Aberrant AHRR, ADAMTS2 and FAM184 DNA Methylation: Candidate Biomarkers in the Oral Rinse of Heavy Smokers.

OBJECTIVE: To identify DNA methylation patterns of heavy smokers in oral rinse samples. METHODS: Genome-wide DNA methylation data was imported from Gene Expression Omnibus GSE70977 using the GEOquery package. Two independent sets were analyzed: (a) 71 epigenomes of cancer-free subjects (heavy smokers n = 37 vs. non-smokers n = 31); for concordance assessment (b) 139 oral-cancer patients' epigenomes (heavy smokers n = 92 vs. non-smokers n = 47). Differential DNA methylation for CpG positions and at the regional level was determined using Limma and DMRcate Bioconductor packages. The linear model included sex, age, and alcohol consumption. The statistical threshold was set to p < 0.05. Functional gene prioritization analysis was performed for gene-targeted analysis. RESULTS: In individuals without cancer and heavy smokers, the FAM184B gene was found with two CpG positions differentially hypermethylated (p = 0.012 after FDR adjustment), in a region of 48 bp with an absolute methylation difference >10% between groups (p = 1.76 × 10-8). In the analysis corresponding to oral-cancer patients, we found AHRR differentially hypomethylated cancer patients, but also in subjects without oral cancer in the targeted analyses. Remarkably, ADAMTS2 was found differentially hypermethylated in heavy smokers without a diagnosis of cancer in two consecutive probes cg05575921 (p = 3.13 × 10-7) and cg10208897 (p = 1.36 × 10-5). CONCLUSIONS: Differentially methylated AHRR, ADAMTS2, and FAM184B genes are biomarker candidates in oral rinse samples.

Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis.

Cry11 proteins are toxic to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of Bacillus thuringiensis and subjected to median-lethal concentration (LC50) tests on first-instar larvae of A. aegypti. LC50 analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL-1), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against A. aegypti. This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.

Generation of Cry11 Variants of Bacillus thuringiensis by Heuristic Computational Modeling.

Directed evolution methods mimic in vitro Darwinian evolution, inducing random mutations and selective pressure in genes to obtain proteins with enhanced characteristics. These techniques are developed using trial-and-error testing at an experimental level with a high degree of uncertainty. Therefore, in silico modeling of directed evolution is required to support experimental assays. Several in silico approaches have reproduced directed evolution, using statistical, thermodynamic, and kinetic models in an attempt to recreate experimental conditions. Likewise, optimization techniques using heuristic models have been used to understand and find the best scenarios of directed evolution. Our study uses an in silico model named HeurIstics DirecteD EvolutioN, which is based on a genetic algorithm designed to generate chimeric libraries from 2 parental genes, cry11Aa and cry11Ba, of Bacillus thuringiensis. These genes encode crystal-shaped δ-endotoxins with 3 conserved domains. Cry11 toxins are of biotechnological interest because they have shown to be effective as biopesticides for disease-spreading vectors. With our heuristic model, we considered experimental parameters such as DNA fragmentation length, number of generations or simulation cycles, and mutation rate, to get characteristics of Cry11 chimeric libraries such as percentage of population identity, truncation of variants obtained from the presence of internal stop codons, percentage of thermodynamic diversity, and stability of variants. Our study allowed us to focus on experimental conditions that may be useful for the design of in vitro and in silico experiments of directed evolution with Cry toxins of 3 conserved domains. Furthermore, we obtained in silico libraries of Cry11 variants, in which structural characteristics of wild Cry families were observed in a review of a sample of in silico sequences. We consider that future studies could use our in silico libraries and heuristic computational models, as the one suggested here, to support in vitro experiments of directed evolution.