Viabilidade de blastocistos de Mus musculus domesticus expostos à alta pressão gasosa e submetidos à criopreservação

A alta pressão gasosa (high gaseous pressure, HGP) tem sido descrita como um agente estressante capaz de induzir resposta ao estresse subletal, fornecendo proteção celular a estresses subsequentes, como a criopreservação, embora os mecanismos celulares envolvidos ainda não estejam elucidados. Este princípio foi investigado em blastocistos murinos expostos a diferentes tratamentos utilizando-se HGP, seguidos ou não de criopreservação. De um total de 427 fêmeas de Mus musculus domesticus superovuladas, 308 (72,1%) produziram 3070 blastocistos viáveis (média de 7,2 ± 11,5 blastocistos/fêmea superovulada e de 10,0 ± 12,5 blastocistos/fêmea responsiva ao tratamento superovulatório) que foram expostos a tratamentos de distintas pressões e tempos de exposição, em quatro grupos experimentais: (a) 20,7 MPa por 2 h (P20.T2); (b) 20,7 Mpa por 4 h (P20.T4); (c) 27,6 Mpa por 2 h (P27.T2); e (d) 34,5 Mpa por 2 h (P34.T2). Todos os grupos foram pareados com controles não expostos à HGP. No primeira parte do experimento, embriões foram expostos à HGP, conforme os grupos experimentais acima, e posteriormente submetidos ao cultivo in vitro por 72 h com o objetivo de verificar a influência dos protocolos de HGP sobre a taxa de eclosão. Não houve diferença nas taxas de eclosão e na morfologia entre os grupos experimentais e os controles. Já na segunda parte do experimento, os embriões foram expostos à HGP, conforme os grupos experimentais acima, e posteriormente criopreservados por curva de congelamento clássica (rápida). Após o descongelamento, os embriões foram cultivados in vitro por 72 h para a avaliação das taxas de eclosão entre os grupos. A taxa de eclosão do Grupo P34.T2 foi significativamente maior do que a dos controles criopreservados não expostos à HGP (70,2% vs. 58,6%, p < 0,05). Conclui-se que a HGP pode ser usada como agente estressor subletal sem perda da viabilidade embrionária, e que o uso da HGP de 34,5 MPa por 2 h prévio à criopreservação modificou positivamente a taxa de sobrevivência in vitro de blastocistos murinos criopreservados.High gaseous pressure (HGP) has been reported as a sublethal stressor able to induce "sublethal stress response”, providing cell protection to subsequent stress, such as cryopreservation, although the involved cellular mechanisms are little understood. This principle was investigated on murine blastocysts exposed to different HGP treatments with and without subsequent cryopreservation. A total of 308 (72.1%) out of 427 superovulated Mus musculus domesticus females produced 3,070 viable blastocysts (7.2 ± 11.5 blastocysts/superovulated mouse and 10.0 ± 12.5 blastocysts/female responsive to superovulation) that were exposed to distinct pressure intensities and exposure times, according to four experimental groups: (a) 20.7 MPa for 2 h (P20.T2); (b) 20.7 MPa for 4 h (P20.T4); (c) 27.6 MPa for 2 h (P27.T2); and (d) 34.5 MPa for 2 h (P34.T2). All groups were compared with non-exposed HGP controls. In the first part of the experiment, embryos were exposed to HGP treatments, according to the experimental groups as above, and subsequently in vitro-cultured for 72 h aiming to compare the effects of the HGP protocols on subsequent hatching rates. No differences in hatching rates and morphology were observed between experimental and control groups. In the second parto of the experiment, embryos were exposed to HGP treatments, according to the experimental groups as above, and subsequently cryopreserved by rapid (classic) freezing. After thawing, embryos were in vitro-cultured for 72 h to compare hatching rates between groups. Hatching rates in the P34.5.T2 group were significantly higher than for the cryopreserved control embryos with no HGP treatment (70.2% vs. 58.6%, p < 0.05). Therefore, we conclude that HGP can be used as a stressor without compromising subsequent in vitro embryo viability, and the use of HGP at 34.5 MPa for 2 h prior to cryopreservation improved the in vitro survival of cryopreserved murine blastocysts compared with control counterparts

Determinação do efeito da alta pressão gasosa sobre oócitos imaturos de Bos taurus taurus e embriões de Mus musculus domesticus na eficiência da produção in vitro de embriões

A alta pressão gasosa (HGP, do inglês high gaseous pressure) tem sido descrita como um agente estressante capaz de induzir respostas ao estresse subletal, fornecendo proteção celular e estímulos positivos a desafios subsequentes, como a criopreservação, produção in vitro de embriões (PIVE) , entre outras biotécnicas da reprodução, embora os mecanismos celulares, a modulação epigenética e as modificações no padrão de expressão gênica envolvidos ainda não estejam totalmente elucidados. Este princípio foi investigado em gametas bovinos e embriões murinos no presente trabalho. Foram realizados dois experimentos independentes. No primeiro, avaliou-se o efeito da exposição de oócitos bovinos no estádio de vesículas germinativa à HGP na eficiência e cinética de desenvolvimento da PIVE por fecundação in vitro (FIV). Os resultados revelaram que oócitos expostos a HGP em temperatura ambiente apresentaram taxa de maturação in vitro (MIV) e cinética de desenvolvimento até o estádio de blastocisto semelhantes ao controle mantido em condições ideais durante o tratamento do grupo experimental, diferentemente do observado no grupo com CCOs mantidos em temperatura ambiente durante a exposição à HGP. Em um segundo experimento, observou-se as alterações no padrão de expressão gênica de blastocistos murinos expostos previamente à alta pressão gasosa no estádio de embrião de 8-células, bem como se este desafio alterava a taxa de sobrevivência destes blastocistos após serem submetidos ao processo de criopreservação. A análise do padrão de transcrição revelou que um gene associado ao estresse oxidativo (Sod2) teve sua expressão aumentada, enquanto genes de proliferação e desenvolvimento (Igf2 e Igf2R) tiveram sua expressão reduzida, quando comparados com os embriões no estádio de 8-células, embora a relação Igf2/Igf2R não demonstrou ser afetada. O tratamento por HGP mostrou-se eficiente no aumento da taxa de sobrevivência de blastocistos após a criopreservação. Os experimentos permitiram concluir que a resposta ao estresse subletal estimulada pela HGP é eficiente na melhora da performance de gametas femininos e embriões submetidos a novas situações estressantes.High gaseous pressure (HGP) has been described as stressor, providing cell protection and positive stimuli to subsequent challenges like cryopreservation, embryo in vitro production (IVP), among other reproduction biotechnologies, although cells mechanisms, epigenetic modulation and transcriptional modifications are not totally elucidated. This principle was investigated in bovine gametes and murine embryos in this report at two independent studies. First, the effect of bovine oocyte exposure to HGP in germinal vesicle stage was evaluated to efficiency and development kinect of IVP by in vitro fertilization (IVF). Results showed that oocytes exposed to HGP at room temperature had in vitro maturation (IVM) rate and developmental kinect to reach the blastocyst stage similar to control group kept in ideal conditions during experimental group treatment, differently that what was observed in the group with COCs just kept at room temperature during HGP exposure. In the second experiment, modifications in transcriptional response were observed in murine blastocysts previously exposed to HGP when embryo 8-cells stage, as well if this challenge modified survival rate of this blastocysts after cryopreservation procedures. The analysis of the transcriptional pattern revealed that a gene associated to oxidative stress (Sod2) was upregulated, while genes of proliferation and development (Igf2 and Igf2R) were downregulated, when compared with 8-cells embryos, even though the ratio Igf2/Igf2R appeared not be affected. HGP treatment showed to be efficient to improve blastocyst survival rate to cryopreservation. The experiments enable inferring that subletal response stimulated by HGP was efficient to enhance performance of female gametes and embryos exposed to new stressor situations

12,500+ and counting: biodiversity of the Brazilian Pampa

Knowledge on biodiversity is fundamental for conservation strategies. The Brazilian Pampa region, located in subtropical southern Brazil, is neglected in terms of conservation, and knowledge of its biodiversity is fragmented. We aim to answer the question: how many, and which, species occur in the Brazilian Pampa? In a collaborative effort, we built species lists for plants, animals, bacteria, and fungi that occur in the Brazilian Pampa. We included information on distribution patterns, main habitat types, and conservation status. Our study resulted in referenced lists totaling 12,503 species (12,854 taxa, when considering infraspecific taxonomic categories [or units]). Vascular plants amount to 3,642 species (including 165 Pteridophytes), while algae have 2,046 species (2,378 taxa) and bryophytes 316 species (318 taxa). Fungi (incl. lichenized fungi) contains 1,141 species (1,144 taxa). Animals total 5,358 species (5,372 taxa). Among the latter, vertebrates comprise 1,136 species, while invertebrates are represented by 4,222 species. Our data indicate that, according to current knowledge, the Pampa holds approximately 9% of the Brazilian biodiversity in an area of little more than 2% of Brazil’s total land The proportion of species restricted to the Brazilian Pampa is low (with few groups as exceptions), as it is part of a larger grassland ecoregion and in a transitional climatic setting. Our study yielded considerably higher species numbers than previously known for many species groups; for some, it provides the first published compilation. Further efforts are needed to increase knowledge in the Pampa and other regions of Brazil. Considering the strategic importance of biodiversity and its conservation, appropriate government policies are needed to fund studies on biodiversity, create accessible and constantly updated biodiversity databases, and consider biodiversity in school curricula and other outreach activitie

Description and performance of track and primary-vertex reconstruction with the CMS tracker

A description is provided of the software algorithms developed for the CMS tracker both for reconstructing charged-particle trajectories in proton-proton interactions and for using the resulting tracks to estimate the positions of the LHC luminous region and individual primary-interaction vertices. Despite the very hostile environment at the LHC, the performance obtained with these algorithms is found to be excellent. For tbar t events under typical 2011 pileup conditions, the average track-reconstruction efficiency for promptly-produced charged particles with transverse momenta of pT > 0.9GeV is 94% for pseudorapidities of |η| < 0.9 and 85% for 0.9 < |η| < 2.5. The inefficiency is caused mainly by hadrons that undergo nuclear interactions in the tracker material. For isolated muons, the corresponding efficiencies are essentially 100%. For isolated muons of pT = 100GeV emitted at |η| < 1.4, the resolutions are approximately 2.8% in pT, and respectively, 10μm and 30μm in the transverse and longitudinal impact parameters. The position resolution achieved for reconstructed primary vertices that correspond to interesting pp collisions is 10–12μm in each of the three spatial dimensions. The tracking and vertexing software is fast and flexible, and easily adaptable to other functions, such as fast tracking for the trigger, or dedicated tracking for electrons that takes into account bremsstrahlung

Alignment of the CMS tracker with LHC and cosmic ray data

© CERN 2014 for the benefit of the CMS collaboration, published under the terms of the Creative Commons Attribution 3.0 License by IOP Publishing Ltd and Sissa Medialab srl. Any further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation and DOI.The central component of the CMS detector is the largest silicon tracker ever built. The precise alignment of this complex device is a formidable challenge, and only achievable with a significant extension of the technologies routinely used for tracking detectors in the past. This article describes the full-scale alignment procedure as it is used during LHC operations. Among the specific features of the method are the simultaneous determination of up to 200 000 alignment parameters with tracks, the measurement of individual sensor curvature parameters, the control of systematic misalignment effects, and the implementation of the whole procedure in a multi-processor environment for high execution speed. Overall, the achieved statistical accuracy on the module alignment is found to be significantly better than 10μm

Climate seasonality limits leaf carbon assimilation and wood productivity in tropical forests

The seasonal climate drivers of the carbon cycle in tropical forests remain poorly known, although these forests account for more carbon assimilation and storage than any other terrestrial ecosystem. Based on a unique combination of seasonal pan-tropical data sets from 89 experimental sites (68 include aboveground wood productivity measurements and 35 litter productivity measurements), their associated canopy photosynthetic capacity (enhanced vegetation index, EVI) and climate, we ask how carbon assimilation and aboveground allocation are related to climate seasonality in tropical forests and how they interact in the seasonal carbon cycle. We found that canopy photosynthetic capacity seasonality responds positively to precipitation when rainfall is  < 2000 mm yr⁻¹ (water-limited forests) and to radiation otherwise (light-limited forests). On the other hand, independent of climate limitations, wood productivity and litterfall are driven by seasonal variation in precipitation and evapotranspiration, respectively. Consequently, light-limited forests present an asynchronism between canopy photosynthetic capacity and wood productivity. First-order control by precipitation likely indicates a decrease in tropical forest productivity in a drier climate in water-limited forest, and in current light-limited forest with future rainfall  < 2000 mm yr⁻¹

Uso de pool de soros como controle interno de qualidade em um laboratório de análises clínicas veterinárias

A confiabilidade dos resultados do laboratório é garantida pela realização do controle de qualidade, que tem como funções básicas análise, pesquisa e prevenção de erros laboratoriais por meio de metodologias que abrangem tanto o controle de qualidade interno quanto o externo. O controle de qualidade interno faz-se mais importante nas análises clínicas veterinárias, pois, com a impossibilidade de se fazer uma correta anamnese do paciente, o exame de laboratório tem papel fundamental no diagnóstico. Este trabalho tem como objetivo propor a implantação de um controle interno de qualidade no Laboratório de Análises Clínicas Veterinárias da Faculdade de Veterinária da Universidade Federal do Rio Grande do Sul. Foram escolhidas as análises de alanina aminotransferase (ALT), creatinina, fosfatase alcalina (FAL) e uréia para início desta implementação. Para a confecção do pool, utilizou-se soros considerados normais e livres de interferências analíticas. Para a aceitação dos valores encontrados pela amostra confeccionada, escolheu-se como referência o calibrador proteico Qualitrol 2H (Labtest Diagnóstica). Os valores encontrados para os analitos foram plotados em gráfico de Levey-Jennings e, para a sua avaliação, utilizou-se as regras múltiplas de Westgard. Após acompanhamento das corridas, não foram observadas violações das regras. Pode-se concluir que os resultados analíticos monitorados através do pool de soros mantiveram-se estáveis, servindo para seu propósito de controle de qualidade interno, e novas análises serão realizadas para estender este controle aos demais exames realizados no setor de bioquímica do laboratório.The reliability of laboratory results is ensured by the implementation of quality control, which has as basic functions analysis, research and prevention of laboratory errors through methodologies that include both internal and external quality controls. The internal quality control becomes more important in veterinary clinical analysis, because, since a correct patient's medical history is difficult to achieve, the laboratory results have a crucial role in the diagnosis. This work aims to propose the establishment of a policy of internal quality control in the Veterinary Clinical Laboratory of the Faculty of Veterinary Medicine of the Federal University of Rio Grande do Sul. We chose the analysis of alanine aminotransferase (ALT), creatinine, alkaline phosphatase (FAL) and urea to begin this implementation. For preparing the pool, we used sera considered analytical interferences free. For the acceptance of the values found for the prepared sample, we chose as reference the protein calibrator QUALITROL 2H (Labtest Diagnostic). The values found for the analyses were plotted on a Levey-Jennings chart, and for their evaluation, we used the Multiple Rules of Westgard. After monitoring the races, violations of the rules were not observed. It can be concluded that the monitored analytical results by serum pool remained stable while serving its purpose of internal quality control, and further analysis will be conducted to extend this control to other tests performed in the biochemistry department of the laboratory

Uso de pool de soros como controle interno de qualidade em um laboratório de análises clínicas veterinárias

A confiabilidade dos resultados do laboratório é garantida pela realização do controle de qualidade, que tem como funções básicas análise, pesquisa e prevenção de erros laboratoriais por meio de metodologias que abrangem tanto o controle de qualidade interno quanto o externo. O controle de qualidade interno faz-se mais importante nas análises clínicas veterinárias, pois, com a impossibilidade de se fazer uma correta anamnese do paciente, o exame de laboratório tem papel fundamental no diagnóstico. Este trabalho tem como objetivo propor a implantação de um controle interno de qualidade no Laboratório de Análises Clínicas Veterinárias da Faculdade de Veterinária da Universidade Federal do Rio Grande do Sul. Foram escolhidas as análises de alanina aminotransferase (ALT), creatinina, fosfatase alcalina (FAL) e uréia para início desta implementação. Para a confecção do pool, utilizou-se soros considerados normais e livres de interferências analíticas. Para a aceitação dos valores encontrados pela amostra confeccionada, escolheu-se como referência o calibrador proteico Qualitrol 2H (Labtest Diagnóstica). Os valores encontrados para os analitos foram plotados em gráfico de Levey-Jennings e, para a sua avaliação, utilizou-se as regras múltiplas de Westgard. Após acompanhamento das corridas, não foram observadas violações das regras. Pode-se concluir que os resultados analíticos monitorados através do pool de soros mantiveram-se estáveis, servindo para seu propósito de controle de qualidade interno, e novas análises serão realizadas para estender este controle aos demais exames realizados no setor de bioquímica do laboratório.The reliability of laboratory results is ensured by the implementation of quality control, which has as basic functions analysis, research and prevention of laboratory errors through methodologies that include both internal and external quality controls. The internal quality control becomes more important in veterinary clinical analysis, because, since a correct patient's medical history is difficult to achieve, the laboratory results have a crucial role in the diagnosis. This work aims to propose the establishment of a policy of internal quality control in the Veterinary Clinical Laboratory of the Faculty of Veterinary Medicine of the Federal University of Rio Grande do Sul. We chose the analysis of alanine aminotransferase (ALT), creatinine, alkaline phosphatase (FAL) and urea to begin this implementation. For preparing the pool, we used sera considered analytical interferences free. For the acceptance of the values found for the prepared sample, we chose as reference the protein calibrator QUALITROL 2H (Labtest Diagnostic). The values found for the analyses were plotted on a Levey-Jennings chart, and for their evaluation, we used the Multiple Rules of Westgard. After monitoring the races, violations of the rules were not observed. It can be concluded that the monitored analytical results by serum pool remained stable while serving its purpose of internal quality control, and further analysis will be conducted to extend this control to other tests performed in the biochemistry department of the laboratory