High-throughput method for detecting DNA methylation

Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related gene may be useful for cancer diagnosis or the detection of recurrence. However, most of the studies have focused on a single gene only and gave little information about the concurrent methylation status of multiple genes. In this study, we attempted to develop a microarray method coupled with linker-PCR for detecting methylation status of multiple genes in the tumor tissue. A series of synthesized oligonucleotides were synthesised and purified to completely match with 16 investigated targets. Then they were immobilized on the aldehyde-coated glass slide to fabricate a DNA microarray for detecting methylation status of these genes. The results indicated that these genes were all methylated in the positive control. However, no methylated was found in these genes for the negative control. Only p16 and p15 genes were methylated in investigated genes for the gastric tumor tissue, whereas others were not methylated. The above results were validated by bisulfite DNA sequencing. Our experiments successfully demonstrated that the DNA microarray could be applied as a high-throughput tool to determine methylation status of the investigated genes.     

Leptin receptor Arg109 homozygotes display decreased total mortality as well as lower incidence of cardiovascular disease and related death

Two leptin receptor single nucleotide polymorphisms, Lys109Arg and Gln223Arg, have been shown to associate with several risk factors for cardiovascular disease. In addition, we have previously shown that Arg109 and Arg223 homozygotes displayed lower intima-media thickness in our well-defined OPERA (Oulu Project Elucidating Risk of Atherosclerosis) study. This current research investigated the impact of these LEPR polymorphisms on cardiovascular events and related death as well as to total mortality in the 19-year follow-up of OPERA. Subjects were randomly selected, middle-aged drug-treated hypertensives and their age- and sex-matched control subjects recruited to the OPERA study between 1990 and 1993. Mortality and hospital events of 1045 subjects were followed up until 2009. A total of 151 coronary heart disease (CHD) and 211 cardiovascular disease (CVD) events or deaths including 58 CHD and 69 CVD deaths occurred. Furthermore, during this follow-up, a total of 165 subjects died. Logistic regression analysis was performed to assess the impact of Lys109Arg and Gln223Arg on the events and death. Further modeling was performed with Cox regression for Lys109Arg. The logistic regression analysis revealed a significant protective impact of Arg109Arg genotype on CHD (OR 0.433; CI 95% 0.217–0.863) and CVD (OR 0.540; CI 95% 0.309–0.942) events or death as well as on total mortality (OR 0.390; CI 95% 0.196–0.775) when adjusted with age, sex and study group. Even after further adjustment with BMI, smoking status, systolic blood pressure and low-density lipoprotein cholesterol, the protective effect of Arg109Arg on CHD events or death and total mortality still remained statistically significant (OR 0.463; CI 95% 0.230–0.931 and OR 0.442; CI 95% 0.218–0.896, respectively). Arg109Arg was also shown to confer protection against CHD mortality (HR 0.224; CI95% 0.055–0.919) and overall mortality (HR 0.413; CI95% 0.218–0.783) also in Cox regression analysis. In conclusion, the Arg109Arg genotype of LEPR seems to be protective from cardiovascular events and death and this phenomenon seems to be independent of the traditional risk factors for atherosclerosis.     

Effects of variation in precipitation on the distribution of soil bacterial diversity in the primitive Korean pine and broadleaved forests

Patterns of precipitation have changed as a result of climate change and will potentially keep changing in the future. Therefore, it is critical to understand how ecosystem processes will respond to the variation of precipitation. However, compared to aboveground processes, the effects of precipitation change on soil microorganisms remain poorly understood. Changbai Mountain is an ideal area to study the responses of temperate forests to the variations in precipitation. In this study, we conducted a manipulation experiment to simulation variation of precipitation in the virgin, broad-leaved Korean pine mixed forest in Changbai Mountain. Plots were designed to increase precipitation by 30 % [increased (+)] or decrease precipitation by 30 % [decreased (?)]. We analyzed differences in the diversity of the bacterial community in surface bulk soils (0–5 and 5–10 cm) and rhizosphere soils between precipitation treatments, including control. Bacteria were identified using the high-throughput 454 sequencing method. We obtained a total 271,496 optimized sequences, with a mean value of 33,242 (±1,412.39) sequences for each soil sample. Being the same among the sample plots with different precipitation levels, the dominant bacterial communities were Proteobacteria, Acidobacteria, Actinobacteria, Planctomycetes, and Chloroflexi. Bacterial diversity and abundance declined with increasing soil depth. In the bulk soil of 0–5 cm, the bacterial diversity and abundance was the highest in the control plots and the lowest in plots with reduced precipitation. However, in the soil of 5–10 cm, the diversity and abundance of bacteria was the highest in the plots of increased precipitation and the lowest in the control plots. Bacterial diversity and abundance in rhizosphere soils decreased with increased precipitation. This result implies that variation in precipitation did not change the composition of the dominant bacterial communities but affected bacterial abundance and the response patterns of the dominant communities to variation in precipitation.