Amniotic fluid metabolomics identifies impairment of glycerophospholipid and amino acid metabolism during congenital Zika syndrome development.

PURPOSE: Our main goal is to identify the alterations in the amniotic fluid (AF) metabolome in Zika virus (ZIKV)-infected patients and their relation to congenital Zika syndrome (CZS) progression. EXPERIMENTAL DESIGN: We applied an untargeted metabolomics strategy to analyze seven AF of pregnant women: healthy women and ZIKV-infected women bearing non-microcephalic and microcephalic fetuses. RESULTS: Infected patients were characterized by glycerophospholipid metabolism impairment, which is accentuated in microcephalic phenotypes. Glycerophospholipid decreased concentration in AF can be a consequence of intracellular transport of lipids to the placental or fetal tissues under development. The increased intracellular concentration of lipids can lead to mitochondrial dysfunction and neurodegeneration caused by lipid droplet accumulation. Furthermore, the dysregulation of amino acid metabolism was a molecular fingerprint of microcephalic phenotypes, specifically serine, and proline metabolisms. Both amino acid deficiencies were related to neurodegenerative disorders, intrauterine growth retardation, and placental abnormalities. CONCLUSIONS AND CLINICAL RELEVANCE: This study enhances our understanding of the development of CZS pathology and sheds light on dysregulated pathways that could be relevant for future studies.

Coordination chemistry applied to anti-doping analysis: Cobalt quantification as its diethyldithiocarbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry.

Cobalt was included on the World Anti-Doping Agency Prohibited List in 2015 due to its effect on stimulus of erythropoiesis via stabilization of hypoxia-inducible factor. Although it has proven benefits for performance enhancement, the unavailability of inductively coupled plasma-mass spectrometry on routine of the accredited laboratories is a factor that reduces its applicability in anti-doping analysis. Therefore, an analytical method for quantification of urinary cobalt as its diethyldithiocarbamate complex by liquid chromatography coupled with high-resolution tandem mass spectrometry was developed and validated. Palladium was proposed as internal standard and rhodium as a complexation control. A microwave-assisted acid digestion of the urine samples was essential, not only to eliminate the matrix effect but mainly to avoid the non-specific bond of cobalt to endogenous molecules. A linear method was obtained over the studied range from a negative urine control to a spiked concentration of 25 ng/mL, with an estimated limit of quantification of 2.5 ng/mL, and an adequate combined standard uncertainty of 11.4%. Considering that all reagents are commercially available, the proposed strategy is feasible to be included on routine sample preparation. Monitoring urinary cobalt concentrations globally opens the perspective to support the anti-doping system to define a suitable threshold value and to understand its potential misuse by athletes seeking for performance improvement.

Monitoring casbene synthase in Jatropha curcas tissues using targeted proteomics.

BACKGROUND: Casbene synthase (CS) is responsible for the first committed step in the biosynthesis of phorbol esters (PE) in the Euphorbiaceae. PE are abundant in the seeds of the biofuel crop Jatropha curcas and its toxicity precludes the use of the protein-rich cake obtained after oil extraction as an animal feed and the toxicity of the fumes derived from burning PE containing biofuel is also a matter of concern. This toxicity is a major hindrance to exploit the potential of this crop as a source of raw material to produce biodiesel. For this reason, the current research on J. curcas is mainly focused on the understanding of the biosynthesis and site of synthesis of PE, as an avenue for the development of genotypes unable to synthesize PE in its seeds. RESULTS: Here, we present targeted proteomics assays (SRM and PRM) to detect and quantify CS in leaves, endosperm, and roots of two J. curcas genotypes with contrasting levels of PE. These assays were based on the use of reference isotopic labeled synthetic peptides (ILSP) predicted from 12 gene models of CS from the J. curcas genome. CONCLUSION: Our targeted proteomics methods were able to detect and quantify, for the first time, CS gene products and demonstrate the distribution of CS isoforms only in roots from J. curcas genotypes with a high and low concentration of PE. These methods can be expanded to monitor CS, at the protein level, in different tissues and genotypes of J. curcas.

Quantitative Subcellular Proteomics of the Orbitofrontal Cortex of Schizophrenia Patients.

Schizophrenia is a chronic disease characterized by the impairment of mental functions with a marked social dysfunction. A quantitative proteomic approach using iTRAQ labeling and SRM, applied to the characterization of mitochondria (MIT), crude nuclear fraction (NUC), and cytoplasm (CYT), can allow the observation of dynamic changes in cell compartments providing valuable insights concerning schizophrenia physiopathology. Mass spectrometry analyses of the orbitofrontal cortex from 12 schizophrenia patients and 8 healthy controls identified 655 protein groups in the MIT fraction, 1500 in NUC, and 1591 in CYT. We found 166 groups of proteins dysregulated among all enriched cellular fractions. Through the quantitative proteomic analysis, we detect as the main biological pathways those related to calcium and glutamate imbalance, cell signaling disruption of CREB activation, axon guidance, and proteins involved in the activation of NF-kB signaling along with the increase of complement protein C3. Based on our data analysis, we suggest the activation of NF-kB as a possible pathway that links the deregulation of glutamate, calcium, apoptosis, and the activation of the immune system in schizophrenia patients. All MS data are available in the ProteomeXchange Repository under the identifier PXD015356 and PXD014350.

Zebrafish (Danio rerio) water tank model for the investigation of drug metabolism: Progress, outlook, and challenges.

Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability. As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described.