GamaComet: A Deep Learning-Based Tool for the Detection and Classification of DNA Damage from Buccal Mucosa Comet Assay Images.

Comet assay is a simple and precise method to analyze DNA damage. Nowadays, many research studies have demonstrated the effectiveness of buccal mucosa cells usage in comet assays. However, several software tools do not perform well for detecting and classifying comets from a comet assay image of buccal mucosa cells because the cell has a lot more noise. Therefore, a specific software tool is required for fully automated comet detection and classification from buccal mucosa cell swabs. This research proposes a deep learning-based fully automated framework using Faster R-CNN to detect and classify comets in a comet assay image taken from buccal mucosa swab. To train the Faster R-CNN model, buccal mucosa samples were collected from 24 patients in Indonesia. We acquired 275 comet assay images containing 519 comets. Furthermore, two strategies were used to overcome the lack of dataset problems during the model training, namely transfer learning and data augmentation. We implemented the proposed Faster R-CNN model as a web-based tool, GamaComet, that can be accessed freely for academic purposes. To test the GamaComet, buccal mucosa samples were collected from seven patients in Indonesia. We acquired 43 comet assay images containing 73 comets. GamaComet can give an accuracy of 81.34% for the detection task and an accuracy of 66.67% for the classification task. Furthermore, we also compared the performance of GamaComet with an existing free software tool for comet detection, OpenComet. The experiment results showed that GamaComet performed significantly better than OpenComet that could only give an accuracy of 11.5% for the comet detection task. Downstream analysis can be well conducted based on the detection and classification results from GamaComet. The analysis showed that patients owning comet assay images containing comets with class 3 and class 4 had a smoking habit, meaning they had more cells with a high level of DNA damage. Although GamaComet had a good performance, the performance for the classification task could still be improved. Therefore, it will be one of the future works for the research development of GamaComet.

Alteration of mucosa cell maturation pattern after exposure to different radiographic imaging methods

Aim: Dental imaging has been widely used for diagnosis in dentistry. However, dental X-ray may induce cytotoxicity leading to apoptosis in oral mucosa cells. The present study aimed to observe the maturation pattern of buccal and gingival cells after exposure to X-ray radiation from analog/digital panoramic scanning and cone beam computed tomography (CBCT). Methods: The research samples were 40 subjects who fulfilled the inclusion and exclusion criteria. The subjects were divided into the exposed (patients who received analog/digital panoramic radiography or CBCT) and controlled (patients who had no radiography examinations) groups, with 10 subjects in each group. Exfoliative cytology smears were obtained from buccal mucosa and gingiva before exposure (or on day 0 for the control group) and 10 days later. The cells were stained with the Papanicolaou method. Then, the superficial, intermediate, and parabasal cells were counted in each glass slide. Results: No significant differences (p > 0.05) were observed among all cell types between day 0 and 10 in the control group. Meanwhile, after exposure to three kinds of radiography examinations, the frequency of intermediate cells in buccal mucosa and gingiva increased (p < 0.05), but that of superficial cells decreased (p < 0.05) significantly. No significant difference was found in the parabasal cells (p > 0.05). The frequency differences between intermediate and superficial cells showed no significant difference between the buccal mucosa and gingiva. Conclusion: Analog/digital panoramic radiography and CBCT exposure can induce cytotoxicity by altering the maturation pattern of buccal mucosa cells and gingiva, so it is strongly recommended to only perform these procedures if necessary and avoid repeated exposure to the same patient

Comparison of radiation risk perception and knowledge of radiation between Indonesian and Japanese dental students.

The purpose of the study was to obtain basic data to identify problems in radiation education in a situation where confidence in nuclear power has fluctuated over time and fear of nuclear power has increased globally due to nuclear power plant disasters at Chernobyl and Fukushima. We conducted a questionnaire survey on understanding and risk perception of radiation and atomic power, before and after lectures, for 107 Japanese and 137 Indonesian dental students. Thirty-six phrases were extracted from two supplementary texts about radiation created by the Japanese Ministry of Education, Culture, Sports, Science and Technology, and 30 events commonly used in research on risk perception were used. The students were asked to rate their level of understanding of 36 phrases and risk perception of 30 events. Moreover, the students were asked to answer 6 general questions about radiation. For Japanese students, understanding of radiation increased and risk perception for both nuclear power and X-rays decreased after lectures (p < 0.05). Concerning nuclear power, the risk-value declined as the level of understanding increased (p < 0.01). However, for Indonesian students, who had lectures on only radiation excluding nuclear power in dental radiology, risk perception increased for X-ray after lectures (p < 0.05). This indicates that thought and custom, in the absence of knowledge, are influenced by lectures. In general, it is said that increase in knowledge will lower risk perception, but even if radiation education is imparted, risk perception may rise if the lectures are not understood properly. It was concluded that educators need to incorporate sufficient knowledge in their teachings, and correct thinking, to mitigate the risk of future radiation education giving the opposite of the intended effect.

Sp6 regulation of Rock1 promoter activity in dental epithelial cells.

Sp6 is a transcription factor of the SP/KLF family and an indispensable regulator of the morphological dynamics of ameloblast differentiation during tooth development. However, the underlying molecular mechanisms remain unclear. We have previously identified one of the Sp6 downstream genes, Rock1, which is involved in ameloblast polarization. In this study, we investigated the transcriptional regulatory mechanisms of Rock1 by Sp6. First, we identified the transcription start sites (TSS) and cloned the 5'-flanking region of Rock1. Serial deletion analyses identified a critical region for Rock1 promoter activity within the 249-bp upstream region of TSS, and chromatin immunoprecipitation assays revealed Sp6-binding to this region. Subsequent transient transfection experiments showed that Rock1 promoter activity is enhanced by Sp6, but reduced by Sp1. Treatment of dental epithelial cells with the GC-selective DNA binding inhibitor, mithramycin A, affected Rock1 promoter activity in loss of enhancement by Sp6, but not repression by Sp1. Further site-directed mutagenesis indicated that the region from -206 to -150 contains responsive elements for Sp6. Taken together, we conclude that Sp6 positively regulates Rock1 transcription by direct binding to the Rock1 promoter region from -206 to -150, which functionally distinct from Sp1.

Ctip2-mediated Sp6 transcriptional regulation in dental epithelium-derived cells.

Tooth development relies on the interaction between the oral ectoderm and underlying mesenchyme, and is regulated by a complex genetic cascade. This transcriptional cascade is regulated by the spatiotemporal activation and deactivation of transcription factors. The specificity proteins 6 (Sp6) and chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (Ctip2) were identified in loss-of-function studies as key transcription factors required for tooth development. Ctip2 binds to the Sp6 promoter in vivo; however, its role in Sp6 expression remains unclear. In this study, we investigated Sp6 transcriptional regulation by Ctip2. Immunohistochemical analysis revealed that Sp6 and Ctip2 colocalize in the rat incisor during tooth development. We examined whether Ctip2 regulates Sp6 promoter activity in dental epithelial cells. Cotransfection experiments using serial Sp6 promoter-luciferase constructs and Ctip2 expression plasmids showed that Ctip2 significantly suppressed the Sp6 second promoter activity, although the Sp6 first promoter activity was unaffected. Ctip2 was able to bind to the proximal region of the Sp6 first promoter, as previously demonstrated, and also to the novel distal region of the first, and second promoter regions. Our findings indicate that Ctip2 regulates Sp6 gene expression through direct binding to the Sp6 second promoter region. J. Med. Invest. 61: 126-136, February, 2014.

Possible linkage of SP6 transcriptional activity with amelogenesis by protein stabilization.

Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis) are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.