Dissection of miRNA Pathways Using Arabidopsis Mesophyll Protoplasts

MicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.Fundação para a Ciência e Tecnologia fellowships: (SFRH/BD/33563/2008, SFRH/BPD/47280/2008, SFRH/BPD/79255/2011) and grant: (PTCD/BIA-BCM/107924/2008); EMBO fellowship & EMBO Installation program; Deutsche Forschungsgemeinschaft grant: (SPP1530); Max Planck Society grant

Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases

The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems.Fundação para a Ciência e Tecnologia fellowships (SFRH/BPD/79255/2011,SFRH/BPD/47280/2008, SFRH/BD/51627/2011, SFRH/BD/33563/2008 ); EMBO Installation Program, Marie Curie Actions FP7-People- 2010-ITN (MERIT)

Avaliação horizontal da distribuição de sementes de milho em função de mecanismos dosadores e velocidade operacional

The type of operation of the seed drill metering mechanism and the operating speed are determinant in the crop's productivity. Thus, the objective was to evaluate the longitudinal distribution of maize seeds with different seed metering mechanisms at different displacement speeds. The study was carried out at the Experimental Farm of Agricultural Sciences - FAECA of the Federal University of Grande Dourados (UFGD), in the municipality of Dourados, Mato Grosso do Sul (MS). The experimental design used was completely randomized in a 2 x 4 factorial scheme, with two seed dosers (pneumatic and mechanical) and four sowing displacement speeds (5, 7, 8 and 10 km-1). The longitudinal distribution of seeds was evaluated by checking the spacing between acceptable, flawed and multiple seedlings. The data were submitted to analysis of variance and the means were compared by the Tukey test at 5%. There was no significant difference up to the speed of 8 km h-1 for the mechanical feeder in the analysis of the performance of each mechanism within the different speeds. However, the pneumatic feeder achieved superior performance for faulty and double spacing. In general, for both metering mechanisms, the increase in sowing speed increased the number of faulty and double spacings. Therefore, the pneumatic metering mechanism showed a better response in the number of acceptable spaces with increasing speed.O tipo de funcionamento do mecanismo dosador das semeadoras e a velocidade de operação são determinantes na produtividade da cultura. Assim, objetivou-se avaliar a distribuição longitudinal de sementes de milho com diferentes mecanismos dosadores de sementes em diferentes velocidades de deslocamento. O estudo foi realizado na Fazenda Experimental de Ciências Agrárias - FAECA da Universidade Federal da Grande Dourados (UFGD), no município de Dourados, Mato Grosso do Sul (MS). O delineamento experimental utilizado foi inteiramente casualizado em esquema fatorial 2 x 4, sendo dois dosadores de sementes (pneumático e mecânico) e quatro velocidades de deslocamento da semeadura (5, 7, 8 e 10 km-1). Foi avaliada a distribuição longitudinal de sementes por meio da verificação do espaçamento entre plântulas em aceitável, falho e múltiplo. Os dados foram submetidos à análise de variação e as médias comparadas pelo teste de Tukey a 5%. Não houve diferença significativa até a velocidade de 8 km h-1 para o dosador mecânico na análise do desempenho de cada mecanismo dentro das diferentes velocidades. No entanto, o dosador pneumático obteve desempenho superior para espaçamentos falhos e duplos. De modo geral, para ambos os mecanismos dosadores o aumento da velocidade de semeadura proporcionou acréscimo no número de espaçamentos falhos e duplos. Portanto, o mecanismo dosador pneumático apresentou melhor resposta no número de espaçamento aceitável de distribuição com o incremento da velocidade

Characterization of Systemic Disease Development and Paw Inflammation in a Susceptible Mouse Model of Mayaro Virus Infection and Validation Using X-ray Synchrotron Microtomography

Mayaro virus (MAYV) is an emerging arthropod-borne virus endemic in Latin America and the causative agent of arthritogenic febrile disease. Mayaro fever is poorly understood; thus, we established an in vivo model of infection in susceptible type-I interferon receptor-deficient mice (IFNAR−/−) to characterize the disease. MAYV inoculations in the hind paws of IFNAR−/− mice result in visible paw inflammation, evolve into a disseminated infection and involve the activation of immune responses and inflammation. The histological analysis of inflamed paws indicated edema at the dermis and between muscle fibers and ligaments. Paw edema affected multiple tissues and was associated with MAYV replication, the local production of CXCL1 and the recruitment of granulocytes and mononuclear leukocytes to muscle. We developed a semi-automated X-ray microtomography method to visualize both soft tissue and bone, allowing for the quantification of MAYV-induced paw edema in 3D with a voxel size of 69 µm3. The results confirmed early edema onset and spreading through multiple tissues in inoculated paws. In conclusion, we detailed features of MAYV-induced systemic disease and the manifestation of paw edema in a mouse model extensively used to study infection with alphaviruses. The participation of lymphocytes and neutrophils and expression of CXCL1 are key features in both systemic and local manifestations of MAYV disease

Indications for head computed tomography in children with mild traumatic brain injury

The Evidence Based Telemedicine - Trauma and Emergency Surgery (TBE-CITE) performed a critical appraisal of the literature and selected the three most relevant and recent publications on the indications for head computed tomography (CT) scan in pediatric patients with mild traumatic brain injury (TBI). The first study identified patients with mild TBI, high and low risk factors for intracranial injuries detected on CT scan and need for neurosurgical intervention. The second study evaluated the guidelines of the National Institute of Clinical Excellence for pediatric patients with TBI. The outcome of this study was either performing a head CT scan or hospital admission. The last study identified and analyzed the patients in whom the CT scan is not necessary and consequently should not be routinely indicated. Based on the critical appraisal of the literature and expert discussion, the opinion of the TBE-CITE was to favor the adoption of the PECARN guidelines, proposing CT scans for children with GCS of 14, altered level of consciousness and palpable skull fracture, or when the physician experience, multiple findings or worsening symptoms warrant it.A reunião de revista Telemedicina Baseada em Evidência - Cirurgia do Trauma e Emergência (TBE-CiTE) realizou uma revisão crítica da literatura e selecionou os três artigos mais relevantes e atuais sobre a indicação de tomografia de crânio em pacientes pediátricos com trauma craniencefálico leve (TCE). O primeiro trabalho identificou pacientes vítimas de TCE leve com fatores de alto e baixo risco de apresentarem lesões intracranianas vistas à tomografia computadorizada (TC) de crânio e com necessidade de intervenção neurocirúrgica. O segundo trabalho avaliou o uso das recomendações do National Institute of Clinical Excellence em pacientes pediátricos com TCE, e utilizou como variáveis de desfecho a realização de TC ou internação hospitalar. O último artigo analisou e identificou os pacientes onde a TC de crânio seria desnecessária e, portanto, não deve ser feita rotineiramente. Baseado nessa revisão crítica da literatura e a discussão com especialistas, o TBE-CiTE concluiu que é importante evitar a exposição desnecessária de crianças com TCE leve à radiação ionizante da TC de crânio. O grupo favoreceu a utilização do guideline do PECARN onde ECG de 14, alteração do nível de consciência ou fratura do crânio palpável são indicações de TC de crânio, ou quando a experiência do médico, achados múltiplos ou piora dos sintomas ocorrerem.51551

Foreign Body Aspiration In Children: Clinical Aspects, Radiological Aspects And Bronchoscopic Treatment.

To describe the clinical manifestations and bronchoscopic treatment of foreign body aspiration in children under 14 years of age, correlating the clinical aspects with the bronchoscopic findings. A retrospective, descriptive study analyzing data related to children under 14 years of age undergoing bronchoscopy due to clinical suspicion of foreign body aspiration at the State University at Campinas Hospital das Clinicas from January of 2000 to December of 2005. The sample consisted of 69 patients, ranging in age from 8 months to 12 years/7 months (75.4% under 3 years of age), 62.3% of whom were male. The principal complaint was sudden-onset cough (75.4%), auscultation was abnormal in 74%, and dyspnea was observed in 29%. Radiological abnormalities were seen in 88% of the cases. Aspirations were primarily into the right lung (54.8%), and 30.7% of the foreign bodies were of vegetal origin (principally beans and peanuts). In the follow-up period, 29% presented complications (most commonly pneumonia), which were found to be associated with longer aspiration time (p = 0.03). Mechanical ventilation was required in 7 children (10.1%), and multiple bronchoscopies were performed in 5 (7.2%). A history of sudden-onset choking and cough, plus abnormal auscultation and radiological findings, characterizes the profile of foreign body aspiration. In such cases, bronchoscopy is indicated. Longer aspiration time translates to a higher the risk of complications. The high prevalence of foreign bodies of vegetal origin underscores the relevance of prevention programs aimed at children younger than 3 years of age.3474-8

Foreign body aspiration in children: clinical aspects, radiological aspects and bronchoscopic treatment

OBJECTIVE: To describe the clinical manifestations and bronchoscopic treatment of foreign body aspiration in children under 14 years of age, correlating the clinical aspects with the bronchoscopic findings. METHODS: A retrospective, descriptive study analyzing data related to children under 14 years of age undergoing bronchoscopy due to clinical suspicion of foreign body aspiration at the State University at Campinas Hospital das Clinicas from January of 2000 to December of 2005. RESULTS: The sample consisted of 69 patients, ranging in age from 8 months to 12 years/7 months (75.4% under 3 years of age), 62.3% of whom were male. The principal complaint was sudden-onset cough (75.4%), auscultation was abnormal in 74%, and dyspnea was observed in 29%. Radiological abnormalities were seen in 88% of the cases. Aspirations were primarily into the right lung (54.8%), and 30.7% of the foreign bodies were of vegetal origin (principally beans and peanuts). In the follow-up period, 29% presented complications (most commonly pneumonia), which were found to be associated with longer aspiration time (p = 0.03). Mechanical ventilation was required in 7 children (10.1%), and multiple bronchoscopies were performed in 5 (7.2%). CONCLUSION: A history of sudden-onset choking and cough, plus abnormal auscultation and radiological findings, characterizes the profile of foreign body aspiration. In such cases, bronchoscopy is indicated. Longer aspiration time translates to a higher the risk of complications. The high prevalence of foreign bodies of vegetal origin underscores the relevance of prevention programs aimed at children younger than 3 years of age.Descrever manifestações clínicas e tratamento broncoscópico da aspiração de corpo estranho em crianças menores de 14 anos de idade, correlacionando com achados broncoscópicos. MÉTODOS: Estudo retrospectivo, descritivo analisando prontuários de todas as crianças menores de 14 anos de idade atendidas no Hospital das Clinicas da Universidade Estadual de Campinas de janeiro de 2000 a dezembro de 2005, submetidas à broncoscopia por suspeita clínica de aspiração de corpo estranho. RESULTADOS: Foram analisados 69 pacientes, com idade entre 8 meses e 12 anos/7 meses (75,4% abaixo de 3 anos), dos quais 62,3% eram do sexo masculino. A principal queixa foi tosse súbita (75,4%). Em 74% dos casos houve alteração de ausculta pulmonar e dispnéia foi observada em 20 crianças (29%). Um total de 88% apresentou alteração radiológica. A aspiração ocorreu predominantemente em pulmão direito (54,8%), com material de origem vegetal, destacando feijão e amendoim (30,7%). Complicações ocorreram em 29% dos pacientes, sendo pneumonia a mais comum, e foram associadas ao tempo maior de aspiração (p = 0,03). Um total de 7 pacientes (10,1%) necessitaram ventilação mecânica, e 5 (7,2%) foram submetidos a mais de uma broncoscopia. CONCLUSÃO: História clínica com início súbito de engasgo e tosse, anormalidades na ausculta pulmonar e na radiografia de tórax caracterizam o quadro clínico de aspiração e são indicativas de broncoscopia. Quanto maior o tempo de aspiração, maior o risco de complicações. A alta prevalência de corpos estranhos de origem vegetal alerta para a necessidade de programas preventivos dirigidos aos menores de 3 anos3427482To describe the clinical manifestations and bronchoscopic treatment of foreign body aspiration in children under 14 years of age, correlating the clinical aspects with the bronchoscopic findings. METHODS: A retrospective, descriptive study analyzing data related to children under 14 years of age undergoing bronchoscopy due to clinical suspicion of foreign body aspiration at the State University at Campinas Hospital das Clinicas from January of 2000 to December of 2005. RESULTS: The sample consisted of 69 patients, ranging in age from 8 months to 12 years/7 months (75.4% under 3 years of age), 62.3% of whom were male. The principal complaint was sudden-onset cough (75.4%), auscultation was abnormal in 74%, and dyspnea was observed in 29%. Radiological abnormalities were seen in 88% of the cases. Aspirations were primarily into the right lung (54.8%), and 30.7% of the foreign bodies were of vegetal origin (principally beans and peanuts). In the follow-up period, 29% presented complications (most commonly pneumonia), which were found to be associated with longer aspiration time (p = 0.03). Mechanical ventilation was required in 7 children (10.1%), and multiple bronchoscopies were performed in 5 (7.2%). CONCLUSION: A history of sudden-onset choking and cough, plus abnormal auscultation and radiological findings, characterizes the profile of foreign body aspiration. In such cases, bronchoscopy is indicated. Longer aspiration time translates to a higher the risk of complications. The high prevalence of foreign bodies of vegetal origin underscores the relevance of prevention programs aimed at children younger than 3 years of ag

Prevalência de inatividade física e fatores associados em adolescentes

OBJETIVO: Estimar a prevalência de inatividade física em adolescentes (14 a 18 anos) da cidade de Maringá/PR e explorar sua associação com variáveis demográficas, socioeconômicas, comportamental e indicadores de estado nutricional. MÉTODOS: Estudo transversal com uma amostra representativa de escolares do ensino médio da cidade incluindo 991 (54,5% moças) de 12 colégios públicos e privados selecionados por meio de amostragem em múltiplos estágios. O nível de atividade física habitual foi verificado por meio do IPAQ modificado para adolescentes, usando como referência a última semana. A inatividade física foi definida 4 h/dia), estado nutricional e obesidade abdominal. RESULTADOS: A prevalência de inatividade física em adolescentes foi de 56,9% (moças= 57,9%, rapazes= 55,7%, p=0,46). Os fatores de risco associados à inatividade física foram pertencer ao nível socioeconômico mais baixo, estudar em escolas públicas e ser obeso. CONCLUSÃO: Encontramos uma alta prevalência de inatividade física no estudo. Faz-se urgentemente necessário o desenvolvimento de estratégias, que visem o aumento da atividade física, o que pode ser obtido por meio de desenvolvimento de conteúdos escolares que promovam estilos de vida saudável.OBJECTIVE: To estimate the prevalence of physical inactivity among adolescents (14-18 years) in the city of Maringá/PR and to explore its association with demographic, socioeconomic and behavioral, indicators of nutritional status variables. METHODS: Cross-sectional study with a representative sample of 991 adolescents (54.5% girls) from both public and private high schools selected through multi stage random sampling. The level of habitual physical activity was verified through the IPAQ modified for adolescents, using the last week as a reference period. Physical inactivity was defined as 4 h/d), nutritional status and abdominal obesity. RESULTS: Prevalence of physical inactivity among adolescents was 56.9% (girls = 57.9%, boys = 55.7%, p =. 046). The risk factors associated with physical inactivity were belonging to the lower socioeconomic level, studying at public schools and being obese. CONCLUSION: We found a high prevalence of physical inactivity in the study. It is urgently necessary to develop strategies aimed at increasing physical activity, which can be achieved by development of an educational content that promotes healthy life styles