Lipidomic and metabolomic characterization of a genetically modified mouse model of the early stages of human type 1 diabetes pathogenesis

The early mechanisms regulating progression towards beta cell failure in type 1 diabetes (T1D) are poorly understood, but it is generally acknowledged that genetic and environmental components are involved. The metabolomic phenotype is sensitive to minor variations in both, and accordingly reflects changes that may lead to the development of T1D. We used two different extraction methods in combination with both liquid- and gas chromatographic techniques coupled to mass spectrometry to profile the metabolites in a transgenic non-diabetes prone C57BL/6 mouse expressing CD154 under the control of the rat insulin promoter (RIP) crossed into the immuno-deficient recombination-activating gene (RAG) knockout (−/−) C57BL/6 mouse, resembling the early stages of human T1D. We hypothesized that alterations in the metabolomic phenotype would characterize the early pathogenesis of T1D, thus metabolomic profiling could provide new insight to the development of T1D. Comparison of the metabolome of the RIP CD154 × RAG(−/−) mice to RAG(−/−) mice and C57BL/6 mice revealed alterations of >100 different lipids and metabolites in serum. Low lysophosphatidylcholine levels, accumulation of ceramides as well as methionine deficits were detected in the pre-type 1 diabetic mice. Additionally higher lysophosphatidylinositol levels and low phosphatidylglycerol levels where novel findings in the pre-type 1 diabetic mice. These observations suggest that metabolomic disturbances precede the onset of T1D. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-015-0889-1) contains supplementary material, which is available to authorized users

Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal.

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells

A detailed mechanistic investigation of the exoglycanase from Cellulomonas fimi

The exoglycanase from Cellulomonas fimi catalyses the hydrolysis of cello oligosaccharides to cellobiose as well as the hydrolysis of xylan and aryl β-glycosides (Gilkes et al (1984) J. Biol. Chem. 259, 10455). Its mechanism of action is thought to involve a double displacement reaction which is investigated here through detailed kinetic studies of the native enzyme and point mutants with a range of aryl β-glycosides, and through inactivation studies with 2-deoxy- and 2-deoxyfluoro-glycoside mechanism-based inactivators and the affinity label, N-bromoacetyl cellobiosylamine. A pH study of the native enzyme revealed ionisations of PKa = 4.1 and 7.7 in the free enzyme, likely corresponding to the catalytic nucleophile and the acid-base catalyst, respectively. The large secondary deuterium kinetic isotope effects measured on both steps for the glucosides and on the deglycosylation step for the cellobiosides reveal significant oxocarbonium ion character at the corresponding transition states, thus suggesting substantial C-O bond cleavage and little nucleophilic preassociation. By contrast, the relatively small secondary deuterium kinetic isotope effect and the small Broensted constant measured on the glycosylation step for the cellobiosides suggest that the cellobiosylation transition state is less highly charged than the glucosylation transition state. These studies suggest that the primary function of the distal glucosyl moiety of the cellobiosides is to increase the rate of glycosylation, likely through improved acid catalysis and greater nucleophile preassociation, without affecting its rate of deglycosylation. The greater rates of hydrolysis of the xylo-sugars, relative to those for the gluco-sugars, indicate that the substrate preference of C. fimi exoglycanase increases in the order glucosides <xylosides <cellobiosides <xylobiosides and that the C-5 hydroxymethyl group is slightly inhibitory to catalysis. The role of the C-2 hydroxyl group was probed using 2,4-dinitrophenyl 2-deoxy-2-fluoro cellobioside (2F-DNPC) and cellobial (a 2-deoxycellobiose analogue). Rates of hydrolysis of the 2-deoxyfluorocellobiosyl- and 2-deoxycellobiosyl-enzymes are 10⁷ and10⁶-fold lower respectively, than that for the cellobiosyl-enzyme, indicating that the C-2 hydroxyl group is necessary for catalysis and that it contributes a minimum of -9 kcal/mole of stabilisation energy to the transition state. Electrospray ionisation mass spectrometry (ESI-MS) of the 2F-DNPC-inactivated enzyme provided evidence for the covalent nature of the glycosyl-enzyme intermediate while ¹⁹FNMR analysis of this 2FCb-enzyme and the 2-deoxy-2-fluoro 4-O-(f-glucosyl)- β-mannosyl fluoride (2F-GMF) -inactivated enzyme provided evidence for the α-anomeric stereochemistry of the intermediate. The catalytic nucleophile involved in C. fimi exoglycanase-catalysed hydrolysis of the cellobiosides was identified as Glu 233 by use of tandem MS techniques and 2F-DNPC and cellobial. Kinetic analysis of the Glu233Asp mutant revealed that pulling the catalytic nucleophile 1 Å away from the reacting anomeric centre reduces the rates of glycosylation and deglycosylation —4 x10³-fold. ESI-MS analysis of N-bromoacetyl cellobiosylamine-inactivated C. fimi exoglycanase reveals that one mole of N-acetyl cellobiosylamine is incorporated per mole of enzyme. The labeled residue was identified as Glu 127 by use of a combination of MS techniques. This residue has recently been suggested to be the acid-base catalyst based on kinetic analysis of mutants (MacLeod et al (1994) Biochemistry 33, 6571). More detailed kinetic analysis of the Glul27Ala mutant revealed rate reductions of 200-300 fold on the deglycosylation step while the rate reductions on the glycosylation step are dependent on the leaving group ability of the phenolate. The larger Broensted constant seen with the Glul27Ala mutant compared to that for the native enzyme reflects greater negative charge accumulation on the leaving phenolate at the glycosylation transition state for the Glul27Ala mutant. These results are consistent with the role of Glu 127 as the acid-base catalyst. These structural findings are completely consistent with the recently solved X-ray crystal structure of the catalytic domain of C.fimi exoglycanase (White et al (1994) Biochemistry 33, 12546).Science, Faculty ofChemistry, Department ofGraduat

An investigation of the mechanism of the Cellulomonas fimi exoglucanase

The exoglucanase from Cellulomonas fimi catalyses the hydrolysis of cellobiose units from the non-reducing terminus of cello-oligosaccharides with overall retention of anomeric configuration. Its mechanism of action is therefore thought to involve a double displacement reaction, involving as the first step, formation of a glycosyl-enzyme intermediate (glycosylation) and as a second step, the hydrolysis of this intermediate (deglycosylation). This mechanism is investigated here through the study of the kinetics of hydrolysis of aryl β-glucosides and aryl β-cellobiosides and by employing the mechanism-based irreversible inactivators, 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2F-DNPG) and 2", 4"-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside (2F-DNPC). The study with the aryl β-glucosides revealed that this enzyme is indeed active on glucosides, a feature that had previously been undetected. A linear relationship was found to exist between the logarithm of Vmax for hydrolysis and the phenol pKa as well as between the logarithm of Vmax/Krn and me phenol pKa, showing that glycosylation is both the rate determining step and the first irreversible step for all substrates. The reaction constant calculated, ρ = 2.21, indicates a considerable amount of charge build up at the transition state of glycosylation. The linear free energy relationship study of the aryl β-cellobiosides revealed no significant dependence of the logarithm of Vmax on the pKa of the phenol, indicating that deglycosylation is rate determining. However, the slight downward trend in this Hammett plot at higher pKa values may suggest that the rate determining step is changing from deglycosylation to glycosylation. However, the logarithm of Vmax/Km does correlate with the pKa of the phenol, thus showing that the first irreversible step is glycosylation. The reaction constant (ρ = 0.60) which reflects the development of charge at the glycosylation transition state for the cellobiosides is less than that calculated for the glucosides, thus suggesting a glycosylation transition state with either a greater degree of acid catalysis or less C-O bond cleavage than that for the glucosides. The inactivators, 2F-DNPC and 2F-DNPG, are believed to inactivate the exoglucanase by binding to the enzyme and forming covalent glycosyl-enzyme intermediates. The inactivated-enzyme was stable in buffer but reactivated in the presence of a suitable glycosyl-acceptor such as cellobiose, presumably via a transglycosylation reaction. These results indicate that covalent 2F-glycosyl-exoglucanase intermediates are stable and are catalytically competent to turn over to product, thus supplying further evidence for the Koshland mechanism. The exoglucanase is inactivated more rapidly by 2F-DNPC than by 2F-DNPG. However, both inactivated forms of the enzyme reactivated at comparable rates in the presence of cellobiose, showing that the second glucosyl unit present on the cellobiosides increases the rate of glycosylation relative to that found for the glucosides but not the rate of deglycosylation. The stable covalent nature of the 2F-glycosyl-enzyme intermediates provided an excellent opportunity to identify the enzymic nucleophile. This was accomplished by radiolabelling the exoglucanase with a tritiated analogue of 2F-DNPG cleaving the protein into peptides and purifying the radiolabelled peptides. Sequencing of this peptide resulted in the identification of the active site nucleophile as glutamic acid residue 274. This residue was found to be highly conserved in this family of β-glycanases, further indicating its importance in catalysis.Science, Faculty ofChemistry, Department ofGraduat

Talaromyces marneffei simA Encodes a Fungal Cytochrome P450 Essential for Survival in Macrophages

ABSTRACT Fungi are adept at occupying specific environmental niches and often exploit numerous secondary metabolites generated by the cytochrome P450 (CYP) monoxygenases. This report describes the characterization of a yeast-specific CYP encoded by simA ("survival in macrophages"). Deletion of simA does not affect yeast growth at 37°C in vitro but is essential for yeast cell production during macrophage infection. The ΔsimA strain exhibits reduced conidial germination and intracellular growth of yeast in macrophages, suggesting that the enzymatic product of SimA is required for normal fungal growth in vivo. Intracellular ΔsimA yeast cells exhibit cell wall defects, and metabolomic and chemical sensitivity data suggest that SimA may promote chitin synthesis or deposition in vitro. In vivo, ΔsimA yeast cells subsequently lyse and are degraded, suggesting that SimA may increase resistance to and/or suppress host cell biocidal effectors. The results suggest that simA synthesizes a secondary metabolite that allows T. marneffei to occupy the specific intracellular environmental niche within the macrophage. IMPORTANCE This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth

Ribose-cysteine protects against the development of atherosclerosis in apoE-deficient mice.

Ribose-cysteine is a synthetic compound designed to increase glutathione (GSH) synthesis. Low levels of GSH and the GSH-dependent enzyme, glutathione peroxidase (GPx), is associated with cardiovascular disease (CVD) in both mice and humans. Here we investigate the effect of ribose-cysteine on GSH, GPx, oxidised lipids and atherosclerosis development in apolipoprotein E-deficient (apoE-/-) mice. Female 12-week old apoE-/- mice (n = 15) were treated with 4-5 mg/day ribose-cysteine in drinking water for 8 weeks or left untreated. Blood and livers were assessed for GSH, GPx activity and 8-isoprostanes. Plasma alanine transferase (ALT) and lipid levels were measured. Aortae were quantified for atherosclerotic lesion area in the aortic sinus and brachiocephalic arch and 8-isoprostanes measured. Ribose-cysteine treatment significantly reduced ALT levels (p50% in both the aortic sinus and brachiocephalic branch (p<0.05). Ribose-cysteine promotes a significant GSH-based antioxidant effect in multiple tissues as well as an LDL-lowering response. These effects are accompanied by a marked reduction in atherosclerosis suggesting that ribose-cysteine might increase protection against CVD

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

Liquid chromatography&ndash;mass spectrometry (LC&ndash;MS) methods using either aqueous normal phase (ANP) or reversed phase (RP) columns are routinely used in small molecule or metabolomic analyses. These stationary phases enable chromatographic fractionation of polar and non-polar compounds, respectively. The application of a single chromatographic stationary phase to a complex biological extract results in a significant proportion of compounds which elute in the non-retained fraction, where they are poorly detected because of a combination of ion suppression and the co-elution of isomeric compounds. Thus coverage of both polar and non-polar components of the metabolome generally involves multiple analyses of the same sample, increasing the analysis time and complexity. In this study we describe a novel tandem in-line LC&ndash;MS method, in which compounds from one injection are sequentially separated in a single run on both ANP and RP LC-columns. This method is simple, robust, and enables the use of independent gradients customized for both RP and ANP columns. The MS signal is acquired in a single chromatogram which reduces instrument time and operator and data analysis errors. This method has been used to analyze a range of biological extracts, from plant and animal tissues, human serum and urine, microbial cell and culture supernatants. Optimized sample preparation protocols are described for this method as well as a library containing the retention times and accurate masses of 127 compounds