�����ƽ��

As a heterogeneous autoimmune disorder, systemic lupus erythematosus (SLE) involves poorly characterized etiological mechanisms and disease pathways. While glycosylation’s impact on immune homeostasis and disease pathogenesis has become a focal point in contemporary research, delineating the specific contribution of associated genes to SLE requires expanded investigation. This study uses bioinformatics methods to explore the potential diagnostic value of glycosylation-related differentially expressed genes (GRDEGs) in SLE and verify their differential expression in peripheral blood between patients and healthy individuals through RT-qPCR. Data were obtained from several GEO datasets, specifically GSE50772, GSE81622, and GSE20864. Through expression differential analysis, a total of 26 GRDEGs were detected. The diagnostic utility of these genes was assessed using receiver operating characteristic (ROC) curves, while enrichment evaluations for Gene Ontology and KEGG pathways were conducted to elucidate their functional properties. Additionally, a predictive model for SLE that integrated 14 GRDEGs was developed using logistic regression, LASSO regression, and support vector machines (SVM), and its performance was evaluated on both the training dataset and an external validation cohort. Finally, RT-qPCR was utilized to verify the expression levels of RNASE2, PTGDS, CXCL2, TNFRSF21, and LAMP3 in peripheral blood mononuclear cells collected from SLE subjects and normal donors. Employing differential expression profiling, 26 GRDEGs were identified. Subsequently, their capacity for distinguishing SLE from controls was demonstrated using ROC curves, with discriminative power reflected in AUC scores between 0.7 and 0.9. Functional assessments of these GRDEGs using Gene Ontology and KEGG pathway annotation chiefly indicated association with immune functions, modulation of phagocytic processes, and inflammatory cascades, including but not limited to interleukin-17 and tumor necrosis factor signal transduction pathways. The SLE diagnostic model, constructed using logistic regression, SVM, and LASSO regression, incorporates 14 GRDEGs and demonstrates high robustness in both the training dataset and external validation. Furthermore, RT-qPCR results showed significant expression differences of RNASE2, PTGDS, CXCL2, TNFRSF21, and LAMP3 in SLE patients compared to healthy individual. This study identifies glycosylation-related gene candidates and establishes a preliminary diagnostic model for SLE, utilizing integrated bioinformatics analysis and experimental validation. These findings provide foundational insights for further exploration of SLE’s molecular mechanisms and diagnostic advancement.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-24401-0.

BackgroundSterol-O acyltransferase 1 (SOAT1) functions by converting cholesterol and acyl-CoA into cholesterol ester and CoA-SH. SOAT1 inhibition suppresses tumor growth. A recent study has shown that inhibiting cholesterol esterification in T cells using genetic manipulation or drug treatment of SOAT1 boosted the cytotoxicity of CD8+ T cells. To better understand the role of SOAT1 in the tumor immune microenvironment of ovarian cancer (OC) and to optimize combined immunotherapy strategies, we examined the effect of SOAT1 manipulation and drug inhibition in OC cells on CD8+ T cell-mediated immune response in vitro.ResultsCorrelation analysis results obtained using GEPIA2 showed that SOAT1 expression was positively correlated with cytotoxic CD8+ T cell (CTL) infiltration levels and effector CD8+ T cell signature in OC. Additionally, the survival plot from the GSE26712 dataset indicated that CTLs provided clinical benefit in OC patients with high SOAT1 expression but not in those with low expression. The study findings also revealed that SOAT1 knockdown or avasimibe (SOAT1 inhibitor) treatment in OC cells resulted in the downregulation of IFN-γ secretion by CD8+ T cells in vitro. Interestingly, IL-6 and IL-8, two immunosuppressive cytokines known to promote CD8+ T cell dysfunction, were upregulated in SOAT1-silenced and avasimibe-treated OC cells.ConclusionIn conclusion, the present study suggested that SOAT1 inhibition in OC cells could impair the cytotoxic capability of CD8+ T cells in vitro, probably through the increased secretion of IL-6 and IL-8 in OC cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13048-025-01832-x.

Alterations in gut microbiota composition have been implicated in various diseases, including cancer. Recent evidence suggests that intestinal microbiota may influence the efficacy of immunotherapy. In this study, we investigated the relationship between gut dysbiosis and NK cell exhaustion in Mexican patients with cervical cancer (CC), a connection not previously explored. This cross-sectional study included newly diagnosed CC patients, a separate cohort of post-radio-chemotherapy (RCT) patients, and healthy donors (HD). Fecal microbiota profiles were assessed using 16S rRNA sequencing, while peripheral NK cell immune checkpoint expression was analyzed by multiparametric flow cytometry. CC patients exhibited significant gut dysbiosis, marked by reduced α-diversity, enrichment of pro-inflammatory taxa (Escherichia-Shigella, Prevotella), depletion of short-chain fatty acid (SCFA)-producing bacteria (Ruminococcus, Christensenellaceae), and enrichment of microbial metabolic pathways related to inflammation, oxidative stress, nutrient limitation, and immune suppression. Dysbiosis was more pronounced in patients after RCT, with further enrichment of Phascolarctobacterium. In parallel, NK cells displayed a putative exhausted phenotype, with elevated expression and co-expression of PD-1, LAG-3, TIM-3, TIGIT, BTLA, and NKG2A. A dysbiosis score and an NK exhaustion score were developed, revealing a significant positive correlation between microbial imbalance and NK cell exhaustion. Machine learning analysis identified the Escherichia/Ruminococcus ratio and PD-1+CD56bright NK cells as predictive markers of CC. Moreover, both dysbiosis and NK cell exhaustion markers were significantly associated with reduced patient survival. This is the first study to demonstrate a link between gut microbiota alterations and NK cell exhaustion in CC. Our findings suggest that gut dysbiosis may contribute to impaired anti-tumor immunity. This study supports the rationale for microbiota-targeted interventions as adjunctive strategies in CC, although prospective validation is required.

A challenge in gene editing for hematopoietic stem and progenitor cells (HSPCs) is achieving efficient editing while preserving long-term engraftment and clonal diversity. Tracking edited clones with high resolution is essential to understand the impact of editing on hematopoiesis. We developed a barcoded AAV6 donor template (BC-AAV) to precisely monitor the fate of edited HSPCs following transplantation. Our findings reveal that, despite initial barcode diversity in vitro, human hematopoiesis generated by edited HSPCs transplanted in immunodeficient mice is driven by a limited number of dominant clones. The engraftment of gene-edited cells follows an oligo/polyclonal pattern, indicating that editing does not alter clonal dynamics in this model. Using BC-AAV, we optimized a gene editing protocol for correcting the PKLR gene, responsible for pyruvate kinase deficiency, a rare disorder that causes severe anemia due to red blood energy imbalance. We implemented key improvements. GMP-grade StemSpan AOF medium and StemRegenin-1 increased clonal diversity while maintaining hematopoietic potential. NHEJ inhibitor AZD-7648, significantly boosted editing efficiency in vitro, and a shorter transduction period enhanced engraftment and clonal balance without compromising editing outcomes. This refined strategy for gene editing in human HSPCs optimizes both efficiency and long-term polyclonal dynamics and has important implications for clinical applications. Graphical abstract Using a barcoded AAV clonal tracking system, the authors show that short post-editing culture, SR1 supplementation, optimized cell concentration, and culture media significantly influence clonal diversity in gene-edited human HSPCs. These findings help refine protocols to improve the safety and long-term efficacy of gene therapy approaches.

SummaryLong intergenic noncoding RNAs (lincRNAs) regulate biological processes in health and disease. Recent findings highlight the importance of lincRNAs in regulating T cell development and function. Here, we identified a lincRNA, LINC01871, which is highly induced upon CD4+ T cell activation and is predominantly located in the cytoplasm. The anti-inflammatory cytokine TGF-β was found to suppress its expression. Silencing LINC01871 led to a modest decrease in IL-2 secretion. Notably, LINC01871 expression was highly specific to NK cells and T cells in several cross-tissue single cell RNA-seq atlases. These data suggest that LINC01871 is specifically expressed in T and NK cells and may contribute to T cell-mediated immunity in humans. Graphical abstract Highlights•LINC01871 is a cytoplasmic lincRNA induced upon CD4+ T cell activation•TGF-β negatively regulates LINC01871 expression•Expression of LINC01871 is restricted to effector and memory T and NK cells Immunology; Molecular biology; Cell biology

Background/Objectives: β-Carotene oxygenase-2 (BCO2) is a mitochondrial carotenoid-cleaving enzyme expressed in multiple tissues, including the lungs. While BCO2 regulates carotenoid handling, its role in shaping pulmonary immune architecture and antiviral responses is unknown. We hypothesized that BCO2 deficiency reprograms epithelial–innate circuits and alters antiviral outcomes. Methods: BCO2-knockout (KO) and C57BL/6J wild-type (WT) mice underwent lung single-cell RNA sequencing (scRNA-seq), immunoblotting, and intranasal SARS-CoV-2 challenge to assess cell-type heterogeneity, pathway programs (by gene set variation analysis, GSVA), and antiviral responses. Results: scRNA-seq resolved 14 major lung cell populations with cell-type-specific pathway shifts. Compared with WT, BCO2 KO lungs showed increased conventional dendritic cells and natural killer (NK) cells, with reductions in macrophages, B cells, and endothelial cells. In KO alveolar type II cells, GSVA indicated a stress-adapted metabolic program. Ciliated epithelium exhibited vitamin-K-responsive and axoneme-remodeling signatures with attenuated glucocorticoid and very-low-density lipoprotein remodeling. Innate lymphoid type 2 cells favored fatty acid oxidation and chromatin dynamics with reduced mitochondrial activity. NK cells were biased toward constitutive chemokine/cytokine secretion and counter-inflammatory signaling. Immunoblotting confirmed the elevated level of interferon regulatory factor-3 protein in BCO2-KO lungs. Functionally, BCO2-KO mice had improved outcomes after intranasal SARS-CoV-2 exposure. Conclusions: Loss of BCO2 reconfigures the pulmonary immune landscape and enhances antiviral responsiveness in mice. These findings identify BCO2 as a nutrient-linked enzyme with immunomodulatory impact and highlight cell-state changes as candidate mechanisms for improved antiviral tolerance.

Simple SummaryEndometrial cancer (EC) develops in an environment strongly modulated by the immune system, and changes in dendritic cells (mDCs and pDCs), and monocytes (MO) expressing PD-L1 and PD-L2 may influence disease progression. Our study demonstrated that EC patients have lower percentages of PD-L1-positive MO and pDCs, as well as PD-L2-positive MO and mDCs, compared to the control group. We also found changes in plasma levels of soluble forms of PD-1, PD-L1, and PD-L2, which correlated with tumor PD-L2 expression and clinical characteristics, such as BMI and disease FIGO stage. AbstractBackground: Endometrial cancer is one of the most common female genital cancers and poses a significant clinical problem due to its increasing incidence and variable prognosis depending on the stage of the disease. The development of EC is largely dependent on interactions with the immune system, including immune checkpoints (ICPs) such as PD-1, PD-L1, and PD-L2. The aim of our study was to evaluate the PD-1/PD-L1/PD-L2 pathway in EC and its clinical significance. Methods: The analysis was performed by flow cytometry on myeloid and plasmacytoid dendritic cells and monocytes (MO) in peripheral blood (PB). The concentration of sPD-1, sPD-L1, and sPD-L2 in plasma was determined by ELISA. Additionally, PD-L1 and PD-L2 gene expression levels in tumor tissue (TT) were assessed using real-time polymerase chain reaction (qPCR). The obtained results were correlated with clinical data of EC patients. Results: Patients with EC had lower percentages of PD-L1-positive MO and pDCs, as well as PD-L2-positive MO and mDCs, compared with the control group. We observed accumulation of sPD-1 and lower levels of sPD-L1 and sPD-L2 in EC patients compared to the control group, with sPD-L2 correlating with PD-L2 gene expression level in the TT. Conclusions: The study results indicate a difference in the distribution of mDCs, pDCs, and MO with PD-L1/PD-L2 expression in EC patients. Reduced percentages of MO and DCs expressing PD-L1 and PD-L2, altered concentrations of soluble forms of these IPCs, and correlations with gene expression in TT suggest that dysregulation of this pathway may influence disease progression. Furthermore, the relationships between immunological parameters and clinical features such as BMI and FIGO stages suggest the potential use of these factors as diagnostic and prognostic biomarkers and the possibility of incorporating them into future therapeutic strategies. However, further studies are necessary to validate this hypothesis.

Oral squamous cell carcinoma (OSCC) is marked by profound differences in survival between the localized and disseminated disease, estimated to result in a 70% and less than a 40% five-year survival rate with surgical and/or radiation approaches (in localized cases) and chemotherapy (in metastatic cases), respectively. Given the suboptimal efficacy of current management options, new therapeutic approaches are needed to supplement existing chemotherapies and improve outcomes. One emerging therapeutic option is naltrexone (NTX), an opioid antagonist that has shown promising outcomes at low doses in other forms of cancer. This study sought to determine the effectiveness of intermittent dosing of naltrexone on oral cancer cell survival, either as a single agent or in combination with traditional chemotherapy. Two human OSCC lines (locally invasive SCC-25 and metastatic Detroit 562) were cultured. Cells were exposed to 1 µM and 10 µM NTX alone, using intermittent (5 h once, 5 h daily, 5 h every other day) or constant 72 h exposure. Cells were exposed to combination therapy with cisplatin or docetaxel under three NTX regimens (5 h, 24 h, and continuous). Cell viability was determined using Sulphorhodamine B (SRB) assay and Cell Counting Kit-8 (CCK-8). Differences across treatments were assessed using ANOVA (p < 0.05). The effect of low-dose NTX alone, across varying treatment regimens, did not yield significant, consistent changes in OSCC cell survival. Combination with cytotoxic drugs reduced cell viability more efficiently than chemotherapy alone at select doses, particularly through intermittent short-term pretreatment schedules, but the full dose response demonstrated antagonism between NTX and chemotherapy, independent of the dosing schedule. These results contrast with previous findings in other cancers, and, thus, further study and optimization will be needed to determine the clinical benefit and reproducibility of these findings.

Mammalian hearing loss is typically permanent due to the inability to replace damaged cochlear hair cells. However, the neonatal mice inner ear demonstrates regenerative capacity, with cochlear floor cells proliferating and differentiating into organoids containing new hair cells and supporting cells, yet the governing molecular mechanisms remain poorly understood. Here, we isolated extracellular vesicles (EVs) from inner ear organoids at proliferation and differentiation stages, characterized their EV miRNA profiles through sequencing, and validated findings using public transcriptomic datasets to elucidate miRNA-mediated regulatory mechanisms during inner ear development. Inner ear organoids were successfully developed from cochlear duct cells, expressing otic progenitor marker SOX2 and hair cell marker Myo7A and demonstrating functional mechano-transduction activity through FM1-43 uptake. Small RNA sequencing identified 35 differentially expressed EV miRNAs between developmental stages. Integrated analysis with public transcriptome datasets revealed 18 genes with significant differential expression, leading to identification of three key regulatory genes—Trp53, Ezh2, and Zbtb4—that exhibited dynamic spatiotemporal expression during inner ear maturation. Pathway analysis demonstrated that these genes are associated with DNA Repair, P53, and Wnt/β-Catenin signaling with remarkable cell-type specificity. Our results demonstrate that EV miRNAs are temporally regulated during organoid development, with predominant downregulation during differentiation. These findings provide crucial insights into developmental mechanisms that could optimize organoid-based models and guide EV miRNA-based therapeutic strategies for hearing restoration.

Chronic hemophilic synovitis (CHS), driven by hemosiderin-laden macrophages from recurrent hemarthrosis, is a major cause of joint damage in hemophilia. Platelet-rich plasma (PRP) is a promising regenerative therapy for joint diseases. This study investigated PRP’s ability to modulate macrophage polarization from a pro-inflammatory (M1) to a pro-resolving, tissue-repairing (M2) phenotype in CHS. We analyzed synovial fluid (SF) from CHS patients (N = 22), both pre- and post-PRP treatment. Ex vivo analysis revealed a predominant M1 profile with an increased proportion of CD11+CD14+CD64hi compared with CD206+ or CD163+ M2 macrophages in CHS SF. In vitro experiments showed that CHS SF skewed monocyte-derived macrophages toward an M1 inflammatory program, evaluated by flow cytometry, qPCR, and ELISA. However, adding PRP significantly modulated the pro-inflammatory macrophage program, promoting an M2 tissue repair profile. Furthermore, a random forest machine learning algorithm, applied to public scRNAseq data, confirmed PRP’s macrophage reprogramming effect. Functional assays also showed increased TGF-β secretion and macrophage fusion when challenged with neutrophil extracellular traps (NETs). A small patient follow-up cohort treated with intra-articular PRP showed similar results, including normalization of cellular content and reduced CD64/CD206 expression. These findings indicate that PRP treatment effectively shifts SF-associated M1 macrophages to an M2-like phenotype, highlighting its potential as a therapeutic strategy for CHS.

Autophagy is a lysosome-mediated self-degradation process of eukaryotic cells which is critical for the elimination of cellular damage. Its capacity progressively declines with age, and this change can lead to the development of various neurodegenerative pathologies including Spinocerebellar ataxia type 1 (SCA1). SCA1 is mainly caused by mutations in the polyglutamine region of Ataxin 1 protein. In patients affected by the disease, Purkinje neurons of the cerebellum frequently undergo demise and eventually become lost. Here we tested whether two well-characterized autophagy-enhancing small molecules, AUTEN-67 and -99, which antagonize the autophagy complex Vps34 through blocking the myotubularin-related lipid phosphatase MTMR14/EDTP, have the capacity to ameliorate SCA1 symptoms. We found that in a Drosophila model of SCA1, only AUTEN-67 exerts positive effects including improvement in climbing ability and extending life span. Based on these results, we hypothesized that the two compounds influence autophagy in the brain in a neuron-specific manner. Indeed, according to data we obtained, AUTEN-67 and -99 exhibit shared and unique functional domains in the Drosophila brain. AUTENs enhance autophagy in GABAergic and dopaminergic neurons. In addition, AUTEN-67 also affect autophagy in cholinergic neurons, while AUTEN-99 trigger the process in glutaminergic neurons and motoneurons. We also observed varying efficiencies between the two AUTENs among different subtypes of cultured hippocampal neurons of mice. These data suggest that the two compounds display neuron-specific differences in exerting autophagy-enhancing effects, and may lead to a better understanding of which types of neurons autophagy could potentially be activated to treat SCA1 in human patients.

Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) offer a promising model system for understanding motor neuron diseases (MNDs) and advancing drug discovery. However, variability in differentiation outcomes presents a major barrier to reproducibility and model reliability. This study evaluates a widely adopted small molecule protocol for MN differentiation to quantify variability and identify its sources within an industrial setting. Analysing data from 15 differentiation sets across 8 cell lines, we found that non-genetic factors – particularly induction set and operator – were the predominant sources of variability, outweighing the contribution from cell line genetics. We further demonstrated that iPSC genomic instability, as assessed by a targeted RT-qPCR assay for common karyotypic abnormalities, significantly affected differentiation efficiency and purity. Cultures derived from genomically stable iPSCs exhibited reduced variance and improved MN marker expression profiles. These findings support routine genomic assessment of iPSCs as a practical and effective strategy to enhance the reliability of iPSC-derived MN models, thereby improving their utility in preclinical MND research and therapeutic development.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-23378-0.