Characterisation of Aberrant Metabolic Pathways in Hepatoblastoma Using Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS).

Hepatoblastoma (HB) is a rare childhood tumour with an evolving molecular landscape. We present the first comprehensive metabolomic analysis using untargeted and targeted liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS) of paired tumour and non-tumour surgical samples in HB patients (n = 8 pairs). This study demonstrates that the metabolomic landscape of HB is distinct from that of non-tumour (NT) liver tissue, with 35 differentially abundant metabolites mapping onto pathways such as fatty acid transport, glycolysis, the tricarboxylic acid (TCA) cycle, branched-chain amino acid degradation and glutathione synthesis. Targeted metabolomics demonstrated reduced short-chain acylcarnitines and a relative accumulation of branched-chain amino acids. Medium- and long-chain acylcarnitines in HB were similar to those in NT. The metabolomic changes reported are consistent with previously reported transcriptomic data from tumour and non-tumour samples (49 out of 54 targets) as well as metabolomic data obtained using other techniques. Gene set enrichment analysis (GSEA) from RNAseq data (n = 32 paired HB and NT samples) demonstrated a downregulation of the carnitine metabolome and immunohistochemistry showed a reduction in CPT1a (n = 15 pairs), which transports fatty acids into the mitochondria, suggesting a lack of utilisation of long-chain fatty acids in HB. Thus, our findings suggest a reduced metabolic flux in HB which is corroborated at the gene expression and protein levels. Further work could yield novel insights and new therapeutic targets.

Pre-sleep Protein Ingestion Increases Mitochondrial Protein Synthesis Rates During Overnight Recovery from Endurance Exercise: A Randomized Controlled Trial.

BACKGROUND: Casein protein ingestion prior to sleep has been shown to increase myofibrillar protein synthesis rates during overnight sleep. It remains to be assessed whether pre-sleep protein ingestion can also increase mitochondrial protein synthesis rates. Though it has been suggested that casein protein may be preferred as a pre-sleep protein source, no study has compared the impact of pre-sleep whey versus casein ingestion on overnight muscle protein synthesis rates. OBJECTIVE: We aimed to assess the impact of casein and whey protein ingestion prior to sleep on mitochondrial and myofibrillar protein synthesis rates during overnight recovery from a bout of endurance-type exercise. METHODS: Thirty-six healthy young men performed a single bout of endurance-type exercise in the evening (19:45 h). Thirty minutes prior to sleep (23:30 h), participants ingested 45 g of casein protein, 45 g of whey protein, or a non-caloric placebo. Continuous intravenous L-[ring-13C6]-phenylalanine infusions were applied, with blood and muscle tissue samples being collected to assess overnight mitochondrial and myofibrillar protein synthesis rates. RESULTS: Pooled protein ingestion resulted in greater mitochondrial (0.087 ± 0.020 vs 0.067 ± 0.016%·h-1, p = 0.005) and myofibrillar (0.060 ± 0.014 vs 0.047 ± 0.011%·h-1, p = 0.012) protein synthesis rates when compared with placebo. Casein and whey protein ingestion did not differ in their capacity to stimulate mitochondrial (0.082 ± 0.019 vs 0.092 ± 0.020%·h-1, p = 0.690) and myofibrillar (0.056 ± 0.009 vs 0.064 ± 0.018%·h-1, p = 0.440) protein synthesis rates. CONCLUSIONS: Protein ingestion prior to sleep increases both mitochondrial and myofibrillar protein synthesis rates during overnight recovery from exercise. The overnight muscle protein synthetic response to whey and casein protein does not differ. CLINICAL TRIAL REGISTRATION: NTR7251 .

Acute effects of prior dietary fat ingestion on postprandial metabolic responses to protein and carbohydrate co-ingestion in overweight and obese men: A randomised crossover trial.

BACKGROUND: Obesity and insulin resistance are associated with an impaired sensitivity to anabolic stimuli such as dietary protein (anabolic resistance). Omega-3 polyunsaturated fatty acids (n-3 PUFA) may be protective against the deleterious effects of saturated fatty acids (SFA) on insulin resistance. However, the contribution of excess fat consumption to anabolic and insulin resistance and the interaction between SFA and n-3 PUFA is not well studied. AIM: The primary aim of this study was to investigate the effects of an oral fat pre-load, with or without the partial substitution of SFA with fish oil (FO)-derived n-3 PUFA, on indices of insulin and anabolic sensitivity in response to subsequent dietary protein and carbohydrate (dextrose) co-ingestion. METHODS: Eight middle-aged males with overweight or obesity (52.8 ± 2.0 yr, BMI 31.8 ± 1.4 kg·m-2) ingested either an SFA, or isoenergetic SFA and FO emulsion (FO), or water/control (Con), 4 h prior to a bolus of milk protein and dextrose. RESULTS: Lipid ingestion (in particular FO) impaired the early postprandial uptake of branched chain amino acids (BCAA) into the skeletal muscle in response to protein and dextrose, and attenuated the peak glycaemic response, but was not accompanied by differences in whole body (Matsuda Index: Con: 4.66 ± 0.89, SFA: 5.10 ± 0.94 and FO: 4.07 ± 0.59) or peripheral (forearm glucose netAUC: Con: 521.7 ± 101.7; SFA: 470.2 ± 125.5 and FO: 495.3 ± 101.6 µmol·min-1·100 g lean mass·min [t = 240-420 min]) insulin sensitivity between visits. Postprandial whole body fat oxidation was affected by visit (P = 0.024) with elevated rates in SFA and FO, relative to Con (1.85 ± 0.55; 2.19 ± 0.21 and 0.65 ± 0.35 kJ·h-1·kg-1 lean body mass, respectively), however muscle uptake of free fatty acids (FFA) was unaffected. CONCLUSION: Oral lipid preloads, consisting of SFA and FO, impair the early postprandial BCAA uptake into skeletal muscle, which occurs independent of changes in insulin sensitivity. CLINICAL TRIAL REGISTRY NUMBER: ClinicalTrials.gov Identifier NCT03146286.

Safety and tolerability of hydroxychloroquine in health care workers and first responders for the prevention of COVID-19: WHIP COVID-19 Study.

BACKGROUND: Health care workers (HCW) are among the highest risk groups for acquisition of COVID-19 because of occupational exposures. The WHIP COVID-19 Study aimed to evaluate the safety and efficacy of hydroxychloroquine (HCQ) as chemoprophylaxis for SARS-CoV-2 infection in this population. METHODS: HCW, first responders, and other occupationally high-risk participants were enrolled in a randomized, placebo-controlled clinical study of HCQ from April to October 2020. The trial compared daily versus weekly HCQ with placebo and with a prospective cohort on HCQ for autoimmune diseases. Participants were followed for 8 weeks. Serology or a positive polymerase chain reaction test was used to determine laboratory confirmed clinical cases. RESULTS: A total of 624 participants were randomized to placebo (n = 200), weekly HCQ (n = 201), daily HCQ (n = 197). For the primary safety end point, 279 (44.7%) participants experienced adverse event (AE) level II or lower (total AEs n = 589), similar rates in all randomized groups (P = .188) with no hospitalizations or interventions required. Only 4 laboratory confirmed COVID-19 cases occurred, with 2 in the placebo arm and one in each HCQ randomized arm. CONCLUSIONS: This randomized placebo-controlled trial was able to demonstrate the safety of HCQ outpatient chemoprophylaxis in high-risk groups against COVID-19. Future studies of chemoprophylaxis for SARS-CoV-2 are needed as the epidemic continues worldwide.

Two weeks of early time-restricted feeding (eTRF) improves skeletal muscle insulin and anabolic sensitivity in healthy men.

BACKGROUND: Altering the temporal distribution of energy intake (EI) and introducing periods of intermittent fasting (IF) exert important metabolic effects. Restricting EI to earlier in the day [early time-restricted feeding (eTRF)] is a novel type of IF. OBJECTIVES: We assessed the chronic effects of eTRF compared with an energy-matched control on whole-body and skeletal muscle insulin and anabolic sensitivity. METHODS: Sixteen healthy males (aged 23 ± 1 y; BMI 24.0 ± 0.6 kg·m-2) were assigned to 2 groups that underwent either 2 wk of eTRF (n = 8) or control/caloric restriction (CON:CR; n = 8) diet. The eTRF diet was consumed ad libitum and the intervention was conducted before the CON:CR, in which the diet was provided to match the reduction in EI and body weight observed in eTRF. During eTRF, daily EI was restricted to between 08:00 and 16:00, which prolonged the overnight fast by ∼5 h. The metabolic responses to a carbohydrate/protein drink were assessed pre- and post-interventions following a 12-h overnight fast. RESULTS: When compared with CON:CR, eTRF improved whole-body insulin sensitivity [between-group difference (95% CI): 1.89 (0.18, 3.60); P = 0.03; η2p = 0.29] and skeletal muscle uptake of glucose [between-group difference (95% CI): 4266 (261, 8270) µmol·min-1·kg-1·180 min; P = 0.04; η2p = 0.31] and branched-chain amino acids (BCAAs) [between-group difference (95% CI): 266 (77, 455) nmol·min-1·kg-1·180 min; P = 0.01; η2p = 0.44]. eTRF caused a reduction in EI (∼400 kcal·d-1) and weight loss (-1.04 ± 0.25 kg; P = 0.01) that was matched in CON:CR (-1.24 ± 0.35 kg; P = 0.01). CONCLUSIONS: Under free-living conditions, eTRF improves whole-body insulin sensitivity and increases skeletal muscle glucose and BCAA uptake. The metabolic benefits of eTRF are independent of its effects on weight loss and represent chronic adaptations rather than the effect of the last bout of overnight fast. This trial was registered at clinicaltrials.gov as NCT03969745.

Effect of acute and short-term dietary fat ingestion on postprandial skeletal muscle protein synthesis rates in middle-aged, overweight, and obese men.

Muscle anabolic resistance to dietary protein is associated with obesity and insulin resistance. However, the contribution of excess consumption of fat to anabolic resistance is not well studied. The aim of these studies was to test the hypothesis that acute and short-term dietary fat overload will impair the skeletal muscle protein synthetic response to dietary protein ingestion. Eight overweight/obese men [46.4 ± 1.4 yr, body mass index (BMI) 32.3 ± 5.4 kg/m2] participated in the acute feeding study, which consisted of two randomized crossover trials. On each occasion, subjects ingested an oral meal (with and without fat emulsion), 4 h before the coingestion of milk protein, intrinsically labeled with [1-13C]phenylalanine, and dextrose. Nine overweight/obese men (44.0 ± 1.7 yr, BMI 30.1 ± 1.1 kg/m2) participated in the chronic study, which consisted of a baseline, 1-wk isocaloric diet, followed by a 2-wk high-fat diet (+25% energy excess). Acutely, incorporation of dietary amino acids into the skeletal muscle was twofold higher (P < 0.05) in the lipid trial compared with control. There was no effect of prior lipid ingestion on indices of insulin sensitivity (muscle glucose uptake, pyruvate dehydrogenase complex activity, and Akt phosphorylation) in response to the protein/dextrose drink. Fat overfeeding had no effect on muscle protein synthesis or glucose disposal in response to whey protein ingestion, despite increased muscle diacylglycerol C16:0 (P = 0.06) and ceramide C16:0 (P < 0.01) levels. Neither acute nor short-term dietary fat overload has a detrimental effect on the skeletal muscle protein synthetic response to dietary protein ingestion in overweight/obese men, suggesting that dietary-induced accumulation of intramuscular lipids per se is not associated with anabolic resistance.

Demonstration of therapeutic window of Cerebrolysin in embolic stroke: A prospective, randomized, blinded, and placebo-controlled study.

Background and aims In an effort to characterize the effects of Cerebrolysin for treatment of stroke that are essential for successful clinical translation, we have demonstrated that Cerebrolysin dose dependently enhanced neurological functional recovery in experimental stroke. Here, we conduct a prospective, randomized, placebo-controlled, blinded study to examine the therapeutic window of Cerebrolysin treatment of rats subjected to embolic stroke. Methods Male Wistar rats age 3-4 months (n = 100) were subjected to embolic middle cerebral artery occlusion. Animals were randomized to receive saline or Cerebrolysin daily for 10 consecutive days starting 4, 24, 48, and 72 h after middle cerebral artery occlusion. Neurological outcome was measured weekly with a battery of behavioral tests (adhesive removal test, modified neurological severity score (mNSS), and foot-fault test). Global test was employed to assess Cerebrolysin effect on neurological recovery with estimation of mean difference between Cerebrolysin and control-treated groups and its 95% confidence interval in the intent-to-treat population, where a negative value of the mean difference and 95% confidence interval < 0 indicated a significant treatment effect. All rats were sacrificed 28 days after middle cerebral artery occlusion and infarct volume was measured. Results Cerebrolysin treatment initiated within 48 h after middle cerebral artery occlusion onset significantly improved functional outcome; mean differences and 95% confidence interval were -11.6 (-17.7, -5.4) at 4 h, -7.1 (-13.5, -0.8) at 24 h, -8.4 (-14.2, -8.6) at 48 h, and -4.9 (-11.4, 1.5) at 72 h. There were no differences on infarct volume and mortality rate among groups. Conclusions With a clinically relevant rigorous experimental design, our data demonstrate that Cerebrolysin treatment effectively improves stroke recovery when administered up to 48 h after middle cerebral artery occlusion.

Cerebrolysin dose-dependently improves neurological outcome in rats after acute stroke: A prospective, randomized, blinded, and placebo-controlled study.

BACKGROUND: Cerebrolysin is a mixture of neuropeptides and free amino acids that is clinically used for the treatment of stroke. To further standardize treatment schemes, we assessed the dose response of Cerebrolysin on sensorimotor outcome in a rat model of ischemic stroke. METHODS: This study was a prospective, blinded, placebo-controlled, preclinical experiment. Male and female Wistar rats, subjected to embolic middle cerebral artery occlusion, were randomly treated with Cerebrolysin doses of 0.8, 2.5, 5.0, 7.5 ml/kg or placebo, 4 h after middle cerebral artery occlusion for a total of 10 consecutive days. RESULTS: The primary outcome was neurologic improvement at day 28, lesion volume, mortality, and animal weight were secondary and safety outcomes, respectively. There was a significant (p < 0.001) dose effect of Cerebrolysin on neurological outcome. Cerebrolysin at a dose of ≥ 2.5 ml/kg significantly (p < 0.001) improved neurological outcome (Mean Estimate (95% CL): 0.8 ml/kg: 6.2 (-6.0/18.4), 2.5 ml/kg: -28.9 (-41.6/-16.2), 5.0 ml/kg: -33.4 (-45.0/-21.7), 7.5 ml/kg: -36.3 (-48.2/-24.4). Higher doses (≥ 2.5 ml/kg) resulted in better recovery; however, differences between effective doses were not significant. Treatment with 5 ml/kg reduced lesion volume (p = 0.016). No treatment gender interactions were found and there were no differences in death or weight loss. CONCLUSION: Collectively, these data on Cerebrolysin efficacy demonstrate the feasibility of a preclinical study setup following a randomized, placebo-controlled, and blinded design with a clinical relevant treatment scheme. Cerebrolysin at doses of ≥ 2.5 ml/kg improved functional outcome and at a dose of 5 ml/kg reduced infarct volume.