Biology subjects

Lee, D.

Publications and source records attributed to Lee, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Sulfidogenic Bacteria and the Risk of Colorectal Cancer

BackgroundSulfur-rich dietary patterns have been associated with colorectal cancer risk. Certain gut bacteria metabolize dietary sulfur compounds into hydrogen sulfide, which can damage the intestinal epithelium and promote carcinogenesis. We evaluated whether sulfur-metabolizing enzymes are enriched in colorectal cancer and whether associations differ by age. ResultsAcross eleven metagenomic cohorts, several sulfur-metabolizing enzymes were more prevalent in individuals with colorectal cancer than in healthy controls. Enzymes involved in sulfur reduction and detoxification, including sulfolactaldehyde reductase, peptide-methionine sulfoxide reductase, dimethylsulfoxide reductase, glutathione transferase, hydroxyacylglutathione hydrolase, and arylsulfatase, were consistently enriched in colorectal cancer. In contrast, enzymes involved in cysteine and methionine synthesis were more common in healthy controls. Age-stratified analyses showed minimal effect modification. Fusobacterium nucleatum, Intestinimonas butyriciproducens, and Bilophila wadsworthia carried more sulfur-metabolizing genes in colorectal cancer samples. Early-onset colorectal cancer samples were enriched for these genes in Citrobacter, Klebsiella, and Raoultella, whereas healthy controls showed greater representation of detoxification-associated genes in Bifidobacterium species. ConclusionsIndividuals with colorectal cancer harbor a greater abundance of sulfur-metabolizing enzymes, supporting a potential role for microbial conversion of dietary sulfur into hydrogen sulfide in colorectal carcinogenesis. Because these genes are distributed across many taxa, microbial function may better explain disease risk than individual species. Associations were consistent across age groups, suggesting that prolonged sulfur-rich dietary exposure may foster a microbial environment capable of generating carcinogenic metabolites. Microbial sulfur metabolism may therefore represent a modifiable pathway for prevention and further mechanistic study.

microbiology↗

bioRxiv2026

Midgut damage triggers thromboxane A2-dependent hemocyte recruitment in Culex pipiens molestus

Mosquitoes transmit diverse pathogens through repeated blood feeding, a process that subjects the midgut to significant mechanical stress and cellular damage. While hemocyte association with the midgut is known to occur following injury, the mechanisms promoting their recruitment remain poorly defined. Given the conserved roles of eicosanoid signaling in injury responses, we hypothesized that thromboxane A2 (TXA2) mediates hemocyte recruitment to the damaged midgut. Here, we demonstrate that chemically induced midgut damage significantly increases both the number of hemocytes attached to the midgut and those in circulation. Pharmacological inhibition of cyclooxygenase suppresses hemocyte recruitment whereas supplementation with a stable TXA2 analog restores the response, indicating that TXA2 signaling is required for this process. To identify candidate enzymes involved in TXA2 biosynthesis, we performed in silico docking analyses and identified two cytochrome P450 (CYP) candidates. Among these, CYP6D3 was shown to be strongly upregulated in hemocyte populations following midgut damage. RNA interference-mediated knockdown of CYP6D3 significantly reduced both hemocyte recruitment to the midgut and systemic TXB2 levels, supporting its role in the eicosanoid-mediated immune response. Together, our findings demonstrate that TXA2 signaling drives hemocyte recruitment to the damaged mosquito midgut and suggest a conserved lipid-mediated mechanism underlying insect tissue-associated immune responses that may influence vector competence.

biochemistry↗

bioRxiv2026

A Modular T7 RNAP Expression Architecture for Orthogonal Multigene Expression in Yeast

Orthogonal gene expression systems based on bacteriophage-derived T7 RNA polymerase (T7 RNAP) offer a promising strategy for decoupling engineered transcription from host regulatory networks. Although recent advances have enabled productive T7 RNAP-mediated expression in Saccharomyces cerevisiae, strategies for independently controlling multiple genes within a shared T7 transcriptional framework remain limited. Here, we developed a modular pYTK-compatible T7 RNAP expression architecture that separates overall transcriptional capacity from gene-specific control of protein output. We established interchangeable T7 promoter, terminator, polyadenylation signal, and 5 untranslated region (UTR) parts for combinatorial assembly. Analysis of expression cassette dosage showed that transcript abundance and expression output scale with cassette copy number, with high-copy constructs producing transcript levels comparable to those driven by the GAL1 promoter. To enable gene-specific tuning, we constructed a library of fourteen 18 nt UTR elements that generated a [~]38-fold range of protein output from a common T7 promoter. Application of this library to a three-gene {beta}-carotene biosynthesis pathway enabled [~]20-fold variation in product titer through UTR assignment alone, without altering promoter identity or gene copy number. Together, these results establish a modular T7 RNAP expression framework in which cassette dosage defines overall transcriptional capacity while interchangeable UTR elements enable gene-specific tuning of expression output, providing a practical strategy for multigene expression control in yeast.

Synthetic Biology↗

bioRxiv2026

Beta-Adrenergic Stimulation and MYH7 G256E Mutant Gene Dosage Drive Hypertrophic Cardiomyopathy Phenotype Penetrance

AimsHypertrophic cardiomyopathy (HCM) is the most prevalent genetic heart disorder, characterized by significant phenotypic variability even among individuals with identical MYH7 mutations. This study aims to elucidate factors contributing to this variability and identify drivers of phenotype penetrance. We compared the baseline phenotypes of a highly penetrant MYH7 H251N mutation and the variably penetrant MYH7 G256E mutation and investigated the impact of adding beta-adrenergic stimulation and homozygosity on disease phenotype penetrance using cardiomyocytes from an isogenic line of human induced pluripotent stem cells (hiPSC-CMs). Methods and ResultsIsogenic hiPSCs with MYH7 H251N and MYH7 G256E mutations were generated using CRISPR/Cas9 technology and differentiated into cardiomyocytes (CMs). Single-cell RNA sequencing (scRNAseq) and functional analysis of contractile function revealed consistent HCM phenotype presentation in H251N CMs, whereas G256E CMs exhibited a subtle and more variable phenotype. Beta-adrenergic stimulation induced a distinct metabolic stress response in G256E CMs, characterized by impaired mitochondrial ATP upregulation. Increasing mutant gene dosage from hetero- to homozygosity led to consistent increase in hypertrophic and structural gene expression changes in G256E CMs at RNA and protein levels. These changes were distinct from the changes observed with stress response. Importantly, homozygous G256E CMs exhibited a hypercontractile functional and disorganized structural phenotype. Across multiple experimental conditions, we identified consistent increase in cardiomyocyte specific transcriptomic markers such as NPPB, APOE, PDLIM3 and ANKRD1. ConclusionsOur study highlights the use of a variably penetrant MYH7 mutation to investigate factors that influence HCM phenotype penetrance. Specifically, we found that mutant gene dosage and beta-adrenergic stimulation induce distinct HCM disease phenotypes, providing novel insights into mechanisms that may contribute to variable disease expression in HCM. Translational PerspectiveHCM is characterized by significant phenotypic variability, complicating both diagnosis and clinical management. This study explores the factors driving HCM phenotype penetrance using isogenic hiPSC-CMs with MYH7 mutations. We demonstrate that beta-adrenergic stimulation and increased mutant gene dosage significantly impact HCM disease penetrance. Beta-adrenergic stimulation triggers metabolic stress responses, while increased gene dosage leads to a hypercontractile and structurally disorganized phenotype. These findings provide insight into how specific modifiers can shape disease-associated phenotypes in HCM model systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/729411v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@16a72d7org.highwire.dtl.DTLVardef@1999ca6org.highwire.dtl.DTLVardef@1fb8752org.highwire.dtl.DTLVardef@7148b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

The genomic basis of evolutionary stasis in the 500-million-year-old red seaweed genus Ahnfeltia

The red seaweed genus Ahnfeltia is an ancient lineage that has persisted for over 500 million years with remarkably limited diversification despite a global distribution in cold-temperate intertidal habitats. Compared to the highly diverse sister lineage, Rhodymeniophycidae, Ahnfeltia provides a unique system for investigating long-term evolutionary persistence in marine macroalgae. Here, we generated chromosome-scale genomes from five populations across three species and combined population genomics with paleogeographic niche modelling. Our results reveal remarkable genomic conservation, strong geographic isolation with limited gene flow, high homozygosity, and evidence of purifying selection. Niche projections indicate long-term stability and spatial connectivity of suitable cold-temperate habitats. These findings suggest that Ahnfeltias persistence and limited diversification are linked to genomic constraints and stable ecological niches over geological timescales. This study provides new insights into the genomic basis of evolutionary stasis in ancient marine lineages and highlights potential vulnerability to ongoing climate change affecting cold-water coastal ecosystems.

evolutionary biology↗

bioRxiv2026

A flow cytometry-based assay to quantify the binding of transmembrane ligands to their cognate receptors using fluorescent virus-like particles

The binding of transmembrane (TM) ligands to their cognate TM receptors on neighboring cells governs intercellular adhesion and direct cell-cell communication. However, these interactions are difficult to study in vitro because they depend on membrane presentation, ligand orientation, receptor clustering, and avidity, features often not captured by soluble recombinant ligands or cell-free assays. Here, we describe a flow cytometry-based assay using fluorescent, lentiviral-derived virus-like particles (VLPs) displaying TM ligands to quantify binding to their receptors on target cells. Fluorescent VLPs are generated in-house by plasmid transfection in HEK293T cells and enable direct fluorescent detection without fluorochrome-conjugated secondary antibodies. The system is modular and readily accommodates engineered ligand constructs, including patient-derived variants. We applied this platform to generate ICAM-1-displaying fluorescent VLPs and to study human LFA-1 function in patient-derived leukocytes. This protocol provides a detailed workflow for VLP production and in vitro binding assays, offering a simple, quantitative, and cost-effective approach for studying TM ligand-receptor interactions in a membrane context. The system is well suited for mechanistic studies, functional assessment of patient-derived variants, and direct binding assays using patient-derived cells. Integrating the assay into multicolor flow cytometry panels enables simultaneous immunophenotyping and quantification of up to four ligand-receptor interactions at single-cell resolution. Key featuresO_LIQuantifies TM ligand-receptor binding in a membrane context using fluorescent VLPs and flow cytometry. C_LIO_LIFully in-house, modular system based on plasmid transfection in HEK293T cells, without reliance on recombinant ligands or fluorochrome-conjugated secondary antibodies. C_LIO_LISupports testing of engineered ligand variants, including patient-derived alleles, and direct functional studies on patient-derived cells. C_LIO_LICompatible with multicolor flow cytometry panels, enabling simultaneous immunophenotyping and quantification of up to four ligand-receptor interactions at single-cell resolution. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/725198v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@a43069org.highwire.dtl.DTLVardef@166491borg.highwire.dtl.DTLVardef@49c7d4org.highwire.dtl.DTLVardef@1de36a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

A self-limiting orexin-habenula circuit for stress resilience

Resilience requires neural systems that mobilise active coping during stress while limiting its persistence to preserve homeostasis under sustained challenge1-4. Here we identify a self-limiting orexin-habenula circuit in which lateral hypothalamic orexin neurons engage aromatic L-amino acid decarboxylase-expressing D-neurons (encoded by Ddc) in the lateral habenula via orexin receptor type 2 (OX2R)5-7. Activation of this pathway increased nucleus accumbens dopamine and promoted active coping and positive valence. Optotagging revealed rapid stress-evoked recruitment followed by post-stress suppression. In lateral habenula neurons, orexin peptides exerted dissociable effects: orexin-A engaged an OX2R-dependent inhibitory programme superimposed on a parallel inward current, whereas orexin-B did not reproduce this inhibitory profile and instead exerted a distinct membrane effect. Chronic stress disrupted this buffering system through coordinated inflammatory activation, promoter methylation and erosion of D-neuron identity and orexin responsiveness. Restoring orexin-A reversed behavioural and molecular deficits through OX2R-dependent suppression of nuclear factor kappa B signalling, preservation of Tet2 expression, and demethylation-linked maintenance of the Ddc programme. Together, these findings define a self-limiting orexin-habenula resilience circuit that enables adaptive coping while constraining stress-induced vulnerability.

neuroscience↗

bioRxiv2026

Metabolic signals regulate resuscitation speed of antibiotic persister bacteria during infection

All living organisms adjust their metabolism in response to environmental changes. Under unfavorable conditions, organisms enter a state of dormancy by halting metabolism, enabling survival. Dormant bacteria become highly tolerant to antibiotics-a phenomenon called persistence. Here, we demonstrate that selective metabolic reprogramming controls the resuscitation speed of persister after antibiotic exposure. Using multi-omics and in silico modeling, we found that dormant bacteria reprogram metabolic pathways to modulate persister awakening. Accumulation of L-serine and reduction of arginine drive rapid resuscitation. L-serine promotes cysteine biosynthesis and motility while reducing energy metabolism to facilitate rapid resuscitation. In contrast, arginine slows regrowth from dormancy by enhancing ethanol-aldehyde and energy metabolism. L-serine and arginine can, respectively, promote or inhibit the regrowth of antibiotic persister cells in macrophages and mouse models, and regulate the awakening speed of Salmonella, E. coli, and methicillin-resistant Staphylococcus aureus (MRSA). These findings suggest new strategies to target chronic bacterial infections. TeaserL-serine speeds and arginine slows the awakening of antibiotic persisters, revealing targets for chronic infection.

microbiology↗

bioRxiv2026

Designed Minibinders Rewire Receptor Signaling to Enable Functional Human Myogenic Reprogramming

Sarcopenia, loss of muscle mass is a considerable health burden that demands immediate societal attention. Direct myogenic somatic cell reprogramming, a potential muscle regeneration method is constrained by an inability to control the signaling logic that governs cell fate. Here, we show that this barrier can be overcome using AI-designed receptor modulators. Screening de novo minibinders, we identify a synthetic protein cocktail, C6-DPC, that drives efficient human fibroblast-to-muscle transdifferentiation with robust structural and metabolic maturation. C6-DPC reprograms extracellular signaling by activating pro-myogenic FGFR1/2c pathways while suppressing anti-myogenic inputs through ALK1 and TGFBR2; targeted depletion of ALK1 is sufficient to lower the reprogramming barrier. Inflammatory signaling via gp130 emerges as a dominant checkpoint, and its inhibition further enhances conversion. Engineered tissues generate high twitch and tetanic forces in both wild-type and dystrophin-deficient human cells. These findings demonstrate that programmable synthetic ligands can rewrite receptor-level signaling to direct cell fate and enable functional tissue regeneration.

biochemistry↗

bioRxiv2026

Cohesin and NuRD Antagonistically Drive Alternative Neuronal Fates via PLZF Transcription Factors

Diverse genetic and epigenetic factors cooperate to specify cellular fates during development. Establishing these fates is especially critical in the nervous system, which comprises diverse neuronal cell types. How genomic architecture interfaces with epigenetic regulators to drive transcriptional programs underlying neuronal fates remains poorly understood. Here we show that cohesin, a protein complex that shapes genomic architecture, promotes GABAergic fate specification in a subset of neurons in the nematode Caenorhabditis elegans. This process is facilitated by EOR-1, a homolog of the human promyelocytic leukemia zinc finger (PLZF) transcription factor. The nucleosome remodeling and deacetylase (NuRD) complex and TRA-4, another PLZF homolog, promote tyraminergic fate in the normally GABAergic neurons when cohesin or EOR-1 function is lost, revealing an antagonistic mechanism determining alternative neuronal fates. These findings highlight a critical interplay among genome architecture, epigenetic remodeling and transcriptional regulation in neuronal fate specification and, given the evolutionary conservation of these factors, suggest a mechanism underlying neural development across species. TeaserGenomic architecture, epigenetic and genetic factors interact to determine specific neuronal fates during development.

developmental biology↗

bioRxiv2026

Multiomic screening platform uncovers the impact of histone mutations on chromatin and cell fate

Somatic missense mutations in histone genes, often referred to as oncohistones, have been identified in diverse types of human cancers. The functional and mechanistic impact of most oncohistones remains unknown. To address this gap, we developed CHANCLA, a modular platform for high-throughput functional screening of oncohistones using multiomic phenotypic readouts. We used CHANCLA to systematically measure the impact of 303 human oncohistones on cellular proliferation, differentiation, histone-specific post-translational modifications, and chromatin accessibility. Integrative multiomic analyses revealed discrete oncohistone molecular classes that promote proliferation, block lineage-specific differentiation, and physically remodel the chromatin landscape by altering specific histone modifications and reducing nucleosome stability. Structural mapping and computational modeling studies uncovered that functionally convergent mutations are clustered at key nucleosome interfaces, particularly H2B-H4, and that chromatin accessibility-promoting mutations are linked to mono-nucleosome destabilization. Leveraging this multiomic resource, we discovered that the H3.3-Q5H mutant histone is a bona fide human oncohistone that accelerates lung adenocarcinoma growth in vivo. Mechanistically, we found that H3.3-Q5H expression leads to suppression of promoter-associated H3K4me3 and expansion of repressive H3K27me3 domains, resulting in increased KRAS signaling and gene expression programs associated with epithelial-to-mesenchymal transition. Together, this work provides a multiomic functional atlas of cancer-associated histone mutations, identifies structural and mechanistic principles governing chromatin reprogramming by oncohistones, and establishes CHANCLA as a modular platform for systematic discovery of mechanisms and vulnerabilities associated with these genetic lesions.

cancer biology↗

bioRxiv2026

Genetic analysis of bone morphometry and ivory vertebrae in threespine stickleback

Previous genetic studies of skeletal variation in threespine stickleback fish (Gasterosteus aculeatus) have focused primarily on striking morphological differences. Here, we examine the largely unexplored genetic architecture of internal bone microstructural variation between marine and freshwater stickleback. {micro}CT X-ray analysis revealed differences in the porosity, bone thickness, and bone volume fraction within armor plates and vertebrae from a marine and freshwater stickleback. Quantitative trait locus mapping in F2 progeny from a marine x freshwater stickleback cross identified a significant locus on chromosome 4 influencing multiple aspects of armor plate internal microstructure. This locus overlaps the well-characterized Eda region previously known to control armor plate number and size. Co-mapping of bone microstructure could be due to pleiotropic effects of Eda on multiple aspects of plate development or to changes in closely linked genes including Itm2a, which also plays a role in bone formation. Most bone microstructure traits in vertebrae showed weak or no genetic signal, consistent with a polygenic architecture. However, we identified a highly significant locus on chromosome 17 that is strongly associated with abnormally thickened "ivory vertebrae" that occurred in 8.4% of F2 offspring. This phenotype resembles Pagets disease in humans, and the major locus region contains Tnfrsf1b, the stickleback ortholog of a human Pagets disease susceptibility gene TNFRSF11A. Together, our findings identify genetic loci underlying natural variation in bone microstructure in wild fish and reveal a candidate gene associated with a disease-like skeletal phenotype, highlighting stickleback as a model for studying both evolutionary and pathological bone biology.

genetics↗

bioRxiv2026

Quantifying Scientific Consensus in Biomedical Hypotheses via LLM-Assisted Literature Screening

Systematic literature reviews are labor-intensive tasks in biomedical research. While Large Language Models (LLMs) using Retrieval-Augmented Generation (RAG) techniques have enhanced information accessibility, the inherent complexity of biological systems--characterized by high context dependency and conflicting data--remains a primary driver of LLM hallucinations. This imposes a structural constraint that limits the precision of evidence synthesis. To address these limitations, we propose an automated framework designed for the exhaustive identification of supporting and contradictory evidence within a target literature set. Rather than relying on a models pre-trained knowledge, our system requires the LLM to review each paper individually to determine its alignment with a specific research hypothesis. By evaluating semantic context, the framework captures subtle contradictions that are often overgeneralized by conventional methods. The frameworks performance was validated using the BioNLI task, where it demonstrated high classification accuracy in distinguishing whether evidence supports or contradicts a given hypothesis. Notably, the implementation of an ensemble approach provided superior stability and slightly higher precision compared to individual models. Furthermore, the framework exhibited robust performance across several well-established biological hypotheses, confirming its practical utility and reliability in real-world research. This approach provides a rigorous basis for biomedical discovery by enabling the precise, systematic analysis of biological literature and the robust collection of evidence.

bioinformatics↗

bioRxiv2026

Global genomic diversity of the selfing nematode Caenorhabditis tropicalis correlates with geography

Self-fertilization reduces genetic diversity compared to outcrossing and hypothetically decreases the ability to adapt to diverse environments. Among Caenorhabditis nematodes, self-fertilization evolved three times independently in Caenorhabditis elegans, Caenorhabditis briggsae, and the more recently discovered Caenorhabditis tropicalis. To survey C. tropicalis genetic relatedness, the influence of geography and niche on species-wide variation, and the signatures of selection, we collected 785 wild strains, sequenced their genomes, and identified 622 distinct genotypes (isotypes). In contrast to C. elegans and C. briggsae, C. tropicalis relatedness shows substantial association with geography and no transcontinental selective sweeps or broadly sampled isotypes. Populations from the Hawaiian Islands or Taiwan harbor more genetic variation than populations from the Caribbean or Americas, suggesting a Pacific species origin similar to other members of the Elegans subclade. Punctuated genomic regions of extreme genetic variation pervade the genome. These hyper-divergent regions (HDRs) comprise less than 6% of the reference genome in any given strain despite harboring 73% of all variant sites and are enriched for genes likely involved in environmental adaptation. HDRs represent a shared genomic feature of self-fertilizing Caenorhabditis nematodes despite their independent evolutionary origins and suggest a mechanism to explain worldwide distributions despite low species-wide levels of genetic variation.

genomics↗

bioRxiv2026

BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

Precise control over protein secretion is essential for programming intercellular communication and coordinating complex physiological responses. However, conventional methods relying on transcriptional regulation or chemical induction often lack the spatiotemporal precision and reversibility required to mimic endogenous signaling dynamics. Here, we present the Blue Light-Assisted Secretion Toolkit (BLAST), a genetically encoded system that orchestrates protein release from the endoplasmic reticulum via light-tunable protein-protein interactions. BLAST comprises two complementary modules utilizing both light-induced iLID/SspB association (a-BLAST) and LOV2/Zdk1 dissociation (d-BLAST). Both modules harness the highly conserved RXR motif to enforce strict ER confinement in the dark state. Most importantly, by utilizing non-destructive steric masking rather than enzymatic cleavage, BLAST achieves unprecedented temporal resolution with strict reversibility. We demonstrate that both systems can be repeatedly toggled ON and OFF, instantaneously arresting cargo release upon light withdrawal to generate highly controlled, pulsatile secretion profiles. Leveraging this dynamic control, we successfully achieved the rapid, robust, and light-triggered secretion of complex therapeutic proteins, including insulin and interleukin-12. By bypassing transcriptional delays and irreversible activation steps, BLAST provides a generalized, plug-and-play platform for the on-demand delivery of therapeutic proteins, significantly expanding the optogenetic toolbox for synthetic biology and cell-based therapies.

synthetic biology↗

bioRxiv2026

GYDE: A collaborative drug discovery platform for AI-powered protein design and engineering

As computational tools and machine learning models for protein sciences continue to advance and proliferate, bench scientists face increasing technical challenges adopting these tools for specific applications such as drug discovery. Here we present GYDE (Guide Your Design and Engineering), an open-source, versatile, and web-based collaboration platform designed to make computational analyses of proteins and antibodies easily accessible to bench scientists. GYDE enables the exploration of sequence-structure-function relationships through a tightly integrated visual interface, offering researchers a comprehensive exploration of protein functional determinants either via real assay data or computational tools. GYDEs intuitive interface facilitates seamless access to cutting-edge AI models for protein and antibody structure prediction, design, and downstream analyses. The flexible and easy addition of new tools and models is facilitated by the use of the Slivka compute API. The platform supports saved sessions that enable researchers to easily share their findings with other users, fostering a more collaborative scientific community. GYDE is freely available for protein scientists in academia and industry to build drug discovery analytics platforms customized to their needs.

bioinformatics↗

bioRxiv2026

Microenvironmental Determinants of Reaction Kinetics in Biomolecular Condensates Probed with Protein Ligation

Cells utilize liquid-liquid phase separation to organize biochemical reactions within biomolecular condensates, which function as membraneless organelles. Although these assemblies are known to enhance reaction rates by concentrating reactants, the mechanisms beyond simple mass-action effects remain poorly understood. Here, we examined how the physicochemical microenvironment within condensates modulates reaction kinetics using spontaneous protein ligation as a model reaction, conducting a systematic analysis across various condensates, ranging from structured scaffolds (PRM-SH3 systems) to intrinsically disordered protein (IDP)-based scaffolds such as LAF, TAF, and FUS. We designed a FRET-based proximity-sensitive client probe to quantify increases in effective local concentration arising from excluded-volume effects. In parallel, we measured internal hydrophilicity and water activity, revealing them as additional key determinants of reaction acceleration. Together, the findings presented here elucidate how phase-separated compartments regulate biochemical reactions through the interplay of physical (effective concentration) and chemical (hydrophilicity and water activity) microenvironments and provide mechanistic insights for engineering condensates with tunable reactivity.

biochemistry↗

bioRxiv2026

Mitochondrial metabolic remodeling drives innate immune activation in Drosophila hemocytes

Innate immune cells rapidly reprogram their metabolism upon activation, yet the metabolic basis of this flexibility in invertebrate systems remains largely unexplored. Here, we investigate the metabolic landscape of Drosophila larval hemocytes, functional analogs of vertebrate myeloid cells, across developmental stages, genotypes, and immune activation states, by combining metabolic flux measurements with single-cell transcriptomics. Under homeostatic conditions, hemocytes rely predominantly on mitochondrial oxidative phosphorylation for ATP production, with minimal glycolytic contribution. Immune activation, particularly lamellocyte differentiation, drives enhanced mitochondrial respiration and metabolic flexibility, accompanied by structural remodeling of the mitochondrial network. Mechanistically, functional lamellocytes require Drp1-mediated mitochondrial fission and utilize glucose and trehalose as primary carbon sources to sustain mitochondrial respiration, which is essential for effective immune responses. Overall, these findings establish that mitochondrial metabolic reprogramming is a conserved feature of innate immune activation in myeloid-like immune cells and reveal an evolutionarily ancient link between mitochondrial dynamics and immune cell activation, with implications for understanding metabolic regulation of innate immunity in invertebrate models and beyond.

developmental biology↗