Biology subjects

Atkinson, M.

Publications and source records attributed to Atkinson, M..

6 recordsLinked to original sources

bioRxiv2025

The SpyBLI cell-free pipeline for the rapid quantification of binding kinetics from crude samples

Accurate measurements of binding kinetics, encompassing equilibrium dissociation constant (KD), association rate (kon), and dissociation rate (ko,), are critical for the development and optimisation of high-affinity binding proteins. However, such measurements require highly purified material and precise ligand immobilisation, limiting the number of binders that can be characterised within a reasonable timescale and budget. Here, we present the SpyBLI method, a rapid and cost-effective biolayer interferometry (BLI) pipeline that leverages the SpyCatcher003-SpyTag003 covalent association, eliminating the need for both binder purification and concentration determination. This approach allows for accurate binding-kinetic measurements to be performed directly from crude mammalian-cell supernatants or cell-free expression mixtures. We also introduce a linear gene fragment design that enables reliable expression in cell-free systems without any PCR or cloning steps, allowing binding kinetics data to be collected in under 24 hours from receiving inexpensive DNA fragments, with minimal hands-on time. We demonstrate the methods broad applicability using a range of nanobodies and single-chain antibody variable fragments (scFvs), with affinity values spanning six orders of magnitude. By minimising sample preparation and employing highly controlled, ordered sensor immobilisation, our workflow delivers reliable kinetic measurements from crude mixtures without sacrificing precision. We expect that the opportunity to carry out rapid and accurate binding measurements in good throughput should prove especially valuable for binder engineering, the screening of next-generation sequencing-derived libraries, and computational protein design, where large numbers of potential binders for the same target must be rapidly and accurately characterised to enable iterative refinement and candidate selection.

biophysics↗

bioRxiv2024

ENTPD3-specific CAR Regulatory T cells for Local Immune Control in T1D

Despite advances in Type 1 Diabetes (T1D) management such as hybrid closed loop systems, patients still face significant morbidity, reduced life expectancy, and impaired glucose regulation compared to healthy individuals or those with pancreas transplants. Here we developed beta cell-specific Chimeric Antigen Receptors (CAR) targeting the antigen ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) using a novel cell-based phage display methodology. ENTPD3 is highly expressed on beta cells of both early and progressed T1D patients. ENTPD3 CAR regulatory T cells (Tregs) homed, expanded and persisted in pancreatic islets in a T1D mouse model (NOD) and completely prevented disease progression. Human ENTPD3 CAR Tregs displayed a stable regulatory phenotype, strong activation, and suppression. Importantly, ENTPD3 CAR T cells recognised and were fully activated by human islets. This approach holds great promise as a durable treatment option for patients with prediabetes, new-onset diabetes, or those undergoing beta cell replacement therapy.

immunology↗

bioRxiv2021

Alpha cell dysfunction in early type 1 diabetes

Multiple islet autoantibodies (AAb) predict type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in just [~]15% of single AAb+ (generally against glutamic acid decarboxylase, GADA+) individuals; hence the single GADA+ state may represent an early stage of T1D amenable to interventions. Here, we functionally, histologically, and molecularly phenotype human islets from non-diabetic, GADA+ and T1D donors. Similar to the few remaining beta cells in T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, alpha cell glucagon secretion was dysregulated in both T1D and GADA+ islets with impaired glucose suppression of glucagon secretion. Single cell RNA sequencing (scRNASeq) of GADA+ alpha cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of PKIB, providing a molecular basis for the loss of glucose suppression and the increased effect of IBMX observed in GADA+ donor islets. The striking observation of a distinct early defect in alpha cell function that precedes beta cell loss in T1D suggests that not only overt disease, but also the progression to T1D itself, is bihormonal in nature.

physiology↗

bioRxiv2021

geneBasis: an iterative approach for unsupervised selection of targeted gene panels from scRNA-seq.

The problem of selecting targeted gene panels that capture maximum variability encoded in scRNA-sequencing data has become of great practical importance. scRNA-seq datasets are increasingly being used to identify gene panels that can be probed using alternative molecular technologies, such as spatial transcriptomics. In this context, the number of genes that can be probed is an important limiting factor, so choosing the best subset of genes is vital. Existing methods for this task are limited by either a reliance on pre-existing cell type labels or by difficulties in identifying markers of rare cell types. We resolve this by introducing an iterative approach, geneBasis, for selecting an optimal gene panel, where each newly added gene captures the maximum distance between the true manifold and the manifold constructed using the currently selected gene panel. We demonstrate, using a variety of metrics and diverse datasets, that our approach outperforms existing strategies, and can not only resolve cell types but also more subtle cell state differences. Our approach is available as an open source, easy-to-use, documented R package (https://github.com/MarioniLab/geneBasisR).

bioinformatics↗

bioRxiv2020

Development of early-stage type 1 diabetes in germ-free interleukin-10 deficient mice

Several experimental models demonstrate a role for gut microbiota in the progression of type 1 diabetes (T1D) in genetically prone hosts. While the association between disturbances in gut microbiota, or microbial dysbiosis, and complex immune diseases such as inflammatory bowel diseases (IBD) are well established, less is known about its role in T1D pathogenesis. In IBD-prone interleukin-10 deficient (IL-10 KO) mice, the absence of gut microbiota under germ-free (GF) conditions prevents IBD development. However, in aged GF IL-10 KO mice (>6-months of age), polyuria and pancreatic lymphocytic infiltration resembling T1D lesions was observed. Approximately 50% of male and female mice above 6-months of age develop pancreatic immune cell infiltration, as compared to none in conventionally-raised and fecal microbiota transplanted (FMT) IL-10 KO counterparts. Immunofluorescence staining of islet infiltrates was positive for adaptive and innate immunological markers, including lymphoid and myeloid cell markers, which typically characterize autoimmune T1D lesions. A subset of GF IL-10 KO mice was also positive for insulin autoantibodies (IAA), but the majority of mice did not become diabetic. Our findings of early stage lymphocytic infiltrates in the pancreas and IAA in the absence of overt diabetes in GF IL-10 KO mice embody the early stages of T1D pathogenesis. As such, we propose that the presence of gut microbiota play a protective role against immune infiltration in the pancreas of genetically prone hosts. Moreover, our model provides an opportunity to better understand the role of the microbiota in the early stages of immune pathogenesis and perhaps conceive the development of microbe-mediated prophylactic strategies to treat or even prevent T1D.

physiology↗

bioRxiv2019

Cohort Profile: HealthWise Wales. A research register and data collection and analysis platform with linkage to NHS datasets in Wales

PurposeRecruitment and follow-up in epidemiological studies is challenging, time-consuming and expensive. Combining online data collection with a register of individuals who agree to be contacted with information on research opportunities provides an efficient, cost-effective platform for population-based research. HealthWise Wales (HWW) aims to support researchers by recruiting a cohort of \"research-ready\" individuals; advertising relevant studies to these participants; providing access to cohort data for secondary analyses; and supporting data collection on specific topics that can be linked with healthcare data.\n\nParticipantsAdults (aged 16 and above) living or receiving their healthcare in Wales are eligible for inclusion. Participants consent to be followed-up every 6 months; for their details to be used to access their routinely-collected NHS records for research purposes; to be contacted about research projects in which they could participate; and to be informed about involvement or engagement opportunities. Data are collected using a web-based application, with new questionnaires added every six months. Data collection on socio-demographic and lifestyle factors is repeated at two-to-three year intervals. Recruitment is ongoing, with 21,779 active participants (alive and currently registered).\n\nFindings to date99% of participants have complete information on age and sex, and 64% have completed questionnaires on socio-demographic and lifestyle factors. These data can be linked with national health databases within the Secure Anonymised Information Linkage (SAIL) databank, with 93% of participants matching a record in SAIL. HWW has facilitated recruitment of 43,826 participants to 15 different studies.\n\nFuture plansThe medium-term goal for the project is to enrol at least 50,000 adults. Recruitment strategies are being devised to achieve a study sample that closely models the population of Wales, with sufficient numbers in socio-demographic subgroups to allow for the selection of populations for research from those groups. Potential bio-sampling methods are also currently being explored.

epidemiology↗