Biology subjects

Wilkinson, D. J.

Publications and source records attributed to Wilkinson, D. J..

5 recordsLinked to original sources

bioRxiv2025

Proteostasis Modelling using Deuterated Water Metabolic Labeling and Data-Independent Acquisition Mass Spectrometry

We present the first application of a deuterated water metabolic labeling workflow coupled with data-independent acquisition (DIA) tandem mass spectrometry (MS/MS) for quantifying label enrichment in MS/MS to study protein turnover. The approach automates the turnover rate determination from combined precursor and fragment ions. The truncation of the observed isotope distributions of fragments is overcome by implementing an approach to determining the label enrichment from two mass isotopomers. The high redundancy of fragment ions provides a confident assessment for deuterium enrichment. The DIA approach provides increased proteome coverage and depth for quantifying protein turnover rates compared to the traditional data-dependent (DDA) approach. This novel approach is validated in a murine myotube model of muscle hypertrophy and atrophy through the treatment with insulin-like growth factor 1 (IGF-1) and dexamethasone (Dex), respectively.

bioinformatics↗

bioRxiv2025

Rapamycin exerts its geroprotective effects in the ageing human immune system by enhancing resilience against DNA damage

mTOR inhibitors such as rapamycin are among the most robust life-extending interventions known, yet the mechanisms underlying their geroprotective effects in humans remain incompletely understood. At non-immunosuppressive doses, these drugs are senomorphic, i.e. they mitigate cellular senescence, but whether they protect genome stability itself has been unclear. Given that DNA damage is a major driver of immune ageing, and immune decline accelerates whole-organism ageing, we tested whether mTOR inhibition enhances genome stability. In human T cells exposed to acute genotoxic stress, we found that rapamycin and other mTOR inhibitors suppressed senescence not by slowing protein synthesis, halting cell division, or stimulating autophagy, but by directly reducing DNA lesional burden and improving cell survival. Ex-vivo analysis of aged immune cells from healthy donors revealed a stark enrichment of markers for DNA damage, senescence, and mTORC hyperactivation, suggesting that human immune ageing may be amenable to intervention by low-dose mTOR inhibition. To test this in vivo, we conducted a placebo-controlled experimental medicine trial in older adults administered with low-dose rapamycin. p21, a marker of DNA damage-induced senescence, was significantly reduced in immune cells from the rapamycin compared to placebo group. These findings reveal a previously unrecognised role for mTOR inhibition: direct genoprotection. This mechanism may help explain rapamycins exceptional geroprotective profile and opens new avenues for its use in contexts where genome instability drives pathology, ranging from healthy ageing, clinical radiation exposure, and even the hazards of cosmic radiation in space travel.

immunology↗

bioRxiv2024

Domain Specific AI Segmentation of IMPDH2 Rod/Ring Structures in Mouse Embryonic Stem Cells

BackgroundInosine monophosphate dehydrogenase 2 (IMPDH2) is an enzyme that catalyses the rate limiting step of guanine nucleotides. In mouse embryonic stem cells (ESCs) IMPDH2 is held as large multi-protein complexes known as rod-ring (RR) structures that dissociate when ESCs differentiate. Manual analysis of RR structures from confocal microscopy images, although possible, is not feasible on a large scale due to the quantity of RR structures present in each field of view. To address this analysis bottleneck, we have created a fully automatic RR image classification pipeline to segment, characterise and measure feature distributions of these structures in ESCs. ResultsWe find that this model can automatically segment images with a Dice score of over 80% for both rods and rings for in-domain images compared to expert annotation, with a slight drop to 70% for datasets out of domain. Important feature measurements derived from these segmentations show high agreement with the measurements derived from expert annotation, achieving an R2 score of over 90% for counting the number of rings and rods over the dataset. ConclusionsWe have established for the first time a quantitative baseline for RR distribution in pluripotent ESCs and have made a pipeline available for training to be applied to other models in which RR remain an open topic of study.

bioinformatics↗

bioRxiv2021

Motor unit recruitment strategies of the vastus lateralis according to sex

AimDespite men exhibiting greater muscle strength and fatigibility than women, it remains unclear if there are sex-based differences in muscle recruitment strategies e.g. motor unit (MU) recruitment and modulation of firing rate (FR) at normalised forces and during progressive increases in force. MethodsTwenty-nine healthy male and thirty-one healthy female participants (18-35 years) were studied. Intramuscular electromyography was used to record individual motor unit potentials (MUPs) and near fibre MUPs from the vastus lateralis (VL) during 10% and 25% maximum isometric voluntary contractions (MVC), and spike-triggered averaging was used to obtain motor unit number estimates (MUNE) of the VL. Multilevel mixed-effects linear regression models were used to investigate the effects of sex at each contraction level. ResultsMen exhibited greater muscle strength (p<0.001) and size (p<0.001) than women, with no difference in force steadiness at 10% or 25% MVC. Women had smaller MUs and higher FR at 10% MVC (both p<0.02), similar to that at 25% MVC in MU size (p=0.062) and FR (p=0.031). However, both sexes showed similar increases in MU size and FR when moving from low-to mid-level contractions. There were no sex differences in any near fibre MUP parameters or in MUNE. ConclusionIn the vastus lateralis, women produce muscle force via different neuromuscular recruitment strategies to men which is characterised by smaller MUs discharging at higher rates. However, similar strategies are employed to increase force production from low to moderate contractions. These findings of similar proportional increases between sexes support the use of mixed sex cohorts in studies of this nature. Key pointsO_LIIncreases in muscle force production are mediated by motor unit (MU) recruitment, and MU firing rate (FR). C_LIO_LIWomen are underrepresented in studies of human neuromuscular research and markedly differ to men in a number of aspects of neuromuscular function, yet little is known of the recruitment strategies of each. C_LIO_LIHere we demonstrate men and women have similar vastus lateralis MU number estimates, yet women recruit smaller MUs with higher FR than men at normalised contraction levels. However, increases in force are achieved via similar trajectories of MU recruitment and MU FR in men and women. C_LIO_LIAlthough men and women exhibit divergent neuromuscular recruitment strategies to achieve normalised forces, increases in force are achived similarly and support the inclusion of mixed sex cohorts in studies of this nature. C_LI

physiology↗

bioRxiv2017

Systematic analysis of the effects of the DNA damage response network in telomere defective budding yeast

Functional telomeres are critically important to eukaryotic genetic stability. Budding yeast is a powerful model organism for genetic analysis and yeast telomeres are maintained by very similar mechanisms to human telomeres. Scores of proteins and pathways are known to affect telomere function. Here, we report a series of related genome-wide genetic interaction screens performed on budding yeast cells with acute or chronic telomere defects. We examined genetic interactions in cells defective in Cdc13 and Stn1, affecting two components of CST, a single stranded DNA (ssDNA) binding complex that binds telomeric DNA. We investigated genetic interactions in cells with defects in Rfa3, affecting the major ssDNA binding protein, RPA, which has overlapping functions with CST at telomeres. We also examined genetic interactions in cells lacking EXO1 or RAD9, affecting different aspects of the DNA damage response in a cdc13-1 background. Comparing fitness profiles across the data sets allows us build up a picture of the specific responses to different types of dysfunctional telomeres. Our results show that there is no universal response to telomere defects. To help others engage with the large volumes of data we make the data available via two interactive web-based tools: Profilyzer and DIXY. Among numerous genetic interactions we found the chk1{Delta} mutation improved fitness of cdc13-1 exo1{Delta} cells more than other checkpoint mutations (ddc1{Delta}, rad9{Delta}, rad17{Delta}, rad24{Delta}), whereas in cdc13-1 cells the effects of all checkpoint mutations were similar. We find that Chk1 stimulates resection at defective telomeres, revealing a new role for Chk1 in the eukaryotic DNA damage response network.

genetics↗