Biology subjects

Liu, Y.

Publications and source records attributed to Liu, Y..

At least 523 records · Page 29Linked to original sources

bioRxiv2025

Scalable spike sorting across thousands of neurons by modeling neural dynamics with NeuroSort

Neural circuit research requires resolving the functional coding mechanisms at the single-neuron scale. However, neural signals captured from extracellular recordings are inherently complex, shaped both by electrode properties and the spatially distributed neural population activity across recording sites. These factors introduce strong signal mixing and waveform variation, making it diffcult for existing spike sorting methods to reliably isolate individual neurons from large-scale recodings. To address this, we develop NeuroSort, which fuses spatiotemporal information through a contrastive learning strategy to adaptively model neural dynamics. NeuroSort is a GPU-enabled, highly efficient spike sorting framework, enabling the isolating of up to thousands of neurons. We validated NeuroSort on in vivo extracellular recordings, including Utah array data from rhesus macaques performing motor tasks and Neuropixels 1.0 probe data from a rat during rest. Moreover, in a recording collected by the 1,024-channel Neuroscroll probe, NeuroSort sorted the largest number of well-isolated neurons from different brain areas while achieving a 2-8 times speedup over other methods. Taken together, NeuroSort generalizes across recording conditions, providing an interpretable and scalable paradigm for whole-brain dynamic analysis.

neuroscience↗

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

Oxygen-adaptive plasticity of Asgard archaea dependent on terminal oxidase and globin

The oxygenation of ancient Earth is thought to have driven eukaryogenesis, beginning with the endosymbiosis of an aerobic alphaproteobacterium (proto-mitochondria) with an archaeal host. Given that the archaeal host likely evolved from within Asgard archaea (phylum Promethearchaeota), the metabolic traits of Asgard archaea could provide key insights into eukaryotic origins. Although Asgard archaea cultured to date are obligate anaerobes, their genomes encode oxygen-adaptive proteins, suggesting they might be oxygen-tolerant. Here, we demonstrate that some Asgard archaea, in particular, Hodarchaeales, the closest known relatives of eukaryotes, and Kariarchaeaceae, exhibit oxygen adaptation mediated by terminal oxidase and globin. Phylogenetic analysis reveals long-term vertical evolution of terminal oxidases in Asgard archaea, suggesting ancient adaptation to molecular oxygen. By contrast, globin was likely acquired by Asgard archaea via horizontal gene transfer from facultative aerobic Chloroflexales bacteria. Heterologous expression of the Asgard globin enhances aerobic growth of Haloarchaea and Escherichia coli in the presence of terminal oxidase-dependent electron transfer chain, suggesting that Asgard growth benefits from ambient oxygen. The Asgard globin gene is embedded in an oxygen-sensitive bidirectional promoter region, with one promoter driving oxygen-induced globin expression, and the other anaerobically activating expression of two enzymes, PdxS and PdxT, involved in a pyridoxal 5-phosphate biosynthesis. The Asgard globin and promoter region exhibit high functional robustness across archaea and bacteria, and could contribute to the symbiosis between the Asgard and aerobic bacterial partners. These findings highlight the oxygen-adaptive plasticity of Asgard archaea and its potential contribution to eukaryogenesis.

evolutionary biology↗

bioRxiv2025

Virus induced transgene- and tissue-culture free heritable genome editing in tomato

Genome editing has emerged as a powerful tool for genome manipulation and trait improvement in crops. However, most commonly used approaches rely on tissue culture and transgenic materials, which are time-consuming, labor-intensive, and often strongly genotype-dependent. Here, we developed a tobacco rattle virus (TRV)-based system to deliver the compact ISYmu1 TnpB endonuclease, coupled with in planta shoot regeneration, to achieve somatic and heritable genome editing across different tomato cultivars without tissue culture. By targeting SlPDS, we successfully generated virus-free homozygous mutant progeny in a single generation. Furthermore, we extended this system to the functional analysis of the previously uncharacterized SlDA1 locus, revealing its involvement in organ size regulation, and recovered transgene-free SlDA1 mutants displaying enlarged fruits. Given the wide host range of TRV, our system should be broadly applicable for rapid, non-transgenic and less genotype-dependent heritable genome editing, thereby advancing both functional genomics and crop improvement. SignificanceEfficient genome editing without the need for transgenesis or tissue culture remains a major challenge in crop breeding. Here, we establish a simple single-step system for transgene- and tissue culture-free genome editing in tomato based on Tobacco Rattle Virus-mediated delivery of the compact RNA-guided TnpB enzyme ISYmuI and its guide RNA. This strategy enabled somatic editing of de novo shoots and heritable transmission of targeted mutations to the next generation. Notably, editing an agronomically relevant gene produced tomato plants with larger fruits, highlighting the potential of this system for crop improvement.

plant biology↗

bioRxiv2025

Microcompartments in Hodarchaeales: a bioenergetic engine that could have fuelled eukaryogenesis

Eukaryotic intracellular compartmentalization is a key innovation in the evolution of complex cellular life. While microcompartments enable metabolic specialization in many bacteria, to our knowledge, no analogous systems have been identified in Archaea. Here, we report the discovery of archaeal microcompartments (AMCs) in Hodarchaeales, an order within the phylum Promethearchaeati (Asgard archaea) that includes the closest known archaeal relatives of eukaryotes. Phylogenetic and structural analyses indicate that these catabolic AMCs, which are specialized for sugar-phosphate metabolism, were acquired by horizontal gene transfer from deep-rooted bacteria of the phylum Myxococcota. The shell pentamers of AMCs are fused to lysine/arginine-rich intrinsically disordered regions that capture cytosolic DNA, facilitating nutrient scavenging. Reaction-diffusion modelling predicts that enzyme colocalization and substrate channelling within AMCs can increase the NADH flux approximately 100-fold. Thus, the AMCs substantially boost energy production in the cell and might have primed the archaeal host for eukaryogenesis.

microbiology↗

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

Dysregulation of U12-Type Splicing in Lupus Neutrophils

ObjectiveNeutrophil dysfunction is a hallmark of systemic lupus erythematosus (SLE), but its molecular basis remains unclear. This study explores transcriptional and post-transcriptional changes in low-density granulocytes (LDGs), a proinflammatory neutrophil subset expanded in SLE, focusing on NADPH oxidase (Nox) function and minor intron splicing. MethodsLDGs and normal-density neutrophils (NDGs) were isolated from SLE patients and healthy controls (HCs). CYBA (P22phox) expression was evaluated at transcript and protein levels. Nox activity was measured using luminol assays. Bulk RNA sequencing and rMATS software were used to assess alternative splicing, particularly of U12-type intron-containing genes. ResultsCYBA expression was reduced in SLE LDGs (n=11) compared to SLE NDGs and HCs (n=6), with levels resembling those in chronic granulomatous disease neutrophils. SLE LDGs exhibited impaired Nox activity (n=7 SLE, n=12 HC). CYBA is a U12 intron-containing gene, and transcriptomic analysis revealed broad downregulation of this gene class in SLE LDGs, suggesting minor spliceosome dysfunction. rMATS analysis showed increased U12-type intron retention and widespread splicing defects-- including exon skipping and mutually exclusive exon use--in genes such as GBP5, MAEA and STX10. These abnormalities were validated in an independent long-read RNA-seq dataset from SLE PBMCs. Importantly, splicing disruptions correlated with disease activity and autoantibody profiles. ConclusionImpaired U12-dependent splicing may contribute to neutrophil dysfunction in SLE, potentially via defective oxidative burst and altered immune regulation. These findings highlight the minor spliceosome as a novel player in lupus pathogenesis.

immunology↗

bioRxiv2025

Development and Genome-level Microevolution of Oral Microbiome during Surface Colonization

The oral microbiome is essential to human health, yet its de novo ecological succession and microevolutionary dynamics remain poorly understood due to the absence of tractable in vivo models. Dental implants provide a unique opportunity to investigate these processes by establishing a defined time-zero for initial bacterial attachment. In this cohort study, we performed shotgun metagenomics on 95 subgingival plaque samples from 19 participants, including peri-implant sites at weeks 1-4 after crown placement and adjacent teeth as controls. Peri-implant and adjacent periodontal microbiomes exhibited distinct taxonomic and functional profiles. Even the same taxa had different functional potential across the two sites. These findings indicated that oral microbiome development is a niche-specific process with selective colonization rather than passive microbial translocation from adjacent sites. Longitudinal analysis identified three microbial community modules driving the succession of oral microbiome, including pioneer colonizers, constitutive species, and late commensals. Each module followed distinct temporal abundance patterns and played unique ecological roles throughout the succession process. Strain-level resolution revealed divergent microevolutionary trajectories: pioneer colonizers and late commensals exhibited higher cumulative mutation rates and greater strain heterogeneity overtime, with enrichment of nonsynonymous single nucleotide variants in genes related to virulence and metabolism, whereas constitutive species remained evolutionarily stable by contrast. Our study reveals that the development, succession, and microevolution of the oral microbiome is structured, niche-dependent, and modulated by inter-species facilitation and selective genomic adaptation. These findings advance the understanding of oral microbiome ecology and provide a conceptual foundation for manipulating microbial succession in health and disease.

microbiology↗

bioRxiv2025

Estrogen Dependent Variation in the Contributions of TRPM4 and TRPM5 to Fat Taste

Sex differences in physiology have garnered significant interest of late; however, comparatively little is known about the effects of sex on the function of the peripheral taste system. Previously, we have shown that fat taste functions in a sexually dimorphic manner using molecular, cellular, and behavioral assays, and that a subtype of estrogen receptor (ER) proteins are highly expressed in Type II (receptor) cells. The underlying mechanisms of estrogens action, though, remain unknown. Here, we sought to better understand estrogens role in fat taste transduction at the molecular level by initially focusing on the transient receptor potential channels types M4 (Trpm4) and M5 (Trpm5), which we have shown to play roles in estrogen-sensitive fatty acid signaling in taste cells. That is, using Trpm5-deficient mice, both males and females in the estrus phase showed significantly reduced FA responses, whereas females in the proestrus phase did not, suggesting that there may be E2-dependent TRPM5-independent FA signaling in Type II cells. During periods of high levels of circulating estrogen, there was no significant difference in cellular responses to fatty acid (FA) stimuli between Trpm5-/- mice and their wild-type counterparts. Moreover, supplemental estradiol enhanced linoleic acid (LA)-induced TRPM5- mediated taste cell activation. Finally, while Type II cells depend on TRPM4 and TRPM5 for FA taste cell activation, proestrus (high estrogen) females showed a greater dependence on a TRPM5-independent pathway for fatty acid responsiveness. Together, these results underscore the substantial regulatory role of estrogen in the taste system, particularly for fatty acid signaling. Given that the taste system guides food preferences and intake, these findings may have important implications for understanding sex-specific differences in diet and, ultimately, metabolic health. SUMMARYThe current manuscript shows sex differences in fat taste signaling and identifies functional variations in specific transduction elements in taste cells that respond to sex hormones, such as estrogen, that may mediate differences in peripheral fat taste pathways.

neuroscience↗

bioRxiv2025

A multi-tissue atlas of genetic regulatory effects in sheep

Characterizing the impact of genomic variants on genome function and ultimately complex traits in livestock is essential for the development of sustainable precision agriculture and comparative genomics. Here, as part of the Farm animal Genotype-Tissue Expression (FarmGTEx) project, we present the pilot phase of the SheepGTEx resource through analyzing 6,761 RNA-sequencing samples of 51 primary tissues in a multi-breed population of sheep. We identify millions of regulatory variants associated with seven types of molecular phenotypes, and fine-map 322,468 primary and 114,017 non-primary effects, revealing a high degree of regulatory allelic heterogeneity. We systematically characterize the pleiotropic effects of these variants on molecular phenotypes, assess their context-specific regulatory patterns across tissues, breeds, sexes, and developmental stages, as well as explore their evolutionary constraints across mammals. Finally, we demonstrate the substantial potential of the SheepGTEx resource (https://sheepgtex.farmgtex.org), by providing examples of regulatory mechanisms underpinning 34 complex traits, population divergence between European and Asian breeds, and adaptive evolution in sheep over the past ten millennia.

genomics↗

bioRxiv2025

Dangling Ends of Third Strand and Duplex Drive Nucleic Acid Triplex Stabilization through Bimodal Association

Nucleic acid triplexes are crucial structural motifs in gene regulation and biotechnology, yet the kinetic principles governing their formation remain poorly understood. While a stability hierarchy of RNA*DNA-DNA > DNA*DNA-DNA > RNA*RNA-RNA, with no DNA*RNA-RNA triplex forming, is known, the kinetic roles of terminal residues remain poorly understood. Here, we employ bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy to demonstrate that dangling ends from both the third strand (triplex-forming oligonucleotide, TFO) and the duplex dramatically enhance triplex stability. Kinetic analysis reveals this stabilization is primarily driven by a marked increase in the association rate (k{square}{square}). Crucially, creating a single-base-pair dangling end at either terminus of the duplex enhanced triplex stability more effectively than blunt ends. For example, DNA TFO dTFO5 binding to d(HP5+TA) was enhanced compared to dHP5, and similarly RNA TFO rTFO5 binding to RNA duplex r(HP5+UA) and DNA duplex d(HP5+TA) showed stronger affinity and faster association than to blunt-ended rHP5 and dHP5. Interestingly, removal of a terminal base pair from the blunt-end duplex, generating a TFO dangling end, also enhances binding affinity and association rate. This indicates that both duplex and TFO dangling ends provide critical nucleation platforms, while blunt-ended terminal triples are dynamic and contribute minimally to stability. Thus, our work establishes that optimal triplex formation requires strategic optimization of both TFO and duplex terminal structures through a fundamental kinetic principle (bimodal nucleation). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/686076v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1dc6950org.highwire.dtl.DTLVardef@1861387org.highwire.dtl.DTLVardef@16c24a5org.highwire.dtl.DTLVardef@da0bf0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDangling ends at both third strand and duplex termini enhance triplex stability by accelerating association C_LIO_LITriplex formation can be nucleated from either end with a bimodal association mechanism C_LIO_LITerminal blunt-end base triples are dynamic and contribute minimally to stability compared to tailored overhangs C_LI

biophysics↗

bioRxiv2025

Transforming a Fragile Protein Helix into an Ultrastable Scaffold via a Hierarchical AI and Chemistry Framework

The rational design of proteins that maintain structural integrity under concurrent thermal, mechanical, and chemical stress remains a challenge in molecular engineering. We present a hierarchical framework that transforms an -helical domain into an ultrastable scaffold by integrating AI-guided design with foundational chemical principles. This approach progresses from global architectural reinforcement, using multiple AI tools to create a stabilized four-helix bundle, to local chemical tuning, where AlphaFold3 guides the installation of salt bridges and metal-coordination motifs. A computational pipeline using physics-based screening such as molecular dynamics (MD) simulations efficiently distilled millions of designs into a minimal candidate set. The resulting -helical proteins exhibit unprecedented multi-axis stability, with mechanical unfolding forces exceeding 200 pN, thermal resilience >100 {degrees}C, and high resistance to chemical denaturants. By systematically dissecting the contributions of hydrophobic packing, electrostatics, and metal coordination, we establish a general blueprint for imparting extreme robustness. This work bridges AI-driven structural generation with chemical precision, advancing the creation of durable proteins for mechanistic studies and synthetic biology.

biophysics↗

bioRxiv2025

Graphene nanoedge electronics for monolithic and chronic recording of local microcircuits at neuronal density

High-density single-unit recording among closely spaced neurons over long durations is crucial for understanding the cellular-level functional architecture of the brain. Existing brain probes, however, either laterally sample neurons far more sparsely than neuronal density in the same cortical layer or suffer from long-term instability due to electrode modification, preventing precise neuron-to-neuron interrogation in local microcircuits. Here, we report a monolithic graphene-edge probe (NeuroEdge) that achieves single-unit recording at neuronal density (16 electrodes within 100 {micro}m diameter). We fabricate NeuroEdge using self-assembled reduced graphene oxide nanoflakes to form an electrochemically active tip consisting of exposed graphene nanoedges and electrolyte-filling nanotunnels, achieving an ultralow specific impedance of 20 M{Omega} {micro}m2. In vivo experiments over 5 months demonstrate recording at a high signal-to-noise ratio (>20 dB) and reliable interrogation of neighboring neurons. We also show that NeuroEdge can record from a single auditory cortical layer and reveal heterogeneities in the acoustic frequency response and dynamic connectivity among neighboring neurons. NeuroEdge provides a tool for precisely interrogating local microcircuits at the density of neurons in the brain.

bioengineering↗

bioRxiv2025

Data-driven model reveals increased stability of CAG-expanded huntingtin RNA due to MID1 binding

RNA-binding proteins (RBP) are important regulators of RNA metabolism. In neurode-generative disorders such as Huntingtons Disease (HD), disrupted RBP-RNA interactions contribute to neuronal dysfunction. One such RBP, Midline 1 (MID1), has been shown to aberrantly associate with mutant huntingtin (Htt) RNA, enhancing its translation, yet the mechanism driving this effect remains unknown. Here, we develop a computational model to understand the role of MID1. Based on previously published data, our model predicts that MID1 increases the stability of the Htt RNA. We experimentally validate this prediction, showing that overexpression of MID1 significantly prolongs the half-life of mutant Htt RNA. Furthermore, we evaluate model refinements, including clustering of MID1-bound RNA, which allow capturing all key observations in the data. Together, we provide a data-driven framework that underlines the importance of RBP-RNA interaction in post-transcriptional regulation. This framework also shows how individual molecular reactions jointly determine RNA stability and protein levels in HD.

systems biology↗

bioRxiv2025

Phage display-mediated immuno-PCR to detect low-abundance secreted proteins in Drosophila

Circulating hormones, that mediate communications across organs to maintain physiological balance, are commonly detected and quantified using enzyme-linked immunosorbent assays (ELISAs). However, while ELISA is well-suited for organisms where sample blood can be readily obtained, its application is considerably more challenging in smaller organisms, particularly Drosophila, which has gained widespread use in recent years for physiological studies. Here, we present sensitive phage display-mediated immuno-PCR (PD-iPCR) to detect Drosophila hemolymph proteins via two approaches: 1) by identifying high-affinity nanobodies through phage-display library screening and subsequent affinity maturation and 2) by generating a knock-in fly line producing secreted proteins tagged with tandem NanoTags composed of VHH05 and 127D01. Using these approaches, we successfully established PD-iPCR to detect insulin-binding ImpL2 protein in fly hemolymph. Notably, the tandem NanoTag-based sandwich PD-iPCR enabled highly sensitive detection of tagged antigens, allowing us to quantify elevated ImpL2 levels in the hemolymph of starved flies and those bearing Yki-induced gut tumors. Collectively, our results demonstrate that PD-iPCR enables detection of endogenous, low-abundance circulating hormones in Drosophila, providing a powerful tool for studying interorgan communication. Significance statementHormones and other secreted factors orchestrate organism-wide physiology, yet their routine quantification in Drosophila has been limited by the limited volume of hemolymph available for assays like enzyme-linked immunosorbent assay (ELISA). Here, we present phage display-mediated immuno-PCR (PD-iPCR) as a novel sensitive platform for quantifying secreted proteins in flies in vivo. Using ImpL2 as an example, we successfully detected nanomolar level of circulating ImpL2 and monitored its physiological changes during starvation and tumorigenesis using PD-iPCR. This approach can be readily expanded to multiplexed quantification of secreted proteins in vivo by leveraging the multiple available nanobodies and the vast collection of epitope-tagged Drosophila lines. This work opens the door to systematic endocrine phenotyping across developmental stages and diverse physiological conditions.

genetics↗

bioRxiv2025

Orchestrating Microbiome Analysis with Bioconductor

The expansion of microbiome research has led to the accumulation of interlinked datasets encompassing versatile taxonomic and functional assays. The analysis of increasingly large and heterogeneous multi-modal microbiome data would benefit from unified approaches supporting the design of modular data science workflows through interoperable methods. The Bioconductor project has recently developed an optimized statistical programming framework for multi-assay data integration. Building on this foundation, we introduce a community-developed open source ecosystem for microbiome data science. In contrast to the previous alternatives, the methodology is specifically designed to support joint analysis of hierarchical, interlinked, and heterogeneous multi-table datasets that are increasingly common in modern microbiome research. This data science ecosystem encompasses open data, methods, tutorials, and an active online community. These resources support standardized and reproducible data wrangling, joint analysis, and reporting. We have detailed the functionality and usage in the online book https://microbiome.github.io/OMA, which offers guidance for prospective users and contributors.

bioinformatics↗

bioRxiv2025

Structure-based Predictions of Conformational B Cell Epitopes by Protein Language Model and Deep Learning

Mapping conformational B-cell epitopes remains a central challenge for antibody discovery: experiments are costly and most computational tools trained on generic protein-protein interfaces transfer poorly to antibody-antigen recognition. We introduce a patch-centric framework that predicts epitopes directly on antigen structures. Each surface "patch" is defined as a triad of neighboring residues, capturing the smallest local unit that encodes both shape and chemistry. We evaluate two classifiers: (i) a protein language model (PLM) approach that averages ESM-2 embeddings over each triad and scores them with a small multilayer perceptron [1], and (ii) a convolutional baseline that consumes a hand-crafted 15x20 feature matrix summarizing amino-acid identity, secondary structure, solvent accessibility, and shape index. Trained with five-fold cross-validation on 1,151 AbDb antibody-antigen complexes, the PLM model markedly outperforms the CNN at the patch level (e.g., F1{approx} 0.986, ROC-AUC{approx} 0.998). Aggregating patch scores to residues with an ensemble over all folds yields robust residue-wise performance, surpassing the CNN (ROC-AUC 0.689{+/-}0.072 vs. 0.548{+/-}0.018). Against widely used sequence- and structure-based tools on AbDb, our PLM achieves the best summary metrics (ROC-AUC 0.67, PR- AUC 0.56) with full coverage of all antigens. On five external complexes unseen during development, the model generalizes well (ROC-AUC 0.663) and accurately localizes binding regions qualitatively. The method converts PLM representations into interpretable epitope likelihood maps, offering a practical aid for antigen prioritization, antibody engineering, and vaccine design.

bioinformatics↗

bioRxiv2025

Macrophage EHD1 promotes inflammation and stabilizes sortilin to accelerate atherosclerosis

BackgroundMacrophages are key players in the pathogenesis of atherosclerosis. They trigger immune responses through their cell-surface receptors. However, how macrophages regulate those receptors in response to pro-inflammatory stimuli is not completely understood. Endocytic membrane trafficking involving receptor internalization, followed by endosomal transport and recycling of the internalized receptors, plays essential roles in balancing cell-surface receptors to meet cellular needs. Here, we explored the role of the endocytic regulator EHD1 in immune responses in macrophages and determined its contribution to atherosclerosis progression. MethodsEHD1 expression profiles in mouse and human plaques were determined by single-cell RNA sequencing (scRNA-seq) and immunofluorescence staining. Bone marrow transplantation (BMT) by transplanting bone marrow cells from Ehd1-/- or littermate wild-type mice to irradiated Ldlr-/- mice was performed to determine the effect of EHD1 deletion on atherosclerosis progression. In vitro mechanistic studies including inflammation signaling and endocytosis assays were performed in bone marrow-derived macrophages. ResultsEHD1 expression in macrophages is enhanced as atherosclerosis progresses in both mice and humans. Histological analysis of aortic root sections from BMT mice showed that EHD1 deletion reduces lesion size. ScRNA-seq of aortic CD45+ cells demonstrated that EHD1 deletion attenuates pro-inflammatory responses and cell-cell interactions. Mechanistic studies revealed that EHD1 accelerates the endocytic recycling of TNFR2 and activates NF-kB, leading to increased expression of inflammatory cytokines. Moreover, EHD1 interacts with retromer and stabilizes sortilin, a retrograde cargo of retromer and a risk factor for atherosclerosis. ConclusionsEHD1 promotes inflammation by enhancing TNFR2-NF-kB signaling and stabilizing sortilin, leading to accelerated atherosclerosis. Our study reveals novel roles for EHD1-mediated membrane trafficking in macrophage function and paves the way to innovative therapeutic strategies that aim to address dysregulated membrane trafficking in atherosclerosis.

cell biology↗