Biology subjects

Zhang, M.

Publications and source records attributed to Zhang, M..

At least 91 records · Page 5Linked to original sources

bioRxiv2026

A fast-to-faithful transition shapes the DNA repair landscape during embryogenesis

Accurate and timely repair of DNA double-strand breaks (DSBs) is essential for genome maintenance in all cells. Embryos are particularly vulnerable to DSBs. During zebrafish development, a single fertilized cell undergoes rapid divisions to form an embryo of 50,000 cells in the first 24 hours, subjecting its genome to intense replication stress and the inevitable formation of genomic DSBs. While we know that failure to repair these breaks can result in embryonic lethality, the kinetics, fidelity, and pathway choice of DSB repair during embryogenesis are not well understood. Here, we used light-activated CRISPR to generate targeted genomic DSBs across zebrafish embryo development. Importantly, DSB induction occurs within seconds after light stimulation, enabling precise measurements of repair kinetics within a single cell cycle. We found that DSBs were repaired within 15 minutes during the early, rapid-division stages. At later stages, the pace of DNA repair declines as the cell cycle slows. By leveraging mathematical modeling and mutants that disrupt DNA repair pathways, we uncovered a developmental transition from error-prone microhomology-mediated end joining to more faithful non-homologous end joining that correlates with the gradual shift in repair kinetics. To our knowledge, this is the first study to resolve DSB repair dynamics with high temporal resolution during embryo development. Our study establishes a framework for systematically interrogating the cellular responses to DNA damage in living model organisms. SIGNIFICANCE STATEMENTThis study reveals how developing embryos rapidly repair dangerous DNA breaks while balancing speed and accuracy. Using a light-activated CRISPR system, the authors precisely control when DNA damage occurs and measure repair in real time in living zebrafish embryos. They show that early embryos prioritize extremely fast but error-prone repair, then transition to slower, more accurate mechanisms as development progresses. This "fast-to-faithful" shift explains how embryos protect their genomes during rapid cell division. The work provides a new framework for studying DNA repair in vivo and has implications for understanding developmental disorders, genome editing outcomes, and how cells manage DNA damage under changing biological conditions.

genetics↗

bioRxiv2026

Distinct neural architectures of focus: Memory-related engagement is associated with control disengagement

Prevailing models propose that sustained focus is a product of top-down control that amplifies task-relevant representations while suppressing distraction. We challenge this view, demonstrating that the neural signatures of mental focus differ across cognitive modes. In two neuroimaging studies, we used a paradigm that creates conflict between external task goals and self-generated thought and modelled neural activity as a function of trial-by-trial subjective focus to identify how people stay on task in contexts varying in memory engagement. In Study 1, on-task thoughts were associated with distinct neural architectures in arithmetic and comprehension: arithmetic-related focus recruited visual-motor regions, while comprehension-related focus was characterized by the recruitment of memory systems and reduced activation of control regions. Study 2 extended these findings, with both externally directed comprehension and internal memory retrieval associated with a shared neural profile of reduced control engagement, despite reliance on distinct memory systems. Furthermore, focus covaried with activity in heteromodal semantic regions across both reading and listening, suggesting semantic engagement is a primary marker of being on-task during comprehension. Our findings indicate that sustained focus during memory-guided cognition is characterized by mutual disengagement between control and memory networks. This discovery challenges the view that top-down supervision is a universal requirement for task focus, since sustained attention is associated with distinct neural configurations in different cognitive modes.

neuroscience↗

bioRxiv2026

CHORD: a framework for cross-species single-cell integration across gene, cell and cell-type levels

Quantifying cross-species relationships among cell types from single-cell transcriptomic data can reveal both conserved and divergent patterns of cell-type hierarchies. However, existing cross-species integration methods can be limited in modeling genes beyond orthologs by leveraging cell-type-resolved transcriptional context, or in learning explicit type-level representations. Here we present CHORD, a cross-species integration framework that jointly learns representations of genes, cells and cell types. We demonstrate that CHORD can integrate cross-species single-cell atlases and support cell-type annotation with unknown cell-type detection. In the frog-zebrafish embryogenesis and mammalian motor cortex atlases, CHORD infers cell-type trees that place conserved cell types from different species in relative proximity and summarize hierarchical relationships among cell types. CHORD also supports cross-species comparison of continuous phenotypic variation by placing embryonic cells along an aligned developmental timeline. CHORD further yields gene embeddings that capture orthologous and functional relationships, and gene importance scores linking genes to cell types.

bioinformatics↗

bioRxiv2026

Role of Alanine Transaminase in Retinal Metabolic Homeostasis: Potential therapeutic target in retinal diseases

PurposeAlanine transaminases (ALT), encoded by the GPT gene, catalyzes the reversible conversion of pyruvate and glutamate to alanine and alpha-ketoglutarate, thereby correlating carbohydrate and amino acid metabolism. However, its role in the human neural retina remains unclear. This study aimed to explore the expression, localization, and metabolic function of ALT in the human neural retina and its potential involvement in retinal diseases. MethodsALT1 and ALT2 expression and localization were examined in the retinas of healthy and diabetic retinopathy (DR) donors via immunoblotting and immunofluorescence. ALT function was assessed in ex vivo human retinal explants using pharmacological inhibition with beta-chloro-L-alanine (BCLA), followed by the analyses of enzyme activity, tissue injury, and transcriptomic responses. Stable-isotope tracing with 13C-and 15N-labelled substrates combined with GC-MS was used to define ALT-dependent carbon and nitrogen fluxes in macular and peripheral retinas. Redox level (NADPH/NADP+) was also evaluated under tert-butyl hydroperoxide-induced oxidative stress. ResultsALT1 and ALT2 were both expressed in the human neural retina, with prominent localization in Muller glia and photoreceptor inner segments. ALT1 displayed a diffuse cytoplasmic distribution, whereas ALT2 demonstrated a punctate pattern consistent with mitochondrial localization. In DR retinas, ALT1 expression was spatially disorganized and heterogeneous, while ALT2 remained comparatively preserved. Inhibition of ALT with BCLA markedly reduced ALT activity without causing overt cytotoxicity or major transcriptional changes. Isotope tracing demonstrated that retinal ALT predominantly channels pyruvate-derived carbon into alanine, whereas alanine was minimally contributed to pyruvate production under basal conditions. ALT inhibition suppressed alanine synthesis and release, redirected nitrogen flux towards glutamate, glutamine, and aspartate, and uncovered distinct metabolic adaptations in macular but not peripheral retinas. Under oxidative stress, ALT inhibition induced the decrease of NADP+/NADPH ratio and LDH release, indicating improved redox balance and reduced tissue injury. ConclusionsALT is previously unrecognized as a regulator of carbon and nitrogen partitioner in the human neural retina, contributing to redox homeostasis under stress. The altered distribution of ALT1 in DR retina and the protective metabolic effects of ALT inhibition suggest ALT as a potential contributor to retinal metabolic vulnerability and a candidate therapeutic target in retinal diseases.

neuroscience↗

bioRxiv2026

Emergence of rigidity percolation and critical behavior in tunable protein condensates

Multivalent proteins are known to form complex networks within biomolecular condensates, yet the mechanisms governing the emergence and evolution of these networks remain poorly understood. Here, we utilize a synthetic protein chimera system with tunable interactions to investigate the transition from liquid-like droplets to networked condensates. By employing single-amino-acid substitutions, we generated a series of mutants with varying protein-binding strengths. As the interaction strength increases, the condensates undergo a sharp rigidity percolation transition, characterized by a more than 200-fold increase in both elastic modulus and viscosity. Near this transition, we identify a critical scaling relation in the condensate elasticity, providing robust evidence for a percolation-driven assembly mechanism. Furthermore, we demonstrate that this network architecture is fundamentally linked to biological functions. The introduction of disease-associated mutations disrupts the network, significantly softening the condensates and rendering them fluid-like. Our findings reveal that network percolation and criticality in protein condensates can be sensitively regulated by single-amino-acid substitutions, underscoring their essential role in maintaining structural integrity and supporting physiological function.

biophysics↗

bioRxiv2026

Auxin promotes GPI-anchored protein-mediated trafficking of ABP1 to enable cell-surface auxin signaling

Rapid auxin responses are triggered by Auxin-Binding Protein 1 (ABP1) signaling; however, the coupling of hormone perception to signaling activation is unclear. ABP1, a cell surface auxin receptor, is localized to endoplasmic reticulum (ER), raising a fundamental question of where auxin is perceived and how signaling-competent receptors are mobilized to cell surface. Herein, we established a dark-induced auxin synthesis system in Arabidopsis hypocotyl; abp1 mutants showed pronounced elongation. Auxin dose-dependently stimulates LORELEI-like glycosyl-phosphatidylinositol-anchored protein 1 (GPI-AP, LLG1) interaction with ABP1, driving their outward trafficking from ER. However, this complex dissociated in acidic apoplast, facilitating H+-ATPase-mediated auxin signaling. Our findings reveal how intracellular auxin perception is converted into rapid cell-surface signaling, establishing a mechanistic framework for fast auxin responses and uncovering a GPI-AP-mediated trafficking mechanism for ER-retained receptors.

plant biology↗

bioRxiv2026

A PTM Regulatory Enzyme Co expression Code Defines Microglial Functional Heterogeneity in Cerebral Ischemia Reperfusion Injury

BackgroundCerebral ischemia-reperfusion injury (CIRI) is a major determinant of poor outcome after recanalization therapy in acute ischemic stroke. Microglial functional heterogeneity underpins neuroinflammation, yet the molecular mechanisms governing microglial phenotypic transitions remain incompletely understood. Metabolite-driven post-translational modifications (PTMs) have emerged as key regulators of microglial metabolism and inflammation, but whether PTM regulatory enzymes form co-expression modules that define microglial states is unknown. MethodsWe analyzed single-cell RNA-seq datasets from five GEO studies (GSE174574, GSE227651, GSE245386, GSE267240, GSE319237) covering tMCAO reperfusion and permanent ischemia models. Microglia were purified using double filtration (P2ry12/Tmem119/Cx3cr1+, Cd68/Adgre1/Ly6c-). PTM enzyme co-expression modules were identified by non-negative matrix factorization (NMF). Spatiotemporal dynamics were assessed by module projection across timepoints (Sham, 1d, 3d, 7d) and pseudotime analysis. Independent validation was performed in an additional tMCAO dataset (GSE245386). Sex differences were explored in a mixed-sex permanent ischemia dataset (GSE267240). ResultsThree robust PTM enzyme co-expression modules were identified: Metabolic stress-associated (M1), Pro-inflammatory-associated (M2), and Reparative-associated (M3). M1 was enriched in TCA cycle enzymes, M2 in inflammatory pathways (leukocyte activation, chemotaxis), and M3 in vascular development and translation. Module proportions and scores showed dynamic transitions: M1 decreased after reperfusion, M2 peaked at day 1-3, and M3 slightly increased at day 7. Independent validation in GSE245386 yielded high module conservation (cosine similarity = 0.874). Sex-specific differences in module distribution were observed in permanent ischemia ({chi}2 = 14.98, p = 0.00056). ConclusionsPTM enzyme co-expression modules delineate metabolic, pro-inflammatory, and reparative microglial states in CIRI with distinct spatiotemporal dynamics. This transcriptional framework supports the "PTM enzyme code" hypothesis and provides stage-specific targets for stroke therapy.

neuroscience↗

bioRxiv2026

Endogenous mutational mechanisms and metabolic context shape endometrial cancer

Uterine corpus endometrial carcinoma (UCEC) is a common gynecologic malignancy with rising mortality, yet its genome-wide mutational architecture remains incompletely understood. Here we analyze deep whole-genome sequencing data from 440 UCEC tumors from The Cancer Genome Atlas, integrated with transcriptomic and clinical data, to define subtype-specific mutational processes and genomic architectures. We uncover pronounced molecular subtype-specific differences in endogenous mutational mechanisms, retrotransposition activity, structural variation, and chromosomal instability. LINE-1 retrotransposition emerges as a key contributor to genome instability in UCEC, acting as a prominent source of structural variation in copy-number stable tumors and associating with chromothripsis and ecDNA-mediated oncogene amplification in copy-number high tumors. Mutational signature SBS28 contributes significantly to mutations in POLE-deficient tumors and is strongly correlated with SBS10a and SBS10b. Mismatch repair-deficient tumors exhibit a previously unrecognized doublet base substitution signature and a defining imbalance between ID2 and ID1 indel processes, reflecting pervasive DNA template-strand replication slippage and associated with increased tumor immunogenicity. Copy-number low tumors follow a distinct low-mutagenesis evolutionary trajectory characterized by reduced replication stress, low proliferative activity, genomic stability and enrichment of SBS18 associated with oxidative damage. Notably, we identify a consistent inverse association between body mass index and tumor mutational burden in UCEC, suggesting that host metabolic state may influence fundamental cellular processes governing mutation accumulation, promoting tumor development through non-mutagenic mechanisms rather than elevated genomic instability. Together, these findings establish a mutagenesis-centric framework for UCEC that links endogenous mutational mechanisms to tumor architecture and host metabolic context, uncovering previously unrecognized subtype-specific genomic features with important implications for refined risk stratification and therapeutic strategies.

genomics↗

bioRxiv2026

Celastrol alleviates SGLT2 inhibitor-induced diabetic hyperketonemia by inhibiting hepatic ketogenesis

SGLT2 inhibitor (SGLT2i)-induced diabetic hyperketonemia is a life-threatening acute complication of diabetes. While Celastrol has been reported to exert beneficial effects on obesity; its potential role in ketogenesis remains unclear. In this study, Celastrol administration significantly attenuates the fasting-induced elevation of blood {beta}-hydroxybutyrate. Moreover, a 7-day course of Celastrol (1 mg/kg/day) leads to reductions in body weight and fat mass. Mechanistically, Celastrol specifically downregulates HMGCS2 expression and suppressess hepatic ketogenesis through inhibiting PPAR expression in the short term ([≤] 2 days). However, after prolonged treatment for 7 days, Celastrol modulates both PPARand serum free fatty acids (FFAs) levels. Furthermore, anti-ketogenic effect of Celastrol is abolished in Ppar{square} /{square} mice. Importantly, Celastrol effectively ameliorates SGLT2i-induced hyperketonemia. In summary, Celastrol curbs hepatic ketone overproduction in a PPAR-dependent manner, indicating its protective potential against SGLT2i-induced hyperketonemia.

molecular biology↗

bioRxiv2026

A rugged binding landscape unifies static and dynamic paradigms in protein-protein interactions

Predicting protein-protein interaction (PPI) affinities from structural data remains a challenge. Although binding funnel theory describes the formation of native complexes, the topography of the funnel bottom and its influence on affinity are often overlooked. Using two well-controlled nanobody-antigen datasets as model systems, we demonstrate that PPI can exist in both static and dynamic binding paradigms. These two nanobody series adopt nearly identical binding poses toward their respective antigens yet exhibit diverse affinities, each representing a distinct paradigm: a static paradigm in which affinity can be ranked by Rosetta scoring of co-crystal structures alone, and a dynamic paradigm in which affinity can only be ranked using molecular dynamics-sampled ensembles. The two paradigms differ in interfacial dynamics. In the dynamic series, the relative motion between binding partners ({Delta}RMSF) is temperature-sensitive, with optimal affinity correlation at 298 K. In the static series, {Delta}RMSF is minimal and insensitive to temperature. Local frustration analysis establishes a mechanistic bridge between interfacial dynamics and landscape topology. Dynamic interfaces exhibit increased frustration upon thermal sampling, facilitating sampling of functionally relevant microstates, whereas static interfaces show minimal frustration changes across temperatures. The temperature-dependent frustration difference mirrors {Delta}RMSF sensitivity, confirming local frustration as a determinant of interfacial dynamics. Furthermore, static interfaces are characterized by a higher density of canonical hotspot residues, while dynamic interfaces utilize interfacial ruggedness to modulate affinity. Together, these results demonstrate that conserved binding modes can encode different energy landscapes and provide a practical framework for determining when ensemble-based sampling is required for accurate affinity prediction.

biophysics↗

bioRxiv2026

T-bet Expressing B cells are Key Determinants of Protective Immunity Against Norovirus Infection

The gastrointestinal tract (GI) is the largest environmental mucosal interface and is exposed to diverse commensal and pathogenic microbes. B cells are a prevalent immune component of the GI tract and its associated secondary lymphoid organs, yet we know little about the diversity and stability of distinct transcriptional programs that modulate B cell responses against different classes of pathogens or environmental perturbations. A subset of B cells defined by expression of the transcription factor T-bet, has been canonically associated with antiviral immunity through IgG production. However, the role of T-bet expressing B cells in mucosal tissues, where IgA responses predominate, is poorly understood. Here, we identify a population of intestinal T-bet+ B cells that, in the absence of overt perturbation, constitutes a minor fraction of intestinal associated B cells and undergoes continuous turnover. In contrast, during enteric viral infection with murine norovirus (MNV), T-bet B cells undergo a marked expansion, with T-bet expression stably maintained in the majority of virus-specific B cells, including IgG2c and IgA switched B cells. Moreover, virus-reactive IgG2c and IgA B cells arise independently rather than through sequential switching, and B cell intrinsic T-bet expression is required for effective germinal center responses but dispensable for IgA class switching. Moreover, T-bet expressing B cells are required for the generation of all MNV-specific circulating IgG and mucosal IgA, and for protection upon re-encounter with the virus. Together, these findings establish T-bet expressing B cells as a specialized B cell subset essential for mucosal immunity and protection against norovirus infection.

immunology↗

bioRxiv2026

Purifying selection and phylogenetic discord among microneme proteins in Toxoplasma gondii

In Toxoplasma gondii, microneme proteins (MICs) are secreted components of the apical complex that play central roles in motility, host cell attachment, and invasion. Because proteins at the host-parasite interface are often predicted to evolve rapidly, MICs have been suggested as candidates for adaptive diversification. We tested this expectation using comparative analyses of three relatively understudied microneme proteins, MIC13, MIC12, and MIC16. Coding sequences were assembled from GenBank and ToxoDB, aligned by translation, and analyzed using maximum-likelihood phylogenetics, codon-based tests of selection, and predicted protein structure. MIC13 was represented by 51 sequences, MIC12 by 30, and MIC16 by 34, spanning multiple T. gondii haplogroups and including Hammondia hammondi and Neospora caninum as outgroups. All three genes were highly conserved among T. gondii strains, but their phylogenetic trees were topologically incongruent, indicating that individual MICs do not recover a single shared strain history. Contrary to expectation, no positively selected codons were detected in any gene. Instead, purifying selection was detected at one site in MIC13 and 15 sites in MIC12, while no significant codon-specific selection was detected in MIC16. Several constrained MIC12 sites overlapped annotated EGF and calcium-binding EGF-like domains, consistent with structural conservation of extracellular adhesion modules. AlphaFold prediction of MIC13 supported two sialic acid-binding micronemal adhesive repeat regions, but the single constrained MIC13 site did not overlap these motifs. Together, these results indicate that MIC13, MIC12, and MIC16 are shaped more by sequence conservation and heterogeneous gene histories than by strong recurrent positive selection. These findings refine expectations for microneme evolution in T. gondii and highlight conserved domains that may be important for parasite invasion and future functional study.

evolutionary biology↗

bioRxiv2026

A Conserved Metabolic Oxidative Axis Underlies Immune Cell Cryo-vulnerability

Immunotherapy has emerged as a transformative approach for treating cancer and other diseases, yet its widespread deployment requires effective cryopreservation strategies to enable scalable global distribution. However, many immune cell types remain acutely vulnerable to freeze-thaw stress, and the underlying mechanisms of this cryo-vulnerability are not well defined. In this study, we combined metabolic flux analysis, ROS quantification, lipidomics, and preclinical xenograft models to investigate how metabolic state influences cryopreservation outcomes. We found that immune cell activation induces a metabolic shift characterized by elevated glucose utilization and excessive ROS production, leading to profound post-thaw loss of viability and function, as demonstrated by a [~]25% survival rate in natural killer (NK) cells. Targeted pretreatments--including inhibitors of glucose metabolism, antioxidants, and suppression of lipid peroxidation--restored post-thaw recovery to nearly 90% while preserving effector activity and antitumor efficacy. Similar protective effects were observed across {beta} T cells, {gamma}{delta} T cells, and macrophages, defining a conserved metabolic-oxidative pathway of cryo-vulnerability and offering applicable strategies to enhance immune cell preservation.

immunology↗

bioRxiv2026

Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

Protein interactions with canonical B-form DNA are well-characterized, yet the effect on these interactions of torsionally constrained DNA--ubiquitous in cells--remains underexplored. Using cryo-electron tomography (cryo-ET), we 3D-reconstructed entire negatively supercoiled DNA substrates bound to active RNA polymerase (RNAP), revealing diverse DNA supercoiling conformations and their interplay with transcription. RNAP preferentially localizes at plectoneme apices in a swiveled, pause-prone state. RNAP, along with other DNA-melting proteins like dCas9, can act as torsional roadblocks that segregate "twin-supercoiling domains" during active transcription, independent of external DNA/RNAP tethering. Co-transcribing RNAPs further intensify this domain separation: tandem oriented RNAPs relieve negative supercoiling more effectively than opposing ones, promote greater RNAP accumulation and enhanced elongation, both in vitro and in vivo. Topoisomerase I relieves torsional stress and facilitates RNAP escape from apical stalls, thereby supporting apical transcription regulation. Together, these findings support a load-and-release mechanism at plectoneme apices that may underlie supercoiling-dependent transcriptional bursting.

biophysics↗

bioRxiv2026

Isoform-resolved spatial transcriptomics on a lab-made high-density array via a single-chip NGS-TGS workflow

High-resolution spatial transcriptomics is still cost-prohibitive and dominated by short-read sequencing, limiting in situ detection of transcript structures. Here we present a low-cost, lab-made, high-density bead-in-microwell chip assembled by routine centrifuge and decoded by a tri-part combinatorial long-barcode strategy. The design provides 5.6x107 barcode combinations across 4.1x10{square} capture sites, reducing collisions while remaining tolerant to third-generation sequencing raw errors. Using a single-chip, dual-platform workflow, we split full-length cDNA for next-generation and third-generation sequencing within the same spatial coordinates. In an incompatible tomato-pepper graft, we identified interface-associated splicing reprogramming, including thousands of unannotated isoforms, enriched novel CaGRP1 isoforms, and SlPIP2 intron retention, supported by individual long-read sequences. In mouse embryos, we further demonstrate cross-species applicability by resolving unannotated Col1a2 isoforms in oligodendrocyte progenitors and spatially restricted Dalrd3 intron retention. This cost-efficient system broadens access to spatial full-length transcriptomics and may support diverse biological studies.

plant biology↗

bioRxiv2026

QUANTIFYING GLYCOGEN AND LIPID DROPLET SYNTHESIS IN OVARIAN AND CERVICAL CANCER CELLS USING DEUTERATED RAMAN PROBES WITH STIMULATED RAMAN SCATTERING MICROSCOPY

Epithelial ovarian cancer remains one of the most lethal malignancies among women, with late-stage diagnoses yielding 5-year survival rates below 30%. The metabolic heterogeneity of the tumor microenvironment (TME) highlights the need for methods capable of rapid, chemically specific phenotyping. Stimulated Raman scattering (SRS) microscopy when combined with deuterium labeled metabolites enables the non-invasive high contrast interrogation of cellular metabolic pathways. In this study, we used SRS microscopy to profile fatty acid and glycogen metabolism in epithelial ovarian cancer (SKOV-3) and cervical cancer (HeLa) cell models. Deuterium labeled glucose revealed striking differences in glycogen synthesis and intracellular distribution, with SKOV-3 cells exhibiting markedly greater single-cell heterogeneity than HeLa. Complementary measurements of lipid droplet (LD) synthesis and turnover under nutrient starvation further revealed cell-line-specific metabolic strategies, identifying LD and glycogen dynamics as a potential diagnostic marker of cancer metabolic phenotypes. These results demonstrate that SRS microscopy in the Raman silent region, paired with metabolic labeling, can sensitively resolve metabolic diversity across cancer cell subpopulations. Such metabolic phenotyping may inform both early diagnostic strategies and therapeutic approaches that combine cytotoxic treatment with targeted metabolic disruption.

bioengineering↗

bioRxiv2026

EnsAgent: a tool-ensemble multiple Agent system for robust annotation in spatial transcriptomics

MotivationAutomated domain annotation in spatially resolved transcriptomics (SRT) remains challenging since it depends on gene expression, morphology, and clinical conventions, which vary across cohorts and platforms. While Large Language Model (LLM)-driven agents show promise, current approaches typically condition semantic reasoning on static, single-method partitions. This reliance makes annotation pipelines fragile to upstream partition errors and prone to hallucinations when molecular evidence is ambiguous. A robust framework integrating ensemble intelligence with iterative, evidence-based reasoning is required to ensure reproducibility and accuracy. ResultsWe introduce EnsAgent, a tool-ensemble multi-agent system designed for robust SRT annotation. Uniquely, EnsAgent decouples structural partitioning from semantic labeling via a Consultation-Review workflow. A Tool-Runner Agent orchestrates a diverse portfolio of clustering algorithms via the Model Context Protocol (MCP), generating a consensus partition optimized by a multimodal Scoring Agent. Subsequently, a Proposer-Critic feedback loop coordinates four specialized experts (Marker, Pathway, Spatiality, and Visual) to formulate annotations with explicit evidence trails and uncertainty estimates. Benchmarking on three SRT datasets demonstrates that EnsAgent effectively neutralizes batch effects and resolves subtle tumor microenvironment niches missed by single-paradigm baselines, delivering state-of-the-art accuracy and interpretability. Availability and ImplementationEnsAgent is available at github.com/keviccz/ensAgent. Contactdongqishi@sztu.edu.cn, kexiao@sztu.edu.cn Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2026

Myristoylation licenses disordered viral VP4 protein to anchor to and perforate the membrane through phase separation

The VP4 protein of enteroviruses, such as Coxsackievirus B3, is a small, intrinsically disordered protein essential for perforating the host cell membrane during viral entry. A key feature of VP4 is its N-terminal myristoylation, which is required for infectivity in some enteroviruses but dispensable in others, suggesting a complex and context-dependent role that is not fully understood. The precise biophysical mechanisms by which this lipid anchor enables a disordered protein to breach a membrane remain unresolved. Here, using Coxsackievirus B3 VP4 as a model system and integrating multi-scale molecular dynamics simulations with confocal microscopy, we demonstrate that myristoylation is not a simple membrane tether but a multi-functional regulator that orchestrates VP4 activity through distinct, hierarchical roles. First, it provides the necessary hydrophobic anchor to recruit the disordered VP4 to the membrane interface. Second, the myristoyl group acts as a key molecular driver that promotes the liquid-liquid phase separation of VP4, leading to the formation of dynamic condensates on the membrane surface. These condensates actively remodel the membrane, generating substantial curvature that, in turn, lowers the free energy barrier for VP4 penetration. Furthermore, we find evidence that the myristoyl group plays a third role in stabilizing the final transmembrane pore. Our findings establish a novel paradigm where a single lipid modification empowers a disordered viral protein to form a functionally potent condensate that mechanically primes and physically breaches the target membrane, a mechanism that may explain the conditional myristoylation requirement across enteroviruses.

biophysics↗