dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model.

cDNA-AFLP analysis for transcript profiling has been successfully applied to study many plant biological systems, particularly plant-microbe interactions. However, the separation of cDNA-AFLP fragments by gel electrophoresis is reported to be labor-intensive with only limited potential for automation, and the collection of differential bands for gene identification is even more cumbersome. In this work, we present the use of dHPLC (denaturing high performance liquid chromatography) and automated DNA fragment collection using the WAVE(®) System to analyze and recover cDNA-AFLP fragments. The method is successfully applied to the Malus-Venturia inaequalis interaction, making it possible to collect 66 different transcript-derived fragments for apple genes putatively involved in the defense response activated by the HcrVf2 resistance gene. The results, validated by real time quantitative RT-PCR, were consistent with the plant-pathogen interaction under investigation and this further supports the suitability of dHPLC for cDNA-AFLP transcript profiling. Features and advantages of this new approach are discussed, evincing that it is an almost fully automatable and cost-effective solution for processing large numbers of samples and collecting differential genes involved in other biological processes and non-model plants.

Rapid peat accumulation favours the occurrence of both fen and bog microbial communities within a Mediterranean, free-floating peat island.

The unique environment of a 4m-thick, free-floating peat island within the Posta Fibreno lake (Central Italy) was analyzed using DNA-based techniques to assess bacterial and fungal community members identity and abundance. Two depths were sampled at 41 and 279 cm from the surface, the former corresponding to an emerged portion of Sphagnum residues accumulated less than 30 yrs ago, and the latter mainly consisting of silty peat belonging to the deeply submerged part of the island, dating back to 1520-1660 AD. The corresponding communities were very diverse, each of them dominated by a different member of the Delta-proteobacteria class for prokaryotes. Among Eukaryotes, Ascomycota prevailed in the shallow layer while Basidiomycota were abundant in the deep sample. The identity of taxa partitioning between acidic surface layer and neutral core is very reminiscent of the differences reported between bogs and fens respectively, supporting the view of Posta Fibreno as a relic transitional floating mire. Moreover, some microbial taxa show an unusual concurrent species convergence between this sub-Mediterranean site and far Nordic or circumpolar environments. This study represents the first report describing the biotic assemblages of such a peculiar environment, and provides some insights into the possible mechanisms of its evolution.

Methylation profiling by bisulfite sequencing analysis of the mtDNA Non-Coding Region in replicative and senescent Endothelial Cells.

The regulation and function of Mitochondrial DNA (mtDNA) cytosine methylation (5 mC) are largely unexplored. Mitochondria, Endothelial Cell (EC) senescence, and cardiovascular dysfunction are closely related. We extensively investigated the mtDNA Non-Coding Region (NCR) methylation pattern and its variations in EC replicative senescence. We observed previously undescribed 5 mC clusters and a biased distribution of 5 mC among DNA sites and throughout the NCR. The methylation pattern in senescent EC showed non-random variations, including the hypo-methylation of mtDNA replication regulatory sites. Additional experiments opened to a possible role for 5 mC in D-loop formation, rather than in mitochondrial gene expression.

Hemolytic crisis in a G6PD-deficient infant after ingestion of pumpkin.

A 8 month-old infant presented with acute onset of severe jaundice, anemia requiring transfusion and Glucose-6-Phosphate Dehydrogenase deficiency. The infant did not take drugs, he did not consume fava beans, but fava beans DNA was found on pumpkin he consumed the day before jaundice onset. This is the first case of hemolysis triggered by ingestion of food cross-contaminated with fava beans.

Microbiological features and bioactivity of a fermented manure product (preparation 500) used in biodynamic agriculture.

The fermented manure derivative known as Preparation 500 is traditionally used as a field spray in biodynamic agriculture for maintaining and increasing soil fertility. This work aimed at characterizing the product from a microbiological standpoint and at assaying its bioactive properties. The approach involved molecular taxonomical characterization of the culturable microbial community; ARISA fingerprints of the total bacteria and fungal communities; chemical elemental macronutrient analysis via a combustion analyzer; activity assays for six key enzymes; bioassays for bacterial quorum sensing and chitolipooligosaccharide production; and plant hormonelike activity. The material was found to harbor a bacterial community of 2.38 × 10(8) CFU/g dw dominated by Grampositives with minor instances of Actinobacteria and Gammaproteobacteria. ARISA showed a coherence of bacterial assemblages in different preparation lots of the same year in spite of geographic origin. Enzymatic activities showed elevated values of beta-glucosidase, alkaline phosphatase, chitinase, and esterase. The preparation had no quorum sensing-detectable signal, and no rhizobial nod gene-inducing properties, but displayed a strong auxin-like effect on plants. Enzymatic analyses indicated a bioactive potential in the fertility and nutrient cycling contexts. The IAA activity and microbial degradation products qualify for a possible activity as soil biostimulants. Quantitative details and possible modes of action are discussed.