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Cell fractionation can be used to determine the localization and trafficking of proteins between cellular compartments such as the cytosol, mitochondria, and nuclei. Subcellular fractionation is usually performed immediately after tissue dissection because freezing may fragment cell membranes and induce organellar cross-contamination. Mitochondria are especially sensitive to freezing/thawing and mechanical homogenization. We proposed a protocol to improve the retention of soluble proteins in the mitochondrial fraction obtained from small amounts of frozen skeletal muscle. Fifty milligrams of the red portion of gastrocnemius muscle from Wistar rats were immediately processed or frozen in liquid nitrogen and stored at -80 °C for further processing. We compared the enrichment of subcellular fractions from frozen/fresh samples obtained with the modified protocol with those obtained by standard fractionation. Western blot analyses of marker proteins for cytosolic (alpha-tubulin), mitochondrial (VDAC1), and nuclear (histone-H3) fractions indicated that all of the procedures resulted in enriched subcellular fractions with minimal organellar cross-contamination. Notably, the activity of the soluble protein citrate synthase was higher in the mitochondrial fractions obtained with the modified protocol from frozen/fresh samples compared with the standard protocol. Therefore, our protocol improved the retention of soluble proteins in the mitochondrial fraction and may be suitable for subcellular fractionation of small amounts of frozen skeletal muscle samples.

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Purpose: This study aimed to investigate changes in muscle damage during the course of a 217-km mountain ultramarathon (MUM). In an integrative perspective, inflammatory response and renal function were also studied. Methods: Six male ultra-runners were tested four times: pre-race, at 84 km, at 177 km, and immediately after the race. Blood samples were analyzed for serum muscle enzymes, acute-phase protein, cortisol, and renal function biomarkers. Results: Serum creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) increased significantly throughout the race (P < 0.001, P < 0.001; P = 0.002, respectively), and effect size (ES) denoted a large magnitude of muscle damage. These enzymes increased from pre-race (132 ± 18, 371 ± 66, and 28 ± 3 U/L, respectively) to 84 km (30, 1.8, and 3.9-fold, respectively); further increased from 84 to 177 km (4.6, 2.9, and 6.1-fold, respectively), followed by a stable phase until the finish line. Regarding the inflammatory response, significant differences were found for C-reactive protein (CRP) (P < 0.001) and cortisol (P < 0.001). CRP increased from pre-race (0.9 ± 0.3 mg/L) to 177 km (243-fold), cortisol increased from pre-race (257 ± 30 mmol/L) to the 84 km (2.9-fold), and both remained augmented until the finish line. Significant changes were observed for creatinine (P = 0.03), urea (P = 0.001), and glomerular filtration rate (GFR) (P < 0.001), and ES confirmed a moderate magnitude of changes in renal function biomarkers. Creatinine and urea increased, and GFR decreased from pre-race (1.00 ± 0.03 mg/dL, 33 ± 6 mg/dL, and 89 ± 5 ml/min/1.73 m2, respectively) to 84 km (1.3, 3.5, and 0.7-fold, respectively), followed by a plateau phase until the finish line. Conclusion: This study shows evidence that muscle damage biomarkers presented early peak levels and they were followed by a plateau phase during the last segment of a 217-km MUM. The acute-phase response had a similar change of muscle damage. In addition, our data showed that our volunteers meet the risk criteria for acute kidney injury from 84 km until they finished the race, without demonstrating any clinical symptomatology.

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The use of small-sided games (SSG) for soccer training has increased considerably in recent years. Tactical principles can influence the intensity and the movement of soccer players during training. The aim of this study was to evaluate the influence of maintaining possession of the ball on the intensity and the time of motion characteristics of players during training in games. Eleven athletes of the U-20 category of a professional club in São Paulo, Brazil, participated in this study. To check the influence of the tactical principle, we used a game control and an experimental game in three different field dimensions and number of players. The results show that possession of the maintenance rule influences the intensity of the games (p <0.05). However, it is concluded that the rule is fundamental for training through games.

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Conceptual small-sided games (CSSGs) may be interesting as a methodology for training soccer players given its connection to the unpredictability that is inherent to soccer. Our aim was investigate, through videogrammetry, if the technical and tactical principles promoted through the adoption of distinct rules from two distinct CSSGs (maintaining ball possession; and progression to the target) would actually be achieved. The study included 24 athletes assigned to 6-player teams. Our data showed that the CSSGs' organising principles create situations with differing levels of difficulty that obey the propositions of maintaining ball possession and progression to the target, i.e., CSSGs permit systematic training on technical and tactical components in order to emphasize the concepts adopted in this study in games context. Our data credit the CSSGs for teaching technical and tactical lessons that, when coupled with adequate physical conditioning, can facilitate a player's capacity to merge thoughts and events in different situations.

A utilização de jogos reduzidos conceituais (JRC) pode ser uma metodologia de treino interessante para treinar a imprevisibilidade inerente ao jogo de futebol. Nosso objetivo no presente estudo foi investigar por videogrametria os princípios técnico-táticos adotados em resposta as respectivas regras de dois jogos reduzidos conceituais distintos (manutenção de posse de bola e progressão ao alvo). Participaram do estudo 24 atletas separados em equipes de 6 jogadores. Nossos dados comprovaram que os princípios organizadores dos JRC criam situações distintas com graus de dificuldade diferentes. Ou seja, é possível sistematizar o treinamento dos componentes técnicos e táticos de forma a enfatizar os conceitos adotados neste estudo e inseridos no contexto do jogo. Nossos dados credenciam os JRC para o aprendizado de situações técnico táticas que, aliadas ao condicionamento físico adequado podem contribuir para produzir um jogador treinado para aproximar cada vez mais pensamentos e ações em situações diversificadas.

El uso de los juegos reducidos conceptuales (JRC) puede ser una interesante metodología para entrenar a la imprevisibilidad inherente en el juego de fútbol. Nuestro objetivo en este estudio fue investigar por videogrametría los principios técnico-tácticos adoptadas en respuesta a las respectivas reglas de dos JRC distintos (posesión del balón y la progresión a la meta). El estudio incluyó a 24 atletas divididos en equipos de 6 jugadores. Nuestros datos muestran que los principios de organización de los JRC crean diferentes situaciones con diferentes grados de dificultad. Es decir, es posible sistematizar el desarrollo de los componentes técnicos y tácticos de una manera a enfatizar los conceptos adoptados para este estudio en el contexto del juego. Nuestros datos acreditan JRC para el aprendizaje de situaciones técnicas tácticas que, junto con la preparación física adecuada puede ayudar a producir jugadores capacitados a unir cada vez más pensamientos y acciones en diferentes situaciones.

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Introdução: Há métodos diretos e indiretos que são utilizados pelos clubes de futebol para avaliar, acompanhar e determinar o VO2max dos jogadores, sendo este muito importante para o rendimento e a recuperação dos atletas durante uma partida. Objetivo: Avaliar o nível de correlação entre as medidas de VO2max por meio da análise direta de gases e do teste de campo Yo-Yo Intermitente Recovery Level 1 (Yo-YoIR1). Métodos: Participaram do estudo 24 jogadores de futebol da categoria SUB-20 de um clube do estado de São Paulo, Brasil, com estatura 1,72±0,08 m e massa corporal 61,17±9,18 kg, com no mínimo cinco anos de prática na modalidade. Os atletas realizaram o teste de análise direta dos gases em esteira ergométrica e após 48 horas foi realizado o Yo-Yo IR1. Resultados: Os resultados apontaram uma correlação significante entre os testes (r=0,524; p<0,01), porém o Yo-Yo IR1 subestimou as medidas de análise direta dos gases em laboratório (44,98ml/kg/min e 48,14ml/kg/min, respectivamente). Conclusão: Os resultados apontaram uma correlação moderada entre as medidas de VO2max, dessa forma pode-se utilizar ambos os testes para análise da potência aeróbia dos jogadores de futebol, desde que seja repetido o mesmo protocolo nas avaliações subsequentes. .

Introduction: There are direct and indirect methods that are used by soccer clubs to assess, monitor and determine the VO2max of soccer players, which is very important for the performance and recovery of athletes during a match. Objective: The aim of this study was to assess the level of the correlation between measures of VO2max by direct gas analysis and by the field test Yo-Yo Intermittent Recovery Level 1 (Yo-Yo IR1). Methods: The study included 24 Brazilian male soccer players from under 20 category, of a club in the State of São Paulo, Brazil with stature of 1.72 ± 0.08 meter and body mass of 61.17 ± 9.18 kg, with at least five years of practice in the sport. The athletes performed the direct analysis of gases test on a treadmill 48 hours later the Yo-Yo IR1 was performed. Results: The results revealed a significant correlation between tests (r=0.524; p<0.01), but Yo-Yo IR1 underestimated measures of direct analysis of gases in the laboratory (44.98ml/kg/min and 48.14ml/kg/min, respectively). Conclusion: The results showed a moderate correlation between measures of VO2max, that way both tests can be used to analyze the aerobic power of soccer players, since the same protocol is repeated in subsequent evaluations. .

Introducción: Los métodos directos e indirectos son utilizados por los clubes de fútbol para evaluar, monitorear y determinar el VO2max de los jugadores, lo que es muy importante para el rendimiento y la recuperación de los atletas durante un partido. Objetivo: Evaluar el nivel de correlación entre las medidas de VO2max de análisis directo de gases y la prueba de campo Yo-Yo Intermitente Recovery Nivel 1 (Yo-YoIR1). Métodos: El estudio incluyó a 24 futbolistas categoría SUB-20 de un club en el Estado de São Paulo, Brasil, con estatura de 1,72 ± 0,08 metro y masa corporal de 61,17 ± 9,18 Kg, con al menos cinco años de práctica en la modalidad. Los atletas realizaron el análisis directo de los gases en una caminadora y después de 48 horas se realizó el Yo-Yo IR1. Resultados: Los resultados revelaron una correlación significativa entre los ensayos (r = 0,524, p < 0,01), pero el Yo-Yo IR1 ha subestimado las mediciones de laboratorio del análisis directo de gases (44,98 ml/Kg/min. y 48,14 ml/Kg/min., respectivamente). Conclusión: Los resultados indicaron una correlación moderada entre las medidas de VO2max, de esta manera se pueden utilizar ambas las pruebas para analizar la capacidad aeróbica de los jugadores de fútbol, siempre que se repita el mismo protocolo en las evaluaciones posteriores. .

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Objetivo: Este trabalho consistiu em avaliar biomarcadores de estresse oxidativo em portadores de insuficiência respiratória crônica (IResC) em três momentos: antes, após 7 e ao longo de 270 dias de oxigenoterapia domiciliar prolongada (ODP). Métodos: Foram medidas as atividades das enzimas catalase (CAT) e glutationa redutase (GR) no hemolisado, as concentrações de hemoglobina, lactato e ácido úrico (AU) no sangue total e as concentrações de grupamentos sulfidrilas totais (GST) e proteínas carboniladas (PC) no plasma desses pacientes por método espectrofotométrico. A saturação de oxigênio (SpO2) no sangue foi medida através de oxímetro de pulso. Comparamos, num primeiro momento indivíduos fumantes com pacientes com IResC e ambos com o grupo-controle, de não fumantes. Resultados: Observou-se que, embora os dois grupos possuam níveis aumentados de estresse oxidativo, este foi muito maior no grupo IResC, representado principalmente pela diminuição nas atividades das enzimas CAT, GR e concentração plasmática de GST. Após sete dias de tratamento com oxigênio houve um aumento na SpO2 (P < 0,05), CAT, GR e AU (P < 0,05). Por outro lado, a concentração de GST se manteve diminuída nesse período (P < 0,05). Os dados referentes aos pacientes submetidos a ODP ao longo de 270 dias mostraram que somente a atividade da GR se apresentou significativamente diminuída nesse período (P < 0.05). Conclusão: A hipoxemia crônica produz efeitos prejudiciais que não são revertidos com a administração prolongada de oxigênio, que não é capaz, portanto, de impedir a evolução clínica da doença...

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The aim of this study was to establish reference intervals (RIs) for the hemogram and iron status biomarkers in a physically active population. The study population included male volunteers (n = 150) with an average age of 19 ± 1 years who had participated in a regular and controlled exercise program for four months. Blood samples were collected to determine hematological parameters using a Sysmex XE-5000 analyzer (Sysmex, Kobe, Japan). Iron, total iron-binding capacity (TIBC), transferrin saturation and ferritin, and high-sensitivity C-reactive protein (CRP) concentrations in serum samples were measured using commercial kits (Roche Diagnostics, GmbH, Mannheim, Germany) and a Roche/Hitachi 902 analyzer. The RIs were established using the RefVal program 4.1b. The leucocyte count, TIBC, and CRP and ferritin concentrations exhibited higher RIs compared with those in a nonphysically active population. Thirty volunteers (outliers) were removed from the reference population due to blood abnormalities. Among the outliers, 46% exhibited higher CRP concentrations and lower concentrations of iron and reticulocyte hemoglobin compared with the nonphysically active population (P < 0.001). Our results showed that it is important to establish RIs for certain laboratory parameters in a physically active population, especially for tests related to the inflammatory response and iron metabolism.

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Tendons adapt to different mechanical stimuli through a remodeling process involving metalloproteinases (MMPs) and collagen synthesis. The purpose of this study was to investigate the activities of MMP-2 and MMP-9 and the collagen content in tendons after exhaustive acute exercise sessions over the course of 1, 3, or 6 days, with 1-hr or 3-hr rest periods between each session. Wistar rats were grouped into control (C), trained with 1-hr (groups 1d1h, 3d1h, and 6d1h) and trained with 3-hr (groups 1d3h, 3d3h and 6d3h) groups with rest periods between the treadmill running sessions, for 1, 3, and 6 days. The analysis of MMP-2 showed a larger presence of the latent isoform in the 1d3h group and a larger presence of the active isoform in the 6d3h group compared to the control. No differences were detected for MMP-9. A lower concentration of hydroxyproline was found in the 6d3h group compared to the 6d1h group. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed more prominent collagen bands in the 6d3h group, which was confirmed by Western blotting for collagen type I. A higher concentration of glycosaminoglycans was observed in the 3d3h group compared to the 3d1h group, and the 6d3h group presented the highest value for non-collagenous proteins compared to other groups. In conclusion, different rest periods between exercise sessions had different effects on the composition of the calcaneal tendon because a greater activation of MMP-2 and a reduction of total collagen were observed on day 6 of exercise with 3-hr rest periods compared to 1-hr rest periods.

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The use of saliva in the diagnosis of pathologies and/or monitoring of athletes in competitions or trainings is an attractive alternative due to the fact that samples are easily obtained and it is mostly a less invasive method in comparison with venous blood collection. The saliva is a hypotonic fluid in relation to plasma, containing compounds produced in the salivary glands (immunoglobulin A [IgA] and α-amylase) as well as compounds diffused in the plasma (water, electrolytes, proteins, metabolites and hormones). It plays a pivotal role in the protection of oral mucosa against microbes and in food digestion. Its production and composition depend on the sympathetic and parasympathetic nervous system activity, whose antagonistic action may result in different saliva volumes with distinct ionic and protein profiles. The aim of this review was to present a critical analysis of the potential and limitations of saliva as a diagnostic tool in sports medicine. Although there are studies that have deployed it to monitor athletes in training and doping, the standardization of some preanalytical variables are still required, among which the following ones are worth mentioning: the accurate choice of collection system, which allows the easy quantification of volume with adequate sample recovery; well-defined collection schedules in accordance with the circadian variations of the analyte; prevention of sample contamination with blood from oral mucosa lesions. Another key point for its application in sports is the establishment of reference intervals for analytes quantified in the saliva, collected from a population that comprises healthy individual that exercise regularly and systematically, with physical activity progression.

A utilização de saliva como alternativa para o diagnóstico de patologias e/ou monitoramento de atletas em competições ou treinos é muito atrativa devido à facilidade de obtenção da amostra e, principalmente, pela natureza menos invasiva que a coleta de sangue venoso. A saliva é um fluído hipotônico em relação ao plasma; contém compostos produzidos localmente nas glândulas salivares (imunoglobulina A [IgA] e α-amilase), além de compostos difundidos do plasma (água, eletrólitos, proteínas, metabólitos e hormônios). A saliva desempenha funções importantes na proteção da mucosa oral contra microrganismos e na digestão dos alimentos. Sua produção e sua composição são dependentes da atividade do sistema nervoso autônomo simpático e parassimpático, cuja ação antagônica pode resultar em diferentes volumes de saliva com perfis proteico e iônico distintos. O objetivo da presente revisão é apresentar uma análise crítica das potencialidades e limitações da utilização da saliva como ferramenta diagnóstica para a medicina esportiva. Embora existam estudos que a utilizam para o monitoramento de atletas em situações de exercício e doping, ainda é necessário padronizar algumas variáveis pré-analíticas, como a escolha correta do melhor sistema de coleta, que permite quantificar facilmente o volume, com boa recuperação de amostra; os horários de coleta bem definidos, de acordo com as possíveis variações circadianas do analito; e a contaminação da saliva com sangue proveniente de lesões da mucosa oral, que tem de ser evitada. Outro ponto fundamental para aplicação no esporte é o estabelecimento de valores de referência para analitos quantificados na saliva, obtidos de uma população composta de sujeitos saudáveis e exercitados de forma constante e sistematizada, com progressão de cargas de esforço.

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The purpose of this study was to observe the time course of muscle damage and inflammatory responses following an eccentric overload resistance-training (EO) program. 3 females (23.8 ± 2.6 years; 70.9 ± 12.7 kg; 1.6 ± 0.08 m) and 5 males (23.8 ± 2.6 years; 75.1 ± 11.2 kg; 1.8 ± 0.1 m) underwent thirteen training sessions (4 × 8-10 eccentric-only repetitions-80% of eccentric 1RM, one-minute rest, 2x week(-1), during 7 weeks, for three exercises). Blood samples were collected prior to (Pre) and after two (P2), seven (P7), nine (P9), eleven (P11), and thirteen (P13) sessions, always 96 hours after last session. The reference change values (RCV) analysis was employed for comparing the responses, and the percentual differences between the serial results were calculated for each subject and compared with RCV(95%). Four subjects presented significant changes for creatine kinase at P2, and another two at P13; six for C-reactive protein at P2, and three at P11; two for neutrophils at P2, P4, and P13, respectively; and only one for white blood cells at P2, P4, P7, and P9, for lymphocyte at P7, P9, and P13, and for platelet at P4. We conclude that EO induced high magnitude of muscle damage and inflammatory responses in the initial phase of the program with subsequent attenuation.

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