Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on MCF-7 Breast Cancer Cells

The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damage-inducing and DNA methylation-changing abilities. We demonstrate that small modifications in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, with PK9320 and PK9323 being eligible candidates as “anticancer compounds” and “anticancer epi-compounds” and PK083 a “damage-corrective” compound on human breast adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents and chemical probes.

Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40

To further explore the epigenetic changes in nasopharyngeal carcinoma (NPC), methylation-sensitive arbitrarily primed PCR was performed on NPC biopsies and nontumor nasopharyngeal samples. We have shown mainly two DNA fragments that appeared to be differentially methylated in NPCs versus nontumors. The first, defined as hypermethylated, corresponds to a CpG island at the 5′-end of the tetratricopeptide repeat domain 40 (TTC40) gene, whereas the second, defined as hypo-methylated, is located on repetitive sequences at chromosomes 16p11.1 and 13.1. Thereafter, the epigenetic alteration on the 5′-TTC40 gene was confirmed by methylation-specific PCR, showing a significant aberrant methylation in NPCs, compared to nontumors. In addition, the bisulfite sequencing analysis has shown a very high density of methylated cytosines in C15, C17, and X666 NPC xenografts. To assess whether TTC40 gene is silenced by aberrant methylation, we examined the gene expression by reverse transcription-PCR. Our analysis showed that the mRNA expression was significantly lower in tumors than in nontumors, which is associated with 5′-TTC40 gene hypermethylation. In conclusion, we found that the 5′-TTC40 gene is frequently methylated and is associated with the loss of mRNA expression in NPCs. Hypermethylation of 5′-TTC40 gene might play a role in NPC development; nevertheless, other studies are needed.

Pacemaker lead infection and related bacteraemia caused by normal and small colony variant phenotypes of Bacillus licheniformis

Here, we report what we believe to be the first case of bacteraemia with small colony variants of Bacillus licheniformis related to a pacemaker lead infection by B. licheniformis displaying the normal phenotype. Arbitrarily primed PCR analysis showed a clonal strain. The infection was cured after the removal of the infected device.

Longitudinal Analysis of the Prevalence, Maintenance, and IgA Response to Species of the Order Bacteroidales in the Human Gut

ABSTRACTBacteroidalesspecies are the most abundant Gram-negative bacteria of the human intestinal microbiota. These bacteria evolved to synthesize numerous capsular polysaccharides (PS) that are subject to phase variation. InBacteroides fragilis, PS synthesis is regulated so that only one of the eight PS biosynthesis loci is transcribed at a time in each bacterium. To determine if the bacteria evolved this unusual property to evade a host IgA response, we directly studied the human fecal ecosystem. We performed a longitudinal analysis of the abundantBacteroidalesspecies from 15 healthy adults at four intervals over a year. For this study, we used bacterial culture to perform analyses not accurate with DNA-based methods, including quantification of total viableBacteroidalesbacteria, strain maintenance, and IgA responses. AbundantBacteroidalesisolates were identified to the species level using multiplex PCR and 16S rRNA gene sequencing. Arbitrarily primed PCR was used for strain typing. IgA responses to endogenous strains carried over the year were analyzed, and the orientations of the invertible PS locus promoters from the ecosystem were quantified. Subjects consistently harbored from 5 × 108to 8 × 1010Bacteroidalesbacteria/g of feces. Within the cohort, 20 differentBacteroidalesspecies were detected at high concentrations.Bacteroides uniformiswas the most prevalent; however, abundantBacteroidalesspecies varied between subjects. Strains could be maintained over the year within the ecosystem at high density. IgA responses were often not induced and did not correlate with the elimination of a strain or major changes in the orientations of the capsular PS locus promoters.

Rapid differentiation of phenotypically and genotypically similar Synechococcus elongatus strains by PCR fingerprinting.

PCR amplification techniques viz., repetitive DNA element PCR (REP-PCR), short tandemly repeated repetitive PCR (STRR-PCR) and arbitrarily primed PCR (RAPD-PCR) were used for the taxonomic discrimination among the strains of the unicellular cyanobacterium Synechococcus elongatus collected across the coastal regions of the Indian subcontinent. These strains showed similar phenotypic and genotypic characteristics. Data obtained from genomic fingerprinting were used to perform cluster analysis and demonstrated ability to differentiate strains at intra-specific level. Polymorphisms of different PCR amplification products can serve as strain-specific molecular fingerprints. In comparison with the STRR and RAPD, the REP primer set generates fingerprints of lower complexity, but still the phenogram clearly differentiated the strains. In conclusion, described PCR fingerprinting methods can be considered as promising tools for the differentiation at the strain level of cyanobacteria from the same species.

Genotypic and phenotypic analysis of S. mutans isolated from dental biofilms formed in vivo under high cariogenic conditions

The oral cavity harbors several Streptococcus mutans genotypes, which could present distinct virulence properties. However, little is known about the diversity and virulence traits of S. mutans genotypes isolated in vivo under controlled conditions of high cariogenic challenge. This study evaluated the genotypic diversity of S. mutans isolated from dental biofilms formed in vivo under sucrose exposure, as well as their acidogenicity and aciduricity. To form biofilms, subjects rinsed their mouths with distilled water or sucrose solution 8 times/day for 3 days. S. mutans collected from saliva and biofilms were genotyped by arbitrarily-primed PCR. Genotypes identified in the biofilms were evaluated regarding their ability to lower the suspension pH through glycolysis and their acid susceptibility and F-ATPase activity. Most subjects harbored only one genotype in saliva, which was detected in almost all biofilm samples at high proportions. Genotypes isolated only in the presence of sucrose had higher acidogenicity than those isolated only in the presence of water. Genotypes from biofilms formed with sucrose were more aciduric after 30 and 60 min of incubation at pH 2.8 and 5.0, respectively. The present results suggest that biofilms formed under high cariogenic conditions may harbor more aciduric and acidogenic S. mutans genotypes.