Search Results (3,437)

Background: Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality, and although PD-1/PD-L1 immune checkpoint blockade has improved outcomes in some patients, therapeutic responses remain heterogeneous. Tumor-intrinsic heterogeneity within malignant epithelial populations is increasingly recognized as a critical determinant of disease progression and therapy response. Methods: Here, we constructed a comprehensive single-cell atlas of NSCLC by integrating 650,461 cells from 216 tumor and normal samples. Tumor-derived epithelial cells were reclustered to identify transcriptionally distinct subpopulations. Pseudotime analysis, functional experiments, and in vivo validation using a humanized xenograft model were performed to investigate the role of COL3A1. Results: Reclustering of tumor-derived epithelial cells revealed 25 transcriptionally distinct subpopulations. Among these, a high-risk cluster exhibited coordinated activation of epithelial–mesenchymal transition (EMT) and angiogenesis programs and was associated with poor patient survival. Within this aggressive subpopulation, Collagen type III alpha 1 (COL3A1) emerged as a tumor-intrinsic gene associated with extracellular matrix remodeling and angiogenic signaling. Pseudotime analysis indicated that COL3A1⁺ cells represent a late-stage, poorly differentiated malignant state. Functional experiments demonstrated that COL3A1 knockdown impaired NSCLC cell proliferation, migration, and invasion. Virtual knockout further suggested that COL3A1 may be associated with transcriptional programs involved in PD-L1 upstream signaling pathways, indicating a potential indirect link between tumor-intrinsic states and immune regulatory networks. Consistently, in vivo silencing of COL3A1 enhanced the antitumor efficacy of PD-L1 blockade. Conclusions: Collectively, our study identifies COL3A1 as a tumor-intrinsic gene enriched in malignant epithelial cells with mesenchymal features and a potential therapeutic target. These findings provide a rationale for exploring combinatorial strategies integrating tumor-intrinsic pathway inhibition with immune checkpoint blockade in NSCLC. Full article

The marble industry relies on proprietary commercial names rather than objective visual categories, creating market inefficiencies for stakeholders who select stones based on appearance. Supervised classification perpetuates this problem by replicating inconsistent commercial labels instead of discovering intrinsic visual structure. We propose an unsupervised pipeline combining a two-stage training strategy: A pure self-supervised pretraining followed by cluster-aware fine-tuning of a DINO Vision Transformer, with empirically selected dimensionality reduction and agglomerative hierarchical clustering. Systematic ablation studies on 1480 marble images spanning 10 commercial varieties validate each design choice: cluster-aware training at k = 10 yields geometrically improved embeddings over the self-supervised baseline (mean Silhouette Score 0.693 ± 0.053 vs. 0.660 ± 0.030; mean Davies–Bouldin Index 0.386 ± 0.075 vs. 0.569 ± 0.012; N = 9 independent evaluations across 3 data partitions × 3 training initializations). The resulting taxonomy reveals three phenomena invisible to commercial classification: cross-category merging of visually indistinguishable stones carrying different market names, intra-category splitting of heterogeneous sub-populations within single varieties, and coherent grouping where commercial and visual boundaries coincide, with all three confirmed in every independent run. We further demonstrate that standard extrinsic metrics are misaligned with unsupervised taxonomy objectives when reference labels encode the inconsistencies the method aims to resolve. Validating this methodology across diverse stone types, larger datasets, and varied acquisition conditions represents a natural and necessary next step toward establishing its cross-domain generalizability. Full article

Background: Chimerism analysis is a key tool for monitoring donor-cell engraftment and the risk of relapse and graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT). The advantage of lineage-specific chimerism assessment, and its dynamics in patients receiving post-transplant cyclophosphamide (PTCy)-based GVHD prophylaxis, remains unclear. Objective: This review summarizes the current state of the art on chimerism analysis in patients with myeloid neoplasms undergoing allo-HCT with PTCy, with emphasis on lineage-specific testing and modern methodologies. Methods: A structured literature review was conducted to assess chimerism dynamics in whole blood (WB), bone marrow, and peripheral blood (PB) subpopulations, including T-cells, CD34⁺, myeloid, B, and NK (natural killer) cells, and their association with clinical outcomes following PTCy. Results: Lineage-specific PB chimerism, particularly in T-cells, myeloid lineage and CD34⁺ cells, is more sensitive than WB chimerism for predicting relapse. Declining donor myeloid chimerism or persistent myeloid mixed donor chimerism (MDC) may precede hematologic relapse and provide an early signal of graft instability or ineffective graft-versus-leukemia activity. T-cell MDC has been associated with an increased risk of relapse and a lower risk of GVHD, although persistent T-cell MDC in some patients may instead indicate immune tolerance. Declining CD34⁺ donor chimerism correlates with a higher risk of relapse and inferior survival outcomes and may therefore complement measurable residual disease testing. Data regarding B-cell and NK-cell chimerism remain inconsistent, likely influenced by delayed immune reconstitution. Compared to anti-thymocyte globulin, PTCy may promote higher donor T-cell chimerism, though findings across studies are variable. Next-generation sequencing (NGS) enables more sensitive detection of microchimerism and relapse prediction. Conclusions: Chimerism analysis, particularly when lineage-specific and NGS-based, offers valuable prognostic insight in allo-HCT with PTCy. Further prospective studies are needed to standardize testing and guide personalized post-HCT strategies. Full article

Extracellular vesicles (EVs) are promising biomarkers for liquid biopsy, but their clinical application is limited by intrinsic heterogeneity and the lack of methods capable of resolving functionally distinct EV subpopulations at the single-vesicle level. Conventional bulk analyses obscure rare but clinically relevant EV subsets, while most single-EV approaches focus on physical properties or surface markers, with limited access to intravesicular functional information. Here, we report a fusion-enabled EV detection strategy at the single-particle level for functional profiling of macrophage-derived EVs. Liposomal probes encapsulating L-arginine, NADPH, and a nitric oxide (NO)-responsive fluorescent dye are engineered to fuse with EV membranes, delivering substrates into the vesicle lumen. In macrophage-derived EVs, inducible nitric oxide synthase (iNOS) catalyzes NO production, activating the fluorescent probe and generating a localized signal within individual vesicles. Signal generation is confined to vesicle-restricted reactions, ensuring specificity and minimizing background. The formation of hybrid vesicles further facilitates optical detection using conventional fluorescence microscopy. Full article

Background and Objectives: The incidence of colorectal cancer (CRC) in developed Western countries is constantly growing. CRC represents the third most common cancer and the second leading cancer-related cause of death worldwide. Innate and adaptive immunity play a pivotal role in the tumor response, but many of these interactions are still not well understood. Granulysin (GNLY) is an effector, cytolytic molecule, present in human cytotoxic granules of different lymphocyte subpopulations, mainly in cytotoxic T cells and NK cells. Pore-forming proteins GNLY, perforin and granzymes play a key role in cell-mediated immune responses against tumors and infections. Materials and Methods: We aimed to analyze perforin and GNLY-mediated cytotoxicity in the peripheral blood of patients with CRC by flow cytometry. Simultaneously, the cells were labeled with monoclonal antibodies against perforin, GNLY and different surface antigens (CD3, CD4, CD8 and CD56). Phenotypes of lymphocyte subpopulation and expression of perforin and GNLY were analyzed using intracellular and surface immunofluorescence. Results: Total perforin and GNLY expressions in peripheral blood mononuclear cells (PBMC) were significantly lower than in the control group. Statistically significant differences were observed in the distribution of perforin and GNLY expression in different stages of tumors classified according to Dukes’, indicating that the percentage of total perforin and GNLY was significantly diminished in accordance with tumor progression. Perforin and GNLY expression were significantly reduced in NK and NKT cells, accompanied by reduced cytolytic potential in patients with CRC and a consequent reduction in their ability to eliminate tumors and infected cells. Conclusions: The determination of cytotoxic potential may provide a valuable assessment of a patient’s immune status and represent a novel therapeutic target. Patients with CRC exhibit markedly impaired perforin- and GNLY-mediated cytotoxicity that correlates with disease progression. Assessment and restoration of cytolytic potential may therefore serve as indicators of immune competence and promising therapeutic strategies to improve perioperative and oncologic outcomes. Full article

Head and neck mucosal melanoma (HNMM) arises in the nasal and oral cavities and has the propensity to metastasize to local and distant body sites. HNMM is also notable for its resistance to available therapeutics. The rarity of this disease makes it difficult to conduct large-scale clinical studies to develop standard treatment protocols. In contrast to cutaneous melanoma, c-Kit-dependent pathways are well studied in HNNMM and provide a potential therapeutic target. We identified and isolated genetically distinct subpopulations with stem cell characteristics in HNMM samples bearing Kit wild-type and mutations. Functional analysis of these subpopulations reveals that, in addition to expressing the stem cell marker proteins CD20, CD117, CD133, and CD166, these subpopulations are characterized by self-renewal potential, migratory capacity, and resistance to Kit inhibitors such as Imatinib. Immunofluorescence staining and inhibition experiments demonstrate that the maintenance and resistance of HHMM subpopulations to Kit inhibitors is mediated by the Kit signal to the PI3K signaling pathway. The KIT signal to the PI3K signaling pathway does not result exclusively from a KIT mutation localized to Exon 17, but can also be triggered by mutations localized to Exons 11 and 13. In the present study, we identify and characterize an HNMM subpopulation with stemness properties in patients with c-Kit wild-type and mutation, and demonstrate for the first time the mechanisms by which the CD117⁺/CD133⁺ HNMM subpopulations survive and confer resistance to the specific inhibitor of c-Kit mutation. Full article

When the data at hand are suspected to stem from several latent subpopulations, Statisticians commonly speak of “unobserved heterogeneity”. While the presence and importance of this phenomenon is commonly acknowledged, there is relatively little guidance on how to carry out correct inferences under unobserved heterogeneities, even in relatively simple scenarios such as the linear regression model. In this work, bootstrap algorithms for the computation of standard errors are investigated in the context of a mixture-based regression approach which accounts for the clustered nature of the data. Of interest is both the accuracy of the standard errors (evidenced by confidence interval coverage rates) and the relative reduction in standard errors achieved in comparison to a naïve linear model fit. Simulations and a real data example are provided. Full article

Understanding the genetic diversity and population structure of Cucurbita species is essential for effective germplasm conservation and the development of improved cultivars. This study aimed to evaluate the genetic diversity, population structure, and genetic relationships among accessions of C. pepo, C. moschata and C. maxima and their interspecific hybrids (Tetsukabuto hybrid C. maxima × C. moschata). A total of 92 accessions were analyzed using 22 polymorphic simple sequence repeat (SSR) markers selected from previous studies due to their high polymorphic information content (PIC). Genetic diversity parameters were estimated, and population structure was inferred using Bayesian clustering, complemented by dendrogram and principal component analysis (PCA). All markers were successfully amplified in C. pepo, C. moschata, C. maxima, and the hybrids, with polymorphic information content (PIC) values ranging from 0.191 (CMTm232) to 0.448 (CMTm48) and average of 0.274. The AMOVA analysis showed that 50% of the total variation was attributed to differences both within and among groups. PCA revealed clear genetic differentiation among the analyzed species, with C. maxima and hybrid accessions clustering closely and exhibiting lower genetic dissimilarity. In contrast, C. pepo displayed greater genetic divergence, supporting its distinct evolutionary trajectory. According STRUCTURE analysis the accessions can be divided into four subpopulations, which are closely related to the species. PCA and dendrogram showed similar results for genetic structure of Cucurbita germplasm; C. maxima and hybrid accessions clustering closely and C. pepo as a distinct group. These findings provide valuable insights for breeding programs, germplasm management, and conservation strategies aimed at preserving genetic diversity and exploiting interspecific variation. Full article

Background/Objectives: Mycoplasma genitalium is an emerging cause of sexually transmitted infections (STIs) and is increasingly recognized for its association with cervicitis and pelvic inflammatory disease. However, prevalence data in specific Japanese subpopulations, particularly comparing pregnant and non-pregnant women, remains limited. This study aimed to determine the prevalence of M. genitalium and its co-infection rates with Chlamydia trachomatis and Neisseria gonorrhoeae among Japanese women. Methods: A cross-sectional study was conducted using vaginal swab specimens collected between April 2021 and November 2022 from patients visiting two clinics in Gifu, Japan. The study population comprised 2138 non-pregnant women presenting with urogenital symptoms or sexual contact history, and 236 pregnant women undergoing routine antenatal screening. Detection was performed using real-time polymerase chain reaction assays on the cobas^® 8800 system (Roche Diagnostics). Results: Among non-pregnant women, the overall prevalence was 3.8% (82/2138) for M. genitalium, 3.4% (72/2138) for C. trachomatis, and 0.4% (9/2138) for N. gonorrhoeae. Co-infection rates were low; M. genitalium and C. trachomatis co-infection was observed in 0.2% of cases. Among pregnant women, the prevalence was 3.8% (9/236) for both M. genitalium and C. trachomatis, and 0.4% (1/236) for N. gonorrhoeae. No statistically significant differences in prevalence were observed between pregnant and non-pregnant women for any pathogen. Conclusions: The prevalence of M. genitalium in this Japanese cohort was comparable to that of C. trachomatis in both pregnant and non-pregnant women, highlighting its significance as a major STI pathogen. These findings underscore the importance of including M. genitalium in routine STI screening panels for symptomatic women and antenatal care to prevent reproductive health complications. Given the high rates of antimicrobial resistance documented in Japanese M. genitalium strains, specific diagnostic testing is essential to enable targeted, resistance-guided therapy. Full article

With the rapid development of cancer treatment, immunotherapy has revolutionized renal cell carcinoma (RCC) treatment, yet patient responses remain heterogeneous. Here, a computational pipeline was constructed by integrating single-cell and bulk RNA sequencing data to identify immune-related candidate driver genes and characterize their impact on RCC immunotherapy. Based on gene regulatory networks (GRN), 25 immune-related candidate driver genes were identified, leading to the stratification of patients into three clusters (C1–C3). Compared to the C2/C3 cluster, the C1 cluster exhibited elevated immune infiltration, tumor mutation burden and checkpoint expression, which may represent immunotherapy responders. Dynamic analysis of GRNs revealed the critical role of candidate driver genes in predicting the efficacy of immunotherapy. IRF1, IRF9 and STAT1 in lymphoid cells of C1 participated in anti-tumor immune response by impacting target genes CD8A, HLA-A/E, TAP1 and PD-1. JUN, FOS, STAT3, JUND and NR2F1 were up-regulated in clusters C2 and C3, leading to tumor progression and immune evasion by influencing target genes HSPA1A, CXCL9 and PDGFR. In conclusion, integration of the transcriptome with molecular networks provided a network-based framework to uncover immune-related candidate driver genes for stratifying RCC patients, thereby serving as potential therapeutic targets to improve the outcome of RCC immunotherapy. Full article

Pasteuria penetrans (Pp) is a mycelial and endospore-forming bacterium that parasitizes Meloidogyne spp. A single Pp population may contain multiple genotypes that differ in their spore-attachment specificity. Consequently, a subpopulation within a Pp isolate, which can attach to one Meloidogyne species, may fail to attach to another. Repeated culturing of that Pp isolate, on different Meloidogyne species, may therefore lead to shifts in host specificity. We tested this hypothesis using M. luci and M. arenaria, both of which are quite poor hosts of the Pp3 isolate maintained on M. javanica. Using relatively high spore concentrations (10⁶ spores/mL), low levels of attachment and infection were obtained, and after three successive selection cycles, Pp3 sub-isolates adapted to M. luci and M. arenaria were generated. This selection process was associated with a fitness cost, expressed as reduced spore attachment on M. javanica. The shift in host specificity proved reversible. When the adapted Pp3 M. arenaria and Pp3 M. luci sub- isolates were subsequently selected on M. javanica, for two generations, they regained the ability to attach on M. javanica but with a corresponding fitness cost, of spore attachment on M. arenaria and M. luci. These results demonstrate that Pp host specificity is plastic and capable of rapid selection-driven changes in attachment patterns, although such shifts are accompanied by fitness trade-offs. Full article

CAR-T cell therapy has shown substantial efficacy in haematological malignancies, but its application to solid tumours remains limited by poor effector-cell infiltration, functional exhaustion, antigenic heterogeneity, and an immunosuppressive microenvironment. In this study, we develop a new spatiotemporal mathematical model of CAR-T therapy for solid tumours that integrates these resistance mechanisms within a single reaction–diffusion framework. The model is formulated as a system of partial differential equations describing functional and exhausted CAR-T cells, antigen-positive and antigen-low tumour subpopulations, and chemokine, immunosuppressive, and hypoxic fields. Steady-state analysis and finite-difference simulations showed that therapeutic outcome is governed by the interplay between CAR-T cell infiltration, exhaustion, and antigen escape. The model reproduces partial tumour regression followed by residual tumour persistence, therapy-driven enrichment of antigen-low cells, and reduced efficacy under stronger immunosuppressive and hypoxic conditions. In the combination therapy scenario considered here, repeated simulated CAR-T cell administration together with attenuation of the suppressive microenvironment improves tumour control. The proposed model provides a mechanistic basis for analysing resistance and for future optimisation studies of CAR-T therapy in solid tumours. Full article

Skeletal muscle differentiation is tightly regulated by membrane potential dynamics and voltage-dependent ion channel activity. Potassium (K⁺) and calcium (Ca²⁺) currents cooperate to orchestrate the transition of myoblasts into fusion-competent myotubes, and alterations in this process are associated with dystrophic phenotypes. Here, we investigated the electrophysiological remodeling accompanying C2C12 myogenesis and the modulatory effects of the polyphenol resveratrol (RES) on calcium voltage-gated channel subunit alpha 1 S (CACNA1S, Cav1.1, L-type) currents. Whole-cell patch-clamp recordings were performed in proliferating and differentiating C2C12 cells to characterize the temporal expression of K⁺ currents and voltage-dependent Ca²⁺ channels (VDCCs). During differentiation, three electrophysiological subpopulations were identified according to K⁺ current profiles: SK4+/EAG−/Kir−, SK4−/EAG+/Kir−, and SK4−/EAG+/Kir+. This sequence paralleled a progressive membrane hyperpolarization from −20 mV to −70 mV, consistent with the physiological maturation of myogenic cells. In C2C12 myocytes, nimodipine-sensitive L-type currents were the only Ca²⁺ conductance observed. Their activation threshold (~−30 mV) and half-activation voltage (V/2 ≈ −12 mV) indicated the co-expression of embryonic and adult Cav1.1 isoforms. Exposure to RES (30 µM, 48 h) produced a depolarizing shift in activation (ΔV/2 ≈ +9 mV) and a reduction in current amplitude across all voltages, consistent with a transition toward the adult splice variant of Cav1.1. These findings suggest that RES promotes electrophysiological maturation of skeletal muscle cells by modulating calcium channel expression and gating behavior. Given its known ability to correct splicing abnormalities in CACNA1S and related genes, resveratrol emerges as a promising pharmacological agent for restoring calcium homeostasis in neuromuscular disorders such as myotonic dystrophy type 1 (DM1). Full article

Background: The prevalence of severe aortic stenosis (AS) is increasing, in accordance with a longer life expectancy. Aortic valve calcification is a multifactorial pathological process involving a complex interplay between different types of regenerative cellular and genetic factors. Among these cells, endothelial progenitor cells (EPCs) and their osteoblastic phenotype subpopulation (EPC-OCNs) have been implicated in vascular remodeling and disease progression. Objectives: To assess longitudinal changes in EPC and EPC-OCN levels in patients with severe symptomatic AS undergoing transcatheter aortic valve implantation (TAVI). Methods: In this prospective observational study, 65 patients with severe AS undergoing TAVI were enrolled. Circulating EPC and EPC-OCN levels were quantified by flow cytometry before the procedure, at 4 ± 1 days, and at 90 ± 29 days after TAVI. EPCs were defined by expression of CD133, CD34, and VEGFR-2. Results: Circulating EPC levels remained unchanged throughout the follow-up. In contrast, circulating EPC-OCNs increased significantly over time. Specifically, CD133+/VEGFR-2+/OCN+ cells rose from 2.50% to 6.25%, CD34+/VEGFR-2+/OCN+ from 2.04% to 4.05%, and VEGFR-2+/OCN+ from 1.46% to 3.01% (all p < 0.01). This suggests an osteogenic response to TAVI, while classical endothelial repair mechanisms were not systemically activated. Conclusions: EPC-OCNs increased significantly following TAVI, possibly reflecting ongoing tissue remodeling or calcification processes. In contrast, the stability of classical EPCs levels suggests limited systemic endothelial regeneration. These observations underscore the potential role of EPC-OCNs as markers or modulators of pre- and post-TAVI vascular remodeling. Full article

Intermediate monocytes (CD14⁺⁺CD16⁺), a highly pro-inflammatory subset, are linked to endothelial activation, thrombus formation, and poor outcomes in acute coronary syndrome (ACS), suggesting a role in the transition to plaque vulnerability. MicroRNA-92a (miR-92a) promotes vascular inflammation by repressing the transcription factors Kruppel-like factors (KLFs) 2/4, thereby inducing endothelial dysfunction and increasing leukocyte adhesion. Because both intermediate monocytes and miR-92a contribute to plaque instability, their expression profiles appear relevant in acute ischemia. We investigated whether miR-92a is differentially regulated in monocyte subpopulations in ACS compared to chronic coronary syndrome (CCS). Patients with ACS (STEMI/NSTEMI) undergoing urgent coronary angiography and patients with CCS were enrolled. Blood samples were collected peripherally (T_0P) and from the culprit coronary artery (T_0C) during catheterization. Additional peripheral samples were collected 48 h after intervention (T₁) and at the 3-month follow-up (T₂). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density-gradient centrifugation. Monocytes were sorted by fluorescence-activated cell sorting (FACS) into classical (CD14⁺⁺CD16^–), intermediate (CD14⁺⁺CD16⁺), and non-classical (CD14⁺CD16⁺⁺) subsets. MiR-92a expression was measured using real-time PCR and analyzed across predefined time points. In classical and non-classical monocytes, miR-92a levels remained stable throughout the observation period and did not differ between ACS and CCS patients. No spatial expression gradient was observed between intracoronary and peripheral samples at baseline. In contrast, intermediate monocytes in the ACS cohort showed a transient increase in miR-92a expression at T₁ compared with baseline (T_0p) and the 3-month follow-up (T₂). No comparable temporal changes were observed in CCS patients. These findings indicate a temporary alteration of miR-92a expression in intermediate monocytes during the early post-interventional phase following ACS. However, given the exploratory nature of this study and the limited sample size, the biological significance of this observation requires confirmation in larger cohorts. Full article