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Background: Physical activity and vitamin D receptor (VDR) have been associated with type 2 diabetes mellitus (T2DM). However, the associations of VDR methylation with T2DM and physical activity remained unknown. We aimed to investigate whether VDR methylation was a link between physical activity and T2DM. Methods: A 1:1 matching case‐control study was designed based on the Henan Rural Cohort Study, including 272 pairs of T2DM patients and nonpatients. Physical activity level was assessed using the International Physical Activity Questionnaire. The high‐resolution melt method was applied to determine the methylation level of the promoter region of VDR. The association between physical activity and T2DM was analyzed with a conditional logistic regression model. The effect modification of VDR methylation levels on the association between physical activity and T2DM was conducted. A multivariate correlation analysis model was applied to investigate correlations of VDR methylation with insulin sensitivity. Results: Physical activity level was associated with T2DM risk (crude model: odds ratio [OR] 0.611; 95% CI, 0.416‐0.897; adjusted model: OR 0.619; 95% CI, 0.418‐0.917). In effect modification analysis, the effects of physical activity on T2DM were stronger for low VDR methylation levels than for high (P =.025). Moreover, VDR methylation levels were associated with insulin (r = −0.089, P =.039) as well as homeostatic model assessment of insulin resistance (r = −0.098, P =.022). Conclusions: The methylation status of the VDR promoter is associated with the secretion and sensitivity of insulin. VDR methylation attenuates the association between physical activity and T2DM, indicating that proactively physical activity may reduce the risk of T2DM, especially in people with low VDR methylation level. [ABSTRACT FROM AUTHOR]

This work aimed to develop an ultrasensitive and specific immunosorbent assay for simultaneous detection of double DNA methylation marks. Being considered the most important indicators in disease diagnosis, clinical treatment, and prognosis, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were chosen as the proof-of-concept targets. The described strategy consisted of Phos-tag Biotin anchoring at streptavidin-magnetic nanoparticles, specific immune recognition of anti-5mC antibody and anti-5hmC antibody and labeling of Barcode-antibody, signal amplification of immune PCR and digital PCR machine. Under optimal conditions, the digital immuno-PCR assay showed a board dynamic range from 2.7 × 10−13 mol/L to 2.7 × 10−9 mol/L and the detection limits were 61.7 fmol/L for 5mC, and of 0.111 pmol/L for 5hmC. A 16-fold and 186-fold improvement of LOD were obtained by the proposed approach for 5mC and 5hmC detection compared with real-time immune PCR. The approach also showed ideal specificity, repeatability and stability. The recovery test demonstrated that the digital immuno-PCR assay is a promising platform for the simultaneous determination of the two DNA methylation marks in human serum sample. In this work, a simple and sensitive digital immune PCR assay was developed for simultaneous determination of 5-methylcytosine and 5-hydroxymethylcytosine in human serum. This simultaneous detection method can effectively save serum sample and improve efficiency. [Display omitted] • Simultaneous detection of 5mC and 5hmC were of great importance for physiological and pathological study. • We successfully developed a novel digital immuno-PCR assay for simultaneous detection of 5mC and 5hmC in human serum. • The LOD of digital immuno-PCR signal readout for 5mC was 6.17 × 10−14 mol/L (61.7 fmol/L) and for 5hmC was 1.11 × 10−13 mol/L (0.111 pmol/L). [ABSTRACT FROM AUTHOR]