Biology subjects

Jin, S.

Publications and source records attributed to Jin, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Gene-centric metagenomic analyses reveal microbiome functional insights into diseases

The microbiome encodes millions of genes; however, understanding their role in human health remains challenging. Here, we developed MetaGEAR, a gene-centric analysis framework for metagenomic data. MetaGEAR constructs cohort-specific databases for efficient retrieval of gene annotations, abundances, and co-localization, while providing enhanced taxonomic resolution by integrating reference- and assembly-based approaches. This is combined with a Metagenomic Assembled Graph (MAGraph) capturing gene neighborhood information. Using MetaGEAR, we built a multi-cohort database comprising >33 million gene families to investigate microbiome functionality across 24 cohorts of inflammatory bowel disease, colorectal cancer, and healthy populations and identified disease signature genes. Furthermore, the MAGraph revealed mobile genetic elements acting as hubs for tetracycline resistance spreading among Enterococcus, Streptococcus, and Veillonella species. Also, a duplicated nitrate reduction operon in Klebsiella pneumoniae was linked to differential gene expression under stress and virulence-inducing conditions. In summary, gene-centric metagenomic analyses reveal important insights into microbiome functionality in diseases.

microbiology↗

bioRxiv2025

Directed evolution of a compact TranC11a system for efficient genome editing

The recently discovered TranC systems represent programmable RNA-guided DNA endonucleases of transposon origin with compact protein sizes ideal for therapeutic delivery. However, their editing efficiency in human and plant cells is limited. Here, we evolved TranC11a and engineered its sgRNA to enhance overall editing efficiencies. TranC11a systems exhibit up to 9.2-fold higher editing activity than its parent and achieves efficiency comparable to SpCas9 across multiple human genome endogenous sites, significantly outperforming compact editors NovaIscB and enTnpB1c. TranC11a enables efficient editing of disease-relevant genes in human cells and breeding traits in maize. With its high editing activity and compact size (574 aa), TranC11a demonstrates strong potential for future in vivo genome editing and crop engineering.

synthetic biology↗

bioRxiv2025

Microbial oral-gut translocation in advanced chronic liver disease is linked to exacerbation of intestinal barrier dysfunction and hepatic fibrosis

While microbiome perturbations are associated with advanced chronic liver disease (ACLD), microbial disease mechanisms are poorly understood. Using multi-omics analyses of paired saliva and faecal samples from an ACLD cohort, we identified next-to-identical oral and gut bacterial strains (including Veillonella and Streptococcus spp.) which increased in absolute abundance in the gut of ACLD patients. These translocators uniquely encoded a collagenase-like proteinase (prtC) with the potential for gut barrier disruption and prtC faecal abundance was a robust ACLD biomarker (auPR=0.91). CCl4-treated mice inoculated with Veillonella and Streptococcus prtC-encoding patient isolates showed exacerbation of gut barrier impairment and hepatic fibrosis. Furthermore, faecal collagenase activity was increased in ACLD patients and experimentally confirmed for the prtC gene from translocating Veillonella parvula. Overall, our study establishes mechanistic links between oral-gut translocation and ACLD pathobiology, and identifies the oral microbiome as an important contributing factor with potential for microbial diagnostics and therapeutics.

microbiology↗

bioRxiv2025

ADAMTS3 as a promising novel biomarker for the diagnosis of hepatocellular carcinoma

Early diagnosis of hepatocellular carcinoma (HCC) still represents a significant challenge. The rising of obesity and non-viral liver disease associated HCC further limits the effectiveness of conventional screening approaches such as ultrasonography and alpha-fetoprotein (AFP) testing. In this context, the identification of potential effective biomarkers for rapid and precise diagnosis of HCC still represents an extremely important task. Here, we identify ADAMTS3 as a novel mechanistic serum biomarker for HCC that addresses this shifting etiological landscape. Transcriptome analysis of four independent cohorts-including HCC patients, individuals with obesity-or non-viral liver disease, and healthy controls- reveals four secreted proteins associated with HCC progression. Among them, ADAMTS3 demonstrates strong diagnostic performance by ROC (receiver operating characteristic) and LASSO (least absolute shrinkage and selection operator) analysis. High ADAMTS3 expression correlates with aggressive molecular signature, characterized by enhanced proliferative signaling, suppressed immune effector responses, and a reprogrammed immune microenvironment marked by reduced NK cell infiltration and increased accumulation of immunosuppressive tumor-associated macrophages, driven by extracellular matrix stiffening. Knockdown of ADAMTS3 in cell lines significantly reduced proliferation, and clonogenic potential. Reintroduction of ADAMTS3 partially rescued both phenotypes, further confirming its role in promoting HCC cell growth. Optical tweezer-based measurements further reveal reduced cell stiffness upon ADAMTS3 deficiency, highlighting its role in extracellular matrix (ECM) remodeling. Finally, to provide a simple strategy for HCC diagnosis based on this novel biomarker, we demonstrated a DNA origami-based SERS biosensor capable of detecting ADAMTS3 at 10-11 M. Together, these findings identify ADAMTS3 as a mechanistic biomarker and demonstrate a translational sensing strategy for non-invasive HCC early screening.

cancer biology↗

bioRxiv2025

Evolutionary innovation within conserved gene regulatory networks underlying biomineralized skeletons in Bilateria

Biomineralized skeletons have evolved convergently across animals, yet exhibit remarkable diversity in structure and development. However, the evolutionary origins of gene regulatory networks underlying biomineralized skeletons remains elusive. Here, we report comprehensive developmental profiling of transcriptomic and chromatin dynamics in a bivalve mollusc, Crassostrea nippona. We provide evidence for a biphasic regulatory program orchestrating larval and adult shell formation, involving the coordinated activity of ancient transcription factors and dynamic chromatin remodeling. Comparative analyses suggest a conserved developmental toolkit was co-opted for larval exoskeleton formation in the common lophotrochozoan ancestor. In contrast, limited regulatory conservation was observed between lophotrochozoans and echinoderms with regards to the formation of biomineralized skeletons, despite both relying on a heterochronic activation of ancestral regulators. Together, our findings support a hierarchical model where dynamic chromatin decouples rapidly evolving effectors from deeply conserved regulators, allowing modular innovations within conserved gene regulatory networks. This study highlights how epigenetic dynamics bridge evolutionary conservation and novelty, offering a framework for understanding the independent evolution of biomineralization across Bilateria through combinatorial regulatory evolution.

evolutionary biology↗

bioRxiv2025

A small cationic probe for accurate, punctate discovery of RNA tertiary structure

RNA molecules fold into intricate three-dimensional tertiary structures that are central to their biological functions. Yet reliably discovering new motifs that form true tertiary interactions remains a major challenge. Here we show that RNA tertiary folding occasionally generates electronegative motifs that react selectively with the small, positively-charged probe trimethyloxonium (TMO). Sites with enhanced reactivity to TMO, compared with the neutral reagent dimethyl sulfate (DMS), are indicative of tertiary structure and define T-sites. These positions share a structural signature in which a reactive nucleobase is adjacent to non-bridging phosphate oxygens, creating localized regions of negative charge. T-sites consistently map to the cores of higher-order structural interactions and functional centers across diverse RNAs, including distinct states in conformational ensembles. In the 10,723-nt dengue virus genome, three strong T-sites were detected, each within a complex structure required for viral replication. Cation-based covalent chemistry enables high-confidence discovery and analysis of functional RNA tertiary motifs across long and complex RNAs, opening new opportunities for transcriptome-wide structural analysis.

biochemistry↗

bioRxiv2025

The Nox2 NADPH oxidase regulates neutrophilic inflammation in the oral cavity

The leukocyte NADPH oxidase 2 (NOX2) is an important regulator of inflammatory responses, independent of its antimicrobial activity. Inactivating mutations in NOX2 cause chronic granulomatous disease (CGD), a severe immunodeficiency associated with recurrent infections and dysregulated neutrophilic inflammation. Recurrent oral ulcers, stomatitis, gingivitis, and other inflammatory issues affecting the oral mucosa have been observed in patients with CGD; however, the underlying mechanisms are not known. Here, we present evidence that the extensive inflammatory destruction of oral mucosal tissues observed in Nox2-deficient or CybbKOmice was not caused by impaired antimicrobial surveillance against oral pathobionts but instead resulted from a cell-intrinsic dysregulation of neutrophil inflammatory responses. Transcriptional and cellular profiling of oral tissues isolated from wild-type and CybbKO mice showed a dominant neutrophil signature, which was accompanied by a significant upregulation of several bone-resorbing, tissue-degrading inflammatory cytokines and a reduced expression of nuclear factor erythroid 2-related factor 2 (Nrf2) regulated genes. Mechanistically, hyperinflammatory responses were mitigated by restoring Nrf2 transcriptional activity using a synthetic agonist. Thus, our studies show that Nox2 oxidase and its derived reactive oxygen species are crucial for balanced recruitment and cell-intrinsic regulation of neutrophil inflammatory responses within oral tissues in an Nrf2-dependent manner.

immunology↗

bioRxiv2025

A naturalistic reinforcement learning task uncovers historical neural representations

Exploration is essential for reinforcement learning (RL) in human development, facilitating cognitive and behavioral adaptation. However, conventional RL paradigms in neuroimaging studies often rely on highly constrained search spaces and artificial, non-naturalistic scenarios, limiting their capacity to reflect the complexity of real-world human exploration and decision- making. To address this gap, we developed a novel, naturalistic RL paradigm called Photographer, which involves a photograph-taking task set in a virtual street-view environment. Participants navigated city scenes and attempted to infer the paradigms covert goal solely from feedback, defined as the conceptual similarity between their photograph and a hidden target sentence. Representational similarity analysis revealed that feedback history, an encoding of recent trial attempts, was robustly represented in the middle orbital gyrus and inferior frontal gyrus. These neural representations were consistently observed across two independent participant groups. Moreover, although the two regions coactivated during exploration, each was associated with a distinct functional network.

neuroscience↗

bioRxiv2025

Non-disruptive 3D profiling of combinations of epigenetic marks in single cells

Recent advancements in single-cell sequencing and spatial omics technologies have enhanced our understanding of diverse cellular identities, compositions, architectures, and functions. However, the single-cell three-dimensional (3D) organization of the epigenome is still not well understood, due to an absence of spatial single-cell methods that allow high-resolution, locus-specific detection of combinations of epigenetic marks while maintaining the 3D organization of the genome. Here, we develop Epigenetic Proximity Hybridization Reaction (Epi-PHR), a non-disruptive image-based single-cell spatial epigenetic profiling technology. Epi-PHR enables locus-specific and high-resolution in situ detection of combinations of epigenetic marks at hundreds of single gene targets within the same individual cells, while retaining the 3D organization of the genome. Dual-mark Epi-PHR in hippocampus tissue sections revealed region-specific epigenetic profiles. Phased Epi-PHR combined with chromatin tracing simultaneously detects allele-specific epigenetic states and chromatin conformations of a paternally imprinted Meg3 gene cluster in single cells, revealing associations between specific epigenetic mark enrichment and chromatin folding features for the distinct alleles from different parental origins. Surprisingly, we found that H3K9me3 abundance is positively associated with a chromatin domain boundary at the Meg3 locus among maternal copies. We expect Epi-PHR will be broadly applicable in research requiring single-cell spatial epigenetic information, and will help extend our understanding of the combinatorial epigenetic code and its relationship with chromatin organization.

genetics↗

bioRxiv2025

Reference-guided genome assembly at scale using ultra-low-coverage high-fidelity long-reads with HiFiCCL

Population genomics using short-read resequencing captures single nucleotide polymorphisms and small insertions and deletions but struggles with structural variants (SVs), leading to a loss of heritability in genome-wide association studies. In recent years, long-read sequencing has improved pangenome construction for key eukaryotic species, addressing this issue to some extent. Sufficient-coverage high-fidelity (HiFi) data for population genomics is often prohibitively expensive, limiting its use in large-scale populations and broader eukaryotic species and creating an urgent need for robust ultra-low coverage assemblies. However, current assemblers underperform in such conditions. To address this, we propose HiFiCCL, the first assembly framework specifically designed for ultra-low-coverage high-fidelity reads, using a reference-guided, chromosome-by-chromosome assembly approach. We demonstrate that HiFiCCL improves ultra-low-coverage assembly performance of existing assemblers and outperforms the state-of-the-art assemblers on human and plant datasets. Tested on 45 human datasets ([~]5x coverage), HiFiCCL combined with hifiasm reduces the length of misassembled contigs relative to hifiasm by an average of 21.19% and up to 38.58%. These improved assemblies enhance germline structural variant detection, reduce chromosome-level mis-scaffolding, enable more accurate pangenome graph construction, and improve the detection of rare and somatic structural variants based on the pangenome graph under ultra-low-coverage conditions.

bioinformatics↗

bioRxiv2025

Structural Heterogeneity of Proteoform-Ligand Complexes in AMP-Activated Protein Kinase Uncovered by Integrated Top-Down Mass Spectrometry

AMP-activated protein kinase (AMPK) is a heterotrimeric complex ({beta}{gamma}) that serves as a master regulator of cellular metabolism, making it a prominent drug target for various diseases. Post-translational modifications (PTMs) and ligand binding significantly affect the activity and function of AMPK. However, the dynamic interplay of PTMs, non-covalent interactions, and higher-order structures of the kinase complex remains poorly understood. Herein, we report the structural heterogeneity of the AMPK complex arising from ligand binding and proteoforms--protein products derived from PTMs, alternative splicing, and genetic variants--using integrated native and denatured top-down mass spectrometry (TDMS). The fully intact AMPK heterotrimeric complex exhibits heterogeneity due to phosphorylation and multiple adenosine monophosphate (AMP) binding states. Native TDMS delineates the subunit composition, AMP binding stoichiometry, and higher-order structure of AMPK complex, whilst denatured TDMS comprehensively characterizes the proteoforms and localizes the phosphorylation site. This is the first study to structurally characterize AMPK proteoform-ligand complexes. Notably, by integrating native TDMS and AlphaFold, we elucidate a flexibly connected regulatory region of AMPK {beta} subunit that has been difficult to visualize with traditional structural biology tools. Our findings uncover previously unresolvable structural features of AMPK, offer new perspectives on protein kinase regulation, and establish a versatile framework for comprehensive characterization of proteoform-ligand complexes.

biochemistry↗

bioRxiv2025

Native Top-Down Proteomics of Endogenous Protein Complexes Enabled by Online Two-Dimensional Liquid Chromatography

Protein complexes are essential for virtually all biological processes, yet their structural characterization remains a major challenge due to their heterogeneous, dynamic nature and the complexity of the proteome. Native top-down mass spectrometry (nTDMS) has emerged as a powerful tool for comprehensive structural characterization of purified protein complexes, but its application to endogenous protein complexes in the proteome is challenging and typically requires labor-intensive and time-consuming prefractionation. Here, for the first time, we develop a nondenaturing online two-dimensional liquid chromatography (2D-LC) method for native top-down proteomics (nTDP), enabling high-throughput structural analysis of endogenous protein complexes. The automated, online interfacing of size-exclusion and mixed-bed ion-exchange chromatography achieves high coverage of endogenous protein complexes. We further develop a multistage nTDMS approach that enables comprehensive structural characterization within the chromatographic timescale, capturing intact non-covalent complexes, released subunits/cofactors, and backbone fragments. Our analysis detected 133 native proteoforms and endogenous protein complexes (up to 350 kDa) from human heart tissue in less than two hours. Such technological leaps in high-throughput structural characterization of endogenous protein complexes will advance large-scale nTDP studies in health and disease.

biochemistry↗

bioRxiv2025

Rational design of a foldon-derived heterotrimer guided by quantitative native mass spectrometry

Designing stable hetero-oligomeric protein complexes with defined inter-subunit stoichiometries remains a significant challenge. In this study, we report the design of a highly selective heterotrimeric assembly derived from the well-known foldon homotrimer. We generated an aab heterotrimer by introducing the Q11E modification to destabilize the homotrimer and a compensatory modification, either V14A or V14L, to stabilize the hydrophobic core of the heterotrimer. Native mass spectrometry (MS) was essential for guiding the design process, enabling precise characterization of oligomeric states and their equilibrium distributions. The heterotrimer structure was validated by x-ray crystallography. Our findings highlight the effectiveness of combining rational design with native MS to develop specific hetero-oligomeric assemblies.

biochemistry↗

bioRxiv2025

Sequencing-free whole genome spatial transcriptomics at molecular resolution in intact tissue

Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, compositions, interactions, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution. Leading spatial-capture or spatial-tagging transcriptomics techniques that rely on in-vitro sequencing offer whole-transcriptome coverage, in principle, but at the cost of lower spatial resolution compared to image-based techniques. In contrast, high-performance image-based spatial transcriptomics techniques, which rely on in situ hybridization or in situ sequencing, achieve single-molecule spatial resolution and retain sub-cellular morphologies, but are limited by probe libraries that target only a subset of the transcriptome, typically covering several hundred to a few thousand transcript species. Together, these limitations hinder unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here we develop a new image-based spatial transcriptomics technology termed Reverse-padlock Amplicon Encoding FISH (RAEFISH) with whole-genome level coverage while retaining single-molecule spatial resolution in intact tissues. We demonstrate image-based spatial transcriptomics targeting 23,000 human transcript species or 22,000 mouse transcript species, including nearly the entire protein-coding transcriptome and several thousand long-noncoding RNAs, in single cells in cultures and in tissue sections. Our analyses reveal differential subcellular localizations of diverse transcripts, cell-type-specific and cell-type-invariant tissue zonation dependent transcriptome, and gene expression programs underlying preferential cell-cell interactions. Finally, we further develop our technology for direct spatial readout of gRNAs in an image-based high-content CRISPR screen. Overall, these developments provide the research community with a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNA species in many biomedical contexts.

genetics↗

bioRxiv2025

DNA polymerase actively and sequentially displaces single-stranded DNA-binding proteins

Single-stranded DNA-binding proteins (SSBs) play a crucial role in stabilizing and protecting transiently exposed single-stranded DNA (ssDNA), yet the mechanisms governing their displacement by DNA polymerase (DNAp) during replication remain largely unexplored. Using bacteriophage T7 DNAp and its SSB, T7 gp2.5, we investigated the molecular mechanisms and visualized the dynamic process underlying SSB displacement. Our single-molecule force spectroscopy demonstrates that T7 SSB modulates DNA replication in an ssDNA conformation-dependent manner, regulated by tension applied to the DNA template. By integrating dual-color single-molecule imaging, we observe that T7 SSB remains stationary as DNAp approaches, indicating that SSB molecules are sequentially displaced rather than pushed forwards. Molecular dynamics (MD) simulations revealed reduced energy barriers for SSB dissociation in the presence of DNAp. This finding, combined with the detected FRET signals when DNAp approaches an SSB-bound ssDNA region and observations of faster replication rates compared to relative slow intrinsic SSB dissociation, collectively support an active displacement mechanism. Using both ensemble and single-molecule analyses, we demonstrated that SSB saturation of ssDNA is critical for optimal replication efficiency, with each SSB molecule contributing positively to the process. Taken together, the uncovered spatial-temporal coordination between SSB and DNAp is necessary for resolving molecular collisions during DNA replication, and may represent a universal strategy employed by other DNA translocating motors to ensure genomic integrity.

biophysics↗

bioRxiv2025

Tyrosine phosphorylation coupling of one carbon metabolism and virulence in an endogenous pathogen

Endogenous pathogens can constrain virulence to ensure survival in the host. Pathogenic state can be controlled by metabolic responses to the prevailing microenvironment; however, the coupling and effector mechanisms are not well understood. Flux through the One Carbon Metabolism (OCM) pathway can modulate virulence of the oral pathobiont Porphyromonas gingivalis, and here we show that this is controlled by tyrosine phosphorylation-dependent differential partitioning of gingipain proteases. The OCM essential precursor pABA inhibits the low molecular weight tyrosine phosphatase Ltp1, and consequently relieves inhibition of its cognate kinase, Ptk1. We found that in the absence of pABA, reduced Ptk1 kinase activity blocks extracellular release of gingipains. Surface retention of gingipains confers resistance to neutrophil mobilization and killing, and virulence in animal models of disease is elevated. Reciprocally, Ptk1 and gingipains are required for maximal flux through OCM, and Ptk1 can phosphorylate the OCM pathway enzymes GlyA and GcvT. Further, ALP, an alkaline phosphatase involved in synthesis of DHPPP, which combines with pABA to make DHP, is phosphorylated and activated by Ptk1. We propose, therefore, that although the primary function of Ptk1 is to maintain OCM balance, it mechanistically couples metabolism with tunable pathogenic potential through directing the location of proteolytic virulence factors.

microbiology↗

bioRxiv2025

High-Throughput Antibody Neutralization Screening in Massively Parallel Droplet Arrays

Neutralizing antibodies provide rapid immune defense against infectious diseases, but are difficult to discover at scale because neutralization assays require live reporter cells and soluble monoclonal antibodies. Here we report Droplet Reporter Cell Testing for Neutralization (DrReCT-Neutralization) to screen antibody gene libraries for their ability to neutralize viral infections. We established the necessary engineered cell lines and validated the DrReCT screening platform using synthetic oligoclonal libraries, followed by an example discovery campaign that demonstrated scalable functional antibody data collection against viral diseases.

immunology↗

bioRxiv2025

Multicilia dynamically transduce Shh signaling to regulate choroid plexus functions

Choroid plexus is a major site for cerebrospinal fluid (CSF) production, characterized by a multiciliated epithelial monolayer that regulates CSF production. We demonstrate that defective choroid plexus ciliogenesis or Intraflagellar transport yields neonatal hydrocephalus, at least in part, due to increased water channel Aqp1 and ion transporter Atp1a2 expression. We demonstrate choroid plexus multicilia as sensory cilia, transducing both canonical and non-canonical Shh signaling. Interestingly, it is the non-canonical Shh signaling that represses Aqp1 and Atp1a2 expression by Smo/Gi/cAMP pathway. Choroid plexus multicilia exhibit unique ciliary ultrastructure, carrying features of both primary and motile cilia. Unlike most cilia that elongate during maturation, choroid plexus ciliary length decreases during development, causing a decline of Shh signaling intensity in developing choroid plexus, a derepression of Aqp1 and Atp1a2, and ultimately, an increased CSF production. Hence, developmental dynamics of choroid plexus multicilia dampens the Shh signaling intensity to promote CSF production.

developmental biology↗