Biology subjects

Walker, G.

Publications and source records attributed to Walker, G..

14 recordsLinked to original sources

bioRxiv2026

Proteolytic control of the SARS-CoV-2 furin cleavage site defines the phenotypic evolution from the pandemic to endemic state

The successive emergence of SARS-CoV-2 variants with altered tissue tropism has progressively decoupled transmissibility from lower respiratory tract pathogenicity. Omicron lineages transmit exceptionally well whilst limiting severe lung disease. Here, we demonstrate that this phenotype has evolved through two temporally distinct tissue-specific proteolytic controls at the Spike (S) furin cleavage site (FCS). At the virion level, cell-type-dependent furin-mediated FCS hyper-cleavage depletes S in lung but not nasal epithelial cells. At the infected cell membrane during cell-cell spread, TMPRSS2 mediates S FCS cleavage but is negatively regulated in the presence of ACE2. Tissue-specific solute carriers SLC6A19 and SLC6A20 sequester ACE2, thereby relieving inhibition of TMPRSS2 and enabling S FCS cleavage during cell-cell spread. Critically, whilst all SARS-CoV-2 lineages benefit from this latter pathway, Omicron variants have evolved exclusive dependence on it, a strategic consolidation that focuses S proteolytic activation to TMPRSS2 alone. The combined outcomes of S protein regulation across viral and cell membranes reveal how tissue-specific proteolytic optimisation drives Omicron's transmission fitness advantage in the upper respiratory tract but at the cost of heavy attenuation in the lower respiratory tract.

microbiology↗

bioRxiv2026

Species and strain diversity in Staphylococcus drive divergent host responses in human skin

The skin microbiome regulates key skin processes, yet the functional diversity of a dominant genus, Staphylococcus, remains poorly resolved at the strain level for multiple species across its pathogenic and commensal continuum. It is likely that Staphylococcus effects on skin are diverse at these finest taxonomic resolutions, but current skin models lack the physiological relevance and scalability needed to profile this diversity. Using an organotypic 3D human skin model (reconstructed human epidermis, RHE), we profiled skin responses to 187 Staphylococcus strains across seven dominant species. Canonically pathogenic species (e.g., S. aureus) induced broad inflammatory responses, whereas prototypical commensal species (e.g., S. hominis) elicited more nuanced effects on innate immune and skin barrier responses. Strikingly, S. epidermidis displayed pronounced strain-level heterogeneity, with subsets inducing either commensal or pathogen-like responses despite lacking canonical virulence factors, suggesting pleiotropic effects. Comparative genomics, dual-transcriptomics, untargeted metabolomics, and growth phenotyping revealed species- and strain-specific traits underlying these differential effects on RHE, including the presence of select cell surface proteins and differential arginine metabolism. Together, our study provides the first high-throughput, species- and strain-resolved analysis of skin-Staphylococcus interactions, offering mechanistic insights and a platform for microbiome-based strategies to modulate skin inflammation and diseases. One-line summaryHigh-throughput profiling of Staphylococcus in a human skin model shows that species- and strain-level diversity underlies a continuum of host barrier and immune responses.

microbiology↗

bioRxiv2026

Heterologous Rieske non-heme iron monooxygenases enable efficient microbial conversion of lignin guaiacol to adipic acid

Adipic acid (1,6-hexanedioic acid) is a key building block for nylon-6,6, a widely used polymer in the global chemical industry. Current industrial production relies on petrochemical feedstocks and nitric acid oxidation of cyclohexane/cyclohexanol mixtures, releasing nitrous oxide, a potent greenhouse gas. Biotechnological routes offer sustainable alternatives but have been limited by low yields or reliance on multi-strain systems. Here we report a one-pot, single-strain microbial process for the efficient conversion of guaiacol - a lignin derived aromatic - into adipic acid. By integrating heterologous Rieske non-heme iron monooxygenases from Cupriavidus necator N-1 with systematic process optimisations in engineered Escherichia coli, we achieve near-quantitative conversion with 97% yield and titres of 1.5 g/L in aqueous, lab-scale reactions. This work demonstrates a novel and efficient strategy for lignin valorisation through engineered microbial synthesis, providing a new sustainable and scalable route to adipic acid.

synthetic biology↗

bioRxiv2025

A painless nerve growth factor variant uncouples nociceptive and neurotrophic TrkA signaling

Nerve growth factor (NGF) binding to the receptor tyrosine kinase, TrkA, drives neurotrophic signaling essential for neuronal development and survival. This interaction simultaneously drives peripheral pain, making this pathway an attractive but complicated therapeutic target for chronic pain. By integrating single-molecule microscopy, structural and electrophysiology analyses, with a human NGF variant, NGFpainless, which retains neurotrophic effects but abolishes pain, we delineate the molecular mechanisms that bias TrkA signaling towards neurotrophic functions without triggering nociception. We show that, unlike wild-type NGF, NGFpainless fails to sensitize TRPV1 channels to capsaicin, thus disengaging TrkA from the nociceptive pathway. We further show that this selective loss of nociceptive TrkA signaling by NGFpainless results from its reduced ability to activate PLC{gamma}1 and trigger calcium release compared to NGF, while still preserving the ERK and AKT signaling essential for neurotrophic functions. This biased signaling arises from reduced electrostatic complementarity at the TrkA:NGFpainless complex interface, which shortens the lifetime of this functional complex on native membranes. Mutations in TrkA that restore the electrostatic complementarity at the TrkA:NGFpainless interface eliminate biased signaling. This mechanistic understanding of TrkA binding by NGFpainless, and how it differs from NGF, will spur the development of two therapeutic classes of molecules - one that selectively suppresses nociceptive signaling while preserving neurotrophic functions in chronic pain, and another that enhances neurotrophic activity without evoking peripheral pain in neurodegenerative conditions.

neuroscience↗

bioRxiv2025

Lipid Nanoparticle Delivery of Mesenchymal Stromal Cell-Derived microRNA 187-3p as a First-in-Class Therapy for Myocardial Dysfunction in Sepsis

BackgroundSepsis-induced myocardial dysfunction is a common and critical complication of sepsis. Extracellular vesicles (EVs) from clonally expanded immortalized mesenchymal stromal cells (ciMSCs) contain microRNAs that may be exploited as therapy. MethodsIn mouse models of septic cardiomyopathy induced by caecum ligation and puncture, cardiac function was determined by invasive and echocardiographic assessment. Primary cardiomyocytes derived from foetal murine and human adult ventricular tissue, as well as murine hearts were used for mechanistic studies. Studies using post-mortem human hearts or patient plasma, and clinical and echocardiographic measurements were used to establish translational relevance. ResultsIn preclinical models of sepsis, intravenous administration of either MSCs or ciMSC-EVs, given after the induction of sepsis, prevented a decrease in myocardial ejection fraction, ventricular inflammation, and mortality compared to placebo or platelet-derived control EVs. EV-microRNA sequencing identified enrichment for microRNA-187a-3p (miR-187) in ciMSC-EVs. miR-187 is anti-inflammatory; with interleukin-6 (IL-6) as its major target. Intravenous delivery of lipid nanoparticle (LNP) encapsulated miR-187 improved cardiac function, reduced inflammation, and enhanced survival of septic mice. In cardiomyocytes and in murine hearts, LNP-miR-187 reduces inflammation and expression of myocardial transcription factors linked to fetal gene reactivation in failing septic hearts. In human septic hearts, low circulating miR-187 levels correlate with reduced cardiac function and high sequential organ failure assessment (SOFA) scores. ConclusionThese findings support the development of first-in-class, cell-free, miRNA-based therapy as a novel approach to treat sepsis-induced cardiomyopathy to address a critical gap in sepsis care. One Sentence SummarymiR-based therapy for sepsis The Clinical PerspectiveA. What is NEW? Sepsis accounts for 1 in 5 deaths worldwide. Here, we demonstrate that sepsis-induced myocardial dysfunction represents a discrete, targetable sepsis-trait -- a distinct biological abnormality characterized by cardiomyocyte inflammation and fetal gene reactivation. This component contributes to the propagation of organ dysfunction and overall mortality and may respond to focused epigenetic-based interventions. B. What are the Clinical implications? Currently, there are no effective treatments to reduce, limit, or reverse the immune dysfunction component of sepsis that contributes to multiorgan failure, such as sepsis-induced cardiomyopathy. We identify miR-187 as a clinically relevant post-transcriptional regulator of cardiac inflammation and cardiomyocyte gene expression. Intravenous delivery of miR-187 encapsulated in a lipid nanoparticle (LNP) represents a fundamentally distinct, effective and pathogen-agnostic approach to correcting sepsis-induced cardiac dysfunction through modulation of cardiomyocyte inflammatory and metabolic pathways.

molecular biology↗

bioRxiv2025

Missense3D-PTMdb an interactive web resource to explore and visualize genetic variants and post-translational modifications sites (PTMs) using AlphaFold 3D models

Only a fraction of the >11 million missense variants identified in the human population has a known damaging or tolerated clinical impact. Post-translational modifications (PTMs), such as phosphorylation, glycosylation and ubiquitination, are key regulators of protein function and structure, and are critical for protein localisation, stability and interactions with other molecules. The ability of a protein to undergo PTMs, is subject to a correctly folded protein structure, and the recognition and binding of enzymes to specific amino acid motifs in close proximity to residues that undergo PTMs [PTM residue]. AlphaFold models provide an unprecedented opportunity to perform sequence-structure mapping of variants, which are in close linear or spatial proximity to a PTM site and should have their impact on protein function experimentally investigated. We present Missense3D-PTMdb, a "one-stop-shop" interactive web tool that provides a user-friendly sequence-structure mapping of 20,235 human proteins to 11,544,303 naturally occurring human missense variants, 203,775 PTM sites and their neighbours in sequence and 3D structure space using AlphaFold generated 3D models of the human proteome. Additionally, the sequence-structure mapping tool allows visualization and exploration of any human variant not currently stored in the database. Missense3D-PTMDb is freely available at https://missense3d.bc.ic.ac.uk/ptmdb.

genetics↗

bioRxiv2024

A proteome-wide quantitative guide for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs

The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access database that quantifies the polymer-specific extraction variability for 2065 unique mammalian MPs and provides the most optimized condition for each of them. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform, we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to efficiently capture membrane nano-scoops containing a target MP and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

biophysics↗

bioRxiv2023

FVIII interacts with cell surface to regulate endothelial cell functionality

Haemophilia A (HA) is a rare bleeding disorder caused by factor 8 (F8) mutations. Clinical manifestations are spontaneous bleedings that primarily consist of hemarthrosis and intracranial haemorrhages. To date, the impairment of vessel stability in HA patients and the correlation between FVIII and endothelial functionality is poorly understood. Here we show that FVIII plays a role in endothelial cell functionality. Blood Outgrowth endothelial cells (BOECs) knockout generated by CRISPR/Cas9, HA BOECs and HA iPSCs-derived ECs showed alteration of vessel-formation, endothelial cell migration, and vessel permeability. Importantly, the impaired EC phenotype was rescued by treatment with recombinant human FVIII or by lentiviral vector (LV) expressing FVIII. The FVIII function on endothelium was confirmed in vivo in a mouse model of severe HA which showed that an altered angiogenesis and vesselpermeability could be treated by exogenous FVIII. BOECstranscriptomic profiles revealed that FVIIIregulates the expression of endothelial basement membrane and extracellular matrix genes. Furthermore, exogenous expression of Nidogen2, identified as a FVIII regulated gene, restored the extracellular matrix integrity and EC functionality of HA ECs. In conclusion, FVIII is not only a coagulation factor but also an endothelial cell autocrine factor which promotes vessel stability.

molecular biology↗

bioRxiv2023

Determination of oligomeric organization of membrane proteins from native membranes at nanoscale-spatial and single-molecule resolution

The oligomeric organization of membrane proteins in native cell membranes is a critical regulator of their function. High-resolution quantitative measurements of oligomeric assemblies and how they change under different conditions are indispensable to the understanding of membrane protein biology. We report a single-molecule imaging technique (Native-nanoBleach) to determine the oligomeric distribution of membrane proteins directly from native membranes at an effective spatial resolution of [~]10 nm. We achieved this by capturing target membrane proteins in "native nanodiscs" with their proximal native membrane environment using amphipathic copolymers. We established this method using structurally and functionally diverse membrane proteins with well-established stoichiometries. We then applied Native-nanoBleach to quantify the oligomerization status of a receptor tyrosine kinase (TrkA) and a small GTPase (KRas) under conditions of growth-factor binding or oncogenic mutations, respectively. Native-nanoBleach provides a sensitive, single-molecule platform to quantify membrane protein oligomeric distributions in native membranes at an unprecedented spatial resolution.

biophysics↗

bioRxiv2022

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells (PBMCs) of convalescent patients using SARS-CoV-2 receptor binding domains (RBDs) carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and class 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (mAb S309) by orders of magnitude. They also provide potent prophylactic and therapeutic in vivo protection of hACE2 mice against viral challenge. Our results indicate that exposure to Wuhan SARS-CoV-2 induces antibodies that maintain potent and broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

immunology↗

bioRxiv2021

Dominant Effect Of Host Genetics On Skin Microbiota Composition In Homeostasis And Wound Healing

Animal microbiota have complex interactions with hosts and environment that determines its composition. Yet the ability of hosts to determine their microbiota composition is less well studied. In this study, to investigate the role host genetics in determining skin microbiota, we used 30 different mouse strains from the recombinant inbred panel, the Collaborative Cross. Murine skin microbiota composition was strongly dependent on murine strain with > 50% of the variation explained by murine strain. In particular, a quantitative trait locus on chromosome 4 associates both with Staphylococcus abundance and principal-component multi-trait analyses. Additionally, excisional wound associated changes in microbiota composition were not uniform across mouse strains and were host-specific, the genetic background accounting for about 40% of the variation in microbiota. Genetic background also had the highest effect on the healing speed of wounds accounting for over 50% of the variation while mouse age and microbiota composition change accounted only for 20% and 5% of the healing speed despite reaching statistical significance. In conclusion, host genetics has a significant impact on the skin microbiota composition during both homeostasis and wound healing. These findings have long reaching implications in our understanding of associations between microbiota dysbiosis and disease.

systems biology↗

bioRxiv2020

Receptor-independent membrane mediated pathways of serotonin action

Serotonin is a neurotransmitter as well as a somatic signaling molecule, and the serotonergic system is a major target for psychotropic drugs. Serotonin, together with a few related neurotransmitters, has recently been found to exhibit an unexpectedly high lipid membrane affinity1–3. It has been conjectured that extrasynaptic serotonin can diffuse in the lipid membrane to efficiently reach remote receptors (and receptors with buried ligand-binding sites)4, providing a mechanism for the diffuse ‘volume’ neurotransmission that serotonin is capable of5–10. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, independent of its receptor-mediated actions. Atomic force microscopy shows that serotonin binding makes artificial lipid bilayers softer. It induces nucleation of liquid disordered domains inside the raft-like liquid-ordered domains in a ternary bilayer displaying phase separation. Solid-state NMR spectroscopy corroborates this data, revealing a rather homogeneous decrease in the order parameter of the lipid chains in the presence of serotonin. In the RN46A immortalized serotonergic neuronal cell line, extracellular serotonin enhances transferrin receptor endocytosis, an action exerted even in the presence of both broad-spectrum serotonin receptor and transporter inhibitors. Similarly, it increases the binding and internalization of Islet Amyloid Polypeptide (IAPP) oligomers, suggesting a connection between serotonin, which is co-secreted with IAPP by pancreatic beta cells, and the cellular effects of IAPP. Our results uncover a hitherto unknown serotonin-bilayer interaction that can potentiate key cellular processes in a receptor-independent fashion. Therefore, some pathways of serotonergic action may escape potent pharmaceutical agents designed for serotonin transporters or receptors. Conversely, bio-orthogonal serotonin-mimetics may provide a new class of cell-membrane modulators.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Disease Risk Analysis in sea turtles: A baseline study to inform conservation efforts

The impact of a range of different threats has resulted in the listing of six out of seven sea turtle species on the IUCN Red List of endangered species. Disease risk analysis (DRA) tools are designed to provide objective, repeatable and documented assessment of the disease risks for a population and measures to reduce these risks through management options. To the best of our knowledge, DRAs have not previously been published for sea turtles, although disease is reported to contribute to sea turtle population decline. Here, a comprehensive list of health hazards is provided for all seven species of sea turtles. The possible risk these hazards pose to the health of sea turtles were assessed and "One Health" aspects of interacting with sea turtles were also investigated. The risk assessment was undertaken in collaboration with more than 30 experts in the field including veterinarians, microbiologists, social scientists, epidemiologists and stakeholders, in the form of two international workshops and one local workshop. The general finding of the DRA was the distinct lack of knowledge regarding a link between the presence of pathogens and diseases manifestation in sea turtles. A higher rate of disease in immunocompromised individuals was repeatedly reported and a possible link between immunosuppression and environmental contaminants as a result of anthropogenic influences was suggested. Society based conservation initiatives and as a result the cultural and social aspect of interacting with sea turtles appeared to need more attention and research. A risk management workshop was carried out to acquire the insights of local policy makers about management options for the risks relevant to Queensland and the options were evaluated considering their feasibility and effectiveness. The sea turtle DRA presented here, is a structured guide for future risk assessments to be used in specific scenarios such as translocation and head-starting programs.

ecology↗

bioRxiv2018

HLA-DQA1*05 is associated with the development of antibodies to anti-TNF therapy

BackgroundAnti-tumour necrosis factor (anti-TNF) therapies are the most widely used biologic therapies for treating immune-mediated diseases. Their efficacy is significantly reduced by the development of anti-drug antibodies which can lead to treatment failure and adverse reactions. The biological mechanisms underlying antibody development are unknown but the ability to identify subjects at higher risk would have significant clinical benefits.\n\nMethodsThe PANTS cohort consists of Crohns disease patients recruited prior to first administration of anti-TNF, with serial measurements of anti-drug antibody titres. We performed a genome-wide association study across 1240 individuals from this cohort to identify genetic variants associated with anti-drug antibody development.\n\nFindingsThe Human Leukocyte Antigen allele, HLA-DQA1*05, carried by approximately 40% of Europeans, significantly increased the rate of anti-drug antibody development (hazard ratio [HR], 1.90; 95% confidence interval [CI], 1.60 to 2.25; P=5.88x10-13). This association was consistent for patients treated with adalimumab (HR, 1.89; 95% CI, 1.32 to 2.70) and infliximab (HR, 1.92; 95% CI, 1.57 to 2.33), and for patients treated with mono-(HR, 1.75; 95% CI, 1.37 to 2.22) or combination therapy with immunomodulators (HR, 2.0; 95% CI, 1.57 to 2.58).\n\nInterpretationHLA-DQA1*05 is significantly associated with an increased rate of anti-drug antibody formation in patients with Crohns disease treated with infliximab and adalimumab. Pre-treatment HLA-DQA1*05 genetic testing may help personalise the choice of anti-TNF therapy and allow the targeted use of immunomodulator therapy to minimise risk and maximise response.

genomics↗