Biology subjects

Hunter, B.

Publications and source records attributed to Hunter, B..

12 recordsLinked to original sources

bioRxiv2025

Targeting multidrug-resistant bacteria with genetic-information-free protein-only phages

Bacteriophages offer advantages over small-molecule antibiotics, including host specificity and general compatibility with the human phagenome. However, their evolvability as replicating biological entities introduces therapeutic unpredictability and risks of phage-bacteria co-evolution. Here, we retain the targeting benefits of phages while avoiding genetic replication by engineering genetic-information-free, protein-only phages (POPs). These genome-free particles self-assemble in a cell-free protein synthesis system from modular, de novo gene fragments encoding only structural and antimicrobial proteins. Using Enterobacteria phage T7 and its susceptible bacterial host as a model, we test the hypothesis that POPs stochastically encapsulate small antimicrobial proteins during self-assembly and deliver them into bacteria during adsorption via an ejectome-mediated injection mechanism. A computational survey of T7 small proteins revealed early and mid-genome enrichments of hypothetical proteins and capsid volume sufficient to accommodate multiple small proteins in the absence of the [~]40-kb genome. In time-series antimicrobial susceptibility assays (48-72 h), POPs produced initial growth inhibition comparable to wild-type T7 at the highest doses with a linear dose-effect relationship and a minimum inhibitory concentration-like threshold. These results establish the feasibility of genetic-information-free POPs as protein-based antimicrobials that couple phage receptor specificity with minimal biosafety risks, supporting the development of more stable and predictable phage-inspired therapeutics.

pharmacology and toxicology↗

bioRxiv2025

Spatial profiling of longitudinal glioblastoma reveals consistent changes in cellular architecture, post-treatment

Glioblastoma (GBM), the most aggressive adult brain cancer, comprises a complex tumour microenvironment (TME) with diverse cellular interactions driving progression and pathobiology. How these spatial patterns and interactions evolve with treatment remains unclear. Here, we apply imaging mass cytometry to analyse protein-level changes in paired pre- and post-treatment GBM samples from five patients. We find a significant post-treatment increase in normal brain cells alongside a reduction in vascular cells. Moreover, despite minimal overall change in cellular diversity, interactions among astrocytes, oligodendrocytes, and vascular cells increase post-treatment, suggesting reorganisation of the TME. The GBM TME cells form spatially organized layers driven by hypoxia pre-treatment, but this influence diminishes post-treatment, giving way to less organised layers with organisation driven by reactive astrocytes and lymphocytes. These findings provide insight into treatment-induced shifts in GBMs cellular landscape, highlighting aspects of the evolving TME that appear to facilitate recurrence and are, therefore, potential therapeutic targets. Key pointsO_LISpatial organisation in primary GBM consist of layers driven by the presence of hypoxia C_LIO_LIThe layers in recurrent GBM appear are driven more by the presence of reactive astrocytes C_LIO_LIIncreased cellular cross-talk in recurrent GBM presents novel therapeutic targets C_LI

cancer biology↗

bioRxiv2024

Kinesin-8 motors dimerize by folding their proximal tail domain into a compact helical bundle

Kinesin-8 motor proteins help align and segregate chromosomes during mitosis by regulating the dynamics of kinetochore-attached microtubules and the length and position of the mitotic spindle. Some kinesin-8 isoforms accomplish these roles by operating as multifunctional mechanoenzymes that can traverse microtubules, accumulate at the microtubule plus-ends, and then remove terminal {beta}-tubulin subunits. While these activities are mainly powered by the motor domain, whose unique structure-function relationships have been recently reported, the non-motor tail domain contains integral functional elements that have not been structurally illuminated. Using the Candida albicans Kip3 protein as a kinesin-8 model system, we present an X-ray crystal structure and hydrodynamic data showing how the motor domain-proximal segment of the tail directs the assembly of two kinesin-8 polypeptides into a homodimer that forms the stalk of this motor. Unlike the extended coiled coil-forming helices of most other motile kinesin stalks, the proximal tail of CaKip3 folds into a compact 92 [A]-long four-helix bundle that dimerizes. The first and third helices provide most of the surface area for the dimer interface, while the other two helices brace the folded stalk structure. The upper and lower lobules of the helical bundle are separated by a flexible hinge that gives the exterior faces of the stalk slightly different shapes when bent. We propose that these unique characteristics provide structural rigidity to the kinesin-8 stalk, as well as sites for transient interactions with kinesin-8-associated proteins or other regulatory regions of the motor.

biochemistry↗

bioRxiv2024

Nanopore sequencing from protozoa to phages: decoding biological information on a string of biochemical molecules into human-readable signals

Biological information is encoded in a sequence of biochemical molecules such as nucleic acids and amino acids, and nanopore sequencing is a long-read sequencing technology capable of directly decoding these molecules into human-readable signals. The long reads from nanopore sequencing offer the advantage of obtaining contiguous information, which is particularly beneficial for decoding complex or repetitive regions in a genome. In this study, we investigated the efficacy of nanopore sequencing in decoding biological information from distinctive genomes in metagenomic samples, which pose significant challenges for traditional short-read sequencing technologies. Specifically, we sequenced blood and fecal samples from mice infected with Trypanosoma brucei, a unicellular protozoan known for its hypervariable and dynamic regions that help it evade host immunity. Such characteristics are also prevalent in other host-dependent parasites, such as bacteriophages. The taxonomic classification results showed a high proportion of nanopore reads identified as T. brucei in the infected blood samples, with no significant identification in the control blood samples and fecal samples. Furthermore, metagenomic de novo assembly of these nanopore reads yielded contigs that mapped to the reference genome of T. brucei in the infected blood samples with over 96% accuracy. This exploratory work demonstrates the potential of nanopore sequencing for the challenging task of classifying and assembling hypervariable and dynamic genomes from metagenomic samples.

bioengineering↗

bioRxiv2024

A pangenome analysis of ESKAPE bacteriophages: the underrepresentation may impact machine learning models

Bacteriophages are the most prevalent biological entities in the biosphere. However, limitations in both medical relevance and sequencing technologies have led to a systematic underestimation of the genetic diversity within phages. This underrepresentation not only creates a significant gap in our understanding of phage roles across diverse biosystems but also introduces biases in computational models reliant on these data for training and testing. In this study, we focused on publicly available genomes of bacteriophages infecting high-priority ESKAPE pathogens to show the extent and impact of this underrepresentation. First, we demonstrate a stark underrepresentation of ESKAPE phage genomes within the public genome and protein databases. Next, a pangenome analysis of these ESKAPE phages reveals extensive sharing of core genes among phages infecting the same host. Furthermore, genome analyses and clustering highlight close nucleotide-level relationships among the ESKAPE phages, raising concerns about the limited diversity within current public databases. Lastly, we uncover a scarcity of unique lytic phages and phage proteins with antimicrobial activities against ESKAPE pathogens. This comprehensive analysis of the ESKAPE phages underscores the severity of underrepresentation and its potential implications. This lack of diversity in phage genomes may restrict the resurgence of phage therapy and cause biased outcomes in data-driven computational models due to incomplete and unbalanced biological datasets.

bioinformatics↗

bioRxiv2023

Characterizing the microbial metagenome of calcareous stromatolite formations in the San Felipe Creek in Anza Borrego Desert

Here we describe the metagenome composition, community functional annotation, and diversity of prokaryotic microbial species derived from calcareous stromatolite formations discovered in the dry stream bed of the open-canopy, ephemeral San Felipe Creek in the Anza Borrego Desert. In this environment, resident microbes must be able to adapt to the harsh conditions of extreme heat, high UV light, desiccation and fluctuating solubilization/precipitation and hydration/evaporation. Metagenomic analysis revealed a community capable of carrying out complete nitrogen fixation and assimilatory nitrate reduction, forming biofilms and quorum sensing, and potentially forming thick-walled akinetes as desiccation-resistant stages. Nitrogen cycling is likely to play a fundamental role in mediating both the structure of the stromatolite microbial community and the mineral precipitation/dissolution. The viruses present in the stromatolites, particularly Nodularia and Mycobacterium phages are also likely to impact community population dynamics and activity. Stromatolite community members possess different morphological and physiological strategies to cope with desiccation stress. Metagenomic signatures were found for scytonemin, carotenoids, synthesis of potential microsporine-like amino acids; genes involved in microalgal desiccation tolerance, including those encoding aquaporins, chaperones, antioxidants; and enzymes responsible for the synthesis of trehalose, sucrose, and polyamines. The stromatolite ecosystem provides a diverse array of microniches where different functional guilds can develop complex metabolite exchange with the substrate supporting their life in extreme conditions. Metagenome analyses revealed several genes that might enable a specialized and unique group of endolithic cyanobacteria including Chroococcidiopsis, Hyella, Myxosarcina, and Pleurocapsa to derive metals and important nutrients from rocks, being potentially destructive for the calcareous formations. Our study revealed environmental adaptations of freshwater microbial communities in desert stream stromatolites which may provide valuable insights into Precambrian paleoenvironments, which are little known.

genomics↗

bioRxiv2023

OPTIMAL: An OPTimsed Imaging Mass cytometry AnaLysis framework for Segmentation and Data Exploration

Analysis of Imaging Mass Cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single cell segmentation and sub-optimal approaches for data visualisation and exploration. This can lead to inaccurate identification of cell phenotypes, states or spatial relationships compared to reference data from single cell suspension technologies. To this end we have developed the "OPTIMAL" framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualisation/clustering and spatial neighbourhood analysis. Using a panel of 27 metal-tagged antibodies recognising well characterised phenotypic and functional markers to stain the same FFPE human tonsil sample Tissue Microarray (TMA) over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, five different dimensionality reduction algorithms and two clustering methods. Finally we assessed the optimal approach for performing neighbourhood analysis. We found that single cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using "classical" bi-variate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximised the statistical separation between negative and positive signal distributions and a simple Z-score normalisation step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing Phenograph in terms of cell type identification. We also found that neighbourhood analysis was influenced by the method used for finding neighbouring cells with a "disc" pixel expansion outperforming a "bounding box" approach combined with the need for filtering objects based on size and image-edge location. Importantly OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates .FCS files from the segmentation output, allows for single cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

bioinformatics↗

bioRxiv2022

GBMdeconvoluteR accurately infers proportions of neoplastic and immune cell populations from bulk glioblastoma transcriptomics data

The biological and clinical impact of neoplastic and immune cell type ratios in the glioblastoma (GBM) tumour microenvironment is being realised. Characterising and quantifying cell types within GBMs at scale will facilitate a better understanding of the association between the cellular landscape and tumour phenotypes or clinical correlates. This study aimed to develop a tool that can deconvolute immune and neoplastic cells within the GBM tumour microenvironment from bulk RNA sequencing data. We developed an IDH wild-type (IDHwt) GBM specific single immune cell reference dataset, from four independent studies, consisting of B cells, T cells, NK cells, microglia, tumour associated macrophages, monocytes, mast and DC cells. We used this alongside an existing neoplastic single cell-type dataset consisting of astrocyte-like, oligodendrocyte- and neuronal-progenitor like and mesenchymal GBM cancer cells to create both marker and gene signature matrix-based deconvolution tools. We then applied single-cell resolution imaging mass cytometry (IMC) to ten IDHwt GBM samples, five paired primary and recurrent tumours, in parallel with these tools to determine which performed best. Marker based gene expression deconvolution using GBM tissue specific markers, which we have packaged as GBMdeconvoluteR, gave the most accurate results. The correlation between immune cell quantification by IMC and by GBMdeconvoluteR for primary IDHwt GBM samples was 0.52 (Pearsons P=7.8x10-3) and between neoplastic cell quantification by IMC and by GBMdeconvoluteR was 0.75 (Pearsons P=1.2x10-3). We applied GBMdeconvoluteR to bulk GBM RNAseq data from The Cancer Genome Atlas (TCGA) and were able to recapitulate recent findings from multi-omics single cell studies with regards associations between mesenchymal GBM cancer cells and both lymphoid and myeloid cells. Furthermore, we were able to expand upon this to show that these associations are stronger in patients with worse prognosis. GBMdeconvoluteR is accessible online at https://gbmdeconvoluter.leeds.ac.uk. Key pointsGBMdeconvoluteR is a glioblastoma-specific cellular deconvolution tool. When applied to bulk GBM RNAseq data, it accurately quantifies the neoplastic and immune cells in that tumour. It is available online at https://gbmdeconvoluter.leeds.ac.uk

cancer biology↗

bioRxiv2022

Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape

Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 [A]) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unbound CaKip3-ADP crystal structure (2.0 [A]). Together with biochemical analyses of CaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

biochemistry↗

bioRxiv2022

A highly sensitive LC-MS/MS method for quantitative determination of 7 vitamin D metabolites in mouse brain tissue

Despite its critical role in neurodevelopment and brain function, vitamin-D (vit-D) homeostasis, metabolism and kinetics within the central nervous system remain largely undetermined. Thus, it is of critical importance to establish an accurate, highly sensitive and reproducible method to quantitate vit-D in brain tissue. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method and for the first time, demonstrate detection of seven major vit-D metabolites in brain tissues of C57BL/6J wild-type mice, namely: 1,25(OH)2D3, 3-epi-1,25(OH)2D3, 1,25(OH)2D2, 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, and 24,25(OH)2D2. Chromatographic separation was achieved on a pentaflurophenyol column 3 mM ammonium formate with water/methanol [A] and methanol/isopropanol [B] phases. Detection was by positive-ion electrospray tandem mass spectrometry. We used calibration standards of each metabolite prepared in brain matrices to validate the detection range, precision, accuracy and recovery. Isotopically labelled analogues, 1,25(OH)2D3-d3, 25(OH)D3-C5 and 24,25(OH)2D3-d6, served as the internal standards for the closest molecular related metabolite in all measurements. The calibration range was between 1 fg/mL to 10 ng/mL with an LLOD and LLOQ of 10 fg/mL and 3 fg/mL, respectively. The intra-/inter-day precision and accuracy for measuring brain vit-D metabolites ranged between 0.12-11.53% and 0.28-9.11%, respectively. Recovery ranged between 99.06% and 106.9% for all metabolites. Collectively, the sensitivity and efficiency of our method supersedes previously reported protocols used to measure vit-D and to our knowledge, the first protocol to reveal the abundance of 25(OH)D2, 1,25(OH)D2 and 24,25(OH)2D2, in brain tissue of any species. This technique may be important in supporting the future advancement of pre-clinical research into the function of vit-D in neurophysiological, neuropsychiatric, and neurodegeneration.

physiology↗

bioRxiv2019

Transposable element over-accumulation in autopolyploids results from relaxed purifying selection and provides variants for rapid local adaptation

Polyploidization is frequently associated with increased transposable element (TE) content. However, what drives TE dynamics following whole genome duplication (WGD) and the evolutionary implications remain unclear. Here, we leveraged whole-genome resequencing data of [~]300 individual Arabidopsis arenosa plants, a well characterized natural diploid-autotetraploid species, to address these questions. Based on 43,176 polymorphic TE insertions we detected in these genomes, we demonstrate that relaxed purifying selection rather than transposition bursts is the main driver of TE over-accumulation after WGD. Furthermore, the increased pool of TE insertions in tetraploids is especially enriched within or near abiotic and biotic stress response genes. Notably, we uncovered one such insertion in a major flowering-time repressor gene and found that the resulting allele is specific to the rapid-cycling tetraploid lineage that colonized mainland railways. Together, our findings indicate that tetrasomy by itself leads to an enhanced tolerance to accumulating genic TE variants, some of which can potentially contribute to local adaptation.

genomics↗

bioRxiv2017

A ticket to ride - Allele delivery by rail in secondary ruderal colonization by Arabidopsis arenosa

Human-generated ruderal habitats are abundant, but challenging for plants. Some ruderal habitats, however, provide networked corridors (e.g. roadsides and railways) that can facilitate rapid long-distance spread of successfully adapted variants. Here we use transcriptomic and genomic analyses, coupled with genetic mapping and transgenics to understand adaptation to railways in Arabidopsis arenosa. We show normally perennial A. arenosa switched to rapid cycling, a common adaptation for ruderal plants, at least twice upon railway colonization. We further show substantial gene flow from a widely distributed railway colonist likely contributed to secondary colonization by a non-ruderal type, highlighting how connectivity can affect adaptability. We find loss of expression of the reproductive repressor FLOWERING LOCUS C (FLC) is likely primarily responsible for rapid cycling in the widely distributed railway variant. However, a second railway colonist in the Alps also cycles rapidly, but retains high FLC. Some alleles in this population encode non-functional proteins, suggesting FLC has started to decay, but most are functional. Instead, this population likely circumvents FLC via a derived allele of CONSTANS (CO), which shows strong evidence of selection in this population. Importantly, we find this CO allele arrived via gene flow from the widespread ruderal, where it was also previously under selection. This suggests ruderal adaptation may have been progressive, perhaps in both cases, with FLC-circumvention arising first, and FLC loss arising later but ultimately obscuring its earlier circumvention. These snapshots of railway adaptation highlight that gene flow from widespread ruderals can provide opportunities for subsequent adaptation by local genotypes.

evolutionary biology↗