RESUMO
Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms. We recently described the prediction of some members of the large-pore channel family in kinetoplastids, consisting of proteins called unnexins, which share several structural features with innexin and pannexin proteins. Here, we demonstrated that the unnexin1 protein (Unx1) is delivered to the cell membrane, displaying a topology consisting of four transmembrane domains with C and N termini on the cytoplasmic side and form large-pore channels that are permeable to small molecules. Low extracellular Ca2+/Mg2+ levels or extracellular alkalinization, but not mechanical stretching, increases channel activity. The Unx1 channel mediates the influx of Ca2+ and does not form intercellular dye coupling between HeLa Unx1 transfected cells. Unx1 channel function was further evidenced by its ability to mediate ionic currents when expressed in Xenopus oocytes. Downregulation of Unx1 mRNA with morpholine contains Trypanosoma cruzi invasion. Phylogenetic analysis revealed the presence of Unx1 homologs in other protozoan parasites, suggesting a conserved function for these channel parasites in other protists. Our data demonstrate that Unx1 forms large-pore membrane channels, which may serve as a diffusional pathway for ions and small molecules that are likely to be metabolic substrates or waste products, and signaling autocrine and paracrine molecules that could be involved in cell invasion. As morpholinos-induced downregulation of Unx1 reduces the infectivity of trypomastigotes, the Unx1 channels might be an attractive target for developing trypanocide drugs.
Assuntos
Subunidades Proteicas , Filogenia , Membrana Celular , Citoplasma , MorfolinosRESUMO
CONDIÇÃO CLÍNICA: A distrofia muscular de Duchenne (DMD) faz parte do espectro das distrofinopatias. As distrofinopatias são um grupo de doenças neuromusculares com herança ligada ao cromossomo X, causadas por mutações no gene que codifica a distrofina (DMD), uma proteína estrutural das células musculares. Em quadros leves das distrofinopatias pode-se notar o aumento assintomático da concentração sérica de creatina fosfoquinase (CK), cãibras musculares e mioglobinúria. Enquanto nos casos graves estão as doenças musculares progressivas, especialmente DMD. A distrofia muscular de Becker, apesar de também ser progressiva e grave, é uma forma de distrofinopatia mais branda comparada à DMD. Ela é caracterizada por fraqueza muscular esquelética de início tardio com gravidade e/ou curso variáveis. TRATAMENTO: Não há cura para a DMD, e as intervenções são baseadas na prevenção e tratamento das complicações. Atualmente, a terapia padrão geralmente é baseada em corticosteróides. Os corticosteróides são capazes de retardar o declínio da força e da função muscular, proporcionando uma redução do risco de escoliose e estabilização da função pulmonar. Pode haver benefícios para a função cardíaca, embora as evidências sejam limitadas. De acordo com uma revisão sistemática Cochrane não há consenso sobre a idade ou estágio funcional para o início do tratamento com corticosteroides sendo necessários mais estudos para determinar as condições ideias para iniciá-lo, ainda que haja autores que defendam que deveria ocorrer antes do declínio físico substancial, normalmente aos 5 anos de idade. .ESTRATÉGIA DE BUSCA: Uma busca foi realizada no banco de dados eletrônico ClinicalTrials.gov, em 14 de julho de 2021. O termo empregado foi: "Duchenne Muscular Dystrophy". Foram considerados os estudos dos medicamentos a partir da fase 1/2 de pesquisa clínica, com a DMD como alvo e sem registro para essa indicação terapêutica no Brasil. Dos 314 cadastros de estudos, 157 atendiam ao critério de elegibilidade referente à fase da pesquisa. Destes, apenas os estudos em andamento ou concluídos nos últimos 5 anos foram selecionados, restando 79 protocolos. A fim de mapear os resultados publicados para esses ensaios clínicos, utilizaram-se os códigos de registro do ClinicalTrials.gov nas bases de dados MEDLINE (via PubMed), EMBASE e Google Acadêmico. Na ausência de resultados, buscas complementares foram feitas com o nome do medicamento associado à indicação. Pipelines de desenvolvimento de medicamentos para DMD também foram revisados. Por fim, os sítios eletrônicos da Agência Nacional de Vigilância Sanitária (Anvisa, Food and Drug Administration (FDA, EUA) e European Medicines Agency (EMA) foram consultadas com o intuito de verificar os registros. O levantamento de resultados publicados e do registro dos medicamentos aconteceu entre 14 de junho e 17 de julho de 2021. MEDICAMENTOS: Ainda assim, os avanços na tecnologia devem ser reconhecidos. De 1998 a 2021, treze oligonucleotídeos antisense foram aprovados pela FDA, sendo quatro deles para DMD. Dos treze, dez receberam aprovação a partir de 2016. Esses resultados, somados aos ensaios clínicos em andamento e aos esforços para amadurecimento das tecnologias, consolidam a perspectiva desta abordagem terapêutica no tratamento da DMD. Por fim, é importante destacar que o pequeno número de pacientes incluídos nos estudos de doenças raras é agravado no caso das estratégias terapêuticas baseadas no genótipo. No caso da DMD, cada uma das tecnologias incluídas neste informe aplica-se a subgrupos com 8-15% do total de pacientes com a doença. INFORMAÇÕES ADICIONAIS: Uma série de tratamentos emergentes para DMD foram identificados durante o mapeamento de tecnologias para este informe, incluindo terapias com oligonucleotídeos antisense, substituição de genes mediada por vírus adeno-associado recombinante (AAVr) e diversos medicamentos para o tratamento sintomático da doença. No entanto, a maior parte encontra-se nas primeiras fases de desenvolvimento, não apresenta resultados publicados ou apenas resultados preliminares foram divulgados. Diante deste cenário, este informe abordou exclusivamente os medicamentos voltados à causa-base da doença e que foram recém-registrados nas agências FDA e/ou EMA. CONSIDERAÇÕES FINAIS: Os quatro oligômeros fosforodiamidato morfolino que receberam aprovação acelerada da FDA para o tratamento de DMD apresentaram aumento da produção de distrofina e parecem ser bem tolerados. No entanto, conforme previsto no Programa de Aprovação Acelerada da FDA, a continuação das aprovações depende da verificação de um benefício clínico em ensaios de confirmação. O eteplirsen e o golodirsen são os únicos oligômeros fosforodiamidato morfolino que apresentam avaliações publicadas acerca do seu efeito funcional. Até a última atualização deste informe, os benefícios clínicos desses medicamentos não estão bem estabelecidos, uma vez que houve declínio na deambulação e função pulmonar dos pacientes tratados, ainda que a maior parte dos valores tenham sido inferiores àqueles encontrados nos controles e na história natural publicada, especialmente para TC6. Para que ocorra a oferta desses medicamentos no SUS, é necessária a análise pela Conitec, conforme disposto na Lei nº 12.401/2011, que alterou a Lei nº 8.080/1990. Os relatórios de recomendação da Conitec levam em consideração as evidências científicas sobre eficácia, a acurácia, a efetividade e a segurança do medicamento, e, também, a avaliação econômica comparativa dos benefícios e dos custos em relação às tecnologias já incorporadas e o impacto da incorporação da tecnologia no SUS.
Assuntos
Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Morfolinos/uso terapêutico , Brasil , Eficácia , Análise Custo-Benefício/economia , Projetos de Desenvolvimento Tecnológico e InovaçãoRESUMO
Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the elongation of fatty acids facilitates the formation of complex lipids. The aim of this work was to investigate the involvement of the elongation of very long chain fatty acid enzyme 5 (ELOVL5) in embryonic development and lipid determination. Bovine embryos were produced in vitro using a standard protocol and randomly divided to receive one of three treatments at Day 4: morpholino (Mo) gene expression knockdown assay for ELOVL5 (ELOVL5-Mo), Mo antisense oligonucleotides for the thalassemic ß-globulin human mRNA (technical control Mo), and placebo (biological control). The phenotypes of embryonic development, cell number, ELOVL5 protein abundance, lipid droplet deposits, and lipid fingerprint were investigated. No detrimental effects (p > 0.05) were observed on embryo development in terms of cleavage (59.4 ± 3.5%, 63.6 ± 4.1%, and 65.4 ± 2.2%), blastocyst production (31.3 ± 4.2%, 28.1 ± 4.9%, and 36.1 ± 2.1%), and blastocyst cell number (99.6 ± 7.7, 100.2 ± 6.2, 86.8 ± 5.6), respectively, for biological control, technical control Mo, and ELOVL5-Mo. ELOVL5 protein abundance and cytoplasmic lipid droplet deposition were increased (p < 0.05) in ELOVL5-Mo-derived blastocysts compared with the controls. However, seven lipid species, including phosphatidylcholines, phosphatidylethanolamines, and triacylglycerol, were downregulated in the ELOVL5-Mo-derived blastocysts compared with the biological control. Therefore, ELOVL5 is involved in the determination of embryonic lipid content and composition. Transient translational blockage of ELOVL5 reduced the expression of specific lipid species and promoted increased cytoplasmic lipid droplet deposition, but with no apparent deleterious effect on embryonic development and blastocyst cell number.
Assuntos
Blastocisto/metabolismo , Membrana Celular/química , Citoplasma/química , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Animais , Blastocisto/química , Bovinos , Desenvolvimento Embrionário , Elongases de Ácidos Graxos/antagonistas & inibidores , Feminino , Técnicas de Silenciamento de Genes , Humanos , Metabolismo dos Lipídeos , Morfolinos/farmacologia , Gravidez , Globinas beta/antagonistas & inibidores , Globinas beta/genéticaRESUMO
Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.
Assuntos
Characidae/genética , Edição de Genes , Animais , Evolução Biológica , Cavernas , Feminino , Masculino , Morfolinos , FenótipoRESUMO
The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.
Assuntos
Hemangioblastos/metabolismo , Proteínas de Membrana/genética , Peixe-Zebra/embriologia , Animais , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Desenvolvimento Embrionário , Hematopoese , Morfolinos/farmacologia , Família Multigênica , Peixe-Zebra/sangue , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Treacher Collins Syndrome (TCS) is a congenital disease characterized by defects in the craniofacial skeleton and absence of mental alterations. Recently we modelled TCS in zebrafish (Danio rerio) embryos through the microinjection of Morpholino® oligonucleotides blocking the translation of the ortholog of the main causative gene (TCOF1). We showed that Cnbp, a key cytoprotective protein involved in normal rostral head development, was detected in lower levels (without changes in its mRNA expression) in TCS-like embryos. As previous reports suggested that Cnbp is degraded through the proteasomal pathway, we tested whether proteasome inhibitors (MG132 and Bortezomib (Velcade®, Millennium laboratories)) were able to ameliorate cranial skeleton malformations in TCS. Here we show that treatment with both proteasome inhibitors produced a robust craniofacial cartilage phenotype recovery. This recovery seems to be consequence of a decreased degradation of Cnbp in TCS-like embryos. Critical TCS manifestations, such as neuroepithelial cell death and cell redox imbalance were attenuated. Thus, proteasome inhibitors may offer an opportunity for TCS molecular and phenotypic manifestation's prevention. Although further development of new safe inhibitors compatible with administration during pregnancy is required, our results encourage this therapeutic approach.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Disostose Mandibulofacial/metabolismo , Morfolinos/efeitos adversos , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Disostose Mandibulofacial/patologia , Fosfoproteínas/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Ric-8A is a pleiotropic guanine nucleotide exchange factor involved in the activation of various heterotrimeric G-protein pathways during adulthood and early development. Here, we sought to determine the downstream effectors of Ric-8A during the migration of the vertebrate cranial neural crest (NC) cells. We show that the Gα13 knockdown phenocopies the Ric-8A morphant condition, causing actin cytoskeleton alteration, protrusion instability, and a strong reduction in the number and dynamics of focal adhesions. In addition, the overexpression of Gα13 is sufficient to rescue Ric-8A-depleted cells. Ric-8A and Gα13 physically interact and colocalize in protrusions of the cells leading edge. The focal adhesion kinase FAK colocalizes and interacts with the endogenous Gα13, and a constitutively active form of Src efficiently rescues the Gα13 morphant phenotype in NC cells. We propose that Ric-8A-mediated Gα13 signalling is required for proper cranial NC cell migration by regulating focal adhesion dynamics and protrusion formation.
Assuntos
Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Crista Neural/citologia , Transdução de Sinais , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Adesões Focais/efeitos dos fármacos , Modelos Biológicos , Morfolinos/farmacologia , Crista Neural/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Xenopus/embriologia , Quinases da Família src/metabolismoRESUMO
OBJECTIVE: To describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen. METHODS: Clinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen. RESULTS: Reverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001). CONCLUSION: Taken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.
Assuntos
Distrofina/biossíntese , Éxons/genética , Morfolinos/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Biópsia , Criança , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Resultado do TratamentoRESUMO
Adenosinergic signaling has important effects on brain function, anatomy, and physiology in both late and early stages of development. Exposure to caffeine, a non-specific blocker of adenosine receptor, has been indicated as a developmental risk factor. Disruption of adenosinergic signaling during early stages of development can change the normal neural network formation and possibly lead to an increase in susceptibility to seizures. In this work, morpholinos (MO) temporarily blocked the translation of adenosine receptor transcripts, adora1, adora2aa, and adora2ab, during the embryonic phase of zebrafish. It was observed that the block of adora2aa and adora2aa + adora2ab transcripts increased the mortality rate and caused high rate of malformations. To test the susceptibility of MO adora1, MO adora2aa, MO adora2ab, and MO adora2aa + adora2ab animals to seizure, pentylenetetrazole (10 mM) was used as a convulsant agent in larval and adult stages of zebrafish development. Although no MO promoted significant differences in latency time to reach the seizures stages in 7-day-old larvae, during the adult stage, all MO animals showed a decrease in the latency time to reach stages III, IV, and V of seizure. These results indicated that transient interventions in the adenosinergic signaling through high affinity adenosine receptors during embryonic development promote strong outcomes on survival and morphology. Additionally, long-term effects on neural development can lead to permanent impairment on neural signaling resulting in increased susceptibility to seizure.