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The observation of a weak proton-emission branch in the decay of the 3174-keV 53mCo isomeric state marked the discovery of proton radioactivity in atomic nuclei in 1970. Here we show, based on the partial half-lives and the decay energies of the possible proton-emission branches, that the exceptionally high angular momentum barriers, [Formula: see text] and [Formula: see text], play a key role in hindering the proton radioactivity from 53mCo, making them very challenging to observe and calculate. Indeed, experiments had to wait decades for significant advances in accelerator facilities and multi-faceted state-of-the-art decay stations to gain full access to all observables. Combining data taken with the TASISpec decay station at the Accelerator Laboratory of the University of Jyväskylä, Finland, and the ACTAR TPC device on LISE3 at GANIL, France, we measured their branching ratios as bp1 = 1.3(1)% and bp2 = 0.025(4)%. These results were compared to cutting-edge shell-model and barrier penetration calculations. This description reproduces the order of magnitude of the branching ratios and partial half-lives, despite their very small spectroscopic factors.
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Limb-Girdle Muscular Dystrophy Type R1 (LGMDR1; formerly LGMD2A), characterized by progressive hip and shoulder muscle weakness, is caused by mutations in CAPN3. In zebrafish, capn3b mediates Def-dependent degradation of p53 in the liver and intestines. We show that capn3b is expressed in the muscle. To model LGMDR1 in zebrafish, we generated three deletion mutants in capn3b and a positive-control dmd mutant (Duchenne muscular dystrophy). Two partial deletion mutants showed transcript-level reduction, whereas the RNA-less mutant lacked capn3b mRNA. All capn3b homozygous mutants were developmentally-normal adult-viable animals. Mutants in dmd were homozygous-lethal. Bathing wild-type and capn3b mutants in 0.8% methylcellulose (MC) for 3 days beginning 2 days post-fertilization resulted in significantly pronounced (20-30%) birefringence-detectable muscle abnormalities in capn3b mutant embryos. Evans Blue staining for sarcolemma integrity loss was strongly positive in dmd homozygotes, negative in wild-type embryos, and negative in MC-treated capn3b mutants, suggesting membrane instability is not a primary muscle pathology determinant. Increased birefringence-detected muscle abnormalities in capn3b mutants compared to wild-type animals were observed following induced hypertonia by exposure to cholinesterase inhibitor, azinphos-methyl, reinforcing the MC results. These mutant fish represent a novel tractable model for studying the mechanisms underlying muscle repair and remodeling, and as a preclinical tool for whole-animal therapeutics and behavioral screening in LGMDR1.
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Distrofia Muscular de Cinturas , Distrofia Muscular de Duchenne , Animales , Pez Cebra/genética , Distrofia Muscular de Cinturas/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/patologíaRESUMEN
Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.
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Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Edición Génica/métodos , Pez Cebra/genética , Animales , Microinyecciones , Mutagénesis Sitio-Dirigida , ARN Guía de Sistemas CRISPR-CasRESUMEN
BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder characterized by a lack of blood vessel growth to the periphery of the retina with secondary fibrovascular proliferation at the vascular-avascular junction. These structurally abnormal vessels cause leakage and hemorrhage, while the fibroproliferative scarring results in retinal dragging, detachment and blindness. Mutations in the FZD4 gene represent one of the most common causes of FEVR. METHODS: A loss of function mutation resulting from a 10-nucleotide insertion into exon 1 of the zebrafish fzd4 gene was generated using transcription activator-like effector nucleases (TALENs). Structural and functional integrity of the retinal vasculature was examined by fluorescent microscopy and optokinetic responses. RESULTS: Zebrafish retinal vasculature is asymmetrically distributed along the dorsoventral axis, with active vascular remodeling on the ventral surface of the retina throughout development. fzd4 mutants exhibit disorganized ventral retinal vasculature with discernable tubular fusion by week 8 of development. Furthermore, fzd4 mutants have impaired optokinetic responses requiring increased illumination. CONCLUSION: We have generated a visually impaired zebrafish FEVR model exhibiting abnormal retinal vasculature. These fish provide a tractable system for studying vascular biology in retinovascular disorders, and demonstrate the feasibility of using zebrafish for evaluating future FEVR genes identified in humans.
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Receptores Frizzled/fisiología , Retina/patología , Vasos Retinianos/patología , Remodelación Vascular/genética , Proteínas de Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Modelos Animales de Enfermedad , Embrión no Mamífero , Vitreorretinopatías Exudativas Familiares/diagnóstico , Vitreorretinopatías Exudativas Familiares/genética , Vitreorretinopatías Exudativas Familiares/patología , Estudios de Factibilidad , Receptores Frizzled/genética , Humanos , Neovascularización Patológica/embriología , Neovascularización Patológica/genética , Neovascularización Patológica/fisiopatología , Retina/diagnóstico por imagen , Retina/embriología , Retina/metabolismo , Enfermedades de la Retina/genética , Enfermedades de la Retina/patología , Vasos Retinianos/embriología , Vasos Retinianos/fisiología , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Histamine (HA) is a neurotransmitter in arthropod photoreceptors. It is recycled via conjugation to ß-alanine to form ß-alanylhistamine (carcinine). Conjugation occurs in epithelial glia that surround photoreceptor terminals in the first optic neuropil, and carcinine (CA) is then transported back to photoreceptors and cleaved to liberate HA and ß-alanine. The gene Inebriated (Ine) encodes an Na+/Cl--dependent SLC6 family transporter translated as two protein isoforms, long (P1) and short (P2). Photoreceptors specifically express Ine-P2 whereas Ine-P1 is expressed in non-neuronal cells. Both ine1 and ine3 have significantly reduced head HA contents compared with wild type, and a smaller increase in head HA after drinking 1% CA. Similarly, uptake of 0.1% CA was reduced in ine1 and ine3 mutant synaptosomes, but increased by 90% and 84% respectively for fractions incubated in 0.05% ß-Ala, compared with wild type. Screening potential substrates in Ine expressing Xenopus oocytes revealed very little response to carcinine and ß-Ala but increased conductance with glycine. Both ine1 and ine3 mutant responses in light-dark phototaxis did not differ from wild-type. Collectively our results suggest that Inebriated functions in an adjunct role as a transporter to the previously reported carcinine transporter CarT.
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INTRODUCTION: Genome editing using CRISPR/Cas9 has advanced very rapidly in its scope, versatility and ease of use. Zebrafish (Danio rerio) has been one of the vertebrate model species where CRISPR/Cas9 has been applied very extensively for many different purposes and with great success. In particular, disease modeling in zebrafish is useful for testing specific gene variants for pathogenicity in a preclinical setting. Here we describe multiple advances in diverse species and systems that can improve genome editing in zebrafish. OBJECTIVE: To achieve temporal and spatial precision of genome editing, many new technologies can be applied in zebrafish such as artificial transcription factors, drug-inducible or optogenetically-driven expression of Cas9, or chemically-inducible activation of Cas9. Moreover, chemically- or optogenetically- inducible reconstitution of dead Cas9 (catalytically inactive, dCas9) can enable spatiotemporal control of gene regulation. In addition to controlling where and when genome editing occurs, using oligonucleotides allows for the introduction (knock-in) of precise modifications of the genome. CONCLUSION: We review recent trends to improve the precision and efficiency of oligo-based point mutation knock-ins and discuss how these improvements can apply to work in zebrafish. Similarly to how chemical mutagenesis enabled the first genetic screens in zebrafish, multiplexed sgRNA libraries and Cas9 can enable the next revolutionary transition in how genetic screens are performed in this species. We discuss the first examples and prospects of approaches using sgRNAs as specific and effective mutagens. Moreover, we have reviewed methods aimed at measuring the phenotypes of single cells after their mutagenic perturbation with vectors encoding individual sgRNAs. These methods can range from different cell-based reporters to single-cell RNA sequencing and can serve as great tools for high-throughput genetic screens.
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Sistemas CRISPR-Cas , Edición Génica/métodos , Genoma/genética , Genómica/métodos , Animales , Mutación , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Nitric oxide gas acts as a short-range signaling molecule in a vast array of important physiological processes, many of which include major changes in gene expression. How these genomic responses are induced, however, is poorly understood. Here, using genetic and chemical manipulations, we show that nitric oxide is produced in the Drosophila prothoracic gland, where it acts via the nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with its heterodimer partner, Drosophila hormone receptor 3 (DHR3). Manipulation of these interactions leads to gross alterations in feeding behavior, fat deposition, and developmental timing. These neuroendocrine interactions and consequences appear to be conserved in vertebrates.
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Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Óxido Nítrico/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/farmacología , Proteínas de Drosophila/genética , Proteínas de Drosophila/farmacología , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Ecdisona/farmacología , Conducta Alimentaria/fisiología , Depuradores de Radicales Libres/farmacología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Larva , Metabolismo de los Lípidos , Metamorfosis Biológica/genética , Metamorfosis Biológica/fisiología , Óxido Nítrico/farmacología , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/farmacologíaRESUMEN
Establishment of the Drosophila dorsal-ventral axis depends upon the correct localization of gurken mRNA and protein within the oocyte. gurken mRNA becomes localized to the presumptive dorsal anterior region of the oocyte, but is synthesized in the adjoining nurse cells. Normal gurken localization requires the heterogeneous nuclear ribonucleoprotein Squid, which binds to the gurken 3' untranslated region. However, whether Squid functions in the nurse cells or the oocyte is unknown. To address this question, we generated genetic mosaics in which half of the nurse cells attached to a given oocyte are unable to produce Squid. In these mosaics, gurken mRNA is localized normally but ectopically translated during the dorsal anterior localization process, even though the oocyte contains abundant Squid produced by the wild type nurse cells. These data indicate that translational repression of gurken mRNA requires Squid function in the nurse cells. We propose that Squid interacts with gurken mRNA in the nurse cell nuclei and, together with other factors, maintains gurken in a translationally silent state during its transport to the dorsal anterior region of the oocyte. This translational repression is not required for gurken mRNA localization, indicating that the information repressing translation is separable from that regulating localization.
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Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Oocitos , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Factor de Crecimiento Transformador alfa/genética , Animales , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/fisiología , Células Germinativas/citología , Células Germinativas/fisiología , Ribonucleoproteínas Nucleares Heterogéneas/genética , Mosaicismo , Oocitos/citología , Oocitos/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
INTRODUCTIONFluorescent in situ hybridization (FISH) is commonly used to analyze the three-dimensional distribution of RNAs in intact embryos and tissues. Tyramide signal amplification (TSA) significantly increases the sensitivity and resolution of FISH probe signals. This protocol includes optimized TSA-FISH procedures for Drosophila embryos, ovaries, and larval tissues. Instructions are given for the preparation of RNA probes, the collection and fixation of tissues, and the hybridization and TSA-mediated detection of probes, including options for high-throughput processing in 96-well plates. Variations of the procedure for RNA-RNA and RNA-protein costaining are also described.
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The asymmetric localization of gurken mRNA and protein in the developing Drosophila oocyte defines both the anteroposterior and dorsoventral axes of the future embryo. Understanding the origin of these asymmetries requires knowledge of the source of gurken transcripts. During oogenesis most transcripts in the oocyte are produced by the associated nurse cells, but it has been proposed that gurken is an exceptional oocyte-derived transcript. Using a novel application of a standard mitotic recombination technique, we generated mosaic egg chambers in which the nurse cells, but not the oocyte, could produce gurken. Gurken was properly localized in these mosaics and oocyte axial polarity was established normally, indicating that the nurse cells synthesize gurken and that their contribution is sufficient for Gurken function. Our data demonstrate the existence of a mechanism for transport of gurken from the nurse cells and its subsequent localization within the oocyte.