SCN3B) plays a crucial role in electrically excitable cells and conduction tissue in the heart. Some previous studies have established that genetic modification in sodium voltage-channel genes encoding for the cardiac β-subunits, such as SCN1B, SCN2B, SCN3B and SCN4B, can result in atrial fibrillation (AF). In the current study, we identified two rare variants in 5′UTR  of SCN3B in two unrelated lone AF patients. Our further functional studies discovered that one of them, the A allele of c.-324C>A (rs976125894), can improve transcriptional activity and may raise SCN3B expression levels. The A allele of c.-324C>A (rs976125894) has higher transcriptional activity when it interacts with GATA4, as we confirmed transcription factor GATA4 is a regulator of SCN3B. To the best of our knowledge, the current study is the first to demonstrate that the gain-of-function mutation of SCN3B can produce AF and the first to link a mutation occurring in the non-coding 5′UTR region of SCN3B to lone AF. The work also offers empirical proof that GATA4 is a critical regulator of SCN3B gene regulation. Our findings may serve as an encyclopedia for AF susceptibility variants and can also provide insight into the investigation of the functional mechanisms behind AF variants discovered by genetic methods.The sodium voltage-gated channel beta subunit 3 ( Atrial Fibrillation (AF), characterized by a rapid and chaotic rhythm in atria, including the absence of P waves, irregular RR intervals and a fast-atrial rate of up to 300 beats per minute, is the most common clinically sustained cardiac arrhythmia . AF occuAF is often associated with complications, such as coronary artery disease (CAD), hypertension, valvular heart disease, hyperthyroidism, heart failure and structural heart diseases. In the Framingham cohort and after age adjustment, hypertension, diabetes mellitus, coronary artery disease and smoking were the main risk factors for non-valvular AF . The comKCNQ1 was first reported by Chen et al. in 2003 [NPPA and NUP155, were found in some patients with lone AF [TTN gene are enriched among individuals with early-onset AF  versus control subjects ) [Epidemiological studies revealed that while numbers of prevalent cardiovascular disorders are risk factors for developing AF, genetic components have also been proven to be involved, particularly in the case of lone AF . Accordi in 2003 . Later,  lone AF ,9. Mutat lone AF ,11. Loss4–2.97)) . In addiV1.5) and AF-causing variants have been reported in both the α-subunit (encoded by the SCN5A gene) and associated β-subunits (Navβ) (encoded by the genes SCN1B–SCN4B) [SCN1B, the gene encoding for the β1-subunit and SCN2B, the gene encoding for the β2-subunit, have been identified as susceptibility genes for AF [SCN1B and SCN3B that encode for the β3-subunit have been identified as susceptibility genes of Brugada Syndrome (BrS), while SCN4B has been linked to the congenital long-QT syndrome (LQTS) 3, ventricular tachycardia and familial AF [SCN3B was found to induce lone AF and Olesen et al. also showed that R6K, L10P and M161T mutation of SCN3B was associated with early-onset lone AF [SCN3B mutation to AF.Studies have linked cardiac arrhythmogenesis to mutations in the genes encoding the cardiac sodium channel (NaB–SCN4B) ,15,16,17s for AF . SCN1B ailial AF . In our  lone AF ,20; howeSCN3B gene in 355 lone AF patients and found that two patients carried rare variants in 5′UTR  of SCN3B and our further functional studies revealed that the rare variant c.-324C>A (rs976125894), which is located in the promoter region and enhanced the transcriptional activity, may regulate the expression level of SCN3B through interacting with the GATA4 transcription factor.In the current study, we carried out mutation screening of the The study subjects included in the present study were enrolled from the GeneID database. All the study patients are of Han ethnic origin by self-description.AF diagnosis was performed by a panel of cardiologists based on the data from electrocardiograms (ECG) and/or Holter ECG and based on the criteria of the ACC/AHA/ESC AF guidelines. The ECG characteristics of AF include the absence of P waves, replacement of P waves by rapid oscillations of fibrillation waves that varied in size, shape and timing (referred to as “f” waves) and irregular ventricular response  with abnormal atrioventricular conduction. Patients with other types of cardiac arrhythmias , thyroid dysfunction, congenital heart disease, cardiomyopathies and valvulopathies were excluded. An AF patient was diagnosed as being affected with “lone AF” if he/she had no CAD, congestive heart failure, hypertension or diabetes and was below the age of 55 years at the first diagnostics of AF.Demographic and clinical information on age, gender, CAD, hypertension and diabetes mellitus was obtained from medical records. CAD was defined as ongoing therapy of CAD, 70% luminal narrowing in at least one vessel by coronary angiography, percutaneous coronary angioplasty, coronary artery bypass graft and MI. Hypertension was defined as ongoing medication for hypertension, systolic blood pressure of ≥140 mmHg or diastolic blood pressure of ≥90 mmHg. Diabetes mellitus was defined as ongoing therapy of diabetes or a fasting plasma glucose level of ≥7.0 mmol/L.The study protocol was approved by Ethics Committee on Human Subject Research at Huazhong University of Science and Technology (HUST) and Liyuan Hospital Affiliated to Tongji Medical College of HUST and also conforms to the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all subjects.SCN3B (NM_018400), including the coding region, 5′ untranslated region, 3′ untranslated region and the boundaries of intron–exon were amplified by polymerase chain reactions (PCR) from AF patient DNA samples and sequenced. The primers for PCR are shown in SCN3B gene was performed in all study subjects to identify a novel mutation responsible for AF. A variant was defined as a mutation if present in less than 0.01% in the general population according to the public database.Genomic DNA was isolated from peripheral blood samples using standard protocols with the Wizard Genomic DNA Purification Kit . Direct Sanger DNA sequencing was used for screening mutations as described previously. All the six exons of SCN3B in unrelated controls was checked by high-resolution melt analysis (HRM) utilizing a Rotor-Gene 6000 High-Resolution Melt system , as previously disclosed by us, to validate the mutation frequency in the Chinese common population. Primers for PCR of HRM are shown in The sequencing of http://fathmm.biocompute.org.uk/ accessed on 5 August 2022), GERP++ (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html accessed on 5 August 2022) and regulatory Mendelian mutation (ReMM) (https://charite.github.io/software-remm-score.html accessed on 5 August 2022).The deleterious potential of the identified mutations was measured using Functional Analysis through Hidden Markov Models (FATHMM) (http://asia.ensembl.org/ accessed on 5 August 2022) and ENCODE data of UCSC genome browser (https://genome.ucsc.edu accessed on 5 August 2022). The Human Transcription Factor Database (http://bioinfo.life.hust.edu.cn/HumanTFDB#!// accessed on 15 August 2022) and JASPAR (https://jaspar.genereg.net accessed on 15 August 2022) were applied to analyze and predict the potential transcription factors binding to the area of the SNP.The possible regulatory features of the variants in the non-coding region were predicted by Ensembl database (To further explore whether the genomic region overlapping > (rs976125894) or c.-303C>T (rs1284768362) has regulatory activity and is a potential promoter, the wild-type 858 bp length genomic fragment overlapping rs976125894 and rs1284768362 was generated by PCR analysis using human genomic DNA as a template. The Prime STAR HS DNA polymerase  was applied and PCR primers were SCN3B-Kpn I-F 5′-GATTCCCAGGGCTGACAGCACACACGG-3′ and SCN3B-HindⅢ-R 5′-AAGCTTGAAGCCGCCAGCCCCAGAAG-3′. The wild-type 858 bp length PCR products were digested with restriction enzyme KpnI and HindIII  and then sub-cloned into the multiple cloning site of the pGL3-basic luciferase vector , resulting in pGL3-SCN3B-WT.The two variants, c.-324 C>A (rs976125894) and c.-303 C>T (rs1284768362), were introduced into the pGL3-SCN3B-WT plasmid by using PCR-based site-directed mutagenesis, referred to as pGL3-Mut-324A and pGL3-Mut-303T, respectively. The primer sequences for c.-324 C>A were 5′-GAGCGAGGCAGGGGAGCGAGTGGAAGCTGG-3′ (forward) and 5′-CCAGCTTCCACTCGCTCCCCTGCCTCGCTC (reverse). The primer sequences for c.-303 C>T were 5′-AGTGGAAGCTGGAGTTCTGGGGTGGGCGGGGAGGC-3′ (forward) and 5′-GCCTCCCCGCCCACCCCAGAACTCCAGCTTCCACT-3′ (reverse). The PCR products were digested by DpnⅠ  in a 37 °C incubator for 6 h.The 5′ UTR contained wild-type c.-324C or mutant A, plus the whole CDS (delete the stop codon) of SCN3B was amplified and digested by EcoRI and BamHI  and then sub-cloned into pEGFP-N1 vector (named pEGFP-N1-WT-324C and pEGFP-N1-Mut-324A) before the ORF of EGFP to validate whether the mutant can regulate the expression of SCN3B. The primer sequences are 5′-GGCGACCGGTGGATCCCGTTCCTCCACTGGTACCGCAGAG-3′ (forward) and 5′-CTCAAGCTTCGAATTCTGCAGGGCTGACAGCACAC-3′ (reverse).pcDNA3.1, pcDNA3.1-GATA4, pcDNA3.1-IKZF1 and pcDNA3.1-WT1 were reserved in the plasmid library from our laboratory. All constructions were confirmed by Sanger sequencing.5 cells per well and transfected at 37 °C with 5% CO2 of 24 h seeding in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . Further, 25ng Renilla luciferase control vector was co-transfected with (1) 200ng pGL3-basic, (2) 200ng pGL3-WT, (3) 200ng pGL3-Mut-324A or 200ng pGL3-Mut-303T. For transcription factor co-transfection experiments, 25ng Renilla luciferase control vector was co-transfected with (1) 100ng pcDNA3.1 + 100ng pcDNA3.1-GATA4 or pcDNA3.1-IKZF1 or pcDNA3.1-WT1, (2) 100ng pGL3-WT + 100ng pcDNA3.1-GATA4 or pcDNA3.1-IKZF1 or pcDNA3.1-WT1, (3) 100ng pGL3-Mut-324A + 100ng pcDNA3.1-GATA4 or pcDNA3.1-IKZF1 or pcDNA3.1-WT1. Lipofectamine 2000  was used. Cells were harvested in 48 h. Firefly luciferase signal intensities and Renilla luciferase activities were measured by using the Dual-Glo Luciferase Assay System  on GloMax 20/20 . Renilla luciferase activity was normalized by relative luciferase intensity. Independent experiments were performed in triplicate at minimum for wild types and mutant types.Human embryonic kidney 293T cells (HEK293T) and rat cardiomyoblast cells (H9C2) were cultured in 24-well plates, at a density of 102. After 24 h seeding in DMEM with 10% of FBS, at a density of 105 cells per well, cells were transfected with (1) 1 μg pEGFP-N1, (2) 1 μg pEGFP-N1-WT-324C, (3) 1 μg pEGFP-N1-Mut-324A, (4) 500 ng pEGFP-N1 + 500 ng pcDNA3.1-GATA4, (5) 500 ng pEGFP-N1-WT-324-C + 500 ng pcDNA3.1-GATA4 and (6) 500 ng pEGFP-N1-Mut-324A + 500 ng pcDNA3.1-GATA4. Lipofectamine 2000 was used. Cells were collected using cell lysis buffer after 48 h feeding. SCN3B  and GAPDH  antibodies were used for Western blotting.HEK293T cells were plated in 12-well plates in a humidified incubator at 37 °C and 5% COTotal RNA was isolated with RNAiso Plus TRIZOL  and was reverse transcribed using HiScriptⅢ RT SuperMix for qPCR , according to the manufacturer’s protocol. qRT-PCR analysis was conducted using MonAmp qPCR SYBR Green Mix  in an Applied Biosystems instrument . qPCR-primers are listed in t-test. A value of p < 0.05 was considered statistically significant. Data analysis was performed using GraphPad Prism software version 6 .For analysis of promoter activity, data were presented as means ± standard deviations from at least three independent experiments. Statistical multiple-group comparisons were evaluated with a one-way ANOVA test with Turkey’s post hoc test and the difference between two groups was tested using Student’s unpaired SCN3B in 355 lone AF sporadic patients selected from the GeneID database to confirm whether SCN3B is the causative gene for AF. The basic clinical characteristics of the study patients are shown in We performed a mutation screening of SCN3B gene in two unrelated patients clinically diagnosed as lone AF (SCN3B mutation carriers in order to determine if the mutations were de novo or germline inherited. While DNA from the parents of subjects who carried s c.-303C>T mutation (rs1284768362) was not accessible, we were successful in getting genomic DNA from the parents of the subject who carried the c.-324C>A (rs976125894) mutation. The results demonstrated that the mutation c.-324C>A (rs976125894) originated de novo as the parents showed an homozygous wild-type genotype and without AF , we identified two susceptibility rare variants, c.-324C>A  and c.-303C>T , in the first exon and the 5′UTR region of  lone AF a,b. The thout AF c. The EC−6) and the TOPMED database , the frequency of the mutation A allele of the c.-324C>A (rs976125894) variant was incredibly low. For the c.-303C>T (rs1284768362) variant, there was no record in the gnomAD database and we found only two carriers in the TOPMED database (2/125568). We also performed validation of the frequency of the two variants in the Chinese Han population and the results indicated that none of the mutations were present in control group containing 500 subjects without AF.According to the public database, both variants were found in exon 1 and were incredibly rare in the general population. In the public databases, such as the gnomAD database  may be deleterious based on the fathmm MKL non-coding score (0.993), GERP++ RS rankscore (0.997) and ReMM score of 0.984. Only fathmm MKL score revealed deleterious effects (0.915) for the c.-303C>T (rs1284768362) variant, but not in others.SCN3B mRNA. The regulatory feature records in the Ensembl database indicated that both rs1284768362 and rs976125894 were active promoters in several tissues and cell types, including cardiac muscle (Ensembl regulatory feature ENSR00000445310). We next assess whether the mutant alleles of these two 5′-UTR rare variants alter the SCN3B promoter activity by in vitro cell transfection studies using luciferase reporter assays in HEK293T cells and H9C2 cells. We cloned the whole exon 1 of SCN3B (858 bp) into the pGL3-basic plasmid and then it was used as a template to construct a c.-303 C>T (rs1284768362) plasmid , carrying a reference C allele, has promoter activity relative to the pGL3-basic vector. As to the mutant A allele carriers, the relative luciferase intensity is significantly enhanced, compared to that of the wild-type C carriers. For the c.-303 C>T (rs1284768362) variant, there is no significant discrepancy between the reference C allele carriers and the alternative T allele carriers .SCN3B promoter, suggesting that the variant c.-324C>A variant (rs976125894) is further suspected as disease inducing.Further Western blot analysis was then performed to evaluate whether the c.-324C>A (rs976125894) regulates the expression level of SCN3B in HEK293T cells. Compared with the wild-type allele C, the promoter region containing the mutant A allele is able to promote higher protein expression level of SCN3B , which iSCN3B, we overexpressed these three transcription factors in HEK293T cells. As shown in SCN3B were upregulated by GATA4 (p < 0.05), while the other two transcription factors, IKZF1 and WT1, did not result in a significant difference. The putative binding site of GATA4 was predicted by JASPAR  is affected by the transcription factors that bind on the surrounding of the c.-324 C>A, bioinformatics-based algorithms were applied to analyze and predict the potential transcription factors. The prediction results suggest that the alteration of C to A may change the motif of GATA4, WT1 and IKZF1. We overexpressed the above-mentioned three transcription factors in HEK293T cells and H9C2 cells, as shown in y JASPAR f. To dety JASPAR g–i. ThesSCN3B and in a total of 355 AF patients, we found two rare variations, c.-324C>A (rs976125894) and c.-303C>T (rs1284768362), in the 5′UTR. We next examined whether the mutant alleles altered the regulatory features using bioinformatics and in vitro functional analyses. We discovered that variant c.-324C>A (rs976125894) in the SCN3B promoter region interacted with the transcription factor GATA4. The mutant A allele of the c.-324C>A (rs976125894) variant increased the promoter activity and may have resulted in abnormal overexpression of SCN3B when compared to the wild-type C allele. The most recent ACMG  guideline classifies the c.-324C>A (rs976125894) variant as pathogenic when taken as a whole based on the extremely low frequency in the population database (PM2 Supporting), de novo via trio analysis (PS2), harmful prediction (PP3) and functional analysis (PS3). The disease-causing potential of the variant c.-303C>T (rs1284768362) is uncertain according to its ACMG categorization of variant of uncertain significance (VUS).In the present investigation, we carried out mutational screening for all exons and exon–intron boundaries of SCN3B is linked to lone AF and may be related to an increase in the expression level of the β3-subunit of the cardiac sodium ion channel (NaV1.5). Together, these results provide strong genetic support for SCN3B’s role as an AF susceptibility gene. The data also support the idea that rare mutations with major impacts play a significant role in the genetic underpinnings of AF.Four non-synonymous mutations  showed a decrease in the cardiac sodium current density and were discovered to be related to lone AF in previous studies ,20. HoweSCN3B—A130V, R6K, L10P and M161T—were electrophysiologically classified as loss-of-function mutations because they all decreased the cardiac sodium channel peak current density and impaired sodium current function. SCN3B A130V mutation might prevent the sodium channel Nav1.5 from trafficking to plasma membranes, R6K exerted a negative influence on steady-state inactivation, M161T caused a decrease in the peak current density and L10P affected both the steady-state inactivation and the sodium peak current density [SCN3B gene knockout mice model (SCN3B−/−) was applied to study atrial electrophysiology by Hakim et al. Compared with WT mice, the SCN3B−/− mice exhibited abnormal ECG characteristics and irregular spontaneous atrial activity. Furthermore, SCN3B−/− mice demonstrated a conduction abnormality and dysfunction of sinus node since the hearts of SCN3B−/− mice show significantly longer sinus node recovery time than that of the WT hearts [SCN3B mutation V54G in a 20-year-old patient suffering from idiopathic ventricular fibrillation (IVF) and the mutation then caused a functional defect of SCN3B and reduced the sodium current, possibly via the mechanism of interfering with the normal localization of chaperone and cell membrane of SCN3B, thus, confirming that SCN3B, indeed, performs a significantly important role in maintaining the electric stability of the heart [SCN3B for the atrial function and conduction. Taken together, loss-of-function mutations in SCN3B may cause AF via shortening the cardiac refractory period or affecting the electric stability of the heart, indicating that SCN3B is AF causing.More than 100 rare variants have been reported in patients with lone AF. All four previously described rare non-synonymous mutations in  density ,19,20. AT hearts . Valdivihe heart . The aboSCN3B or that gain of function can increase vulnerability to AF. The mutant A allele of the c.-324C>A variant (rs976125894) displayed an increased promoter activity and was positively controlled by GATA4. Together, the findings of the current study’s variant in the 5′UTR and the previously reported rare non-synonymous mutations showed that both gain-of-function and loss-of-function variants can provide a proarrhythmogenic substrate and increase the risk of AF. The same circumstances were seen in the SCN5A and SCN1B studies. In 2008, Ellinor and colleagues discovered that the SCN5A N1986K mutation led to a significant loss of sodium channel function and a prolongation of the atrial APD [SCN5A gain-of-function mutations with heightened vulnerability to AF lend credence to this theory. An SCN5A gain-of-function mutation (K1493R) that can increase cellular excitability and reduce the AP threshold has been found in AF patients, according to Li and colleagues [SCN5A, M1851V, has been linked to ventricular arrhythmias and early-onset atrial fibrillation [SCN1B T189M mutation was found in patients with paroxysmal lone AF and when NaV1.5 and the T189M mutant NaVβ1 subunit were co-expressed, electrophysiological analysis revealed that the expressed current density was significantly higher than that seen with the WT NaVβ1 subunit [SCN1B. Additionally, it was discovered that the PITX2c M207V mutation caused familial AF and reporter gene assays showed that M207V increased PITX2c’s transactivation activity by 3.1-fold in comparison to its wild-type counterpart. This gain of function increased the mRNA levels of KCNH2 (2.6-fold), SCN1B (1.9-fold), GJA5 (3.1-fold), GJA1 (2.1-fold) and KCNQ1 in the homozygous form (1.8-fold) in the HL-1-immortalized mouse atrial cell line when the variant was co-expressed with wild-type PITX2c [Our findings may indicate, for the first time, that the variant increases the expression level of rial APD . Althougnt rs9761894 displ subunit . Pilsicae PITX2c . Taken tSCN3B; however, functional evidence about the mechanisms linking to non-coding variants with SCN3B or the incidence of AF is limited [Genetic analysis, such as GWAS and candidate gene-based analysis, has identified a panel of SNPs associated with the risk of AF and most of them were found located within intergenic or intronic genomic regions . Previou limited ,20. Rece limited . TherefoSCN3B caused an increase in transcriptional activity via interacting with transcription factor GATA4. GATA4 expression is seen throughout cardiac morphogenesis and is essential for normal heart development [SCN3B expression levels.Growing data suggest that a number of transcription factors, including GATA4, TBX5 and NKX2.5, play critical roles in cardiogenesis, recently conferring risk to AF or lone AF ,30,31. Ielopment ,33. Patielopment ,35,36. Ielopment . These fSCN3B expression in the myocardium because we were unable to obtain the patients’ human tissue. We examined man eQTL databases, including GTEX and eQTLGen, which can find the correlation between genetic variants and gene expression level in human tissues and obtained no results because of the extremely low frequency of these two variations and the lack of records for these two variants in these databases. Another limitation is that our functional work is preliminary and further investigation, such as functional electrophysiological analysis in induced pluripotent stem cells (iPS) or Crispr-Cas9 base-edited cells, will be required to establish the mechanism by which mutations in the 5′UTR of SCN3B cause atrial fibrillation.The current study has some limitations. First, it is unknown if the variants in the current investigation impact the level of SCN3B in patients with lone AF and discovered that one of them, the A allele of c.-324C>A (rs976125894), can improve transcriptional activity and may raise SCN3B expression levels. The A allele of c.-324C>A (rs976125894) has higher transcriptional activity when it interacts with GATA4, as we confirmed that the transcription factor GATA4 is a regulator of SCN3B. To the best of our knowledge, the current study is the first to demonstrate that the gain-of-function mutation of SCN3B can produce AF and the first to link a mutation occurring in the non-coding 5′UTR region of SCN3B to lone AF. The work also offers empirical proof that GATA4 is a critical regulator of SCN3B gene regulation. Our findings may serve as an encyclopedia for AF susceptibility variants and can also provide insight into the investigation of the functional mechanisms behind AF variants discovered by genetic methods.In conclusion, we identified two rare variants in the 5′UTR and promoter region of


Background: Given the role of the P2X7 receptor (P2X7R) in inflammatory bowel diseases (IBD), we investigated its role in the development and progression of colitis-associated colorectal cancer (CA-CRC). Methods: CA-CRC was induced in P2X7R+/+ and P2X7R−/− mice with azoxymethane (AOM) combined with dextran sodium sulfate (DSS). In a therapeutic protocol, P2X7R+/+ mice were treated with a P2X7R-selective inhibitor (A740003). Mice were evaluated with follow-up video endoscopy with endoluminal ultrasound biomicroscopy. Colon tissue was analyzed for histological changes, densities of immune cells, expression of transcription factors, cytokines, genes, DNA methylation, and microbiome composition of fecal samples by sequencing for 16S rRNA. Results: The P2X7R+/+ mice displayed more ulcers, tumors, and greater wall thickness, than the P2X7R−/− and the P2X7R+/+ mice treated with A740003. The P2X7R+/+ mice showed increased accumulation of immune cells, production of proinflammatory cytokines, activation of intracellular signaling pathways, and upregulation of NLRP3 and NLRP12 genes, stabilized after the P2X7R-blockade. Microbial changes were observed in the P2X7R−/− and P2X7R+/+-induced mice, partially reversed by the A740003 treatment. Conclusions: Regulatory mechanisms activated downstream of the P2X7R in combination with signals from a dysbiotic microbiota result in the activation of intracellular signaling pathways and the inflammasome, amplifying the inflammatory response and promoting CA-CRC development. One of the most feared complications resulting from chronic colonic inflammation in ulcerative colitis (UC) and Crohn’s disease (CD), collectively known as inflammatory bowel disease (IBD), is the development of colitis-associated colorectal cancer (CA-CRC). Although improvements in endoscopic surveillance protocols have allowed the earlier detection of dysplasia and subsequent CA-CRC , the diaWhile the development of sporadic CRC is well established and characterized by histological and genetic changes known as the adenoma-carcinoma sequence, CA-CRC develops through a different sequence. Chronic inflammation of IBD induces mutations and creates a regenerative background resulting in a distinctive selective pressure that may lead to CA-CRC . AlthougExtracellular adenosine triphosphate (ATP) released from tissue injury resulting from infection or cellular stress is regarded as a damage-associated molecular pattern (DAMP) capable of regulating several physiological cell functions through the interaction with the purinoreceptor P2X7 (P2X7R), an extracellular ATP-gated cation channel expressed on both epithelial and immune effector cells . The modGiven the already mentioned information regarding the ATP-P2X7R pathway and our previous findings demonstrating site-specific modulation of P2X7 in intestinal epithelial cells , we hypo+/+ wild-type mice displayed mucosal granularity and friability, and most of them also had ulcers and bleeding. The P2X7R+/+ wild-type mice treated with A740003 did not present the entire range of abnormalities observed in the untreated animals. However, in some cases, the colonic mucosa showed friability, and edema was also corroborated by the ultrasonic images showing focal increases in the wall thickness. Similar changes were observed among the AOM/DSS-induced P2X7R−/− animals, while no change was detected among the P2X7R−/− control animals. After the third cycle of DSS (week 9), the colonic abnormalities were strongly aggravated in the AOM/DSS-treated P2X7R+/+ animals. The colonic mucosa showed advanced inflammatory changes but also the formation of polyps. The ultrasonic images revealed wall thickening of the hyper- and hypoechoic layers, lacking a clear distinction among each other. Some of the induced wild-type mice treated with A740003 showed mucosal edema, vascular changes, and discrete elevations in the colonic mucosa without clear inflammatory abnormalities; however, the well-defined layers under the ultrasonic images were preserved, indicating superficial changes only. Most P2X7R−/− mice showed a normal colon during the whole experiment, but in some cases, mild inflammatory changes could be detected at the end of the protocol; however, there were no ulcers or polyps  in the AOM/DSS-induced P2X7R+/+ mice compared to the control P2X7R+/+ mice. Upon treatment termination at the end of week 8 (Day 56), body weight changed even more in the AOM/DSS-induced P2X7R+/+ mice than in the A740003-treated and control P2X7R+/+ mice . In the AOM/DSS-treated mice, 90% of the lesions were adenomas, and 10% were adenocarcinomas. In contrast, in the A740003-treated animals, no adenocarcinomas were detected, with increases in lesions that were either hyperplastic polyps (50%) or adenomas (50%).As shown in +/+ mice D. The cose group E,F. Furto tumors G. Polypo+/+ mice compared with the control mice and with the AOM/DSS-induced mice treated with A740003. However, no tumor formation and practically no inflammatory response were observed in the AOM/DSS-induced P2X7R−/− mice (−/− mice and the P2X7R+/+ mice treated with the P2X7R antagonist (A740003) develop less inflammation and consequently less hyperplasia and tumors following AOM/DSS induction.The histological assessment confirmed significantly increased inflammation and the development of hyperplastic changes, including tumors, in the AOM/DSS-induced P2X7R−/− mice . These d+/+ mice compared with the control mice and with the AOM/DSS-induced mice treated with A740003. In the epithelial compartment of the colon, the density of mucous-secreting goblet cells was significantly lower in the AOM/DSS-induced P2X7R+/+ mouse samples than in the samples from the normal control group or the AOM/DSS-induced mice treated with A740003. No significant changes were detected between the P2X7−/− groups . The reads were delineated into 493 operational taxonomy units (OTUs). Estimators of alpha diversity were calculated according to the Shannon index. Although no significant difference was detected among the groups, we observed a trend toward a decreased diversity in the AOM/DSS-induced P2X7R+/+ group (−/− (triangles) away from P2X7R+/+ samples (circles). Among the P2X7R+/+ samples, unique structural changes in the fecal microbiota appear to have occurred after P2X7R blockade with A740003, as most samples shifted away from AOM/DSS-induced P2X7R+/+ . However, the P2X7R+/+ controls were distributed unevenly, revealing the heterogeneity within that group, which might have prevented a more comprehensive and accurate analysis. In summary, we found a dissimilarity in the fecal microbiota between the A740003-treated and untreated AOM/DSS-induced P2X7R+/+ mice and between the P2X7R+/+ and P2X7−/− controls (+/+ (53%) and P2X7−/− (44%) groups  and Tenericutes (0–0.4% vs. 0.1–1.4%), whereas Fusobacterium was exclusively identified in the AOM/DSS-induced P2X7R+/+ group  had a higher relative abundance in the AOM/DSS-induced P2X7R+/+ group than in the P2X7+/+ control group. In contrast, compared to the P2X7R+/+ control group, the P2X7R−/− group presented a trend toward a higher relative abundance of Cyanobacteria (0–1.9% vs. 0.3–4.3%) and Spirochaetes (0–0 vs. 0.1–0.4)  after Deblur quality control clean-up from 36 samples (P2X7R/+ group A. Princicontrols B. The Ve) groups C. As exp) groups . At the /+ group D. Furthe0.1–0.4) . The Fir0.1–0.4) .−/− mice did not develop CA-CRC. In particular, we demonstrated that prophylactic treatment with the P2X7R antagonist stabilized mucosal cell turnover and substantially attenuated morphologic and molecular abnormalities underlying the chronic inflammatory process of the colon. Moreover, we detected subtle changes in intestinal microbial components and the methylation patterns of genes related to CRC.In this study, we investigated whether purinergic signaling through P2X7R could play a role in the development of tumors in a murine model of CA-CRC. The results showed that chemical P2X7R blockade markedly abrogated the development of tumors, whereas the P2X7RCurrent data regarding the role of P2X7R in human CRC derive basically from descriptive studies associating the increased expression of P2X7R in tumors with the poorer survival and prognosis of patients ,22,23. PThe effect of P2X7R activation by ATP in CRC in vitro with cancer cell lines and in vivo using xenograft tumor models has been investigated recently. In one of the studies, the investigators concluded that ATP-P2X7R activation promotes the migration and invasion of CRC cells, possibly through the activation of the STAT3 pathway . In anot−/− mice and treating animals with the selective PX7R antagonists A438079 and A740003, the investigators achieved opposing results. More tumors were detected among the P2X7R−/− mice and the animals treated with P2X7R antagonists, including a large number of adenocarcinomas [However, in contrast to our findings, in a previous study using the AOM/DSS model including P2X7Rrcinomas . It is p+/+, but not in the P2X7R−/− mice, and at significantly lower rates in the P2X7R+/+-induced mice treated with the selective blockade of P2X7R. Similar to previous studies reporting the effects of P2X7R activation in the intestine, the nondysplastic areas of the colon of the AOM/DSS-induced P2X7R+/+ animals displayed characteristic chronic inflammatory changes combined with epithelial cell loss [Similar to previous publications using the AOM/DSS model ,33, thisell loss , includiell loss , and accell loss ,37, decrell loss , and uprell loss ,31. A prell loss , which cell loss . In addiell loss ,41.+/+ AOM/DSS-induced mice. NF-kappa B represents a well-established proinflammatory and protumor pathway both in humans and in experimental IBD [+/+ AOM/DSS-induced mice shows upregulation of MAPK14 expression and overexpression of extracellular signal-regulated kinase (ERK). The upregulation of both MAPK14 and ERK expression was blocked in the P2X7R−/− mice and in the mice treated with the P2X7R antagonist. ERK is a downstream protein belonging to the Ras-Raf-MEK-MAPK/ERK signaling pathway and commonly shows upregulated expression in tumorigenesis [Regarding the activation of intracellular signaling pathways, this study investigated several molecules usually involved in tumorigenesis, including CRC development, such as STAT3, p53, Akt1, and Bcl2. Nevertheless, the analysis showed that the levels of NF-kappa B and mitogen-activated protein kinases (MAPK) are upregulated in the colon of the P2X7Rntal IBD ,43 and wntal IBD . In addintal IBD . Concernigenesis ,47. Simiigenesis . Charact+/+ AOM/DSS-induced mice. Interestingly, the cytosolic recognition of microbial components resulting in NLRP3 activation was promoted by pannexin-1 in the presence of ATP [Although the participation of the inflammasome in the pathogenesis of IBD and CRC has been investigated in recent years, its intricate mechanisms have resulted in some contradictory results in both humans and experimental models . Regardie of ATP . Taken te of ATP , in vitre of ATP . Moreovee of ATP . AlthougEnterobacteriaceae, Bacteroidaceae, and Clostridium spp. [Bacteroides and a decreased abundance of Prevotella [Akkermansia in ethanol-fed mice [+/+ mice and between the P2X7R+/+ and P2X7−/− controls. In addition, bacteria belonging to the Mycoplasma and Mucispirillum genera had a higher relative abundance, whereas Fusobacterium was exclusively identified in the AOM/DSS-induced P2X7R+/+ group. Similar to our results, the phylum Tenericutes was positively associated with CA-CRC incidence and tumor burden in the AOM/DSS experimental model [Mycoplasma has also been recently associated with CRC, suggesting a potential role in colonic tumorigenesis [Mucispirillum has also been associated with the development of CA-CRC [−/− samples, suggesting a possible protective role against the development of CA-CRC. However, the lack of statistical significance and the only partial restoration with the chemical antagonist of P2X7R suggest that bacterial changes observed in this study may represent a secondary phenomenon and are not tumorigenic per se in isolation. In fact, the results appear to reinforce the fundamental role of the ATP/P2X7R pathway in the underlying context of tumor development in this model.The mechanism by which DSS induces colitis in mice is still poorly understood; however, studies have identified several factors, including changes in the intestinal microbiota , an effeium spp. . In contevotella . In a prfed mice . This fial model . Among tigenesis . Corrobof CA-CRC . Here, wp53, p16, MLH1, Igfbp3, and APC were less consistent than those of Gja9 and did not allow clear differentiation among the experimental groups. In turn, Gja9 was shown to be relatively hypermethylated in the AOM/DSS-induced P2X7R+/+ mice. Gja9 belongs to the connexin family of proteins that constitute gap junctions, often compromised in cancers, including CRC [APC function [Selective hypermethylation of the promoter regions of tumor suppressor genes with hypomethylation of the tumor genome has been previously reported as a common epigenetic modification in CRC. Moreover, mutations involved in the pathogenesis of CRC are usually regarded as similar to those underlying CA-CRC, but the order in which they arise is rather different . In thisding CRC . Howeverfunction . The relfunction . Taken tAlthough this study presents a successful approach for the development of CA-CRC and its association with the ATP/P2X7R pathway, important limitations should be acknowledged. First, the number of experiments and animals per experiment was relatively small, mainly owing to technical difficulties regarding the care of animals and the long-term follow-up. However, the overall results were broadly consistent and showed several significant differences. Therefore, the successful data generated in this study should serve, at least in part, as a pilot for further investigations. Future studies on the subject should also consider other dosages and other P2X7R antagonists, different animal models, and protocols, including a collection of samples throughout the study, to evaluate the dynamic changes that might occur in the parameters analyzed, particularly in the microbiota. Finally, antibiotics could provide additional relevant information on the exact role of the microbiota in the model and possibly confirm its potential synergism with the ATP/P2X7R pathway for the activation of the inflammasome and the full development of colitis and CA-CRC.The ethics statement regarding animal experiments is provided in the +/+ and C57BL/6 P2X7−/− mice  were maintained under specific pathogen-free conditions on a 14-h/10-h light and dark cycle in a temperature-controlled room (20–25 °C) with a relative humidity of 44–55%.Age-matched male 6-week-old (18–20 g) C57BL/6 P2X7+/+ mice were treated with intraperitoneal injections of A-740003 , a P2X7R-selective antagonist, 1 h prior to the second and third cycles of DSS. The mice were weighed weekly and euthanized between Days 56 and 57 by inhalation of carbon dioxide (CO2). Additional details on the care of animals and study protocols are presented in After an acclimation period of 1 week, mice were randomly assigned to 1 of 5 groups of 3–5 animals each (in 5 experiments). CA-CRC was induced with a single intraperitoneal (i.p.) injection of 12.5 mg/kg azoxymethane (AOM) . After seven days, the animals were given drinking water containing 2.5% dextran sodium sulfate (DSS) salt, 40–50 kDa , for seven days in three cycles, with two intervals of 14 days each (between cycles 1 and 2 and between cycles 2 and 3) with drinking water without DSS. In a therapeutic protocol, P2X7RThe details regarding the colonoscopy assessment are provided in The distal 2 cm of the colon was divided into portions to perform all the experimental procedures. Colon samples were fixed in 40 g/L formaldehyde saline, embedded in paraffin, and 5 μm sections were then stained with H&E, periodic acid of Schiff (PAS), and phosphomolybdic acid picrosirius red dye and examined microscopically by two independent observers. Details regarding the histologic assessment are provided in The details regarding immunohistochemistry and evaluation of apoptosis are provided in the The details of the quantitative analysis of tissue sections are described in Cytokine concentrations in 24 h culture supernatants of colon explants were measured via flow cytometry using the BD™ Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit .Gene expression was assessed by real-time reverse-transcription (RT) polymerase chain reaction (PCR). The procedures are described in the The details regarding the methylation studies are described in Details regarding the sequencing of the variable V3–V4 regions of the 16S rRNA gene are described in The details regarding the statistical analysis are provided in the Taken together, these findings support the participation of the ATP-P2X7R pathway in establishing an inflammatory microenvironment favoring the development of CA-CRC. These regulatory mechanisms activated downstream of P2X7R, in combination with signals from a dysbiotic microbiota, probably contribute to the maintenance and amplification of the inflammatory response resulting from the crosstalk of converging intracellular signaling pathways and the inflammasome. In addition, these mechanisms conveyed by P2X7R activation may be involved in the disruption of immune surveillance against tumor cells. Thus, we speculate that targeting P2X7R in IBD may constitute a potential new approach not only for preventing and treating inflammation but also for preventing and even contributing to the treatment of CA-CRC.


Compared to wild-type hNET, neither of Y467H or Y467F altered Bmax and Kd values of [3H]WIN35,428 binding, whereas Y467H but not Y467F decreased the Bmax of [3H]nisoxetine binding without changes in Kd. Y467H also increased the affinity of nisoxetine for inhibiting [3H]dopamine uptake relative to wild-type hNET. Recombinant Tat1-86 (140 nM) induced a significant reduction of [3H]dopamine uptake in wild-type hNET, which was attenuated in both Y467H and Y467F. Compared to wild-type hNET, neither Y467H or Y467F altered [3H]dopamine efflux in CHO cells expressing WT hNET and mutants, whereas Y467F but not Y467H decreased [3H]MPP+ efflux. These results demonstrate tyrosine467 as a functional recognition residue in the hNET for Tat-induced inhibition of dopamine transport and provide a novel insight into the molecular basis for developing selective compounds that target Tat-NET interactions in the context of HAND.Dysregulation of dopaminergic transmission induced by the HIV-1 transactivator of transcription (Tat) has been implicated as a central factor in the development of HIV-1 associated neurocognitive disorders (HAND). We have demonstrated that the tyrosine470 residue of the human dopamine transporter (hDAT) plays a critical role in Tat-hDAT interaction. Based on the computational modeling predictions, the present study sought to examine the mutational effects of the tyrosine467 residue of the human norepinephrine transporter (hNET), a corresponding residue of the hDAT tyrosine470, on Tat-induced inhibition of reuptake of dopamine through the hNET. Mutations of the hNET tyrosine467 to a histidine (Y467H) or a phenylalanine (Y467F) displayed similar kinetic properties of reuptake of [ Despite the success of combinatorial antiretroviral therapy (cART) in controlling peripheral HIV infection and improving the lives of HIV patients, an estimated 50% of the 38 million HIV-positive patients have neurophysiological dysfunction and HIV-associated neurocognitive disorders (HAND) , 2. MostClinical observations revealed that DA levels are increased in the CSF of therapy naïve HIV patients in asymptomatic infection  and HIV-1-86 transgenic mice DA  and [3H] norepinephrine (specific activity 14.5 Ci/mmol) were purchased from American Radiolabeled Chemicals, Inc . [3H]WIN 35,428 , [3H]Nisoxetine (specific activity 82.0 Ci/mmol), and [3H]MPP+ (82.9 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences . Recombinant HIV-1 transactivator of transcription (rTat1–86) protein was purchased from ImmunoDX . Antibodies recognizing anti-hNET , anti-mouse IgG horseradish peroxidase , and anti-Calnexin  were purchased from Mab Technology , Cell Signaling Technology  and Santa Cruz Biotechnology, Inc. , respectively. PC12 cells (ATCC® CRL-1721.1), Chinese hamster ovarian cells , and RPMI-1640 and F-12K cell medium were purchased from American Type Culture Collection . Fetal bovine serum was purchased from Atlanta Biologicals . Horse serum, penicillin/streptomycin, and trypsin/EDTA were purchased from Fisher Scientific . D-Glucose, L-ascorbic acid, cocaine, nisoxetine, WIN35,428, nomifensine, desipramine, DMSO, bovine serum albumin, pyrocatechol, α-D-glucose, HEPES, isopropanol, sucrose, 2- and Tween 20 were purchased from Sigma-Aldrich .Dopamine ([3H]DA) or [3H]Norepinephrine ([3H]NE) uptake was evaluated in PC12 cells transfected with WT hNET, Y467H, or Y467F using a procedure modified from our previous reports DA uptake) or 10 μM desipramine (for [3H]NE uptake) to determine nonspecific binding. Following the 10 min incubation, cells were incubated for an additional 8 min at room temperature in the presence of one of six concentrations of unlabeled DA or NE  combined with a fixed concentration of [3H]DA  or [3H]NE , respectively. Specific hNET-mediated DA or NE uptake was calculated by subtracting the non-specific uptake (in the presence of nomifensine or desipramine) from the total uptake. The reaction was terminated by the removal of solution from the wells followed quickly by three washes with ice-cold KRH buffer. Cells were then lysed in 500 μL of 1% SDS for one hour and radioactivity was measured using a liquid scintillation counter .The maximal velocity . To assess whether the Y467H or Y467F mutations could attenuate the inhibitory effects of HIV-1 Tat on hNET, we performed [3H]DA uptake in PC12 cells transfected with WT hNET or mutants in the presence or absence of HIV-1 Tat protein. Cells were detached from 10 cm culture dishes with trypsin/EDTA (0.25%/0.1%), resuspended in culture medium, and incubated at room temperature for 10 min. The cell suspensions were pelleted by centrifugation at 400 × g for 5 min at 4°C and then washed once with phosphate-buffered saline followed by additional 5 min centrifugation . The resulting cell pellets were then resuspended in KRH assay buffer. The cell suspensions from WT hNET, Y467H, or Y467F mutants were then preincubated with or without rTat1–86  at room temperature for 20 min followed by additional 8 min incubation with mixed [3H]DA  as reported previously [3H]DA uptake was determined in the presence of 10 μM desipramine. The reaction was terminated by immediate filtration through Whatman GF/B glass filters (presoaked for 2 h with 1 mM pyrocatechol) followed by three washes with ice-cold KRH buffer containing pyrocatechol using a Brandel cell harvester . Radioactivity was determined as described above.To determine whether Y467H or Y467F alter the affinity of NET substrates or inhibitors, the competitive inhibition of DA or NE uptake via hNET was determined in intact PC12 cells transfected with WT hNET or mutants as described above. Cells were preincubated with a series of final concentrations of DA (1 nM– 1 mM), nisoxitine (0.1 nM– 100 μM), or cocaine (1 nM– 100 μM) at room temperature for 10 min followed by an additional 8 min incubation with a fixed concentration of WIN 35,428 and [3H]Nisoxetine represent binding sites on hNET with different kinetic profiles [max and Kd) of [3H]WIN 35,428 or [3H]Nisoxetine in PC12 cells transfected with WT hNET or mutants. Cell suspensions were prepared as described above. Following cell suspension preparation, the final cell pellets were resuspended in 1 mL of assay buffer . For the [3H]WIN 35,428 binding assay, 25 μL of cell suspension was added to tubes containing 200 μL of assay buffer with or without 10 μM desipramine (for nonspecific binding) and preincubated for 5 min at room temperature. Following the preincubation, 25 μL of one of six concentrations of unlabeled WIN35,428  combined with a fixed concentration of [3H]WIN 35,428  was added to each tube  and incubated for an additional 15 min at room temperature. The reaction was terminated by immediate filtration through Whatman GF/B glass filters (presoaked for 2 h with 1 mM pyrocatechol) followed by three washes with ice-cold assay buffer using a Brandel cell harvester  and radioactivity was determined as described above. The [3H]Nisoxetine binding assays were performed identically to the [3H]WIN 35,428 binding assays with the following modifications: (1) The same concentrations of unlabeled and [3H]Nisoxetine  were used instead of [3H]WIN 35,428, and (2) instead of being resuspended in 1 mL assay buffer, for the [3H]Nisoxetine binding assay, the cells were counted after being suspended in 1 mL assay buffer, and then diluted to a concentration of 100,000 cells/ 25 μL.DA  for 20 minutes or [3H]MPP+  for 30 minutes at room temperature. After the incubation period, cells were then washed 2 times with 1 × KRH buffer before collecting fractional release samples. Total uptake of [3H]DA or [3H]MPP+ at zero time point was estimated from four wells per genotype that were lysed immediately with 1% SDS after the incubation period. Nonspecific uptake of [3H]DA or [3H]MPP+ at zero time point was estimated from four wells per genotype that were incubated with the hNET inhibitors desipramine and nomifensine  and lysed immediately with 1% SDS after the incubation period. To collect fractional release, 500 μL of KRH buffer was added into a set of four wells and transferred to scintillation vials after 0 minutes as the initial fractional efflux. Fresh 500 μL of KRH buffer was added into the wells and incubated for 10 minutes then transferred to scintillation vials. Fractional efflux was collected every 10 minutes for 90 minutes ([3H]DA) or 110 minutes ([3H]MPP+). After the last fractional efflux was collected, the cells were lysed and samples collected as leftover [3H]DA or [3H]MPP+ remaining in the cells.To determine whether the mutated Y467 in hNET alters transporter conformational transients, DA or MPPeviously . Intact n represents the number of independent experiments for each experiment group. Kinetic parameters  were determined from saturation curves by nonlinear regression analysis using a one-site model with variable slope. IC50 values for substrate and inhibitors for [3H]DA or [3H]NE uptake were determined from inhibition curves by nonlinear regression analysis using a one-site model with variable slope. For experiments involving comparisons between unpaired samples, unpaired Student’s t tests were used to assess any difference in the kinetic parameters  between WT and hNET mutants; log-transformed values of IC50, Km or Kd were used for the statistical comparisons. One way ANOVA with repeat measurement was used for analyzing the efflux data. Significant differences between samples were analyzed with separate ANOVAs followed by post-hoc tests, as indicated in the Results Section of each experiment. All statistical analyses were performed using IBM SPSS Statistics version 28, and differences were considered significant at p < 0.05.Results are presented as mean ± SEM, and The computationally modeled hNET-Tat binding structure is depicted in 3H]DA or [3H]NE uptake was performed in PC12 cells transfected with WT hNET, Y467A, or Y467F. As shown in max: 57.0 ± 6.3 fmol/min/106 cells, Km: 0.219 ± 0.063 μM), neither Vmax or Km of [3H]DA uptake was altered in Y467H  and Y467F . For [3H]NE uptake, no significant difference in Vmax was observed among WT hNET (52.6 ± 15 fmol/min/106), Y467H (74.4 ± 16 fmol/min/106), and Y467F (86.2 ± 30 fmol/min/106). Compared to WT hNET (0.179 ± 0.018 μM), Km was not significantly altered in Y467H (0.597 ± 0.23 μM), but there was a 2-fold increase in Y467F  = 4.7; p < 0.01, unpaired Student’s t test]). This change suggests that mutating the Y to F mutation decreases the affinity of NE to its specific uptake site on hNET.To determine the functional influence of Tyr467 mutations on hNET function, kinetic analysis of WIN 35,428 or [3H]Nisoxetine binding to WT hNET, Y467H, and Y467F. As shown in 6 cells), the Bmax values of [3H]WIN35,428 binding were not altered in Y467H (0.209 ± 0.034 pmol/106 cells) or Y467F (0.322 ± 0.078 pmol/106 cells). Compared to WT hNET (24.7 ± 3.1 nM), the Kd value was not altered in Y467H (31.0 ± 3.1 nM), but increased in Y467F . For [3H]Nisoxetine binding (6 cells), the Bmax value was decreased in Y467H  but not altered in Y467F (0.273 ± 0.031 pmol/106 cells). Additionally, no changes to the Kd value of [3H]Nisoxetine binding were found between WT hNET (8.86 ± 1.0 nM) and Y467H (9.44 ± 1.3 nM) or Y467F (9.61 ± 1.2 nM). Overall this data suggests that mutating Y467 to either Y467H or Y467F results in modest yet detectable alterations in transporter conformational states, which may affect the binding of substrates to the transporter.We next examined the B binding , compare3H]DA or [3H]NE uptake in WT hNET and mutants (50) values for DA or cocaine inhibiting [3H]DA uptake were not altered in Y467H , or Y467F . Compared to WT hNET (26.7 ± 4.0 nM), the IC50 for nisoxetine inhibiting [3H]DA uptake was significantly reduced in Y467H  but not altered in Y467F (21.1 ± 5.1 nM). Compared to WT hNET , the IC50 values for NE, nisoxetine, and cocaine inhibiting [3H]NE uptake were not altered in Y467H  or Y467F . The selective decrease in the IC50 of [3H]DA uptake, which was only observed in response to nisoxetine in the Y467H mutant, is in accordance with subtle differences between the nisoxetine and cocaine binding sites on hNET, and indicate that this mutation may specifically affect the nisoxetine site.To further determine whether Tyr467 mutations influence selective binding sites of substrates or inhibitors on hNET, we determined the ability of substrate (DA or NE) and NET inhibitors (nisoxetine and cocaine) to inhibit [ mutants . Compare3H]DA uptake in WT hNET and the Tyr467 mutants in the presence or absence of 140 nM rTat1-86. As shown in 1-86 on DA uptake via WT hNET, Y467H, and Y467F are presented as the ratio of [3H]DA uptake for each respective mutant in the presence of rTat1-86 compared to [3H]DA uptake in the absence of Tat. Addition of 140 nM rTat1-86 induced a 26.4% reduction of the specific [3H]DA uptake in WT hNET  relative to the control , whereas Tat did not significantly alter the specific [3H]DA uptake in Y467H [in DPM: Tat (2848 ± 680) vs control (2784 ± 732)] or Y467F [in DPM: Tat (3416 ± 145) vs control (3568 ± 433)]. This finding indicates that the Y467 residue on hNET is crucial to HIV-1 Tat-mediated inhibition of DA uptake, suggesting that preventing HIV-1 Tat’s interaction with this residue may restore physiological DA levels in HIV-1 infected patients.Our computational modeling predicted that mutation of Tyr467 to either Y467H or Y467 would considerably weaken the favorable NET-Tat binding . We vali3H]DA and [3H]MPP+ in WT hNET and its mutants. As shown in 3H]DA, cells were washed and initial fractional efflux samples were immediately collected as 0 time point. While the efflux peak of [3H]DA was observed at 10 min in WT hNET and mutants, there was no difference in the efflux between WT hNET and mutants. With regards to the efflux of [3H]MPP+  = 5.4; p < 0.05), time  = 140.8; p < 0.001), and time × mutation  = 2.0, p < 0.05) for Y467F and Y467H when compared to WT hNET. Post-hoc analysis with a simple comparison for Y467F vs WT hNET revealed significant main effects of mutation  = 20.8, p < 0.05), time  = 110.5, p < 0.001), and time × mutation  = 3.527, p < 0.005) suggesting that overall efflux in Y467F was significantly reduced across 110 min. No difference in the efflux was observed between Y467H and WT hNET. These data support our findings that Y467F alters the conformation of the transporter, and additionally suggest that the Y467F mutation reduces the capability of the transporter to undergo efficient conformational transitions.To further determine the effects of the mutations of Tyr467 residue on hNET on transporter conformational transitions, we examined the fractional efflux levels of [[3H]MPP+ , a two-wmax of DA or NE uptake but attenuate Tat-induced inhibition of DA transport, which provide molecular insight into the modeled binding structure of the hNET-Tat complex.Our previous studies identified key residues in hDAT which are critical for Tat-induced inhibition of DAT and transporter conformational transitions –49, 64. Considering that Tat protein binds to the recognition residues in hDAT and hNET, this study evaluated the mutational effects of Tyr467 on basal hNET function as well as their ability to disrupt hNET-Tat interaction. Although the the Tyr467 residue of hNET is identical to Tyr470 in hDAT, where the mutants Y470H and Y470F decrease and retain DAT-mediated DA uptake, respectively , both Y4We have demonstrated that Tat-induced inhibition of hDAT is mediated by allosteric binding site(s) on hDAT, not by interaction with the DA binding site , 66, 67.max of [3H]Nisoxetine binding without changing the Bmax of [3H]WIN 35,428 binding, and potentiates the affinity of nisoxetine inhibiting [3H]DA uptake. This suggests that mutating Tyr467 to Tyr467His may alter the nisoxetine binding site in hNET, which could be induced by a transporter confirmation change. Our data is also consistent with a previous report showing that mutation of Tyr467 reduced the Bmax of [3H]Nisoxetine and suggested it was due to decreased transporter expression  DA uptake by assessing functional substrate efflux. We found that the DA efflux was not altered in Y467H and Y467F compared to WT hNET. However, the efflux of MPP+ was significantly reduced in Y467F, compared to WT hNET. Considering that the Y467 residue is critical for Tat interaction with hNET, these findings significantly highlight the impact of the Y467F mutant on the allosteric modulatory Tat interaction with hNET. While both DA and MPP+ are a substrate for NET [+ has less diffusive properties than DA in heterologous expression systems [The HIV-1 Tat protein has been shown to interact with DAT allosterically evidenced by altering zinc-regulation of DA uptake and WIN35,428 binding , 67. Sin for NET , 78, MPP systems . The cur systems . Thus, fIn conclusion, we provide novel evidence that inhibition of DA uptake by HIV-1 Tat extends beyond the hDAT and affects DA uptake via the hNET as well. Attenuation of HIV-1 Tat-induced inhibition of hNET reuptake via mutation of hNET Tyr467 supports our computational modeling prediction of the HIV-1 Tat/NET interaction and suggests that this interaction may be similar to our previously reported HIV-1 Tat-hDAT interaction. Future studies will aim to investigate the contribution of multiple residues on hNET which have been predicted to be involved in the HIV-1 Tat-hNET interaction, which may provide molecular insight into the development of allosteric hDAT/hNET modulators that are capable of attenuating the inhibitory effects of HIV-1 Tat on DA reuptake.S1 File(DOCX)Click here for additional data file.


Escherichia phage vB_EcoM-S1P5QW, a T4-like bacteriophage that was isolated from manures collected from cattle farms in Maine. Escherichia phage vB_EcoM-S1P5QW can infect Escherichia coli O26:H11 strains and is devoid of virulence, antibiotic resistance, and lysogeny-associated genes, which may be meaningful for further biocontrol studies.Here, we report a complete genome sequence of  Escherichia coli (STEC) O26:H11 (STEC O26) has contributed to recent outbreaks associated with prepacked sandwiches in the United Kingdom and raw flour in the United States (Escherichia phage vB_EcoM-S1P5QW (S1P5QW), a T4-like bacteriophage that was isolated from manure samples and can infect and lyse STEC O26:H11, as shown by plaque formation and the presence of lysis-protein-encoding genes based on whole-genome analysis. In brief, S1P5QW was isolated by enrichment (overnight at 37°C) of manure samples from an anonymous farm in central Maine with STEC O26:H11 strains in tryptic soy broth with 10% CaCl2 and was purified and concentrated using Amicon filters  and CsCl density gradient centrifugation, respectively  using SPAdes v3.13.0 on the KBase server . The genomic features of S1P5QW are highly similar to those of T4 or T4-like phages. A total of 256 ORFs were found; 127 of those were annotated as encoding functional proteins, including 39 ORFs encoding DNA replication and regulation proteins, DNA helicase, DNA polymerase, clamp, reductase, and DNA primase. PhageTerm indicated that the S1P5QW phage genome was circularly permuted with random termini, but it was unable to determine the packaging mechanism. An inner membrane subunit of spanin shares 91.5% amino acid identity with its counterpart in phage T4 (GenBank accession number NP_049837). Lysis proteins (657-bp holin and 495-bp endolysin) confirmed the lytic activity of S1P5QW against STEC O26:H11. S1P5QW does not possess stx, lysogeny-associated, and antibiotic resistance genes, which is meaningful for STEC O26 biocontrol applications.S1P5QW, with morphology belonging to the analysis , the phaEscherichia phage vB_EcoM-S1P5QW has been deposited in GenBank under the accession number OL956808. The sequencing raw reads have been deposited under the BioSample accession number PRJNA790824. The version of the phage genome described in this paper is the first version.The complete genome sequence of


H3F3A gene occurs frequently in Diffuse Intrinsic Pontine Gliomas (DIPGs), the most aggressive pediatric glioma. As H3K27M mutation is necessary but insufficient to cause DIPGs, it is accompanied by additional mutations in tumors. However, how H3K27M alone increases vulnerability to DIPG tumorigenesis remains unclear.Neurodevelopmental disorders increase brain tumor risk, suggesting that normal brain development may have protective properties. Mutations in epigenetic regulators are common in pediatric brain tumors, highlighting a potentially central role for disrupted epigenetic regulation of normal brain development in tumorigenesis. For example, lysine 27 to methionine mutation (H3K27M) in the Here, we used human embryonic stem cell models with this mutation, in the absence of other DIPG contributory mutations, to investigate how H3K27M alters cellular proliferation and differentiation. We found that H3K27M increased stem cell proliferation and stem cell properties. It interfered with differentiation, promoting anomalous mesodermal and ectodermal gene expression during both multi-lineage and germ layer-specific cell specification, and blocking normal differentiation into neuroectoderm. H3K27M mutant clones exhibited transcriptomic diversity relative to the more homogeneous wildtype population, suggesting reduced fidelity of gene regulation, with aberrant expression of genes involved in stem cell regulation, differentiation, and tumorigenesis. These phenomena were associated with global loss of H3K27me3 and concordant loss of DNA methylation at specific genes in H3K27M-expressing cells.Together, these data suggest that H3K27M mutation disrupts normal differentiation, maintaining a partially differentiated state with elevated clonogenicity during early development. This disrupted response to early developmental cues could promote tissue properties that enable acquisition of additional mutations that cooperate with H3K27M mutation in genesis of DMG/DIPG. Therefore, this work demonstrates for the first time that H3K27M mutation confers vulnerability to gliomagenesis through persistent clonogenicity and aberrant differentiation and defines associated alterations of histone and DNA methylation.The online version contains supplementary material available at 10.1186/s12915-022-01324-0. Pluripotent stem cells differentiate to form every complex tissue structure of multicellular life. The progression of pluripotent stem cells to a differentiated cell state is mediated by epigenetic modification of DNA and histone proteins, including methylation or acetylation of the lysine 27 position on histone H3 (H3K27). H3K27 trimethylation (H3K27me3) modifications of chromatin increase during differentiation, resulting in wide-scale gene repression and heterochromatization , 2. ConcOne critical example of this may be the mechanisms by which lysine 27 to methionine mutation of histone H3 drives the genesis of Diffuse Midline Gliomas (DMG), including Diffuse Intrinsic Pontine Gliomas (DIPGs). DIPGs are the leading cause of brain tumor-associated mortality in the pediatric population , 5. Not While it is known how H3K27M affects the epigenetic state of mutant cells, it is currently unknown how this altered epigenetic state contributes to tumorigenesis. H3K27M mutation is necessary but not sufficient to cause cancer, and several studies have shown links between this histone mutation and changes in expression of genes that regulate neural differentiation , 10. TheHere, we utilize pairs of isogenic human embryonic stem cell lines with or without the H3K27M mutation to investigate mechanisms by which H3K27M mutation drives tumorigenesis. We assessed the effect of H3K27M on multiple stem cell differentiation paradigms, including trilineage, ecto-, endo-, and mesodermal differentiation, and neural differentiation. This revealed some common features of gene dysregulation that occur in cells with H3K27M mutation. We further linked a failure of normal neural specification and differentiation to alterations of histone and DNA methylation. In this work, H3K27M increased stem cell proliferation and stem cell properties and interfered with differentiation, resulting in loss of most H3K27me3 marks and in anomalous onset of expression of developmental genes during multilineage or directed differentiation. These data provide key support for the hypothesis that the H3K27M mutation confers vulnerability to gliomagenesis through persistent clonogenicity and aberrant differentiation.H3F3A gene, resulting in a K to M amino acid substitution at position 27 in the protein (H3K27M). Multiple clones were derived and the A>T mutation was confirmed by Sanger sequencing . The prevalence of H3.1K27M is ~20% in histone H3 mutant DMG cases whereas, H3.3K27M is ~80% , 13–17. We first assessed whether introduction of the H3K27M mutation altered clonogenic cell activity using an extreme limiting dilution assay  in neural induction media for two days, followed by plating on poly-L-ornithine (PLO)/Laminin for 3 days as described Fig. ”. ConsisTo assess whether the H3K27M mutation might also alter the capacity for hESCs to be specified as a range of other cell types, we evaluated the capacity of the hESCs to differentiate into the three germ layers by specification as ectoderm, mesoderm, or endoderm in defined media, as described in the “To further assess how the H3K27M mutation may have altered regulation of gene expression and cell fate acquisition, we allowed undirected cell specification to occur in KnockOut Serum Replacement (KOSR) media, as shown, providing conditions that were generally permissive for differentiation into the three germ layers  to assess how DNA methylation was altered in H3K27M versus WT cells at day 0 and day 5. Relative to WT cells, H3K27M cells predominantly lost DNA methylation and this trend was seen predominantly in day 5 cells Fig. C, D. HowWe examined browser views for marker genes that were dysregulated in the mutant cells in Figs. We further integrated these data to examine enriched GO terms in the subset of genes that exhibited more than one of these trends, including genes that exhibited increased expression in mutant versus WT cells and were associated with H3K27me3 peaks  lines were grown under feeder-free conditions on Matrigel (Corning) in Essential 8™ Flex Medium  and regularly passaged as aggregates with ReLeSR . hESC colonies exhibiting altered morphology indicating initiation of differentiation were manually removed from the culture; increased propensity to differentiate when grown in stem cell media was a particular characteristic of ESC lines carrying the H3K27M mutation. Experiments adhered to protocols approved by the Washington University hESC Research Oversight Committee (protocol #12-002). As this work was designated nonhuman subjects research by Washington University’s Institutional Review Board, informed consent was not required. Cells were regularly checked for Mycoplasma contamination by using the Mycoalert Mycoplasma Detection Kit, Lonza (Cat. # LT07-118).6 H1ESC cells were resuspended in P3 primary buffer and electroporated using a 4D-Nucleofector (Lonza) with 1 μg of H3F3a-K27M-KI-Puromyocin donor plasmid (GeneArt) and 1 μg gRNA SM928.H3F3a.g37 target oligo (5′- atgctggtaggtaagtaagg agg-3′) and 1.2 μg Cas9 expression vector. Nucleofected cells were plated in media containing Rock inhibitor (Y-27632) for 24 h. post nucleofection and then returned to regular E8-Flex media. Following nucleofection, cells were screened with PCR, using primer sets specific to the 5′ and 3′ junctions of the donor oligo and the target gene. After confirmation of donor oligo knock-in in the nucleofected pool, the cells were treated with 0.2 μg/ml of Puromycin for 72 h. Surviving cells were then passaged and treated with 0.5 μg/ml of Puromycin for 72 h and Sanger sequencing was used to confirm the heterozygous knock-in of the K27M mutation in the 5' junction PCR product of the subclones. Karyotyping of the H3F3A-K27M lines was performed by Cell Line Genetics. Five clonal hESC lines with the K27M were utilized for experiments, with all findings documented by 3-6 biological replicate experiments conducted using at least two clonal lines, except for the chromatin immunoprecipitation sequencing (ChIP-seq) analysis, which involved 4 biological replicate experiments performed using one clonal line; the clonal line used for each biological replicate experiment is described in Additional file The H3F3a-K27M-Puro cell line was generated from the H1 human embryonic stem cell (H1 hESC) line (obtained from WiCell) by the Genome Engineering and iPSC Center (GEiC), Washington University in St. Louis. Prior to transfection, the cells were treated with 10 μM Rock (Y-27632) inhibitor for at least 1 h. Approximately 1.5 × 10Histones were extracted from hESCs and their differentiated derivatives by the acid extraction method, using Abcam’s histone extraction protocol for western blot. Cells were washed with room-temperature phosphate-buffered saline (PBS) and lysed in Triton extraction buffer  and protease inhibitors  for 10 min at 4°C. Nuclei were pelleted at 2000 rpm for 10 min at 4°C and washed in TEB before extracting the histones with 0.2 N HCl overnight at 4°C and collecting the supernatant after centrifugation at 2000 rpm for 10 min. Histones were quantified by the colorimetric DC protein assay , according to the manufacturer’s instructions. For Western blotting, 5 μg of protein mixed with NuPAGE LDS sample buffer  and supplemented with 100 mM DTT was separated on a NuPAGE 4-12 % Bis-Tris protein gel  in 1X MES SDS running buffer . Proteins were transferred to nitrocellulose membranes on a trans-blot turbo system and then blocked in Odyssey blocking buffer  for 1 h. at room temperature. The blots were then incubated with primary antibody overnight at 4°C with gentle agitation and following four washes in 0.1 % TBST buffer at room temperature, secondary antibodies Donkey anti-Rabbit IRDye 800CW  and Donkey anti-Mouse IRDye 600CW  diluted in Odyssey blocking buffer were added at room temperature for 1 h. with gentle agitation. Blots were then washed thoroughly with 0.1 % TBST four times and analyzed using the Odyssey Infrared Imaging System (Li-COR). Primary antibodies used for Western blotting were rabbit α-H3K27M  and mouse α-H3 (pan histone) .Acta Neuropathol., 2018 , 1× high-salt buffer , 500 mM NaCl), 2× LiCl buffer , 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris, pH 8.0). Bead pellets were resuspended in 125 μl of elution buffer  and incubated at 65 °C for 10 min. The supernatant was collected in a new tube, and the elution was repeated again. DNA was reverse cross-linked  at 65°C overnight followed by Proteinase K digestion (250 μg Proteinase K) at 37°C for 1 h. The DNA samples were purified using the NucleoSpin gel/PCR purification kit (Takara). DNA sizing was determined using Agilent Bioanalyzer, and concentration was determined with Qubit assay. Library preparation was performed with up to 10 ng of DNA. DNA was blunt ended, had an “A” base added to the 3′ ends, and then had Illumina sequencing adapters ligated to the ends. Size selection was performed with Ampure XP beads (Beckman Coulter) to select for fragments between 150 and 700bp. Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired-end reads extending 150 bases.ChIP-seq data analysis was performed as described previously . BrieflyAdditional file 1: Figure S1. Derivation of hESC lines carrying the H3K27M mutation. (A) CRISPR-Cas9-mediated genome engineering was used for knock-in of a single base mutation (A>T) into one allele of the H3F3A gene in the H1 hESC line, resulting in cells heterozygous for mutant H3F3A, which encodes an H3.3 protein with a K to M amino acid substitution at position 27 (H3K27M). Multiple independent clonal lines carrying this mutation were derived, with sequence analysis of three clonal lines (C1-3) shown. (B) Sanger sequencing was used to confirm that this mutation was present in each clone. (C) Clonal hESC lines with H3K27M mutation were confirmed to have a normal karyotype. (D) Clonal hESC lines were all demonstrated to express H3K27M protein, by western blotting isolated histones with antibodies specific to the mutated variant of H3.3 (H3K27M) or to pan-H3 (as a loading control). (E) Original and uncropped images of the western blot gels presented in panel (D).Additional file 2: Table S1. Biological replicates and clonal lines used. (A) Biological replicate experiments performed, and clones used for each biological replicate experiment are shown. Results obtained for the wild type (WT) H1 hESC line were compared with at least two different clonal H1 derivatives with CRISPR-mediated introduction of the H3K27M mutation, across 3-6 biological replicate experiments, except for ChIP-seq, which involved H3K27M clone 1 versus WT comparisons. (B) ELDA assay: biological replicate experiments performed, and clones used for each biological replicate experiment as well as individual data values are shown. (C) Titer-Glo assay: biological replicate experiments performed, and clones used for each biological replicate experiment as well as individual data values are shown. (D-L) Protein assay: biological replicate experiments performed, and clones used for each biological replicate experiment as well as individual data values are shown. (M-O) Scorecard assay: biological replicate experiments performed, and clones used for each biological replicate experiment as well as individual data values are shown. (P) RT-qPCR assay: biological replicate experiments performed, and clones used for each biological replicate experiment as well as individual data values are shown. (Q) Positive and negative control primer pairs used for DNA methylation analysis.Additional file 3: Figure S2. NeuN and H3K27M immunostaining. Representative immunostaining image for the indicated proteins in RCAS DMG tumor tissue sections are shown. Scale bar=50μm.Additional file 4: Table S2. Transcriptomic analysis of differential gene expression in the K27M (mutant) versus wild type cells. Differentially expressed genes in clusters a-e are shown, with their fold changes and p-value/adjusted p-value. Final sheet shows differential gene expression (log2 fold change) and adjusted p-values for each pairwise sample comparison.Additional file 5: Figure S3. RT-qPCR validation of findings from RNA-seq. Genes from Figure p<0.0001 was determined by two tailed student's t-test. Clones used for each biological replicate experiment and numbers of biological replicate experiments performed are described in Additional file Additional file 6: Table S3. Pathway analysis of differentially expressed genes defined enriched gene ontology (GO) terms. DEGs from clusters a-e were analyzed. (PPTX 41 kb)Additional file 7: Figure S4. Networks of genes with elevated expression in the H3K27M cells, relative to WT, at day 5. Analysis of differentially expressed genes in Figure Additional file 8: Table S4. ChIP-seq analysis demonstrates widespread loss of H3K27me3 in H3K27M mutant cells. (A) Numbers of peaks meeting each criterion, (B) all peaks obtained in either H3K27M mutant or WT cells, and those peaks that were unique (non-intersecting) between the two sample types. (C) Genes associated with H3K27me3 peaks that were unique to the WT sample. (D-F) GO terms associated with genes that were unique to the WT sample. (G) Genes associated with H3K27me3 peaks that were unique to the H3K27M mutant sample. (H-J) GO terms associated with genes that were unique to the mutant sample.Additional file 9: Table S5. Analysis of differential DNA methylation in H3K27M mutant versus WT cells. (A) DNA methylation peaks gained in the H3K27M mutant versus WT samples at day 0. (B-D) GO terms associated with day 0 H3K27M mutant gained peak-associated genes. (E) DNA methylation peaks lost in the mutant vs WT samples at day 0. (F-H) GO terms associated with day 0 mutant lost peak-associated genes. (I) DNA methylation peaks gained in the mutant vs WT samples at day 5. (J-L) GO terms associated with day 5 mutant gained peak-associated genes. (M) DNA methylation peaks lost in the mutant vs WT samples at day 5. (N-P) GO terms associated with day 5 mutant lost peak-associated genes.Additional file 10: Table S6. GO terms associated with genes with increased expression in the mutant versus WT at day 5 and H3K27me3 at day 0. Enriched biological processes, cellular components, molecular functions, and MeSH disease terms are shown with the p-value/adj. p-value (FDR), genes observed versus expected, and term-associated genes.Additional file 11: Table S7. GO terms associated with genes with increased expression in the mutant versus WT at day 5, H3K27me3 at day 0, and loss of DNA methylation in the mutant at day 5. Enriched biological processes, cellular components, molecular functions, and MeSH disease terms are shown with the p-value/adj. p-value (FDR), genes observed versus expected, and term-associated genes.Additional file 12: Table S8. Concordance in gene expression patterns between our human embryonic stem cells with H3K27M mutation and DIPG tumors involving H3K27M mutation. (A) Pathway enrichment analysis for concordant differentially regulated genes in H3K27M versus WT cells at day 5 and DIPG tumor versus normal brain tissue samples. (B) List of concordant differentially regulated genes (139 genes) in H3K27M versus WT cells at day 5 and DIPG tumor versus normal brain tissue samples.


However, the differences in metabolic phenotypes between B6J and B6N mice are not coherent, and genotypic differences in metabolically important tissues have not been well studied. The phenotypic differences between B6J and B6N substrains have often been attributed to the role of the nicotinamide nucleotide transhydrogenase (Nnt) gene, whereby B6J has a spontaneous missense mutation of Nnt. Nevertheless, phenotypic differences between the two cannot be explained by Nnt mutations alone, especially in metabolic traits. Therefore, we aimed to investigate the genetic cause of the phenotypic differences between B6J and B6N mice. Determining consistent genetic differences across multiple tissues involved in metabolic traits such as subcutaneous and visceral white adipose tissues, brown adipose tissue, skeletal muscle, liver, hypothalamus, and hippocampus, may help explain phenotypic differences in metabolism between the two substrains. We report candidate genes along with comparative data on body weight, tissue weight, blood components involved in metabolism, and energy balance of B6J and B6N mice. Insulin degrading enzyme, adenylosuccinate synthase 2, and ectonucleotide triphosphate diphosphohydrolase 4 were highly expressed in B6J mice compared with those in B6N mice, and Nnt, WD repeat and FYVE domain containing 1, and dynein light chain Tctex-type 1 were less expressed in B6J mice compared with those in B6N mice in all seven tissues. Considering the extremely wide use of both substrains and their critical importance in generating transgenic and knock-out models, these findings guide future research across several interrelated fields.C57BL/6J (B6J) and C57BL/6N (B6N) mice are the most frequently used substrains in C57BL/6 (B6) inbred mice, serving as physiological models for  However, the B6N substrain has recently become more common and standardized because it was used as the embryonic stem cell line for large-scale knockout generation and phenotyping projects , generating more than 5,000 targeted mutant mouse lines that are currently available to researchers worldwide . Total carbohydrate consumption = 4.585 × VCO2 − 3.226 × VO2 [mg.min−1]. Fat consumption = 1.695 × VO2 − 1.701 × VCO2 [mg.min−1]. Data were normalized to body weight. Locomotor activity was estimated based on the number of infrared beams broken in both the x- and y-directions using an activity monitoring system combined with a food intake recording system .Oxygen consumption . Quality analysis of RNA samples was performed using TapeStation  and RNA ScreenTape (Agilent Technologies).Minced tissues were homogenized with Sepasol RNAI solution  using a TissueLyser LT instrument  set at 50 strokes/s for 5 min. The homogenate from adipose tissues was centrifuged at 3000 × Libraries were generated with the NEBNext Ultra RNA Library Prep Kit for Illumina . mRNA was enriched from total RNA (250 ng) using magnetic poly-T beads. First- and second-strand cDNAs were synthesized using random hexamer primers, M-MuLV reverse transcriptase, DNA polymerase I, and RNase H, followed by the conversion of overhangs to blunt ends. DNA fragments were ligated with NEBNext adaptors and size-fractionated with the AMPure XP system  before treatment with the USER enzyme (New England Biolabs) and polymerase chain reaction (PCR) amplification with universal and index primers using Phusion high-fidelity DNA polymerase. PCR products were purified using the AMPure XP system, and the quality of the library was assessed using the TapeStation system (Agilent Technologies). Pooled libraries were sequenced on an Illumina HiSeq 2500 platform to obtain 50 bp single-end reads.Reads were mapped to genes in the reference mouse genome (UCSC mm9) and assembled into transcripts, whose abundance was estimated as the expected number of fragments per kilobase per million base pairs sequenced (FPKM) using Cufflinks (v 1.3.0). Bowtie (v 0.12.7) was used to build an index of the reference genome, and TopHat (v 1.4.0) was used to align the reads.p-values, followed by multiple testing corrections using the Benjamini and Hochberg method to acquire the corrected p-value (q). Comparing the significance of differences in gene expression levels between groups was analyzed using Tukey’s post hoc test on normalized FPKM.Data were analyzed using Strand NGS . DESeq was used to compare pairs of sample groups that included five biological replicates for each group. All genes were tested to obtain the corresponding t-test. A comparison of data with two and three factors was performed using an analysis of variance ANOVA.Statistical analyses were performed using GraphPad Prism 8 software. Quantitative two-group data were analyzed using an unpaired two-tailed There were considerable differences in body weight between the B6J and B6N groups, regardless of whether they were fed ND or HF . Regressp<0.0001 for both iWAT and eWAT) less than those of B6N mice, when measured in absolute weight (p = 0.011 for B6J and p = 0.003 for B6N) reduction in the muscle in both B6J and B6N mice (p = 0.002 for B6J and p = 0.013 for B6N) increased liver in both B6J and B6N mice (p = 0.0003 for iWAT and p = 0.025 for BAT) increased iWAT and BAT weights, but only in B6J mice, not in B6N mice (p<0.0001) less in the HF group than in the ND group  of ND-fed B6J mice were significantly  lower than those of B6N mice  in B6J mice than in B6N fed ND  lower in B6J mice than in B6N mice under the ND fed condition (p = 0.010) lower in HF-fed B6J mice than in HF-fed B6N mice (p = 0.003) lower than in mice fed the ND diet, with less reduction in B6N mice , lipidic , and liver enzymes . The only blood components that differed between B6J and B6N mice were insulin and FFA . Insulinondition . FFA wasB6N mice . T-Cho . Values are presented per diet and per light/dark phase . Although the overall differences between B6J and B6N mice were not large, some marked differences were observed such as energy expenditure, with B6J mice tending to expend more energy than B6N mice during the active phase . Similarp<0.05) in the iWAT, eWAT, BAT, muscle, liver, hypothalamus, and hippocampus between B6J and B6N mice. Comparable numbers of DEGs were upregulated and downregulated in each tissue in the ND and HF groups, respectively. The gene names of the DEGs in each tissue are listed in p-values, and corrected p-value (q). Among the DEGs that were highly expressed in B6J mice compared to B6N mice, three genes: insulin degrading enzyme (Ide), adenylosuccinate synthase 2 (Adss2), and ectonucleotide triphosphate diphosphohydrolase 4 (Entpd4) in the ND group and five genes: Ide, Adss2, Entpd4, B-TFIID TATA-box binding protein associated factor 1 (Btaf1), and transmembrane protein 267 (TMEM267) in the HF group overlapped in all seven tissues examined in the present study. Likewise, among the DEGs that showed lower expression in B6J mice compared with in B6N mice, four genes: Nnt, WD repeat and FYVE domain containing 1 (Wdfy1), dynein light chain Tctex-type 1 (Dynlt1), and RAB4A, member RAS oncogene family (Rab4A) in the ND group and three genes: Nnt, Wdfy1, and Dynlt1 in the HF group were identified. Of these overlapping genes, Wdfy1 showed high FC and was significantly  in the adrenal, testes, and heart has shown that there were no common DEGs across tissues when comparing the B6J and B6JBAC mice, despite the presence of far more DEGs in both compared with in B6N mice, suggesting a very modest effect of NNT status on transcriptome regulation and phenotype differences [Nnt mutation alleles are present in either B6J or B6N substrains.Phenotypic differences among B6 substrains have been reported in many studies in a variety of fields, including metabolism , 31, 33,B6J mice –24, inclB6J mice . HoweverB6J mice  in the pB6J mice , as repoB6N mice . Moreoveferences . Given tNnt, there are common genes across tissues regulating metabolic phenotypes that may explain the substrain differences. As a result, six DEGs, including Nnt, overlapped in all seven tissues regardless of dietary conditions  more expressed in B6J mice than in B6N mice, whereas three DEGs: Nnt, Wdfy1, and Dynlt1 were significantly (p<0.05) less expressed in B6J mice compared with B6N mice.We investigated DEGs between B6J and B6N substrains in seven tissues involved in metabolic function: iWAT, eWAT, BAT, muscle, liver, hypothalamus, and hippocampus tissues, assuming that, besides nditions . Three DWdfy1 and Entpd4 have been reported to be differentially expressed in the brain and pancreas between B6J and B6N [Wdfy1 expression in the brain of B6N is speculated to be related to reduced alcohol intake in B6N mice [Wdfy1 expression in the pancreas of B6J mice is associated with the progression of chronic pancreatitis in B6J mice [Entpd4 [Previously,  and B6N , 53; thuB6N mice , whereas [Entpd4 , 53, itsIde locus in B6J mice results in increased Ide expression [Ide expression between B6J and B6N observed in this study. The lists of B6N substrains of protein-inactivating sequence variations  that referenced the B6J mouse genome [Ide, Adss2, Entpd4, Wdfy1, and Dynlt1 genes identified in this study. Therefore, CNVs in B6J and B6N mice should be investigated in addition to indels and SNPs [Ide expression level in B6J mice is consistent with the lower blood insulin concentration  at the pression . The CNVe genome  did not and SNPs . Additiontration . These r disease ; thus, cNUCB2), a precursor of nesfatin involved in appetite regulation, exhibit insulin resistance and high Wdfy1 expression in their visceral macrophages [Wdfy1 may be also involved in insulin signaling. However, since the genetic background of the floxed mice used in the NUCB2 paper [Wdfy1 expression was simply due to comparing B6N, which has high Wdfy1 expression, with B6J, which has low expression. Similarly, the use of mice with a mixed background of B6J and B6N in immunological studies leads to confounding result interpretations. For example, the effect of granzyme A (GZMA) on viral arthritis remains inconclusive because the phenotypes observed in experiments using GZMA knockout mice vary from paper to paper [GZMA knockout mice was found to be a mix of B6J and B6N, indicating that viral arthritis ameliorated in GZMA knockout mice was not the consequence of loss of GZMA expression, but rather of the genetic background of B6N, including Nnt [A previous study has reported that mice lacking nucleobindin 2 Click here for additional data file.S2 TableP-values were calculated using the moderated t-test, followed by multiple testing corrections using the Benjamini-Hochberg method to acquire the corrected p-value (q). Fold change (FC) values were calculated as log2 of B6J relative to B6N.(XLSX)Click here for additional data file.S3 TableP-values were calculated using the moderated t-test, followed by multiple testing corrections using the Benjamini-Hochberg method to acquire the corrected p-value (q). Fold change (FC) values were B6J relative to B6N. ND, normal diet; HF, high-fat diet; iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue; Bat, brown adipose tissue; Muscle, skeletal muscle; Hyt, hypothalamus; Hic, hippocampus.(TIF)Click here for additional data file.S1 FigNnt alleles. DNA was obtained from the tail of B6J and B6N mice. The amplification products were 579 bp and 743 bp for the wild-type and mutant alleles, respectively.Polymerase chain reaction analysis of (TIF)Click here for additional data file.S2 FigFour groups of animals were used. Colored boxes represent mouse substrain and diet groups; open blue box, normal diet (ND)-fed C57BL/6J (B6J); open red box, ND-fed C57BL/6NCrl (B6N); filled blue box, high-fat diet (HF)-fed B6J; filled red box, HF-fed B6N. Body weight was measured at the ages indicated by arrows. Mice were subjected to an indirect calorimetry system at the time points indicated as “metabolic assessment”. All animals were sacrificed and samples were taken at 38 weeks of age.(TIF)Click here for additional data file.S3 Figp<0.0001; F = 38.56; Df = 16). There was a significant effect of strain on iWAT  independent of diet, and significant effect of diet on iWAT , BAT  and liver  independent of strain. Individual means were compared within groups by unpaired Student’s t-test. Asterisks (*) and daggers (†) indicate significant differences between B6J and B6N substrain groups, and ND and HF diet groups, respectively . J, B6J substrain; N, B6N substrain; ND, normal diet; HF, high-fat diet; open circles, ND-fed B6J; filled circles, ND-fed B6N; open squares, HF-fed B6J; filled squares, HF-fed B6N.(A) iWAT, inguinal white adipose tissue; (B) eWAT, epididymal white adipose tissue; (C) BAT, brown adipose tissue; (D) Muscle, skeletal muscle; (E) Liver. Bars are representative of means + SEM (n = 5) for the B6J (blue color) and B6N (red color) groups. Two-way ANOVA indicated a significant interaction between substrain and diet for eWAT ((TIF)Click here for additional data file.