Fast Sampling of the Cellular Metabolome.

Obtaining meaningful snapshots of the metabolome of microorganisms requires rapid sampling and immediate quenching of all metabolic activity, to prevent any changes in metabolite levels after sampling. Furthermore, a suitable extraction method is required ensuring complete extraction of metabolites from the cells and inactivation of enzymatic activity, with minimal degradation of labile compounds. Finally, a sensitive, high-throughput analysis platform is needed to quantify a large number of metabolites in a small amount of sample.

Human Lectins, Their Carbohydrate Affinities and Where to Find Them.

Lectins are a class of proteins responsible for several biological roles such as cell-cell interactions, signaling pathways, and several innate immune responses against pathogens. Since lectins are able to bind to carbohydrates, they can be a viable target for targeted drug delivery systems. In fact, several lectins were approved by Food and Drug Administration for that purpose.

Can the adrenergic system be implicated in the pathophysiology of bladder pain syndrome/interstitial cystitis? A clinical and experimental study.

Aims: To evaluate sympathetic system activity in bladder pain syndrome/interstitial cystitis (BPS/IC) patients and to investigate if chronic adrenergic stimulation in intact rats induces BPS/IC-like bladder modifications.

Methods: Clinical study--In BPS/IC patients and aged and body mass index matched volunteers TILT test was undertaken and catecholamines were measured in plasma and 24 hr urine samples. Experimental study--Phenylephrine was injected subcutaneously (14 days) to female Wistar rats.

Inferring differences in the distribution of reaction rates across conditions.

Elucidating changes in the distribution of reaction rates in metabolic pathways under different conditions is a central challenge in systems biology. Here we present a method for inferring regulation mechanisms responsible for changes in the distribution of reaction rates across conditions from correlations in time-resolved data. A reversal of correlations between conditions reveals information about regulation mechanisms.

Fast sampling of the cellular metabolome.

Obtaining meaningful snapshots of the metabolome of microorganisms requires rapid sampling and immediate quenching of all metabolic activity, to prevent any changes in metabolite levels after sampling. Furthermore, a suitable extraction method is required ensuring complete extraction of metabolites from the cells and inactivation of enzymatic activity, with minimal degradation of labile compounds. Finally a sensitive, high-throughput analysis platform is needed to quantify a large number of metabolites in a small amount of sample.

Development of quantitative metabolomics for Pichia pastoris.

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters.

Global test for metabolic pathway differences between conditions.

In many metabolomics applications there is a need to compare metabolite levels between different conditions, e.g., case versus control.

Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry.

A sensitive, selective, and comprehensive method for the quantitative determination of tryptophan and 18 of its key metabolites in serum, urine, and cell culture supernatants was developed. The analytes were separated on a C18 silica column by reversed-phase liquid chromatography and detected by electrospray ionization tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) mode, except for indoxyl sulfate which was measured in negative ion MRM mode in a separate run. The limits of detection and lower limits of quantification were in the range of 0.

An in vivo data-driven framework for classification and quantification of enzyme kinetics and determination of apparent thermodynamic data.

Kinetic modeling of metabolism holds great potential for metabolic engineering but is hindered by the gap between model complexity and availability of in vivo data. There is also growing interest in network-wide thermodynamic analyses, which are currently limited by the scarcity and unreliability of thermodynamic reference data. Here we propose an in vivo data-driven approach to simultaneously address both problems.

Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains.

The field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory.

Measuring enzyme activities under standardized in vivo-like conditions for systems biology.

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions.

Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics.

Accurate determination of intracellular metabolite levels requires well-validated procedures for sampling and sample treatment. Several methods exist for metabolite extraction, but the literature is contradictory regarding the adequacy and performance of each technique. Using a strictly quantitative approach, we have re-evaluated five methods (hot water, HW; boiling ethanol, BE; chloroform-methanol, CM; freezing-thawing in methanol, FTM; acidic acetonitrile-methanol, AANM) for the extraction of 44 intracellular metabolites (phosphorylated intermediates, amino acids, organic acids, nucleotides) from S.

A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC-IDMS.

A gas chromatography isotope dilution mass spectrometry (GC-IDMS) method was developed for the quantification of the metabolites of the non-oxidative part of pentose phosphate pathway (PPP). A mid-polar GC column (Zebron ZB-AAA, 10m, film composition 50% phenyl 50% dimethyl polysiloxane) was used for the chromatographic separation of the intermediates. The optimized GC-MS procedure resulted in improved separation performances and higher sensitivities compared to previous methods.

Dynamics of glycolytic regulation during adaptation of Saccharomyces cerevisiae to fermentative metabolism.

The ability of baker's yeast (Saccharomyces cerevisiae) to rapidly increase its glycolytic flux upon a switch from respiratory to fermentative sugar metabolism is an important characteristic for many of its multiple industrial applications. An increased glycolytic flux can be achieved by an increase in the glycolytic enzyme capacities (V(max)) and/or by changes in the concentrations of low-molecular-weight substrates, products, and effectors. The goal of the present study was to understand the time-dependent, multilevel regulation of glycolytic enzymes during a switch from fully respiratory conditions to fully fermentative conditions.

Quantitative analysis of the high temperature-induced glycolytic flux increase in Saccharomyces cerevisiae reveals dominant metabolic regulation.

A major challenge in systems biology lies in the integration of processes occurring at different levels, such as transcription, translation, and metabolism, to understand the functioning of a living cell in its environment. We studied the high temperature-induced glycolytic flux increase in Saccharomyces cerevisiae and investigated the regulatory mechanisms underlying this increase. We used glucose-limited chemostat cultures to separate regulatory effects of temperature from effects on growth rate.

Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions.

The coenzyme NAD plays a major role in metabolism as a key redox carrier and signaling molecule but current measurement techniques cannot distinguish between different compartment pools, between free and protein-bound forms and/or between NAD(H) and NADP(H). Local free NAD/NADH ratios can be determined from product/substrate ratios of suitable near-equilibrium redox reactions but the application of this principle is often precluded by uncertainties regarding enzyme activity, localization and coenzyme specificity of dehydrogenases. In Saccharomyces cerevisiae, we circumvented these issues by expressing a bacterial mannitol-1-phosphate 5-dehydrogenase and determining the cytosolic free NAD/NADH ratio from the measured [fructose-6-phosphate]/[mannitol-1-phosphate] ratio.