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1.
Curr Urol Rep ; 20(10): 63, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31478109

RESUMO

PURPOSE OF REVIEW: Although still considered experimental, focal irreversible electroporation (IRE) as a primary treatment for prostate cancer (PCa) is considered one of the most promising ablative technologies for focal therapy. This review provides a description of the principle of IRE for the treatment of PCa, combined with an overview of the recent research. RECENT FINDINGS: It has been almost a decade since the first human studies of focal IRE for PCa were trying to demonstrate its feasibility and safety, and recently new data are emerging regarding the functional and oncological outcomes. It was shown that the expected ablation efficacy of IRE is dependent on increased safety margins of > 9 mm and an uninterrupted IRE procedure, but these findings need further investigation in larger cohorts and randomized control trials (RCT). Recent data from larger cohorts with a longer follow-up of up to 12 months prove that focal IRE as primary treatment for localized PCa is indeed safe, has effective short-term oncological control in selected patients, and it has good functional outcomes by retaining urinary function and causing only mild erectile dysfunction.


Assuntos
Técnicas de Ablação/métodos , Eletroporação/métodos , Neoplasias da Próstata/terapia , Disfunção Erétil/etiologia , Humanos , Masculino , Margens de Excisão , Neoplasias da Próstata/patologia , Recuperação de Função Fisiológica
2.
World J Microbiol Biotechnol ; 35(8): 119, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332541

RESUMO

The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 µg mL-1 of zeocin or 5 µg mL-1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10-8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11 × 10-6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well. High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.


Assuntos
Eletroporação , Água Doce/microbiologia , Microalgas/metabolismo , Estramenópilas/metabolismo , Acetatos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microalgas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Estramenópilas/genética , Transformação Genética
3.
Cancer Immunol Immunother ; 68(8): 1235-1243, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31214732

RESUMO

Off-target toxicity due to the expression of target antigens in normal tissue or TCR cross-reactivity represents a major risk when using T cell receptor (TCR)-engineered T cells for treatment of solid tumours. Due to the inherent cross-reactivity of TCRs it is difficult to accurately predict their target recognition pre-clinically. It has become evident that direct testing in a human being represents the best evaluation of the risks. There is, therefore, a clear unmet need for assessing the safety of a therapeutic TCR in a more controllable manner than by the injection of permanently modified cellular products. Using transiently modified T cells combined with dose escalation has already been shown feasible for chimeric antigen receptor (CAR)-engineered T cells, but nothing is yet reported for TCR. We performed a preclinical evaluation of a therapeutic TCR transiently expressed in T cells by mRNA electroporation. We analyzed if the construct was active in vitro, how long it was detectable for and if this expression format was adapted to in vivo efficacy assessment. Our data demonstrate the potential of mRNA engineered T cells, although less powerful than permanent redirection, to induce a significant response. Thus, these findings support the development of mRNA based TCR-therapy strategies as a feasible and efficacious method for evaluating TCR safety and efficacy in first-in-man testing.


Assuntos
Vacinas Anticâncer/imunologia , Neoplasias Colorretais/terapia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Animais , Neoplasias Colorretais/imunologia , Reações Cruzadas , Citotoxicidade Imunológica , Eletroporação , Células HCT116 , Humanos , Camundongos , Camundongos SCID , Neoplasias Experimentais , RNA Mensageiro/genética , Receptores de Antígenos Quiméricos/genética , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Protoc ; 14(6): 1926-1943, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101906

RESUMO

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Epitopos/imunologia , Neoplasias/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Eletroporação/métodos , Epitopos/genética , Humanos , Imunoterapia/métodos , Neoplasias/terapia , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/imunologia , Transfecção/métodos
5.
Analyst ; 144(11): 3581-3589, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065636

RESUMO

The microalgal cell wall is a natural barrier that limits the efficiency of gene delivery in algae genetic engineering. Here, we report the role of hard-uptake nanoparticles (huNPs) in microalgae cell electroporation to enhance the delivery of genes in Chlamydomonas reinhardtii. This role can be divided into two categories: (i) a 'transient state' for short-term behavior under confocal visualization and (ii) a 'steady state' for long-term behavior in cell culture. First, the 'transient' role of gene-huNP complexes was investigated after washing for clear confocal imaging to observe the location of huNPs after electroporation. Second, the 'steady-state' role of the gene-huNP complexes was examined after electroporation by transferring cells to a fresh, medium-rich culture environment without washing to obtain a stable cell culture. For selection of the huNPs, we used two types of nanoparticles (NPs, 250 nm and 530 nm) larger than the threshold size of electroporation uptake to avoid unwanted endocytic uptake of NPs. In the transient state, the visualization results indicate that gene-NP (250 nm) complexes were positioned on the cells and helped to deliver more genes than did the 530 nm NPs. In the steady state, the gene-NP (530 nm) complexes helped stably deliver more genes to the cells by precipitation of NPs due to gravity. We believe that these findings illustrate how gene-NP complexes function in microalgae cell electroporation and could help set up a protocol for enhanced microalgae applications associated with NPs such as environmental waste removal and biofuel production.


Assuntos
DNA/farmacocinética , Técnicas de Transferência de Genes , Nanopartículas/química , Sobrevivência Celular/efeitos dos fármacos , Chlamydomonas reinhardtii , DNA/genética , Eletroporação/métodos , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Proteínas de Fluorescência Verde/genética , Microalgas , Nanopartículas/toxicidade , Oxazinas/química , Oxazinas/toxicidade , Tamanho da Partícula , Poliestirenos/química , Poliestirenos/toxicidade , RNA Guia/genética
6.
Methods Mol Biol ; 1966: 151-161, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041745

RESUMO

The overexpression of a specific protein is a common method for investigating the specific biological function of the substance and the mechanism of action. In vivo electrotransfer has been confirmed to be one of the most reliable, efficient and cost-effective way to overexpress a protein in a select biological tissue. Typically, this technique involves a physical injection of plasmid DNA followed by electric pulses across the injection site. Here, we introduce this method that we used to transfect green fluorescent protein (GFP)-tagged PGC-1α plasmid DNA into mouse tibialis anterior (TA) muscle, which attained high transfection efficiency with no muscle damage. To quantify the transfection efficiency, we also demonstrate the visualization of plasmid DNA transfected fibers via immunohistochemical staining on muscle cross sections.


Assuntos
Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Transfecção/métodos , Animais , Eletroporação , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde , Camundongos , Plasmídeos
7.
BMC Cancer ; 19(1): 394, 2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31029111

RESUMO

BACKGROUND: Locally advanced pancreatic cancer (LAPC) represents more than one third of pancreatic cancers and owns poor survival after the standard chemotherapy. Irreversible electroporation (IRE) is a novel method and has been recently used in LAPC. The aim of this study was to compare the efficacy of IRE and radiotherapy after induction chemotherapy for patients with LAPC. METHODS: From August 2015 to August 2017, a total of 76 patients with biopsy proven LAPC and who had received IRE or radiotherapy after chemotherapy were included. Thirty-two pairs of patients were selected through propensity score matching (PSM) analysis and the efficacy of two treatments was compared. RESULTS: Before PSM analysis, after induction chemotherapy, patients with LAPC benefited more in terms of overall survival (OS) and progression free survival (PFS) from IRE, compared with radiotherapy (2-year OS rates, 53.5% vs 26.9%, p = 0.039; 2-year PFS rates, 28.4% vs 13.3%, p = 0.045). After PSM analysis, the survival benefits of OS and PFS of patients after induction chemotherapy followed by IRE were more obvious than those of patients treated with radiotherapy (2-year OS rates, 53.5% vs 20.7%, p = 0.011; 2-year PFS rates, 28.4% vs 5.6%, p = 0.004). Multivariate Cox regression analysis indicated that IRE after induction chemotherapy was identified as a significant favourable factor for both OS and PFS in both the whole and matched cohort. CONCLUSIONS: Induction chemotherapy followed by IRE is superior to induction chemotherapy followed by radiotherapy for treating LAPC. A randomized clinical trial comparing the efficacy of IRE and radiotherapy after the induction chemotherapy is therefore considerable.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Eletroporação/métodos , Neoplasias Pancreáticas/terapia , Radioterapia/métodos , Adulto , Idoso , Terapia Combinada , Feminino , Humanos , Quimioterapia de Indução/métodos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pâncreas/efeitos da radiação , Neoplasias Pancreáticas/patologia , Intervalo Livre de Progressão , Pontuação de Propensão
8.
IET Nanobiotechnol ; 13(1): 58-65, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30964039

RESUMO

Electroporation facilitates loading of cells with molecules and substances that are normally membrane impermeable. Flow cytometry is used in this study to examine the effects of the application of electroporation-level monopolar electric field pulses of varying electrical field strength on Ishikawa endometrial adenocarcinoma cells. Analysis of the fluorescence versus forward scatter plots corroborates the well-recognised threshold and cell size dependence characteristics of electroporation, but also shows the progression of cell lysis and generation of particulate material. Two 500 µs monopolar rectangular pulses ranging from 1.0 × 105 to 2.5 × 105 V/m were used to electroporate the cells. Electroporation yields (fraction of viable cells exhibiting significant propidium iodide uptake) ranged from 0 to 97%, with viability ranging between 78 and 34% over the electric field strength range tested. The higher electric field strength pulses not only reduced cell viability, but also generated a substantial amount of sub-cellular sized particulate material indicating cells have been physically disrupted enough to create these particles.


Assuntos
Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Eletroporação/métodos , Citometria de Fluxo/métodos , Linhagem Celular Tumoral , Tamanho Celular , Humanos
9.
Methods Mol Biol ; 1976: 55-70, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977065

RESUMO

For decades, the quail-chick system has been a gold standard approach to track cells and their progenies over complex morphogenetic movements and long-range migrations as well as to unravel their dialogue and interplays in varied processes of cell induction. More specifically, this model became decisive for the systematic explorations of the neural crest and its lineages and allowed a tremendous stride in understanding the wealth and complexity of this fascinating cell population. Much of our knowledge on craniofacial morphogenesis and vertebrate organogenesis was first gained in avian chimeras and later extended to mammalian models and humans. In addition, this system permits tissue and gene manipulations to be performed at once in the same cell population. Through the use of in ovo electroporation, this model became tractable for functional genomics, hence being even more resourceful for functional studies. Due to the ease of access and the possibility to combine micromanipulation of tissue anlagen and gene expression, this model offers the prospect of decrypting instructive versus permissive tissue interactions, to identify and crack the molecular codes underlying cell positioning and differentiation, with an unparalleled spatiotemporal accuracy.


Assuntos
Crista Neural/citologia , Animais , Galinhas , Eletroporação , Desenvolvimento Embrionário , Genômica , Codorniz
10.
Methods Mol Biol ; 1976: 71-82, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977066

RESUMO

In ovo electroporation enables transfection of non-viral plasmid DNA and/or morpholinos to fluorescently label and/or perturb gene function in cells of interest. However, targeted electroporation into specific subregions of the embryo can be challenging due to placement and size limitations of the electrodes. Here we describe the basic techniques for in ovo electroporation in the chick embryo and suggest parameters to electroporate cells within different target tissues that with some modifications may be applicable to a wide range of developmental stages and other embryo model organisms.


Assuntos
Eletroporação/métodos , Morfolinos/metabolismo , Plasmídeos/genética , Animais , Embrião de Galinha , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento
11.
Methods Mol Biol ; 1976: 107-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977069

RESUMO

Mammalian development occurs in utero, which makes it difficult to study the diverse morphogenetic events of neural crest cell development in vivo. Analyses of fixed samples in conjunction with histological methods to evaluate the spatiotemporal roles of specific genes of interest only provide snapshots of mammalian neural crest cell development. This chapter describes methods for isolating and culturing mouse embryos and their organs in vitro, outside the mother, to facilitate real-time imaging and functional analyses of the dynamics of neural crest cell development.


Assuntos
Crista Neural/citologia , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Eletroporação , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Camundongos
12.
Nihon Yakurigaku Zasshi ; 153(4): 167-171, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-30971656

RESUMO

In the developing mammalian cerebral cortex, newly generated neurons migrate toward the pial surface to form a mammalian-specific six-layered cerebral cortex. Genetic studies of human neurological diseases have suggested the involvement of several molecules in cortical neuronal migration. In vivo electroporation is another powerful tool for understanding the molecular mechanisms of neuronal migration. By using these techniques, however, it is difficult to understand molecular basis of time-dependent changes of neuronal morphologies. Here, we introduce a pharmacological approach to cerebral cortical development. Major advantages of the pharmacological approach include the transient suppression of molecules of interest and analyzing time-dependent changes of neuronal morphologies. It also allows us to search molecules regulating neuronal migration with comparative ease. We propose the complementarity between the pharmacological approach and genetics or in vivo electroporation experiments.


Assuntos
Córtex Cerebral , Neurogênese , Neurônios , Animais , Movimento Celular , Eletroporação , Humanos
13.
Bioelectrochemistry ; 128: 148-154, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31003053

RESUMO

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.


Assuntos
Apoptose/efeitos dos fármacos , Candida/efeitos dos fármacos , Caspases/metabolismo , Eletroporação/métodos , Formiatos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Candida/classificação , Candida/citologia , Candida/enzimologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Especificidade da Espécie
14.
Bioelectrochemistry ; 128: 175-185, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31004911

RESUMO

The treatment of seeds and plants by electrically generated cold atmospheric pressure plasma can accelerate seed germination and radicle growing rates. The plasma generated reactive oxygen and nitrogen species, UV photons, and high frequency electromagnetic fields can penetrate into seed coats and modify their surface properties. Atomic force microscope data shows that cold helium or argon plasma induces strong corrugation of pumpkin seed coats, produces pores and surface defects. These structural deformations and poration enhance water uptake by seeds during the imbibing process, accelerate seeds germination, and increase seed growth. The cold atmospheric pressure plasmas treatment of pumpkin seeds also decreases the apparent contact angle between a water drop and the seed surface, thereby improving the wetting properties of seeds surfaces. Magnetic resonance imaging studies show acceleration of water uptake in pumpkin seeds exposed to a cold plasma jet. Reactive nitrogen and oxygen species, high frequency electromagnetic fields and photons emitted by the plasma jets accelerate germination of pumpkin seeds both independently and synergistically. These results show that cold plasma can be used in agriculture for acceleration of seed germination, increasing growth of plants seedlings, poration and corrugation of the bio-tissue surfaces.


Assuntos
Cucurbita/embriologia , Eletroporação/métodos , Gases em Plasma , Sementes/química , Cucurbita/crescimento & desenvolvimento , Campos Eletromagnéticos , Germinação , Imagem por Ressonância Magnética , Microscopia de Força Atômica , Raios Ultravioleta , Molhabilidade
15.
In Vitro Cell Dev Biol Anim ; 55(4): 237-242, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30820813

RESUMO

The application of CRISPR/Cas9 strategy promises to rapidly increase the production of genetically engineered animals since it yields stably integrated transgenes. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine-matured oocytes and putative zygotes. Effects of pulse number (3-7 pulse repetitions) during electroporation on the embryonic development and mutation efficiency were also investigated. Our results showed that the cleavage rate of matured oocytes with electroporation treatment significantly decreased as compared with electroporated putative zygotes (p < 0.05). Moreover, the rates of blastocyst formation from oocytes/zygotes electroporated with more than 5 pulses decreased. Mutation efficiency was then assessed after sequencing the target sites in individual blastocysts derived from oocytes/zygotes electroporated by 3 and 5 pulses. No bi-allelic mutations in all examined blastocysts were observed in this study. There were no differences in the mutation rates (50-60%) between blastocysts derived from matured oocytes electroporated by 3 and 5 pulses, irrespective of targeting gene. In the targeting MSTN gene, however, the mutation rate (12.5%) of blastocysts derived from putative zygotes electroporated by 3 pulses tended to be lower than that (60%) from 5-pulsed electroporated putative zygotes. These data indicate that the type of eggs may influence not only their development after electroporation treatment but also the mutation rate in the resulting blastocysts.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Diferenciação Celular , Eletroporação/métodos , Edição de Genes , Genoma , Mutação/genética , Oócitos/metabolismo , Zigoto/metabolismo , Animais , Blastocisto/metabolismo , Desenvolvimento Embrionário , Taxa de Mutação , RNA Guia/metabolismo , Suínos
16.
Methods Mol Biol ; 1961: 127-134, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912044

RESUMO

CRISPR/Cas9 is an effective and easy-to-use tool for editing the genome of many human cancer cell lines. However, in some hard-to-transfect cell lines and primary cells, gene editing is more challenging. This protocol details an electroporation-based protocol for the delivery of Cas9 protein from Streptococcus pyogenes complexed with chemically modified sgRNAs. We have found this protocol to work very efficiently in numerous cell lines and primary cells that are difficult to transfect by conventional chemical-based transfection methods.


Assuntos
Proteína 9 Associada à CRISPR/genética , RNA Guia/genética , Sistemas CRISPR-Cas/genética , Eletroporação , Edição de Genes , Humanos
17.
Methods Mol Biol ; 1961: 249-254, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912050

RESUMO

Programmable nucleases like CRISPR/Cas9 enable to edit the mouse genome directly in the zygote. Several methods have been successfully used for this. Here we describe injection into one of the pronuclei of the zygote and electroporation of zygotes. Alternative methods will be mentioned.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Animais , Eletroporação , Camundongos , RNA Guia/genética , Zigoto/metabolismo
18.
J Ultrasound ; 22(1): 53-58, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30843171

RESUMO

PURPOSE: To report our first results on sixteen patients affected by liver and abdominal malignant tumors, unfit for surgery or thermal ablation, treated with US-guided percutaneous irreversible electroporation (IRE). METHODS: From June 2014 to December 2016, all patients meeting the inclusion criteria (malignant hepatic or abdominal tumors not eligible for resection or thermal ablation) and not meeting the exclusion criteria (heart arrhythmia, pro-hemorrhagic hematological alterations, tumor size > 8 cm, presence of a biliary metallic stent) referred to our institutions were prospectively enrolled to undergo percutaneous US-guided irreversible electroporation (IRE). Sixteen patients (age range 59-68 years, mean 63; 7 females) with 18 tumors (diameter range 1.3-7.5 cm) fulfilled the inclusion criteria and were included in the study. Data concerning efficacy (tested by a 1-week CEUS and a 4-week enhanced CT and/or enhanced MRI) and safety were recorded during a 18-month follow up. RESULTS: All patients completed a 35-50-min procedure without complications. One patient with 6 cm Klatskin tumor also underwent a second session for 1 month. A 1-week CEUS and a 4-week e-CT and/or e-MRI arterial phase contrast enhancement analysis showed an overall reduction of arterial flow with confirmation of unenhanced lesions for seven nodules. After 1-18 months of follow up, no major complications were recorded and no tumor-related death occurred. The lesions of two patients disappeared 3 and 6 months after their treatment, respectively. CONCLUSIONS: IRE is a promising ablation modality in the treatment of malignant hepatic and abdominal tumors unsuitable for resection or thermal ablation.


Assuntos
Neoplasias Abdominais/terapia , Eletroporação , Neoplasias Hepáticas/terapia , Ultrassonografia de Intervenção , Neoplasias Abdominais/diagnóstico por imagem , Idoso , Feminino , Seguimentos , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Carga Tumoral
19.
Zhongguo Zhong Yao Za Zhi ; 44(1): 77-81, 2019 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-30868815

RESUMO

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.


Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional , Pogostemon/microbiologia , Ralstonia solanacearum/genética , Eletroporação , Genes Bacterianos , Virulência
20.
Curr Protoc Neurosci ; 87(1): e65, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30861320

RESUMO

Interneurons in the olfactory bulb are generated from neuronal precursor cells migrating from the anterior subventricular zone (SVZa) throughout the embryonic and postnatal life of mammals. This article describes basic methods for in vivo electroporation to label SVZa cells of both embryonic and postnatal rats. In addition, it describes three methods for tracing SVZa progenitors and following their migration pathway and differentiation, including immunohistochemistry, time-lapse live imaging in slice culture, and time-lapse imaging following transplantation in slice culture. These methods may be applied to all strains of rats and mice, including reporter mice. They may also be combined with methods such as BrdU labeling, tamoxifen injection, and electrophysiology, allowing one to observe proliferation or control gene expression at specific times and for specific neuronal functions. With time-lapse live imaging, details of labeled cells can be studied, including morphology, motility pattern, differentiation, and crosstalk between cells. © 2019 by John Wiley & Sons, Inc.


Assuntos
Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Eletroporação , Imagem com Lapso de Tempo , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Movimento Celular/fisiologia , Eletroporação/métodos , Interneurônios/fisiologia , Camundongos , Neurônios/fisiologia , Ratos , Células-Tronco/citologia , Imagem com Lapso de Tempo/métodos
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