbx20-TLE-mediated repression may serve as a primary mechanism by which Tbx20 regulates cardiac gene expression.The cardiac defects in the Tbx20 mutants . These mEH1Mut and to identify the subset of cardiac genes repressed by Tbx20, we performed transcriptomics analysis. We intersected our dataset with ChIP-seq data to identify putative direct Tbx20 targets  were upregulated in Tbx20EH1Mut hearts. Although regulation of the RA genes by Tbx20 was not demonstrated previously, Tbx5 and Tbx1, two additional T-box family members, are known regulators of the RA signaling genes  are substantially increased in Tbx20EH1Mut embryos, indicating that the Tbx20-TLE repression complex regulates transcriptional programs in FHF and SHF progenitors. We hypothesize that impaired heart tube formation in mutants is due in part to compromised progenitor specification, which results in uncoordinated differentiation and deployment. For example, disrupted cardiac specification could account for the decrease in FHF/left ventricle-specific gene expression in Tbx20EH1Mut hearts. We also propose that the reduction of the SHF pool, paralleled with the increase in the proportion of SHF-derived cells in the heart tube at E9.5, suggests premature deployment of SHF progenitors. Taken together, our study suggests that impaired heart development in Tbx20EH1Mut embryos results from misspecification of cardiac progenitors and perturbed progenitor function, including premature deployment of the SHF cells. Finally, although SHF cells successfully incorporate into the heart tube, they arrest in development and are unable to differentiate further 26Sortm14(CAG-tdTomato)Hze (stock 007194), Mef2c-AHF-cre  followed by in vitro transcription and silica spin column purification. Functional testing was performed by in vitro Cas9 cleavage assay. Each gRNA was incubated with recombinant Cas9 protein  and PCR-amplified target region DNA, followed by gel electrophoresis to detect cleavage of the target site. Two guide RNAs were selected for genome editing in embryos: Tbx20-sg97B (protospacer sequence 5′-GGACATAAGCGCGGCGA-3′) and Tbx20-sg83B (protospacer sequence 5′-AAGCGCGGCGATGGAGA-3′). A donor oligonucleotide was designed to facilitate homologous recombination to introduce the F18L (TTC to TTa) and S19I (TCC to atC) mutations. The donor sequence was 5′-ATGGAGTTCACGGCGTCGCCCAAGCCCCAGCTCTCCTCTCGAGCCAATGCCTTaatCATCGCCGCGCTTATGTCCAGCGGCGGCCCCAAGGAGAAGGAGGCAGCAGAG -3′.Cas9 guide RNAs (gRNAs) targeting the in vitro fertilization of C57BL/6J oocytes with sperm from a Tbx20Avi/+ male. Embryos were divided into two groups and each group was microinjected with recombinant Cas9 protein, donor oligo and one of the two guide RNAs. Microinjected embryos were implanted in pseudopregnant recipient females and resulting pups were screened by PCR and sequencing for the presence of the F18L-S19I allele. The first microinjection session was performed with 400 nM Cas9, 50 ng/μl gRNA and 50 ng/μl donor oligonucleotide. The session yielded three pups from 444 injected embryos, with none of the pups showing mutations at the target site. A second microinjection session was performed with 200 nM Cas9, 25 ng/μl gRNA and 50 ng/μl donor oligonucleotide. The Tbx20-sg97B gRNA mix yielded two pups from 188 injected embryos, with one animal showing insertion/deletion (indel) mutations at the target site. The Tbx20-sg83B yielded 11 pups from 291 injected embryos. One male (#7) was found to have the desired F18L-S19I amino acid changes. The animal did not have the Tbx20-avitag, indicating that the F18L-S19I mutation was inserted in an otherwise wild-type Tbx20 allele. The positive founder was transferred to the client for breeding. Two additional animals with indel mutations were also identified in this group. A third injection session was performed with the same reagent concentrations as the second, yielding three pups from the Tbx20-sg97B gRNA and nine pups from the Tbx20-sg83B gRNA. One male (#21) from the sg83B injection had a small amount of the F18L-S19I mutation in addition to an indel, and was positive for the Tbx20-avitag. The founder was mated to C57BL/6J females for transmission of the mutant alleles. Approximately 50% of the pups from this founder had a 16 bp deletion mutation at the F18-S19 target area in cis with the avitag insertion. The other pups were wild type at the F18-S19 target region, and no transmission of the F18L-S19I mutation was detected. The resulting pups were screened by PCR and sequenced for the presence of the mutation allele. Male founders with the correct mutation were mated to wild-type C57BL/6J females for germline transmission of the mutated allele. Lines were back-crossed for at least three generations.Embryos were produced by SEM was performed as previously described . BrieflyFor histology and immunohistochemistry, embryos were fixed in 4% paraformaldehyde/PBST (PBS + 0.1% Tween) overnight at 4°C. Embryos were then processed for either paraffin embedding or frozen in OCT. For histology, paraffin sections were dewaxed and stained with Hematoxylin and Eosin, according to standard protocols . Histoloin situ hybridization experiments (in situ protocol was performed. The RNAscope Multiplex Fluorescent reagent Kit V2 (323100) was used according to the manufacturer's protocol with the following adaptations: the air-drying step was omitted, for embryos younger than E9.5 the protease digestion step was omitted, probes were hybridized overnight at 40°C, 0.2× SSCT  was used for all wash steps and embryos were counterstained overnight with DAPI. Embryos were mounted onto coverslips and imaged in 1× PBST. For analysis of the FHF and SHF the entirety of the heart fields was imaged using a Zeiss LSM 900 laser scanning confocal microscope  according to the manufacturer's protocol, with the following adaptations: slides were thawed at room temperature for 5 min, the protease digestion step was omitted and probes were hybridized overnight at 40°C. The following RNAScope probes (ACDBio) were used in this study, Hcn4 (421271), Isl1 (451931-C3), Tbx1 (481911), Tbx5 (519581-C2) and Aldh1a2 (540221-C3).We adapted a previously published protocol for the whole-mount eriments . Brieflycroscope . For in +/+) (N=3) and Tbx20EH1mut (Tbx20EH1Mut/EH1Mut) (N=4) embryos. These sections represent the entirety of the heart . The mitotic index was calculated by dividing the total cells positive for TMY and pHH3 by the total cells positive for TPM1. Statistical analysis was performed using Welch's t-test.For quantification of cardiomyocyte proliferation, embryos were processed as described above (see ‘Histology and immunohistochemistry’). Cryosections were stained for TPM1, pHH3 and DAPI. For quantitative analysis, 11-16 sections were analyzed from control  software. Images were converted to Imaris file format and were masked to include only the CC. Surface volume renderings were performed using the Surface tool. The Spots tool was used to calculate the number of cells per surface and the number of + cardiomyocytes, embryos were processed as described above (see ‘Histology and immunohistochemistry’). Cryosections were co-stained for Isl1, SMA and DAPI. A total of seven sections corresponding to the anterior, middle and posterior positions of the heart were analyzed from wild-type (N=3) and Tbx20EH1mut (N=3) embryos. Percentage Isl1+ cardiomyocytes were calculated by dividing total cells positive for SMA and Isl1 by total cells positive for SMA. To quantify the mitotic index of Isl1+ cells in the SHF, cryosections were co-stained with Isl1, pHH3 and DAPI. A total of 5-7 sections corresponding to anterior, middle and posterior positions of the SHF were analyzed from wild-type (N=4) and Tbx20EH1mut (N=4) embryos. The mitotic index was calculated by dividing the total SHF cells positive for Isl1 and pHH3 by the total SHF cells positive for Isl1. Statistical analysis was performed using Welch's t-test.For quantification of Isl1EH1Mut embryos. Four biological replicates were performed (three hearts were pooled for each replicate). RNA was isolated using the RNAqueous micro kit (Ambion) as per the manufacturer's protocol. Poly-A selected RNA-seq libraries preparation, sequencing reactions, and initial bioinformatic analysis were conducted at GENEWIZ, LLC. Samples were run on a HiSeq2500 (Illumina) with 2×150 bp paired-end reads.E9.5 hearts were collected from 12 wild-type and 12 Tbx20P-value<0.05 and a log2(fold change)>0.5 in either direction were considered differentially expressed. Canonical pathway and upstream regulator analysis were performed using Ingenuity Pathway Analysis (IPA) . Tbx20 ChIP-seq data from E11.5 hearts was obtained from previously published data available from the European Molecular Biology Laboratory–European Bioinformatics Institute (EMBL-EBI) database (accession number E-MTAB-3967).Genes with an adjusted EH1Mut (Tbx20EH1MUT-GFP) protein and Nkx2-5-V5 were washed twice with cold PBS , harvested by scraping with a cell lifter and centrifuged at 350 g for 10 min at 4°C. Cell pellets were resuspended in 600 µl lysis buffer  with protease , phosphatase inhibitors  and nuclease . The cell suspension was then incubated on ice for 30 min with pipetting every 10 min and then pelleted at 20,000 g for 10 min at 4°C. Then, 900 µl of dilution buffer  was added to cleared lysate. Diluted lysate was incubated with 50 µl equilibrated GFP-Trap magnetic beads by rotating for 1 h at 4°C. Beads were washed three times with 500 μl of dilution buffer. GFP complexes were eluted from beads in 45 µl 6× Laemmli Sample Buffer at 95°C for 10 min. Protein complexes were examined by Western blotting.TBX20 protein co-immunoprecipitation was performed using GFP-Trap magnetic beads (Chromotek) following the manufacturer's protocols with slight modification. Briefly, HEK293 cells expressing either wild-type (Tbx20-GFP) or Tbx20Click here for additional data file.10.1242/develop.201677_sup1Supplementary informationClick here for additional data file.EH1Mut hearts at E9.5 from RNA-Seq analysis.Table S1. Dataset of differentially expressed genes in wild-type and Tbx20Click here for additional data file.


Type-1 diabetes mellitus (T1DM) is a complex multifactorial disease with an autoimmune etiology and is thought to result from an interaction between genetic and non-genetic factors. Cytokines play a crucial role in the pathogenesis of autoimmune diseases due to their effector and regulatory functions in immune responses. Interleukin-4 (IL4) and Interleukin-13 (IL13) are anti-inflammatory cytokines and are considered as important mediators in pathology of the autoimmune diseases.IL4 gene promoter polymorphism , IL13 gene polymorphism p. and human leukocyte antigen, HLA-DQ and DR genotypes in Kuwaiti children with T1DM to investigate their role in genetic susceptibility. This study included 261 Kuwaiti children with T1DM and 214 healthy controls. The genotypes for IL4 (−590C/T) and IL13 p.(Arg130Glu) gene polymorphisms were detected by PCR-RFLP methods. HLA-DQ and DR genotypes were determined by sequence-specific PCR methods.We have determined the genotype frequency of IL4 gene polymorphism (−590C/T) was significantly related to the risk for T1DM in Kuwaiti patients (OR 1.64). The homozygous AA (QQ) and heterozygous AG (RQ) genotypes of IL13 gene polymorphism p.(Arg130Glu), also manifested a statistically significant association with T1DM (OR 2.92 and 4.79). In 55% T1DM patients, the HLA genotype was either DQ2/DQ2 or in combination with a DQ8 allele. Collectively, 91% Kuwaiti T1DM patients had either DQ2 or DQ8 alleles in different combinations highlighting them as the high risk-genotypes in comparison to the controls. In the case of HLA-DR, the genotypes DR3/DRB5, DR3/DR4, DR3/DR7 and DR4/DR4 showed highest frequency amongst the Kuwaiti T1DM patients and thus can be considered as high-risk genotypes when compared to the controls. A high degree of co-inheritance (>80%) was detected between IL4 and IL13 gene polymorphism genotypes (CC and QQ) and the high-risk HLA-DQ and DR genotypes amongst the Kuwaiti T1DM patients.The CC genotype of IL4 and IL13 gene polymorphisms with susceptibility to T1DM in Kuwaiti children and the co-inheritance of these polymorphisms with high-risk HLA genotypes. The findings may contribute to early identification of childhood diabetes.We have identified the association of  CTLA4) and the protein tyrosine phosphatase non-receptor type 22 (PTPN22) loci have all been proven important in the pathogenesis of autoimmunity globally, whereas insulin (INS) gene is a disease-specific T1DM susceptibility locus (Type-1 diabetes mellitus (T1DM) is an auto-immune, multi-factorial disease which is thought to result from T cell-mediated destruction of the pancreatic beta cells . T1DM acty locus . Most ofty locus . A numbety locus –11. Thesty locus . It has ty locus , 11.IL4 gene has been suggested as one of the genetic risk factors in autoimmune diseases. Several previous reports have explored the relationship between this single nucleotide polymorphism (SNP) and genetic susceptibility of autoimmune diseases in different populations but the results from these studies remain inconclusive . It has been proposed that IL13 downregulates the inflammatory process by suppressing the production of Th1 cytokines (Interleukin 13 (IL13) is an anti-inflammatory cytokine that is produced predominantly by CD4+ T cells with Th2 characteristics . IL13 plytokines .IL4 and IL13 gene polymorphisms in a number of autoimmune diseases prompted us to investigate their role along with HLA-DQ and DR genotypes in genetic susceptibility of T1DM in a completely different population (Kuwaiti Arab children) which has a high incidence rate of T1DM.Association studies focusing on candidate genes involved in immune responses, such as those encoding elements of the T cell activation pathway, represent a useful approach for finding the T1DM susceptibility genes. IL4 and IL13 have been shown to protect against the disease development in rodent models of T1DM , suggest(i)Diagnosis by a physician as diabetic(ii)Placed on a daily dose of insulin before the 15th birthday(iii)A Kuwaiti national resident in the area at the time of the first insulin administration.The ISPAD Guidelines . The inclusion criteria used for T1DM patients was based on combined recommendations of the WHO Multinational Project for Childhood Diabetes (DiaMond Project) and the ISPAD Guidelines—International Society for Pediatric and Adolescent Diabetes Clinical Practice Consensus Guidelines 2014 :(i)Diagidelines  providedThe glycemic status in all the study subjects (T1DM patients and controls) was ascertained by determining the HbA1c levels. All the control subjects had their HbA1C below 5.7% in accordance with the ISPAD Guidelines . The cliTotal genomic DNA was extracted from the peripheral leukocytes using a standard method . The genIL4 gene -C590T, rs2243250 promoter polymorphism, have been described earlier  genotype.The primers and procedures used for determination of  earlier . The C→TIL13 gene polymorphism p. was identified by PCR-RFLP method by the procedure described earlier (IL13 gene. Homozygous AA genotype codes for Glutamine (Q) and homozygous GG genotype encodes for Arginine (R) residue at codon 130. It is a common practice to use QQ and RR to represent the resulting amino acids from this polymorphism. The PCR products were cleaved with restriction enzyme NlaIV (0.5 U) at 37 °C for 3 h. The cleavage products were analyzed by Agarose gel electrophoresis and detected under UV light after staining with Ethidium bromide. The cleavage products were 210 bp and 26 bp in the case of AA (QQ) genotype and in individuals with GG (RR) genotype the product sizes obtained were 178 bp, 32 bp and 26 bp. The heterozygous individuals with AG (QR) genotype had the products with sizes, 210 bp, 178 bp, 32 bp and 26 bp respectively.The  earlier . This poHLA-DQ and DR genotypes were identified by using a polymerase chain reaction-sequence specific primers method . DYNALP < 0.05 (two-tailed). The P values were corrected for multiple comparisons by Bonfferoni's correction. Fisher's Exact test was used to determine statistical significance of the differences between genotype and allele frequency between the T1DM patients and the controls. For calculation of the statistical significance in co-dominant and dominant genetic models, the genotype frequency in homozygous TT (IL4 gene) and RR subjects (IL13 gene) and the “T” and “R” allele frequency was considered as reference [as per previous reports . The frequencies of various genotypes and alleles detected in T1DM patients and controls were calculated by direct counting. The confidence interval (CI) was set at 95% and statistical significance was at  reports –31]. In P = 0.50; The characteristics of T1DM patients and controls are presented in IL4 gene  polymorphism and IL13 gene polymorphism p. were detected by PCR-RFLP method as described in Methods section. Examples of the typical results of these analyses are presented in The IL4 gene -C590T polymorphism genotypes and alleles are presented in IL4 gene (-C590T) polymorphism (CC) was significantly higher in T1DM patients compared to that in the controls in Kuwaiti children , statistically significant difference was detected in the frequency of homozygous QQ (AA) and heterozygous RQ (AG) genotypes , DQ2/DQ2 , DQ2/DQ8  and DQ8/DQ8  respectively (P 0.003), DR3/DR4 , DR3/DR7  and DR4/DR4  respectively  polymorphism genotypes with HLA-DQ genotypes in Kuwaiti T1DM patients is presented in IL4 gene polymorphism. When the homozygous (CC) and heterozygous (CT) genotypes  were considered together, they were detected in 95% of the T1DM patients with high risk DQ2/DQ2 genotype  polymorphism with HLA-DR genotypes in Kuwaiti T1DM patients is presented in The frequency of co-inheritance of IL13 gene p. polymorphism with HLA-DQ genotypes in Kuwaiti T1DM patients is presented in The frequency of co-inheritance of IL13 gene p.  polymorphism with HLA-DR genotypes in Kuwaiti T1DM patients is presented in The frequency of co-inheritance of IL4 gene  polymorphism in Kuwaiti T1DM patients. This is in sharp contrast to the data from Saudi Arabian population  polymorphism were significantly more susceptible to develop T1DM . The heterozygous (CT) genotype was not significantly associated with T1DM in the study from Saudi Arabia  polymorphism and susceptibility to autoimmune diseases was published in 2014  polymorphism, which was found to be associated with RA and MS. However, in that meta-analysis, no association was reported in the case of SLE or GD  polymorphism and T1DM in Kuwaiti children  may contribute to the onset of T1DM (Another interesting finding in this study is that a significant association was detected between the AA (Q) genotype and “Q” allele of children . The potchildren . IL4 haschildren . It has children . Also, tchildren . It has children , 40. Rec of T1DM .IL4 gene CC) together with a normal Th1 profile may result in a net pro-inflammatory cytokine expression pattern (IL4 gene CC genotype), will result in a relative Th1 cytokine overload, possibly resulting in destructive insulitis  and IL13 gene polymorphism genotype (AA/Q) as high-risk genotypes in the Kuwaiti T1DM patients. This study also report a significant co-inheritance of the CC genotype of IL4 gene polymorphism , and the (AA/Q) genotype of IL13 p.(Arg130Glu) gene polymorphism with the high-risk HLA-DQ and DR genotypes and highlights their significant role in genetic susceptibility of the T1DM in Kuwaiti Arab children.In this study, we have identified


MEF2A) is a member of the myocyte enhancer factor 2 family. MEF2A is widely distributed in various tissues and organs and participates in various physiological processes. This study aimed to investigate the effect of MEF2A expression on the proliferation and apoptosis of bovine myoblasts. CCK8, ELISA, cell cycle, and apoptosis analyses were conducted to assess cell status. In addition, the mRNA expression levels of genes associated with bovine myoblast proliferation and apoptosis were evaluated using RT-qPCR. The results showed that the upregulation of MEF2A mRNA promoted the proliferation rate of myoblasts, shortened the cycle process, and increased the anti-apoptotic rate. Furthermore, the RT-qPCR results showed that the upregulation of MEF2A mRNA significantly increased the cell proliferation factors MyoD1 and IGF1, cell cycle factors CDK2 and CCNA2, and the apoptotic factors Bcl2 and BAD (p < 0.01). These results show that the MEF2A gene can positively regulate myoblast proliferation and anti-apoptosis, providing a basis for the analysis of the regulatory mechanism of the MEF2A gene on bovine growth and development.Myocyte enhancer factor 2A ( Muscle yield and the quality of livestock are key indicators of the quality of livestock products. The growth and development of skeletal muscle and genetic characteristics significantly influence muscle yield and the quality of livestock . MeanwhiMEF2A, MEF2B, MEF2C, and MEF2D. However, this family is a single gene in Drosophila and nematodes [MEF2A activates many muscle-specific, growth factor-induced, and stress-induced genes. Moreover, MEF2A participates in life processes, such as cell proliferation and differentiation, apoptosis, and morphological changes [MEF2A is located downstream of the signalling pathway regulating the expression of musculogenic genes. MEF2A functions as a regulatory factor and a structural protein carrier. Moreover, MEF2A performs a transcriptional activity not carried out by any other factors in the family [MEF2A gene specifically binds to the Gtl2/Dio3 locus, upregulates transcriptional cofactor CITED2, and promotes cardiomyocyte proliferation [MEF2A gene is one of the first genes detected during muscle regeneration in mice. Liu et al. showed that toxin injection in mouse tibialis anterior muscle can significantly downregulate MEF2A and MEF2C on day 2, and significantly upregulate MEF2A during later muscle regeneration [MEF2A expression can promote atherosclerotic lesions in apoE knockout mice [MEF2A can regulate vascular endothelial cell migration and anti-apoptotic cell death [MEF2A in myogenesis, MEF2A is also involved in lipid metabolic pathways and plays a role in various biological processes. Moreover, MEF2A regulates muscle tissue growth and development, depending on tissue type [Myocyte enhancer factor 2 (MEF2), belonging to the MADS transcription factor superfamily, can regulate muscle-specific gene expression . The MEFematodes ,10. MEF2ematodes . The MADematodes ,13. MEF2ematodes . MEF2A a changes ,16. MEF2e family . Clark eferation . The MEFneration . Zhou etout mice . Studiesll death . Besidessue type ,23.MEF2A in large domestic animals. Furthermore, systematic MEF2A studies on cattle are lacking. Guanling cattle are a unique breed in Guizhou Province, China, which have the advantages of strong physique and disease resistance, but their growth is slow and meat yield is low. In this study, Guanling cattle were used as experimental subjects with the aim of investigating the effects of changes in MEF2A gene expression on the proliferation and apoptosis of bovine myoblasts. Therefore, this study can help to investigate the regulation mechanism of beef qualitative traits and provide basic theoretical references for bovine growth and development.However, no study has evaluated Animal experiments were approved by the Laboratory Animal Ethics of Guizhou University .MEF2A (NM_001083638.2) was amplified and ligated to the PMD-19T vector. The recombinant MEF2A vector and pEGFP-C1 vector were double-enzymatically digested using EcoR Ⅰ and Sal Ⅰ. The recombinant digest product was obtained to obtain the MEF2A overexpression vector and negative control (NC) was supplied by our laboratory. Four interfering group target sequences and one NC group sequence were set based on the sequence of the coding region of MEF2A and shRNA design principles. The vector with the best interfering effect was selected for further analysis. The primer sequences were synthesized by GEMA Ltd. . The detailed shRNA target sequences are shown in The full-length CDS region sequence of bovine Myoblasts were isolated and cultured from three healthy 3-day-old Guanling calves. The calves were born in Guanling Cattle Industrial Park, Anshun  then slaughtered. The longest dorsal muscle tissue was obtained, cut, and digested with type II collagenase. The filtrate was centrifuged, and the supernatant was discarded. The cells were then resuspended in DMEM-F/12  containing 12% foetal bovine serum and 1.5% penicillin-streptomycin. The purity of the myocytes was determined using immunofluorescence. α-actin  was used as primary antibody and Alexa Fluor594  as secondary antibody. Cells were inoculated into 6-well cell plates. Each vector was transfected into cells using Lipofectamine 3000  transfection reagent when cell density reached about 80%. The cells were harvested after 48 h of transient transfection for subsequent assays, including CCK8, ELISA, RT-qPCR, flow cytometry, cell cycle, and apoptosis analyses.MEF2A overexpression and the expression interference on the proliferative capacity of adult myoblasts was examined using CCK8 reagent . Briefly, myoblasts were inoculated in 96-well plates and incubated at 37 °C in 5% CO2 for 0, 6, 12, 24, 48, and 72 h, followed by the addition of CCK8 reagent (10 μL) to each well. An enzyme marker  was used to detect absorbance at 450 nm.The effect of First, each vector was transfected into myoblasts for 48 h. The cells were washed with PBS, then 180 μL of RIPA lysis solution  was added, and they were lysed on ice for 5 min. Total protein was collected by vortexing at 12,000 rpm for 15 min. Absorbance was measured at 450 nm using an enzyme marker  according to the instructions of the GH and INS kits. Linear regression equations were calculated for the standards based on OD values.Cells were cultured in 6-well plates, and each vector was transfected into myoblasts for 48 h. The cells were collected via centrifugation after trypsin digestion, followed by the addition of 2 mL of 70% ethanol, then fixed at 4 °C overnight. the fixed cells were centrifuged and the supernatant was discarded. The cells were washed with PBS, resuspended with 100 μL of RNase A, and placed in a 37 °C water bath for 30 min. Then, 500 μL of PI staining solution was added, and the sample was left to react for 30 min at 4 °C away from light. The DNA content of the different transfected and control groups was measured via flow cytometry .Cells were collected and prepared, as described above. The cells were centrifuged and washed with precooled PBS, resuspended with 100 μL binding buffer, and incubated with propidium iodide (PI) solution and FITC Annexin V (Thermo Fisher Scientific) at room temperature for 15 min while away from light. The samples were analysed via flow cytometry.MEF2A expression changes on the expression levels of cell cycle factors CDK2 and CCNA2, apoptosis factors Bcl2 and BAD, and cell growth factors IGF1 and MyoD1 were detected via RT-qPCR analysis. qRT-PCR primer information is shown in ® SuperMix, 0.5 μL each of upstream and downstream primers, 0.5 μL of cDNA, and 4 μL of ddH2O. Each sample had three triplicates. The reaction program consisted of pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, and annealing for 5 s (39 cycles). GAPDH was used as the internal reference gene. Gene expression was calculated via the 2Ct−ΔΔ method.Total RNA was extracted using TRIZOL reagent . The total RNA was reverse-transcribed using StarScrip II First Strand cDNA Kit  to obtain cDNA. The effects of p < 0.05.Data are expressed as mean ± SD of three biological replicates. One-way analysis of variance (ANOVA) and the independent t-test were used to assess differences between groups via SPSS 18.0 software . Statistical significance was analysed at The cells were identified via indirect immunofluorescence . MyocyteMEF2A gene and the efficiency of the interfering vector were analysed using qRT-PCR. The gene expression of the overexpressed OE-MEF2A was about 100 times (p < 0.01) higher than that of the control OE-NC (p < 0.01). The sh-RNA3 vector (sh-MEF2A) was selected for subsequent experiments.The expression of the ol OE-NC A. Moreovol OE-NC B(interfeThe CCK8 results showed that OE-MEF2A significantly promoted the proliferative activity of myogenic cells after 6 h compared with OE-NC A. HoweveMEF2A overexpression slightly increased GH content (p < 0.01) and INS content in myoblasts (p > 0.05) compared with OE-NC (MEF2A interference decreased GH and INS content in myoblasts (p < 0.01) (MEF2A gene can promote cellular activity.The ELISA results showed that th OE-NC A. Howeve < 0.01) B, suggesMEF2A overexpression and interference on myoblast cell cycle and apoptosis was examined via flow cytometry. Compared with OE-NC, the number of OE-MEF2A myoblast cells was significantly reduced in the G1 phase (p < 0.05). Furthermore, the number of S-phase myoblast cells was significantly increased (p < 0.01). Furthermore, G2/M was not significantly changed in the S-phase (p > 0.05) (p < 0.01) (p < 0.05) in the G1 phase and significantly decreased (p < 0.01) in the S and G2/M phases (MEF2A gene (p < 0.01). Furthermore, the inhibition of the MEF2A gene significantly increased the apoptosis rate of myoblast cells (p < 0.01)  A, while  < 0.01) B. ComparM phases C. Moreov < 0.01) D. These MEF2A overexpression significantly increased the expression of the MyoD1, IGF1, CCNA2, CDK2, and Bcl2 genes (p < 0.01) and significantly decreased the expression of the BAD gene (p < 0.05) in myoblasts , significantly reduced the expression of the CDK2 gene (p < 0.05), and slightly upregulated the BAD gene (p > 0.05) (MEF2A gene on myoblast cells.yoblasts A. In con > 0.05) B. These MEF2A promotes myofibril formation and participates in the regeneration and differentiation of skeletal muscle stem cells and myoblasts cells [MEF2A-MEG3/DIO3-PP2A axis promotes bovine muscle regeneration [MEF2A can promote postnatal muscle growth and development in goat kids. Moreover, MEF2A significantly increases muscle fibre diameter in the loin [CDK2 is a cell cycle regulator that promotes the transition from the G1 to the S phase by forming a complex with CCNA2, thereby regulating cell cycle progression [MEF2A upregulation or downregulation changed CDK2 and CCNA2 expression. MEF2A overexpression significantly decreased the number of G1-phase cells and significantly increased the number of S-phase cells, thus greatly shortening the G1-S phase process. Moreover, MEF2A interference did not affect the G1-S phase process, indicating that MEF2A accelerates the cell cycle process, consistent with the study of Wang et al. [The protein encoded by MEF2A can participate in various cellular processes as homo/heterodimers, including muscle development, cell growth control, and apoptosis. The proliferation and differentiation of myoblast cells are crucial in muscle tissue growth through cell proliferation and protein deposition . MEF2A pts cells . Wang etneration . Chen etthe loin . CDK2 isgression . In thisg et al. .MyoD1 positively regulates skeletal muscle growth and development, thus affecting the development and repair of muscle tissue [IGF1 can play a synergistic role with growth hormone (GH) to accelerate cell proliferation and differentiation and other physiological functions [MEF2A upregulation significantly increased the viability of myoblast cells. Other assays also showed that MEF2A upregulation increased the expression of the MyoD1 and IGF1 genes, while MEF2A inhibition decreased these expressions. Growth hormone promotes growth, cell division, and proliferation, thus accelerating metabolism [MEF2A overexpression significantly increased GH levels and slightly increased INS levels. Moreover, MEF2A inhibition significantly decreased both INS and GH levels.e tissue . IGF1 caunctions . Herein,tabolism . Insulintabolism . In thisMEF2A expression in apoE−/− mice increased the levels of pro-inflammatory cytokines, such as IL-6, MCP-1, TNF-α, and MMP-8, and atherosclerosis in apoE−/−mice [MEF2A in VECs, thereby preventing apoptosis-induced oxidative stress [MEF2A upregulation increased the activity of myogenic cells and had an anti-apoptotic effect. RT-qPCR also showed that MEF2A overexpression inhibited the expression of the pro-apoptogenic gene BAD and increased the expression of the anti-apoptogenic gene Bcl2. Furthermore, MEF2A inhibition increased the rate of apoptosis. One study reported that MEF2A reduced the expression level of Hspb7 in mouse skeletal muscle, which was effective in preventing muscle atrophy [MEF2A overexpression can disrupt myoblast differentiation patterns and apoptosis [Zhou et al. reported that siRNA inhibition of E−/−mice . Lu et aE−/−mice . Resverae stress . In this atrophy . Howeverpoptosis .MEF2A, MEF2C, and MEF2D have synergistic roles in muscle growth and development, especially MEF2A [MEF2A are defective in terms of skeletal muscle regeneration [MEF2A, or MEF2A knockout, can significantly cause a disease phenotype in mice [MEF2A has an irreplaceable function. Wu et al. found that MEF2A is evolutionarily higher on the list than MEF2C through phylogenetic analysis [MEF2A gene among the MEF2 family members.In addition, several studies have shown that ly MEF2A ,38. Synd in mice ,40, sugganalysis . These fMEF2A can positively regulate the proliferation and anti-apoptosis of bovine myoblasts through the regulation of key genes of the cycle , growth , and apoptosis . These results provide a basic theoretical reference for analysing the mechanisms through which MEF2A regulates bovine growth and development.In conclusion,


Escherichia coli isolates G2M6U and G6M1F, two multidrug-resistant strains that were isolated from mammary tissue and fecal samples, respectively, from mice with induced mastitis. The complete genomes of G2M6U and G6M1F consist of chromosomes of 4.4 Mbp and 4.6 Mbp, respectively.We sequenced the genomes of  Escherichia coli is a rod-shaped, flagellated, facultative aerobic, nonsporulating, Gram-negative bacterium of the family Enterobacteriaceae. It is considered one of the most important causes of clinical and subclinical mastitis in dairy cows  and the nutrition status and immune responses of affected cows  and humans  overnight at 37°C, and the harvested cultures were used for DNA extraction with a QIAamp DNA minikit . The DNA purity and concentration were checked using a NanoDrop 2000 UV-visible spectrophotometer . The Nextera DNA Flex library preparation kit  was used to generate libraries from 1 ng DNA, and whole-genome sequencing of the prepared libraries was performed using a MiSeq sequencer (Illumina) with a 2 × 250-bp protocol. The paired-end raw reads  were trimmed using Trimmomatic v0.39  to remove Illumina adapters, known Illumina artifacts, and phiX reads  of the Bangabandhu Sheikh Mujibur Rahman Agricultural University  reviewed and approved the experimental procedures of this study (reference number FVMAS/AREC/2023/6679 [16 January 2023]). Mammary tissues and fecal samples from mice in which mastitis was induced  were plated on nutrient agar for overnight incubation at 37°C. Pure colonies were isolated and subsequently streaked on eosin methylene blue (EMB) agar  plates and incubated at 37°C for 24 h  and ST58 according to seven-gene multilocus sequence typing (MLST)  with a core genome MLST (cgMLST) scheme. The RAST FIGfams v70 annotations revealed that the G2M6U genome contained 367 metabolic features in SEED subsystems, with 32% coverage, 4,343 protein-coding sequences (CDSs), and 68 RNA genes; whereas 378 SEED subsystem features, with 32% coverage, 4,607 CDSs, and 76 RNA genes, were predicted in the G6M1F genome. Seventy-eight ARGs , conferring resistance to multiple antibiotics and metals, and 276 virulence factor-related genes  were detected in the draft genomes. One plasmid replicon like IncY  was identified in the G6M1F genome (with 95% identity and 60% coverage), while the G2M6U genome harbored none.The draft assembly sizes of G2M6U and G6M1F were 4.4 Mbp and 4.6 Mbp, respectively. Further sequencing and assembly statistics are given in PRJNA946194. Individual accession numbers are listed in Sequencing data were deposited in GenBank and the NCBI Sequence Read Archive (SRA) under BioProject accession number


Adolescents with 46,XY disorders of sex development (DSD) face additional medical and psychological challenges. To optimize management and minimize hazards, correct and early clinical and molecular diagnosis is necessary.NR5A1) [c.64G > T (p.G22C)] was identified in the patient. In vitro functional analyses of the novel variant suggested no impairment to NR5A1 mRNA or protein expression relative to wild-type, and immunofluorescence confirmed similar localization of NR5A1 mutant to the cell nucleus. However, we observed decreased DNA-binding affinity by the NR5A1 variant, while dual-luciferase reporter assays showed that the mutant effectively downregulated the transactivation capacity of anti-Müllerian hormone. We described a novel NR5A1 variant and demonstrated its adverse effects on the functional integrity of the NR5A1 protein resulting in serious impairment of its modulation of gonadal development.We report a 13-year-old Chinese adolescent with absent Müllerian derivatives and suspected testis in the inguinal area. History, examinations, and assistant examinations were available for clinical diagnosis of 46,XY DSD. The subsequent targeting specific disease‐causing genes, comprising 360 endocrine disease-causing genes, was employed for molecular diagnosis. A novel variation in nuclear receptor subfamily 5 group A member 1 (NR5A1 variant to the pool of pathogenic variants and enriches the adolescents of information available about the mutation spectrum of this gene in Chinese population.This study adds one novel The online version contains supplementary material available at 10.1186/s12887-023-03974-7. NR5A1 c.64G > T (p.G22C).46,XY disorders/differences of sex development (DSD) occur with a frequency of approximately 1:20,000 . A greatNR5A1, also known as SF-1, AD4BP and FTZF1) is a key transcription factor that determines gonadal development and regulates coordinates endocrine functions [NR5A1 variants occur in ~ 15–20% of patients and are regarded as a common genetic pathogeny of 46,XY DSD [. [NR5A1 variants in individuals with 46,XY DSD who presented primary adrenal insufficiency and complete gonadal dysgenesis. Additionally, Pedace et al. [NR5A1 variants among 81 cases with 46,XY DSD in 2014, while Fabbri-Scallet et al. [NR5A1 in 238 cases with 46,XY DSD in 2020. These studies revealed that pathogenic variants are reported in 35–45% of individuals with 46,XY DSD [NR5A1 variant; in vitro functional study of this novel variant will expand our knowledge of the NR5A1 mutational spectrum.Nuclear receptor subfamily 5 group A member 1  heterozygous and missense variant in NR5A1 that was absent in the parents  NR5A1 variant. The diagnostic process is shown in Fig. To obtain the molecular diagnosis, the subsequent genetic sequencing was performed. As chromosome microdeletion and microduplication are pathogenic factors associated with DSD, the copy number variation sequencing (CNV-seq) was performed firstly and the results indicated that there was no pathogenic variation in the patient at the chromosome level Fig. A. SecondG > T p.G2C heteroFollowing the molecular diagnosis, further investigations are warranted to determine a more accurate phenotype. The three-day human chorionic gonadotropin (hCG) stimulation test and seven-day human menopausal gonadotropin (HMG) stimulation test were performed to further assess gonad function. Negative results Table  indicateSubsequent issues involved treatment and gender assignment/reassignment. Psychological assessment revealed that the gender identity of the patient was female, which coincided with the social gender. The multi-disciplinary treatment team of DSD in our hospital conducted extensive and in-depth counseling with the adolescent and parents. Following the family agreeing to surgery, exploration results revealed absent Müllerian derivatives with blind vagina and poorly developed testicular tissue in the inguinal region without spermatogenic cells, which were confirmed by intraoperative frozen pathology and postoperative microscopic biopsy Fig. . With thth to 90th percentile of height for girls of the same age and the same ethnic group); weight of 64.3 kg (> 90th centile); BMI of 24.8; bone age of 12.5-years old. The blood pressure was normal at 111/73 mmHg. The patient was treated with a low dose of oral progynova . The breasts developed to Tanner stage 2, and estrogen levels increased after ERT. The patient received regular follow-up and ERT for > 9 months with no adverse drug reactions.The patient returned to our department for estrogen-replacement therapy (ERT) nearly 4 months after the surgery. We performed selective examinations of the patient at age 13 . Despite the G22C substitution, both WT Gly22 and NR5A1-Mut Cys22 form hydrogen bonds with threonine at position 29 (Thr29)  variation in 2C variatNR5A1 were conducted. 293 T cells , Myc-NR5A1 WT (WT) and Myc-p.G22C (NR5A1-Mut) plasmids, were used to conduct the NR5A1-overexpression system. qRT-PCR analysis showed no difference in NR5A1 mRNA levels between cells transfected with WT or NR5A1-Mut plasmids  variation in NR5A1 variant. Advanced genetic detection is applied as a first line of investigation for a molecular diagnosis and helpful to the etiologic diagnosis of patients with DSD and progressively improves the etiologic diagnostic rate [Although the diagnosis of 46,XY DSD is rare, the impact on the life quality of the affected adolescents and their families is significant even devastating . Physicitic rate . Followitic rate , 19, whiNR5A1 mutational site using TRS in a Chinese adolescent with 46,XY DSD. This novel c.64G > T (p.G22C) variant is a heterozygous variant, similar to most reported NR5A1 variants [NR5A1 variants in human [The emergence of new genetic techniques strongly influences the rate of correct diagnoses and reduces diagnostic delay . The preG > T p.GC variantvariants ; howeverNR5A1 variants include missense and small deletions and insertions [NR5A1 variants [NR5A1 transcription, with the first two variations resulting in altered subcellular localization [AMH transcription.To date, the reported sertions . The novvariants . The p.Gvariants , 24–26. variants . Additiolization . Moreovelization . In the Interestingly, the site of the amino acid substitution reported here (G22C) is the same as that described by Sudhakar et al.  (G22S); Furthermore, although previous studies suggest that some NR5A1 variants alter the 3D structure of the protein , 28, we NR5A1 variant may affect its function of promoting the secretion of AMH. In the human fetus at 9 weeks, Müllerian ducts have nearly totally disappeared [AMH secreted by Sertoli cells immediately after testicular differentiation, is responsible for the regression of Müllerian ducts in the male fetus . The staappeared . It suggThis patient was diagnosed with 46,XY DSD during adolescent years. The foreign and sudden disorders is undoubtedly an alarming and traumatic event for the adolescent and families. They have to face with some problems, including impaired fertility, medical treatment and possibly gonadal or vaginal surgery . SexualiNR5A1 as a pathogenic variant that resulted in the NR5A1-mediated dysregulation of gonadal development. This study adds one more NR5A1 variant to the long list of previously published data for this gene and enriches the adolescents of information available about the NR5A1 mutation spectrum in Chinese population. Genetic analysis of more samples of NR5A1 variants and functional studies would be of great significance for understanding the mechanism of gonadal development and sex determination.In summary, we described the diagnostic process for a 13-year-old Chinese patient with partial gonadal dysgenesis, including clinical and molecular diagnoses. Functional analysis identified a novel c.64G > T (p.G22C) variant in Additional file 1: Supplementary Fig 1. Timeline of the diagnostic process for the patient.Additional file 2: Supplementary Fig 2. Conformational changes in the NR5A1 mutant. The residual at position 22 changes from a hydrophobic to hydrophilic amino acid with the G22C substitution; both the WT and NR5A1-Mut proteins show the formation of a hydrogen bond with Thr29.Additional file 3: Supplementary Fig 3. The raw figures of gels (EMSA) and the blots (Western blot) (A) the bands of the internal reference (β-actin). From left to right, these bands are non-transfected , transfected with an empty vector (Vector), Myc-tagged WT (WT), and c.64G>T (p. G22C) NR5A1-Mut (Mut) vectors; (B) NR5A1 expression in 293T cells according to western blot analysis. (same grouping as A); (C) ESMAs results showing altered DNA binding by the NR5A1 mutant (same group as A).Additional file 4: Supplementary Table 1. List of 360 endocrine-related genes detected by TRS.Additional file 5: Supplementary Table 2. Prediction of the impact of the c.64G>T (p.G22C) NR5A1 variant.


These procedures were reassessed to meet the strict requirements associated with the further implementation of RFBs, including fast and affordable synthetic procedures with high reaction yields. In contrast to the lengthy and complicated synthetic approaches published to date, the optimized synthesis reported in this work enables the isolation of the pure crystalline salt of the 12CoW anion with a 75% reduction of the time of the whole reaction procedure, eliminating tedious steps such as the recrystallization and including a 20% increased yield. The control of the stoichiometry, fine-tuning of reaction conditions, and the identification of intermediate species, as well as the acidic equilibria taking place during the process, were monitored via thermal, spectroscopic, and structural analyses. In the case of the 11CoSiW anion, its preparation was based on a simple and highly efficient procedure. Moreover, promising electrochemical properties were observed with the use of the one-pot synthetic approach, in which the stoichiometric amounts of the starting reagents are dissolved in the supporting electrolyte to be directly implemented as the electrolyte for a RFB.New energy storage materials are an object of study within the framework of the global energy transition. The development of renewable sources is being boosted thanks to stationary energy storage systems such as redox flow batteries (RFBs). This work reports the synthesis of the cobalt-containing Keggin-type polyoxometalates [CoW After the careful optimization of the electrolyte composition, they exhibited a suitable operational potential as well as high reversibility of the redox processes in an appropriate medium, namely 1M acetic acid/lithium acetate (H/LiAc) buffer solution ·17H2O (2W11K-Co) was obtained. Yield: 96% based on W. UV-Vis (H2O) λmax (nm): 600. FT-IR −1): 929 (s), 860 (s), 781 (s), 660 (m), 540 (w), 459 (m).The terature . To an a2W11K-Co salt was dissolved in acidic medium with a 1 g:10 mL 1M HCl ratio. After five days, needle-like dark blue crystals of K4H2[CoW12O40]·12H2O were isolated after slow evaporation of the final solution at room temperature. Overall yield: 88% based on W. Elemental analyses: Co 1.77%, K 4.75%, W 67.29%. Calculated for CoH26K4O52W12: Co 1.82%, K 4.82%, W 67.96%. UV-Vis (H2O) λmax (nm): 624. FT-IR −1): 943 (s), 881 (s), 736 (s), 447 (s). Powder XRD: ICSD-89706 6− (11CoSiW) polyanion. After filtering any solid out, the electrolyte was ready for the electrochemical tests. Yield: 78% determined by UV-Vis (1M H/LiAc), λmax (nm): 546.Na12O40]6− was first isolated in 1956 by Baker et al. [2(H2O)W11O39]8− (2W11Co) anion on the basis of elemental analyses. In order to obtain the desired [CoW12O40]6− (12CoW), it was necessary to dissolve the intermediate in highly acidic conditions (1M HCl) and to let it evaporate at room temperature until dark blue needle-like crystals were formed. The product was obtained at a 70% yield, but four recrystallization processes were needed to obtain a pure crystalline product. Although a short reaction time can be advantageous, the whole procedure takes several weeks to complete because up to four recrystallizations are needed to obtain a pure compound. This synthetic procedure has been reproduced over the years with slight modifications 10− paratungstate-A anion on the basis of FT-IR spectroscopy (6[{Co(H2O)4}2(H2W12O42)] Co 3.84%, W 72.93%; Anal. Co 3.85%, W 82.12%) ·17H2O: Co 3.46%, W 59.45%; Anal. Co 2.69%, W 57.45%). The vibrational analysis of the compound via FT-IR spectroscopy showed the most representative signals in the wavenumber range below 1000 cm−1. The assignment was performed based on previous results for these specific polyanions ·15H2O (ICSD-89706) 10− paratungstate anion  precursor, in which one of the tungstate octahedral is removed in comparison to the plenary species [2O)SiW11O39]6− (11CoSiW) anions. It is worth highlighting that the pure crystalline compound is obtained within less than one week with a high yield and without further purification steps. In fact, cobalt(II)-substituted POMs have been an object of study in diverse applications such as the electroreduction of CO2 to CO 6− (12CoW) and monosubstituted [Co(H2O)SiW11O39]6− (11CoSiW) anions. During the optimization procedure, stoichiometry, starting materials, pH, and reaction time, among other parameters, were carefully evaluated in order to improve the yield and decrease the total cost of the reaction. With the aim of creating an efficient POM-based RFB, the optimization of the synthesis of two electroactive species is reported in this work. The species are the cobalt(II)-containing Keggin-type plenary [CoW4H2[CoW12O40]·12H2O revealed critical steps of the reaction. First, specific pH conditions (7.5) and a 1:6 Co:W molar ratio of the starting reagents are crucial for the efficient isolation of the K8[Co2W11O39]·17H2O intermediate. Side products of this process were first identified using FT-IR, XRF, and PXRD and then avoided. Finally, the transformation to 12CoW was found to be optimal when the reaction medium was acidified with aqueous HCl to pH = 0. These modifications involved an increase of up to 20% of the total reaction yield in comparison to reported procedures and the whole reaction time was reduced five-fold. It was also proven that an electrolyte based on 11CoSiW cobalt(II)-monosubstituted tungstosilicate anions can be rapidly obtained by reacting the metal sources in a suitable supporting electrode such as the aqueous 1M H/LiAc buffer. The resulting one-pot electrolyte displayed suitable conductivities and redox properties, and the total cost and duration of the electrolyte production was decreased considerably. The detailed study of the synthesis of K


ZAP70 gene cause a combined immunodeficiency characterized by low or absent CD8+ T cells and nonfunctional CD4+ T cells. Most deleterious missense ZAP70 mutations in patients are located in the kinase domain but the impact of mutations in the SH2 domains, regulating ZAP-70 recruitment to the TCR, are not well understood.ZAP-70, a protein tyrosine kinase recruited to the T cell receptor (TCR), initiates a TCR signaling cascade upon antigen stimulation. Mutations in the ZAP70 mutations was developed. The impact of SH2 domain mutations was evaluated by biochemical and functional analyses as well as by protein modeling.Genetic analyses were performed on four patients with CD8 lymphopenia and a high resolution melting screening for ZAP70 gene . A distantly related second patient was found to be compound heterozygous for the R170C variant and a 13bp deletion in the ZAP70 kinase domain. While the R170C mutant was highly expressed, there was an absence of TCR-induced proliferation, associated with significantly attenuated TCR-induced ZAP-70 phosphorylation and a lack of binding of ZAP-70 to TCR-ζ. Moreover, a homozygous ZAP-70 R192W variant was identified in 2 siblings with combined immunodeficiency and CD8 lymphopenia, confirming the pathogenicity of this mutation. Structural modeling of this region revealed the critical nature of the arginines at positions 170 and 192, in concert with R190, forming a binding pocket for the phosphorylated TCR-ζ chain. Deleterious mutations in the SH2-C domain result in attenuated ZAP-70 function and clinical manifestations of immunodeficiency.Genetic characterization of an infant who presented with pneumocystis pneumonia, mycobacterial infection, and an absence of CD8 T cells revealed a novel homozygous mutation in the C-terminal SH2 domain (SH2-C) of the  Inborn errors of immunity (IEI), also known as primary immunodeficiencies (PID), comprise a heterogeneous group of disorders, generally resulting from genetic mutations that negatively impact immune cell development and function. Within this group of diseases, severe combined immunodeficiencies (SCID) are characterized by defective T and B lymphocyte differentiation that are almost universally fatal in infancy. In these patients, replacement of the mutant hematopoietic stem cells (HSCs), either by HSC transplantation or by genetic correction of these progenitors, represents a critical therapeutic approach . While tZAP70 protein tyrosine kinase (PTK) gene, at least during the neonatal period  the majority of patients exhibit a complete loss of expression and/or function of the ZAP-70 protein; 2) a smaller group present with hypomorphic mutations; and 3) a single patient with compound heterozygous mutations resulting in both loss-and gain-of-function mutations has been reported (ZAP-70 is recruited to the T cell receptor (TCR) following Lck-induced phosphorylation of the immunoreceptor tyrosine–based activation motifs (ITAMs) in the TCRζ chain , 9. Bindζ chain –12 . She did though present with autoimmune symptoms including alopecia areata as well as papules and nodules predominantly on the face and limbs. She died from infectious complications at 7 years of age.Third degree cousins, the product of consanguineous and non-related parents, respectively . Lymphocyte immunophenotyping revealed decreased T lymphocyte numbers in Patient 1 but not Patient 2 with normal levels of CD4A third female patient, the product of consanguineous parents, presented at Texas Children’s Hospital with fever and respiratory distress at 16 months of age. Her symptoms had started four months after receiving her first dose of the MMR vaccine. She had a maculopapular rash on her face and extremities with an unusual clustering of the rash, palatal ulcers, and autoimmunity manifested as bilateral hand arthritis, with inflammation at the proximal, intermediate and distal interphalangeal joints of both hands. She had progressive deterioration of her lung function and expired. Microbiology and pathology studies demonstrated pneumonitis and hepatitis induced by the measles vaccine strain, and ruled out other pathogens. Immunologic evaluation uncovered a severe T-cell lymphopenia with nearly absent CD8 T cells , resulting in an Arg to Cys mutation at position 170 of the C-terminal SH2 domain  technique has been used to scan for mutations across several important genes. Here, we evaluated the detection of the single C>T mutation (R170C) and the 13bp deletion using primers designed to amplify a 163bp and 139bp qPCR products, respectively. Initial analyses of the R170C melting curve by the Tm calling analysis were not able to distinguish patient and healthy donor curves but a normalization melting curve representation was able to discriminate the homozygous and heterozygous C>T mutation in patient 1 and 2, respectively -stimulated proliferation of the CD4 T cell subset in patients 1 and 2 was compared to CD4 T cells from a healthy control. T cells were labeled with the CFSE proliferation dye and stimulation of control CD4 T cells with either anti-CD3/anti-CD28 mAbs or PHA with IL-2 resulted in at least 5 divisions. In contrast, there was a complete absence of proliferation of patient CD4 T cells in response to CD3/CD28 or PHA/IL-2  that weakens the autoinhibitory conformation of ZAP-70 , 42. IndWe report a novel mutation in the C-terminal SH2 domain of ZAP-70, resulting in a homozygous arginine-170 to cysteine mutation and compound heterozygosity with a previously described 13bp deletion leading to a translational frameshift after residue 503 (K504_P508delfsX35) . A seconZAP70 might not present with TREC numbers that are below the threshold for diagnosis  or at Texas Children’s Hospital, USA (family harboring the R192W mutation). Samples were obtained after signed informed consent and approval by the IRBs of the two hospitals. T cells were purified by centrifugation on a Ficoll gradient  following incubation with the RosetteSep T Cell Enrichment kit (StemCells Technologies) according to the manufacturer’s instructions. Primary T cells were cultured in RPMI 1640 medium supplemented with 10%FCS and 1% Penicillin-Streptomycin for functional analysis.-/-/Syk-/- p116  and ZAP-70-/- p116  Jurkat cT cells (10e6) were lysed and RNA were extracted using the RNA Easy mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was obtained by elongation using oligo-dT primers with the M-MuLV Reverse Transcriptase Reaction kit (Qiagen) and PCR-amplified exons were sequenced by Sanger’s Method.MluI restriction enzyme (1U) for 30min at 37°C to assess the mutation-induced disruption of this restriction enzyme recognition site. MluI-digested DNA fragments were separated on a 1.8% agarose gel and the image was captured using an UV-illuminator (Ingenius SynGene).Genomic DNA (10ng), extracted with the QiaAMP DNA Blood Mini kit (Qiagen), was amplified using the Sybr Green I Master Mix (Roche) with exon 4 primers specific for the R170C mutation  using the following program; 5 sec 95°C, 10 sec 60°C, 10 sec 72°C, for 30 cycles. Products from the R170C qPCR were purified using the Nucleospin Gel PCR cleanup kit (Macherey-Nagel) and digested with the Genomic DNA was extracted as described above. Detection of the R170C mutation and the 13bp deletion was performed by Real-Time qPCR using the LightCycler 480 High Resolution Melting Master mix (Roche) on the LightCycler480 machine (Roche). Briefly, genomic extract (10ng) was amplified with exon 4 primers specific for the R170C mutation  and exon 12 primers specific for the 13bp deletion  using the following program; 5 sec 95°C, 10 sec 60°C, 10 sec 72°C, for 30 cycles. An extensive HRM curve  was analyzed using LightCycler 480 II software.202Y204-phosphorylated ERK1/2 , and T180/Y182-phosphorylated p38  were detected after cell fixation and permeabilization .To detect cell surface markers, cells were incubated with the appropriate fluorochrome-conjugated mAbs and expression was monitored in comparison with isotype controls. Antibodies against CD3, CD4, CD8, CD25, HLA-DR, CD19, CD20, and CD56 were from Beckman Coulter. Intracellular ZAP-70 was detected using PE-conjugated anti ZAP-70 . T6) were then activated in 100 µl RPMI with anti-CD3  and anti-CD4  mAbs for 1 min, and crosslinked by goat anti–mouse Fab’2 fragment (1:100) for 2 min. Cells were assessed on a FACS-CantoII or BD LSRII-Fortessa (BD Biosciences) and data were analyzed using Diva (BD Biosciences) or FlowJo (Tree Star) software.For lymphocyte subset analysis, 100 µl of whole blood was incubated with mAbs against surface markers for 20 minutes in the dark at room temperature. Red cells were then lysed and washed before acquisition. For evaluation of TCR signaling, Jurkat cells transduced with the indicated vectors were starved overnight in RPMI media containing only 1% FCS. Cells  for 3 min and washed twice with PBS 2% FCS. T cells (1x106) were subsequently plated into anti-CD3/anti-CD28 coated wells  or stimulated with PHA (1μg/ml-Sigma) and rhIL2 (100IU/ml-Proleukin). Proliferation was evaluated at day 5 following staining with anti-CD4 APC (clone 13B8.2) and anti-CD8 PeCy7 (clone SFC121Thy2D3) mAbs and data acquired on a FACSCanto II Flow Cytometer (Becton Dickinson). Analyses were performed using FlowJo Software (v10–10.6.2).Proliferation was monitored as a function of CFSE dilution. CD3BamHI/BstEII fragment of the WT ZAP-70 cDNA from a previously reported lentiviral vector containing the EGFP reporter gene (DpnI restriction enzyme (1U) for 1h at 37°C. After sequencing, the BamHI/BstEII fragment harboring the R170C mutant was reinserted into the original pRRL PGK ZAP-70 vector and sequence verified.Site-directed R170C c>t mutagenesis was performed by amplification of the ter gene . Amplifi-/-/Syk-/- p116 cell line. Transduced p116 cells were sorted on the basis of EGFP expression on a BD FACSAria (BD Biosciences).Self-inactivating HIV-1-derived viruses were generated by transient co-transfection of 293T cells with the PsPax2 packaging vector, encoding Gag, Pol, Rev and Tat, a VSV-G envelope glycoprotein plasmid (pCMV-VSV-G), and the HIV-1 derived SIN vector encoding either the EGFP reporter gene alone, or WT ZAP-70 or the R170C ZAP-70 mutant with EGFP downstream of an IRES site . Viral s6) were stimulated with anti-CD3 (clone OKT3 (1 µg/ml) and anti-CD4 (clone 13B8.2 (1 µg/ml) mAbs for 1 min at 37°C, followed by a cross-linking with goat anti–mouse Ig (1:100) for 2 min. Cells were then lysed in 1% NP-40 detergent buffer  containing 5μg/ml aprotinin (Sigma Aldrich), 1mM sodium orthovanadate (Sigma Aldrich) and 1mM sodium fluoride (Sigma Aldrich).To assess expression of ZAP-70 and protein phosphorylation, T cells  Buffer  supplemented with 1mM sodium orthovanadate. Lysates were incubated with an anti-TCR ζ antibody  for 1h at 4°C, followed by a 1h incubation with magnetic protein AG beads (Ademtech) at 4°C. Immunoprecipitated proteins were eluted and analyzed by immublotting as described below.For immunoprecipitation of the TCR ζ chain in the different Jurkat clones, 1x10319ZAP-70/P-Tyr352Syk and P-T202Y204 ERK1/2 (CST), and then revealed by incubation with HRP-coupled secondary antibodies at room temperature for 1h (1:10.000 in PBS-Tween 1% milk). Immunoreactive bands were visualized by enhanced chemiluminescence .SDS-reduced samples were separated on a 4-20% polyacrylamide gel under SDS-denaturing conditions, and transferred electrophoretically to PVDF. Membranes were incubated overnight at 4°C with primary antibodies against ZAP-70 (clone 2F3.2), P-Tyr-/-/Syk-/- p116 cells (2x106) expressing EGFP, WT ZAP70, or R170C ZAP70 were loaded with 1µM of the ratiometric calcium indicator Indo-1 (Molecular Probes) for 30min at 37°C. Cells were then washed and analyzed; after 1 min of acquisition on a BD FACSAria (Becton Dickinson), cells were activated by addition of anti-CD3 (clone UCHT1) and anti-CD4 (clone 13B8.2) mAbs for 1 min followed by crosslinking using an anti-mouse Fab’2 fragment for 2 min. Calcium flux was analyzed as a function of the ratio of the green (filter BP 530/30nm) and violet emissions (filter BP 450/50nm) of Indo-1 using FlowJo software.ZAP-70Predictions of the structure of the SH2 domains of ZAP-70, with or without the kinase domain (PDB 2OQ1), in association with the phosphorylated ITAMs of the TCR-ζ chain were determined using PyMOL, an open source molecular visualization system , as wellThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Coimbra Hospital and University Centre, Coimbra, Portugal; Baylor College of Medicine IRB, Texas. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.CM, RV, NN, EF, JC, VZ, and NT conceived the study. CM, RV, NN, VC, and VZ performed experiments. LN, JC, and EF provided patient care, collected samples and clinical data, and analyzed patient data. JC, VZ, and NT supervised the study. All authors participated in data analysis and discussions. CM, JC, VZ, and NT wrote the manuscript and all authors critically reviewed the manuscript and approved the final version.