Dissolved organic metabolite extraction from high-salt media.

We describe considerations and strategies for developing a nuclear magnetic resonance (NMR) sample preparation method to extract low molecular weight metabolites from high-salt spent media in a model coculture system of phytoplankton and marine bacteria. Phytoplankton perform half the carbon fixation and oxygen generation on Earth. A substantial fraction of fixed carbon becomes part of a metabolite pool of small molecules known as dissolved organic matter (DOM), which are taken up by marine bacteria proximate to phytoplankton. There is an urgent need to elucidate these metabolic exchanges due to widespread anthropogenic transformations on the chemical, phenotypic, and species composition of seawater. These changes are increasing water temperature and the amount of CO2 absorbed by the ocean at energetic costs to marine microorganisms. Little is known about the metabolite-mediated, structured interactions occurring between phytoplankton and associated marine bacteria, in part because of challenges in studying high-salt solutions on various analytical platforms. NMR analysis is problematic due to the high-salt content of both natural seawater and culture media for marine microbes. High-salt concentration degrades the performance of the radio frequency coil, reduces the efficiency of some pulse sequences, limits signal-to-noise, and prolongs experimental time. The method described herein can reproducibly extract low molecular weight DOM from small-volume, high-salt cultures. It is a promising tool for elucidating metabolic flux between marine microorganisms and facilitates genetic screens of mutant microorganisms.

NMR Precision Metabolomics: Dynamic Peak Sum Thresholding and Navigators for Highly Standardized and Reproducible Metabolite Profiling of Clinical Urine Samples.

Metabolomics, especially urine-based studies, offers incredible promise for the discovery and development of clinically impactful biomarkers. However, due to the unique challenges of urine, a highly precise and reproducible workflow for NMR-based urine metabolomics is lacking. Using 1D and 2D non-uniform sampled (NUS) 1H-13C NMR spectroscopy, we systematically explored how changes in hydration or specific gravity (SG) and pH can impact biomarker discovery. Further, we examined additional sources of error in metabolomics studies and identified Navigator molecules that could monitor for those biases. Adjustment of SG to 1.002-1.02 coupled with a dynamic sum-based peak thresholding eliminates false positives associated with urine hydration and reduces variation in chemical shift. We identified Navigator molecules that can effectively monitor for inconsistencies in sample processing, SG, protein contamination, and pH. The workflow described provides quality assurance and quality control tools to generate high-quality urine metabolomics data, which is the first step in biomarker discovery.

Growth-stage-related shifts in diatom endometabolome composition set the stage for bacterial heterotrophy.

Phytoplankton-derived metabolites fuel a large fraction of heterotrophic bacterial production in the global ocean, yet methodological challenges have limited our understanding of the organic molecules transferred between these microbial groups. In an experimental bloom study consisting of three heterotrophic marine bacteria growing together with the diatom Thalassiosira pseudonana, we concurrently measured diatom endometabolites (i.e., potential exometabolite supply) by nuclear magnetic resonance (NMR) spectroscopy and bacterial gene expression (i.e., potential exometabolite uptake) by metatranscriptomic sequencing. Twenty-two diatom endometabolites were annotated, with nine increasing in internal concentration in the late stage of the bloom, eight decreasing, and five showing no variation through the bloom progression. Some metabolite changes could be linked to shifts in diatom gene expression, as well as to shifts in bacterial community composition and their expression of substrate uptake and catabolism genes. Yet an overall low match indicated that endometabolome concentration was not a good predictor of exometabolite availability, and that complex physiological and ecological interactions underlie metabolite exchange. Six diatom endometabolites accumulated to higher concentrations in the bacterial co-cultures compared to axenic cultures, suggesting a bacterial influence on rates of synthesis or release of glutamate, arginine, leucine, 2,3-dihydroxypropane-1-sulfonate, glucose, and glycerol-3-phosphate. Better understanding of phytoplankton metabolite production, release, and transfer to assembled bacterial communities is key to untangling this nearly invisible yet pivotal step in ocean carbon cycling.

Resource partitioning of phytoplankton metabolites that support bacterial heterotrophy.

The communities of bacteria that assemble around marine microphytoplankton are predictably dominated by Rhodobacterales, Flavobacteriales, and families within the Gammaproteobacteria. Yet whether this consistent ecological pattern reflects the result of resource-based niche partitioning or resource competition requires better knowledge of the metabolites linking microbial autotrophs and heterotrophs in the surface ocean. We characterized molecules targeted for uptake by three heterotrophic bacteria individually co-cultured with a marine diatom using two strategies that vetted the exometabolite pool for biological relevance by means of bacterial activity assays: expression of diagnostic genes and net drawdown of exometabolites, the latter detected with mass spectrometry and nuclear magnetic resonance using novel sample preparation approaches. Of the more than 36 organic molecules with evidence of bacterial uptake, 53% contained nitrogen (including nucleosides and amino acids), 11% were organic sulfur compounds (including dihydroxypropanesulfonate and dimethysulfoniopropionate), and 28% were components of polysaccharides (including chrysolaminarin, chitin, and alginate). Overlap in phytoplankton-derived metabolite use by bacteria in the absence of competition was low, and only guanosine, proline, and N-acetyl-D-glucosamine were predicted to be used by all three. Exometabolite uptake pattern points to a key role for ecological resource partitioning in the assembly marine bacterial communities transforming recent photosynthate.

Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum.

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite's development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.

Metabolic dependency of chorismate in Plasmodium falciparum suggests an alternative source for the ubiquinone biosynthesis precursor.

The shikimate pathway, a metabolic pathway absent in humans, is responsible for the production of chorismate, a branch point metabolite. In the malaria parasite, chorismate is postulated to be a direct precursor in the synthesis of p-aminobenzoic acid (folate biosynthesis), p-hydroxybenzoic acid (ubiquinone biosynthesis), menaquinone, and aromatic amino acids. While the potential value of the shikimate pathway as a drug target is debatable, the metabolic dependency of chorismate in P. falciparum remains unclear. Current evidence suggests that the main role of chorismate is folate biosynthesis despite ubiquinone biosynthesis being active and essential in the malaria parasite. Our goal in the present work was to expand our knowledge of the ubiquinone head group biosynthesis and its potential metabolic dependency on chorismate in P. falciparum. We systematically assessed the development of both asexual and sexual stages of P. falciparum in a defined medium in the absence of an exogenous supply of chorismate end-products and present biochemical evidence suggesting that the benzoquinone ring of ubiquinones in this parasite may be synthesized through a yet unidentified route.

Dysfunctional glycosynapses in schizophrenia: disease and regional specificity.

BACKGROUND: Our previous lipidomics studies demonstrated elevated sulfatides, plasmalogens, and N-acylphosphatidylserines in the frontal cortex of schizophrenia subjects. These data suggest that there may be an abnormal function of glycosynapses in schizophrenia. We further examined the disease and anatomical specificity of these observations. METHODS: We undertook a targeted lipidomics analysis of plasmalogens, sulfatides, and N-acyl-phosphatidylserines in the frontal cortex obtained from schizophrenia, bipolar, and ALS subjects and the cerebellum of schizophrenia subjects. RESULTS: We demonstrate that sulfatides, plasmalogens, and N-acyl-phosphatidylserines are significantly elevated in the frontal cortex of patients suffering from schizophrenia and bipolar depression but not in ALS patients. These lipids were unchanged in the cerebellum of subjects with schizophrenia. CONCLUSIONS: Our data suggest that dysfunction of oligodendrocyte glycosynapses may be specific to limbic circuits in schizophrenia and that this dysfunction is also detected in bipolar depression, suggesting that these disorders possess several common pathophysiological features.