Exequibilidade do DDRT-PCR para análise da expressão gênica diferencial em células de medula óssea murina

Model of study: Experimental study. Introduction: Recently, stem cell research has generated greatinterest due to its applicability in regenerative medicine. Bone marrow is considered the most importantsource of adult stem cells and the establishment of new methods towards gene expression analysisregarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the geneexpression of different stem cells in distinct situations.Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in globalgene expression of mice bone marrow cells under two conditions.Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week withoutmedium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to geneexpression analyses by DDRT-PCR.Results: Initially, it was possible to observe in one week-cultured bone marrow cells, changes in morphology (oval cells to fibroblastic-like cells) and protein profile, which was seen through differences inband distribution in SDS-Page gels. Finally through gene expression analysis, we detected three bands(1300, 1000 and 225 bp) exclusively expressed in the fresh bone marrow group and two bands (400 and300 bp) expressed specifically in the cultivated bone marrow cell group.Conclusions: In summary, the DDRT-PCR method was proved efficient towards the identification ofsmall differences in gene expression of bone marrow cells in two defined conditions. Thus, we expectthat DDRT-PCR can be fast and efficiently designed to analyze differential gene expression in severalstem cell types under distinct conditions.Modelo do estudo: Estudo Experimental. Introdução: Atualmente a pesquisa com células-tronco temgerado grande interesse devido a sua aplicabilidade no campo na medicina regenerativa. A medulaóssea é considerada a maior fonte de células-tronco adultas e o estabelecimento de novos métodospara a análise da expressão gênica torna-se estritamente necessário. Desse modo, o "DifferentialDisplay Reverse Transcription Polymerase Chain Reaction (DDRT-PCR)", pode ser uma ferramentaacessível para a investigação de pequenas diferenças no nível de expressão gênica em diferentes tiposcelulares, sob distintas condições.Objetivo: Neste presente trabalho nós investigamos a exequibilidade do DDRT-PCR na identificação dediferenças no nível de expressão gênica global em células da medula óssea de camundongos sobduas condições. Métodos: Primeiramente, a medula óssea foi isolada frescamente e uma secundaparte foi cultivada por uma semana sem troca de meio. Posteriormente, as células da medula (fresca ecultivada) foram submetidas a análise da expressão gênica, seguindo a metodologia de DDRT-PCR.Resultados: Inicialmente, foi possível identificar em células da medula óssea com uma semana decultivo, pequenas alterações morfológicas (células ovais para fibroblastóides) e no perfil de proteínas,por meio da visualização de bandas em SDS-Page gel. Finalmente, a análise da expressão gênica porDDRT-PCR, mostrou uma expressão diferencial com a presença de três bandas (1300, 1000 and 225pb) exclusivamente expressas na medula óssea fresca e mais duas bandas (400 and 300 pb) presentes somente nas células de medula cultivadas.Conclusões: Em suma, a metodologia de DDRT-PCR mostrou-se eficiente para a identificação depequenas diferenças no nível de expressão gênica em células da medula óssea sob duas definidascondições. Portanto, nós acreditamos que o DDRT-PCR possa ser designado de forma rápida e eficiente para a análise diferencial de expressão gênica em diferentes tipos de células-tronco, sob diferentescondições

In search of mechanisms associated with mesenchymal stem cell-based therapies for acute kidney injury

Acute kidney injury (AKI) is classically described as a rapid loss of kidney function. AKI affects more than 15% of all hospital admissions and is associated with elevated mortality rates. Although many advances have occurred, intermittent or continuous renal replacement therapies are still considered the best options for reversing mild and severe AKI syndrome. For this reason, it is essential that innovative and effective therapies, without side effects and complications, be developed to treat AKI and the end-stages of renal disease. Mesenchymal stem cell (MSC) based therapies have numerous advantages in helping to repair inflamed and damaged tissues and are being considered as a new alternative for treating kidney injuries. Numerous experimental models have shown that MSCs can act via differentiation-independent mechanisms to help renal recovery. Essentially, MSCs can secrete a pool of cytokines, growth factors and chemokines, express enzymes, interact via cell-to-cell contacts and release bioagents such as microvesicles to orchestrate renal protection. In this review, we propose seven distinct properties of MSCs which explain how renoprotection may be conferred: 1) anti-inflammatory; 2) pro-angiogenic; 3) stimulation of endogenous progenitor cells; 4) anti-apoptotic; 5) anti-fibrotic; 6) anti-oxidant; and 7) promotion of cellular reprogramming. In this context, these mechanisms, either individually or synergically, could induce renal protection and functional recovery. This review summarises the most important effects and benefits associated with MSC-based therapies in experimental renal disease models and attempts to clarify the mechanisms behind the MSC-related renoprotection. MSCs may prove to be an effective, innovative and affordable treatment for moderate and severe AKI. However, more studies need to be performed to provide a more comprehensive global understanding of MSC-related therapies and to ensure their safety for future clinical applications

Sildenafil improves microvascular O-2 delivery-to-utilization matching and accelerates exercise O-2 uptake kinetics in chronic heart failure

Sperandio PA, Oliveira MF, Rodrigues MK, Berton DC, Treptow E, Nery LE, Almeida DR, Neder JA. Sildenafil improves microvascular O-2 delivery-to-utilization matching and accelerates exercise O-2 uptake kinetics in chronic heart failure. Am J Physiol Heart Circ Physiol 303: H1474-H1480, 2012. First published September 28, 2012; doi:10.1152/ajpheart.00435.2012.-Nitric oxide (NO) can temporally and spatially match microvascular oxygen (O-2) delivery (QO(2mv)) to O-2 uptake (VO2) in the skeletal muscle, a crucial adjustment-to-exercise tolerance that is impaired in chronic heart failure (CHF). To investigate the effects of NO bioavailability induced by sildenafil intake on muscle QO(2mv)-to-O-2 utilization matching and VO2 kinetics, 10 males with CHF (ejection fraction = 27 +/- 6%) undertook constant work-rate exercise (70-80% peak). Breath-by-breath VO2, fractional O-2 extraction in the vastus lateralis {similar to deoxy-genated hemoglobin + myoglobin ([deoxy-Hb + Mb]) by near-infrared spectroscopy}, and cardiac output (CO) were evaluated after sildenafil (50 mg) or placebo. Sildenafil increased exercise tolerance compared with placebo by similar to 20%, an effect that was related to faster on-and off-exercise VO2 kinetics (P 0.05). On-exercise [deoxy-Hb + Mb] kinetics were slowed by sildenafil (similar to 25%), and a subsequent response overshoot (n = 8) was significantly lessened or even abolished. in contrast, [deoxy-Hb + Mb] recovery was faster with sildenafil (similar to 15%). Improvements in muscle oxygenation with sildenafil were related to faster on-exercise VO2 kinetics, blunted oscillations in ventilation (n = 9), and greater exercise capacity (P < 0.05). Sildenafil intake enhanced intramuscular QO(2mv)-to-VO2 matching with beneficial effects on VO2 kinetics and exercise tolerance in CHF. the lack of effect on CO suggests that improvement in blood flow to and within skeletal muscles underlies these effects.Universidade Federal de São Paulo, Dept Med, Div Resp Dis, Pulm Funct & Clin Exercise Physiol Unit, BR-04020050 São Paulo, BrazilQueens Univ, Dept Med, Div Resp & Crit Care Med, Kingston, ON K7L 3N6, CanadaUniversidade Federal de São Paulo, Dept Med, Div Cardiol, BR-04020050 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Div Resp Dis, Pulm Funct & Clin Exercise Physiol Unit, BR-04020050 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Div Cardiol, BR-04020050 São Paulo, BrazilWeb of Scienc

Urochloa ruziziensis cover crop increases the cycling of soil inositol phosphates

Ruzigrass (Urochloa ruziziensis) is a cover crop that is commonly used in Brazil and exudes high concentrations of organic acids from its roots, and is therefore expected to mobilize soil organic P such as inositol phosphates. However, it is not known if this can occur only under P deficient conditions. Specifically, we aimed to test the hypothesis that the degradation of inositol phosphates is increased by growing ruzigrass at two different P levels. To investigate this, we studied soil organic P in a 9-year-old field experiment, with treatments consisting of ruzigrass or fallow during the soybean (Glycine max) off-season, with or without P addition. Organic P was extracted in NaOH-EDTA, followed by colorimetric quantification of organic P hydrolysable by phytase, and myo-inositol hexakisphosphate by hypobromite oxidation and HPLC separation. Ruzigrass dry matter yield increased by about 80% with P application. Ruzigrass reduced the concentration of phytase labile P and myo-inositol hexakisphosphate, but only in soil receiving P. A corresponding increase in unidentified inositol phosphates, presumably representing lower-order esters, was also observed after ruzigrass in soil with P application. We deduce that the degradation of inositol phosphates under ruzigrass with P application is due to greater ruzigrass productivity in the more fertile treatment, increasing the release of root exudates that solubilize inositol phosphates and promote their decomposition by phytase. We conclude that ruzigrass cover cropping can promote the cycling of recalcitrant soil organic P, but only when fertility is raised to a sufficient level to ensure a productive crop. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature

Globalização, segurança e defesa no Atlântico Sul

Brasil e Portugal: o caminho da influência militar na política via golpe de Estado ou Eleições Utilizando uma estrutura conceptual que requer análise do controle civil democrático e a eficácia militar, o artigo compara e contrasta Brasil e Portugal. Em Portugal, as instituições foram criadas para alcançar o controlo civil democrático e a eficácia militar, principalmente na manutenção da paz internacional e assistência humanitária. Em contraste, no Brasil não foram estabelecidas instituições que garantam o controlo civil democrático nem a eficácia militar. O artigo argumenta que as diferenças na cultura estratégica são fundamentais para entender a experiência contrastante destes dois países.Por entre a narrativa crescente de uma crise do multilateralismo internacional, instru‑ mentos e arcabouços coletivos são cada vez mais questionados em toda a linha. Como pensar tais desenvolvimentos em termos do Atlântico e como compreender este espaço do ponto de vista multilateral mais a sul? Este artigo argumenta que existem especificidades próprias, que ajudam a melhor compreender a evolução da oferta institucional nos últimos anos. Com um enfoque nos desafios e oportu‑ nidades associados a dinâmicas informais de cooperação entre as duas margens, são analisados mecanismos de governação para o Atlântico Sul com pressupostos, alcances e resultados distintos. Conclui‑se pela necessidade de atender quer à multiplicidade existente da oferta multilateral quer à irregularidade operacional recorrente, por forma a conseguir‑se caracterizar devidamente este espaço regional.Portugal inscreveu no seu Conceito Estratégico de Defesa Nacional (CEDN) que a unidade estratégica do Atlântico é do seu interesse por motivos relacionados com questões de segurança e também pelo facto de sete dos nove membros da CPLP aí se encontrarem. Já o Brasil diverge desta posição empenhando‑se apenas no Atlântico Sul com ênfase em organizações como a ZOPACAS e no relacionamento com os Estados da região. Serão estas duas visões incompatíveis? Ou poderão ser compatibilizadas no quadro da nova administração Bolsonaro e da sua aproximação aos EUA? Tentaremos responder a estas e outras questões neste artigo, onde também se tentará percecionar qual a nova posição estratégica brasileira sob o impulso de Jair Bolsonaro.A conjuntura internacional relacionada com os espaços marítimos apresenta, ao final desta segunda década do século XXI, duas tendências: a crescente importância econômica e militar dos oceanos, e o incremento das tensões derivadas do processo de demarcação das fronteiras marítimas. Essas duas tendências se desenvolvem na moldura de uma importante mudança na segurança internacional que é o retorno da competição entre as grandes potências, anunciado oficialmente na Estratégia de Segurança Nacional dos EUA publicada em 2017. O retorno da política de poder e da competição entre as grandes potências incrementa a impor‑ tância dos oceanos nas conceções estratégicas no sentido clássico, inclusive no que diz respeito à competição por recursos derivados do mar e o combate a ameaças como pirataria. Este artigo analisa essas tendências, relacionando‑as, principalmente, com o Atlântico Sul. Nesse sentido, são apresentados inicialmente alguns conceitos, tais como o poder capacita‑ dor dos oceanos, poder marítimo e poder na‑ val. Em seguida, são mostrados alguns dados que reafirmam a importância dos mares na globalização comercial e financeira mundial. O terceiro movimento apresenta as principais tensões relacionadas com os espaços maríti‑ mos. A quarta e última parte trata do papel do Atlântico Sul na globalização e na segurança e defesa.info:eu-repo/semantics/publishedVersio

Caloric Restriction Is More Efficient than Physical Exercise to Protect from Cisplatin Nephrotoxicity via PPAR-Alpha Activation

The antineoplastic drug cisplatin promotes renal injury, which limits its use. Protocols that reduce renal cisplatin toxicity will allow higher doses to be used in cisplatin treatment. Here, we compare physical exercise and caloric restriction (CR) as protocols to reduce cisplatin renal injury in mice. Male C57BL/6 were divided into four groups: Control, cisplatin, exercise + cisplatin, and 30% CR + cisplatin. Animals were injected with a single dose of cisplatin (20 mg/kg i.p.) and sacrificed 96 h after injection. Quantitative real time PCR, histological analyses, immunohistochemistry, and biochemical measurements were performed to investigate renal injury, necrosis, apoptosis, and inflammatory mechanisms. Both protocols protected against cisplatin renal injury, but CR was more effective in reducing uraemia and renal necrosis. The CR + Cisplatin group exhibited reduced serum IL-1 beta and INF-alpha levels. No differences were noted in the renal mRNA expression of cytokines. Both interventions reduced apoptosis, but only the CR + Cisplatin group decreased TNFR2 protein expression. PPAR-ci was activated in mice after CR. An antagonist of PPAR-alpha blocked the protective effect of CR. Both interventions attenuated the nephrotoxicity caused by cisplatin injection, but CR + Cisplatin showed a better response by modulating TNFR2. Moreover, part of the CR benefit depends on PPAR-alpha activation.FAPESP (Fundacao de Apoio a Pesquisa do Estado de Sao Paulo)CAPES/DAADUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Med, Disciplina Nefrol, Sao Paulo, BrazilUniv Sao Paulo, Inst Ciencias Biomed, Dept Immunol, Sao Paulo, BrazilUniv Sao Paulo, Dept Clin Med, Sao Paulo, BrazilUniv Fed Pelotas, Escola Nutr, Dept Nutr, Pelotas, BrazilMax Delbruck Ctr Mol Med, Berlin, GermanyUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Med, Disciplina Nefrol, Sao Paulo, BrazilFAPESP: 2013/06207-6FAPESP: 2015/20082-7CAPES/DAAD: 427/15Web of Scienc

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Differentiation of the Dimorphic Fungal Species Paracoccidioides brasiliensis and Paracoccidioides lutzii

Isolates of Paracoccidioides brasiliensis and Paracoccidioides lutzii, previously characterized by molecular techniques, were identified for the first time by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). All isolates were correctly identified, with log score values of >2.0. Thus, MALDI-TOF MS is a new tool for differentiating species of the genus Paracoccidioides.Univ São Paulo, Fac Med, Hosp Clin, Cent Lab Div LIM03, São Paulo, BrazilUniv São Paulo, Inst Med Trop, Hosp Clin FMUSP, Lab Med Mycol LIM53, São Paulo, BrazilUniv São Paulo, Dept Biophys, Escola Paulista Med, São Paulo, BrazilUniv Estadual Paulista, Dept Microbiol & Immunol, Inst Biociencias Botucatu, Botucatu, SP, BrazilUniv São Paulo, Dept Biophys, Escola Paulista Med, São Paulo, BrazilWeb of Scienc

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a "swim ergometer" by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL∙min 1∙kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.info:eu-repo/semantics/publishedVersio