Association between serum miRNAs and gingival gene expression in an obese rat model.

INTRODUCTION: Recent studies have reported a relationship between periodontitis and obesity; however, the mechanisms of obesity's effects on periodontitis are not well understood. On the other hand, microRNAs (miRNAs) are known to play key roles in the post-transcriptional regulation gene expression by suppressing translation and protein synthesis. We examined the association between obesity-related miRNAs and gene expression in gingival tissue using miRNA-messenger RNA (mRNA) pairing analysis in an obese rat model. METHODS: Sixteen male Wistar rats aged 8 weeks old were divided into two groups: the control group was fed a normal powdered food for 8 weeks, and the obesity group was fed a high-fat diet for 8 weeks. Distance from the cement-enamel junction to the alveolar bone crest of the first molars was measured. miRNA microarray analysis was performed on samples of serum and gingival tissue; the resulting data were used to calculate fold changes in miRNA levels in the obesity group relative to the control group, and miRNA-mRNA pairing analysis was performed to identify mRNAs potentially targeted by miRNAs of interest. RESULTS: Alveolar bone loss in the obesity group exceeded that in the control group (p = .017). miRNA-mRNA pairing analysis identified an association between 4 miRNAs (miR-759, miR-9a-3p, miR-203b-3p, and miR-878) that were differentially expressed in the obesity and control groups and 7 genes (Ly86, Arid5b, Rgs18, Mlana, P2ry13, Kif1b, and Myt1) expressed in gingival tissue. CONCLUSION: This study revealed that several miRNAs play an important role in the mechanism of periodontal disease progression induced by the obesity.

Living with Family Is Directly Associated with Regular Dental Checkup and Indirectly Associated with Gingival Status among Japanese University Students: A 3-Year Cohort Study.

Although some studies showed that lifestyle was associated with oral health behavior, few studies investigated the association between household type and oral health behavior. The aim of this prospective cohort study was to investigate the association between household type, oral health behavior, and periodontal status among Japanese university students. Data were obtained from 377 students who received oral examinations and self-questionnaires in 2016 and 2019. We assessed periodontal status using the percentage of bleeding on probing (%BOP), probing pocket depth, oral hygiene status, oral health behaviors, and related factors. We used structural equation modeling to determine the association between household type, oral health behaviors, gingivitis, and periodontitis. At follow-up, 252 students did not live with their families. The mean ± standard deviation of %BOP was 35.5 ± 24.7 at baseline and 32.1 ± 25.3 at follow-up. In the final model, students living with their families were significantly more likely to receive regular dental checkup than those living alone. Regular checkup affected the decrease in calculus. The decrease in calculus affected the decrease in %BOP over 3 years. Living with family was directly associated with regular dental checkups and indirectly contributed to gingival status among Japanese university students.

Caries Increment and Salivary Microbiome during University Life: A Prospective Cohort Study.

The purpose of this 3-year prospective cohort study was to explore the relationship between an increase in dental caries and oral microbiome among Japanese university students. We analyzed 487 students who volunteered to receive oral examinations and answer baseline (2013) and follow-up (2016) questionnaires. Of these students, salivary samples were randomly collected from 55 students at follow-up and analyzed using next-generation sequencing. Students were divided into two groups: increased group (Δdecayed, missing, and filled teeth (ΔDMFT) score increased during the 3-year period) and non-increased group (ΔDMFT did not increase). Thirteen phyla, 21 classes, 32 orders, 48 families, 72 genera, and 156 species were identified. Microbial diversity in the increased group (n = 14) was similar to that in the non-increased group (n = 41). Relative abundances of the family Prevotellaceae (p = 0.007) and genera Alloprevotella (p = 0.007) and Dialister (p = 0.039) were enriched in the increased group compared with the non-increased group. Some bacterial taxonomic clades were differentially present between the two groups. These results may contribute to the development of new dental caries prevention strategies, including the development of detection kits and enlightenment activities for these bacteria.

Effects of Coffee Intake on Oxidative Stress During Aging-related Alterations in Periodontal Tissue.

BACKGROUND/AIM: The purpose of this study was to determine the anti-aging effects of coffee intake on oxidative stress in rat periodontal tissue and alveolar bone loss. MATERIALS AND METHODS: Male Fischer 344 rats (8 weeks old) were randomized to four groups; the baseline group immediately sacrificed, the control group fed with normal powdered food for 8 weeks, and the experimental groups fed with powdered food containing 0.62% or 1.36% coffee components for 8 weeks. RESULTS: Alveolar bone loss and gingival level of 8-hydroxydeoxyguanosine were significantly lower in the 1.36% coffee group than in the control group. Nuclear factor erythroid 2-related factor 2 translocation to the nucleus was significantly higher in the 1.36% coffee group than in the control group. CONCLUSION: Continuous intake of 1.36% coffee could prevent age-related oxidative stress in the periodontal tissue and alveolar bone loss, possibly by up-regulating the Nrf2 signaling pathway.

Relationship of Salivary Microbiome with the Worsening of the Periodontal Health Status in Young Adults: A 3-Year Cohort Study.

The purpose of this prospective cohort study was to investigate the influence of the salivary microbiome on the worsening of the periodontal health status among Japanese young adults. We assessed the data of systemically healthy and non-smoking young (18-22 years) university students (n = 457) from Okayama University at baseline (2013) and follow-up (2016). The worsening group was defined based on an increase in the percentage of bleeding on probing (%BOP) or an increase in probing pocket depth (PPD) from <4 mm to ≥4 mm. Unstimulated saliva samples were randomly collected from 69 students for microbiome analysis at follow-up. The salivary microbiome was assessed through 16S rRNA metagenomic sequencing. The type of community in the salivary microbiome clustered by statistical analysis and diversity was not significantly associated with the worsening of the periodontal health status in cases of increasing %BOP and PPD (p > 0.05). The prevalence of some species was significantly higher in the worsening group than in the non-worsening group (p < 0.05) in both cases. The worsening of the periodontal health status was associated with some species, but not the type of community and diversity in the salivary microbiome among Japanese young adults.

Detection of Serum miRNAs Affecting Liver Apoptosis in a Periodontitis Rat Model.

BACKGROUND/AIM: miRNA molecules have been attracting attention as genetic modifiers between organs. We examined the relationship between serum miRNA and targeted liver mRNA profiles in a periodontitis rat model, and the influence of periodontitis on the liver. MATERIALS AND METHODS: Male Wistar rats (n=16, 8 weeks old) were randomly divided into two groups (8 rats each): control and periodontitis (ligature placement for 4 weeks). Serum miRNA and liver mRNA profiles were compared. RESULTS: Periodontal destruction and hepatocyte apoptosis were induced in the periodontitis group. Microarray analysis indicated that 52 serum miRNAs and 33 liver mRNAs were expressed with a >1.5-fold change (FC) and a >2.0-FC (p<0.05), respectively, between the two groups. From the miRNA target genes, 12 genes equivalented to liver mRNAs with a >2.0-FC, among which, Hyou1, Chac1, and Bloc1s3 have apoptotic functions in our model. miRNAs upstream of these 3 mRNAs are miR-3591, miR-181a-2-3p and miR-6321. CONCLUSION: miR-3591, miR-181a-2-3p and miR-6321 induced hepatocyte apoptosis in our periodontitis rat model.

Awareness of Clenching and Underweight are Risk Factors for Onset of Crowding in Young Adults: A Prospective 3-Year Cohort Study.

Bruxism is a parafunctional activity that can seriously affect quality of life. Although bruxism induces many problems in the oral and maxillofacial area, whether it contributes to the onset of malocclusion remains unclear. The purpose of this prospective cohort study was to investigate the association between the onset of malocclusion and awareness of clenching during the daytime in young adults. Among 1,092 Okayama University students who underwent normal occlusion at baseline, we analysed 238 who had undergone a dental examination and had complete data after 3 years (2013⁻2016). We also performed subgroup analysis to focus on the association between awake bruxism and the onset of crowding (n = 216). Odds ratios (ORs) were calculated using multivariate logistic regression analyses. The incidences of malocclusion and crowding were 53.8% and 44.5%, respectively. In multivariate logistic regression, awareness of clenching was a risk factor for crowding (OR: 3.63; 95% confidence interval [CI]: 1.08⁻12.17). Moreover, underweight (body mass index < 18.5 kg/m²) was related to the onset of malocclusion (OR: 2.34; 95%CI: 1.11⁻4.92) and crowding (OR: 2.52, 95%CI: 1.25⁻5.76). These results suggest that awareness of clenching during the daytime and underweight are risk factors for the onset of crowding in young adults.

Relationship Between Renal Dysfunction and Oral Mucositis in Patients Undergoing Concurrent Chemoradiotherapy for Pharyngeal Cancer: A Retrospective Cohort Study.

BACKGROUND/AIM: The aim of this retrospective cohort study was to investigate the association between renal dysfunction (RD) and the development of oral mucositis (OM) in patients undergoing concurrent chemoradiotherapy (CCRT) for pharyngeal cancer including radiation to the oral cavity. PATIENTS AND METHODS: Of 130 patients diagnosed as having pharyngeal cancer who received CCRT at the Okayama University Hospital Head and Neck Cancer Center, 44 were finally selected. RESULTS: During the observation period, 24 (54.5%) patients experienced severe OM (grade 3). The Cox proportional hazards regression model demonstrated that RD (hazard ratio(HR)=2.45, 95% confidence interval(CI)=1.067-6.116, p=0.035) and nasopharynx/oropharynx as center of the irradiated area (HR=2.56, 95% CI=1.072-5.604, p=0.034) were significantly associated with the incidence of severe OM (grade 3). CONCLUSION: In patients with pharyngeal cancer treated with CCRT including radiation to the oral cavity, RD at baseline can be a risk factor for developing severe OM.

Topical application of ointment containing 0.5% green tea catechins suppresses tongue oxidative stress in 5-fluorouracil administered rats.

OBJECTIVE: The purpose of this study was to investigate the preventive effects of topical application of green tea catechins on tongue oxidative stress induced by 5-fluorouracil (5-FU) administration in rats. DESIGN: Male Wistar rats (n=28, 8 weeks old) were divided into four groups of seven rats each: a negative control group (saline administration and application of ointment without green tea catechins), a positive control group (5-FU administration and application of ointment without green tea catechins), and two experimental groups (5-FU administration and application of ointment containing 0.1% or 0.5% green tea catechins). Topical application of each ointment to the ventral surface of the tongue was performed once a day for 5days. The level of 8-hydroxydeoxyguanosine (8-OHdG) was determined to evaluate oxidative stress. Fluorescence staining was also performed to confirm nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus. RESULTS: After the experimental period, the ratios of 8-OHdG-positive cells in the ventral tongue tissue were higher in the positive control group than in the negative control group (P<0.05). On the other hand, those in the 0.5% green tea catechin group, but not in the 0.1% green tea catechin group, were lower than the positive control group (P<0.05). In addition, Nrf2 translocation to the nucleus was greater in the 0.5% green tea catechin group than in the positive control group (P<0.05). CONCLUSIONS: Topical application of ointment containing 0.5% green tea catechins could prevent tongue oxidative stress in 5-FU administered rats, via up-regulation of the Nrf2 signaling pathway.

Expression of Salivary miR-203a-3p Was Related with Oral Health-Related Quality of Life in Healthy Volunteers.

Oral health-related quality of life (OHRQoL) is a multidimensional construct that involves subjective evaluation of an individual's oral health. Although it is difficult to evaluate OHRQoL biologically, recently, it has been reported that circulating microRNAs (miRNAs) in several body fluids could reflect various health conditions. The aim of this pilot study was to investigate whether salivary miRNAs expression differs according to OHRQoL in healthy volunteers. Forty-six volunteers (median age, 23.0 years) were recruited, and their OHRQoL was assessed using the Japanese version of the Oral Health Impact Profile (OHIP-J). Then, we compared salivary microRNA profiles of the high-OHRQoL group (≤25th percentile score of OHIP-J) and the low-OHRQoL group (≥75th percentile score of OHIP-J) using the polymerase chain reaction (PCR) array and the quantitative real-time PCR. There were no significant differences between the two groups in terms of oral health status. In the PCR array, miR-203a-3p and miR-30b-5p were significantly more expressed in the low-OHRQoL group (p < 0.05). Quantitative real-time PCR assay also showed that miR-203a-3p was more highly expressed in the low-OHRQoL group than in the high-OHRQoL group (p < 0.05). These observations suggest that expression of salivary miR-203a-3p was related with OHRQoL in healthy volunteers.

Generation of viable fish from cryopreserved primordial germ cells.

An increasing number of wild fish species are in danger of extinction, often as a result of human activities. The cryopreservation of gametes and embryos has great potential for maintaining and restoring threatened species. The conservation of both paternal and maternal genetic information is essential. However, although this technique has been successfully applied to the spermatozoa of many fish species, reliable methods are lacking for the long-term preservation of fish eggs and embryos. Here, we describe a protocol for use with rainbow trout (Oncorhynchus mykiss) primordial germ cells (PGCs) and document the restoration of live fish from gametes derived from these cryopreserved progenitors. Genital ridges (GRs), which are embryonic tissues containing PGCs, were successfully cryopreserved in a medium containing 1.8 M ethylene glycol (EG). The thawed PGCs that were transplanted into the peritoneal cavities of allogenic trout hatchlings differentiated into mature spermatozoa and eggs in the recipient gonads. Furthermore, the fertilization of eggs derived from cryopreserved PGCs by cryopreserved spermatozoa resulted in the development of fertile F1 fish. This PGC cryopreservation technique represents a promising tool in efforts to save threatened fish species. Moreover, this approach has significant potential for maintaining domesticated fish strains carrying commercially valuable traits for aquaculture purposes.

Manipulation of fish germ cell: visualization, cryopreservation and transplantation.

Germ-cell transplantation has many applications in biology and animal husbandry, including investigating the complex processes of germ-cell development and differentiation, producing transgenic animals by genetically modifying germline cells, and creating broodstock systems in which a target species can be produced from a surrogate parent. The germ-cell transplantation technique was initially established in chickens using primordial germ cells (PGCs), and was subsequently extended to mice using spermatogonial stem cells. Recently, we developed the first germ-cell transplantation system in lower vertebrates using fish PGCs and spermatogonia. During mammalian germ-cell transplantation, donor spermatogonial stem cells are introduced into the seminiferous tubules of the recipient testes. By contrast, in the fish germ-cell transplantation system, donor cells are microinjected into the peritoneal cavities of newly hatched embryos; this allows the donor germ cells to migrate towards, and subsequently colonize, the recipient genital ridges. The recipient embryos have immature immune systems, so the donor germ cells can survive and even differentiate into mature gametes in their allogeneic gonads, ultimately leading to the production of normal offspring. In addition, implanted spermatogonia can successfully differentiate into sperm and eggs, respectively, in male and female recipients. The results of transplantation studies in fish are improving our understanding of the development of germ-cell systems during vertebrate evolution.

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae.

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.

Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry.

A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67: 91-100, 2004.

Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter.

Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.