RESUMO
Epigenomics is a fast-evolving field of research that has lately attracted considerable interest, mainly due to the reversibility of epigenetic marks. Clinically, among solid tumors, the field is still limited. In cholangiocarcinoma (CCA) it is well known that the epigenetic landscape is deregulated both during carcinogenesis and disease progression as a consequence of aberrant mechanisms leading to genome instability. In this article, we will briefly review the molecular alterations that have been described in the transformation of normal cholangiocytes into malignant derivatives, focusing on the role of non-coding RNA (ncRNA) interactions, DNA methylation, post-translational modifications (PTMs) of histones and chromatin remodeling complexes.
Assuntos
Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares/citologia , Ductos Biliares/patologia , Transformação Celular Neoplásica/genética , Colangiocarcinoma/patologia , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA/genética , Progressão da Doença , Células Epiteliais/patologia , Instabilidade Genômica , Histonas/genética , Humanos , Processamento de Proteína Pós-Traducional/genética , RNA não Traduzido/genéticaRESUMO
Human rhinovirus type C (HRV-C) is a newly discovered enterovirus species frequently associated with exacerbation of asthma and other acute respiratory conditions. Until recently, HRV-C could not be propagated in vitro, hampering in-depth characterization of the virus replication cycle and preventing efficient testing of antiviral agents. Herein we describe several subgenomic RNA replicon systems and a cell culture infectious model for HRV-C that can be used for antiviral screening. The replicon constructs consist of genome sequences from HRVc15, HRVc11, HRVc24, and HRVc25 strains, with the P1 capsid region replaced by a Renilla luciferase coding sequence. Following transfection of the replicon RNA into HeLa cells, the constructs produced time-dependent increases in luciferase signal that can be inhibited in a dose-dependent manner by known inhibitors of HRV replication, including the 3C protease inhibitor rupintrivir, the nucleoside analog inhibitor MK-0608, and the phosphatidylinositol 4-kinase IIIß (PI4K-IIIß) kinase inhibitor PIK93. Furthermore, with the exception of pleconaril and pirodavir, the other tested classes of HRV inhibitors blocked the replication of full-length HRVc15 and HRVc11 in human airway epithelial cells (HAEs) that were differentiated in the air-liquid interface, exhibiting antiviral activities similar to those observed with HRV-16. In summary, this study is the first comprehensive profiling of multiple classes of antivirals against HRV-C, and the set of newly developed quantitative HRV-C antiviral assays represent indispensable tools for the identification and evaluation of novel panserotype HRV inhibitors.
Assuntos
Antivirais/farmacologia , Rhinovirus/efeitos dos fármacos , Resfriado Comum/tratamento farmacológico , Resfriado Comum/virologia , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Técnicas In Vitro , Isoxazóis/farmacologia , Oxidiazóis/farmacologia , Oxazóis , Fenilalanina/análogos & derivados , Piperidinas/farmacologia , Piridazinas/farmacologia , Pirrolidinonas/farmacologia , RNA Viral/genética , Replicon/efeitos dos fármacos , Rhinovirus/genética , Tubercidina/análogos & derivados , Tubercidina/farmacologia , Valina/análogos & derivados , Replicação Viral/efeitos dos fármacosRESUMO
Influenza polymerase is a heterotrimer protein with both endonuclease and RNA-dependent RNA polymerase (RdRp) activity. It plays a critical role in viral RNA replication and transcription and has been targeted for antiviral drug development. In this study, we characterized the activity of recombinant RdRp purified at 1:1:1 ratio in both ApG-primed RNA replication and mRNA-initiated RNA transcription. The heterotrimer complex showed comparable activity profiles to that of viral particle derived crude replication complex, and in contrast to the crude replication complex, was suitable for detailed mechanistic studies of nucleotide incorporation. The recombinant RdRp was further used to examine distinct modes of inhibition observed with five different nucleotide analog inhibitors, and the apparent steady-state binding affinity Kapp was measured for selected analogs to correlate antiviral activity and enzymatic inhibition with substrate efficiency.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Influenza A/enzimologia , Nucleotídeos/metabolismo , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Animais , Antivirais/farmacologia , Biocatálise/efeitos dos fármacos , Bioensaio , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Cães , Eletroforese em Gel de Ágar , Vírus da Influenza A/efeitos dos fármacos , Concentração Inibidora 50 , Cinética , Células Madin Darby de Rim Canino , Transcrição Gênica/efeitos dos fármacosRESUMO
Mouse embryonic stem cells (ESC) make cell fate decisions based on intrinsic and extrinsic factors. The decision of ESC to differentiate to multiple lineages in vitro occurs during the formation of embryoid bodies (EB) and is influenced by cell-environment interactions. However, molecular mechanisms underlying cell-environmental modulation of ESC fate decisions are incompletely understood. Since adhesion molecules (AM) influence proliferation and differentiation in developing and adult tissues, we hypothesized that specific AM interactions influence ESC commitment toward hematopoietic and endothelial lineages. Expression of AM in the adherens, tight and gap junction pathways in ESC subpopulations were quantified. E-cadherin (E-cad), Claudin-4 (Cldn4), Connexin-43 (Cx43), Zona Occludens-1 (ZO-1) and Zona Occludens-2 (ZO-2) transcript levels were differentially expressed during early stages of hematopoietic/endothelial commitment. Stable ESC lines were generated with reduced expression of E-cad, Cldn4, Cx43, ZO-1 and ZO-2 using shRNA technology. Functional and phenotypic consequences of modulating AM expression were assessed using hematopoietic colony forming assays, endothelial sprouting assays and surface protein expression. A decrease in E-cad, Cldn4, Cx43 and ZO-1 expression was associated with less commitment to the hematopoietic lineage and increased endothelial differentiation as evidenced by functional and phenotypic analysis. A reduction in ZO-2 expression did not influence endothelial differentiation, but decreased hematopoietic commitment two-fold. These data indicate that a subset of AM influence ESC decisions to commit to endothelial and hematopoietic lineages. Furthermore, differentially expressed AM may provide novel markers to delineate early stages of ESC commitment to hematopoietic/endothelial lineages.