RESUMO
⢠The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. ⢠We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. ⢠We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. ⢠Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.
Assuntos
Glomeromycota/genética , Micorrizas/genética , Simbiose/genética , Transcriptoma/genética , Sequência de Bases , Contagem de Colônia Microbiana , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Genes Fúngicos/genética , Glomeromycota/crescimento & desenvolvimento , Meiose/genética , Micélio/genética , Micorrizas/crescimento & desenvolvimento , Plantas/microbiologia , Polimorfismo de Nucleotídeo Único/genética , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
After more than 1500 gene therapy clinical trials in the past two decades, the overall conclusion is that for gene therapy (GT) to be successful, the vector systems must still be improved in terms of delivery, expression and safety. The recent development of more efficient and stable vector systems has created great expectations for the future of GT. Impressive results were obtained in three primary immunodeficiencies and other inherited diseases such as congenital blindness, adrenoleukodystrophy or junctional epidermolysis bullosa. However, the development of leukemia in five children included in the GT clinical trials for X-linked severe combined immunodeficiency and the silencing of the therapeutic gene in the chronic granulomatous disease clearly showed the importance of improving safety and efficiency. In this review, we focus on the main strategies available to achieve physiological or tissue-specific expression of therapeutic transgenes and discuss the importance of controlling transgene expression to improve safety. We propose that tissue-specific and/or physiological viral vectors offer the best balance between efficiency and safety and will be the tools of choice for future clinical trials in GT of inherited diseases.
Assuntos
Doenças Genéticas Inatas/terapia , Terapia Genética/métodos , Vetores Genéticos , Endotélio , Expressão Gênica , Marcação de Genes , Humanos , Fígado , MicroRNAs , Especificidade de Órgãos , Reparo Gênico Alvo-Dirigido/métodos , TransgenesRESUMO
BACKGROUND AND PURPOSE: Recombinant IFN-ß is one of the first-line treatments in multiple sclerosis (MS), despite its lack of efficacy in some patients. In this context, mesenchymal stem cells (MSCs) represent a promising therapeutic alternative due to their immunomodulatory properties and multipotency. Moreover, by taking advantage of their pathotropism, these cells can be genetically modified to be used as carriers for delivering or secreting therapeutic drugs into injured tissues. Here, we report the therapeutic effect of systemic delivery of adipose-derived MSCs (AdMSCs), transduced with the IFN-ß gene, into mice with experimental autoimmune encephalomyelitis (EAE). EXPERIMENTAL APPROACH: Relapsing-remitting and chronic progressive EAE were induced in mice. Cells were injected i.v. Disease severity, inflammation and tissue damage were assessed clinically, by flow cytometry of spleens and histopathological evaluation of the CNS respectively. KEY RESULTS: Genetic engineering did not modify the biological characteristics of these AdMSCs (morphology, growth rate, immunophenotype and multipotency). Furthermore, the transduction of IFN-ß to AdMSCs maintained and, in some cases, enhanced the functional properties of AdMSCs by ameliorating the symptoms of MS in EAE models and by decreasing indications of peripheral and central neuro-inflammation. CONCLUSION AND IMPLICATIONS: Gene therapy was found to be more effective than cell therapy in ameliorating several clinical parameters in both EAE models, presumably due to the continuous expression of IFN-ß. Furthermore, it has significant advantages over AdMSC therapy, and also over systemic IFN-ß treatment, by providing long-term expression of the cytokine at therapeutic concentrations and reducing the frequency of injections, while minimizing dose-limiting side effects.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Terapia Genética/métodos , Interferon beta/genética , Transplante de Células-Tronco Mesenquimais/métodos , Esclerose Múltipla Recidivante-Remitente/terapia , Tecido Adiposo/citologia , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Citometria de Fluxo , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla Crônica Progressiva/genética , Esclerose Múltipla Crônica Progressiva/fisiopatologia , Esclerose Múltipla Crônica Progressiva/terapia , Esclerose Múltipla Recidivante-Remitente/genética , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Índice de Gravidade de DoençaRESUMO
Changes induced by arbuscular mycorrhizal (AM) formation in the plasma membrane polypeptide pattern of tomato roots have been assessed by 2D-PAGE analysis. Plasma membrane fractions were isolated by aqueous two-phase partitioning from control and mycorrhizal tomato root microsomes. Analysis of 2D-PAGE gels revealed that AM colonization induces at the plasma membrane level two major changes in protein synthesis: down-regulation of some constitutive polypeptides and synthesis of new polypeptides or endomycorrhizins. A comparison of changes induced by two different levels of AM colonization showed that 16 polypeptides were differentially displayed at both AM colonization stages, while some others were transiently regulated. Five of the differentially displayed plasma membrane polypeptides at both AM colonization stages were selected for N-terminal amino acid sequencing. Reliable sequences were obtained for two of the selected spots. Sequence alignment search indicated that one of the sequenced polypeptides showed 75% identity to the N-terminal sequence of the 69 kDa catalytic subunit of the vacuolar type H(+)-ATPase of several plants. The possible significance of these findings is discussed in relation to the functioning of the AM symbiosis.