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1.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2202-2205, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018444

RESUMO

In-vitro transfection of cells by electroporation is a widely used approach in cell biology and medicine. The transfection method is highly dependent on the cell culture's electrical resistance, which is strongly determined by differences in the membranes, but also on the morphology of the electrodes. Microneedle (MN)-based electrodes have been used to concentrate the electrical field during electroporation, and therefore maximize its effect on cell membrane permeability. So far, the methods used for the fabrication of MN electrodes have been relatively limited with respect to the needle design. In this work, we provide a method to fabricate MNs using 3D printing, which is a technology that provides a high degree of flexibility with respect to geometry and dimensions. Pyramidal-shaped MN designs were fabricated and tested on HCT116 cancer cells. Customization of the tips of the pyramids permits tailoring of the electrical field in the vicinity of the cell membranes. The fabricated device enables low-voltage (2 V) electroporation, eliminating the need for the use of specialized chemical buffers. The results show the potential of this method, which can be exploited and optimized for many different applications, and offer a very accessible approach for in-vitro electroporation and cell studies. The MNs can be customized to create complex structures, for example, for a multi-culture cell environment.


Assuntos
Sistemas de Liberação de Medicamentos , Eletroporação , Agulhas , Impressão Tridimensional , Transfecção
2.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2235-2238, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018452

RESUMO

Electroporation is a well-established technique used to stimulate cells, enhancing membrane permeability. Although the biological phenomena occurring after the poration process have been widely studied, the physical mechanisms of pore formation are not clearly understood. In this work we investigated by means of molecular dynamics simulations the kinetics of pore formation, linking the different stages of poration to specific arrangements of lipid membrane domains.Clinical Relevance-The approach followed in this study aims to shed light on the molecular mechanisms at the basis of the electroporation technique, nowadays used to enhance the entrance of poorly permeant anticancer drugs into tumor cells, for gene electrotransfer and all the other applications exploiting the modulation of cell membrane properties.


Assuntos
Fenômenos Biológicos , Bicamadas Lipídicas , Eletroporação , Cinética , Simulação de Dinâmica Molecular
3.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 3424-3427, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018739

RESUMO

We present MAPSYNE, a miniaturized and automated system combining a high-density microelectrode array (HD-MEA) and a movable micropipette for studying, monitoring, and perturbing neurons in vitro. The system involves an all-electrical approach to automatically move a glass micropipette towards a target location on the HD-MEA surface, without the need for a microscope. Two methods of performing blind navigation are employed, (i) stop-measure-go approach wherein the pipette moves for a predefined distance before measuring its location then the process is repeated until the pipette reaches its destination, and (ii) predictive approach wherein the pipette is continuously tracked and moved. This automated system can be applied for unsupervised single-cell manipulation of neurons in a network, such as electroporation and local delivery of compounds.


Assuntos
Eletroporação , Neurônios , Microeletrodos
4.
Nat Commun ; 11(1): 3481, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661226

RESUMO

Color vision is essential for an animal's survival. It starts in the retina, where signals from different photoreceptor types are locally compared by neural circuits. Mice, like most mammals, are dichromatic with two cone types. They can discriminate colors only in their upper visual field. In the corresponding ventral retina, however, most cones display the same spectral preference, thereby presumably impairing spectral comparisons. In this study, we systematically investigated the retinal circuits underlying mouse color vision by recording light responses from cones, bipolar and ganglion cells. Surprisingly, most color-opponent cells are located in the ventral retina, with rod photoreceptors likely being involved. Here, the complexity of chromatic processing increases from cones towards the retinal output, where non-linear center-surround interactions create specific color-opponent output channels to the brain. This suggests that neural circuits in the mouse retina are tuned to extract color from the upper visual field, aiding robust detection of predators and ensuring the animal's survival.


Assuntos
Visão de Cores/fisiologia , Retina/fisiologia , Animais , Eletroporação , Feminino , Luz , Masculino , Camundongos , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Campos Visuais/fisiologia , Vias Visuais/fisiologia
5.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510510

RESUMO

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.


Assuntos
Comportamento Animal , Eletroporação/métodos , Técnicas Genéticas , Animais , Estudos de Viabilidade , Camundongos , Camundongos Transgênicos , Plasmídeos/genética
6.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510512

RESUMO

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.


Assuntos
Astrócitos/citologia , Córtex Cerebral/citologia , Eletroporação/métodos , Animais , Cor , Feminino , Camundongos , Neurogênese
7.
Anim Sci J ; 91(1): e13386, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32512638

RESUMO

This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.


Assuntos
Proteína 9 Associada à CRISPR , Eletroporação/métodos , Eletroporação/veterinária , Desenvolvimento Embrionário/genética , Edição de Genes , Marcação de Genes/veterinária , Miostatina/genética , RNA Guia , Suínos/embriologia , Suínos/genética , Zigoto , Animais , Blastocisto , Proteína 9 Associada à CRISPR/farmacologia , Relação Dose-Resposta a Droga
8.
J Surg Oncol ; 122(3): 407-411, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32483842

RESUMO

BACKGROUND AND OBJECTIVES: Irreversible electroporation (IRE) is a nonthermal electrical tumor ablative strategy for unresectable tumors. IRE is relatively safe around critical structures but may induce cardiac arrhythmia when its delivery is not synchronized to the cardiac cycle. We performed a systematic literature review to determine rates of arrhythmia when IRE was utilized with or without cardiac synchronization. METHODS: An online literature search was conducted with additional hand selection of articles. Data were extracted and pooled analyses were performed. RESULTS: Twelve articles were included in analysis. IRE was performed for 481 patients; 46% hepatic tumors (n = 223), 36% pancreatic lesions (n = 168), and multiple other locations including prostate. Synchronization was performed on 422 patients. Arrhythmias were noted in 3.7% of cases (n = 18/481); cardiac synchronization: 1.2% (n = 5/422) vs unsynchronized: 22.0% (n = 13/59), P < .0001. These events occurred in every organ except the prostate. CONCLUSIONS: IRE remains a potent technology for unresectable tumors, but arrhythmia is a clinical concern. This literature review confirms that cardiac gating should be used in all cases outside of prostate to prevent this potentially serious adverse event.


Assuntos
Técnicas de Ablação/estatística & dados numéricos , Arritmias Cardíacas/epidemiologia , Eletroporação/estatística & dados numéricos , Neoplasias Hepáticas/cirurgia , Neoplasias Pancreáticas/cirurgia , Técnicas de Ablação/efeitos adversos , Arritmias Cardíacas/diagnóstico por imagem , Arritmias Cardíacas/etiologia , Técnicas de Imagem de Sincronização Cardíaca/métodos , Humanos
9.
Arch Virol ; 165(9): 1959-1968, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32519007

RESUMO

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV) strains, has been one of the most problematic diseases affecting the poultry industry worldwide. Conventional vaccines provide effective protection for birds to survive ND outbreaks, but they may not completely suppress NDV shedding. NDV strains circulate on farms for a long time after the initial infection and cause potential risks. A new vaccine with fast clearance ability and low viral shedding is needed. In this study, we used interleukin-12 (IL-12) as an adjuvant and electroporation (EP) as an advanced delivery system to improve a DNA vaccine candidate. The fusion (F) protein gene from an NDV strain of the prevalent genotype VII.1.1 was cloned to prepare the vaccine. Chickens immunized with the F gene DNA vaccine co-delivered with an IL-12-expressing plasmid DNA showed higher neutralizing antibody levels and stronger concanavalin-A-induced lymphocyte proliferation than those treated with the F gene DNA vaccine alone. The co-delivered vaccine provided 100% protection, and less viral shedding and a shorter release time were observed in challenged chickens than when the F gene DNA vaccine was administered alone. The use of F gene DNA combined with IL-12 delivered by electroporation is a promising approach for vaccination against ND.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Interleucina-12/imunologia , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Galinhas , Eletroporação , Interleucina-12/administração & dosagem , Doença de Newcastle/imunologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/fisiologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Eliminação de Partículas Virais
10.
J Vis Exp ; (160)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32597854

RESUMO

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.


Assuntos
Encéfalo/metabolismo , DNA/administração & dosagem , Eletroporação/métodos , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , Análise Espaço-Temporal , Animais , Encéfalo/citologia , DNA/genética , DNA/metabolismo , Embrião de Mamíferos/citologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Neurogênese , Plasmídeos/administração & dosagem , Gravidez
11.
J Cancer Res Ther ; 16(2): 280-285, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474514

RESUMO

Context: The safety and efficacy of irreversible electroporation (IRE) for locally advanced pancreatic carcinoma (LAPC) are well established. However, whether adjuvant chemoradiotherapy after IRE increases, the survival rate remains unknown. Therefore, this study evaluated the effect of chemoradiotherapy combined with IRE in patients with LAPC. Subjects and Methods: We retrospectively analyzed 42 patients with LAPC between July 2015 and December 2016 at PLA General Hospital treated with IRE or IRE combined with radiation and/or chemotherapy. These patients were divided into the IRE group and the combined-therapy group. All patients underwent computed tomography (CT), magnetic resonance imaging, and positron-emission tomography-CT and no signs of metastases were found. The prognosis of these patients was observed. Results: The times after operation and after diagnosis in the combined-therapy group (304.20 ± 118.54) and (334.40 ± 114.07) days, respectively, were better those than in the IRE group (214.36 ± 95.68) and (244.68 ± 110.61) days, respectively. Moreover, patients in the combined-therapy group had a significantly better survival rate than the IRE group (80 vs. 45.45%, P < 0.05). Conclusions: IRE combined with radiotherapy or chemotherapy was superior to IRE alone for the treatment of LAPC, as it prolonged the survival time and improved the survival rate, making it worthy of wide dissemination and clinical application.


Assuntos
Quimiorradioterapia Adjuvante/mortalidade , Eletroporação/métodos , Neoplasias Pancreáticas/terapia , Quimiorradioterapia Adjuvante/métodos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Tomografia por Emissão de Pósitrons/métodos , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Tomografia Computadorizada por Raios X/métodos
12.
Tech Vasc Interv Radiol ; 23(2): 100678, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32591190

RESUMO

Image-guided percutaneous thermal ablation plays an increasingly important role in the multidisciplinary management of musculoskeletal lesions. Established indications for ablation in this setting include the treatment of osteoid osteomas, palliation of painful skeletal metastases, local control of oligometastatic disease, and consolidation of bone tumors at risk for fracture. Emerging indications include the treatment of symptomatic soft tissue masses such as extra-abdominal desmoid tumors and abdominal wall endometriosis. This review will discuss considerations in patient selection and preprocedural workup, ablation technology and techniques, strategies to avoid complications, and expected outcomes of ablation in the musculoskeletal system.


Assuntos
Neoplasias Ósseas/cirurgia , Criocirurgia/tendências , Eletroporação/tendências , Micro-Ondas/uso terapêutico , Ablação por Radiofrequência/tendências , Neoplasias de Tecidos Moles/cirurgia , Cirurgia Assistida por Computador/tendências , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Tomada de Decisão Clínica , Criocirurgia/efeitos adversos , Humanos , Micro-Ondas/efeitos adversos , Seleção de Pacientes , Complicações Pós-Operatórias/etiologia , Ablação por Radiofrequência/efeitos adversos , Fatores de Risco , Neoplasias de Tecidos Moles/diagnóstico por imagem , Neoplasias de Tecidos Moles/patologia , Cirurgia Assistida por Computador/efeitos adversos , Resultado do Tratamento
13.
Tech Vasc Interv Radiol ; 23(2): 100675, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32591191

RESUMO

Several minimally invasive image guided tumor ablation techniques have been added to the treatment spectrum for locally advanced pancreatic cancer (LAPC). Irreversible electroporation (IRE) might have a significant additive value in the management of this difficult-to-treat disease. As opposed to thermal ablative techniques, IRE induces cell death by the delivery of high-voltage electrical pulses. The electrical energy disrupts the cellular membrane integrity, causes loss of cellular homeostasis and ultimately results in cell death. The extracellular matrix of connective tissue in surrounding delicate structures such as bile ducts, bowel wall, and larger blood vessels is spared. The preservation of these structures makes IRE attractive for the treatment of pancreatic cancers that are unresectable due to their anatomical location (ie, LAPC and local recurrence after surgical resection). In addition to its cytoreductive abilities, evidence is emerging on IRE's capability to induce systemic immunomodulation through active in vivo vaccination against pancreatic cancer cells. These effects in combination with immunotherapy may offer a new treatment paradigm for tumors with low immunogenic potential like pancreatic ductal adenocarcinoma (PDAC). This review discusses several practical and technical issues of IRE for LAPC: clinical evaluation, indications, patient preparations, procedural steps, imaging characteristics, clinical results, and "tricks of the trade" used to improve the safety and efficacy of the treatment. Future directions such as the combination of IRE with immunotherapy will be shortly addressed.


Assuntos
Técnicas de Ablação , Carcinoma Ductal Pancreático/cirurgia , Eletroporação , Neoplasias Pancreáticas/cirurgia , Cirurgia Assistida por Computador , Técnicas de Ablação/efeitos adversos , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/patologia , Tomada de Decisão Clínica , Humanos , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Seleção de Pacientes , Complicações Pós-Operatórias/etiologia , Fatores de Risco , Cirurgia Assistida por Computador/efeitos adversos , Resultado do Tratamento
14.
Tech Vasc Interv Radiol ; 23(2): 100674, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32591194

RESUMO

Renal cell carcinoma is most commonly diagnosed in the sixth or seventh decade of life. Historically, surgical extirpation was the gold standard treatment option for small renal masses. However, given the comorbidities in this elderly population, not all patients are candidates for surgery. The development of minimally invasive ablative therapies has solved the surgical dilemma in this patient population. Furthermore, the 2017 American Urological Association guidelines recommends consideration of percutaneous image guided thermal ablation as a treatment option for masses smaller than 3 cm even in healthy individuals. Percutaneous image guided thermal ablation is an attractive treatment option providing excellent local tumor control, fewer complications, better preservation of the renal functions, faster recovery and shorter hospital stay. Various ablative modalities are available in clinical practice. This includes radiofrequency ablation, cryoablation, microwave ablation, irreversible electroporation, high intensity focused ultrasound, and laser ablation. In this review, we focus on the most commonly used modalities including radiofrequency ablation and cryoablation and to a lesser extent microwave ablation and irreversible electroporation.


Assuntos
Carcinoma de Células Renais/cirurgia , Criocirurgia , Eletroporação , Neoplasias Renais/cirurgia , Micro-Ondas/uso terapêutico , Ablação por Radiofrequência , Cirurgia Assistida por Computador , Carcinoma de Células Renais/diagnóstico por imagem , Carcinoma de Células Renais/patologia , Criocirurgia/efeitos adversos , Criocirurgia/instrumentação , Humanos , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/patologia , Micro-Ondas/efeitos adversos , Complicações Pós-Operatórias/terapia , Ablação por Radiofrequência/efeitos adversos , Ablação por Radiofrequência/instrumentação , Fatores de Risco , Cirurgia Assistida por Computador/efeitos adversos , Cirurgia Assistida por Computador/instrumentação , Resultado do Tratamento , Carga Tumoral
15.
J Vis Exp ; (159)2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32449731

RESUMO

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.


Assuntos
Eletroporação/métodos , Furões/metabolismo , Neocórtex/citologia , Células-Tronco Neurais/citologia , Animais , Feminino , Histerectomia , Neurônios/metabolismo , Gravidez
16.
Nat Struct Mol Biol ; 27(5): 489-499, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32367067

RESUMO

Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1-Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1-Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1-Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex (PSC) has a surprisingly short mean lifetime. Remarkably, transcription effectively resolves the PSC, and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Enterococcus faecalis/enzimologia , Integrases/metabolismo , Eletroporação , Enterococcus faecalis/genética , Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Integrases/genética , Cinética , Microrganismos Geneticamente Modificados , Mutação , Transcrição Genética
17.
J Vis Exp ; (158)2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32421010

RESUMO

Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes. In vitro 3D culture systems simulate the in vivo tumor growth and preserve the biological characteristics of tumors more accurately than conventional monolayer cell cultures. We describe a protocol for the development of 3D tumor organoids using conjunctival melanoma (CM) and uveal melanoma (UM) cell lines as well as the use of hand-held customized electrodes, suitable for in vitro ECT in the culture well without destruction of the tumor environment. This protocol analyzes the culture and growth of 3D CM and UM spheroids and their reaction to bleomycin (2.5 µg/mL) alone, electroporation (EP) (750 Volts/cm, 8 pulses, 100 µs, 5 Hz) alone, and ECT as a combination of EP and bleomycin. The drug concentration and the EP settings used in this protocol were established as preferred ECT conditions according to previous experiments. The assay used to determine the spheroid viability was conducted 3-7 days following treatment. The effect on viability and growth of the 3D tumor spheroids was significant only after ECT. The customized electrodes are described in detail in order to facilitate the application of pulses in the culture well. This novel treatment of 3D UM and CM spheroids sets a steppingstone for future clinical application.


Assuntos
Eletroquimioterapia/instrumentação , Eletroquimioterapia/métodos , Neoplasias Oculares/tratamento farmacológico , Melanoma/tratamento farmacológico , Antineoplásicos/administração & dosagem , Bleomicina/administração & dosagem , Bleomicina/uso terapêutico , Linhagem Celular Tumoral , Eletrodos , Eletroporação , Neoplasias Oculares/patologia , Humanos , Melanoma/patologia , Esferoides Celulares/efeitos dos fármacos
18.
PLoS One ; 15(5): e0233151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469893

RESUMO

INTRODUCTION: Intramuscular electroporation (IM/EP) is a vaccine delivery technique that improves the immunogenicity of DNA vaccines. We evaluated the acceptability and tolerability of electroporation among healthy African study participants. METHODS: Forty-five participants were administered a DNA vaccine (HIV-MAG) or placebo by electroporation at three visits occurring at four week-intervals. At the end of each visit, participants were asked to rate pain at four times: (1) when the device was placed on the skin and vaccine injected, before the electrical stimulation, (2) at the time of electrical stimulation and muscle contraction, and (3) at 10 minutes and (4) 30 minutes after the procedure was completed. For analyses, pain level was dichotomized as either "acceptable" (none/slight/uncomfortable) or "too much" (Intense, severe, and very severe) and examined over time using repeated measures models. Optional brief comments made by participants were summarized anecdotally. RESULTS: All 45 participants completed all three vaccination visits; none withdrew from the study due to the electroporation procedure. Most (76%) reported pain levels as acceptable at every time point across all vaccination visits. The majority of "unacceptable" pain was reported at the time of electrical stimulation. The majority of the participants (97%) commented that they preferred electroporation to standard injection. CONCLUSION: Repeated intramuscular electroporation for vaccine delivery was found to be acceptable and feasible among healthy African HIV vaccine trial participants. The majority of participants reported an acceptable pain level at all vaccination time points. Further investigation may be warranted into the value of EP to improve immunization outcomes. ClinicalTrials.gov NCT01496989.


Assuntos
Vacinas contra a AIDS/administração & dosagem , Eletroporação , Contração Muscular , Músculo Esquelético , Vacinação , Vacinas de DNA/administração & dosagem , Adolescente , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
19.
Am J Physiol Endocrinol Metab ; 318(6): E943-E955, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32369414

RESUMO

Myokines, such as irisin, have been purported to exert physiological effects on skeletal muscle in an autocrine/paracrine fashion. In this study, we aimed to investigate the mechanistic role of in vivo fibronectin type III domain-containing 5 (Fndc5)/irisin upregulation in muscle. Overexpression (OE) of Fndc5 in rat hindlimb muscle was achieved by in vivo electrotransfer, i.e., bilateral injections of Fndc5 harboring vectors for OE rats (n = 8) and empty vector for control rats (n = 8). Seven days later, a bolus of D2O (7.2 mL/kg) was administered via oral gavage to quantify muscle protein synthesis. After an overnight fast, on day 9, 2-deoxy-d-glucose-6-phosphate (2-DG6P; 6 mg/kg) was provided during an intraperitoneal glucose tolerance test (2 g/kg) to assess glucose handling. Animals were euthanized, musculus tibialis cranialis muscles and subcutaneous fat (inguinal) were harvested, and metabolic and molecular effects were evaluated. Muscle Fndc5 mRNA increased with OE (~2-fold; P = 0.014), leading to increased circulating irisin (1.5 ± 0.9 to 3.5 ± 1.2 ng/mL; P = 0.049). OE had no effect on protein anabolism or mitochondrial biogenesis; however, muscle glycogen was increased, along with glycogen synthase 1 gene expression (P = 0.04 and 0.02, respectively). In addition to an increase in glycogen synthase activation in OE (P = 0.03), there was a tendency toward increased glucose transporter 4 protein (P = 0.09). However, glucose uptake (accumulation of 2-DG6P) was identical. Irisin elicited no endocrine effect on mitochondrial biogenesis or uncoupling proteins in white adipose tissue. Hindlimb overexpression led to physiological increases in Fndc5/irisin. However, our data indicate limited short-term impacts of irisin in relation to muscle anabolism, mitochondrial biogenesis, glucose uptake, or adipose remodeling.


Assuntos
Fibronectinas/genética , Músculo Esquelético/metabolismo , Gordura Subcutânea/metabolismo , Animais , Desoxiglucose/metabolismo , Óxido de Deutério , Eletroporação , Fibronectinas/metabolismo , Expressão Gênica , Glucose/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4/genética , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Membro Posterior , Masculino , Proteínas de Desacoplamento Mitocondrial/genética , Biogênese de Organelas , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ratos
20.
Zhongguo Yi Liao Qi Xie Za Zhi ; 44(2): 172-178, 2020 Feb 08.
Artigo em Chinês | MEDLINE | ID: mdl-32400994

RESUMO

Irreversible electroporation (IRE) is an emerging tissue ablation technique. Compared with thermal ablation technique such as radiofrequency, IRE can achieve focal ablation in a shorter time without heat sink effect while sparing the tissue scaffold. IRE has been demonstrated to be a feasible therapeutic modality for the liver, pancreatic, and prostatic cancer. In recent years, several studies regarding of catheter-directed IRE for digestive tract, bronchus, urinary tract, and myocardium have been performed, which preliminarily demonstrated the safety and efficacy of IRE for tissue ablation under endoscopic or interventional technique. This study summarized the research progress of catheter-directed IRE for tissue ablation. The critical technique and future direction of catheter-based IRE are prosp.


Assuntos
Ablação por Cateter , Eletroporação , Cateteres , Endoscopia , Humanos
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