Biomedicines

Neurodegenerative diseases are a major threat to older adults and represent a growing global health burden as the elderly population continues to expand. The complexity of neurodegenerative diseases and the incomplete understanding of their pathophysiology limit the development of effective therapeutics. To study common neurodegenerative mechanisms and accordingly potential drug targets across Alzheimers, Parkinsons, and Huntingtons diseases, transcriptomic profiles of patients with each disease were analyzed to detect common differentially expressed genes and common enriched pathways. Common differentially expressed genes involved in the shared pathways were identified as key drug targets and validated in silico to study the impact of their dysregulation. Three pathways were enriched and upregulated across the three diseases, along with 274 common differentially expressed genes. Two of the shared pathways were involved in activation of the transcription factor nuclear factor kappa B (NF-{kappa}B), indicating inflammatory signaling. The third pathway involved regulation of BCL2L11 transcription by RUNX3, which contributes to the protective effect of neurodegenerative diseases against cancer. Five key drug targets were identified: NFKBIA, NFKB1, RELA, TRIM4, and SMAD4. They were significantly upregulated across all three diseases and involved in the shared pathways. Drugs that target the expression of these genes and previously approved by Food and Drug Administration were reported for treatment repurposing for the three neurodegenerative diseases. The resultant drug list included the conventional and commonly safe antihyperlipidemics, antihypertensives, antidiabetics, analgesics, diuretics, antiparkinsons, and antipsychotics.

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

Electrical stunning is a humane euthanasia method for larval zebrafish, and a suitable euthanasia protocol has recently been established. Here, we assess the associated stress response and tissue integrity. When compared to MS-222 overdose and hypothermia, electrical stunning caused no significant Hsp70 increase. Brain tissue was preserved and muscle tissue affected only slightly. Our results support the suitability of electrical stunning for humane euthanasia and artifact-free post-mortem analyses.

Objective: Preclinical studies suggest that irradiation combined with anti-PD-1 antibody (iRT) exacerbates cardiac injury in mice, partly through CD8 T-cell-mediated mechanisms. We investigated radiation-induced PD-L1 expression in cardiomyocytes and explored its potential role in iRT-associated cardiac injury. Methods: AC16 cells were exposed to different doses of irradiation. PD-L1 expression was assessed at different time points by flow cytometry, qRT-PCR, western blotting, and immunofluorescence. Cell viability, apoptosis, and cytokine levels were evaluated. PD-L1 was knocked down using siRNA to assess its function. PBMCs or CD8 T cells were co-cultured with irradiated or non-irradiated AC16 cells at different E:T ratios, with or without PD-1 blockade. A C57BL/6J mouse model of iRT-induced myocardial injury was established, and myocardial PD-L1 expression and T-cell infiltration were evaluated. Results: Irradiation upregulated PD-L1 expression in AC16 cells, with increased membrane and cytoplasmic signals at 48 h and prominent nuclear-associated fluorescence at 72 h. Irradiation increased IL-6, MCP-1, CCL5, and CXCL10 release, decreased IL-10, reduced cell viability, and increased apoptosis. PD-L1 knockdown further increased inflammatory cytokine and chemokine release but did not significantly alter apoptosis or further reduce viability in irradiated AC16 cells. In the presence of a high density of activated CD8 T cells, disruption of PD-1/PD-L1 signaling was associated with a further reduction in AC16 cell viability. In vivo, myocardial PD-L1 expression was increased, whereas CD8 T-cell infiltration remained limited and focal. Conclusion: PD-L1 upregulation in cardiomyocytes may exert an adaptive protective and immunoregulatory role during iRT-associated injury, potentially by limiting inflammatory mediator release and modulating susceptibility to activated CD8 T-cell mediated injury. CD8 T-cell density and activation are the key issues.

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.

A fraction of patients treated with radiotherapy are known to be more sensitive to ionizing radiations. Skin fibroblasts from such radiosensitive individuals exhibit a higher cellular toxicity after irradiation and a delay in DNA repair. Deciphering the molecular mechanisms underlying these cellular defects is thus of major importance. We previously observed that the transcription factor NFATc2 is expressed at a reduced level in fibroblasts from radiosensitive patients. The present work aimed to elucidate the role of NFATc2 in the regulation of DNA repair, particularly the repair of radiation-induced double-strand breaks. We demonstrate an interaction of NFATc2 with the NHEJ repair protein Ku80 and observe that the NFATc2 RHD domain is necessary and sufficient for this interaction. Moreover, we show that NFATc2-Ku80 complexes are not colocalized to DNA double-strand breaks sites suggesting an involvement upstream of the DNA repair pathway. The silencing of NFATc2 impairs the NHEJ repair activities by delaying Ku70-Ku80 interaction in the early steps of this pathway. Finally, stable over-expression of NFATc2 in patients fibroblasts partially rescues their defective DNA repair phenotype, especially in the most radiosensitive cells. Altogether, our data reveal that NFATc2 is a regulator of DNA repair in skin fibroblasts and therefore a potential modulator of cellular radiosensitivity.

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: Endoscopic submucosal dissection (ESD) of superficial esophageal squamous cell carcinoma (SESCC), with or without the esophageal gland duct involvement (DI or NDI), has been reported in small case series, but ESD-related safety, efficacy, and outcomes remain unknown. We conducted a retrospective study of those issues in consecutive 748 patients treated at our center in China. Material and Methods: Over the study period from January 2014 to January 2019, we identified 748 eligible SESCC patients who were divided into the DI (22.6%, 169/748) and NDI (77.4%, 579/748) groups, based on histopathologic reports. Following the European and Japanese guidelines on endoscopic resection of SESCC, we investigated and statistically compared characteristics of clinicopathology and ESD-related safety, efficacy, and prognosis between DI and NDI groups. Results: The mean age of patients was 64.9 years for the cohort and significantly older in the DI (66.1) than in the NDI (64.5) group (P<0.05). There was no significant difference in smoking/alcohol abuse and comorbidity. Endoscopically, the DI group showed significantly larger tumor size and deeper invasion than the NDI group. There was no ESD-related death in the cohort. Although the en bloc resection rate was 100%, the complete and curative resection rates were significantly lower in the DI (88.2% and 61.2%, respectively) than in the NDI (96.4%, and 86.9%, respectively) group (P<0.01). ESD-related infection (0.7%) and bleeding (2.3%) rates were very low and no significant difference was found between the two groups. Only one perforation (0.1%) occurred in the NDI group. Post-ESD stenosis was 11.9% for the cohort and significantly more common in the DI (20.7%) than in the NDI (9.3%) group (P<0.01). The overall survival rate was 95.7% for the cohort and significantly lower in the NDI (94.6%) than in the DI (100.0%) group (P<0.05). However, there was no significant difference in disease-specific death, recurrence-free survival, distant metastasis, and the requirement for subsequent chemoradiation therapy and surgery between the two groups. Conclusion: In our cohort, there was no ESD-associated death; ESD-related infection and bleeding were minimal; and perforation was rare. Post-ESD stenosis was 11.9% in prevalence and successfully managed by endoscopic dilation. The overall survival rate was 95.7%. Compared to the NDI group, the DI group showed older age, larger tumor size, deeper invasion, lower complete and curative resection rates, higher prevalence of post-ESD stenosis; there was no significant difference in disease-specific death. Taken together, ESD was safe with excellent efficacy and prognosis for endoscopic resection of SESCC with or without esophageal gland duct involvement. Keywords: superficial esophageal squamous cell carcinoma, endoscopic submucosal dissection, ductal involvement, survival, postoperative stenosis.

Background: Spontaneous cervical artery dissection (sCeAD) is a rare vasculopathy whose pathophysiology remains incompletely understood. Impaired vascular extracellular matrix integrity, including elastic fibers, may contribute to its development. We investigated whether serum fibrillin-1 and soluble elastin fragments (sELF) differ between patients with sCeAD and controls during the acute and chronic stages. Methods: Patients with acute sCeAD were prospectively enrolled at four German stroke centers. Blood samples were collected at baseline and after 6{+/-}1 months. Patients with a first acute ischemic stroke unrelated to sCeAD and healthy individuals served as controls. Serum fibrillin-1 and sELF concentrations were measured using enzyme-linked immunosorbent assays. Results: 61 patients with sCeAD, 53 patients with first non-CeAD ischemic stroke, and 79 healthy controls were included. After sex-matching, serum fibrillin-1 concentrations were significantly lower in patients with acute sCeAD than in healthy controls (97 [60; 192] vs. 176 [113; 269] ng/mL; p=0.009). Fibrillin-1 concentrations were also lower in both male and female patients with sCeAD than in respective healthy controls. In patients with sCeAD, fibrillin-1 concentrations increased significantly after 6 months compared with baseline (171 [130; 270] vs. 104 [67; 205] ng/mL; p=0.021). Serum fibrillin-1 concentrations were higher in men than in women across all study groups. No significant differences in sELF concentrations were observed between groups or time points. Discussion: Serum fibrillin-1 concentrations were lower during acute sCeAD and increased significantly during follow-up, whereas sELF concentrations remained unchanged. These findings support an association between circulating fibrillin-1 and acute sCeAD and warrant further investigation of its role in sCeAD pathophysiology. Pronounced sex-related differences in fibrillin-1 concentrations highlight the importance of sex-specific analyses in future.

Background: Chronic lowgrade inflammation in long standing rheumatoid arthritis (RA) contributes not only to joint damage but also to metabolic dysregulation, endothelial dysfunction, and elevated cardiovascular (CV) risk. Although combination disease modifying anti rheumatic drugs (DMARDs) remain the mainstay of therapy, their long term efficacy in controlling systemic inflammation and preventing metabolic complications appears limited. Phytochemicals such as resveratrol, a polyphenolic compound widely used in traditional and complementary medicine, possess anti inflammatory and immunomodulatory properties. Objectives: To investigate whether resveratrol can complement the immunomodulatory effects of combination DMARDs in long duration RA patients by modulating inflammatory cytokines and the JNK-IRS-Akt insulin signaling axis. Methods: This study enrolled early and late rheumatoid arthritis patients to assess disease activity, vascular markers, and ex vivo PBMC responses. PBMCs were isolated for cytotoxicity testing and resveratrol treatment, followed by ELISA and Western blot analysis. Statistical comparisons evaluated immunomodulatory effects and alterations in inflammatory signaling. Results: Longitudinal follow up of RA patients showed significant first year improvement in disease activity and atherosclerotic markers, correlated with MTX dose. An early versus late RA comparison revealed elevated cytokines and enhanced JNK mediated stress signaling in longstanding disease. Resveratrol maintained PBMC viability, reduced LPS induced TNF&alpha and adipokine levels, and downregulated pJNK and GSK&beta, indicating targeted anti inflammatory modulation independent of Akt activation. Conclusion: Chronic RA showed persistent inflammatory and metabolic dysregulation driven by JNK-NF&kappaB activation. Resveratrol reduced cytokines, corrected adipokine imbalance, and selectively inhibited JNK, suggesting adjunct therapeutic value alongside DMARDs for improving immunometabolic disturbances in long-standing RA.

The effect of microcurrents on facultative microbial pathogens remains controversial. Solid cultures in Sabouraud Glucose Agar of the ubiquitous mold Aspergillus fumigatus were repeatedly treated with a commercially available device performing wireless conductive microelectrostimulation by 3.5 A microcurrent routed by spraying negatively charged air particles onto solid cultures in modified petri dishes. The treated cultures displayed increased growth compared to standard ones, but only as a function of mycelial density and total surface; the radial growth rate of the mycelium remained unaltered. The increased growth was positively related to the duration of the treatment. At the same time, secondary development (new mycelial loci within the dish) was greatly upheld due to treatment, as the spraying created microairstreams dislocating the fungal spores. These results imply perplexed kinetics of mycelial growth both with and without treatment, since the folding of the mycelial mat is observed regularly. Both the fungus response to the ES and the possible revision of growth kinetics create prospects for biotechnological and bioremediation applications but also imply biomedical considerations, regarding infection dynamics of mycelial fungi and their in situ resistance to immune responses and treatment.

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.

Therapy of ischemic stroke is currently limited to pharmacological and/or mechanical recanalization. There are no neuroprotective therapies approved for use during the rehabilitative phase of ischemic stroke, which is characterized by neurodegenerative changes. Thus, the search for neuroprotective compounds capable of preventing neuronal death caused by pathogenetic cascades triggered during hypoxia is an urgent task. In this study, we demonstrate increased culture viability following pre- and post-incubation with salicyl-carnosine (SC) in a model of oxygen glucose deprivation on a primary culture of rat cortical neurons. Its neuroprotective properties were greater than that of acetylsalicylic acid and carnosine, and it was effective in lower concentrations. In addition, SC protected the culture from NMDA-induced excitotoxicity. We also showed the passage of SC into neurons, and the presence of its direct antioxidant activity in a model of paraquat-induced oxidative stress. The neuroprotective effects of SC are associated with a decrease in the level of pro-apoptotic protein Bak and a decrease in the activation of kinase p38, as well as an increase in the activation of kinase ERK1/2. The acquired data suggests that SC is a promising neuroprotective compound, and warrants further investigation in vivo.

The LRRK2 kinase has emerged as a priority therapeutic target due to its well-documented role in Parkinson-s diseases (PD). One of LRRK2s key partners is the phosphatase PP1, which dephosphorylates LRRK2. Therefore, modulating this protein/protein interaction represents a promising therapeutic strategy for PD. We have developed a bi-functional peptide, PEP 3, capable of crossing the blood-brain barrier (BBB) and disrupting the LRRK2/PP1 interaction. In vitro competition assays confirmed that PEP 3 specifically targets this interaction. The in vivo imaging demonstrated that the peptide remains detectable in the mouse brain up to 6 hours or retro-orbital vein post-injection. The PEP 3 peptide does not show chronic toxicity in CD1 mice and is resistant to degradation by mouse, human, dog and monkey serum proteases. In mouse models overexpressing alpha-synuclein, repeated intraperitoneal injections of PEP 3 for 15 and 30 days were well tolerated. Immunohistochemistry revealed a significant reduction of dopaminergic cell death in the substantia nigra, and quantification of phosphorylated pathological alpha-synuclein showed decreased levels in the PEP 3-treated group compared to controls. These findings support the potential of PEP 3 as a therapeutic agent for Parkinsons disease.

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.

Accurate, noninvasive biomarkers for diagnosis and longitudinal monitoring of metabolic dysfunction-associated steatohepatitis (MASH) remain an urgent unmet clinical need. Here, we describe an electrochemical immunoassay that involves capturing hepatic extracellular vesicles (EVs) on antibody-modified electrodes followed by detecting EV-borne biomarkers using redox active gold nanoparticles (AuNPs). The intensity of electrochemical redox signal represents a quantitative measure of biomarker abundance. Three MASH-associated biomarkers-S100A11, TNF-, and TGF-{beta}1 were analyzed on hepatic EVs to assess steatotic, inflammatory, and fibrotic states of the diseased liver. The electrochemical signals for individual biomarkers were added into a composite EVMASH score. Plasma samples from 28 patients with MASH, 22 healthy controls, and 27 patients with non-MASH liver disease were scored in this manner, revealing 100% accuracy in the training cohort and >90% accuracy in an independent validation cohort. Our electrochemical immunosensor represents a minimally invasive liquid liver biopsy, offering a promising alternative to solid liver biopsies for screening and monitoring of MASH.

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.

Increased platelet microparticle (PMP) levels in individuals with risk factors for cardiovascular disease correlate with clinical outcomes in these patient groups. PMPs promote thrombosis through enhancing platelet aggregation and binding to the sub-endothelial matrix following vascular injury. Thus, PMPs behave as soluble ligands and adhesive substrates for platelets, and may drive cardiovascular disease progression. Nitric oxide (NO) is released continually from the endothelium as a potent regulator of platelet activation that is crucial to the balance between haemostasis and thrombosis. However, it is unknown if NO regulates PMP-induced platelet activation. In this study we isolated platelets and PMPs from whole blood and measured their interactions in adhesion assays and by flow cytometry. Platelet activation was analysed by ELISA for ADP and thromboxane-B2; both secondary platelet agonists released by activated platelets which enhance thrombosis. The affinity upregulation of the principal platelet integrin receptor responsible for platelet aggregation, integrin IIb{beta}3, was measured using the antibody PAC-1. Our data show that PMP induced platelet adhesion was associated with, and partially dependent upon, platelet ADP release, TxA2 production and IIb{beta}3 upregulation. Crucially, NO dose-dependently reduced these events through cGMP dependent signalling. This is the first report that NO signalling can regulate PMP induced platelet activation and may open an avenue of exploration for clinically targeting PMP driven cardiovascular disease processes.

Aims: Arrhythmogenic cardiomyopathy (ACM) is a genetic disease defined by arrhythmias and myocardial fibrosis with impaired cardiac function and increased risk of sudden cardiac death. Pathogenic variants are mostly identified in desmosomal genes such as desmoglein-2 (DSG2). We identified a novel disease phenotype in patients homozygous for the DSG2 variant c.523+2T>C (splice site of exon 5/intron 5), characterized by cardioembolic events in addition to classical ACM features. Here, we evaluate this new thromboembolic phenotype by comparing the clinical data to specific murine disease models. Methods and Results: We describe three unrelated patients presenting with an embolic event and/or left ventricular thrombus. Clinical evaluation revealed a shared right ventricular ACM phenotype characterized by arrhythmias, impaired function, and fibrotic remodeling. In addition, patients exhibited localized fibrotic changes of the left ventricular apex with formation of an aneurysm and predisposition to thrombus formation. Genetic analysis identified the DSG2 variant c.523+2T>C as a founder variant from the "Bernese Oberland". To elucidate the variant's functional impact, a mouse model deficient for Dsg2 exon 5 (Dsg2{Delta}ex5) was established and compared to a model carrying the adhesion-deficient Dsg2-W2A variant. Echocardiography, ECG, and histology in Dsg2{Delta}ex5 mice revealed similar disease patterns to patients and a loss of DSG2 expression. Importantly, these animals exhibited left apical fibrosis with aneurysm formation and left ventricular thrombus formation. In contrast, the Dsg2-W2A model presented with a biventricular ACM-phenotype but without left ventricular thrombi. Conclusions: We identified a novel ACM phenotype in patients homozygous for the DSG2 founder variant c.523+2T>C characterized by left ventricular apical fibrosis. Dsg2{Delta}ex5 mice recapitulate the patients' phenotype suggesting a causative link between left ventricular aneurysm due to DSG2 deficiency and thrombus formation with subsequent embolism. This highlights a novel pathological feature of ACM and the need for variant and phenotype-specific therapy.