New tools are desirable to examine the metabolic state of individual cells within tissues. We proposed a fluorescence-based procedure consisting of acridine orange staining and fast green ...counterstaining (AO-FG) to improve the selectivity of the former for nucleic acids (acridine orange stains both DNA and RNA with different fluorescence colors), with no interference from proteins. We compared this test with the biochemical quantification of the relative amounts of RNA and DNA in selected rat ventral prostate samples and PC3 cells. The epithelium of the prostate gland is highly active metabolically for the production of secretions. Differences in AO-RNA staining were revealed and correlated with the metabolic state of the epithelium. Specificity was confirmed by RNase A. To assess how AO-FG staining correlates with the metabolic state of the cell, we cultured PC3 cells in different concentrations of glucose and measured the ratios between the amounts of RNA and DNA. In parallel, similar cultures were subjected to AO-FG, and the staining pattern correlated closely (r2=0.886) with the obtained biochemical results. The results confirmed that the combined use of AO and FG is useful for detecting DNA and RNA simultaneously, as well as for assessing quantitatively the transcriptional activity of individual cells and their changes in response to experimental manipulation.
Preclinical tests for evaluating potential drug candidates using conventional protocols can be exhaustive and high-cost processes. Microfluidic technologies that can speed up this process and allow ...fast screening of drugs are promising alternatives. This work presents the design, concept, and operational conditions of a simple, modular, and reversible sealing microdevice useful for drug screening. This microdevice allows for the operation of 4 parallel simultaneous conditions and can also generate a diffusive concentration gradient in sextuplicates. We used laminated polydimethylsiloxane (PDMSLAM) and glass as building materials as proof of concept. The PDMSLAM parts can be reused since they can be easily sterilized. We cultured MCF-7 (Michigan Cancer Foundation-7) breast cancer cells. Cells were exposed to a doxorubicin diffusive concentration gradient for 3 h. They were monitored by automated microscopy, and after data processing, it was possible to determine cell viability as a function of doxorubicin concentration. The reversible sealing enabled the recovery of the tested cells and image acquisition. Therefore, this microdevice is a promising tool for drug screening that allows assessing the cellular behavior in dynamic conditions and the recovery of cells for afterward processing and imaging.
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•The microdevice was designed to analyze different cell lines or different drugs simultaneously.•The operation allows four different parallel conditions in sextuplicates.•Diffusive concentration gradient using doxorubicin and kinetic death evaluation using MCF-7 cells.•Cells inside the microchambers were monitored by automated microscopy and image analysis.•The reversible sealing enabled cell recovery and SEM image acquisition.
O presente artigo tem por objetivo compreender a formação da mentalidade agrícola nos relatórios produzidos por professoras primárias rurais no estado de Sergipe, abrangendo os anos de 1948 e 1950. ...Os relatórios foram produzidos para fins de avaliação dos cursos de técnicas agrícolas realizados no Aviário Dom Pedro II, por solicitação do professor José Ribeiro Filho, mas acabaram por relevar aspectos sobre a formação das professoras rurais. Para o desenvolvimento desta narrativa histórica, optou-se pela pesquisa documental e pela pesquisa bibliográfica, a serem desenvolvidas por meio de localização e análise de fontes documentais, bem como de leitura de bibliografia especializada. Por fim, conclui-se que os relatórios foram produzidos em um período histórico marcado por iniciativas em moldar a escola primária em consonância com o meio rural para a construção de uma escola especificamente rural, ou seja, com desígnios, infraestrutura, programas de ensino e professores especializados.
Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor drugable by agonists approved for treatment of type 2 diabetes, but also inhibits carcinogenesis and cell ...proliferation in vivo. Activating mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene mitigate these beneficial effects by promoting a negative feedback-loop comprising extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated kinase kinase 1/2 (MEK1/2)-dependent inactivation of PPARγ. To overcome this inhibitory mechanism, we searched for novel post-translational regulators of PPARγ. Phosphoinositide phosphatase Myotubularin-Related-Protein-7 (MTMR7) was identified as cytosolic interaction partner of PPARγ. Synthetic peptides were designed resembling the regulatory coiled-coil (CC) domain of MTMR7, and their activities studied in human cancer cell lines and C57BL6/J mice. MTMR7 formed a complex with PPARγ and increased its transcriptional activity by inhibiting ERK1/2-dependent phosphorylation of PPARγ. MTMR7-CC peptides mimicked PPARγ-activation in vitro and in vivo due to LXXLL motifs in the CC domain. Molecular dynamics simulations and docking predicted that peptides interact with the steroid receptor coactivator 1 (SRC1)-binding site of PPARγ. Thus, MTMR7 is a positive regulator of PPARγ, and its mimicry by synthetic peptides overcomes inhibitory mechanisms active in cancer cells possibly contributing to the failure of clinical studies targeting PPARγ.
Many markers can be used in digestibility assays for swine, but needs to be evaluated before its use. An assay was carried out with the objective of measuring the coefficients of ileal apparent ...(CIAD) and standardized digestibility (CISD) of amino acids in meat and bone meal for swine using different markers. Eight crossbred barrows were equipped with T-cannulas in the distal ileum. Four animals received a free protein diet and another four received a diet containing meat and bone meal as the only protein source in the diet. The treatments consisted in evaluating the chromic oxide, acid-insoluble ash naturally occurring in the diet, cellulose and lignin as markers to determine the apparent and standardized digestibility of the amino acids contained in the meat and bone meal. All data were submitted to analysis of variance and means were compared using the Tukey test, with a significance of 5%. The CIAD, obtained using chromic oxide as external marker, were lower (P<0.05) than those obtained when acid insoluble ash, lignin and cellulose were used as markers, observing differences for all the amino acids evaluated. Similarly, the CISD from all the studied amino acids were lower (P<0.05) when the chromic oxide was used for determining the indigestibility factor. It is concluded that chromic oxide used as a external marker, to determine the coefficients of apparent and standardized ileal digestibility of the amino acids of the meat and bone meal, is most appropriate than the acid insoluble ash naturally occurring in diets, lignin and cellulose.
Muitos indicadores podem ser utilizados em ensaios de digestibilidade para suínos, mas precisam ser avaliados antes de serem utilizados. O experimento foi realizado com o objetivo de determinar os coeficientes de digestibilidade ileal aparentes (CDIA) e estandardizados (CDIE) dos aminoácidos da farinha de carne e ossos, para suínos, utilizando diferentes indicadores. Oito suínos, machos castrados, foram submetidos à cirurgia para aimplantação de cânulas T simples, no íleo distal. Quatro animais receberam uma dieta isenta de proteína e outros quatro uma dieta contendo farinha de carne e ossos como única fonte protéica na dieta. Os tratamentos consistiram em avaliar o óxido crómico, cinza insolúvel em ácido naturalmente contido na dieta, celulose e lignina como indicadores para determinar os CDIA e CDIE dos aminoácidos da farinha de carne e ossos. Os dados foram submetidos a análise de variância e as médias foram comparadas pelo teste de Tukey, ao nível de 5% de significância. Os CDIA, obtidos usando o óxido crômico como indicador externo, foram menores (P<0,05) em relação aos obtidos utilizando cinza insolúvel em ácido, lignina e celulose como indicadores, observando-se diferenças para todos os aminoácidos avaliados. O CDIE de todos os aminoácidos estudados foram menores (P<0,05) quando o óxido crômico foi utilizado para a determinação do fator de indigestibilidade. Conclui-se que o óxido de cromo utilizado como indicador externo, para determinar os coeficientes de digestibilidade ileal aparente e standarizado dos aminoácidos da farinha de carne e ossos, é mais apropriado do que a lignina, celulose e a cinza insolúvel em ácido naturalmente encontrada na dieta.
Many markers can be used in digestibility assays for swine, but needs to be evaluated before its use. An assay was carried out with the objective of measuring the coefficients of ileal apparent ...(CIAD) and standardized digestibility (CISD) of amino acids in meat and bone meal for swine using different markers. Eight crossbred barrows were equipped with T-cannulas in the distal ileum. Four animals received a free protein diet and another four received a diet containing meat and bone meal as the only protein source in the diet. The treatments consisted in evaluating the chromic oxide, acid-insoluble ash naturally occurring in the diet, cellulose and lignin as markers to determine the apparent and standardized digestibility of the amino acids contained in the meat and bone meal. All data were submitted to analysis of variance and means were compared using the Tukey test, with a significance of 5%. The CIAD, obtained using chromic oxide as external marker, were lower (P<0.05) than those obtained when acid insoluble ash, lignin and cellulose were used as markers, observing differences for all the amino acids evaluated. Similarly, the CISD from all the studied amino acids were lower (P<0.05) when the chromic oxide was used for determining the indigestibility factor. It is concluded that chromic oxide used as a external marker, to determine the coefficients of apparent and standardized ileal digestibility of the amino acids of the meat and bone meal, is most appropriate than the acid insoluble ash naturally occurring in diets, lignin and cellulose.Muitos indicadores podem ser utilizados em ensaios de digestibilidade para suínos, mas precisam ser avaliados antes de serem utilizados. O experimento foi realizado com o objetivo de determinar os coeficientes de digestibilidade ileal aparentes (CDIA) e estandardizados (CDIE) dos aminoácidos da farinha de carne e ossos, para suínos, utilizando diferentes indicadores. Oito suínos, machos castrados, foram submetidos à cirurgia para aimplantação de cânulas T simples, no íleo distal. Quatro animais receberam uma dieta isenta de proteína e outros quatro uma dieta contendo farinha de carne e ossos como única fonte protéica na dieta. Os tratamentos consistiram em avaliar o óxido crómico, cinza insolúvel em ácido naturalmente contido na dieta, celulose e lignina como indicadores para determinar os CDIA e CDIE dos aminoácidos da farinha de carne e ossos. Os dados foram submetidos a análise de variância e as médias foram comparadas pelo teste de Tukey, ao nível de 5% de significância. Os CDIA, obtidos usando o óxido crômico como indicador externo, foram menores (P<0,05) em relação aos obtidos utilizando cinza insolúvel em ácido, lignina e celulose como indicadores, observando-se diferenças para todos os aminoácidos avaliados. O CDIE de todos os aminoácidos estudados foram menores (P<0,05) quando o óxido crômico foi utilizado para a determinação do fator de indigestibilidade. Conclui-se que o óxido de cromo utilizado como indicador externo, para determinar os coeficientes de digestibilidade ileal aparente e standarizado dos aminoácidos da farinha de carne e ossos, é mais apropriado do que a lignina, celulose e a cinza insolúvel em ácido naturalmente encontrada na dieta.
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