Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

BackgroundA multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program.AimsThe objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA.MethodsSeven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets.ResultsMost of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10² fg/μl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR₂₂₀) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol.ConclusionsAll laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.     

Metallothionein responses in the Asiatic clam (Corbicula fluminea) after exposure to trivalent arsenic

The main objective of this work was to evaluate arsenic effects on metallothionein (MT) induction by exposing a freshwater Asiatic clam (Corbicula fluminea) to different concentrations of this metalloid. The presence of MT-like proteins was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and compared with a standard rabbit MT. In addition, the polarographic response showed good correspondence between standard MT and MT-like curves from C. fluminea, allowing MT quantification. The results show that clams exposed to different concentrations of arsenic are able to induce significant levels of MTs. Although variability was found in MT induction, significant differences in MT levels were found after 28 days of exposure in all treatments in comparison with the controls, suggesting that exposure to arsenic induced MT-like proteins in C. fluminea.     

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.     

Differentially expressed proteins during an incompatible interaction between common bean and the fungus Pseudocercospora griseola

The common bean (Phaseolus vulgaris L.) is the main source of protein and an important source of minerals in several countries around the world. Angular leaf spot, caused by the fungus Pseudocercospora griseola, is one of the major diseases of the common bean. In this work, we used two-dimensional gel electrophoresis and mass spectrometry to analyze alterations in the proteome of common bean leaves challenged with an incompatible race of P. griseola. Twenty-three differentially expressed proteins were detected in leaves of cultivar AND 277 collected at 12, 24 and 48 h after inoculation. The proteins were digested with trypsin and submitted to MALDI-TOF/TOF and MicrOTOF-Q electrospray mass spectrometry. Nineteen of them were identified upon MS/MS fragmentation. Most of these proteins are involved with amino acid metabolism, terpenoid metabolism, phenylpropanoid biosynthesis, antioxidant systems, vitamin and cofactor metabolism, plant–pathogen interaction, carbohydrate metabolism, photosynthesis, or genetic information processing, showing that the interaction in this pathosystem affects different genes from various metabolic pathways and processes.     

Building bridges: an integrated strategy for sustainable food production throughout the value chain

The food production and processing value chain is under pressure from all sides—increasing demand driven by a growing and more affluent population; dwindling resources caused by urbanization, land erosion, pollution and competing agriculture such as biofuels; and increasing constraints on production methods driven by consumers and regulators demanding higher quality, reduced chemical use, and most of all environmentally beneficial practices ‘from farm to fork’. This pressure can only be addressed by developing efficient and sustainable agricultural practices that are harmonized throughout the value chain, so that renewable resources can be exploited without damaging the environment. Bridges must, therefore, be built between the diverse areas within the food production and processing value chain, including bridges between different stages of production, between currently unlinked agronomic practices, and between the different levels and areas of research to achieve joined-up thinking within the industry, so that the wider impact of different technologies, practices and materials on productivity and sustainability is understood at the local, regional, national and global scales. In this article, we consider the challenges at different stages and levels of the value chain and how new technologies and strategies could be used to build bridges and achieve more sustainable food/feed production in the future.     

Stability-Based Comparison of Class Discovery Methods for DNA Copy Number Profiles

Motivation

Array-CGH can be used to determine DNA copy number, imbalances in which are a fundamental factor in the genesis and progression of tumors. The discovery of classes with similar patterns of array-CGH profiles therefore adds to our understanding of cancer and the treatment of patients. Various input data representations for array-CGH, dissimilarity measures between tumor samples and clustering algorithms may be used for this purpose. The choice between procedures is often difficult. An evaluation procedure is therefore required to select the best class discovery method (combination of one input data representation, one dissimilarity measure and one clustering algorithm) for array-CGH. Robustness of the resulting classes is a common requirement, but no stability-based comparison of class discovery methods for array-CGH profiles has ever been reported.

Results

We applied several class discovery methods and evaluated the stability of their solutions, with a modified version of Bertoni''s -based test [1]. Our version relaxes the assumption of independency required by original Bertoni''s -based test. We conclude that Minimal Regions of alteration (a concept introduced by [2]) for input data representation, sim [3] or agree [4] for dissimilarity measure and the use of average group distance in the clustering algorithm produce the most robust classes of array-CGH profiles.

Availability

The software is available from http://bioinfo.curie.fr/projects/cgh-clustering. It has also been partly integrated into "Visualization and analysis of array-CGH"(VAMP)[5]. The data sets used are publicly available from ACTuDB [6].     

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase,Is Involved in Cell Wall Architecture and Virulence

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.     

Maternal Malnutrition and Offspring Sex Determine Juvenile Obesity and Metabolic Disorders in a Swine Model of Leptin Resistance

The present study aimed to determine, in a swine model of leptin resistance, the effects of type and timing of maternal malnutrition on growth patterns, adiposity and metabolic features of the progeny when exposed to an obesogenic diet during their juvenile development and possible concomitant effects of the offspring sex. Thus, four groups were considered. A CONTROL group involved pigs born from sows fed with a diet fulfilling their daily maintenance requirements for pregnancy. The treated groups involved the progeny of females fed with the same diet but fulfilling either 160% or 50% of pregnancy requirements during the entire gestation (OVERFED and UNDERFED, respectively) or 100% of requirements until Day 35 of pregnancy and 50% of such amount from Day 36 onwards (LATE-UNDERFED). OVERFED and UNDERFED offspring were more prone to higher corpulence and fat deposition from early postnatal stages, during breast-feeding; adiposity increased significantly when exposed to obesogenic diets, especially in females. The effects of sex were even more remarkable in LATE-UNDERFED offspring, which had similar corpulence to CONTROL piglets; however, females showed a clear predisposition to obesity. Furthermore, the three groups of pigs with maternal malnutrition showed evidences of metabolic syndrome and, in the case of individuals born from OVERFED sows, even of insulin resistance and the prodrome of type-2 diabetes. These findings support the main role of early nutritional programming in the current rise of obesity and associated diseases in ethnics with leptin resistance.