Biomedicines

Chemobrain, characterized by cognitive dysfunction, has emerged as a significant health concern following cancer treatment. Doxorubicin, an FDA-approved chemotherapeutic agent, is widely used to treat various cancers. However, its adverse effects raise concerns regarding toxicity in non-cancerous cell types. Microglia, the resident immune cells of the brain, play a central role in maintaining neural homeostasis. Despite this, the mechanisms by which microglia become dysfunctional following doxorubicin exposure remain poorly understood. Understanding the transcriptomic alterations induced by doxorubicin in microglia could provide critical insights into mechanisms underlying its neurotoxic effects. In this study, the transcriptomic response of human microglia to doxorubicin using mRNA sequencing combined with bioinformatic analysis was investigated. Exposure to doxorubicin significantly altered microglial morphology, promoting a shift toward a process-bearing phenotype in human HMC3 cells. This phenotypic change was accompanied by elevated levels of DNA damage and was associated with the induction of cellular senescence. At the gene regulation level, the Illumina-based transcriptomic analysis provided 25,834 gene expression profiling in which 5,669 were differentially expressed. The future analyses revealed that 3,431 (upregulated) and 2,238 (downregulated) were differentially expressed under the treatment of doxorubicin. Genes associated with the p53 signaling pathway and inflammatory responses were upregulated, whereas genes involved in translation, DNA replication, and cell-cycle progression were downregulated. This transcriptional dysregulation was accompanied by differential expression of key transcription factors. This study provides transcriptomic evidence elucidating the genotoxic response to doxorubicin in microglial cells, offering mechanistic insights into chemobrain and highlighting key molecular pathways as potential targets for therapeutic intervention.

ABSTRACT Background: The esophageal String Test (EST) is emerging as a minimally invasive alternative for assessing disease activity in eosinophilic esophagitis (EoE), yet the molecular composition of its protein content remains unknown. Objective: To comprehensively characterize the esophageal EST proteome, define an EoE-associated luminal protein signature, and evaluate its biological and clinical relevance. Methods: EST samples were obtained from patients with active EoE, healthy controls (C) and patients with gastroesophageal reflux disease (GERD). Label-free proteomic profiling was performed by liquid chromatography-tandem mass spectrometry. Differential protein accumulation, principal component analysis (PCA), pathway annotation, comparison with published esophageal tissue and salivary proteomes, receiver operating characteristic (ROC) analyses and correlations with clinical disease activity were performed. Results: More than 3,000 proteins were identified in esophageal EST samples. Global proteomic profiling clearly separated patients with EoE from both C and GERD, whereas only limited differences were observed between the latter two groups. This enabled the identification of an EoE-associated luminal protein signature comprising inflammatory, epithelial, and tissue remodeling proteins. Comparison with published proteomic datasets indicated that the EST samples a complex esophageal luminal protein microenvironment containing proteins represented in esophageal mucosa and saliva, together with inflammatory and extracellular components. Several individual proteins showed high discriminatory performance in this exploratory cohort for distinguishing EoE from non-EoE subjects, and multiple proteins correlated with eosinophil counts and endoscopic measures of disease activity. Conclusions: This study demonstrates that the EST provides access to a complex esophageal luminal protein microenvironment and establishes a molecular framework for its minimally invasive characterization in EoE. KEYWORDS: eosinophilic esophagitis, esophageal string test, esophageal luminal protein signature

Background Cardiac amyloidosis is an increasingly recognized cause of heart failure in older adults, yet Colombian data, and data from the southwest of the country in particular, remain scarce. We describe the clinical, diagnostic, imaging and outcome profile of patients with cardiac amyloidosis at two high-complexity institutions in Cali. Methods Observational, descriptive, retrospective, two-center study. Patients were identified in the institutional registries of both centers using ICD-10 codes for amyloidosis, and clinical data were abstracted manually from medical records. Continuous variables are reported as median (interquartile range) and categorical variables as counts and percentages over the number of patients with available data. Results Thirty-two patients were analyzed (median age 77 years; 68.8% men). Heart failure (90.6%), atrial fibrillation (46.9%) and carpal tunnel syndrome (31.2%) were frequent. The etiological classification depended on the test considered: bone scintigraphy was positive (Perugini 2-3) in 23 of 26 patients studied (88.5%), but a monoclonal protein was screened for in only 22, and the full non-invasive criteria for ATTR were met in 16 of 32 (50.0%). Genetic testing was positive in 9 of 18 tested, and the variant was Val142Ile in all 8 patients in whom it was characterized. The working diagnosis was ATTR in 26 patients (81.2%) and AL in 6 (18.8%). Tafamidis was used in 37.5%. Eleven of the 31 patients with known vital status died (35.5%), and NT-proBNP was markedly higher among those who died (6640 vs 1186 pg/mL; p=0.003). Conclusions In this southwestern Colombian cohort, cardiac amyloidosis presented as the classic transthyretin phenotype, with the Val142Ile variant in all 8 patients in whom it was characterized. Bone scintigraphy was frequently positive, but screening for a monoclonal protein was often omitted, so only half the cohort met the full non-invasive criteria for ATTR. Mortality was high and closely associated with NT-proBNP.

PURPOSEPancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with poor prognosis. A transcriptomic-based algorithm called Purity Independent Subtyping of Tumors (PurIST) relies on expression profiling of a defined set of genes to stratify PDAC into classical and basal-like molecular subtypes and assigns a confidence level to the subtypes as either "strong," "likely," or "lean." In this study, we apply PurIST to Appalachian and non-Appalachian patient cohorts and assess demographic variables between groups that may influence treatment outcomes. PATIENTS AND METHODSThis 13-site study includes 780 PDAC patients. Patients were divided into Appalachian and non-Appalachian cohorts according to Appalachian Regional Commission guidelines, and differences in PurIST classification, tobacco use, and alcohol use were analyzed. RESULTSIndividuals from Appalachian regions were more likely to receive a subtype classification from PurIST with "strong" confidence as opposed to "likely" or "lean" (P=0.0175; odds ratio=1.66). We did not observe any significant difference between basal and classical subtype classification in Appalachia (P=0.1457), yet an exploratory analysis showed that individuals aged [≥]85 were significantly more likely to have basal-like tumor classifications (P=0.005817; odds ratio=3.79). Appalachian patients were more likely to have a history of smoking (59.6% vs 49.9%; P=0.01756) and less likely to report alcohol consumption (50.5% vs 65.6%; P=0.000229). CONCLUSIONWe conducted the largest comparison of the PurIST algorithm in Appalachia to date and found PurIST is more likely to have higher confidence in molecular subtype classifications for Appalachian PDAC tumors compared to non-Appalachian tumors. If PurIST is to be considered as a prognostic or decision-making tool for PDAC patients, we must account for variability in different populations and model the likelihood that tumors will have a strong molecular signature.

Background: Epidemiological studies have highlighted an inverse association between cancer and Alzheimers disease (AD), but the mechanisms underlying this association are not currently understood. Amyloid precursor protein (APP) and its cleavage may represent one potential mechanism. Its importance has been heavily researched due to it giving rise to the production of amyloid-beta, a pathological hallmark of AD. APP, however, has not been extensively investigated in cancer. Methods: FFPE prostate tumour and adjacent benign tissue immunohistochemically stained for APP, androgen receptor and the insulin-like growth factor-I receptor (IGF1R). Normal prostate PNT2, androgen sensitive LNCaP and androgen insensitive PC3 in vitro were used to assess the effects of androgen, IGF-I and their inhibitors on APP and its cleavage; changes in protein abundance were measured by western blot; interactions between proteins using co-immunoprecipitation and effects on cell proliferation, apoptosis and cell signalling of APP silenced cells using a BrdU proliferation assay, Muse Cell Analyser and by TMT proteomic analysis respectively. Results: Data showed that APP was upregulated in prostate tumour tissue compared to adjacent benign. Furthermore, in cancer cell models, in contrast to normal prostate epithelial cells, it was observed that APP was critical for prostate cancer cell survival by interacting with androgen and IGF-I signalling pathways. Conclusions: Drugs that target APP have already been developed for AD and our data suggests they offer an exciting opportunity for repurposing in the treatment of prostate cancer.

Background: Growth differentiation factor 15 (GDF-15) has emerged as a prognostic biomarker in patients with non-ST-segment-elevation acute coronary syndrome (NSTE-ACS). Our study evaluated the temporal evolution of GDF-15 in the acute phase and its association with recurrent ischemic events. Methods: Serum samples were collected from patients with NSTE-ACS at admission and at 1 and 3 hours after admission. GDF-15 levels, along with other prognostic biomarkers, NT-proBNP and hs-cTnT, were measured. Spearman rank-correlation coefficients and linear regression models were used to identify the variables associated with GDF-15 levels at admission and with their temporal changes. Associations between GDF-15 and clinical outcomes were evaluated using Cox proportional hazards models. To assess incremental prognostic value, the areas under the curve (AUC) were calculated for each model. Results: GDF-15 levels showed a slight increasing trend within 3 hours of admission. The 1-hour change in GDF-15 was correlated with hs-cTnT changes. Increased 1-hour GDF-15 levels, compared with the admission levels, were associated with a higher risk of recurrent ischemic events during the 1-year follow-up (1.9% vs. 17.5%, P =0.019) and were an independent predictor after adjusting for established clinical variables and biomarkers (adjusted hazard ratio, 1.71 (1.28 to 228.75), P =0.03). Furthermore, serial measurements of GDF-15 provided additional prognostic information beyond traditional risk factors. Conclusions: Serial measurements of GDF-15 of NSTE-ACS patients provided independent and incremental prognostic value for predicting recurrent ischemic events. These findings support the potential utility of GDF-15 as a dynamic biomarker for early risk stratification in the acute management of NSTE-ACS.

Background: Determining the cause of death in electrocution is often challenging at autopsy, particularly when characteristic external electrical marks are absent. Reliable internal markers are scarce, and the neuropathological changes associated with electrocution remain underexplored. Objective: To identify and characterize histopathological alterations in the brain in cases of fatal electrocution and evaluate their diagnostic utility. Methods: A prospective observational study was conducted on 12 autopsy cases, including 7 confirmed electrocution deaths and 5 non-electrocution deaths (control). Brain tissue samples from multiple regions were processed using H&E and Golgi Cox staining. Histopathological parameters, including neuronal morphology, vascular changes, haemorrhage, and dendritic architecture, were evaluated. Results: Electrocution cases demonstrated neuronal destruction, axonal ballooning, focal hemorrhages, and dendritic disruption. Cerebellar involvement with Purkinje cell damage. Conclusion: Comprehensive neurohistological technique may helpful in evaluation neuronal damage in electrocution death. Neuropathological findings, particularly neuronal and dendritic injury, may serve as supportive evidence in determining cause of death when external signs are absent. Keywords: Electrocution, Neuropathology, Golgi Cox stain, Forensic histopathology, death.

Introduction: In hypertrophic cardiomyopathy (HCM), wall stress and subclinical myocardial injury related in part to pathological hypercontractility, which in turn are associated with worse outcomes. Cell-free DNA (cfDNA) is released from cardiomyocytes (cfDNA-CM) through irreversible cellular injury and death. We investigated the feasibility of quantifying cfDNA-CM and its relationship to HCM patient characteristics. Methods: We analyzed 57 plasma samples from stable patients with HCM and 10 plasma samples from patients with acute myocardial infarction (AMI). Plasma cfDNA was isolated from both groups. Whole genome bisulfite sequencing (WGBS) libraries were generated and sequenced with short-read sequencing. Methylation patterns were used for tissue deconvolution analysis and cell-specific cfDNA quantification. Results: Yields of cfDNA (p = 0.003) and cfDNA-CM (p = 0.002) fractions were higher in AMI controls than HCM, supporting the validity of the assay. Within the HCM cohort, the median cfDNA yield was 12.22 ng [IQR: 7.77, 18.94]. The cfDNA-CM fraction in this cohort ranged from 0% to 6.4% of total cfDNA. Higher cfDNA-CM tertiles were associated with higher body mass index (BMI, p=0.001), a greater prevalence of BMI 30 kg/m2 (p<0.001), and a higher prevalence of obstructive sleep apnea (p=0.038). No significant differences were observed in other demographic, genetic, and laboratory characteristics. Higher cfDNA-CM tertile was numerically associated with higher New York Heart Association (NYHA) functional class and left ventricular outflow tract (LVOT) obstruction, while circulating biomarkers of wall stress (NT-proBNP) and myocardial injury (troponin) did not differ across tertiles (p=0.83 and p=0.29, respectively). Conclusion: Isolation and quantification of cfDNA as well as detection of cfDNA-CM in chronic stable patients with HCM are feasible. Higher cfDNA-CM fraction was associated with increasing BMI and cardiometabolic comorbidity burden. The absence of association with traditional circulating biomarkers suggests that cfDNA-CM may reflect a distinct domain of myocardial health, injury, and adverse remodeling.

Roseoloviruses, notably human herpesviruses 6A and 6B (HHV-6A and HHV-6B), are neurotropic viruses implicated as agents in neurological disorders, including: epilepsy, multiple sclerosis, and chronic fatigue syndrome. However, the effects of roseolovirus infection on neuronal signaling and network activity are not characterized. This is, in part, due to the complexities of monitoring electrical activity in individual neurons and across neuronal connections during viral infection. This protocol describes a human induced pluripotent stem cell (iPSC)-derived neuronal culture system that employs multi-electrode array (MEA) recordings to study neurophysiological changes during viral infection. Two culture platforms are described: (a) NGN2-induced forebrain neuronal cultures; and, (b) progenitor cell-derived neuron-astrocyte mixed cultures. Cell composition in cultures is validated by immunofluorescence staining using neuronal, glial, and viral markers. Functional activity is assessed using extracellular recordings detected via the MEA2100 system. Mean firing rate, inter-spike interval, single-electrode bursting, and network burst activity are characterized between roseolovirus-infected versus uninfected (control) states. Pharmacological treatment with bicuculline, gabazine, and nicotine in conjunction with immunofluorescence is used to confirm functional responsiveness of defined neuronal neurotransmitter chemotypes. Here, we show that HHV-6A infection changes neuronal firing compared to uninfected controls. The methods/workflows described provide a strategy to study how virus infection alters neuronal excitability and may be adapted to compare the effects of different viruses on nerve cell function, infection time courses, different multiplicities of infection (MOI), and therapeutic interventions.

Cytosolic phospholipase A2 (cPLA2), encoded by PLA2G4A plays a crucial role in platelet activation through thromboxane A2 biosynthesis. Defects in this pathway can lead to bleeding disorders; however, the precise role of cPLA2 in Glanzmann thrombasthenia (GT) or GT-like syndromes remains poorly understood. The current study emerged from a fortuitous discovery of novel PLA2G4A variants, p.Asp455Val (D455V) and p.Glu119Lys (E119K), among 3 GT patients from two unrelated families in south India. We envisaged a hypothesis to identify a hitherto unknown role of cPLA2 that could be attributed to GT-like bleeding symptoms in these patients. An extensive computational workflow, including variant classification, conservation analysis, stability prediction, molecular docking, molecular dynamics simulations, principal component analysis, and binding free energy (MMPBSA) calculations, were employed to assess mutation-induced alterations in the cPLA2 protein. The current findings depicted that both variants affected conformational dynamics, ligand recognition patterns, and local interaction networks, without causing any drastic global structural destabilization. In particular, the D455V partially disrupted the catalytic domain, potentially reducing substrate (PAPC) stabilization and hampering interactions with catalytic products. Likewise, the E119K variant partially affected the C2 domain, potentially altered electrostatic interactions and enhanced regional flexibility, which may compromise product (AA) stabilization. Further analyses revealed mutation-induced changes in collective motions, protein-ligand interactions, solvent accessibility, and secondary structural elements. Overall, our findings promote the hypothesis that both E119K and D455V variants may partially alter phospholipid-associated interactions, and cPLA2-mediated downstream signaling. Moreover, this study underscores cPLA2 as a potential modifier of platelet function and bleeding severity.

Chronic intermittent hypobaric hypoxia is an occupational exposure experienced by workers who alternate between high-altitude worksites and lower-altitude resting locations. Its effects on cardiac autonomic modulation and electrocardiographic parameters remain insufficiently characterized in Andean workers. This study aimed to compare heart rate variability and electrocardiographic parameters between workers exposed to chronic intermittent hypobaric hypoxia and controls working permanently at lower altitude in Arequipa, Peru. An observational cross-sectional study was conducted in 96 male workers aged 20-60 years. The exposed group included 53 workers performing rotational shifts at altitudes [&ge;]3,000 m above sea level with rest periods at <2,500 m, while the control group included 43 workers working permanently at <2,500 m. Heart rate variability was assessed during the morning rest period in Arequipa, at approximately 2,300 25 m above sea level, using a Polar(R) H10 heart rate sensor after 15 minutes of rest. Participants also underwent standard 12-lead electrocardiography. Vital signs were clinically similar between groups. No statistically significant differences were observed in PR interval, corrected QT interval, time-domain heart rate variability parameters, or frequency-domain heart rate variability parameters. In this sample of apparently healthy male workers evaluated during their rest period at moderate altitude, exposure to chronic intermittent hypobaric hypoxia was not associated with significant differences in resting heart rate variability or standard electrocardiographic parameters.

Objective To estimate the mortality proportion among patients with severe traumatic brain injury (sTBI) and to assess variation in mortality according to geographical region, study characteristics, patient populations, and mortality assessment time points. Design Systematic review and meta-analysis. Data sources PubMed/MEDLINE, Embase, Scopus, Web of Science, Google Scholar, and Cochrane Library were searched from database inception to January 2026. Eligibility criteria Studies reporting mortality among patients with severe TBI were eligible. Severe TBI was defined as a Glasgow Coma Scale (GCS) score of [&le;]8, equivalent to GCS <9 or GCS 3-8. Studies involving mixed TBI severity were included only when data for patients with severe TBI could be separately extracted. Data extraction and synthesis Two reviewers independently screened studies, extracted data, and assessed methodological quality. Mortality proportions were pooled using a random-effects model. Statistical heterogeneity was assessed using Cochran's Q and the I2 statistic. Subgroup and sensitivity analyses were undertaken where sufficient data were available. Results Twenty-five studies comprising 12,829 patients with severe TBI were included in the systematic review. Across studies, 5,550 deaths were reported. Mortality proportions ranged from 17.5% to 71.1%. The pooled mortality proportion was 40.6% (95% CI 35.7% to 45.7%), with substantial heterogeneity (I2=96.8%, p<0.001). Mortality varied considerably according to geographical region and timing of outcome assessment, with higher estimates generally observed in African settings and at longer follow-up periods. Conclusion Mortality among patients with severe TBI remains substantial, with approximately two in five patients dying across the included studies. However, the very high heterogeneity indicates that the pooled estimate should not be interpreted as a universal mortality risk. Differences in healthcare resources, patient characteristics, injury severity, management practices, and mortality assessment time points may contribute to the observed variation.

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Acute radiation syndrome remains a major consequence of high-dose ionizing radiation exposure, and effective pharmacological countermeasures are limited. Nitro-fatty acids possess anti-inflammatory and cytoprotective properties, suggesting potential effectiveness in radiation injury. Here, we investigated the radioprotective effects of 10-nitro-oleic acid (10-NO2OA) in mouse models of total body irradiation (TBI). C57BL/6 mice were exposed to either sublethal (4 Gy) or LD50-level (7.5 Gy) gamma irradiation and treated with 10-NO2OA according to pre-irradiation, post-irradiation, or combined treatment regimens. Survival, hematological responses, bone marrow cells (BMCs)/colony-forming units (CFU), plasma granulocyte colony-stimulating factor (G-CSF), NRF2-responsive signaling, and intestinal responses were evaluated. Administration of 10-NO2OA before and after irradiation significantly improved 30-day survival following 7.5 Gy TBI (57% vs. 20% in vehicle-treated controls). In contrast, post-irradiation treatment alone was ineffective, whereas pre-irradiation treatment alone produced a non-significant trend toward improved survival. The protective effect was absent in BALB/c mice, indicating strain dependence. Although 10-NO2OA did not prevent long-term radiation-induced cytopenia, it increased neutrophil and platelet counts, elevated plasma G-CSF levels shortly after irradiation, and transiently enhanced BMC counts. In irradiated BMCs, 10-NO2OA activated NRF2-responsive gene expression in vitro and in vivo. Exploratory intestinal analyses revealed improved macroscopic appearance and partial restoration of irradiation-suppressed cytokine and growth factor production. In conclusion, 10-NO2OA improves survival following LD50-level gamma irradiation and is associated with modulation of hematopoietic, cytoprotective, and intestinal responses. These findings identify 10-NO2OA as a promising candidate radioprotective agent and support a multifactorial mechanism of action.

Nerve Growth Factor (NGF) supports neuronal survival, plasticity, and growth by binding to p75NTR and TrkA receptors. There is a strong link between NGF and the cholinergic hippocampus, and changes in NGF pathways are observed in Alzheimer's disease (AD). Research shows NGF's neuroprotective and regenerative effects on cholinergic neurons, with positive results in AD models and clinical studies. However, NGF therapy faces challenges: limited blood-brain barrier crossing and dose-dependent pain. To address pain, a mutated NGF called CHF6467 was developed. Thanks to the R100E mutation, it prevents binding to p75NTR while maintaining TrkA affinity, which helps reduce pain side effects. Understanding CHF6467's mechanism remains a challenge. In this context, untargeted metabolomics is effective for uncovering unforeseen responses, making it suitable for assessing CHF6467's overall effects. Building on our previous research and recognizing the importance of amyloid-{beta} peptide as a critical pathological marker of AD, we used NMR-metabolomics to study the effect of CHF6467 on SH-SY5Y cells. The study examined metabolomic profiles of cells treated with CHF6467, with and without A{beta}(1-42), using NMR. Results show CHF6467 influences the metabolic changes caused by A{beta}(1-42), inducing biochemical effects consistent with the promotion of phospholipid precursors, reduced glutamate excitotoxicity, and restored energy metabolism.

Abstract Background: Chronic subdural hematoma (cSDH) remains a significant and increasingly prevalent cause of morbidity in older adults. Disease progression is sustained by de novo formed highly vascularized pseudomembranes and a persistent cycle of fluid accumulation and hematoma expansion. Although inflammatory and angiogenic pathways have been implicated in cSDH pathogenesis, the endothelial mechanisms underlying persistent vascular leak remain poorly understood. We investigated the cSDH vascular microenvironment in human patients and examined the role of vascular endothelial cadherin (VECAD) remodeling in cSDH endothelial dysfunction. Methods: Human cSDH membranes and fluid were evaluated by multiplex profiling, histology, immunofluorescence, immunoprecipitation, western blotting and exploratory bulk RNA sequencing. A human brain endothelial cell (HBEC) line was exposed to patient-derived cSDH fluid to assess VECAD remodeling, endothelial barrier permeability, and proliferation. Results: Multiplex profiling of patient-derived cSDH fluid detected mediators associated with endothelial cell (EC) activation, extracellular matrix remodeling, inflammation, and angiogenesis. In patient pseudomembranes, discontinuous VECAD coincided with erythrocyte extravasation. Two hyperdense (high-risk) specimens showed more extensive disruption than the hypodense (low-risk) specimen while vessel counts appeared similar. Bulk RNA sequencing identified an angiogenic environment and enrichment in cadherin regulation. A soluble VECAD (sVECAD) isoform was detected in cSDH fluid. Exposure of HBECs to patient-derived cSDH fluid increased EC proliferation, cytoplasmic VECAD (cVECAD) accumulation, and EC barrier permeability. Concurrent dexamethasone or minocycline treatment attenuated VECAD cleavage. Conclusions: VECAD disruption is a prominent feature of cSDH and is associated with EC barrier dysfunction. These findings support further evaluation of soluble VECAD as a candidate biomarker and of junction-preserving pathways as potential therapeutic targets.

An Ischemic stroke is one of the main causes of death and long-term disability around the world. There are very few treatment options available, especially for the cases where the short time period to give clot dissolving medicine has passed (Powers et al., 2019). While single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for understanding cell-to-cell differences within at issue (cellular heterogeneity) (Tang et al., 2009). However translating these high dimensional datasets into clinically useful biomarkers remains challenging (Rajko-mar et al., 2019). A major problem with current biomarker studies is their reliance on a single machine learning model, where results may reflect model bias rather than true biological signals (Lopez-Rincon et al., 2020). Methods: We developed a multi-model consensus framework integrating eight different machine learning methods from three groups: traditional models (logistic regression, random forest, XGBoost), a hybrid deep learning model (CNN-XGBoost), and transformer-inspired models (scGPT, scBERT, sc-Former, Geneformer). The proposed framework was applied to scRNA-seq data from a mouse middle cerebral artery occlusion (MCAO) model (GSE174574; n=6 mice, 54,599 cells). To prevent data leakage, we implemented rigorous subject-wise train/test splitting, ensuring all cells from individual animals remained either in the training sets or in the testsets. Results: All eight models achieved excellent performance on the independent test data, with AUC scores between 0.9778 and 0.9975. From six models that allowed gene level importance extraction, we identified 63 consensus genes (threshold: [&ge;] 4/6 models), with 17 genes selected by all 6 models. Pathway enrichment analysis showed significant enrichment of interferon signaling, with a novel dual-interferon pattern. Twelve of the consensus genes respond to interferon, and five of them (Ifitm6, Ifitm1, Gbp2, Igtp, Tgtp1) respond to both Type I (IFN-/{beta}) and Type II (IFN-{gamma}) interferons. Notably the consensus included Il1rn (interleukin-1 receptor antagonist), the target of Anakinra -- an FDA-approved drug that has completed Phase II stroke trials with promising safety profiles (Smith et al., 2018; Emsley et al., 2005). Conclusions: The Multi-model consensus framework, proposed in this paper, provides a robust and reliable approach to biomarker discovery that goes beyond the limitations of any single method and reduces individual algorithm bias. The convergence of classical, ensemble, and deep learning methods on interferon and IL-1 pathway genes supports their biological validity as stroke biomarkers. Our findings provide a prioritized gene list for further clinical validation and highlight interferon signaling as a possible treatment strategy warranting further investigation. Keywords: ischemic stroke; machine learning; single-cell RNA sequencing; biomarker discovery; consensus analysis; interferon signaling; neuroinflammation

BACKGROUND: Radiofrequency catheter ablation (RFCA) uses rigid metallic electrodes whose fixed geometry limits tissue contact, lesion continuity, and safe energy delivery near vulnerable structures. We evaluated a moldable, tissue-conforming conductive hydrogel as an RFCA electrode ex vivo. METHODS: Poly(ether urethane diacrylamide) hydrogels with saline-tuned conductivity were molded into plugs, disks, and bars and affixed to an irrigated 8-Fr catheter. Ablations were performed on excised porcine tissue in circulated saline at 37 C under fixed contact force and irrigation. Lesion geometry, intramural temperatures, and steam pops were compared with bare-metal (BM) electrodes and size-matched metallic analogs; an atrioesophageal stack model assessed collateral injury. Lesions were delineated using triphenyltetrazolium chloride staining. RESULTS: Hydrogel bars produced wide, ellipsoidal lesions (width 10.51 {+/-} 1.40 versus 8.71 {+/-} 1.65 mm for BM; P=0.031; aspect ratio 1.72); size-matched metal bars produced none. Hydrogel disks produced lesions statistically indistinguishable from BM in depth (4.54 {+/-} 0.90 versus 4.84 {+/-} 0.55 mm) and width (9.47 {+/-} 0.89 versus 11.41 {+/-} 2.39 mm), whereas metal disks produced significantly shallower lesions (2.30 {+/-} 1.70 mm; P=0.015 versus BM). In the atrioesophageal model, hydrogel plugs achieved transmural atrial lesions with a reduced esophageal lumen temperature (56.33 {+/-} 3.80 versus 61.30 {+/-} 5.14 C; P=0.046), total lesion depth (4.03 {+/-} 1.25 versus 7.58 {+/-} 0.78 mm; P<0.001), and esophageal lesion width (3.05 {+/-} 2.35 versus 8.70 {+/-} 2.69 mm; P<0.001) compared with BM. Hydrogel electrodes reduced steam-pop risk (relative risk 0.34; 95% CI 0.13-0.88). Plug characterization confirmed saline-dependent conductivity and attenuated energy delivery, with lower subsurface peak temperatures and shallower lesions than BM. CONCLUSIONS: Moldable, conductivity-tunable hydrogels shape RF lesions and may improve ablation safety, supporting evaluation for contiguous or linear lesion sets and ablation near sensitive structures.

BackgroundThe progression of abdominal aortic aneurysm (AAA) is closely associated with the phenotypic switch of vascular smooth muscle cells (VSMC). While members of the Serine/Arginine-rich splicing factor (SRs) superfamily are increasingly acknowledged for their pivotal role in modulating cell phenotype, the specific contribution of these factors to AAA development has remained underexplored. MethodsThrough integrated analyses the whole transcriptome of human and murine AAA tissues derived from clinical samples and animal models, we identified serine-arginine repeat matrix 4 (SRRM4) as a candidate gene regulating AAA formation. To investigate the role of SRRM4 in AAA formation, we used global and VSMC-specific SRRM4-knockout mice to construct AAA mouse models in two different ways. The impact of SRRM4 on AAA was further investigated in in vivo and in vitro models ResultsElevated levels of SRRM4 mRNA and protein were observed in VSMC from both human and murine AAA tissues. Targeted silencing of SRRM4 in VSMC (but not in endothelial or myeloid cells) significantly attenuated AAA formation in both AngII- and CaCl2-induced mouse models. Our results identify the RE1 silencing transcription factor (REST) as a critical target gene of SRRM4 in AAA, a master regulator of cellular phenotypic stability. We show that SRRM4 modulates VSMC phenotype through alternative splicing of the REST gene, resulting in a non-functional REST4 isoform. Furthermore, our study shows that REST directly represses STAT1, a mechanism by which SRRM4 influences VSMC phenotypic transition. Regarding clinical significance, inhibition of both STAT1 expression and function was shown to be beneficial in preventing AAA formation and slowing disease progression in mouse models. ConclusionsThis investigation uncovers a previously unrecognized role of SRRM4-mediated splicing of the REST gene in the progression of AAA and identifies a novel REST/STAT1 axis critical in the phenotypic modulation of VSMC. The findings of this study may provide insights for the development of innovative therapeutic strategies for AAA. What is New?O_LIOur study demonstrates that the RNA splicing factor SRRM4 (serine/arginine repetitive matrix 4) expression is increased in human and murine abdominal aortic aneurysm (AAA) tissues and unveils a previously undescribed role of SRRM4 in regulating AAA development. C_LIO_LISRRM4 promotes AAA development and progression through inducing splicing of REST (RE1 silencing transcription factor) gene and upregulating STAT1 (signal transducer and activator of transcription 1) expression, which result in extracellular matrix degradation, inflammation, and VSMC phenotypic switching. C_LIO_LIOur study provides proof-of-concept evidence indicating the therapeutic value of STAT1 inhibition in AAA prevention and treatment. C_LI What Are the Clinical Implications?O_LIOur study defining a novel mechanism underlying the SRRM4/REST/STAT1 axis is crucial for developing new therapeutic strategies for AAA. C_LIO_LIBased on these findings, we have initiated a prospective study (REST4-AAA; ClinicalTrials.gov Identifier: NCT07056491) to evaluate REST4 expression levels in serum of AAA patients and explore its potential as a serum biomarker for AAA prediction C_LIO_LIOur findings indicate that fludarabine, a clinically available agent reported to selectively suppress STAT1, effectively attenuates AAA progression in mice, suggesting its potential for repurposing as a therapeutic strategy for AAA. C_LI

Introduction: Cerebral ischemia induces a reduction in brain pH. GPR4, a proton-sensing G protein-coupled receptor is highly expressed in vascular endothelial cells. Pharmacological inhibition of GPR4 with NE52-QQ57 (NEQQ) reduces ischemia-induced blood-brain barrier (BBB) disruption and brain injury. To facilitate targeting GPR4 in vivo, it is important to better define the effect of NEQQ on post-ischemia signaling and the pharmacokinetics of NEQQ in vivo. Methods: We performed biochemical analysis following transient middle cerebral artery occlusion (tMCAO). We used Liquid Chromatography-Mass spectrometry (LC-MS/MS) to quantify the concentration of NEQQ in serum, brain, and peripheral tissues. Results: NEQQ administration decreased the phosphorylation of PKA substrates, PKC substrates, ERK1/2, and CREB. LC-MS/MS analysis detected NEQQ in both ipsilateral and contralateral hemisphere of the brain at 5 h and 24 h after reperfusion, demonstrating that NEQQ crosses the BBB. In peripheral tissues, NEQQ exhibited highest level in kidney and lowest in the heart. In sham controls, average serum concentrations were ~4 and 0.9 g/g at 5 and 24 h, respectively. Following tMCAO, ipsilateral brain exhibited higher levels as compared to the contralateral side at 5 h while the difference diminished at 24 h. Conclusions: NEQQ attenuated the ischemia-induced activation of multiple signaling pathways. NEQQ exhibited broad distribution in peripheral organs, and its brain level was sufficient for GPR4 inhibition at 24 h after injection. These findings support the use of NEQQ as a pharmacological inhibitor of GPR4-mediated signaling following ischemic stroke.