Optimization of the 503 antigen induction strategy of Leishmania infantum chagasi expressed in Escherichia coli M15

Abstract

Leishmaniosis is a complex of diseases that can be fatal, if not given proper attention. Despite its relevance in the public health system, there is no vaccine capable of preventing the disease in humans so far and its treatment is expensive and aggressive to human health. The present study aims to optimize the induction parameters of the 503 Leishmania i. chagasi antigen expressed in recombinant Escherichia coli M15. The induction at different cell densities was evaluated in order to analyze the influence of the induction time on the yield of the protein of interest. In this segment, lactose and isopropyl-β-d-thiogalactopyranoside (IPTG) were used as inducer molecules, using various concentrations: 0.1?g/L, 1.0?g/L, and 10?g/L for lactose and 20?μM, 100?μM, 500?μM, and 1000?μM for IPTG. The results presented that the concentration of IPTG that obtained the higher antigen levels was that of 100?μM (0.087?g/L), a 10-fold lower concentration than was being previously used in this type of system and for lactose, it was 1?g/L (0.016?g/L). Thus, the induction with 100?μM allowed obtaining the antigen with a concentration 5.6 times higher than the lactose induction maximum concentration.     

Robust Demographic Inference from Genomic and SNP Data

We introduce a flexible and robust simulation-based framework to infer demographic parameters from the site frequency spectrum (SFS) computed on large genomic datasets. We show that our composite-likelihood approach allows one to study evolutionary models of arbitrary complexity, which cannot be tackled by other current likelihood-based methods. For simple scenarios, our approach compares favorably in terms of accuracy and speed with , the current reference in the field, while showing better convergence properties for complex models. We first apply our methodology to non-coding genomic SNP data from four human populations. To infer their demographic history, we compare neutral evolutionary models of increasing complexity, including unsampled populations. We further show the versatility of our framework by extending it to the inference of demographic parameters from SNP chips with known ascertainment, such as that recently released by Affymetrix to study human origins. Whereas previous ways of handling ascertained SNPs were either restricted to a single population or only allowed the inference of divergence time between a pair of populations, our framework can correctly infer parameters of more complex models including the divergence of several populations, bottlenecks and migration. We apply this approach to the reconstruction of African demography using two distinct ascertained human SNP panels studied under two evolutionary models. The two SNP panels lead to globally very similar estimates and confidence intervals, and suggest an ancient divergence (>110 Ky) between Yoruba and San populations. Our methodology appears well suited to the study of complex scenarios from large genomic data sets.     

Assembling a xylanase–lichenase chimera through all-atom molecular dynamics simulations

Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase–lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (k_cat/K_m). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes.     

Antitumor Activity of Hierridin B,a Cyanobacterial Secondary Metabolite Found in both Filamentous and Unicellular Marine Strains

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.     

Recycling of the High Valence States of Heme Proteins by Cysteine Residues of Thimet-Oligopeptidase

The peptidolytic enzyme THIMET-oligopeptidase (TOP) is able to act as a reducing agent in the peroxidase cycle of myoglobin (Mb) and horseradish peroxidase (HRP). The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme. EPR (electron paramagnetic resonance) analysis using DBNBS spin trapping revealed that TOP also prevented the formation of tryptophanyl radical in Mb challenged by H₂O₂. The oxidation of TOP thiol groups by peroxidases did not promote the inactivating oligomerization observed in the oxidation promoted by the enzyme aging. These findings are discussed towards a possible occurrence of these reactions in cells.     

Contribution of Multiparameter Flow Cytometry Immunophenotyping to the Diagnostic Screening and Classification of Pediatric Cancer

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56^hi/GD2⁺/CD81^hi), primitive neuroectodermal tumors (CD271^hi/CD99⁺), Wilms tumors (>1 cell population), rhabdomyosarcoma (_nuMYOD1⁺/_numyogenin⁺), carcinomas (CD45⁻/EpCAM⁺), germ cell tumors (CD56⁺/CD45⁻/NG2⁺/CD10⁺) and eventually also hemangiopericytomas (CD45⁻/CD34⁺). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.     

Climate Change and Genetic Structure of Leading Edge and Rear End Populations in a Northwards Shifting Marine Fish Species,the Corkwing Wrasse (Symphodus melops)

One mechanism by which marine organisms may respond to climate shifts is range shifts. The corkwing wrasse (Symphodus melops) is a temperate fish species, inhabiting the coasts of Europe, that show strong indications of current as well as historical (ice-age) range shifts towards the north. Nine neutral microsatellite DNA markers were screened to study genetic signatures and spatial population structure over the entire geographic and thermal gradient of the species from Portugal to Norway. A major genetic break (F _ST  = 0.159 average among pairs) was identified between Scandinavian and more southern populations, with a marked reduction (30% or more) in levels of genetic variability in Scandinavia. The break is probably related to bottleneck(s) associated with post-glacial colonization of the Scandinavian coasts, and indicates a lack of present gene flow across the North Sea. The lack of gene flow can most likely be attributed to the species’ need for rocky substrate for nesting and a relatively short pelagic larval phase, limiting dispersal by ocean currents. These findings demonstrate that long-distance dispersal may be severely limited in the corkwing wrasse, and that successful range-shifts following present climate change may be problematic for this and other species with limited dispersal abilities, even in the seemingly continuous marine environment.     

(3,3’-Methylene)bis-2-hydroxy-1,4-naphthoquinones induce cytotoxicity against DU145 and PC3 cancer cells by inhibiting cell viability and promoting cell cycle arrest

Molecular Biology Reports - We developed a novel method for the synthesis of bis-naphthoquinones (BNQ), which are hybrids of lawsone (2-hydroxy-1,4-naphthoquinone) and 3-hydroxy-juglone...     

Taurine supplementation in conjunction with exercise modulated cytokines and improved subcutaneous white adipose tissue plasticity in obese women

Amino Acids - Interventions that can modulate subcutaneous white adipose tissue (scWAT) function, such as exercise training and nutritional components, like taurine, modulate the inflammatory...