RESUMO
The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.
Assuntos
Técnicas de Cultura de Células/métodos , Endotélio Corneano/citologia , Adolescente , Adulto , Movimento Celular , Proliferação de Células , Senescência Celular , Criança , Pré-Escolar , Biologia Computacional , Transplante de Córnea , Feminino , Humanos , Masculino , Fenótipo , Transdução de Sinais , Transcriptoma , Adulto JovemRESUMO
The zinc finger e-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT), and of the reverse mesenchymal to epithelial transition (MET) processes. ZEB1 plays an integral role in mediating cell state transitions during cell lineage specification, wound healing and disease. EMT/MET are characterized by distinct changes in molecular and cellular phenotype that are generally context-independent. Posterior polymorphous corneal dystrophy (PPCD), associated with ZEB1 insufficiency, provides a new biological context in which to understand and evaluate the classic EMT/MET paradigm. PPCD is characterized by a cadherin-switch and transition to an epithelial-like transcriptomic and cellular phenotype, which we study in a cell-based model of PPCD generated using CRISPR-Cas9-mediated ZEB1 knockout in corneal endothelial cells (CEnCs). Transcriptomic and functional studies support the hypothesis that CEnC undergo a MET-like transition in PPCD, termed endothelial to epithelial transition (EnET), and lead to the conclusion that EnET may be considered a corollary to the classic EMT/MET paradigm.
Assuntos
Endotélio Corneano/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Córnea/metabolismo , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição/metabolismo , Transcriptoma , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Unbiased genome-wide screens combined with imaging data on brain function may identify novel molecular pathways related to human cognition. Here we performed a dense genome-wide screen to identify episodic memory-related gene variants. A genomic locus encoding the brain-expressed beta-catenin-like protein 1 (CTNNBL1) was significantly (P=7 × 10(-8)) associated with verbal memory performance in a cognitively healthy cohort from Switzerland (n=1073) and was replicated in a second cohort from Serbia (n=524; P=0.003). Gene expression studies showed CTNNBL1 genotype-dependent differences in beta-catenin-like protein 1 mRNA levels in the human cortex. Functional magnetic resonance imaging in 322 subjects detected CTNNBL1 genotype-dependent differences in memory-related brain activations. Converging evidence from independent experiments and different methodological approaches suggests a role for CTNNBL1 in human memory.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Encéfalo/irrigação sanguínea , Encéfalo/fisiologia , Expressão Gênica/genética , Memória/fisiologia , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Reguladoras de Apoptose/metabolismo , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Proteínas Nucleares/metabolismo , Oxigênio/sangue , RNA Mensageiro/metabolismo , Sérvia , Suíça , Aprendizagem Verbal/fisiologiaRESUMO
Pyrantel embonate and febantel are both constituents of Drontal Plus and Drontal Puppy broad spectrum anthelmintics for dogs. The effects of pyrantel and the febantel metabolite fenbendazole were investigated against Toxocara canis in-vitro by studying changes in worm motility and tissue damage. Pyrantel and fenbendazole were added to worms incubated in media for 8 h at the following concentrations: pyrantel: 12.2 microg, 25 microg, or 50 microg; fenbendazole: 50 microg, 100 microg or 200 microg; mixture of pyrantel and fenbendazole: 12.2 microg p + 50 microg f, 25 microg p + 100 microg f, 50 microg p + 200 microg f. Following this 8 h incubation period, one group of the worms was immediately fixed and studied by light- and electron microscopical examination. Other groups have been observed for further 8 h periods up to 56 hours and then studied in the same way.
