Male sex, early life chemical exposure and the brain aromatase enzyme have been implicated in autism spectrum disorder (ASD). In the Barwon Infant Study birth cohort (n = 1074), higher prenatal maternal bisphenol A (BPA) levels are associated with higher ASD symptoms at age 2 and diagnosis at age 9 only in males with low aromatase genetic pathway activity scores. Higher prenatal BPA levels are predictive of higher cord blood methylation across the CYP19A1 brain promoter I.f region (P = 0.009) and aromatase gene methylation mediates (P = 0.01) the link between higher prenatal BPA and brain-derived neurotrophic factor methylation, with independent cohort replication. BPA suppressed aromatase expression in vitro and in vivo. Male mice exposed to mid-gestation BPA or with aromatase knockout have ASD-like behaviors with structural and functional brain changes. 10-hydroxy-2-decenoic acid (10HDA), an estrogenic fatty acid alleviated these features and reversed detrimental neurodevelopmental gene expression. Here we demonstrate that prenatal BPA exposure is associated with impaired brain aromatase function and ASD-related behaviors and brain abnormalities in males that may be reversible through postnatal 10HDA intervention. Prenatal bisphenol A exposure is associated with an increased risk of ASD in boys through a mechanism involving aromatase suppression. These resulting ASD-related behaviors and brain abnormalities may be reversed through postnatal intervention with 10HDA in mice.

It is known that dietary factors within the gestational and nursing period affect early life and stably affect later life traits in animals. However, there is very little understanding of whether dietary factors within the early life period from post-nursing to adulthood affect traits in adulthood. To address this, we conducted studies on male C57Bl/6J mice fed from 3 weeks (immediately post-nursing) until 12 weeks (full maturity) using nine different diets varying in all three major macronutrients to parse out the effects of individual macronutrients. Early life macronutrient balance affected body composition and glucose homeostasis in early adulthood, with dietary protein and fat showing major effects. Despite this, mice showed rapid reversal of the effects on body composition and glucose homeostasis of early life diet feeding, upon standard diet feeding in adulthood. However, some traits were persistent, with early life low dietary protein levels stably affecting lean and muscle mass, and early life dietary fat levels stably affecting serum and liver triglyceride levels. In summary, macronutrient balance in the post-nursing early life period does not stably affect adiposity or glucose homeostasis but does impact muscle mass and lipid homeostasis in adulthood, with prominent effects of both protein and fat levels. KEY POINTS: Early life dietary low protein and high fat levels lowered and heightened body mass, respectively. These effects did not substantially persist into adulthood with rapid catch-up growth on a normal diet. Early life protein (negative) and fat (positive) levels affected fat mass. Early life low protein levels negatively affected lean mass. Low protein effects on lower lean and muscle mass persisted into adulthood. Early life macronutrient balance effects did not affect later life glucose homeostasis but early life high fat level affected later life dyslipidaemia. Effects of dietary carbohydrate levels in early and later life were minor.

Preeclampsia is a multisystem hypertensive disorder of pregnancy that remains one of the leading causes of maternal and perinatal morbidity and mortality worldwide. The widespread maternal endothelial dysfunction that underlies preeclampsia is thought to arise from excessive placental production of various factors combined with enhanced oxidative stress. While previous studies have reported elevated activin A in women diagnosed with preeclampsia, whether activin A can cause vascular dysfunction has not yet been thoroughly investigated. Here, we demonstrated that different subtypes of activin A receptors were localised to the endothelial and smooth muscle cells of mouse and human aortae. Then, the aorta of healthy female C57Bl6J mice (n = 8) were incubated for 24 h in various concentrations of recombinant activin A to mimic early pregnancy (5 ng/mL), late pregnancy (20 ng/mL) and preeclampsia (50 ng/mL). Vascular reactivity as assessed by wire myography revealed that only the preeclamptic level of activin A impaired agonist-mediated endothelium-dependent relaxation by reducing the vasodilator prostanoid contribution to relaxation. However, agonist-mediated endothelium-independent mechanisms were unaffected. Further investigations carried out on human aortic endothelial cells suggested that the impairment of aorta relaxation could also be driven by increased endothelial cell permeability, and decreased cell viability, adherence and proliferation. This is the first direct evidence to show that activin A can induce endothelial dysfunction in whole blood vessels, suggesting that at high circulating levels it may contribute to the widespread endothelial dysfunction in women with preeclampsia.

Nuclear factor erythroid 2-related factor-2 (Nrf2), and the less well characterised proteins Nrf1 and Nrf3, are member of the cap ‘n’ collar family of transcription factors. Nrf proteins regulate the expression of endogenous antioxidant enzymes and have recently become the targets for various therapeutic treatments. Recently, Nrf proteins have been of particular interest as a target in placental-derived oxidative stress induced pregnancy disorders. Here, we report the presence of Nrf1, Nrf2 and Nrf3 proteins in both human primary trophoblast and human trophoblast choriocarcinoma cell line (BeWo). We also detail the steps taken to successfully silence all Nrf proteins in both human primary trophoblast cells and BeWo via detection of mRNA and protein using quantitative PCR, and SDS-PAGE and Western Blotting respectively.

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 show that systemic alanine metabolism is linked to glycaemic control. We find that expression of alanine aminotransferases is increased in the liver in mice with obesity and diabetes, as well as in humans with type 2 diabetes. Hepatocyte-selective silencing of both alanine aminotransferase enzymes in mice with obesity and diabetes retards hyperglycaemia and reverses skeletal muscle atrophy through restoration of skeletal muscle protein synthesis. Mechanistically, liver alanine catabolism driven by chronic glucocorticoid and glucagon signalling promotes hyperglycaemia and skeletal muscle wasting. We further provide evidence for amino acid–induced metabolic cross-talk between the liver and skeletal muscle in ex vivo experiments. Taken together, we reveal a metabolic inter-tissue cross-talk that links skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.

Abstract Introduction Sleep and affect are closely related. Late adolescence and emerging adulthood are associated with unique sleep patterns and risk for mood disturbances. This daily study examined whether dysfunctional beliefs and attitudes about sleep (DBAS), a modifiable cognitive vulnerability factor, moderated daily sleep-affect associations. Methods 421 community adolescents (n=205, 54.1% females, M±SDage=16.9±0.87) and emerging adults (n=216, 73.1% females, M±SDage=21.31±1.73) self-reported sleep and affect (adapted 12-item PANAS) and wore an actigraphy device for 7–28 days, providing >5000 daily observations. Linear mixed models tested whether DBAS moderated daily associations between self-reported and actigraphic sleep duration (total sleep time), sleep efficiency, and next-day affect on between and within-person levels. Both valence (positive/negative) and arousal (high/low) dimensions of affect were examined. Covariates included age, gender, ethnicity, day of week, and previous-day affect. Results DBAS significantly moderated associations between average sleep and next-day positive, but not negative, affect. Individuals with higher DBAS had significantly lower high arousal positive affect as average sleep duration (actigraphic: p=.002; self-reported: p=.014) and efficiency (actigraphic: p=.014) decreased. Similar moderation was found for average self-reported sleep duration and low arousal positive affect (p=.032). No significant results emerged on the within-person level. Previous-day affect significantly predicted next-day affect across models and outcomes (all p<.001). Discussion Adolescents and emerging adults with more negative views about sleep may experience dampened positive affect in shorter, or poorer, sleep periods. DBAS may constitute a modifiable factor increasing affective vulnerability on a global but not day-to-day level, and a therapeutic target for sleep-related affect disturbances in youths.

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.

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, 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. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.

Stress predicts sleep, and theoretically, coping moderates the stress response. Yet, few studies examine whether coping buffers the effects of stress on sleep, especially on a daily basis. This study tested whether coping moderated the stress-sleep relationship. 98 healthy, young adults (62% female) aged 22.5 ± 4.3 years (M±SD) reported perceived stress (0–10 scale) and coping strategies (problem-focused, emotional-approach, emotional-avoidance) each evening for 12 days. Actigraphy and sleep diaries measured total sleep time (TST), sleep onset latency (SOL), and wake after sleep onset (WASO). Analyses controlled for baseline (age, sex, education, race/ethnicity, body mass index, smoking, alcohol use) and daily (study day, day of week) covariates, and previous-night sleep to provide a strong test of directionality. Cross-lagged multilevel models tested both within-person (deviations from individuals’ own average) and between-person (deviations from the sample mean) effects. For actigraphy-assessed sleep, within-person problem-focused coping moderated the effects of stress on TST (p<.05). Higher stress predicted shorter TST on days with high (p<.001) but not low (p=.82) problem-focused coping. Between-person emotional-avoidance coping moderated the effects of stress on actigraphy-SOL (p<.001). Higher stress predicted longer SOL (p=.01) for individuals with high emotional-avoidance, but shorter SOL (p=.01) for those with low emotional-avoidance. For self-reported sleep, between-person emotional-avoidance moderated the effects of stress on TST (p<.05). Higher stress predicted shorter TST for those with low (p=.008) but not high (p=.61) emotional-avoidance. Between-person emotional-approach coping moderated the effects of stress on SOL (p<.05). Higher stress predicted longer SOL for those with low (p=.053), but not high (p=.17), emotional-approach. This study is the first to demonstrate that daily coping moderates the effects of stress on sleep using rigorous, intensive longitudinal methods. Emotional-approach coping may protect sleep quality by reducing the impact of stress on SOL, whereas emotional-avoidance may exacerbate these effects. Interventions that reduce emotional-avoidance (e.g., acceptance, ‘sitting with feelings’) and enhance emotional-approach (e.g., emotional awareness) may improve sleep quality. Problem-focused coping (e.g., problem-solving) may reduce sleep duration in the context of stress, and should be done during the day to protect sleep. Support (If Any):

Research shows that higher stress predicts poorer sleep. However, the temporal order and whether stress is an outcome of poor sleep remain unclear. This study aimed to test the bidirectional relations and temporal sequence between daily stress and sleep across 12 days, using both actigraphy-assessed and self-reported measures of sleep. Participants were 98 healthy, young adults (62% female) aged 22.5 ± 4.3 years (M±SD). Total sleep time (TST), sleep onset latency (SOL), and wake after sleep onset (WASO) were measured for 12 continuous days using actigraphy and sleep diary. Perceived stress (rated from 0–10) was measured daily in the morning, afternoon, and evening. Covariates were assessed daily (study day, day of week) and at baseline (age, sex, education, race/ethnicity, body mass index, smoking, alcohol use) and were controlled in all analyses. To provide a strong test of directionality, all relationships were tested prospectively (e.g., evening stress predicting subsequent sleep; sleep predicting next-day stress), while controlling for previous-day outcomes. Within-person (deviations from individuals’ own average) and between-person (deviations from the sample mean) effects were tested in cross-lagged multilevel models. Significant relations were found for within-, but not between-person stress and actigraphy-assessed or self-reported sleep. On the within-person level, two-units higher evening stress predicted 6.0-minute shorter actigraphy-assessed TST (p<.05). Conversely, one-hour shorter actigraphy-assessed and self-reported TST both predicted 0.2-unit higher stress the following day (both p<.001). Longer self-reported SOL (p<.05) and WASO (p<.001) both predicted higher next-day stress. There were large interindividual differences in the relations observed, such that there were strong within-person stress-sleep associations in some participants but not in others. Using one of the strongest test of directionality, this study demonstrated a strong within-individual bidirectional relationship between stress and sleep. Interpreted in the context of weak between-person stress-sleep relations, results highlighted the importance of considering each individual’s own day-to-day experiences. From a clinical perspective, these findings also raised the possibility that addressing stress on a specific day may benefit sleep at night, and vice versa. Support (If Any):

Mitochondrial dysfunction, ubiquitin-proteasomal system impairment and excitotoxicity occur during the injury and death of neurons in neurodegenerative conditions. The aim of this work was to elucidate the cellular mechanisms that are universally altered by these conditions. Through overlapping expression profiles of rotenone-, lactacystin- and N-methyl-d-aspartate-treated cortical neurons, we have identified three affected biological processes that are commonly affected; oxidative stress, dysfunction of calcium signalling and inhibition of the autophagic–lysosomal pathway. These data provides many opportunities for therapeutic intervention in neurodegenerative conditions, where mitochondrial dysfunction, proteasomal inhibition and excitotoxicity are evident.

Glutaredoxin1 (GRX1) is a glutathione (GSH)-dependent thiol oxidoreductase. The GRX1/GSH system is important for the protection of proteins from oxidative damage and in the regulation of protein function. Previously we demonstrated that GRX1/GSH regulates the activity of the essential copper-transporting P1B-Type ATPases (ATP7A, ATP7B) in a copper-responsive manner. It has also been established that GRX1 binds copper with high affinity and regulates the redox chemistry of the metallochaperone ATOX1, which delivers copper to the copper-ATPases. In this study, to further define the role of GRX1 in copper homeostasis, we examined the effects of manipulating GRX1 expression on copper homeostasis and cell survival in mouse embryonic fibroblasts and in human neuroblastoma cells (SH-SY5Y). GRX1 knockout led to cellular copper retention (especially when cultured with elevated copper) and reduced copper tolerance, while in GRX1-overexpressing cells challenged with elevated copper, there was a reduction in both intracellular copper levels and copper-induced reactive oxygen species, coupled with enhanced cell proliferation. These effects are consistent with a role for GRX1 in regulating ATP7A-mediated copper export, and further support a new function for GRX1 in neuronal copper homeostasis and in protection from copper-mediated oxidative injury.

Mitochondrial dysfunction and oxidative stress are currently considered two key mechanisms contributing to pathobiology in neurodegenerative conditions. The current study investigated the temporal molecular events contributing to programmed cell death after treatment with the mitochondrial complex I inhibitor rotenone. Microarray analysis was performed using cultured neocortical neurons treated with 10nM rotenone for 8, 15, and 24h. Genes showing at least ±1.2-fold change in expression at one time point were considered significant. Transcriptomic analysis of the 4178 genes probes revealed major changes to nine biological processes, including those eliciting mitochondrial dysfunction, activation of calcium signaling, increased expression of apoptotic genes, and downplay of chaperones/co-chaperones, ubiquitin–proteasome system and autophagy. These data define targets for intervention where mitochondrial complex I dysfunction plays a substantial role, most notably Parkinson’s disease.

Excitotoxicity, induced by the aberrant rise in cytosolic Ca2+ level, is a major neuropathological process in numerous neurodegenerative disorders. It is triggered when extracellular glutamate (Glu) concentration reaches neuropathological levels resulting in dysregulation and hyper-activation of ionotropic glutamate receptor subtype (iGluRs). Even though all three members of the iGluRs, namely N-methyl-d-aspartate (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and kainate (KAR) receptors are implicated in excitotoxicity, their individual contributions to downstream signaling transduction have not been explored. In this study, we report a comprehensive description of the recruitment of cellular processes in neurons upon iGluR activation during excitotoxicity through temporal (5 h, 15 h, and 24 h) global gene profiling of AMPA, KA, NMDA, and Glu excitotoxic models. DNA microarray analyses of mouse primary cortical neurons treated with these four pharmacological agonists are further validated via real-time PCR. Bi-model analyses against Glu model demonstrate that NMDARs and KARs play a more pivotal role in Glu-mediated excitotoxicity, with a higher degree of global gene profiling overlaps, as compared to that of AMPARs. Comparison of global transcriptomic profiles reveals aberrant calcium ion binding and homeostasis, organellar (lysosomal and endoplasmic reticulum) stress, oxidative stress, cell cycle re-entry and activation of cell death processes as the main pathways that are significantly modulated across all excitotoxicity models. Singular profile analyses demonstrate substantial transcriptional regulation of numerous cell cycle proteins. For the first time, we show that iGluR activation forms the basis of cell cycle re-activation, and together with oxidative stress fulfill the “two-hit” hypothesis that accelerates neurodegeneration.

AimsWith the identification of hypochlorous acid (HOCl) as a biomarker in diseased brains and endogenous detection of its modified proteins, HOCl might be implicated in the development of neurodegenerative disorders. However, its effect on neuronal cell death has not yet been investigated at gene expression level.Main methodsTherefore, DNA microarray was performed for screening of HOCl-responsive genes in primary mouse cortical neurons. Neurotoxicity caused by physiological relevant HOCl (250μM) exhibited several biochemical markers of apoptosis.Key findingsThe biological processes affected during HOCl-mediated apoptosis included cell death, response to stress, cellular metabolism, and cell cycle. Among them, mRNAs level of cell death and stress response genes were up-regulated while expression of metabolism and cell cycle genes were down-regulated.SignificanceOur results showed, for the first time, that HOCl induces apoptosis in cortical neurons by upregulating apoptotic genes and gene expression of stress response such as heat shock proteins and antioxidant proteins were enhanced to provide protection. These data form a foundation for the development of screening platforms and define targets for intervention in HOCl neuropathologies where HOCl-mediated injury is causative.

Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2α) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2α mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2α was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2α can modulate HCC cell growth.

Nitric oxide (NO), ubiquitously expressed in the central nervous system, has been perceived to be a potential neuromodulator. Employing cultured murine primary cortical neurons, NO resulted in an inhibition of the ubiquitin–proteasome system (UPS) with a dose- and time-dependent decrease in cell viability. This is consistent with a previous study that reported a dysfunction of UPS with consequential apoptotic death in macrophage cell with NO treatment. However, it cannot be unclear if the drop in UPS efficiency is directly imposed on by NO. Therefore by using microarray analysis, our study revealed an early down-regulation or non-significant differential expression of genes encoding UPS proteins in NOC-18 (NO donor)-treated neurons as compared to an observed elevation of corresponding gene expression genes in lactacystin (classical proteasome inhibitor)-treated neurons (conducted earlier). Furthermore, time-course analysis of proteasome activity in NOC-18-treated neurons demonstrated a late onset of reduction. This is intriguing as it is well established that in an exclusive proteasome dysfunction-induced cell death, a compensatory feedback mechanism will be activated with an initial and concerted up-regulation of genes encoding proteins involved in UPS as seen when neurons were treated with lactacystin. Thus, it is highly suggestive that NO-triggered neuronal death takes on a different signaling cascade from that of a classical proteasome inhibitor, and that the late reduction of proteasome activity is a downstream event following the activation of apoptotic cellular signaling cascade. In intracellular condition, the proteasome is not NO preferred primary target responsible for the trigger of the cell death machinery. In conclusion, we presented novel findings that shed light into NO-induced cell death signaling cascade, which would be important in understanding the pathogenesis of neurodegenerative disorders such as Parkinson’s disease.

Oxidative stress has been implicated as playing a role in neurodegenerative disorders, such as ischemic stroke, Alzheimer's, Huntington's, and Parkinson's disease. Persuasive evidences have shown that microglial-mediated oxidative stress contributes significantly to cell loss and accompanying cognitive decline characteristic of the diseases. Based on the facts that (i) levels of catalytically active myeloperoxidase are elevated in diseased brains and (ii) myeloperoxidase polymorphism is associated with the risk of developing neurodegenerative disorders, HOCl as a major oxidant produced by activated phagocytes in the presence of myeloperoxidase is therefore suggested to be involved in neurodegeneration. Its association with neurodegeneration is further showed by elevated level of 3-chlorotyrosine (bio-marker of HOCl in vivo) in affected brain regions as well as HOCl scavenging ability of neuroprotectants, desferrioxamine and uric acid. In this review, we will summary the current understanding concerning the association of HOCl and neuronal cell death where production of HOCl will lead to further formation of reactive nitrogen and oxygen species. In addition, HOCl also causes tissue destruction and cellular damage leading cell death.