Genic silencing by RNA in Moniliophthora perniciosa

Orientadores: Michel Georges Albert Vincentz, Júlio Cézar de Mattos CascardoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O fungo Moniliophthora perniciosa, agente causal da doença "vassoura-de-bruxa" (VB) do cacaueiro (Theobroma cacao L.) é responsável pela diminuição da produção de cacau no Brasil e em outras regiões da América do Sul e Central. Esse decréscimo está diretamente associado a graves problemas sociais e ambientais, particularmente nas regiões amazônica e Sul da Bahia. A interação cacau-M. perniciosa envolve mecanismos genéticos complexos e pouco estudados em nível molecular. Portanto, este trabalho objetivou estabelecer uma metodologia de silenciamento gênico por RNA de interferência (RNAi) em M. perniciosa, visando análises funcionais de genes relacionados à sua patogenicidade. RNAi é uma eficiente ferramenta para reprimir a expressão gênica experimentalmente, portanto, uma alternativa tecnológica muito promissora. Devido ao fato de RNAi ser um mecanismo pós-transcricional que promove o silenciamento de genes com alta especificidade e de modo sistêmico é considerado uma ferramenta versátil para o estudo de fungos filamentosos heterocarióticos. O trabalho foi conduzido em três etapas integradas: 1) estabelecimento e avaliação das condições adequadas para eletroporação e regeneração de protoplastos de M. perniciosa, como método alternativo para a transfecção de DNA e de dsRNAs nos experimentos de transformação e de silenciamento do fungo, respectivamente; 2) produção de uma linhagem transgênica de M. perniciosa que expresse o gene heterólogo gfp e o estabelecimento da metodologia de silenciamento por RNAi, usando o gene gfp como modelo; 3) silenciamento mediado por dsRNAs de dois genes endógenos de M. perniciosa, vinculados a detoxificação de ROS e a produção de corpos de frutificação. Os parâmetros que promoveram a maior freqüência de regeneração dos protoplastos eletroporados foram a aplicação de um pulso de 1.5 kV. Em relação à transformação de M. perniciosa, o gene repórter gfp foi estavelmente introduzido no genoma do fungo e posteriormente silenciado por transfecção de gfpdsRNA sintetizado in vitro, comprovando a operacionalidade do mecanismo de RNAi em M. perniciosa. Em adição, os genes endógenos MpPRX1 e MpHYD que codificam para peroxiredoxina e hidrofobina, respectivamente, foram silenciados, usando dsRNAs específicos e apresentaram níveis reduzidos de mRNAs que variaram de 18% a 98% quando comparados aos controles. O silenciamento dos genes gfp e MpPRX1 foi corroborado pela redução da fluorescência de GFP e da atividade da peroxidase, bem como da sobrevivência do micélio submetido a H2O2, respectivamente. O silenciamento de gfp e de MpPRX1 persistiu por aproximadamente quatro meses, indicando silenciamento sistêmico. O estabelecimento da técnica de silenciamento gênico por RNAi em M. perniciosa abre novos caminhos para explorar funcionalmente o genoma deste fitopatógeno, bem como o desenvolvimento de mecanismos que bloqueiem ou diminuam a ação desteAbstract: The fungus Moniliophthora perniciosa, causal agent of cacao (Theobroma cacao L.) witches' broom (WB) disease, is responsible for the decrease in cacao production in Brazil and other regions of South and Central America. This decrease is directly associated to severe social and ecological problems, particularly in the Amazon and Southern Bahia regions. The cacao-M. perniciosa interaction involves complex genetic mechanisms little studied at the molecular level. Therefore, this study aimed to establish a methodology of gene silencing by RNA interference (RNAi) in M. perniciosa, aiming functional analyses of genes related to his pathogenicity. RNAi is a powerful tool to experimentally suppress or regulate gene expression, therefore, a very promising technological alternative. Because RNAi is a post-transcriptional event that promotes gene silencing with high specificity and in a systemic way, it is considered a versatile tool for the study of heterokaryotic fungi. The work was conducted in three integrated steps: 1) establishment and evaluation of appropriate conditions for electroporation and regeneration of M. perniciosa protoplasts, as an alternative method for transfection of transgenic DNA and dsRNAs in transformation and fungus silencing experiments, respectively; 2) production of a transgenic strain of M. perniciosa that expresses the heterologous gene gfp and the establishment of methodology for silencing by RNAi, using the gfp gene as a model; 3) silencing mediated by dsRNAs of two endogenous M. perniciosa genes linked to detoxification of ROS and the production of fruiting bodies. The parameters that promoted the highest frequency of regeneration of electroporated protoplasts were the application of one pulse of 1.5 kV. Regarding M. perniciosa transformation, the gfp reporter gene was stably inserted into the genome of the fungus and subsequently silenced by transfection of gfpdsRNA synthesized in vitro, demonstrating the operationality of the RNAi mechanism in M. perniciosa. In addition, the endogenous genes MpPRX1 and MpHYD, coding for peroxiredoxin and hydrophobin, respectively, were silenced using specific dsRNAs showing reduced levels of mRNAs ranging from 18% to 98% when compared to controls. Silencing of gfp and MpPRX1 was validated by experiments that showed correlation between reduced levels of GFP fluorescence and peroxidase activity, as well as survival of mycelium subjected to H2O2, respectively. The silencing of gfp and MpPRX1 persisted for approximately four months, indicating systemic silencing. The establishment of the gene silencing technique by RNAi in M. perniciosa opens new paths to functionally explore the genome of this pathogen and the development of mechanisms that block or decrease his actionDoutoradoGenetica de MicroorganismosDoutor em Genetica e Biologia Molecula

Algas marinhas bentônicas do municipio de Ilhéus, Bahia, Brasil

In this qualitative survey of benthic algae, were identified 77 laxa distributed into 22 Chlorophyta, 15 Phaeophyta and 40 Rhodophyta. Pterosiphonia pennata (C. Agardh) Falkenberg and Caulerpa taxifolia (H. West in Vahl) C. Agardh were recorded in the first time for Bahia State.Levantamento qualitativo das algas marinhas bentônicas, totalizando 77 táxons distribuídos em 22 Chlorophyta, 15 Phaeophyta e 40 Rhodophyta. Pterosiphonia pennata (C. Agardh) Falkenberg e Caulerpa taxtfolia (H. West in Vahl) C. Agardh constituem-se em novas citações para o Estado da Bahia.

HOXB7 siRNA Delivered by Hybrid Nanoparticles and the Co-Therapy with Tamoxifen: Promising Strategy against Hormone Receptor-Positive Breast Cancer

Breast cancer is the most common type of cancer that affects and kills women annually in the world. It impacts more than two million women and is responsible for the death of approximately 25% of them. Almost 70% of breast cancer diagnoses are positive for hormone receptor and have a good prognosis. However, resistance to drugs used in hormone therapy, such as tamoxifen, is usual and about 40% of recurrences do not respond to it. In some cases, the overexpression of the HOXB7 gene is related to this mechanism and its silencing can reverse the response to tamoxifen. Here, we used copolymer-coated calcium phosphate nanoparticles to deliver HOXB7 siRNA and restore the sensitization of MCF7 cells to tamoxifen. Nanoparticle synthesis and characterization were performed, and cell viability and gene expression were evaluated. Hybrid nanoparticle presented a Z-average diameter of 83 nm and polydispersity index (PdI) of 0.07, while showing good entrapment of siRNA molecules. We also observed a decrease in HOXB7 gene expression (~65%) promoted by the siRNA molecule delivered by the nanoparticles. The gene silencing has good correlation to the cell viability assay: a reduction in breast cancer viability was observed in 48 (31%) and 72 (38%) hours. As for the co-treatment with tamoxifen, cell viability started dropping after 15 h, which did not occur in the treatment only with Tamoxifen at the same concentration. This result indicates that the biological effect was possibly related to RNAi effect and suggests that HOXB7 may be promoting cell sensitization to tamoxifen without reducing cell viability. Overall, these results suggest that the nanostructured system was effective in promoting gene silencing and that the co-therapy can be a promising tool for the treatment of hormone receptor-positive breast cancers

Revista Brasileira de Parasitologia Veterinária

p. 43-48O estudo avaliou a atividade anti-helmíntica do extrato aquoso das folhas de Zanthoxylum rhoifolium em dois experimentos. O teste in vitro utilizou cultivos de fezes de caprinos tratados com diferentes concentrações do extrato:(134,5 a 335,0 mg.mL–1). No teste in vivo, utilizaram-se 20 ovelhas: G1 tratado durante quatro dias (0,63 g.kg–1 PV); G2, mesma dose, por oito dias; G3, ivermectina (200 μg.kg–1); e G4, sem tratamento. In vitro, observou-se redução de larvas de Haemonchus, Trichostrongylus e Oesophagostomum superior a 95% nas concentrações de 193,7 a 335,0 mg.mL–1. In vivo, a redução de ovos por grama de fezes foi de 51, 56 e 90%, respectivamente, para G1, G2 e G3, no oitavo dia de tratamento, enquanto para os estágios imaturos e adultos variou de 0 a 91% no G1 e 26 a 94% no G2. A eficácia da ivermectina alcançou 99% para L4 e L5 de H. contortus e 100% para as demais espécies de nematoides. Parâmetros clínicos e bioquímicos permaneceram na faixa de normalidade, e as análises histopatológicas não revelaram alterações sugestivas de toxicidade. Embora altamente efetivo in vitro, o extrato aquoso de folhas de Z. rhoifolium foi pouco eficaz in vivo na redução de nematoides gastrintestinais

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data