Biology subjects

Kent, C.

Publications and source records attributed to Kent, C..

7 recordsLinked to original sources

bioRxiv2026

Methodological Evaluation and Data Resource for Andes Virus Sequencing Preparedness

As part of preparedness activities supporting pathogens classified under the UK High Consequence Infectious Diseases (HCID) framework, we previously evaluated both a whole-genome tiling amplicon sequencing scheme and a pan-viral hybridisation capture approach (TWIST-CVRP) for sequencing Andes virus (ANDV). In light of the recent outbreak, we make available viral sequencing datasets generated using a historical ANDV isolate (Chile, 1997). In addition, we provide an evaluation of tiling amplicon scheme performance and present recommended primer updates informed by in silico comparison with the recently released outbreak genome. These datasets are intended to support benchmarking, validation, and optimisation of bioinformatic pipelines across the community.

genomics↗

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

Reducing Supply Chain Dependencies for Viral Genomic Surveillance: Get by with a Little HELP from Commercial Enzymes already in your Lab Freezer

The COVID-19 pandemic exposed vulnerabilities in global laboratory supply chains, disrupting genomic surveillance efforts essential to epidemic response. To address this challenge, we developed ARTIC HELP (Homebrew Enzymes for Library Preparation), a practical, open-source adaptation of the widely adopted ARTIC nanopore sequencing protocol for viral genomic surveillance. We describe generic, cost-effective alternatives to all enzyme mixes used in tiling multiplex RT-PCR amplification of the virus genome, and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL), making it broadly applicable to any laboratory. Through systematic evaluation, we identified a wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases as effective alternatives when standard reagents are unavailable due to high cost or limited supply: B-family Pfu-based polymerases with a fused Sso7d DNA-binding domain, and blends combining A-family (Taq-based) and B-family (Pfu-based) polymerases. Validation on clinical samples of SARS-CoV-2 and Norovirus GII confirmed that the HELP workflow achieves genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct [≤]28), the wild-type M-MLV RT combined with selected Pfu or A+B polymerases, along with optimised HELP mixes (EP, BL, AL), achieved genome coverage of 84.0-99.6%. For Norovirus GII (Ct [≤]32), the HELP workflow using one of the Pfu polymerases achieved genome coverage of >85% for six out of eight genotypes tested. Notably, several of the other polymerases tested showed reduced performance at higher Ct values. However, they still achieved strong coverage at Ct <24, supporting their use as emergency alternatives in rapid outbreak-response sequencing when viral input is high and RNA quality is sufficient. Our approach, ARTIC HELP, provides a framework which can be implemented to address supply chain disruptions, while maintaining robust genomic sequencing capabilities. A cost analysis highlights the well-known significant global disparities in reagent pricing, driven not by protocol differences but by import fees and supply barriers. Thus, our findings highlight the need for fairer global pricing models and support for local sourcing strategies like HELP, to promote equity in genomic research and ensure preparedness for future public health challenges.

genomics↗

bioRxiv2024

PrimalScheme: open-source community resources for low-cost viral genome sequencing

Viral genome sequencing using the ARTIC protocol has been a vital tool for understanding the spread of epidemics including Ebola, Zika, Covid-19 and Mpox and has seen widespread adoption due to its low cost and high sensitivity. Here, we describe PrimalScheme, an open-source toolkit and website that allows users to easily design primer schemes for amplicon sequencing of viruses, and has generated over 67,000 primer schemes for the global community since 2017. In January 2020, PrimalScheme was used to rapidly generate a primer scheme for SARS-CoV-2, with primer pools distributed to researchers from 44 countries to help scale-up genomic surveillance efforts. Overall, these primers were used to generate an estimated 18M genome sequences and the protocols were viewed online [~]250K times. To complement PrimalScheme, we have built PrimalScheme Labs, a scheme repository which allows users to find and share primer schemes as well as establishing a set of data standards. Through improvements to the primer design process, including the use of discrete primer clouds, we have expanded the use of amplicon sequencing to include diverse virus species. We demonstrate the utility of this approach through a high diversity pan-genotype Measles virus (MeV) scheme. We also demonstrate its use on a high sensitivity, short amplicon Monkeypox virus (MPXV) scheme with over 1000 primers, showing high genome recovery on low-titre clinical samples. These developments have implications for sequencing from samples such as wastewater, for genomic surveillance of endemic pathogens and in preparing for future pandemics.

genomics↗

bioRxiv2023

White matter hyperintensity genetic risk factor TRIM47 regulates autophagy in brain endothelial cells

White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. GWAS identified TRIM47 at 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found selectively expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially-induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we identified the highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing Trim47 significantly increased autophagy with ULK1 phosphorylation induction, transcription and vacuole formation. Together, we confirm that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation. SUMMARY STATEMENTTRIM47, top genetic risk factor for white matter hyperintensity formation, is a negative regulator of autophagy in brain endothelial cells and implicates a novel cellular mechanism for age-related cerebrovascular changes.

cell biology↗

bioRxiv2020

Angioplasty-induced epigenomic remodeling entails BRD4 and EZH2 hierarchical regulations

Atherosclerosis is commonly treated with angioplasty which, however, evokes neointimal hyperplasia (IH) and recurrent stenotic diseases. Epigenomic investigation was lacking on post-angioplasty IH. The histone acetylation reader BRD4 and H3K27me3 writer EZH2 are potent epigenetic factors; their relationship is little understood. Through genome-wide survey in the rat angioplasty model, we studied BRD4 and EZH2 functional regulations involved in IH. We performed chromatin immunoprecipitation sequencing (ChIPseq) using rat carotid arteries. While H3K27me3 ChIPseq signal prevalently intensified in balloon-injured (vs uninjured) arteries, BRD4 and H3K27ac became more enriched at Ezh2. Indeed, BRD4-siRNA or CRISPR-deletion of BRD4-associated enhancer abated the smooth muscle cell (SMC) expression of EZH2, and SMC-specific BRD4 knockout in BRD4fl/fl; Myh11CreERT2 mice reduced both H3K27me3 and IH in wire-injured femoral arteries. In accordance, post-angioplasty IH was exacerbated and mitigated, respectively, by lentiviral expression and pharmacological inhibition of EZH2/1; EZH2 (or EZH1) loss- and gain-of-function respectively attenuated and aggravated pro-IH SMC proliferative behaviors. Furthermore, while H3K27me3 ChIPseq signal magnified at P57 and ebbed at Ccnd1 and Uhrf1 after injury, silencing either EZH2 or EZH1 in SMCs up-regulated P57 and down-regulated Ccnd1 and Uhrf1. In summary, our results reveal an upsurge of EZH2/H3K27me3 after angioplasty, BRD4s control over EZH2 expression, and non-redundant EZH2/1 functions. As such, this study unravels angioplasty-induced loci-specific H3K27me3/ac redistribution in the epigenomic landscape rationalizing BRD4/EZH2-governed pro-IH regulations.

pathology↗

bioRxiv2019

BRD2 regulation of sigma-2 receptor expression upon cytosolic cholesterol deprivation

Traditionally a pharmacologic target for antipsychotic treatment, the sigma-2 receptor (S2R) was recently implicated in cholesterol homeostasis. Here we investigated the transcriptional regulation of S2R by the Bromo/ExtraTerminal epigenetic reader family (BETs, including BRD2, 3, 4) upon cholesterol perturbation.\n\nCytosolic cholesterol deprivation was induced using an export blocker of lysosomal cholesterol in ARPE19 cells. This condition upregulated mRNA and protein levels of S2R, and of SREBP2 but not SREBP1, transcription factors key to cholesterol/fatty acid metabolism. Silencing BRD2 but not BRD4 (though widely deemed as a master regulator) or BRD3 prevented S2R upregulation induced by cholesterol deprivation. Silencing SREBP2 but not SREBP1 diminished S2R expression. Furthermore, BRD2 co-immunoprecipitated with the SREBP2 transcription-active N-terminal domain, and chromatin immunoprecipitation-qPCR showed a BRD2 occupancy at the S2R gene promoter.\n\nIn summary, this study reveals a novel BRD2/SREBP2 cooperative regulation of S2R transcription in response to cytosolic cholesterol deprivation, thus shedding new light on epigenetic control of cholesterol biology.

biochemistry↗