Neutrophil dysregulation is one of the main features of periodontitis (PD). This study delineated neutrophil heterogeneity and predicted potential communication of mast cells within the microenvironment of PD using computational single-cell transcriptomics. We analyzed a public scRNA-seq dataset (GSE171213) from gingival tissues of healthy controls, PD patients, and post-treatment patients. Data processing, clustering, and annotation were performed using Seurat and Harmony packages. Cell-cell communication was computationally inferred using CellChat and NicheNet packages, and transcriptional regulatory programs were predicted via SCENIC. HMC-1.2 mast cells were treated with TGFB1 and assessed for proliferation, migration, the level of VEGFA, IL-6, and TNF-α. Ten cell types in PD were identified. Two distinct transcriptional states of neutrophils were identified based on gene expression profiles. Cell-cell communication analysis predicted that Subtype 2, which was enriched for inflammatory and effector genes, exhibited stronger putative interactions with mast cells via ligand-receptor pairs such as IL6-(IL6R+IL6ST) and SEMA4D- PLXNB2. NicheNet-based analysis further inferred that neutrophil-derived TGFB1, OSM, and IL1B were associated with mast-cell target gene programs related to proliferation, migration, angiogenesis, and inflammatory responses. Finally, in vitro cell experiments indicated that TGFB1 inhibited proliferation but promoted migration and the expression of VEGFA, IL-6, and TNF-α. However, all effects were reversed by SB431542, confirming TGFBR1 dependence. This study delineated neutrophil transcriptional heterogeneity in PD and identified neutrophil- mast cell communication axes as a contributor to the inflammatory microenvironment in PD. Through computational analysis of single-cell transcriptomics, this study described two neutrophil states and their predicted interaction network with mast cells in PD, providing testable hypotheses for future mechanistic and translational studies.

As an engulfment adapter for apoptotic clearance, GULP1 is related to tumor progression, but its role in ovarian cancer (OVCA) remains unclear. GULP1 expression, copy number variations (CNVs), and their correlations were analyzed using datasets collected from The Cancer Genome Atlas (TCGA)-OVCA and the Gene Expression Profiling Interactive Analysis (GEPIA). Gene set enrichment analysis (GSEA), immune infiltration assessment, and drug sensitivity prediction were conducted to identify pathways enriched by GULP1 and to explore its potential association with drug sensitivity. The binding affinity of candidate drugs to GULP1 was evaluated through molecular docking. Finally, cellular assays were performed to explore the biological functions of GULP1 in OVCA cells. GULP1 was downregulated in OVCA, particularly in advanced stages. The GULP1 expression was associated with enrichment of hypoxia, EMT, angiogenesis, and TGFβ signaling, which were correlated with immune evasion. LFM-A13 demonstrated binding affinity for GULP1 (ΔG = -5.85 kcal/mol). Overexpression of GULP1 inhibited migration and invasion of A2780 and SK-OV-3 cells. Conversely, GULP1 knockdown exerted the opposite effects. This study explored the role of GULP1 in OVCA and analyzed its correlation with the immune microenvironment and key pathways. Molecular docking predicted LFM-A13 as a potential therapeutic agent for OVCA; however, its efficacy remains to be clinically verified. The current findings reveal GULP1 as a novel biomarker for OVCA progression and immune escape, highlighting its role in modulating the tumor microenvironment (TME). Targeting GULP1 with LFM-A13 may offer a potential strategy for OVCA precision therapy.

To devise a bioactive surface functionalization approach for 3D-printed Ti- 6Al-4V scaffolds that influences macrophage polarization towards the pro-reparative M2 phenotype, therefore enhancing immunomodulation and facilitating good implant-soft tissue integration. Porous Ti-6Al-4V scaffolds were produced by selective laser melting and then covered with a polydopamine-multi-element-doped hydroxyapatite-type I collagen (PDA-mHA-Col I) composite. The scaffolds' physicochemical characteristics were characterized. Murine RAW264.7 macrophages were cocultured with uncoated (T) or coated (TPMC) scaffolds. Cell viability, proliferation, apoptosis, adhesion, and polarization were assessed via CCK-8 tests, EdU staining, flow cytometry, phalloidin staining, ELISA, and qRT-PCR. The NF-κB, PI3K/Akt, and STAT6 signaling pathways were examined using Western blotting and targeted inhibitors. The PDA-mHA-Col I coating improved surface hydrophilicity while maintaining mechanical characteristics. XPS verified effective collagen immobilization, exhibiting a surface nitrogen concentration of 13.03%. The coating demonstrated stability after 7 days in PBS, retaining a nitrogen content of 11.74% and negligible titanium exposure. In comparison to the T group, the TPMC scaffold markedly enhanced macrophage adhesion, proliferation, and spreading, while diminishing apoptosis. It prompted M2 polarization, as shown by reduced expression of M1 markers (iNOS, CD86) and pro-inflammatory cytokines (TNF-α, IL-6), with elevated expression of M2 markers (Arg-1, CD206) and anti-inflammatory cytokines (IL-10, TGF-β1). The TPMC scaffold suppressed the phosphorylation of NF-κB p65 while simultaneously activating PI3K/Akt and STAT6 signaling pathways. The inhibition of PI3K or STAT6 somewhat mitigated the increase of M2 markers. The coating created a pro-healing milieu by inhibiting inflammatory signals and stimulating pro-reparative pathways, thus tackling a significant obstacle in oral and maxillofacial bone repair. The PDA-mHA-Col I composite coating facilitates macrophage M2 polarization by concurrently inhibiting NF-κB and activating PI3K/Akt/STAT6 signaling, presenting a viable immunomodulatory approach for oral and maxillofacial bone restoration.

Osteoporosis (OP) and osteoarthritis (OA) are two skeletal disorders characterized by disrupted bone homeostasis. Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) has emerged as a promising therapeutic strategy for both conditions; however, the precise molecular mechanisms mediating their beneficial effects remain poorly defined. The GSE147287 dataset containing scRNA-seq data from BM-MSCs from OA and OP patients was obtained. Dimensionality reduction and cell clustering were performed using the Seurat R package, pseudotime trajectory analysis was carried out with the Monocle 2 package, and transcription factor (TF)-target gene regulatory networks were inferred using the GENIE3 R package. RT-qPCR quantified mRNA levels in a rat OP model, which was established via bilateral ovariectomy. Nine distinct BM-MSC subtypes were classified. OP samples had higher osteocytes and neutrophils and lower macrophages, chondroblasts, monocytes, and plasma B cells than OA samples. Chondroblasts (4 clusters), 2/3 linked to autophagy, may drive OA-to-OP progression. Osteoblasts (the largest OP-OA difference) showed reduced osteoblast differentiation, downregulated Wnt pathway genes, and upregulated ossification genes in late stages. In the rat model, CAT, CHRDL1, RUNX1, ETS1, FOXO3, and TAL1 were dysregulated. OP and OA exhibit distinct BM-MSC lineage heterogeneity. OA-to-OP progression involves enhanced oxidative phosphorylation and reduced autophagy. Downregulated RUNX1 (inhibiting NF-κB/IL-6) and Wnt pathway in OP were consistent with previous findings, showing the potential to serve as biomarkers for predicting disease progression and therapy response. This study preliminarily examined BM-MSC lineage heterogeneity in OP and OA, clarifying the dynamic development, transcriptional regulation, and biological functions of chondrocytes and osteoblasts in these two bone diseases.

Intrahepatic cholangiocarcinoma (iCCA) is characterized by heterogeneity and poor survival. It remains unclear how telomere maintenance programs influence the prognosis and immune microenvironment in iCCA, and clinically applicable, telomere‑anchored transcriptomic tools are currently lacking. Transcriptomes and survival data from TCGA-CHOL, GSE107943, and E-MTAB-6389 cohorts were integrated (n=135). Telomere maintenance gene (TMG) scores were calculated using GSVA and subsequently used for WGCNA module identification. Functional enrichment was analyzed with clusterProfiler (FDR-adjusted P < 0.05). A prognostic model was constructed using univariate Cox and LASSO-Cox regression analyses, and validated by Kaplan-Meier (KM) curve and time-dependent ROC. Immune features were inferred by MCPcounter, CIBERSORT, and ssGSEA algorithms. Drug sensitivity was predicted by pRRophetic, and the resulting IC50 estimates were then correlated with the RiskScore. The TMG score was inversely correlated with the ESTIMATE score (Spearman ρ=-0.2439, P=0.0044). WGCNA identified a TMG-associated module enriched for cellcycle/ mitotic and microtubule functions. Three key TMGs (PTTG1, TSPYL5, PLLP) were integrated to develop a RiskScore that can consistently stratify overall survival (OS) in both the integrated set and all external cohorts, demonstrating a robust accuracy for 1-, 3-, and 5-year prognostic prediction. High-RiskScore tumors exhibited reduced immune infiltration across multiple deconvolution frameworks. The RiskScore was negatively correlated with predicted IC50 for several agents (e.g., pyrimethamine, GNF-2, NSC-87877, CGP-082996, KIN001-135), suggesting higher drug sensitivity in high-risk cases. We identified a three-gene, telomere-related risk score for iCCA, which can effectively predict patients' survival and distinguish immune-cold, high-proliferation phenotypes. Potential targeted drugs for high-risk patients were predicted and supported by mechanistic validation. A compact telomere-anchored three-gene RiskScore was developed to predict the prognosis, immune contexture, and therapy sensitivity for iCCA.

In advanced Prostate Cancer (PCa), metastatic spread and the inevitable emergence of enzalutamide resistance represent major clinical hurdles. Although apolipoprotein L3 (APOL3) is linked to oncogenesis, its precise mechanistic role in PCa progression and antiandrogen resistance, particularly its regulation of the STAT3-DAB2IP axis, remains largely unexplored. Publicly available clinical datasets were analyzed to evaluate APOL3 expression and its prognostic value. The functional consequences of modulating APOL3 and DAB2IP levels were assessed using in vitro and in vivo PCa models, including an established enzalutamide-resistant cell line (C4-2R). Mechanistic insights into cellular proliferation, motility, angiogenesis, and drug response were derived from RNA sequencing, reciprocal co-immunoprecipitation (Co-IP), and dual-targeting phenotypic assays. APOL3 is significantly upregulated in PCa, strongly correlating with elevated Gleason scores, advanced stage, TP53 mutational status, and poor prognosis. Functionally, APOL3 promotes PCa proliferation, metastasis, and angiogenesis. Mechanistically, APOL3 sustains STAT3 phosphorylation and suppresses the tumor suppressor DAB2IP. Notably, Co-IP assays revealed a direct, bidirectional physical interaction between APOL3 and DAB2IP. Furthermore, we discovered that elevated APOL3 drives enzalutamide resistance not by enhancing classical Androgen Receptor (AR) activity, but by directly binding the Glucocorticoid Receptor (GR). This APOL3-GR complex activates a bypass signaling pathway entirely independent of the AR. While restoring DAB2IP resensitized cells to enzalutamide, it triggered a compensatory upregulation of APOL3. Consequently, concurrent APOL3 knockdown and DAB2IP overexpression yielded a powerful synergistic effect, profoundly dismantling malignant phenotypes, suppressing pro-metastatic markers (p-STAT3, VEGF, SNAIL, MMP2), and restoring enzalutamide sensitivity. These findings establish APOL3 as a central driver of prostate cancer metastasis and enzalutamide resistance. APOL3 drives these aggressive phenotypes by directly binding and suppressing DAB2IP to sustain oncogenic STAT3 signaling, and by activating an AR-independent bypass pathway through its physical interaction with the Glucocorticoid Receptor (GR). The enrichment of APOL3 in TP53-mutated and resistant tumors underscores its critical role in tumor plasticity. Consequently, synergistically co-targeting APOL3 alongside DAB2IP restoration represents a highly promising therapeutic strategy to overcome adaptive antiandrogen resistance and halt metastatic progression. APOL3 is a central driver of PCa aggressiveness and enzalutamide resistance, functioning via the direct modulation of the DAB2IP/STAT3 axis and the activation of the GR bypass pathway. Cotargeting APOL3 alongside DAB2IP restoration represents a highly promising, synergistic therapeutic strategy to circumvent adaptive resistance and halt metastatic progression in advanced castration-resistant prostate cancer.

Members of the Homeobox (HOX) gene family, particularly HOXCs, may be implicated in the development and prognosis of prostate cancer (PCa), but the specific mechanisms remain unclear. The clinical and transcriptomic data from TCGA-PRAD and GSE70770 were collected to examine the impact of HOXC family members on progression-free survival (PFS) in PCa. Meanwhile, the mechanisms of HOXC family members in PCa and their relationship with patients' prognosis were systematically investigated by performing survival analysis, enrichment analysis, and COX regression analysis. This study also developed a prognostic model. Additionally, a qPCR assay was conducted to determine the mRNA level of HOXC family members in PCa cells. Upregulated expressions of HOXC4, HOXC5, HOXC12, and HOXC13 in PCa tissues were found to be correlated with a worse prognosis. A prognostic model incorporating HOXC4 expression level, T stage, and Gleason score was constructed, exhibiting excellent performance in predicting 1-, 3-, and 5-year PFS (average AUC > 0.7). Enrichment analysis showed that HOXC4, which was highly expressed in PCa cells, may be involved in the progression and invasion of PCa through pathways such as oxidative phosphorylation, PLK1 signaling, cell cycle, and DNA methylation. Further, ssGSEA showed that HOXC4 may affect immune cell infiltration in PCa tumors. HOXC4 was related to immune cell infiltration and pathway activation in PCa and confirmed to be a risk factor for the cancer prognosis. However, the specific molecular mechanisms of HOXC4 in PCa development still require further investigation. The HOXC4-based prognostic model may be an effective tool for assessing the prognosis of PCa patients.

Autism Spectrum Disorder (ASD) is a highly heterogeneous neurodevelopmental condition. Single-cell RNA sequencing (scRNA-seq) has revealed transcriptional disruptions, particularly in interneurons, yet their subtypes and molecular signatures remain poorly understood. It was analyzed scRNA-seq data from the human Prefrontal Cortex (PFC). Key cell types were identified using Scissor and ROGUE methods, followed by secondary clustering for subtype annotation. A signature matrix was established using CIBERSORTx to deconvolute the bulk transcriptomes and estimate cell type-specific proportions. Differential subtype proportions between ASD and control samples were compared to identify key cell subtypes. Differentially Expressed Genes (DEGs) from both the key subtype and bulk data were intersected to determine subtypespecific biomarkers, which were further assessed via molecular docking. Interneurons were identified as the most heterogeneous cell population in ASD-affected PFC and were further categorized into five subtypes. A signature matrix was then developed with CIBERSORTx to reflect the proportion of each cell type and subtype. Among the cell subtypes, synaptic membrane-integrating interneurons (SMI-IN) emerged as the key subtypes, which exhibited notable distinctions between the ASD and control samples. Furthermore, five potential biomarkers (PRELID2, MYO1B, LRCH2, LIFR, and RERG) were identified from the SMI-IN subtype. Finally, quercetin and coumestrol were predicted as potential therapeutic compounds targeting these biomarkers. Nevertheless, as all findings were obtained via computational analysis, further cellular and clinical experiments are required to validate these identified biomarkers and candidate compounds. This study focused on interneurons and identified SMI-IN as a key cell subtype and its potential biomarkers (PRELID2, MYO1B, LRCH2, LIFR, and RERG). The present findings provided new insights for ASD intervention.

A comprehensive study of Cell-In-Cell (CIC) structures in stomach adenocarcinoma (STAD) may facilitate the development of therapeutic strategies. Enrichment analysis was conducted using ssGSEA. WGCNA was used to identify hub genes, and Differentially Expressed Genes (DEGs) were screened by the DESeq2 package. Gene selected by both LASSO regression (glmnet package) and SVM-RFE algorithms (e1071 package) were intersected to develop a diagnostic model for STAD using the rms package. Immune infiltration of STAD samples was analyzed using CIBERSORT and ESTIMATE algorithms. Single-cell data were processed by Seurat package. Cell subpopulations were identified using the FindClusters function, and their marker genes were subsequently determined by FindAllMarkers, followed by in vitro functional validation. Five genes (MSR1, PDGFRB, COL8A1, PLA2G7, and FCGR3A) with AUC > 0.8 were identified as potential biomarkers for STAD and combined into a five-gene diagnostic model. Immune infiltration analysis showed that these genes were associated with T-cell and macrophage infiltration. Among the ten cell types identified by single-cell analysis, the five biomarkers were found to be highly expressed specifically in macrophages and fibroblasts. In vitro functional assays confirmed that these markers were upregulated in HGC-27 and AGS cells, and that MSR1 regulated STAD cell proliferation, migration, and invasion. The five biomarkers, which showed a specifically high expression in macrophages and fibroblasts, were closely associated with heterotypic CIC formation. Further analysis suggested that these biomarkers contributed to STAD pathogenesis, potentially via immune, stromal, and metabolic crosstalk. These findings help clarify the interactions between CIC biology and the STAD tumorimmune microenvironment. The five biomarkers provide valuable insights for the early screening, diagnosis, and treatment of STAD.

To evaluate the prognostic value of complement and coagulation cascades (CCC) in lung adenocarcinoma (LUAD). Transcriptomic and clinical data of LUAD were retrieved from public databases. Differentially Expressed Genes (DEGs) were identified via the limma R package, and WGCNA was used to screen gene modules strongly correlated with CCC enrichment scores. Functional enrichment analysis was conducted with clusterProfiler. Univariate and LASSO Cox regression analyses were applied to build a CCC-related prognostic risk model, and its performance was validated using Kaplan-Meier survival analysis and ROC curves. ESTIMATE, MCPcounter, GSVA, and pRRophetic algorithms assessed immune infiltration and drug sensitivity, while CCK-8, Transwell, and scratch-healing assays verified the regulatory effects of biomarkers on cancer cells. WGCNA identified brown modules that were significantly correlated with CCC scores; enrichment analysis showed that these genes mainly participated in NF-κB, B-cell receptor signaling, and T-cell differentiation pathways. Six key genes (CPS1, EPHB2, LARGE2, MS4A1, OAS3, S100P) were selected to construct the risk model, with the high-risk group exhibiting poorer overall survival and lower T/B-cell infiltration. Risk scores were positively correlated with Rapamycin and Phenformin IC50s but negatively correlated with FTI-277, BMS-509744, etc. EPHB2 inhibition suppressed lung cancer cell viability, migration, and invasion. This study systematically characterized the molecular features and prognostic significance of CCC-associated genes in LUAD, established a CCC-related risk model, and confirmed their tight association with immune infiltration and drug sensitivity. The findings provide a theoretical foundation for precise prognostic evaluation and personalized treatment of LUAD.