63 research outputs found
Diversidad patogénica dentro de poblaciones de jopo de girasol (O. cumana)
Resúmes del XII Congreso Nacional de la Sociedad Española de FitopatologíaPeer reviewe
Analysis of in vivo Function of Predicted Isoenzymes-A Metabolomic Approach
Isoenzymes occur in all organisms. Often, they are regulated with respect to environmental perturbations to assure metabolic flexibility. In bioinformatics-driven functional genome annotations, the presence of isoenzymes is frequently predicted. It is desirable to verify the functions of putative isoenzymes experimentally for a correct estimation of the metabolic capacities in a given organism. Using metabolome analysis, we investigated two knockout mutants of putative shikimate dehydrogenases (SDH) in Corynebacterium glutamicum (C. glutamicum). Here, we show that different metabolic responses to defined gene deactivations of the putative SDHs clearly indicate different functions of these enzymes. Our investigation revealed that the gene product of open reading frame Cg1835 is the essential SDH in C. glutamicum, whereas it is more likely that the gene product of the open reading frame Cg1283 belongs to the SDH-like enzyme family (SDH-L). The results of the metabolome analysis are verified with two independent methods. First, by in vitro characterization of kinetic constants after heterologous expression, and second, by measurement of SDH activity in raw cell extracts
Unprecedented sensitivity of the planar yeast estrogen screen by using a spray-on technology
The planar yeast estrogen screen (p-YES) can serve as a highly valuable and sensitive screening tool for the detection of estrogenic compounds in various sample matrices such as water and wastewater, personal care products and foodstuff. The method combines the separation of sample constituents by thin layer chromatography with the direct detection of estrogenic compounds on the surface of the HPTLC-plate. The previous protocol using the immersion of a normal phase silica HPTLC-plate in a cell suspension for bio-autography resulted in blurred signals due to the accelerated diffusion of compounds on the wet surface of the HPTLC-plate. Here, the application of the yeast cells by spraying on the surface of the HPTLC-plate is described as an alternative approach. The presented method for the hyphenation of normal phase thin layer chromatography with a yeast estrogen screen results in much sharper signals compared to reports in previous publications. Satisfying results were achieved using cultures with cell densities of 1000 FAU. Due to the reduced signal broadening, lower limits of quantification for estrogenic compounds were achieved (Estrone (E1) = 2 pg/zone, 17β-estradiol (E2) = 0.5 pg/zone, 17α-ethinylestradiol (EE2) = 0.5 pg/zone and Estriol (E3) = 20 pg/zone). As demonstrated, it is possible to characterize profiles of estrogenic activity of wastewater samples with high quality and reproducibility. The improved sensitivity opens the stage for applications using native samples from waste- or even surface water directly applied on HPTLC-plates without the need for prior sample treatment by e.g. solid phase extraction
Coupling High-Performance Thin-Layer Chromatography with Bacterial Genotoxicity Bioreporters
We present an innovative technological
platform for monitoring
the direct genotoxicity of individual components in complex environmental
samples, based on bioluminescent <i>Escherichia coli</i> genotoxicity bioreporters, sprayed onto the surface of a high-performance
thin-layer chromatography (HPTLC) plate. These sensor strains harbor
plasmid-borne fusions of selected gene promoters of the <i>E.
coli</i> SOS DNA repair system to the <i>Photorhabdus luminescens
luxABCDE</i> gene cassette, and mark by increased luminescence
the presence of potentially DNA-damaging sample components separated
on the plate. We demonstrate an “on plate” quantifiable
dose-dependent response to several model genotoxicants (without metabolic
activation). We further demonstrate the applicability of the system
by identifying as genotoxic specific components of HPTLC-separated
influent and effluent samples of wastewater treatment plants, thereby
alleviating the need for a comprehensive chemical analysis of the
sample
UV aged epoxy coatings ̶ Ecotoxicological effects and released compounds
Organic coatings can guarantee long-term protection of steel structures due to causing a physical barrier against water and oxygen. Because of their mechanical properties and resistances to heat and chemicals, epoxy resin-based coatings are widely used for corrosion protection. Despite of the aromatic backbone and the resulting susceptibility to UV degradation, epoxy resins are frequently used as binding agent in top layers of anti-corrosion coating systems. Consequently, these organic polymers are directly exposed to sunlight and thus UV radiation. The present study was designed to investigate if toxic effects of epoxy resin-based-coatings are changed by UV-A irradiation. For this purpose, two epoxide-based top coatings were examined with and without UV aging for their bacterial toxicity and estrogenicity. In addition, chemical analyses were performed to identify released compounds as well as photolytic degradation products and to assign toxic effects to individual substances. UV-A irradiation of epoxy resin based top coatings resulted in an overall decrease of acute and specific ecotoxicological effects but as well to the formation of toxic transformation products. Both, in leachates of untreated and UV-A irradiated coatings, 4tBP was identified as the main driver of estrogenicity and toxicity to luminescent bacteria. BPA and structural analogs contributing to estrogenic effects in leachates were formed by UV-A irradiation. The combination of HPTLC coupled bioassays and LC-MS analyses supported the identification of bioactive compounds in terms of an effect-directed analysis. The present findings indicate that epoxide-based coatings are less suitable for the application as top coatings and more UV stable coatings like aliphatic polyurethanes should be preferred
Deriving bio-equivalents from in vitro bioassays: Assessment of existing uncertainties and strategies to improve accuracy and reporting
Bio-equivalents (e.g., 17β-estradiol or dioxin equivalents) are commonly employed to quantify the in vitro effects of complex human or environmental samples. However, there is no generally accepted data analysis strategy for estimating and reporting bio-equivalents. Therefore, the aims of the present study are to 1) identify common mathematical models for the derivation of bio-equivalents from the literature, 2) assess the ability of those models to correctly predict bio-equivalents, and 3) propose measures to reduce uncertainty in their calculation and reporting. We compiled a database of 234 publications that report bio-equivalents. From the database, we extracted 3 data analysis strategies commonly used to estimate bio-equivalents. These models are based on linear or nonlinear interpolation, and the comparison of effect concentrations (ECX). To assess their accuracy, we employed simulated data sets in different scenarios. The results indicate that all models lead to a considerable misestimation of bio-equivalents if certain mathematical assumptions (e.g., goodness of fit, parallelism of dose-response curves) are violated. However, nonlinear interpolation is most suitable to predict bio-equivalents from single-point estimates. Regardless of the model, subsequent linear extrapolation of bio-equivalents generates additional inaccuracy if the prerequisite of parallel dose-response curves is not met. When all these factors are taken into consideration, it becomes clear that data analysis introduces considerable uncertainty in the derived bio-equivalents. To improve accuracy and transparency of bio-equivalents, we propose a novel data analysis strategy and a checklist for reporting Minimum Information about Bio-equivalent ESTimates (MIBEST). © 2013 SETAC
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