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1.
J Biotechnol ; 342: 79-91, 2021 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-34751134

RESUMEN

GR15 is a short molecule or peptide composed of aliphatic amino acids and possesses to have antioxidant properties. The GR15, 1GGGAFSGKDPTKVDR15 was identified from the protein S-adenosylmethionine synthase (SAMe) expressed during the sulfur departed state of Arthrospira platensis (spirulina or cyanobacteria). The in-silico assessment and the structural features of GR15 showed its antioxidant potency. Real-time PCR analysis found the up-regulation of ApSAMe expression on day 15 against oxidative stress due to 10 mM H2O2 treatment in A. platensis (Ap). The antioxidant activity of GR15 was accessed by the cell-free antioxidant assays such as ABTS, SARS, HRAS and NO; the results showed dose-dependent antioxidant activity. The toxicity assay was performed in both in vitro and in vivo models, in which peptide does not exhibit any toxicity in MDCK cell and zebrafish embryos. The intercellular ROS reduction potential of GR15 peptide was also investigated in both in vitro and in vivo models including LDH assay, antioxidant enzymes (SOD and CAT), and fluorescent staining assay (DCFDA, Hochest and Acridine orange sting) was performed; the results showed that the GR15 peptide was effectively reduced the ROS level. Further, RT-PCR demonstrated that GR15 enhanced the antioxidant property and also up-regulated the antioxidant gene, thus reduced the ROS level in both in vitro and in vivo models. Based on the results obtained from this study, we propose that GR15 has the potential antioxidant ability; hence further research can be directed towards the therapeutic product or drug development against disease caused by oxidative stress.


Asunto(s)
Antioxidantes , Spirulina , Animales , Antioxidantes/farmacología , Perros , Peróxido de Hidrógeno/toxicidad , Larva/metabolismo , Células de Riñón Canino Madin Darby , Estrés Oxidativo , Péptidos/metabolismo , S-Adenosilmetionina , Spirulina/metabolismo , Pez Cebra/metabolismo
2.
J Vis Exp ; (175)2021 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-34633379

RESUMEN

The pancreatic ß-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ß-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, ß-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying ß-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how ß-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate ß-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate ß-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in ß-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect ß-cell function.


Asunto(s)
Calcio , Células Secretoras de Insulina , Animales , Calcio/metabolismo , Glucosa/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Pez Cebra/metabolismo
3.
Molecules ; 26(20)2021 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-34684799

RESUMEN

The quantification of steroid hormones of individual zebrafish (Danio rerio) provides perspective to understand endogenous hormone function. A UPLC-TOF-MS method was developed to provide a reproducible, sensitive, and efficient assay to determine the concentration of steroid hormones, including cortisol, testosterone, androstenedione, 11-deoxycortisol, 11-deoxycorticosterone, and 17-hydroxyprogesterone in whole-body homogenates of each zebrafish. Solid-phase extraction was used to sample matrix clean-up and acquired a recovery from 89.7% to 107.9%. The analytes were separated on an Aquity BEH C18 column using gradient elution. Mass spectrometric analysis was performed by single reaction monitoring (SRM) using positive electrospray ionization mode. The total running time was 6 min, which was greatly shortened compared with a previously reported method. The developed method exhibited excellent linearity for all the analytes, with regression coefficients higher than 0.99. The limit of detection varied between 0.1 and 0.5 ng/L and the limit of quantification was 0.5-1.7 ng/L for all analytes. The precision of the method was assessed on replicate measurements and was found to be in the ranges of 1.9 % to 6.6% and 4.3% to 8.6%, for intra- and inter-day analysis, respectively. This method was validated according to FDA guidance and applied to determine steroid hormone levels in the tissue homogenate of zebrafish acutely treated with caffeine and ethanol.


Asunto(s)
Esteroides/análisis , Pez Cebra/metabolismo , Animales , Vías Biosintéticas , Cafeína/administración & dosificación , Cromatografía Liquida/métodos , Cromatografía Liquida/estadística & datos numéricos , Etanol/administración & dosificación , Femenino , Masculino , Modelos Animales , Extracción en Fase Sólida/métodos , Extracción en Fase Sólida/estadística & datos numéricos , Esteroides/biosíntesis , Estrés Fisiológico/efectos de los fármacos , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas en Tándem/estadística & datos numéricos
4.
Nat Commun ; 12(1): 6094, 2021 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-34667153

RESUMEN

Zygotic genome activation (ZGA) initiates regionalized transcription underlying distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, the direct mechanistic link between the onset of ZGA and the tissue-specific transcription remains unclear. Here, we have addressed the involvement of chromatin organizer Satb2 in orchestrating both processes during zebrafish embryogenesis. Integrative analysis of transcriptome, genome-wide occupancy and chromatin accessibility reveals contrasting molecular activities of maternally deposited and zygotically synthesized Satb2. Maternal Satb2 prevents premature transcription of zygotic genes by influencing the interplay between the pluripotency factors. By contrast, zygotic Satb2 activates transcription of the same group of genes during neural crest development and organogenesis. Thus, our comparative analysis of maternal versus zygotic function of Satb2 underscores how these antithetical activities are temporally coordinated and functionally implemented highlighting the evolutionary implications of the biphasic and bimodal regulation of landmark developmental transitions by a single determinant.


Asunto(s)
Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Factores de Transcripción/metabolismo , Vertebrados/embriología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Factores de Transcripción/genética , Transcriptoma , Vertebrados/genética , Vertebrados/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Cigoto/metabolismo
5.
PLoS One ; 16(10): e0257774, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34624042

RESUMEN

Previously we have shown that trypsin, a protein typically involved in digestion, is released from gills of both fresh and saltwater fishes into surrounding water under stress or injury. We have also shown that each species produces trypsin with different specific activities. In this report, using zebrafish as a model, we identified that trypsin induces an aversive response in zebrafish larvae and adult zebrafish. Since Protease-Activated Receptor 2 (PAR2) responds to trypsin, we tested whether the aversive response is dependent on the activation of PAR2 located on the zebrafish skin cells. Zebrafish larvae treated separately with neomycin and zinc sulfate also showed aversive response indicating neuromast, and olfactory cells are not involved in this aversion. Cultured keratinocytes from zebrafish showed a response to trypsin. Zebrafish larvae subjected to knockdown of par2a also exhibited reduced escape response. Similarly, par2a-deficient mutant larvae displayed no response to trypsin. Since it has been shown that stress activates PAR2 and sends signals to the brain as shown by the increased c-fos expression, we tested c-fos expression in adult zebrafish brains after trypsin treatment of adults and found enhanced c-fos expression by qRT-PCR. Taken together, our results show that the trypsin activates PAR2 on keratinocytes signaling the brain, and this pathway of trypsin-induced escape response will provide a unique communication mechanism in zebrafish. Furthermore, since PAR2 activation also occurs in pain/pruritus sensing, this model might be useful in elucidating components of signaling pathways in pain/pruritus.


Asunto(s)
Receptor PAR-2/genética , Piel/metabolismo , Tripsina/metabolismo , Pez Cebra/genética , Animales , Línea Celular , Branquias/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Larva/efectos de los fármacos , Larva/genética , Neomicina/farmacología , Receptor PAR-2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Piel/efectos de los fármacos , Tripsina/efectos adversos , Pez Cebra/metabolismo , Sulfato de Zinc/farmacología
6.
Aquat Toxicol ; 240: 105994, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34656894

RESUMEN

This paper investigates the effect of lanthanum (La) on lipid deposition and Wnt10b signaling in the liver of male zebrafish with exposure of 0, 10, 20, and 30 µmol/L La. It suggests that La can be accumulated in liver, and its treatments decrease the activities and gene expression of enzymes related to fatty acid synthesis. The levels of total cholesterol (TC), triglyceride (TG), and nonesterified fatty acids (NEFA) as well as the size of lipid droplets are decreased by La treatments. Moreover, La treatments affect the composition of fatty acids and the content of nutrient elements. Meanwhile, they also induce the gene expression of wnt10b, ß-catenin, pparα, and pparγ, but inhibit gsk-3ß gene expression in liver. Further study on the result of wnt10b gene interference shows that Wnt10b/ß-catenin signaling plays a crucial role in the regulatory process of hepatic lipid deposition. Taken together, our observations suggest that La accumulation affects lipid deposition in the liver of male zebrafish, and Wnt10b signaling pathway may be involved in this process.


Asunto(s)
Contaminantes Químicos del Agua , Pez Cebra , Animales , Ácidos Grasos/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Lantano/metabolismo , Lantano/toxicidad , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Transducción de Señal , Contaminantes Químicos del Agua/toxicidad , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
7.
J Hazard Mater ; 416: 125969, 2021 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-34492880

RESUMEN

In the present study the effects of sublethal concentrations of polystyrene microplastics (PS-MPs) on zebrafish were evaluated at multiple levels, related to fish activity and oxidative stress, metabolic changes and contraction parameters in the heart tissue. Zebrafish were fed for 21 days food enriched with PS-MPs (particle sizes 3-12 µm) and a battery of stress indices like DNA damage, lipid peroxidation, autophagy, ubiquitin levels, caspases activation, metabolite adjustments, frequency and force of ventricular contraction were measured in fish heart, parallel to fish swimming velocity. In particular, exposure to PS-MPs caused significant decrease in heart function and swimming competence, while enhanced levels of oxidative stress indices and metabolic adjustments were observed in the heart of challenged species. Among stress indices, DNA damage was more vulnerable to the effect of PS-MPs. Our results provide evidence on the multiplicity of the PS-MPs effects on cellular function, physiology and metabolic pathways and heart rate of adult fish and subsequent effects on fish activity and fish fitness thus enlightening MPs characterization as a potent environmental pollutant.


Asunto(s)
Microplásticos , Contaminantes Químicos del Agua , Animales , Estrés Oxidativo , Plásticos , Poliestirenos/metabolismo , Poliestirenos/toxicidad , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/metabolismo
8.
Ecotoxicol Environ Saf ; 225: 112715, 2021 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-34500382

RESUMEN

As a natural heme protein catalyzing the oxidation of sulfides to sulfoxides without sulfone formation, chloroperoxidase (CPO) is well suited for the degradation of sulfur mustard (HD), a persistent chemical warfare agent that has been widely disposed since World War II and continuously leaks into aquatic environments. Herein, we report the first systematic investigation of CPO-catalyzed degradation of HD and the potential application of CPO in destroying chemical weapons under mild conditions. The related Michaelis-Menten parameters (Km=0.17 mM, Vmax=0.06 mM s-1 (R2 =0.935), and kcat= 2717 s-1) indicated nearly a prominent enzymatic efficiency. Under optimal conditions, 80% of HD was transformed to bis(2-chloroethyl) sulfoxide as identified by mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Other metabolites were also generated during the decontamination process. A plausible oxidation mechanism was proposed based on the degradation products, NMR titration experiments, and molecular dynamics simulations. CPO also promoted the degradation of other chemical weapon agents, namely, Lewisite (L) and venomous agent X (VX), thereby exhibiting a broad substrate scope. The high potential of the developed system for the decontamination of aquatic environments was demonstrated by the successful hatching of zebrafish embryos after HD degradation and the survival of zebrafish (Danio rerio, AB strain) larvae after the degradation of Agent Yellow (L+HD).


Asunto(s)
Cloruro Peroxidasa , Gas Mostaza , Animales , Catálisis , Gas Mostaza/toxicidad , Estrés Oxidativo , Pez Cebra/metabolismo
9.
Molecules ; 26(17)2021 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-34500575

RESUMEN

Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies are; therefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/patología , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Pronóstico , Pez Cebra/metabolismo
10.
An Acad Bras Cienc ; 93(suppl 3): e20201938, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34550207

RESUMEN

Triclosan (TCS) is an antimicrobial and antimycotic agent widely used in personal care products. In aquatic environments, both TCS and its biomethylated more persistent form, methyl-triclosan (MeTCS), are usually detected in wastewater effluents and rivers, where are commonly adsorbed to suspended solids and sediments. The aim of this study was to evaluate biochemical and physiological effects in Danio rerio after a short term (2 days) and prolonged (21 days) exposures to sediment spiked with TCS acting as the source of the pollutant in the assay. The activities of catalase (CAT), glutathione-s transferase (GST) and superoxide dismutase (SOD), lipid peroxidation levels (LPO), total capacity against peroxyl radicals (ACAP), and acetylcholinesterase enzymatic activity (AChE) were measured in liver, gills, and brain. Most of TCS on the spiked sediment was biotransformed to MeTCS and promoted different adverse effects on D. rerio. Gills were the most sensitive organ after 2 day-exposure, showing lipid damage and increased SOD activity. After 21 days of exposure, liver was the most sensitive organ, showing lower ACAP, increased LPO levels, and SOD and CAT activities. This is the first study reporting the effects on biochemical markers in D. rerio from a MeTCS sink resulting from sediment spiked with TCS.


Asunto(s)
Triclosán , Contaminantes Químicos del Agua , Acetilcolinesterasa , Animales , Catalasa/metabolismo , Estrés Oxidativo , Superóxido Dismutasa/metabolismo , Triclosán/toxicidad , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/metabolismo
11.
Mar Biotechnol (NY) ; 23(5): 821-835, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34490548

RESUMEN

The human sarcomeric myosin heavy chain gene MYH14 contains an intronic microRNA, miR-499. Our previous studies demonstrated divergent genomic organization and expression patterns of myh14/miR-499 among teleosts; however, the regulatory mechanism is partly known. In this study, we report the regulation of myh14 expression in zebrafish, Danio rerio. Zebrafish myh14 has three paralogs, myh14-1, myh14-2, and myh14-3. Detailed promoter analysis suggested that a 5710-bp 5'-flanking region of myh14-1 and a 5641-bp region of myh14-3 contain a necessary regulatory region to recapitulate specific expression during embryonic development. The 5'-flanking region of zebrafish myh14-1 and its torafugu ortholog shared two distal and a single proximal conserved region. The two distal conserved regions had no effect on zebrafish myh14-1 expression, in contrast to torafugu expression, suggesting an alternative regulatory mechanism among the myh14 orthologs. Comparison among the 5'-flanking regions of the myh14 paralogs revealed two conserved regions. Deletion of these conserved regions significantly reduced the promoter activity of myh14-3 but had no effect on myh14-1, indicating different cis-regulatory mechanisms of myh14 paralogs. Loss of function of miR-499 resulted in a marked reduction in slow muscle fibers in embryonic development. Our study identified different cis-regulatory mechanisms controlling the expression of myh14/miR-499 and an indispensable role of miR-499 in muscle fiber-type specification in zebrafish.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Cadenas Pesadas de Miosina/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Embrión no Mamífero/metabolismo , MicroARNs/genética , Cadenas Pesadas de Miosina/genética , Takifugu/genética , Pez Cebra/genética
12.
Elife ; 102021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34550876

RESUMEN

Eukaryotes generally display a circadian rhythm as an adaption to the reoccurring day/night cycle. This is particularly true for visual physiology that is directly affected by changing light conditions. Here we investigate the influence of the circadian rhythm on the expression and function of visual transduction cascade regulators in diurnal zebrafish and nocturnal mice. We focused on regulators of shut-off kinetics such as Recoverins, Arrestins, Opsin kinases, and Regulator of G-protein signaling that have direct effects on temporal vision. Transcript as well as protein levels of most analyzed genes show a robust circadian rhythm-dependent regulation, which correlates with changes in photoresponse kinetics. Electroretinography demonstrates that photoresponse recovery in zebrafish is delayed in the evening and accelerated in the morning. Functional rhythmicity persists in continuous darkness, and it is reversed by an inverted light cycle and disrupted by constant light. This is in line with our finding that orthologous gene transcripts from diurnal zebrafish and nocturnal mice are often expressed in an anti-phasic daily rhythm.


Asunto(s)
Ritmo Circadiano/efectos de la radiación , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Animales , Arrestinas/genética , Arrestinas/metabolismo , Oscuridad , Electrorretinografía , Femenino , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Luz , Fototransducción , Masculino , Ratones , Modelos Animales , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Visión Ocular/efectos de la radiación , Pez Cebra/genética , Pez Cebra/metabolismo
13.
Elife ; 102021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34550070

RESUMEN

Parkinson's disease (PD) is a common neurodegenerative disorder without effective disease-modifying therapeutics. Here, we establish a chemogenetic dopamine (DA) neuron ablation model in larval zebrafish with mitochondrial dysfunction and robustness suitable for high-content screening. We use this system to conduct an in vivo DA neuron imaging-based chemical screen and identify the Renin-Angiotensin-Aldosterone System (RAAS) inhibitors as significantly neuroprotective. Knockdown of the angiotensin receptor 1 (agtr1) in DA neurons reveals a cell-autonomous mechanism of neuroprotection. DA neuron-specific RNA-seq identifies mitochondrial pathway gene expression that is significantly restored by RAAS inhibitor treatment. The neuroprotective effect of RAAS inhibitors is further observed in a zebrafish Gaucher disease model and Drosophila pink1-deficient PD model. Finally, examination of clinical data reveals a significant effect of RAAS inhibitors in delaying PD progression. Our findings reveal the therapeutic potential and mechanisms of targeting the RAAS pathway for neuroprotection and demonstrate a salient approach that bridges basic science to translational medicine.


Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Antiparkinsonianos/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Sistema Renina-Angiotensina/efectos de los fármacos , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Animales Modificados Genéticamente , Antiparkinsonianos/uso terapéutico , Estudios de Casos y Controles , Bases de Datos Factuales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Sistema Renina-Angiotensina/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
14.
Exp Cell Res ; 408(1): 112831, 2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34547256

RESUMEN

Angiogenesis is the process by which new blood vessels form from preexisting vessels and regulates the processes of embryonic development, wound healing and tumorigenesis. HMGA2 is involved in the occurrence of several cancers, but its biological role and the exact downstream genes involved in vascular development and sprouting angiogenesis remain largely unknown. Here, we first found that HMGA2 knockdown in zebrafish embryos resulted in defects of central artery formation. RNA sequencing revealed that IGFBP2 was significantly downregulated by interference with HMGA2, and IGFBP2 overexpression reversed the inhibition of brain vascular development caused by HMGA2 deficiency. In vitro, we further found that HMGA2 knockdown blocked the migration, tube formation and branching of HUVECs. Similarly, IGFBP2 protein overexpression attenuated the impairments induced by HMGA2 deficiency. Moreover, the promotion of angiogenesis by HMGA2 overexpression was verified in a Matrigel plug assay. We next found that HMGA2 bound directly to a region in the IGFBP2 promoter and positively regulated IGFBP2 expression. Interestingly, the mRNA expression levels of HMGA2 and IGFBP2 were increased significantly in the peripheral blood of hemangioma patients, indicating that overexpression of HMGA2 and IGFBP2 results in vessel formation, consistent with the results of the in vivo and in vitro experiments. In summary, our findings demonstrate that HMGA2 promotes central artery formation by modulating angiogenesis via IGFBP2 induction.


Asunto(s)
Proteína HMGA2/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Morfogénesis/fisiología , Neovascularización Patológica/metabolismo , Animales , Carcinogénesis/metabolismo , Desarrollo Embrionario/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Neoplasias/metabolismo , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Pez Cebra/genética , Pez Cebra/metabolismo
15.
Cells ; 10(9)2021 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-34571837

RESUMEN

Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.


Asunto(s)
Plaguicidas/farmacología , Transducción de Señal/efectos de los fármacos , Testículo/diagnóstico por imagen , Animales , Diferenciación Celular/efectos de los fármacos , Células Germinativas/efectos de los fármacos , Células Germinativas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Reproducción/efectos de los fármacos , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/metabolismo , Espermatogénesis/efectos de los fármacos , Testículo/metabolismo , Hormonas Tiroideas/metabolismo , Pez Cebra/metabolismo
16.
Nat Commun ; 12(1): 5284, 2021 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-34489414

RESUMEN

Cell death is a critical process that occurs normally in health and disease. However, its study is limited due to available technologies that only detect very late stages in the process or specific death mechanisms. Here, we report the development of a family of fluorescent biosensors called genetically encoded death indicators (GEDIs). GEDIs specifically detect an intracellular Ca2+ level that cells achieve early in the cell death process and that marks a stage at which cells are irreversibly committed to die. The time-resolved nature of a GEDI delineates a binary demarcation of cell life and death in real time, reformulating the definition of cell death. We demonstrate that GEDIs acutely and accurately report death of rodent and human neurons in vitro, and show that GEDIs enable an automated imaging platform for single cell detection of neuronal death in vivo in zebrafish larvae. With a quantitative pseudo-ratiometric signal, GEDIs facilitate high-throughput analysis of cell death in time-lapse imaging analysis, providing the necessary resolution and scale to identify early factors leading to cell death in studies of neurodegeneration.


Asunto(s)
Técnicas Biosensibles , Muerte Celular/genética , Regulación del Desarrollo de la Expresión Génica , Enfermedades Neurodegenerativas/genética , Neuronas/metabolismo , Animales , Calcio/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Embrión no Mamífero , Colorantes Fluorescentes/química , Genes Reporteros , Ácido Glutámico/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Larva/citología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Ratones , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/citología , Neuronas/efectos de los fármacos , Cultivo Primario de Células , Ratas , Ratas Long-Evans , Análisis de la Célula Individual/métodos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
17.
FASEB J ; 35(10): e21915, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34496088

RESUMEN

During development, erythroid cells are generated by two waves of hematopoiesis. In zebrafish, primitive erythropoiesis takes place in the intermediate cell mass region, and definitive erythropoiesis arises from the aorta-gonad mesonephros. TALE-homeoproteins Meis1 and Pbx1 function upstream of GATA1 to specify the erythroid lineage. Embryos lacking Meis1 or Pbx1 have weak gata1 expression and fail to produce primitive erythrocytes. Nevertheless, the underlying mechanism of how Meis1 and Pbx1 mediate gata1 transcription in erythrocytes remains unclear. Here we show that Hif1α acts downstream of Meis1 to mediate gata1 expression in zebrafish embryos. Inhibition of Meis1 expression resulted in suppression of hif1a expression and abrogated primitive erythropoiesis, while injection with in vitro-synthesized hif1α mRNA rescued gata1 transcription in Meis1 morphants and recovered their erythropoiesis. Ablation of Hif1α expression either by morpholino knockdown or Crispr-Cas9 knockout suppressed gata1 transcription and abrogated primitive erythropoiesis. Results of chromatin immunoprecipitation assays showed that Hif1α associates with hypoxia-response elements located in the 3'-flanking region of gata1 during development, suggesting that Hif1α regulates gata1 expression in vivo. Together, our results indicate that Meis1, Hif1α, and GATA1 indeed comprise a hierarchical regulatory network in which Hif1α acts downstream of Meis1 to activate gata1 transcription through direct interactions with its cis-acting elements in primitive erythrocytes.


Asunto(s)
Células Eritroides/metabolismo , Eritropoyesis , Factor de Transcripción GATA1/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Inmunoprecipitación de Cromatina , Eritrocitos/citología , Eritrocitos/metabolismo , Células Eritroides/citología , Eritropoyesis/genética , Factor de Transcripción GATA1/genética , Regulación del Desarrollo de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/deficiencia , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Factor de Transcripción 1 de la Leucemia de Células Pre-B/deficiencia , Factor de Transcripción 1 de la Leucemia de Células Pre-B/genética , Transcripción Genética , Pez Cebra/sangre , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética
18.
Chem Biol Interact ; 349: 109674, 2021 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-34562440

RESUMEN

We have assessed the molecular role of Rutin and rutin-Zn(II) complex on osteoblast differentiation and mineralization in human dental pulp cells and zebrafish model. The biocompatibility of the rutin-Zn(II) complex was determined using MTT and chick embryotoxicity assays. Alizarin red staining and ALP measurements were performed to study the osteogenic role of Rutin and rutin-Zn(II) complex at the cellular level in hDPSCs. At molecular level, following rutin and rutin-Zn(II) exposure, the mRNA expression profile of osteoblast markers such Runx2, type 1 col, OC, and ON were investigated. In addition to this, the expression of negative regulators of osteoblast development such Smad7, Smurf1, and HDAC7 waere studied by Real time RT-PCR analysis. The osteogenic role of prepared complex under in vivo was studied by an in-house zebrafish scale model followed by osteoblast differentiation markers expression profiling and Ca:P level measurement by ICP-MS. Rutin and the rutin-Zn(II) complex were found to be non-toxic till 10 µM and increased the expression of osteoblast differentiation marker genes. It also enhanced calcium deposition in both in vitro and in vivo models. Osteogenic property of rutin-Zn(II) in hDPSCs was found be mediated by Smad7, Smurf1, and HDAC7 and enhancing Runx2 expression. Our study warrants the possible use of rutin-Zn(II) as naïve agent or in combination with other bone scaffolding systems/materials for bone tissue engineering applications.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Complejos de Coordinación/farmacología , Osteogénesis/efectos de los fármacos , Rutina/química , Zinc/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Pulpa Dental/citología , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Células Madre/citología , Células Madre/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Pez Cebra/metabolismo
19.
Ecotoxicol Environ Saf ; 226: 112809, 2021 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-34592523

RESUMEN

Dinotefuran is a widely used neonicotinoid pesticides in agriculture and it has certain ecological toxicity to aquatic organisms. Studies on the potential toxicological effects of dinotefuran on fish are limited. In the present study, 96 h acute toxicity test indicated that enantiomers of R-(-)-dinotefuran had a greater toxic effect than Rac-dinotefuran on zebrafish, and S-(+)-dinotefuran was the least. In chronic assay, R-(-)-dinotefuran exerted more effects on the development of zebrafish than S-(+)-dinotefuran, and dinotefuran also had enantioselective effect on oxidative stress. Significant changes were observed in the superoxide dismutase (SOD), glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities and malondialdehyde (MDA) contents, which demonstrated dinotefuran induced oxidative stress in zebrafish. Besides, through an ultra-performance liquid chromatography quadrupole-TOF mass spectrometry (UPLC-Q-TOF-MS)-based metabolomics method was used to evaluate the enantioselectivity of dinotefuran enantiomers in zebrafish. The results indicated that R-(-)-dinotefuran caused greater disturbances of endogenous metabolites. Phenylalanine metabolic pathways, glycine, serine and threonine metabolic pathways are only involved in zebrafish exposed to R-(-)-dinotefuran; whereas phenylalanine, tyrosine and tryptophan biosynthesis was only involved in zebrafish exposed to S-(+)-dinotefuran. This study provides a certain reference value for assessing the environmental risks of dinotefuran enantiomers to aquatic organisms, and has practical significance for guiding the ecologically and environmentally safety use of dinotefuran.


Asunto(s)
Contaminantes Químicos del Agua , Pez Cebra , Acetilcolinesterasa , Animales , Guanidinas , Neonicotinoides/toxicidad , Nitrocompuestos , Estrés Oxidativo , Estereoisomerismo , Superóxido Dismutasa/metabolismo , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/metabolismo
20.
BMC Genomics ; 22(1): 658, 2021 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-34517816

RESUMEN

BACKGROUND: Zebrafish is a popular animal model used for high-throughput screening of chemical hazards, however, investigations of transcriptomic mechanisms of toxicity are still needed. Here, our goal was to identify genes and biological pathways that Aryl Hydrocarbon Receptor 2 (AHR2) Activators and flame retardant chemicals (FRCs) alter in developing zebrafish. Taking advantage of a compendium of phenotypically-anchored RNA sequencing data collected from 48-h post fertilization (hpf) zebrafish, we inferred a co-expression network that grouped genes based on their transcriptional response. RESULTS: Genes responding to the FRCs and AHR2 Activators localized to distinct regions of the network, with FRCs inducing a broader response related to neurobehavior. AHR2 Activators centered in one region related to chemical stress responses. We also discovered several highly co-expressed genes in this module, including cyp1a, and we subsequently show that these genes are definitively within the AHR2 signaling pathway. Systematic removal of the two chemical types from the data, and analysis of network changes identified neurogenesis associated with FRCs, and regulation of vascular development associated with both chemical classes. We also identified highly connected genes responding specifically to each class that are potential biomarkers of exposure. CONCLUSIONS: Overall, we created the first zebrafish chemical-specific gene co-expression network illuminating how chemicals alter the transcriptome relative to each other. In addition to our conclusions regarding FRCs and AHR2 Activators, our network can be leveraged by other studies investigating chemical mechanisms of toxicity.


Asunto(s)
Proteínas de Pez Cebra , Pez Cebra , Animales , Secuencia de Bases , Embrión no Mamífero/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transcriptoma , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética
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