Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Lab Chip ; 14(3): 592-601, 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24322338

RESUMO

Gene transfection is an important technology for various biological applications. The exogenous DNA is commonly delivered into cells by using a strong electrical field to form transient pores in cellular membranes. However, the high voltage required in this electroporation process may cause cell damage. In this study, a dielectrophoretically-assisted electroporation was developed by using light-activated virtual microelectrodes in a new microfluidic platform. The DNA electrotransfection used a low applied voltage and an alternating current to enable electroporation and transfection. Single or triple fluorescence-carrying plasmids were effectively transfected into various types of mammalian cells, and the fluorescent proteins were successfully expressed in live transfected cells. Moreover, the multi-triangle optical pattern that was projected onto a photoconductive layer to generate localized non-uniform virtual electric fields was found to have high transfection efficiency. The developed dielectrophoretically-assisted electroporation platform may provide a simpler system for gene transfection and could be widely applied in many biotechnological fields.


Assuntos
DNA/metabolismo , Luz , Técnicas Analíticas Microfluídicas , Transfecção/instrumentação , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Microeletrodos , Plasmídeos/metabolismo
2.
PLoS One ; 8(12): e81882, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312597

RESUMO

The peroxide regulator (PerR) is a ferric uptake repressor-like protein, which is involved in adaptation to oxidative stress and iron homeostasis in group A streptococcus. A perR mutant is attenuated in surviving in human blood, colonization of the pharynx, and resistance to phagocytic clearance, indicating that the PerR regulon affects both host environment adaptation and immune escape. Sda1 is a phage-associated DNase which promotes M1T1 group A streptococcus escaping from phagocytic cells by degrading DNA-based neutrophil extracellular traps. In the present study, we found that the expression of sda1 is up-regulated under oxidative conditions in the wild-type strain but not in the perR mutant. A gel mobility shift assay showed that the recombinant PerR protein binds the sda1 promoter. In addition, mutation of the conserved histidine residue in the metal binding site of PerR abolished sda1 expression under hydrogen peroxide treatment conditions, suggesting that PerR is directly responsible for the sda1 expression under oxidative stress. Our results reveal PerR-dependent sda1 expression under oxidative stress, which may aid innate immune escape of group A streptococcus.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Desoxirribonuclease I/genética , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/virologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Metais/metabolismo , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Streptococcus pyogenes/genética , Sítio de Iniciação de Transcrição
3.
Biomed Microdevices ; 15(2): 339-52, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23315192

RESUMO

Ovarian cancer is the second most common of the gynecological cancers in Taiwan. It is challenging to diagnose at an early stage when proper treatment is the most effective. It is well recognized that the detection of tumor cells (TCs) is critical for determining cancer growth stages and may provide important information for accurate diagnosis and even prognosis. In this study, a new microfluidic platform integrated with a moving-wall micro-incubator, a micro flow cytometer and a molecular diagnosis module performed automated identification of ovarian cancer cells. By efficiently mixing the cells and immunomagnetic beads coated with specific antibodies, the target TCs were successfully isolated from the clinical samples. Then counting of the target cells was achieved by a combination of the micro flow cytometer and an optical detection module and showed a counting accuracy as high as 92.5 %. Finally, cancer-associated genes were amplified and detected by the downstream molecular diagnosis module. The fluorescence intensity of specific genes (CD24 and HE4) associated with ovarian cancer was amplified by the molecular diagnosis module and the results were comparable to traditional slab-gel electrophoresis analysis, with a limit of detection around 10 TCs. This integrated microfluidic platform realized the concept of a "lab-on-a-chip" and had advantages which included automation, disposability, lower cost and rapid diagnosis and, therefore, may provide a promising approach for the fast and accurate detection of cancer cells.


Assuntos
Biomarcadores Tumorais/análise , Contagem de Células/instrumentação , Separação Celular/instrumentação , Citometria de Fluxo/instrumentação , Técnicas de Diagnóstico Molecular/instrumentação , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/metabolismo , Linhagem Celular Tumoral , Desenho de Equipamento , Análise de Falha de Equipamento , Feminino , Humanos , Imunoensaio/instrumentação , Separação Imunomagnética/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de Sistemas
4.
Mol Cell Proteomics ; 10(9): M110.007013, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21636680

RESUMO

Streptococcus pyogenes is a human pathogen that causes various diseases. Numerous virulence factors secreted by S. pyogenes are involved in pathogenesis. The peroxide regulator (PerR) is associated with the peroxide resistance response and pathogenesis, but little is known about the regulation of the secretome involved in virulence. To investigate how PerR regulates the expression of the S. pyogenes secretome involved in virulence, a perR deficient mutant was used for comparative secretomic analysis with a wild-type strain. The conditioned medium containing secreted proteins of a wild-type strain and a perR deficient mutant at the stationary phase were collected for two-dimensional gel electrophoresis analysis, where protease inhibitors were applied to avoid the degradation of extracellular proteins. Differentially expressed protein spots were identified by liquid chromatography electrospray ionization tandem MS. More than 330 protein spots were detected on each gel. We identified 25 unique up-regulated proteins and 13 unique down-regulated proteins that were directly or indirectly controlled by the PerR regulator. Among these identified proteins, mitogen factor 3 (MF3), was selected to verify virulence and the expression of gene products. The data showed that MF3 protein levels in conditioned medium, as measured by immunoblot analysis, correlated well with protein levels determined by two-dimensional gel electrophoresis analysis. We also demonstrated that PerR bound to the promoter region of the mf3 gene. The result of an infection model showed that virulence was attenuated in the mf3 deficient mutant. Additional growth data of the wild-type strain and the mf3 deficient mutant suggested that MF3 played a role in digestion of exogenous DNA for promoting growth. To summarize, we conclude that PerR can positively regulate the expression of the secreted protein MF3 that contributes to the virulence in S. pyogenes. The analysis of the PerR-regulated secretome provided key information for the elucidation of the host-pathogen interactions and might assist in the development of potential chemotherapeutic strategies to prevent or treat streptococcal diseases.


Assuntos
Proteínas de Bactérias , Desoxirribonucleases , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Proteômica/métodos , Proteínas Repressoras , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultivo Condicionados/química , Desoxirribonucleases/deficiência , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Eletroforese em Gel Bidimensional , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Deleção de Genes , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peróxidos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica/genética , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Infecções Estreptocócicas/mortalidade , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Taxa de Sobrevida , Fatores de Virulência/química , Fatores de Virulência/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...