Sampling for metabolome analysis of microorganisms

In the present work we investigated the most commonly applied methods used for sampling of microorganisms in the field of metabolomics in order to unravel potential sources of error previously ignored but of utmost importance for accurate metabolome analysis. To broaden the significance of our study, we investigated different Gram-negative and Gram-positive bacteria, i.e., Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Gluconobacter oxydans, Pseudomonas putida, and Zymononas mobilis, and analyzed metabolites from different catabolic and anabolic intracellular pathways. Quenching of cells with cold methanol prior to cell separation and extraction led to drastic loss (>60%) of all metabolites tested due to unspecific leakage. Using fast filtration, Gram-negative bacteria also revealed a significant loss (>80%) when inappropriate washing solutions with low ionic strength were applied. Adapting the ionic strength of the washing solution to that of the cultivation medium could almost completely avoid this problem. Gram-positive strains did not show significant leakage independent of the washing solution. Fast filtration with sampling times of several seconds prior to extraction appears to be a suitable approach for metabolites with relatively high intracellular level and low turnover such as amino acids or TCA cycle intermediates. Comparison of metabolite levels in the culture supernatant and the cell interior revealed that the common assumption of whole broth quenching protocols attributing the metabolites found exclusively to the intracellular pools may not be valid in many cases. In such cases a differential approach correcting for medium-contained metabolites is required.     

Common pattern of gene expression in response to hypoxia or cadmium in the gills of the European glass eel (Anguilla anguilla)

European eel (Anguilla anguilla) populations are in decline. Glass eel recruitment has fallen 10-fold since the early 1980s. Estuaries play a fundamental role in the life history of eels because glass eels must pass through them to reach freshwater ecosystems. Unfortunately, because of their geographical position at the upstream basin slopes, estuaries accumulate metals like cadmium and are important sites of hypoxia events. In this context, we studied the effect of the oxygen level on the ventilation of the glass eel. In parallel, glass eels were submitted to different dissolved cadmium concentrations (0, 2, and 10 microg L(-1)) under two oxygen levels (normoxia PO2 = 21 kPa and Hypoxia PO2 = 6 kPa). The expression level of various genes involved in the mitochondrial respiratory chain, in the cellular response to metal and oxidative stresses, was investigated. Our results showed that hypoxia enhances (1) ventilation of the postlarval stage and (2) Cd accumulation in gills only at the lowest metal water concentration tested (2 microg Cd L(-1)). At the gene level, Cd exposure mimics the effect of hypoxia since we observed a decrease in expression of genes involved in the respiratory chain and in the defense against oxidative stress.     

Controlling test case explosion in test generation from B formal models

BZ‐TESTING‐TOOLS (BZ‐TT) is a tool set for automated test case generation from B and Z specifications. BZ‐TT uses boundary and cause–effect testing on the basis of the formal model. It has been used and validated on several industrial applications in the domain of critical software, particularly smart card and transport systems. This paper presents the test coverage criteria supported by BZ‐TT. On the one hand, these correspond to various classical structural coverage criteria, but specialized to the case of B abstract machines. The paper gives algorithms for these in Prolog. On the other hand, BZ‐TT introduces new coverage criteria for complex data structures, based on boundary analysis: this paper defines weak and strong state‐boundary coverage, input‐boundary coverage and output‐boundary coverage. Finally, the paper describes how BZ‐TT presents a unified view of these criteria to the validation engineer, and allows him or her to control the test case explosion on a coarse basis (choosing from a range of coverage criteria) as well as a fine basis (selecting options for each state or input variable). Copyright © 2004 John Wiley & Sons, Ltd.     

Cross-influence of membrane polyunsaturated fatty acids and hypoxia-reoxygenation on α- and β-adrenergic function of rat cardiomyocytes

The purpose of the present investigation was to determine whether the beneficial effects of polyunsaturated fatty acids (PUFA) may influence ischemia-reperfusion-induced alterations of myocardial α- and β-adrenoceptor (α-AR, β-AR) responsiveness. This study was carried out using monolayer cultures of neonatal rat ventricular myocytes in a substrate-free, hypoxia-reoxygenation model of ischemia. The cardiomyocytes (CM) were incubated during 4 days in media enriched either with n−6 PUFA (arachidonic acid, AA) or with n−3 PUFA (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA). The n−6/n−3 ratio in n−3 CM was close to 1.2, compared to 20.1 in n−6 CM. The contractile parameters of n−6 CM and n−3 CM were similar in basal conditions as well as during hypoxia and reoxygenation. In basal conditions, the phospholipid (PL) enrichment with long chain n−3 PUFA resulted in an increased chronotropic response to isoproterenol (ISO) and to phenylephrine (PHE). After posthypoxic reoxygenation, the chronotropic response to β-AR activation in n−6 CM was significantly enhanced as compared with the control response in normoxia. In opposition, the ISO-induced rise in frequency in n−3 CM in control normoxia and after reoxygenation was similar. In these n−3 CM, the changes in contractile parameters, which accompanied the chronotropic response, were also similar in reoxygenation and in normoxic periods, although the rise in shortening velocity was slightly increased after reoxygenation. In response to PHE addition, only the chronotropic effect of n−6 CM appeared significantly enhanced after hypoxic treatment. These results suggested that increasing n−3 PUFA in PL reduced the increase in α- and β-AR functional responses observed after hypoxia-reoxygenation. This effect may partly account for the assumed cardiac protective effect of n−3 PUFA, through the attenuation of the functional response to catecholamines in the ischemic myocardium.     

Optical characterization of an unknown single layer: Institut Fresnel contribution to the Optical Interference Coatings 2004 Topical Meeting Measurement Problem

We present the characterizations performed at the Institut Fresnel for the Measurement Problem of the Optical Interference Coatings 2004 Topical Meeting. A single layer coated on a fused-silica substrate of unknown composition and parameters is analyzed in terms of optogeometrical parameters, uniformity, and scattering. We determine the refractive index and the average thickness of the coating, then provide the localized determination of the thickness with a 2 mm spatial resolution. Topography measurements include atomic force microscopy and angle-resolved scattering measurements. These results are completed thanks to a Taylor Hobson noncontact 3D surface profiler.     

Study of viscoelastic effect on the frequency shift of microcantilever chemical sensors

Microcantilevers coated with a chemically sensitive layer are increasingly being used in chemical detection systems. The sensitive coating, often a polymer, absorbs specific molecules, which can be detected by monitoring the shift in the mechanical resonant frequency. Usually, the frequency shift resulting from molecular absorption is interpreted as a mass loading effect. However, mass loading is not the only effect that has an impact on the frequency shift; the viscoelastic properties of the sensitive coating also are affected by the sorption process. Sorption-induced modulus changes are typically difficult to characterize. However, it is known that the sorption of analyte molecules in a polymer coating results in the plasticization of the coating. In most cases, the polymer becomes more rubbery with increasing concentration of analyte molecules, i.e., the coating becomes softer with increasing loss modulus, and the storage modulus decreases. Using a new analytical model developed for the resonant frequency expression of a hybrid microcantilever (elastic base and viscoelastic layer), the effects of the modification of the storage and loss moduli of the sensitive layer on the resonant frequency are examined. The main conclusion of this analytical study is that, even if the sensitive coating moduli are small compared to the base cantilever's Young's modulus, the effect of the change in the viscoelastic coating properties could contribute significantly to the overall frequency shift (8-23% in the simulations depending on the coating thickness, with even higher contributions for other sets of problem parameters).     

Combining Fourier transform-ion cyclotron resonance/mass spectrometry analysis and Kendrick plots for silicon speciation and molecular characterization in petroleum products at trace levels

A new method combining FT-ICR/MS analysis and Kendrick plots for the characterization of silicon species at trace levels in light petroleum products is presented. The method provides efficient instrumental detection limits ranging from 80 ng/kg to 5 μg/kg and reliable mass accuracy lower than 0.50 ppm for model silicon molecules in spiked gasoline. More than 3000 peaks could be detected in the m/z 50-500 range depending on the nature of the gasoline sample analyzed. An in-house software program was used to calculate Kendrick plots. Then, an algorithm searched, selected, and represented silicon species classes (O(2)Si, O(3)Si, and O(4)Si classes) in Kendrick plots by incorporating model molecules' information (i.e., exact mass and intensity). This procedure allowed the complete characterization of more than 50 new silicon species with different degrees of unsaturation in petroleum products.     

Feedforward-feedback control of a solid oxide fuel cell power system

One of the main challenges for wide-spread utilization of the solid oxide fuel cell (SOFC) power systems is how to achieve high electrical efficiency without increasing the degradation rate of the fuel cells. To run the SOFC power system at high efficiency over a long period of time, properly designed controllers are indispensable.Although a number of various approaches to control SOFC have been proposed so far, it seems that the design of control system, along with simple tuning procedure, has not been treated in a consistent manner. This issue is addressed in the present paper resulting in a feedforward-feedback control structure. The feedforward part is based on the stoichiometry of electro-oxidation, reforming and combustion reactions, which allow immediate response to variable current demand. The feedback part performs additional fine adjustment of fuel and air supply in order to minimize the undesired system temperatures variations. The selection of pairings of manipulated and controlled variables for control is based on physical knowledge of the system. Input/output pairing for single-loop feedback control is assessed by the relative gain analysis. An efficient procedure for tuning the parameters of the feedback controllers is suggested, relying on simple open-loop step responses of the system.The proposed low-level control is assessed on a detailed physical model of a 2.5 kW SOFC power system by simulating two nonstationary load regimes. Simulations show that the control provides a robust operation under large load variations while meeting the operating constraints. Due to its simplicity, the control is feasible for implementation on a real SOFC system.     

Selective Oxidation of Chromium by O2 Impurities in CO2 During Initial Stages of Oxidation

This study shows that the corrosion behaviour of 12 wt% Cr steel in CO₂ at 550 °C is determined in the first stage of oxidation by reaction with O₂ impurities. Depending on the amount of theses impurities and the thermal ramp rate, selective oxidation of chromium could lead to the formation of a protective chromium-rich oxide. An oxidation model describing qualitatively the nature of the oxide layer formed in the initial period of oxidation is presented. From these observations, surface engineering processes for protecting 9–12 wt% chromium steels from fast corrosion rate have emerged.