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bioRxiv2026

Iron-Flavonoid-Peroxidase Signaling Triggers Seed-Coat Remodeling for Rapid Germination

Seed germination and survival depend on a sustained water supply. We previously observed that iron (Fe) supply accelerates germination in tomato. Using Arabidopsis thaliana, we show here that increased Fe in the spermosphere acts as an extrinsic signal that increases seed coat permeability. Fe triggers structural remodeling of the cuticle covering the endosperm, as evidenced by loss of Auramine O staining in the testa-ruptured seeds. Fe-triggered germination phenotypes are compromised in the flavonoid (tt4 and tt7) and prx17 mutants, and in peroxidase inhibitor-treated wild-type seeds. We hypothesize that PRX17-driven peroxidase activity is governed by a maternal repressor-derepressor mechanism in which seed coat flavonoids constitutively repress enzyme activity. This repression is relieved by chelation of Fe by flavonoids, allowing PRX17 to remodel the cuticle and accelerate germination. To test this model, we examined the interaction between Fe and peroxidases with quercetin, a major seed coat flavonoid. Upon imbibition, quercetin is deglycosylated and inhibits PRX17 activity. Formation of Fe-quercetin complexes at the chalaza reduces the level of active quercetin, thereby restoring PRX17 activity in a dose-dependent manner. This study suggests that elevated Fe levels in oxygen-deprived, submerged soils prime seeds for rapid germination, a novel control mechanism mediated by seed coat biochemistry.

plant biology↗

bioRxiv2026

MicroGrowAgents: An Agentic AI System for Microbial Cultivation Engineering

Microbial cultivation optimization remains labor-intensive and inefficient, requiring extensive experimental screening to identify suitable growth conditions. Traditional one-factor-at-a-time approaches are particularly ineffective for exploring complex, multidimensional nutrient parameter spaces. We present MicroGrowAgents, an agent-assisted system for auditable design of candidate growth media through integration of knowledge graphs, metabolic modeling, and optimal experimental design. The system comprises 29 documented agents implementing 58 skills across seven functional categories that query structured biological knowledge (KG-Microbe: 864,363 validated species), Bakta-annotated genomes (667,000+ annotated features), curated organism-specific FACTS sheets, and a DOI-linked publication corpus, and apply this evidence to three ends: recommending candidate ingredients and concentration ranges, specifying the factors of a statistically optimal MaxPro experimental design, and interpreting cultivation outcomes against known biochemistry. We applied the approach to Methylorubrum extorquens AM1 (formerly Methylobacterium extorquens; reclassified per LPSN) by cultivating 69 designed media plus a default media baseline (70 total tested conditions) in quadruplicate and assessing two concurrent objectives: biomass turbidity (740 nm) and apparent residual-Nd depletion (residual Nd by arsenazo III). Monte-Carlo resampling of the replicate-level uncertainty (1000 iterations) identified MPOB_058 as the single MC-stable Pareto-optimal condition (membership frequency 0.922); paired-control biology analysis flags MPOB_058 as chemistry-confounded and nominates MPOB_008 as the cleaner biological-signal anchor (its lower abiotic-drift contribution to the residual-Nd measurement makes it the better candidate for confirming biological lanthanide handling in a follow-up round), with MPOB_019 borderline-stable, providing a prioritized anchor set for confirmation in subsequent rounds rather than a single declared optimum. The integration of chemical similarity search (208,000+ embeddings), metabolic gap analysis, and multi-modal reasoning enables evidence-based hypothesis generation that reduces experimental burden while accelerating discovery of growth-promoting conditions. On the Biolog Odin platform, the MC-stable composite candidate MPOB_058 grew 79% more integrated biomass (area under the 740 nm growth curve) and 46% faster (maximum specific growth rate _max; Gompertz fits, R{superscript 2} > 0.99) than the standard MP base medium for this organism (the unsupplemented starting-point recipe carried as the on-plate control). This biomass and growth-rate advantage is a direct kinetic measurement; the conditions apparent residual-Nd depletion ranking, by contrast, awaits confirmation in a subsequent round, because MPOB_058s apparent Nd depletion is partly abiotic. Unlike general-purpose AI co-scientists, MicroGrowAgents grounds every recommendation in inspectable evidence -- structured provenance manifests with per-session input checksums, schema- and ontology-validated outputs, and 90.5% literature citation coverage (143 of 158 curated DOIs with evidence-supports-claim verification) -- separating deterministic design and analysis from agentic interpretation so that recommendations are transparent, explainable, and auditable, while passing 7 of 9 bbop-skills agentic-system criteria in a Claude-Code self-audit (the two unmet criteria are MCP-standardized tool exposure and full input-data cryptographic hashing, both tracked roadmap items). The Bigger PictureIdentifying growth conditions for poorly characterized microbes is one specific instance of a recurring problem in the natural sciences: how to integrate heterogeneous prior knowledge -- structured databases, published literature, mechanistic models -- with statistically efficient experimental design so that limited wet-lab time is spent where it most reduces the search. We show that a multi-agent AI system can perform that integration from organism input to wet-lab design, with every recommendation traceable from its source evidence through to the experimental design that tested it. The same architecture (specialized agents over structured knowledge bases, mechanistic models, and statistical design, with checksummed provenance recorded as the unit of evidence) is in principle transferable to other DBTL loops against under-explored search spaces such as catalyst discovery, materials formulation, cell-line engineering, drug-combination screens, or environmental remediation. We demonstrate it here only for microbial cultivation and flag cross-domain transfer as future work rather than a demonstrated capability. Our cultivation results on Methylorubrum extorquens AM1 are a concrete demonstration that this pattern works on real laboratory data: an uncertainty-aware shortlist of candidate media emerged from Monte-Carlo stability analysis of two competing objectives, with the leading composite candidate growing 79% more integrated biomass and 46% faster than the standard MP base medium for this organism; this biomass and growth-rate advantage is directly measured, while its apparent residual-Nd depletion advantage awaits confirmation in a subsequent round, since the apparent Nd depletion is partly abiotic. But the lesson we want to port across research domains is the architecture and its provenance discipline, not the specific organism. MicroGrowAgents is released under BSD-3 with checksummed YAML provenance manifests so that any group adopting these agents inherits a workflow designed to be re-runnable from its recorded inputs.

synthetic biology↗

bioRxiv2026

Comparing Machine Learning Approaches for Predicting CFD-Derived Stroke Risk Indicators in Atrial Fibrillation Patients

Non-valvular atrial fibrillation (AF) is associated with a five-fold increased risk of stroke, mainly due to impaired contractility of the left atrium (LA) leading to blood stasis and subsequent thrombus formation within the left atrial appendage (LAA). Current AF stroke risk stratification schemes, such as the CHA2DS2-VASc/ CHA2DS2-VA score, use comorbidities and do not capture mechanistic factors like blood flow dynamics and hypercoagulability. To address this, we developed a multiphase computational fluid dynamics (CFD) model of the LA, incorporating patient-specific geometries; modelling of the coagulation cascade; and non-Newtonian blood behaviour within the LAA. Using 84 simulation cases generated via Latin Hypercube Sampling of physiological blood parameters and 21 patient-derived LA anatomies, we trained surrogate machine learning models, including Ridge regression, XGBoost, Gaussian Process Emulators (GPEs), and deep learning networks, to predict CFD outputs such as blood viscosity in and fibrin concentrations in the LAA. Deep learning achieved R{superscript 2} values up to 0.90, with the accuracy increasing when both physiological parameters and the raw CT image were included. Other models showed uneven performance with R2 values below 0.7, highlighting the role of nonlinearities between parameters. The study presents a novel CFD model that captures the transition from blood stasis to clot formation, representing the full thrombotic continuum underlying stroke risk in AF, and a deep learning approach to enable efficient prediction of mechanistic outputs of clinical value for stroke risk stratification in AF patients. Author SummaryAtrial fibrillation is a common heart rhythm disorder that greatly increases the risk of stroke. In many patients, blood can pool inside a small pouch of the heart called the left atrial appendage, where clots may form and later travel to the brain. Current clinical tools used to estimate stroke risk mainly rely on a patients medical history and do not directly assess the mechanistic processes that lead to clot formation. In this study, we developed a computer model that simulates how blood flows and clots inside the heart using patient-specific heart anatomies derived from medical imaging. Our model combines blood flow, blood biochemistry, and the changing physical properties of blood during clot formation. We then used machine learning methods to predict these complex simulation results more efficiently. Deep learning models performed best, particularly when both clinical parameters and heart imaging data were included. Our work provides a new way to study the full process linking abnormal blood flow to clot formation in atrial fibrillation. In the future, this approach could support more personalised and mechanistic assessment of stroke risk and help guide treatment decisions.

bioengineering↗

bioRxiv2026

RetroMol: Parsing a shared encoding from natural products and their biosynthetic gene clusters

Natural products such as polyketides and nonribosomal peptides (NRPs) are important sources of bioactive compounds, including many antibiotics. Many of them are assembled by modular enzyme complexes and further modified and diversified by tailoring reactions encoded by biosynthetic gene clusters (BGCs). Although natural products and their coding BGCs describe different data modalities of the same biochemical process, a unified language to jointly describe their biochemistry is lacking. Here we introduce a sequence-based representation of the core biosynthesis of modular natural products, which we call primary sequences, that bridges chemical structures and BGCs. We also present RetroMol, an algorithm that parses either natural product structures or their encoding BGCs into their primary sequences of natural product building blocks. RetroMol allows for similarity scoring between natural products and BGCs, enabling the retrieval of compounds, BGCs, and a combination of the two, based on their biosynthetic similarity. This can, for instance, be used to retrieve biosynthetically similar but structurally dissimilar compounds, or link natural products to candidate coding BGCs in large experimental datasets. We demonstrate the latter by rediscovering the nocardichelin B BGC as a proof of principle. We also exemplify the utility of biosynthetic similarity by showing various pairs of biosynthetically similar compounds with low structural similarity. Together, these results establish primary sequences as a shared biosynthetic encoding for natural product comparison and BGC prioritization.

bioinformatics↗

bioRxiv2026

Cytopenias and Functional Defects in a Novel Murine Model of VPS45 Severe Congenital Neutropenia

Mutations in the VPS45 gene are associated with a rare form of severe congenital neutropenia (SCN5), a life-threatening inherited error of immunity. We developed and characterized a novel mouse model of SCN5 by CRISPR/Cas9-mediated knock-in of pathogenic VPS45 E238K and T224N mutations. Both Vps45 mutations led to decreased protein expression in bone marrow cells. In vivo phenotyping demonstrated a non-Mendelian genetic distribution with reduced numbers of knock-in homozygotes Vps45E238K. Vps45E238K knock-in homozygous mice showed reduced body weight, reduced body condition with age, and increased mortality. As in human SCN5, Vps45E238K knock-in homozygotes demonstrated neutropenia and lymphopenia. Functionally, Vps45E238K knock-in homozygote neutrophils exhibited increased lipopolysaccharide-induced apoptosis and decreased peroxide production, phagocytic capacity and in vivo cell migration, phenocopying the functional defects reported in patients. Vps45T224N knock-in homozygous mice showed a milder phenotype or no abnormalities. In conclusion, this mouse model phenocopies, in part, human SCN5. It provides a novel platform for future studies of the pathophysiology of defects in neutrophil number and function in human SCN5, potential therapies for the disease, and the biochemistry and cell biology of VPS45. Summary statementWe report a mouse model of severe congenital neutropenia due to VPS45 missense mutations. It represents the first animal model of human neutropenia due to a defect in intracellular trafficking.

immunology↗

bioRxiv2026

Divergent specificity of PatA, GabT, and IlvE defines the branched transamination of Nε-carboxymethyllysine and its metabolite Nε-carboxymethylcadaverine in Escherichia coli

Thermal food processing generates N{varepsilon}-carboxymethyllysine (CML), a key advanced glycation end product (AGE) and marker of the Maillard reaction in food. Escherichia coli utilizes CML as a nitrogen source. While SpeC initiates degradation by decarboxylating CML to N-carboxymethylcadaverine (CM-Cad), the enzymes liberating the nitrogen remained unknown. Here, we identify PatA, GabT, and IlvE as the glutamate-dependent transaminases responsible for CML and CM-Cad transamination. Our results reveal a branched metabolic network rather than a linear pathway: PatA shows specificity towards both substrates, while GabT and IlvE selectively process CM-Cad and CML, respectively. We further demonstrate that the carboxymethyl piperideinium ion (CM-Pip) is formed spontaneously following CM-Cad transamination and reveal the previously unknown carboxymethyl-tetrahydropicolinic acid (CM-THPA) as novel metabolite in CML metabolism. Combining molecular microbiology, biochemistry, and analytical chemistry, we demonstrate that these transaminases are essential for integrating dietary CML into bacterial nitrogen metabolism, providing a model for microbial AGE processing via underground metabolism.

microbiology↗

bioRxiv2026

Stable Hrd1 tetramers at the heart of the retrotranslocon in living cells

Terminally misfolded or damaged proteins in the endoplasmic reticulum (ER) are degraded by the proteasome in a process termed endoplasmic reticulum-associated degradation (ERAD). To reach the proteasome in the cytosol, cargo proteins must be dislocated across the ER lipid bilayer. Retrograde transport is performed by a set of membrane-spanning multi-protein complexes that are notoriously difficult to study. Despite decades of genetic and biochemical analysis, there is little consensus regarding its molecular mechanism. Here, we characterize the dynamic assembly of the mammalian Hrd1 complex, one of the most thoroughly studied mammalian dislocons, using in situ multicolor single-particle tracking. Surprisingly, quantitative dual-color tracking reveals that the majority of Hrd1 is assembled into stable homo-tetramers in situ. Using a herein developed single-molecule assay based on binding competition, we show that this tetramerization is driven by a short stretch (Hrd1479-530, HAF-H) within the cytosolic domain of Hrd1. Combining classical purification assays, quantitative imaging, and structural predictions, we demonstrate that Hrd1479-530 forms a highly stable tetrameric helix bundle via a conserved hydrophobic coiled-coil motif. While higher order assemblies of Hrd1 have been previously implicated, this work offers direct evidence of their role in dislocation, demonstrating that quantitative single-molecule imaging can yield information on species previously hidden from classical biochemistry.

Cell Biology↗

bioRxiv2026

A substrate recursion principle for biological information, with empirical anchoring through a templating-mode taxonomy

Biological inheritance can be treated as a class of catalytic templating reactions in which a daughter molecule, generated by a kinetic kernel acting on a parent template, is itself a substrate for the next round of the same catalysis. We give the physicochemical conditions under which such a reaction can support unbounded heritable molecular distinguishability. Four conditions on the template-operator pair organize the analysis: nonzero per-site information content under the activesite recognition kernel (R1), a count of independently variable recognized positions that grows without bound as the reaction extends (R2), catalytic closure under iteration, possibly through a reversible involution such as Watson-Crick complementation (R3), and stochastic drift of the kernel in its recognition alphabet (R4). A fifth, scope-defining condition (R5) restricts the principle to kernels that are intrinsic physicochemistry rather than externally optimized search. These conditions are necessary for two distinct outcomes, separable as two necessity results. The capacity theorem states that linear scaling of substrate Shannon capacity with reaction extent requires R1, R2, and R3 but not R4: a perfect copier transmits an exponentially large configurational ensemble while producing no novelty. The generation theorem states that diversification of the heritable configuration set beyond the deterministic closure of a finite initial repertoire additionally requires R4, because branching trajectories in the recognized alphabet are what produce innovation. Populationlevel kinetics follow as a corollary that organizes six attested templating reactions into a taxonomy, and as a finite-population, finite-horizon proposition tested over five inheritance kinetic schemes, in which only individual-level stochastic drift reaches the target within the model class. We test the framework on the recently characterized bacterial defense system Drt3b, which makes alternating poly(AC) DNA without using a nucleic acid template. The framework classifies Drt3b as a cyclic two-state catalytic templating channel with a 1-bit capacity ceiling, and predicts that the Glu26-to-Gln active-site mutant incorporates dG at 10% probability at the dA-selecting state; the published biochemistry reports 10.16%. Across 1,232 Drt3b homologs, the framework predicts and recovers a 15.7-fold elevation of dG misincorporation in six clades carrying the natural Glu26-to-Asp substitution at this gate. Substitutions at two universal gate residues, Arg253 (architectural) and Gly248 (selectivity), provide single-experiment site-directed mutagenesis tests of the frameworks predictions. POPULAR SUMMARYA bacterial defense protein called Drt3b, recently characterized in E. coli, synthesizes DNA with a strict alternating ACAC pattern without copying any template. Two conserved active-site residues, Glu26 and Arg253, are modeled as enforcing an alternating two-state catalytic cycle that selects which nucleotide enters at each step. This is sequence without a sequence template, and it does not fit the textbook picture of inheritance. We treat inheritance as a class of catalytic templating reactions and ask which chemistries can support openended evolution. Two distinct requirements emerge. Capacity, the ability to transmit exponentially many distinct heritable configurations, requires three conditions on the template-catalyst pair: more than one monomer state recognized at each position, a position count that grows unboundedly with the reaction extent, and applicability of the catalysis to its own product. Generation of novel heritable configurations beyond what is already present requires a fourth condition: stochastic drift of the catalysis in its recognition alphabet. A perfect copier has capacity but cannot innovate; a drifting copier has both. Drt3b fails the second capacity condition, because its cycle has two states regardless of product length. The framework classifies six attested biological templating reactions as instances or partial instances of the same chemical specification, and it identifies two single-residue substitutions at Drt3bs active site whose measured effects would test its predictions.

biophysics↗

bioRxiv2026

The TARGET OF RAPAMYCIN (TOR) kinase controls shade-mediated hypocotyl elongation responses in Arabidopsis

Plants perceive neighboring vegetation through an enrichment of far-red light (shade) in the environment. These changes in light quality trigger molecular and physiological responses aimed at outgrowing competitors, collectively known as the shade avoidance syndrome. In this study, we identify the TARGET OF RAPAMYCIN (TOR) complex 1 (TORC1), a major growth-regulating hub in eukaryotes, as a driver of shade-mediated growth responses in plants. Combining physiology, genetics, biochemistry, and proteomics, we show that TOR activity is rapidly enhanced upon shade perception and is required for proper shade responses, as TOR inhibition severely impairs shade-mediated elongation. Furthermore, we found that the control of shade-mediated elongation by TOR involves auxin-dependent mechanisms, requires efficient translation activity, and is closely linked to epidermal cell elongation capacity. Altogether, our work identifies TOR as a key integrator of light quality signals to control adaptive growth responses. Finally, we further highlight the conservation of shade-mediated TOR activation in tomato, with potential implications for engineering crop cultivars better suited to high-density planting.

Plant Biology↗

bioRxiv2026

Transcription-induced coacervation accelerates and sensitizes cell-free biosensing

Cell-free biosensors leverage in vitro gene expression reactions to detect chemicals. While inexpensive, modular, and distributable, these platforms are constrained by slow readouts at ambient temperatures, precluding practical field operation. In cells, phase separation accelerates biochemical reactions; however, recapitulating these gains in vitro has remained challenging for complex biochemistries. Here, we report the first self-assembling coacervate system that accelerates in vitro transcription. Prepared by simple mixing, coacervation with spermine and polyacrylic acid occurs dynamically in response to NTP consumption and co-localizes DNA templates and RNA polymerase to accelerate transcription, mimicking intracellular phenomena. We exploit this discovery to accelerate the cell-free biosensing of six ligands, demonstrating that coacervation can preserve platform modularity, improve sensitivity, retain lyophilization compatibility, function in field matrices, and reduce ambient-temperature time-to-signal by hours. This work contributes to a growing understanding of phase separation in biology and advances the use of membrane-less organization for real-world applications.

synthetic biology↗

bioRxiv2026

A quantum interface with mitochondrial bioenergetics

Summary ParagraphRecent work has shown that genetically engineered proteins can serve as quantum bits in living systems. These quantum bits arise from the photochemistry of protein-bound flavins: blue-light excitation drives electron transfer to form a spin-correlated radical pair whose coherent singlet-triplet interconversion makes the proteins fluorescence sensitive to weak magnetic fields. Because this radical-pair reaction depends on the redox state of the flavin--itself a central electron carrier in cellular metabolism--the magneto-fluorescence of a biological qubit is intrinsically coupled to the biochemistry around it. This suggests a powerful application of fundamental significance in biology, until now an unsolved problem in the field of quantum sensing. Here we show a new class of quantum sensor, mtMagLOV2, that interfaces directly to a defining feature of life itself: the bioenergetic state of the cell. We genetically engineer flavin mononucleotide (FMN)-containing, magnetic-field-sensitive fluorescent proteins ("biological qubits") to be expressed and translocated into the key bioenergetic machinery of the cell: the mitochondrial matrix. Using confocal and super-resolution microscopy, mtMagLOV2 localizes to the mitochondrial cristae, home of the electron transport chain complexes I-V and ATP synthase--the site of oxidative phosphorylation (OXPHOS). By pharmacological manipulation of OXPHOS, we show that the sensors magneto-fluorescence tracks the redox (oxidation-reduction) state of the mitochondrial flavins, providing a quantum readout of redox status. The response differs between cancer cells (which rely heavily on glycolysis) and cardiomyocytes (which rely predominantly on OXPHOS), demonstrating "quantum bioenergetic profiling". Together, these results establish biological qubits as quantum sensors capable of probing mitochondrial bioenergetics, opening a quantum window into the energetic machinery of living cells. More broadly, we anticipate that coupling quantum redox sensitivity to specific biochemical targets will extend the reach of quantum technologies across the life sciences.

biophysics↗

bioRxiv2026

Beyond Bisulfite Sequencing: Resolving 5-hmC with Nanopore Sequencing Unmasks the True-5mC Methylation Entropy Landscape

DNA methylation dynamically regulates cellular function and phenotype. At the tissue level, stochasticity in methylation patterns, as measured by methylation entropy, drives plasticity, development, cancer, and aging. Modulation of methylation patterns is facilitated by erasure of 5-methylcytosine (5mC) via the oxidized intermediate 5-hydroxymethylcytosine (5hmC). Bisulfite sequencing cannot distinguish the two modifications, labeling them both as 5-mC. We quantitatively analyze the effect of this historical conflation on the genome-wide distribution of methylation levels and methylation entropy. Using nanopore sequencing with direct 5mC and 5hmC calling, we compare True-mC to bisulfite-like analysis in two model systems, kidney cancer and the mouse medial prefrontal cortex. We show that bisulfite sequencing introduces systematic, tissue-specific shifts in methylation distribution that affect the mechanistic interpretation of the underlying biology. The distortion scaled with endogenous 5hmC content: substantial in brain, where over half of the entropy-associated gene ontology terms recovered under bisulfite-like analysis were absent under True-mC, and minimal in the low-5hmC kidney cancer sample, which delimits the regime in which bisulfite-derived entropy remains interpretable. Together, these findings establish that True-5mC-based methylation entropy redefines the physical mapping of certain epigenomes, demonstrating that in some contexts, what has previously been interpreted as stochastic maintenance failure is frequently the structured signature of distinct and mechanistically interpretable cytosine biochemistry.

bioinformatics↗

bioRxiv2026

Nutritional Anthelmintics: Chicory Reconfigures the Equine Holobiont Across Microbial, Parasitic, and Host Scales

Anthelmintic resistance in cyathostomins is escalating worldwide, threatening equine health and highlighting the need for sustainable, ecology-based parasite control strategies. Chicory (Cichorium intybus, Puna II) has emerged as a promising antiparasitic forage, yet its broader effects on the equine holobiont, parasites, microbiota, and host physiology remain poorly understood. We conducted a 32-day longitudinal grazing trial in young horses to assess how chicory affects parasitological outcomes, gut microbial ecology, nemabiome composition, behaviour, and host physiological and immune responses. Twenty-six naturally infected Anglo-Arabian horses were monitored weekly, with 13 grazing a chicory-based sward and 13 grazing a permanent pasture. Clinical parameters, body weight, and serum biochemistry remained stable across treatments, indicating that chicory was well tolerated. Immune profiles showed limited variation, although IL-10 increased in chicory-fed horses, suggesting subtle immune modulation. Behavioural observations revealed no signs of discomfort and indicated slightly enhanced social interactions in the chicory group. Chicory grazing produced a marked reduction in cyathostomin egg excretion, accompanied by species-specific shifts in nemabiome composition. Several cyathostomin taxa, including Cylicocyclus ashworthi, C. leptostomus, and C. nassatus, declined in chicory-fed horses, whereas certain Cylicostephanus spp increased, indicating differential sensitivity rather than uniform suppression. Concomitantly, chicory induced profound ecological changes in the gut microbiota, including reduced alpha diversity, increased beta dispersion, and destabilised individual microbial trajectories. Several bacterial lineages, particularly Oscillospiraceae, Clostridiaceae, Lachnospiraceae, and Bacteroidales, were differentially enriched, reflecting a functional reorganisation of the intestinal ecosystem. Together, these findings demonstrate that chicory reduces parasite fitness, reshapes nemabiome composition, and alters gut microbial ecology while maintaining host physiological stability. Chicory thus emerges as a promising ecological tool for parasite control, capable of modulating the equine holobiont in ways that complement and potentially reduce reliance on conventional anthelmintic strategies. However, because its effects on gut microbial ecology remain uncertain, and may include shifts resembling dysbiosis, future studies are needed to monitor microbial dynamics more closely and clarify the long-term ecological consequences of chicory grazing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/737212v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@7b7367org.highwire.dtl.DTLVardef@d5914aorg.highwire.dtl.DTLVardef@135ad65org.highwire.dtl.DTLVardef@10b0322_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2026

Glucose and methylglyoxal alter plasma protein dynamics, immune traits and glucose homeostasis in a sex- and season-dependent manner in zebra finches

Birds maintain unusually high circulating glucose levels compared with other vertebrates without developing the diabetic complications observed in mammals, yet the mechanisms underlying this resistance remain unclear. We investigated the effects of chronic glucose and methylglyoxal supplementation on physiological condition in zebra finches (Taeniopygia guttata), with particular emphasis on sex- and season-dependent variation in plasma biochemistry, haematology and immune traits. Ninety zebra finches (45 males, 45 females) were randomly assigned to control, glucose-supplemented (50 g/L), or methylglyoxal (8.33 g/L) drinking treatments. Over one year, we analysed plasma proteins, metabolites (glucose, uric acid, bile acids), tissue damage markers (AST, CK), electrolytes, and immune parameters (leukocyte profiles). Both supplementations increased plasma glucose concentrations, with methylglyoxal producing the strongest effect. More importantly, both treatments disrupted seasonal plasma protein dynamics, preventing the increase in total proteins and globulins normally observed in females during the reproductive period, which suggests alterations in reproductive-related protein metabolism. Glucose supplementation elevated the heterophil-to-lymphocyte (H/L) ratio in May and August, consistent with elevated physiological stress. In contrast, methylglyoxal supplementation reduced the H/L ratio in November and unexpectedly lowered plasma AST and CK concentrations in May, suggesting context-dependent protective effects on tissue integrity despite its well-established pro-oxidative properties, potentially through hormetic mechanisms. Supplementation also modified the calcium/phosphate balance, further supporting treatment effects on seasonal (reproductive) physiology. Overall, our findings demonstrate that glucose and methylglyoxal reshape physiological regulation in zebra finches in a strongly sex- and season-dependent manner rather than simply inducing generalized metabolic damage. These results provide new insights into avian resistance to glucose-associated physiological challenges and highlight the importance of considering both biological context and standardized haematological reference values when investigating glucose metabolism in birds.

physiology↗

bioRxiv2026

Hologenomics of xylotrophic bivalves reveals a minimalist, remote-acting evolutionary strategy of wood digestion

Wood constitutes the largest reservoir of biogenic carbon on Earth, yet remarkably few animals can exploit it. While terrestrial wood-feeders like termites rely on highly diverse gut microbiomes, xylotrophic marine bivalves have evolved a fundamentally different approach: a spatially segregated system where intracellular gill symbionts produce enzymes that act remotely within a nearly sterile cecum. However, the genetic and evolutionary basis of this unique symbiosis remains largely elusive. Here, we integrate hologenomics, transcriptomics, and biochemistry of a shallow-water shipworm (Teredo navalis) and a deep-sea borer (Xyloredo sp.). We find that despite diverging approximately 147 million years ago and occupying drastically different habitats, these bivalves maintain a strictly conserved ancestral karyotype and a shared genomic architecture for wood digestion. Our models reveal a clear host-symbiont division of labor. The host genome is specialized for lignin modification and targeted enzyme transport, whereas a highly streamlined symbiont community is responsible for core polysaccharide degradation. Central to this minimalist strategy is a lineage-specific GH5-GH6 dual-catalytic enzyme. By sharing amino acids across proximal binding pockets, this fusion protein unites endo- and exo-cellulase activities, enabling highly synergistic cellulose cleavage without the need for complex microbial communities. Ultimately, our comparative analysis with terrestrial models demonstrates that these marine invertebrates achieve efficient biomass degradation not through microbial expansion, but through extreme functional streamlining and molecular innovation, offering a distinct evolutionary paradigm for marine carbon cycling.

evolutionary biology↗

bioRxiv2026

CRISPR/Cas9 gene editing in the heterotrophic protist Acanthamoeba castellanii

Members of the amoebozoan genus Acanthamoeba are unicellular heterotrophic protists that inhabit a wide range of terrestrial and aquatic environments. They are opportunistic pathogens known to host disease-causing bacteria as well large DNA viruses. While Acanthamoeba is commonly used to study cellular locomotion, phagocytosis, and disease, genetic tools for precise editing of the Acanthamoeba genome are still in their infancy. Here we have developed CRISPR/Cas9 tools for genetically engineering the model acanthamoebid Acanthamoeba castellanii strain Neff. In proof-of-principle experiments, we targeted the myosin-II heavy chain gene. Evidence for editing was found using reverse transcriptase PCR, microscopy, and DNA sequencing, which also identified unique alleles for the target locus. Collectively, we demonstrate that CRISPR can be employed to edit Acanthamoeba genes using a knock-in approach. This work serves as a foundation to further develop A. castellanii as a model system with which to study diverse questions in cell and molecular biology, biochemistry and evolution.

molecular biology↗

bioRxiv2026

Structural basis for alternative 3' splice site selection in the human spliceosome active center

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/741260v1_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@279388org.highwire.dtl.DTLVardef@46d636org.highwire.dtl.DTLVardef@997672org.highwire.dtl.DTLVardef@12f40ee_HPS_FORMAT_FIGEXP M_FIG C_FIG Key findingsO_LICryo-EM reveals SDE2 as a novel factor stabilizing the spliceosome active center C_LIO_LISDE2 {Delta}N structures visualize a stalled C* state with impaired docking-factor engagement. C_LIO_LISDE2, Prp18, and FAM32A read a cis-code to stabilize weaker proximal 3'-ss. C_LIO_LISDE2 {Delta}N destabilizes 3'-ss docking and rescues BRCA1 and CFTR mis-splicing in vivo. C_LI Accurate alternative splicing requires discrimination between adjacent 3' splice sites (3'-ss) during catalysis and is disrupted by pathogenic AG-gain mutations that create competing 3'-ss. Here, we present cryo-EM structures of human spliceosomes assembled on native-sequence pre-mRNAs, revealing how the catalytic core controls alternative 3'-ss selection. SDE2 is a previously unrecognized active-center component that promotes a docking-competent spliceosome conformation. Machine learning, in vivo transcriptomics, and in vitro biochemistry show how SDE2 cooperates with FAM32A and Prp18 to act as readers of a cis-regulatory code that governs 3'-ss selection during catalysis. These factors promote weaker, proximal site use by counteracting an intrinsic distal bias generated by active-site interactions with the distal-site -4 nucleotide. Structural or genetic perturbation of these exon-ligation factors destabilizes proximal 3'-ss docking and restores canonical splicing in disease-relevant CFTR and BRCA1 AG-gain alleles. Our work establishes the spliceosome active center as a tunable regulatory hub for alternative splicing.

molecular biology↗

bioRxiv2026

Molecular orchestration of global actin remodelling by INF2

The actin cytoskeleton rapidly reorganizes in response to intracellular calcium signals, driving cellular morphogenesis and wound healing. Among actin regulators, the formin INF2 uniquely mediates the "Calcium-mediated Actin Reset" (CaAR) reaction, orchestrating transient and global actin remodeling upon calcium influx. Excessive INF2 activity is linked to kidney and neuronal diseases, underscoring the need for its tight control. Combining live cell imaging with single molecule tracking, biochemistry and structural analysis we discover that INF2 activity is tightly controlled by two interlinked mechanisms: canonical intramolecular autoinhibition and binding of the INF2 N-terminus to the side of actin filaments. Side-binding limits actin elongation and supports re-establishment of autoinhibition. Disruption of this negative feedback prolongs INF2 activity, affecting plasma membrane organization and repair as well as transcriptional control. Our findings uncover a novel product-inhibition mechanism that limits INF2 function and offer important insight into disease mechanisms linked to actin dysregulation.

cell biology↗