Biology subjects

Zhao, J.

Publications and source records attributed to Zhao, J..

At least 109 records · Page 6Linked to original sources

bioRxiv2026

BRD8 Disruption Unlocks Retrotransposon-Driven Tumor-Intrinsic Immunogenicity via Redistribution of Histone Acetylation in Liver Cancer

Overall response rate for immune checkpoint blockade (ICB) therapy remains limited in advanced hepatocellular carcinoma (HCC). Epigenetic perturbation has been shown to enhance tumor-intrinsic immunogenicity and thus reverse resistance to immunotherapy in multiple cancer types. Here, through domain-focused CRISPR/Cas9 genetic screens targeting chromatin regulators, we identified the bromodomain-containing protein 8 (BRD8) as a novel epigenetic factor that functions to suppress tumor-intrinsic immunity through BRD8/EP400 chromatin remodeling complex in HCC. Depletion of BRD8 potently induces anti-tumor immune responses and sensitizes tumors to ICB therapy in mouse tumor models. Mechanistically, BRD8 knockout leads to partial redistribution of H3K27ac to repetitive elements, activating double-stranded RNA (dsRNA) accumulation and subsequent type I interferon (IFN) response, leading to tumor rejection via remodeling the tumor microenvironment. The first-in-class selective chemical probe DN02 targeting the bromodomain of BRD8 also exhibits immunomodulatory and anti-tumor effects. In line with these findings, low BRD8 expression correlated with enriched interferon signatures, elevated immune cell infiltration, and prolonged survival in HCC patients. Together, this study reveals BRD8 as a selective epigenetic vulnerability for promoting tumor immunogenicity, and its bromodomain as a druggable target to overcome immunotherapy resistance in liver cancer patients. SignificanceThe induction of viral mimicry by epigenetic interference could trigger tumor-intrinsic immune response and enhance immunotherapy efficacy. Here, through domain-focused CRISPR/Cas9 screening, we identify BRD8 complex as a regulator involved in the suppression of repetitive elements by controlling H3K27ac distribution and propose BRD8 as a novel target to enhance anti-tumor immunity.

cancer biology↗

bioRxiv2026

Optimizing broadly neutralizing antibodies via all-atom interaction modeling and pre-trained language models

Antibody optimization is a fundamental challenge, and the identification of antibody-antigen interactions is crucial in the optimization process. However, current methods cannot accurately predict antibody-antigen interactions, providing limited functional guidance to improve the time-consuming and costly traditional optimization techniques. Here, we present InterAb and InterAb-Opt, a unified computational framework that integrates all-atom modeling with antibody language models to predict antibody-antigen interactions and enable antibody optimization. Leveraging the proposed all-atom modeling approach, AtomInter, and pre-trained antibody language models, InterAb outperforms existing methods in predicting antibody specificity and antibody-antigen binding affinity. InterAb successfully identified influenza A virus-binding antibodies from an antibody library and accurately detected high-affinity antibodies in the AIntibody competition. Empowered by the robust functional insights from InterAb, InterAb-Opt was developed to optimize broadly neutralizing antibodies. For R1-32 antibody, biolayer interferometry results reveal that 85%, 80%, 90%, and 67.5% of the 40 InterAb-Opt-optimized antibodies exhibit enhanced binding affinities to wild-type SARS-CoV-2, Lambda, BQ.1.1, and EG.5.1, respectively, with a maximum improvement of up to 96-fold. For the newly emerging BA.2.86 and KP.3, 55% and 52.5% of the optimized antibodies notably transition from non-binding to binding. Neutralization assays demonstrated that the optimized antibodies exhibited enhanced neutralization activity across multiple targets, highlighting the capability of InterAb-Opt in engineering broadly neutralizing antibodies. This technology enables precise analysis of antibody-antigen interactions and optimization of broadly neutralizing antibodies, holding promise for addressing challenges in immune evasion and vaccine design.

bioinformatics↗

bioRxiv2026

Covalent Leader Peptide Probes Enable Proteome-Level Mapping of RiPP Enzymes beyond Biosynthetic Gene Clusters

Genome-based analyses have enabled widespread discovery and biosynthetic annotation of ribosomally synthesized and post-translationally modified peptides (RiPPs) pathways, yet often fail to capture all participating enzymes, particularly those encoded outside canonical biosynthetic gene clusters. In this study, leveraging the central role of leader peptides (LPs) in RiPP enzyme-substrate recognition, we developed a substrate-guided chemical-proteomic strategy using covalent LP probes to directly identify LP-binding enzymes from native proteomes. Using the LctA-LctM and PatE-LynD systems, we demonstrate that LP probes carrying a photocrosslinker engage enzymes at their authentic LP-recognition sites while preserving full catalytic competence. Application of this strategy to a model strain Streptomyces sparsogenes uncovered lanthipeptide synthetases encoded outside canonical biosynthetic gene clusters and revealed graded cross-cluster activities among multiple RiPP pathways. This work establishes a proof-of-concept model platform for proteome-level discovery of LP-binding RiPP enzymes and highlights cross-cluster enzymatic crosstalk as a latent mechanism for structural diversification.

biochemistry↗

bioRxiv2026

Fluorescence-lifetime optical electrophysiology in contracting cardiomyocytes

Precise monitoring of cardiac electrophysiology in vitro is crucial to understanding heart function and cardiac disease. However, high-throughput, contact-free methods for directly measuring excitation-contraction coupling remain limited. Here, we introduce a new paradigm for quantitative electrophysiological imaging that combines fluorescence lifetime and intensity information to capture dynamic cardiac signals with high fidelity. We show that lifetime measurements are intrinsically decoupled from motion artifacts and provide calibrated calcium concentration and membrane potential readouts across wide fields of view. Using a gated single-photon avalanche diode camera, we acquire fluorescence lifetime images at up to 200 frames per second with sufficient signal-to-noise ratio such that each frame contains meaningful lifetime information without temporal averaging. This approach yields spatially resolved maps of absolute voltage and calcium values across contracting cardiomyocyte monolayers, revealing heterogeneous cell behaviors within individual assays and uncovering previously unreported dynamics during late-phase repolarization for real-time analysis of excitation-contraction coupling.

bioengineering↗

bioRxiv2026

lncRNAs contribute to caste differentiation as a regulatory layer in ants

Caste differentiation in ants represents one of the major evolutionary transitions and provides a unique model for studying developmental mechanisms underlying division of labor. Genetically identical individuals follow divergent epigenetically-regulated developmental trajectories that give rise to morphologically distinct phenotypes and specialized roles. While long non-coding RNAs (lncRNAs) are emerging as key epigenetic regulators across diverse biological systems, their specific contributions to caste development in social insects remain largely unexplored. Here, we conducted comprehensive transcriptomic analyses across major developmental stages of two evolutionarily and ecologically distinct ants Monomorium pharaonis and Acromyrmex echinatior, identifying over 10,000 lncRNAs. We demonstrated that lncRNAs exhibit dynamic, caste-specific expression patterns throughout development. We also identified a subset of lncRNAs displaying canalized expression patterns, characterized by progressively increasing caste bias and decreasing within-caste variation as development proceeds. Co-expression analyses revealed that canalized lncRNAs are functionally linked to canalized protein-coding genes, which are crucial regulators for caste differentiation. These canalized lncRNAs show striking tissue-specific enrichment consistent with canalized protein-coding genes. Functional validation through RNA interference revealed that canalized lncRNAs directly regulate caste-specific traits. Furthermore, juvenile hormone treatment capable of redirecting worker lncRNA expression profiles toward gyne-like patterns, with similar expression changes and tissue specificity to JH-responsive protein-coding genes, linking lncRNA regulation to established hormonal pathways controlling caste fate. Our findings establish lncRNAs as active architects of caste differentiation in social insects, demonstrating that these rapidly-evolving regulatory molecules contribute to the evolution and maintenance of social phenotypes through tissue-specific regulation of caste-associated developmental programs.

developmental biology↗

bioRxiv2026

Uncompetitive Allosteric Inhibitor of Mitochondrial Creatine Kinase Prevents Binding and Release of Creatine by Stabilization of Loop Closure

Mitochondrial creatine kinase (MtCK) is a key enzyme in energy buffering and homeostasis in cells. It catalyzes transfer of phosphoryl group from ATP to creatine. Overexpression of MtCK occurs in many cancer cells to meet elevated energy demands, which is associated with poor prognosis. This suggests that MtCK may be a promising target for cancer therapeutics. We sought to discover first-in-class selective inhibitors of MtCK with diverse mechanisms of action using high-throughput screening, biochemical characterization and cryo-EM studies. Through these studies, we identified diverse types of compounds that modulate activity of MtCK in vitro, including fast-equilibrium and time-dependent orthosteric and allosteric inhibitors. Select hits were subjected to in vitro enzymatic and binding assays to assess MtCK inhibition and binding. A subset of inhibitors was advanced into structural studies using cryo-EM resulting in the molecular structure of MtCK with and without bound substrates in complex with an allosteric uncompetitive inhibitor that we discovered. These studies identified compounds unique binding pocket on MtCK and established the molecular steps manifesting in the apparent uncompetitive mode of inhibition. We demonstrate that the compound stabilizes an active site loop in a closed conformation restricting access of the creatine substrate to and the release of phosphocreatine product from its binding pocket. These findings establish chemical tools suitable for validation of MtCK as a promising breast cancer target with high therapeutic potential and build a foundation for future structure-guided optimization of the hit compounds of MtCK we identified and de novo rational design of novel MtCK inhibitors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/696683v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1fd9870org.highwire.dtl.DTLVardef@230e31org.highwire.dtl.DTLVardef@178d8fdorg.highwire.dtl.DTLVardef@edeca6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Perturbing H-NS function reveals roles in restricting virulence heterogeneity and pathogen adaptation

Bacterial pathogens must balance rapid expression of virulence genes in host niches with tight repression when not needed to avoid fitness costs and ensure survival. Integration of virulence gene regulatory networks with the conserved global repressor H-NS was critical to achieve this balance. H-NS-mediated repression of virulence genes in non-inducing environments is essential for maintaining virulence genes and has shaped pathogen evolution. However, the role of H-NS-mediated repression in virulence gene activation and pathogen evolution in virulence-inducing conditions is less clear. For instance, although virulence gene expression is often heterogeneous, whether relief of H-NS repression contributes to this heterogeneity remains unknown. Furthermore, whether H-NS repression shapes pathogen evolution in virulence-inducing environments is unclear. Here, we use a Salmonella strain with reduced H-NS DNA-binding affinity to investigate the role of H-NS in virulence gene expression in individual bacteria and pathogen adaptation. We find that reduced H-NS repression increases the fraction of virulence gene expressing cells without eliminating bimodality, resulting in enhanced epithelial cell infection in vitro. Using experimental evolution, we demonstrate that the hns genotype constrains adaptive mutations and that disabling virulence gene expression is a common path to improved fitness in intracellular-like environments. Our results expand the role of H-NS-mediated repression from silencing virulence genes in non-inducing conditions to regulating heterogeneity in inducing conditions and demonstrate that evolutionary conservation of H-NS constrains adaptive strategies in intracellular-like environments.

systems biology↗

bioRxiv2025

Extracellular matrix regulates lineage plasticity in prostate cancer through YAP/TEAD

Treatment-related neuroendocrine prostate cancer (NEPC) is an increasingly frequent mechanism of resistance to androgen receptor pathway inhibitor (ARPI) therapy in prostate adenocarcinoma (PRAD). This lineage transition is dependent on upregulation of the NE-specifying transcription factor ASCL1, typically in a genetic background of RB1 and TP53 loss. Here we identify extracellular matrix-integrin-YAP1/TEAD signaling as a critical brake on NEPC lineage transition. Deletion of Itgb1, the shared B1 subunit required for collagen and laminin-mediated integrin activation, is sufficient to induce ASCL1 and NE lineage gene expression, by activating LATS1/2 kinases with subsequent inactivation of YAP1/TEAD signaling. Conversely, restoration of YAP1/TEAD signaling by pharmacological LATS1/2 inhibition, or by expression of constitutively active YAP1/TAZ mutants, prevents or reverts NEPC lineage transition. NOTCH and AR cooperate with YAP/TEAD to repress ASCL1, such that combined inhibition leads to complete reprograming of PRAD into NEPC in vitro, providing a dynamic platform to dissect the molecular events responsible for lineage transition over time. We find that lineage transition is accompanied by a redistribution of FOXA1 and TEAD cistromes from PRAD to NEPC-specific enhancers and requires the pioneering activity of FOXA1. Thus, extracellular matrix/integrin signaling in the PRAD tumor microenvironment restrains NE lineage plasticity, highlighting a potential path for pharmacological inhibitors in modulating treatment-induced lineage change.

cancer biology↗

bioRxiv2025

A stem cell knockout village reveals lineage rewiring and a non-canonical islet cell fate in monogenic diabetes

Genetics studies have identified a core set of regulators essential for pancreatic {beta} cell development, many of which are mutated in monogenic diabetes. However, how these mutations alter developmental trajectories to produce pathological cell states remains elusive. Here we introduce a knockout village framework that enables longitudinal scRNA-seq profiling of 79 human pluripotent stem cell mutant lines targeting 30 developmental regulators, including 15 diabetes genes, across five islet differentiation stages. We show that loss of lineage regulators impairs {beta} cell formation in a stage-specific manner and rewires developmental trajectories towards competing lineages. Notably, several monogenic diabetes gene mutations drive a shift from {beta} cells to enterochromaffin (EC)-like cells, a recently recognized non-canonical islet cell fate. These EC-like cells exhibit incomplete activation of hormone regulation programs, along with elevated neuron signatures. Leveraging the diversity of cell fate outcomes across mutants, we predicted and experimentally validated ISL1 as a key downstream effector of PDX1 and PAX6 that safeguards {beta} cell identity against an EC-like fate. Together, our findings reveal cell fate rewiring as a widespread, previously underappreciated pathological mechanism in monogenic diabetes and establish a scalable platform for uncovering developmental vulnerabilities in human genetic disorders.

developmental biology↗

bioRxiv2025

Comparative Study of BBB-Targeting AAV Capsids on Central Nervous System Delivery Efficiency

The efficacy of adeno-associated virus (AAV)-mediated systemic gene therapy for central nervous system (CNS) diseases is often limited by the blood-brain barrier (BBB). This study systematically evaluated the tissue distribution of three BBB-crossing AAV capsid variants (PHP.eB, CNSRCV300, and BI-hTFR1) following intravenous injection in mice, using either a constitutive promoter (CAG) or a neuron-specific promoter (hSyn) to drive EGFP reporter expression. Compared with AAV9, both PHP.eB and CNSRCV300 demonstrated significantly enhanced BBB penetration and brain transduction efficiency. While the use of the hSyn promoter led to reduced transgene expression in the brain compared with the CAG promoter, and substantially decreased visible reporter expression in peripheral organs, viral deposition in the liver could still be detected via immunohistochemistry. Overall, CNSRCV300 exhibited the most favorable balance between brain-targeting efficiency and biosafety, highlighting its potential as a promising delivery vector. In summary, both the capsid and promoter jointly influence AAV-mediated expression in vivo, and although cell type-specific promoters can reduce off-target expression, residual viral deposition in non-target tissues remains a potential safety concern.

neuroscience↗

bioRxiv2025

Eosinophil-derived COX-2 protects against experimental colitis through the PGE2-IL-22 axis

Inflammatory bowel disease (IBD) is driven by a breakdown in immune regulation and epithelial barrier function, yet the contribution of eosinophils to this process has remained poorly defined and controversial. While eosinophils infiltrate the intestinal mucosa during both flares and remission, their role in shaping disease outcomes is unclear. Our RNA-seq analyses of colonic eosinophils isolated from dextran sulfate sodium (DSS)-treated mice revealed a significant upregulation of cyclooxygenase (Cox)-2 (gene name, Ptgs2). Eosinophil-specific deletion of Cox-2 (Ptgs2fl/fleoCre+/-) reduced IL-22 production and exacerbated DSS- and trinitrobenzene sulfonic acid (TNBS)-induced colitis, characterized by greater weight loss, higher disease activity, colon shortening, and epithelial injury. Administration of recombinant IL-22 reversed these phenotypes. Mechanistically, eosinophil-derived COX-2 enhanced IL-22 production by type 3 Innate lymphoid cells (ILC3s) through prostaglandin E2 (PGE2) signaling. Consistently, Ptgs2fl/fleoCre+/-mice exhibited reduced colonic PGE2 levels, while PGE2 analog treatment restored IL-22 production and mucosal protection. Our findings identify eosinophil-derived COX-2 and PGE2 as a critical regulator of IL-22 production during colitis, uncovering a previously unrecognized eosinophil-ILC3 crosstalk that safeguards the intestinal barrier and represents a promising therapeutic target in IBD. Significance StatementThe role of eosinophils in inflammatory bowel disease (IBD) has remained unresolved. We identify eosinophil-derived COX-2 as the main source of prostaglandin E2 (PGE2) in colitis, which drives IL-22 production by ILC3s to maintain epithelial protection. This discovery defines a protective eosinophil/PGE2/IL-22 circuit and reveals a therapeutic target to strengthen barrier defense in IBD.

immunology↗

bioRxiv2025

AI Identifies Broadly Neutralizing Antibodies from an IGHV1-69 Public Antibody Class Exerting Continued Selection Over SARS-CoV-2

R1-32-like public antibodies, characterized by shared IGHV1-69/IGLV1-40 usage, are elicited in more than 50% of individuals with COVID-19 and have been implicated in driving recurrent mutations at L452SARS2 and F490SARS2 within their convergent epitope in the SARS-CoV-2 spike receptor-binding domain. These mutations effectively mediate escape from non-affinity-matured R1-32-like antibodies with germline-like sequences. Here, we characterize four affinity-matured R1-32-like antibodies, C092, C807, BD56-104, and BD56-597, that tolerate L452SARS2 and F490SARS2 mutations. We show that this tolerance arises from residues introduced by somatic hypermutation at convergent positions across multiple CDR loops and surrounding regions, thereby creating additional contacts that reinforce epitope binding. An unusual N354SARS2 glycosylation site, which emerged in BA.2.86 and became fixed in its descendants, is linked to escape from affinity-matured R1-32-like antibodies, implying ongoing selection by this public antibody class. Using an AI model trained on integrated structural, neutralization, and binding data, we further identified ZL525, an ultrapotent R1-32-like antibody with pan-SARS-CoV-2 variant activity, including against the highly evasive KP.3 variant carrying the N354SARS2 glycosylation, and broad sarbecovirus cross-reactivity extending to SARS-CoV-1. Together, these findings show how affinity maturation enables public antibodies to adapt to viral antigenic drift, reveal their role in shaping SARS-CoV-2 antigenic evolution, and demonstrate the potential of AI-empowered strategies for discovering broadly neutralizing antibodies.

immunology↗

bioRxiv2025

LDLR variant classification through activity-normalized prime editing screening

BackgroundInherited variants in the LDL receptor (LDLR) gene are the most common cause of familial hypercholesterolemia (FH), significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt intervention with lipid-lowering therapies; however, the majority of LDLR variants observed in the population have uncertain or absent clinical classifications, limiting the potential to improve clinical management. MethodsWe developed an innovative, activity-normalized prime editing screening pipeline to measure the impact of 5,184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Through pairing a genotypic outcome reporter with every prime editing guide RNA (pegRNA), we adjust phenotypic measurements to account for variable editing efficiency, extending activity normalization to prime editing for the first time at this scale. Further, we use a statistical estimation approach that leverages measurements for all missense variants at a given position to denoise the resulting scores. ResultsWe show that prime editing-mediated reporter editing correlates with endogenous variant installation frequency, allowing activity normalization to improve imputation of LDLR variant effect. Our optimized prime editing assay identifies a broad, continuous spectrum of variant functional effects. We achieve robust separation of pathogenic vs. benign ClinVar variants and concordance between experimentally derived functional scores and LDL-C levels measured in UK Biobank participants. Further, when calibrating the strength of evidence provided by this functional screening data to align with the ACMG/AMP variant interpretation guidelines, and integrating additional sources of evidence, a majority of currently unclassified rare LDLR variants meet evidence thresholds for reclassification. We use the broad coverage of this screen to gain insight into how apolipoproteins bind to LDLR. In particular, we identify and characterize rare LDLR variants that enhance LDL-C uptake through increased interaction with apolipoprotein B. Finally, we compare prime editing-based functional scores with those derived from recent base editing and cDNA-based LDLR variant screens, showing that these approaches all show robust correlation with clinically observed LDL-C levels and computational scores, while prime editing identifies candidate splice-altering coding variants that are not modeled by cDNA screening. ConclusionsAltogether, our approach demonstrates the power of prime editing to significantly improve understanding of how variants in LDLR impact function and contribute to FH.

genetics↗

bioRxiv2025

Decoding Neoantigen-encoding Tumor-Specific Transcripts Unveils a Shared Target Reservoir for Immunotherapy in Hepatocellular Carcinoma

Background and AimsPrimary liver cancer, predominantly hepatocellular carcinoma (HCC), has limited therapeutic options. While mutation-derived neoantigen vaccine holds promise, its success is hindered by low antigen availability. This study explores transcriptome-derived neoantigens (neoantigen-encoding tumor-specific transcripts, neoTSTs) in HCC, characterizing their features, generation mechanisms, and therapeutic potential. Approach and ResultsWe analyzed RNA-seq data from 1,013 liver cancer patients and constructed a multi-layered reference dataset. Using a customized pipeline, we identified an average of 60 neoTSTs per patient, significantly surpassing mutation-derived neoantigens (neoMuts). NeoTSTs exhibited higher population frequencies, with 73.1% providing multiple epitopes, and were validated through mass spectrometry and HLA transgenic mouse models. Mechanistically, neoTSTs were generated via retained introns, transposable element activation, HNF4A-regulated alternative promoters, and de novo transmembrane domain (TMD) generation. Single-cell analysis revealed neoTSTs cover >75% of tumor cells and identified antigen-presenting cancer-associated fibroblasts (apCAFs) that enriched in immunotherapy responders and amplified CD4 T-cell responses via MHC-II presentation. In murine HCC models, neoTST vaccination outperformed neoMuts, inducing dual MHC-I/II activation and significant tumor growth inhibition. ConclusionsNeoTSTs represent a superior neoantigen source in HCC, compensating for the limitations of mutation-derived targets. Their abundance, sharedness, and dual MHC pathway activation highlight their potential for personalized immunotherapy, particularly in low-TMB tumors. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/692877v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1bedfedorg.highwire.dtl.DTLVardef@5f6c95org.highwire.dtl.DTLVardef@d1deb5org.highwire.dtl.DTLVardef@743e85_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

AI recognizes convergent somatic hypermutation signatures to allow the discovery of variant-resilient broadly neutralizing antibodies

Elucidating the mechanisms by which broadly neutralizing antibodies (bnAbs) develop to confer durable immunity in humans is pivotal for the rational design of next-generation vaccines and therapeutics. VH3-53/3-66-encoded public antibodies are widely elicited in the population after the COVID-19 pandemic. Here, we isolated 15 VH3-53/3-66-encoded bnAbs from elite neutralizers who experienced sequential SARS-CoV-2 Omicron breakthrough infections. These bnAbs exhibit exceptional potency and breadth against circulating variants and, as members of a long-lived public antibody lineage, likely contribute to durable humoral protection. Genetic analysis of VH3-53/3-66-encoded bnAbs reveals a convergent somatic hypermutation pattern at seven positions in and around the CDR loops that emerges with repeated viral exposures and distinguishes them from non-bnAbs. Grafting these combined mutations onto clinically escaped antibodies broadens their breadth and restores neutralization activity. Structural analysis shows that these convergent mutations cooperatively remodel CDRs to bind and tolerate virus receptor-binding domain mutations. Using an AI-based antibody language model trained on data linking somatic hypermutations to binding affinity and neutralization, the model is able to identify mutational patterns predictive of breadth, enabling the discovery of a rare bnAb with protective activity against the latest variants from early-pandemic antibody repertoires. This demonstrates the feasibility of establishing an AI-empowered pipeline to identify mutation-tolerant bnAbs from early-pandemic antibody repertoires to fight fast-evolving newly introduced viruses.

immunology↗

bioRxiv2025

A haplotype-resolved T2T genome assembly of Indigofera pseudotinctoria reveals the genetic basis of flavonoid biosynthesis in Chinese Indigo

Indigofera pseudotinctoria, commonly known as Chinese Indigo, is a multifunctional leguminous shrub widely distributed in East Asia, valued for its medicinal, ecological, and forage importance, and naturally adapted to drought and nutrient-poor soils. However, the absence of genomic resources has hindered insights into the genetic basis of its bioactive metabolites and adaptive traits. Here, we present a haplotype-resolved, telomere-to-telomere (T2T) reference genome of I. pseudotinctoria, which represents the first complete genome within the genus Indigofera. By integrating PacBio HiFi sequencing, Oxford Nanopore ultra-long reads, and Hi-C chromatin conformation sequencing technologies, we successfully constructed two gap-free haplotypes assemblies (647.38 Mb and 657.52 Mb), characterized by high BUSCO completeness and QV scores, and fully captured all telomeric sequences. Comparative analysis between haplotypes revealed extensive synteny but notable structural heterozygosity but notable structural heterozygosity. Approximately 46% of structural variations overlap with genic or regulatory regions, leading to allele-specific expression divergence. Between the two haplotypes, we identified 2,607 and 2,331 haplotype-specific genes, reflecting a complementary functional specialization in defense and repair versus metabolism and growth. Integrative transcriptomic and metabolomic profiling across six tissues reconstructed the flavonoid biosynthetic network and identified MYB, bHLH, NAC, WRKY, and ERF transcription factors regulating five key pharmacologically active flavonoids (Calycosin, Butein, Sulfuretin, Chrysoeriol, and Genistin). These results collectively uncover the haplotype-specific regulatory and structural basis of Indigofera pseudotinctoria and establish a high-quality genomic framework for evolutionary, medicinal, and metabolic engineering studies in leguminous plants, and provide a framework for molecular breeding and bioactive compound discovery in medicinal legumes.

genomics↗

bioRxiv2025

Characterisation of SpoT-disruption induced metabolic shift in bacteria

SpoT, a pivotal stringent response regulatory enzyme, serves a dual function of both synthesizing and hydrolyzing the stress alarmones ppGpp and pppGpp, collectively known as (p)ppGpp. These alarmones act as global regulators, governing bacterial metabolic and physiological adaptation to diverse environmental stresses. Our previous investigation revealed that multidrug-resistant Acinetobacter baumannii notably upregulates SpoT in response to polymyxin treatment. Intriguingly, disrupting spoT gene enhances polymyxin killing. To comprehend SpoT metabolic regulatory function in mediating polymyxin tolerance, we conducted untargeted metabolomics, comparing metabolic perturbations upon polymyxin B (PMB) treatment between a spoT-disrupted A. baumannii mutant and its wild-type counterpart. Depletion of guanine-based purines GTP and GDP in the PMB-treated spoT-disrupted mutant suggests impaired (p)ppGpp hydrolysis, potentially leading to lethal accumulation of these molecules. PMB also induced more pronounced depletion of carbon sources (i.e., phosphoenolpyruvate, succinate, coenzyme A), energy metabolites (i.e., NADH biosynthesis pathway, ADP), amino acids, and the antioxidative system (i.e., glutathione, gamma-L-glutamyl-L-cysteine, (R)-S-lactoylglutathione) in the spoT-disrupted mutant. Interestingly, a distinctive time-dependent perturbation of fatty acyls was also observed following polymyxin treatment, with multiple fatty acyl conjugates significantly elevated in the spoT-disrupted mutant at 1 hour. However, this situation reversed at 4 hours, with more elevated fatty acyl groups in the wild-type compared to the spoT-disrupted mutant, indicating greater and more rapid PMB-induced membrane disruption in the spoT-disrupted mutant. Collectively, our findings suggest a potential role for SpoT in intricately coordinating A. baumannii energy expenditure and metabolite repertoire to ensure optimal functionality in stress tolerance and repair machineries (e.g., glutathione system, fatty acid regulation) following polymyxin treatment. IMPORTANCEPolymyxins, the last-resort antibiotics for multidrug-resistant Gram-negative bacteria, face increasing challenges due to growing tolerance. To tackle Acinetobacter baumannii polymyxin tolerance, we turned to SpoT, a (p)ppGpp synthetase/hydrolase protein. Our study focused on spoT-disrupted mutant, which exhibited heightened polymyxin antibacterial killing, yet the underlying mechanisms remained elusive. Through metabolomics, we unraveled key insights. Enhanced polymyxin killing in spoT-disrupted mutant likely resulted from lethal accumulation of stress alarmones (p)ppGpp, perturbations in carbon and energy metabolism, thiol-based antioxidant system depletion, and disrupted lipidic membrane repair. These findings illuminate the intricate metabolic signaling networks driving polymyxin tolerance in A. baumannii. By untangling pathogen-directed stress tolerance mechanisms, we deepen our understanding and identify potential therapeutic targets. Our discoveries offer new avenues to combat polymyxin resistance, paving the way for more effective treatments against superbugs. Through continued exploration, we can harness this knowledge to develop innovative strategies and overcome the challenges posed by polymyxin tolerance.

microbiology↗

bioRxiv2025

Rapid ecosystem collapse and biofilter formation following seabed methane leakage

Globally, vast amounts of methane are trapped within subseafloor gas hydrates. Recent evidence suggests that climate- and human-induced perturbations can destabilize gas hydrates and trigger methane release, yet the response of deep-sea ecosystems to these changes remains poorly understood. Here we show multi-year in situ monitoring of seabed ecosystem following methane leakage induced by hydrate exploration activities. Within just two years, benthic microbial and eukaryotic diversity in the affected areas declined significantly, while microbial and macrofaunal abundance increased. Integrated geochemical and omics analyses reveal the rapid successional shift of seabed ecosystem to a novel chemosynthetic system. Aerobic methanotrophs (Methyloprofundus) co-established with unexpectedly fast growing anaerobic methanotrophic archaea (ANME-3), accompanied by a rapid recruitment of opportunistic polychaetes that bioirrigated the seabed to >50 cm depths. The active animal-microbe interactions sustained exceptionally high rates of methane oxidation that utilized diverse electron acceptors. We demonstrate that methane hydrate destabilization can trigger rapid collapse of native seabed ecosystem while driving the formation of an effective methane biofilter consuming this potent greenhouse gas much faster than previously estimated. Understanding seabed ecosystem response and feedback is critical for predicting benthic methane cycling under ongoing global change and for developing sustainable strategies for methane hydrate resource management.

ecology↗