Biology subjects

Le, S.

Publications and source records attributed to Le, S..

At least 19 recordsLinked to original sources

bioRxiv2026

High-resolution photocatalytic crosslinking of protein neighborhoods using contact-dependent antibody mapping (contactMAP)

Photo-proximity labeling proteomics (PLP) has emerged as a powerful method for rapid and temporal mapping of transient and fragile protein-protein interactions, especially those in membranes. Numerous catalysts can trigger highly reactive diffusive biotinylated probes to label protein neighborhoods at various length scales. Photocatalysts can also trigger protein crosslinking, predominantly between neighboring tyrosines or between histidine and lysine residues. We have exploited this sidechain directed crosslinking for photo-PLP, in a method we call contactMAP. Using biotinylated antibody binders to photocrosslink Her2 or EGFR neighborhoods, contactMAP enriched high resolution maps of these cancer-associated protein neighborhoods in a manner that rivals or outperforms established photo-PLP methods. ContactMAP is an extraordinarily simple and democratic photo-PLP labeling method with broad applications for probing biomolecular interactions in complex mixtures.

Cancer Biology↗

bioRxiv2026

Pseudomonas aeruginosa CRISPR-Cas primes a minimal proline-codon toxin to abort anti-CRISPR phages

Pseudomonas aeruginosa typically carries type I-F CRISPR-Cas systems, which are targeted by diverse (pro)phage-encoded anti-CRISPR (Acr) proteins. Here, we show that [~]26% of P. aeruginosa Cas effectors are reprogrammed by a regulatory RNA guide (CreA) to transcriptionally silence a conserved small RNA toxin (CreT) that arrests cell growth upon Cas inactivation. This toxin unprecedentedly employs two consecutive proline codons, thus termed "proline-codon toxin". Combined phylogenetic and genetic analyses unraveled that CreTA has forced P. aeruginosa to reject prophages whose Acrs disrupt Cas-DNA binding, which is essential for CreT repression. We further show that Acr-armed lytic phages designed to overcome CRISPR adaptive immunity can be instead aborted by CreTA-mediated defense in vitro and in a mouse model. Our findings unravel intra-genomic arms races between CreTAs and anti-CRISPR phages, and underscore the necessity of rational engineering of strain-specific therapeutic phages to penetrate the layered CRISPR and TA barriers of multidrug-resistant P. aeruginosa.

microbiology↗

bioRxiv2026

Distinct senescent β-cell senotypes differentially drive islet aging and dysfunction

Biological aging greatly impacts the bodys ability to handle glucose, and represents a major risk factor the development and progression of type 2 diabetes (T2D). Nonetheless, despite advances in cellular senescence research and the development of new senolytic therapies, the heterogeneity of cellular senescence in the human endocrine pancreas, as well as its roles in normal aging, remains to be elucidated at the single-cell level. Here, we performed single-cell-resolved spatial proteomics and transcriptomics on intact pancreas from 26 donors (ages 20-80) and multiplexed single-cell RNA sequencing and functional assays on dispersed islets from 14 donors (ages 34-69). We identify two discrete SnC subpopulations distinguished by relative expression of CDKN1A and CDKN2A. CDKN1A senescent cells (SnCs) exhibit loss of {beta}-cell identity, impaired insulin secretion, and a proinflammatory SASP associated with increased islet immune infiltration. In contrast, CDKN2A SnCs retain transcriptional identity and functional competence, with lower inflammatory signaling. Together, these findings identify heterogeneous and functionally divergent senotypes in the human pancreas, distinguishing an adaptive (CDKN2A) from a maladaptive (CDKN1A) senescence program, thus providing a mechanism-guided framework for senescence-targeted therapies in T2D.

cell biology↗

bioRxiv2026

Optimization of a liver Trm cell-inducing mRNA vaccine by reduction of type I interferon response

CD8+ tissue-resident memory T cells provide rapid frontline protection at pathogen invasion sites, making them attractive targets for vaccine-mediated immunity. We previously developed an NKT cell-adjuvanted mRNA lipoplex vaccine capable of inducing liver Trm cells and sterile protection against malaria in mice. Here, we show that type I interferon (IFN-I) signalling through dendritic cells -- not T cells -- is a key brake on liver Trm induction by this vaccine. Optimising mRNA manufacturing to reduce immunostimulatory contaminants substantially dampened IFN-I production, boosted antigen expression in the lymphoid tissues, and drove significantly greater Trm accumulation. These enhanced responses translated into superior protection against parasite challenge. Our findings identify DC-intrinsic IFN-I signalling as a tractable target, and mRNA manufacturing quality as a critical and underappreciated lever, for maximising Trm-based vaccine efficacy.

immunology↗

bioRxiv2026

Dormant Bacteria's Fatal Attraction to RNA Bacteriophages

Phages are the most abundant biological entities on Earth, yet RNA phages are strikingly scarce compared to their DNA counterparts--a long-standing mystery in phage biology. Here, we use dsRNA phage phiYY as a model to demonstrate that while RNA phages efficiently infect growing bacteria, they are gradually eliminated inside dormant bacteria through weak and time-dependent ROS-mediated damage. The RNA phages decline over days rather than through immediate clearance inside dormant bacteria. Accordingly, scavenging ROS with mannitol or overexpressing ROS degradation enzymes AhpB/TrxB2 rescues RNA phages from elimination. Crucially, because the underlying ROS-mediated RNA damage is minimal, RNA phage survival hinges on genomic redundancy. In single-phage infections, the lone RNA genome is highly vulnerable to cumulative damage and is eventually inactivated. In contrast, during co-infection by multiple RNA phages, the presence of multiple genome copies provides functional redundancy, thereby allowing a fraction of RNA phages to survive inside dormant bacteria. Given that most environmental bacteria are dormant and subject to heterogeneous phage infection, this copy number-dependent vulnerability offers a possible explanation for the scarcity, but not perish, of RNA phages in nature.

microbiology↗

bioRxiv2026

Innate immune sensing of cell traversal by Plasmodium sporozoites drives protective T cell responses

Attenuated Plasmodium sporozoites elicit robust adaptive immune responses that protect against parasite challenge, yet how they engage the innate immune system remains poorly understood. To obtain sterile preparation of sporozoites we used flow cytometry to isolate pure GFP+ P. berghei and P. falciparum sporozoites, which were sorted onto murine bone marrow-derived macrophages (BMDMs) or human PBMCs to assess innate immune activation. Sporozoites induced a distinctive activation pattern resembling cell wounding responses, consistent with their ability to traverse host cells en route to the liver. Traversal-deficient sporozoites failed to activate BMDMs and showed impaired priming of protective CD8+ T cell immunity. This wounding response was independent of inflammasome or Toll-like receptor signaling but required {gamma}{delta} T cells, which are known to support CD8+ T cell responses against Plasmodium. These findings reveal a previously unappreciated innate sensing mechanism triggered by cell traversal that underpins the potent immunogenicity of Plasmodium sporozoites.

immunology↗

bioRxiv2026

An Aggregation Prone Region (APR) in talin controls talin self-interactions to regulate integrin adhesion complex dynamics

The tight regulation of the integrin family of extracellular matrix (ECM) receptors is essential for the coordination of most cellular processes. Talin regulates integrin function by binding to the cytoplasmic tail of the integrin beta subunit. Two integrin-binding sites (IBS) in talin have been reported, one in the talin head domain (IBS1), and a second in the C-terminal rod region of talin (IBS2), mapped to the R11-R12 domains. Whilst the structural details of integrin binding to IBS1 are well understood, IBS2s mode of binding integrin is less clear, and the biochemical details of this site have been elusive. Here we report that talin R11 contains a cryptic high affinity talin-binding site. We show that mechanical unfolding of R11 exposes an Aggregation Prone Region (APR) in Helix50 that oligomerizes with other talin R11s. Our data support a model whereby the process of mechanically unfolding one R11, exposes and maintains the APR in a high affinity conformation that drives unfolding of other R11 domains and oligomerisation. Atomic Force Microscopy confirms that the APR region alone forms micron long amyloid-like fibres, via a beta-sheet amyloid arrangement which can be abolished using a canonical "gatekeeper" point mutation, V2078K, that prevents APR interactions. Introducing this gatekeeper mutant in talin eliminates recruitment of talin IBS2 to integrin adhesion sites. The APR region in talin overlaps with a vinculin-binding site in Helix50, and we show a novel role for vinculin as a mechano-chaperone for talin, able to resolve the talin large species by binding to the VBS in Helix50. In light of this new information, we propose that the ability of R11-R12 to target to integrin adhesion complexes and enhance integrin activation can be better explained by a model where functional aggregation of talin serves to recruit and activate more talin molecules at the adhesion site leading to enhanced integrin activation. Whilst further work is required to fully exclude integrin binding to R11 we suggest that the name IBS2 might be a misnomer and propose the name Aggregation Prone Region 1 (APR1) for this talin oligomerisation motif.

cell biology↗

bioRxiv2025

LAMP2A-dependent chaperone-mediated autophagy enhances oxidative stress resistance in gastric cancer cells through selective degradation of accumulated oxidized DJ-1

Reactive oxygen species (ROS) function as potent activators of chaperone-mediated autophagy (CMA). While previous investigations have demonstrated that CMA facilitates cancer cell survival through selective degradation of oxidatively damaged proteins, the precise molecular mechanisms remain poorly defined. Hence, it is important to investigate the correlation between CMA and the antioxidative stress within tumor cells and elucidate its specific mechanism. LAMP2A expression profiles in gastric carcinoma cell lines and clinical specimens were quantified via qPCR, WB and immunohistochemical analysis. An in vitro oxidative stress model was established through 24-hour hydrogen peroxide (H2O2) exposure. Gastric cancer cell models with knockdown and overexpression of LAMP2A were established. Apoptotic indices and proliferative capacity were assessed through high-throughput flow cytometry and CCK-8 viability assays. The interaction between LAMP2A and DJ-1 was investigated via co-immunoprecipitation and confocal microscopy. This study demonstrates that LAMP2A is upregulated in gastric cancer and further induced by oxidative stress. Down-regulated LAMP2A impaired CMA functionality, sensitizing gastric cancer cells to oxidative cytotoxicity and significantly augmenting apoptosis rates. Elevated DJ-1 levels in gastric cancer cells are further amplified by oxidative stress, with severe stress enhancing LAMP2A-DJ-1 colocalization. Mechanistically, CMA inhibition precipitated accumulation of hyperoxidized DJ-1 isoforms, concomitant with pro-apoptotic BAX upregulation and anti-apoptotic BCL-2 downregulation. We found hyperoxidized DJ-1 as a novel CMA substrate. The LAMP2A-DJ-1 regulatory axis represents a critical adaptive mechanism whereby CMA activation maintains redox homeostasis in gastric malignancies. Specifically, CMA-mediated clearance of oxidized DJ-1 prevents pro-apoptotic protein cascade activation, thereby conferring oxidative stress resistance and promoting tumor cell survival.

cancer biology↗

bioRxiv2025

Mechanical Responses of the Giant Nesprin-2

The nesprin protein family serves as a critical physical bridge between the cytoskeleton-a fundamental structural scaffold and mechanotransduction hub of the cell, and the nucleus-an intriguing and emerging mechano-responsive element. Due to the external mechanical cues and the nucleo-cytoskeletal dynamics, the nesprins are physiologically under forces. However, the dynamics of nesprins within physiological forces and loading rates remain largely unexplored. In this study, we employ magnetic tweezers based single-molecule manipulation alongside molecular dynamic simulations and AlphaFold structural predictions to comprehensively investigate the dynamics of force-bearing spectrin repeat (SR) domains of the giant nesprin-2 protein. Through direct quantification, we unveil that the numerous SRs undergo mechanical unfolding and refolding dynamics with distinct transition rates within several pN scale. Furthermore, we show that the giant nesprin-2 could act as an effective molecular absorber adeptly maintaining forces on the nucleoskeleton and cytoskeleton linkage within a few pN across displacement spans exceeding {micro}m. Notably, our findings imply that subtle pN-level mechanical forces intricately modulate nesprin-protein interactions via the dynamics of domain folding and unfolding. Collectively, our study offers a comprehensive understanding of the mechanical characteristics of giant nesprin-2, shedding light on its pivotal role in nucleoskeleton-cytoskeleton mechanotransduction.

biophysics↗

bioRxiv2025

A human and mouse subpopulation of senescent β-cells induces pathologic dysfunction through targetable paracrine signaling

Cellular senescence is a stress response mechanism marked by irreversible growth arrest, upregulation of antiapoptotic pathways, loss of cellular function, and remodelling of the cellular secretory profile. In both humans and mice, pancreatic {beta}-cells undergo senescence with age and insulin resistance. Targeted removal of senescent cells in mouse models of diabetes improves glucose homeostasis, demonstrating the role {beta}-cell senescence in diabetes progression. In contrast, {beta}-cell senescence also promotes immune surveillance, promoting {beta}-cell survival and function. Thus, a better understanding of senescent cells phenotypic and functional heterogeneity is needed to develop effective therapeutic strategies. Herein, we show that subpopulations of senescent {beta}-cells in mice and humans, which were identified through the expression of Cdkn1a (encoding p21Cip1) and Cdkn2a (encoding p16Ink4a) by single-cell RNA sequencing (scRNA-seq), flow cytometry, spatial transcriptomics, and spatial proteomics, exhibit distinct transcriptional and functional identities. The predominant senescent {beta}-cell subpopulation expressed Cdkn1a and was characterized by a lack of glucose responsiveness, high basal insulin secretion, and transcription of canonical SASP factors. The SASP of Cdkn1a-expressing {beta}-cells had non-cell autonomous effects on neighbouring cells. A subset of four SASP factors from Cdkn1a+ cells was sufficient to induce secondary senescence and {beta}-cell dysfunction in vitro. JAK inhibitors (JAK1/2 and JAK1/3) counteracted secondary senescence induction and restored {beta}-cell function in high-fat diet-fed mice and human islets from donors with or without type 2 diabetes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/648438v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@9b8addorg.highwire.dtl.DTLVardef@1b9c4eborg.highwire.dtl.DTLVardef@12f2964org.highwire.dtl.DTLVardef@1468297_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

Bacteriophage protein Dap2 inhibits bacterial type III secretion system and synergizes with Dap1 to evade anti-phage immunity

The evolutionary arms race between bacterial immunity and phages has driven the emergence of sophisticated anti-defense systems (ADSs). However, certain ADSs exhibit incomplete suppression of their cognate defense systems, suggesting functional cooperation between multiple ADSs targeting the same bacterial safeguard. In this study, we characterize Dap2, a protein encoded by a Pseudomonas aeruginosa phage PaoP5, which directly binds to the Lon protease to prevent the degradation of the phage-encoded HNH endonuclease. Deletion of dap2 in PaoP5 exhibits significantly impaired genome packaging due to insufficient levels of HNH. Strikingly, Dap2 synergizes with its genomically adjacent partner Dap1, a previously identified HNH-binding protein providing partial Lon resistance, to achieve complete protection of HNH. Beyond anti-defense activity, Dap2 disrupts host virulence by sequestering the type III secretion system (T3SS) transcriptional activator ExsA, suppressing bacterial pathogenicity while redirecting metabolic resources toward phage progeny production. This study unveils a dual functional ADS that simultaneously modulates bacterial virulence and anti-phage immunity, both aimed at ensuring phage survival and maximizing progeny production. Furthermore, it elucidates a novel mechanism whereby phages employ synergistic ADS pairs (Dap1/Dap2) to achieve complete neutralization of a bacterial defense system, when individual components provide only partial protection. These findings significantly enhance our understanding of the intricate evolutionary arms race between phages and their bacterial hosts.

microbiology↗

bioRxiv2025

Chemical inhibition of a bacterial immune system

The rise of antibiotic resistance motivates a revived interest in phage therapy. However, bacteria possess dozens of anti-bacteriophage immune systems that confer resistance to therapeutic phages. Chemical inhibitors of these anti-phage immune systems could be employed as adjuvants to overcome resistance in phage-based therapies. Here, we report that anti-phage systems can be selectively inhibited by small molecules, thereby sensitizing phage-resistant bacteria to phages. We discovered a class of chemical inhibitors that inhibit the type II Thoeris anti-phage immune system. These inhibitors block the biosynthesis of a histidine-ADPR intracellular alarm signal by ThsB and prevent ThsA from arresting phage replication. These inhibitors promiscuously inhibit type II Thoeris systems from diverse bacteria--including antibiotic-resistant pathogens. Chemical inhibition of the Thoeris defense improved the efficacy of a model phage therapy against a phage-resistant strain of P. aeruginosa in a mouse infection, suggesting a therapeutic potential. Furthermore, these inhibitors may be employed as chemical tools to dissect the importance of the Thoeris system for phage defense in natural microbial communities.

biochemistry↗

bioRxiv2025

Infectome Analysis of Small Mammals in Southern China Reveals Ecological Associations and Emerging Threats from Diverse Pathogens

Small mammals harbor a diverse array of zoonotic pathogens. To date, however, metagenomic surveys of these species have primarily focused on viral diversity, with limited attention paid to bacteria and eukaryotic pathogens. Additionally, the ecological determinants of pathogen diversity within these mammals have not been systematically examined. Herein, we employed a metatranscriptomics approach to survey the pathogen infectome of 2,408 individual samples, representing lung, liver, and gut tissues from 858 animals collected throughout Guangdong province, China, with a study design that accounted for host species, tissue, season, and geographic location. We identified 76 pathogen species, including 29 RNA viruses, 12 DNA viruses, 5 bacteria, and 30 eukaryotic pathogens, 33 of which were newly discovered. Tissue distribution analysis revealed distinct organotropisms, suggesting varied transmission routes, while host distribution analysis showed that each animal carried an average of one pathogen, with 10 pathogens widely distributed among mammalian orders. Our characterization of the geographic and seasonal patterns revealed that pathogen richness was primarily influenced by region, host species, and season, while pathogen composition was largely shaped by host genetic distance. Collectively, these data provide the first comprehensive insight into the dynamics of the pathogen infectome in these key mammalian disease reservoirs, highlighting the major factors driving pathogen diversity and transmission.

microbiology↗

bioRxiv2025

Tissue-specific effects of dietary protein on cellular senescence are mediated by branched-chain amino acids

Dietary protein is a key regulator of healthy aging in both mice and humans. In mice, reducing dietary levels of the branched-chain amino acids (BCAAs) recapitulates many of the benefits of a low protein diet; BCAA-restricted diets extend lifespan, reduce frailty, and improve metabolic health, while BCAA supplementation shortens lifespan, promotes obesity, and impairs glycemic control. Recently, high protein diets have been shown to promote cellular senescence, a hallmark of aging implicated in many age-related diseases, in the liver of mice. Here, we test the hypothesis that the effects of high protein diets on metabolic health and on cell senescence are mediated by BCAAs. We find that reducing dietary levels of BCAAs protects male and female mice from the negative metabolic consequences of both normal and high protein diets. Further, we identify tissue-specific effects of BCAAs on cellular senescence, with restriction of all three BCAAs - but not individual BCAAs - protecting from hepatic cellular senescence while potentiating cell senescence in white adipose tissue. We find that the effects of BCAAs on hepatic cellular senescence are cell-autonomous, with lower levels of BCAAs protecting cultured cells from antimycin-A induced senescence. Our results demonstrate a direct effect of a specific dietary component on a hallmark of aging and suggest that cellular senescence may be highly susceptible to dietary interventions.

physiology↗

bioRxiv2024

Vgamma1+ gammadelta T cell-derived IL-4 initiates CD8 T cell immunity

Dendritic cells (DC) are pivotal for initiating adaptive immunity, a process triggered by the activation of DC via pathogen products or damage. Here, we describe an additional layer to this process, essential when pathogen-derived signals alone cannot directly achieve full DC activation. Immunisation with sporozoites from Plasmodium leads to CD8 T cell priming in a complex response that is initiated by a collaboration between conventional type 1 DC (cDC1) and {gamma}{delta} T cells. We unveil a pivotal initiating role for V{gamma}1+ {gamma}{delta} T cells, as they directly supply IL-4 to DC and CD8 T cells. IL-4 synergises with a CD4 T cell-derived CD40L signal to induce IL-12 production by cDC1. Both IL-12 and IL-4 then directly signal CD8 T cells, with synergy between these cytokines driving enhanced IL-12 receptor expression and expansion of responding CD8 T cells. This study reveals a key role for V{gamma}l+ {gamma}{delta} T cells in initiating CD8 T cell immunity to Plasmodium. More broadly, it shows that responses to some pathogens require help from innate-like T cells to pass an initiation threshold and further amplify the response in a process underscored by IL-4 production.

immunology↗

bioRxiv2023

In-situ Single-Molecule Investigations of the Impacts of Biochemical Perturbations on Conformational Intermediates of Monomeric α-Synuclein

-Synuclein aggregation is a common trait in synucleinopathies, including Parkinsons disease. Being an unstructured protein, -synuclein exists in several distinct conformational intermediates, contributing to both its function and pathogenesis. However, the regulation of these monomer conformations by biochemical factors and potential drugs has remained elusive. In this study, we devised an in-situ single-molecule manipulation approach to pinpoint kinetically stable conformational intermediates of monomeric -synuclein and explore the effects of various biochemical factors and drugs. We uncovered a partially folded conformation located in the non-amyloid-{beta} component (NAC) region of monomeric -synuclein, which is regulated by preNAC region. This conformational intermediate is sensitive to biochemical perturbations and small-molecule drugs that influencing -synucleins aggregation tendency. Our findings reveal that this partially folded intermediate may play a role in -synuclein aggregation, offering fresh perspectives for potential treatments aimed at the initial stage of higher-order -synuclein aggregation. The single-molecule approach developed here can be broadly applied to the study of disease-related intrinsically disordered proteins.

biophysics↗

bioRxiv2023

Mutant Huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure

Expansion of the glutamine tract (poly-Q) in the protein Huntingtin (HTT) causes the neurodegenerative disorder Huntingtons disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. 70Q introduction caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics and elevated resting energy expenditure. Elimination of the poly-Q tract of HTT normalized CHCHD2 expression and mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early intervention target for HD.

neuroscience↗

bioRxiv2022

Gap-free nuclear and mitochondrial genomes of Fusarium verticillioides strain HN2

Fusarium ear rot (FER) and Fusarium stalk rot (FSR) caused by the filamentous fungus Fusarium verticillioides have become increasingly serious around the world. Additionally, fumonisins produced by F. verticillioides threaten food and feed security. By adding the contribution of genomic resources to better understand the pathosystem including the mechanisms of F. verticillioides-maize interactions, and further improving the quality of the F. verticillioides genome, the gap-free nuclear genome and mitochondrial genome of F. verticillioides strain HN2 were sequenced and assembled. Using Oxford Nanopore long reads and next-generation sequencing short reads, the final 42.81-Mb genome was assembled into 12 contigs (N50 = 4.16-Mb). A total of 13,466 protein-coding genes were annotated, including 1,076 secreted proteins that contain 342 candidate effectors. In addition, we assembled the complete 53,764 bp mitochondrial genome. F. verticillioides strain 7600 genome assemblies are fragmented and high-quality reference genomes were needed. The genomes presented here will serve as an important resource for F. verticillioides research.

genomics↗