RESUMEN
Candida glabrata (Nakaseomyces glabratus) is an opportunistic fungal pathogen that has become a significant concern in clinical settings due to its increasing resistance to antifungal treatments. Understanding the genetic basis of its pathogenicity and resistance mechanisms is crucial for developing new therapeutic strategies. One powerful method of studying gene function is through targeted gene deletion. This paper outlines a comprehensive protocol for the deletion of genes in C. glabrata, encompassing primer design, preparation of electrocompetent cells, transformation, and finally confirmation of the gene deletion. The protocol begins with the identification and design of primers necessary for generating deletion constructs, involving the precise targeting of up- and downstream regions flanking the gene of interest to ensure high specificity and efficiency of homologous recombination. Followed is the preparation of electrocompetent cells, a critical step for successful transformation. Transformation of the competent cells is achieved through electroporation, facilitating the introduction of exogenous DNA into the cells. This is followed by the selection and confirmation of successfully transformed colonies. Confirmation involves the use of colony PCR to verify the correct integration of the NAT resistance cassette and deletion of the target gene. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Primer design for gene deletion in C. glabrata Basic Protocol 2: Preparing competent C. glabrata cells Basic Protocol 3: Transforming C. glabrata using electroporation Basic Protocol 4: Confirming deletion strains with colony PCR.
Asunto(s)
Candida glabrata , Eliminación de Gen , Candida glabrata/genética , Candida glabrata/patogenicidad , Electroporación , Transformación Genética , Recombinación Homóloga , Cartilla de ADN/genéticaRESUMEN
The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture Basic Protocol 4: Electroporation of Neisseria gonorrhoeae Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5 Basic Protocol 7: Chemical mutagenesis Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome Alternate Protocol 1: Complementation with replicating plasmids Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies.
Asunto(s)
Neisseria gonorrhoeae , Neisseria , Transformación Bacteriana , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria/genética , Neisseria/efectos de los fármacos , Electroporación , Gonorrea/microbiología , Gonorrea/tratamiento farmacológico , HumanosRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)-based editing tools have transformed the landscape of genome editing. However, the absence of a robust and safe CRISPR delivery method continues to limit its potential for therapeutic applications. Despite the emergence of various methodologies aimed at addressing this challenge, issues regarding efficiency and editing operations persist. We introduce a microfluidic gene delivery system, called droplet cell pincher (DCP), designed for highly efficient and safe genome editing. This approach combines droplet microfluidics with cell mechanoporation, enabling encapsulation and controlled passage of cells and CRISPR systems through a microscale constriction. Discontinuities created in cell and nuclear membranes upon passage facilitate the rapid CRISPR-system internalization into the nucleus. We demonstrate the successful delivery of various macromolecules, including mRNAs (~98%) and plasmid DNAs (~91%), using this platform, underscoring the versatility of the DCP and leveraging it to achieve successful genome engineering through CRISPR-Cas9 delivery. Our platform outperforms electroporation, the current state-of-the-art method, in three key areas: single knockouts (~6.5-fold), double knockouts (~3.8-fold), and knock-ins (~3.8-fold). These results highlight the potential of our platform as a next-generation tool for CRISPR engineering, with implications for clinical and biological cell-based research.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Humanos , Microfluídica/métodos , Técnicas de Transferencia de Gen , Electroporación/métodos , Células HEK293 , Plásmidos/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
The phenomenon known as bipolar cancellation is observed when biphasic nanosecond electric field pulses are used, which results in reduced electroporation efficiency when compared to unipolar pulses of the same parameters. Basically, the negative phase of the bipolar pulse diminishes the effect of the positive phase. Our study aimed to investigate how bipolar cancellation affects Ca2+ electrochemotherapy and cellular response under varying electric field intensities and pulse durations (3-7 kV/cm, 100, 300, and 500 ns bipolar 1 MHz repetition frequency pulse bursts, n = 100). As a reference, standard microsecond range parametric protocols were used (100 µs × 8 pulses). We have shown that the cancellation effect is extremely strong when the pulses are closely spaced (1 MHz frequency), which results in a lack of cell membrane permeabilization and consequent failure of electrochemotherapy in vitro. To validate the observations, we have performed a pilot in vivo study where we compared the efficacy of monophasic (5 kV/cm × ↑500 ns × 100) and biphasic sequences (5 kV/cm × ↑500 ns + ↓500 ns × 100) delivered at 1 MHz frequency in the context of Ca2+ electrochemotherapy (B16-F10 cell line, C57BL/6 mice, n = 24). Mice treated with bipolar pulses did not exhibit prolonged survival when compared to the untreated control (tumor-bearing mice); therefore, the bipolar cancellation phenomenon was also occurrent in vivo, significantly impairing electrochemotherapy. At the same time, the efficacy of monophasic nanosecond pulses was comparable to 1.4 kV/cm × 100 µs × 8 pulses sequence, resulting in tumor reduction following the treatment and prolonged survival of the animals.
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Electroquimioterapia , Melanoma Experimental , Animales , Electroquimioterapia/métodos , Ratones , Línea Celular Tumoral , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/terapia , Proyectos Piloto , Electroporación/métodos , Ratones Endogámicos C57BL , Calcio/metabolismoRESUMEN
Natural Killer (NK) cells are exciting candidates for cancer immunotherapy with potent innate cytotoxicity and distinct advantages over T cells for Chimeric Antigen Receptor (CAR) therapy. Concerns regarding the safety, cost, and scalability of viral vectors has ignited research into non-viral alternatives for gene delivery. This review comprehensively analyses recent advancements and challenges with non-viral genetic modification of NK cells for allogeneic CAR-NK therapies. Non-viral alternatives including electroporation and multifunctional nanoparticles are interrogated with respect to CAR expression and translational responses. Crucially, the link between NK cell biology and design of drug delivery technologies are made, which is essential for development of future non-viral approaches. This review provides valuable insights into the current state of non-viral CAR-NK cell engineering, aimed at realising the full potential of NK cell-based immunotherapies.
Asunto(s)
Ingeniería Celular , Técnicas de Transferencia de Gen , Inmunoterapia Adoptiva , Células Asesinas Naturales , Receptores Quiméricos de Antígenos , Células Asesinas Naturales/inmunología , Humanos , Receptores Quiméricos de Antígenos/genética , Animales , Inmunoterapia Adoptiva/métodos , Ingeniería Celular/métodos , Nanopartículas/química , Neoplasias/terapia , Neoplasias/inmunología , Electroporación/métodos , Inmunoterapia/métodosRESUMEN
The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.
Asunto(s)
Sistemas CRISPR-Cas , Electroporación , Fertilización In Vitro , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Ribonucleoproteínas , Cigoto , Animales , Edición Génica/métodos , Electroporación/métodos , Cigoto/metabolismo , Fertilización In Vitro/métodos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Ovinos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Miostatina/genética , Femenino , Animales Modificados GenéticamenteRESUMEN
Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.
Asunto(s)
Búfalos , Sistemas CRISPR-Cas , Electroporación , Edición Génica , Técnicas de Transferencia Nuclear , Transfección , Animales , Búfalos/genética , Electroporación/veterinaria , Electroporación/métodos , Femenino , Embarazo , Edición Génica/métodos , Edición Génica/veterinaria , Transfección/veterinaria , Transfección/métodos , Técnicas de Transferencia Nuclear/veterinaria , Miostatina/genética , Cigoto/metabolismoRESUMEN
Electroporation-based procedures employing nanosecond bipolar pulses are commonly linked to an undesirable phenomenon known as the cancelation effect. The cancellation effect arises when the second pulse partially or completely neutralizes the effects of the first pulse, simultaneously diminishing cells' plasma membrane permeabilization and the overall efficiency of the procedure. Introducing a temporal gap between the positive and negative phases of the bipolar pulses during electroporation procedures may help to overcome the cancellation phenomenon; however, the exact thresholds are not yet known. Therefore, in this work, we have tested the influence of different interphase delay values (from 0 ms to 95 ms) using symmetric bipolar nanoseconds (300 and 500 ns) on cell permeabilization using 10 Hz, 100 Hz, and 1 kHz protocols. As a model mouse hepatoma, the MH-22a cell line was employed. Additionally, we conducted in vitro electrochemotherapy with cisplatin, employing reduced interphase delay values (0 ms and 0.1 ms) at 10 Hz. Cell plasma membrane permeabilization and viability dependence on a variety of bipolar pulsed electric field protocols were characterized. It was shown that it is possible to minimize bipolar cancellation, enabling treatment efficiency comparable to monophasic pulses with identical parameters. At the same time, it was highlighted that bipolar cancellation has a significant influence on permeabilization, while the effects on the outcome of electrochemotherapy are minimal.
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Permeabilidad de la Membrana Celular , Electroquimioterapia , Electroquimioterapia/métodos , Animales , Ratones , Permeabilidad de la Membrana Celular/efectos de los fármacos , Línea Celular Tumoral , Electroporación/métodos , Cisplatino/farmacología , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Antineoplásicos/farmacologíaRESUMEN
Exposing cells to intense and brief electric field pulses can modulate cell permeability, a phenomenon termed electroporation. When applied in medical treatments of diseases like cancer and cardiac arrhythmias, depending on level of cellular destruction, it is also referred to as irreversible electroporation (IRE) or Pulsed Field Ablation (PFA). For ablation device testing, several pulse parameters need to be characterized in a comprehensive manner to assess lesion boundary and efficacy. Overly aggressive voltages and application numbers increase animal burden. The potato tuber is a widely used initial model for the early testing of electroporation. The aim of this study is to characterize and refine bench testing for the ablation outcomes of PFA in this simplistic vegetal model. For in vitro assays, several pulse parameters like voltage, duration, and frequency were modulated to study effects not only on 2D ablation area but also 3D depth and volume. As PFA is a relatively new technology with minimal thermal effects, we also measured temperature changes before, during, and after ablation. Data from experiments were supplemented with in silico modeling to examine E-field distribution. We have estimated the irreversible electroporation threshold in Solanum Tuberosum to be at 240 V/cm. This bench testing platform can screen several pulse recipes at early stages of PFA device development in a rapid and high-throughput manner before proceeding to laborious trials for IRE medical devices.
Asunto(s)
Electroporación , Solanum tuberosum , Electroporación/métodos , Técnicas de Ablación/métodos , Técnicas de Ablación/instrumentación , Temperatura , Simulación por Computador , Terapia de Electroporación IrreversibleRESUMEN
Background: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. Methods: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Results: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. Conclusions: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.
Asunto(s)
Antígenos CD19 , Elementos Transponibles de ADN , Inmunoterapia Adoptiva , Leucemia Mieloide Aguda , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva/métodos , Antígenos CD19/inmunología , Antígenos CD19/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/genética , Células Nutrientes , Linfoma de Células B/terapia , Linfoma de Células B/inmunología , Linfoma de Células B/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Electroporación , Células Alogénicas/inmunologíaRESUMEN
Electroporation (EP) of the normal cell and cancer cell both in single-cell and multicellular models was investigated by the meshed transport network method (MTNM) in this paper. The simulation results suggest that the cancer cell undergoes faster and more significant local EP than that of the corresponding normal cell induced by nanosecond pulsed electric fields (nsPEFs) both in single-cell and multicellular models. Furthermore, the results of the multicellular model indicate that there is a unidirectional neighboring effect in the multicellular model, meaning that cells at the center are affected and their pore formation is significantly reduced, but this effect is very weak for cells at the edges of the system. This means that the electric field selectively kills cells in different distribution locations. This work can provide guidance for the selection of parameters for the cancer cell EP process.
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Electroporación , Modelos Biológicos , Neoplasias , Electroporación/métodos , Humanos , Neoplasias/patología , Neoplasias/terapia , Simulación por ComputadorRESUMEN
The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure.
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Ectodermo , Electroporación , Regulación del Desarrollo de la Expresión Génica , Tubo Neural , Animales , Ectodermo/metabolismo , Ectodermo/embriología , Ratones , Tubo Neural/embriología , Tubo Neural/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Electroporación/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Embrión de Mamíferos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodermo/metabolismo , Endodermo/embriología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neurulación/genética , Vectores Genéticos/genética , EmbarazoRESUMEN
Advances in synthetic biology allow the design and manipulation of DNA from the scale of genes to genomes, enabling the engineering of complex genetic information for application in biomanufacturing, biomedicine and other areas. The transfer and subsequent maintenance of large DNA are two core steps in large scale genome rewriting. Compared to small DNA, the high molecular weight and fragility of large DNA make its transfer and maintenance a challenging process. This review outlines the methods currently available for transferring and maintaining large DNA in bacteria, fungi, and mammalian cells. It highlights their mechanisms, capabilities and applications. The transfer methods are categorized into general methods (e.g., electroporation, conjugative transfer, induced cell fusion-mediated transfer, and chemical transformation) and specialized methods (e.g., natural transformation, mating-based transfer, virus-mediated transfection) based on their applicability to recipient cells. The maintenance methods are classified into genomic integration (e.g., CRISPR/Cas-assisted insertion) and episomal maintenance (e.g., artificial chromosomes). Additionally, this review identifies the major technological advantages and disadvantages of each method and discusses the development for large DNA transfer and maintenance technologies.
Asunto(s)
Bacterias , ADN , Hongos , Animales , Hongos/genética , Bacterias/genética , ADN/genética , Humanos , Electroporación , Técnicas de Transferencia de Gen , Mamíferos/genética , Biología Sintética/métodos , Sistemas CRISPR-Cas/genéticaRESUMEN
Dendrite morphology and dendritic spines are key features of the neuronal networks in the brain. Abnormalities in these features have been observed in patients with psychiatric disorders and mouse models of these diseases. In utero electroporation is an easy and efficient gene transfer system for developing mouse embryos in the uterus. By combining with the Cre-loxP system, the morphology of individual neurons can be clearly and sparsely visualized. Here, we describe how this labeling system can be applied to visualize and evaluate the dendrites and dendritic spines of cortical neurons.
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Espinas Dendríticas , Electroporación , Neuritas , Animales , Electroporación/métodos , Ratones , Femenino , Neuritas/metabolismo , Espinas Dendríticas/metabolismo , Embarazo , Útero/citología , Técnicas de Transferencia de Gen , Neuronas/citología , Neuronas/metabolismoRESUMEN
During the development of mammalian brains, pyramidal neurons in the cerebral cortex form highly organized six layers with different functions. These neurons undergo developmental processes such as axon extension, dendrite outgrowth, and synapse formation. A proper integration of the neuronal connectivity through dynamic changes of dendritic branches and spines is required for learning and memory. Disruption of these crucial developmental processes is associated with many neurodevelopmental and neurodegenerative disorders. To investigate the complex dendritic architecture, several useful staining tools and genetic methods to label neurons have been well established. Monitoring the dynamics of dendritic spine in a single neuron is still a challenging task. Here, we provide a methodology that combines in vivo two-photon brain imaging and in utero electroporation, which sparsely labels cortical neurons with fluorescent proteins. This protocol may help elucidate the dynamics of microstructure and neural complexity in living rodents under normal and disease conditions.
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Neuronas , Animales , Ratones , Neuronas/citología , Neuronas/metabolismo , Electroporación/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Células Piramidales/metabolismo , Células Piramidales/citología , Femenino , Corteza Cerebral/citología , Dendritas/metabolismoRESUMEN
To investigate the cell behavior underlying neuronal differentiation in a physiologically relevant context, differentiating neurons must be studied in their native tissue environment. Here, we describe an accessible protocol for fluorescent live imaging of differentiating neurons within ex vivo embryonic chicken spinal cord slice cultures, which facilitates long-term observation of individual cells within developing tissue.
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Diferenciación Celular , Electroporación , Neuronas , Médula Espinal , Animales , Electroporación/métodos , Médula Espinal/citología , Médula Espinal/embriología , Embrión de Pollo , Neuronas/citología , Neuronas/metabolismo , Pollos , NeurogénesisRESUMEN
The use of time-lapse live imaging enables us to track the dynamic changes in neurites during their formation. Ex vivo live imaging with acute brain slices provides a more physiological environment than cultured cells. To accomplish this, a certain method of labeling is necessary to visualize and identify neurite morphology. To understand the dynamics of neurite structure at early stages of neurite formation, we describe in this chapter ex vivo live imaging using a confocal microscope at P0 in combination with in utero electroporation (IUE).
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Encéfalo , Electroporación , Neuritas , Animales , Electroporación/métodos , Neuritas/metabolismo , Encéfalo/citología , Encéfalo/embriología , Encéfalo/diagnóstico por imagen , Ratones , Femenino , Microscopía Confocal/métodos , Imagen de Lapso de Tiempo/métodos , Embarazo , NeurogénesisRESUMEN
PURPOSE: This study evaluates the clinical efficacy and safety of irreversible electroporation (IRE) therapy combined with chemotherapy in patients with stage IV pancreatic cancer. METHODS: Between September 2021 and November 2023, we enrolled 38 patients with stage IV pancreatic cancer, with 20 receiving IRE plus chemotherapy and 18 receiving only chemotherapy. We recorded the general information of the patients and regularly followed up postoperative IRE-related adverse reactions. Progression-free survival (PFS) and overall survival (OS) were evaluated during follow-up. RESULTS: Median OS was longer in the IRE group than in the chemotherapy group. Median PFS was slightly extended with IRE compared to chemotherapy alone. The mean hospital stay for the IRE group was 5.90 ± 0.75 days. Four serious adverse events occurred after IRE. Postoperative pain scores were significantly lower than preoperative scores. CONCLUSION: IRE combined with chemotherapy showed clinical effectiveness in stage IV pancreatic cancer treatment, offering potential pain reduction benefits with fewer adverse effects and shorter hospital stays.
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Protocolos de Quimioterapia Combinada Antineoplásica , Electroporación , Estadificación de Neoplasias , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/mortalidad , Masculino , Femenino , Persona de Mediana Edad , Electroporación/métodos , Anciano , Terapia Combinada , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Resultado del Tratamiento , Tasa de Supervivencia , Estudios de Seguimiento , Tiempo de Internación/estadística & datos numéricos , Adulto , Supervivencia sin ProgresiónRESUMEN
Prostate cancer (PCa) is currently the second most common malignancy in men worldwideï¼and its incidence rate is on the rise. Most cases of PCa are treated by radical prostatectomy, but with the development of medical imaging and innovation in therapeutic theories and technology, focal therapy has shown better application prospects in the treatment of PCa. Compared with radical prostatectomy, focal therapy yields satisfactory results in terms of effectiveness and reduction of complications in addition to avoidance of overtreatment and treatment-related financial burden. This article reviews the strategies of focal therapy for PCa, including cryoablation, high-intensity focused ultrasound, irreversible electroporation, and photodynamic therapy, with an analysis of the clinical trials in recent years.
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Criocirugía , Fotoquimioterapia , Neoplasias de la Próstata , Humanos , Neoplasias de la Próstata/terapia , Masculino , Fotoquimioterapia/métodos , Criocirugía/métodos , Ultrasonido Enfocado de Alta Intensidad de Ablación/métodos , Prostatectomía/métodos , Electroporación/métodosRESUMEN
Efficient drinking water disinfection methods are critical for public health. Locally enhanced electric field treatment (LEEFT) is an antimicrobial method that uses sharp structures, like metallic nanowires, to enhance the electric field at tips and cause bacteria inactivation. Electroporation is the originally designed mechanism of LEEFT. Although oxidation is typically undesired due to byproduct generation and electrode corrosion, it can enhance the overall disinfection efficiency. In this work, we conduct an operando investigation of LEEFT, in which we change the electrical parameters to tune the mechanisms between electrophysical electroporation and electrochemical oxidation. Pure electroporation (i.e., without detectable oxidation) could be achieved under a duty cycle of ≤0.1% and a pulse width of ≤2 µs. Applying 2 µs pulses at 7-8 kV/cm and 0.1% duty cycle results in 80-100% bacteria inactivation with pure electroporation. A higher chance of oxidation is found with a higher duty cycle and a longer pulse width, where the antimicrobial efficiency could also be enhanced. For water with a higher conductivity, a higher antimicrobial efficiency can be achieved under the same treatment conditions, and electrochemical reactions could be induced more easily. The findings shown in this work improve the fundamental understanding of LEEFT and help optimize the performance of LEEFT in real applications.