An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells.

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza's Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.

Efecto de la insulina humana sobre el crecimiento y expresión de proteínas celulares de Klebsiella pneumoniae aislada de pie diabético / Effect of human insulin on growth and cellular protein expression of Klebsiella pneumoniae isolated from diabetic foot infections

Algunos microorganismos patógenos aumentan su proliferación al cultivarlos en presencia de insulina, por lo tanto, la hiperinsulinemia podría influir sobre las infecciones de pacientes diabéticos. En este trabajo se determinó el efecto de la insulina sobre el crecimiento y expresión de proteínas celulares de Klebsiella pneumoniae aislada de pie diabético. Las bacterias se cultivaron en medio Luria-Bertani en presencia o en ausencia de insulina humana. El crecimiento se determinó midiendo la DO600 de los cultivos hasta alcanzar la fase estacionaria; se colectaron bacterias a diferentes tiempos para extraer sus proteínas celulares (extractos) y analizarlas mediante electroforesis en geles de poliacrilamida-SDS y densitometría cuantitativa. En presencia de insulina (0,5 U/mL) el crecimiento bacteriano se incrementó en 40% respecto al control en las fases logarítmica temprana y media. Todos los perfiles electroforéticos de los extractos mostraron 27 bandas polipeptídicas (rango 150-9 kDa). La expresión del 41% de estas bandas aumentó en los extractos de las bacterias cultivadas con insulina, respecto a los controles, mientras que la expresión del 22% disminuyó. Los resultados indicaron que la insulina incrementó la proliferación y moduló la expresión genética de K. pneumoniae, sugiriendo que la hiperinsulinemia podría favorecer la severidad de infecciones en pacientes diabéticos.

Some pathogens increase their proliferation when cultured in the presence of insulin, therefore, hyperinsulinemia may influence diabetic infections. In this work we determine the effect of insulin on growth and cellular protein expression from Klebsiella pneumoniae isolated from diabetic foot infections. Bacteria were grown in Luria-Bertani medium in the presence and absence of human insulin. Growth was determined by measuring OD600 of the cultures until reached the stationary phase. In order to extract cellular proteins, bacteria were collected at different times to obtain extracts that were analyzed by electrophoresis on SDS-polyacrylamide gels and quantitative densitometry. In the presence of insulin (0.5 U/mL) bacterial growth increased by 40% compared with control in the early and middle logarithmic phase. All electrophoretic profiles of the extracts showed 27 polypeptide bands (range 150-9 kDa). The expression of 41% of these bands increased in extracts from bacteria grown with insulin when compared with controls, whereas protein expression decreased by 22%. The results indicated that insulin increased K. pneumoniae proliferation and modulated gene expression, suggesting that hyperinsulinemia could contribute to the severity of the infections in diabetic patients.

Conduta dos agentes comunitários de saúde diante de casos de violência familiar / The conduct of community health agents in cases of family violence / Conducta de los agentes comunitarios de salud ante casos de violencia familiar

Este estudo teve como objetivo identificar a conduta dos agentes comunitários de saúde (ACS) inseridos na Estratégia de Saúde da Família diante da suspeita ou detecção dos casos de violência familiar. Trata-se de pesquisa descritiva de natureza qualitativa realizada junto a ACS, utilizando entrevistas. Os dados foram submetidos à análise de conteúdo, o que resultou em duas categorias. A primeira se refere ao rastreamento da violência doméstica à mulher. Nela os agentes comunitários de saúde relatam as suspeitas ou a detecção de casos e a forma que estes assumiram. Na segunda categoria são referenciadas quais as condutas adotadas, emergindo as possibilidades de abordagens às vítimas e as limitações do serviço. Os resultados mostram que a violência doméstica é identificada pelos agentes comunitários de saúde, indicando a necessidade de estabelecer estratégias que contribuam para uma melhor atenção à mulher e para a prevenção da violência familiar...

The objective of this study was to identify the conduct of community health agents (CHA) of the Family Health Strategy in cases of suspected or confirmed family violence. This qualitative, descriptive study was performed by interviewing CHAs. The content analysis of the data revealed two categories. The first refers to tracking domestic violence against women. In this category, community health agents reported suspected or confirmed cases and their form. The second category consists of reports regarding the conducts that were adopted, revealing the possible approaches used with victims and the limitations of the service. Results showed that community health agents identify domestic violence, thus indicating the need to establish strategies that contribute with improving women's care and preventing family violence...

Estudio que objetivó identificar la conducta de agentes comunitarios de salud (ACS) desempeñándose en la Estrategia Salud de la Familia ante la sospecha o detección de casos de violencia familiar. Investigación descriptiva, cualitativa, realizada junto a Agentes Comunitarios de Salud, utilizándose entrevistas. Datos sometidos a análisis de contenido, resultando dos categorías. La primera se refiere al rastreo de la violencia doméstica a la mujer. En ella, los Agentes Comunitarios de Salud informan las sospechas o la detección de casos y la forma que dichos casos asumen. En la segunda categoría son explicitadas las conductas adoptadas, emergiendo las posibilidades de abordaje a las víctimas y las limitaciones del servicio. Los resultados demuestran que la violencia doméstica es identificada por los Agentes Comunitarios de Salud, indicando la necesidad de establecer estrategias que contribuyan a una mejor atención de la mujer y a la prevención de la violencia familiar...

Effects of short-term and long-term treatment with medium- and long-chain triglycerides ketogenic diet on cortical spreading depression in young rats.

The ketogenic diet (KD) is a high fat and low carbohydrate and protein diet. It is used in the clinical treatment of epilepsy, in order to decrease cerebral excitability. KD is usually composed by long-chain triglycerides (LCT) while medium-chain triglycerides (MCT) diet is beginning to be used in some clinical treatment of disorders of pyruvate carboxylase enzyme and long-chain fatty acid oxidation. Our study aimed to analyze the effects of medium- and long-chain KD on cerebral electrical activity, analyzing the propagation of the phenomenon of cortical spreading depression (CSD). Three groups of weaned rats (21 days old) received, for 7 weeks, either a control (AIN-93G diet), or a MCT-KD (rich in triheptanoin oil), or a LCT-KD (rich in soybean oil). They were compared to another three groups (21 days old) receiving the same diets for just 10 days. CSD propagation was evaluated just after ending the dietary treatments. Results showed that short-term KD treatment resulted in a significant reduction of the CSD velocity of propagation (control group: 4.02+/-1.04mm/min; MCT-KD: 0.81+/-1.46mm/min and LCT-KD: 2.26+/-0.41mm/min) compared to the control group. However, long-term treatment with both KDs had no effect on the CSD velocity (control group: 3.10+/-0.41mm/min, MCT-KD: 2.91+/-1.62mm/min, LCT-KD: 3.02+/-2.26mm/min) suggesting that both short-term KDs have a positive effect in decreasing brain cerebral excitability in young animals. These data show for the first time that triheptanoin has an effect on central nervous system.