Nanoscale analysis of extracellular DNA (eDNA) that is present on the surface of cells in trace biological samples can provide insight into the understanding of DNA transfer through touch, and thereby, the role of eDNA is a biologically and forensically relevant phenomenon. While various bulk scale tools and DNA analysis can be used to quantitatively obtain this information, obtaining a three dimensional (3D) visualization of the eDNA can provide a unique look into the spatial and temporal dynamics at the cellular level. In this study, we show how atomic force microscopy (AFM) can be integrated with optical microscopy to visualize the distribution of surface associate eDNA at a single cell level. Using a nucleic acid fluorophore such as Diamond™ Dye, the surface eDNA can be observed and quantified using fluorescence microscopy. This informational channel can then be overlaid with surface topography and cellular elasticity to provide structural visualization. Finally, chemical force spectroscopy can be used to obtain the distribution of surface-associated eDNA on the cell surface at the molecular level. Such integrated techniques can enhance understanding of the biological role of eDNA, and can also be potentially valuable for investigating challenging trace samples, containing very few cells for various analyses.

It is unclear how running modality influences telomere length (TL). This single laboratory visit study compared the TL of master sprinters and endurance runners with their young counterparts. The correlation between leukocyte and buccal cell TL in athletes was also explored. Participants consisted of 11 young controls, 11 young sprinters, 12 young endurance runners, 12 middle-aged controls, 11 master sprinters, and 12 master endurance runners. Blood and buccal samples were collected and randomized for analysis of TL by quantitative polymerase chain reaction. Young endurance runners displayed longer telomeres than master athletes (p < .05); however, these differences were not significant when controlled for covariates (p > .05). A positive correlation existed between leukocyte and buccal cell TL in athletes (r = .567, p < .001). In conclusion, young endurance runners possess longer telomeres than master endurance runners and sprinters, a consequence of lower body mass index and visceral fat.

UNASSIGNED: Buccal cells are an ideal surrogate tissue for studying biologic effects of carcinogens or drugs, however inherent fragility and salivary RNAses limit RNA yield. We conducted healthy volunteer trials to optimize collection conditions.
UNASSIGNED: We conducted: (a) a single-arm crossover study evaluating four test conditions on RNA yield by buccal cytobrush; (b) a single-arm prospective study evaluating RNA yield by investigator vs self-collection.
UNASSIGNED: Antecedent toothbrushing, time of day, and number of cytobrush strokes did not significantly impact RNA yield. RNA yield was doubled by using 2 vs 1 cytobrush per buccal surface (P = .0054). Self-collection of buccal cells for RNA was feasible; 36 of 50 (72%) samples passed quality control.
UNASSIGNED: RNA yield was doubled by using two cytobrushes per buccal surface. Healthy volunteers can self-collect sufficient buccal RNA for gene expression studies. Techniques from these pragmatic trials could enhance availability of a limited tissue for serial biomarker measurements.
UNASSIGNED: 1b-Prognosis Study (Individual prospective cohort study).

Background: Azo compounds, containing naphthol and diazonium salts, are synthetic dyes widely used in the batik industry. Azo compounds are considered toxic when they are exposed to human tissue. The purpose of this study was to analyze buccal cell DNA exposed to azo compounds in batik workers. Methods: A cross-sectional study involving 20 male subjects divided into two groups (n=10 group), namely azo-exposed and non-exposed (control group). Inclusion criteria were batik workers of the colouring division who have been exposed to azo for at least 5 years. Buccal cells were taken using cytobrush then DNA were isolated from buccal cell. DNA isolation was done by buccal DNA kit, while the purity and concentration of the DNA was determined using spectrophotometer and electrophoresis. Results: The azo-exposed group revealed higher purity DNA than those in the control group. The purity of the DNA in the azo-exposed group and control group was 0.61±0.93 and 0.21±0.09, respectively, while the concentration of DNA was of 59.02 and 19.35 ng/UL, respectively. The ratio at 260/280 nm was 1.84-1.94 (azo-exposed) and 1.85-1.92 (control). Principal component analysis using the first principle component (PC1) and second principle component (PC2) could successfully classify subjects in the control and azo-exposed groups. Conclusion: Characteristics of DNA could be used as an indication of exposure to azo compounds in workers of batik industries.

OBJECTIVE: This prospective clinical study comparatively investigated the effects of tobacco smoking on global methylation and hydroxymethylation in oral epithelial cells.
METHODS: Buccal cells from the inside of the cheeks were collected from 47 individuals, including smokers, former smokers, and never smokers. DNA was extracted using dedicated kits. Methylated and hydroxymethylated DNA fractions were measured using assays similar to enzyme-linked immunosorbent assays. The levels of methylation and hydroxymethylation were compared among groups using unpaired two-tailed t-tests or the Mann-Whitney U test; P < 0.05 was considered statistically significant.
RESULTS: There was no statistically significant difference in the average number of cigarettes between smoker and former smoker groups. Although methylation levels were lower for smokers (3.1%) and former smokers (2.16%), compared with never smokers (4.16%), these differences were not statistically significant. There was a two-fold increase in hydroxymethylation level in never smokers, compared with smokers.
CONCLUSIONS: Our findings suggest that smoking leads to global reductions in both methylation and hydroxymethylation levels in oral epithelial cells in a manner influenced by the intensity and length of exposure to tobacco smoke.

The micronucleus (MN) assay, applied in different surrogate tissues, is one of the best validated cytogenetic techniques for evaluating chromosomal damage in humans. The cytokinesis-block micronucleus cytome assay in peripheral blood lymphocytes (L-CBMNcyt) is the most frequently used method in biomonitoring human populations to evaluate DNA damage caused by exposure to genotoxic agents, micronutrient deficiency or excess and genetic instability. Furthermore, recent scientific evidence suggests an association between an increased MN frequency in lymphocytes and risk of cancer and other age-related degenerative diseases. The micronucleus cytome assay applied in buccal exfoliated cells (BMNCyt), provides a complementary method for measuring DNA damage and cytotoxic effects in an easily accessible tissue not requiring ex vivo/in vitro culture. The protocol for L-CBMNcyt described here, refers to the use of ex vivo whole blood method, involving 72 h of culture with the block of cytokinesis starting at 44 h. BMNCyt protocol reports the established method for sample collection, processing, slide preparation and scoring.

OBJECTIVE: The adherence of oral pathogenic microorganisms to host tissues is the initial step for successful process of oral diseases. This study aimed to determine the effect of the Rhodomyrtus tomentosa leaf extract and rhodomyrtone, an antibacterial compound from R. tomentosa leaf, on adhesion of some oral pathogens to polystyrene plastic surface and human buccal epithelial cells.
METHODS: The minimum inhibitory concentration (MIC) was evaluated using broth microdilution method. The microbial adhesion to the plastic surface and buccal cells was determined using microtiter plate method and microscopy technique.
RESULTS: The ethanol extract of leaf demonstrated antibacterial activity against oral microorganisms including Staphylococcus aureus ATCC 25923, Streptococcus mutans (clinical isolate), and Candida albicans ATCC 90028 with the MIC values of 31.25, 15.62, and 1000μg/ml, respectively. Rhodomyrtone displayed activity with the MIC values of 0.78 and 0.39μg/ml against S. aureus ATCC 25923 and S. mutans, respectively. The MIC value of the compound against C. albicans ATCC 90028 was more than 100μg/ml which was the highest test concentration. All pathogenic microorganisms treated with the extract and rhodomyrtone at their subinhibitory concentrations resulted in a decrease in their adherence ability to both plastic surface and buccal cells.
CONCLUSIONS: It is suggested that R. tomentosa extract and rhodomyrtone may be useful in therapy or as prophylaxis in infections involving oral pathogens.