Biology subjects

Wilkinson, S.

Publications and source records attributed to Wilkinson, S..

16 recordsLinked to original sources

bioRxiv2026

A Context-Aware Single-Cell Proteomics Analysis pipeline.

Single-cell proteomics (SCP) by mass spectrometry can now quantify hundreds to thousands of proteins per cell, but the field still lacks standardised analytical pipelines that accommodate the diversity of instruments, sample preparation workflows and biological contexts encountered in practice. Existing workflows, largely adapted from single-cell transcriptomics, do not account for the informative missingness, pervasive ambient protein contamination and limited feature space that distinguish proteomic from transcriptomic data. In addition, cell type annotation remains a manual bottleneck that is subjective, difficult to reproduce and hard to scale. Here we present an end-to-end pipeline that integrates adaptive quality control, entropy-guided iterative batch correction, multi-modal marker discovery that exploits detection patterns unique to proteomics, and context-aware annotation by large language models (LLMs) coupled to structured contradiction reasoning and orthogonal data-driven validation. Benchmarking on published single-cell proteomic datasets from developing human brain and glioblastoma-associated neutrophils revealed systematic LLM failure modes, including context-insensitive marker vocabulary and misinterpretation of phagocytic or lytic cell states. We addressed these errors using a three-round prompt architecture that combines general biological principles with auto-generated dataset-specific constraints. In held-out validation on a skin tumour dataset acquired, the pipeline showed high concordance with FACS-sorted ground truth. In the caerulein-injured pancreas, orthogonal immunohistochemistry further supported annotations of macrophage, stellate and immune populations. The pipeline is fully automated under fixed settings, and available as Context-Aware Single-Cell Proteomics Analysis (CASPA), providing SCP laboratories and facilities with a reproducible workflow that delivers interpretable, confidence-quantified annotations suitable for downstream expert review.

bioinformatics↗

bioRxiv2026

It is not just about the science - the impact of undergraduate research projects and COVID-19 on graduate attributes and employability.

Over the past two decades, Higher Education Institutions have increasingly prioritised transferrable skills to enhance graduate employability. Graduate Attributes (GAs) now act as key indicators of student competencies for both learners and employers. Final-year research projects, typically high in credit value, represent capstone experiences that promote subject expertise and GA development through research, written work, and oral presentations. This study analyses pre- and post-project survey data from RQF Level 6 biomedical and biomolecular science students at a Russell Group University over four years (2019-2023). Most projects were laboratory-based, though the 2020-2021 cohort completed theirs remotely due to COVID-19. Students reflected on expectations and experiences of GA development, subject knowledge, and employability. Initial responses revealed anxiety and uncertainty, particularly among the 2020-2021 cohort, but most anticipated gains in skills and employability. Post-project feedback confirmed this, identifying critical thinking, confidence, resilience, collaboration, and future focus as key outcomes. Digital capability was notably strengthened, especially during remote delivery. The findings emphasise the importance of a shared understanding of GAs in bioscience education and the value of embedding structured reflection and preparatory support to help students recognise and articulate their evolving skills.

scientific communication and education↗

bioRxiv2025

ER-liquid condensate contacts sequester FAM134B/C and RhoA to govern cell morphology

ER-phagy receptors have elusive physiological functions beyond ER remodelling. Here, we use proximity biotinylation to identify their cytoplasmic interactomes. Secondary CRISPR/Cas9 screening reveals regulators of the prototypical FAM134B/C receptors, which include PRKAR1A, canonically known as a subunit of PKA. PRKAR1A directly binds an amphipathic helix in the otherwise disordered cytoplasmic domain of FAM134B. Super-resolution, FRAP and CLEM imaging reveal novel interorganellar contacts between liquid-like condensates of cAMP-bound PRKAR1A and the ER. Condensates promote clustering of ER-embedded FAM134B/C with LC3B, independently of regulation of PKA by PRKAR1A. Proteomics reveal that cytoplasmic RhoA interacts with FAM134B/C clusters and that sequestration of both these molecules occurs within lysosomes embedded within the proximal condensates. This results in reduced actomyosin contractility and ER-condensate interactions thusly determine cell morphology and cancer cell invasion modality. In summary, ER-condensate contacts mediated by FAM134B/C are novel cellular degradation hubs that coordinate ER and cellular remodelling.

cell biology↗

bioRxiv2025

Human outbreak detection and best practice MPXV analysis and interpretation with squirrel

High numbers of reported mpox cases and recent identification of multiple sustained human outbreaks of MPXV have highlighted the need for robust, best-practice genomic surveillance tools. In light of the SARS-CoV-2 pandemic, many labs across the globe developed the capacity to do virus genome sequencing, however MPXV presents additional analytical challenges due to its large genome size, tracts of low-complexity or repeat regions, genetically distinct clades, and the need to perform bespoke APOBEC3-mutation reconstruction. We present squirrel (Some Quick Reconstruction to Resolve Evolutionary Links), an open source bioinformatic tool that can perform clade-aware alignment, mutation quality assessment, phylogenetic inference and automated APOBEC3-mutation classification on branches of the phylogeny. Squirrel can be run on the command line or launched through the EPI2ME GUI through the squirrel-nf workflow, enabling robust analysis without need for the command line. With the interactive output report produced and publication-ready APOBEC3-reconstruction visualisation, squirrel enables researchers to distinguish between zoonotic and sustained human outbreaks and help accurately inform public health responses.

evolutionary biology↗

bioRxiv2025

Dysproteostasis primes pancreatic epithelial state changes in KRAS-mediated oncogenesis

Pre-malignant transformation of pancreatic acinar cells by oncogenic Kras is dependent upon stochastic emergence of metaplastic cell states, through unknown mechanisms. We reveal that an early, transcriptionally-mediated effect of Kras is sporadic failure of proteostatic ER-phagy. Genetically-altered mice deficient in ER-phagy demonstrate that this event co-operates with Kras to drive acinar-ductal metaplasia (ADM) and subsequent cancer. Mechanistically, proteomics and high-resolution imaging uncover pathologic aggregation of a subset of ER proteins, including the injury marker REG3B, resulting from failure to physically interact with the ER-phagy receptor CCPG1. Spatial transcriptomics demonstrate that the appearance of sporadic intracellular aggregates upon Kras activation marks rare acinar cells existing in an injured, ADM-primed state. Importantly, engineered mutants of REG3B establish that aggregate formation is sufficient to directly engender this epithelial cell state. Pancreatic cancer can thus arise from stochastic pathologic protein aggregates that are influenced by and co-operate with an oncogene.

cancer biology↗

bioRxiv2025

Chronic lysosome damage boosts interferon responses to Palbociclib through a mitochondrial signalling axis

Acute lysosome damage triggers the endolysosome damage response (ELDR) in order to co-ordinate vesicle repair or removal by autophagy (lysophagy). However, it is unclear whether persistent damage, as occurs after chronic challenge to lysosome integrity, triggers wider cellular responses. Here, we show that longitudinal treatment with a lysosomotropic cancer therapeutic, the CDK4/6 inhibitor Palbociclib, invokes chronic lysosome damage in breast and lung cancer cells. Autophagy ameliorates but does not avert this phenotype, which persists over days. Damaged lysosomes form contacts with mitochondria, which correlates with mitochondrial stress and cytosolic efflux of immunostimulatory mitochondrial nucleic acids. Importantly, mitochondrial nucleic acid release is necessary for the anti-cancer interferon response to Palbociclib. In conclusion, chronic lysosome damage rewires cellular signalling responses in a mitochondrion-dependent manner and this effect should be considered when assessing the cellular actions of cancer therapeutics. Summary statementBozic et al suggest that lysosome damage can trigger interferon responses dependent upon mitochondrial release of immunogenic nucleic acid. This is associated with damaged lysosome-mitochondrion contacts and is prevented by autophagy.

cell biology↗

bioRxiv2025

Overcoming preservation challenges to enable single-cell proteomics of fixed cell and tissue samples with retained proteome integrity

The ability to assay the molecular composition of biological systems with single-cell resolution has revolutionised our understanding of tissue heterogeneity and function. Recent advances in single-cell proteomics (SCP) now enable the unbiased quantification of the proteome to a depth of several thousand proteins across hundreds of cells. Yet the broader adoption beyond specialised groups remains limited due to the need for specific equipment and expertise. A major challenge in making these analyses more broadly available is sample preservation for transporting biological material to SCP-capable facilities. To address this issue and provide practical solutions; we first evaluated various cell preservation methods from monolayer culture samples, then tested our optimised methodology on both cultured cells and, for the first time, preserved animal tissue from an in vivo mouse model. Our findings highlight the feasibility of SCP analyses in preserved tissues, significantly expanding its current applicability. By optimising upstream processing, our approach enables robust single-cell proteome analysis of both cells and tissues, making SCP more accessible to the wider scientific community. Ultimately, this advancement expands the potential applications of SCP, particularly in disciplines where analysing rare or heterogeneous populations is beneficial.

biochemistry↗

bioRxiv2024

NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress

WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity ligation approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation and mass spectrometry and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a R{Phi}-motif binding conserved C-terminal (CCT) domain, similar to the CCT domains in WNKs, SPAK and OXSR1. Knockdown or knock-out of NRBP1 markedly inhibited sorbitol-induced activation of WNK1 and downstream components. We demonstrate recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modelling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 R{Phi}-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicates NRBP1 functions as an upstream activator of the WNK pathway. TeaserNRBP1 functions as a scaffolding component regulating the assembly of a multi-subunit complex, required for the activation of the WNK Lysine Deficient Protein Kinase family in response to osmotic stress. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/628181v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5b37eborg.highwire.dtl.DTLVardef@b4661corg.highwire.dtl.DTLVardef@19bc3e9org.highwire.dtl.DTLVardef@15b052e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Upscaling production of immunogenic poliovirus virus-like particles in Pichia Pastoris by controlled fermentation

The success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses a significant threat to maintaining a polio-free world. Recombinant virus-like particles (VLPs) that lack the viral genome remove this risk. Here, we demonstrate the production of PV VLPs for all three serotypes by controlled fermentation using Pichia pastoris. We determined the cryo-EM structure of a new PV-2 mutant, termed SC5a, in comparison to PV-2 SC6b VLPs described previously and investigated the immunogenicity of PV-2 SC5a VLPs. Finally, a trivalent immunogenicity trial using bioreactor-derived VLPs of all three serotypes in the presence of Alhydrogel adjuvant, showed that these VLPs outperform the current IPV vaccine in the standard vaccine potency assay, offering the potential for dose-sparing. Overall, these results provide further evidence that yeast-produced VLPs have the potential to be a next-generation polio vaccine in a post-eradication world.

microbiology↗

bioRxiv2022

Defining cellular population dynamics at single cell resolution during prostate cancer progression

Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigated prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in vivo. We observe a dramatic expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients. Moreover, transformed epithelial cells and associated fibroblasts create a microenvironment conducive to pro-tumorigenic immune infiltration, which is in part androgen responsive. Androgen independent prostate cancer leads to significant diversification of intermediate luminal cell populations characterized by a range of androgen signaling activity inversely correlated with proliferation and mRNA translation. Accordingly, distinct epithelial populations are exquisitely sensitive to translation inhibition which leads to epithelial cell death, loss of pro-tumorigenic signaling, and decreased tumor heterogeneity. Our findings reveal a complex tumor environment largely dominated by castration-resistant luminal cells and immunosuppressive infiltrates.

cancer biology↗

bioRxiv2021

Toll-like receptor 2 orchestrates a potent anti-tumor response in non-small cell lung cancer

Targeting early-stage lung cancer is vital to improve overall survival. We previously identified Toll-like receptor 2 (TLR2) as a regulator of oncogene-induced senescence (OIS) and the senescence-associated secretory phenotype (SASP), both key for tumor suppression. Here, we demonstrate that TLR2 is widely expressed in human lung tumor epithelium where it correlates with improved survival and clinical regression. Using genetically engineered mouse models of lung cancer we have shown that Tlr2 is a tumor suppressor in lung cancer initiation via regulation of proliferation and the SASP. The SASP is integral in the regulation of immune surveillance of premalignant cells, and we observe impaired myeloid derived immune surveillance following Tlr2 loss. Lastly, we show that administration of a synthetic Tlr2 agonist significantly reduces preinvasive lung tumor growth. Our data highlight an unexpected tumor surveillance pathway in early-stage lung cancer with therapeutic potential. Statement of significanceLung cancer is a major cancer of unmet need. This study identifies a novel tumor suppressor mechanism in lung cancer. Not only does this highlight a potential therapeutic target for early-stage disease but also multiple secreted candidate biomarkers that could be exploited to augment lung cancer screening approaches.

cancer biology↗

bioRxiv2021

Comparison of Approaches to Transcriptomic Analysis in Multi-Sampled Tumors

Intratumoral heterogeneity is a well-documented feature of human cancers associated with outcome and treatment resistance. However, a heterogeneous tumor transcriptome contributes an unknown level of variability to analyses of differentially expressed genes that may contribute to phenotypes of interest, including treatment response. Although current clinical practice and the vast majority of research studies use a single sample from each patient, decreasing costs in sequencing technologies and computing costs have made repeated-measures analyses increasingly economical. Repeatedly sampling the same tumor increases the statistical power of differentially expressed gene analysis that is indispensable towards downstream analysis and also increases ones understanding of within-tumor variance that may affect conclusions. Here, we compared five different methods for analyzing gene expression profiles derived from repeated sampling of human prostate tumors in two separate cohorts of patients. We also benchmarked the sensitivity of generalized linear models to linear mixed models for identifying differentially expressed genes contributing to relevant prostate cancer pathways based on a ground truth model.

bioinformatics↗

bioRxiv2021

Targeted gene sequencing with Nanopore enables rapid and accurate confirmatory diagnostic of Tilapia lake virus

Infectious diseases represent one of the major challenges to sustainable aquaculture production. Rapid, accurate diagnosis and genotyping of emerging pathogens during early-suspected disease cases is critical to facilitate timely response to deploy adequate control measures and prevent or reduce spread. Currently, most laboratories use PCR to amplify partial pathogen genomic regions, occasionally combined with sequencing of PCR amplicon(s) using conventional Sanger sequencing services for confirmatory diagnosis. The main limitation of this approach is the lengthy turnaround time. Here, we report an innovative approach using a previously developed specific PCR assay for pathogen diagnosis combined with a new Oxford Nanopore Technologies (ONT)-based amplicon sequencing method for pathogen genotyping. Using fish clinical samples, we applied this approach for the rapid confirmation of PCR amplicon sequences identity and genotyping of tilapia lake virus (TiLV), a disease-causing virus affecting tilapia aquaculture globally. The consensus sequences obtained after polishing exhibit strikingly high identity to references derived by Illumina and Sanger methods (99.83-100%). This study suggests that ONT-based amplicon sequencing is a promising platform to deploy in regional aquatic animal health diagnostic laboratories in low and medium income countries, for fast identification and genotyping of emerging infectious pathogens from field samples within a single day.

molecular biology↗

bioRxiv2019

MEIS1 down-regulation by MYC mediates prostate cancer development through elevated HOXB13 expression and AR activity

Localized prostate cancer develops very slowly in most men, with the androgen receptor (AR) and MYC transcription factors amongst the most well-characterized drivers of prostate tumorigenesis. Canonically, MYC up-regulation in luminal prostate cancer cells functions to oppose the terminally differentiating effects of AR. However, the effects of MYC up-regulation are pleiotropic and inconsistent with a poorly proliferative phenotype. Here we show that increased MYC expression and activity are associated with the down-regulation of MEIS1, a HOX-family transcription factor. Using RNA-seq to profile a series of human prostate cancer specimens laser capture microdissected on the basis of MYC immunohistochemistry, MYC activity and MEIS1 expression were inversely correlated. Knockdown of MYC expression in prostate cancer cells increased expression of MEIS1 and increased occupancy of MYC at the MEIS1 locus. Finally, we show in laser capture microdissected human prostate cancer samples and the prostate TCGA cohort that MEIS1 expression is inversely proportional to AR activity as well as HOXB13, a known interacting protein of both AR and MEIS1. Collectively, our data demonstrate that elevated MYC in a subset of primary prostate cancers functions in a negative role in regulating MEIS1 expression, and that this down-regulation may contribute to MYC-driven development and progression.

cancer biology↗

bioRxiv2019

Low abundance of circulating tumor DNA in localized prostate cancer

Despite decreased screening-based detection of clinically insignificant tumors, most diagnosed prostate cancers are still indolent, indicating a need for better strategies for detection of clinically significant disease prior to treatment. We hypothesized that patients with detectable circulating tumor DNA (ctDNA) were more likely to harbor aggressive disease. We applied ultra-low pass whole genome sequencing to profile cell-free DNA from 112 patients diagnosed with localized prostate cancer and performed targeted resequencing of plasma DNA for somatic mutations previously identified in matched solid tumor in nine cases. We also performed similar analyses on patients with metastatic prostate cancer. In all cases of localized disease, even in clinically high-risk patients who subsequently recurred, we did not detect ctDNA by either method in plasma acquired before surgery or before recurrence. In contrast, ctDNA was detected from patients with metastatic disease. Our findings demonstrate clear differences between localized and advanced prostate cancer with respect to the dissemination and detectability of ctDNA. Because allele-specific alterations in ctDNA are below the threshold for detection in localized prostate cancer, other approaches to identify cell-free nucleic acids of tumor origin may demonstrate better specificity for aggressive disease.

genomics↗

bioRxiv2017

CardioClassifier: demonstrating the power of disease- and gene-specific computational decision support for clinical genome interpretation

PurposeInternationally-adopted variant interpretation guidelines from the American College of Medical Genetics and Genomics (ACMG) are generic and require disease-specific refinement. Here we developed CardioClassifier (www.cardioclassifier.org), a semi-automated decision-support tool for inherited cardiac conditions (ICCs).\n\nMethodsCardioClassifier integrates data retrieved from multiple sources with user-input case-specific information, through an interactive interface, to support variant interpretation. Combining disease- and gene-specific knowledge with variant observations in large cohorts of cases and controls, we refined 14 computational ACMG criteria and created three ICC-specific rules.\n\nResultsWe benchmarked CardioClassifier on 57 expertly-curated variants and show full retrieval of all computational data, concordantly activating 87.3% of rules. A generic annotation tool identified fewer than half as many clinically-actionable variants (64/219 vs 156/219, Fishers P=1.1x10-18), with important false positives; illustrating the critical importance of disease and gene-specific annotations.\n\nCardioClassifier identified putatively disease-causing variants in 33.7% of 327 cardiomyopathy cases, comparable with leading ICC laboratories. Through addition of manually-curated data, variants found in over 40% of cardiomyopathy cases are fully annotated, without requiring additional user-input data.\n\nConclusionCardioClassifier is an ICC-specific decision-support tool that integrates expertly curated computational annotations with case-specific data to generate fast, reproducible and interactive variant pathogenicity reports, according to best practice guidelines.

genomics↗