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.

Site-Directed Mutants of Parasporin PS2Aa1 with Enhanced Cytotoxic Activity in Colorectal Cancer Cell Lines.

Parasporin 2 has cytotoxic effects against numerous colon cancer cell lines, making it a viable alternative to traditional treatments. However, its mechanism of action and receptors remain unknown. In this study, site-directed mutagenesis was used to obtain PS2Aa1 mutants with variation in domain I at positions 256 and 257. Variants 015, 002, 3-3, 3-35, and 3-45 presented G256A, G256E, G257A, G257V, and G257E substitutions, respectively. Cytotoxicity tests were performed for the cell viability of cell lines SW480, SW620, and CaCo-2. Mutants 3-3, 3-35, and 3-45 efficiently killed the cell lines. It was found that the activated forms of caspase-3 and PARP were in higher abundance as well as increased production of γH2AX when 3-35 was used to treat CaCo-2 and SW480. To assess possible membrane-binding receptors involved in the interaction, an APN receptor blocking assay showed reduced activity of some parasporins. Hence, we performed molecular docking and molecular dynamics simulations to analyze the stability of possible interactions and identify the residues that could be involved in the protein-protein interaction of PS2Aa1 and APN. We found that residues 256 and 257 facilitate the interaction. Parasporin 3-35 is promising because it has higher cytotoxicity than PS2Aa1.

Genetic Modification Approaches for Parasporins Bacillus thuringiensis Proteins with Anticancer Activity.

Bacillus thuringiensis (Bt) is a bacterium capable of producing Cry toxins, which are recognized for their bio-controlling actions against insects. However, a few Bt strains encode proteins lacking insecticidal activity but showing cytotoxic activity against different cancer cell lines and low or no cytotoxicity toward normal human cells. A subset of Cry anticancer proteins, termed parasporins (PSs), has recently arisen as a potential alternative for cancer treatment. However, the molecular receptors that allow the binding of PSs to cells and their cytotoxic mechanisms of action have not been well established. Nonetheless, their selective cytotoxic activity against different types of cancer cell lines places PSs as a promising alternative treatment modality. In this review, we provide an overview of the classification, structures, mechanisms of action, and insights obtained from genetic modification approaches for PS proteins.

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.

Softepigen: Primers Design Web-Based Tool for MS-HRM Technique.

Polymerase Chain Reaction (PCR) based techniques for DNA methylation techniques includes the MS-HRM technique. Methylation Sensitive High-Resolution Melting (MS-HRM) primer-design requires a set of necessary recommendations for such DNA methylation assessment. However, there were not any available software that allows an automatic design of this kind primers. We present Softepigen, the first complete MS-HRM primer design software. Softepigen allows to search for primers in a genomic region following Wojdacz's recommendations and targets primer binding regions with high linguistic complexity sequences that increase the specificity of the converted sequence of the human genome. We performed in-silico PCR analysis through BiSearch ePCR tool to validate the specificity of the of the primers designed using Softepigen. Softepigen for MS-HRM performance in our genomic regions of interest show satisfactory specificity measurements, and we implemented it for freely available use in the web-based interface at www.soft-epigen.com.

Computer Aided Design of Non-toxic Antibacterial Peptides.

Antimicrobial resistance is increasing at an alarming rate and the number of new antibiotics developed and approved has decreased in the last decades, basically for economic and regulatory obstacles. Pathogenic bacteria that are resistant to multiple or all available antibiotics are isolated frequently. Hence, new antibacterial agents are urgently needed and antimicrobial peptides are being considered as a potential solution to this important threat. These molecules are small host defense proteins that are part of the immune systems of most living organisms such as plants, bacteria, invertebrates, vertebrates, and mammals. These peptides are found in those parts of organisms that are exposed to pathogens and they are active against multiple organisms such as virus, bacteria, and parasites, among others. This review shows different strategies in the computational design of new antibacterial peptides, the physicochemical properties that are considered as the most relevant for the antibacterial activity and toxicity, and it suggests guidelines in order to help in the finding of new non-toxic antibacterial peptides through the development of computational models.