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1.
Int J Mol Sci ; 22(24)2021 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-34948305

RESUMO

Radial glial cells are a distinct non-neuronal cell type that, during development, span the entire width of the brain walls of the ventricular system. They play a central role in the origin and placement of neurons, since their processes form structural scaffolds that guide and facilitate neuronal migration. Furthermore, glutamatergic signaling in the radial glia of the adult cerebellum (i.e., Bergmann glia), is crucial for precise motor coordination. Radial glial cells exhibit spontaneous calcium activity and functional coupling spread calcium waves. However, the origin of calcium activity in relation to the ontogeny of cerebellar radial glia has not been widely explored, and many questions remain unanswered regarding the role of radial glia in brain development in health and disease. In this study we used a combination of whole mount immunofluorescence and calcium imaging in transgenic (gfap-GCaMP6s) zebrafish to determine how development of calcium activity is related to morphological changes of the cerebellum. We found that the morphological changes in cerebellar radial glia are quite dynamic; the cells are remarkably larger and more elaborate in their soma size, process length and numbers after 7 days post fertilization. Spontaneous calcium events were scarce during the first 3 days of development and calcium waves appeared on day 5, which is associated with the onset of more complex morphologies of radial glia. Blockage of gap junction coupling inhibited the propagation of calcium waves, but not basal local calcium activity. This work establishes crucial clues in radial glia organization, morphology and calcium signaling during development and provides insight into its role in complex behavioral paradigms.


Assuntos
Sinalização do Cálcio/fisiologia , Cerebelo/metabolismo , Cerebelo/fisiologia , Neuroglia/metabolismo , Neuroglia/fisiologia , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Cálcio/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia
2.
PLoS One ; 16(10): e0258830, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34673817

RESUMO

Rett syndrome (RTT) is a neurodevelopmental disorder with X-linked dominant inheritance caused mainly by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. The effects of various Mecp2 mutations have been extensively assessed in mouse models, but none adequately mimic the symptoms and pathological changes of RTT. In this study, we assessed the effects of Mecp2 gene deletion on female rats (Mecp2+/-) and found severe impairments in social behavior [at 8 weeks (w), 12 w, and 23 w of age], motor function [at 16 w and 26 w], and spatial cognition [at 29 w] as well as lower plasma insulin-like growth factor (but not brain-derived neurotrophic factor) and markedly reduced acetylcholine (30%-50%) in multiple brain regions compared to female Mecp2+/+ rats [at 29 w]. Alternatively, changes in brain monoamine levels were relatively small, in contrast to reports on mouse Mecp2 mutants. Female Mecp2-deficient rats express phenotypes resembling RTT and so may provide a robust model for future research on RTT pathobiology and treatment.


Assuntos
Acetilcolina/metabolismo , Encéfalo/metabolismo , Cognição , Locomoção , Memória/fisiologia , Proteína 2 de Ligação a Metil-CpG/fisiologia , Comportamento Social , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Modelos Animais de Doenças , Feminino , Aprendizagem , Ratos
3.
Dev Cell ; 56(18): 2649-2663.e6, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34529940

RESUMO

The nuclear lamina is typically associated with transcriptional silencing, and peripheral relocation of genes highly correlates with repression. However, the DNA sequences and proteins regulating gene-lamina interactions are largely unknown. Exploiting the developmentally timed hunchback gene movement to the lamina in Drosophila neuroblasts, we identified a 250 bp intronic element (IE) both necessary and sufficient for relocation. The IE can target a reporter transgene to the lamina and silence it. Endogenously, however, hunchback is already repressed prior to relocation. Instead, IE-mediated relocation confers a heritably silenced gene state refractory to activation in descendent neurons, which terminates neuroblast competence to specify early-born identity. Surprisingly, we found that the Polycomb group chromatin factors bind the IE and are required for lamina relocation, revealing a nuclear architectural role distinct from their well-known function in transcriptional repression. Together, our results uncover in vivo mechanisms underlying neuroblast competence and lamina association in heritable gene silencing.


Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Neurais/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Sistema Nervoso Central/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios/metabolismo
4.
Sci Rep ; 11(1): 16078, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373563

RESUMO

Neurons expressing the neuropeptide hypocretin/orexin (Hcrt) in the hypothalamus promote reward-related behaviors including alcohol consumption and are shown in rodents and zebrafish to be stimulated by embryonic exposure to ethanol (EtOH). We used here in zebrafish three-dimensional analyses of the entire population of Hcrt neurons to examine how embryonic EtOH exposure at low-moderate concentrations (0.1% or 0.5% v/v) alters these neurons in relation to behavior. We found that EtOH in the water for 2 h (22-24 h post fertilization) increases the number of Hcrt neurons on the left but not right side of the brain through a stimulation of cell proliferation, this is accompanied by a decrease in locomotor activity under novel conditions but not after habituation, and these effects are evident in both larvae and adults indicating they are long lasting. Our analyses in adults revealed sexually dimorphic effects, with females consuming more EtOH-gelatin and exhibiting more freezing behavior along with an asymmetric increase in Hcrt neurons and males exhibiting increased aggression with no change in Hcrt. These findings suggest that a long lasting, asymmetric increase in Hcrt neurons induced by EtOH results from an asymmetric increase in proliferation specific to Hcrt and contributes to behavioral changes in females.


Assuntos
Comportamento Animal/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Etanol/farmacologia , Neurônios/efeitos dos fármacos , Orexinas/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Caracteres Sexuais
5.
Development ; 148(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34397091

RESUMO

Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.


Assuntos
Proliferação de Células/fisiologia , Miócitos Cardíacos/fisiologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Calpaína/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Células Cultivadas , Citocinese/fisiologia , Feminino , Coração/fisiologia , Masculino , Mamíferos/metabolismo , Mamíferos/fisiologia , Miócitos Cardíacos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Regeneração/fisiologia , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445111

RESUMO

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs-/- larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.


Assuntos
Proteínas de Choque Térmico/metabolismo , Fármacos Neuroprotetores/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Ataxia/metabolismo , Ataxia Cerebelar/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Espasticidade Muscular/metabolismo , Mutação/genética , Fenótipo , Células de Purkinje/metabolismo , Ataxias Espinocerebelares/congênito , Ataxias Espinocerebelares/metabolismo
7.
Cell Mol Life Sci ; 78(17-18): 6143-6160, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34322715

RESUMO

Amyotrophic lateral sclerosis (ALS) is a rare, devastating disease, causing movement impairment, respiratory failure and ultimate death. A plethora of genetic, cellular and molecular mechanisms are involved in ALS signature, although the initiating causes and progressive pathological events are far from being understood. Drosophila research has produced seminal discoveries for more than a century and has been successfully used in the past 25 years to untangle the process of ALS pathogenesis, and recognize potential markers and novel strategies for therapeutic solutions. This review will provide an updated view of several ALS modifiers validated in C9ORF72, SOD1, FUS, TDP-43 and Ataxin-2 Drosophila models. We will discuss basic and preclinical findings, illustrating recent developments and novel breakthroughs, also depicting unsettled challenges and limitations in the Drosophila-ALS field. We intend to stimulate a renewed debate on Drosophila as a screening route to identify more successful disease modifiers and neuroprotective agents.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Drosophila/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Ataxina-2/genética , Ataxina-2/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo
8.
Sci Rep ; 11(1): 11432, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075074

RESUMO

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.


Assuntos
Animais Geneticamente Modificados , Comportamento Animal/efeitos dos fármacos , Doenças Genéticas Inatas , Retinite Pigmentosa , Rodopsina , Visão Ocular , Peixe-Zebra , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Humanos , Mutação , Células Fotorreceptoras Retinianas Bastonetes , Retinite Pigmentosa/tratamento farmacológico , Retinite Pigmentosa/genética , Retinite Pigmentosa/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Transgenes , Visão Ocular/efeitos dos fármacos , Visão Ocular/imunologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
9.
Biochim Biophys Acta Mol Basis Dis ; 1867(10): 166203, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34146705

RESUMO

Amyloid ß (Aß), a product of APP, and SNCA (α-synuclein (α-syn)) are two of the key proteins found in lesions associated with the age-related neurodegenerative disorders Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. Previous clinical studies uncovered Aß and α-syn co-expression in the brains of patients, which lead to Lewy body dementia (LBD), a disease encompassing Dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD). To explore the pathogenesis and define the relationship between Aß and α-syn for LBD, we established a C. elegans model which co-expresses human Aß and α-syn with alanine 53 to threonine mutant (α-syn(A53T)) in pan-neurons. Compared to α-syn(A53T) single transgenic animals, pan-neuronal Aß and α-syn(A53T) co-expression further enhanced the thrashing, egg laying, serotonin and cholinergic signaling deficits, and dopaminergic neuron damage in C. elegans. In addition, Aß increased α-syn expression in transgenic animals. Transcriptome analysis of both Aß;α-syn(A53T) strains and DLB patients showed common downregulation in lipid metabolism and lysosome function genes, suggesting that a decrease of lysosome function may reduce the clearance ability in DLB, and this may lead to the further pathogenic protein accumulation. These findings suggest that our model can recapitulate some features in LBD and provides a mechanism by which Aß may exacerbate α-syn pathogenesis.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Caenorhabditis elegans/metabolismo , Doença por Corpos de Lewy/metabolismo , Neurônios/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Doença de Alzheimer/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Encéfalo/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Lisossomos/metabolismo , Pessoa de Meia-Idade , Doença de Parkinson/metabolismo , Transcriptoma/fisiologia
10.
Sci Rep ; 11(1): 12099, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103614

RESUMO

In the intestine, the host must be able to control the gut microbiota and efficiently absorb transiently supplied metabolites, at the risk of enormous infection. In mammals, the inflammatory cytokine interleukin (IL)-17A/F is one of the key mediators in the intestinal immune system. However, many functions of IL-17 in vertebrate intestines remain unclarified. In this study, we established a gene-knockout (KO) model of IL-17 receptor A1 (IL-17RA1, an IL-17A/F receptor) in Japanese medaka (Oryzias latipes) using genome editing technique, and the phenotypes were compared to wild type (WT) based on transcriptome analyses. Upon hatching, homozygous IL-17RA1-KO medaka mutants showed no significant morphological abnormality. However, after 4 months, significant weight decreases and reduced survival rates were observed in IL-17RA1-KO medaka. Comparison of gene-expression patterns in WT and IL-17RA1-KO medaka revealed that various metabolism- and immune-related genes were significantly down-regulated in IL-17RA1-KO medaka intestine, particularly genes related to mevalonate metabolism (mvda, acat2, hmgcs1, and hmgcra) and genes related to IL-17 signaling (such as il17c, il17a/f1, and rorc) were found to be decreased. Conversely, expression of genes related to cardiovascular system development, including fli1a, sox7, and notch1b in the anterior intestine, and that of genes related to oxidation-reduction processes including ugp2a, aoc1, and nos1 in posterior intestine was up-regulated in IL-17RA1-KO medaka. These findings show that IL-17RA regulated immune- and various metabolism-related genes in the intestine for maintaining the health of Japanese medaka.


Assuntos
Animais Geneticamente Modificados , Regulação para Baixo , Proteínas de Peixes , Mucosa Intestinal/metabolismo , Oryzias , Receptores de Interleucina-17 , Perda de Peso/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Técnicas de Inativação de Genes , Oryzias/genética , Oryzias/metabolismo , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo
11.
Nat Cell Biol ; 23(5): 497-510, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33972730

RESUMO

The Drosophila trachea, as the functional equivalent of mammalian blood vessels, senses hypoxia and oxygenates the body. Here, we show that the adult intestinal tracheae are dynamic and respond to enteric infection, oxidative agents and tumours with increased terminal branching. Increased tracheation is necessary for efficient damage-induced intestinal stem cell (ISC)-mediated regeneration and is sufficient to drive ISC proliferation in undamaged intestines. Gut damage or tumours induce HIF-1α (Sima in Drosophila), which stimulates tracheole branching via the FGF (Branchless (Bnl))-FGFR (Breathless (Btl)) signalling cascade. Bnl-Btl signalling is required in the intestinal epithelium and the trachea for efficient damage-induced tracheal remodelling and ISC proliferation. Chemical or Pseudomonas-generated reactive oxygen species directly affect the trachea and are necessary for branching and intestinal regeneration. Similarly, tracheole branching and the resulting increase in oxygenation are essential for intestinal tumour growth. We have identified a mechanism of tracheal-intestinal tissue communication, whereby damage and tumours induce neo-tracheogenesis in Drosophila, a process reminiscent of cancer-induced neoangiogenesis in mammals.


Assuntos
Transformação Celular Neoplásica/metabolismo , Hipóxia/metabolismo , Oxigênio/metabolismo , Regeneração/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/genética
12.
Methods Mol Biol ; 2272: 281-318, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009621

RESUMO

5-methylcytosine (5mC) is a gene-regulatory mark associated with transcriptional repression. 5mC can be erased through the catalytic action of Ten-eleven translocation (TET) methylcytosine dioxygenases (TET1, TET2, TET3), which oxidize 5mC resulting in its removal from the genome. In vertebrates, TET enzymes facilitate DNA demethylation of regulatory regions linked to genes involved in developmental processes. Consequently, TET ablation leads to severe morphological defects and developmental arrest. Here we describe a system that can facilitate the study of relationships between TET enzymes, 5mC, and embryo development. We provide detailed descriptions for the generation of F0 zebrafish tet1/2/3 knockouts using CRISPR/Cas9 technology and elaborate on the strategies to assess the impact of TET loss by reduced representation bisulfite sequencing (RRBS).


Assuntos
Animais Geneticamente Modificados/metabolismo , Metilação de DNA , Dioxigenases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Dioxigenases/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
13.
J Med Chem ; 64(11): 7422-7433, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34048659

RESUMO

Neurotoxic aggregation of ß-amyloid (Aß) peptides is a hallmark of Alzheimer's disease and increased reactive oxygen species (ROS) is an associated process. In the present study, we report the neuroprotective effects of disulfide-rich, circular peptides from Clitoria ternatea (C. ternatea) (butterfly pea) on Aß-induced toxicity in transgenic Caenorhabditis elegans. Cyclotides (∼30 amino acids long) are a special class of cyclic cysteine knot peptides. We show that cyclotide-rich fractions from different plant tissues delay Aß-induced paralysis in the transgenic CL4176 strain expressing the human muscle-specific Aß1-42 gene. They also improved Aß-induced chemotaxis defects in CL2355 strain expressing Aß1-42 in the neuronal cells. ROS assay suggests that this protection is likely mediated by the inhibition of Aß oligomerization. Furthermore, Aß deposits were reduced in the CL2006 strain treated with the fractions. The study shows that cyclotides from C. ternatea could be a source of a novel pharmacophore scaffold against neurodegenerative diseases.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Caenorhabditis elegans/metabolismo , Clitoria/metabolismo , Dissulfetos/química , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Animais , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Caenorhabditis elegans/fisiologia , Quimiotaxia/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Fármacos Neuroprotetores/uso terapêutico , Paralisia/tratamento farmacológico , Paralisia/patologia , Fragmentos de Peptídeos/genética , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
Aquat Toxicol ; 234: 105813, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33812311

RESUMO

The production of large amounts of synthetic industrial and biomedical compounds, together with environmental pollutants, poses a risk to our ecosystem and induces negative effects on the health of wildlife and human beings. With the emergence of the global problem of chemical contamination, the adverse biological effects of these chemicals are gaining attention among the scientific communities, industry, governments, and the public. Among these chemicals, endocrine disrupting chemicals (EDCs) are regarded as one of the major global issues that potentially affecting our health. There is an urgent need of understanding the potential hazards of such chemicals. Zebrafish have been widely used in the aquatic toxicology. In this review, we first discuss the strategy of transgenic lines that used in the toxicological studies, followed by summarizing the current omics approaches (transcriptomics, proteomics, metabolomics, and epigenomics) on toxicities of EDCs in this model. We will also discuss the possible transgenerational effects in zebrafish and future prospective of the integrated omics approaches with customized transgenic organism. To conclude, we summarize the current findings in the field, and provide our opinions on future environmental toxicity research in the zebrafish model.


Assuntos
Epigenômica , Metabolômica , Proteômica , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Ecossistema , Disruptores Endócrinos/toxicidade , Modelos Animais , Peixe-Zebra/crescimento & desenvolvimento
15.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800168

RESUMO

Transgenic silkworm expression systems have been applied for producing various recombinant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a P25 gene knockout silkworm. The hEGF gene regulated by the P25 gene promoter was integrated into a silkworm's genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a P25 gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a P25 gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, P25 gene knockout silkworms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials.


Assuntos
Animais Geneticamente Modificados , Bombyx , Fator de Crescimento Epidérmico , Fibroínas/genética , Técnicas de Silenciamento de Genes , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bombyx/genética , Bombyx/metabolismo , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Biochim Biophys Acta Gene Regul Mech ; 1864(6-7): 194704, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895310

RESUMO

BACKGROUND: RNA interference (RNAi) is a cellular mechanism used to fight various threats, including transposons, aberrant RNAs, and some types of viruses. This mechanism relies on the detection of dsRNA molecules, which through a pathway involving Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), produces small interfering RNAs (siRNAs) that bind to the complementary RNAs triggering their degradation. METHODS: Using the cockroach Blattella germanica as a model, we examined AGO2 activity by depleting its mRNA using RNAi and analyzing the phenotypes produced. RESULTS: Depleting AGO2 expression had no remarkable effect on nymphal development or reproduction. dsRNA treatment triggered an immediate and transitory increase in AGO2 expression, independently of Dcr-2 action. In addition, we analyzed the siRNAs generated after injecting a heterologous dsRNA in control and AGO2-depleted animals. The results revealed that obtained siRNAs mapped non-uniformly along the dsRNA sequence. In AGO2-depleted animals, the proportion of 22 nucleotide reads was higher and accumulations of reads appeared in areas less well-represented in the controls. We also detected a preference for cytosine as the first nucleotide in controls that was significantly attenuated in AGO2-depleted individuals. CONCLUSIONS/GENERAL SIGNIFICANCE: The siRNAs produced from a dsRNA mapped heterogeneously along the length of the dsRNA and this arrangement depends on the dsRNA sequence. AGO2 exerts its role as nuclease on the siRNA duplexes independently of its action on the corresponding mRNA. This study sheds light on an extremely useful process for reverse genetics in laboratories, in addition to the design of more effective, specific, and eco-friendly pest-control strategies.


Assuntos
Animais Geneticamente Modificados , Proteínas Argonauta/deficiência , Blattellidae , Inativação Gênica , Proteínas de Insetos/deficiência , RNA Interferente Pequeno , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas Argonauta/genética , Blattellidae/genética , Blattellidae/metabolismo , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
17.
Cells ; 10(2)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669308

RESUMO

Parkinson's disease (PD) is the second most common neurodegenerative disease, afflicting ~10 million people worldwide. Although several genes linked to PD are currently identified, PD remains primarily an idiopathic disorder. Neuronal protein α-synuclein is a major player in disease progression of both genetic and idiopathic forms of PD. However, it cannot alone explain underlying pathological processes. Recent studies demonstrate that many other risk factors can accelerate or further worsen brain dysfunction in PD patients. Several PD models, including non-mammalian eukaryotic organisms, have been developed to identify and characterize these factors. This review discusses recent findings in three PD model organisms, i.e., yeast, Drosophila, and Caenorhabditis elegans, that opened new mechanisms and identified novel contributors to this disorder. These non-mammalian models share many conserved molecular pathways and cellular processes with humans. New players affecting PD pathogenesis include previously unknown genes/proteins, novel signaling pathways, and low molecular weight substances. These findings might respond to the urgent need to discover novel drug targets for PD treatment and new biomarkers for early diagnostics of this disease. Since the study of neurodegeneration using simple eukaryotic organisms brought a huge amount of information, we include only the most recent or the most important relevant data.


Assuntos
Animais Geneticamente Modificados/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Animais , Animais Geneticamente Modificados/genética , Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Humanos
18.
Aquat Toxicol ; 234: 105786, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33735685

RESUMO

The aryl hydrocarbon receptor (AHR) has endogenous functions in mammalian vascular development and is necessary for mediating the toxic effects of a number of environmental contaminants. Studies in mice have demonstrated that AHR is necessary for the formation of the renal, retinal, and hepatic vasculature. In fish, exposure to the prototypic AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of the AHR biomarker cyp1a throughout the developing vasculature and produces vascular malformations in the head and heart. However, it is not known whether the vascular structures that are sensitive to loss of AHR function are also disrupted by aberrant AHR activation. Here, we report that TCDD-exposure in zebrafish disrupts development of 1) the subintestinal venous plexus (SIVP), which vascularizes the developing liver, kidney, gut, and pancreas, and 2) the superficial annular vessel (SAV), an essential component of the retinal vasculature. Furthermore, we determined that TCDD exposure increased the expression of bmp4, a key molecular mediator of SIVP morphogenesis. We hypothesize that the observed SIVP phenotypes contribute to one of the hallmarks of TCDD exposure in fish - the failure of the yolk sac to absorb. Together, our data describe novel TCDD-induced vascular phenotypes and provide molecular insight into critical factors producing the observed vascular malformations.


Assuntos
Dibenzodioxinas Policloradas/toxicidade , Veia Retiniana/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fígado/irrigação sanguínea , Veia Retiniana/crescimento & desenvolvimento , Veias/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
Nat Biomed Eng ; 5(8): 805-814, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33782573

RESUMO

Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.


Assuntos
Animais Geneticamente Modificados/metabolismo , Músculo Esquelético/metabolismo , Proteína MyoD/genética , Fator Regulador Miogênico 5/genética , Fatores de Regulação Miogênica/genética , Animais , Blastômeros/citologia , Blastômeros/metabolismo , Linhagem da Célula , Reprogramação Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/deficiência , Fatores de Regulação Miogênica/metabolismo , Suínos , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
20.
Cell ; 184(7): 1757-1774.e14, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33761328

RESUMO

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.


Assuntos
Granuloma/patologia , Imunidade/fisiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Células Epitelioides/citologia , Células Epitelioides/imunologia , Células Epitelioides/metabolismo , Granuloma/imunologia , Granuloma/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Interferon gama/metabolismo , Interleucina-12/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/isolamento & purificação , Mycobacterium marinum/fisiologia , Necrose , RNA Guia/metabolismo , Receptores de Interleucina-4/antagonistas & inibidores , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/metabolismo , Fator de Transcrição STAT6/antagonistas & inibidores , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
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