Biology subjects

Liu, M.

Publications and source records attributed to Liu, M..

At least 91 records · Page 5Linked to original sources

bioRxiv2026

The GTPase activating protein Gyp6 binds Retromer and inactivates Rab7/Ypt7 to coordinate the formation of endosomal carriers

The Retromer coat is conserved in all eukaryotes and is crucial for the correct intracellular sorting of many transmembrane receptors and lysosomal hydrolases. Retromer is an effector of the late endosomal small GTPase RAB7 and is also implicated in its inactivation required for proper endosomal maturation. Here, we explore the role of controlled GTP hydrolysis by the RAB7 ortholog Ypt7 in the formation of Retromer-coated membrane carriers in yeast. Proximity labelling and genetic ablation identify the GTPase Activating Protein (GAP) Gyp6 as a critical regulator of Ypt7 activity in the context or Retromer. Structural studies show that Retromer recruits Gyp6 through its Vps29 subunit, which recognises a specific PL motif and a secondary binding site in the C-terminal domain of Gyp6. This interaction does not occur with other yeast GAPs. Ablation of the Gyp6-Retromer interface or the catalytic activity of Gyp6 leads to the accumulation of tubular structures on endo-lysosomal compartments and to increased association of Ypt7 with Retromer and its cargo Vps10. These results support a model in which Gyp6 controls the switch from Ypt7-dependent Retromer coat assembly and cargo collection to the departure of the carrier through membrane fission and uncoating.

cell biology↗

bioRxiv2026

AlphaGenome -enabled analysis of non-coding regulatory variants underlying RHD Expression

Systematic identification of functional non-coding regulatory variants remains a major challenge in human genetics. Conventional approaches such as large-scale CRISPR screening and genome-wide association studies (GWAS) are powerful but often prohibitively expensive, time-consuming, and experimentally intensive, limiting their scalability for locus-specific mechanistic studies. Recent advances in artificial intelligence offer the potential to partially replace or substantially augment these approaches by prioritizing regulatory variants with high functional likelihood. The RHD antigen, a major contributor to red blood cell alloimmunization, hemolytic transfusion reactions, and hemolytic disease of the fetus and newborn, serves as an excellent model for this paradigm, since coding variants alone do not fully account for differences in RHD expression. Here, we present an integrated artificial intelligence (AI)-guided and experimental framework to identify and validate functional non-coding regulatory variants governing RHD expression. We first applied AlphaGenome (AG), a deep-learning model released in 2025 for non-coding variant impact prediction, to systematically interrogate the RHD locus. By integrating multi-omics datasets, AG prioritized regulatory regions within the promoter, 5' untranslated region (5'UTR), and intragenic regions. In silico deletion- and Single Nucleotide Polymorphism (SNP)-based perturbation analyses consistently predicted that variants within the promoter and its proximal regions, as well as within intragenic regions, exert strong suppressive effects on RHD expression. To experimentally validate these predictions, we performed CRISPR-mediated base editing in K562 cells at AG-prioritized non-coding SNP sites. Editing of a high-score predicted variant (chr1:25272434 G>A) achieved efficient base conversion and was accompanied by additional nearby edits, all predicted by AG to downregulate RHD expression. In contrast, editing of low-score predicted sites (chr1:25272422 C>T) produced much smaller functional effects. Quantitative polymerase chain reaction (qPCR) analysis of full-length RHD transcripts, together with flow cytometry-based analysis of RHD expression, confirmed strong concordance between AI-based predictions and transcriptional as well as phenotypic outcomes. Taken together, our results demonstrate that the combination of AI-guided regulatory variant prioritization and targeted base editing provides a potentially scalable and cost-effective alternative to traditional CRISPR screening for decoding functional non-coding variants in blood group genes, with direct implications for genomics-based RHD typing and transfusion medicine. To our knowledge, this study also represents the first validation of AlphaGenome predictions at the phenotypic level using wet-lab experiments.

genomics↗

bioRxiv2026

Natural H5N1 immunity in dairy cows is durable and cross-protective but non-sterilizing

Ongoing transmission of influenza A virus (H5N1) in U.S. dairy cattle threatens both animal and human health, underscoring the need to understand the durability of host immunity against reinfection with evolving genotypes. We challenged naive and convalescent cows, infected one year prior with H5N1 genotype B3.13, with either homologous B3.13 or heterologous D1.1 genotype virus. Homologous rechallenge resulted in complete clinical protection with no infectious viral shedding. Conversely, heterologous rechallenge led to transient clinical disease and limited infectious viral shedding. Convalescent cows experienced significantly milder disease than naive cows, which developed severe illness with high viral shedding and required early euthanasia, regardless of the strain. These findings indicate that naturally acquired immunity offers strong protection against severe illness but may allow silent transmission of divergent strains. Therefore, natural herd immunity alone is unlikely to eliminate the virus; controlling H5N1 in cattle will likely require vaccination strategies that address viral evolution.

immunology↗

bioRxiv2026

INS-fMRI reveals a mesoscale limbic organization associated with medial pulvinar in primates

The experience of emotion comprises not only the feelings (happiness, sadness), but also behavioral expression, internal awareness, and the bodys physiological response. However, it remains unclear how the brain integrates these disparate aspects of emotion. Here, to examine functional connectivity in the limbic system of macaque monkeys, we combined focal Infrared Neural Stimulation of a sensory gateway (medial pulvinar PM), with ultra-high-field 7T functional magnetic resonance imaging (INS-fMRI). We find connected sites are mesoscale (millimeter-scale) in size and arranged in patchy patterns across cingulate, insula, and amygdala. Non-overlapping connections evoked from three sequential stimulation sites in PM form clusters of multi-site integration, and appear related to known functional organization within these limbic regions. We suggest these mesoscale functional connections link the limbic axes of motor expression (cingulate), interoception (insula), and emotion-related processing (amygdala), and that, much like visual system, the limbic system is fundamentally quite orderly at mesoscale. Our results underscore the importance of millimeter-scale precision and organization in diagnosis and treatment of affective disorders.

neuroscience↗

bioRxiv2025

Alignment of Artificial Neural Networks with Human Eye Movement Patterns in Facial Expression Recognition

Artificial neural networks (ANNs) have become powerful tools for modeling human perception and hehavior, yet it remains unclear if they resemble the sequential eye movement strategies humans employ during visual processing. Human studies demonstrate that the first two fixations play distinct roles in face processing: the initial fixation (Fix I) captures global configurational information, while the second (Fix II) targets local diagnostic details, with the two working collaboratively to support recognition. In this study, we tested if such properties emerge in ANNs trained for facial expression recognition. We fine-tuned a convolutional neural network (VGG16) and a Vision Transformer (ViT) on multiple facial expression datasets and evaluated their performance on the same images used in a human eye-tracking experiment. Human participants (N = 28) achieved a mean accuracy of 84.2%, VGG reached 82.9%, and ViT outperformed both at 90.0%. Perturbation analyses showed that both models aligned with the independent functions of Fix I and Fix II, but only ViT displayed a synergistic effect when information from both fixations was combined. Layer-wise analyses revealed stronger correspondence with Fix II than Fix I in both models, while ViT uniquely captured the statistical dependencies of fixation transitions. These findings suggest that VGG primarily reflects feedforward, detail-based processing, whereas ViT supports feedback-like integration across sequential fixations. Our results indicate that current ANNs can approximate human eye movement patterns in face processing, and highlight the potential of transformer-based architectures to model sequential strategies in human visual exploration.

neuroscience↗

bioRxiv2025

Antibacterial Activity of the Natural Product Elaiophylin against Streptococcus suis and its Biofilm Eradication Effect

The multidrug resistance (MDR) problem in Streptococcus suis(S. suis) is becoming increasingly severe, necessitating the development of novel antibacterial agents and strategies. In this study, seven Elaiophylin derivatives were isolated from Streptomyces sp. WS-30248, and the antibacterial activity and mechanism of action of the principal compound, Elaiophylin, against S. suis were systematically evaluated for the first time. Through comprehensive approaches including in vitro efficacy assays, biofilm inhibition and eradication tests, bacterial membrane integrity analysis, reactive oxygen species (ROS) level detection, and a mouse infection model, Elaiophylin was found to exhibit significant antibacterial activity against multiple clinically MDR S. suis strains. Its minimum inhibitory concentration (MIC) was as low as 0.5 g/mL, and complete bactericidal activity was achieved within 24 hours at 4xMIC. The compound effectively inhibited and eradicated bacterial biofilms, directly killing embedded cells within the biofilm matrix. Mechanistic studies revealed that Elaiophylin functions through multiple synergistic pathways, including disruption of bacterial membrane integrity and induction of massive ROS accumulation, thereby interfering with the proton motive force (PMF), depleting intracellular ATP, and blocking energy metabolism. In the mouse infection model, the Elaiophylin-treated group showed a significantly increased survival rate of 60% and effectively reduced bacterial loads in tissues. This study demonstrates that Elaiophylin is a highly promising natural candidate drug with multi-target synergistic effects, offering a new strategy to combat MDR S. suis and biofilm-associated infections.

microbiology↗

bioRxiv2025

Impaired Chylomicron Secretion Results in Reduced Body Fat and Increased Intestinal Fatty Acid Oxidation by Activation of Autophagy

Intestinal absorption of dietary lipid is essential for systemic lipid homeostasis; however, elevated postprandial plasma lipid levels are associated with obesity and increased risk for atherosclerotic cardiovascular disease. In humans and rodents, biological sex impacts dietary triglyceride absorption and males tend to have higher postprandial triglyceride levels compared to females, but the physiological basis for this is not well understood. Here, we show that the gene DENND5B is associated with body composition in humans and mice and that genetic deletion of Dennd5b in mice prevents postprandial plasma triglyceride elevations in both sexes. Our findings establish a role for this protein in intestinal chylomicron secretion and reveal a biological sex-biased differential impact of its deletion on body composition, dietary fatty acid uptake, and intestinal metabolism. Mechanistically, our findings implicate autophagy and mitochondrial beta oxidation in coping with enterocyte lipid accumulation due to impaired chylomicron secretion. This work extends the emerging concept that the intestinal epithelium may play a prominent role in oxidation of diet-derived fatty acids and suggests that this process may contribute to biological sex-based differences in postprandial lipemia and body composition. HighlightsO_LIThe DENND5B gene is required for dietary triglyceride absorption and is associated with metabolic phenotypes in humans and mice. C_LIO_LIDisruption of chylomicron secretion differentially affects body composition, dietary lipid absorption, and intestinal metabolic activity in male and female mice. C_LIO_LIIn mice, genetic disruption of Dennd5b results in lower body fat in both sexes, and increased lean mass only in males. C_LIO_LIMetabolic phenotypes in Dennd5b-deficient mice are driven by reduced absorption of dietary lipid characterized by enterocyte lipid retention and increased fecal lipid excretion. C_LIO_LIImpaired chylomicron secretion in Dennd5b-/- mice induces autophagy-mediated disposal of cellular triglyceride and increased fatty acid oxidation in intestinal tissue. C_LI

physiology↗

bioRxiv2025

Immune-Derived THBS1-CD47 Axis Induces Cellular Senescence and Suppresses Osteogenesis in Diabetic Periosteum

Type 2 diabetes mellitus (T2DM) weakens bone repair and increases fracture risk, but how the periosteal environment contributes to this problem is not fully understood. We found that diabetic mice showed thinning of the periosteum, reduced bone mass, and poor function of bone-forming cells. Using single-cell RNA sequencing, we discovered that immune cells and bone-lineage cells interact more strongly in diabetes through the THBS1-CD47 pathway. THBS1 was mainly produced by macrophages, while its receptor CD47 was abundant in osteogenic cells. Laboratory experiments showed that THBS1 blocked bone gene activity, increased inflammation and cell death, and damaged mitochondria, leading to higher oxidative stress. Knocking down CD47 reversed these effects, restoring bone cell activity and energy balance. In diabetic fracture models, blocking THBS1 improved callus formation and bone healing. These findings identify THBS1-CD47 as a key driver of periosteal dysfunction in T2DM and highlight it as a potential target to improve skeletal repair in diabetic patients.

cell biology↗

bioRxiv2025

Epithelial FOXP3 Drives Pancreatic Fibrosis through O-glycosylated IL-6

BACKGROUND & AIMSThe transcription factor FOXP3, known for specifying regulatory T cell fate, is unexpectedly expressed in the epithelium of precancerous pancreatic lesions. Here we investigate its non-immune role in initiating pancreatic fibrosis, a process driving therapeutic resistance and organ failure in pancreatic ductal adenocarcinoma (PDAC) with unknown origins in early neoplasia. METHODSUsing human tissues and genetically engineered mouse models, we analysed FOXP3 expression in premalignant lesions. We employed epithelial-specific FOXP3 knockout and knock-in strategies to determine its functional impact on fibrogenesis and neoplasia progression. Mechanistic studies included chromatin immunoprecipitation, glycomic analyses, and signalling assays. RESULTSFOXP3 was consistently expressed in the epithelial compartment of human and murine precancerous pancreas. Epithelial-specific deletion of FOXP3 attenuated pancreatic fibrosis and delayed neoplasia, whereas its knock-in induced spontaneous stromal activation and accelerated PanIN progression. Mechanistically, epithelial FOXP3 directly transactivated the glycosyltransferase GALNT1. GALNT1, in turn, mediated O-glycosylation of interleukin (IL)-6, which was essential for its rapid secretion. CONCLUSIONSOur study establishes epithelial-derived FOXP3 as a master regulator of early pancreatic fibrocarcinogenesis. It drives a glycosylation-dependent amplification loop for IL-6 signalling, orchestrating sustained stromal activation. This pathway represents a promising target for intercepting pancreatic fibrosis and carcinogenesis at its origin.

cancer biology↗

bioRxiv2025

Colorectal cancer relies on an immunosuppressive cellular topography and genomic adaptations for establishing brain metastases

Colorectal cancer (CRC) brain metastases have a poor prognosis and limited treatment options, including resistance to radiation therapy. Little is known about the molecular and cellular mechanisms that enable CRC tumor cells to adapt to the brain and establish a supportive tumor microenvironment. To address this gap we used spatial transcriptomics to analyze 51 CRC brain metastases. A subset had matched primary colon tumors and longitudinally paired metastatic resections before and after radiation treatment. We identified the critical spatial cellular features of the tumor epithelium and the surrounding tumor microenvironment that support metastatic growth in the brain. CRC brain metastases developed a stromal microenvironment with abundant fibroblasts and tumor-associated macrophages. A fibroblast-macrophage cellular neighborhood promoted angiogenesis, extracellular matrix remodeling, and immune suppression. Tumor cells showed local adaptations. In endothelial-rich regions, they were proliferative whereas in macrophage-rich regions, they were more differentiated and immune evasive. Compared with paired primary tumors, CRC brain metastases showed increased chromosomal instability, with activation of RNA-processing, stress response, and junctional remodeling pathways. After radiation treatment, resistant clones had increased epithelial-mesenchymal transition, while the immunosuppressive stroma remained intact. We identified tumor-derived MIF, GDF15, PRSS3 and SEMA3C ligands and macrophage-derived SPP1 that have the potential to affect multiple cell types in the metastatic niche. These ligand-receptor interactions drive angiogenesis, stromal activation and immune suppression. In a macrophage-tumor-fibroblast co-culture model, knockout of SPP1 in macrophages led to reduced expression of lipid-metabolism related genes and disrupted tumor-promoting interactions. Together, these results indicate that CRC growth in the brain is sustained by a specific cellular organization with immunosuppressive multicellular interactions.

cancer biology↗

bioRxiv2025

Agentic Lab: An Agentic-physical AI system for cell and organoid experimentation and manufacturing

Reproducibility in biological research and manufacturing remains constrained by the complexity of multi-step protocols, fragmented data-analysis pipelines, and the intrinsic variability of experimental execution. Here, we present Agentic Lab, an agentic-physical AI platform that unifies large language model and vision language model (LLMs/VLMs)-driven reasoning with real-world laboratory operations. Agentic Lab uses multi-agent orchestration architecture, comprising of specialized subagents for knowledge retrieval, protocol design, multimodal data analysis, and training-free segmentation and representation learning for intrinsically explainable single-cell and organoid phenotyping. These agents operate under the orchestration of a virtual principal investigator MolAgent that is linked to an augmented reality (AR)-based physical AI interface, which can bridge digital reasoning with human physical execution. Agentic Lab perceives real-world experimental activities, provides context-aware instructions, identifying procedural errors in real time for humans to correct, and continuously evolves with its long-term memory database expanding through the accumulation of experimental data logs from human scientists. This interaction allows scientists and AI agents to collaborate and co-evolve dynamically, closing the loop between planning, action, and analysis in the traditional cell and organoid research lifecycle. We demonstrate Agentic Lab in organoid differentiation from human pluripotent stem cells, where it autonomously generates protocols, monitors culture procedures, and identifies subtle morphological heterogeneity linked to growth conditions. The system interprets these phenotypes, grounds them in literature, and proposes targeted instructions for improving differentiation efficiency. By combining multi-agent reasoning with physical laboratory awareness, Agentic Lab transforms experimentation and biomanufacturing from a static workflow into an adaptive, feedback-driven, bidirectional process that integrates agentic AI into the research lifecycle. This framework establishes a foundation for intelligent laboratories that integrate design, execution, and interpretation within a unified agentic-physical system.

bioengineering↗

bioRxiv2025

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

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

microbiology↗

bioRxiv2025

An extensive forward genetic screen identifies the HWS-AGO1 axis as pivotal for target mimicry-mediated miRNA degradation in Arabidopsis

Target RNAs have emerged as key regulators of miRNA stability, thereby influencing development and physiological processes in both plants and animals. The F-box protein HAWAIIAN SKIRT (HWS) has been characterized as a crucial factor in targeted miRNA degradation in plants (pTMD), a process triggered by a specific type of target RNA called target mimicry. However, the precise mechanism by which HWS functions is still poorly understood. We previously established a genetic reporter system based on Short Tandem Target Mimicry of miR160 (STTM160) triggered miR160 degradation, resulting in pleiotropic developmental defects that are easy to monitor. Here, through an extensive forward genetic screen, we identified fourteen additional hws alleles that near-completely restored STTM160-induced developmental defects, highlighting a central role for HWS in pTMD. Intriguingly, we discovered two adjacent amino acid substitutions (R421K and G422D) in the PAZ domain of AGO1 that significantly suppressed the STTM160 phenotype. Similar to HWS dysfunction, the AGO1 R421K substitution caused a significant retention of target mimicry RNAs in AGO1 immunoprecipitates, suggesting that the R421 residue of AGO1 may cooperate with HWS for the clearance of AGO1-miRNA-target mimicry complexes. Although the R/G mutations only impacted a small set of endogenous miRNA levels, they were sufficient to rescue the developmental defects caused by HWS overexpression (HWS-OE), suggesting that the developmental abnormalities caused by HWS-OE largely depend on the AGO1-miRNA pathway. Taken together, our findings provide a solid genetic evidence for the coordination of HWS and AGO1 in pTMD.

plant biology↗

bioRxiv2025

A human forebrain organoid model phenocopies dysregulated RNA and protein homeostasis in ALS/FTD-associated TDP-43 proteinopathies

TAR DNA-binding protein 43 (TDP-43) proteinopathy is a defining pathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet the downstream molecular events linking TDP-43 dysfunction to neurodegeneration remain incompletely understood. Here, we model TDP-43 proteinopathy by introducing the ALS/FTD-associated TARDBP K181E mutation into human induced pluripotent stem cells and differentiating them into three-dimensional forebrain organoids. Organoids carrying the K181E mutation exhibit spontaneous TDP-43 hyperphosphorylation, cytoplasmic p-TDP43 accumulation, RNA dysregulation, pro-inflammatory signaling, and activation of apoptotic pathways without TDP-43 overexpression or external stress. Single-cell transcriptomics and enhanced crosslinking immunoprecipitation reveal that the K181E mutation alters TDP-43 RNA-binding specificity, causing widespread RNA mis-splicing including cryptic exon inclusion. Among affected targets, we identify cryptic exon inclusion in the transcription factor PRDM2. PRDM2 cryptic exon-derived peptide immunoreactivity was detected in ALS spinal motor neurons and associated with p-TDP-43 pathology. These findings identify PRDM2 mis-splicing as a candidate downstream event linking TDP-43 dysfunction relevant to ALS and FTD. One sentence summaryHuman brain organoids reveal how mutant TDP-43 disrupts RNA processing and causes neuronal stress.

neuroscience↗

bioRxiv2025

High-Concordance Validation of Droplet Digital PCR and Next-Generation Sequencing for EGFR Mutation Detection Across Diverse Biospecimens in a Large-scale NSCLC Cohort Study

BackgroundsNext-generation sequencing (NGS) and droplet digital PCR (ddPCR) are both established methods for detecting EGFR mutations in non-small cell lung cancer (NSCLC). However, comprehensive validation of their concordance in mutation detection and variant allele frequency (VAF) quantification across heterogeneous sample types remains limited. Inconsistent results from different sample types (e.g., cfDNA, FFPE) pose a significant challenge to clinical decisions. This is particularly critical for advanced NSCLC patients, who often rely on liquid biopsy, yet the concordance between liquid and tissue-based testing lacks validation in large-scale studies. Another issue in clinical practice is that many tumor samples are limited in quantity, making it difficult to meet testing requirements. Therefore, it is worth exploring whether pre-capture NGS libraries can serve as substitutes for original DNA. MethodsIn this study, we first developed three assays to detect EGFR L858R, exon 19 deletions (Ex19del) and T790M, respectively using ddPCR platform. Their Limit of Detection (LOD) could reach 0.01% at 100 ng of input DNA. Subsequently, we conducted a large retrospective clinical study to systematically compare the detection performance of ddPCR and NGS across three mutation types using approximately 1,000 EGFR-positive samples, including cell-free DNA (cfDNA), pre-capture NGS libraries of cfDNA (cfDNA-prePCR), FFPE-derived DNA (ffpeDNA), pre-capture NGS libraries of FFPE-derived DNA (ffpeDNA-prePCR), fresh tumor tissue DNA (ttDNA), pre-capture NGS libraries of ttDNA (ttDNA-prePCR), pleural effusion supernatants DNA (peDNA), and pre-capture NGS libraries of peDNA (peDNA-prePCR). They were analyzed for detection concordance and VAF correlation. Especially, we made comparisons of the tumor DNA, including ctDNA and tumor tissue DNA, with their paired pre-capture NGS library. ResultsKey findings demonstrated excellent overall agreement between NGS and ddPCR. The mutation detection concordance rates were 98.72% (overall), with subtype-specific rates of 98.93% (L858R), 99.23% (Ex19del), and 97.14% (T790M). VAF measurements between ddPCR and NGS showed exceptional correlation (Pearsons r = 0.975, P<0.001). Notably, pre-capture NGS libraries showed remarkable VAF concordance with their source materials (0.993 for cfDNA libraries vs cfDNA; 0.998 for tumor tissue libraries vs tumor DNA with EGFR L858R; 0.991 for tumor tissue libraries vs tumor DNA with EGFR Ex19del). ConclusionsNGS and ddPCR demonstrate high concordance in EGFR mutation detection and VAF quantification, supporting their complementary roles in clinical testing. Using pre-capture libraries as an alternative to source samples can avoid repeat biopsies and enables subsequent testing for patients with inadequate FFPE sample quantity. These findings establish an evidence base for integrated diagnostic paradigms leveraging NGSs multiplexing power and ddPCRs sensitivity.

cancer biology↗

bioRxiv2025

A functional amyloid matrix underpins the PDIM-architected corded superstructure of the Mycobacterium tuberculosis biofilm

Bacterial biofilms generate collective properties that cannot be explained by individual cells alone. Yet the structural principles that organize these communities and their relationship to drug tolerance remain poorly understood, including in major human pathogens such as Mycobacterium tuberculosis (Mtb). Here we show that submerged Mtb biofilms form organized multicellular architectures in which bacterial cords are integrated with a composite extracellular matrix. The virulence lipid phthiocerol dimycocerosate (PDIM) governs the higher-order organization of cords, proteinaceous matrix interactions support cohesion and attachment, an amyloid-like component contributes to biofilm integrity and establishment, and the ESX-1 secretion system increases matrix biochemical complexity. Biofilm growth conferred antibiotic tolerance across genetic backgrounds, while PDIM-dependent organization provided additional protection against first-line anti-tuberculosis drugs, most clearly isoniazid. Targeting amyloid assembly with epigallocatechin gallate impaired biofilm establishment without inhibiting planktonic growth. Together, these findings establish a cording-centred framework linking bacterial surface composition, cellular organization and extracellular matrix assembly to antibiotic tolerance, and show how resolving higher-order biofilm architecture can reveal processes amenable to chemical perturbation.

microbiology↗

bioRxiv2025

Physics-Diffusion-Driven Multiscale Aggregation for Drug-Target Interaction Prediction

Drug-target interaction prediction is an important task in computational drug discovery. To address the limitations of existing graph neural network approaches in propagation depth and robustness, we propose PDDMA-DTI (Physics-Diffusion-Driven Multiscale Aggregation for Drug-Target Interaction Prediction), a physics-inspired, multiscale adaptive aggregation framework. PDDMA-DTI injects a physics-motivated diffusion operator into a heterogeneous drug-target network: global diffusion models cross-modal information propagation via the graph Laplacian, while local diffusion performs neighborhood smoothing in the low-dimensional embedding space. Both processes are solved with the implicit Euler scheme to ensure numerical stability. For each node, we introduce an adaptive stopping rule based on steady-state convergence and a weighted fusion strategy. This strategy leverages hop counts and node degrees to aggregate multi-step diffusion snapshots into a final representation that preserves original features while capturing multi-scale context. To emphasize strong interaction pathways, we further propose a Physical Interaction Summation Enhancement module that augments pairwise interaction features with a linear plus quadratic energy term, thereby amplifying highly correlated drug-target pairs. Extensive experiments on multiple benchmark datasets show that PDDMA-DTI consistently outperforms representative baselines in accuracy and robustness.

bioinformatics↗

bioRxiv2025

A line attractor maintains aggressiveness during feeding in hangry mice

Aggression evolved to protect resources such as food from competitors, but animals must balance fighting and feeding so that they facilitate rather than hinder re-establishment of energy homeostasis. How this balancing is computed is not well understood. We have approached this problem at the level of neural population-coding by examining the effect of progressive starvation on a hypothalamic line attractor that encodes an internal state of aggressiveness. Moderate fasting yielded "hangry" mice, decreasing attack latency and increasing attack frequency. In parallel, line attractor ramping rate and stability were increased, suggesting that hunger enhances aggressiveness by modifying neural dynamics. In contrast, prolonged starvation inhibited aggression and eliminated the line attractor. In satiated mice, titrated acute chemogenetic activation of arcuate AgRP neurons recapitulated the biphasic effects of progressive starvation, suggesting that a continuous increase in hunger exerts bi-directional influences at different intensities. When confronted with food and an intruder, hangry mice alternated between feeding and fighting. During eating, population neural activity moved out of the line attractor while activity in the attractor dimension remained unchanged. Following feeding, activity rapidly relaxed back into the attractor and aggression resumed. Thus, the line attractor may serve to keep hungry animals primed for aggression during intermittent feeding bouts.

animal behavior and cognition↗