Processo autofágico no reconhecimento de Escherichia coli enteroinvasora: um possível mecanismo da degradação bacteriana por células epiteliais / Autophagy in recognition of Enteroinvasive Escherichia coli: a possible bacterial degradation mechanism by epithelial cells

O reconhecimento de bactérias invasoras pelas células hospedeiras através do processo autofágico é um fator chave na determinação da infecção bacteriana. Escherichia coli enteroinvasora (EIEC) possui uma proteína, denominada IcsB, que em estudos em Shigella, é responsável pela inativação deste processo de degradação bacteriana. Uma vez que EIEC expressa menos IcsB do que S. flexneri, nos propusemos a investigar o processo autofágico na infecção por EIEC, utilizando as técnicas de mutação gênica por inserção, western-blot, microscopia de fluorescência e eletrônica de transmissão e microarray. Verificamos que a proteína IcsB é um fator de virulência importante na camuflagem de EIEC, pois quando pouco ou nada expresso, há um maior reconhecimento da bactéria pelas células hospedeiras, favorecendo sua menor disseminação. Isto corrobora não somente com a transcrição gênica, mas com a importância da sequência de nucleotídeos deste gene, uma vez que a cepa de E. coli SM124/13 complementada com o icsB de Shigella se mostrou mais eficiente na disseminação dentro da célula hospedeira. De forma interessante, IcsB apresentou um papel inédito na regulação da resposta inflamatória das células HeLa, onde a ausência de IcsB em EIEC promoveu uma intensa perturbação na homeostase da célula hospedeira, com aumento da secreção de IL-6, IL-8 e morte celular. Adicionalmente, ficou evidente que a célula eucariótica responde de maneira distinta frente a infecção por EIEC e Shigella flexneri. EIEC provavelmente ativou o processo autofágico em células humanas de forma não canônica. Nossa hipótese seria de que EIEC é reconhecida pelo processo autofágico, podendo ser este um importante fenômeno de reconhecimento bacteriano que colabore para a menor disseminação intracelular de EIEC, e assim tornar sua doença mais branda, quando comparada com a infecção por Shigella

The invasive bacteria recognition by host cells through autophagy is a key factor for determining bacterial infection. Enteroinvasive Escherichia coli (EIEC) express a protein IcsB, which in Shigella, is known for inactivating the bacterial degradation process. Once EIEC showed less expression of icsB when compared to S. flexneri, we proposed to investigate the autophagy caused by EIEC infection, using techniques such as gene mutation by insertion, western blot, fluorescence microscopy, transmission electron microscopy and microarray. Our results showed that IcsB protein is an important virulence factor in EIEC because it causes a camouflage of the bacteria in the eukaryotic cell. When there is a low expression of the protein, the cell recognition of the invasive bacteria is high, decreasing the bacteria dissemination. This found confirms the importance of the gene transcription and the gene sequence, since the strain E. coli SM124/13, complemented with icsB from Shigella, showed higher dissemination efficiency inside of the host cell. Interestingly, IcsB showed a new role on regulating the inflammatory response in Hela cells. The absence of IcsB in EIEC generated an intense disturbance of the cell homeostasis, increased the secretion of IL-6 and IL-8, and caused cell death. Additionally, our results revealed that eukaryotic cell infected by EIEC or Shigella flexneri showed distinguish responses. In EIEC infection, the autophagy was activated in human cells, but not in a conventional mode. Our hypothesis is that EIEC is recognized by autophagy, being an important cell process for bacterial recognition. This process can cause a decrease in the intracellular spread of EIEC making the infection less severe when compared to the infection caused by Shigella

Optimizing in vivo gene transfer into mouse corpus cavernosum by use of surface electroporation

PURPOSE: Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum. MATERIALS AND METHODS: Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 microg/40 microL), different electroporation settings (5-50 V, 8-16 pulses with a duration of 40-100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue. RESULTS: Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis. CONCLUSIONS: We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.

RE: Irreversible Electroporation of a Hepatocellular Carcinoma Lesion Adjacent to a Transjugular Intrahepatic Portosystemic Shunt Stent Graft

No abstract available.

A detailed description of an economical setup for electroporation of chick embryos in ovo

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

Irreversible Electroporation of a Hepatocellular Carcinoma Lesion Adjacent to a Transjugular Intrahepatic Portosystemic Shunt Stent Graft

We report in a 65-year-old man hepatocellular carcinoma adjacent to a transjugular intrahepatic portosystemic shunt stent-graft which was successfully treated with irreversible electroporation (IRE). IRE is a new non-thermal tissue ablation technique which uses electrical pulses to induce cell necrosis by irreversible membrane poration. IRE proved to be more advantageous in the ablation of perivascular tumor with little injury to the surrounding structures.

Fosfoproteômica e proteômica quantitativa de células mesenquimais durante a diferenciação osteoblástica mediada por BMP2, expressão e purificação de diferentes tipos de proteínas morfogenéticas ósseas / Quantitative phosphoproteomics and proteomics of mesenchymal stem cells during BMP2-mediated osteoblastic differentiation, expression and purification of different types of bone morphogenetic proteins

As fraturas e perdas ósseas representam altos riscos para o Sistema público de Saúde (SUS), além de afetar a qualidade de vida do paciente, portanto é necessário o entendimento das bases moleculares que envolvem os mecanismos de reparo ósseo. Citocinas secretadas por células do sistema imune presentes no local da inflamação, como as IL-6, IL-10 e TNFα atuam como fatores quimiotáticos para células mesenquimais, que proliferam e se diferenciam em osteoblastos pela ação autócrina e parácrina de Proteínas Morfogenéticas Ósseas (BMPs), principalmente a BMP2. Embora seja conhecido que a ação de BMP2 ocorra através de sua ligação nos receptores ActRI/BMPR, que ativam proteínas SMADS 1/5/8 efetoras, pouco se sabe sobre os mecanismos intracelulares que participam do processo de diferenciação osteoblástico. Neste estudo propôs-se analisar as diferenças no conteúdo de proteínas totais e de proteínas fosforiladas em células mesenquimais de pele induzidas à osteogênese pelo tratamento com BMP2 por diferentes períodos de tempo, utilizando-se de Isótopos Estáveis de Dimetila acoplado ao LC/MS. A partir de 150µg de material inicial, foi possível identificar 2.264 proteínas, as quais foram quantificadas nos diferentes pontos de indução, sendo que 235 são fosforiladas. Análise de motivos de quinases mostrou que diversos substratos possuem sítios fosforilados correspondentes àqueles dos motivos de fosforilação das quinases Casein Kinase, p38, CDK e JNK. A análise da ontologia gênica mostrou um aumento de processos biológicos relacionados com sinalização e diferenciação após a primeira hora de indução com rhBMP2. Além disso, proteínas envolvidas com o rearranjo do citoesqueleto e com vias de sinalização Wnt e Ras foram encontradas como tendo fosforilação diferencial durante todos os períodos estudados. Os dados revelaram novos substratos intracelulares que são fosforilados nos primeiros momentos do comprometimento com a diferenciação osteoblástica mediada pelo tratamento com rhBMP2 em células mesenquimais derivadas da pele. Além disso, clones celulares que superexpressam as proteínas recombinantes humanas BMP2 e BMP4 foram gerados, e sua atividade verificada in vitro. Paralelamente, a rhBMP7, obtida anteriormente, foi purificada por cromatografia de afinidade utilizando-se uma coluna de Heparina-Sepharose, que foi posteriormente utilizada para ensaios in vitro e in vivo, nos quais se mostrou capaz de gerar osteoblastos e tecido ósseo, respectivamente, o que abre novas possibilidades para o uso destas proteínas como biofármacos no Brasil

Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated human skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. From 150 µg of starting material, 2,264 proteins containing two or more peptides were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during commitment to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. Cell clones overexpressing the human BMP 2 and 4 recombinant proteins were also generated, and their biological activity was confirmed in vitro. In parallel, chromatography-affinity purified rhBMP7, obtained using heparin-Sepharose columns, was used for in vivo and in vitro assays to evaluate the ability of this purified protein to generate osteoblasts and bone tissue, respectively, opening new avenues for the use of these proteins as biopharmaceuticals in Brazil

Observation the electroporation of blood cells in vitro by scanning electron microscope / 法医学杂志

OBJECTIVE@#To observe changes on cell membrane in blood cells after they were been electrified.@*METHODS@#Blood were electrified for 5, 10, 20, 30 s, 1 min respectively, and Scanning electron microscope was used to detect the changes on their cell membranes.@*RESULTS@#Pores were detected both on electrified erythrocytes and leukocytes with round or ellipse shapes. The erythrocytes often have one or more pores while the leukocytes often have more pores looked like cribble. The rates of perforated cells were increased with the prolonging time of electrification, 5 s with 6% and 1 min increased to 40%.@*CONCLUSIONS@#Alternating current can cause the cell perforating, and the rates of perforated cell were increased with the prolonging time of electrification.

Low-dose radiation response of the p21(WAF1/CIP1) gene promoter transduced by adeno-associated virus vector

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (<1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.

Administração do gene da proteína verde fluorescente no músculo tireoaritenóideo do rato pela técnica de eletroporação / Administration of the green fluorescent protein gene into the rat thyroarytenoid muscle using the electroporation technique

A eletroporação é uma técnica de administração de pulsos elétricos no tecido que promove a entrada de genes na célula. Utilizou-se o gene do marcador da proteína fluorescente verde, o qual foi injetado no músculo tireoaritenóideo previamente à aplicação dos pulsos. Foram estudadas 32 laringes de ratos, divididas em grupos de alta voltagem, baixa voltagem e associação de alta e baixa voltagem. Cortes histológicos foram analisados e os grupos comparados quanto a número, intensidade da fluorescência, distribuição das fibras musculares marcadas e infiltrado inflamatório. O grupo de alta e baixa voltagens foi o mais eficiente, de acordo com os parâmetros estudados.Electroporation is a technique for the application of electric pulses in a tissue that allows the entrance of genes into a cell. The gene Enhanced green fluorescent protein was injected into the thyroarythenoid muscle before the pulse application. Thirty- two rat larynges were divided into groups of high voltage, low voltage and high and low voltage together. Histological sections were analized and the groups were compared in relation to the number, fluorescent signal, distribution of the marked muscular fibers and the inflammatory pattern. The group of high and low voltage pulses had the best performance according to these parameters...

Promotores de permeaçäo para a liberaçäo transdérmica de fármacos: uma nova aplicaçäo para as ciclodextrinas / Permeation enhancers in transdermal drug delivery systems: a new aplication of cyclodextrins

No presente trabalho é feita uma breve revisäo sobre promotores de permeaçäo cutânea, descrevendo-se os seus mecanismos de açäo e alguns exemplos. Abordam-se as vias de permeaçäo de fármacos através da pele e liberaçäo transdérmica. Säo também focadas as ciclodextrinas e seu derivados, a sua estrutura e propriedades físico-químicas, formaçäo de complexos de inclusäo e o seu papel como excipientes em sistemas transdérmicos. As ciclodextrinas constituem um grupo de excipientes que têm um papel de grande importância em formulaçäo farmacêutica. Uma das mais extradionárias propriedades destas moléculas é a sua capacidade de incrementar a liberaçäo de fármacos através da pele sem, no tentanto, afetar a sua funçäo barreira...

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression in-creased as cation concentration of vehicle dec-reased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression in-creased as cation concentration of vehicle dec-reased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.