Effects of adenovirus-mediated shRNA down-regulates PTEN expression on fibril-binding proteins vinculin, filamin A and cortactin in activated hepatic stellate cells / 中华肝脏病杂志

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.

Construction of an adenovirus vector expressing engineered splicing factor for regulating alternative splicing of YAP1 in neonatal rat cardiomyocytes / 南方医科大学学报

OBJECTIVE@#To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes.@*METHODS@#The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag.@*RESULTS@#The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05).@*CONCLUSION@#We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.

Maternidade atrás das grades: em busca da cidadania e da saúde. Um estudo sobre a legislação brasileira / Motherhood behind bars: the struggle for citizens' rights and health for women inmates and their children in Brazil / La maternidad entre rejas: en búsqueda de la ciudadanía y de la salud. Un estudio sobre la legislación brasileña

Este estudo analisa as conexões entre saúde, direitos, legislação e políticas públicas a partir da pesquisa documental realizada no âmbito federal e nos estados do Rio Grande do Sul, Mato Grosso, Paraná e São Paulo, acerca das garantias legais das mulheres e seus filhos que vivem no cárcere. Busca instrumentalizar uma atuação garantista dos agentes públicos e dar visibilidade à problemática, diante das extremas vulnerabilidades e invisibilidade jurídica e administrativa da questão. Foram identificadas 33 normas legais, com pontos de tensão, como a possibilidade de prisão domiciliar e as disparidades quanto a prazos e condições de permanência das crianças no sistema penitenciário. A garantia legal constitucional do direito à amamentação é refletida nas regulamentações identificadas. Mas constatam-se ausências de outros aspectos relativos à maternidade na prisão, que se traduzem em dupla penalidade às mulheres, arbitrariamente estendida aos seus filhos. É necessária a ampliação e efetivação da regulamentação existente para prevenir e coibir as violações de direitos apontadas.

This study analyzes the links between health, rights, legislation, and public policies based on document research on legal safeguards for women and their children residing in prison. The research was conducted at the Federal level and in four States of Brazil: Rio Grande do Sul, Mato Grosso, Paraná, and São Paulo. The study aims to back measures by public agencies to guarantee such rights and to raise awareness of the problem, given the extreme vulnerability of women inmates and their children and the issue's legal and administrative invisibility. The authors identified 33 different legal provisions as points of tension, such as the possibility of house arrest and disparities in the terms and conditions for children to remain inside the prison system. Various provisions cite the Constitutional guarantee of women inmates' right to breastfeed in prison. Meanwhile, the study found gaps in other issues pertaining to motherhood in prison, expressed as dual incarceration (imprisonment arbitrarily extended to their children). It is necessary to expand and enforce the existing legislation to prevent such violations of rights.

Este estudio analiza las conexiones entre la salud, derechos humanos, legislación y políticas públicas, partiendo de una investigación documental, realizada a nivel federal y en los estados de Río Grande do Sul, Mato Grosso, Paraná y São Paulo, sobre las garantías jurídicas de las mujeres presas y sus hijos. El estudio pretende instrumentalizar una actuación garantista de los agentes públicos y dar visibilidad a esta problemática, frente a la extrema vulnerabilidad e invisibilidad jurídica y administrativa existente. Se identificaron 33 normas legales, con puntos de tensión, como la posibilidad de arresto domiciliario y disparidades en cuanto a los términos y condiciones de la estancia de los niños en el sistema penitenciario. La garantía constitucional del derecho a la lactancia materna se refleja en las regulaciones identificadas. No obstante, hay ausencias de otros aspectos de la maternidad en la cárcel, que se traduce en una doble pena para las mujeres, extendida arbitrariamente a sus hijos. Es necesaria la ampliación y ejecución efectiva de las regulación existente para prevenir y frenar las violaciones de los derechos.

Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.

Anti-tumor immunity elicited by adenovirus encoding AdhTrp2 or AdmTrp2 without vitiligo / 华中科技大学学报（医学）（英德文版）

To compare the difference in tumor immunity and autoimmunity elicited by adenovirus (Ad) encoding human or murine tyrosinase-related protein 2 (AdhTRP2 or AdmTRP2), and to find the most effective way to induce immunity by AdhTRP2 or AdmTRP2, C57BL/6 mice were immunized with AdhTRP2 or AdmTRP2 intramuscularly at different doses of 10(5), 10(6), 10(7) and 10(8) separately (10 mice for each dose). Two weeks after the immunization, in vivo CTL assay and intracellular staining (ICS) of IFN-gamma were carried out to analyze the dose-effect relationship. Tumor growth and vitiligo (as an sign of autoimmunity) were observed until 3 months after challenge with 10(5) B16F10 tumor cells. The results showed that Ad encoding AdmTrp2 induced weak tumor immune response. Similar immunization with AdhTrp-2 elicited stronger protective immunity. CTL activity and IFN-gamma-produced CD8+T cells were directly proportional to dose of AdhTrp2 or AdmTrp2. Moreover, AdhTrp2 group showed tumor rejection in 100% of challenged mice till the end of 3rd month while 60% of mice immunized with AdmTrp2 were protected against tumor. In the whole process of this experiment, no vitiligo was observed in mice immunized either with AdhTrp2 or AdmTrp2. It is concluded that anti-melanoma responses induced by genetic vaccination expressing xenoantigens breaks immune tolerance effectively and is able to elicit strong antigen-specific cytotoxic T cell response without vitiligo.

Frecuencia de rotavirus y adenovirus entre octubre 2005-agosto 2006 en el Instituto Médico La Floresta: Caracas-Venezuela / Frequency of rotavirus and adenovirus from october 2005-august 2006 at the Instituto Medico La Floresta: Caracas-Venezuela

La gastroenteritis clásica por rotavirus y adenovirus ocurre principalmente en niños entre 6 y 24 meses, pero es común en todos los grupos etarios, incluyendo adultos. Generalmente, los adultos han sido infectados, como lo demuestra la presión de anticuerpos séricos, sin embargo, no están protegidos contra nuevos episodios. Determinar la frecuencia de infección por rotavirus y adenovirus en el Instituto Médico La Florestas (IMLF), octubre 2005-agosto 2006. Estudio descriptivo, retrospectivo de datos obtenidos de la detección de rota/ adenovirus en heces por técnica inmunocromatografía del IMFL. Se procesaron 465 muestras de heces para rotavirus y adenovirus en todos los grupos etarios. De éstas, 162 (34,84 por ciento) muestras fueron positivas para ambos virus y sólo en 52 (32,10 por ciento) fue posible precisar si provenían de adultos o niños. En los niños, la frecuencia fue 80,77 por ciento, más elevada para adenovirus 42,31 por ciento, que para rotavirus 38,64 por ciento. En los adultos, fue 19,23 por ciento; con 13,46 por ciento y 5,77 por ciento para rotavirus y adenovirus respectivamente. La coinfección por ambos virus se encontró en 6 casos (11,54 por ciento. La infección por rotavirus, suele ser más leve en adultos; sin embargo, han sido reportadas muertes; y puede ser severa en inmunocomprometidos como lo reportado en esta casuística con shock hipolémico en un paciente trasplantado renal e insufiencia renal aguda y, trombocitopenia, cilindruria y proteinuria en un paicente adulto infectado por VIH. La afectación familiar con un niño como caso índice se reporta en dos grupos de familias. La infección por rotavirus y adenovirus se debe considerar como diagnóstico diferencial en pacientes adultos con diarrea independientemente de su condición inmunológica. Se reporta la casuística en un grupo pequeño de un centro privado. Se recomienda vigilancia epidemiológica nacional a todo nivel para conocer la frecuencia a mayor escala en todos los grupos etarios.