RESUMEN
Dietary restriction in the form of fasting is a putative key to a healthier and longer life, but these benefits may come at a trade-off with reproductive fitness and may affect the following generation(s). The potential inter- and transgenerational effects of long-term fasting and starvation are particularly poorly understood in vertebrates when they originate from the paternal line. We utilised the externally fertilising zebrafish amenable to a split-egg clutch design to explore the male-specific effects of fasting/starvation on fertility and fitness of offspring independently of maternal contribution. Eighteen days of fasting resulted in reduced fertility in exposed males. While average offspring survival was not affected, we detected increased larval growth rate in F1 offspring from starved males and more malformed embryos at 24 h post-fertilisation in F2 offspring produced by F1 offspring from starved males. Comparing the transcriptomes of F1 embryos sired by starved and fed fathers revealed robust and reproducible increased expression of muscle composition genes but lower expression of lipid metabolism and lysosome genes in embryos from starved fathers. A large proportion of these genes showed enrichment in the yolk syncytial layer suggesting gene regulatory responses associated with metabolism of nutrients through paternal effects on extra-embryonic tissues which are loaded with maternal factors. We compared the embryo transcriptomes to published adult transcriptome datasets and found comparable repressive effects of starvation on metabolism-associated genes. These similarities suggest a physiologically relevant, directed and potentially adaptive response transmitted by the father, independently from the offspring's nutritional state, which was defined by the mother.
Asunto(s)
Yema de Huevo , Embrión no Mamífero , Padre , Pez Cebra , Animales , Masculino , Humanos , Pez Cebra/genética , Regulación de la Expresión Génica , Expresión GénicaRESUMEN
In the past decades, the zebrafish has become a disease model with increasing popularity owing to its advantages that include fast development, easy genetic manipulation, simplicity for imaging, and sharing conserved disease-associated genes and pathways with those of human. In parallel, studies of disease mechanisms are increasingly focusing on non-coding mutations, which require genome annotation maps of regulatory elements, such as enhancers and promoters. In line with this, genomic resources for zebrafish research are expanding, producing a variety of genomic data that help in defining regulatory elements and their conservation between zebrafish and humans. Here, we discuss recent developments in generating functional annotation maps for regulatory elements of the zebrafish genome and how this can be applied to human diseases. We highlight community-driven developments, such as DANIO-CODE, in generating a centralised and standardised catalogue of zebrafish genomics data and functional annotations; consider the advantages and limitations of current annotation maps; and offer considerations for interpreting and integrating existing maps with comparative genomics tools. We also discuss the need for developing standardised genomics protocols and bioinformatic pipelines and provide suggestions for the development of analysis and visualisation tools that will integrate various multiomic bulk sequencing data together with fast-expanding data on single-cell methods, such as single-cell assay for transposase-accessible chromatin with sequencing. Such integration tools are essential to exploit the multiomic chromatin characterisation offered by bulk genomics together with the cell-type resolution offered by emerging single-cell methods. Together, these advances will build an expansive toolkit for interrogating the mechanisms of human disease in zebrafish.
Asunto(s)
Genómica , Pez Cebra , Animales , Humanos , Pez Cebra/genética , Genómica/métodos , Genoma , Cromatina , Regeneración/genéticaRESUMEN
(analítico) Se exploran las prácticas institucionales que facilitan u obstaculizan la protección de los derechos de niños, niñas y adolescentes en el sistema de protección de la niñez en El Salvador. Partiendo de un diseño de etnografía institucional, se realizaron 61 entrevistas a trabajadores pertenecientes al sistema de protección. Como resultado, se identificó la ausencia de manuales que establezcan prácticas concretas en la aplicación de la Ley de Protección Integral de la Niñez y Adolescencia, obstruyendo su óptimo funcionamiento. A través del uso de la teoría del interaccionismo simbólico, se explora cómo la interpretación discrecional o no entendimiento de la ley forma instituciones aisladas del sistema; también se analiza el grado en que las dinámicas socioeconómicas del país ponen en desventaja a los sectores rurales para acceder a estos servicios.
(analytical) This study explores the institutional practices that support or hinder the rights of children and young people in the child protection system in El Salvador. Using an institutional ethnography approach, 61 individuals who worked directly or indirectly in the child protection system were interviewed. The findings highlight a lack of manuals that establish concrete practices in accordance with the application of the Law of Comprehensive Protection for Children and Youth, which reduces the effectiveness of the country's child protection system. Through an analytical approach based on symbolic interactionism, this study explored how the individual interpretations of the law, or lack thereof, combine with socioeconomic disadvantages to create difficulties for rural child protection institutions in terms of accessing operational resources.
(analítico) O presente estudo explora as práticas institucionais que apoiam ou dificultam os direitos de crianças e jovens no sistema de proteção infantil em El Salvador. Utilizando um desenho de etnografia institucional, foram entrevistados 61 indivíduos que trabalhavam ou trabalham no sistema de proteção à criança. Os achados indicam que faltam manuais que estabeleçam práticas concretas em consonância com a aplicação da Lei de Proteção Integral da Infância e Juventude o que dificulta a efetividade do sistema de proteção. Por meio de um entendimento baseado no interacionismo simbólico, este estudo explorou como as interpretações individuais da lei, ou a falta dela, e a desvantagem socio-econômica das instituições colocam os setores rurais em desvantagem no acesso aos recursos.
RESUMEN
Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.
Asunto(s)
Bases de Datos Genéticas , Regulación del Desarrollo de la Expresión Génica , Genoma , Genómica , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas de Pez Cebra , Pez Cebra , Animales , Cromatina/genética , Genoma/genética , Humanos , Ratones , Anotación de Secuencia Molecular , Organogénesis/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Objective: To compile the screening tools used to study temporomandibular disorders (TMD), classify and analyze their potential application in the field of physiotherapy.Methods: All gathered data included randomized clinical trials on humans over 18 years of age pulled from three databases up to November 2019.Results: Nineteen articles were retained, in which the subjects included 1095 women and 385 men. The 32 valuation systems reported were classified as follows: direct, requiring observation and/or palpation in situ by a clinician, which can be subdivided into protocols and instrumental systems; and indirect, requiring neither observation nor palpation by a clinician, which can be subdivided into questionnaires and scales.Conclusion: In order to evaluate TMD, the best choice is to combine direct and indirect assessment methodologies. The valuation of pressure pain threshold with an algometer and Fonseca's Anamnestic Index in combination with Anamnestic Questionnaire CMD, respectively, seems to provide the best results.
RESUMEN
In many animal models, primordial germ cell (PGC) development depends on maternally deposited germ plasm, which prevents somatic cell fate. Here, we show that PGCs respond to regulatory information from the germ plasm in two distinct phases using two distinct mechanisms in zebrafish. We demonstrate that PGCs commence zygotic genome activation together with the somatic blastocysts with no demonstrable differences in transcriptional and chromatin opening. Unexpectedly, both PGC and somatic blastocysts activate germ-cell-specific genes, which are only stabilized in PGCs by cytoplasmic germ plasm determinants. Disaggregated perinuclear relocalization of germ plasm during PGC migration is regulated by the germ plasm determinant Tdrd7 and is coupled to dramatic divergence between PGC and somatic transcriptomes. This transcriptional divergence relies on PGC-specific cis-regulatory elements characterized by promoter-proximal distribution. We show that Tdrd7-dependent reconfiguration of chromatin accessibility is required for elaboration of PGC fate but not for PGC migration.
Asunto(s)
Diferenciación Celular , Cromatina/genética , Células Germinativas/citología , Ribonucleoproteínas/metabolismo , Transcriptoma , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Movimiento Celular , Cromatina/química , Epigénesis Genética , Genoma , Células Germinativas/metabolismo , Ribonucleoproteínas/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: The abuse of opioids, such as morphine and phentanyl or other drugs as heroin is a social and health problem that affects an increasing number of people each year. The activation of the mu opioid receptor triggers several molecular changes that alter the expression of diverse genes, including miRNAs. The dysregulation of these molecules could explain some of the developmental alterations that are induced after drug intake. In addition, the Notch signaling cascade has also been related to alterations on these processes. METHODS: Zebrafish embryos and SH-SY5Y cells were used to assess the effects of opioid and Notch signaling on the expression on miR-29a and miR-212/132 by qPCR and ChIP-qPCR. Notch1 expression was analyzed using in situ hybridization on 24 hpf zebrafish embryos. In addition, OPRM1 and NICD levels were measured using western blot on the cultured cells to determine the cross-talk between the two pathways. RESULTS: We have observed changes in the levels of miR-212/132 after administrating DAPT to zebrafish embryos indicating that this pathway could be regulating mu opioid receptor expression. In addition, the ISH experiment showed changes in Notch1 expression after morphine and DAPT administration. Moreover, morphine affects the expression of miR-29a through NF-κB, therefore controlling the cleavage and activation of Notch through ADAM12 expression. CONCLUSIONS: This study shows that these two pathways are closely related, and could explain the alterations triggered in the early stages of the development of addiction. GENERAL SIGNIFICANCE: Opioid and Notch pathway are reciprocally regulated by the miRNAs 212/132 and 29a.
Asunto(s)
MicroARNs/metabolismo , Péptidos Opioides/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal/genética , Animales , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Receptor Notch1/genética , Pez Cebra/embriologíaRESUMEN
The opioid system is well conserved among species and plays a critical role in pain and addiction systems. The use of zebrafish as an experimental model to study development and genetics is extraordinary and has been proven to be relevant for the study of different diseases. The main drawback to its use for the analysis of different pathologies is the lack of protein tools. Antibodies that work in other models are not suitable for zebrafish due to the low degree of homology that exists among the opioid receptor protein sequences in different species. Here we report the successful generation and characterization of antibodies against the mu, delta 1 and delta 2 opioid receptors in zebrafish. The antibodies obtained, which are specific for each receptor due to the use of the C-terminus as antigens, work for Western blotting and immunohistochemistry. In addition, the antibodies against mu and delta 1 opioid receptors, but not those against delta 2, are able to immunoprecipitate the corresponding receptor from zebrafish lysates. The development of opioid receptor antibodies is an asset to the further study of the endogenous opioid system in zebrafish.
Asunto(s)
Anticuerpos/metabolismo , Receptores Opioides/inmunología , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Femenino , Células HEK293 , Humanos , Larva/metabolismo , Conejos , Receptores Opioides/química , Receptores Opioides delta/metabolismo , Alineación de SecuenciaRESUMEN
BACKGROUND: Morphine is used as an analgesic although it causes important secondary effects. These effects are triggered by several mechanisms leading to the dysregulation of gene expression. Here we aimed to study these alterations on neural stem cells (NSC) during CNS development. METHODS: AB strain and tg nestin:GFP zebrafish embryos, zebrafish primary neuron culture and mouse embryonic stem cells were used to assess the effect of morphine by qPCR, time lapse microscopy and western blot. ChIP-qPCR and bisulfite conversion assay were performed to determine the changes exerted by morphine in a Nestin candidate enhancer. RESULTS: Morphine increases GFP in nestin:GFP embryos and overexpresses the NSC marker Nestin. Morphine also exerts a hyperacetylation effect on H3K27 and decreases DNA methylation within a region located 18 Kb upstream nestin transcription starting site. Here, a binding site for the transcription factor complex Sox2/Oct4/Nanog was predicted. These factors are also upregulated by morphine. Besides, morphine increases the histone acetyl transferase p300. The inhibition of p300 activity decreases Nestin. CONCLUSIONS: Morphine facilitates Nestin increase by several mechanisms which include hyperacetylation of H3K27, decreased DNA methylation and the overexpression of the transcription factors sox2, oct4 and nanog. It has also been demonstrated that nestin levels depend on p300 activity. The facilitated Nestin expression delays the normal differentiation of neural stem cells. GENERAL SIGNIFICANCE: The present work provides novel evidence of the effects induced by morphine in the normal differentiation of NSCs, altering Nestin through changes on p300, H3K27ac, DNA methylation and Oct4, Sox2, and Nanog.