Biology subjects

Thomas, H.

Publications and source records attributed to Thomas, H..

At least 19 recordsLinked to original sources

bioRxiv2026

A Transferable Genomic Language Model Framework for Fungal Gene Essentiality Prediction

Predicting biological function from genomic sequence remains a major challenge in computational and systems biology. Here, we tested whether the genomic language model Evo2, which encodes context-dependent DNA sequence patterns into embeddings, enables prediction of essential genes in fungi, a phenotype central to fungal biology and antifungal target discovery. We found that model performance was constrained not by the type or complexity of the downstream classifier, but by the biological information contained in Evo2 DNA embeddings. Specifically, the information recoverable from these embeddings progressively declined for biological features further downstream of DNA sequence, revealing a bottleneck for predicting higher-order cellular phenotypes. We alleviated this bottleneck by integrating Evo2 embeddings with two sequence-informed, system-level features: ortholog-based essentiality and protein-protein interactions. The multimodal framework demonstrated consistent performance both within and across three evolutionarily divergent yeasts (Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe), and its predictions were supported by published experimental evidence when transferred to the filamentous mold Aspergillus fumigatus. These results establish our framework as a transferrable tool for predicting essential genes across fungal genomes, including species with limited or no experimentally determined essentially data.

bioinformatics↗

bioRxiv2026

PIGSTI: a modular, reproducible pipeline for detecting species identity, pathogens, and microbes from animal palaeogenomic data

Ancient genomics has enabled discovery of diverse pathogens across various time periods, host species, and material types. However, existing palaeogenomic pipelines predominantly focus on screening data from human hosts, or do not incorporate microbial screening methodologies. We present PIGSTI (Pathogen anImal Genome Sequence ToolkIt), a bioinformatic pipeline specifically designed for both the initial screening and subsequent detection of pathogens in shotgun sequencing data from ancient animal remains. PIGSTI's integrated Snakemake workflow performs both host detection, genome mapping and pathogen identification, generating outputs suitable for population genetics and phylogenetic analyses. Testing on 952 newly sequenced and publicly available animal palaeogenomic datasets, we identified ~15 ancient zoonotic and animal pathogens with high confidence, including the first documented case of Rickettsia felis and Leptospira borgpetersenii in an ancient animal. Our results demonstrate PIGSTI's utility for screening pathogen diversity in ancient animal hosts and reconstructing historical host-pathogen relationships.

genomics↗

bioRxiv2026

A hierarchical regulatory cascade defines the temporal window of natural transformation in Staphylococcus aureus

Natural transformation (NT), a major mechanism of horizontal gene transfer, promotes bacterial genome plasticity and contributes to the dissemination of antibiotic resistance. NT requires the development of competence, a transient and tightly regulated physiological state that is induced by environmental cues. Although competence has recently been demonstrated in the human pathogen Staphylococcus aureus, the regulatory architecture and temporal coordination governing its development remain poorly understood. Using a highly sensitive luciferase-based transcriptional reporter, we demonstrate that NT gene expression in S. aureus is transient, thereby defining a temporal window during which DNA uptake and recombination can occur. We further identify two classes of NT genes with distinct regulatory requirements and activation kinetics: Class I genes require both SigH and ComK1 and are activated approximately 2.5 hours later than Class II genes, which are regulated exclusively by ComK1. Mechanistically, we demonstrate that ComK1 activates sigH transcription, establishing a hierarchical regulatory cascade that explains its essential role in Class I gene expression. Finally, time-resolved transcriptomic analyses identify the global regulator CodY as an early regulator of competence that differentially controls comK1 and sigH transcription, thereby linking metabolic adaptation to competence development. Together, our findings define the regulatory architecture that governs competence development in S. aureus and reveal how a hierarchical cascade integrates environmental and metabolic cues to restrict horizontal gene transfer to a precise developmental window. This work provides a mechanistic framework for understanding how bacteria coordinate physiological adaptation with genome diversification through NT. Author summaryBacteria constantly evolve by acquiring new genetic information from their environment. One important mechanism driving this process is natural transformation, which allows bacterial cells to take up and integrate exogenous DNA into their genomes. However, this process is not continuously active: bacteria must first enter a specialized physiological state called competence. How pathogens control the development of competence remain poorly understood. In this study, we investigate how the human pathogen Staphylococcus aureus regulates natural transformation. Using a highly sensitive reporter system that allows us to monitor gene expression over time, we show that competence is activated only during a limited period, creating a defined window during which bacteria can acquire new DNA sequences. We uncover a hierarchical regulatory cascade involving the transcription factors ComK1 and SigH that controls the sequential activation of natural transformation genes. We further identify the metabolic regulator CodY as an upstream regulator that connects bacterial physiological state with the initiation of competence. Our findings reveal that natural transformation is not a random or continuous process, but a precisely timed developmental program controlled by multiple regulatory layers. By defining how S. aureus coordinates environmental sensing, metabolism, and horizontal gene transfer, this work provides new insight into how bacterial pathogens generate genetic diversity and adapt to changing environments, including conditions that may promote the emergence of antibiotic resistance.

microbiology↗

bioRxiv2025

Nek family members regulate Rad54 during homologous recombination in developing mice

Homologous recombination (HR) represents an important pathway for repairing DNA double-strand breaks (DSBs) but HR factors, including RAD51, also serve to protect and restart stalled replication forks. RAD54 functions during DSB repair where it removes RAD51 from duplex DNA including heteroduplex DNA which is formed during D-loop formation. This allows subsequent DNA repair synthesis and completion of the HR process. We have previously suggested that RAD54s activity is regulated by never-in-mitosis-gene A (NIMA) related kinase 1 (NEK1) in a cell cycle-specific manner to promote RAD51 removal and HR in G2 phase without interfering with RAD51s fork stabilization role during S phase. Here, we establish that Nek1 regulates the phosphorylation of Rad54 at Ser572 (S572) to promote HR in vivo in adult mice and in vitro in fibroblasts derived from such mice. In contrast, embryonic mice and fibroblasts derived from them do not require Nek1 for HR. We further show that HR requires Rad54 phosphorylation at S572 both in embryonic and adult fibroblasts and that this is mediated in embryonic fibroblasts by Nek3 and Nek5 instead of Nek1. Thus, our work identifies a developmental change in the regulation of HR and uncovers two new factors involved in this process.

cell biology↗

bioRxiv2025

Arhgef18 is a component of the outer limiting membrane required for retinal homeostasis

Biallelic ARHGEF18 mutations cause human adult-onset retinal degeneration. We now find that Arhgef18 associates with the retinal outer limiting membrane (OLM), an adherens junction between Muller glia and photoreceptors. Arhgef18 knockout in Muller glial cells led to OLM disruption and vision loss by P60. While mice developed morphologically normal retinas, retinal rosettes started to form by P8, and retinas then progressively degenerated with OLM disintegration, retinal thinning, and vascular leakage. ARHGEF18/p114RhoGEF depletion in Muller cells in culture confirmed disruption of junctional recruitment of OLM proteins. Depletion also induced activation of NF-{kappa}B and {beta}-catenin signalling, activation of the multifunctional kinase Tank-binding kinase 1 (TBK1) and reduced mitochondrial activity. TBK1 inhibition or directly supporting mitochondrial activity with nicotinamide attenuated NF-{kappa}B and {beta}-catenin signalling and rescued mitochondrial activity. Thus, Arhgef18 is essential for OLM maintenance, and its disruption leads to activation of mechanisms that are targetable for possible therapeutic approaches.

cell biology↗

bioRxiv2025

Absolute Quantification of Fluorescent Protein Fusions by Proteomics

Fusions with fluorescence proteins (FPs) play a pivotal role in experimental biology because of their sensitive and spatially precise visualization by spectroscopy. However, observed fluorescence is not always proportional to their molar abundance. Only a fraction of the fusion protein containing the mature fluorescence chromophore is detectable by spectroscopy and there is no generic method for estimating its molar abundance. We developed a fluorescence-independent mass-spectrometry based approach for accurate absolute sub-femtomole quantification of FP-fusions that also estimates the protein faction having fully matured chromophore. The method exploits an isotopically labelled 68 kDa recombinant protein standard containing peptide proxies for 6 prototypical FPs (mCherry, mScarlet-I, mKate2, EGFP, mNeonGreen and Dendra2) and two self-labelling (Halo- and SNAP-) tags. It enables quantification of proteins fused to any of more than 70 FPs or self-labelling tags. It is versatile, robust, precise and can be used broadly for the absolute quantification of fluorescent fusions in in-vivo and in-vitro cellular systems. As case study we combined mass spectrometry with fluorescence spectroscopy to study expression kinetics of FP fusions in cell-free systems. Molar concentrations of the expressed fusion protein, its fraction containing the mature fluorescent chromophore and of the fluorescent protein were integrated into a mathematical model to obtain kinetic rates of translation, chromophore maturation and folding.

biochemistry↗

bioRxiv2025

A probiotic bacterium modulates antitumor γδ T-cell responses in lung cancer

The link between the intestinal microflora and cancer outcomes has been recognized for over a decade. Several recent studies have demonstrated that the gut microbiome is associated with the efficiency of T-cell checkpoint blockade therapy for cancer raising interest in strategies to harness this effect via consumption of live microorganisms (probiotics). The probiotic Clostridium butyricum strain MIYAIRI 588 (CBM588) enhances both response rates and overall survival in patients receiving immune checkpoint blockade therapy for non-small cell lung cancer and metastatic renal cell carcinoma but the mechanism underpinning this benefit remains unclear. Here, we show that CBM588 spores induce a population of V{gamma}9V{delta}2 T-cells from the peripheral blood of healthy donors and lung cancer patients. A subset of these T-cells responded to, and directly lysed, cancer cell lines via a butyrophilin 3A-dependant mechanism. In patients taking CBM588 alongside checkpoint blockade, peripheral V{delta}2 T-cells expressed the activation marker CD69 more frequently than those receiving checkpoint blockade alone and the frequency of V{delta}2CD69 cells increased following initiation of CBM588 treatment (p = 0.0041). Our findings provide a potential mechanism by which the manipulation of the intestinal microflora might directly contribute to cancer prognosis by activating immune effector cells with intrinsic anticancer properties.

immunology↗

bioRxiv2025

Functional synaptic connectivity of engrafted spinal cord neurons with locomotor circuitry in the injured spinal cord

Spinal cord injury (SCI) results in significant neurological deficits, with no currently available curative therapies. Neural progenitor cell (NPC) transplantation has emerged as a promising approach for neural repair, as graft-derived neurons (GDNs) can integrate into the host spinal cord and support axon regeneration. However, the mechanisms underlying functional recovery remain poorly understood. In this study, we investigate the synaptic integration of NPC-derived neurons into locomotor circuits, the projection patterns of distinct neuronal subtypes, and their potential to modulate motor circuit activity. Using transsynaptic tracing in a mouse thoracic contusion SCI model, we found that NPC-derived neurons form synaptic connections with host locomotor circuits, albeit at low frequencies. Furthermore, we mapped the axon projections of V0C and V2a interneurons, revealing distinct termination patterns within host spinal cord laminae. To assess functional integration, we employed chemogenetic activation of GDNs, which induced muscle activity in a subset of transplanted animals. However, NPC transplantation alone did not significantly improve locomotor recovery, highlighting a key challenge in the field. Our findings suggest that while GDNs can integrate into host circuits and modulate motor activity, synaptic connectivity remains a limiting factor in functional recovery. Future studies should focus on enhancing graft-host connectivity and optimizing transplantation strategies to maximize therapeutic benefits for SCI.

neuroscience↗

bioRxiv2024

Deep-learning and analytical models give distinct results for the brain structure-function relationship in health and in psychosis

Understanding the intricate relationship between brain structure and function is a cornerstone challenge in neuroscience, critical for deciphering the mechanisms that underlie healthy and pathological brain function. In this work, we present a comprehensive framework for mapping structural connectivity measured via diffusion-MRI to resting-state functional connectivity measured via magnetoencephalography, utilizing a deep-learning model based on a Graph Multi-Head Attention AutoEncoder. We compare the results to those from an analytical model that utilizes shortest-path-length and search-information communication mechanisms. The deep-learning model outperformed the analytical model in predicting functional connectivity in healthy participants at the individual level, achieving mean correlation coefficients higher than 0.8 in the alpha and beta frequency bands. Our results imply that human brain structural connectivity and electrophysiological functional connectivity are tightly coupled. The two models suggested distinct structure-function coupling in people with psychosis compared to healthy participants (p < 2 x 10-4 for the deep-learning model, p < 3 x 10-3 in the delta band for the analytical model). Importantly, the alterations in the structure-function relationship were much more pronounced than any structure-specific or function-specific alterations observed in the psychosis participants. The findings demonstrate that analytical algorithms effectively model communication between brain areas in psychosis patients within the delta and theta bands, whereas more sophisticated models are necessary to capture the dynamics in the alpha and beta band.

neuroscience↗

bioRxiv2024

Optimised proteomic analysis of insulin granules from MIN6 β-cells identifies Scamp3, a novel regulator of insulin secretion and content

Pancreatic {beta}-cells in the islets of Langerhans are key to maintaining glucose homeostasis, by secreting the peptide hormone insulin. Insulin is packaged within vesicles named insulin secretory granules (ISGs), that have recently been considered to have intrinsic structures and proteins that regulate insulin granule maturation, trafficking, and secretion. Previously, studies have identified a handful of novel ISG-associated proteins using different separation techniques. Here, this study combines an optimized ISG isolation technique and mass spectrometry-based proteomics, with an unbiased protein correlation profiling and targeted machine learning approach to uncover 211 ISG-associated proteins. Five of these proteins: Syntaxin-7, Synaptophysin, Synaptotagmin-13, Zinc transporter ZIP8 and SCAMP3 have not been previously ISG-associated. Through colocalization analysis of confocal imaging we validate the association of these proteins to the ISG in MIN6 and human {beta}-cells. We further validate the role for one (SCAMP3) in regulating insulin storage and secretion from {beta}-cells for the first time. SCAMP3 knock-down INS-1 cells show a reduction in insulin content and dysfunctional insulin secretion. These data provide the basis for future investigation into {beta}-cell biology and the regulation of insulin secretion. Article HighlightsO_LIWhy did we undertake this study? We undertook this study to optimize insulin granule isolation techniques alongside enhanced proteomics analyses to establish the first published murine insulin granule proteome. C_LIO_LIWhat is the specific question(s) we wanted to answer? We aimed to specifically answer and investigate what proteins are present on insulin granules from MIN6 cells to further our understanding of insulin granule biogenesis, trafficking, and secretion. C_LIO_LIWhat did we find? We find and validate the presence of 5 novel insulin granule-associated proteins. C_LIO_LIWhat are the implications of our findings? An extensive proteomics analysis of MIN6 insulin granules and implicate Scamp3 as a novel protein that regulates insulin content and secretion in beta-cells. C_LI

cell biology↗

bioRxiv2023

CZ CELLxGENE Discover: A single-cell data platform for scalable exploration, analysis and modeling of aggregated data

Hundreds of millions of single cells have been analyzed to date using high throughput transcriptomic methods, thanks to technological advances driving the increasingly rapid generation of single-cell data. This provides an exciting opportunity for unlocking new insights into health and disease, made possible by meta-analysis that span diverse datasets building on recent advances in large language models and other machine learning approaches. Despite the promise of these and emerging analytical tools for analyzing large amounts of data, a major challenge remains the sheer number of datasets and inconsistent format, data models and accessibility. Many datasets are available via unique portals platforms that often lack interoperability. Here, we present CZ CellxGene Discover (cellxgene.cziscience.com), a data platform that provides curated and interoperable data. This single-cell data resource, available via a free-to-use online data portal, hosts a growing corpus of community contributed data that spans more than 50 million unique cells. Curated, standardized, and associated with consistent cell-level metadata, this collection of interoperable single-cell transcriptomic data is the largest of its kind. A suite of tools and features enables accessibility and reusability of the data via both computational and visual interfaces to allow researchers to rapidly explore individual datasets and perform cross-corpus analysis. This functionality is enabling meta-analyses of tens of millions of cells across studies and tissues and providing global views of human cells at the resolution of single cells.

cell biology↗

bioRxiv2023

Tuberculosis susceptibility in genetically diverse mice reveals functional diversity of neutrophils

Tuberculosis (TB) is a heterogenous disease in humans with individuals exhibiting a wide range of susceptibility. This heterogeneity is not captured by standard laboratory mouse lines. We used a new collection of 19 wild-derived inbred mouse lines collected from diverse geographic sites to identify novel phenotypes during Mycobacterium tuberculosis (Mtb) infection. Wild derived mice have heterogenous immune responses to infection that result in differential ability to control disease at early timepoints. Correlation analysis with multiple parameters including sex, weight, and cellular immune responses in the lungs revealed that enhanced control of infection is associated with increased numbers of CD4 T cells, CD8 T cells and B cells. Surprisingly, we did not observe strong correlations between IFN-{gamma} production and control of infection. Although in most lines high neutrophils were associated with susceptibility, we identified a mouse line that harbors high neutrophils numbers yet controls infection. Using single-cell RNA sequencing, we identified a novel neutrophil signature associated with failure to control infection.

immunology↗

bioRxiv2023

The impacts of contemporary logging after 250 years of deforestation and degradation on forest-dependent threatened species

Despite the importance of safeguarding forests and woodlands for achieving global climate and biodiversity agendas, logging continues across most forested countries. Forestry advocates often claim logging has minimal impacts, but rarely consider the cumulative threat deforestation and degradation has had, and continue to have, on species. Using New South Wales (Australia) as a case study, we quantify the extent of deforestation and degradation from 1750 - current. Using these estimates of overall loss as a baseline, we then quantify the relative extent of contemporary (2000 - 2022) logging and the condition of the remaining native forest and woodland (quantified by measuring the similarity of a current ecosystem to a historical reference state with high ecological integrity). Using these data, we measure the impacts on distinct vegetation types and on 484 terrestrial forest-dependent now-threatened species. We show that more than half (29 million ha) of pre-1750 (pre-European colonization of Australia) native forest and woodland vegetation in NSW has been lost. Of the remaining 25 million ha, 9 million ha is degraded. We found contemporary degradation from logging affected 244 forest-dependent now-threatened species that had already been affected by this historical deforestation and degradation, but the impacts varied across species and vegetation types. We found that 70 now-threatened species that were impacted by historical deforestation and degradation and continue to be impacted by logging, now have [≤]50% of their pre-1750 extent remaining that is intact (with three species now having <20%). By quantifying the historical impacts of deforestation and degradation, our research sets the impact of contemporary degradation from logging in perspective and highlights shortfalls in current environmental assessments that fail to consider appropriate baselines when reporting on overall impact. Future land management decisions need to consider not only the extent of remaining habitat based on pre-1750 extents, but also its condition. Article impact statementThe impact of logging needs to be placed in perspective by considering past losses and degradation due to human land use decisions.

ecology↗

bioRxiv2023

Extra-islet expression of islet antigen boosts T-cell exhaustion to prevent autoimmune diabetes

Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (TPEX) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear if and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using non-obese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs. To examine the impact of antigen on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen presenting cells. Antigen exposure in the extra-islet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce IFN{gamma}, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes.

immunology↗

bioRxiv2022

Integrated PET and confocal imaging informs a functional timeline for the dynamic process of vascular reconnection during grafting.

Grafting is a widely used agricultural technique that involves the physical joining of separate plant parts so they form a unified vascular system, enabling beneficial traits from independent genotypes to be captured in a single plant. This simple, yet powerful tool has been used for thousands of years to improve abiotic and biotic stress tolerance, enhance yield, and alter plant architecture in diverse crop systems. Despite the global importance and ancient history of grafting, our understanding of the fundamental biological processes that make this technique successful remains limited, making it difficult to efficiently expand on new genotypic graft combinations. One of the key determinants of successful grafting is the formation of the graft junction, an anatomically unique region where xylem and phloem strands connect between newly joined plant parts to form a unified vascular system. Here, we use an integrated imaging approach to establish a spatiotemporal framework for graft junction formation in the model crop Solanum lycopersicum (tomato), a plant that is commonly grafted worldwide to boost yield and improve abiotic and biotic stress resistance. By combining Positron Emission Tomography (PET), a technique that enables the spatio-temporal tracking of radiolabeled molecules, with high-resolution laser scanning confocal microscopy (LSCM), we are able to merge detailed, anatomical differentiation of the graft junction with a quantitative timeline for when xylem and phloem connections are functionally re-established. In this timeline, we identify a 72-hour window when anatomically connected xylem and phloem strands regain functional capacity, with phloem restoration typically preceding xylem restoration by about 24-hours. Furthermore, we identify heterogeneity in this developmental and physiological timeline that corresponds with microvariability in the physical contact between newly joined rootstock-scion tissues. Our integration of PET and confocal imaging technologies provides a spatio-temporal timeline that will enable future investigations into cellular and tissue patterning events that underlie successful versus failed vascular restoration across the graft junction.

plant biology↗

bioRxiv2022

Inhibition of Phosphodiesterase 12 results in Antiviral Activity against several RNA Viruses including SARS-CoV-2.

Phosphodiesterase 12 (PDE12) is a negative regulator of the type 1 interferon (IFN) response and here we show that PDE12 inhibitors (lead compounds 63 and 17) are associated with increased RNAseL activity, are well tolerated at the therapeutic range and inhibit, both in vitro and in vivo, the replication of several RNA viruses including hepatitis C virus (HCV), dengue virus (DENV), West Nile Virus (WNV) and SARS-CoV-2.

microbiology↗

bioRxiv2022

Anatomical and biophysical characterization of intergenic graft-incompatibility within the Solanoideae

Interspecies grafting is a technique that allows beneficial shoot and root combinations from separate species to be combined into a single organism. Despite its relevance to agricultural production, little is known about the determinants of graft compatibility. One hypothesis for compatibility revolves around the taxonomic degree of relatedness between the two plants. To test how phylogenetic distance affects interspecific graft compatibility within the economically important Solanaceae sub-family, Solanoideae, we characterized the anatomical and biophysical integrity of graft junctions for graft combinations made between four species: tomato (Solanum lycopersicum), eggplant (Solanum melongena), pepper (Capsicum annuum), and groundcherry (Physalis pubescens). We analyzed the survival, growth, and junction integrity via bend tests, and imaged the cellular composition of the graft junctions in order to deduce the status of vascular connectivity across the junction. Utilizing these techniques, we were able to quantitatively assess the degree to which each interspecific combination exhibits compatibility. Despite the fact that most of our graft combinations exhibited high survival rates, we show that only tomato and eggplant heterografts are truly compatible. Unlike incompatible grafts, the formation of reconnected vascular tissue within the tomato and eggplant heterografts contributed to biophysically stable grafts that were resistant to snapping. Furthermore, we identified 10 graft combinations that show delayed incompatibility, providing a useful, economically relevant system to pursue deeper work into genetic and genomic determinants of graft compatibility. This work provides new evidence indicating that graft compatibility may be limited to intrageneric combinations within the Solanoideae subfamily. Further research using more extensive graft combinations amongst Solanaceous species can be used to test whether our hypothesis broadly applies to this family.

plant biology↗

bioRxiv2022

Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR

Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex, a first-in-class molecular glue. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809).

biochemistry↗