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1.
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
2.
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
3.
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
4.
Anticancer Res ; 40(5): 2771-2775, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366423

RESUMO

BACKGROUND/AIM: Irreversible electroporation (IRE) has recently been used as an experimental treatment for cancers including locally advanced pancreatic cancer. There is very limited data on IRE in pancreatic cancer that is locally recurrent after surgical resection. The aim of this study was to evaluate the safety and efficacy of IRE in this setting. PATIENTS AND METHODS: Ten patients with locally recurrent pancreatic cancer without distant metastases after surgical resection were included and treated with ultrasound-guided percutaneous IRE. RESULTS: Two patients had severe complications, of whom one died. Median disease-free survival was 3.3 months and overall median survival after IRE and resection was 16.5 and 42.7 months, respectively. Two patients are alive 42.1 and 23.9 months after the IRE without signs of local recurrence. CONCLUSION: Percutaneous IRE in locally recurrent pancreatic cancer following curative resection is feasible, but should be regarded as a high-risk procedure that, at present, cannot be recommended outside of clinical trials. Further research is needed to select patients who might benefit from this treatment.


Assuntos
Eletroporação , Recidiva Local de Neoplasia/diagnóstico por imagem , Recidiva Local de Neoplasia/terapia , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/cirurgia , Ultrassonografia , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
5.
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
6.
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
7.
Science ; 368(6487): 181-186, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32273467

RESUMO

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Microscopia Intravital/métodos , Crista Neural , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Divisão Celular , Movimento Celular , Quimera/embriologia , Quimera/fisiologia , Eletroporação , Feminino , Técnicas de Transferência de Genes , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica , Crista Neural/irrigação sanguínea , Crista Neural/citologia , Crista Neural/embriologia , Placenta/fisiologia , Gravidez , Retina/embriologia , Retina/fisiologia , Transmissão Sináptica , Útero
8.
Proc Natl Acad Sci U S A ; 117(17): 9413-9422, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291340

RESUMO

Astrogenesis is repressed in the early embryonic period and occurs in the late embryonic period. A variety of external and internal signals contribute to the sequential differentiation of neural stem cells. Here, we discovered that immune-related CD93 plays a critical negative role in the regulation of astrogenesis in the mouse cerebral cortex. We show that CD93 expression is detected in neural stem cells and neurons but not in astrocytes and declines as differentiation proceeds. Cd93 knockout increases astrogenesis at the expense of neuron production during the late embryonic period. CD93 responds to the extracellular matrix protein Multimerin 2 (MMRN2) to trigger the repression of astrogenesis. Mechanistically, CD93 delivers signals to ß-Catenin through a series of phosphorylation cascades, and then ß-Catenin transduces these signals to the nucleus to activate Zfp503 transcription. The transcriptional repressor ZFP503 inhibits the transcription of glial fibrillary acidic protein (Gfap) by binding to the Gfap promoter with the assistance of Grg5. Furthermore, Cd93 knockout mice exhibit autism-like behaviors. Taken together, our results reveal that CD93 is a negative regulator of the onset of astrogenesis and provide insight into therapy for psychiatric disorders.


Assuntos
Astrócitos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Transtorno Autístico , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Eletroporação , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inflamação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Subfamília D de Receptores Semelhantes a Lectina de Células NK/genética , Proteínas do Tecido Nervoso/genética , Neurogênese , Neuroglia , Gravidez
9.
Invest Ophthalmol Vis Sci ; 61(4): 21, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32301972

RESUMO

Purpose: Human corneal endothelial cells (hCECs) have limited regenerative capacity in vivo. Reduced hCEC density results in bullous keratopathy requiring corneal transplantation. This study reveals the role of transcription factor 4 (TCF4) in hCEC diseases and suggests that TCF4 may be a molecular target for hCEC regeneration. Methods: Cell shape, cell proliferation rates, and proliferation-associated proteins were evaluated in normal or senescent hCECs. TCF4 was blocked by siRNA (si-TCF4) or activated using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 activation systems (pl-TCF4). The corneal endothelium of six-week-old Sprague-Dawley (SD) rats was transfected by electroporation followed by cryoinjury. Results: Cell proliferation rates and TCF4 levels were reduced in senescent cells. TCF4 CRISPR activation enhanced corneal endothelial wound healing. TCF4 regulated mitochondrial functions including mitochondrial membrane potential, mitochondrial superoxide levels, and energy production. The percentage of cells in the S-phase was reduced with si-TCF4 and increased with pl-TCF4. Cell proliferation and cell cycle-associated proteins were regulated by TCF4. Autophagy was induced by si-TCF4. In vivo transfection of CRISPR/dCas9 activation systems (a-TCF4) induced regeneration of corneal endothelium. Conclusions: Corneal endothelial diseases are associated with TCF4 reduction; TCF4 may be a potential target for hCEC diseases. Gene therapy using TCF4 CRISPR/dCas9 may be an effective treatment for hCEC diseases.


Assuntos
Epitélio Posterior/fisiologia , Regeneração/fisiologia , Fator de Transcrição 4/fisiologia , Cicatrização/fisiologia , Adulto , Animais , Western Blotting , Proteína 9 Associada à CRISPR/metabolismo , Proliferação de Células , Forma Celular , Células Cultivadas , Eletroporação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasmídeos , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
10.
Radiology ; 295(2): 254-272, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32208094

RESUMO

This review summarizes the use of high-voltage electrical pulses (HVEPs) in clinical oncology to treat solid tumors with irreversible electroporation (IRE) and electrochemotherapy (ECT). HVEPs increase the membrane permeability of cells, a phenomenon known as electroporation. Unlike alternative ablative therapies, electroporation does not affect the structural integrity of surrounding tissue, thereby enabling tumors in the vicinity of vital structures to be treated. IRE uses HVEPs to cause cell death by inducing membrane disruption, and it is primarily used as a radical ablative therapy in the treatment of soft-tissue tumors in the liver, kidney, prostate, and pancreas. ECT uses HVEPs to transiently increase membrane permeability, enhancing cellular cytotoxic drug uptake in tumors. IRE and ECT show immunogenic effects that could be augmented when combined with immunomodulatory drugs, a combination therapy the authors term electroimmunotherapy. Additional electroporation-based technologies that may reach clinical importance, such as gene electrotransfer, electrofusion, and electroimmunotherapy, are concisely reviewed. HVEPs represent a substantial advancement in cancer research, and continued improvement and implementation of these presented technologies will require close collaboration between engineers, interventional radiologists, medical oncologists, and immuno-oncologists.


Assuntos
Eletroporação/métodos , Oncologia/métodos , Neoplasias/terapia , Antineoplásicos/administração & dosagem , Fusão Celular/métodos , Terapia por Estimulação Elétrica/métodos , Eletroquimioterapia/métodos , Técnicas de Transferência de Genes , Humanos , Imunoterapia/métodos
12.
J Vis Exp ; (156)2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32150173

RESUMO

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.


Assuntos
Neoplasias Colorretais/patologia , Eletroporação/métodos , Engenharia Genética/métodos , Neoplasias Hepáticas/patologia , Organoides/patologia , Neoplasias Pancreáticas/patologia , Plasmídeos/administração & dosagem , Neoplasias Gástricas/patologia , Humanos , Técnicas de Cultura de Órgãos , Plasmídeos/genética , Transfecção
13.
Lett Appl Microbiol ; 70(5): 388-393, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32077122

RESUMO

Four Agrobacterium tumefaciens strains AGL-1, C58C1, EHA105 and LBA4404 were tested for the effects of strain types on the transformation efficiency in Mortierella alpina. Results showed that AGL-1, C58C1 and EHA105 transformed M. alpina successfully. Among them, A. tumefaciens EHA105 was first proven successful transformation of M. alpina. AGL-1 and EHA105 had the highest transformation efficiency among the four strains, while LBA4404 failed to transform M. alpina. The reason leading to the transformation efficiency difference among the four strains was explored by determining transcription levels of the virulence (vir) gene in the induction medium. Results showed that the expressions of virD1, virD2, virD4 and virE1 genes were obviously induced by acetosyringone in all the strains, and their transcriptional levels as well as virA's of AGL-1, C58C1 and EHA105 were higher than that of LBA4404, suggesting high transcriptional levels of vir genes were important for successful transformation. The study selected A. tumefaciens with high transformation efficiency of M. alpina, and would accelerate the genetic management of M. alpina. SIGNIFICANCE AND IMPACT OF THE STUDY: Oleaginous filamentous fungus Mortierella alpina is a commercial strain for the production of arachidonic acid. Genetic manipulation of M. alpina requires highly efficient transformation method. In this study, we explore the effect of Agrobacterium tumefaciens strain types on the transformation efficiency of M. alpina and select A. tumefaciens with the highest transformation efficiency, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of virulence genes in A. tumefaciens were proven to play an important role for successful transformation.


Assuntos
Agrobacterium tumefaciens/genética , Mortierella/genética , Transformação Genética , Eletroporação , Microbiologia Industrial , Microrganismos Geneticamente Modificados , Plasmídeos , Virulência , Fatores de Virulência/genética
14.
PLoS One ; 15(2): e0229214, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084220

RESUMO

INTRODUCTION: The application of brief high voltage electrical pulses to tissue can lead to an irreversible or reversible electroporation effect in a cell-specific manner. In the management of ventricular arrhythmias, the ability to target different tissue types, specifically cardiac conduction tissue (His-Purkinje System) vs. cardiac myocardium would be advantageous. We hypothesize that pulsed electric fields (PEFs) can be applied safely to the beating heart through a catheter-based approach, and we tested whether the superficial Purkinje cells can be targeted with PEFs without injury to underlying myocardial tissue. METHODS: In an acute (n = 5) and chronic canine model (n = 6), detailed electroanatomical mapping of the left ventricle identified electrical signals from myocardial and overlying Purkinje tissue. Electroporation was effected via percutaneous catheter-based Intracardiac bipolar current delivery in the anesthetized animal. Repeat Intracardiac electrical mapping of the heart was performed at acute and chronic time points; followed by histological analysis to assess effects. RESULTS: PEF demonstrated an acute dose-dependent functional effect on Purkinje, with titration of pulse duration and/or voltage associated with successful acute Purkinje damage. Electrical conduction in the insulated bundle of His (n = 2) and anterior fascicle bundle (n = 2), was not affected. At 30 days repeat cardiac mapping demonstrated resilient, normal electrical conduction throughout the targeted area with no significant change in myocardial amplitude (pre 5.9 ± 1.8 mV, 30 days 5.4 ± 1.2 mV, p = 0.92). Histopathological analysis confirmed acute Purkinje fiber targeting, with chronic studies showing normal Purkinje fibers, with minimal subendocardial myocardial fibrosis. CONCLUSION: PEF provides a novel, safe method for non-thermal acute modulation of the Purkinje fibers without significant injury to the underlying myocardium. Future optimization of this energy delivery is required to optimize conditions so that selective electroporation can be utilized in humans the treatment of cardiac disease.


Assuntos
Eletroporação , Ventrículos do Coração/citologia , Animais , Cães , Estudos de Viabilidade , Masculino , Células de Purkinje/citologia , Segurança , Sobrevivência de Tecidos , Função Ventricular
16.
Biochim Biophys Acta Biomembr ; 1862(5): 183213, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057755

RESUMO

Picosecond pulse trains (psPTs) are emerging as a new characteristic diagnostic and therapeutic tool in biomedical fields. To specifically determine the stimulus provided to cells, in this article, we use a molecular dynamics (MD) model to show the molecular mechanisms of electroporation induced by symmetrical bipolar psPTs and predict a bipolar cancellation for the studied picosecond pulses. Electric field conditions that do not cause electroporation reveal that the interfacial water molecules continuously flip and redirect as the applied bipolar psPT reverses, and the molecules cannot keep moving in one direction or leave the lipid-water interface. Based on our simulation results, we determine the threshold for electroporation with symmetrical bipolar psPTs. For a fixed electric field intensity, a lower repetition frequency leads to more rapid electroporation. For a fixed repetition frequency, a higher electric field intensity leads to more rapid electroporation. We found that the water dipole relaxation time decreases as the electric field magnitude increases. Additionally, the influences of the symmetrical bipolar psPT intensity and frequency on the pore formation time are presented. Discrete nanoscale pores can form with the applied psPT at terahertz (THz) repetition frequency. When the psPT amplitude increases or the frequency decreases, the number of water bridges will increase. Moreover, for the first time, the molecular mechanism of bipolar cancellation for the studied picosecond pulse is discussed preliminarily. Our results indicate that the influence of the unipolar picosecond pulse on the interfacial water dipoles will accumulate in one direction, but the bipolar picosecond pulse does not cause this effect.


Assuntos
Eletroporação/métodos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Eletricidade , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Fosfatidilcolinas/fisiologia
17.
Development ; 147(4)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32001442

RESUMO

In chickens, the embryonic ovary differentiates into two distinct domains before meiosis: a steroidogenic core (the female medulla), overlain by the germ cell niche (the cortex). The differentiation of the medulla is a cell-autonomous process based on chromosomal sex identity (CASI). In order to address the extent to which cortex differentiation depends on intrinsic or extrinsic factors, we generated models of gonadal intersex by mixing ZW (female) and ZZ (male) cells in gonadal chimeras, or by altering oestrogen levels of ZW and ZZ embryos. We found that CASI does not apply to the embryonic cortex. Both ZW and ZZ cells can form the cortex and this can happen independently of the phenotypic sex of the medulla as long as oestrogen is provided. We also show that the cortex-promoting activity of oestrogen signalling is mediated via estrogen receptor alpha within the left gonad epithelium. However, the presence of a medulla with an 'intersex' or male phenotype may compromise germ cell progression into meiosis, causing cortical germ cells to remain in an immature state in the embryo.


Assuntos
Estrogênios/fisiologia , Oogênese , Ovário/embriologia , Animais , Embrião de Galinha , Galinhas/genética , Cromossomos/genética , Eletroporação , Células Epiteliais/citologia , Feminino , Células Germinativas/citologia , Gônadas/citologia , Masculino , Meiose , Mitose , Fenótipo , Cromossomos Sexuais , Diferenciação Sexual/genética , Transdução de Sinais
18.
Gene ; 730: 144318, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-31917231

RESUMO

Although the chicken embryo has been a classical model for developmental studies, the lack of straightforward technologies for chicken transgenesis limited the usefulness of this animal model. Here, we assessed electroporation and lipofection approaches for in ovo transfection of Sleeping Beauty transposon system in stage X-XII chicken embryos. Electroporation of chicken embryos could transfect the trophectodermal cells. Then, a mixture of transposon lipoplexes and high concentrated carboxymethylcellulose (HCC) solution was injected into the subgerminal cavity of day 0 embryos. The lipoplex-HCC mixture substantially increased the number of trophectodermal cells expressing the reporter. Importantly, the fluorescent reporter was detected in cells inside of the embryos as well as circulation cells in the bloodstream during days 3-4 of incubation. This study provided evidence for direct in ovo transfection of early chicken embryos, though the long-term outcome of this approach warrants further studies.


Assuntos
Eletroporação/métodos , Transfecção/métodos , Transposases/genética , Animais , Animais Geneticamente Modificados , Carboximetilcelulose Sódica , Embrião de Galinha , Galinhas/genética , Elementos de DNA Transponíveis/genética , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Transferência de Genes
19.
PLoS Biol ; 18(1): e3000589, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31922526

RESUMO

Electroporation is a basic yet powerful method for delivering small molecules (RNA, DNA, drugs) across cell membranes by application of an electrical field. It is used for many diverse applications, from genetically engineering cells to drug- and DNA-based vaccine delivery. Despite this broad utility, the high cost of electroporators can keep this approach out of reach for many budget-conscious laboratories. To address this need, we develop a simple, inexpensive, and handheld electroporator inspired by and derived from a common household piezoelectric stove lighter. The proposed "ElectroPen" device can cost as little as 23 cents (US dollars) to manufacture, is portable (weighs 13 g and requires no electricity), can be easily fabricated using 3D printing, and delivers repeatable exponentially decaying pulses of about 2,000 V in 5 ms. We provide a proof-of-concept demonstration by genetically transforming plasmids into Escherichia coli cells, showing transformation efficiency comparable to commercial devices, but at a fraction of the cost. We also demonstrate the potential for rapid dissemination of this approach, with multiple research groups across the globe validating the ease of construction and functionality of our device, supporting the potential for democratization of science through frugal tools. Thus, the simplicity, accessibility, and affordability of our device holds potential for making modern synthetic biology accessible in high school, community, and resource-poor laboratories.


Assuntos
Eletroporação/instrumentação , Técnicas de Transferência de Genes/instrumentação , Análise Custo-Benefício , Eletricidade , Eletroporação/economia , Desenho de Equipamento/economia , Escherichia coli , Técnicas de Transferência de Genes/economia , Humanos , Laboratórios/economia , Manufaturas/economia , Áreas de Pobreza , Impressão Tridimensional , Transformação Bacteriana , Transportes
20.
J Vasc Interv Radiol ; 31(3): 482-491.e4, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31956003

RESUMO

PURPOSE: To determine the safety and feasibility of percutaneous high-frequency irreversible electroporation (HFIRE) for primary liver cancer and evaluate the HFIRE-induced local immune response. MATERIALS AND METHODS: HFIRE therapy was delivered percutaneously in 3 canine patients with resectable hepatocellular carcinoma (HCC) in the absence of intraoperative paralytic agents or cardiac synchronization. Pre- and post-HFIRE biopsy samples were processed with histopathology and immunohistochemistry for CD3, CD4, CD8, and CD79a. Blood was collected on days 0, 2, and 4 for complete blood count and chemistry. Numeric models were developed to determine the treatment-specific lethal thresholds for malignant canine liver tissue and healthy porcine liver tissue. RESULTS: HFIRE resulted in predictable ablation volumes as assessed by posttreatment CT. No detectable cardiac interference and minimal muscle contraction occurred during HFIRE. No clinically significant adverse events occurred secondary to HFIRE. Microscopically, a well-defined ablation zone surrounded by a reactive zone was evident in the majority of samples. This zone was composed primarily of maturing collagen interspersed with CD3+/CD4-/CD8- lymphocytes in a proinflammatory microenvironment. The average ablation volumes for the canine HCC patients and the healthy porcine tissue were 3.89 cm3 ± 0.74 and 1.56 cm3 ± 0.16, respectively (P = .03), and the respective average lethal thresholds were 710 V/cm ± 28.2 and 957 V/cm ± 24.4 V/cm (P = .0004). CONCLUSIONS: HFIRE can safely and effectively be delivered percutaneously, results in a predictable ablation volume, and is associated with lymphocytic tumor infiltration. This is the first step toward the use of HFIRE for treatment of unresectable liver tumors.


Assuntos
Técnicas de Ablação/veterinária , Carcinoma Hepatocelular/veterinária , Doenças do Cão/cirurgia , Eletroporação/veterinária , Neoplasias Hepáticas/veterinária , Animais , Complexo CD3/imunologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Doenças do Cão/imunologia , Doenças do Cão/patologia , Cães , Estudos de Viabilidade , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Linfócitos do Interstício Tumoral/imunologia , Masculino , Estudo de Prova de Conceito , Sus scrofa
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