RESUMEN
Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXOEAA-PMO). EXOEAA-PMO elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXOEAA-PMO elicited therapeutic levels of dystrophin restoration and functional improvements in mdx mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.
Asunto(s)
Exosomas , Distrofia Muscular de Duchenne , Animales , Ratones , Distrofina/genética , Ratones Endogámicos mdx , Exosomas/metabolismo , Morfolinos/metabolismo , Morfolinos/farmacología , Morfolinos/uso terapéutico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Oligonucleótidos/metabolismo , Oligonucleótidos/uso terapéuticoRESUMEN
BACKGROUND: Pituitary adenylate cyclase-activating polypeptide is a neuropeptide with diverse roles in biological processes. Its involvement in the blood coagulation cascade is unclear. OBJECTIVES: This study unraveled adcyap1b's role in blood coagulation using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 in zebrafish. Effects were validated via adcyap1b knockdown. Gene expression changes in adcyap1b mutants were explored, linking them to clotting disorders. An analysis of proca gene splicing illuminated its role in adcyap1b-related anticoagulation deficiencies. METHODS: Zebrafish were genetically modified using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to induce adcyap1b knockout. Morpholino-mediated gene knockdown was employed for validation. Expression levels of coagulation factors, anticoagulant proteins, and fibrinolytic system genes were assessed in adcyap1b mutant zebrafish. Alternative splicing of proca gene was analyzed. RESULTS: Adcyap1b mutant zebrafish exhibited severe hemorrhage, clotting disorders, and disrupted blood coagulation. Morpholino-mediated knockdown replicated observed phenotypes. Downregulation in transcripts related to coagulation factors V and IX, anticoagulation protein C, and plasminogen was observed. Abnormal alternative splicing of the proca gene was identified, providing a mechanistic explanation for anticoagulation system deficiencies. CONCLUSION: Adcyap1b plays a crucial role in maintaining zebrafish blood coagulation and hemostasis. Its influence extends to the regulation of procoagulant and anticoagulant pathways, with abnormal alternative splicing contributing to observed deficiencies. These findings unveil a novel aspect of adcyap1b function, offering potential insights into similar processes in mammalian systems.
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Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Morfolinos/genética , Morfolinos/metabolismo , Coagulación Sanguínea/genética , Factor V/metabolismo , Hemorragia , Anticoagulantes/metabolismo , Mamíferos/metabolismoRESUMEN
Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.
Asunto(s)
Morfolinos , Oligonucleótidos Antisentido , Precursores del ARN , Ribonucleoproteína Nuclear Pequeña U1 , Morfolinos/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/farmacología , Poliadenilación , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Precursores del ARN/metabolismo , Humanos , Células HeLa , Técnicas de Silenciamiento del Gen , Factor de Especificidad de Desdoblamiento y Poliadenilación , Factor de Estimulación del Desdoblamiento/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Work in the catshark Scyliorhinus canicula has shown that the evolutionary origin of postnatal neurogenesis in vertebrates is earlier than previously thought. Thus, the catshark can serve as a model of interest to understand postnatal neurogenic processes and their evolution in vertebrates. One of the best characterized neurogenic niches of the catshark CNS is found in the peripheral region of the retina. Unfortunately, the lack of genetic tools in sharks limits the possibilities to deepen in the study of genes involved in the neurogenic process. Here, we report a method for gene knockdown in the juvenile catshark retina based on the use of Vivo-Morpholinos. To establish the method, we designed Vivo-Morpholinos against the proliferation marker PCNA. We first evaluated the possible toxicity of 3 different intraocular administration regimes. After this optimization step, we show that a single intraocular injection of the PCNA Vivo-Morpholino decreases the expression of PCNA in the peripheral retina, which leads to reduced mitotic activity in this region. This method will help in deciphering the role of other genes potentially involved in postnatal neurogenesis in this animal model.
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Tiburones , Animales , Tiburones/genética , Tiburones/metabolismo , Morfolinos/genética , Morfolinos/farmacología , Morfolinos/metabolismo , Técnicas de Silenciamiento del Gen , Antígeno Nuclear de Célula en Proliferación/genética , Retina/metabolismoRESUMEN
Pregnenolone (P5) is a steroid that functions in the brain and in zebrafish embryogenesis. It is synthesized from cholesterol via the enzymatic activity of P450scc, encoded by CYP11A1. P5 exerts its function by activating CLIP1, which in turn promotes microtubule assembly necessary for many biological processes including embryogenesis. To examine the functional relatedness of CYP11A1 and CLIP1, we ablated the embryonic expression of both genes in zebrafish, i.e. cyp11a1 and clip1a. Two cyp11a1 knockout fish lines were generated. Both homozygous cyp11a1 knockout lines appeared normal. But the development of fish embryos was delayed and embryonic cell migration was reduced when cyp11a1 function was depleted of by morpholinos. This discrepancy in phenotypes by two different gene depletion methods was also observed for clip1a. While clip1a morphants are defective in embryogenesis, clip1a knockout fish appeared normal. The phenotypes depend on the methods that create gene depletion. While knockout fish lines do not have expected phenotypic defects, clip1a and cyp11a1 morpholinos both reduce embryonic cell migration. We have evaluated the usefulness of both methods of gene ablation, and conclude that CYP11A1 and CLIP1 function in the same pathway to promote embryogenesis.
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Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Pez Cebra , Animales , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Pregnenolona/metabolismo , Morfolinos/metabolismo , Desarrollo Embrionario/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Interleukin-1ß (IL-1ß) is a potent proinflammatory cytokine that plays a vital role in the innate immune system. To observe the innate immune response in vivo, several transgenic zebrafish lines have been developed to model IL-1ß-induced inflammation and to visualize immune cell migration and proliferation in real time. However, our understanding of the IL-1ß response in zebrafish is limited due to an incomplete genome annotation and a lack of functional data for the cytokine receptors involved in the inflammatory process. Here, we use a combination of database mining, genetic analyses, and functional assays to identify zebrafish Interleukin-1 receptor, type 1 (Il1r1). We identified putative zebrafish il1r1 candidate genes that encode proteins with predicted structures similar to human IL1R1. To examine functionality of these candidates, we designed highly effective morpholinos to disrupt gene expression in a zebrafish model of embryonic Il-1ß-induced systemic inflammation. In this double transgenic model, ubb:Gal4-EcR, uas:il1ßmat , the zebrafish ubiquitin b (ubb) promoter drives expression of the modified Gal4 transcription factor fused to the ecdysone receptor (EcR), which in turn drives the tightly-regulated expression and secretion of mature Il-1ß only in the presence of the ecdysone analog tebufenozide (Teb). Application of Teb to ubb:Gal4-EcR, uas:il1ßmat embryos causes premature death, fin degradation, substantial neutrophil expansion, and generation of reactive oxygen species (ROS). To rescue these deleterious phenotypes, we injected ubb:Gal4-EcR, uas:il1ßmat embryos with putative il1r1 morpholinos and found that knockdown of only one candidate gene prevented the adverse effects caused by Il-1ß. Mosaic knockout of il1r1 using the CRISPR/Cas9 system phenocopied these results. Taken together, our study identifies the functional zebrafish Il1r1 utilizing a genetic model of Il-1ß-induced inflammation and provides valuable new insights to study inflammatory conditions specifically driven by Il-1ß or related to Il1r1 function in zebrafish.
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Receptores de Interleucina-1 , Pez Cebra , Animales , Humanos , Receptores de Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Pez Cebra/genética , Morfolinos/metabolismo , Inflamación/inducido químicamente , Inflamación/genéticaRESUMEN
Two pairs of unprecedented ß-carboline-phenylpropanoid heterogeneous alkaloids, (±)-pheharmines A-B (1-4), characterized by a morpholino[4,3,2-hi]ß-carboline core with two chiral centers, were isolated from the roots of Peganum harmala. The structures, including their absolute configurations, were identified using spectroscopic analyses and electronic circular dichroism (ECD) calculations. The biosynthetic hypothesis for the formation of pheharmines A-B was proposed. Compounds 1-4 exhibited moderate cytotoxic activities against HL-60 cell lines.
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Alcaloides , Peganum , Humanos , Peganum/química , Peganum/metabolismo , Morfolinos/análisis , Morfolinos/metabolismo , Semillas , Estructura Molecular , Alcaloides/farmacología , Alcaloides/química , Carbolinas/farmacología , Carbolinas/químicaRESUMEN
This work reports on the reactive oxygen species (ROS) generation and the therapeutic activities of new triphenyl-phosphonium-labelled phthalocyanines (Pcs), the 2,9,16,23-tetrakis(N-(N-butyl-4-triphenyl-phosphonium)- pyridine-4-yloxy) Zn(II) Pc (3) and 2,9,16,23-tetrakis-(N-(N-butyl-4-triphenyl-phosphonium)-morpholino) Zn(II) Pc (4) upon exposure to light, ultrasound and the combination of light and ultrasound. Two types of ROS were detected: the singlet oxygen (1O2) and hydroxyl radicals. For light irradiations, only the 1O2 was detected. An increase in the ROS generation was observed for samples treated with the combination of light and ultrasound compared to the light and ultrasound mono-treatments. The in vitro anticancer activity through photodynamic (PDT) and sonodynamic (SDT) therapy for the Pcs were also determined and compared to the photo-sonodynamic combination therapy (PSDT). The two cancer cell lines used for the in vitro studies included the Michigan Cancer Foundation-7 (MCF-7) breast cancer and Henrietta Lacks (HeLa) cervical cancer cell lines. The SDT treatments showed improved therapeutic efficacy on the cancer cells for both the Pcs compared to PDT. PSDT showed better therapeutic efficacy compared to both the PDT and SDT mono-treatments.
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Neoplasias de la Mama , Fotoquimioterapia , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Células HeLa , Humanos , Indoles/metabolismo , Indoles/farmacología , Células MCF-7 , Michigan , Mitocondrias/metabolismo , Morfolinos/metabolismo , Piridinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Oxígeno Singlete/metabolismoRESUMEN
Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities.
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Distrofia Muscular de Cinturas , Sarcoglicanopatías , Niño , Humanos , Morfolinos/genética , Morfolinos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/patología , Mutación , Sarcoglicanopatías/metabolismoRESUMEN
BACKGROUND: The cadherin-associated protein p120 catenin regulates convergent extension through interactions with cadherin proteins, Cdc42, and Rac1, as we previously showed in zebrafish (Danio rerio). Phosphorylation of p120 catenin changes the nature of its activity in vitro but is virtually unexplored in embryos. We used our previously developed antisense RNA splice-site morpholino targeted to endogenous p120 catenin-δ1 to cause defects in axis elongation probing the functions of three p120 catenin tyrosine-phosphorylation sites in gastrulating zebrafish embryos. RESULTS: The morpholino-induced defects were rescued by co-injections with mouse p120 catenin-δ1-3A mRNAs mutated at residues Y228 and Y217 to a non-phosphorylatable phenylalanine (F) or mutated at residue Y335 to a phosphomimetic glutamic acid (E). Co-injection of the complementary mutations Y228E, Y217E, or Y335F mRNAs partially rescued embryos whereas dual mutation to Y228E-Y217E blocked rescue. Immunopurification showed Y228F mutant proteins preferentially interacted with Rac1, potentially promoting cell migration. In contrast, the phosphomimetic Y228E preferentially interacted with E-cadherin increasing adhesion. Both Y228F and Y335F strongly bind VAV2. CONCLUSIONS: p120 catenin serves dual roles during gastrulation of zebrafish. Phosphorylation and dephosphorylation of tyrosine residues Y217, Y228, and Y335 precisely balance cell adhesion and cell migration to facilitate somite compaction and axis elongation.
Asunto(s)
Gastrulación , Pez Cebra , Ratones , Animales , Pez Cebra/metabolismo , Fosforilación , Morfolinos/metabolismo , Cateninas/genética , Cateninas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular/fisiología , Tirosina/genética , Tirosina/metabolismo , Fosfoproteínas/metabolismo , beta Catenina/metabolismoRESUMEN
Axonal connections between the two sides of the brain are essential for processing sensorimotor functions, especially in animals with bilateral symmetry. The anterior commissure and postoptic commissure are two crucial axonal projections that develop early in the zebrafish central nervous system. In this study, we characterized the function of collapsin response mediator protein 2 (CRMP2) and CRMP4 in patterning the development of the anterior and postoptic commissures by analyzing morpholino-knockdown zebrafish morphants and CRISPR/Cas9-edited gene-knockout mutants. We observed a loss of commissural structures or a significant reduction in axon bundles connecting the two hemispheres, but the defects could be largely recovered by co-injecting CRMP2 or CRMP4 mRNA. Loss of both CRMP2 and CRMP4 function resulted in a synergistic increase in the number of commissural defects. To elucidate the mechanism by which CRMP2 and CRMP4 provide guidance cues for the development of the anterior and postoptic commissures, we included neuropilin 1a (Nrp1a) morphants and double morphants (CRMP2/Nrp1a and CRMP4/Nrp1a) for analysis. Our experimental results indicated that CRMP2 and CRMP4 might mediate their activities through the common semaphorin 3/Nrp1a signaling pathway.
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Semaforinas , Pez Cebra , Animales , Morfolinos/metabolismo , Morfolinos/farmacología , Neuropilinas/metabolismo , Prosencéfalo/metabolismo , ARN Mensajero/metabolismo , Semaforina-3A/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Pez Cebra/metabolismoRESUMEN
Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described "formative" pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms 1/2 (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 messenger RNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the "formative" state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression toward a primed pluripotent state.
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Células Madre Pluripotentes , Piruvato Quinasa , Animales , Diferenciación Celular/genética , Ratones , Morfolinos/metabolismo , Músculos , Células Madre Pluripotentes/metabolismo , Isoformas de Proteínas , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismoRESUMEN
Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafish (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafish has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune homeostasis, and recognition of microbial pathogens. Galectins are ß-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), that constitute an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic inflammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafish, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.
Asunto(s)
Galectinas , Pez Cebra , Animales , Galectinas/metabolismo , Técnicas de Silenciamiento del Gen , Mamíferos/genética , Morfolinos/genética , Morfolinos/metabolismo , ARN/metabolismo , Pez Cebra/metabolismoRESUMEN
Sea urchin larval skeletons are produced by skeletogenic primary mesenchyme cells (PMCs), which migrate to form two ventrolateral clusters (VLCs) at the sites where biomineralization is initiated. Both PMC migration and biomineralization are controlled by VEGF signals emitted from lateral ectodermal cells. In mammals, VEGF signaling can be activated by hypoxia-inducible factor alpha (HIFα), an oxygen-sensitive transcription factor. Our previous study showed that the sea urchin maternal HIFα is involved in regulating gene expression along the dorsoventral axis. In this study, we discovered that zygotic hifα is expressed in PMCs, and at the late gastrula stage, hifα transcripts display a graded pattern, with stronger signal in the ventral PMCs than in the dorsal PMCs. We further showed that PMCs are hypoxic, which is a condition typically required for HIFα function. In embryos injected with a splice-blocking morpholino against hifα, elongation of the skeleton was impaired, and expression of vegfr-10-Ig (encodes VEGF receptor; VEGFR) was significantly reduced. This morpholino-caused defect could be partially rescued by injection of vegfr-10-Ig mRNA. Expression patterns of transcription factor and biomineralization genes, such as alx1, tbr, msp130, and the sm30 family, were affected when HIFα was knocked down or when VEGF signaling was inhibited. These results suggest that zygotic HIFα acts upstream or in parallel with VEGF signaling to regulate skeletogenic gene expression and participate in spicule elongation. Our study therefore links HIFα with the known role of VEGF signaling in sea urchin biomineralization.
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Embrión no Mamífero , Factor A de Crecimiento Endotelial Vascular , Animales , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hipoxia/metabolismo , Mamíferos/genética , Morfolinos/genética , Morfolinos/metabolismo , Morfolinos/farmacología , Erizos de Mar/genética , Erizos de Mar/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Aminoacyl tRNA synthetases (ARSs) are a group of proteins, acting as transporters to transfer and attach the appropriate amino acids onto their cognate tRNAs for translation. So far, 18 out of 20 cytoplasmic ARSs are reported to be connected to different neuropathy disorders with multi-organ defects that are often accompanied with developmental delays. Thus, it is important to understand functions and impacts of ARSs at the whole organism level. Here, we systematically analyzed the spatiotemporal expression of 14 ars and 2 aimp genes during development in zebrafish that have not be previously reported. Not only in the brain, their dynamic expression patterns in several tissues such as in the muscles, liver and intestine suggest diverse roles in a wide range of development processes in addition to neuronal function, which is consistent with potential involvement in multiple syndrome diseases associated with ARS mutations. In particular, hinted by its robust expression pattern in the brain, we confirmed that aimp1 is required for the formation of cerebrovasculature by a loss-of-function approach. Overall, our systematic profiling data provides a useful basis for studying roles of ARSs during development and understanding their potential functions in the etiology of related diseases.
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Aminoacil-ARNt Sintetasas/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Aminoacil-ARNt Sintetasas/clasificación , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Embrión no Mamífero , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Intestinos/crecimiento & desarrollo , Intestinos/metabolismo , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Anotación de Secuencia Molecular , Morfolinos/administración & dosificación , Morfolinos/genética , Morfolinos/metabolismo , Músculos/metabolismo , Proteínas del Tejido Nervioso/clasificación , Proteínas del Tejido Nervioso/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/clasificación , Proteínas de Pez Cebra/metabolismoRESUMEN
In the zebrafish embryo, the onset of blood flow generates fluid shear stress on endocardial cells, which are specialized endothelial cells that line the interior of the heart. High levels of fluid shear stress activate both Notch and Klf2 signaling, which play crucial roles in atrioventricular valvulogenesis. However, it remains unclear why only individual endocardial cells ingress into the cardiac jelly and initiate valvulogenesis. Here, we show that lateral inhibition between endocardial cells, mediated by Notch, singles out Delta-like-4-positive endocardial cells. These cells ingress into the cardiac jelly, where they form an abluminal cell population. Delta-like-4-positive cells ingress in response to Wnt9a, which is produced in parallel through an Erk5-Klf2-Wnt9a signaling cascade also activated by blood flow. Hence, mechanical stimulation activates parallel mechanosensitive signaling pathways that produce binary effects by driving endocardial cells toward either luminal or abluminal fates. Ultimately, these cell fate decisions sculpt cardiac valve leaflets.
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Endocardio/metabolismo , Mecanotransducción Celular , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente/metabolismo , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Desarrollo Embrionario , Endocardio/citología , Válvulas Cardíacas/crecimiento & desarrollo , Válvulas Cardíacas/metabolismo , Válvulas Cardíacas/patología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Morfolinos/metabolismo , Receptores de Neurotransmisores/antagonistas & inhibidores , Receptores de Neurotransmisores/genética , Receptores de Neurotransmisores/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeated DNA onto the 3' ends of linear chromosomes. Telomerase inhibition was broadly used for cancer therapeutics. Here, six antisense oligonucleotides were designed to regulate TERT mRNA alternative splicing and protein translation. To pursue a better stability in vitro, we chemically modified the oligonucleotides into phosphorothioate (PS) backbone and 2'-O-methoxyethyl (2'-MOE PS) version and phosphoroamidate morpholino oligomer (PMO) version. The oligonucleotides were transfected into HEK 293T cells and HeLa cells, and the mRNA expression, protein level and catalytic activity of telomerase were determined. We found the Int8 notably promoted hTERT mRNA exon 7-8 skipping, which greatly reduced telomerase activity, and the 5'-UTR treatment led to an obvious protein translation barrier and telomerase inhibition. These results demonstrate the potential of antisense oligonucleotide drugs targeting hTERT for antitumor therapy. Moreover, two specific antisense oligonucleotides were identified to be effective in reducing telomerase activity.
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Morfolinos/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Fosforotioatos/genética , ARN Mensajero/genética , Telomerasa/genética , Empalme Alternativo/efectos de los fármacos , Antineoplásicos/farmacología , Células HEK293 , Células HeLa , Humanos , Morfolinos/síntesis química , Morfolinos/metabolismo , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/síntesis química , Oligonucleótidos Fosforotioatos/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismoRESUMEN
The BH3-only molecule Bad regulates cell death via its differential protein phosphorylation, but very few studies address its effect on early embryonic development in vertebrate systems. In this work, we examined the novel role of zebrafish Bad in the initial programmed cell death (PCD) for brain morphogenesis through reducing environmental stress and cell death signaling. Bad was considered to be a material factor that because of the knockdown of Bad by morpholino oligonucleotides, PCD was increased and the reactive oxygen species (ROS) level was enhanced, which correlated to trigger a p53/caspase-8 involving cell death signaling. This Bad knockdown-mediated environmental stress and enhanced cell dying can delay normal cell migration in the formation of the three germ layers, especially the ectoderm, for further brain development. Furthermore, Bad defects involved in three-germ-layers development at 8 hpf were identified by in situ hybridization approach on cyp26, rtla, and Sox17 pattern expression markers. Finally, the Bad knockdown-induced severely defected brain was examined by tissue section from 24 to 48 h postfertilization (hpf), which correlated to induce dramatic malformation in the hindbrain. Our data suggest that the BH3-only molecule Bad regulates brain development via controlling programmed cell death on overcoming environmental stress for reducing secondary cell death signaling, which suggests that correlates to brain developmental and neurological disorders in this model system.
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Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Desarrollo Embrionario , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Animales , Apoptosis , Encéfalo/patología , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Genes p53 , Morfolinos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína Letal Asociada a bcl/genéticaRESUMEN
The assembly pathways of mitochondrial respirasome (supercomplex I+III2+IV) are not fully understood. Here, we show that an early sub-complex I assembly, rather than holo-complex I, is sufficient to initiate mitochondrial respirasome assembly. We find that a distal part of the membrane arm of complex I (PD-a module) is a scaffold for the incorporation of complexes III and IV to form a respirasome subcomplex. Depletion of PD-a, rather than other complex I modules, decreases the steady-state levels of complexes III and IV. Both HEK293T cells lacking TIMMDC1 and patient-derived cells with disease-causing mutations in TIMMDC1 showed accumulation of this respirasome subcomplex. This suggests that TIMMDC1, previously known as a complex-I assembly factor, may function as a respirasome assembly factor. Collectively, we provide a detailed, cooperative assembly model in which most complex-I subunits are added to the respirasome subcomplex in the lateral stages of respirasome assembly.
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Complejo III de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/genética , Complejo I de Transporte de Electrón/genética , Mitocondrias/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/genética , Animales , Linfocitos B , Línea Celular Transformada , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/deficiencia , Morfolinos/genética , Morfolinos/metabolismo , Hipotonía Muscular/genética , Hipotonía Muscular/metabolismo , Hipotonía Muscular/patología , Fosforilación Oxidativa , Pez CebraRESUMEN
BACKGROUND: We developed a preclinical protocol for the screening of candidate drugs able to control myopia and prevent its progression. The protocol uses zebrafish, C57BL/6 mice, and golden Syrian hamster models of myopia. METHODS: A morpholino (MO) targeting the zebrafish lumican gene (zlum) was injected into single-cell zebrafish embryos, causing excessive expansion of the sclera. A library of 640 compounds with 2 matrix metalloproteinase (MMP) inhibitors (marimastat and batimastat), which have the potential to modulate scleral remodelling, was screened to identify candidates for mitigating scleral diameter expansion in zlum-MO-injected embryos. The myopia-prevention ability of compounds discovered to have superior potency to inhibit scleral expansion was validated over 4 weeks in 4-week-old C57BL/6 mice and 3-week-old golden Syrian hamsters with form-deprivation myopia (FDM). Changes in the refractive error and axial length were investigated. Scleral thickness, morphology of collagen fibrils in the posterior sclera, messenger RNA (mRNA) expressions, and protein levels of transforming growth factor-ß2 (TGF-ß2), tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-7, MMP-9, and collagen, type I, alpha 1 (collagen Iα1) were investigated in C57BL/6 mice, and MMP-2, MMP-9, and MMP activity assays were conducted in these mice. FINDINGS: In the zebrafish experiment, atropine, marimastat, batimastat, doxycycline, and minocycline were the drugs that most effectively reduced expansion of scleral equatorial diameter. After 28-day treatment in diffuser-wearing mice and 21-day treatment in lid-sutured hamsters, myopic shift and axial elongation were significantly mitigated by eye drops containing 1% atropine, 50 µM marimastat, 5 µM batimastat, or 200 µM doxycycline. MMP-2 mRNA expression in mouse sclera was lower after treatment with atropine, marimastat, batimastat, or doxycycline. The protein levels and activity of MMP-2 and MMP-7 were significantly reduced after treatment with atropine, marimastat, batimastat, doxycycline, and minocycline. Furthermore, scleral thickness and collagen fibril diameter were not lower after treatment with atropine, marimastat, batimastat, or doxycycline than those of occluded eyes. INTERPRETATION: Stepwise drug screening in a range of models from zlum-MO-injected zebrafish to rodent FDM models identified effective compounds for preclinical myopia control or prevention. On the basis of the 640 compounds that were screened, MMP inhibitors may offer alternatives for clinical trials. FUNDING: This research was supported by grants from Taiwan's Ministry of Science and Technology and Ministry of Health and Welfare.