Results of a collaborative study on DNA identification of aged bone samples.

AIM: A collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. METHODS: Eighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. RESULTS: Although the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. CONCLUSION: The laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized.

Dual roles of reactive oxygen species and NADPH oxidase RBOHD in an Arabidopsis-Alternaria pathosystem.

Arabidopsis (Arabidopsis thaliana) NADPH oxidases have been reported to suppress the spread of pathogen- and salicylic acid-induced cell death. Here, we present dual roles of RBOHD (for respiratory burst oxidase homolog D) in an Arabidopsis-Alternaria pathosystem, suggesting either initiation or prevention of cell death dependent on the distance from pathogen attack. Our data demonstrate that a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola. However, the cellular patterns of reactive oxygen species accumulation and cell death are fundamentally different in the AtrbohD mutant compared with the wild type. Functional RBOHD causes marked extracellular hydrogen peroxide accumulation as well as cell death in distinct, single cells of A. brassicicola-infected wild-type plants. This single cell response is missing in the AtrbohD mutant, where infection triggers spreading-type necrosis preceded by less distinct chloroplastic hydrogen peroxide accumulation in large clusters of cells. While the salicylic acid analog benzothiadiazole induces the action of RBOHD and the development of cell death in infected tissues, the ethylene inhibitor aminoethoxyvinylglycine inhibits cell death, indicating that both salicylic acid and ethylene positively regulate RBOHD and cell death. Moreover, A. brassicicola-infected AtrbohD plants hyperaccumulate ethylene and free salicylic acid compared with the wild type, suggesting negative feedback regulation of salicylic acid and ethylene by RBOHD. We propose that functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels.

Evolution of the indole alkaloid biosynthesis in the genus Hordeum: distribution of gramine and DIBOA and isolation of the benzoxazinoid biosynthesis genes from Hordeum lechleri.

Two indole alkaloids with defense related functions are synthesized in the genus Hordeum of the Triticeae. Gramine (3(dimethyl-amino-methyl)-indole) is found in H. spontaneum and in some varieties of H. vulgare, the benzoxazinoid 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) is detected in H. roshevitzii, H. brachyantherum, H. flexuosum, H. lechleri. Biosynthesis of DIBOA and of gramine was found to be mutually exclusive in wild Hordeum species, indicating that there was selection against simultaneous expression of both pathways during evolution. The full set of genes required for DIBOA biosynthesis in H.lechleri was isolated and the respective enzyme functions were analyzed by heterologous expression. The cytochrome P450 genes Bx2-Bx5 demonstrate a monophyletic origin for H. lechleri, Triticum aestivum and Zea mays. HlBx2-HlBx5 share highest homology to the orthologous genes of T. aestivum. In contrast, the branch point enzyme of the DIBOA pathway, the indole-3-glycerol phosphate lyase BX1, might have evolved independently in H. lechleri. In all Hordeum species that synthesize DIBOA, DNA sequences homologous to Bx genes are found. In contrast, these sequences are not detectable in the genomes of H. vulgare and H. spontaneum that do not synthesize benzoxazinoids.