Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic arrhythmic syndrome caused by mutations in the sarcoplasmic reticulum (SR) calcium (Ca[2+]) release channel RyR2 or its accessory proteins, including calsequestrin-2 (CASQ2). RyR2 dysfunction or leak is the hallmark feature of CPVT and directly triggers arrhythmias. Despite this electrical instability, CPVT hearts are typically anatomically normal and lack pathological remodelling. Therefore, we hypothesized that introducing additional stressors, such as metabolic stress, would worsen cardiac arrhythmias and potentially trigger pathological remodelling. To test this hypothesis, metabolic stress was introduced by crossbreeding a CPVT model of CASQ2[-/-] mice with the Akita mouse, a genetic model of diabetes that also lacks cardiac remodelling. We found that dysregulation of myocyte Ca[2+] handling was exacerbated in Akita-CASQ2[-/-] cells, which exhibited slower Ca[2+] transient decay kinetics and increased arrhythmogenic Ca[2+] waves. Akita-CASQ2[-/-] mice also displayed more severe arrhythmias, diastolic dysfunction, and cardiac hypertrophy, but without evidence of fibrosis, cell death, or worsened metabolic dysfunction. Finally, mitochondrial reactive oxygen species production was increased in Akita-CASQ2[-/-] cardiomyocytes, which may contribute to the more severe pathological phenotype. Overall, our results suggest that metabolic stress can aggravate dysregulated myocyte Ca[2+] handling and promote cardiac dysfunction in CPVT hearts.

The insidious malignant transformation of oral leukoplakia (OLK) into oral squamous cell carcinoma (OSCC) represents a critical clinical bottleneck in head and neck oncology. Grounded in the cancer immunoediting theory of "elimination-equilibrium-escape," this review systematically reconstructs the spatiotemporal continuum of OLK transitioning toward malignant conversion. We comprehensively dissect the localized immunosuppressive network centered on T cell exhaustion, Natural killer cell inactivation, M2 macrophage polarization, antigen-presenting cell defects, and the dual roles of fibroblasts, elucidating the interactive mechanisms that drive mucosal barrier collapse and immune escape. Accordingly, this paper focuses on the translational pathways of leveraging multimodal deep learning algorithms and big data oncology to integrate heterogeneous omics data, thereby constructing chairside precision decision-making systems. This review ultimately aims to offer a new perspective on the clinical management of oral premalignant lesions, rendering the transition from traditional "reactive surveillance" to algorithm-driven "precision cancer interception" clinically feasible.

It has been almost four decades since CD28 was identified as a major player (co-stimulator) of T cell activation and has been recognized as one of the major milestones in the T cell field. However, the exact underlying mechanism involved in the activation of CD28 still remains poorly understood. Most importantly, the depletion of CD28 in mice did not lead to a major defect in T cell activation response, even when combined with the deletion of ICOS, a homolog of CD28. Here, we propose that CD28 is just a survival receptor, which works together with CD45 to maintain T cell survival. It does not participate in the activation of the T-cell activation process by antigen-presenting cells (APCs) in vivo. Furthermore, we propose that CD28 acts like CD4/CD8 to recruit LCK to activate the PI3 kinase pathway.

Vibrio parahaemolyticus is a Gram-negative pathogen widely distributed in marine environments and can cause infections in humans and aquatic animals. Type III secretion system 1 (T3SS1) accessory protein VscW is homologous to YscW from Yersinia, which contributes to the stability of the T3SS outer membrane secretin YscC and facilitates its oligomerization and localization to the outer membrane. However, the specific function of vscW in V. parahaemolyticus remains poorly understood. In this study, a vscW deletion mutant (ΔvscW-P2C) was constructed by homologous recombination. Compared with the wild-type strain (WT-P2C), ΔvscW-P2C exhibited 41.1% and 45.0% reductions in swimming motility and biofilm formation, respectively. N-phenyl-1-naphthylamine (NPN) fluorescence intensity increased by 60.7% in ΔvscW-P2C, indicating enhanced outer membrane permeability. Pathogenicity examination showed that the cumulative mortality of Litopenaeus vannamei decreased from 66.7% to 41.7% after deletion of the vscW gene, representing an approximate 37.5% reduction in mortality. Proteomic analysis identified 243 differentially expressed proteins, mainly involving T3SS-associated proteins (VscC and VopB), T6SS-associated proteins (Hcp, VgrG, VasG, ImpC, and ImpL), c-di-GMP-associated proteins (GGDEF and EAL-domain-containing proteins), biofilm-associated proteins (VpsT-like, GlnA, and GST), motility- and chemotaxis-associated proteins (FliC and MCP), and putative virulence-associated proteins (PAAR-like-AHH, TcpQ, and VesC). Together, these phenotypic and proteomic findings suggest that vscW deletion is associated with alterations in T3SS1 and other virulence-related functional networks. Collectively, our findings support a contribution of VscW to virulence-associated phenotypes and pathogenicity in V. parahaemolyticus and provide a basis for further investigation of the underlying regulatory mechanisms.

In the present study, bioactive lignans from Zanthoxylum armatum fruits have been investigated for their anti-inflammatory properties and evaluated using dendritic cell-based immunological assays and network pharmacology. The ethyl acetate fraction was subjected to bioassay-guided fractionation, chromatography, and HPLC to isolate pure compounds, characterised by NMR, LC-QTOF-MS, and single-crystal x-ray diffraction. Biological activities were evaluated using DPPH/ABTS radical scavenging assays, albumin denaturation and haemolysis inhibition assays, and flow cytometric analysis of IL-12p40/70 and CD80 expression in CpG-stimulated MuTuDC1940 dendritic cells. Sesamin exhibited the strongest antioxidant activity, while sesamin and fargesin showed notable anti-inflammatory effects. In CpG-stimulated MuTuDC1940 cells, sesamin reduced the proportion of IL-12-positive cells from 63.85% in the CpG-treated group to 52.67%, whereas fargesin (63.23%) showed little change and eudesmin (65.88%) showed a slight increase. Nevertheless, intracellular IL-12 MFI was significantly reduced by eudesmin, sesamin, and fargesin compared with CpG stimulation (p < 0.01), suggesting modulation of IL-12 expression intensity rather than a uniform reduction in the IL-12-positive cell population. Network pharmacology, PPI, GO, and KEGG analyses indicated involvement of PI3K-Akt, NF-κB, JAK-STAT, toll-like receptor, Rap1, and VEGF signalling pathways. These findings suggest these compounds may serve as potential natural therapeutics for inflammatory and immune-mediated diseases.

Endosomal Sorting Complexes Required for Transport (ESCRT) machinery drives membrane remodeling and scission events essential for multivesicular body (MVB) biogenesis. The accessory ESCRT-III subunit Vps60 (mammalian CHMP5) shares structural features with the major polymerizing subunit Snf7, including the capacity to form homopolymers in vitro. Here, we demonstrate in Saccharomyces cerevisiae that Vps60 recruitment and polymerization require conditions that permit Snf7 polymer turnover: Vps60 is largely excluded when Snf7 assembly is absent and when Snf7 disassembly is blocked, consistent with recruitment of Vps60 late in the ESCRT-III cycle to Snf7 assemblies that undergo normal turnover. Structure-function analysis reveals that Vps60 regulation diverges fundamentally from core ESCRT-III subunits: while occlusion of the Vps60 C terminus by a GFP tag drives constitutive membrane association and polymer assembly, C-terminal truncation impairs endosomal recruitment, indicating that the Vps60 C terminus is required for membrane recruitment, in contrast to the autoinhibitory C-terminal domains of core ESCRT-III subunits. A C-terminally tagged Vps60 that constitutively associates with membranes phenocopies loss of Vps60 function in both intralumenal vesicle (ILV) formation and cargo sorting. These findings indicate that despite structural similarity to Snf7, Vps60 functions as a late-arriving accessory factor whose recruitment and assembly are tightly coupled to Snf7 dynamics. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

After encountering allogeneic nonself, monocytes differentiate to DCs, which in turn activate the adaptive immune system that drives transplant rejection. The downstream mechanisms of allorecognition are largely unknown. We analyzed scRNA-seq data sets to identify transcriptional changes in monocytes occurring after allostimulation. Hspa1a, which encodes HSP70, was upregulated in monocytes after allostimulation in contrast to syngeneic controls in mice lacking T, B, and NK cells. Similar findings were seen in scRNA-seq data derived from kidney biopsies of rejecting transplant patients. To validate the role of HSP70 in innate allorecognition and transplantation, we performed allogeneic bone marrow plug and kidney transplantation into WT, Hspa1a/Hspa1b-/-, and DSG-treated (HSP70 inhibitor) B6 mice and examined the graft immune infiltrate. Histology, T cell infiltration, and survival were assessed in kidney transplanted mice. In both models, DSG-treated or HSP70-KO recipients displayed significantly reduced infiltration by monocyte-derived DCs (mo-DC). Chronic rejection of Balb/c kidney grafts in WT B6 recipients was attenuated in DSG-treated and HSP70-KO recipients as indicated by a reduction in Banff score. Further experiments demonstrated that in vitro allostimulated immature HSP70-KO BMDC showed less maturation compared with WT BMDC. Targeting HSP70 in innate immune cells offers a novel approach to reduce chronic kidney graft rejection.

Juvenile idiopathic arthritis (JIA) is a common inflammatory rheumatic disease of childhood, yet the mechanisms driving immune activation in affected joints remain unclear. In this study, we analyzed synovial fluid (SF) immune cells from patients with JIA using two complementary methodologies, single-cell multiomics and flow cytometry. SF displayed a markedly altered immune landscape compared with blood. Both myeloid and plasmacytoid dendritic cells were enriched in SF, and SF monocytes exhibited signs of macrophage differentiation. B cells were strikingly rare in SF, and the few B cells present were predominantly CD21[lo] age-associated B cells. In contrast, CD56[hi]CD16[-] NK cells and CD161[-] γδ T cells were strongly enriched in SF. Among SF CD4[+] T cells, regulatory T cells, T peripheral helper cells, and CD161[+] T cells were most abundant, while CD8[+] T cells exhibited a tissue-resident memory phenotype with a broad expression of different granzymes. Immune cell frequencies in SF correlated poorly with matched blood samples, and no clear association between SF immune cell composition and JIA subtype or selected clinical parameters was detected. This comprehensive characterization of the SF immune environment provides a foundation for future studies aimed at defining the roles of specific immune cell subsets in JIA pathogenesis.

Mast cells (MCs) are increasingly recognized as modulators of intestinal homeostasis and inflammation; however, their precise contribution during colitis remains incompletely defined. Here, we investigated the role of MCs in dextran sulfate sodium (DSS)-induced colitis using a Kit-independent MC-deficient model (Cpa3-Cre; Mcl1[fl/fl]), in which selective deletion of the anti-apoptotic gene Mcl1 ablates MCs. Despite efficient and tissue-wide MC depletion, MC-deficient mice exhibited macroscopic and histological disease severity comparable to control mice. However, MC deficiency significantly altered epithelial and immune responses during acute intestinal injury. Specifically, MC-deficient mice displayed increased colonic MUC2 expression, together with decreased tight-junction integrity. The impaired epithelial barrier was associated with increased bacterial infiltration and selective accumulation of myeloid cells, including neutrophils, dendritic cells, and macrophages, alongside increased TNF-α and reduced VEGF levels. Systemically, in DSS-treated mice, MC deficiency was associated with splenomegaly and increased splenic neutrophils and dendritic cells. Importantly, adoptive transfer of bone marrow-derived MCs restored the epithelial barrier-associated features and reduced neutrophil accumulation. Collectively, these findings identify MCs as key regulators of epithelial-myeloid crosstalk contributing to the maintenance of barrier integrity and limiting excessive myeloid accumulation and bacterial exposure rather than acting as primary drivers of tissue injury.

The endolysosomal degradation assay mimics endolysosomal processing in antigen-presenting cells in vitro and can be used to evaluate protein immunogenicity and T cell epitopes. As previous endolysosomal degradation results demonstrated significant variability, we developed an optimized protocol to ensure the reproducibility and reliability of the assay.

Cancer immunotherapy, particularly immune checkpoint blockade, has substantially changed the treatment landscape for multiple malignancies, but clinical benefit remains limited by low response rates, primary tolerance, and acquired resistance. Tumor-associated macrophages (TAMs) are abundant and highly plastic myeloid cells in the tumor immune microenvironment. They contribute to tumor progression and immunotherapy resistance by suppressing T-cell recruitment and effector function, promoting angiogenesis, remodeling extracellular matrix, regulating metabolism, and expressing immune-checkpoint-related molecules. TAMs exhibit overlapping functional states beyond a fixed M1/M2 dichotomy, shaped by cytokines, chemokines, pattern-recognition receptors, kinase-transcription factor networks, hypoxia, lactate, and redox signals. Reprogramming immunosuppressive TAMs toward inflammatory, antigen-presenting, and T-cell-supporting phenotypes has therefore become an important pharmacological strategy to enhance antitumor immunotherapy. Smart nanodelivery systems can improve the in vivo stability, tumor accumulation, TAM exposure, and spatiotemporal release of immunomodulators, small-molecule inhibitors, nucleic acid therapeutics, cytokines, and macrophage immune-checkpoint modulators, thereby improving efficacy while reducing systemic toxicity. This review summarizes the rationale for targeting TAMs in cancer immunotherapy, the mechanisms regulating TAM functional states, the design principles of smart nanodelivery systems, nanomedicine-mediated reprogramming strategies, pharmacodynamic outcomes, pharmacokinetic and safety issues, preclinical evaluation methods, translational barriers, and future directions.

CD28 costimulation contributes to pathogenic T cell responses in inflammatory bowel disease (IBD), but current B7-directed blockade also limits CTLA-4 signaling. Using a sensitive NanoBiT split-luciferase screening platform, we identified and optimized CA-23, a small molecule antagonist that directly binds human and mouse CD28 without measurable binding to CD80, CD86, or CTLA-4. CA-23 inhibited CD28-B7 engagement and CD28-dependent T cell activation without agonist activity in human whole blood and peripheral blood mononuclear cells. CA-23 achieved exposure in the colon and mesenteric lymph nodes and reduced disease severity, histologic injury, and pathogenic Th1 and Th17 responses in a T cell transfer model of colitis. In PBMCs from donors with ulcerative colitis or Crohn's disease, CA-23 suppressed inflammatory cytokine production and T cell activation to a degree matching or exceeding Abatacept. In human intestinal epithelial-PBMC co-cultures, CA-23 preserved Treg suppressive activity and epithelial barrier integrity, whereas Abatacept reduced Treg function. CA-23 did not alter CD80 or CD86 expression on autologous antigen-presenting cells and showed no substantial off-target activity in the tested selectivity panel. These findings support direct CD28 antagonism as a mechanistically differentiated alternative to B7-directed co-stimulation blockade for suppressing pathogenic T cell responses in preclinical models of IBD.

The HIV-1 accessory protein Nef plays a central role in viral pathogenesis by enhancing viral replication, modulating cellular signaling, and evading immune recognition, making it a compelling target for therapeutic intervention. Nef lacks enzymatic activity and instead functions through diverse interactions with host cell proteins including the Src-family tyrosine kinase, Hck. Kinase activation may requires Nef homodimerization, as mutations disrupting the Nef dimer interface impair kinase activation as well as many other Nef functions. In the present study, we investigated the structural consequences of dimer interface mutations and their impact on Nef interactions with Hck regulatory domains. Using size-exclusion chromatography, multi-angle light scattering and crystallography, we found that mutations at dimer interface residues Leu112 and Phe121 abolish recombinant Nef protein dimerization while preserving the overall Nef fold, resulting in monomeric 1:1 complexes with Hck SH3 or SH3-SH2 domain proteins. These findings demonstrate that the broad phenotypic effects of interface mutations arise from loss of Nef dimerization rather than global misfolding or perturbation of SH3 binding. We also investigated the effects of small molecule Nef inhibitors on homodimer formation. These compounds, like the dimerization-defective mutations, suppress kinase activation, viral replication and restore immune recognition of HIV-infected cells. Using a SplitFAST fluorescence complementation assay, we provide direct evidence that these inhibitors disrupt Nef homodimer formation in solution. Co-crystallization of a wild-type Nef:SH3 complex with an inhibitor also prevented homodimer formation. Computational docking identified a shared pocket for six active Nef inhibitors formed by the Nef dimer interface but lost in the monomer. Together, our findings support homodimerization as a structural feature essential for many Nef functions and validate disruption of this interface as a promising therapeutic strategy against HIV-1.

DNA double-strand breaks (DSBs) are primarily repaired by non-homologous end joining (NHEJ), which tethers and ligates DNA ends within a synaptic complex. PAXX is an NHEJ accessory factor related to XRCC4 and XLF, but its mechanistic role and genetic interaction with XLF are unclear. Using biochemical assays and single-molecule imaging within Xenopus laevis egg extracts and human cell assays, we show that PAXX bridges DNA ends by using its disordered tails to bind opposing Ku molecules. This PAXX tether substantially extends long-range synaptic complex (LRSC) lifetime and modestly stabilizes the short-range synaptic complex (SRSC). LRSC stabilization increases XLF residence and promotes the LRSC-to-SRSC transition. Using domain-swapped chimeras, we demonstrate that PAXX's tails provide synaptic stabilization, whereas XLF's heads interact with XRCC4 to drive the LRSC-to-SRSC transition: an XLF head-PAXX tail chimera recapitulates the functions of both proteins, revealing complementary, sequential roles for PAXX and XLF in NHEJ.