Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays ...revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.
To improve a DNA vaccine containing the truncated dengue virus serotype 2 (DENV-2) envelope (E) protein and evaluate the influence of precursor membrane (prM) glycoprotein polymorphism on E protein ...immunogenicity, two vaccine candidates have been constructed by upstream insertion of the DENV-2 and DENV-3 prM genes into the DENV-2 E gene, named pCID2EtD2prM and pCID2EtD3prM, respectively. Both constructs were able to induce antibody production, which were neutralizing against DENV-2 in a murine model. Splenocytes of immunized groups, when challenged with virus, demonstrated Th1 cytokine pattern and proliferation, in addition to the increase of specific T cells. Vaccine candidates pCID2EtD2prM and pCID2EtD3prM confer 70% and 90% protection against DENV-2, respectively. The pCID2EtD3prM plasmid conferred only 40% protection in the lethal challenge with DENV-2. The results demonstrate that DENV-3 prM has a greater influence on the immunogenicity of the E protein and, probably due to its role as a chaperone, these results may be related to the correct folding and, consequently, an increase in the presentation efficiency of produced transcripts.
Mayaro virus (MAYV) is a neglected arthropod-borne virus found in the Americas. MAYV infection results in Mayaro fever, a non-lethal debilitating disease characterized by a strong inflammatory ...response affecting the joints and muscles. MAYV was once considered endemic to forested areas in Brazil but has managed to adapt and spread to urban regions using new vectors, such as
, and has the potential to cause serious epidemics in the future. Currently, there are no vaccines or specific treatments against MAYV. In this study, the antiviral activity of a series of synthetic cyclic ketones were evaluated for the first time against MAYV. Twenty-four compounds were screened in a cell viability assay, and eight were selected for further evaluation. Effective concentration (EC
) and selectivity index (SI) were calculated and compound 9-(5-(4-chlorophenylfuran-2-yl)-3,6-dimethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2))-dione (9) (EC
= 21.5 µmol·L
, SI = 15.8) was selected for mechanism of action assays. The substance was able to reduce viral activity by approximately 70% in both pre-treatment and post-treatment assays.
The multidrug-resistant bacteria have become a serious problem to public health. In this scenery the antimicrobial peptides (AMPs) derived from animals and plants emerge as a novel therapeutic ...modality, substituting or in addition to the conventional antimicrobial. The anurans are one of the richest natural sources of AMPs. In this work several cycles of cDNA cloning of the skin of the Brazilian treefrog Hypsiboas semilineatus led to isolation of a precursor sequence that encodes a new AMP. The sequence comprises a 27 residue signal peptide, followed by an acidic intervening sequence that ends in the mature peptide at the carboxy terminal. The AMP, named Hs-1, has 20 amino acids residues, mostly arranged in an alpha helix and with a molecular weight of 2144.6 Da. The chemically synthesized Hs-1 showed an antimicrobial activity against all Gram-positive bacteria tested, with a range of 11–46 μM, but it did not show any effect against Gram-negative bacteria, which suggest that Hs-1 may have a selective action for Gram-positive bacteria. The effects of Hs-1 on bacterial cells were also demonstrated by transmission electron microscopy. Hs-1 is the first AMP to be described from H. semilineatus.
•We report the identification and characterization of a new AMP named Hs-1.•Hs-1 is the first AMP described for the Brazilian endemic frog Hypsiboas semilineatus.•Hs-1 is selective against Gram-positive bacteria, especially against MRSA.•Hs-1 has a direct antibacterial killing by membrane disruption.•Hs-1 is predominantly structured as an amphipathic alpha helix.
This study aimed to evaluate the potential of aqueous extract from Mitracarpus frigidus aerial parts (MFAq) in the treatment of inflammation and oxidative stress, as well as to characterize its ...chemical constituents.
Total phenolic and flavonoid contents were determined, and phytoconstituents were detected by ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-QTOF-MS). The antioxidant activity was evaluated by DPPH, TAC and β-carotene/linoleic acid assays. In-vitro anti-inflammatory activity, cell viability and cell cycle were performed in J774A.1 cell line. In-vivo anti-inflammatory activity was investigated by two ear oedema assays (croton oil and phenol).
Chlorogenic acid, clarinoside, quercetin-hexosylpentoside, rutin, kaempferol-3-O-rutinoside, kaempferol-rhamnosylhexoside, quercetin-pentosylrhamnosylhexoside, harounoside, 2-azaanthraquinone and sucrose were identified by UFLC-QTOF-MS. MFAq showed antioxidant activity, which was positively correlated to the content of phenolic compounds. MFAq significantly inhibited the production of nitric oxide, did not decrease viability in MTT assay (all concentrations) and showed no changes in membrane permeability and cell cycle of J774A.1 cell line. Furthermore, MFAq showed a reduction in ear oedema in all tested doses.
MFAq was effective in some antioxidant and inflammatory parameters, in the experimental conditions that were used in the study. This is the first report of chemical composition and bioactivities from this extract.
Oil refineries are known for the large volume of water used in their processes, as well as the amount of wastewater generated at the end of the production chain. Due to strict environmental ...regulations, the recycling of water has now become a viable alternative for refineries. Among the many methods available to treat wastewater for reuse, the use of membranes in reverse osmosis systems stands out due to several economic and environmental benefits. However, these systems are vulnerable to contamination and deposition of microorganisms, mainly because of the feedwater quality. In this study, the microbial diversity of feedwater and reverse osmosis membranes was investigated using a combination of culture-dependent and culture-independent methods in order to characterize the microorganisms colonizing and deteriorating the membranes. In total, 37 bacterial isolates, 17 filamentous fungi and approximately 400 clones were obtained and analyzed. Among the bacterial genera identified, the most represented were Sphingobium, Acidovorax, Microbacterium, Rhizobium and Shinella. The results revealed genera that acted as candidate key players in initial biofilm formation in membrane systems, and provided important information concerning the microbial ecology of oligotrophic aquatic systems.
Antimicrobial peptides have been suggested as an alternative to classical antibiotics in livestock production and bacteriocin-producing bacteria could be added to animal feeds to deliver bacteriocins ...in the gastrointestinal (GI) tract of ruminant and monogastric animals. In this study, viable (V) and heat-killed (HK) Streptococcus bovis HC5 cells were orally administered to pre-sensitized mice in order to assess the effects of a bacteriocin-producing bacteria on histological parameters and the immune response of the GI tract of monogastric animals. The administration of V and HK S. bovis HC5 cells during 58 days to BALB/c mice did not affect weight gain, but an increase in gut permeability was detected in animals receiving the HK cells. Viable and heat killed cells caused similar morphological alterations in the GI tract of the animals, but the most prominent effects were detected in the small intestine. The oral administration of S. bovis HC5 also influenced cytokine production in the small intestine, and the immune-mediated activity differed between V and HK cells. The relative expression of IL-12 and INF-γ was significantly higher in the small intestine of mice treated with V cells, while an increase in IL-5, IL-13 and TNF-α expression was only detected in mice treated with HK cells. Considering that even under a condition of severe challenge (pre-sensitization followed by daily exposure to the same bacterial immunogen) the general health of the animals was maintained, it appears that oral administration of S. bovis HC5 cells could be a useful route to deliver bacteriocin in the GI tract of livestock animals.
Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from ...cauliflower (Brassica oleracea ssp. botrytis – BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H2O2 and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of ∼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of H2O2 and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal.
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.
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.
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