Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Dynamic alterations of DNA methylation and transcriptome in adaptation to and recovery from the space environment

Space exploration presents tremendous health challenges. Here, we report time series of multi-omic and phenotypic profiles of seventeen astronauts from six China Manned Space missions with continuous spaceflight durations ranging from 13 to 180 days. We revealed a key role of DNA methylation regulation in reshaping gene expression patterns to adapt to the space environment. Long-duration spaceflight showed more alterations in epigenetic modifications correlated with alternative splicing and protein acetylation. During recovery, an "overrange rebound" phenomenon was observed, furthermore, a mathematical model was established to describe this implying important phenomenon. Moreover, we revealed the correlations between molecular alterations and phenotypic changes such as coagulation activation and bone intensity loss. Additionally, we performed ground-based simulation experiments to estimate the impacts of individual stressors in the space environment on DNA methylation. In summary, our study highlights the importance and complexity of epigenetic regulation in adaptation to and recovery from the space environment.

genomics↗

bioRxiv2025

Synthetic Modular Cyanobacterial Consortium Enables Carbon-Negative Production of Glycerol and Derivatives

Cyanobacteria offer a direct route for converting solar energy and CO2 into valuable chemicals, yet the metabolic burden of complex heterologous pathways often limits their productivity and stability. Here, we present a modular co-culture strategy that uses glycerol as an efficient mediator metabolite to link cyanobacterial carbon fixation with downstream bioconversion. We first engineered Synechococcus elongatus UTEX 2973 (Syn2973) for high-level glycerol biosynthesis by optimizing synthase selection, eliminating competing pathways, and improving CO2-supply conditions, achieving a production of 14.53 g{middle dot}L-1. In parallel, we constructed glycerol conversion modules in Syn2973 and Gluconobacter oxydans enabling production of 1,3-propanediol (1,3-PDO), 3-hydroxypropionic acid, and dihydroxyacetone, with exogenous glycerol yielding up to 30.07 g{middle dot}L-1 1,3-PDO. Integrating the two modules resulted in a fully autotrophic co-culture that produced all three target chemicals directly from CO2, including semi-continuous 1,3-PDO production at 6.02 g{middle dot}L-1{middle dot}day-1, corresponding to a net carbon fixation efficiency of 3.43 kg CO2 eq/kg 1,3-PDO.

synthetic biology↗

bioRxiv2025

The Human Hippocampus Is Involved in Auditory Short-Term Memory Trace Formation

The human hippocampus has been claimed to play an important role in long-term memory, or episodic memory, but its contribution to short-term memory remains debated. Here, we demonstrate human hippocampal involvement in auditory short-term memory (ASTM) trace formation. We used a classic oddball paradigm and intracranial recordings across various human brain areas. High frequency activities evoked by deviant stimuli in subjects indicate the generation of ASTM trace. Analysis of auditory response latencies across the hippocampus, insula, and temporal, parietal, and frontal lobes revealed the early processing of ASTM trace at a pre-attention stage. Granger causality analysis further showed that ASTM trace processing engages hierarchical cortical areas and clarified their interactions. Bottom-up signals flow from the insula and auditory temporal regions to the frontal lobe early on, followed by top-down information from the frontal lobe to the hippocampus, inferior temporal lobe, and insula at a later pre-attentive stage. Critically, Bayesian model selection of dynamic causal modeling revealed that deviant stimuli selectively enhanced the effectiveness of top-down connections to the hippocampus, thereby facilitating ASTM trace formation. These results provide compelling evidence that the human hippocampus contributes to auditory short-term memory at a pre-attention stage and suggests a challenge to entrenched beliefs in the classification of memory systems.

neuroscience↗

bioRxiv2025

Learning Invariant Graph Representations for Cox Survival Modeling under Distribution Shifts

Survival prediction from high-dimensional biomedical data is frequently compromised by distribution shifts across multi-center cohorts, where models trained on specific populations often rely on spurious correlations that fail to generalize to new environments. While recent independence-driven reweighting techniques attempt to mitigate this, they typically treat patients as isolated instances, neglecting the intrinsic topological structures and biological pathways shared within patient populations. To address this limitation, we propose InvGraphCox (Invariant Graph Cox), a novel framework that integrates graph-structured representation learning with robust survival modeling. InvGraphCox constructs a k-nearest-neighbor patient graph to capture local manifold structures and employs a Variational Graph Autoencoder (VGAE) combined with a cohort-wise alignment mechanism to learn low-dimensional patient embeddings that are invariant to site-specific biases. We comprehensively evaluate the framework across three distinct experimental settings: the Curated Top-100 Gene Benchmark for stable biomarker identification, large-scale, high-dimensional transcriptomic datasets (Ovarian and Breast Cancer) for unsupervised representation learning, and clinical datasets (Breast and Lung Cancer) involving mixed-type covariates. Experimental results demonstrate that InvGraphCox consistently outperforms state-of-the-art baselines in terms of discrimination, calibration, and risk stratification, confirming its ability to extract robust, biologically meaningful representations in heterogeneous healthcare settings.

bioinformatics↗

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

IgG-Bridging-Seeded Synergistic Aggregation of SARS-CoV-2 Spikes Underlies Potent Neutralization by A Low-Affinity Antibody

Mechanistic studies of viral neutralization typically prioritize high-affinity antibodies, relegating low-affinity binders to the sidelines. We report P5-1C8, a Class 1 SARS-CoV-2 antibody that exemplifies this underexplored "low-affinity yet high-potency" phenotype, retaining strong neutralization of Omicron JN.1 despite markedly weakened trimer binding (KD = 225 nM; IC50 = 0.06 nM). Structural and biophysical analyses reveal that P5-1C8 engages WT and BA.1 spikes through canonical intra-spike bivalency, but with JN.1 it induces aggregation. Using virion-like nanoparticles displaying multiple spikes, we show that IgG remains bound with no detectable dissociation and triggers pronounced aggregation. Coarse-grained molecular dynamics delineate the stepwise pathway in which weak IgG-spike contacts seed aggregation via transient inter-spike bridging. Together, these findings establish the first mechanistic framework demonstrating how weak-binding antibodies can nonetheless achieve potent neutralization through higher-order aggregation, thereby expanding the conceptual landscape of antibody function and opening new directions for antibody evaluation and design. Graphical AbstractLow-affinity antibodies are frequently disregarded in discovery pipelines. This work reports P5-1C8, a Class 1 SARS-CoV-2 antibody with weak trimer binding (KD-to-IC50 > 3,700-fold) yet potent neutralization of Omicron JN.1. Structural, biophysical, functional and coarse-grained simulations collectively demonstrate that transient inter-spike IgG bridging seeds higher-order aggregation, which in turn drives neutralization and provides a mechanistic framework. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/691454v1_ufig1.gif" ALT="Figure 1000"> View larger version (79K): org.highwire.dtl.DTLVardef@1d4f132org.highwire.dtl.DTLVardef@1274400org.highwire.dtl.DTLVardef@e186a6org.highwire.dtl.DTLVardef@4f1870_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Emergence of hypervirulent Klebsiella pneumoniae ST111 with a novel virulence plasmid causing pyogenic liver abscess

Hypervirulent Klebsiella pneumoniae (hvKp)-associated pyogenic liver abscess is typically linked to K1/K2 capsule serotypes and canonical lineages such as ST23, ST65, and ST86. Cases outside these lineages are rare but pose significant public health concerns. We investigated an unusual liver abscess in an immunocompetent patient caused by hvKp sequence type 111 (ST111). Five isolates (KP1-KP5) were recovered, all belonging to the same clonal lineage. Genomic analysis revealed a 181 kb IncHI1B/IncFIB virulence plasmid encoding rmpADC and the salmochelin cluster (iroBCDN), but lacking aerobactin (iuc) and rmpA2 gene loci. Notably, this plasmid also carried the yersiniabactin locus (ybt), which is typically located on the chromosome. Comparative genomics indicated that similar ST111 strains have been reported only in China, suggesting clonal expansion of a previously unrecognized hvKp lineage. Functional assays demonstrated that plasmid curing abolished the hypermucoviscous phenotype, reduced capsule production, and completely attenuated virulence in murine models, confirming the plasmid as the major driver of hypervirulence. Interestingly, plasmid loss enhanced biofilm formation and relieved growth burden, highlighting fitness trade-offs associated with virulence. This study documents a rare case of hvKp ST111 liver abscess and identifies a novel plasmid conferring hypervirulence. Our findings expand the known diversity of hvKp and underscore that non-canonical lineages can acquire potent virulence determinants enabling severe disease. Continued clinical and genomic surveillance is essential to detect emerging hvKp clones before widespread dissemination.

microbiology↗

bioRxiv2025

INVA8001, a Novel and Highly Selective Chymase Inhibitor, Ameliorates Liver Inflammation, Fibrosis, and Hyperplasia in Mdr2 Knockout Mice

Background and aimsMast cells (MCs) play a significant role in autoimmune diseases by mediating inflammatory responses, innate and adaptive responses, angiogenesis, and various pathological processes. MC numbers are significantly increased in chronic liver disease and liver cancer, and degranulation leads to a release of MC mediators, including chymase, which increases inflammation, activates hepatic stellate cells (HSCs), ultimately leading to fibrosis. Primary sclerosing cholangitis (PSC) is a chronic, progressive cholestatic liver disease characterized by inflammation, fibrosis, ductular reaction, and eventual liver failure. MCs and their mediators, particularly chymase, have been implicated in PSC pathogenesis; however, targeting chymase therapeutically has remains largely unexplored. In this study, we aimed to evaluate the efficacy of INVA8001, a highly selective chymase inhibitor, on PSC pathogenesis in the Mdr2 knockout mouse (Mdr2-/- mice) model of PSC. Approach and ResultsWe evaluated the levels of chymase and other MC markers in liver biopsy samples collected from late-stage PSC patients. The effect of chymase inhibition was evaluated using a highly selective and potent small molecule chymase inhibitor, INVA8001, in Mdr2-/- mice. Ten-week-old male Mdr2-/- mice were injected intraperitoneally (IP) with 20 mg/kg of INVA8001 daily for two weeks and varying parameters of disease pathogenesis, including MC activation, inflammation, fibrosis, biliary pathology, and cholestasis were evaluated. In addition, histological, immunohistochemical, biochemical, and molecular analyses were conducted to evaluate the effects of INVA8001 treatment in Mdr2-/- mice. The liver biopsies of PSC patients showed an increased number of chymase-positive cells compared with control samples (collected from non-diseased patients). INVA8001 treatment resulted in a reduction of MC accumulation, inflammation, histological damage, fibrosis, ductular reaction, and biliary senescence in Mdr2-/- mice. The current data show the pathophysiological role of chymase in PSC and the impact of a selective chymase inhibitor on PSC disease pathogenesis. This can further be extrapolated to various MC-driven diseases such as chronic urticaria, atopic dermatitis, asthma, metabolic dysfunction-associated steatohepatitis (MASH), and eosinophilic gastrointestinal diseases, among others, where chymase is central to the disease pathogenesis. ConclusionsOur findings identify chymase as a key driver of PSC pathogenesis establishing INVA8001 as a promising new therapeutic candidate for hepatobiliary disorders, including PSC. Chymase inhibition simultaneously targets MC activation, inflammation, fibrosis, and biliary senescence, and offers a multifaceted approach to treating PSC and other MC-related disorders.

pathology↗

bioRxiv2025

Emergence of mutHV76G among longitudinal carbapenem resistant Klebsiella pneumoniae causing long-term colonization and recurrent infection disrupts DNA mismatch repair and results in a hypermutator phenotype

Although hypermutation due to Mut protein mutations that disrupt DNA mismatch repair has been characterized in some bacteria, its mechanisms and consequences in Klebsiella pneumoniae remain poorly defined. We analyzed 11 longitudinal KPC-3 carbapenemase-producing, ST258 K. pneumoniae isolates collected over [~]4 years from an immunocompromised patient with chronic colonization and recurrent infections. After [~]3.3 years, isolates developed ceftazidime-avibactam (CZA)-resistance with restored carbapenem susceptibility, coinciding with emergence of a V76G substitution in a highly-conserved motif in the core of MutH endonuclease. Compared with earlier isolates, mutHV76G-carrying isolates showed greater within-host genomic diversification (69-179 vs. 2-12 SNP differences) and acquired blaKPC-3L169P, encoding an KPC {Omega}-loop substitution that mediates CZA resistance and re-establishes carbapenem susceptibility. mutHV76G isolates exhibited stepwise increases in meropenem-vaborbactam (MVB) and cefiderocol minimum inhibitory concentrations, plausibly linked to substitutions in KPC, OmpK36 porin, CirA iron transporter and/or EnvZ kinase. Clinical mutHV76G isolates and CRISPR-engineered mutHV76G mutants were hypermutators based on rifampin mutational frequencies. Using isogenic mutant and parent strains, we confirmed that mutHV76G accelerated evolution of CZA and MVB resistance in vitro and in vivo, promoted transfer and uptake of resistance plasmids, and improved bacterial fitness during mouse infections. Resistance evolution in mice recapitulated clinical trajectories, with blaKPC-3 and ompK36 mutations emerging under CZA and MVB exposure, respectively. Phenotypes of mutHV76G and mutH-null strains were comparable, indicating that the V76G substitution largely abrogates MutH function. Our findings reveal MutH-mediated hypermutation as an adaptive mechanism in K. pneumoniae, enabling rapid antibiotic resistance development and plasmid acquisition without fitness cost. ImportanceHypermutator bacteria pose a formidable clinical threat by rapidly evolving antibiotic resistance and adapting within the human host. Klebsiella pneumoniae is a major cause of multidrug-resistant infections, yet the contribution of hypermutation to its evolution remains poorly characterized. Analyzing K. pneumoniae isolates collected over [~]4 years from a chronically infected/colonized patient, we demonstrate that emergence of a mutation in mutH (mutHV76G), a DNA mismatch repair gene, results in hypermutation phenotypes and rapid accumulation of gene mutations. Both clinical and lab-engineered mutHV76G mutant strains rapidly acquire resistance or reduced susceptibility to new antibiotics like ceftazidime-avibactam, meropenem-vaborbactam and cefiderocol, due to mutations in carbapenemase (blaKPC-3), porin (ompK36) and other genes. mutHV76G-driven hypermutation also enhances horizontal transfer of resistance plasmids and improves K. pneumoniae fitness during mouse infections. This study is important for understanding K. pneumoniae hypermutation as a potent mediator of antibiotic resistance and other phenotypes relevant to human infections.

microbiology↗

bioRxiv2025

Isolation of functional supramolecular attack particles (SMAPs)

High density cultures of the GMP compatible NK-92 natural killer cell line release heterogeneous extracellular particles (EP), notably extracellular vesicles (EV) and non-vesicular extracellular particles (NVEP). The NVEP include a small proportion of supramolecular attack particles (SMAPs), the direct cytotoxic potential of which suggests therapeutic promise, yet scalable enrichment and functional evaluation alongside other extracellular particles (EPs) have been lacking. Here, we develop a high-resolution serial size-exclusion liquid chromatography (SELC) workflow that resolves EPs into 4 fractions F1-F4, in which EV are enriched in F2 and SMAPs are enriched in F3. Multi-modal characterization using Nanoparticle Tracking Analysis (NTA), Nano Flow Cytometry (Nano FCM), Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRFM) and Proteomics) demonstrated that F2 NK-92 EVs are [~]130-150 nm particles enriched for complement proteins, whereas SMAPs (F3) are 100-110 nm particles enriched for cytotoxic proteins (PRF1, GZMB) and SMAP shell components (e.g., THBS1, THBS4). Functionally, Ca2+-stabilized SMAPs (F3) trigger caspase-3-dependent apoptosis in tumor cell lines with robust dose responses, while F2 lacks direct cytotoxicity in vitro. Ex vivo, F3 SMAPs kill patient-derived breast cancer and chronic lymphocytic leukaemia cells (CLL) in a concentration-dependent manner. In NSG mice, intra-tumoral treatment with SMAPs (F3) restrains the growth of aggressive B16F10 melanoma and PANC-1 pancreatic cancer, confirming in vivo functionality. These results establish a scalable method to isolate and compare NK-derived particle classes and provide a foundation for targeted SMAP engineering as a promising approach for treatment of solid tumours. Significance StatementCell therapies face barriers in solid tumours, including lack of entry and suppression. NK cells release extracellular particles (EPs), including supramolecular attack particles (SMAPs), that can traffic into tumour tissue. We define a practical, scalable chromatography process that resolves NK-derived SMAPs from other EPs and demonstrate that SMAPs induce caspase mediated apoptosis in vitro and engage in cytotoxic lymphocyte independent anti-tumour activity in vivo. These insights open a route to engineering and testing SMAP-based therapeutics to overcome limitations of whole-cell approaches in solid cancers.

immunology↗

bioRxiv2025

pGGTOX: a versatile plasmid platform for recombination-based genome editing across Enterobacteriaceae

Precise genome editing in Enterobacteriaceae is essential for studying gene function, pathogenesis, and antimicrobial resistance, yet many current systems face host-specific and efficiency limitations. We developed pGGTOX, a modular plasmid platform that enables efficient homologous recombination-mediated genome editing across diverse Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Enterobacter intestinihominis. The system integrates a rhamnose-inducible toxin (MqsR) for stringent counterselection, a sfGFP reporter for visual tracking of recombination events, Golden Gate cloning for rapid assembly of homologous arms, an FRT-flanked resistance cassette for marker removal, and an oriT sequence for conjugative transfer. Together with the companion plasmid pCP20-oriT, pGGTOX supports precise, marker-free genomic modification. Using pGGTOX, we achieved targeted deletions of dapA in E. coli and mrkCD in carbapenem-resistant K. pneumoniae, both with 100% efficiency. The dapA mutant exhibited diaminopimelate auxotrophy, while mrkCD deletion markedly reduced biofilm formation, consistent with the loss of function associated with these genes. pGGTOX also enabled deletion of a 43.1-kb type IV secretion gene cluster (tra) from an IncN/FII plasmid in E. intestinihominis and insertion of a 10-kb CRISPR-Cas9 plasmid-curing module (pCasCure) into an S. enterica IncX1 plasmid. Deletion of the tra gene cluster resulted in a substantial reduction in plasmid conjugation efficiency. Conjugative transfer of the engineered IncX1-pCasCure plasmid into K. pneumoniae facilitated CRISPR-mediated curing of blaKPC, sensitizing carbapenem resistance to susceptibility. In summary, pGGTOX provides a versatile, efficient, and broadly applicable platform for genome engineering and CRISPR delivery in Enterobacteriaceae, expanding the toolkit for bacterial genetics and translational antimicrobial research. ImportancePrecise genetic manipulation in Enterobacteriaceae remains a major technical challenge, particularly for non-model or multidrug-resistant strains. We developed pGGTOX, a versatile and broadly applicable plasmid platform that enables efficient, marker-free genome editing through homologous recombination. By integrating stringent counterselection, visual screening, modular cloning, and conjugative transfer, pGGTOX simplifies construction and streamlines editing across multiple clinically relevant species. We demonstrate its utility in deleting chromosomal and plasmid-borne loci, inserting large genetic modules, and delivering CRISPR-Cas9 systems for targeted elimination of antibiotic resistance genes. This platform expands the molecular toolkit for functional genomics and provides a powerful new strategy for dissecting bacterial virulence, resistance, and plasmid biology.

microbiology↗

bioRxiv2025

Distinct and Shared Impacts of Virulence Plasmids on the Phenotype and Transcriptome in Convergent Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae

The global rise of convergent carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) represents a major clinical challenge, yet the role of virulence plasmids (pVirs) in shaping bacterial physiology and pathogenicity remains incompletely understood. Using a CRISPR-Cas9-based curing system, we precisely eliminated pVirs from two clinical CR-hvKp strains with distinct genetic backgrounds, ST23-KL1 (blaNDM-1) and ST11-KL64 (blaKPC-2). Loss of pVir conferred fitness advantages in vitro, reduced capsule production and hypermucoviscosity, and promoted biofilm formation, while markedly attenuating virulence in murine sepsis models. Despite this reduction, the pVir-cured ST23-KL1 strain retained higher virulence than the pVir-cured ST11-KL64 strain, underscoring the contribution of chromosomal background to pathogenic potential. Transcriptomic profiling revealed both shared and strain-specific transcriptional responses to pVir deletion, with broader perturbations observed in the ST11-KL64 strain. pVir removal had limited effects on antibiotic MICs. Complementation experiments further demonstrated differential regulatory roles of the rmpADC and rmpA2D2 operons in capsule expression and hypermucoviscosity across the two strains. Together, these findings establish pVirs as central determinants of CR-hvKp virulence and highlight complex host-plasmid interactions that influence bacterial adaptation and pathogenicity. IMPORTANCEThe emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) poses a critical threat to global health, yet the contribution of virulence plasmids (pVirs) to bacterial fitness and pathogenicity remains poorly defined. By employing a CRISPR-Cas9-based curing strategy, we dissected the role of pVirs in two genetically distinct CR-hvKp strains and uncovered their multifaceted impact on capsule production, hypermucoviscosity, biofilm formation, and virulence. Our findings reveal that pVir loss confers fitness advantages in vitro while attenuating virulence in vivo, with strain-specific transcriptional responses and differential regulation by rmp operons. These results underscore the complex interplay between plasmid-encoded and chromosomal determinants in shaping CR-hvKp pathogenicity and adaptation, offering mechanistic insights that may inform future therapeutic strategies targeting plasmid-mediated virulence.

microbiology↗

bioRxiv2025

mdBIRCH for Fast, Scalable, Online Clustering of Molecular Dynamics Trajectories

We present mdBIRCH, an online clustering method that adapts the BIRCH CF-tree to molecular dynamics (MD) data by applying a merge test calibrated directly to RMSD. Each arriving frame is routed to the nearest leaf microcluster and merged only if the post-merge centroid-based spread, computed from the cluster feature (CF) summaries, remains within a user-supplied threshold{tau} . This enables incremental, memory-bounded operation without constructing pairwise distance matrices, with a physically interpretable parameter controlling structural granularity. We evaluate mdBIRCH on {beta}-heptapeptide and HP35 systems and propose two practical strategies to make the threshold selection easier: (a) RMSD-anchored runs that use controlled structural edits to define interpretable operating points and (b) a blind sweep that tracks how cluster counts, occupancies, and coverage evolve with the threshold. Across both systems, increasing this threshold produces predictable consolidation into fewer, higher-occupancy states, and broader RMSD-to-centroid distributions. We further analyze the effect of the CF-tree capacity through the branching factor, quantify sensitivity to data ordering, and compare dominant-state representatives against batch clustering workflows to contextualize the resulting partitions. Finally, because decisions rely only on cluster summaries, mdBIRCH scales near-linearly with the number of frames on standard CPU hardware, offering a practical combination of speed and interpretability for large-scale trajectory analysis.

biophysics↗

bioRxiv2025

Bridging Language Markers and Pathology: Correlations Between Digital Speech Measures and Surrogate CSF Biomarkers in Alzheimer's Disease

BackgroundDigital language markers show promise in detecting early cognitive impairment related to Alzheimers disease (AD), yet their relationship with cerebrospinal fluid (CSF) biomarkers of AD pathology remains unclear mainly due to the lack of data with both CSF and language markers. ObjectiveThis study aims to build links between digital language markers and fluid biomarkers through surrogate CSF biomarkers. MethodsUsing NACC clinical data as anchor variables, language makers in the I-CONECT study were linked to NACC CSF data. Surrogate CSF biomarkers were created for I-CONECT subjects using machine learning models from common NACC clinical variables. Correlations assessed associations between CSF and language markers. ResultsLower predicted amyloid-{beta} correlated significantly with reduced syntactic complexity and shorter speech responses. Higher predicted total tau and phosphorylated tau correlated with reduced syntactic complexity. ConclusionsThis study demonstrates novel links between language markers and fluid biomarkers, highlighting conversational language as a potential accessible, non-invasive approach for early detection and monitoring of Alzheimers pathology.

bioinformatics↗

bioRxiv2025

The Structural Basis of Pacs1-Wdr37 Complex Assembly and Stability

Phosphofurin acidic cluster sorting protein 1 (Pacs1) is a multidomain adaptor proposed to bind transmembrane cargo proteins to facilitate their intracellular trafficking. Pacs1 also forms a complex with WD-repeat protein 37 (Wdr37), which is essential for lymphocyte homeostasis. Despite numerous proposed binding partners, a validated structure for Pacs1-containing protein complexes is lacking. Here, we present the cryo-electron microscopy structure of the Pacs1-Wdr37 complex. Pacs1 binds Wdr37 through a conserved interface within its furin-binding region (FBR), the domain previously linked to cargo recognition. This interaction stabilizes Wdr37 and is critical for the expression of both proteins. A gain-of-function mutation in PACS1 (R203W) causes a highly penetrant neurodevelopmental syndrome. This pathogenic mutation lies on a solvent-exposed surface of the FBR and does not disrupt complex formation. Instead, Pacs1-R203W remains dependent on Wdr37 for stability and its levels can be reduced through targeted Wdr37 proteolytic degradation. Structural homology of the FBR to synaptotagmin C2 domains reveals a previously unrecognized ability of Pacs1 to bind negatively charged phospholipids through a unique positively charged cleft. Together these findings define the structural basis for Pacs1-Wdr37 complex assembly and stability, present potential strategies for Pacs1-mediated neurodevelopmental disease, and suggest novel Pacs1 functions in membrane association.

biochemistry↗

bioRxiv2025

Clair-Mosaic: A deep-learning method for long-read mosaic small variant calling

Mosaic variants, defined as postzygotic mutations occurring during an organisms development from zygote to adult, play critical roles in developmental biology, aging, and diseases such as cancer and neurological disorders. However, their accurate detection remains challenging due to low abundance in the genome and low variant allelic fractions (VAF). While current mosaic variant callers are primarily designed for short-read sequencing, no method is available for long-read sequencing, which can generate tens of kilobase-long reads to cover complex genomic regions inaccessible to short reads. To fill the gap, we present Clair-Mosaic, a deep-learning-based method for detecting mosaic small variants from long-read data. Clair-Mosaic was trained on hundreds of millions synthetic variants encompassing diverse read coverages and allelic fractions, enabling it to detect low-VAF mosaic variants with high sensitivity in paired-sample and single-sample modes. In addition to neural network prediction, Clair-Mosaic distinguishes genuine mosaic variants from sequencing artifacts by leveraging their inherent haplotype relationship in phased long reads. Furthermore, a Bayesian mosaic-germline discriminator is introduced to distinguish mosaic variants from germline variants. It also employs multiple post-calling filters, including a mosaic variant database and multiple germline population resources, to tag common germline and mosaic variants. Comprehensive benchmarking on synthetic datasets and real samples demonstrated Clair-Mosaics outstanding performance in ONT and PacBio. Clair-Mosaic is also applicable to short-read data and outperforms methods like MosaicHunter, MosaicForecast, DeepMosaic, and DeepSomatic. Clair-Mosaic is open-source and available at https://github.com/HKU-BAL/Clair-Mosaic.

bioinformatics↗

bioRxiv2025

Using the simple telegraph model to decipher transcriptional burst regulation across genome-wide data

Gene transcription is a stochastic bursting process, with burst frequency and burst size as core parameters. Deciphering its genome-wide regulation is challenging, as complex models (capturing detailed biology) are impractical for large-scale use due to unstable inference and high computation. We proposed a simple telegraph model-based framework to analyze genome-wide scRNA-seq and smFISH datasets, addressing these limitations. When sample size [≥] 500 and burst parameter change [≥] 3-fold, its inferred burst frequency- and size-dominated variations reliably proxy true regulation. Analyses across mouse cells (CAST/C57 alleles, fibroblasts vs. embryonic stem cells) and healthy/hypertrophic cardiomyopathy (HCM) human heart tissues revealed key rules: 1) Burst frequency- or size-dominated regulation was primary (over 70% of variable genes), with more bursty-regulated genes in HCM; 2) TATA-initiator synergy (enhancing burst size-dominated regulation) was lost in HCM; 3) Burst frequency-dominated genes enriched in genome stability/cell cycle/apoptosis (via TFs like Foxo4/Mcm2), while burst size-dominated ones in signaling/metabolism/proteostasis (via TFs like Zfp322a/Smchd1/Ppargc1a), and their interdependent dysregulation accelerated HCM. This study establishes the simple telegraph model as a scalable framework linking transcriptional burst dynamics to cell fate and pathology.

molecular biology↗

bioRxiv2025

VRC01 Selects Rare HIV Escape Mutations After Acquisition in Antibody-Mediated Prevention Trials

Broadly neutralizing antibodies (bnAbs) show promise in HIV prevention, yet viral escape remains a challenge. In the Antibody Mediated Prevention (AMP) trials, the CD4 binding site (CD4bs) bNAb VRC01 blocked acquisition by VRC01-sensitive strains. However, its influence on viral evolution post-acquisition is not fully understood. Here we analyzed >12,000 HIV env sequences from 47 participants from the AMP trials, identifying VRC01-mediated de novo escape mutations in 8 of 26 VRC01-treated participants but none in 21 placebo participants. These mutations were found at very low frequency (<1%) in global viruses. Escape mutations, primarily located in the Loop-D and {beta}23/V5 regions of Env, conferred cross-resistance to several CD4bs bnAbs, while more potent CD4bs bnAbs like N6 and 1-18 largely retained their activity. Our findings demonstrate that prophylactic VRC01 can select for viral escape after infection, underscoring the need for next-generation bnAbs with improved breadth and potency to enhance durability and efficacy of antibody-based HIV prevention.

microbiology↗