Biology subjects

Garcia, M.

Publications and source records attributed to Garcia, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Carboxymethyl Dextran Coated Iron Oxide Magnetic Nanoparticles as Vehicles for CRISPR Ribonucleoproteins

The efficient and safe intracellular delivery of CRISPR ribonucleoproteins (RNPs) remains a major challenge for the development of genome editing strategies. Magnetic iron oxide nanoparticles (MNPs) offer an attractive and biocompatible delivery platform. However, their use for the delivery of functional CRISPR RNPs remains comparatively underexplored. Here, we developed carboxymethyl dextran (CMD)-coated MNPs as non-viral carriers for Cas9 and Cas12a RNPs. Nanocomplexes were formed through electrostatic interactions, without the need for chemical coupling reagents or additional surface functionalization. Both RNPs efficiently associated with MNPs, and the resulting nanocomplexes exhibited stable physicochemical properties and retained their cargo in serum-supplemented culture medium for up to 24 hours. Cellular uptake was confirmed by microscopy, while biocompatibility assays revealed no overt cytotoxicity. Functional genome editing was demonstrated at reporter and endogenous gene loci in HEK-293T cells, as well as in primary human dermal fibroblasts. Finally, we explored the therapeutic potential of this platform for recessive dystrophic epidermolysis bullosa (RDEB) by targeting COL7A1 exon 80, with the aim of removing the prevalent c.6527insC mutation while preserving the reading frame. Cas9 RNP nanocomplexes enabled exon 80 deletion. These findings establish CMD-coated MNPs as a versatile platform for the delivery of Cas9 and Cas12a RNPs and support further investigation of magnetic nanoparticle-mediated genome editing for inherited skin disorders.

molecular biology↗

bioRxiv2026

Tryptophan Restores Recovery Competence Following Starvation in the Absence of Lysosomal Amino Acid Sensing

How organisms recover from starvation to nutrient-replete physiology remains poorly understood. In C. elegans, we previously observed that transcription factor hlh-30 or a combination of glucose and linoleic acid in its absence are essential for survival with refeeding following starvation, i.e. recovery competence. A forward genetic screen to evaluate mechanisms identified depd-5, which encodes a GATOR1 subunit, loss of which is expected to impair amino acid sensing with unrestrained raga-1 and mTOR activation. Surprisingly, glucose refeeding reduced recovery competence in depd-5(lf, loss-of-function) animals, which was rescued by amino acids, specifically tryptophan. Increasing tryptophan levels in depd-5(lf) worms through supplementation, reduced tryptophan catabolism with tdo-2 ablation and prolonged starvation restored recovery competence. raga-1(gf, gain-of-function) recapitulated whereas raga-1(lf) rescued the impaired recovery competence in starved depd-5(lf) worms. An analogous DEPDC5 mutation in mammalian cells impaired starvation-induced mTOR inactivation. We conclude that tryptophan counters unrestrained mTOR activity to restore recovery competence following starvation.

cell biology↗

bioRxiv2026

Cell State Transitions Drive Transcriptome-Proteome Alignment in a Minimal Photoautotroph

The cyanobacterium Prochlorococcus marinus MED4 is a minimal photoautotroph that maintains circadian regulation despite its reduced circadian regulatory toolkit relative to other cyanobacteria. Moreover, MED4, like other small prokaryotes, has low transcript counts per cell and poor transcriptome-proteome alignment. A systems-level understanding of how MED4 uses its minimal regulatory machinery to achieve a well-coordinated circadian cycle is currently lacking. We address this knowledge gap by analyzing global proteomics, transcriptomics, metabolomics, and lipidomics collected across a circadian day. Integrating these data with statistical analyses, enzyme-constrained metabolic modeling, and stochastic mathematical modeling allows us to reveal and characterize six major population-level cell states. Here we present evidence that high-light response regulator RpaB plays an important night-time regulatory role in MED4 by inhibiting RpaA, the master transcriptional activator of dusk genes and HimA, a regulatory target of RpaA previously shown to influence DNA conformation, is highly correlated with global transcriptome-proteome alignment (R2 = 0.67, p < 0.001). We construct stochastic differential equations consistent with genome-wide promotion of transcription by HimA as a plausible mechanism. Our HimA-driven model recapitulates alignment dynamics (R2 = 0.59, p < 0.01) as one plausible explanation. Collectively, our results elucidate how a minimal circadian regulatory program achieves robust coordination across transcriptional, translational, and metabolic levels

systems biology↗

bioRxiv2026

Visualizing micro- nanoplastics in the human brain: Early evidence for roles in microvascular pathology

Our environment has become progressively contaminated with non-biological materials, especially synthetic carbon polymers, plastics. Unsurprisingly some of these make it into the human body as detected by chemical analyses, yet it is unknown whether they cause harm or are innocent bystanders. Building on our observations of glossy deposits and unusual non-biological fluorescent particles in blood vessels, we aimed to determine whether these objects represented plastics. To do this we prepared plastics-enriched pellets from brain and subjected them to pyrolysis gas-chromatography/mass spectroscopy (py-GC/MS), electron microscopy and confocal laser scanning microscopy. Py-GC/MS confirmed the presence of 10 different plastics. Contents of pellets were examined by thin-section EM. We then used laser scanning confocal microscopy to acquire hyperspectral profiles of each type of plastic in suspensions. We found fluorescent particles in all pellets from brains. All 12 plastics in the calibration standard fluoresced. We obtained hyperspectral profiles of single species plastics from industry: Polyethylene, polypropylene and polystyrene. Controls included chemical analysis of brain storage buffer, which had no detectable plastics; and imaging water only and areas on slides lacking tissue. Abundant particles with emission profiles similar to polyethylene and polypropylene decorated the walls of both arterioles and venules. These particles were coincident with glossy deposits we first observed in white matter, suggesting that both features represent plastics. In summary our results demonstrate that synthetic polymers exhibit detectable fluorescence, and particles with similar spectral properties are visible in histologic sections. This opens the door to investigating correlations between plastics and pathology.

pathology↗

bioRxiv2026

Parallel and Divergent Evolution in Pseudomonas aeruginosa Under Constant and Fluctuating Predator-Mediated Selection

Environmental predation is a major driver of bacterial evolution and may indirectly influence virulence through coincidental selection. However, how sustained versus fluctuating predator pressure shapes long-term evolutionary trajectories remains poorly understood. Here, we used experimental evolution to investigate the genetic and phenotypic responses of Pseudomonas aeruginosa to continuous, absent, or fluctuating exposure to the protozoan predator Tetrahymena thermophila over 180 days. Whole-population and isolate-level shotgun metagenomic sequencing revealed fewer mutations over time but increasing frequencies of surviving mutations, consistent with selection, extensive gene-level parallel evolution, and signatures of both positive and purifying selection. Recurrently mutated genes encompassed diverse functional pathways, reflecting both shared and treatment-specific adaptive responses. Despite this parallelism, historical contingency was evident, with starting conditions influencing subsequent evolutionary trajectories. We also observed the emergence of hypermutator lineages, which are frequently recovered from chronic lung infections, suggesting that repeatedly evolving elevated mutation rates may represent a common adaptive strategy of P. aeruginosa across environmental and host-associated settings. Fluctuating predation repeatedly reshaped the adaptive landscape, leading to greater temporal turnover of mutations and a higher accumulation of mutations that ultimately reached fixation than in constant environments. Phenotypic assays revealed widespread divergence in fitness, motility, biofilm formation, siderophore production, protease activity, hemolysis, and cell size, whereas virulence in an invertebrate host model varied among treatments but did not differ significantly. Together, these findings demonstrate that variation in predator-mediated selection reshapes the dynamics and genetic targets of bacterial adaptation, highlighting the roles of ecological context, historical contingency, and hypermutability in driving the evolutionary trajectories of opportunistic pathogens. Significance StatementEnvironmental predators are drivers of bacterial evolution, yet their effects on adaptation remain poorly understood. We used experimental evolution to show that constant and fluctuating protozoan predation produce evolutionary trajectories in Pseudomonas aeruginosa, altering tempo, predictability, and targets of adaptation. Adaptation to predator-present or predator-absent environments shaped evolutionary trajectories, demonstrating importance of historical contingency. Fluctuating predation promoted turnover of mutations as populations adapted to selective pressures. We also observed repeated emergence of hypermutator lineages, a hallmark of chronic infections, suggesting that elevated mutation rates represent a favored adaptive strategy across environmental and host-associated settings. These findings provide insight into the environmental origins of genetic changes commonly associated with opportunistic pathogens, while showing that these changes do not necessarily increase virulence.

evolutionary biology↗

bioRxiv2026

Characterisation of the new microalgal protein xATPA related to the F-type ATP synthase α subunit, from the ecosystem to the molecule

Microalgal metabolism relies on their chloroplasts, and involves both nucleus and plastidial-encoded proteins of various evolutionary origins. The plastidial ATP synthase complex is a key player in photosynthesis, and has been extensively studied in plants. However, our knowledge in other photosynthetic eukaryotes remains limited, despite their importance in marine environments. Here, we report the characterisation of a novel homologue of the F-type ATP synthase alpha subunit, hereby named xATPA, widespread in microalgae but absent from other photosynthetic organisms. Comparisons of xATPA sequences and predicted structures revealed a specific feature, the bump domain, and highlighted the absence of an ATP-binding site. We assessed xATPA prevalence in microalgae in the global ocean using environmental data from Tara Oceans, with a particular focus on diatoms, and demonstrate that its expression is associated with polar summer conditions. Using a reverse genetic approach in the model diatom Phaeodactylum tricornutum, we show that xATPAPt has a plastidial localisation, and that xATPA KO mutants exhibit growth deficiencies in a combination of low temperature, low salinity and constant light, consistent with environmental analysis. Surprisingly, both RNAseq and physiological assays suggest that xATPA is not involved in ATP synthase functions. On the other hand, xATPA interacts with other F1 ATP synthase subunits in vitro, which we suggest forms transient unassembled complexes. This study hence represents a comprehensive analysis of a novel protein from the environment to the lab, and reveals a new player in the plastidial physiology of eukaryotic microalgae.

plant biology↗

bioRxiv2026

Extreme genome reduction selectively retains modular regulatory architecture in Prochlorococcus MED4: conserved transcriptional modules reveal core physiological regulatory programs

Prochlorococcus MED4 is a minimal photoautotroph whose extreme genome streamlining extends to its transcriptional regulatory architecture, yet it dominates high-light oligotrophic surface waters and drives marine carbon cycling. Despite ecological significance, MED4 remains genetically intractable, lacking molecular tools to characterize regulatory mechanisms and construct a transcriptome-wide regulatory map. To address this, we assembled an RNA-seq compendium of 253 samples, including 207 new samples capturing transcriptional responses across three classes of experiments: diverse environmental perturbations, a 24-hour circadian cycle, and phage infection. Using independent component analysis (ICA) applied to 247 quality-filtered samples, we identify 32 independently regulated gene set modules in MED4 (iModulons). By comparison, we previously identified 78 iModulons in the model cyanobacterium Synechococcus elongatus PCC 7942, revealing how dramatically genome reduction has simplified MED4s regulatory architecture. Of the 32 iModulons in MED4, 13 are conserved modules that correlate with experimentally validated transcriptional regulons in PCC 7942, identifying regulatory programs that resisted elimination under extreme selective pressure. These conserved modules reveal regulatory programs governing photosynthesis and light responses (RpaB), circadian rhythms (RpaA), and nutrient assimilation (NtcA, PhoB). Known regulator-specific DNA-binding motifs upstream of genes in conserved modules independently support their identification as regulatory targets. Notably, RpaA governs circadian rhythms through three temporally distinct modules in MED4 versus one in PCC 7942, and the RpaB photoprotection module similarly splits into two. This work uncovers the minimal regulatory core governing photosynthesis, circadian rhythms, and C/N/P metabolism in a globally critical but genetically intractable photoautotroph. This approach offers a generalizable framework for regulatory inference beyond model organisms. ImportanceThe minimal photoautotroph Prochlorococcus MED4 possesses only 28 transcriptional regulators, versus 150+ found in most cyanobacteria, reflecting a genome streamlined by billions of years of natural selection. This streamlining minimized metabolic costs, enabling MED4 to dominate nutrient-depleted oceans today. This raises a fundamental question: which regulatory programs did nature choose to keep? The answer matters, but MED4 cannot be studied by conventional genetics, placing its molecular machinery beyond direct experimental reach. Instead, we use large-scale computational methods to define groups of co-regulated genes in MED4. By comparing MED4 with a genetically tractable model cyanobacterium, we can distinguish which regulatory programs nature preserved from those it discarded. This work reveals the minimal regulatory architecture sufficient to sustain the smallest oxygenic photoautotroph on Earth. The principles uncovered here, distinguishing essential from dispensable regulatory programs in a naturally streamlined organism, inform the design of minimal photosynthetic platforms for biotechnology.

systems biology↗

bioRxiv2026

A critical signaling role for diacylglycerol in phagocytosis of M. tuberculosis

Mycobacterium tuberculosis (Mtb) establishes infection by entering host phagocytes through phagocytosis. While host lipids are known to influence this process, the specific contribution of the signaling lipid diacylglycerol (DAG) remains poorly defined. Here, we identify DAG as a critical regulator of phagocytosis. Disruption of DAG production, through inhibition or genetic deletion of adipose triglyceride lipase (ATGL) and phospholipase C gamma 2 (PLC{gamma}2), two major pathways that generate cellular DAG pools, markedly reduced uptake of both Mtb and zymosan-coated beads. Notably, loss of ATGL or PLC{gamma}2 did not impair receptor trafficking to the cell surface or cargo binding, indicating that DAG is not required for phagocytic recognition or initiation, but instead for a later step in phagosome formation. Mechanistically, cells lacking ATGL or PLC{gamma}2 displayed constitutive phosphoinositide 3-kinase (PI3K) phosphorylation, suggesting that dysregulated intracellular signaling prevents completion of phagocytosis. These findings uncover a previously unappreciated role for DAG biosynthesis in coordinating intracellular signaling required for phagocytosis and provide new insight into host pathways that govern Mtb entry.

microbiology↗

bioRxiv2026

The ITCC-P4 PDX platform of pediatric cancers for preclinical testing

Cancer is the leading cause of disease-related deaths among children in high-income countries. Tumor heterogeneity and lack of mechanism-of-action-based therapeutic options are key challenges to overcome in order to improve pediatric cancer patients survival. Here, we report the EU-IMI-2 funded public-private partnership "ITCC-Pediatric Preclinical Proof-of-Concept Platform" (ITCC-P4), which has built a large repertoire of patient-derived xenograft (PDX) models, representing all major solid pediatric cancer types, for in vivo drug testing. Three-hundred-fifty-three PDX models from diagnostic and relapsed pediatric cancers have been established and molecularly characterized, together with matched germline/tumor samples. As proof-of-concept, we present in vivo drug screening data in neuroblastoma and rhabdomyosarcoma models. PDX data, accessible at http://r2platform.com/itcc-p4, allow the selection of models based on oncogenic drivers and/or potential biomarkers for preclinical testing. Operated by a non-profit entity (www.itccp4.com), this sustainable platform aids academic and industrial researchers in developing and prioritizing innovative therapies for pediatric cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/703023v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@195ba30org.highwire.dtl.DTLVardef@f2c2d9org.highwire.dtl.DTLVardef@1d63f4dorg.highwire.dtl.DTLVardef@d60027_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

Mapping SARS-CoV-2 Nucleocapsid Function with Nanobodies

The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the proteins structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential. ImportanceSARS-CoV-2 and emerging coronaviruses remain a major global health threat, yet critical gaps persist in our understanding of their molecular pathogenesis. The nucleocapsid (N) protein, the most abundantly expressed SARS-CoV-2 antigen, plays essential roles beyond genome packaging, including immune evasion and intracellular organization. Here, we generate and characterize a panel of domain-specific nanobodies (VHHs) that enable precise dissection of Ns functional architecture. Using integrated biochemical, structural, and virological approaches, we uncover distinct mechanisms of viral inhibition, including disruption of phase condensation through a conserved linker region. These findings address long-standing knowledge gaps about a multifunctional viral protein and establish VHHs as powerful, modular tools for probing coronavirus biology, with broad potential for therapeutic, diagnostic, and cell biology applications.

microbiology↗

bioRxiv2026

Spatiotemporal 4D Whole-cell Modeling of a Minimal Autotroph Reveals Central Carbon Metabolism Regulated Locally by Protein Megacomplexes via Post-translational Modifications under Light Disturbance

Photosynthetic microorganisms rely on multiple pathways in central carbon metabolism to adapt to fluctuating light and energy availability across diel cycles. Mechanistic insight into the regulatory dynamics of this adaptation requires integrating processes spanning disparate timescales, from rapid redox-dependent post-translational modifications (PTMs) to slower changes in protein expression and metabolic pathway usage. To address this complexity beyond genome-based inference and traditional modeling, we develop a whole-cell four-dimensional (3D + time) model of the marine cyanobacterium Prochlorococcus marinus MED4 that explicitly represents the spatial organization of enzymatic and molecular processes in central carbon metabolism under light perturbation. We employ a perturbation-based research design to experimentally generate time-series, multi-omics measurements that provide molecular descriptors and cryo-ET images as constraints for this dynamic 4D framework. The integration of experiments and modeling across defined light regimes enables quantitative validation of system-level responses and forecasting under distinct light disturbances. We test the hypothesis that light-dependent redox PTMs regulating the structural assembly of a protein megacomplex, the "dark complex," modulate metabolic flux at a conserved regulatory node of the Calvin-Benson cycle (CBC) in cyanobacteria. Our model shows that subcellular spatial organization buffers rapid light-induced changes in thylakoid reaction rates, which are followed by redox-PTM-mediated sequestration or release of CBC enzymes in the dark complex, ultimately impacting carbon fixation dynamics within carboxysomes. Comparison with an equivalently parameterized well-mixed stochastic model demonstrates that post-translational regulation not only buffers transcriptional noise and diffusion-driven fluctuations but also stabilizes phenotypic outcomes, underscoring the importance of spatial heterogeneity in phenotypic robustness. This ability to probe adaptive, spatiotemporally resolved mechanisms in photosynthetic machinery and central carbon metabolism addresses a critical gap in genotype-to-phenotype inference and expands modeling and design capabilities for understudied or genetically intractable autotrophs such as P. marinus MED4. Significance StatementThis work advances 4D whole-cell modeling by presenting the first spatiotemporal simulation of a photosynthetic autotroph using the Lattice Microbes platform. Using Prochlorococcus marinus MED4, we show that subcellular spatial organization of organelles, diffusion constraints, and redox regulation collectively shape central carbon metabolism across orders of magnitude in space and time. Through a perturbation-based strategy that generates multi-omics data sets over time, we construct and validate a spatially and temporally resolved model of MED4, constrained by high-resolution (10 nm) cryo-electron tomography. Our results highlight the importance of localized biochemical reactions and redox-dependent post-translational modification of enzymes in regulating carbon fixation in a noisy environment under light disturbance. This study establishes a spatiotemporal, whole-cell physiology modeling framework as a transformative tool for uncovering multiscale regulatory responses to environmental gradients.

systems biology↗

bioRxiv2026

PARP1 recruits SPRTN to DNA-protein crosslinks through a conserved poly-ADP-ribose binding domain.

DNA-protein crosslinks (DPCs) are toxic DNA lesions formed by the covalent attachment of proteins to DNA. Failure to resolve DPCs leads to genomic instability, premature aging, and cancer predisposition. Although multiple proteases and the 26S proteasome degrade DPCs, how these lesions are detected and marked for proteolysis remains unclear. Here, we show that poly-(ADP-ribose) polymerases (PARP1/2) sense DPCs and modify them with poly(ADP-ribose) (PAR) to promote repair via a SPRTN-Tdp1 axis. We discovered a Nudix homology domain (NHD) in SPRTN that mediates direct non-covalent PAR binding and is important for DPC repair. Loss of PARP1/2 activity or mutation of the SPRTN NHD leads to sustained DPCs. Single-molecule analysis revealed that SPRTN does not bind efficiently to the DPC, however after the addition of PARP1 in the presence of NAD+, SPRTN binding to the DPC was significantly increased. Our findings establish PARP1/2 enzymes as immediate DPC sensors, reveal PARylation as a signal marking DPCs for SPRTN-dependent degradation, and identify SPRTN as the first PARP-directed protease.

molecular biology↗

bioRxiv2026

Shining a light on the dark Nt-acetylome by integrating omics data

N-terminal acetylation, catalysed by N-terminal acetyltransferases (Nats), is one of the most prevalent protein modifications and is implicated in human diseases. Yet, despite extensive COFRADIC-based proteomics, only [~]5-10% of the proteome has been interrogated, leaving the majority of the Nt-acetylome unexplored. Here, we combined all major COFRADIC datasets with sel-TRAP, a high-sensitivity, orthogonal approach for profiling co-translational Nat targets via selective ribosome purification. This integrated analysis refined the substrate specificities of NatA, NatB, and NatC/E/F and provided the most comprehensive view to date of the canonical human and yeast Nt-acetylomes. Importantly, we also uncovered hundreds of cryptic Nat substrates arising from alternative translation initiation, with unexpected Nt-proteoforms constituting a previously underappreciated source of Nat targets. Collectively, our results revealed the complex landscape of a "dark" Nt-acetylome, the characterization of which, including its functional roles in regulating protein function and in disease, remains a major challenge for future research.

genomics↗

bioRxiv2025

Host and microbial factors influence bacterial colonization of the honey bee gut

The guts of many animals are colonized by host-specific microbes, yet the extent to which host filtering (host-derived constraints) shapes microbial colonization and host specificity remains poorly understood. Here, we used gnotobiotic honey bees (Apis mellifera) as a model system to systematically assess the colonization potential of a phylogenetically and ecologically diverse panel of 56 bacterial strains, spanning native symbionts, opportunistic bee-associated taxa, gut microbes from other bee species, and non-bee environmental isolates. Bacterial load and colonization frequency were quantified by strain-specific qPCR seven days post-inoculation, in monocolonization and in the presence of a synthetic community composed of native honeybee core bacteria. Bacterial load was highest for native strains and declined with increasing phylogenetic distance from native symbionts. Co-colonization with the synthetic community reduces load across all groups, but native strains were least affected. Across strains, completeness of KEGG metabolic pathways correlated with bacterial load in some ecological groups; however, metabolic capacity alone did not fully explain colonization patterns, either in monocolonization or under competitive conditions. A key finding was that in vitro sensitivity to antimicrobial peptide (AMPs; apidaecin, abaecin, defensins, hymenoptaecin) varied widely among strains and was highest in closely related bee-associated bacteria. Notably, even highly successful colonizers such as Gilliamella and Snodgrassella were AMP-sensitive. AMP sensitivity showed a negative correlation with bacterial load, but not with the frequency of host colonization. These findings suggest that AMPs modulate symbiont abundance rather than acting as strict barriers to colonization. Overall, our results reveal that host filtering in the bee gut is multifaceted, integrating immune-mediated barriers, microbial traits, and competitive interactions.

microbiology↗

bioRxiv2025

A Two-Component Regulatory System Mediates Quorum Sensing-Dependent Morphology and Motility Transitions in the Archaeon Haloferax volcanii

Quorum sensing (QS) enables microorganisms--including bacteria, eukaryotes, and viruses--to coordinate collective behaviors in response to population density. Despite their ecological and evolutionary significance, QS mechanisms in Archaea remain poorly characterized. The halophilic archaeon Haloferax volcanii provides a model for archaeal QS, transitioning from motile rods to non-motile disks in a density-dependent response to a secreted disk-forming signal (DFS). To identify components of the DFS regulatory network, we screened for spontaneous mutants that retained motility in DFS-containing soft-agar medium. One candidate, HVO_1357, encodes a predicted response regulator located adjacent to a histidine kinase (HVO_1356) and a second response regulator (HVO_1358), consistent with an extended two-component regulatory system (TCS). Based on our results, these genes encode quorum-sensing associated regulators (Qar), therefore, we propose rename them qarA (HVO_1357), qarB (HVO_1356), and qarC (HVO_1358). Deletion of qarA enabled cells to swim on DFS-containing soft-agar plates and conferred hypermotility on standard soft-agar media; however, these phenotypes were not due to changes in motility-related parameters, but a reduced sensitivity to DFS for induction of the non-motile, disk-shaped state. In contrast, {Delta}qarB and {Delta}qarC strains were non-motile and exhibited premature disk formation during normal growth. Suppressor mutations that restored motility to {Delta}qarB and {Delta}qarC mapped exclusively to qarA, suggesting QarA is the central regulator of this system. Transcriptomic analyses revealed that qarA deletion leads to upregulation of genes involved in motility and rod-shape formation. Together, these findings reveal qarABC as a DFS-responsive regulatory module and represent the first TCS in archaea shown to control QS-dependent behavior. IMPORTANCEArchaea are ubiquitous and play key roles across diverse ecosystems--including human microbiomes--yet little is known about how they communicate with one another and with other organisms, or how these interactions shape their ecological impact. Such intercellular communication, including quorum sensing (QS), allows microorganisms to coordinate behaviors critical for survival, adaptation, and community organization. In this study, we identify the first archaeal two-component regulatory system that is involved in QS-dependent regulation, providing a foundation for understanding how organisms in this domain sense and respond to population cues. By revealing a previously unknown aspect of archaeal biology, this work represents an important step toward understanding how archaeal communication shapes both their physiology and their interactions within complex microbial communities.

microbiology↗

bioRxiv2025

Monomer binding modes of small molecules that modulate the kinetics of hIAPP amyloid formation

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils that accumulate in pancreatic {beta}-cells of Type II Diabetes (T2D) patients. Recently discovered small molecules that modulate the kinetics of hIAPP amyloid formation could serve as starting points for developing T2D therapeutics, but no structural or mechanistic rationale exists to explain their binding mechanisms or effects on hIAPP aggregation pathways. Here, we utilize all-atom molecular dynamics computer simulations to elucidate the binding mechanisms of an hIAPP aggregation inhibitor (YX-I-1) and an aggregation accelerator (YX-A-1) to disordered monomers of wild-type hIAPP and the naturally occurring pathogenic S20G hIAPP variant associated with early-onset T2D. We observe that the inhibitor exhibits substantially higher affinity for monomeric wild-type hIAPP than the accelerator, consistent with previously reported biophysical experiments. We dissect the interactions that stabilize binding of each molecule to wild-type and S20G hIAPP and characterize conformational changes that occur upon ligand binding. In all ligand-bound ensembles, distinct fragments of YX-I-1 and YX-A-1 are sequestered from solvent upon binding while other fragments remain solvent-exposed. Based on our simulations we hypothesize that buried ligand moieties confer hIAPP monomer binding affinity while solvent-exposed ligand moieties modulate the kinetics of intermolecular association of bound hIAPP into higher-order oligomeric intermediates on amyloid aggregation pathways.

biophysics↗

bioRxiv2025

The pH gradient contributes to persistence in Mycobacterium tuberculosis

Tuberculosis (TB) remains difficult to cure due in part to poorly defined drug-tolerant persister cells formed by Mycobacterium tuberculosis (Mtb), which survive antibiotic treatment without evidence of genetic resistance. To better define this phenotype, we screened 2,336 FDA-approved drugs for compounds that target persistence. Unexpectedly, we identified a strong inducer of drug tolerance -- the antiparasitic niclosamide (NCA), which is known to disrupt proton motive force. In contrast to earlier reports that it harbors promising anti-TB activity, we found that NCA protected Mtb from bactericidal doses of isoniazid, rifampicin, and other standard TB drugs. Investigating further, we showed that disruption of the pH gradient and consequent intracellular acidification is needed to induce tolerance, while disruption of membrane potential is not, and also that protection is tunable by external pH. Transcriptomic analysis of these chemically-induced persister (CIP) cells implicated specific genes in this phenotype, and targeted knockdowns confirmed roles for three genes in either promoting or mitigating the tolerance state. These findings highlight that chemical disruption of the pH gradient is a facile and rapid means to induce drug tolerance, offering a potentially useful tool to probe persister biology in TB and other infectious diseases.

microbiology↗

bioRxiv2025

The Global Canopy Atlas: analysis-ready maps of 3D structure for the world's woody ecosystems

Woody canopies regulate exchanges of energy, water and carbon, and their three-dimensional (3D) structure supports much of terrestrial biodiversity. Remote sensing technologies such as airborne laser scanning (ALS) now enable the 3D mapping of entire landscapes. However, we lack the large, harmonized and geographically representative ALS collections needed to build a global picture of woody ecosystem structure. To address this challenge, we developed the Global Canopy Atlas (GCA): 3,458 ALS acquisitions transformed into standardized and analysis-ready maps of canopy height and elevation at 1 m2 resolution. The GCA covers 56,554 km2 across all major biomes. 19% of this area has been scanned multiple times, and 87% of all GCA products are openly available, covering 95% of the total area. To showcase its wide range of applications, we applied the GCA in three case studies. First, we validated three global satellite-derived canopy height maps, finding poor performance at native resolution (1-30 m, R2 < 0.38) and moderate performance at 250 m resolution (R2 < 0.65). Second, analyzing global patterns in canopy gap size frequency we discovered an unexpectedly large variation of power law exponents from branch to stand level ( = 1.52 to 2.38), pointing to a fundamental scale-dependence of forest structure. Third, we developed a framework to standardize forest turnover quantification from multi-source, multi-temporal ALS. In a temperate forest in North America it revealed that 21% of canopy gaps closed within 12 years of opening and would thus be missed by infrequent monitoring. As demonstrated by these case studies, the GCA provides a novel data source for ecologists, foresters, remote sensing scientists and the ecosystem modelling community that substantially advances our ability to understand the structure and dynamics of woody ecosystems at global scales.

ecology↗