Science Translational Medicine

ObjectiveTo determine whether quantitative ultrasound (QUS), which characterizes tissue microstructure using radiofrequency data, can identify regional heterogeneity within seminiferous tubules that corresponds to localized spermatogenesis in men with non-obstructive azoospermia (NOA). DesignTwo-cohort study using a biological extremes cohort to establish plausibility of a QUS biomarker, followed by an independent NOA-biopsy cohort with site-matched imaging and tissue sampling. SettingAcademic male fertility referral center. PatientsThe biological extremes cohort included fertile men with presumed intact spermatogenesis (n=15) and men with NOA and subsequent negative microdissection testicular sperm extraction (mTESE; n=10). The NOA-biopsy cohort consisted of 27 men with NOA undergoing site-matched testicular biopsy via testicular sperm aspiration (TESA) or testicular sperm extraction (TESE), yielding 12 sperm-positive and 36 sperm-negative biopsy sites. InterventionsHigh-frequency testicular ultrasound (36 MHz) with acquisition of raw radiofrequency data, allowing objective, quantitative analysis of tissue scattering patterns beyond conventional grayscale imaging. Regions of interest were manually annotated and, in the NOA-biopsy cohort, spatially matched to biopsy locations. Main Outcome MeasuresAssociation between sperm presence at biopsy sites and a pre-specified QUS measure of local tissue heterogeneity: the 75th percentile of a sliding window coefficient of variation map of the Nakagami k-factor within the superficial testicular parenchyma (K_Zone1_CV). This metric reflects the upper range of local variability in ultrasound backscatter, which is influenced by the underlying organization of seminiferous tubules. ResultsIn the biological extremes cohort, K_Zone1_CV distinguished fertile controls (median 1.79, IQR 1.64-1.85) from NOA men with globally negative mTESE (median 1.51, IQR 1.42-1.58; P < 0.001), with an area under the receiver operating characteristic curve (AUC) of 0.91 (95% CI 0.79-1.00). In the independent NOA-biopsy cohort, K_Zone1_CV discriminated sperm-positive from sperm-negative biopsy sites with an AUC of 0.93 (95% CI 0.85-0.99). At a threshold of 1.60, sensitivity was 100%, specificity was 86.1%, positive predictive value was 70.6%, and negative predictive value was 100%. Serum hormone levels, testicular volumes, and biopsy technique did not differ significantly between groups. ConclusionsRegional testicular tissue heterogeneity measured by quantitative ultrasound is associated with localized spermatogenesis in men with NOA. At the selected threshold, no sperm-positive biopsy site was misclassified as negative. These findings support the hypothesis that QUS can noninvasively detect the focal seminiferous tubule heterogeneity that predicts sperm retrieval success. This imaging approach could inform future image-guided sperm retrieval strategies. Further validation in larger cohorts and assessment of intra-patient variability are needed.

Sepsis is a dysregulated host response to infection and a leading cause of global mortality, yet effective targeted therapies remain lacking. Here, we applied a proteogenomic framework integrating large-scale human genetics with circulating proteomics to identify therapeutic targets. In a meta-analysis of genome-wide association studies of 60,314 sepsis cases and 1,464,733 controls, we identified four genome-wide significant loci, including a missense variant in SERPINA1, encoding alpha-1 antitrypsin (AAT), that was also associated with 30-day sepsis mortality in the UK Biobank. Mendelian randomization (MR) and colocalization analyses supported a causal and protective effect of higher genetically predicted circulating AAT levels on sepsis risk. The protective association was highly specific to acute infectious phenotypes, including pneumonia, and was not observed for non-infectious traits. In two independent cohorts (UK Genomic Advances in Sepsis and the Biobanque Quebecois sur la COVID-19), circulating AAT increased markedly during acute illness but was significantly attenuated among missense variant carriers in a dose-dependent manner, consistent with impaired protease-antiprotease balance. MR of the AAT-regulated proteome recapitulated findings from prior sepsis trials, both negative and positive, providing orthogonal genetic support for therapeutic modulation of this pathway. Together, these findings provide the first human genetic evidence for AAT's causal role in sepsis, positioning SERPINA1 as a high-priority candidate for drug repurposing and targeted therapeutic interventions.

Management of peripheral nervous system (PNS) neuropathies, such as traumatic peripheral nerve injury (PNI), relies on accurate assessment of muscle denervation and recovery. Yet, the current gold-standard clinical test, needle electromyography (EMG), has multiple shortcomings that can complicate surgical treatment. Here, we introduce a noninvasive method for holistic evaluation of muscle denervation by utilizing positron emission tomography (PET) to quantify expression of prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), within muscles. We identified that GCPII is persistently over-expressed in denervated muscles and that expression normalizes with muscle reinnervation. Leveraging this phenomenon, we used two PSMA/GCPII-PET agents that are FDA-approved for prostate cancer imaging, [18F]DCFPyL and [68Ga]PSMA-11, to detect muscle denervation and subsequent reinnervation in experimental models of PNI. We found that denervated muscle had approximately twice the uptake as innervated muscle on GCPII-PET/magnetic resonance (MR) imaging and GCPII-PET/computed tomography (CT), which persisted for at least 16 weeks after nerve injury without repair in rats and swine. GCPII-targeted uptake also declined to near baseline levels with muscle reinnervation after nerve repair. To assess clinical feasibility, we performed [18F]DCFPyL PET/CT in a patient who had sustained a unilateral radial nerve injury 15 weeks prior, and we observed elevations in denervated muscle uptake that mirrored our preclinical findings. Our consistent findings across species of increased GCPII-PET uptake in chronically denervated muscle and its decline with muscle reinnervation, along with the established safety profile of available GCPII-PET agents, support the promise of GCPII-PET as a rapidly translatable strategy for characterization and longitudinal monitoring of PNIs and non-traumatic PNS neuropathies.

ABSTRACT Background: The UroLume endoprosthesis (AMS/Endo-care), commercially available 1988-2007 and FDA-approved in 1996, was positioned as a permanent minimally invasive solution for recurrent bulbar urethral stricture and benign prostatic hyperplasia (BPH). Despite early procedural success, long-term data revealed a catastrophic complication profile - including irreversible urethral destruction, spongiofibrosis, MDR infections, chronic kidney disease, and severe psychological morbidity - culminating in the clinical entity termed UroLume Cripple Syndrome. No systematic epidemiological analysis of surviving patients in 2026 currently exists. Objectives: To synthesise four decades of evidence on UroLume pathophysiology, complications, surgical management hierarchy, psychological burden, and cumulative multimorbidity; to perform a pooled meta-analysis of primary complication endpoints; and to present an original epidemiological model estimating surviving patients globally and in Greece in 2026. Methods: PRISMA 2020-compliant systematic review and meta-analysis of PubMed, Embase, and Cochrane Library (all dates to March 2026). Inclusion: peer-reviewed studies of UroLume implantation, explantation, or post-UroLume reconstruction; minimum 12-month follow-up; series n >= 10. Random-effects meta-analysis (DerSimonian-Laird estimator) was performed for three primary complication endpoints across all 43 included studies. An original bottom-up sequential filter epidemiological model was constructed integrating WHO 2021 actuarial tables, published explantation rates, multimorbidity excess mortality, age distributions, complete epithelialisation prevalence, and reconstruction failure rates. Results: Forty-three studies met inclusion criteria (n=3,847 patients). Pooled meta-analysis yielded: restenosis/tissue ingrowth 37.9% (95% CI 36.1%-39.8%, I2=0%); stent explantation 8.7% (95% CI 7.7%-9.8%, I2=0%); urinary incontinence 9.7% (95% CI 8.7%-10.9%, I2=0%). Complete epithelialisation, irreversible after 12 months, affects approximately 8-13% of long-term survivors and defines the UroLume Cripple endpoint. Post-UroLume buccal mucosa graft urethroplasty achieves 76.7% success at 5 years when explantation is feasible. Our epidemiological model estimates 2,500-5,000 surviving patients globally with UroLume in situ in 2026, reducing to fewer than 100 clinically active patients aged <60 years following full multimorbidity adjustment. A six-filter sequential model for Greece converges to a final estimate of 1 surviving patient aged <60 years with complete epithelialisation following failed reconstruction. Conclusions: UroLume Cripple Syndrome is a chronic iatrogenic disease with distinct pathophysiological, reconstructive, psychological, and social dimensions that has received insufficient recognition as a defined clinical entity. The surviving patient population is small but institutionally invisible: no registry exists, no dedicated follow-up protocol has been established, and specialist reconstructive capacity is confined to approximately eight centres worldwide. Registry creation, EAU guideline extension, and specialist referral pathways are the minimum adequate institutional responses. This preprint has been deposited on medRxiv simultaneously with journal submission.

Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is a major contributor to stroke, heart failure, and mortality worldwide. Although AF affects both men and women at a similar rate, accumulating experimental and clinical evidence indicates that its underlying mechanisms, disease progression, and treatment responses differ by sex. However, current antiarrhythmic drug development and clinical management of AF remains largely sex neutral, likely contributing to limited efficacy and increased adverse effects. To address this gap, we developed a computational drug-screening pipeline based on experimentally constrained, sex-specific human atrial cardiomyocyte models to predict and evaluate sex-specific pharmacological strategies for AF. The pipeline integrates multivariable regression with mechanistic modeling to systematically test multi-target combinations of ion channel inhibitors and Ca2+ handling modulators and identify interventions that reduce arrhythmia vulnerability by restoring sex-specific electrophysiological and Ca2+ handling properties toward normal sinus rhythm (nSR). Application of this approach revealed a greater number of successful inhibitory drug combinations in males than in females. In males, optimal recovery to nSR primarily required inhibition of Na+ and K+ channels to prolong repolarization and refractoriness, increase Ca2+ transient amplitude (CaTAmp), and reduce susceptibility to action potential duration (APD) alternans. In females, modulation of Ca2+-related pathways was additionally required to suppress delayed afterdepolarizations (DADs). Forward single-cell simulations confirmed the predictions of the drug-analysis pipeline, demonstrating recovery of APD, CaTAmp, and arrhythmia vulnerability indices without introducing instabilities. Importantly, extension of these interventions to two-dimensional atrial tissue simulations demonstrated that sex-specific drug strategies reduce vulnerability to triggered activity, while suppression of reentry was most effective when combined with partial recovery of cell-cell coupling. Our results establish a multiscale computational pipeline for identifying sex-informed, multi-target antiarrhythmic therapies, amenable to experimental validation and translation to the clinic. Clinical PerspectiveO_ST_ABSWhat is KnownC_ST_ABSO_LIAtrial fibrillation arises from multiple interacting multiscale mechanisms, which limit the effectiveness of single-target therapies. C_LIO_LICurrent single-target antiarrhythmic drugs for atrial fibrillation show more limited efficacy and higher adverse event rates in women than in men C_LI What the Study AddsO_LIThis study demonstrates that effective pharmacologic strategies require different combinations of ion channel and calcium handling modulation in males versus females with persistent (chronic) atrial fibrillation. C_LIO_LIIn males, coordinated Na+ and K+ channel inhibition most effectively improves electrical stability, whereas in females additional targeting of Ca2+ handling is required to suppress triggered activity. C_LIO_LISex-specific multi-target drug strategies including partial recovery of intercellular coupling suppress triggered activity and reentry in atrial tissue while preserving conduction. C_LI

There is no readily accessible, scalable cure for HIV infection. Trials of HIV-specific broadly neutralising antibodies (bNAbs) demonstrate inhibition of viral replication and reduction of the reservoir of latently-infected cells, potentially offering new strategies for HIV eradication. Animal and human studies suggest bNAbs have multiple activities, including a direct antiviral action and a secondary induction of T cell responses, the vaccinal effect. The RIO trial assessed HIV-specific cell-mediated immunity after dosing with two long-acting bNAbs (10-1074-LS and 3BNC117-LS) in people treated with antiretroviral therapy (ART) since early stage HIV followed by treatment interruption. BNAbs resulted in sustained viral suppression with 75% of participants controlling off ART after 20 weeks. Here we show that HIV-specific T cell immunity was enhanced for at least 36 weeks after bNAbs in aviraemic participants. Gag-specific T cell immune responses predicted virological outcomes. Baseline CD8+ AIM responses predicted longer times to rebound; baseline CD8+ proliferative responses were additionally protective in participants without baseline bNAb resistance mutations. Baseline ELISpot responses were associated with faster rebound. These data highlight the complex interplay between bNAbs and T cells, identify a post-bNAb protective T cell-driven vaccinal effect, and reinforce the role of immune-based interventions as part of HIV cure strategies.

Inflammatory diseases cause significant morbidity and mortality, but their pathobiology is often difficult to dissect due to complex genetic-environmental interactions. Genetic forms of heterotopic ossification, such as fibrodysplasia ossificans progressiva (FOP), reduce genetic variability, allowing careful dissection of non-genetic drivers of inflammation. While >95% of FOP patients harbor the ACVR1R206H mutation, patients exhibit significant variability in disease progression, suggesting a role of environmental drivers. Here, we identify the gut microbiome as a regulator of inflammation-driven HO in FOP. Metagenomic profiling of cohabitating FOP/unaffected sibling pairs revealed a pathogenic gut microbiome profile in FOP patients (Bray-Curtis, p < 0.05). In Pdgfr-Cre/Acvr1R206H (FOP) mice, gut microbiome ablation by antibiotics reduced spontaneous HO formation (47.4% reduction, p < 0.05) and reduced plasma IL-1 pathway activity. IL-1{beta} blockade in FOP mice suppressed trauma-induced HO formation. These findings identify a gut microbiome-IL-1-HO axis with modifiable targets for developing treatments for HO and related inflammatory conditions. One Sentence SummaryAntibiotic disruption of the gut microbiome reduces HO in FOP mice via an IL-1 mediated pathway.

Oropouche virus (OROV) is an emerging arbovirus responsible for large outbreaks of febrile illness in Central and South America, with increasing reports of severe neurological disease and fatal outcomes. Despite its growing public health impact, no approved antiviral therapies or vaccines are currently available. Here, we show that favipiravir, a broad-spectrum nucleoside analogue, robustly suppresses OROV replication and disease in vivo. In a lethal Syrian hamster model, favipiravir treatment provided complete protection against OROV infection, preventing viral dissemination to peripheral organs and the central nervous system, and remained highly effective when administration was initiated after infection. In contrast, insufficient antiviral control resulted in viral neuroinvasion and fatality. To define host responses associated with OROV pathogenesis and their modulation by antiviral therapy, we performed transcriptomic profiling of liver and brain tissues. OROV infection induced interferon-driven inflammatory programs accompanied by marked disruption of metabolic and tissue homeostatic pathways, whereas these transcriptional signatures were largely abrogated by favipiravir treatment. Together, our findings identify favipiravir as a potent antiviral candidate against OROV and provide the first in vivo, tissue-resolved transcriptomic framework of OROV infection, linking effective viral suppression with the prevention of neuroinvasion and pathogenic host responses. These results highlight antiviral intervention as a viable strategy to mitigate OROV-associated disease and mortality.

Dementia affects tens of millions of people worldwide, yet disease-modifying treatments remain strikingly limited. Although the recombinant zoster vaccine Shingrix has been associated with reduced dementia incidence, its potential influence on individuals already living with dementia is unknown. Here, we followed a propensity-score matched cohort of 68,960 US dementia patients using a nationwide electronic health record network, comparing Shingrix recipients within two years of diagnosis to recipients of any other vaccine. Shingrix was associated with substantially reduced all-cause mortality across the first three years of follow-up (hazard ratios 0.74, 0.88, and 0.89; P[&le;]0.006), robust across multiple sensitivity analyses. Furthermore, within-individual subgroup analyses of repeated Mini-Mental State Examinations conducted 3-6 years apart revealed significantly divergent cognitive decline rates across groups (time-by-group interaction P=0.002). Interval vaccination was associated with more stable cognition, contrasting with steeper declines in unvaccinated individuals. These findings support prospective evaluation of recombinant zoster vaccination as a potential strategy to improve outcomes in patients with established dementia.

The mitochondrial permeability transition pore (mPTP) is a voltage- and calcium-regulated channel located in the inner mitochondrial membrane whose activity critically influences cellular fate. While prolonged pore opening leads to mitochondrial depolarization, matrix swelling, and cell death, brief and reversible opening events, referred to as flickering, enable controlled release of calcium and reactive oxygen species and serve essential physiological functions. Emerging evidence indicates that restoring physiological mPTP flickering, rather than suppressing pore activity, may be beneficial in disorders characterized by impaired pore dynamics, including hereditary spastic paraplegia type 7 (SPG7). However, no approved therapies are currently available to promote controlled mPTP pore opening. To identify pharmacological modulators of flickering, we performed a high-content screening of 2,000 FDA and EMA-approved compounds using a validated fluorescence-based assay coupled with automated image analysis. Thirteen compounds increased both the frequency and the area of flickering events while preserving cellular and mitochondrial integrity. Validation in fibroblasts derived from two SPG7 patients and healthy controls confirmed reproducible activity across distinct genetic backgrounds. Among the prioritized candidates, berberine emerged as the most robust modulator, consistently enhancing mPTP flickering independently of SPG7 mutation status. Notably, berberine selectively increased the proportion of small-size flickering events, indicative of physiological pore activity. These findings identify berberine as a promising modulator of mPTP dynamics and support pharmacological restoration of physiological flickering as a potential therapeutic strategy for SPG7 and other disorders associated with impaired mitochondrial permeability transition pore regulation.

Myotonic dystrophy type 1 (DM1) lacks human in vitro models that directly link RNA toxicity to mature skeletal muscle function, particularly myotonia. Here, we engineer contractile 3D human skeletal muscle tissues from immortalized myoblasts derived from three DM1 patients representing juvenile, adult, and late-onset subtypes. These tissues reproduce key molecular features of DM1, including nuclear RNA foci, MBNL1 sequestration, and widespread mis-splicing. Functionally, DM1 tissues exhibit impaired calcium handling, subtype-dependent weakness, rapid fatigue, and a fiber-type distribution characterized by increased slow type I fibers and pathological MyHC-I/IIx hybrids. Notably, the 3D environment enables expression and complete pathogenic mis-splicing of CLCN1--undetectable in matched 2D cultures--accompanied by myotonia-like delayed relaxation. Using this model, we assessed therapeutic responses of candidate small-molecule modulators. Phenylbutazone reduced RNA foci and MBNL1 sequestration but failed to rescue spliceopathy or function. In contrast, calcitriol induced coordinated transcriptomic remodeling and robustly rescued myotonia-like relaxation despite persistent CLCN1 mis-splicing. These findings establish a functionally mature human DM1 muscle model and highlight compensatory network activation as a strategy to improve muscle function in DM1.

Metabotropic glutamate receptor 5 (mGlu5) is a class C GPCR crucial for neuronal development and synaptic transmission. mGlu5 is a potential therapeutic target in pain management and modulates pain-associated gene expression and signaling pathways. Although mGlu5 inhibitors have shown promise in treating pain, none have translated to the clinic. Up to 90% of neuronal mGlu5 expression is intracellular, although the precise locations and function of different mGlu5 intracellular pools remains unclear. Building on recent evidence showing the importance of endosome-mediated nociceptive signaling by other GPCRs, we hypothesized that endosomal pools of mGlu5 contribute to pain transmission, and that targeted inhibition of intracellular mGlu5 signaling results in superior analgesia. Using calcium mobilization assays and genetically encoded resonance energy transfer biosensors, we report that upon its activation mGlu5 recruits Gq/11 and Gs to the plasma membrane. Conversely, internalized mGlu5 in endosomes recruits only Gq/11 proteins. mGlu5 signaling is highly dependent on receptor trafficking to endosomes, with sustained nuclear ERK1/2 signaling requiring both receptor internalization and active glutamate transport into the cell. We generated pH responsive nanoparticles loaded with the mGlu5 negative allosteric modulator VU0366058 (DIPMA-VU058), enabling endosome-targeted inhibition of mGlu5. Nanoparticle encapsulation of VU0366058 enhanced inhibition of both acute and sustained nuclear ERK1/2 signaling, and significantly reduced neuronal excitability in nociceptive circuits in spinal cord slices from rats with neuropathic pain. Intrathecal administration of DIPMA-VU058 achieved superior analgesia in both inflammatory and neuropathic models of pain in mice compared to free VU0366058 and the reference compound fenobam. These studies demonstrate the importance of endosome-associated receptors for the complete mGlu5 signaling response. Furthermore, we show that manipulating the cellular distribution of an allosteric modulator can engender location-biased pharmacological effects. Together, we have revealed new and unappreciated roles for endosome-specific mGlu5 signaling and demonstrate that endosome-selective targeting may offer an alternative therapeutic approach for modulating mGlu5 activity.

Aging is characterized by a gradual decline in function, partly due to accumulated molecular damage. Human skin undergoes both chronological aging and environmental degradation, particularly UV-induced photoaging. Detrimental structural and physiological changes caused by aging include epidermal thinning due to stem cell depletion and dermal atrophy associated with decreased collagen production. Here, we present a comprehensive single-cell atlas of skin aging, analyzing samples from young, middle-aged, and elderly individuals, including both sun-exposed and sun-protected areas. This atlas reveals age-related changes in cellular composition and function across various skin cell types, including epidermal stem cells, fibroblasts, hair follicles, and endothelial cells. Using our atlas, we have uncovered basal stem cells as a highly variable population across aging, more so than other skin cell populations, such as fibroblasts. In basal stem cells, we identified ATF3 as a novel regulator of skin aging. ATF3 is a transcriptional factor for genes involved in the aging process, with its expression reduced by 20% during aging. Based on this discovery, we developed an innovative mRNA-based treatment to mitigate the effects of skin aging. After treatment with ATF3 mRNA, cell senescence decreased 25%, and we observed an over 20% increase in proliferation in treated basal stem cells. Importantly, we also found communication between keratinocytes and fibroblasts as a critical component of therapeutic interventions, with ATF3 mRNA rescue of basal cells significantly enhancing fibroblast collagen production by approximately 200%. Furthermore, we validated the efficacy of ATF3 mRNA treatment in ex vivo human skin and in vivo mouse models. In ex vivo human skin, microneedle-mediated delivery of ATF3 mRNA induced robust rejuvenation, expanding the basal stem-cell population and enhancing dermal ECM reconstruction. In a wound healing mouse model, ATF3 mRNA reduced scarring, demonstrating strong regenerative potential for reversing age-related skin decline. We conclude that ATF3 mRNA treatment effectively reverses the effects of skin aging by modulating specific cellular mechanisms, offering a novel, targeted approach to human skin rejuvenation.

Haemorrhage is the leading preventable cause of trauma death, primarily through ischaemic consequences that current treatments cannot adequately address. We combined human transcriptomic data (n=458) with a controlled porcine model of haemorrhagic shock to identify treatment-responsive molecular mechanisms. Using latent factorisation, we prioritised distinct molecular signatures of the human shock response, including stress signalling, neutrophil activation, and cytotoxic lymphocyte programmes. We assessed the behaviour of these pathways in the experimental porcine system, revealing that shock-initiated immune trajectories are not immutable: blood resuscitation normalised maladaptive transcriptomic changes whilst noradrenaline exacerbated them. While resuscitation modulated neutrophil, heat-shock, and barrier defence programmes, interferon and coagulation pathways were neither mortality-predictive nor treatment-responsive. A factor representing p38-MAPK/AP-1 stress signalling emerged as the dominant mortality-predictive pathway. An in silico small molecule screen identified p38 inhibitors as leading candidates for reversing shock-induced transcriptomic signatures. Our framework identifies modifiable pathways in trauma shock, prioritising p38-MAPK inhibition for therapeutic development and providing a systematic approach for trauma drug repurposing. One sentence summary: Human-porcine cross-analysis reveals treatment-modifiable molecular signatures of trauma shock, identifying potential therapeutic strategies.

Sudden unexpected death in epilepsy (SUDEP) is the most devastating complication of epilepsy, yet the molecular features distinguishing individuals at risk remain poorly defined. Although epilepsy and SUDEP share substantial genetic overlap, fatal outcomes may arise when shared risk genes are differentially deployed across neuronal and cardiac systems. Here, we identify tissue- and isoform-level regulation as a key determinant of divergence between epilepsy and SUDEP risk. We performed a large-scale integrated analysis of genetic variants reported in epilepsy and SUDEP across 419 sequencing-based studies encompassing 35,659 individuals, and quantified gene-level burden using a Bayesian Poisson-Gamma rate ratio framework. This analysis revealed preferential enrichment of genes related to cardiac electrophysiology and contractile function in SUDEP, whereas epilepsy was dominated by genes involved in neuronal excitability and synaptic signaling. To determine how shared genetic loci are deployed across tissues, we integrated GTEx-based tissue expression profiles with long-read single-cell transcriptomic datasets from human heart and brain to resolve isoform-level expression patterns. These analyses revealed pronounced tissue-specific transcript architectures. Cardiac-associated genes, including HCN4, KCNH2, KCNE1, MYH6, MYO18B, and ATP1A2, showed heart-restricted isoform expression, whereas neuronal genes such as ADGRV1, CACNA1A, GRIN2B, HCN1, HCN2, KCNA1, SCN1A, SCN2A, and SCN8A. Importantly, several shared genes exhibited tissue-partitioned isoform expression, with distinct transcript repertoires in heart and brain, particularly across pathways related to ion transport, signaling, metabolism, and structural organization. Consistent patterns were observed in iPSC-derived cardiomyocytes and neurons, indicating that lineage-dependent deployment of shared genes is preserved in controlled systems. Together, these findings suggest that tissue-specific isoform regulation provides a mechanistic basis linking shared epilepsy genetics to SUDEP susceptibility, whereby the same genetic loci contribute to neuronal dysfunction in epilepsy and to cardiac vulnerability in SUDEP. This positions SUDEP as a neuro-cardiac interface disorder shaped by isoform-level regulatory divergence.

Dengue is the canonical viral disease for which immune history predicts protective versus pathogenic responses and immunogenicity. Yet, due to limitations in animal models and clinical presentation after peak viremia, how pre-infection and early immune responses affect dengue outcomes is not confirmed. We conducted a phase 1 clinical trial with 45 healthy adults to test if secondary infection challenge with a heterotypic, full-length, attenuated virus increases viremia and immunogenicity compared to primary and tertiary infection. Viremia was associated with more, but still mild, clinical signs and symptoms, and secondary infection predicted greater viremia and neutralizing antibodies. However, those with the highest baseline enhancing antibodies experienced delayed inflammatory and adaptive activation, the highest viremia, strong acute immune responses, but waning of potent CD8+ T cells and antibodies. Baseline antibodies to non-structural protein 1 of multiple serotypes predicted early interferon and balanced immune activation, viremia control, and development of enduring, potent B and T cells, revealing how vaccines can induce broad long-lasting protection. Finally, these antibody and T cell profiles at baseline predicted sterilization of infection. We demonstrate that controlled human challenge can delineate coordinated versus dysregulated acute responses and effects on immunogenicity, informing therapeutic and vaccine strategies for dengue and other viral diseases.

Vacuole, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome (VS) is a severe autoinflammatory disease driven by somatic mutations in ubiquitin-like modifier activating enzyme 1 (UBA1), for which disease activity biomarkers and therapeutic targets are needed. We conducted longitudinal deep phenotyping of patients with VS and found that ribonuclease 1 (RNASE1) expression is most strongly correlated with the disease activity score measured by the VEXAS Current Activity Form (VEXASCAF). Single-cell RNA-sequencing showed that RNASE1 was upregulated in VS monocytes. We generated human monocytic cell lines harboring UBA1 mutations (p.Met41Val, p.Met41Leu, and p.Met41Thr). These cells exhibited varying degrees of impaired ubiquitination and subsequent pathological features such as the unfolded protein response, increased pro-inflammatory cytokine production, cell death, and RNASE1 expression, mirroring the genotype-phenotype associations observed in patients with VS. Multi-omics analyses revealed that genotypes linked to greater clinical severity were enriched in pro-inflammatory, interferon, and necroptosis signatures. Notably, functional interrogation demonstrated that inhibition of receptor-interacting protein kinase 3 (RIPK3), a key regulator of necroptosis, markedly suppressed all these pathological features, including the elevated RNASE1 expression. These results identify the RIPK3-RNASE1 axis in VS, highlighting RNASE1 as a potential biomarker and RIPK3 as an attractive therapeutic target.

Disease-modifying therapies (DMTs) for relapsing-remitting multiple sclerosis (RRMS) act through distinct immunological mechanisms, yet the within-patient molecular response programs associated with these therapies remain incompletely defined. Here, we reanalyzed publicly available PBMC miRNA microarray data (GSE230064) using a longitudinal, robustness-focused framework to compare therapy-associated miRNA response patterns following cladribine versus ocrelizumab treatment. Baseline (t0) and 6-month post-treatment (t1) samples were paired within individuals and technical replicates consolidated prior to analysis, yielding a final paired cohort of 4 cladribine-treated and 6 ocrelizumab-treated patients. Within each treatment arm, we quantified per-patient {Delta}-miRNA (t1-t0) values and prioritized therapy-associated response features using a multi-evidence framework integrating effect direction, magnitude, directional consistency across individuals, and leave-one-out sensitivity. Cladribine treatment was associated with a highly coordinated, directionally concordant upregulation of five miRNAs including hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-503-5p, hsa-miR-148a-3p, and hsa-miR-26a-5p, all exhibiting 100% directional stability across patients and mean {Delta}-expression values ranging from +0.77 to +1.38. These miRNAs target pathways relevant to MS pathophysiology, including Th17/Treg balance, Wnt-{beta}-catenin signaling, macrophage polarization, and epigenetic immune regulation. In contrast, ocrelizumab elicited a more selective response pattern, with five miRNAs including hsa-miR-100-5p, hsa-miR-410-3p, hsa-miR-432-5p, hsa-miR-296-5p, and hsa-miR-485-3p showing moderate directional stability (83%) and greater inter-individual heterogeneity, consistent with the more targeted mechanism of CD20+ B-cell depletion. Notably, the two treatment-associated signatures were non-overlapping, with hsa-miR-27b-3p representing the only miRNA shared with prior cross-sectional analyses of this dataset. The identified ocrelizumab-associated miRNAs implicate pathways including mTOR/IGF1R signaling, NF-{kappa}B regulation, RNA editing, and mitochondrial biogenesis, several of which are dysregulated in progressive MS. Together, these findings demonstrate that cladribine and ocrelizumab induce distinct, treatment-specific miRNA response architectures that reflect their divergent immunological mechanisms. This work establishes a stability-aware analytic template for extracting reproducible longitudinal miRNA signals from small paired RRMS cohorts and provides a ranked set of biologically plausible candidate miRNAs for prospective validation and mechanistic investigation.

Chronic constipation is highly prevalent, and cases refractory to treatment are particularly challenging to manage. High-resolution colonic manometry (HRM) is used to further evaluate these patients to identify cases of intrinsic motor dysfunction (underlying myopathy or neuropathy). However, HRM is invasive and resource-intensive, limiting uptake and clinical utility. This study presents Body Surface Colonic Mapping (BSCM), a non-invasive cutaneous electrical recording technique, as a clinical alternative. Simultaneous recordings from HRM (36-channel) and BSCM (8x8 electrode array) were performed in 10 patients with chronic refractory constipation. Lower gut symptom scores were also tracked patients over the duration of the recording. Motility was assessed during meal and bisacodyl challenges. We optimized BSCM signal processing specifically to detect high-amplitude propagating contractions (HAPCs) evoked by bisacodyl. Analysis included time-frequency quantification of motility indices and blinded visual assessment by domain experts to classify the presence or absence of motor responses. BSCM motility indices showed strong correlation with HRM for both meal (r = 0.86) and bisacodyl (r = 0.69) responses. Expert visual analysis yielded concordant classification between BSCM and HRM in the majority (87.5 {+/-} 9.6%) of cases. Furthermore, BSCM identified distinct, patient-specific symptom-motility associations during the meal response. BSCM accurately detects meal- and stimulant-induced increases in colonic motility with high fidelity to invasive HRM. As a non-invasive method that is easy to apply with minimal resource and time requirements, BSCM is well-positioned for clinical translation as a scalable diagnostic tool to elucidate symptom-motility associations and guide personalized management in refractory chronic constipation.

Human platelets change over their 7-10 day lifespan, yet the molecular mechanisms underlying platelet aging remain poorly defined. Using two independent RNA sequencing datasets of fluorescence-activated cell sorted young and old human platelets, we developed a unified transcriptomic model to characterize RNA metabolism across the platelet lifespan, which we termed platelet molecular maturation. This was applied to RNA sequencing data from room-temperature stored platelets (up to 7 days) and cold-stored platelets (7, 14, or 21 days). We identified highly concordant aging signatures, including 6,015 shared expressed genes and 2,008 shared differentially expressed genes (DEGs) with strongly correlated fold changes, demonstrating a conserved platelet aging program. Nucleotide-level analyses revealed preferential 3'-directed degradation among downregulated transcripts during endogenous platelet aging and room-temperature storage, supporting an organized RNA decay process that was correlated with platelet function changes. Room-temperature storage recapitulated platelet molecular maturation, showing concordance with aging-related gene expression changes and enrichment of downregulated gene sets. In contrast, cold-storage significantly attenuated platelet molecular maturation and 3'-directed degradation. A total of 669 genes were consistently differentially expressed between room-temperature and cold-stored platelets, while no DEGs were detected during cold-storage, indicating transcriptional stability. Platelet transcript stability in cold-storage correlated with preserved platelet hemostatic function. These findings establish platelet molecular maturation as a conserved, functionally relevant model linking endogenous platelet aging to platelet storage lesions and providing mechanistic insight into preserved platelet hemostatic function in cold-storage. This atlas of platelet RNA metabolism supports biomarker discovery and strategies to improve storage. KEY POINTSO_LIBy integrating multiple high-quality RNA sequencing datasets with novel analytic approaches tailored to the biology of anucleate platelets, we show that platelet aging is not a passive process of transcript decay, but follows a structured and reproducible molecular trajectory both endogenously and in storage, which we term platelet molecular maturation. C_LIO_LIStorage temperature emerged as a dominant modifier of this trajectory, with cold-storage markedly slowing RNA metabolic kinetics and preserving transcripts associated with younger, more hemostatically competent platelets. C_LIO_LITogether, these findings provide mechanistic insight into the platelet storage lesion and identify transcriptomic features that may serve as biomarkers or therapeutic targets to extend platelet shelf life. C_LI