Determination of Denitrification Genes Abundance in Environmental Samples

Abstract Diversity of microorganisms involved in the biogeochemical N cycle is of fundamental interest in microbial ecology. Denitrification is a key step in the cycle by which nitrate is reduced to dinitrogen gas via the soluble nitrite and the gaseous compounds nitric oxide and nitrous oxide. The process is carried out by the sequential activity of the nitrate, nitrite, nitric oxide, and nitrous oxide reductase enzyme, respectively. The fluorescence-based quantitative real-time polymerase chain reaction (qPCR) is widely used for quantification of nucleic acids in samples obtained from numerous, diverse sources. Here, we provide a well-proven methodology for isolation of DNA from environmental samples and describe relevant experimental conditions for utilization of qPCR to assay the 16S rRNA and nar/nap, nirK/nirS, c-nor/qnor, and nos denitrification genes that encode synthesis of denitrifying enzymes. The ISO 11063 standard method and MIQUE guidelines are considered with the aim to increase experimental transparency

Cellular localisation of VvRops and VvRabA5e, small GTPases developmentally regulated in grape berries

VvRops, in particular VvRop9, and VvRabA5e are small GTPases which are developmentally regulated in grape berries. In an attempt to help elucidate the role of these proteins during fruit development and ripening, we investigated their localisation in the fruit by immunocytofluorescence. These proteins were observed at a perinuclear location, at cell periphery and around vesicles. In particular VvRops were found to be located in the nucleus and likely on the plasma membrane. VvRop9 and VvRabA5e cDNAs were introduced separately into S. cerevisiae mutants with RHO1 and YPT31/YPT32 defective genes respectively. Neither cDNAs could complement these temperature-sensitive mutants, suggesting that the functions of the VvRop9 and VvRabA5e genes in grapevine likely differ from the functions of RHO1 and YPT31/YPT32 genes in yeast.

Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations

MoSfl1 Is Important for Virulence and Heat Tolerance in Magnaporthe oryzae

The formation of appressoria, specialized plant penetration structures of Magnaporthe oryzae, is regulated by the MST11-MST7-PMK1 MAP kinase cascade. One of its downstream transcription factor, MST12, is important for penetration and invasive growth but dispensable for appressorium formation. To identify additional downstream targets that are regulated by Pmk1, in this study we performed phosphorylation assays with a protein microarray composed of 573 M. oryzae transcription factor (TF) genes. Three of the TF genes phosphorylated by Pmk1 in vitro were further analyzed by coimmunoprecipitation assays. One of them, MoSFL1, was found to interact with Pmk1 in vivo. Like other Sfl1 orthologs, the MoSfl1 protein has the HSF-like domain. When expressed in yeast, MoSFL1 functionally complemented the flocculation defects of the sfl1 mutant. In M. oryzae, deletion of MoSFl1 resulted in a significant reduction in virulence on rice and barley seedlings. Consistent with this observation, the Mosfl1 mutant was defective in invasive growth in penetration assays with rice leaf sheaths. In comparison with that of vegetative hyphae, the expression level of MoSFL1 was increased in appressoria and infected rice leaves. The Mosfl1 mutant also had increased sensitivity to elevated temperatures. In CM cultures of the Mosfl1 and pmk1 mutants grown at 30°C, the production of aerial hyphae and melanization were reduced but their growth rate was not altered. When assayed by qRT-PCR, the transcription levels of the MoHSP30 and MoHSP98 genes were reduced 10- and 3-fold, respectively, in the Mosfl1 mutant. SFL1 orthologs are conserved in filamentous ascomycetes but none of them have been functionally characterized in non-Saccharomycetales fungi. MoSfl1 has one putative MAPK docking site and three putative MAPK phosphorylation sites. Therefore, it may be functionally related to Pmk1 in the regulation of invasive growth and stress responses in M. oryzae

Habilidades comunicativas e lexicais de crianças com Síndrome de Down: reflexões para inclusão escolar

Resumo: OBJETIVO: verificar o desempenho comunicativo e lexical expressivo de crianças com Síndrome de Down e refletir sobre como a compreensão de fatores interferentes no processo de aprendizagem pode contribuir para uma melhor adaptação dessas crianças no ambiente escolar. MÉTODOS: a amostra proposta foi de 60 crianças, porém, após análise dos critérios de inclusão, participaram 20 crianças, 10 com Síndrome de Down e 10 com neurodesenvolvimento típico, de idade entre 36 a 62 meses, pareadas quanto ao gênero, idade cronológica e nível socioeconômico. Procedimentos: entrevista com familiares, Observação do Comportamento Comunicativo e Teste de Linguagem Infantil ABFW-Vocabulário Parte B. A análise dos dados foi realizada por meio de estatística descritiva e aplicação do Teste "t" Student (p≤ 0,05). RESULTADOS: indicaram diferença estatisticamente significante para produção de palavras e frases, narrativa, tempo de atenção, designação verbal usual e não designação. Para processos de substituição a análise estatística não acusou diferença estatisticamente significante. Apenas para profissões e locais, nesta categoria, houve diferença estatisticamente significante entre os grupos. Como são avaliados nove campos conceituais, este dado não interferiu na análise estatística da somatória dos valores de todos os campos. CONCLUSÃO: o desempenho comunicativo e lexical expressivo de crianças com Síndrome de Down é inferior quando comparado com crianças com neurodesenvolvimento típico. A escola tem importante papel em proporcionar um ambiente estimulador, por meio de práticas pedagógicas adequadas às necessidades de aprendizagem destas crianças

First record of Aluterus monoceros (Linnaeus, 1758) off the Iberian Peninsula (Pisces, Monacanthidae)

3 páginas, 2 figuras, 1 tabla.[EN] Aluterus monoceros (L., 1758) is cited off the Iberian Peninsula for the firt time, on the basis of one specimen collected in coastal waters off Chipiona (Cádiz). Its biometric and meristic characters are compared with those described by other authors.[ES] Aluterus monoceros (L., 1758) es citado por vez primera para la península Ibérica a partir de un ejemplar capturado en la costa de Chipiona (Cádiz). Sus caracteres biométricos y merísticos son comparados con los descritos por otros autores.Peer reviewe