Biology subjects

Gao, X.

Publications and source records attributed to Gao, X..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Development and application of a genotyping by target sequencing SNP array panel in Salix suchowensis

Salix suchowensis is an important species of Salix, known for its rapid growth property and wide application in environmental construction, ecological restoration, wicker production, and biomass energy production. Due to its significance as a sustainable biological resource, S. suchowensis has been the centre of intensive breeding. However, rapid improvement of growth and biomass has been hindered by a lack of genomic resources. To address this limitation, we designed a genotyping by target sequencing SNP array panel to facilitate efficient genotyping. Using whole-genome resequencing data, a total of 39,076 SNPs were selected for the array panel, consisting of trait-associated SNPs, intragenic SNPs, and intergenic SNPs. This panel was validated by genotyping 550 new samples, demonstrating high call rates and effective capture of the population structure. Genome-wide association analysis identified 72 SNPs associated with plant height and ground diameter. Additionally, the array panel shows a high potential for genomic selection, with high prediction accuracy for various traits. These results highlight the efficiency of this panel in capturing genomic variations that are highly valuable for future genetic research and breeding applications.

genetics↗

bioRxiv2025

A synergistic generative-ranking framework for tailored design of therapeutic single-domain antibodies

Single-domain antibodies (sdAbs) have emerged as powerful therapeutic agents due to their small size, high stability, and superior tissue penetration. However, unlike conventional monoclonal antibodies (mAbs), sdAbs lack an Fc domain, limiting their functional versatility and manufacturability. To address this challenge, we developed TFDesign-sdAb, a deep learning-based generative-ranking framework that enables rational engineering of sdAbs with tailored functionalities. Our framework integrates a structure-aware diffusion model (IgGM) for large-scale candidate generation and a fine-tuned sorter (A2binder) that evaluates and prioritizes them based on predicted functionality. Unlike traditional CDR-focused approaches, TFDesign-sdAb optimizes both complementarity-determining regions (CDRs) and framework regions (FRs), allowing sdAbs to acquire new functional properties while maintaining antigen specificity. We validated our approach by conferring Protein A binding to human VHs and nanobodies that originally lacked this feature, achieving high expression rates, strong binding affinities, and successful purification via industry-standard Protein A affinity chromatography. High-resolution structural characterization (1.49 [A] and 2.0 [A]) of the redesigned sdAb-Protein A complexes revealed conserved FR-mediated binding motifs that recapitulate natural Fc-Protein A interactions, validating the accuracy of our model. Furthermore, our pipeline streamlined the antibody engineering process, achieving a 100% success rate in generating Protein A-binding sdAbs while maintaining their original antigen-binding affinity. This work demonstrates the power of AI-driven design in overcoming long-standing limitations in antibody engineering and presents a scalable, generalizable solution for enhancing sdAb functionality.

bioinformatics↗

bioRxiv2025

3D X-ray microscopy lights up nanoparticles in plants

The discovery of novel plant fertilization strategies heavily relies on our capabilities to probe physiological processes in living plants with sub-cellular precision. State-of-the-art microscopy techniques are in general limited to surface investigation or they require elaborated tissue preparation and often destruction. X-ray microscopy has the potential to resolve some of these limitations by generating micro-to nanometer-scale 3D images deep into the tissue. We introduce experimental designs and the quantitative analysis methodologies, pioneering nanoscale ({approx}150 nm resolution) in-vivo 3D microscopy of plant tissue. We show the first direct in-vivo visualization of foliar-applied untagged nanoparticulate fertilizers deep under the leaf surface, not accessible by other microscopy methods. Ultimately, our approach provides the means for a direct observation of nanoparticle transport and dissolution in living plant tissue, a step critical for developing sustainable plant fertilization approaches.

plant biology↗

bioRxiv2025

Adaptive Asymmetric Adjustment in Affective Evaluation and Reciprocity for Altruistic Behaviors from Exogenous Uncertainty to Certainty

In direct reciprocity, environmentally imposed exogenous uncertainty frequently decouples benefactors altruistic intentions from final outcomes. How do cooperative bonds remain resilient against this inevitable "noise"? Adopting a comprehensive levels-of-analysis framework across eight experiments, we integrate interpersonal paradigms, computational modeling, multivariate fMRI decoding, and third-party social evaluations to uncover a robust "asymmetric adjustment" in beneficiarys affective evaluation and reciprocity under exogenous uncertainty, systematically delineating its cognitive algorithmic rules, neural representations, and adaptive functions in sustaining human cooperation. Specifically, beneficiarys reciprocity amplifies for positive deviations from expectations while buffering against reciprocal reductions from unintended shortfalls. Modeling reveals this computational resilience is governed by an asymmetric intrinsic prosocial bias, extending beyond traditional frameworks of outcome/intention-based evaluation, prediction-error-based social learning, or loss aversion. Supported by theory-of-mind system, this "asymmetric adjustment" is perceived as morally superior to other calculations, thereby serving a critical adaptive function to sustain cooperation amidst the volatility of an uncertain world.

animal behavior and cognition↗

bioRxiv2025

Low level of viral intra-host diversity is associated with severe SFTS disease

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus that can cause severe clinical symptoms and high fatality. However, the dynamics of SFTSV intra-host diversity during infection and its association with disease severity remain unexplored. Here, we characterized SFTSV iSNVs in longitudinal clinical samples from patients with distinct clinical outcomes. Our analyses revealed that SFTSV intra-host diversity was associated with disease severity and showed distinct patterns in severe and mild cases. Severe cases exhibited paradoxically lower SFTSV intra-host diversity despite higher viral loads, likely driven by reduced selection pressure. Notably, iSNVs linked to viral replication emerged in severe cases, while iSNVs associated with immunity invasion arose in mild cases. Machine learning models showed that intra-host diversity can contribute to classification of SFTS by severity. Our findings, through the lens of within-host viral populations, highlight variations in viral-host interactions during SFTSV infection and provide potential biomarkers for predicting SFTS severity.

pathology↗

bioRxiv2025

SCRIPT: predicting single-cell long-range cis-regulation based on pretrained graph attention networks

Single-cell cis-regulatory relationships (CRRs) are essential for deciphering transcriptional regulation and understanding the pathogenic mechanisms of disease-associated non-coding variants. Existing computational methods struggle to accurately predict single-cell CRRs due to inadequately integrating causal biological principles and large-scale single-cell data. Here, we present SCRIPT (Single-cell Cis-regulatory Relationship Identifier based on Pre-Trained graph attention networks) that infers single-cell CRRs from transcriptomic and chromatin accessibility data. SCRIPT incorporates two key innovations: graph causal attention networks supported by empirical CRR evidence, and representation learning enhanced through pretraining on atlas-scale single-cell chromatin accessibility data. Validation using cell-type-specific chromatin contact data demonstrates that SCRIPT achieves a mean AUC of 0.9, significantly outperforming state-of-the-art methods (AUC: 0.68). Notably, SCRIPT obtains a threefold improvement in predicting long-range CRRs (>100 Kb) compared to existing methods. Applying SCRIPT to Alzheimers disease and schizophrenia, we establish a framework for prioritizing disease-causing variants and elucidating their functional effects in a cell-type-specific manner. By uncovering molecular genetic mechanisms undetected by existing computational methods, SCRIPT provides a roadmap for advancing genetic diagnosis and target discovery.

bioinformatics↗

bioRxiv2025

Mapping the functional differentiation and interactions among the inferior, medial frontal and posterior temporal cortex in semantic control

Semantic control refers to the ability to flexibly retrieve and manipulate stored knowledge to support context-appropriate behavior. A left-lateralized network comprising the left inferior frontal gyrus (IFG), posterior middle temporal gyrus (pMTG), and dorsal medial prefrontal cortex (dmPFC) has been consistently implicated in this process. While previous studies have established the necessity of the IFG and pMTG in semantic control, the causal role of the left dmPFC remains unclear. Additionally, it is unknown whether each of these three regions exhibits internal functional differentiation and how they interact to support semantic control. To address these questions, we combined task-based functional magnetic resonance imaging (fMRI) with fMRI-guided transcranial magnetic stimulation (TMS). We found that dmPFC, like IFG and pMTG, is causally involved in semantic control. All three regions exhibited a consistent anterior-posterior functional gradient: anterior subregions were selectively engaged during high-demand semantic processing, whereas posterior subregions responded to both easy and hard tasks. Furthermore, combined activation patterns of these regions better predicted the behavioral differences between hard and easy semantic tasks compared to the activation patterns of any single region. Semantic control modulated both the autoinhibition within individual regions and the functional connectivity among them, suggesting these regions operate in a coordinated network rather than in isolation. These findings advance our understanding of the neural architecture supporting flexible semantic behavior. Significance StatementUnderstanding how the brain supports flexible semantic behavior is critical for both cognitive neuroscience and clinical neuropsychology. While prior studies have consistently implicated the left IFG, pMTG, and dmPFC in semantic control, the causal contribution of the dmPFC and the functional dynamics among these regions have remained unclear. This study provides the first causal evidence for the dmPFCs role in semantic control, reveals functional differentiation within the IFG and pMTG, and shows that these regions interact as an integrated network. These findings challenge the notion of functionally homogeneous control nodes and highlight a topographically organized, interactive system underlying controlled semantic retrieval. This work refines our mechanistic understanding of semantic control and may inform clinical models of language and conceptual deficits.

neuroscience↗

bioRxiv2025

Establishing Comprehensive Transthoracic Echocardiography Reference Ranges for Mouse Models: Insights into the Impact of Anesthesia, Sex, and Age

Mouse models play a critical role in cardiology research, offering valuable insights into the molecular mechanisms, genetics, and potential treatments for cardiovascular diseases. However, the ability to transfer findings in mice between studies is limited by the absence of standardized protocols and valid reference values for the assessment of normal cardiac function in mice. This study aims to establish comprehensive transthoracic echocardiography (TTE) reference ranges for mice, particularly focusing on C57BL/6N wildtype controls. The study, which includes data from over 15,000 mice through the International Mouse Phenotyping Consortium (IMPC), highlights how variables such as sex, age, body weight, and anesthesia affect TTE parameters. The findings showed that anesthesia is the primary predictor of variability in cardiac function. Isoflurane and tribromoethanol anesthetized mice presented with modified cardiac function compared to conscious mice. Additionally, we observed minimal sex differences in cardiac morphology and function, except for small variations influenced by anesthesia. The effects of aging on cardiac function were modest, characterized by a decrease in heart rate and subtle changes in ventricular dimensions without evidence of pathological remodeling, likely attributable to disease-free cardiovascular aging. Validation of the reference ranges across multiple mouse strains showed that these values provide a reliable baseline for experiments involving cardiac function in mice. The data underscore the importance of using anesthesia-specific reference values when interpreting TTE results, ensuring robust comparisons in genetic and pharmacological studies. These reference ranges serve as quality assurance tools for future cardiac studies in mice, offering insights into typical TTE parameter values, supporting the detection of experimental perturbations, and contributing to more effective translation of findings from mouse to human.

genetics↗

bioRxiv2025

THLANet: A Deep Learning Framework for Predicting TCR-pHLA Binding in Immunotherapy Applications

Adaptive immunity is a targeted immune response that enables the body to identify and eliminate foreign pathogens, playing a critical role in the anti-tumor immune response. Tumor cell expression of antigens forms the foundation for inducing this adaptive response. However, the human leukocyte antigens (HLA)-restricted recognition of antigens by T-cell receptors (TCR) limits their ability to detect all neoantigens, with only a small subset capable of activating T-cells. Accurately predicting neoantigen binding to TCR is, therefore, crucial for assessing their immunogenic potential in clinical settings. We present THLANet, a deep learning model designed to predict the binding specificity of TCR to neoantigens presented by class I HLAs. THLANet employs evolutionary scale modeling-2 (ESM-2), replacing the traditional embedding methods to enhance sequence feature representation. Using scTCR-seq data, we obtained the TCR immune repertoire and constructed a TCR-pHLA binding database to validate THLANets clinical potential. The models performance was further evaluated using clinical cancer data across various cancer types. Additionally, by analyzing divided complementarity-determining region (CDR3) sequences and simulating alanine scanning of antigen sequences, we unveiled the 3D binding conformations of TCRs and antigens. Predicting TCR-neoantigen pairing remains a significant challenge in immunology, THLANet provides accurate predictions using only the TCR sequence (CDR3{beta}), antigen sequence, and class I HLA, offering novel insights into TCR-antigen interactions. Author summaryT-cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes is fundamental to immune responses. However, predicting their binding poses a significant challenge due to the intricate dynamics of their interactions. We developed THLANet, a novel deep learning model, to address this challenge by integrating the ESM-2 and Transformer-Encoder modules. This approach enhances sequence feature encoding, improving the models generalization capability and enabling accurate predictions of TCR-pHLA binding in clinical datasets. Using data processed from open-source databases, THLANet outperformed existing methods, such as PanPep and pMTnet, in precision-recall metrics across multiple epitopes. Additionally, THLANet demonstrates superior capability in identifying critical binding sites within 3D structures, providing structural insights into TCR-pHLA interactions. THLANet offers a robust framework for advancing immunotherapy research, with potential applications in the development of personalized medicine.

bioinformatics↗

bioRxiv2025

DICAR-JP Regulates Ribosome Migration, a New Theory of Mitochondrial Protein Production

BackgroundDICAR plays a cardioprotection of diabetic models. DICAR-JP, a short sequence of DICAR, may represent DICARs active functional domain. NAC is necessary for the heart tissue development. Addtionally, OGDHL is a metabolism regulator. In this study, we examine a new mechanism of DICAR via NAC/OGDHL on regulating cardiomyocyte metabolism in DCM. We also sought to elucidate the function of DICAR/DICAR-JP in modulating NAC-dependent production of OGDHL nascent peptides and their mitochondrial translocation, and we also aimed to optimize the DICAR-JP sequence to be better therapy on DCM. MethodsSPR was to investigate the binding interaction between DICAR-JP and NAC. The function of DICAR-JP and DICAR-JP45 on OGDHL nascent peptide transfection was detected by in vitro translation approach. Biotin-DICAR-JP was transfected to study the function of DICAR-JP, NAC and OGDHL. Untargeted metabolomics was utilized to characterize the metabolic reprogramming of cardiomyocytes. AAV9-DICAR-JP and DICAR-JP45 were constructed and administered to db/db mice for 1-2 months to assess their cardioprotective effects detected by Echocardiography confirmed mouse cardiac function. ResultsOur findings demonstrated that DICAR-JP be the functional domain that interacts with NAC to regulate OGDHL nascent peptide expression. Disruption of OGDHL expression reversed the metabolic reprogramming observed in diabetic cardiomyocytes, highlighting its crucial role in maintaining cardiac metabolic homeostasis. DICAR-JP facilitated the translocation of OGDHL nascent peptides from the cytoplasm to the mitochondria, leading us to hypothesize that DICAR-JP plays a key role in regulating ribosomal migration from the endoplasmic reticulum to the mitochondria. We refer to this process as the Ribosome Migration. In our studies using AAV9-mediated DICAR-JP and DICAR-JP45 overexpression in heart tissue. DICAR-JP and DICAR-JP45 both exhibited significant cardioprotective effects against diabetic cardiomyopathy (DCM), comparable to those observed with Empagliflozin. ConclusionsIn a conclusion, we propose a new endogenous nucleic acid candidate drug library, and a new molecular framework, the Ribosome Migration, which implicates the DICAR-JP/NAC/OGDHL nascent peptide axis in metabolic reprogramming related to DCM. This theory constructs a new mitochondrion protein from nuclear original protein. Moreover, DICAR-JP45 shows strong potential as a nucleic acid-based therapeutic candidate for treating DCM. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSDICAR is a new protective circular noncoding RNA for diabetic cardiomyopathy. NAC is located in ribosome and regulates new peptide production, which is also a key factor in heart tissue development. OGDHL can regulate different energy metabolism in mitochondria. What New Information Does This Article Contribute?DICAR-JP is the functional domain that interacts with NAC to regulate OGDHL nascent peptide expression. There is an interaction between DICAR-JP and NAC. DICAR-JP sequence is protected by NAC and NAC function is mediated by DICAR-JP. We called this RNA functional domain. OGDHL expression reversed the metabolic reprogramming observed in diabetic cardiomyocytes, highlighting its crucial role in maintaining cardiac metabolic homeostasis. DICAR-JP takes part in tanscription and translocation of OGDHL nascent peptides from the cytoplasm to the mitochondria via plasma ribosome. We call this process the Ribosome Migration Theory. DICAR-JP and DICAR-JP45 both exhibited significant cardioprotective effects against diabetic cardiomyopathy (DCM), comparable to those observed with Dapagliflozin (DAPA). Our study revealed a novel molecular framework, the Ribosome Migration Theory, which implicates the DICAR-JP/NAC/OGDHL nascent peptide axis in metabolic reprogramming related to DCM. Moreover, DICAR-JP45 shows strong potential as a nucleic acid-based therapeutic candidate for treating DCM.

pharmacology and toxicology↗

bioRxiv2025

Cold-activated hepatocyte derived exosomes promote BAT thermogenesis through miR-293-5p

The liver-adipose axis serves as a fundamental regulatory network governing systemic lipid homeostasis, with hepatic-derived signals orchestrating adipose tissue plasticity through multimodal mechanisms. Comprehensive elucidation of these bidirectional crosstalk mechanisms may reveal novel therapeutic strategies for metabolic disorders. Our investigation demonstrates that cold exposure induces hepatic secretion of exosomes that potentiate adipose thermogenic activation in both in vitro and in vivo models. This thermogenic potentiation is mechanistically linked to cold-induced upregulation of hepatocyte-derived exosomal miR-293-5p. Notably, pharmacological administration of miR-293-5p agomir significantly attenuates diet- induced obesity and associated metabolic dysregulation in murine models. Through mechanistic interrogation, we identified Tet1 as a direct downstream target of miR- 293-5p, where ectopic Tet1 expression disrupts brown adipose tissue (BAT) thermogenic programming independently of miR-293-5p modulation. Our findings establish cold-activated hepatocyte exosomes as endocrine signaling mediators containing thermogenic microRNA cargos, with miR-293-5p emerging as a regulatory nexus coordinating mitochondrial bioenergetics and systemic energy equilibrium through liver-adipose cross-talk.

genetics↗

bioRxiv2025

Unveiling the pathogenicity of Cunninghamella echinulata SMH-1 and its bioactive metabolites through metabolomics: Dual-action disruption of Solenopsis invicta mitochondrial bioenergetics and cuticular integrity

The invasive red imported fire ant, Solenopsis invicta, poses significant ecological and economic challenges, necessitating sustainable alternatives to conventional pest control. This study evaluates the biocontrol potential of Cunninghamella echinulata isolate SMH-1, an entomopathogenic fungus, through integrated morphological, biochemical, and metabolomic analyses. Light and scanning electron microscopy elucidated infection dynamics, revealing conidial germination, cuticular penetration, and systemic hyphal proliferation, culminating in complete cadaver colonization. At 1x10 conidia/mL, SMH-1 achieved 100% mortality in S. invicta workers within 7 days, with virulence maintained across thermal gradients (10-30{degrees}C). Acetone extracts of SMH- 1 induced dose-dependent lethality, reaching 100% mortality at 1,000 mg/L. Nineteen significantly differentiated metabolites were identified from phospholipids category through UPLC/MS analysis of isolate SMH-1. The derived compounds also showed strong potential against S. invicta, among which dihydrocoumarin caused 100% mortality over 12-days of exposure. The bioactivity of isolate SMH-1 (Conidia), (extracts) and (dihydrocoumarin) against S. invicta revealed significantly differentiated metabolites as (Sulfolithocholylglycine, PE(18_1(9Z)_0_0), CE(12:0), LysoPI(18:1(9Z)/0:0) and Hydroxydestruxin B), (Tropine, lignoceric acid, 9-deoxy-9-methylene-16,16-dimethyl-PGE2, Flecainide and Jubanine B), and (3-O-alpha-D-Glucopyranuronosyl-D-xylose, Rilmakalim, 2-Amino-3-methyl-1-butanol, 2,4-Dimethyl-1,3-oxazole-5-carboxylic acid and Caffeinol), respectively. Citrate cycle (TCA) was significantly impacted by differential expression of pyruvate, pyruvic acid, succinic acid and fumaric acid. These findings underscore SMH-1s multifaceted biocontrol potential, combining direct pathogenicity, thermo-tolerance, and metabolite-mediated toxicity. By delineating host-pathogen interactions and metabolic disruptions, this work advances C. echinulata isolate SMH-1 as a sustainable candidate for integrated S. invicta management, offering an eco-friendly alternative to synthetic insecticides. Author SummaryInvasive fire ants (S. invicta) threaten ecosystems and agriculture worldwide, and current chemical controls often harm the environment. In this study, we explored a natural alternative: a soil-dwelling fungus called Cunninghamella echinulata (strain SMH-1). We discovered that this fungus can kill fire ants with remarkable efficiency. When applied directly, its spores caused 100% ant mortality within a week, even under varying temperatures. We also identified key fungal compounds, including dihydrocoumarin, which are lethal to ants. By studying how the fungus invades and disrupts the ants biology-such as breaking down their protective outer layers and interfering with their energy production, we revealed why SMH-1 is so effective. Our findings highlight SMH-1s potential as a safe, eco-friendly biocontrol agent. This fungus could reduce reliance on synthetic pesticides, offering farmers and communities a sustainable tool to combat fire ant invasions while protecting biodiversity. Our work bridges lab discoveries and real-world pest management, paving the way for greener solutions in agriculture and ecosystem conservation.

microbiology↗

bioRxiv2025

Phase separation of Polycomb-like (PCL) proteins drive PRC2 complex condensates to regulate gene expression

Polycomb group (PcG) proteins function as two major multicomponent protein complexes: Polycomb repressive complex 1 (PRC1) and 2 (PRC2) to repress key developmental genes and maintain epigenetic memory of cell identity during development. Phase-separation has long been implicated in PRC1 mediated gene silencing, but it is still unclear whether PRC2 also utilize a similar mechanism to regulate gene expression. Here we report that Polycomb-like (PCL) proteins, PRC2 accessory factors, can phase-separate in vitro and generate dynamic puncta in vivo. Biochemical and cellular analyses reveal that PCL proteins (hereafter referred to as PCLs) have intramolecular interaction between N- and C-terminal domains, which make PCLs into more compact conformations. The intramolecular interaction not only controls the size of the PCLs phase separation droplets, but also affects the chromatin association of PRC2. Finally, we show that CpG islands, key DNA regulatory elements in mammalian promoters, disrupt the N-C intramolecular interaction of PCLs to expose their middle intrinsically disordered regions (IDRs), which in turn trigger PCLs driven PRC2 condensates formation. Together, this study provides a new perspective on the regulation of PRC2 by PCLs, implicating PCLs read CpG islands to fine-tune their oligomerization to overcome the threshold for PRC2 recruitment to corresponding genomic loci via multivalent interaction with CpG islands chromatin.

biochemistry↗

bioRxiv2025

Resilience and vulnerabilities of tumor cells under purine shortage stress

Purine metabolism is a promising therapeutic target in cancer; however how cancer cells respond to purine shortage,particularly their adaptation and vulnerabilities, remains unclear. Using the recently developed purine shortage-inducing prodrug DRP-104 and genetic approaches, we investigated these responses in prostate, lung and glioma cancer models. We demonstrate that when de novo purine biosynthesis is compromised, cancer cells employ microtubules to assemble purinosomes, multi-protein complexes of de novo purine biosynthesis enzymes that enhance purine biosynthesis efficiency. While this process enables tumor cells to adapt to purine shortage stress, it also renders them more susceptible to the microtubule-stabilizing chemotherapeutic drug Docetaxel. Furthermore, we show that although cancer cells primarily rely on de novo purine biosynthesis, they also exploit Methylthioadenosine Phosphorylase (MTAP)-mediated purine salvage as a crucial alternative source of purine supply, especially under purine shortage stress. In support of this finding, combining DRP-104 with an MTAP inhibitor significantly enhances tumor suppression in prostate cancer (PCa) models in vivo. Finally, despite the resilience of the purine supply machinery, purine shortage-stressed tumor cells exhibit increased DNA damage and activation of the cGAS-STING pathway, which may contribute to impaired immunoevasion and provide a molecular basis of the previously observed DRP-104-induced anti-tumor immunity. Together, these findings reveal purinosome assembly and purine salvage as key mechanisms of cancer cell adaptation and resilience to purine shortage while identifying microtubules, MTAP, and immunoevasion deficits as therapeutic vulnerabilities.

cancer biology↗

bioRxiv2025

The architecture, assembly, and evolution of a complex flagellar motor

Bacterial flagella drive motility in many species, likely including the last bacterial common ancestor 1,2. Knowledge of flagellar assembly and function has mainly come from studies of Escherichia coli and Salmonella enterica, which have simple flagellar motors 3-7. However, most flagellated bacteria possess complex motors with unique, species-specific adaptations whose mechanisms and evolution remain largely unexplored 8-10. Here, we deploy a multidisciplinary approach to build a near-complete model of the flagellar motor in Campylobacter jejuni, revealing its remarkable complexity in architecture and composition. We identify an E-ring around the MS-ring, a periplasmic cage with two distinctive conformations, and an intricate interaction network between the E-ring and cage. These scaffolds play critical roles in stabilizing and regulating 17 torque-generating stator complexes for optimal motility. In-depth evolutionary analyses uncover the ancient origin and prevalence of the E-ring in flagellated species of the domain Bacteria as well as a unique exaptation of type IV pili components PilMNOPQF in the ancestral motor of the phylum Campylobacterota. Collectively, our studies reveal novel mechanisms of assembly and function in complex flagellar motors and shed light on the evolution of flagella and modern bacterial species.

microbiology↗

bioRxiv2025

Modular DNA Barcoding of Nanobodies Enables Multiplexed in situ Protein Imaging and High-throughput Biomolecule Detection

Current immunodetection methods using antibody-DNA conjugates enable multiplexed target detection through orthogonal DNA barcodes, but existing conjugation approaches are labor-intensive and often compromise antibody function. Here we present a modular, site-specific, and cost-efficient DNA tagging strategy -- multiplexed and modular barcoding of antibodies (MaMBA). Utilizing nanobodies as modular adaptors, MaMBA enables direct site-specific labeling of off-the-shelf IgG antibodies with a one-component design. We first applied MaMBA to develop the misHCR method for highly multiplexed in situ protein imaging via orthogonal hybridization chain reaction (HCR). Its cleavable variant, misHCRn, achieves simultaneous visualization of 12 different targets within the same mouse brain sections through iterative probe use. We further extended the cleavable MaMBA to develop the barcode-linked immunosorbent assay (BLISA) for multiplexed and high-throughput biomolecule detections. By combining BLISA with next-generation sequencing, we successfully measured SARS-CoV-2 IgG and HBV-associated antigens in a large number of human serum samples. Additionally, we demonstrated a small-scale drug screen by using BLISA to simultaneously detect eight protein targets. In conclusion, MaMBA offers a highly modular and easily adaptable approach for antibody DNA-barcoding, which can be broadly applied in basic research and clinical diagnostics.

bioengineering↗

bioRxiv2025

easyEWAS: a flexible and user-friendly R package for Epigenome-Wide Association Study

MotivationRapid advancements in high-throughput sequencing technologies especially the Illumina DNA methylation Beadchip greatly fueled the surge in epigenome-wide association study (EWAS), providing crucial insights into intrinsic DNA methylation modifications associated with environmental exposure, diseases, and health traits. However, current tools are complex and less user-friendly to accommodate appropriate EWAS designs and make downstream analyses and result interpretations complicated, especially for clinicians and public health professionals with limited bioinformatic skills. ResultsWe integrated the current state-of-the-art EWAS analysis methods and tools to develop a flexible and user-friendly R package easyEWAS for conducting DNA methylation-based research using Illumina DNA methylation Beadchips. With easyEWAS, we provide a battery of statistical methods to support differential methylation position analysis across various scenarios, as well as differential methylation region analysis based on the DMRcate method. To facilitate result interpretation, we provide comprehensive functional annotation and result visualization functionalities. Additionally, a bootstrap-based internal validation was incorporated into easyEWAS to ensure the robustness of EWAS results. Evaluation in asthma patients as the example demonstrated that easyEWAS could simplify and streamline the conduction of EWAS and corresponding downstream analyses, thus effectively advancing DNA methylation research in public health and clinical settings. Availability and implementationeasyEWAS is implemented as an R package and is available at https://github.com/ytwangZero/easyEWAS. Contactxu.gao@pku.edu.cn, ytwang@pku.edu.cn

bioinformatics↗

bioRxiv2025

A novel human blood-brain barrier model reveals pericytes as critical regulators of viral neuroinvasion

The blood-brain barrier (BBB) plays a vital role in regulating the passage of biomolecules between the bloodstream and the central nervous system (CNS) while also protecting the CNS from pathogens. Pericytes reside at the interface between the endothelial cells that form the vessel walls and the brain parenchyma. These cells are critical for maintaining BBB integrity and play key roles in regulating vessel permeability, blood flow, and immune cell migration. In this study, we developed a novel serum-free protocol to generate neural crest cell-derived pericytes (NCC-PCs) from human pluripotent stem cells (hPSCs). These NCC-PCs enhance BMEC barrier function and can be co-cultured with hPSC-derived brain microvascular endothelial cells (BMECs) in a contact co-culture BBB model that recapitulates the in vivo cellular interactions at the BBB. We used this model to evaluate the pathological consequences of BBB cell infection by highly neuroinvasive flaviviruses. Our results identify a previously undescribed role for NCC-PCs in maintaining BMEC barrier integrity during infection and reducing the spread of viral infection to the CNS.

microbiology↗