Correcting ligands, metabolites, and pathways

BACKGROUND: A wide range of research areas in bioinformatics, molecular biology and medicinal chemistry require precise chemical structure information about molecules and reactions, e.g. drug design, ligand docking, metabolic network reconstruction, and systems biology. Most available databases, however, treat chemical structures more as illustrations than as a datafield in its own right. Lack of chemical accuracy impedes progress in the areas mentioned above. We present a database of metabolites called BioMeta that augments the existing pathway databases by explicitly assessing the validity, correctness, and completeness of chemical structure and reaction information. DESCRIPTION: The main bulk of the data in BioMeta were obtained from the KEGG Ligand database. We developed a tool for chemical structure validation which assesses the chemical validity and stereochemical completeness of a molecule description. The validation tool was used to examine the compounds in BioMeta, showing that a relatively small number of compounds had an incorrect constitution (connectivity only, not considering stereochemistry) and that a considerable number (about one third) had incomplete or even incorrect stereochemistry. We made a large effort to correct the errors and to complete the structural descriptions. A total of 1468 structures were corrected and/or completed. We also established the reaction balance of the reactions in BioMeta and corrected 55% of the unbalanced (stoichiometrically incorrect) reactions in an automatic procedure. The BioMeta database was implemented in PostgreSQL and provided with a web-based interface. CONCLUSION: We demonstrate that the validation of metabolite structures and reactions is a feasible and worthwhile undertaking, and that the validation results can be used to trigger corrections and improvements to BioMeta, our metabolite database. BioMeta provides some tools for rational drug design, reaction searches, and visualization. It is freely available at provided that the copyright notice of all original data is cited. The database will be useful for querying and browsing biochemical pathways, and to obtain reference information for identifying compounds. However, these applications require that the underlying data be correct, and that is the focus of BioMeta

A pilot study comparing the metabolic profiles of elite-level athletes from different sporting disciplines

Background: The outstanding performance of an elite athlete might be associated with changes in their blood metabolic profile. The aims of this study were to compare the blood metabolic profiles between moderate- and high-power and endurance elite athletes and to identify the potential metabolic pathways underlying these differences. Methods: Metabolic profiling of serum samples from 191 elite athletes from different sports disciplines (121 high- and 70 moderate-endurance athletes, including 44 high- and 144 moderate-power athletes), who participated in national or international sports events and tested negative for doping abuse at anti-doping laboratories, was performed using non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography. Multivariate analysis was conducted using orthogonal partial least squares discriminant analysis. Differences in metabolic levels between high- and moderate-power and endurance sports were assessed by univariate linear models. Results: Out of 743 analyzed metabolites, gamma-glutamyl amino acids were significantly reduced in both high-power and high-endurance athletes compared to moderate counterparts, indicating active glutathione cycle. High-endurance athletes exhibited significant increases in the levels of several sex hormone steroids involved in testosterone and progesterone synthesis, but decreases in diacylglycerols and ecosanoids. High-power athletes had increased levels of phospholipids and xanthine metabolites compared to moderate-power counterparts. Conclusions: This pilot data provides evidence that high-power and high-endurance athletes exhibit a distinct metabolic profile that reflects steroid biosynthesis, fatty acid metabolism, oxidative stress, and energy-related metabolites. Replication studies are warranted to confirm differences in the metabolic profiles associated with athletes’ elite performance in independent data sets, aiming ultimately for deeper understanding of the underlying biochemical processes that could be utilized as biomarkers with potential therapeutic implications

Biomarkers of one-carbon metabolism and B-vitamin status : Targeted metabolomics in rats and humans exploring the effects of PPARα-activation and dietary composition

BACKGROUND: One-carbon metabolism is an overarching term describing central metabolic pathways involved in the transfer of one-carbon units. These include the methionine-homocysteine and folate cycles, as well as the choline oxidation and transsulfuration pathways, all of which depend on B-vitamins as cofactors. Circulating concentrations of several metabolites and intermediates of these metabolic pathways have been linked to chronic disease risk. Thus, extending our knowledge regarding the regulation of these pathways is warranted. Targeted metabolomics offers the opportunity to study the concentration of several metabolites of these pathways simultaneously, and is thus required for the thorough investigation of the effects of diet and other factors on one-carbon metabolism. Diet provides both substrates, cofactors and one-carbon units, influencing the complex regulation of the different metabolic pathways. Furthermore, of particular interest is the activation of the nuclear receptor PPAR (peroxisome proliferator-activated receptor) α, a lipid sensor involved in the regulation of energy metabolism. The overall aim of this thesis was to utilize targeted metabolomics to explore the effect of PPARα-activation and dietary composition on of one-carbon metabolism and B-vitamin status. The effect of pharmacological PPARα-activation was addressed in Paper I and II, taking advantage of studies in laboratory animals. In Paper III, the effect of dietary macronutrient composition was investigated in a cross-sectional analysis of a human cohort. METHODS: Animal models: We utilized data from two animal experiments, where male Wistar rats were treated with PPAR-agonists. In the first experiment, the rats received a pan-PPAR-agonist for 50 weeks. In the second experiment, specific PPARα and γ- agonists were administered for 12 days. In both studies, plasma concentrations of metabolites were compared between treated and control animals. Human study: This was a cross-sectional analysis in a cohort of 1928 patients with stable angina pectoris. Dietary data was derived from a food frequency questionnaire, and associations between dietary composition and plasma metabolite concentrations were assessed with multiple linear regression analyses. In all studies, plasma one carbon metabolites and B-vitamin markers were quantified by applying mass spectrometric methods. RESULTS: Long-term (Paper I) and short-term (Paper II) pharmacological PPARα-activation influenced the one-carbon metabolome, with the strongest effects seen for increased plasma concentration of nicotinamide and methylnicotinamide (vitamin B3), pyridoxal (vitamin B6), methylmalonic acid (marker of vitamin B12 status), dimethylglycine and glycine (choline oxidation pathway metabolites), and reduced flavin mononucleotide (vitamin B2). In humans, the observed effects of macronutrient intake were strongest for protein, where increased intake was associated with higher plasma concentrations of pyridoxal, pyridixal-5-phosphate and pyridoxic acid (vitamin B6), vitamin B12, riboflavin (vitamin B2) and methylnicotinamide. Further, we observed inverse associations between protein intake and plasma homocysteine and methylmalonic acid concentrations. When modeling the substitution of saturated with polyunsaturated fatty acids, we observed higher methylnicotinamide, pyridoxal, pyridixal-5-phosphate, cobalamin and dimethylglycine, as well as lower riboflavin concentrations. CONCLUSION AND IMPLICATION: PPARα-activation and dietary macronutrient composition altered the concentration of circulating biomarkers of one carbon metabolism. The effects of PPARα-activation were consistent across different conditions, and our results strongly suggest a central role for PPARα in the regulation of these metabolic pathways. Estimated effects of substituting saturated with polyunsaturated fatty acids yielded associations with the biomarkers similar to those observed with PPARα-activation in the animal studies. This suggests that the effects of diet on one-carbon metabolism, especially related to dietary fatty acid composition, may be partly mediated through altered PPARα-activity. This is the first metabolomic investigation targeting the majority of the metabolites of the one-carbon metabolism pathways simultaneously. Linking mechanistic studies in animals with observational data in humans provides novel information regarding metabolic regulations. The current investigations extend our understanding of how PPARα-activation and dietary composition influences the one-carbon metabolome. Application to the human situation will offer potential for more individualized dietary advice

Regulation of ENaC-Mediated Sodium Reabsorption by Peroxisome Proliferator-Activated Receptors

Peroxisome proliferator-activated receptors (PPARs) are members of a steroid hormone receptor superfamily that responds to changes in lipid and glucose homeostasis. Peroxisomal proliferator-activated receptor subtype γ (PPARγ) has received much attention as the target for antidiabetic drugs, as well as its role in responding to endogenous compounds such as prostaglandin J2. However, thiazolidinediones (TZDs), the synthetic agonists of the PPARγ are tightly associated with fluid retention and edema, as potentially serious side effects. The epithelial sodium channel (ENaC) represents the rate limiting step for sodium absorption in the renal collecting duct. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. The role of PPARγ agonists on ENaC activity remains controversial. While PPARγ agonists were shown to stimulate ENaC-mediated renal salt absorption, probably via Serum- and Glucocorticoid-Regulated Kinase 1 (SGK1), other studies reported that PPARγ agonist-induced fluid retention is independent of ENaC activity. The current paper provides new insights into the control and function of ENaC and ENaC-mediated sodium transport as well as several other epithelial channels/transporters by PPARs and particularly PPARγ. The potential contribution of arachidonic acid (AA) metabolites in PPAR-dependent mechanisms is also discussed

Impact of Bacterial Metabolites on Gut Barrier Function and Host Immunity:A Focus on Bacterial Metabolism and Its Relevance for Intestinal Inflammation

The diverse and dynamic microbial community of the human gastrointestinal tract plays a vital role in health, with gut microbiota supporting the development and function of the gut immune barrier. Crosstalk between microbiota-gut epithelium and the gut immune system determine the individual health status, and any crosstalk disturbance may lead to chronic intestinal conditions, such as inflammatory bowel diseases (IBD) and celiac disease. Microbiota-derived metabolites are crucial mediators of host-microbial interactions. Some beneficially affect host physiology such as short-chain fatty acids (SCFAs) and secondary bile acids. Also, tryptophan catabolites determine immune responses, such as through binding to the aryl hydrocarbon receptor (AhR). AhR is abundantly present at mucosal surfaces and when activated enhances intestinal epithelial barrier function as well as regulatory immune responses. Exogenous diet-derived indoles (tryptophan) are a major source of endogenous AhR ligand precursors and together with SCFAs and secondary bile acids regulate inflammation by lowering stress in epithelium and gut immunity, and in IBD, AhR expression is downregulated together with tryptophan metabolites. Here, we present an overview of host microbiota-epithelium- gut immunity crosstalk and review how microbial-derived metabolites contribute to host immune homeostasis. Also, we discuss the therapeutic potential of bacterial catabolites for IBD and celiac disease and how essential dietary components such as dietary fibers and bacterial tryptophan catabolites may contribute to intestinal and systemic homeostasis

Microbiome modulates intestinal homeostasis against inflammatory diseases

Eliminating prophylactic antibiotics in food animal production has exerted pressure on discovering antimicrobial alternatives (e.g. microbiome) to reduce elevated intestinal diseases. Intestinal tract is a complex ecosystem coupling host cells with microbiota. The microbiota and its metabolic activities and products are collectively called microbiome. Intestinal homeostasis is reached through dynamic and delicate crosstalk between host immunity and microbiome. However, this balance can be occasionally broken, which results in intestinal inflammatory diseases such as human Inflammatory Bowel Diseases, chicken necrotic enteritis, and swine postweaning diarrhea. In this review, we introduce the intestinal immune system, intestinal microbiome, and microbiome modulation of inflammation against intestinal diseases. The purpose of this review is to provide updated knowledge on host-microbe interaction and to promote using microbiome as new antimicrobial strategies to reduce intestinal diseases

RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes.

Synthetic biology and metabolic engineering seek to re-engineer microbes into living foundries for the production of high value chemicals. Through a design-build-test cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production

Is systems pharmacology ready to impact upon therapy development? A study of the cholesterol biosynthesis pathway.

Background and Purpose An ever-growing wealth of information on current drugs and their pharmacological effects is available from online databases. As our understanding of systems biology increases, we have the opportunity to predict, model and quantify how drug combinations can be introduced that outperform conventional single-drug therapies. Here, we explore the feasibility of such systems pharmacology approaches with an analysis of the mevalonate branch of the cholesterol biosynthesis pathway. Experimental Approach Using open online resources, we assembled a computational model of the mevalonate pathway and compiled a set of inhibitors directed against targets in this pathway. We used computational optimization to identify combination and dose options that show not only maximal efficacy of inhibition on the cholesterol producing branch but also minimal impact on the geranylation branch, known to mediate the side effects of pharmaceutical treatment. Key Results We describe serious impediments to systems pharmacology studies arising from limitations in the data, incomplete coverage and inconsistent reporting. By curating a more complete dataset, we demonstrate the utility of computational optimization for identifying multi-drug treatments with high efficacy and minimal off-target effects. Conclusion and Implications We suggest solutions that facilitate systems pharmacology studies, based on the introduction of standards for data capture that increase the power of experimental data. We propose a systems pharmacology workflow for the refinement of data and the generation of future therapeutic hypotheses

Acute Hypercapnia/Ischemia Alters the Esterification of Arachidonic Acid and Docosahexaenoic Acid Epoxide Metabolites in Rat Brain Neutral Lipids.

In the brain, approximately 90% of oxylipins are esterified to lipids. However, the significance of this esterification process is not known. In the present study, we (1) validated an aminopropyl solid phase extraction (SPE) method for separating esterified lipids using 100 and 500 mg columns and (2) applied the method to quantify the distribution of esterified oxylipins within phospholipids (PL) and neutral lipids (NL) (i.e. triacylglycerol and cholesteryl ester) in rats subjected to head-focused microwave fixation (controls) or CO2 -induced hypercapnia/ischemia. We hypothesized that oxylipin esterification into these lipid pools will be altered following CO2 -induced hypercapnia/ischemia. Lipids were extracted from control (n = 8) and CO2 -asphyxiated (n = 8) rat brains and separated on aminopropyl cartridges to yield PL and NL. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced SPE columns, and analyzed with ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. Method validation showed that the 500 mg (vs 100 mg) aminopropyl columns yielded acceptable separation and recovery of esterified fatty acid epoxides but not other oxylipins. Two epoxides of arachidonic acid (ARA) were significantly increased, and three epoxides of docosahexaenoic acid (DHA) were significantly decreased in brain NL of CO2 -asphyxiated rats compared to controls subjected to head-focused microwave fixation. PL-bound fatty acid epoxides were highly variable and did not differ significantly between the groups. This study demonstrates that hypercapnia/ischemia alters the concentration of ARA and DHA epoxides within NL, reflecting an active turnover process regulating brain fatty acid epoxide concentrations

Prebiotics and the Modulation on the Microbiota-GALT-Brain Axis

It is well known that there exists a bi-directional communication system between the enteric nervous system and central nervous system. Recent research has attempted to understand the influence of intestinal bacteria on the brain and behavior. In this manner, it has been observed that pathogenic bacterial products such as lipopolysaccharides (LPSs) can induce behavioral changes such as acute anxiety, depressive symptoms, cognitive deficits, and increased sensitivity to visceral pain. The modulation of LPS production through probiotics, prebiotics, and symbiotics can prevent these changes. In addition to the neuronal, endocrine, and metabolic pathways, it has been observed that the immune mechanism also exerts an influence on the gut-brain axis. The cells of the immune system can undergo phenotypic changes by the induction of certain bacterial species, which can have an important participation in the development of brain disorders. Although the main effect of prebiotics is through the stimulation of probiotic bacteria, in this chapter, we review the indirect therapeutic potential of prebiotics on the brain through the intestinal microbiota, the gut-associated lymphoid tissue (GALT), and other components of the intestinal lumen. Thus, the objective is to elucidate the mechanisms underlying its effects on the gut-brain axis. Here, we will summarize the possible therapeutic effect of prebiotics on intestinal microbiota, the gut-associated lymphoid tissue (GALT), and brain