Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Here, we present stable isotope cysteine labelling with iodoacetamide (SICyLIA), a mass spectrometry-based workflow to assess proteome-scale cysteine oxidation. SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity. Applying SICyLIA to diverse cellular models and primary tissues provides detailed insights into thiol oxidation proteomes. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys identifies oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies

Pre-Steady-State Decoding of the Bicoid Morphogen Gradient

Morphogen gradients are established by the localized production and subsequent diffusion of signaling molecules. It is generally assumed that cell fates are induced only after morphogen profiles have reached their steady state. Yet, patterning processes during early development occur rapidly, and tissue patterning may precede the convergence of the gradient to its steady state. Here we consider the implications of pre-steady-state decoding of the Bicoid morphogen gradient for patterning of the anterior–posterior axis of the Drosophila embryo. Quantitative analysis of the shift in the expression domains of several Bicoid targets (gap genes) upon alteration of bcd dosage, as well as a temporal analysis of a reporter for Bicoid activity, suggest that a transient decoding mechanism is employed in this setting. We show that decoding the pre-steady-state morphogen profile can reduce patterning errors caused by fluctuations in the rate of morphogen production. This can explain the surprisingly small shifts in gap and pair-rule gene expression domains observed in response to alterations in bcd dosage

Effects of Valproic Acid on Cerebral Nutrient Carriers' Expression in the Rat

Objective: The antiepileptic drug valproate has been shown to affect the expression of carriers for essential compounds and drugs in extracerebral tissues. The aim of the current study was to evaluate in vivo the effect of valproate treatment on the cerebral expression of carriers and selected genes of the blood-brain barrier (BBB) in the rat.Methods: Male Wistar rats were treated daily for 7 days by intraperitoneal injections of valproate (75, 150, or 300 mg/kg/day) or the vehicle. mRNA was isolated from the cerebral cortex and the hippocampus. Transcript levels of 37 genes were measured using a customized gene expression assay. Quantitative histone acetylation was evaluated by western blotting. Glucose6-phosphate (G6P) tissue levels were used as a surrogate of cerebral glucose concentrations.Results: Valproate treatment was associated with significant reduction (up to 22%; P < 0.05) in cortical and hippocampal claudin 5-normalized Slc2a1 (Glut1) mRNA expression. G6P levels were not significantly altered, but were correlated with Slc2a1 transcript levels (r = 0.499; P < 0.02). None of the other 36 screened genes were significantly affected by valproate. Cortical histone hyperacetylation indicated cerebral activity of valproate on a major pathway regulating gene expression (P < 0.02).Significance: The effect of valproate on nutrient carriers appears to be tissue-specific and even brain area-specific. If validated in humans, the changes in Glut1 expression might have clinical implications in positron emission tomography (PET) imaging. Further studies are required for elucidating the relevance of these findings to the clinic

Comparative omics and feeding manipulations in chicken indicate a shift of the endocrine role of visceral fat towards reproduction.

BACKGROUND: The mammalian adipose tissue plays a central role in energy-balance control, whereas the avian visceral fat hardly expresses leptin, the key adipokine in mammals. Therefore, to assess the endocrine role of adipose tissue in birds, we compared the transcriptome and proteome between two metabolically different types of chickens, broilers and layers, bred towards efficient meat and egg production, respectively. RESULTS: Broilers and layer hens, grown up to sexual maturation under free-feeding conditions, differed 4.0-fold in weight and 1.6-fold in ovarian-follicle counts, yet the relative accumulation of visceral fat was comparable. RNA-seq and mass-spectrometry (MS) analyses of visceral fat revealed differentially expressed genes between broilers and layers, 1106 at the mRNA level (FDR ≤ 0.05), and 203 at the protein level (P ≤ 0.05). In broilers, Ingenuity Pathway Analysis revealed activation of the PTEN-pathway, and in layers increased response to external signals. The expression pattern of genes encoding fat-secreted proteins in broilers and layers was characterized in the RNA-seq and MS data, as well as by qPCR on visceral fat under free feeding and 24 h-feed deprivation. This characterization was expanded using available RNA-seq data of tissues from red junglefowl, and of visceral fat from broilers of different types. These comparisons revealed expression of new adipokines and secreted proteins (LCAT, LECT2, SERPINE2, SFTP1, ZP1, ZP3, APOV1, VTG1 and VTG2) at the mRNA and/or protein levels, with dynamic gene expression patterns in the selected chicken lines (except for ZP1; FDR/P ≤ 0.05) and feed deprivation (NAMPT, SFTPA1 and ZP3) (P ≤ 0.05). In contrast, some of the most prominent adipokines in mammals, leptin, TNF, IFNG, and IL6 were expressed at a low level (FPKM/RPKM< 1) and did not show differential mRNA expression neither between broiler and layer lines nor between fed vs. feed-deprived chickens. CONCLUSIONS: Our study revealed that RNA and protein expression in visceral fat changes with selective breeding, suggesting endocrine roles of visceral fat in the selected phenotypes. In comparison to gene expression in visceral fat of mammals, our findings points to a more direct cross talk of the chicken visceral fat with the reproductive system and lower involvement in the regulation of appetite, inflammation and insulin resistance.The study was supported by the Israel Academy of Sciences grants no. 876/ 14 and 1294/17, and Chief Scientist of the Israeli Ministry of Agriculture 0469/14 (to MFE and ES)

Midlife Alcohol Consumption and the Risk of Stroke in the Atherosclerosis Risk in Communities Study

Alcohol consumption is common in the US and may confer beneficial cardiovascular effects at light-to-moderate doses. The alcohol-stroke relationship remains debated. We estimated the relationship between mid-life, self-reported alcohol consumption and ischemic stroke (IS) and intracerebral hemorrhage (ICH) in a biracial cohort

Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability

Influence of cytochrome P450 2D6*10/*10 genotype on the risk for tramadol associated adverse effects: a retrospective cohort study

Background: Tramadol is primarily metabolized by the highly polymorphic CYP2D6 enzyme, leading to a large spectrum of adverse events and clinical response. Ample evidence pointed a reduced CYPD26 activity score in individuals harboring the CYP2D6*10/*10 genotype, nevertheless, there is scarce studies on the impact of CYP2D6*10/*10 genetic polymorphism on long-term tramadol’s adverse effects.Aim: To test the correlation between CYP2D6*10/*10 expression and the risk for tramadol-associated adverse effects.Method: Using a database of Leumit Healthcare Services in Israel, we retrospectively assessed the occurrence of adverse events in patients who were prescribed tramadol. A binary logistic regression model was applied to model the relationship between CYP2D6*10/*10 genotype and the occurrence of adverse effects.Results: Data from four hundred ninety-three patients were included in this study. Only 25 (5.1%) patients were heterozygous for the CYP2D6*10 variant, while 56 patients (11%) were tested positive to the CYP2D6*10/*10 genotype. Compared to carriers of other variants, patients with the CYP2D6*10/*10 variant exhibited a higher occurrence of adverse events (odds ratio [OR] = 6.14, 95% confidence interval 3.18–11.83); the odds ratio for central nervous system adverse events and gastrointestinal adverse events were 5.13 (95% CI 2.84–9.28), and 3.25 (95% CI 1.78–5.93), respectively.Conclusion: Among the different CYP2D6 genotypes, CYP2D6*10/*10 genotype carries the higher risk of tramadol related adverse events. Appreciating the frequency of this specific allele it seems prudent to pharmacogenetically screen patients considered for long term tramadol treatment for better tolerability and efficacy outcomes

The ocean sampling day consortium.

Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world's oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits

Families\u27 healthcare experiences for children with inherited metabolic diseases: Protocol for a mixed methods cohort study

Introduction Children with inherited metabolic diseases (IMDs) often have complex and intensive healthcare needs and their families face challenges in receiving high-quality, family centred health services. Improvement in care requires complex interventions involving multiple components and stakeholders, customised to specific care contexts. This study aims to comprehensively understand the healthcare experiences of children with IMDs and their families across Canada. Methods and analysis A two-stage explanatory sequential mixed methods design will be used. Stage 1: quantitative data on healthcare networks and encounter experiences will be collected from 100 parent/guardians through a care map, 2 baseline questionnaires and 17 weekly diaries over 5-7 months. Care networks will be analysed using social network analysis. Relationships between demographic or clinical variables and ratings of healthcare experiences across a range of family centred care dimensions will be analysed using generalised linear regression. Other quantitative data related to family experiences and healthcare experiences will be summarised descriptively. Ongoing analysis of quantitative data and purposive, maximum variation sampling will inform sample selection for stage 2: a subset of stage 1 participants will participate in one-on-one videoconference interviews to elaborate on the quantitative data regarding care networks and healthcare experiences. Interview data will be analysed thematically. Qualitative and quantitative data will be merged during analysis to arrive at an enhanced understanding of care experiences. Quantitative and qualitative data will be combined and presented narratively using a weaving approach (jointly on a theme-by-theme basis) and visually in a side-by-side joint display. Ethics and dissemination The study protocol and procedures were approved by the Children\u27s Hospital of Eastern Ontario\u27s Research Ethics Board, the University of Ottawa Research Ethics Board and the research ethics boards of each participating study centre. Findings will be published in peer-reviewed journals and presented at scientific conferences