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2.
Int J Mol Sci ; 22(19)2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34638816

RESUMO

Vertebrates generate mature red blood cells (RBCs) via a highly regulated, multistep process called erythropoiesis. Erythropoiesis involves synthesis of heme and hemoglobin, clearance of the nuclei and other organelles, and remodeling of the plasma membrane, and these processes are exquisitely coordinated by specific regulatory factors including transcriptional factors and signaling molecules. Defects in erythropoiesis can lead to blood disorders such as congenital dyserythropoietic anemias, Diamond-Blackfan anemias, sideroblastic anemias, myelodysplastic syndrome, and porphyria. The molecular mechanisms of erythropoiesis are highly conserved between fish and mammals, and the zebrafish (Danio rerio) has provided a powerful genetic model for studying erythropoiesis. Studies in zebrafish have yielded important insights into RBC development and established a number of models for human blood diseases. Here, we focus on latest discoveries of the molecular processes and mechanisms regulating zebrafish erythropoiesis and summarize newly established zebrafish models of human anemias.


Assuntos
Anemia Aplástica , Desenvolvimento Embrionário , Eritrócitos/metabolismo , Eritropoese , Peixe-Zebra/embriologia , Anemia Aplástica/genética , Anemia Aplástica/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Peixe-Zebra/genética
3.
Photochem Photobiol Sci ; 20(10): 1273-1285, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34647278

RESUMO

Sunscreen safety and efficacy is generally evaluated based upon the properties of the individual chemicals in a formulation. However, the photostability of sunscreens has been shown to be highly dependent on the mixture of chemicals present. To better understand how sunscreen formulation influences stability, and to establish a foundation for probing the influence of zinc oxide additives, we formulated five different small-molecule based ultraviolet-filter (UV-filter) mixtures with a Sun Protection Factor (SPF) of 15. These mixtures contained active ingredients approved in either the United States or European Union and were designed to represent formulations of actual products on the market. We evaluated the photostability and toxicity of these mixtures in the absence and presence of zinc oxide after UV exposure for two hours. Changes in UV absorbance were minimal for all five small-molecule-based mixtures without zinc oxide. The presence of either micro- or nano-sized zinc oxide caused significant small-molecule photodegradation and the degraded mixtures exhibited higher levels of toxicity in embryonic zebrafish assays. This study suggests that caution must be taken when formulating sunscreens containing both zinc oxide and small-molecule UV-filters to avoid unintended consequences during use.


Assuntos
Protetores Solares/farmacologia , Protetores Solares/toxicidade , Raios Ultravioleta , Óxido de Zinco/farmacologia , Óxido de Zinco/toxicidade , Animais , Tamanho da Partícula , Protetores Solares/química , Peixe-Zebra/embriologia , Óxido de Zinco/química
4.
Molecules ; 26(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34684803

RESUMO

Honey is prone to be adulterated through mixing with sugars, cheap and low-quality honey, and other adulterants. Consumption of adulterated honey may cause several health issues such as weight gain, diabetes, and liver and kidney dysfunction. Therefore, studying the impact of consumption of adulterated honey on consumers is critical since there is a lack of study in this field. Hence, the aims of this paper were: (1) to determine the lethal concentration (LC50) of adulterated honey using zebrafish embryo, (2) to elucidate toxicology of selected adulterated honey based on lethal dose (LD50) using adult zebrafish, (3) to determine the effects of adulterated honey on histological changes of zebrafish, and (4) to screen the metabolites profile of adulterated honey by using zebrafish blood serum. The LC50 of Heterotrigona itama honey (acacia honey) and its sugar adulterants (light corn sugar, cane sugar, inverted sugar, and palm sugar in the proportion of 1-3% (w/w) from the total volume) was determined by the toxicological assessment of honey samples on zebrafish embryos (different exposure concentrations in 24, 48, 72, and 96 h postfertilization (hpf)). Pure H. itama honey represents the LC50 of 34.40 ± 1.84 (mg/mL) at 96 hpf, while the inverted sugar represents the lowest LC50 (5.03 ± 0.92 mg/mL) among sugar adulterants. The highest concentration (3%) of sugar adulterants were used to study the toxicology of adulterated honey using adult zebrafish in terms of acute, prolong-acute, and sub-acute tests. The results of the LD50 from the sub-acute toxicity test of pure H. itama honey was 2.33 ± 0.24 (mg/mL). The histological studies of internal organs showed a lesion in the liver, kidney, and spleen of adulterated treated-honey groups compared to the control group. Furthermore, the LC-MS/MS results revealed three endogenous metabolites in both the pure and adulterated honey treated groups, as follows: (1) S-Cysteinosuccinic acid, (2) 2,3-Diphosphoglyceric acid, and (3) Cysteinyl-Tyrosine. The results of this study demonstrated that adulterated honey caused mortality, which contributes to higher toxicity, and also suggested that the zebrafish toxicity test could be a standard method for assessing the potential toxicity of other hazardous food additives. The information gained from this research will permit an evaluation of the potential risk associated with the consumption of adulterated compared to pure honey.


Assuntos
Acacia/química , Contaminação de Alimentos/análise , Mel/análise , Mel/toxicidade , Açúcares/análise , Açúcares/toxicidade , Animais , Abelhas , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Dose Letal Mediana , Fígado/efeitos dos fármacos , Fígado/patologia , Malásia , Metaboloma , Baço/efeitos dos fármacos , Baço/patologia , Espectrometria de Massas em Tandem , Testes de Toxicidade Aguda/métodos , Peixe-Zebra/sangue , Peixe-Zebra/embriologia
5.
FASEB J ; 35(11): e21961, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34665878

RESUMO

Many organisms rely on oxygen to generate cellular energy (adenosine triphosphate or ATP). During severe hypoxia, the production of ATP decreases, leading to cell damage or death. Conversely, excessive oxygen causes oxidative stress that is equally damaging to cells. To mitigate pathological outcomes, organisms have evolved mechanisms to adapt to fluctuations in oxygen levels. Zebrafish embryos are remarkably hypoxia-tolerant, surviving anoxia (zero oxygen) for hours in a hypometabolic, energy-conserving state. To begin to unravel underlying mechanisms, we analyze here the distribution of the N-myc Downstream Regulated Gene (ndrg) family, ndrg1-4, and their transcriptional response to hypoxia. These genes have been primarily studied in cancer cells and hence little is understood about their normal function and regulation. We show here using in situ hybridization that ndrgs are expressed in metabolically demanding organs of the zebrafish embryo, such as the brain, kidney, and heart. To investigate whether ndrgs are hypoxia-responsive, we exposed embryos to different durations and severity of hypoxia and analyzed transcript levels. We observed that ndrgs are differentially regulated by hypoxia and that ndrg1a has the most robust response, with a ninefold increase following prolonged anoxia. We further show that this treatment resulted in de novo expression of ndrg1a in tissues where the transcript is not observed under normoxic conditions and changes in Ndrg1a protein expression post-reoxygenation. These findings provide an entry point into understanding the role of this conserved gene family in the adaptation of normal cells to hypoxia and reoxygenation.


Assuntos
Hipóxia Celular , Embrião não Mamífero/metabolismo , Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Oxigênio/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Estresse Oxidativo
6.
Molecules ; 26(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34641329

RESUMO

The antioxidant activity of food compounds is one of the properties generating the most interest, due to its health benefits and correlation with the prevention of chronic disease. This activity is usually measured using in vitro assays, which cannot predict in vivo effects or mechanisms of action. The objective of this study was to evaluate the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, ß-carotene, and astaxanthin) naturally present in foods using a zebrafish embryo model. The zebrafish embryo was pretreated with each of the nine antioxidant compounds and then exposed to tert-butyl hydroperoxide (tBOOH), a known inducer of oxidative stress in zebrafish. Significant differences were determined by comparing the concentration-response of the tBOOH induced lethality and dysmorphogenesis against the pretreated embryos with the antioxidant compounds. A protective effect of each compound, except ß-carotene, against oxidative-stress-induced lethality was found. Furthermore, apigenin, rutin, and curcumin also showed protective effects against dysmorphogenesis. On the other hand, ß-carotene exhibited increased lethality and dysmorphogenesis compared to the tBOOH treatment alone.


Assuntos
Antioxidantes/administração & dosagem , Fatores Biológicos/administração & dosagem , Carotenoides/administração & dosagem , Polifenóis/administração & dosagem , Peixe-Zebra/embriologia , terc-Butil Hidroperóxido/efeitos adversos , Animais , Antioxidantes/farmacologia , Apigenina/administração & dosagem , Apigenina/farmacologia , Fatores Biológicos/farmacologia , Carotenoides/farmacologia , Curcumina/administração & dosagem , Curcumina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Flavanonas/administração & dosagem , Flavanonas/farmacologia , Licopeno/administração & dosagem , Licopeno/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Polifenóis/farmacologia , Xantofilas/administração & dosagem , Xantofilas/farmacologia , beta Caroteno/administração & dosagem , beta Caroteno/efeitos adversos , beta Caroteno/farmacologia
7.
FASEB J ; 35(11): e22002, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34708458

RESUMO

Autophagy is a catabolic process responsible for the removal of waste and damaged cellular components by lysosomal degradation. It plays a key role in fundamental cell processes, including ER stress mitigation, control of cell metabolism, and cell differentiation and proliferation, all of which are essential for cartilage cell (chondrocyte) development and survival, and for the formation of cartilage. Correspondingly, autophagy dysregulation has been implicated in several skeletal disorders such as osteoarthritis and osteoporosis. To test the requirement for autophagy during skeletal development in zebrafish, we generated an atg13 CRISPR knockout zebrafish line. This line showed a complete loss of atg13 expression, and restricted autophagic activity in vivo. In the absence of autophagy, chondrocyte maturation was accelerated, with chondrocytes exhibiting signs of premature hypertrophy. Focussing on the jaw element, autophagy disruption affected joint articulation causing restricted mouth opening. This gross behavioural phenotype corresponded with a failure to thrive, and death in homozygote atg13 nulls within 17 days. Taken together, our results are consistent with autophagy contributing to the timely regulation of chondrocyte maturation and for extracellular matrix formation.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Condrócitos/citologia , Condrogênese , Articulações/embriologia , Peixe-Zebra/embriologia , Animais , Autofagia , Diferenciação Celular
8.
Nutrients ; 13(10)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34684347

RESUMO

Inositol is a natural substance found widely in plants. It is used in therapies for many medical cases. The aim of this study was to determine the toxicity of myo-inositol (MI) and to investigate its potential hepatoprotective character. In the first part of the study, zebrafish embryos were incubated with 5, 10, 20, 40, 60, 80, and 100 mg/mL MI. Endpoints such as survivability, hatching rate, malformation, and mobility were evaluated. Our results demonstrated that the high doses of MI lead to increased mortality and malformations and reduce the hatching rate in comparison to the control group. Moreover, low doses of this compound do not produce a negative effect on zebrafish and even have the ability to increase the hatching rate and mobility. In the second part of the study, the hepatoprotective effect of MI was tested. Zebrafish larvae from the line Tg (fabp10a:DsRed) were incubated for 24 h with 1% and 2% ethanol (EtOH), 5 mg/mL of MI with 1% EtOH, and 5 mg/mL of MI with 2% EtOH. No significant differences between the groups with EtOH and the group treated with EtOH with MI were observed. Our results suggest that MI has no positive benefits on hepatocytes of zebrafish larvae.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Inositol/farmacologia , Fígado/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/efeitos dos fármacos , Etanol , Fluorescência , Inositol/química , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Fígado/patologia , Modelos Animais , Análise de Sobrevida , Testes de Toxicidade
9.
Nat Commun ; 12(1): 5284, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489414

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Morte Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Doenças Neurodegenerativas/genética , Neurônios/metabolismo , Animais , Cálcio/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Corantes Fluorescentes/química , Genes Reporter , Ácido Glutâmico/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Ratos , Ratos Long-Evans , Análise de Célula Única/métodos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
10.
FASEB J ; 35(10): e21915, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34496088

RESUMO

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.


Assuntos
Células Eritroides/metabolismo , Eritropoese , Fator de Transcrição GATA1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína Meis1/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Imunoprecipitação da Cromatina , Eritrócitos/citologia , Eritrócitos/metabolismo , Células Eritroides/citologia , Eritropoese/genética , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína Meis1/deficiência , Proteína Meis1/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/deficiência , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Transcrição Genética , Peixe-Zebra/sangue , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
11.
Nat Commun ; 12(1): 5263, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489457

RESUMO

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeo Hidrolases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase Ialfa/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Evolução Molecular , Células HEK293 , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Lenalidomida/química , Lenalidomida/farmacologia , Camundongos , Organoides , Peptídeo Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
Elife ; 102021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34490846

RESUMO

The spatial organization of gut microbiota influences both microbial abundances and host-microbe interactions, but the underlying rules relating bacterial dynamics to large-scale structure remain unclear. To this end, we studied experimentally and theoretically the formation of three-dimensional bacterial clusters, a key parameter controlling susceptibility to intestinal transport and access to the epithelium. Inspired by models of structure formation in soft materials, we sought to understand how the distribution of gut bacterial cluster sizes emerges from bacterial-scale kinetics. Analyzing imaging-derived data on cluster sizes for eight different bacterial strains in the larval zebrafish gut, we find a common family of size distributions that decay approximately as power laws with exponents close to -2, becoming shallower for large clusters in a strain-dependent manner. We show that this type of distribution arises naturally from a Yule-Simons-type process in which bacteria grow within clusters and can escape from them, coupled to an aggregation process that tends to condense the system toward a single massive cluster, reminiscent of gel formation. Together, these results point to the existence of general, biophysical principles governing the spatial organization of the gut microbiome that may be useful for inferring fast-timescale dynamics that are experimentally inaccessible.


Assuntos
Bactérias/crescimento & desenvolvimento , Fenômenos Fisiológicos Bacterianos , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Animais , Trato Gastrointestinal/embriologia , Géis , Cinética , Modelos Teóricos , Densidade Demográfica , Peixe-Zebra/embriologia , Peixe-Zebra/microbiologia
14.
Mar Biotechnol (NY) ; 23(5): 821-835, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34490548

RESUMO

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.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Cadeias Pesadas de Miosina/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/metabolismo , MicroRNAs/genética , Cadeias Pesadas de Miosina/genética , Takifugu/genética , Peixe-Zebra/genética
15.
PLoS One ; 16(9): e0257073, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495967

RESUMO

BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is due to biallelic variants in ALDH7A1 (PDE-ALDH7A1). ALDH7A1 encodes α-aminoadipic semialdehyde dehydrogenase in lysine catabolism. We investigated the gamma aminobutyric acid (GABA) metabolism and energy production pathways in human PDE-ALDH7A1 and its knock-out aldh7a1 zebrafish model. METHODS: We measured GABA pathway, and tricarboxylic acid cycle metabolites and electron transport chain activities in patients with PDE-ALDH7A1 and in knock-out aldh7a1 zebrafish. RESULTS: We report results of three patients with PDE-ALDH7A1: low paired complex I+II and complex II+III and individual complex IV activities in muscle biopsy in patient 1 (likely more severe phenotype); significantly elevated CSF glutamate in the GABA pathway and elevated CSF citrate, succinate, isocitrate and α-ketoglutarate in the TCA cycle in patient 3 (likely more severe phenotype); and normal CSF GABA pathway and TCA cycle metabolites on long-term pyridoxine therapy in patient 2 (likely milder phenotype). All GABA pathway metabolites (γ-hydroxybutyrate, glutamine, glutamate, total GABA, succinic semialdehyde) and TCA cycle metabolites (citrate, malate, fumarate, isocitrate, lactate) were significantly low in the homozygous knock-out aldh7a1 zebrafish compared to the wildtype zebrafish. Homozygous knock-out aldh7a1 zebrafish had decreased electron transport chain enzyme activities compared to wildtype zebrafish. DISCUSSION: We report impaired electron transport chain function, accumulation of glutamate in the central nervous system and TCA cycle dysfunction in human PDE-ALDH7A1 and abnormal GABA pathway, TCA cycle and electron transport chain in knock-out aldh7a1 zebrafish. Central nervous system glutamate toxicity and impaired energy production may play important roles in the disease neuropathogenesis and severity in human PDE-ALDH7A1.


Assuntos
Aldeído Desidrogenase/genética , Alelos , Metabolismo Energético , Epilepsia/metabolismo , Proteínas de Peixe-Zebra/genética , Animais , Ciclo do Ácido Cítrico , DNA Mitocondrial/genética , Transporte de Elétrons , Embrião não Mamífero , Metabolismo Energético/genética , Peixe-Zebra/embriologia
16.
Int J Mol Sci ; 22(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34502201

RESUMO

BACKGROUND: Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear. METHODS: Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms. RESULTS: CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes. CONCLUSIONS: The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters.


Assuntos
Neoplasias da Mama/patologia , Modelos Animais de Doenças , Células Neoplásicas Circulantes , Peixe-Zebra/embriologia , Animais , Linhagem Celular Tumoral , Embrião não Mamífero , Feminino , Humanos , Células MCF-7 , Camundongos , Metástase Neoplásica
17.
Int J Mol Sci ; 22(18)2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34576264

RESUMO

After the discovery of endogenous dinitrosyl iron complexes (DNICs) as a potential biological equivalent of nitric oxide (NO), bioinorganic engineering of [Fe(NO)2] unit has emerged to develop biomimetic DNICs [(NO)2Fe(L)2] as a chemical biology tool for controlled delivery of NO. For example, water-soluble DNIC [Fe2(µ-SCH2CH2OH)2(NO)4] (DNIC-1) was explored for oral delivery of NO to the brain and for the activation of hippocampal neurogenesis. However, the kinetics and mechanism for cellular uptake and intracellular release of NO, as well as the biocompatibility of synthetic DNICs, remain elusive. Prompted by the potential application of NO to dermato-physiological regulations, in this study, cellular uptake and intracellular delivery of DNIC [Fe2(µ-SCH2CH2COOH)2(NO)4] (DNIC-2) and its regulatory effect/biocompatibility toward epidermal cells were investigated. Upon the treatment of DNIC-2 to human fibroblast cells, cellular uptake of DNIC-2 followed by transformation into protein-bound DNICs occur to trigger the intracellular release of NO with a half-life of 1.8 ± 0.2 h. As opposed to the burst release of extracellular NO from diethylamine NONOate (DEANO), the cell-penetrating nature of DNIC-2 rationalizes its overwhelming efficacy for intracellular delivery of NO. Moreover, NO-delivery DNIC-2 can regulate cell proliferation, accelerate wound healing, and enhance the deposition of collagen in human fibroblast cells. Based on the in vitro and in vivo biocompatibility evaluation, biocompatible DNIC-2 holds the potential to be a novel active ingredient for skincare products.


Assuntos
Materiais Biocompatíveis/química , Fibroblastos/efeitos dos fármacos , Ferro/química , Óxido Nítrico/química , Óxidos de Nitrogênio/química , Pele/efeitos dos fármacos , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Colágeno/química , Córnea/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Embrião não Mamífero/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Olho/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Cinética , Melanócitos/metabolismo , Oxigênio/química , Pigmentação , Cicatrização , Peixe-Zebra/embriologia
18.
Nat Commun ; 12(1): 5415, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518536

RESUMO

Coordinated chromatin interactions between enhancers and promoters are critical for gene regulation. The architectural protein CTCF mediates chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures. In vitro CTCF depletion leads to a loss of TADs but has only limited effects over gene expression, challenging the concept that CTCF-mediated chromatin structures are a fundamental requirement for gene regulation. However, how CTCF and a perturbed chromatin structure impacts gene expression during development remains poorly understood. Here we link the loss of CTCF and gene regulation during patterning and organogenesis in a ctcf knockout zebrafish model. CTCF absence leads to loss of chromatin structure and affects the expression of thousands of genes, including many developmental regulators. Our results demonstrate the essential role of CTCF in providing the structural context for enhancer-promoter interactions, thus regulating developmental genes.


Assuntos
Fator de Ligação a CCCTC/genética , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes/métodos , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Padronização Corporal/genética , Fator de Ligação a CCCTC/deficiência , Sistemas CRISPR-Cas , Cromatina/genética , Cromatina/metabolismo , Embrião não Mamífero/embriologia , Elementos Facilitadores Genéticos/genética , Organogênese/genética , Regiões Promotoras Genéticas/genética , RNA-Seq/métodos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/deficiência
19.
Nat Commun ; 12(1): 5482, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531379

RESUMO

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3'-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3'-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.


Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Xenopus laevis/genética , Peixe-Zebra/genética , Regiões 3' não Traduzidas/genética , Animais , Desenvolvimento Embrionário/genética , Camundongos , Estabilidade de RNA/genética , Xenopus laevis/embriologia , Peixe-Zebra/embriologia
20.
Life Sci ; 284: 119900, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34453946

RESUMO

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits. To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 days post fertilization (dpf) and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.


Assuntos
Autofagia/genética , Eritropoese/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mutação/genética , Vitamina D/farmacologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Autofagia/efeitos dos fármacos , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Embrião não Mamífero/patologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Eritropoese/efeitos dos fármacos , Dosagem de Genes , Hemoglobinas/metabolismo , Inflamação/genética , Transcriptoma/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo
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