Biology subjects

Alexander, S. E.

Publications and source records attributed to Alexander, S. E..

7 recordsLinked to original sources

bioRxiv2026

Combining Anion Exchange and Size Exclusion Chromatography for Extracellular Vesicle Enrichment from Small Volumes of Human and Mouse Plasma for Quantitative Proteomics

Extracellular vesicles (EVs) are promising biomarkers, yet their proteomic analysis from plasma is hampered by low abundance and co-purification of contaminants (e.g., lipoproteins, platelets) and technical variability, particularly in small-volume animal models. We developed and validated a modular protocol integrating Size Exclusion Chromatography (SEC) with Strong Anion Exchange (SEC-SAX) specifically tailored for quantitative LC-MS proteomics from small starting volumes (150 l of plasma). SEC alone successfully removed 99% of Albumin, and the SAX step significantly enriched EVs over contaminating lipoproteins. Downstream single pot solid phase enhanced (SP3) sample prep and STAGE tip solid phase extraction ensured maximum proteome depth. Critical confounding factors were objectively assessed: Platelet Factor 4 (PF4) was confirmed as a highly sensitive platelet marker, confirming the necessity of meticulous plasma preparation. Sample hemolysis impacted the plasma EV proteome data. As such, an objective measure (nanodrop spectrophotometer) of hemolysis and exclusion of hemolysed samples (heme >0.3 mg/ml) is recommended. The protocol is applicable to both human and mouse plasma as demonstrated by EV enrichment and quantification of biomarker proteins associated with neurodegenerative diseases from eight individual mouse plasma samples. Manuscript HighlightsO_LIDevelopmental workflow for a quantitative SEC-SAX protocol for EV proteomics from small plasma volumes (150 l). C_LIO_LIA range of variables tested including SAX beads amount, digestion buffer, digestion time, STAGE tip solid phase extraction, SAX elution buffer and sample filtration. C_LIO_LIThe SAX step significantly enhances EV proteome depth by increasing EV purity in relation to ApoB lipoproteins. C_LIO_LIShows the impact of the major confounding factors of sample hemolysis and platelet contamination on the EV proteome. C_LIO_LIPlatelet contamination increases the number and abundance of proteins detected including known disease biomarkers and sample hemolysis is associated with proteins derived from platelet and red blood cell derived EVs. C_LIO_LIPlatelet Factor 4 (PF4) is identified and confirmed as a sensitive marker for platelet contamination. C_LIO_LIApplicable to both human and mouse plasma. C_LI

molecular biology↗

bioRxiv2024

Molecular Landscape of Modality-Specific Exercise Adaptation in Human Skeletal Muscle through Large-Scale Multi-OMICs Integration

We conducted a large-scale, statistically powered, meta-analysis of exercise adaptations in human skeletal muscles, integrating epigenetic, transcriptomic, transcription factors, and proteomic data across 12 independent cohorts comprising over 1000 participants and 2340 human muscle samples. Our study identified distinctive signatures associated with maximal oxygen consumption (VO2max), and identified five genes robustly intersecting multi-OMIC layers. Notably, transcription factors predominantly functioned as activators across these layers, regulating expression of target genes irrespective of whether DNA methylation levels were low or high, indicating a synergistic effect between TFs and the methylome. Analysis of distinct exercise modalities (aerobic and resistance exercise) revealed unique gene pathways, contrasting with patterns observed in inactivity (muscle disuse) studies. These findings offer a comprehensive understanding of exercise and modality-specific adaptations, shedding light on muscle health and the molecular mechanisms associated with cardiorespiratory fitness, aging, and disease prevention.

molecular biology↗

bioRxiv2024

Total testosterone is not associated with muscle mass, function or exercise adaptations in pre-menopausal females

Key pointsO_LIBioavailable testosterone was positively related to exercise-induced muscle hypertrophy in pre-menopausal females. C_LIO_LINuclear localisation of the androgen receptor was positively related to muscle mass in pre-menopausal females before resistance training, but not with resistance-training induced hypertrophy. C_LIO_LITotal testosterone is not related to muscle mass or strength in pre-menopausal females. C_LIO_LITestosterone treatment induced androgen receptor nuclear translocation, but did not induce mTOR signalling in myocytes from pre-menopausal females. C_LI Testosterone, the major androgen hormone, influences the reproductive and non-reproductive systems in males and females via binding to the androgen receptor (AR). Both circulating endogenous testosterone and muscle AR protein content are positively associated with muscle mass and strength in males, but there is no such evidence in females. Here, we tested whether circulating testosterone levels were associated with muscle mass, function, or the muscle anabolic response to resistance training in pre-menopausal females. Twenty-seven pre-menopausal, untrained females (aged 23.5 {+/-} 4.8) underwent a 12-week resistance training program. Muscle strength, size, power and plasma and urine androgen hormone levels were measured. Skeletal muscle biopsies were collected before and after the training program to quantify the effect of resistance training on AR protein and mRNA content, and nuclear localisation. Primary muscle cell lines were cultured from a subset (n=6) of the participants biopsies and treated with testosterone to investigate its effect on myotube diameter, markers of muscle protein synthesis and AR cellular localisation. Total testosterone was not associated with muscle mass or strength at baseline or with the changes in muscle mass and strength that occurred in response to resistance training. In vitro, supra-physiological doses of testosterone increased myocyte diameter, but this did not occur via the Akt/mTOR pathway as previously suggested. Instead, we show a marked increase in AR nuclear localisation with testosterone administration. In conclusion, we found that, bioavailable testosterone and the proportion of nuclear-localised AR, but not total testosterone, with skeletal muscle mass and strength in pre-menopausal females.

physiology↗

bioRxiv2024

ACTN3 genotype influences androgen response in skeletal muscle

AbstractAndrogens are vital for the maintenance of muscle mass and their anabolic effects are primarily exerted through the androgen receptor (AR). Accumulating evidence in humans and mice suggests that circulating androgens, AR and androgen response are influenced by ACTN3 (- actinin-3), also known as "the gene for speed". One in 5 people worldwide are -actinin-3 deficient due to homozygous inheritance of a common null polymorphism (577X) in ACTN3. In this study, we show that -actinin-3 deficiency decreases baseline AR in skeletal muscles of mice and humans, in both males and females, and that AR expression directly correlates with ACTN3 in a dosage dependent manner. We further demonstrate in Actn3 knockout mice that - actinin-3 deficiency increases muscle wasting induced by androgen deprivation and reduces the muscle hypertrophic response to dihydrotestosterone and this is mediated by differential activation of pathways regulating amino acid metabolism, intracellular transport, MAPK signalling, autophagy, mitochondrial activity and calcineurin signalling. Gene set enrichment and protein analyses indicate that the absence of -actinin-3 results in a failure to coactivate many of these pathways in response to changes in androgens, and relies on leveraging mitochondrial remodelling and calcineurin signalling to restore muscle homeostasis. We further identified 7 genes that are androgen sensitive and -actinin-3-dependent in expression, and whose functions correspond to these processes. Our results highlight the pivotal role of - actinin-3 in various processes associated with the regulation of protein turnover and muscle mass, and suggest that ACTN3 genotype is a genetic modifier of androgen action in skeletal muscle.

genetics↗

bioRxiv2024

The interactive effect of sustained sleep restriction and resistance exercise on skeletal muscle transcriptomics in young females

IntroductionBoth sleep loss and exercise regulate gene expression in skeletal muscle, yet little is known about how the interaction of these stressors affects the transcriptome. The aim of this study was to investigate the effect of nine nights of sleep restriction, with repeated resistance exercise (REx) sessions, on the skeletal muscle transcriptome of young, trained females. MethodsTen healthy females aged 18-35 years undertook a randomised cross-over study of nine nights sleep restriction (SR; 5-h time in bed) and normal sleep (NS; [≥]7 h time in bed) with a minimum 6-week washout. Participants completed four REx sessions per condition (day 3, 5, 7 and 9). Muscle biopsies were collected both pre- and post-REx on days 3 and 9. Gene and protein expression were assessed by RNA sequencing and Western Blot, respectively. ResultsThree or nine nights of sleep restriction had no effect on the muscle transcriptome independently of exercise. However, close to 3000 transcripts were differentially regulated (FDR < 0.05) 48 h post the completion of three resistance exercise sessions in both NS and SR conditions. Only 39% of downregulated and 18% of upregulated genes were common between both conditions, indicating a moderating effect of sleep restriction on the response to exercise. ConclusionSleep restriction and resistance exercise interacted to alter the enrichment of skeletal muscle transcriptomic pathways in young, resistance-trained females. Performing exercise when sleep restricted may not provide the same adaptive response for individuals as if they were fully rested.

genomics↗

bioRxiv2023

Primary human myoblasts display only minor alternative polyadenylation compared to the transformed C2C12 model of muscle differentiation

Alternative polyadenylation has been linked to multiple developmental and disease transitions. The prevailing hypothesis being that differentiated cells use longer 3 UTRs with expended regulatory capacity whereas undifferentiated cells use shorter 3 UTRs. Here, we describe the gene expression and alternative polyadenylation profiles of human primary myoblasts over a time course of differentiation. Contrary to expectations, only minor changes in the 3 end choice were observed. To reconcile this finding with published research, we devised a new bioinformatic method to compare the degree of alternative polyadenylation in the differentiation of primary human and immortalized murine (C2C12) myoblasts. Differentiated human primary myotubes display only half the alternative polyadenylation of the mouse model, with less than 1/10 of the genes undergoing alternative polyadenylation in C2C12 cells showing evidence of alternative processing in human primary muscle differentiation. A global reduction in the expression of cleavage and polyadenylation factors in C2C12, but not in primary human myotubes may explain the lack of alternative polyadenylation in this system. Looking more broadly at transcriptome changes across differentiation shows that less than half of the genes differentially expressed in the immortalized model were recapitulated in primary cells. Of these, important metabolic pathways, such as glycolysis and sterol biosynthesis, showed divergent regulation. Collectively, our data caution against using immortalized cell lines, which may not fully recapitulate human muscle development, and suggest that alternative polyadenylation in the differentiation of primary cells might be less pronounced than previously thought.

molecular biology↗

bioRxiv2020

The effect of acute sleep deprivation on skeletal muscle protein synthesis and the hormonal environment

Chronic sleep loss is a potent catabolic stressor, increasing the risk of metabolic dysfunction and loss of muscle mass and function. To provide mechanistic insight into these clinical outcomes, we sought to determine if acute sleep deprivation blunts skeletal muscle protein synthesis and promotes a catabolic environment. Healthy young adults (N=13; 7 male, 6 female) were subjected to one night of total sleep deprivation (DEP) and normal sleep (CON) in a randomized cross-over design. Anabolic and catabolic hormonal profiles, skeletal muscle fractional synthesis rate and markers of muscle protein degradation were assessed across the following day. Acute sleep deprivation reduced muscle protein synthesis by 18% (CON: 0.072 {+/-} 0.015 vs. DEP: 0.059 {+/-} 0.014 %*h-1, p=0.040). In addition, it increased plasma cortisol by 21% (p=0.030) and decreased plasma testosterone, but not IGF-1, by 22% (p=0.029). A single night of total sleep deprivation is sufficient to induce anabolic resistance and a pro-catabolic environment. These acute changes may represent mechanistic precursors driving the metabolic dysfunction and body composition changes associated with chronic sleep deprivation.

physiology↗