Biology subjects

Chan, A. Y.

Publications and source records attributed to Chan, A. Y..

7 recordsLinked to original sources

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↗

bioRxiv2022

Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq

Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

immunology↗

bioRxiv2021

A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different agedependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2x106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell-type specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR<0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rodshaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

cell biology↗

bioRxiv2020

An endocrine-hepato-muscular metabolic cycle links skeletal muscle atrophy and hyperglycemia in type 2 diabetes

Both obesity and sarcopenia are frequently associated in ageing, and together may promote the progression of related conditions such as diabetes and frailty. However, little is known about the pathophysiological mechanisms underpinning this association. Here we uncover dysregulated systemic alanine metabolism and hyper-expression of the alanine transaminases (ALT) in the liver of obese/diabetic mice and humans. Hepatocyte-selective silencing of both ALT enzymes revealed a clear role in systemic alanine clearance which related to glycemic control. In obese/diabetic mice, not only did silencing both ALT enzymes retard hyperglycemia, but also reversed skeletal muscle atrophy. This was due to a rescue of depressed skeletal muscle protein synthesis, with a liver-skeletal muscle amino acid metabolic crosstalk exemplified by ex vivo experiments. Mechanistically, chronic liver glucocorticoid and glucagon signaling driven liver alanine catabolism promoted hyperglycemia and skeletal muscle wasting. Taken together, here we reveal an endocrine-hepato-muscular metabolic cycle linking hyperglycemia and skeletal muscle atrophy in type 2 diabetes.

physiology↗

bioRxiv2020

Identification of novel, clinically correlated autoantigens in the monogenic autoimmune syndrome APS1 by PhIP-Seq

The identification of autoantigens remains a critical challenge for understanding and treating autoimmune diseases. Autoimmune polyendocrine syndrome type 1 (APS1), a rare monogenic form of autoimmunity, presents as widespread autoimmunity with T and B cell responses to multiple organs. Importantly, autoantibody discovery in APS1 can illuminate fundamental disease pathogenesis, and many of the antigens found in APS1 extend to common autoimmune diseases. Here, we performed proteome-wide programmable phage-display (PhIP-Seq) on sera from an APS1 cohort and discovered multiple common antibody targets. These novel autoantigens exhibit tissue-restricted expression, including expression in enteroendocrine cells and dental enamel. Using detailed clinical phenotyping, we find novel associations between autoantibodies and organ-restricted autoimmunity, including between anti-KHDC3L autoantibodies and premature ovarian insufficiency, and between anti-RFX6 autoantibodies and diarrheal-type intestinal dysfunction. Our study highlights the utility of PhIP-Seq for interrogating antigenic repertoires in human autoimmunity and the importance of antigen discovery for improved understanding of disease mechanisms.

immunology↗

bioRxiv2019

Restriction of essential amino acids dictates the systemic response to dietary protein dilution

Dietary protein dilution (DPD) promotes metabolic remodelling and health but the precise nutritional components driving this response remain elusive. Here we demonstrate that dietary amino acids (AA) are sufficient and necessary to drive the response to DPD. In particular, the restriction of dietary essential AA (EAA) supply, but not non-EAA, drives the systemic metabolic response to total AA deprivation. Furthermore, systemic deprivation of Thr and Trp, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Thr is also potentially limiting in low-protein diet fed humans, and dietary Thr restriction (DTR) retarded the development of obesity-associated metabolic dysfunction in mice. Liver-derived fibroblast growth factor 21 was required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of Thr biosynthetic capacity reversed the systemic response to DTR. Taken together, our studies demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.

physiology↗

bioRxiv2019

Serotonergic treatment normalizes midbrain dopaminergic neuron increase after periaqueductal gray stimulation-induced anticipatory fear in a rat model

BackgroundElectrical stimulation of the dorsolateral periaqueductal gray (dlPAG) in rats has been shown to elicit panic-like behaviour and can be a useful tool for modelling anticipatory fear and agoraphobia.\n\nMethodsIn this study, we further analysed our previous data on the effects of escitalopram (a selective serotonin reuptake inhibitor, SSRI) and buspirone (a 5-HT1A receptor partial agonist) on dlPAG-induced anticipatory fear behaviour in a rat model using freezing as a measure. We then used tyrosine hydroxylase (TH) immunohistochemistry to probe the effects on dopaminergic neurons.\n\nResultsAlthough acute treatment of escitalopram, but not buspirone, was effective in reducing anticipatory freezing behaviour, chronic administrations of both drugs were comparably effective. We found that the number of dopaminergic neurons in the ventral tegmental area (VTA) was lowered in both chronic buspirone and escitalopram groups. We showed a strong correlation between the number of dopaminergic neurons and freezing in the VTA. We further showed positive correlations between dopaminergic neurons in the VTA and substantia nigra pars compacta in escitalopram and buspirone groups, respectively.\n\nLimitationsAlthough our data strongly hint to a role of dopaminergic mechanisms in the dlPAG induced fear response, more in-depth studies with larger sample sizes are needed to understand the neuronal mechanisms underlying the interactions between serotonergic drugs and dopaminergic cell number and fear behavior.\n\nConclusionChronic treatment with an SSRI and a 5-HT1A agonist decrease the number of dopaminergic neurons in the VTA. These effects seem to be associated with reduced dlPAG-induced anticipatory freezing behaviour.\n\nKey PointsO_LIChronic treatment of escitalopram and buspirone was effective in reducing dlPAG induced anticipatory freezing behaviour.\nC_LIO_LIThe number of dopaminergic neurons in the ventral tegmental area (VTA) was lowered in both chronic buspirone and escitalopram groups and was correlated to freezing.\nC_LIO_LIWe found positive correlations between dopaminergic neurons in the VTA and substantia nigra pars compacta in escitalopram and buspirone groups, respectively.\nC_LI

neuroscience↗