A pilot study on the expression of circadian clock genes in the alveolar bone of mice with periodontitis.

This study aimed to investigate the expression of circadian clock genes in mouse alveolar bone, and the possible reasons for these changes. Fifty C57 mice were orally inoculated with P. gingivalis, establishing a model of periodontitis using healthy mice as controls. The alveolar bone of both groups was taken for micro-computed tomography scanning to measure the amount of attachment loss, and the relative expression of mRNA in each clock gene and periodontitis related inflammatory factor was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). After the establishment of the mouse model, the height of alveolar bone in the periodontitis group was significantly lower than that in the normal group (p < 0.05). The relative transcriptional level of Bmal1, Per2, and Cry1 mRNA was in the circadian rhythm in the normal group (p ≤ 0.05), while in the periodontitis group, its circadian rhythm disappeared and the transcriptional level characteristics were changed. Interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), and interferon (IFN-γ) mRNA transcriptional level were elevated in the periodontitis group compared to the normal group. In conclusion, the mRNA transcriptional level of Bmal1, Per2, and Cry1 in alveolar bone of normal mice has circadian rhythm, but the rhythm disappears under the condition of periodontitis, and the cause of its occurrence may be related to inflammatory cytokines.

Discrimination of A1555G and C1494T point mutations in the mitochondrial 12S rRNA gene by on/off switch.

The objective of this study was to apply the "on/off" switch consisting of 3' phosphorothioate-modified allele specific primers and exo(+) polymerase in single base discrimination of A1555G and C1494T mutations in the highly conserved sites of the mitochondrial 12S rRNA. The two point mutations are the hotspot mutations associated with either aminoglycoside antibiotics induced deafness or inherited nonsyndromic hearing loss. The PCR products of mitochondrial DNA (mtDNA) 12S rRNA gene were inserted into the pMD19-T vector for transformation into Escherichia coli JM109 competent cells for preparing wild-type pMD19-T/mt vector. Inverse PCR was carried out for mtDNA 12S rRNA gene C1494T and A1555G mutagenesis and DpnI endonuclease degradating methylated pMD19-T/mt vector existing in the inverse PCR products was carried out to construct the mutation-type pMD19-T/mtM vector. These constructed vectors were confirmed by DNA sequencing. Allelic specific primers targeting wild-type and mutation-type templates were designed with 3' terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. Amplified by exo(+) polymerase, allelic specific primers perfectly matching wild-type allele were extended while no products were produced from primers targeting point-mutated deafness-related allele. Similarly, allelic specific primers perfectly matching point-mutated deafness-related mutation-type allele were extended and no products were yielded from primers targeting wild-type allele. No specific product was observed in the primer extension reaction mediated by on/off switch in screening the mtDNA 12S rRNA gene harboring either C1494T or A1555G mutation in 40 healthy volunteers tested. These data suggest that the "off switch" mediated by exo(+) polymerase is highly reliable in the diagnosis of monogenic diseases and the novel "on/off" switch has enormous applications in systematic and extended screening of the12S rRNA gene A1555G and C1494T mutations. The established assay can be widely used not only for hearing loss patients but also for normal subjects before the use of aminoglycoside antibiotics.