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Arteriosclerosis, Thrombosis, and... Jun 2024CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary...
BACKGROUND
CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of expression in endothelial cells.
METHODS
To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene , we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells.
RESULTS
We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production.
CONCLUSIONS
Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates expression. A better understanding of gene regulation and the role of single-nucleotide polymorphisms in the modulation of expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.
Topics: Humans; Polymorphism, Single Nucleotide; Endothelial Cells; Coronary Artery Disease; Stress, Mechanical; Calcitonin Receptor-Like Protein; Enhancer Elements, Genetic; Heat Shock Transcription Factors; Mechanotransduction, Cellular; Cells, Cultured; Gene Expression Regulation; Protein Binding; Genetic Predisposition to Disease; Binding Sites
PubMed: 38602103
DOI: 10.1161/ATVBAHA.123.318964 -
Frontiers in Immunology 2024Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell...
Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of V11 and V12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.
Topics: Mice; Animals; B-Lymphocyte Subsets; B-Lymphocytes; Immunoglobulin Light Chains; Translocation, Genetic; Immunoglobulin M; Cell Count
PubMed: 38601144
DOI: 10.3389/fimmu.2024.1380641 -
BioDrugs : Clinical Immunotherapeutics,... May 2024Biologic therapy involving anti-tumor necrosis factor-α (anti-TNFα) agents has fundamentally changed the management of patients with immune-mediated inflammatory... (Review)
Review
Biologic therapy involving anti-tumor necrosis factor-α (anti-TNFα) agents has fundamentally changed the management of patients with immune-mediated inflammatory diseases, including rheumatoid arthritis, thus benefiting many patients. Nevertheless, the inability of some patients to achieve low disease activity or clinical remission remains a major concern. To address such concerns, next-generation anti-TNFα agents that differ from the immunoglobulin G-format anti-TNFα agents that have been used to date are being developed using antibody-engineering technology. Their unique design employing novel molecular characteristics affords several advantages, such as early improvement of clinical symptoms, optimization of drug bioavailability, enhancement of tissue penetration, and a reduction in side effects. This holds promise for a new paradigm shift in biologic therapy via the use of next-generation anti-TNFα agents. Ozoralizumab, a next-generation anti-TNFα agent that was recently approved in Japan, comprises a variable region heavy-chain format. It has a completely different structure from conventional therapeutic antibodies, such as a small molecular size, an albumin-binding module, and a unique format that produces an avidity effect. Ozoralizumab exhibited rapid biodistribution into joints, provided attenuation of Fcγ receptor-mediated inflammatory responses, and had a high binding affinity to TNFα in non-clinical studies. In clinical trials, ozoralizumab yielded an early improvement in clinical symptoms, a sustained efficacy for up to 52 weeks, and an acceptable tolerability in patients with rheumatoid arthritis. This review focuses on the results of pre-clinical and clinical trials for ozoralizumab and outlines the progress in next-generation antibody development.
Topics: Humans; Tumor Necrosis Factor-alpha; Arthritis, Rheumatoid; Antibodies, Monoclonal, Humanized; Animals
PubMed: 38584236
DOI: 10.1007/s40259-024-00648-3 -
Nature Communications Apr 2024Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However,...
Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4-3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.
Topics: Humans; Amino Acid Transport Systems; Fusion Regulatory Protein 1, Heavy Chain; Ligands; Molecular Dynamics Simulation
PubMed: 38582862
DOI: 10.1038/s41467-024-47385-3 -
Frontiers in Pharmacology 2024Membrane transporters playing an important role in the passage of drugs, metabolites and nutrients across the membranes of the brain cells have been shown to be involved...
Membrane transporters playing an important role in the passage of drugs, metabolites and nutrients across the membranes of the brain cells have been shown to be involved in pathogenesis of Alzheimer's disease (AD). However, little is known about sex-specific changes in transporter protein expression at the brain in AD. Here, we investigated sex-specific alterations in protein expression of three ATP-binding cassette (ABC) and five solute carriers (SLC) transporters in the prefrontal cortex of a commonly used model of familial AD (FAD), 5xFAD mice. Sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic analysis was applied for absolute quantification of transporter protein expression. We compared the changes in transporter protein expressions in 7-month-old male and female 5xFAD mice versus sex-matched wild-type mice. The study revealed a significant sex-specific increase in protein expression of ABCC1 ( = 0.007) only in male 5xFAD mice as compared to sex-matched wild-type animals. In addition, the increased protein expression of glucose transporter 1 ( = 0.01), 4F2 cell-surface antigen heavy chain ( = 0.01) and long-chain fatty acid transport protein 1 ( = 0.02) were found only in female 5xFAD mice as compared to sex-matched wild-type animals. Finally, protein expression of alanine/serine/cysteine/threonine transporter 1 was upregulated in both male ( = 0.02) and female ( = 0.002) 5xFAD mice. The study provides important information about sex-specific changes in brain cortical transporter expression in 5xFAD mice, which will facilitate drug development of therapeutic strategies for AD targeting these transporters and drug delivery research.
PubMed: 38572427
DOI: 10.3389/fphar.2024.1365051 -
Journal of Veterinary Science Mar 2024Peste des petits ruminants (PPR) is a contagious and fatal disease of sheep and goats. PPR virus (PPRV) infection induces endoplasmic reticulum (ER) stress-mediated...
BACKGROUND
Peste des petits ruminants (PPR) is a contagious and fatal disease of sheep and goats. PPR virus (PPRV) infection induces endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The activation of UPR signaling pathways and their impact on apoptosis and virus replication remains controversial.
OBJECTIVES
To investigate the role of PPRV-induced ER stress and the IRE1-XBP1 and IRE1-JNK pathways and their impact on apoptosis and virus replication.
METHODS
The cell viability and virus replication were assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, immunofluorescence assay, and Western blot. The expression of ER stress biomarker GRP78, IRE1, and its downstream molecules, PPRV-N protein, and apoptosis-related proteins was detected by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. 4-Phenylbutyric acid (4-PBA) and STF-083010 were respectively used to inhibit ER stress and IRE1 signaling pathway.
RESULTS
The expression of GRP78, IRE1α, p-IRE1α, XBP1s, JNK, p-JNK, caspase-3, caspase-9, Bax and PPRV-N were significantly up-regulated in PPRV-infected cells, the expression of Bcl-2 was significantly down-regulated. Due to 4-PBA treatment, the expression of GRP78, p-IRE1α, XBP1s, p-JNK, caspase-3, caspase-9, Bax, and PPRV-N were significantly down-regulated, the expression of Bcl-2 was significantly up-regulated. Moreover, in PPRV-infected cells, the expression of p-IRE1α, p-JNK, Bax, and PPRV-N was significantly decreased, and the expression of Bcl-2 was increased in the presence of STF-083010.
CONCLUSIONS
PPRV infection induces ER stress and IRE1 activation, resulting in apoptosis and enhancement of virus replication through IRE1-XBP1s and IRE1-JNK pathways.
Topics: Sheep; Animals; Peste-des-petits-ruminants virus; Peste-des-Petits-Ruminants; MAP Kinase Signaling System; Caspase 3; Caspase 9; Endoplasmic Reticulum Chaperone BiP; Endoribonucleases; bcl-2-Associated X Protein; Protein Serine-Threonine Kinases; Goats; Apoptosis; Endoplasmic Reticulum Stress; Goat Diseases; Sheep Diseases; Butylamines; Sulfonamides; Thiophenes
PubMed: 38568823
DOI: 10.4142/jvs.23236 -
International Heart Journal 2024Myocarditis, a severe inflammatory disease, is becoming a worldwide public health concern. This study aims to elucidate the effect of Chemokine (C C motif) receptor-like...
Myocarditis, a severe inflammatory disease, is becoming a worldwide public health concern. This study aims to elucidate the effect of Chemokine (C C motif) receptor-like 2 (CCRL2) in experimental autoimmune myocarditis (EAM) occurrence and its potential regulatory mechanisms.EAM was simulated in a mouse model injected with α-myosin-heavy chain. The changes on EAM were assessed through histological staining of heart tissues, including measuring cardiac troponin I (cTnI), proinflammatory cytokines, transferase-mediated dUTP nick end labeling (TUNEL) assay, and cardiac function. Then, the heart tissues from the EAM mouse model and control groups were analyzed through transcriptome sequencing to identify the differential expressed genes (DEGs) and hub genes related to pyroptosis. Downregulation of CCRL2 further verified the function of CCRL2 on EAM and p21-activated kinase 1/NOD-like receptor protein 3 (PAK/NLRP3) signaling pathways in vivo.The EAM model was constructed successfully, with the heart weight/body weight ratio, serum level of cTnI, and concentrations of proinflammatory cytokines elevation. Moreover, cell apoptosis was also significantly increased. Transcriptome sequencing revealed 696 and 120 upregulated and downregulated DEGs, respectively. After functional enrichment, CCRL2 was selected as a potential target. Then, we verified that CCRL2 knockdown improved cardiac function, alleviated EAM occurrence, and reduced PAK/NLRP3 protein expression.CCRL2 may act as a novel potential treatment target in EAM by regulating the PAK1/NLRP3 pathway.
Topics: Animals; Mice; Autoimmune Diseases; Cytokines; Disease Models, Animal; Myocarditis; NLR Family, Pyrin Domain-Containing 3 Protein; NLR Proteins; p21-Activated Kinases
PubMed: 38556341
DOI: 10.1536/ihj.23-527 -
Human Reproduction Open 2024Does ovarian ferroptosis play an active role in the development of polycystic ovary syndrome (PCOS)?
STUDY QUESTION
Does ovarian ferroptosis play an active role in the development of polycystic ovary syndrome (PCOS)?
SUMMARY ANSWER
Increased ovarian ferroptosis was present in PCOS ovaries and the inhibition of ferroptosis with ferrostatin-1 (Fer-1) ameliorated polycystic ovary morphology and anovulation.
WHAT IS KNOWN ALREADY
Programmed cell death plays a fundamental role in ovarian follicle development. However, the types and mechanisms of cell death involved in the ovary are yet to be elucidated. Ferroptosis is a recently discovered iron-dependent programmed cell death. Impaired iron metabolism and cell death have been observed in women with PCOS, the main cause of anovulatory infertility. Additionally, previous studies reported that an abnormal expression of noncoding RNA may promote ferroptosis in immortalized ovarian granulosa cell lines. However, little is known about whether ovarian ferroptosis is increased in PCOS, and there is insufficient direct evidence for a role of ferroptosis in PCOS, and the underlying mechanism. Moreover, the effect of the inhibition of ferroptosis with Fer-1 in PCOS remains unclear.
STUDY DESIGN SIZE DURATION
Ferroptosis was evaluated in human granulosa cells (hGCs) from non-PCOS (n = 6-16) and PCOS (n = 7-18) patients. The experimental study was completed using primary hGCs from women undergoing IVF. Improvements in PCOS indicators following ferroptosis inhibition with Fer-1 were investigated in a dehydroepiandrosterone (DHEA)-induced PCOS rat model (n = 8 per group).
PARTICIPANTS/MATERIALS SETTING METHODS
Ovarian ferroptosis was evaluated in the following ways: by detecting iron concentrations via ELISA and fluorescent probes; measuring malondialdehyde (MDA) concentrations via ELISA; assessing ferroptosis-related protein abundance with western blotting; observing mitochondrial morphology with transmission electron microscopy; and determining cell viability. Primary hGCs were collected from women undergoing IVF. They were treated with dihydrotestosterone (DHT) for 24 h. The effect of DHT on ferroptosis was examined in the presence or absence of small interfering RNA-mediated knockdown of the putative receptor coregulator for signaling molecules. The role of ovarian ferroptosis in PCOS progression was explored in rats. The DHEA-induced PCOS rat model was treated with the ferroptosis inhibitor, Fer-1, and the oocytes and metaphase II oocytes were counted after ovarian stimulation. Additionally, rats were treated with the ferroptosis inducer, RSL3, to further explore the effect of ferroptosis. The concentrations of testosterone, FSH, and LH were assessed.
MAIN RESULTS AND THE ROLE OF CHANCE
Increased ferroptosis was detected in the ovaries of patients with PCOS and in rats with DHEA-induced PCOS. Increased concentrations of Fe (<0.05) and MDA (<0.05), and upregulated nuclear receptor coactivator 4 protein levels, and downregulated ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) proteins were observed in the hGCs in patients with PCOS and ovaries of PCOS rats (<0.05 versus control). DHT was shown to induce ferroptosis via activation of NOCA4-dependent ferritinophagy. The inhibition of ferroptosis with Fer-1 in rats ameliorated a cluster of PCOS traits including impaired glucose tolerance, irregular estrous cycles, reproductive hormone dysfunction, hyperandrogenism, polycystic ovaries, anovulation, and oocyte quality (<0.05). Treating rats with RSL3 resulted in polycystic ovaries and hyperandrogenism (<0.05).
LARGE-SCALE DATA
N/A.
LIMITATIONS REASONS FOR CAUTION
Although ovarian-targeted ferroptosis inhibition may be a more targeted treatment for PCOS, the underlying mechanisms in the cycle between ferroptosis and hyperandrogenism require further exploration. Additionally, since PCOS shows high heterogeneity, it is important to investigate whether ferroptosis increases are present in all patients with PCOS.
WIDER IMPLICATIONS OF THE FINDINGS
Androgen-induced ovarian ferroptosis appears to play a role in the pathogenesis of PCOS, which potentially makes it a promising treatment target in PCOS.
STUDY FUNDING/COMPETING INTERESTS
This study was supported by the National Key R&D Program of China (2023YFC2705500, 2023YFC2705505, 2019YFA0802604), National Natural Science Foundation of China (No. 82130046, 82320108009, 82101708, 82101747, and 82001517), Shanghai leading talent program, Innovative research team of high-level local universities in Shanghai (No. SHSMU-ZLCX20210201, No. SSMU-ZLCX20180401), Shanghai Jiaotong University School of Medicine, Affiliated Renji Hospital Clinical Research Innovation Cultivation Fund Program (RJPY-DZX-003) and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (No. 20161413), Shanghai's Top Priority Research Center Construction Project (2023ZZ02002), and Three-Year Action Plan for Strengthening the Construction of the Public Health System in Shanghai (GWVI-11.1-36). The authors report no competing interests.
PubMed: 38550897
DOI: 10.1093/hropen/hoae013 -
Cancers Mar 2024Chronic lymphocytic leukemia (CLL) exhibits substantial variability in disease course. The mutational status of the B-cell receptor immunoglobulin heavy variable (IGHV)...
Chronic lymphocytic leukemia (CLL) exhibits substantial variability in disease course. The mutational status of the B-cell receptor immunoglobulin heavy variable (IGHV) chain is a critical prognostic factor, categorizing patients into mutated (M-IGHV) and unmutated (U-IGHV) groups. Recently, a third subgroup with borderline mutational status (BL-IGHV) has been identified, comprising approximately 5% of CLL cases. This study retrospectively analyzes the outcomes of 30 BL-IGHV mutated patients among a cohort of 653 CLL patients, focusing on time to first treatment (TTFT) and overall survival (OS). BL-IGHV patients had a short TTFT similar to U-IGHV patients (median 30.2 vs. 34 months; = 0.9). Conversely, the OS of BL-IGHV patients resembled M-IGHV patients (median NR vs. 258 months; = 1). Despite a similar incidence in unfavorable prognostic factors, the TTFT was shorter compared to other published cohorts. However, striking similarities with other experiences suggest that BL-IGHV mutated patients share common biological characteristics, biased IGHV gene usage and BCR subset frequency. These findings also underscore the need for multicentric efforts aggregating data on BL-IGHV CLL in order to elucidate its disease course and optimize therapeutic approaches for this rare subgroup. Until then, predicting outcomes and optimal management of BL-IGHV CLL will remain challenging.
PubMed: 38539430
DOI: 10.3390/cancers16061095 -
Frontiers in Bioscience (Scholar... Feb 2024Hypertrophic cardiomyopathy is the most frequent autosomal dominant disease, yet due to genetic heterogeneity, incomplete penetrance, and phenotype variability, the...
BACKGROUND
Hypertrophic cardiomyopathy is the most frequent autosomal dominant disease, yet due to genetic heterogeneity, incomplete penetrance, and phenotype variability, the prognosis of the disease course in pathogenic variant carriers remains an issue. Identifying common patterns among the effects of different genetic variants is important.
METHODS
We investigated the cause of familial hypertrophic cardiomyopathy (HCM) in a family with two patients suffering from a particularly severe disease. Searching for the genetic variants in HCM genes was performed using different sequencing methods.
RESULTS
A new missense variant, p.Leu714Arg, was identified in exon 19 of the beta-myosin heavy chain gene (). The mutation was found in a region that encodes the 'converter domain' in the globular myosin head. This domain is essential for the conformational change of myosin during ATP cleavage and contraction cycle. Most reports on different mutations in this region describe severe phenotypic consequences. The two patients with the p.Leu714Arg mutation had heart failure early in life and died from HCM complications.
CONCLUSIONS
This case presents a new likely pathogenic variant in and supports the hypothesis that myosin converter mutations constitute a subclass of HCM mutations with a poor prognosis for the patient.
Topics: Humans; Cardiac Myosins; Cardiomyopathy, Hypertrophic; Cardiomyopathy, Hypertrophic, Familial; Mutation; Mutation, Missense; Myosin Heavy Chains; Phenotype
PubMed: 38538344
DOI: 10.31083/j.fbs1601001