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

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

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

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

Metabolomic Profiles of a Midge (Procladius villosimanus, Kieffer) Are Associated with Sediment Contamination in Urban Wetlands

Metabolomic techniques are powerful tools for investigating organism-environment interactions. Metabolite profiles have the potential to identify exposure or toxicity before populations are disrupted and can provide useful information for environmental assessment. However, under complex environmental scenarios, metabolomic responses to exposure can be distorted by background and/or organismal variation. In the current study, we use LC-MS (liquid chromatography-mass spectrometry) and GC-MS (gas chromatography-mass spectrometry) to measure metabolites of the midge Procladius villosimanus inhabiting 21 urban wetlands. These metabolites were tested against common sediment contaminants using random forest models and metabolite enrichment analysis. Sediment contaminant concentrations in the field correlated with several P. villosimanus metabolites despite natural environmental and organismal variation. Furthermore, enrichment analysis indicated that metabolite sets implicated in stress responses were enriched, pointing to specific cellular functions affected by exposure. Methionine metabolism, sugar metabolism and glycerolipid metabolism associated with total petroleum hydrocarbon and metal concentrations, while mitochondrial electron transport and urea cycle sets associated only with bifenthrin. These results demonstrate the potential for metabolomics approaches to provide useful information in field-based environmental assessments

Application of dynamic metabolomics to examine in vivo skeletal muscle glucose metabolism in the chronically high-fat fed mouse.

RATIONALE: Defects in muscle glucose metabolism are linked to type 2 diabetes. Mechanistic studies examining these defects rely on the use of high fat-fed rodent models and typically involve the determination of muscle glucose uptake under insulin-stimulated conditions. While insightful, they do not necessarily reflect the physiology of the postprandial state. In addition, most studies do not examine aspects of glucose metabolism beyond the uptake process. Here we present an approach to study rodent muscle glucose and intermediary metabolism under the dynamic and physiologically relevant setting of the oral glucose tolerance test (OGTT). METHODS AND RESULTS: In&nbsp;vivo muscle glucose and intermediary metabolism was investigated following oral administration of [U-(13)C] glucose. Quadriceps muscles were collected 15 and 60&nbsp;min after glucose administration and metabolite flux profiling was determined by measuring (13)C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates via gas chromatography-mass spectrometry. While no dietary effects were noted in&nbsp;the glycolytic pathway, muscle from mice fed a high fat diet (HFD) exhibited a reduction in labelling in TCA intermediates. Interestingly, this appeared to be independent of alterations in flux through pyruvate dehydrogenase. In addition, our findings suggest that TCA cycle anaplerosis is negligible in muscle during an OGTT. CONCLUSIONS: Under the dynamic physiologically relevant conditions of the OGTT, skeletal muscle from HFD fed mice exhibits alterations in glucose metabolism at the level of the TCA cycle

Mus musculus deficient for secretory antibodies show delayed growth with an altered urinary metabolome

Abstract Background The polymeric immunoglobulin receptor (pIgR) maintains the integrity of epithelial barriers by transporting polymeric antibodies and antigens through the epithelial mucosa into the lumen. In this study, we examined the role of pIgR in maintaining gut barrier integrity, which is important for the normal development in mice. Methods Cohorts of pIgR −/− mice and their wildtype controls were housed under Specific Pathogen Free (SPF) conditions and monitored for weight gain as an indicator of development over time. The general physiology of the gastrointestinal tract was analysed using immunohistochemistry in young (8–12 weeks of age) and aged mice (up to 18 months of age), and the observed immunopathology in pIgR −/− mice was further characterised using flow cytometry. Urinary metabolites were analysed using gas chromatography-mass spectrometry (GC-MS), which revealed changes in metabolites that correlated with age-related increase in gut permeability in pIgR −/− mice. Results We observed that pIgR −/− mice exhibited delayed growth, and this phenomenon is associated with low-grade gut inflammation that increased with ageing. The gross intraepithelial lymphocytic (IEL) infiltration characteristic of pIgR −/− mice was redefined as CD8α+αβ+ T cells, the majority of which expressed high levels of CD103 and CD69 consistent with tissue resident memory T cells (TRM). Comparison of the urinary metabolome between pIgR −/− and wild-type mice revealed key changes in urinary biomarkers fucose, glycine and Vitamin B5, suggestive of altered mucosal permeability. A significant increase in gut permeability was confirmed by analysing the site-specific uptake of sugar probes in different parts of the intestine. Conclusion Our data show that loss of the secretory antibody system in mice results in enhanced accumulation of inflammatory IELs in the gut, which likely reflects ongoing inflammation in reaction to gut microbiota or food antigens, leading to delayed growth in pIgR −/− mice. We demonstrate that this leads to the presence of a unique urinary metabolome profile, which may provide a biomarker for altered gut permeability