搜索结果：Journal of molecular cell biology

Introduction: Lung cancer, the leading cause of cancer-related mortality worldwide, is a heterogeneous malignancy comprising distinct histological and molecular subtypes, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of cases and adenocarcinoma (ADC) representing the most prevalent histotype. An emerging pathological feature of NSCLC, spread through air spaces (STAS)-defined as the extension of tumor cells into the lung parenchyma beyond the main tumor margin-has been associated with worse disease-free and overall survival and has been proposed as a possible predictor of recurrence to guide surgical extent. Concurrently, recent comprehensive genomic profiling of early-stage NSCLC has highlighted the need to interpret multi-omics data and their relationship with pathological variables, including IASLC histological subtypes, to better personalize treatment strategies. In this context, we investigated the overall distribution of STAS and its association with tumor mutational profiles and IASLC histological subtypes in a large real-world cohort of lung adenocarcinoma patients from the LANTERN project. Materials and Methods: In a prospective, multicenter observational study (March 2023-December 2024), 271 NSCLC patients were enrolled, and clinicopathological, immunohistochemical, and genomic data were collected; comprehensive genomic profiling was performed using the TruSight Oncology 500 assay to analyze 523 cancer-related genes, tumor mutational burden (TMB), and microsatellite instability; and STAS was assessed according to IASLC criteria. Adenocarcinoma accounted for roughly 90% of the cases, with a median age of 69 years and a predominance of stage IV disease (49.5%). STAS was evaluable in 162 cases and was detected in 17.9% of tumors. Results: STAS-positive tumors showed a higher trend towards locally advanced and advanced disease; no differences were observed in sex, age, smoking status, tumor mutational burden, or PD-L1 expression. Additionally, STAS-positive tumors showed a higher association with micropapillary, mucinous, and papillary patterns, whereas the acinar pattern was more frequent in STAS-negative tumors. The most frequently mutated genes were TP53, KRAS, EGFR, and STK11, with no significant differences between groups; ROS1 alterations were absent in STAS-negative tumors but detected more frequently in STAS-positive cases. Conclusions: Overall, these findings indicate that STAS positivity is associated with high-risk histological subtypes and advanced disease, suggesting its importance as a marker of tumor aggressiveness and emphasizing the need for its systematic evaluation in lung adenocarcinoma to better guide surgical planning and patient risk assessment.

Human CD4+ T cells utilize nutrients, including lipids, to support their activation and polarization. Considering the pivotal role of lipoproteins in lipid transport, we reasoned that lipoprotein uptake and processing could effect CD4+ T cell function. Here, we demonstrate that activation of human CD4+ T cells induced expression of LDL receptor (LDLR) to facilitate LDLR-mediated endocytosis of LDL. Degradation of surface LDLR on CD4+ T cells with PCSK9 hampered activation and proliferation of the cells. Lipoprotein deprivation or blocking of lysosomal cholesterol egress impaired activation of mechanistic target of rapamycin complex 1 (mTORC1), affecting CD4+ T cell activation and proliferation. Furthermore, lipoprotein deprivation of cultured primary CD4+ T cells lead to reduced expression of c-MAF and FOXP3, key transcription factors for IL-10, accompanied by reduced IL-10 secretion. The pivotal role of LDLR-mediated lipoprotein uptake for mTORC1 activity, c-MAF and FOXP3 expression, and IL-10 secretion was confirmed using LDLR-dysfunctional CD4+ T cells from patients with homozygous familial hypercholesterolemia. Our study offers valuable insights into the lipoprotein metabolism of human CD4+ T cells and their reliance on the LDLR pathway for activation and polarization, a feature that may be leveraged to modulate CD4+ T cell function.

The 2023 iteration of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) estimated prevalence, incidence, and health burden for 375 diseases and injuries, including 12 mental disorders. We assess past, current, and emerging trends in the prevalence and burden of mental disorders across sexes and age groups, for 21 regions, 204 countries and territories, and by Socio-demographic Index (SDI) quintile, from 1990 to 2023. Mental disorders included in GBD 2023 were anxiety disorders, major depressive disorder, dysthymia, bipolar disorder, schizophrenia, autism spectrum disorders, conduct disorder, attention-deficit hyperactivity disorder, anorexia nervosa, bulimia nervosa, idiopathic developmental intellectual disability, and a residual category of other mental disorders. A literature review identified epidemiological data for each disorder. These were analysed via a Bayesian meta-regression to estimate prevalence by disorder, sex, age, location, and year. Disorder-specific prevalence was multiplied by disability weights representing the severity of health loss associated with each disorder to estimate years lived with disability (YLDs). Deaths due to anorexia nervosa were assessed with a Cause of Death Ensemble modelling strategy to estimate deaths by sex, age, location, and year, and then multiplied by the standard life expectancy at age of death to estimate years of life lost (YLLs). YLDs equalled disability-adjusted life-years (DALYs) for all mental disorders except anorexia nervosa (the only mental disorder considered as an underlying cause of death in GBD), for which DALYs represented the sum of YLDs and YLLs. We presented prevalence, deaths, YLDs, YLLs, and DALYs as counts, age-specific rates per 100 000 population, and age-standardised rates per 100 000 population. We estimated 1·17 billion (95% uncertainty interval 1·06-1·31) prevalent cases of mental disorders globally in 2023, equivalent to an age-standardised prevalence rate of 14 210·7 cases (12 849·5-15 940·1) per 100 000 population. These estimates represented a 95·5% (75·0-121·2) increase in prevalent cases and 24·2% (11·4-41·4) increase in age-standardised prevalence rate between 1990 and 2023. All mental disorders showed increases in prevalent cases between 1990 and 2023, while notable increases were seen in age-standardised prevalence rates for anxiety disorders, major depressive disorder, dysthymia, anorexia nervosa, bulimia nervosa, schizophrenia, and conduct disorder. There were an estimated 171 million (127-228) DALYs due to mental disorders globally across sex and age in 2023, equivalent to an age-standardised DALY rate of 2070·5 DALYs (1519·1-2750·5) per 100 000 population. Mental disorders contributed to 6·1% (4·8-7·6) of all-cause DALYs in 2023, making them the fifth leading cause of global DALYs (up from 12th in 1990). DALYs were almost entirely composed of YLDs. Mental disorders were the leading cause of YLDs in 2023 (up from second in 1990), explaining 17·3% (14·8-20·6) of all-cause global YLDs. Leading causes of mental disorder DALYs were anxiety disorders (ranked 11th among the 304 diseases and injuries at Level 4 of the GBD cause hierarchy), major depressive disorder (15th), and schizophrenia (41st). Globally in 2023, mental disorder age-standardised DALY rates were higher among females (2239·6 [1643·7-3014·1] per 100 000) than among males (1900·2 [1399·8-2510·8] per 100 000), and peaked in the 15-19 years age group (2617·3 [1850·6-3696·8] per 100 000). All locations showed increased mental disorder DALY rates in 2023 compared with 1990, ranging across countries and territories from 1302·4 (952·7-1683·7) per 100 000 in Viet Nam to 3555·8 (2661·9-4715·0) per 100 000 in the Netherlands. Across SDI quintiles, DALY rates ranged from 1853·0 (1352·1-2469·3) per 100 000 for middle SDI to 2184·1 (1606·1-2890·3) per 100 000 for high SDI. A significant health burden was imposed by mental disorders in all countries and territories in 2023, irrespective of the health resources available. In some instances, this burden has increased over time and is unevenly distributed across populations. Stronger surveillance systems, particularly in low-income and middle-income countries, are required. Additionally, we need more coordinated and inclusive policies to reduce the burden through early treatment and prevention, tailored to sex and age differences across locations. Responding to the mental health needs of our global population, especially those most vulnerable, is an obligation, not a choice. Gates Foundation, Queensland Health, and University of Queensland.

The chimeric protein p210 BCR-ABL is a major causative factor of chronic myeloid leukemia (CML). Previously, we found that p210 BCR-ABL translocates from the cytosol to the mitochondria upon mitochondrial damage via the interaction of its pleckstrin homology domain (p210-PH) with cardiolipin (CL), a mitochondria-specific phospholipid. However, the precise pathological functions of this event are unknown. Here, using multivalent peptide library screens, we identified a tetravalent peptide, WDD-R4-tet, which binds to the CL-binding region of p210-PH and inhibits the translocation of p210 BCR-ABL to the mitochondria. Notably, WDD-R4-tet induced the apoptosis of CML cells by specifically suppressing the expression of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2), ubiquitin ligases with anti-apoptotic functions, leading to the activation of caspases. Other compounds that inhibited cIAP1/2 also efficiently inhibited the proliferation of CML cells. Thus, WDD-R4-tet might be a novel therapeutic agent for CML, which functions by inhibiting novel cell-survival signaling pathways generated on the mitochondrial outer membrane of CML cells.

To mount a robust T cell-dependent immune response, antigen-specific B lymphocytes require the stimulation of the transmembrane receptor CD40 through immune synapse formation with CD4+ T follicular helper cells. CD40 stimulates the activation of mammalian target of rapamycin complex 1 (mTORC1) and remodels mitochondria to meet the increased bioenergetic and anabolic demands of activated B cells. Here, we found that diacylglycerol kinase ζ (DGKζ) supported mTORC1 activation downstream of CD40 stimulation in mouse B cells. We showed that DGKζ was required for organellar translocation to the CD40-mediated immune synapse and for the recruitment of mTORC1 to lysosomes, the latter of which was necessary for mTORC1 activation and function. The production of phosphatidic acid by DGKζ was crucial for these processes. DGKζ-/- B cells exhibited defects in protein biosynthesis, metabolite transporter expression, and cell cycle progression, together with dysregulation of the transcriptional network that determines B cell fate. To sustain their bioenergetic and metabolic demands, DGKζ-/- B cells enhanced their mitochondrial function. Together, these effects of DGKζ loss led to decreases in germinal center responses and in the generation of long-lived plasma cells and memory B cells in mice. Thus, our data identify DGKζ as an essential mediator of CD40 functions in the B cell immune response.

Perineural invasion (PNI) is an aggressive feature in head and neck squamous cell carcinoma (HNSCC), but its molecular basis and neuroimmune implications, including potential links to immunotherapy response, remain unclear. We performed an integrative multi-omics analysis using public datasets and an independent clinical cohort. Transcriptomic, proteomic, single-cell, and spatial transcriptomic data were jointly analyzed to identify PNI-associated molecular patterns, construct prognostic signatures, and characterize immune infiltration and cell-cell communication. Regulatory elements were further explored by super-enhancer mapping and target-gene prediction. Pharmacologic inhibition experiments using the TLR2 inhibitor C29 were performed in FaDu cells for functional validation. PNI was associated with extracellular matrix and neuroactive signaling changes. A protein-based signature (ADIPOQ, MB, PLIN1, ADH1B) stratified survival risk. PNI-positive/high-risk tumors showed an immune-suppressed phenotype with lower predicted immunotherapy sensitivity and reduced CD8+ T-cell, B-cell, and Tfh-cell infiltration. Spatial analysis showed higher PNI scores at the invasive front, positive correlations with neural programs, and enrichment of TLR2-related signaling. TLR2 expression was associated with the PNI score, neural markers, and immune exclusion. In FaDu cells, C29 suppressed proliferation, migration, and invasion. Super-enhancer analysis identified candidate SE-target genes, including MYL4, CMYA5, and TNNT3, linked to PNI-associated biology. PNI in HNSCC is associated with coordinated extracellular-matrix, neuroimmune, and immune-suppressive remodeling. These findings support PNI-related molecular signatures for risk stratification and identify TLR2-related signaling and SE-associated programs as candidate mechanisms for further study.

Homologous recombination repair (HRR) deficiency is associated with improved immunotherapy responses in non-small cell lung cancer (NSCLC) patients. The HRR genes BRCA1/2 are key regulators of DNA repair, yet their impact on the tumor microenvironment (TME) in lung adenocarcinoma (LUAD) remains unclear. Using single-cell sequencing and multi-omics data, we characterized BRCA1/2 mutation-associated transcriptional programs, immune cell composition, and functional alterations in T cells, investigating the molecular and immune architecture of BRCA-mutant LUAD patients. BRCA1/2 mutations were associated with increased genomic instability and poor prognosis in LUAD patients, but predicted better clinical outcomes following immune checkpoint blockade (ICB) treatment. BRCA1 mutations correlated with an upregulated type I IFN/IFN-γ signature and CD8+ T cell activation. BRCA2 mutations were associated with alveolar/stress/inflammatory responses and enhanced MHC-II antigen presentation, linked to CD4+ T cell differentiation. Both alterations coincided with reduced CD28 co-stimulation and CTL activity, hinting at immune evasion. We identified two tissue-resident memory T cell (Trm) subsets as predictors of clinical outcomes and ICB response. BRCA1 mutations were associated with CD8+ Trm expansion, whereas BRCA2 mutations were linked to tumor CD4+ Trm expansion and peripheral T/NK cell cytotoxicity. Furthermore, a cancer-promoting program activated by BRCA1 mutation was vulnerable to histone deacetylase inhibitors, which inhibited LUAD tumor growth. This study provides a preliminary characterization of the BRCA-mutant TME in LUAD patients, revealing distinct transcriptional and immune patterns that highlight differences in BRCA1/2-associated molecular architecture and offer a framework for improving therapy efficacy in LUAD.

Cells are extraordinary biochemical systems that have evolved over billions of years into highly sophisticated units of life. Bottom-up synthetic biology seeks to reconstruct cell-like systems from non-living molecular components, producing artificial cells that capture essential cellular features while bypassing the complexity and fragility of living organisms. This approach offers a unique perspective on the organizational principles underlying cellular life and provides a platform for diverse biotechnological applications. In this review, we survey recent advances in bottom-up artificial cell design, covering five principal scaffold materials including the newly prominent coacervate-based and hybrid hierarchical compartments, and four canonical functional categories: cascade metabolism, protein synthesis, division, and energy production. We further discuss the expanding application landscape spanning industrial biocatalysis, therapeutic protein delivery, biosensing, and origins of life research. Finally, we critically evaluate the key technical limitations currently facing the field, including module compatibility, operational stability, and regulatory challenges, and outline the directions that must be pursued to advance artificial cells toward practical realization.

The molecular mechanisms underlying metastasis still remain unclear. We previously established a suspension culture using low-attachment culture dishes and demonstrated that cell lines adapted through 2 months suspension culture (termed FL sublines) exhibited higher metastatic potential than their parental counterparts. In this study, we identified the molecules involved in acquiring these phenotypes under low-adhesion conditions. We showed that detached tumor cells in suspension culture formed spheroids that recapitulated tumor cells in the spread-through-air space (STAS), and demonstrated that the anti-adhesion molecule mucin 21 was upregulated in detached lung cancer cells independent of driver mutations. Analyses of both cell lines and primary tumors revealed that mucin 21 is overexpressed in terminal respiratory unit-type lung adenocarcinomas. Mucin 21-knockout cells showed reduced viability and proliferation under adherent and low-attachment conditions, accompanied by enhanced anoikis. Transmission electron microscopy revealed that the intercellular spaces observed in FL sublines during suspension culture were reduced in mucin 21-knockout cells, suggesting impaired acquisition of low adhesive properties. Thus, mucin 21 appears crucial for the survival of terminal respiratory unit-type lung adenocarcinoma cells under both adherent and low-adhesion conditions.

Autophagy, a vital catabolic process, plays a crucial role in maintaining pancreatic β cell function and is disrupted in established type 1 diabetes. However, it is unclear when and how this critical cell process becomes defective during type 1 diabetes pathogenesis. To study the nature of autophagy dysfunction in the context of autoimmune diabetes, we used real-time intravital microscopy to study autophagic flux in vivo. We generated an AAV8-packaged mCherry-eGFP-LC3B biosensor driven by the insulin promoter for β cell-selective expression. For real-time autophagic flux evaluation, fluorescent signals from eGFP and mCherry fluorophores were correlated in space and time to follow the process of autophagosome-lysosome fusion. We observed autophagic flux defects in the β cells of the nonobese diabetic (NOD) mouse model of type 1 diabetes before hyperglycemia onset at both baseline and in response to interferon-α. These defects were still present, although less apparent, in immunodeficient NOD/scid/il2rg (NSG) mice. We also observed heterogeneous autophagic flux in human donor islets transplanted under the kidney capsules of NSG mice. In sum, the ability to visualize autophagic flux in β cells over time in vivo revealed impairments in those β cells that preceded the onset of autoimmune diabetes.

In head and neck squamous cell carcinoma (HNSCC), an immunosuppressive tumor microenvironment (TME) frequently leads to natural killer (NK) cell dysfunction, but the molecular mechanisms underlying this impairment remain unclear. Here, we combined flow cytometry and multiplex immunohistochemistry to evaluate NK cells infiltration and functional status in HNSCC tumors. Bulk RNA-seq datasets from patient cohorts and single-cell RNA-seq data were analyzed to identify transcriptional correlates of NK cells dysfunction. Mechanistic studies in tumor cell lines assessed the role of osteopontin (OPN)-integrin αvβ3/NF-κB signaling in modulating TGF-β1 production and its effects on NK cell activity. NK cells in HNSCC tumors exhibited reduced infiltration and diminished activation marker expression, coinciding with high OPN levels in the TME. Transcriptomic analyses demonstrated a strong inverse correlation between tumor SPP1 (encoding OPN) expression and NK cells abundance. Mechanistically, tumor-derived OPN engaged integrin αvβ3 to activate NF-κB signaling, inducing robust TGF-β1 secretion that suppressed NK cell activation and cytotoxic function. Single-cell RNA-seq analysis further confirmed the exhausted functional states of NK cells within the HNSCC TME. Tumor-derived OPN promotes immune evasion in HNSCC by driving TGF-β1-mediated suppression of NK cell activity. These findings highlight the OPN-TGF-β1 axis as a potential therapeutic target for restoring NK cell-mediated immunity in HNSCC.

Cervical cancer remains one of the most prevalent malignancies among women worldwide, with persistent infection by high-risk human papillomavirus (HPV), particularly types 16 and 18, as the principal etiological factor. The viral oncoproteins E6 and E7 are well-established drivers of cellular transformation and the maintenance of malignant phenotypes. This study aimed to generate T-cell receptor-like antibodies (TCR-like antibodies) capable of recognizing HPV16-derived E6 and E7 peptides presented by the major histocompatibility complex class I molecule HLA-A2402, with the goal of developing a diagnostic platform for early detection of HPV16-associated disease. Peptide-major histocompatibility complex (P-MHC) antigenic complexes were constructed and characterized using molecular docking, followed by experimental validation. A fully human domain antibody phage display library was screened against these complexes through iterative biopanning, supported by polyclonal and monoclonal enzyme-linked immunosorbent assays (ELISA). Structural evaluation and binding affinity analyses were performed using molecular docking, Western blotting, TCR-like antibody ELISA, and cell-based ELISA. Two novel TCR-like antibodies, designated D2\Ab and A12\Ab, were identified and demonstrated strong and specific binding to HPV16 E6 and E7 P-MHC complexes, confirming high affinity and antigen specificity. Functional validation across multiple assays demonstrated selective recognition of target complexes without reactivity toward irrelevant controls. In conclusion, this study demonstrates the feasibility of generating fully human TCR-like antibodies for HPV-associated malignancies and highlights their potential application in early detection and targeted diagnostic strategies for cervical cancer.

Loss of islet beta cell function and mass are critical in the pathogenesis of type 2 diabetes, a disease in which ~90% of individuals exhibit islet amyloid deposition. Amyloid deposits comprise the normal beta cell secretory product, human islet amyloid polypeptide (hIAPP), the aggregation of which is toxic to beta cells. While the underlying mechanism(s) for toxicity remain unknown, it is likely to involve mitochondrial dysfunction. We have shown that the mitochondrial cholesterol transport protein, steroidogenic acute regulatory protein (StAR), is upregulated in beta cells following amyloid deposition. Here, we examined the role of StAR in the toxicity of islet amyloidosis. Human islets from non-diabetic donors were cultured under amyloidogenic conditions and StAR expression was examined. StAR expression was also determined in islets isolated from transgenic mice expressing amyloidogenic hIAPP or non-transgenic littermates expressing non-amyloidogenic islet amyloid polypeptide, cultured under amyloidogenic conditions with or without the addition of an amyloid inhibitor. Total islet cholesterol content, mitochondrial cholesterol content, mitochondrial function and cell viability/death were compared in transgenic hIAPP and non-transgenic islets cultured in amyloidogenic conditions. Additionally, StAR localisation to islet cells, as well as its intracellular localisation, was examined. StAR was present in human islets at the mRNA and protein level, and expression increased significantly with amyloid formation in vitro. Further, in hIAPP transgenic mouse islets, StAR expression was amyloid-dependent. StAR was predominantly expressed in beta cells, and the amyloid-induced increase in StAR protein was found specifically in the mitochondrial fraction. While total and mitochondrial cholesterol content was unchanged between non-transgenic and hIAPP transgenic mouse islets cultured under amyloidogenic conditions, increased StAR expression was associated with decreased mitochondrial glucose-stimulated respiration and increased cell death. These findings are consistent with StAR having a pathophysiological role in the beta cell in type 2 diabetes, where its upregulation under conditions of islet amyloid deposition could contribute to mitochondrial dysfunction.

Paternal environmental factors can influence offspring development and health through sperm-carried epigenetic information, but the underlying mechanisms are still not fully understood. This study aims to investigate whether paternal vitamin D deficiency can modify the sperm-derived miRNAs and whether it affects the testicular development of the offspring, and to explore the potential molecular mechanisms. Four-week-old male C57BL/6J mice were fed with vitamin D-sufficient or -deficient diet for 16 weeks, then mated with healthy female mice to obtain F1 offspring. High-throughput sequencing of paternal sperm miRNAs and bioinformatics analysis were performed. All offspring were raised to adulthood under standard feeding conditions, and their body weight, reproductive organ development, testicular histomorphology and molecular characteristics were systematically evaluated. Paternal vitamin D deficiency induced significant alterations in the sperm miRNA expression profile, with a total of 16 differentially expressed miRNAs being identified. The target genes of these miRNAs were found to be enriched in pathways related to oxidative stress and fibrosis. Compared to the control group, the male offspring of VDD father group exhibited decreased body weight and testicular weight, accompanied by abnormal testicular tissue structure. At the molecular level, the expression of antioxidant-related genes in the offspring testes was down-regulated, while the TGF-β/SMAD2 signaling pathway and fibrosis markers were significantly up-regulated, suggesting enhanced oxidative stress and activation of the fibrotic remodeling. This study suggests that paternal vitamin D deficiency may reshape the oxidative stress and fibrosis-related pathways in offspring's testes accompanied by altering sperm miRNA-mediated epigenetic information.

Periodontal diseases have been associated with several systemic conditions, including certain types of cancer. Chronic inflammation, a characteristic feature of periodontal disease, has been hypothesized to contribute to carcinogenesis and tumor progression. However, the specific relationship between Aggregatibacter actinomycetemcomitans and cancer development remains an area of ongoing research. The aim of the study was to investigate the potential association between A. actinomycetemcomitans, a well-established periodontal pathogen, and the development of head and neck squamous cell carcinoma (HNSCC). Additionally, the objective was to examine differentially expressed genes (DEGs) in A. actinomycetemcomitans-infected human immortalized gingival keratinocytes (HIGKs) and compare their expression in the HNSCC dataset using computational tools. The Gene Expression Omnibus (GEO) dataset GSE9723 was used to retrieve the global gene expression profile of human gingival keratinocytes exposed to A. actinomycetemcomitans. A computational approach was employed using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System for gene ontology analysis, STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) for network analysis and UALCAN (University of ALabama at Birmingham CANcer data analysis) for gene expression and survival analyses. The GEO2R analysis identified numerous DEGs, of which the top 25 were selected for further evaluation using the HNSCC dataset from The Cancer Genome Atlas (TCGA). Among these genes, DDIT4 and RCE1 demonstrated expression patterns consistent with those observed in A. actinomycetemcomitans-infected HIGKs. Further analysis revealed that patients presenting with elevated DDIT4 and RCE1 expression levels demonstrated a marked reduction in overall survival. The findings suggest that DDIT4 and RCE1 can be considered potential therapeutic targets associated with A. actinomycetemcomitans-mediated induction of head and neck cancers. However, further studies are required to validate these observations.

Advanced non-small cell lung cancer (NSCLC), particularly lung adenocarcinoma, remains a leading cause of cancer-related mortality. Although molecular profiling has improved disease stratification, most biomarker assays rely on invasive tissue sampling. Exhaled breath condensate (EBC) is an attractive non-invasive biofluid that may provide clinically relevant molecular information. This study evaluated whether cell-free RNA (cfRNA) obtained from EBC can capture diagnostically and prognostically informative gene-expression changes in advanced lung adenocarcinoma. In this prospective study, 40 patients with histologically confirmed stage IIIB-IV lung adenocarcinoma and 20 healthy controls were enrolled. EBC samples were collected from all participants, and plasma samples were obtained from a subgroup of 10 patients and 10 controls. Expression of EGFR, FGFR2, PIK3CA, PTEN, SMAD4, STK11, and TP53 was quantified by RT-qPCR and normalized to GAPDH using the 2-ΔΔCT method. Receiver operating characteristic (ROC) analysis, correlation testing, and Kaplan-Meier survival analysis were performed. The most clinically relevant findings were observed for PIK3CA, FGFR2, and EGFR. In EBC, PIK3CA emerged as the only gene with significant diagnostic performance for distinguishing patients from healthy controls (AUC = 0.8522; sensitivity 81.58%; specificity 76.92%). In survival analyses, high FGFR2 and high PIK3CA expression were associated with significantly shorter overall survival, supporting their prognostic relevance. In treatment-stratified analysis, low EGFR expression was associated with longer survival in patients receiving chemotherapy alone, suggesting potential predictive value in this subgroup. In plasma, PIK3CA was upregulated approximately five-fold, whereas EGFR was downregulated approximately five-fold. Notably, EGFR expression showed a strong positive correlation between EBC and plasma (R2 = 0.7849, p = 0.0006), indicating cross-matrix consistency for this marker. By contrast, alterations in PTEN, TP53, and SMAD4 should be considered exploratory, as they showed differential expression but lacked comparable diagnostic, prognostic, or treatment-associated strength. EBC-based cfRNA profiling provides a feasible, non-invasive approach for molecular characterization of advanced lung adenocarcinoma. Among the genes examined, PIK3CA showed the strongest diagnostic signal, FGFR2 demonstrated prognostic significance, and EGFR showed the clearest cross-sample concordance and treatment-associated relevance. Larger independent validation studies are required before clinical implementation.

This study is aimed at systematically identifying key genes associated with EGFR mutations and developing a molecular classification model in lung adenocarcinoma (LUAD) using integrative bioinformatics approaches. Multi-omics datasets derived from cBioPortal and The Cancer Genome Atlas (TCGA) LUAD cohort were interrogated to identify genes correlated with EGFR mutational status. A core set of 18 genes exhibiting significant associations with both EGFR mutation frequency and patient prognosis was identified. Based on this gene signature, a two-cluster molecular subtype stratification was established. These subtypes demonstrated statistically significant divergence in overall survival, immune cell infiltration profiles, and predicted responsiveness to immunotherapeutic intervention. Further analyses, including machine learning algorithms, multivariate Cox regression, and molecular docking, identified TRAF2 as a key prognostic gene closely associated with EGFR. In vitro experiments demonstrated that TRAF2 promotes proliferation, migration, and invasion of LUAD cells. Additional analyses suggested that TRAF2 may contribute to tumor progression through epigenetic regulation and associated signaling pathways. Collectively, these findings provide novel insights into the molecular heterogeneity of EGFR-mutant LUAD and offer potential targets for precision prognostic assessment and combination therapeutic strategies.

Diabetic foot ulcers (DFUs) represent a severe chronic complication of diabetes and are characterized by persistent impairment of wound healing, accompanied by defective angiogenesis, chronic inflammation, and dysregulated extracellular matrix remodeling. Although impaired angiogenesis is widely recognized as a key pathological feature of DFUs, its associated molecular alterations have not been systematically characterized at the transcriptomic and cellular levels. In this study, bulk transcriptomic data were analyzed in combination with machine learning-based gene prioritization and single-cell RNA sequencing to investigate molecular features associated with angiogenesis impairment in DFUs. Differential expression analysis was performed using the GSE199939 and GSE134431 datasets, followed by GO and KEGG enrichment analyses. Angiogenesis-related genes were retrieved from the MSigDB HALLMARK_ANGIOGENESIS and GO:0001525, and intersected with the DEGs to generate a candidate gene set. A LASSO logistic regression model was then constructed in the discovery cohort and evaluated in a replication cohort, yielding a five-gene signature consisting of APLN, ENG, FN1, SERPINA5, and TIMP1. Single-cell transcriptomic data were subsequently used to examine the cellular expression patterns of these feature genes. Among the five feature genes, FN1 and TIMP1 showed relatively clear expression localization at the single-cell level. Single-cell RNA sequencing analysis revealed that FN1 was mainly enriched in fibroblasts and stromal-related cell populations, including pericytes/smooth muscle cells, whereas TIMP1 exhibited a multicellular expression pattern, with relatively high expression in fibroblasts, inflammatory myeloid cells, macrophages, and proliferating cells. In vivo experiments further showed that TIMP1 and EGFR mRNA expression levels were significantly decreased, whereas MMP9 mRNA expression was significantly increased in wound tissues from the model group. FN1 mRNA showed a downward trend, although the difference did not reach statistical significance. This integrative bioinformatic analysis provides an exploratory characterization of molecular features potentially related to restricted angiogenesis and impaired repair in DFUs and suggests that TIMP1 may represent a more robust candidate linked to proteolysis-related dysregulation, whereas FN1 may more likely reflect stromal extracellular matrix remodeling.

The emergence of multidrug-resistant fungal pathogens from urinary tract infections (UTIs) poses a growing challenge in clinical settings. Here, we report a case of a complicated UTI caused by Nakaseomyces glabratus (Candida glabrata) that progressed to urosepsis, leading to the emergence of an isolate carrying simultaneous loss-of-function mutations in ERG3 and ERG11, and abrogated ergosterol biosynthesis. Together with a missense mutation in FUR1-likely responsible for 5-fluorocytosine resistance-this constellation confers resistance to all viable UTI antifungals: azoles, amphotericin B, and flucytosine. Engineered ERG3Δ + ERG11Δ strains recapitulated this multidrug resistance and revealed profound fitness costs that come with it, challenging the assumption that high-cost mutations are unlikely to persist during infection. Among fitness trade-offs, we detected collateral sensitivity to nitroxoline, a commonly used urinary tract antibiotic with potent antifungal activity and a unique mechanism of action. This study provides the first clinical evidence of an elusive mechanism of hyper-multidrug resistance in N. glabratus and highlights nitroxoline as a promising repurposing agent for treating multidrug-resistant fungal infections of the urinary tract. Evolutionary theory states that fitness determines survival. In a drug-treatment environment, resistance increases fitness, but it often comes at a cost, such as slower growth or reduced stress tolerance. If these costs are too severe, they can undermine virulence, making resistance unlikely to persist. Our study challenges this assumption. We describe the first clinical case of Nakaseomyces glabratus evolving multidrug resistance through loss-of-function mutations in ERG3 and ERG11, despite severe fitness trade-offs. This case suggests that certain infection niches, such as the urinary tract, can provide conditions where even highly impaired yet resistant strains persist under strong antifungal pressure. Importantly, we show that this extreme resistance induces collateral sensitivity to nitroxoline, a urinary tract infection antibiotic with potent antifungal activity and a unique mechanism of action. These findings open promising therapeutic avenues to counter multidrug-resistant fungal infections of the urinary tract.