Lack of increases in methylation at three CpG-rich genomic loci in non-mitotic adult tissues during aging

<p>Abstract</p> <p>Background</p> <p>Cell division occurs during normal human development and aging. Despite the likely importance of cell division to human pathology, it has been difficult to infer somatic cell mitotic ages (total numbers of divisions since the zygote) because direct counting of lifetime numbers of divisions is currently impractical. Here we attempt to infer relative mitotic ages with a molecular clock hypothesis. Somatic genomes may record their mitotic ages because greater numbers of replication errors should accumulate after greater numbers of divisions. Mitotic ages will vary between cell types if they divide at different times and rates.</p> <p>Methods</p> <p>Age-related increases in DNA methylation at specific CpG sites (termed "epigenetic molecular clocks") have been previously observed in mitotic human epithelium like the intestines and endometrium. These CpG rich sequences or "tags" start unmethylated and potentially changes in methylation during development and aging represent replication errors. To help distinguish between mitotic versus time-associated changes, DNA methylation tag patterns at 8–20 CpGs within three different genes, two on autosomes and one on the X-chromosome were measured by bisulfite sequencing from heart, brain, kidney and liver of autopsies from 21 individuals of different ages.</p> <p>Results</p> <p>Levels of DNA methylation were significantly greater in adult compared to fetal or newborn tissues for two of the three examined tags. Consistent with the relative absence of cell division in these adult tissues, there were no significant increases in tag methylation after infancy.</p> <p>Conclusion</p> <p>Many somatic methylation changes at certain CpG rich regions or tags appear to represent replication errors because this methylation increases with chronological age in mitotic epithelium but not in non-mitotic organs. Tag methylation accumulates differently in different tissues, consistent with their expected genealogies and mitotic ages. Although further studies are necessary, these results suggest numbers of divisions and ancestry are at least partially recorded by epigenetic replication errors within somatic cell genomes.</p

CHRNA4 rs1044396 is associated with smoking cessation in varenicline therapy

The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4 and CHRNB2 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. This cohort study enrolled 483 smoking patients who received behavioral counseling and drug treatment (varenicline, bupropion and/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196) and CHRNB2 (rs2072660 and rs2072661) polymorphisms were genotyped by high resolution melting analysis.Patients with rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (p=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, p=0.02), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. In conclusion, the CHRNA4 rs1044396 is associated with smoking cessation in individuals on varenicline therapy. We suggest that this polymorphism influences the varenicline response, but replications of this finding are needed

Imaging in living cells using ν

The boron-rich cobaltabisdicarbollide (COSAN) and its 8,8′-I2 derivative (I2-COSAN), both of purely inorganic nature, are shown to accumulate within living cells, where they can be detected using νB–H Raman microspectroscopy. This demonstrates an alternative method for cell labelling and detection. The inorganic, boron-based molecule cobaltabisdicarbollide, [3,3′-Co(1,2-C2B9H11)2]−, commonly known as COSAN, comprises of a cobalt atom sandwiched by two carboranyl clusters.1 This structure exhibits both electrostatic interactions, via a dispersed negative ionic charge covering the whole molecule,2 and non-bonding intermolecular interactions between its weakly polarized B–H and C–H bonds.3 This duality imparts the molecular property of being simultaneously hydrophobic and hydrophilic, and makes COSAN soluble in both water and oils. The polarized lipid molecules that make up biological membranes also possess amphiphilic properties, and can assemble into membranes and vesicles formed from lipid bilayers. COSAN has also been shown to form small nano-vesicles and above a critical aggregation concentration (cacvesicle ≈ 0.01 mM), begin to form micelles.4 However, unlike lipid bilayer membranes, the membranes of COSAN vesicles are monolayered. Recently, [3,3′-Co(8-I-1,2-C2B9H10)2]−, I2-COSAN, has also been found to self-assemble into a lyotropic lamellar phase with sufficient curvature to create closed vesicles.5 These similarities in physicochemical properties to biological lipids suggest that COSAN molecules may be compatible with living cells, and could be used to label cells in a similar manner to lipophilic dyes. Here, we use Raman microspectroscopy to show the accumulation of COSAN and I2-COSAN in living cells. As a first step in this work, COSAN or I2-COSAN was added directly to Dulbecco's Modified Eagle medium (DMEM) with Human Embryonic Kidney (HEK293) cells. When the cells were concentrated by low speed centrifugation, washed and incubated in new DMEM medium, visual inspection showed accumulation of COSAN or I2-COSAN (orange in colour) within cell pellets (Fig. 1). This observation indicated that these COSAN molecules could be taken up by living cells