Biology subjects

Asquith, B.

Publications and source records attributed to Asquith, B..

5 recordsLinked to original sources

bioRxiv2026

Large-scale single-cell mapping of concealed mitochondrial ageing

Mitochondrial DNA (mtDNA) mutations have been linked to ageing. We find that the mtDNA mutations detected in routine bulk sequencing are greatly outnumbered by a class of concealed mutations that, despite being in a proliferative tissue, show little negative selection. Combining new scATAC-seq experiments with existing scRNA-seq, we map mtDNA mutations across 2.7 million cells from 311 individuals, covering 11 proliferative cell types and 5 tissues. We find that low-prevalence mutations (in[≤] 1% of assayed cells), concealed from routine bulk sequencing, accumulate markedly with age, with mutations unique to single cells (cryptic) predominating. We find a near universal accumulation rate across cell types and tissues, with, by age 50, approximately 40% of cells having at least one mutation affecting 30% of cellular mtDNA or more. At a correlative level we find that human age can be predicted from these concealed mutations but there is evidence of, possibly physiological, inter-individual variation. These low-prevalence mutations (unlike mutations tracking larger clones) show limited negative selection and coincide with gene-expression changes marking dysfunction.

genomics↗

bioRxiv2024

Inhibitory KIRs decrease HLA class II-mediated protection in Type 1 Diabetes

Inhibitory killer cell immunoglobulin-like receptors (iKIRs) are a family of inhibitory receptors that are expressed by natural killer cells and late-stage differentiated T cells. There is accumulating evidence that iKIRs regulate T cell-mediated immunity. Recently, we reported that T cell-mediated control was enhanced by iKIRs in chronic viral infections. We hypothesized that in the context of autoimmunity, where an enhanced T cell response might be considered detrimental, iKIRs would have an opposite effect. We studied Type 1 diabetes (T1D) as a paradigmatic example of autoimmunity. In T1D, variation in the Human Leucocyte Antigen (HLA) genes explains up to 50% of the genetic risk, indicating that T cells have a major role in T1D etiopathogenesis. To investigate if iKIRs affect this T cell response we asked whether HLA associations were modified by iKIR genes. We conducted an immunogenetic analysis of a case-control T1D dataset (N= 11,961) and found that iKIR genes, in the presence of genes encoding their ligands, have a consistent and significant effect on protective HLA class II genetic associations. Our results were validated in an independent data set. We conclude that iKIRs significantly decrease HLA class II protective associations and suggest that iKIRs regulate CD4+ T cell responses in T1D.

immunology↗

bioRxiv2024

Absolute quantification of rare gene targets in limited samples using crude lysate and ddPCR

BackgroundAccurate quantification of rare genes from limited clinical samples is crucial for research purposes but is technically challenging. In particular, nucleic acid extraction for quantification of gene targets may lead to target loss. Here, we report the development and validation of a novel crude lysate ddPCR assay for the absolute quantification of rare genes, TRECs in our case, from infrequent cells, that removes the need for DNA extraction, hence minimizing the target loss. MethodsThe analytical validation was performed on PBMCs extracted from the blood of healthy donors. Standard ddPCR was first optimized to detect TREC copies/cell and then applied to a crude lysate ddPCR assay. The assay was optimized by varying several steps. The optimised assay was directly compared to standard ddPCR and the performance of the assay quantified. ResultsThe newly developed assay showed good agreement with the standard ddPCR assay in the range from 0.0003 to 0.01 TRECs/cell. The assay had a limit of quantification of <0.0003 TRECs/cell and a limit of detection of <0.0001 TRECs/cell; this performance is favourable compared to standard ddPCR. The intra-assay variation was low. This method can also be applied to fixed and permeabilized cells. ConclusionsThe newly developed crude lysate ddPCR assay for quantifying rare targets from limited samples has high accuracy, specificity, and reproducibility; additionally, it eliminates the need for DNA extraction for absolute quantification. The assay has the potential to be used for quantification of other trace targets from small samples.

biochemistry↗

bioRxiv2024

The Impact of Model Assumptions in Interpreting Cell Kinetic Studies

Author summaryStable isotope labelling is one of the best available methods for quantifying cell dynamics in vivo, particularly in humans where the absence of toxicity makes it preferable over other techniques such as CFSE or BrdU. Interpretation of stable isotope labelling data necessitates simplifying assumptions. Here we investigate the impact of three of the most commonly used simplifying assumptions (that the cell population of interest is closed, that the population of interest is kinetically homogeneous, and that the population is spatially homogeneous) and suggest pragmatic ways in which the resulting errors can be reduced.

immunology↗

bioRxiv2019

The relative contributions of infectious and mitotic spread to HTLV-1 persistence

Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via infectious spread (de novo infection) and mitotic spread (infected cell proliferation), creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). The relative contributions of infectious and mitotic spread to HTLV-1 persistence are unknown, and will determine the efficacy of different approaches to treatment.\n\nThe prevailing view is that infectious spread is negligible in HTLV-1 proviral load maintenance beyond early infection. However, in light of recent high-throughput data on the abundance of HTLV-1 clones, and recent estimates of HTLV-1 clonal diversity that are substantially higher than previously thought (typically between 104 and 105 HTLV-1+ T cell clones in the body of an asymptomatic carrier or patient with HAM/TSP), ongoing infectious spread during chronic infection remains possible.\n\nWe estimate the ratio of infectious to mitotic spread using a hybrid model of deterministic and stochastic processes, fitted to previously published HTLV-1 clonal diversity estimates. We investigate the robustness of our estimates using two alternative methods. We find that, contrary to previous belief, infectious spread persists during chronic infection, even after HTLV-1 proviral load has reached its set point, and we estimate that between 100 and 200 new HTLV-1 clones are created and killed every day. We find broad agreement between all three methods.\n\nThe risk of HTLV-1-associated malignancy and inflammatory disease is strongly correlated with proviral load, which in turn is correlated with the number of HTLV-1-infected clones, which are created by de novo infection. Our results therefore imply that suppression of de novo infection may reduce the risk of malignant transformation.\n\nAuthor SummaryThere are no effective antiretroviral treatments against Human T-lymphotropic virus type-1 (HTLV-1), which causes a range of inflammatory diseases and the aggressive malignancy Adult T-cell Leukaemia/Lymphoma (ATL) in approximately 10% of infected people. Within hosts the virus spreads via infectious spread (de novo infection) and mitotic spread (infected cell division). The relative contributions of each mechanism are unknown, and have major implications for drug development and clinical management of infection. We estimate the ratio of infectious to mitotic spread during the infections chronic phase using three methods. Each method indicates infectious spread at low but persistent levels after proviral load has reached set point, contrary to the prevailing view that infectious spread features in early infection only. Risk of disease in HTLV-1 infection is known to increase with proviral load, via mutations accrued from repeated infected cell division. Our analyses suggest that ongoing infectious spread may provide an additional mechanism whereby chronic infection becomes malignant. Further, because antiretroviral drugs against Human Immunodeficiency Virus (HIV) inhibit HTLV-1 infectious spread, they may reduce the risk of HTLV-1 malignancy.

immunology↗