Protocolo para sexagem fetal a partir da quarta semana de gestação por PCR Real Time utilizando DNA cell-free

Técnicas empregadas nos exames pré-natais para diagnóstico de doenças genéticas e para a determinação do sexo dos fetos são invasivas e oferecem risco ao feto em desenvolvimento. No entanto, utilizando a biologia molecular é possível implantar e implementar rotinas seguras que se baseiem em DNA fetal encontrado no sangue periférico materno (DNA cell-free). O objetivo do presente trabalho foi descrever um protocolo padronizado com sensibilidade suficiente em inferir, com fidedignidade, o sexo fetal a partir das primeiras semanas de gestação. Os dados foram obtidos do sistema de cadastro de dados do Laboratório de Genética Molecular do Instituto de Perícias Científicas de Mato Grosso do Sul. Durante o processo de padronização do exame de sexagem fetal o laboratório convidou voluntárias (n=132) que se comprometeram a retornar com o resultado do exame de ultrassom.  Essas voluntárias (n=132) foram divididos em grupo controle (não gestantes) e grupo experimental (gestantes). Amostras de sangue das voluntárias foram coletadas e o plasma obtido por centrifugação. Após, a extração do DNA ocorreu por meio de um método inédito in house, utilizando tampão de lise à base de tiocianato de guanidina (Tiocianato de guanidina 5mol/L, Tris HCL 11,2g/L, EDTA 7,43 g/L, 7,8mLTriton X-100) e a amplificação em PCR tempo real, utilizando o kit Quantifiler Y™ e 5µL do DNA extraído. Amostras que amplificaram foram consideradas como positivas (feto do sexo masculino), e as que não amplificaram consideradas como negativas (fetos do sexo feminino). A técnica de extração foi eficiente para obtenção do DNA em quantidades suficientes e com grau de pureza adequado. A análise da sexagem fetal das 132 gestantes obteve 100% de concordância com o exame de ultrassom. A extração proporcionou ao teste a sensibilidade necessária para determinar o sexo fetal mesmo nos estágios inicias de gestação, a partir da quarta semana. Esse dado é pioneiro e não havia sido relatado em nenhuma pesquisa, na literatura consultada pelo nosso grupo, o que viabiliza a sexagem fetal a partir da quarta semana de gestação e abre perspectivas para a aplicação dessa extração no diagnóstico de doenças genética, tornando-se uma ferramenta para o diagnóstico pré-natal, evitando assim técnicas invasivas que coloquem em risco o desenvolvimento fetal

Cell culture and variations in the comet assay do not affect the genomic integrity of adipose-derived stem cells

The application of quality control tests, such as the comet assay, are essential when adipose-derived stem cells are cultured for therapeutic purposes. However, the steps involved in the development of this assay should be investigated, in order to reduce their influence on genomic damage in cells. The study aimed to evaluate if the cell culture process causes DNA damage, and if variations in the lysis time and pH of the electrophoresis buffer interfere in the genotoxicity results. Four different comet assay protocols were evaluated, and the effects of lysis time and pH conditions of the electrophoresis buffer solution were stated as follows: 2 hours and pH 12; 24 hours and pH 12; 2 hours and pH ≥ 13 and 24 hours and pH ≥ 13. The tail moment was analyzed, and results indicated that at the time cells were detached from the flasks, there was little damage to the DNA in the adipose-derived stem cells, which was confirmed by evaluation of the expression of mRNA genes involved in damage and repair processes of genetic material. Also, the tail moment values ​​did not show significant differences among the four evaluated protocols (p < 0.05), with no indication of damage when compared to the positive control (p < 0.05). Thus, any of the tested protocols can be applied in genotoxicity tests with adipose-derived stem cells, without causing damage to them

Mapping density, diversity and species-richness of the Amazon tree flora

Using 2.046 botanically-inventoried tree plots across the largest tropical forest on Earth, we mapped tree species-diversity and tree species-richness at 0.1-degree resolution, and investigated drivers for diversity and richness. Using only location, stratified by forest type, as predictor, our spatial model, to the best of our knowledge, provides the most accurate map of tree diversity in Amazonia to date, explaining approximately 70% of the tree diversity and species-richness. Large soil-forest combinations determine a significant percentage of the variation in tree species-richness and tree alpha-diversity in Amazonian forest-plots. We suggest that the size and fragmentation of these systems drive their large-scale diversity patterns and hence local diversity. A model not using location but cumulative water deficit, tree density, and temperature seasonality explains 47% of the tree species-richness in the terra-firme forest in Amazonia. Over large areas across Amazonia, residuals of this relationship are small and poorly spatially structured, suggesting that much of the residual variation may be local. The Guyana Shield area has consistently negative residuals, showing that this area has lower tree species-richness than expected by our models. We provide extensive plot meta-data, including tree density, tree alpha-diversity and tree species-richness results and gridded maps at 0.1-degree resolution

Mapping density, diversity and species-richness of the Amazon tree flora

Using 2.046 botanically-inventoried tree plots across the largest tropical forest on Earth, we mapped tree species-diversity and tree species-richness at 0.1-degree resolution, and investigated drivers for diversity and richness. Using only location, stratified by forest type, as predictor, our spatial model, to the best of our knowledge, provides the most accurate map of tree diversity in Amazonia to date, explaining approximately 70% of the tree diversity and species-richness. Large soil-forest combinations determine a significant percentage of the variation in tree species-richness and tree alpha-diversity in Amazonian forest-plots. We suggest that the size and fragmentation of these systems drive their large-scale diversity patterns and hence local diversity. A model not using location but cumulative water deficit, tree density, and temperature seasonality explains 47% of the tree species-richness in the terra-firme forest in Amazonia. Over large areas across Amazonia, residuals of this relationship are small and poorly spatially structured, suggesting that much of the residual variation may be local. The Guyana Shield area has consistently negative residuals, showing that this area has lower tree species-richness than expected by our models. We provide extensive plot meta-data, including tree density, tree alpha-diversity and tree species-richness results and gridded maps at 0.1-degree resolution

Author correction: One sixth of Amazonian tree diversity is dependent on river floodplains

In the version of the article initially published, the affiliation of Edgardo Manuel Latrubesse was incorrect and has now been amended to Environmental Sciences Graduate Program-CIAMB, Federal University of Goiás, Goiânia, Brazil in the HTML and PDF versions of the article

Mapping density, diversity and species-richness of the Amazon tree flora

Using 2.046 botanically-inventoried tree plots across the largest tropical forest on Earth, we mapped tree species-diversity and tree species-richness at 0.1-degree resolution, and investigated drivers for diversity and richness. Using only location, stratified by forest type, as predictor, our spatial model, to the best of our knowledge, provides the most accurate map of tree diversity in Amazonia to date, explaining approximately 70% of the tree diversity and species-richness. Large soil-forest combinations determine a significant percentage of the variation in tree species-richness and tree alpha-diversity in Amazonian forest-plots. We suggest that the size and fragmentation of these systems drive their large-scale diversity patterns and hence local diversity. A model not using location but cumulative water deficit, tree density, and temperature seasonality explains 47% of the tree species-richness in the terra-firme forest in Amazonia. Over large areas across Amazonia, residuals of this relationship are small and poorly spatially structured, suggesting that much of the residual variation may be local. The Guyana Shield area has consistently negative residuals, showing that this area has lower tree species-richness than expected by our models. We provide extensive plot meta-data, including tree density, tree alpha-diversity and tree species-richness results and gridded maps at 0.1-degree resolution

One sixth of Amazonian tree diversity is dependent on river floodplains

Amazonia’s floodplain system is the largest and most biodiverse on Earth. Although forests are crucial to the ecological integrity of floodplains, our understanding of their species composition and how this may differ from surrounding forest types is still far too limited, particularly as changing inundation regimes begin to reshape floodplain tree communities and the critical ecosystem functions they underpin. Here we address this gap by taking a spatially explicit look at Amazonia-wide patterns of tree-species turnover and ecological specialization of the region’s floodplain forests. We show that the majority of Amazonian tree species can inhabit floodplains, and about a sixth of Amazonian tree diversity is ecologically specialized on floodplains. The degree of specialization in floodplain communities is driven by regional flood patterns, with the most compositionally differentiated floodplain forests located centrally within the fluvial network and contingent on the most extraordinary flood magnitudes regionally. Our results provide a spatially explicit view of ecological specialization of floodplain forest communities and expose the need for whole-basin hydrological integrity to protect the Amazon’s tree diversity and its function

Unraveling Amazon tree community assembly using Maximum Information Entropy: a quantitative analysis of tropical forest ecology

In a time of rapid global change, the question of what determines patterns in species abundance distribution remains a priority for understanding the complex dynamics of ecosystems. The constrained maximization of information entropy provides a framework for the understanding of such complex systems dynamics by a quantitative analysis of important constraints via predictions using least biased probability distributions. We apply it to over two thousand hectares of Amazonian tree inventories across seven forest types and thirteen functional traits, representing major global axes of plant strategies. Results show that constraints formed by regional relative abundances of genera explain eight times more of local relative abundances than constraints based on directional selection for specific functional traits, although the latter does show clear signals of environmental dependency. These results provide a quantitative insight by inference from large-scale data using cross-disciplinary methods, furthering our understanding of ecological dynamics