Polyphosphates have been found in all cell types examined to date and play diverse roles dependin... more Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been investigated mainly in mammalian cells, and only a few studies have addressed arthropods. Pyrophosphatases have been shown to regulate polyphosphate metabolism. However, these studies were restricted to trypanosomatids. Here we focus on the tick Rhipicephalus microplus, a haematophagous ectoparasite that is highly harmful to cattle. We produced a recombinant R. microplus pyrophosphatase (rRmPPase) with the aim of investigating its kinetic parameters using polyphosphates as substrate. Molecular docking assays of RmPPase with polyphosphates were also carried out. The kinetic and Hill coefficient parameters indicated that rRmPPase has a greater affinity, higher catalytic efficiency and increased cooperativity for sodium phosphate glass type 15 (polyP 15) than for sodium tripolyphosphate (polyP 3). Through molecular docking, we found that polyP 3 binds close to the Mg 21 atoms in the catalytic region of the protein, participating in their coordination network, whereas polyP 15 interactions involve negatively charged phosphate groups and basic amino acid residues, such as Lys56, Arg58 and Lys193; polyP 15 has a more favourable theoretical binding affinity than polyP 3 , thus supporting the kinetic data. This study shows, for the first time in arthropods, a pyrophosphatase with polyphosphatase activity, suggesting its participation in polyphosphate metabolism.
33 Resumo: A oxigenação extracorpórea por membrana (ECMO) é uma técnica de suporte cardiopulmonar... more 33 Resumo: A oxigenação extracorpórea por membrana (ECMO) é uma técnica de suporte cardiopulmonar prolongado que objetiva auxiliar os pulmões e/ou o coração quando estes apresentam processos de falência não responsiva aos tratamentos convencionais. Esta técnica representa um desafio para a medicina, que busca aperfeiçoá-la para torná-la um procedimento mais seguro, reduzindo suas complicações. O objetivo desta pesquisa foi avaliar o comportamento de variáveis hemodinâmicas (freqüência cardíaca, pressão arterial média e temperatura corporal) e bioquímicas séricas (glicose e lactato desidrogenase) em cães submetidos a oxigenação extracorpórea por membrana. Para tal utilizou-se uma amostra de 4 cães sem raça definida, de diferentes idades, peso e sexo. Os animais foram submetidos à ECMO por cateterização femoral (artéria e veia) depois de anestesiados, em um desvio caracterizado como artério-venoso (AV). As variáveis foram avaliadas a cada 30 minutos, durante 3 horas. Os resultados for...
To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to car... more To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer n...
The early diagnosis of sepsis increases the chances of its successful treatment. Biomarkers are a... more The early diagnosis of sepsis increases the chances of its successful treatment. Biomarkers are able to distinguish between systemic inflammatory response syndrome and sepsis and are used to monitor pro- and anti-inflammatory changes associated with the host response to pathogens. A total of 11 rats underwent sepsis induction and measured systolic, diastolic and mean arterial blood pressure. Leukocyte counts, procalcitonin, and nitric oxide also were measured 0, 2, and 4 hours after the induction of sepsis using the cecal ligation and puncture method. The animals were divided into two groups: control (SHAM) and induced. Procalcitonin levels remained within the normal range for an inflammatory response throughout the experiment. There was a statistically insignificant increase in nitric oxide levels. All animals showed increased diastolic arterial blood pressure; however, the increase in the induced animals was even more pronounced. Procalcitonin and nitric oxide levels can increase ...
Reactive oxygen species (ROS) are natural byproducts of metabolism that have toxic effects well d... more Reactive oxygen species (ROS) are natural byproducts of metabolism that have toxic effects well documented in mammals. In hematophagous arthropods, however, these processes are not largely understood. Here, we describe that Rhipicephalus microplus ticks and embryonic cell line (BME26) employ an adaptive metabolic compensation mechanism that confers tolerance to hydrogen peroxide (H2O2) at concentrations too high for others organisms. Tick survival and reproduction are not affected by H2O2 exposure, while BME26 cells morphology was only mildly altered by the treatment. Furthermore, H2O2-tolerant BME26 cells maintained their proliferative capacity unchanged. We evaluated several genes involved in gluconeogenesis, glycolysis, and pentose phosphate pathway, major pathways for carbohydrate catabolism and anabolism, describing a metabolic mechanism that explains such tolerance. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by glucose u...
Biochimica et Biophysica Acta (BBA) - General Subjects, 2017
Background: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate int... more Background: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. Methods: In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. Results: Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. Conclusions: Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. General significance: This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status.
It was evaluated 1.576 dogs with the intention to search positivity and to analyze hematological ... more It was evaluated 1.576 dogs with the intention to search positivity and to analyze hematological aspects of dogs infected with Ehrlichia spp. in Campos dos Goytacazes. The research of blood-parasites and complete blood counts had been carried out, to determine the occurrence and hematological aspects of dogs infected for Ehrlichia spp. The diagnosis was based by visualization of the Ehrlichia spp. morulae in the leukocytes, its great majority was in monocyte, being considered infected 219 (13,89%) dogs. Anemia, thrombocytopenia, nuclear left deviation neutrophils, eosinopenia and absolute monocytopenia was been the most hematological alterations found. Key-words: Blood smears, Ehrlichia spp., hematology. Com o propósito de pesquisar a positividade e analisar os aspectos hematológicos de cães infectados com Ehrlichia spp. na cidade de Campos dos Goytacazes, avaliaram-se 1.576 cães. Realizaram-se pesquisa de hemocitozoários e hemograma para se determinar a ocorrência e os aspectos hem...
A oxigenação extracorpórea por membrana com desvio arterio-venoso (AV-ECMO), em seres humanos, é ... more A oxigenação extracorpórea por membrana com desvio arterio-venoso (AV-ECMO), em seres humanos, é uma técnica de suporte à vida capaz de assumir em parte ou totalmente as funções dos pulmões, quando estes já não são capazes de promover uma adequada hematose. Para conhecer a viabilidade deste método em cães (Canis familiaris), o presente estudo aplicou a AV-ECMO em dez indivíduos adultos, sem raça definida, com uma expectativa de três horas de duração para cada experimento. No grupo 1 (n=4), ventilação mecânica e infusão contínua de dopamina foram associadas ao suporte extracorpóreo. No grupo 2 (n=6), nenhuma medida de terapia intensiva foi instituída concomitantemente à AV-ECMO. O comportamento dos parâmetros de gasometria e os níveis de lactato foram avaliados a cada 10 minutos e 1 hora, respectivamente, por meio de da avaliação de amostras sanguíneas arteriais. O suporte inotrópico foi capaz de promover uma melhor perfusão tecidual com sangue oxigenado, sugerida pelos níveis de lac...
Polyphosphates have been found in all cell types examined to date and play diverse roles dependin... more Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been investigated mainly in mammalian cells, and only a few studies have addressed arthropods. Pyrophosphatases have been shown to regulate polyphosphate metabolism. However, these studies were restricted to trypanosomatids. Here we focus on the tick Rhipicephalus microplus, a haematophagous ectoparasite that is highly harmful to cattle. We produced a recombinant R. microplus pyrophosphatase (rRmPPase) with the aim of investigating its kinetic parameters using polyphosphates as substrate. Molecular docking assays of RmPPase with polyphosphates were also carried out. The kinetic and Hill coefficient parameters indicated that rRmPPase has a greater affinity, higher catalytic efficiency and increased cooperativity for sodium phosphate glass type 15 (polyP 15) than for sodium tripolyphosphate (polyP 3). Through molecular docking, we found that polyP 3 binds close to the Mg 21 atoms in the catalytic region of the protein, participating in their coordination network, whereas polyP 15 interactions involve negatively charged phosphate groups and basic amino acid residues, such as Lys56, Arg58 and Lys193; polyP 15 has a more favourable theoretical binding affinity than polyP 3 , thus supporting the kinetic data. This study shows, for the first time in arthropods, a pyrophosphatase with polyphosphatase activity, suggesting its participation in polyphosphate metabolism.
33 Resumo: A oxigenação extracorpórea por membrana (ECMO) é uma técnica de suporte cardiopulmonar... more 33 Resumo: A oxigenação extracorpórea por membrana (ECMO) é uma técnica de suporte cardiopulmonar prolongado que objetiva auxiliar os pulmões e/ou o coração quando estes apresentam processos de falência não responsiva aos tratamentos convencionais. Esta técnica representa um desafio para a medicina, que busca aperfeiçoá-la para torná-la um procedimento mais seguro, reduzindo suas complicações. O objetivo desta pesquisa foi avaliar o comportamento de variáveis hemodinâmicas (freqüência cardíaca, pressão arterial média e temperatura corporal) e bioquímicas séricas (glicose e lactato desidrogenase) em cães submetidos a oxigenação extracorpórea por membrana. Para tal utilizou-se uma amostra de 4 cães sem raça definida, de diferentes idades, peso e sexo. Os animais foram submetidos à ECMO por cateterização femoral (artéria e veia) depois de anestesiados, em um desvio caracterizado como artério-venoso (AV). As variáveis foram avaliadas a cada 30 minutos, durante 3 horas. Os resultados for...
To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to car... more To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer n...
The early diagnosis of sepsis increases the chances of its successful treatment. Biomarkers are a... more The early diagnosis of sepsis increases the chances of its successful treatment. Biomarkers are able to distinguish between systemic inflammatory response syndrome and sepsis and are used to monitor pro- and anti-inflammatory changes associated with the host response to pathogens. A total of 11 rats underwent sepsis induction and measured systolic, diastolic and mean arterial blood pressure. Leukocyte counts, procalcitonin, and nitric oxide also were measured 0, 2, and 4 hours after the induction of sepsis using the cecal ligation and puncture method. The animals were divided into two groups: control (SHAM) and induced. Procalcitonin levels remained within the normal range for an inflammatory response throughout the experiment. There was a statistically insignificant increase in nitric oxide levels. All animals showed increased diastolic arterial blood pressure; however, the increase in the induced animals was even more pronounced. Procalcitonin and nitric oxide levels can increase ...
Reactive oxygen species (ROS) are natural byproducts of metabolism that have toxic effects well d... more Reactive oxygen species (ROS) are natural byproducts of metabolism that have toxic effects well documented in mammals. In hematophagous arthropods, however, these processes are not largely understood. Here, we describe that Rhipicephalus microplus ticks and embryonic cell line (BME26) employ an adaptive metabolic compensation mechanism that confers tolerance to hydrogen peroxide (H2O2) at concentrations too high for others organisms. Tick survival and reproduction are not affected by H2O2 exposure, while BME26 cells morphology was only mildly altered by the treatment. Furthermore, H2O2-tolerant BME26 cells maintained their proliferative capacity unchanged. We evaluated several genes involved in gluconeogenesis, glycolysis, and pentose phosphate pathway, major pathways for carbohydrate catabolism and anabolism, describing a metabolic mechanism that explains such tolerance. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by glucose u...
Biochimica et Biophysica Acta (BBA) - General Subjects, 2017
Background: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate int... more Background: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. Methods: In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. Results: Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. Conclusions: Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. General significance: This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status.
It was evaluated 1.576 dogs with the intention to search positivity and to analyze hematological ... more It was evaluated 1.576 dogs with the intention to search positivity and to analyze hematological aspects of dogs infected with Ehrlichia spp. in Campos dos Goytacazes. The research of blood-parasites and complete blood counts had been carried out, to determine the occurrence and hematological aspects of dogs infected for Ehrlichia spp. The diagnosis was based by visualization of the Ehrlichia spp. morulae in the leukocytes, its great majority was in monocyte, being considered infected 219 (13,89%) dogs. Anemia, thrombocytopenia, nuclear left deviation neutrophils, eosinopenia and absolute monocytopenia was been the most hematological alterations found. Key-words: Blood smears, Ehrlichia spp., hematology. Com o propósito de pesquisar a positividade e analisar os aspectos hematológicos de cães infectados com Ehrlichia spp. na cidade de Campos dos Goytacazes, avaliaram-se 1.576 cães. Realizaram-se pesquisa de hemocitozoários e hemograma para se determinar a ocorrência e os aspectos hem...
A oxigenação extracorpórea por membrana com desvio arterio-venoso (AV-ECMO), em seres humanos, é ... more A oxigenação extracorpórea por membrana com desvio arterio-venoso (AV-ECMO), em seres humanos, é uma técnica de suporte à vida capaz de assumir em parte ou totalmente as funções dos pulmões, quando estes já não são capazes de promover uma adequada hematose. Para conhecer a viabilidade deste método em cães (Canis familiaris), o presente estudo aplicou a AV-ECMO em dez indivíduos adultos, sem raça definida, com uma expectativa de três horas de duração para cada experimento. No grupo 1 (n=4), ventilação mecânica e infusão contínua de dopamina foram associadas ao suporte extracorpóreo. No grupo 2 (n=6), nenhuma medida de terapia intensiva foi instituída concomitantemente à AV-ECMO. O comportamento dos parâmetros de gasometria e os níveis de lactato foram avaliados a cada 10 minutos e 1 hora, respectivamente, por meio de da avaliação de amostras sanguíneas arteriais. O suporte inotrópico foi capaz de promover uma melhor perfusão tecidual com sangue oxigenado, sugerida pelos níveis de lac...
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