Biology subjects

Hu, B.

Publications and source records attributed to Hu, B..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Cardiac oxidative stress monitoring enabled by hierarchical mechanical adaptation

Soft bioelectronics have advanced cardiac monitoring through electrophysiological tracking, yet this alone cannot resolve the metabolic pathology essential to surgical decision-making. However, real-time molecular sensing on beating hearts remains unresolved due to deformation-induced sensor failure and stress-induced metabolite artifacts. This challenge is exemplified by ischemia-reperfusion injury (IRI), a major cardiac surgery complication characterized by reactive oxidative species (ROS) bursts, where true pathological ROS signals being confounded by mechanotransduction-induced ROS artifacts. Herein, we propose an enzymatic cardiac oxidative stress biosensor (E-cardiac) with hierarchical mechanical adaptation: macro-scale biofluid-mediated contact, micro-scale fiber reorganization, and nano-scale enzymatic confinement within gold nanoarches dissipate interfacial stress. This produces ultrathin ([~]460 nm), soft (0.79 kPa) E-cardiac with robust electrochemical stability (100% strain), low detection limit (380 nM), rapid adhesion (<3 s), stable biosensing on beating heart, as well as minimal invasive deployment capability. Mechanical analysis and cellular studies confirm mitigated stress-induced ROS and absent PIEZO channel activation. Validated across cardiomyocytes, ex vivo tissues, multi-species ischemia models (mouse, rat, rabbit, pig), rat ischemia-reperfusion injury, and Langendorff hearts simulating graded perfusion deficits, E-cardiac quantitatively differentiates IRI severity (sham < ischemia < reperfusion) as well as detecting the "ECG blind window". The E-cardiac platform provides real-time metabolic feedback for surgical guidance during cardiac procedures, enabling timely intervention before irreversible damage.

bioengineering↗

bioRxiv2026

Sacituzumab Govitecan as an Effective Strategy for Sensitizing Chemoresistant HNSCC Cells to Senolytic Intervention

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide and is marked by a high tumor relapse due to acquired chemoresistance, requiring alternative strategies to sensitize resistant tumors to treatment. Sacituzumab govitecan (SG), a TROP2-targeting antibody-drug conjugate, has been successful in limiting tumor progression in pretreated patients with triple-negative and hormone-receptor positive HER2-negative breast cancer. However, it has been ineffective as a monotherapy in HNSCC. This may be attributed to the promotion of senescence ultimately leading to tumor relapse. We established that isogenic cisplatin-sensitive and -resistant HNSCC cells undergo senescence following SG treatment. Senolytics have been effective in sensitizing solid tumors to standard of care chemotherapies in preclinical studies. Consequently, we investigated the effectiveness of SG treatment followed by the senolytic, ABT-263, as a two-hit therapeutic strategy against cisplatin-resistant HNSCC. SG followed by ABT-263 sensitized HNSCC cells to apoptosis via a BAK and BAX-dependent mechanism. In vivo studies confirmed that SG treatment followed by ABT-263 limited tumor progression and extended survival without notable toxicity. Thus, SG in combination with senolytic treatment may be an effective strategy for overcoming not only cisplatin-resistance but also SG resistance via targeting the senescent cell population.

cancer biology↗

bioRxiv2026

Circulating MicroRNAs as Potential Diagnostic Biomarkers for Heart Failure: A Systematic Review and Meta-Analysis

BackgroundCurrent heart failure (HF) biomarkers (e.g., BNP/NT-proBNP) have limitations in specificity and performance in HF with preserved ejection fraction (HFpEF). Circulating microRNAs (miRNAs) are promising novel biomarkers. This study aimed to comprehensively evaluate the diagnostic stability of circulating miRNAs for HF, identify novel candidates, and prioritize them for clinical translation. MethodsWe conducted a systematic review and meta-analysis. PubMed, Embase, and Cochrane Central were searched from inception to March 2025. Studies comparing miRNA expression in HF versus control groups using blood or tissue samples were included. Data were extracted, and study quality was assessed using the Newcastle-Ottawa Scale (human) and SYRCLEs tool (animal). A random-effects model pooled log odds ratios (logORs) for each miRNA. Subgroup analyses were based on species, ethnicity, and sample type. Evidence quality was graded using the GRADE framework. ResultsEighty-six studies (61 human, 25 animal) with 3,023 samples were included. Meta-analysis identified 71 consistently dysregulated circulating miRNAs (58 up, 13 down) in HF. Key upregulated miRNAs included miR-21 (logOR=8.15, 95% CI: 7.55-8.74), miR-423-5p, and miR-210. Key downregulated miRNAs included miR-144 and miR-126. Subgroup analyses revealed differences by species, ethnicity (Asian vs. non-Asian), and sample type (serum vs. plasma). GRADE assessment classified five miRNAs (miR-1, miR-21, miR-221, miR-423-5p, miR-148a) as high-quality evidence. ConclusionsThis meta-analysis identifies a panel of circulating miRNAs with stable expression in HF, with miR-21 and miR-423-5p being the most robust. Evidence grading provides a clear priority list (e.g., miR-21, miR-423-5p) for clinical validation. Subgroup heterogeneity highlights the need for standardized protocols and precision diagnostics in future biomarker development.

pharmacology and toxicology↗

bioRxiv2026

Daraxonrasib (RMC-6236) is an effective targeted therapy for RAS-mutant neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Relapsed or refractory (R/R) high-risk (HR) NB tumors continue to exhibit poor outcomes despite intensive and protractive multimodal therapy. Activating mutations in the RAS- mitogen-activated protein kinase (MAPK) pathway are frequently observed in R/R HRNB. The early promise of ALK inhibitors to treat ALK-mutant NB underscores the ability of appropriate targeted therapies to improve outcomes for HRNB patients. While MAPK pathway activation is prominent in HRNB, FDA-approved MEK inhibitors and KRAS G12C inhibitors have failed to demonstrate significant preclinical single-agent activity. Daraxonrasib (RMC-6236), a potent and selective RAS(ON) inhibitor, has demonstrated activity in both preclinical models and early phase clinical trials of RAS-mutant adult cancers. A subset of R/R HRNB tumors is noteworthy for containing diverse RAS-mutations, providing rationale for RMC-6236 investigation. In this study, we evaluated the therapeutic efficacy and oncogenic signaling modulation of RMC-6236 across NB models harboring RAS pathway activation. RMC-6236 as a single-agent treatment led to a significant decrease in cell viability, suppression of downstream MAPK signaling, upregulation of the MAPK pathway effector protein BIM, and increased cell death in RAS-mutant NB models as well as in NF1-mutant NB models. In vivo studies evidenced that RMC-6236 had on-target activity that significantly reduced tumor growth and extended survival in RAS-mutant HRNB mouse models. Furthermore, RMC-6236-induced both BCL-2 and BIM upregulation and enhancement of BIM:BCL-2 complexes in RAS-mutant NB. As such, the BCL-2 inhibitor venetoclax further enhanced RMC-6236-mediated killing by disrupting RMC-6236 enhanced BIM:BCL-2 complexes. These findings demonstrate that RMC-6236 is a rationale targeted therapy for RAS-mutant NB, a subset of NB that is progressively understood as conferring particularly poor outcomes. RMC-6236 is a clinically relevant drug that can successfully target the MAPK pathway in these cancers. This study supports expanded clinical testing of this novel therapy to this important subset of neuroblastoma.

cancer biology↗

bioRxiv2026

Mechano-activation of synovial fibroblasts and macrophages during OA progression in the dynamically stiffening synovial microenvironment

ObjectiveDuring osteoarthritis (OA) progression, the synovial membrane undergoes profound structural and compositional remodeling and fibrosis. We sought to elucidate how evolving synovial microenvironmental mechanics during fibrotic remodeling influence cell behavior and drive the progression of synovial pathology. MethodsSkeletally-mature male C57BL/6J mice were subjected to destabilization of the medial meniscus (DMM). To control for surgical confounders, both sham-operated and unoperated mice were included, with evaluation at 4- and 8-weeks. Synovial micromechanics were quantified via atomic force microscopy (AFM). Single-cell RNA sequencing (scRNA-seq), RNA fluorescence in situ hybridization (FISH), and flow cytometry were employed to investigate cellular heterogeneity, spatial organization, and crosstalk within fibrotic and non-fibrotic synovial niches. ResultsProgressive fibrotic remodeling and marked matrix stiffening were observed in DMM-operated synovium but absent in sham- and un-operated controls. While both sham and DMM joints mounted an acute stromal and immune response to surgery, these changes resolved over time in sham conditions but persisted in DMM synovium. During disease progression, distinct functional subsets of synovial fibroblasts and immune cells emerged, with mechanosignalling pathways and distinct immune cell-fibroblast crosstalk robustly activated within DMM-induced fibrotic microenvironments. ConclusionThis study demonstrates the complex cellular dynamics and crosstalk that differentiate the evolution of the pathological synovial response in the fibrotic DMM condition relative to surgical sham controls. Our findings highlight mechanotransduction as a central mechanism driving OA synovial pathogenesis and underscore the utility of the DMM model as a platform to dissect the molecular underpinnings of synovial fibrosis.

bioengineering↗

bioRxiv2026

CD14-deficiency protects against osteoarthritic subchondral bone sclerosis via enhanced osteoclastogenesis following joint injury

Aberrant bone remodeling is a hallmark of osteoarthritis, the most common arthritis affecting over 27 million US adults. Subchondral bone sclerosis, one sign of aberrant bone remodeling observable by routine x-rays, occurs as the trabeculae thicken, leading to increased bone volume. Toll-like receptors, pattern-recognition receptors of the innate immune system, have been implicated in OA pathogenesis, with TLR ligands, receptors, and co-receptors shown to mediate the severity and progression of OA. We have previously shown that CD14-deficiency protects mice against post-traumatic OA, and specifically reduces subchondral sclerosis post-injury. We hypothesized that depletion of CD14 protects against TLR4-dependent inhibition of osteoclastogenesis and therefore increases OC density in the SCB after injury, mitigating aberrant bone deposition in a murine model of OA. To determine how cellular changes correlate with bone structure derangements post-DMM, we performed MicroCT, Tartrate-resistant acid phosphatase staining, and alkaline phosphatase staining. To establish mechanistic changes in cellular signaling, we isolated WT and CD14-deficient osteoclast precursors and subjected them to LPS, an osteoarthritis-relevant TLR ligand, during differentiation. CD14-deficient mice, as well as WT mice treated with an anti-CD14 monoclonal antibody, show protection from post-injury increases in both bone volume fraction and bone mineral density. CD14-deficient mice had an increased osteoclast presence in the SCB two weeks post-injury, potentially protecting them from increases in bone volume and density. In vitro, CD14-deficient OCPs differentiated faster than WT OCPs, due to reduced Type I Interferon (IFN-I) signaling. In the presence of an LPS challenge, CD14-deficient OCPs were protected against LPS and TLR4-mediated inhibition, likely due to decreased MyD88-dependent TLR4 signaling. This work opens up new potential pathways to therapeutically target aberrant bone remodeling in the setting of joint injury and PTOA. Lay SummaryOsteoarthritis is one of the leading causes of disability worldwide. One of the hallmarks is subchondral sclerosis, or thickening of the bone in and around the joint. In this work, we used a mouse model of osteoarthritis to show that decreasing inflammatory signaling, through removal of CD14, protects against subchondral sclerosis, due to an increased presence of osteoclasts, cells that combat bone thickening. Osteoclasts without CD14 differentiate faster than osteoclasts with CD14, due to decreased Type I Interferon, an inflammatory cytokine. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/705094v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@176bdd5org.highwire.dtl.DTLVardef@a914bborg.highwire.dtl.DTLVardef@902748org.highwire.dtl.DTLVardef@2f9b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Distinct allosteric remodeling of HIV-1 Env dynamics on virions by gp41-directed antibodies reveals two modes of neutralization

HIV-1 envelope glycoprotein (Env), a gp120-gp41 trimer, undergoes coordinated conformational changes that drive membrane fusion and allow immune evasion by transiently concealing neutralization-sensitive epitopes. Most broadly neutralizing antibodies (bNAbs) target gp120, whereas a distinct subset recognizes conserved gp41 regions, such as the fusion peptide and the membrane-proximal external region; however, their impact on Env dynamics and associated neutralization mechanisms remains unclear. Using bioorthogonal tagging for single-molecule FRET, we monitored real-time bNAb-induced conformational sampling of Env on intact virions. Most gp41-directed bNAbs allosterically stabilized the prefusion-closed (PC) state, whereas the bivalent 10E8.4/iMab favored both PC and CD4-bound open (predominant) states. Antibodies redistributed the conformational populations of Env with modest kinetic effects, preserving the sequential transition pathway. These findings reveal two modes of neutralization for gp41-directed antibodies: fixing the prefusion-closed conformation and opening it, in both cases via long-range allosteric control of Env dynamics.

microbiology↗

bioRxiv2026

Functional Stratification Reveals Speed-Independent Gait Impairments Beyond Chronological Age

BackgroundGait is a clinically relevant indicator of functional decline in aging populations. However, most studies classify older adults by chronological rather than functional age, which may obscure early impairments detectable through kinematic profiling. This study examined whether stratifying older adults by functional status using the Short Physical Performance Battery (SPPB) enhances sensitivity in detecting gait abnormalities and instability-related compensatory patterns. MethodsA total of 190 adults completed gait trials on a pressure-sensitive walkway. Twenty-eight spatial, temporal, and variability-based gait parameters were derived. Participants were categorized as young adults or older adults, who were further stratified into high- and low-functioning groups based on SPPB scores. Analysis of covariance (ANCOVA) was performed, adjusting for habitual walking speed to isolate functional effects. FindingsAfter adjusting for speed, the low-functioning group demonstrated longer stance and double-support durations, wider step width, and greater step-to-step variability in both spatial and temporal domains compared with both the high-functioning and young reference groups. These findings indicate a compensatory, instability-driven control strategy that challenges the assumption of a "slower but steady" gait in aging. High-functioning older adults exhibited gait patterns more closely resembling those of younger adults. InterpretationFunctional classification using the SPPB provided greater sensitivity than chronological age in detecting early mobility decline. Gait variability emerged as a salient biomarker of impaired neuromuscular control. Integrating quantitative gait profiling with validated functional assessments may improve early screening, targeted intervention, and fall prevention strategies.

physiology↗

bioRxiv2026

A non-canonical EZH2/TRIM28 epigenetic axis drives heparan sulfate remodeling and melanoma metastasis

Melanoma progression and metastasis are driven not only by oncogenic alterations but also by epigenetic programs that dynamically remodel the tumor microenvironment. Heparan sulfate (HS) proteoglycans are key extracellular matrix components that integrate growth factor signaling, cell-matrix interactions, and migratory behavior by controlling ligand availability and receptor engagement, yet how chromatin-associated factors regulate HS remodeling in cancer remains poorly defined. Here, we identify the histone methyltransferase EZH2 as a key regulator of HS biosynthesis in melanoma. Integrated bioinformatic and genomic analyses revealed enrichment of EZH2 and additional Polycomb Repressive Complex (PRC) factors at regulatory regions of HS biosynthetic genes. CRISPR-mediated loss of EZH2 altered expression of multiple HS-modifying enzymes, most notably the secreted endosulfatases SULF1 and SULF2, resulting in enhanced HS 6-O sulfation and altered ligand binding at the cell surface. Unexpectedly, EZH2 promoted SULF1 expression through a methyltransferase-independent mechanism via a non-canonical interaction with TRIM28, whereas SULF2 was regulated through canonical PRC2-mediated repression. Functionally, SULF1 depletion impaired melanoma cell migration and invasion in vitro and reduced spontaneous metastasis in an orthotopic xenograft model. Together, these findings define an epigenetic axis linking chromatin regulation to extracellular glycan remodeling and identify HS-modifying enzymes as candidate targets to limit melanoma metastasis.

molecular biology↗

bioRxiv2026

Elucidating Odorant Masking Effects in Food Matrices Through Olfactory Receptor-Based Profiling

Dynamic interactions among aroma compounds, especially odorant masking, constitute a key determinant of the intricate flavor profiles of thermally processed foods. However, the molecular and receptor-level mechanisms governing mutual odor suppression remain largely unclear. This study investigated the reciprocal sensory masking between 2,3,5-trimethylpyrazine and L-menthol via systematic sensory evaluation. Olfactory receptor screening identified OR5K1 as the responsive receptor for 2,3,5-trimethylpyrazine (EC50 = 27.67 {+/-} 2.02 mol/L) and OR2W1 as the functional receptor for L-menthol (EC50 = 22.69 {+/-} 3.85 mol/L). Consistently, quantitative cellular response assays further validated that 2,3,5-trimethylpyrazine and L-menthol exert mutual inhibitory effects on their respective receptors, as evidenced by the significant inhibition of receptor signaling. Moreover, molecular modeling predicted that 2,3,5-trimethylpyrazine and L-menthol competitively occupy the same orthosteric pocket via analogous binding conformations. These results provide novel receptor-level insights into odorant inhibitory effects, connecting sensory perception to molecular recognition, and deepen mechanistic understanding of flavor perception.

cell biology↗

bioRxiv2026

Diet-Driven Gut Microbiome Differences Between Cohabiting Han and Yi Ethnic Groups in Liangshan, China

BackgroundWhile ethnic variations in gut microbiota are well-documented, their persistence in genetically distinct populations sharing the same geographic environment remains poorly understood. This study investigates the Han and Yi ethnic groups cohabiting rural Southwest China, where contrasting dietary traditions persist under identical environmental conditions. MethodsWe analyzed fecal samples from 100 healthy adults (50 Yi/50 Han) using 16S rRNA gene sequencing (V3-V4 region). Dietary intake was quantified via three 24-hour recalls validated with plasma biomarkers. Multivariate analyses (DESeq2, PERMANOVA) controlled for age, sex, and BMI covariates. ResultsYi individuals exhibited significantly higher Prevotellaceae (log2FC=1.82, padj=0.028) and Succinivibrionaceae (log2FC=2.15, padj=0.013) abundances, corresponding to their buckwheat-rich diet (58.5 {+/-} 12.3% vs Han 3.3{+/-}1.8%, p<0.001) Han microbiota showed enriched Bacteroidaceae (log2FC=1.24, padj=0.041), associated with higher animal fat intake (23.6% vs Yi 1.8%, p<0.001) Dietary factors (buckwheat/animal fat intake) explained greater microbiota variation than ethnic genetic ancestry (PERMANOVA R{superscript 2}=0.10 vs 0.07, p < 0.05) ConclusionCohabiting Han and Yi populations maintain distinct gut microbiota compositions primarily driven by dietary differences. These findings highlight diet as a stronger determinant than genetic ancestry in shaping microbial communities under shared environments. Future studies should validate these observations with metagenomic sequencing.

microbiology↗

bioRxiv2026

Molecular insights into bulk lipid transport from structural studies of the bridge-like protein VPS13A complexed with the scramblase XKR1

In eukaryotes, bridge-like lipid-transfer proteins (BLTPs) are central in mediating vesicle-independent lipid transfer between organelles. BLTPs span the cytosolic space between organelles at contact sites, featuring hydrophobic channels for lipids to travel between membranes. How BLTPs cooperate with partner proteins to orchestrate lipid delivery remains mysterious. Here we used cryo-electron microscopy to visualize a complex comprising the prototypical BLTP VPS13A and the plasma membrane localized scramblase XKR1 at near-atomic resolution. VPS13A interacts with XKR1 via its PH-domain, priming VPS13As bridge-like lipid-transfer domain to deliver lipids directly to the cytosolic leaflet of the acceptor membrane. In molecular dynamics simulations, such arrangement allows for robust lipid transfer, accelerated by membrane properties. Newly delivered lipids can then be equilibrated between leaflets of the membrane bilayer by the scramblase, allowing for membrane growth. Mechanistic insights regarding lipid delivery by VPS13A are directly applicable to all VPS13 proteins and all BLTP family members more broadly.

cell biology↗

bioRxiv2025

MicroGenomer: A Foundation Model for Transferable Microbial Genome Representations Enabling Multi-scale Genomic Understanding and Ecophysiological Trait Prediction

Microorganisms underpin global biogeochemical cycles and represent a vast, underexplored reservoir for sustainable biotechnology and human health. Their diversity, arising from the interplay of numerous genes within holistic genomic contexts, dictates complex ecophysiological traits across varied environments. To bridge the gap between complex genomic sequences and associated biological functions and phenotypic outcomes, we present MicroGenomer, a foundation model for transferable microbial genome representations enabling multi-scale genomic understanding and ecophysiological trait prediction. MicroGenomer leverages a hierarchical training strategy comprising pre-training on large-scale genomic sequences (234.5 billion base pairs), domain-specific mid-training using the GTDB-curated marker gene set, and task-specific post-training. Despite a streamlined architecture of 470 million parameters, MicroGenomer achieves performance on key tasks competitive with models nearly 85 times its size. Advancing beyond established gene-scale encoders, MicroGenomer generates robust embeddings at the genome scale for downstream modeling. Extensive evaluations demonstrate that MicroGenomer effectively captures phylogenetic structures in species space, excelling in geneand genome-scale understanding as well as ecophysiological trait prediction. The practical utility of these capabilities is further demonstrated by targeted wet-lab validation on newly isolated strains, indicating that MicroGenomers predictions provide reliable guidance for biological experiments. Collectively, MicroGenomer offers a high-performance, resource-efficient framework that transforms raw sequence data into actionable biological insights, providing a powerful foundation for microbiome research and biotechnology.

bioinformatics↗

bioRxiv2025

6mA demethylase Mi-NMAD-1 modulates nematode development and parasitism through the transcription factor Mi-nhr-118

N6-methyladenine (6mA) DNA methylation has emerged as an important epigenetic mark in eukaryotes, but its biological role in plant-parasitic nematodes remains largely unexplored. Here, we demonstrate that 6mA methylation is essential for the embryonic development and parasitic ability of Meloidogyne incognita. Silencing of the 6mA demethylase genes minmad-1 significantly reduced egg hatching, revealing that 6mA-mediated regulation is required for nematode development. Transcriptome analyses showed that minmad-1 knockdown downregulated genes involved in embryogenesis, RNA biosynthesis, and cell differentiation, suggesting a critical role of 6mA in developmental gene regulation. Among the transcription factors affected by minmad-1 silencing, Mi-nhr-118, a nuclear hormone receptor orthologous to C. elegans nhr-118, was identified as a key regulator of embryonic development. Functional assays confirmed that RNAi or host-induced gene silencing (HIGS) of Mi-nhr-118 led to developmental arrest, reduced gall and egg mass formation, and impaired infectivity across multiple Meloidogyne species. ChIP-seq analysis revealed that Mi-nhr-118 directly binds to promoters of genes associated with cellular regulation and developmental signaling. Several downstream targets, including transcription factors (Mi_23621.1, Mi_39647.1) and genes implicated in metabolic and signaling processes (Mi_03067.1, Mi_08398.1), were shown to be essential for both embryogenesis and parasitism. Functional enrichment analyses of Mi-nhr-118 target genes indicated suppression of immune-related signaling, carbohydrate recognition, and glycoprotein biosynthetic pathways following gene silencing. Collectively, these findings establish Mi-nhr-118 as a central regulator linking 6mA-mediated epigenetic control to development and parasitism in M. incognita.

molecular biology↗

bioRxiv2025

Adapting Epigenetic Clocks for Cell-Free DNA High-Throughput Sequencing Data

Background.Circulating cell-free DNA (cfDNA or ccfDNA) methylation sequencing holds promise for developing epigenetic aging clocks in minimally invasive aging assessment applications. However, current clocks--primarily trained on array-based data--do not readily generalize to methylation profiles of cfDNA, due to stochasticity and uncertainty arising from the limited input amounts of cfDNA and technical characteristics of high-throughput sequencing (HTS) platform. Due to lack of training to correct this uncertainty, direct application of legacy clocks to HTS data will inevitably produce unreliable estimates, introduce predictive discordance and undermine the trustworthiness of epigenetic biomarkers. Despite this urgent challenge, a systematic, model-agnostic framework for adapting existing epigenetic clocks to HTS-based cfDNA data remains lacking. Methods.Here, we generated a dedicated benchmark dataset comprising paired cfDNA and genomic DNA (gDNA), which was replicated and profiled across two methylation arrays and two targeted HTS platforms. We evaluated 53 epigenetic clocks for CpG coverage, reproducibility, cross-platform consistency, and age prediction accuracy. Further, we systematically explored and tested multiple adaptation strategies including depth filtering, beta-value imputation, and transfer learning via model distillation aiming at improving performance and clinical concordance of legacy epigenetic clocks on cfDNA HTS datasets. Results.Our results show that inherent technical noise of HTS platforms compromises diagnostic precision, presenting as a significant burden for applying legacy clocks on cfDNA HTS data. However, this technical limitation can be systematically neutralized. By enforcing stringent sequencing depth protocols ([≥]10x ideally 20x), employing robust algorithmic stabilization (L2-heavy clocks and imputation) and transfer learning, we can effectively isolate genuine physiological aging signals from technical artifacts. Ultimately, an adaptation pipeline incorporating all these methods effectively improved age prediction accuracy and, closed the gap between epigenetically induced aging status and clinically assessed realities. Besides, the pipeline demonstrated improved diagnostic sensitivity for neurodegenerative conditions such as amyotrophic lateral sclerosis, establishing a reliable foundation for non-invasive clinical monitoring. Conclusions.This study provided a comprehensive framework and practical guidelines for adapting epigenetic clocks to HTS-based cfDNA data, paved the path for more general application of cfDNA-based aging assessment and liquid biopsies.

bioinformatics↗

bioRxiv2025

Insights into the regulation of VPS13 family bridge-like lipid transfer proteins from the structure of VPS13C.

Bridge-like lipid transfer proteins (BLTPs) play central roles in redistributing lipids from their primary site of synthesis in the endoplasmic reticulum to other organelles. They comprise bridge-domains spanning between organelles at contact sites that allow lipids to transit the cytosol between adjacent membranes. The assembly of BLTPs into complexes with adaptor proteins enables their lipid transfer ability. To address the mechanisms underlying assembly and regulation of BLTP complexes, we used cryo-EM to resolve the structure of one such BLTP, the Parkinsons protein VPS13C, at near-atomic resolution. The structure identifies a lipid-transfer-nonpermissive conformation, where the built-in C-terminal VAB adaptor module blocks the end of the lipid transfer bridge, interfering with lipid delivery. We also identify calmodulin, central to calcium signaling, as a VPS13 partner, suggesting calcium regulation of VPS13 function. Altogether, this structure of intact VPS13C serves as starting point to understand its regulation and, more broadly, that of other BLTPs.

cell biology↗

bioRxiv2025

Targeted Intestinal Cooling Offers Superior Brain Protection in the Mouse Stroke Model

BackgroundIntestinal immune and inflammatory response plays a detrimental role following a stroke. This study aims to evaluate the brain protective efficacies of a novel intestinal cooling (CC) technique relative to the body surface cooling (SC) and the normothermic (NT) condition in a mouse stroke model. MethodsMice were randomly assigned to CC (n=13), SC (n=15), or NT (n=11) groups. They underwent 60 min of middle cerebral artery occlusion (MCAO) followed by 7-day reperfusion. Both head and intra-colon temperatures were maintained at 37{degrees}C for 30 min before, during, and 30 min after MCAO. At 30 min reperfusion, a cooling catheter was placed to maintain intra-colon at 37{degrees}C in NT or 12{degrees}C in CC. The head temperature was maintained at 37{degrees}C in NT and 30{degrees}C in CC. In SC, both intra-colon and head temperatures were maintained at 30{degrees}C. Cooling lasted 3 hours. Bodyweight, behavioral deficits (nesting and pole test), and survival rate were assessed post-MCAO. At day 7 post-MCAO, mice were perfusion-fixed for histopathological analysis. ResultsPost-stroke histopathological brain injury areas and volume were significantly reduced in CC, and appeared reduced though not statistically significant in SC, relative to NT. Compared with NT, body weight, nest building activity, and pole test were all significantly recovered in CC post-MCAO. In SC, only nest building improved significantly, while body weight and pole test showed marginal, nonsignificant trends. Consistent with functional recovery, survival was significantly improved in CC but not in SC, compared with NT. ConclusionIn a murine model, our novel CC technique successfully achieved targeted intestinal cooling while preserving safe upper-body temperatures necessary for normal cardiopulmonary function. Targeted intestinal cooling provides significant benefits superior to SC and NT, including smaller stroke volume, fewer functional deficits, and lower mortality rates, thus supporting the novel concept that the intestines are potential therapeutic targets for stroke management. Graphical Abstracts O_FIG O_LINKSMALLFIG WIDTH=131 HEIGHT=200 SRC="FIGDIR/small/681764v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1aa07cdorg.highwire.dtl.DTLVardef@1e64569org.highwire.dtl.DTLVardef@d6f178org.highwire.dtl.DTLVardef@1871862_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPost-stroke body surface cooling-induced hypothermia demonstrated marginally neuroprotective effects compared to normothermic conditions in the mouse model. C_LIO_LITargeted deep cooling of the intestine after a stroke resulted in a significantly greater reduction in stroke injury areas and volume, as well as lower mortality rates and fewer functional deficits, compared to body surface cooling-induced hypothermia and normothermic conditions. C_LI

neuroscience↗

bioRxiv2025

Stage-resolved Spatial Multi-omics Reveals Myeloid Niches in Human Atherosclerotic Plaques

BackgroundAtherosclerosis, a leading cause of heart attack and stroke, involves intricate immune cell dynamics within arterial plaques, yet their spatial organization and functional roles remain elusive. MethodsWe combined Visium HD spatial transcriptomics, imaging mass cytometry, and single-cell RNA-seq across fatty-streak, advanced, and restenotic plaques to map myeloid architectures and relate them to lesion geography and extracellular matrix features. Slingshot trajectory analysis resolved macrophages differentiation path. In vitro, we tested microenvironmental and lipid cues separately: fibronectin (FN) exposure during PMA-driven THP-1 differentiation and oxidized LDL (oxLDL)-induced foam cell formation in murine bone-marrow derived macrophages (BMDMs). ResultsOur analysis identified seven macrophage subsets and two neutrophil populations with distinct spatial distribution and functional roles. In early lesions, neutrophils expressing MMP9, MPO, p47phox, TGF-{beta}1 and arachidonate 5-lipoxygenase (ALOX5), aligned with proteolysis, inflammatory processes, and endothelial-mesenchymal transition features. In advanced plaques, macrophage subsets exhibit specialized functions: Ki67+ proliferative macrophages localized near necrotic cores, sustaining local population; SPP1+ macrophages, enriched in lipid handling and tissue remodeling, are prone to apoptosis/ferroptosis, potentially promoting necrotic core expansion; and C3aR+ macrophages form antigen-presenting niches with elevated HLA-DR and CD74, engaging T cells possibly through CXCL12-CXCR4 signaling. Slingshot trajectories indicated progression from C3aR toward SPP1 remodeling states concentrated at fibronectin-rich rims. In vitro, FN increased MMP9 and TIMP1 in THP-1-derived macrophages, consistent with FN imprinting remodeling features characteristic of SPP1 macrophages in situ. Concurrently, oxLDL-treated BMDMs showed enhanced lipid-handling and remodeling modules consistent with the SPP1 program. ConclusionsThese findings define conserved myeloid niches and support a microenvironment-imprinting model that links ECM composition and lipid loading to macrophage state transitions, providing a framework for microenvironment-targeted therapies to stabilize plaques and mitigate cardiovascular risk.

pathology↗