Pathogenic old world hantaviruses infect renal glomerular and tubular cells and induce disassembling of cell-to-cell contacts.

Viral hemorrhagic fevers are characterized by enhanced permeability. One of the most affected target organs of hantavirus-induced hemorrhagic fever with renal syndrome is the kidney, and an infection often results in acute renal failure. To study the underlying cellular effects leading to kidney dysfunction, we infected human renal cell types in vitro that are critical for the barrier functions of the kidney, and we examined kidney biopsy specimens obtained from hantavirus-infected patients. We analyzed the infection and pathogenic effects in tubular epithelial and glomerular endothelial renal cells and in podocytes. Both epithelial and endothelial cells and podocytes were susceptible to hantavirus infection in vitro. The infection disturbed the structure and integrity of cell-to-cell contacts, as demonstrated by redistribution and reduction of the tight junction protein ZO-1 and the decrease in the transepithelial resistance in infected epithelial monolayers. An analysis of renal biopsy specimens from hantavirus-infected patients revealed that the expression and the localization of the tight junction protein ZO-1 were altered compared to renal biopsy specimens from noninfected individuals. Both tubular and glomerular cells were affected by the infection. Furthermore, the decrease in glomerular ZO-1 correlates with disease severity induced by glomerular dysfunction. The finding that different renal cell types are susceptible to hantaviral infection and the fact that infection results in the breakdown of cell-to-cell contacts provide useful insights in hantaviral pathogenesis.

Analysis of amino acids without derivatization in barley extracts by LC-MS-MS.

A method has been developed for quantification of 20 amino acids as well as 13 (15)N-labeled amino acids in barley plants. The amino acids were extracted from plant tissues using aqueous HCl-ethanol and directly analyzed without further purification. Analysis of the underivatized amino acids was performed by liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS-MS) in the positive ESI mode. Separation was achieved on a strong cation exchange column (Luna 5micro SCX 100A) with 30 mM ammonium acetate in water (solvent A) and 5% acetic acid in water (solvent B). Quantification was accomplished using d (2)-Phe as an internal standard. Calibration curves were linear over the range 0.5-50 microM, and limits of detection were estimated to be 0.1-3.0 microM. The mass-spectrometric technique was employed to study the regulation of amino acid levels in barley plants grown at 15 degrees C uniform root temperature (RT) and 20-10 degrees C vertical RT gradient (RTG). The LC-MS-MS results demonstrated enhanced concentration of free amino acids in shoots at 20-10 degrees C RTG, while total free amino acid concentration in roots was similarly low for both RT treatments. (15)NO(3) (-) labeling experiments showed lower (15)N/(14)N ratios for Glu, Ser, Ala and Val in plants grown at 20-10 degrees C RTG compared with those grown at 15 degrees C RT.

Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

Direct analysis of underivatized amino acids in plant extracts by LC-MS-MS.

In this chapter, we describe a method for quantification of 20 proteinogenic amino acids as well as 13 (15)N-labeled amino acids by liquid chromatography-mass spectrometry without the need for derivatization and use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5 µm SCX 100 Å) with 30 mM ammonium acetate in water (A) and 5% acetic acid in water (B). Quantification is accomplished by use of d(5)-phenylalanine as internal standard achieving limits of detection of 0.1-3.0 µM. The method was successfully applied for the determination of proteinogenic and (15)N-labeled amino acids in plant extracts.

Metabolomics: current state and evolving methodologies and tools.

In recent years, metabolomics developed to an accepted and valuable tool in life sciences. Substantial improvements of analytical hardware allow metabolomics to run routinely now. Data are successfully used to investigate genotype-phenotype relations of strains and mutants. Metabolomics facilitates metabolic engineering to optimise mircoorganisms for white biotechnology and spreads to the investigation of biotransformations and cell culture. Metabolomics serves not only as a source of qualitative but also quantitative data of intra-cellular metabolites essential for the model-based description of the metabolic network operating under in vivo conditions. To collect reliable metabolome data sets, culture and sampling conditions, as well as the cells' metabolic state, are crucial. Hence, application of biochemical engineering principles and method standardisation efforts become important. Together with the other more established omics technologies, metabolomics will strengthen its claim to contribute to the detailed understanding of the in vivo function of gene products, biochemical and regulatory networks and, even more ambitious, the mathematical description and simulation of the whole cell in the systems biology approach. This knowledge will allow the construction of designer organisms for process application using biotransformation and fermentative approaches making effective use of single enzymes, whole microbial and even higher cells.