The contribution of gut bacterial metabolites in the human immune signaling pathway of non-communicable diseases.

The interaction disorder between gut microbiota and its host has been documented in different non-communicable diseases (NCDs) such as metabolic syndrome, neurodegenerative disease, and autoimmune disease. The majority of these altered interactions arise through metabolic cross-talk between gut microbiota and host immune system, inducing a low-grade chronic inflammation that characterizes all NCDs. In this review, we discuss the contribution of bacterial metabolites to immune signaling pathways involved in NCDs. We then review recent advances that aid to rationally design microbial therapeutics. A deeper understanding of these intersections between host and gut microbiota metabolism using metabolomics-based system biology platform promises to reveal the fundamental mechanisms that drive metabolic predispositions to disease and suggest new avenues to use microbial therapeutic opportunities for NCDs treatment and prevention. Abbreviations: NCDs: non-communicable disease, IBD: inflammatory bowel disease, IL: interleukin, T2D: type 2 diabetes, SCFAs: short-chain fatty acids, HDAC: histone deacetylases, GPCR: G-protein coupled receptors, 5-HT: 5-hydroxytryptamine receptor signaling, DCs: dendritic cells, IECs: intestinal epithelial cells, T-reg: T regulatory cell, NF-κB: nuclear factor κB, TNF-α: tumor necrosis factor alpha, Th: T helper cell, CNS: central nervous system, ECs: enterochromaffin cells, NSAIDs: non-steroidal anti-inflammatory drugs, AhR: aryl hydrocarbon receptor, IDO: indoleamine 2,3-dioxygenase, QUIN: quinolinic acid, PC: phosphatidylcholine, TMA: trimethylamine, TMAO: trimethylamine N-oxide, CVD: cardiovascular disease, NASH: nonalcoholic steatohepatitis, BAs: bile acids, FXR: farnesoid X receptor, CDCA: chenodeoxycholic acid, DCA: deoxycholic acid, LCA: lithocholic acid, UDCA: ursodeoxycholic acid, CB: cannabinoid receptor, COBRA: constraint-based reconstruction and analysis.

Publisher Correction: Menstrual cycle rhythmicity: metabolic patterns in healthy women.

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Menstrual cycle rhythmicity: metabolic patterns in healthy women.

The menstrual cycle is an essential life rhythm governed by interacting levels of progesterone, estradiol, follicular stimulating, and luteinizing hormones. To study metabolic changes, biofluids were collected at four timepoints in the menstrual cycle from 34 healthy, premenopausal women. Serum hormones, urinary luteinizing hormone and self-reported menstrual cycle timing were used for a 5-phase cycle classification. Plasma and urine were analyzed using LC-MS and GC-MS for metabolomics and lipidomics; serum for clinical chemistries; and plasma for B vitamins using HPLC-FLD. Of 397 metabolites and micronutrients tested, 208 were significantly (p < 0.05) changed and 71 reached the FDR 0.20 threshold showing rhythmicity in neurotransmitter precursors, glutathione metabolism, the urea cycle, 4-pyridoxic acid, and 25-OH vitamin D. In total, 39 amino acids and derivatives and 18 lipid species decreased (FDR < 0.20) in the luteal phase, possibly indicative of an anabolic state during the progesterone peak and recovery during menstruation and the follicular phase. The reduced metabolite levels observed may represent a time of vulnerability to hormone related health issues such as PMS and PMDD, in the setting of a healthy, rhythmic state. These results provide a foundation for further research on cyclic differences in nutrient-related metabolites and may form the basis of novel nutrition strategies for women.

Multivariate pharmacokinetic/pharmacodynamic (PKPD) analysis with metabolomics shows multiple effects of remoxipride in rats.

The study of central nervous system (CNS) pharmacology is limited by a lack of drug effect biomarkers. Pharmacometabolomics is a promising new tool to identify multiple molecular responses upon drug treatment. However, the pharmacodynamics is typically not evaluated in metabolomics studies, although being important properties of biomarkers. In this study we integrated pharmacometabolomics with pharmacokinetic/pharmacodynamic (PKPD) modeling to identify and quantify the multiple endogenous metabolite dose-response relations for the dopamine D2 antagonist remoxipride. Remoxipride (vehicle, 0.7 or 3.5mg/kg) was administered to rats. Endogenous metabolites were analyzed in plasma using a biogenic amine platform and PKPD models were derived for each single metabolite. These models were clustered on basis of proximity between their PKPD parameter estimates, and PKPD models were subsequently fitted for the individual clusters. Finally, the metabolites were evaluated for being significantly affected by remoxipride. In total 44 metabolites were detected in plasma, many of them showing a dose dependent decrease from baseline. We identified 6 different clusters with different time and dose dependent responses and 18 metabolites were revealed as potential biomarker. The glycine, serine and threonine pathway was associated with remoxipride pharmacology, as well as the brain uptake of the dopamine and serotonin precursors. This is the first time that pharmacometabolomics and PKPD modeling were integrated. The resulting PKPD cluster model described diverse pharmacometabolomics responses and provided a further understanding of remoxipride pharmacodynamics. Future research should focus on the simultaneous pharmacometabolomics analysis in brain and plasma to increase the interpretability of these responses.

Pharmacometabolomics reveals that serotonin is implicated in aspirin response variability.

While aspirin is generally effective for prevention of cardiovascular disease, considerable variation in drug response exists, resulting in some individuals displaying high on-treatment platelet reactivity. We used pharmacometabolomics to define pathways implicated in variation of response to treatment. We profiled serum samples from healthy subjects pre- and postaspirin (14 days, 81 mg/day) using mass spectrometry. We established a strong signature of aspirin exposure independent of response (15/34 metabolites changed). In our discovery (N = 80) and replication (N = 125) cohorts, higher serotonin levels pre- and postaspirin correlated with high, postaspirin, collagen-induced platelet aggregation. In a third cohort, platelets from subjects with the highest levels of serotonin preaspirin retained higher reactivity after incubation with aspirin than platelets from subjects with the lowest serotonin levels preaspirin (72 ± 8 vs. 61 ± 11%, P = 0.02, N = 20). Finally, ex vivo, serotonin strongly increased platelet reactivity after platelet incubation with aspirin (+20%, P = 4.9 × 10(-4), N = 12). These results suggest that serotonin is implicated in aspirin response variability.

Integrating metabolomics profiling measurements across multiple biobanks.

To optimize the quality of large scale mass-spectrometry based metabolomics data obtained from semiquantitative profiling measurements, it is important to use a strategy in which dedicated measurement designs are combined with a strict statistical quality control regime. This assures consistently high-quality results across measurements from individual studies, but semiquantitative data have been so far only comparable for samples measured within the same study. To enable comparability and integration of semiquantitative profiling data from different large scale studies over the time course of years, the measurement and quality control strategy has to be extended. We introduce a strategy to allow the integration of semiquantitative profiling data from different studies. We demonstrate that lipidomics data generated in samples from three different large biobanks acquired in the time course of 3 years can be effectively combined when using an appropriate measurement design and transfer model. This strategy paves the way toward an integrative usage of semiquantitative metabolomics data sets of multiple studies to validate biological findings in another study and/or to increase the statistical power for discovery of biomarkers or pathways by combining studies.

Disulfide connectivity of recombinant C-terminal region of human thrombospondin 2.

The thrombospondin (TSP) family of extracellular glycoproteins consists of five members in vertebrates, TSP1 to -4 and TSP5/cartilage oligomeric matrix protein, and a single member in Drosophila. TSPs are modular multimeric proteins. The C-terminal end of a monomer consists of 3-6 EGF-like modules; seven tandem 23-, 36-, or 38-residue aspartate-rich, Ca(2+)-binding repeats; and an approximately 230-residue C-terminal sequence. The Ca(2+)-binding repeats and C-terminal sequence are spaced almost exactly the same in different TSPs and share many blocks of identical residues. We studied the C-terminal portion of human TSP2 from the third EGF-like module through the end of the protein (E3CaG2). E3CaG2, CaG2 lacking the EGF module, and Ca2 composed of only the Ca(2+)-binding repeats were expressed using recombinant baculoviruses and purified from conditioned media of insect cells. As previously described for intact TSP1, E3CaG2 bound Ca(2+) in a cooperative manner as assessed by equilibrium dialysis, and its circular dichroism spectrum was sensitive to the presence of Ca(2+). Mass spectrometry of the recombinant proteins digested with endoproteinase Asp-N revealed that disulfide pairing of the 18 cysteines in the Ca(2+)-binding repeats and C-terminal sequence is sequential, i.e. a 1-2, 3-4, 5-6, etc., pattern.

Advances in the mass spectrometry of hemoglobin adducts: global analysis of the covalent binding of butadiene monoxide.

A common method to assess exposure to 1,3-butadiene through both occupational and environmental routes involves the detection of hemoglobin adducts formed by the primary reactive metabolite butadiene monoxide (EB). This assay is a modification of the Edman degradation procedure, which was developed to determine adducts formed specifically at the amine group of the N-terminal valine of hemoglobin. The goals of the current research are to determine the global modification of alpha- and beta-globin chains by EB and to localize the primary reactive residues to specific regions of the globin polypeptides. The degree of modification was monitored by electrospray mass spectrometry, which was used to measure the formation of EB-hemoglobin adducts (up to ten adducts per globin). Structural analysis of these modifications was performed by peptide mapping of globin peptides after trypsin digestion using liquid chromatography-mass spectrometry. These experiments provided information as to the relative reactivity of alpha- and beta-globin towards EB, as well as to the localization of adducts to specific peptide sequences. The results reveal variable reactivities of alpha- and beta-globin towards EB and also show the formation of multiple adducts at several alpha- and beta-globin sites. In addition, it is established that the N-terminal valine residues are not the first to be modified by EB.

A comprehensive structural analysis of hemoglobin adducts formed after in vitro exposure of erythrocytes to butadiene monoxide.

A widely used method for assessing occupational and environmental exposure to 1,3-butadiene involves the detection of hemoglobin adducts formed by the reactive metabolite butadiene monoxide (BMO). This assay employs the N-alkyl Edman method, which was developed to determine adducts formed at the amine group of the N-terminal valine of hemoglobin. Disadvantages of this procedure include its limitation to detecting only one adduct per globin chain, despite the presence of numerous other, and potentially more reactive, nucleophilic amino acids in hemoglobin. The method is also not suitable for determining whether the reaction of BMO occurs at the N-terminal valine of alpha- or beta-globin. The primary goals of the current research are to determine the degree of modification of alpha- and beta-globin chains by BMO and to localize the reactive residues to specific regions of the globin polypeptides. The reaction products after in vitro incubation of C57Bl/6 mouse erythrocytes with BMO were isolated by acid extraction of heme and microprecipitation of globin, followed by the determination of the number and location of adducts by mass spectrometry. The modification degree was monitored by electrospray mass spectrometry, which was used to measure the time- and concentration-dependent formation of BMO-hemoglobin adducts (< or =10 adducts per globin). The results indicate that BMO reacts faster and to a higher degree with alpha-globin than with beta-globin. Structural analysis was performed by peptide mapping of globin peptides after trypsin digestion using liquid chromatography/mass spectrometry. These experiments allowed the localization of BMO-hemoglobin adducts to specific regions within alpha- and beta-globin, and also provided information about their relative reactivity. Interestingly, the initial site of adduct formation on alpha-globin is located near the N-terminal peptide, whereas the initial site on beta-globin is located at the C-terminal region. Collectively, the results establish differences in the reactivities of alpha- and beta-globin toward BMO, demonstrate the formation of multiple adducts at several alpha- and beta-globin sites, and show that the N-terminal valine residues are not the first to be modified by BMO.

New fluorogenic substrates for N-arginine dibasic convertase.

N-Arginine dibasic (NRD) convertase is a recently described peptidase capable of selectively cleaving peptides between paired basic residues. The characterization of this unique peptidase has been hindered by the fact that no facile assay procedure has been available. Here we report the development of a rapid and sensitive assay for NRD convertase, based on the utilization of two new internally quenched fluorogenic peptides: Abz-GGFLRRVGQ-EDDnp and Abz-GGFLRRIQ-EDDnp. These peptides contain the fluorescent 2-aminobenzoyl moiety that is quenched in the intact peptide by a 2, 4-dinitrophenyl moiety. Cleavage by NRD convertase at the Arg-Arg sequence results in an increase of fluorescence. NRD convertase cleaves these peptides efficiently and with high specificity as observed by both HPLC and fluorescence spectroscopy. The rate of hydrolysis of the fluorogenic substrates is proportional to enzyme concentration, and obeys Michaelis-Menten kinetics. The kinetic parameters for the fluorescent peptides (Km values of approximately 1.0 microM, and Vmax values of approximately 1 microM/(min. mg) are similar to those obtained with peptide hormones as substrates.

Probing RegA/RNA interactions using electrospray ionization-fourier transform ion cyclotron resonance-mass spectrometry.

The interactions of bacteriophage T4 regA protein, a unique translational regulator, with RNAs of various size and sequence were studied using electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry. Using very gentle interface conditions, regA/RNA complexes with a 1:1 binding stoichiometry were observed for all four target RNAs studied, consistent with solution binding studies. Competitive binding of target RNAs and their degradation products with regA demonstrated that the loss of a single nucleotide resulted in a dramatic change in binding affinity in some cases. Competitive binding of regA with four target RNAs revealed similar relative binding affinity order to that suggested by previous in vitro repression experiments. The use of sustained off-resonance irradiation for collisionally induced dissociation of a regA/RNA complex suggested the potential for directly obtaining information regarding the regA binding domain.

Electrospray ionization-mass spectrometry characterization of heterotetrameric sarcosine oxidase.

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i.e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 < m/z < 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization.

On-line microdialysis sample cleanup for electrospray ionization mass spectrometry of nucleic acid samples.

A major limitation of electrospray ionization mass spectrometry (ESI-MS) for oligonucleotide analysis arises due to sodium adduction, a problem that increases with molecular weight. Sodium adduction can preclude useful measurements when limited sample sizes prevent off-line cleanup. A novel and generally useful on-line microdialysis technique is described for the rapid (approximately 1-5 min) DNA sample cleanup for ESI-MS. Mass spectra of oligonucleotides of different size and sequence showing no significant sodium adduct peaks were obtained using the on-line microdialysis system with sodium chloride concentrations as high as 250 mM. Signal-to-noise ratios were also greatly enhanced compared to direct infusion of the original samples. By using ammonium acetate as the dialysis buffer, it was also found that the noncovalent association of double-stranded oligonucleotides could be preserved during the microdialysis process, allowing analysis by ESI-MS.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Mass spectrometric characterization of sequence-specific complexes of DNA and transcription factor PU.1 DNA binding domain.

Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent interaction of the 13.5-kDa DNA binding domain of PU.1 (PU.1-DBD) with specific double-stranded DNA (dsDNA) target molecules. Mixtures of PU.1-DBD protein and wild-type target DNA sequence yielded ESI-MS spectra showing only protein-dsDNA complex ions of 1:1 stoichiometry and free dsDNA. When PU.1-DBD protein, wild type target DNA, and a mutant target DNA lacking the consensus sequence were mixed, only the 1:1 complex with the wild-type DNA was observed, consistent with gel electrophoresis mobility shift assay results, demonstrating the observation of sequence-specific protein-dsDNA complexes using ESI-MS.