DNA databases of an important tropical timber tree species Shorea leprosula (Dipterocarpaceae) for forensic timber identification.

International timber trade communities are increasingly demanding that timber in the wood supply chain be sourced from sustainably harvested forests and certified plantations. This is to combat illegal logging activities to prevent further depletion of our precious forests worldwide. Hence, timber tracking tools are important to support law enforcement officials in ensuring only sustainably harvested timbers are traded in the market. In this study, we developed chloroplast DNA (cpDNA) and simple sequence repeat (SSR) databases as tracking tools for an important tropical timber tree species, Shorea leprosula from Peninsular Malaysia. A total of 1410 individual trees were sampled from 44 natural populations throughout Peninsular Malaysia. Four cpDNA regions were used to generate a cpDNA haplotype database, resulting in a haplotype map comprising 22 unique haplotypes derived from 28 informative intraspecific variable sites. This cpDNA database can be used to trace the origin of an unknown log at the regional level. Ten SSR loci were used to develop the SSR allele frequency database. Bayesian cluster analysis divided the 44 populations into two genetic clusters corresponding to Region A and Region B. Based on conservativeness evaluation of the SSR databases for individual identification, the coancestry coefficients (θ) were adjusted to 0.1900 and 0.1500 for Region A and B, respectively. These databases are useful tools to complement existing timber tracking systems in ensuring only legally sourced timbers are allowed to enter the wood supply chain.

Mycobacterial species as case-study of comparative genome analysis.

The genus Mycobacterium represents more than 120 species including important pathogens of human and cause major public health problems and illnesses. Further, with more than 100 genome sequences from this genus, comparative genome analysis can provide new insights for better understanding the evolutionary events of these species and improving drugs, vaccines, and diagnostics tools for controlling Mycobacterial diseases. In this present study we aim to outline a comparative genome analysis of fourteen Mycobacterial genomes: M. avium subsp. paratuberculosis K­10, M. bovis AF2122/97, M. bovis BCG str. Pasteur 1173P2, M. leprae Br4923, M. marinum M, M. sp. KMS, M. sp. MCS, M. tuberculosis CDC1551, M. tuberculosis F11, M. tuberculosis H37Ra, M. tuberculosis H37Rv, M. tuberculosis KZN 1435 , M. ulcerans Agy99,and M. vanbaalenii PYR­1, For this purpose a comparison has been done based on their length of genomes, GC content, number of genes in different data bases (Genbank, Refseq, and Prodigal). The BLAST matrix of these genomes has been figured to give a lot of information about the similarity between species in a simple scheme. As a result of multiple genome analysis, the pan and core genome have been defined for twelve Mycobacterial species. We have also introduced the genome atlas of the reference strain M. tuberculosis H37Rv which can give a good overview of this genome. And for examining the phylogenetic relationships among these bacteria, a phylogenic tree has been constructed from 16S rRNA gene for tuberculosis and non tuberculosis Mycobacteria to understand the evolutionary events of these species.

Discovery of six families of fungal defensin-like peptides provides insights into origin and evolution of the CSalphabeta defensins.

The defensins with a conserved cysteine-stabilized alpha-helix and beta-sheet (CSalphabeta) structural motif are a group of unique antimicrobial polypeptides widely distributed in plants and animals. Recently, one defensin-like peptide (DLP) with high degree of sequence and structural similarity to defensins from ancient arthropods and molluscs has been identified in a saprophytic fungus [Mygind, P.H., Fischer, R.L., Schnorr, K.M., Hansen, M.T., Sönksen, C.P., Ludvigsen, S., Raventós, D., Buskov, S., Christensen, B., De Maria, L., Taboureau, O., Yaver, D., Elvig-Jørgensen, S.G., Sørensen, M.V., Christensen, B.E., Kjaerulff, S.K., Frimodt-Moller, N., Lehrer, R.I., Zasloff, M., Kristensen, H.-H., 2005. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature 437, 975-980], which poses an important question regarding the evolutionary relationships of this class of effectors of innate immunity in three eukaryotic kingdoms. Here, we report the computational identification of six families of fungal DLPs in which three known defensin types (antibacterial ancient invertebrate-type defensins (AITDs), antibacterial classical insect-type defensins (CITDs), and antifungal plant/insect-type defensins (PITDs)) can be clearly assigned. Sharing of these defensin types between animals and fungi supports their closer evolutionary relationship, consistent with the Opisthokonta Hypothesis. Conservation of the PITDs across three eukaryotic kingdoms suggests their earlier origin than the antibacterial defensins, probably preceded plants and Opisthokonta split. Finally, recognition of an early gene duplication event in the Aspergillus terreus genome allows us to establish a paralogous relationship between AITDs and CITDs, which highlights extensive lineage-specific defensin gene loss during evolution.

Extração de DNA de biópsias de pele fixadas em formalina tamponada e embebidas em parafina e amplificação por PCR / DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification

Biópsias de pele oriundas dos serviços de diagnóstico da hanseníase podem ser grande fonte de material para estudos retrospectivos em genética humana e do Mycobacterium leprae. No entanto, os procedimentos de fixação e inclusão em parafina podem dificultar a obtenção de DNA de qualidade para amplificação por PCR. Assim, estas amostras requerem protocolos especiais para a extração do material genético. O objetivo deste trabalho é apresentar um método alternativo para extração de DNA com base na combinação de calor e digestão enzimática. Para tanto, os cortes foram aquecidos a 120º C em solução tampão de pH 9,0, submetidos à digestão enzimática com proteinase K e o DNA foi extraído por meio de solução de fenol:clorofórmio:álcool isoamílico. A amplificação por PCR para as regiões dos genes humanos TNF e LTA foi bem sucedida para 85,4% dos espécimes. Considerando o DNA do M. leprae, obtivemos amplificação em 67,6%, 48,5%, 36,7% e 64,8% para os marcadores TA18, GTA9, TTC e RLEP, respectivamente. Concluímos que este é um método de baixo custo que proporcionou um rendimento satisfatório de DNA de boa qualidade para emprego em PCR a partir de biópsias parafinadas de pele.

Skin biopsies from leprosy diagnostic services can be great sources of material for retrospective studies concerning human and Mycobacterium leprae genetic. However, fixation and paraffin embedding procedures make difficult to obtain good quality DNA to PCR amplification. Thus, paraffin-embedded samples require special protocols to DNA extraction. The aim of this paper is to present an alternative method for DNA extraction based on the combination of heat and enzymatic digestion. The sections were heated at 120ºC at pH 9.0, submitted to enzymatic digestion with proteinase K and the DNA was extracted by using phenol:chlorophorm:isoamilic alcohol. PCR amplification for regions in TNF and LTA human genes was successful for 85.4 % of specimens. Regarding M. lepraeDNA, we obtained amplification in 67.6%, 48.5%, 36.7% and 64.8% for the TA18, GTA9, TTC and RLEP markers, respectively. We conclude that this is an inexpensive method which provided a satisfactory yield of a good quality DNA for PCR from paraffin- embedded skin biopsies.

[Mycobacterium shinshuense and Mycobacterium leprae infections: usefulness of genetical examinations].

Although Mycobacterium shinshuense and M. leprae infections are relatively rare in the fields of dermatology, an early diagnosis is one of the important prognostic factors of these infections. Applications of the genetical examinations such as PCR and 16S rDNA sequencing are helpful in early diagnosis with culture nagative cases. Short target PCR tests are available to detect DNA of M. shinshuense or M. leprae from clinical specimens including formalin fixed-paraffin embedded samples. A partial 16s rDNA sequencing is functional with enough intact bacterial DNA. A similarity search based on the partial 16S rDNA sequences using RIDOM database is an easy and powerful tool to estimate the species of mycobacteria, however, it is not enough for the identification in some cases. For instance, a clinical isolate of M. shinshuense is clearly discriminated from M. leprae (92.75% sequence identity), however, difficult to be identified from M. marinum and M. ulcerans (99.77% sequence identity). The phylogenetic tree based on 16S rDNA sequences is illustrating that both M. leprae (closely related to M. haemophilum) and M. shinshuense (closely related to M. marinum and M. ulcerans, and also M. tuberculosis) are relatively related species and distantly related to rapidly growing species among 30 species of pathogenic mycobacteria which have been isolated in Japan.