Structure-activity relationships in the metabolism of a series of tertiary amines by cytochrome P450

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Species identification using high resolution melting (HRM) analysis with random forest classification

Species identification is an important facet of forensic investigation. In this study, human and non-human species (cow, chicken, pig, sheep, cat, dog, rabbit, fox, kangaroo and wombat) were assayed on the ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) to rapidly screen for their species of origin using the high resolution melt (HRM) analysis targeting the 16S rRNA gene. Classification of HRM difference profiles using the onboard ViiA 7 software resulted in a classification accuracy of�<20%. Derivative profiles (temperature versus negative first derivative of fluorescence, �dF/dT) were classified using random forest algorithms supplemented by bagging and boosting, with either a randomly partitioned test set or a variety of folds of cross-classification, in addition to a range of trees and variables. Random forest classification with bagging conditions (constructed over 500 trees) was found to considerably outperform the ViiA 7 software for species differentiation with 100% classification accuracy for biological material from humans, domestic pets (cat and dog) and consumable meats (chicken and sheep) with an average classification accuracy of 70% across all species. � 2017 Australian Academy of Forensic Science

Cloning of a chitinase gene from Ewingella americana, a pathogen of the cultivated mushroom, Agaricus bisporus

We have isolated a gene encoding a chitinase (EC 3.2.1.14) from Ewingella americana, a recently described pathogen of the mushroom Agaricus bisporus. This gene, designated chiA (EMBL/Genbank/DDBJ accession number X90562), was cloned by expression screening of a plasmid-based E. americana HindIII genomic library in Escherichia coli using remazol brilliant violet-stained carboxymethylated chitin incorporated into selective medium. The chiA gene has a 918-bp ORF, terminated by a TAA codon, with a calculated polypeptide size of 33.2 kDa, likely corresponding to a previously purified and characterised 33-kDa endochitinase from E. americana. The deduced amino acid sequence shares 33% identity with chitinase II from Aeromonas sp. No. 10S-24 and 7.8% identity with a chitinase from Saccharopolyspora erythraeus. Homology to other chitinase sequences was otherwise low. The peptide sequence deduced from chiA lacks a typical N-terminal signal sequence and also lacks the chitin binding and type III fibronectin homology units common to many bacterial chitinases. The possibility that this chitinase is not primarily adapted for the environmental mineralisation of pre-formed chitin, but rather for the breakdown of nascent chitin, is discussed in the context of mushroom disease.<br>O gene que codifica uma quitinase (EC 3.2.1.14) foi isolado de Ewingella americana, recentemente descrita como patógeno do cogumelo Agaricus bisporus. Este gene, denominado chiA (EMBL/Genebank/DDBJ número de acesso X9061), foi clonado e selecionado a partir de livraria genômica construída por digestão do DNA de E. americana com HindIII e ligação em plasmídio de expressão em E. coli, utilizando meio seletivo contendo quitina carboximetilada, corada com "remazol brilliant violet'' para seleção de clones. O gene chiA apresenta uma ORF de 918 bp, código terminador TAA, tendo o tamanho do polipeptídeo sido calculado como 33,2 kDa, o qual corresponde ao tamanho de 33 kDa da endoquitinase previamente purificada de E. americana. A seqüência deduzida de aminoácidos apresenta 33% de identidade com a quitinase II de Aeromonas sp. No. 10S-24 e 7,8% de identidade com quitinase de Saccharopolyspora erythraeus. Baixa homologia com outras quitinases foi observada. A seqüência deduzida de aminoácidos de chiA não apresenta sinal típico de N-terminal e também não apresenta típico sítio de ligação com quitina nem unidades de homologia à fibronectina do tipo III, comuns a muitas quitinases bacterianas. Existe a possibilidade de que esta quitinase não seja primariamente adaptada para mineralização de quitina pré-formada no ambiente, sendo discutida a digestão e quebra de quitina nascente, no contexto de doenças de cogumelos