RESUMEN

The plant microbiota diversity is often underestimated when approaches developed mainly for the identification of cultivable microorganisms are used. High-throughput sequencing allows a deeper understanding of the microbial diversity associated with plants. The amplification of ITS1 was used to analyze fungal diversity in several plant organs and rhizosphere of three common bean (Phaseolus vulgaris) varieties grown in a greenhouse. The fungal diversity diverged between those plant organs and the rhizosphere, with the highest found in the rhizosphere and the lowest in the stem. In each organ different numbers of genus, OTUs were identified, in a total of 283 OTUs evenly distributed among the varieties. In the co-occurrence network, a larger number of positive interactions were found in the organs of the aerial part in all varieties. We observed that the diversity of the endophytic microbiota differed more between plant organs than between common bean varieties. Our results show that the diversity of endophytic fungi can be efficiently accessed with the sequencing of ITS amplicons and that this diversity may vary among distinct plant organs and the rhizosphere of a single plant variety.

Asunto(s)

Micobioma , Phaseolus/anatomía & histología , Phaseolus/microbiología , Rizosfera , Hongos/clasificación , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Raíces de Plantas/microbiología , Microbiología del Suelo

RESUMEN

The West Nile Virus (WNV) NS2B-NS3 protease is an attractive target for the development of therapeutics against this arboviral pathogen. In the present investigation, the screening of a small library of fifty-eight synthetic compounds against the NS2-NB3 protease of WNV is described. The following groups of compounds were evaluated: 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones; eugenol derivatives bearing 1,2,3-triazolic functionalities; and indan-1,3-diones with 1,2,3-triazolic functionalities. The most promising of these was a eugenol derivative, namely 4-(3-(4-allyl-2-methoxyphenoxy)-propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole (35), which inhibited the protease with IC50 of 6.86 µmol L-1. Enzyme kinetic assays showed that this derivative of eugenol presents competitive inhibition behaviour. Molecular docking calculations predicted a recognition pattern involving the residues His51 and Ser135, which are members of the catalytic triad of the WNV NS2B-NS3 protease.

Asunto(s)

Antivirales/farmacología , Endopeptidasas/metabolismo , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Virus del Nilo Occidental/enzimología , Antivirales/química , Dominio Catalítico/efectos de los fármacos , Descubrimiento de Drogas , Endopeptidasas/química , Eugenol/química , Histidina/química , Histidina/metabolismo , Indanos/química , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/química , Serina/química , Serina/metabolismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/química

RESUMEN

BACKGROUND: In recent times, studies have demonstrated that carbon nanotubes are good candidates for use as vehicles for transfection of exogenous material into the cells. However, there are few studies evaluating the behavior of carbon nanotubes as DNA vectors and few of these studies have used multi-walled carbon nanotubes (MWCNTs) or carboxylated MWCNTs. Thus, this study aims to assess the MWCNTs' (carboxylated or not) efficiency in the increase in expression of the tetravalent vaccine candidate (TVC) plasmid vector for dengue virus in vitro using Vero cells, and in vivo, through the intramuscular route, to evaluate the immunological response profile. RESULTS: Multi-walled carbon nanotubes internalized by Vero cells, have been found in the cytoplasm and nucleus associated with the plasmid. However, it was not efficient to increase the messenger ribonucleic acid (mRNA) compared to the pure vaccine candidate associated with Lipofectamine(®) 2000. The in vivo experiments showed that the use of intramuscular injection of the TVC in combination with MWCNTs reduced the immune response compared to pure TVC, in a general way, although an increase was observed in the population of the antibody-producing B cells, as compared to pure TVC. CONCLUSIONS: The results confirm the data found by other authors, which demonstrate the ability of nanotubes to penetrate target cells and reach both the cytoplasm and the cell nucleus. The cytotoxicity values are also in accordance with the literature, which range from 5 to 20 µg/mL. This has been found to be 10 µg/mL in this study. Although the expression levels are higher in cells that receive the pure TVC transfected using Lipofectamine(®) 2000, the nanotubes show an increase in B-cells producing antibodies.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Linfocitos B/efectos de los fármacos , Vacunas contra el Dengue/administración & dosificación , Dengue/prevención & control , Nanotubos de Carbono/química , Transfección/métodos , Vacunación , Animales , Linfocitos B/inmunología , Linfocitos B/virología , Chlorocebus aethiops , Dengue/inmunología , Dengue/virología , Vacunas contra el Dengue/inmunología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/inmunología , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Lípidos/farmacología , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB C , Plásmidos/química , Plásmidos/metabolismo , Células Vero

RESUMEN

KEY MESSAGE: Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.

Asunto(s)

Dengue/diagnóstico , Nicotiana/genética , Proteínas Recombinantes/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Aedes/virología , Agrobacterium tumefaciens/genética , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Estudios de Casos y Controles , Virus del Dengue/inmunología , Virus del Dengue/patogenicidad , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Plantas Modificadas Genéticamente , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismo , Proteínas no Estructurales Virales/metabolismo

RESUMEN

BACKGROUND: Phages infecting spoilage microorganisms have been considered as alternative biocontrol agents, and the study of their genomes is essential to their safe use in foods. UFV-P2 is a new Pseudomonas fluorescens-specific phage that has been tested for its ability to inhibit milk proteolysis. RESULTS: The genome of the phage UFV-P2 is composed of bidirectional modules and presented 75 functionally predict ORFs, forming clusters of early and late transcription. Further genomic comparisons of Pseudomonas-specific phages showed that these viruses could be classified according to conserved segments that appear be free from genome rearrangements, called locally collinear blocks (LCBs). In addition, the genome organization of the phage UFV-P2 was shown to be similar to that of phages PaP3 and LUZ24 which have recently been classified as a Luz24likevirus. CONCLUSIONS: We have presented the functional annotation of UFV-P2, a new Pseudomonas fluorescens phage. Based on structural genomic comparison and phylogenetic clustering, we suggest the classification of UFV-P2 in the Luz24likevirus genus, and present a set of shared locally collinear blocks as the genomic signature for this genus.

Asunto(s)

Bacteriófagos/clasificación , Bacteriófagos/genética , Genoma Viral , Bacteriófago P2/genética , Análisis por Conglomerados , Biología Computacional , Sistemas de Lectura Abierta , Filogenia , Pseudomonas fluorescens/virología , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

Dengue is a major international public health concern. There is no drug to treat dengue virus infections and a vaccine is yet to be licensed. The laboratory diagnosis of dengue virus infection has been greatly improved during the last decade; therefore, the main limiting factor is the production of recombinant viral antigens on a large scale. Domain III of dengue virus envelope protein contains multiplex conformation-dependent neutralizing epitopes, making it an attractive diagnostic candidate. In this work, we have demonstrated the expression of dengue virus type 1 envelope domain III protein (EDIII-D1) in methylotrophic yeast, Pichia pastoris GS115. The recombinant secreted protein (sEDIII-D1) was purified by affinity chromatography and characterized by SDS-PAGE. Purified protein was recognized in immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue-infected human serum samples. In conclusion, secreted expressions of domain III protein can be obtained in P. pastoris by methanol induction. This product has the potential to be used for the diagnosis of dengue infections.

Asunto(s)

Antígenos Virales/genética , Virus del Dengue/genética , Dengue/diagnóstico , Dengue/virología , Pichia/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Antígenos Virales/aislamiento & purificación , Secuencia de Bases , Clonación Molecular/métodos , Dengue/sangre , Dengue/inmunología , Virus del Dengue/química , Virus del Dengue/inmunología , Virus del Dengue/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificación

RESUMEN

Milk proteolysis caused by Pseudomonas fluorescens is a serious problem in the dairy industries as a result of its ability to grow under refrigeration. The use of phages to control contaminants in food has been considered an alternative to traditional methods; therefore, a thorough understanding of such organisms is vital for their use. In this study, we show the complete genome sequence and analysis of a P. fluorescens phage isolated from wastewater of a dairy industry in Brazil.