Biology subjects

Johnstone, J. N.

Publications and source records attributed to Johnstone, J. N..

5 recordsLinked to original sources

bioRxiv2026

Quantifying Somatic Mutation Burden: An Assay Validation Framework and Implementation in SomaticCODEC

Validation of somatic mutation burden assays is fundamentally constrained by the absence of a robust ground truth, limiting the interpretability of performance metrics. To address this, we propose a framework based primarily on relative validation, complemented by a suite of secondary metrics aligned to common failure modes. We implement this approach in SomaticCODEC, a ready-to-run assay for quantifying SNV burden in primary human samples, demonstrating strong linearity across mixtures of sperm and blood samples (R2 = 0.91) and high intra-batch precision (CV = 3.3%). This framework provides a practical approach for validating somatic mutation burden assays without requiring a ground truth.

genomics↗

bioRxiv2024

Transiently restricting individual amino acids protects D. melanogaster against multiple stressors

Nutrition and resilience are linked, though it is not yet clear how diet confers stress resistance or the breadth of stressors that it can protect against. We have previously shown that transiently restricting an essential amino acid can extend lifespan and also protect against nicotine exposure in Drosophila melanogaster, raising the possibility that amino acid restriction is geroprotective because of elevated detoxification capacity. Here, we sought to characterise the nature of this dietary mediated protection, and determine whether it was sex, amino acid, and/or nicotine specific. When we compared between sexes, we found that isoleucine deprivation increases female, but not male, nicotine resistance. Surprisingly, we found that this protection afforded to females was not replicated by dietary protein restriction and was instead specific to individual amino acid restriction. Other studies have documented methionine or leucine restriction conferring stress resistance, though we previously found that individually depriving them did not increase nicotine resistance. We therefore wondered whether reducing the severity of restriction of these amino acids could confer nicotine resistance. This was true for methionine restriction, and we found that flies fed a diet containing 25% methionine for 7 days protected against subsequent nicotine poisoning ([~]30% longer lived than controls with all amino acids). However, when dietary leucine was altered, nicotine resistance changed, but no single diet was protective. To understand whether these beneficial effects of diet were specific to nicotine or were generalisable across stressors, we pre-treated with amino acid restriction diets and exposed them to other types of stress. We did not find any diets that protected against heat stress or infection with the bacterium Enterococcus faecalis. However, we found that some of the diets that protected against nicotine also protected against oxidative and starvation stress, and improved survival following cold shock. Interestingly, we found that a diet lacking isoleucine was the only diet to protect against all these stressors. These data point to isoleucine as a critical determinant of robustness in the face of environmental challenges.

genetics↗

bioRxiv2023

GCN2 mediates access to stored amino acids for somatic maintenance during Drosophila ageing

Many mechanistic theories of ageing argue that a progressive failure of somatic maintenance, the use of energy and resources to prevent and repair damage to the cell, underpins ageing. To sustain somatic maintenance an organism must acquire dozens of essential nutrients from the diet, including essential amino acids (EAAs), which are physiologically limiting for many animals. In Drosophila, adulthood deprivation of each individual EAA yields vastly different lifespan trajectories, and adulthood deprivation of one EAA, phenylalanine (Phe), has no associated lifespan cost; this is despite each EAA being strictly required for growth and reproduction. Moreover, survival under any EAA deprivation depends entirely on the conserved AA sensor GCN2, a component of the integrated stress response (ISR), suggesting that a novel ISR-mediated mechanism sustains lifelong somatic maintenance during EAA deprivation. Here we investigated this mechanism, finding that flies chronically deprived of dietary Phe continue to incorporate Phe into new proteins, and that challenging flies to increase the somatic requirement for Phe shortens lifespan under Phe deprivation. Further, we show that autophagy is required for full lifespan under Phe deprivation, and that activation of the ISR can partially rescue the shortened lifespan of GCN2-nulls under Phe deprivation. We therefore propose a mechanism by which GCN2, via the ISR, activates autophagy during EAA deprivation, breaking down a larvally-acquired store of EAAs to support somatic maintenance. These data refine our understanding of the strategies by which flies sustain lifelong somatic maintenance, which determines length of life in response to changes in the nutritional environment.

molecular biology↗

bioRxiv2023

Early-adult methionine restriction reduces methionine sulfoxide and extends lifespan in Drosophila

Methionine restriction (MetR) extends lifespan in various organisms, but its mechanistic understanding remains incomplete. Whether MetR during a specific period of adulthood increases lifespan is not shown. In Drosophila, MetR is reported to extend lifespan only when amino acid levels are low. Here, by using an exome-matched holidic medium, we show that decreasing Met levels to 10% extends Drosophila lifespan with or without decreasing total amino acid levels. MetR during the first four weeks of adult life robustly extends lifespan. MetR induces the expression of Methionine sulfoxide reductase A (MsrA) in young flies, which reduces the oxidatively-damaged Met. MsrA induction is foxo-dependent and persists for two weeks after cessation of the MetR diet. Loss of MsrA attenuates lifespan extension by early-adult MetR. Our study highlights the age-dependency of the organismal response to specific nutrient and suggests that nutrient restriction at a particular period of life is sufficient for healthspan extension.

physiology↗

bioRxiv2021

Drosophila melanogaster females prioritise dietary sterols for producing high quality eggs

Limiting calories or specific nutrients without malnutrition, otherwise known as dietary restriction (DR), has been shown to extend lifespan across a broad range of taxa. Our recent findings in Drosophila melanogaster show that supplementing flies on macronutrient-rich diets with additional cholesterol can extend lifespan to the same extent as DR. Macronutrient-rich diets drive high levels of egg production and in doing so deplete the mothers of somatic sterols that are essential for survival. Thus, DR may be beneficial for lifespan because it reduces egg production which in turn reduces the mothers demand for sterols. If this is true, mothers must be prioritising their available sterols, whether from the diet or from their own bodies, to sustain high quality egg production. To test this, we measured the quality of eggs laid by mothers fed either cholesterol-sufficient or cholesterol-depleted diets. We found that even when the mothers diet was completely devoid of cholesterol, high quality egg production persisted. Furthermore, we show that sterol-supplemented flies with long lives continue to lay high quality eggs that give rise to healthy offspring. Thus, in our assays, long life does not require a fecundity cost.

evolutionary biology↗