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关键词： Non-classical MHC   L lineage   Salmonids   Molecular evolution  

摘要： Non-classical MHC class I molecules, which are encoded by a diverse gene family, have a wide range of functions that can be immune-or non-immune related. From an evolutionary perspective, different non-classical MHC class I genes are typically specific to smaller phylogenetic groups and are often not well conserved among species within these groups rendering non-classical MHC phylogeny difficult to establish. Here we perform a comparative analysis spanning non-classical MHC class I L lineage gene content, organization, phylogeny and variability across divergent salmonid species, providing insight into L lineage immune system variation and species-specific adaptations. Based on evolutionary relationships and sequence analysis, salmonid L lineage genes can be classified into three clades, each with distinct features. Clade I gene sequences exhibit significant variation within species, comprising multiple haplotypes and null alleles, as well as notable variations between species, including highly flexible gene numbers and patterns of pseudogenization. Clade I genes comprise eleven subclades, each displaying separate evolutionary patterns, characteristic phylogenetic marker motifs and subclade specific promoter regions, indicative of unique regulatory patterns and subclade specific functional roles. Our study reveals two distinct phylogenetic patterns among these genes: some subclades display a high degree of flexibility, as demonstrated by species specific expansions and contractions, whereas others have been maintained as mono-genic subfamilies with remarkably few changes in their nucleotide sequence across divergent species. Comparably, clade II L lineage genes consist of four subclades, among which LIA and LLA genes are full length genes with species specific pseudogenization patterns. We report compelling evidence that the LIA gene lineage is highly conserved and present in all 23 Salmonidae species examined as well as in representative Esociformes. This suggests t

关键词： HLA   HLA‐DRB1*13:371   next‐generation sequencing   novel allele  

摘要： The novel allele HLA-DRB1*13:371 differs from HLA-DRB1*13:02:01:01 by one nucleotide substitution in codon 127 in exon 3.

关键词： Molecular dynamics  

摘要： Interleukin-37b (IL-37b) has emerged as a potential therapeutic agent for inflammatory diseases. However, IL-37b tends to self-assemble into a dimeric form, which compromises its anti-inflammatory efficacy. Furthermore, the currently active monomeric variants of IL-37b are unstable, limiting their clinical utility. To address these challenges, we employed a series of in silico protein engineering strategies to design IL-37b variants with reduced dimerization propensity and enhanced monomeric stability. We identified and analyzed hot-spot residues critical for dimer formation using a double-alanine scanning approach, and subsequently mutated these residues in search of optimal mutations to block self-dimerization. Further, we expanded computational site-saturation mutagenesis to non-binding-site residues and evaluated their impact on monomer stability. Molecular dynamics (MD) simulations and structural analyses were performed to interpret the effects of these variants on stability. In total, we identified 36 mutations that block IL-37b dimer formation and 10 mutations that enhance monomer stability. The mutational effects of the two lists of mutations were independent, enabling potential multi-mutant combinations into dual-functional variants. The systematic in silico design approach presented for IL-37b in this work also provides a generalizable framework for the rational design of other cytokines and proteins.

关键词： AT1-AA   Interleukin-6   BK channel   NF-kappa B nuclear translocation  

摘要： The inflammatory phenotype of vascular smooth muscle cells (VSMCs) is an important factor in triggering vascular disease, and interleukin-6 (IL-6) is one of the earliest inflammatory cytokines upregulated in many inflammatory contexts. Angiotensin II-1 receptor autoantibody (AT1-AA) can promote the phenotypic transformation of VSMCs into macrophage-like cells, then synthesize abundant IL-6 to induce vascular inflammation. Previous studies suggested that abnormal BK channel function on the surface of VSMCs played an important role in the synthesis of IL-6, but the mechanism of abnormal BK channel involving in AT1-AA-induced IL-6 synthesis in VSMCs was unclear. In this study, the agonist NS1619 of the BK channel and the inhibitor Paxilline were used to reverse or exacerbate IL-6 synthesis in AT1-AA-induced VSMCs. It is known that NF-kappa B can enter the nucleus due to increased calcium ion concentration caused by BK channel dysfunction, thereby increasing IL-6 transcription. This study observed that Paxilline pretreatment significantly increased the residence time of AT1-AAinduced NF-kappa B in the nucleus, while NS1619 pretreatment showed the opposite trend. JSH-23 inhibiting NF-kappa B nuclear entry reversed the increase in IL-6 expression in VSMCs induced by AT1-AA. This study found that AT1AA enhanced NF-kappa B nuclear translocation by inhibiting BK channel function, which in turn promoted IL-6 transcription in VSMCs, increased IL-6 synthesis and induced vascular inflammation. This study revealed the importance of BK channel dysfunction in the process of AT1-AA increasing IL-6 synthesis and promoting vascular inflammation, and provided a new idea for alleviating vascular inflammatory diseases from the perspective of improving potassium channel function.

关键词： HLA‐DQB1*03   NGS   nonsynonymous mutation   novel allele  

摘要： Two novel HLA-DQB1*03 alleles, HLA-DQB1*03:581 and HLA-DQB1*03:582, have nonsynonymous mutations in exon 2 and exon 3, respectively.

关键词： Human leukocyte antigen   HLA   Donor-specific antibodies   DSA   Magnetic beads   Eplet   Epitope   Transplantation  

摘要： Sera from patients with previous transplants, transfusions, and pregnancies may contain multiple Human Leukocyte Antigen (HLA)-specific antibodies recognizing various functional epitopes or eplets. The identification and verification of eplets in these high Panel Reactive Antibody (PRA) cases are important for understanding HLA antigenicity and potentially improving transplantation outcomes. For a particular high PRA case, we utilized adsorption and elution techniques using cells and HLA-specific magnetic beads to identify and verify eplets. The resulting eluates confirmed seven established eplet patterns while also uncovering two novel binding patterns against A*01:01 and B*53:01. The isolation of the HLA-specific antibodies allows us to perform competition assays against monoclonal antibodies to confirm the epitope specificity. Using available HLA crystal structures, we proposed that the physicochemical properties of residues 163 and 167 are crucial for the binding of the A*01:01-targeted antibody identified in this study, as well as another A*01:01-targeted antibody previously discovered by Lima et al. [1]. Our studies demonstrate that isolating antibodies and analyzing the physicochemical properties of the eplet will lead to a more comprehensive understanding of HLA immunogenicity.

关键词： IFN-beta   MUC5AC overexpression   EGCG   STAT1   ERK  

摘要： The overexpression of mucin 5AC (MUC5AC) induced by interferon-beta (IFN-(3) plays a critical role in airway mucus hypersecretion and contributes to the mortality associated with coronavirus disease 2019 (COVID-19) patients. Epigallocatechin gallate (EGCG), a polyphenol derived from green tea, has demonstrated potential as a therapeutic agent for the treatment of coronavirus-related diseases. This study aimed to investigate the regulatory effects of EGCG on IFN-(3-induced MUC5AC overexpression and to elucidate its underlying molecular mechanisms. We found that IFN-(3-induced MUC5AC overexpression was associated with the activation of the signal transducer and activator of transcription 1 (STAT1) and extracellular signal-regulated kinase (ERK) signaling pathways. The use of STAT1 or ERK inhibitors significantly attenuated IFN-(3-induced MUC5AC overexpression. EGCG treatment markedly inhibited IFN-(3-induced MUC5AC overexpression and suppressed activation of the STAT1 and ERK signaling pathways. Subsequent research revealed that EGCG binds to Janus kinase 1 (JAK1) and inhibits its kinase activity, leading to reduced phosphorylation of STAT1 and ERK. The use of JAK1 inhibitors reproduced the effects observed with EGCG treatment. Structural analysis indicated that the methyl gallate of the EGCG molecule was crucial for its inhibitory activity. In vivo studies demonstrated that EGCG effectively suppressed IFN-(3-induced mucus hypersecretion and MUC5AC overexpression, along with inhibition of the associated signaling pathways. In conclusion, EGCG inhibits IFN-(3-induced MUC5AC over-expression by suppressing JAK1 kinase activity, thereby blocking activation of the STAT1 and ERK signaling pathways.

关键词： Coptisine   Ulcerative colitis   Intestinal mucosal barrier   AhR  

摘要： Background: Coptisine is a bioactive isoquinoline alkaloid derived from Coptis Chinensis, exhibiting significant pharmacological potential. Emerging evidence suggests its therapeutic efficacy in gastrointestinal disorders through modulating inflammation and maintaining gut homeostasis. Purpose: The study aimed to elucidate the molecular mechanisms underlying coptisine mediated restoration of intestinal barrier integrity in ulcerative colitis (UC) and TNF-alpha challenged intestinal organoids. Methods: A murine UC model was established by dextran sulfate sodium (DSS) induction to evaluate coptisine's therapeutic effects on colonic inflammation and mucosal barrier dysfunction. Complementary in vitro investigations were conducted using TNF-alpha-challenged HT-29 cells and intestinal organoids derived from wild-type mice to assess epithelial barrier repair capabilities. Additionally, we utilized an integrated experimental approach incorporating cellular thermal shift assay, luciferase reporter assays to explore the influence of coptisine on AhR activation. Lastly, specific AhR dependency of coptisine on barrier protective effects were further validated through three independent model systems: shAhR-transfected HT-29 cells, AhR KO murine intestinal organoids, and AhR KO mice subjected to DSS challenge. Results: Coptisine administration significantly attenuated colitis severity in DSS-treated mice, evidenced by reduced histopathological scores, decreased colonic inflammation, and enhanced gut barrier integrity through upregulation of tight junction proteins (TJ proteins). In a barrier dysfunction model of TNF-alpha stimulation in HT29 cells and intestinal organoids, coptisine treatment effectively normalized the expression levels of TJ proteins. Mechanistically, coptisine exhibited potent AhR activation through increased nuclear translocation and transcriptional regulation of CYP1A1. Coptisine dose-dependently inhibited reactive oxygen species (ROS) production and NF-kapp

关键词： Sciaenops ocellatus   Vibrio harveyi   Interleukin-34   NF-kappa B   Inflammation  

摘要： Interleukin (IL)-34 belongs to the short-chain helical hematopoietic cytokine family, which binds to colony stimulating factor 1 receptor (CSF-1R) to activate several signaling pathways, and perform essential roles in immune surveillance, inflammatory responses, and cell proliferation differentiation and migration. In this study, the IL-34, CSF-1R1, and CSF-1R2 gene of Sciaenops ocellatus (named SoIL-34, SoCSF-1R1, and SoCSF-1R2) have been identified, and their evolutionary conservation among vertebrates as well as their immune function were analyzed. The bioinformatics analysis revealed that SoIL-34, SoCSF-1R1, and SoCSF-1R2 exhibited a conserved structure, indicating its evolutionary trajectory to be relatively conserved. The expression profile analysis showed that Gram-negative bacterium Vibrio harveyi and lipopolysaccharide significantly up-regulated SoIL-34, SoCSF-1R1, and SoCSF-1R2 expression. Further functional experiments demonstrated overexpression or knockdown of SoIL-34 could significantly modulate the expression of inflammatory cytokines and SoCSF-1R1 and SoCSF-1R2. Particularly, we revealed that SoIL-34 exhibited robust activation of NF-kappa B reporter activity, as well as IL-8 and IL-1 beta reporter activities. These results indicated that SoIL-34 may function as a positive regulator in S. ocellatus which will offer insights into the role of IL-34 and CSF-1R in innate immunity of teleost fish and significantly enhancing our understanding of complex host-pathogen interactions in lower vertebrates.