Human telomerase protein: Understanding how the catalytic activity is suppressed under single substitutions of some conserved residues. A computational study.

The reverse transcriptase domain in telomerase proteins contains the essential conserved residues to catalyze the addition of a single nucleotide to the ends of DNA strands of most eukaryotic cells. In human telomerase protein, mutations in the conserved residues K902, R631, K626, D712, D868, and D869 are known to suppress catalytic activity. To understand these results, a computational model was constructed to simulate a ternary complex consisting of a model of the protein reverse transcriptase domain, a DNA/RNA double helix, an incoming dNTP, and two Mg2+ ions. Three independent Molecular Dynamics Simulations were performed for the wild type and the mutated K902N, R631Q, D712A, D868A, and D869A complexes. Binding Free Energies and alanine-scanning studies were also performed. Using the two-metal-ion mechanism for the nucleotide addition, deviations from the wild type which stop the activity of the human protein, were identified in each mutated complex. The K902N and R631Q mutations might stop the catalytic activity preventing the exit of the pyrophosphate from the catalytic pocket. Additionally, evidence that the same mechanism probably applies to the K626A, R631A, and K902A mutations is presented. For D712A mutation, the pentavalent intermediate state might not form; therefore, the catalytic reaction might not even begin. For the D868A mutation, the O3'-hydroxyl might lose coordination with the Mg ion and the reaction might not either start. Finally, from the limited sampling carried out in this work, we did not obtain any evidence to identify the mechanism by which the D869A mutation cancels the activity of telomerase.

Non-clinical safety evaluation of repeated intramuscular administration of the AS15 immunostimulant combined with various antigens in rabbits and cynomolgus monkeys.

Combination of tumor antigens with immunostimulants is a promising approach in cancer immunotherapy. We assessed animal model toxicity of AS15 combined with various tumor antigens: WT1 (rabbits), or p501, dHER2 and recPRAME (cynomolgus monkeys), administered in seven or 20 dose regimens versus a saline control. Clinical and ophthalmological examinations, followed by extensive post-mortem pathological examinations, were performed on all animals. Blood hematology and biochemistry parameters were also assessed. Antigen-specific antibody titers were determined by enzyme-linked immunosorbent assay. Additional assessments in monkeys included electrocardiography and immunohistochemical evaluations of the p501 expression pattern. Transient increases in body temperature were observed 4 h or 24 h after injections of recPRAME + AS15 and dHER2 + AS15. Edema and erythema were observed up to 1 week after most injections of recPRAME + AS15 and all injections of dHER2 + AS15. No treatment-related effects were observed for electrocardiography parameters. Mean fibrinogen levels were significantly higher in all treated groups compared to controls, but no differences could be observed at the end of the treatment-free period. Transient but significant differences in biochemistry parameters were observed post-injection: lower albumin/globulin ratios (p501 + AS15), and higher bilirubin, urea and creatinine (dHER2 + AS15). Pathology examinations revealed significant increases in axillary lymph node mean weights (recPRAME + AS15) compared to controls. A 100% seroconversion rate was observed in all treated groups, but not in controls. p501 protein expression was observed in prostates of all monkeys from studies assessing p501 + AS15. These results suggest a favorable safety profile of the AS15-containing candidate vaccines, supporting the use of AS15 for clinical development of potential anticancer vaccines.

Evaluation and comparison of the ability of online available prediction programs to predict true linear B-cell epitopes.

This work deals with the use of predictors to identify useful B-cell linear epitopes to develop immunoassays. Experimental techniques to meet this goal are quite expensive and time consuming. Therefore, we tested 5 free, online prediction methods (AAPPred, ABCpred, BcePred, BepiPred and Antigenic) widely used for predicting linear epitopes, using the primary structure of the protein as the only input. We chose a set of 65 experimentally well documented epitopes obtained by the most reliable experimental techniques as our true positive set. To compare the quality of the predictor methods we used their positive predictive value (PPV), i.e. the proportion of the predicted epitopes that are true, experimentally confirmed epitopes, in relation to all the epitopes predicted. We conclude that AAPPred and ABCpred yield the best results as compared with the other programs and with a random prediction procedure. Our results also indicate that considering the consensual epitopes predicted by several programs does not improve the PPV.

Effect of repetitiveness on the immunogenicity and antigenicity of Trypanosoma cruzi FRA protein.

Repetitive proteins (RP) of Trypanosoma cruzi are highly present in the parasite and are strongly recognized by sera from Chagas' disease patients. Flagelar Repetitive Antigen (FRA), which is expressed in all steps of the parasite life cycle, is the RP that displays the greatest number of aminoacids per repeat and has been indicated as one of the most suitable candidate for diagnostic test because of its high performance in immunoassays. Here we analyzed the influence of the number of repeats on the immunogenic and antigenic properties of the antigen. Recombinant proteins containing one, two, and four tandem repeats of FRA (FRA1, FRA2, and FRA4, respectively) were obtained and the immune response induced by an equal amount of repeats was evaluated in a mouse model. The reactivity of specific antibodies present in sera from patients naturally infected with T. cruzi was also assessed against FRA1, FRA2, and FRA4 proteins, and the relative avidity was analyzed. We determined that the number of repeats did not increase the humoral response against the antigen and this result was reproduced when the repeated motifs were alone or fused to a non-repetitive protein. By contrast, the binding affinity of specific human antibodies increases with the number of repeated motifs in FRA antigen. We then concluded that the high ability of FRA to be recognized by specific antibodies from infected individuals is mainly due to a favorable polyvalent interaction between the antigen and the antibodies. In accordance with experimental results, a 3D model was proposed and B epitope in FRA1, FRA2, and FRA4 were predicted.

Use of coal mining waste for the removal of acidity and metal ions Al (III), Fe (III) and Mn (II) in acid mine drainage.

The coal industry may generate acid mine drainage (AMD) and mining wastes, which may adversely affect the quality of the environment. In this study we propose the use of this waste in the removal of acidity and metal ions, as well as in the reduction of the toxicity of AMD. A physico-chemical analysis of the waste shows the presence of mainly SiO2, Al2O3 and Fe2O3 and a superficial area of 4.316 m2 g(-1). The treatment of AMD with the waste resulted in an increase in pH from 2.6 to 7.8 and removed 100% of the Al (III), 100% of the Fe (III) and 89% of the Mn (II). We also observed that the high toxicity of the AMD towards Daphnia magna (LC50 = 3.68%) and Artemia sp. (LC50 = 4.97%) was completely eliminated after treatment with the waste. The data obtained allow us to propose that the waste can be used in the treatment of AMD, providing an economic use for the waste.