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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2026

Scalable proxiloids enable human-relevant assessment of kidney proximal tubule toxicity

Drug-induced injury to the human proximal tubule (PT) is a leading cause of acute kidney injury and drug attrition, yet remains difficult to predict preclinically. PT toxicity arises from the coupling of transporter-mediated xenobiotic accumulation and high oxidative metabolic demand. Current models lack key aspects of PT physiology or are difficult to scale for toxicity testing. New Approach Methodologies (NAMs) address this challenge through human-relevant in vitro systems. Here we introduce proxiloids, a scalable suspension-based human induced pluripotent stem cell differentiation strategy. Within 14 days, proxiloids form lumenized, polarized tubular organoids enriched for PT identity, with functional transport and oxidative metabolic competence. Proxiloids are compatible with genetically encoded reporters and standard multiwell assays, enabling detection of defined stress responses. They recapitulate aminoglycoside nephrotoxicity with greater sensitivity than matched two-dimensional cultures and detect adefovir-induced mitochondrial toxicity not predicted in rodents. Together, proxiloids provide a scalable, human-relevant NAM for PT nephrotoxicity assessment.

cell biology↗

bioRxiv2026

Extracellular Vacuole-derived bodies (EVacs) mediate RNA secretion in plants

Extracellular RNAs are found in the plant extracellular space, but how they are exported from cells remains unclear. We found that the plant vacuole is a major source of extracellular RNA and identified a class of large extracellular vacuole-derived bodies, which we termed EVacs, that are key mediators of this transport. EVacs are marked by the vacuolar membrane (tonoplast) proteins {gamma}-TIP and V-ATPase and originate as intravacuolar structures formed by inward folding of the tonoplast, encapsulating intact cytoplasmic material, including both RNAs and proteins. These intravacuolar bodies then escape the vacuole and are subsequently released from the plasma membrane of mesophyll cells into the apoplast. These findings provide a novel mechanism for the unconventional secretion of macromolecules in plants.

plant biology↗

bioRxiv2026

Topological Closure Drives Structural Stabilization and Fast Cooperative Dynamics in Crowded Circular Polysomes

In linear polysomes, excluded-volume interactions among ribosomes can induce dimensional reduction of mRNA. Yet linear architectures allow steric stress to relax at open ends-- limiting how strongly crowding can remodel the mRNA's structure and dynamics. Using coarse-grained molecular-dynamics simulations, we compare circular and linear polysomes over a range of ribosome densities. Circular closure selects a predominantly quasi-planar global conformational ensemble, as indicated by a shape dimensionality dshape {approx} 2 over a range of ribosome densities. Crucially, circular topology and ribosome crowding act cooperatively to suppress structural fluctuations. While closure alone or linear crowding reduces relative global size fluctuations ({Delta}Rg/Rg) only to {approx} 0.16, their combined effect drives this fluctuation down to {approx} 0.07. Within this stabilized architecture, increasing ribosome density drives a distinct in-plane reorganization: the ring becomes more isotropic, global size fluctuations are strongly suppressed, and the scaling exponent increases toward {nu} [~=] 0.74 - 0.77, consistent with two-dimensional self-avoiding walk-like value over the accessible finite-size window, 1000 [≤] N [≤] 4969. Closure shortens the radius-of-gyration decorrelation time of circular polysomes by 40-fold relative to matched linear systems, reflecting the topological elimination of free ends. Within this closureselected ensemble, ribosome crowding further reduces the decorrelation time by up to 20% at the highest density. A fluctuation-informed crossover model links the density dependence of the global scaling exponent to inter-ribosomal subchain statistics. These results distinguish the geometric role of circular closure from the density-dependent steric response that it enables, revealing a confined yet dynamically responsive conformational regime for circular polysomes.

biophysics↗

bioRxiv2026

Hidden molecular states of bacterial replicons beyond the chromosome-plasmid dichotomy

Bacterial genomes are organized into autonomous replicons, traditionally classified as either chromosomes or plasmids-a binary framework that underpins genome annotation and evolution models. Yet whether this binary framework captures the full diversity of replicon organization remains unclear. Here we show that bacterial replicons occupy three recurrent organizational states rather than two canonical categories. By integrating quantitative measures of chromosome-plasmid sequence affinity (plasmidness) across more than 72,000 replicons from 21 bacterial genera, we identify a distinct class-intermediate replicons-that occupies a positional and functional middle ground. These replicons are plasmid-sized, harbor substantial chromosomal sequence ancestry, and lack canonical replication signatures typically associated with either class. Multiple complementary molecular properties converge on this same state. Comparative genomic analyses reveal their enrichment near recurrent chromosome remodeling regions and reveal close evolutionary ties to conjugative and antimicrobial resistance plasmids. Metagenomic data further corroborate their presence across natural ecosystems. Together, these findings reveal a previously unrecognized replicon state and redefine bacterial genome organization beyond the chromosome-plasmid dichotomy.

microbiology↗

bioRxiv2026

The Microbiota Dictates Vendor-Derived Differences in a Murine Clostridioides difficile Infection Model

Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated infectious diarrhea and remains a major burden to healthcare systems worldwide. The development of novel therapeutics for CDI requires robust and reproducible preclinical models. However, the microbiota has emerged as a major source of variability in animal studies. Here, we found that genetically similar mice obtained from two commercial vendors, Jackson Laboratory (JAX) and Charles River Laboratories (CRL), exhibited marked differences in susceptibility to CDI, with JAX mice developing fulminant disease and CRL mice remaining resistant. Using full-length 16S rRNA gene sequencing, we show that JAX and CRL mice harboured distinct gut microbiota, and that cohousing susceptible JAX mice with resistant CRL mice was sufficient to shift the JAX microbiota toward the CRL community structure and confer resistance to CDI. Differential abundance analysis identified taxa distinguishing resistant and susceptible mice, providing candidates for future mechanistic investigation. These findings demonstrate that vendor-derived variation in the gut microbiota drives differential susceptibility to CDI in mice, and that this phenotype is transferable via cohousing, highlighting the importance of accounting for the microbiota when designing and interpreting animal models of infectious disease.

microbiology↗

bioRxiv2026

LINC00536 regulates transcriptional repressor TRPS1 in breast cancer

Metastatic breast cancer with complex molecular mechanisms of progression accounts for most cancer related deaths in women. To improve diagnosis and drug development, it is important to identify novel biomarkers and critical molecular pathways involved in tumor initiation and progression. Here, we profiled and analyzed the expression of long non-coding RNAs (lncRNAs) from three distinct stages of tumor initiation and progression (hyperplasia, adenoma, and carcinoma). We performed RNAseq on tumor and mammary epithelial cells derived from ROSAmT/mG tumor and non-tumor mice. We identified 1913 differentially expressed protein coding genes and 324 lncRNAs in breast cancer cells of all stages compared with normal mammary epithelial cells. Pearson correlation analysis correlated 93 differentially expressed lncRNAs with protein coding genes, providing a comprehensive lncRNA-protein coding genes co-expression network. Among them, we focused on Gm19303 which was paired with the differentially expressed protein coding gene, transcriptional repressor GATA binding 1 (Trps1), and identified its human counterpart as LINC00536. Both LINC00536 and TRPS1 are only overexpressed in breast cancer and correlate with poor prognosis of patient from the TCGA and GTEx databases. Single cell RNAseq data from the Atlas of Human breast cancers further confirmed that TRPS1 is upregulated in human breast cancer compared to normal human mammary tissue with highest expression in ER+ subgroup. In summary, our study explored the potential role of lncRNAs in breast cancer initiation and progression. *Implications statement: Our findings imply that human LINC00536/TRPS1 serves as a novel and early biomarker of cancer progression and a potential therapeutic target for breast cancer.

cancer biology↗

bioRxiv2026

A strong-to-weak interaction shift during microbiome succession is coupled to colonizer-dependent antimicrobial resistance

The outcome of ecological succession is often attributed to the characteristics of the invader or the resident community, but rarely to how the community's interaction network reorganizes during assembly. Here, we track intraspecific lineage dynamics and infer time-resolved community interaction networks using Dynamic Covariance Mapping during ecological invasion of the mouse gut by a chromosomally barcoded, spectinomycin-resistant Escherichia coli K12 colonizer. The network is initially dominated by strong, predominantly inhibitory interactions, but as community diversity recovers, the distribution of interaction strengths contracts toward zero, producing a community increasingly dominated by weak and near-neutral interactions. The dominant eigenvalue of the DCM-inferred interaction matrix moves toward marginal stability predicted for dynamically assembling ecological networks. This pattern replicates across eight independent mice in two experimental cohorts, at both inter- and intra-species resolution. The ecological transition coincides with the reproducible resurgence of Paenibacillaceae to high relative abundance and persistent coexistence with E. coli under continued spectinomycin pressure. Whole-genome sequencing of recovered Paenibacillus macerans isolates identifies recurrent mutations in ribosomal protein S5 region associated with spectinomycin binding and strongly implicating this variant in resistance. Strikingly, under antibiotic pressure but without E. coli K12 invasion, resident Paenibacillaceae never blooms, indicating that expansion of the resistant population depends on the ecological context established by the colonizer. These findings show that gut microbiome succession is accompanied by a reproducible transition from strong toward weak interactions and link this network reorganization to the colonizer-dependent ecological benefit of antimicrobial resistance.

ecology↗

bioRxiv2026

Molecular basis of AMPA receptor labeling by ligand-directed acyl imidazole chemistry in living neurons

Rational design of covalent protein-labeling reagents in complex biological environments requires a molecular-level understanding of how the protein microenvironment governs chemical reactivity; yet, such mechanistic details remain inaccessible to experimental methods alone. In living neurons, Ligand-Directed Acyl Imidazole (LDAI) chemistry has been used to label AMPA receptors as a traceless, affinity-based protein labeling method. Although LDAI labeling reagents have been optimized in the lab, the atomic details of their interactions with the protein and the underlying mechanism remain elusive. In this work, we combined Quantum Mechanical (QM) calculations and molecular dynamics (MD) simulations to propose a detailed reaction mechanism for AMPAR labeling by LDAI reagents and to clarify how the protein microenvironment governs reactivity. Although Lys residues are usually protonated at physiological pH and therefore less nucleophilic in water, our QM results show that Lys labeling is energetically more favorable than competing reactions with Ser or water. MD simulations reveal that PFQX ---the LDAI reagent precursor--- binds dynamically to the GluA2 AMPAR as an antagonist, inducing conformational changes that reshape the local environment of the acyl imidazole (AI) warhead, underscoring that ligand identity strongly affects labeling outcomes. We also identified intra and intermolecular hydrogen bond networks that may contribute to further immobilize and pre-organize the LDAI reagent. Moreover, the probe's chemical nature shapes its interactions with the Ligand Binding Domain (LBD), offering a plausible rationale for the previously experimentally observed ligand-dependent fluorescent response. Taken together, our results establish design principles for exploiting the reagent geometry and binding pocket hydrogen-bonding networks for the rational design of LDAI reagents.

biophysics↗

bioRxiv2026

Structural characterization the LlaI anti-phage defense system reveals insights into the evolution of nucleotide specificity and the organization of DNA binding in McrBC restriction complexes

Canonical McrBC enzymes are nucleotide-powered, motor-driven endonucleases that bind and cleave modified bacteriophage DNA. Non-canonical McrBC homologs like LlaI and BsuMI are distinguished by a unique three-gene organization and the ability to target DNA site-specifically. Here, we report the atomic-resolution crystal structures of the DNA-binding module LlaI.R1 and AAA+ motor LlaI.R2 from the Lactococcus lactis LlaI anti-phage defense system. The crystallized LlaI.R2 hexamer traps two distinct active site conformations that correlate to different states of the nucleotide hydrolysis cycle and reveal that the organization of the critical catalytic machinery present in canonical McrB homologs is also conserved in non-canonical R2 proteins. Although canonical McrB homologs are strictly GTP-specific, we find that the R2 proteins from LlaI and BsuMI do not discriminate between different nucleotides, even when in complex with their respective R1 partners. Using mutagenesis, we define surfaces on the LlaI.R1 structure that are critical for DNA-binding and interaction with LlaI.R2. These observations support computational modelling of the assembled LlaI restriction system bound to DNA. Together, our data provide new insights into the evolution of nucleotide specificity in McrBC restriction complexes and the molecular mechanisms governing McrBC-catalyzed DNA translocation and cleavage.

biochemistry↗

bioRxiv2026

The function of human PIF1 in G quadruplex formation and replication stress response at ALT telomeres

Cancers maintain their telomeres through two telomere maintenance mechanisms: 85-90% of cancers rely on telomerase (TEL+), while 10-15% of cancers adopt the Alternative Lengthening of Telomeres (ALT) pathway. The Break-Induced Replication (BIR) pathway plays a critical role in maintaining telomere length in the ALT+ cells. In both yeast and human, PIF1, a 5' to 3' helicase, is required for the robust activity of BIR. However, the extent of human PIF1 (hPIF1) involvement in the ALT pathway remains unknown. Here we showed that hPIF1 can be recruited to damaged telomeres in ALT+ cells. In addition, we demonstrated that inhibition of hPIF1 induced DNA damage and G quadruplex (G4) accumulation at ALT telomeres, leading to a moderate reduction of the mean telomere length. Most interestingly, we demonstrated that inhibition of hPIF1 also attenuates checkpoint activation, BLM recruitment, single-stranded DNA (ssDNA) formation, DNA damage, and G4s at telomeres in the FANCM deficient ALT+ cells. Finally, we showed that inactivation of hPIF1 affects the viability of both ALT+ and TEL+ cancers, suggesting that hPIF1 is a potential drug target for cancer therapy.

molecular biology↗

bioRxiv2026

The Hidden Prior: Variance Constraints Under Data Augmentation

Data augmentation is now a standard device across capture--recapture and occupancy analysis: adding a fixed number M of all-zero encounter histories replaces a model of unknown dimension with one of fixed dimension. Although M is often treated as a computational tuning choice, it also specifies a finite superpopulation and hence a binomial support constraint on the number of undetected individuals. In a Bayesian implementation that constraint appears as an induced prior; in a likelihood implementation it is the same finite-support assumption reached by another route. That the Bernoulli specification for the inclusion indicators induces a binomial prior on abundance is established (Schofield & Barker 2014); our concern is what that choice costs in estimated uncertainty. We develop the argument using a simple closed-population abundance estimation problem. We show that augmented occupancy and Huggins conditional-likelihood analyses give numerically identical point estimates of N once M is sufficiently large. Their uncertainty estimates, however, need not agree. We distinguish two sources of discrepancy. First, when M is small relative to the number of undetected individuals, the finite binomial ceiling truncates the likelihood or posterior and suppresses uncertainty. Second, once that ceiling no longer binds, Taylor-series (Delta-method) approximations still understate variance, because the quantity of interest is a strongly non-linear function of the estimated parameters and local linearization does not reproduce its curvature. Gauss-Hermite quadrature on the unconstrained logit scale recovers much of the shortfall and approaches the MCMC posterior benchmark, though a small residual remains that does not close as M grows, reflecting the distinction between asymptotic likelihood theory and finite-sample Bayesian inference. Neither mechanism is peculiar to abundance estimation: the first follows from the augmented representation itself, the second from any derived quantity that is a non-linear function of estimated parameters. We close with framework-specific guidance for choosing M.

ecology↗

bioRxiv2026

Rate of meristem initiation driven by the MADS-WUS axis contributes to floral survival and inflorescence evolution in grasses

Crop domestication has repeatedly shaped inflorescence architecture to improve floral production, but mechanisms coordinating the rate of floral initiation, maturation and survival remain unclear. Combining morphometry, modelling and molecular genetic analyses, we show that floral production in the indeterminate barley (Hordeum vulgare L.) inflorescence follows an "initiate fast-die young" strategy orchestrated by a main MADS-box gene, SPIKELET INITIATION AND FERTILITY (SIF). SIF accomplishes this duality by coordinately terminating the inflorescence meristem via WUSCHEL and activating the floral meristem via APETALA1 (Vrn-H1). Hereby, the ancestral SIF "slow" allele promotes a timely commitment to floral maturation, whereas the derived "fast" allele permits more floral initiations. Postdomestication selection of SIF alleles thus enables diversified reproductive strategies in barley populations to maintain yield traits in the field. Finally, we show that a lineage-specific SIF duplication contributed to meristem fate transition and inflorescence evolution during Triticeae cold adaptation. Our results establish developmental rate as a key driver of architectural innovation and reproductive success.

plant biology↗

bioRxiv2026

Highly plastic macrophage niches orchestrate acquired quiescence and reactivation in breast-cancer bone metastasis

Recurrence and metastasis remain major causes of cancer mortality, sustained by therapy-resistant micrometastatic cells. Bone is a frequent site of breast-cancer relapse, yet the cues that reawaken disseminated cells remain poorly defined. We identify a previously unrecognized, highly plastic CXCL16 macrophage population that integrates tumor-associated macrophage programs found in distant metastatic sites such as lung and brain with non-tumor disease-associated traits in bone marrow. These CXCL16 macrophages establish a transient niche that restrains disseminated cancer-cell proliferation. Single-cell transcriptomics delineate functional remodeling of myeloid niches within the bone metastatic microenvironment: a CXCL16 macrophage niche that transiently constrains metastatic growth, and G-CSF macrophage and neutrophil niches that reignite tumor outgrowth. In primary tumors, cancer-associated fibroblasts (CAFs) aberrantly secrete G-CSF in response to cancer-cell signals, expanding G-CSF-receptor-positive subset of cancer cells with high metastatic potential. In advanced human bone metastases, CXCL16 macrophages localize to CAF-rich stroma but are excluded from cancer-cell clusters, indicating immune evasion. Together, these findings uncover CAF-bone-marrow cross-talk as a therapeutic target linking stromal inflammation, immune remodeling, and metastatic progression.

cancer biology↗

bioRxiv2026

Breeding cassava for intercropping with cowpea: monoculture selection captures most intercrop selection gain, but targeted testing remains necessary

Intercropping dominates smallholder cassava production in sub-Saharan Africa, yet cassava breeding programs evaluate genotypes exclusively under monoculture. Despite consistently reported system-level yield advantages, cassava yield is reduced by 17 to 51% under intercropping, indicating a need to reduce this competitive disadvantage through breeding. However, the quantitative-genetic foundations of intercrop breeding remain uncharacterized for tropical root crop systems. We hypothesized that monoculture selection would capture most, but not all, genetic merit for intercropping and that a limited tester set would be sufficient if general mixing ability predominated. We evaluated 120 cassava clones previously selected under monoculture in IITA advanced yield trials, testing them under monoculture and in intercrop with two contrasting cowpea varieties across two years at Ibadan, Nigeria. Spatial mixed models quantified genetic variation, genotype by cropping system interaction, cross system genetic relationships, realized selection gain, mixing ability, tester effects, and land equivalent ratio. Intercropping reduced cassava fresh root yield by 19%, but total land equivalent ratios exceeded 1.0 for all clones, confirming a system-level land use advantage. Cassava performance under intercropping was heritable, with estimates of 0.50 to 0.75, and genotype by cropping system interaction was not significant. Genetic correlations between monoculture and intercrop performance were high and approached unity (rg = 0.92 to 0.99), and selection efficiency was 26 to 44% at 10% intensity, confirming that high genetic correlation does not guarantee effective indirect selection. Monoculture selection captured approximately two-thirds of direct intercrop gain. General mixing ability dominated, specific mixing ability was negligible, and producer effects explained 20 to 47% of intercrop variance. The two architecturally and phenologically contrasting cowpea varieties had limited influence on cassava rankings. Here, we show for the first time that cassava breeding for intercropping can retain monoculture selection during early stages while adding two representative cowpea testers at the advanced trial stage. This staged strategy aligns cassava breeding with diversified smallholder systems without creating a separate pipeline.

plant biology↗

bioRxiv2026

GDNF enemas improve epithelial and immune defects in both aganglionic and ganglionic colon of Hirschsprung mice

Hirschsprung disease (HSCR) is a severe birth defect where ganglia of the enteric nervous system (ENS) are missing from distal bowel. The aganglionic segment is also characterized by increased epithelial permeability and pro-inflammatory immune activation. These problems may sequentially lead to translocation of gut microbes into the colon wall and systemic circulation, resulting in enterocolitis and sepsis. Current HSCR treatment via surgical resection of the aganglionic segment is lifesaving but not curative, often leaving patients with persistent gastrointestinal complications including recurrent risk of enterocolitis. As alternative, we are developing a regenerative medicine strategy based on in situ stimulation of tissue-resident ENS progenitors via rectal administration of the neurotrophic factor GDNF. Here, we report that GDNF-based therapy has pleiotropic gastrointestinal effects in a mouse model of short-segment HSCR, beyond its role in ENS regeneration. Interestingly, we found that these protective effects are not restricted to the aganglionic distal colon, also positively impacting the ENS-containing proximal colon. GDNF treatment reduces bacterial translocation both locally and in peripheral organs, and this is associated with recovery of the key epithelial junction proteins CLDN3, ZO1 and DSG2. Furthermore, multiparameter flow cytometry-based analysis of 55 lymphoid and 17 myeloid cell subtypes revealed that GDNF treatment has global anti-inflammatory effects, preferentially affecting innate over adaptive immunity. Overall, these findings highlight a critical role for GDNF treatment in reestablishing proper epithelial and immune cell homeostasis, offering promising therapeutic avenues not only for HSCR but also potentially for other intestinal disorders with overlapping pathophysiology.

developmental biology↗

bioRxiv2026

Low-Density Lipoprotein Modulates Plasma Fibrin Network Architecture and Impairs Fibrinolysis

Low-density lipoprotein (LDL) is a major atherogenic lipoprotein, yet its potential to directly modify the fibrin scaffold of blood clots is incompletely understood. Here, we investigated how LDL alters plasma fibrin network architecture and internal fibrinolysis across defined fibrinogen/thrombin conditions. Pooled normal human plasma was supplemented with LDL and clotted with controlled concentrations of fibrinogen and thrombin. Fibrin architecture was visualized by confocal microscopy and quantified by pore-size analysis; clot formation and lysis were monitored turbidimetrically in the presence of tissue plasminogen activator (tPA). Increasing LDL produced a pronounced reduction in fibrin-network pore size across the tested fibrinogen/thrombin conditions. The LDL dependence of pore diameter was well described by a power-law relationship, D_pore=(6.54 +/- 0.11)[LDL]^(-0.12 +/- 0.02) , (R^2 = 0.90), with a significant negative LDL exponent (p = 4 x 10^5). Increasing LDL also prolonged clot lysis time and altered turbidity kinetics. These findings extend epidemiologic and clinical associations between ApoB-containing lipoproteins and hypofibrinolytic clot phenotypes by demonstrating, in a controlled plasma system, that LDL itself can modify fibrin network architecture and fibrinolytic susceptibility. The results support a structure-function role for LDL within the fibrin biomaterial and motivate direct tests of LDL incorporation, protofibril packing, fibrinolytic-protein binding, and single-fiber mechanics.

biophysics↗

bioRxiv2026

Single-Cell Profiling of Dynamic Epicardial Cell States During Myocardial Infarction

Background: The epicardium is reactivated after myocardial infarction (MI); however, the gene expression profiles of post-MI adult epicardial subpopulations remain incompletely defined. Methods: Single-cell RNA sequencing was performed on lineage-traced Wt1+ epicardial cells from Wt1CreERT2/+; R26tdT/+; PdgfranGFP/+ adult mice after sham surgery or at 7 and 14 days after permanent artery ligation to induce MI. Immunostaining was performed on Wt1-lineage-traced cardiac tissue to validate spatial expression after ischemic injury. Results: Unbiased clustering identified nine transcriptionally distinct epicardial populations, encompassing mesothelial, fibroblast/mesenchymal, transitional, and proliferative phenotypes. Fibroblast-like epicardial cells (Wt1+/Pdgfra+) showed time-dependent expression profiles associated with upregulation of epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) gene programs. At 7 days post-MI, there was notable enrichment of genes related to chemokines and Wnt components. By 14 days post-MI, the expression profile shifted toward immune regulation. In contrast, a Wt1high/Msln+ population showed minimal upregulation of EMT gene programs but enhanced paracrine signaling related to wound healing and semaphorins, suggesting reactivation of reparative and angiogenic functions akin to those of the epicardium during embryonic development. Immunostaining and in situ hybridization fluorescence analyses validated laminar epicardial cell placement after MI, comprising a surface Msln+ sheet, an overlapping Wt1-lineage band, and a subadjacent PDGFR+ and Periostin+ compartment that expands 7-14 days after MI and regresses by day 28 post-ischemia. Conclusions: Our data define epicardial gene programs in which a signaling epithelial cell surface overlays an effector mesenchymal cell stroma to coordinate angiogenesis, leukocyte recruitment, and ECM remodeling. This study presents the first integrated single-cell atlas of epicardial-derived cells across multiple post-ischemic timepoints, offering new insights into their reparative potential and dynamic signaling diversity in the injured adult heart.

molecular biology↗

bioRxiv2026

Elucidating the functional domain architecture of ArCS1, a biomineralizing myosin chitin synthase: I. The role of lipids

In molluscs, chitin synthases are essential for biologically controlled biomineralization, with some variants possessing a myosin motor domain that may link polymer synthesis to the cytoskeleton. Experimentally, we established a reliable workflow for expressing ArCS1_E22TM in Dictyostelium discoideum and developed effective purification methods to reconstitute ArCS1_E22TM in nanodiscs using MSPs and specific lipid composition. MSP1D1deltaH5 proved optimal for nanodisc formation, yielding homogeneous, monodisperse discs (~8.2 nm). Lipids were refined to POPC:POPE:POPG (3:1:1) with 20% cholesterol, improving nanodisc quality and uniformity as observed by negative-stain EM. The full-length ArCS1 and its subdomains were modeled using AlphaFold3; the myosin motor, glycosyltransferase, and transmembrane regions are well-defined internally but loosely constrained relative to one another, suggesting flexible linking and conformational coupling. Modelling with Mg2+ and oleic acid as ligands and comparative analyses with bacterial cellulose synthase and yeast chitin synthase 1 provided insights into substrate binding and a potential mechanism for chitin polymerization and translocation. This research establishes a standard procedure for comprehensive structural analyses of recombinant molluscan chitin synthase in near-native or biomimetic membranes. This sets the stage for high-resolution cryo-electron microscopy to determine the first experimentally resolved structure of a molluscan chitin synthase and to provide insight into the enzyme's architecture and the regulatory mechanisms of biomineralization.

molecular biology↗