Biology subjects

Owens, R.

Publications and source records attributed to Owens, R..

14 recordsLinked to original sources

bioRxiv2026

Using a lanthanide tagged nanobody to visualise Glial fibrillary acidic protein by secondary ion mass spectrometry

Lanthanide-labelled antibodies have been widely used for multiplex imaging of proteins directly from tissue using two approaches, imaging mass cytometry (IMC) and variants of secondary ion mass spectrometry (SIMS). Here we show the same approach can be taken using SIMS, but utilising lanthanide-labelled nanobodies instead. Nanobodies offer several advantages over conventional antibodies for antigen tagging, owing to their small size (12-15 kDa), high stability, and solubility. Their reduced molecular size enables greater diffusion through tissue, facilitating access to target sites and allowing them to reach regions that may be less accessible to larger antibodies. This enhanced accessibility can promote more effective localisation and increase binding to target antigens. In the work presented here, we produced a lanthanide-tagged nanobody and assessed its efficacy and compatibility with mass spectrometry imaging (MSI). We showed successful detection of Glial fibrillary acidic protein (GFAP) in tissue by correlating MSI of a lanthanide-tagged anti-GFAP nanobody with immunofluorescence microscopy imaging (IF). The use of nanobodies is an excellent alternative to current antibody tagging methods for SIMS and IMC.

molecular biology↗

bioRxiv2026

Structural analysis reveals high-order Nipah Virus Fusion Protein assemblies and interactions with neutralising nanobodies

Nipah virus (NiV) is a highly pathogenic emerging henipavirus for which there are no licensed vaccines or therapeutics currently available. Viral entry is mediated by the coordinated action of the attachment (NiVG) and fusion (NiVF) glycoproteins yet the organisation of NiVF on viral particles and its influence on antibody neutralisation is not completely understood. Using cryo-electron tomography of NiV pseudovirions and virus-like particles (VLPs), we directly observe inter-F contacts on viral membranes. These interactions occur frequently but are distributed stochastically, forming variably sized assemblies rather than ordered arrays, consistent with transient or low-affinity associations. We have generated single domain antibodies (termed nanobodies) to the NiVF glycoprotein that bind to an epitope at the interface between neighbouring NiVF trimers and therefore occlude the inter-NiVF contacts. We show that these nanobodies inhibit VLP cell fusion and neutralise NiV pseudoviruses in vitro, which suggests that the interaction between NiVF trimers is important for NiV virus entry. The identification of the inter-NiVF contacts on viral particles adds a previously unrecognised structural feature to the organisation of the NiV entry machinery which could be targeted to prevent viral infection.

molecular biology↗

bioRxiv2025

The pharmacokinetics, bio-distribution and therapeutic efficacy of a trimeric nanobody against SARS-CoV-2 in the Syrian golden hamster COVID-19 model

The rapid emergence of SARS-CoV-2 prompted the development of anti-viral therapies and vaccines to combat the COVID-19 pandemic. The continuous emergence of variants of concern has necessitated the development of platforms that can be rapidly adapted. Nanobodies, offer advantages over conventional monoclonal antibodies, including low molecular weight, high antigen-binding affinity, enhanced tissue penetration, blood-brain barrier permeability, and simplified production. Previous studies have demonstrated the efficacy of nanobodies against respiratory viruses such as SARS-CoV-2, respiratory syncytial virus, and influenza A virus in animal models. We previously reported the protective effects of nanobody trimers against SARS-CoV-2. However, pharmacokinetic and biodistribution data for nanobodies remain limited. To address this, we evaluated the efficacy, biodistribution and longevity of action of a nanobody trimer (A8) administered intranasally (IN) or intraperitoneally (IP) in Syrian golden hamsters. Our findings revealed that the A8 trimer reached peak concentrations shortly after administration and was subsequently cleared from the system via both routes. Importantly, early administration of A8 trimer reduced virus mediated weight loss and viral load compared with untreated controls supporting its potential as a therapeutic candidate for SARS-CoV-2 infection.

microbiology↗

bioRxiv2025

Alpha-BET: Functional labeling of envelope glycoproteins with single domain antibodies for in-virus single molecule imaging

We present Alpha-BET, a structure-guided strategy leveraging AlphaFold to identify optimal ALFA-tag insertion sites for minimally disruptive labeling of viral glycoproteins with high-affinity nanobodies. Applied to HIV-1 Env, SARS-CoV-2 S, and NiV G, Alpha-BET preserves structural integrity and function. For HIV-1 Env, we demonstrate super-resolution DNA-PAINT MINFLUX 3D imaging enabled by tag insertion, showcasing its power for visualizing native trimers in single virions and potential for broader applications in virus research.

biophysics↗

bioRxiv2025

Permissive central tolerance plus defective peripheral checkpoints licence pathogenic memory B cells in CASPR2-antibody encephalitis

Autoimmunity affects 10% of the population. Within this umbrella, autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the developmental pathway of disease-causing B cells and autoantibodies. While such autoreactivities are believed to be generated during germinal centre reactions, the roles of earlier immune checkpoints in autoantigen-specific B cell tolerance are poorly understood. We address this concept in patients with CASPR2-autoantibody encephalitis and healthy controls. In both groups, comparable and high ([~]0.5%) frequencies of unmutated CASPR2-reactive naive B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with heterogenous binding kinetics. These effector molecules possessed epitope-dependent pathogenic effects in vitro neuronal cultures and in vivo. The unmutated common ancestors of these memory B cells showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results are the first to propose mechanisms underlying autoantigen-specific tolerance in humans. We identify permissive central tolerance, defective peripheral tolerance and heterogenous autoantibody binding properties as sequential pathogenic steps which licence CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches in CASPR2-antibody diseases. This paradigm is applicable across autoimmune conditions.

immunology↗

bioRxiv2024

Assessing drug uptake and response differences in 2D and 3D cellular environments using stimulated Raman scattering microscopy

The architecture of cell culture--two-dimensional (2D) versus three-dimensional (3D)--significantly impacts various cellular factors, including cell-cell interactions, nutrient and oxygen gradients, metabolic activity, and gene expression profiles. This can result in different cellular responses during cancer drug treatment, with 3D-cultured cells often exhibiting higher resistance to chemotherapeutic drugs. While various genetic and proteomic analyses have been employed to investigate the underlying mechanisms of this increased resistance, complementary techniques that provide experimental evidence of spatial molecular profiling data are limited. Stimulated Raman scattering (SRS) microscopy has demonstrated its capability to measure both intracellular drug uptake and growth inhibition. In this work, we applied three-band SRS imaging to 2D and 3D cell cultures and provided a comparative analysis of drug uptake and response with the goal of understanding whether the difference in drug uptake explains the drug resistance in 3D culture compared to 2D. Our investigations revealed that despite similar intracellular drug levels in 2D and 3D A549 cells during lapatinib treatment, the growth of 3D spheroids is less impacted, supporting an enhanced drug tolerance in the 3D microenvironment. We further elucidated drug penetration patterns and the resulting heterogeneous cellular responses across different spheroid layers. Additionally, we investigated the role of the extracellular matrix in modulating drug delivery and cell response, and we discovered that limited drug penetration in 3D could also contribute to lower drug response. Our study provides valuable insights into the intricate mechanisms of increased drug resistance in 3D tumor models during cancer drug treatments.

cell biology↗

bioRxiv2023

Recreating the Biological Steps of Viral Infection on a Bioelectronic Platform to Profile Viral Variants of Concern

Viral mutation rates frequently outpace the development of technologies used to detect and identify harmful variants; for SARS Coronavirus-2 (SARS-CoV-2), these are called variants of concern (VOC). Given the continual emergence of VOC, there is a critical need to develop platforms that can identify the presence of a virus and readily identify its propensity for infection. We present an electronic biomembrane sensing platform that recreates the multifaceted and sequential biological cues that give rise to distinct SARS-CoV-2 virus host cell entry pathways and reports the progression of entry steps of these pathways as electrical signals. Within these electrical signals, two necessary entry processes mediated by the viral Spike protein, virus binding and membrane fusion, can be distinguished. Remarkably, we find that closely related VOC exhibit distinct fusion signatures that correlate with trends reported in cell-based infectivity assays, allowing us to report quantitative differences in fusion characteristics among them that inform their infectivity potentials. This cell-free biomimetic infection platform also has a virus-free option that equally reports infectivity potential of the Spike proteins. We used SARS-CoV-2 as our prototype, but we anticipate that this platform will extend to other enveloped viruses and cell lines to quantifiably explore virus/host interactions. This advance should aid in faster determination of entry characteristics and fusogenicities of future VOC, necessary for rapid response.

bioengineering↗

bioRxiv2022

GPC3-Unc5D complex structure and role in cell migration

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass-spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding. Using these tools in vivo, we show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell-guidance, with the potential for wide- ranging biomedical implications in development and cancer biology.

neuroscience↗

bioRxiv2021

Correlative microscopy reveals the nanoscale morphology of E. coli-derived supported lipid bilayers

Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles are an important tool in molecular biology. A breakthrough in the field has come with the use of vesicles derived from cell membranes to form SLBs. These new supported bilayers, consisting both of natural and synthetic components, provide a physiologically relevant system on which to study protein-protein interactions as well as protein-ligand interactions and other lipid membrane properties. These complex bilayer systems hold promise but have not yet been fully characterised in terms of their composition, ratio of natural to synthetic component and membrane protein content. Here, we describe a method of correlative atomic force (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the enhanced resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the atomic scale resolution that the AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, quantify the amount of bacterial membrane incorporated in the bilayer and directly visualise the interaction of these bilayers with bacteria-specific, membrane-binding proteins. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs and establishes correlative AFM/ SIM as a method for characterising complex systems at the nanoscale.

microbiology↗

bioRxiv2021

Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, where cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused-ion beam milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt pre-existing biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.

microbiology↗

bioRxiv2020

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

immunology↗

bioRxiv2020

Hallmarks of basidiomycete soft- and white-rot in wood-decay -omics data of Armillaria

The genus Armillaria spp. (Fungi, Basidiomycota) includes devastating pathogens of temperate forests and saprotrophs that decay wood. Pathogenic and saprotrophic Armillaria species can efficiently colonize and decay woody substrates, however, mechanisms of wood penetration and colonization are poorly known. We assayed the colonization and decay of autoclaved spruce roots using the conifer-specialists Armillaria ostoyae and A. cepistipes using transcriptomic and proteomic data. Transcript and protein levels were altered more extensively in the saprotrophic A. cepistipes than in the pathogenic A. ostoyae and in invasive mycelia of both species compared to their rhizomorphs. Diverse suites of carbohydrate-active enzyme genes (CAZymes), in particular pectinolytic ones and expansins, were upregulated in both species, whereas ligninolytic genes were mostly downregulated. Our gene expression data, together with previous comparative genomic and decay-chemistry analyses suggest that wood decay by Armillaria differs from that of typical white-rot fungi and shows features resembling soft rot. We propose that Armillaria species have modified the ancestral white-rot machinery so that it allows for selective ligninolysis based on environmental conditions and/or host types.

ecology↗

bioRxiv2019

The evolution of parasite host range in genetically diverse host populations

Parasites vary enormously in their host range. Why are some parasites specialists and others generalists? We tested the hypothesis that genetically diverse host populations select for parasites with broader host ranges than genetically homogeneous host populations. To do so, we selected for increased killing ability of the bacterial parasite Serratia marcescens in populations of the host Caenorhabditis elegans that were either diverse (50% mix of genotypes N2 and LTM1) or homogenous (100% N2 or LTM1). We found mixed support for the hypothesis. After 20 generations of selection, parasites selected in diverse host populations maintained a broad host range, as shown by the retention of high killing ability of a novel host genotype. Parasites selected in diverse populations killed N2 hosts equally well as parasites selected in homogenous populations of N2. However, N2-selected parasites lost killing ability against the novel host, consistent with the evolution of narrow host ranges in homogenous environments. In contrast, parasites selected in homogenous LTM1 populations did not specialize: they did not increase in their killing ability on LTM1 and did not lose killing ability against the novel host. Our results argue that the evolution of host range depends upon both the identity and diversity of hosts that a parasite encounters.

evolutionary biology↗

bioRxiv2018

The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleotide selectivity of PNPs

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, named as SmPNP2. Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands. The binding of cytosine was confirmed by isothermal titration calorimetry, with a KD of 27 M, and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of 76.3 M. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystallographic structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capability of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site allowing an alternative binding mode for nucleosides, with a 180{degrees} rotation from the canonical binding mode. The remarkable plasticity of this binding site deepens the understanding of the correlation between structure and nucleotide selectivity, offering new ways to analyses PNP activity.\n\nAuthor SummarySchistosoma mansoni is a human parasite dependent on purine salvage for purine bases supply. Purine nucleoside phosphorylase (PNP) is a key enzyme in this pathway. It carries two PNP isoforms, one previously characterized (SmPNP1) and one unknown (SmPNP2). Here we present the crystallographic structure of SmPNP2 and its complex with cytosine, cytidine, ribose-l-phosphate, adenine, hypoxanthine, and tubercidin. Cytidine and cytosine were identified as ligands of SmPNP2 using a thermofluorescence approach. Binding of cytosine was proven by Isothermal Titration Calorimetry (ITC) and cytidine, inosine, and adenosine kinetic parameters were also obtained. Purine bases showed different binding in the active site, rotated 180{degrees} from the canonical binding mode. Its the first report showing a Low Molecular Mass PNP capable of catalyzing both types of nucleotide bases. The SmPNP2 odd behavior sheds a new light on the Schistosoma mansonis life cycle metabolic adaptation.

biochemistry↗