Expression, purification and characterization of the transcription termination factor Rho from Azospirillum brasilense.

The Transcription Termination factor Rho is a ring-shaped, homohexameric protein that causes transcript termination by interaction with specific sites on nascent mRNAs. The process of transcription termination is essential for proper expression and regulation of bacterial genes. Although Rho has been extensively studied in the model bacteria Escherichia coli (EcRho), the properties of other Rho orthologues in other bacteria are poorly characterized. Here we present the heterologous expression and purification of untagged Rho protein from the diazotrophic environmental bacterium Azospirillum brasilense (AbRho). The AbRho protein was purified to >99% through a simple, reproducible and efficient purification protocol, a two-step chromatography procedure (affinity/gel filtration). By using analytical gel filtration and dynamic light scattering (DLS), we found that AbRho is arranged as an homohexamer as observed in the EcRho orthologue. Secondary structure and enzyme activity of AbRho was also evaluate indicating a properly folded and active protein after purification. Enzymatic assays indicate that AbRho is a RNA-dependent NTPase enzyme.

A novel miniaturized electroanalytical device integrated with gas extraction for the voltammetric determination of sulfite in beverages.

Voltammetry and amperometry are inexpensive and high-performance analytical techniques. However, their lack of selectivity limits their use in complex matrices such as biological, environmental, and food samples. Therefore, voltammetric and amperometric analyses of these samples usually require time-consuming and laborious sample pretreatments. In this study, we present a simple and cost-effective approach to fabricate a miniaturized electrochemical cell that can be easily coupled to a head space-like gas extraction procedure in such a way the sample pretreatment and voltammetric detection are performed in a single step. As a proof of concept, we have used the proposed system to quantify sulfite in beverage samples after its conversion to SO2(g). Despite the simplicity and low cost of the proposed system, it provided good analytical performance and a limit of detection of 4.0 µmol L-1 was achieved after only 10 min of extraction. The proposed system is quite versatile since it can be applied to quantify any volatile electroactive species. Also, the proposed system provides a unique way to assess real-time extraction curves, which are essential to study and optimize new gas extraction procedures. Therefore, the approach described in this study could contribute to both applied and fundamental Analytical Chemistry.

Corrigendum: Neospora Caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

[This corrects the article DOI: 10.3389/fmicb.2016.01456.].

Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii's GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host's innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis.