Project description:Metabolites present in liver provide important clues regarding the physiological state of an organism. The aim of this work was to evaluate a protocol for high-throughput NMR-based analysis of polar and non-polar metabolites from a small quantity of liver tissue. We extracted the tissue with a methanol/chloroform/water mixture and isolated the polar metabolites from the methanol/water layer and the non-polar metabolites from the chloroform layer. Following drying, we re-solubilized the fractions for analysis with a 600 MHz NMR spectrometer equipped with a 1.7 mm cryogenic probe. In order to evaluate the feasibility of this protocol for metabolomics studies, we analyzed the metabolic profile of livers from house sparrow (Passer domesticus) nestlings raised on two different diets: livers from 10 nestlings raised on a high protein diet (HP) for 4 d and livers from 12 nestlings raised on the HP diet for 3 d and then switched to a high carbohydrate diet (HC) for 1 d. The protocol enabled the detection of 52 polar and nine non-polar metabolites in ¹H NMR spectra of the extracts. We analyzed the lipophilic metabolites by one-way ANOVA to assess statistically significant concentration differences between the two groups. The results of our studies demonstrate that the protocol described here can be exploited for high-throughput screening of small quantities of liver tissue (approx. 100 mg wet mass) obtainable from small animals.
Project description:The HypF protein is involved in the maturation and regulation of hydrogenases. The N-terminal domain of HypF (HypF-N) has served as a key model system to study the pathways of protein amyloid formation and the nature of the toxicity of pre-fibrilar protein oligomers. This domain can aggregate into two forms of oligomers having significantly different toxic effects when added to neuronal cultures. Here, NMR assignments of HypF-N backbone resonances are presented in its native state and under the conditions favouring the formation of toxic and non-toxic oligomers. The analyses of chemical shifts provide insights into the protein conformational state and the possible pathways leading to the formation of different types of oligomers.
Project description:Possible sources of variation in non-polar secondary metabolites of Portieria hornemannii, sampled from two distinct regions in the Philippines (Batanes and Visayas), resulting from different life-history stages, presence of cryptic species, and/or spatiotemporal factors, were investigated. PCA analyses demonstrated secondary metabolite variation between, as well as within, five cryptic Batanes species. Intraspecific variation was even more pronounced in the three cryptic Visayas species, which included samples from six sites. Neither species groupings, nor spatial or temporal based patterns, were observed in the PCA analysis, however, intraspecific variation in secondary metabolites was detected between life-history stages. Male gametophytes (102 metabolites detected) were strongly discriminated from the two other stages, whilst female gametophyte (202 metabolites detected) and tetrasporophyte (106 metabolites detected) samples were partially discriminated. These results suggest that life-history driven variations, and possibly other microscale factors, may influence the variation within Portieria species.
Project description:Modern triple quadrupole mass spectrometers provide the ability to detect and quantify a large number of metabolites using tandem mass spectrometry (MS/MS). Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a variety of column chemistries. Recently, the comprehensive optimization of LC-MS metabolomics using design of experiments (COLMeD) approach has been described and used by our group to develop robust LC-MS workflows (Rhoades and Weljie, 2016). The optimized LC-MS/MS method described here has been utilized extensively for metabolomics analysis of polar metabolites. Typically, tissue or biofluid samples are extracted using a modified Bligh-Dyer protocol (Bligh and Dyer, 1959; Tambellini et al., 2013). The protocol described herein describes this workflow using targeted polar metabolite multiple reaction monitoring (MRM) from tissues and biofluids via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This workflow has been utilized extensively for chronometabolic analysis (Krishnaiah et al., 2017), with applications generalized to other types of analyses as well (Sengupta et al., 2017; Sivanand et al., 2017).
Project description:Harmful algal blooms (HABs) are increasing in frequency, magnitude, and duration around the world. Prymnesium parvum is a HAB species known to cause massive fish kills, but the toxin(s) it produces contributing to this acute toxicity to fish have not been confirmed. In the present study, a 2 × 2 factorial design was employed to examine influences of salinity (2.4 or 5 ppt) and nutrient limitation (f/2 or f/8) on P. parvum acute toxicity to fish and produced molecules. Acute toxicity (LC50) of these cultures, following a 48-h mortality assay, ranged from 10,213 to 96,816 cells mL-1. Non-targeted analysis was performed using liquid chromatography high-resolution mass spectrometry (LC-HRMS) to investigate compounds contributing to the differential toxicological responses. When P. parvum elicited toxicity to fish, suspect screening confirmed the presence of several prymnesins, and the peak area of PRM-A (3 Cl; prymnesin2aglycone) was significantly (p < 0.05) and positively related to acute toxicity. In addition, a non-targeted approach to highlighting peaks that differ between two chemical fingerprints was developed, termed a relative difference plot, and used to search for peaks co-varying with P. parvum induced acute toxicity to fish. Several peaks were highlighted along with the prymnesins identified through suspect screening when acute toxicity to fish was observed.
Project description:BackgroundCeliac disease (CeD) is an autoimmune intestinal disorder caused by gluten protein consumption in genetically predisposed individuals. As biopsy sampling is an invasive procedure, finding novel noninvasive serological markers for screening of at-risk CeD population is a priority. Metabolomics is helpful in monitoring metabolite changes in body fluids and tissues. In the present study, we evaluated serum metabolite levels of CeD patients relative to healthy controls with the aim of introducing new biomarkers for population screening.MethodWe compared the serum metabolic profile of CeD patients (n = 42) and healthy controls (n = 22) using NMR spectroscopy and multivariate analysis.Result25 metabolites were identified by serum metabolic profiling. Levels of 3-hydroxyisobutyric acid and isobutyrate showed significant differences in CeD patients' samples compared with healthy controls (p < 0.05). According to pathway analysis, our data demonstrated that changes in nine metabolic pathways were significantly disrupted/affected in patients with CeD. These enriched pathways are involved in aminoacyl-tRNA biosynthesis; primary bile acid biosynthesis; nitrogen metabolism; glutamine and glutamate metabolism; valine, leucine, and isoleucine biosynthesis and degradation; taurine and hypotaurine metabolism; glyoxylate and dicarboxylate metabolism; glycine, serine, and threonine metabolism; and arginine biosynthesis.ConclusionIn summary, our results demonstrated that changes in the serum level of 25 metabolites may be useful in distinguishing CeD patients from healthy controls, which have the potential to be considered candidate biomarkers of CeD.