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Abstract Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three “red complex” bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.

Extracellular vesicles (EVs) have been recognized as a universal method of cellular communications and are reportedly produced in bacteria, archaea, and eukaryotes. Bacterial EVs are often called “Outer Membrane Vesicles” (OMVs) as they were the result of a controlled blebbing of the outer membrane of gram-negative bacteria such as Porphyromonas gingivalis (P. gingivalis). Bacterial EVs are natural messengers, implicated in intra- and inter-species cell-to-cell communication among microorganism populations present in microbiota. Bacteria can incorporate their pathogens into OMVs; the content of OMVs differs, depending on the type of bacteria. The production of distinct types of OMVs can be mediated by different factors and routes. A recent study highlighted OMVs ability to carry crucial molecules implicated in immune modulation, and, nowadays, they are considered as a way to communicate and transfer messages from the bacteria to the host and vice versa. This review article focuses on the current understanding of OMVs produced from major oral bacteria, P. gingivalis: generation, characteristics, and contents as well as the involvement in signal transduction of host cells and systemic diseases. Our recent study regarding the action of P. gingivalis OMVs in the living body is also summarized.

After the publication of this work [1] errors were noticed in Figs. 1a, 6a, and 8a—in which the β-actin bands were mistakenly presented.

To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses’ whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta.

The aim of this prospective cohort study was to examine whether oral hygiene knowledge, and the source of that knowledge, affect oral hygiene behavior in university students in Japan. An oral exam and questionnaire survey developed to evaluate oral hygiene knowledge, the source of that knowledge, and oral hygiene behavior, such as the frequency of tooth brushing and regular dental checkups and the use of dental floss, was conducted on university student volunteers. In total, 310 students with poor tooth brushing behavior (frequency of tooth brushing per day [≤ once]), 1,963 who did not use dental floss, and 1,882 who did not receive regular dental checkup during the past year were selected. Among these students, 50, 364, and 343 in each respective category were analyzed in over the 3-year study period (follow-up rates: 16.1%, 18.5%, and 18.2%, respectively). The odds ratios (ORs) and 95% confidence intervals (CIs) for oral hygiene behavior were calculated based on oral hygiene knowledge and the source of that knowledge using logistic regression models. The results showed that dental clinics were the most common (> 50%) source of oral hygiene knowledge, and that a more frequent use of dental floss was significantly associated with dental clinics being a source of oral hygiene knowledge (OR, 4.11; 95%CI, 1.871-9.029; p < 0.001). In addition, a significant association was seen between dental clinics being a source of oral hygiene knowledge and more frequent regular dental checkups (OR, 13.626; 95%CI, 5.971-31.095; p < 0.001). These findings suggest the existence of a relationship between dental clinics being the most common source of oral hygiene knowledge and improved oral hygiene behavior in Japanese university students.

Objective Sleep bruxism, a major sleep disorder that causes serious harm to oral health, is considered a multifactorial disease. Sleep bruxism can be induced by secondhand smoke (SHS), which adversely affects sleep quality. The objective of present study was to clarify the associations between sleep bruxism, sleep quality, and SHS exposure. Methods To assess the prevalence of sleep bruxism, sleep quality, and SHS exposure, we conducted oral examinations and self-report questionnaires on university students in Japan. The inclusion criteria were age 18 or 19 years, non-smokers and non-alcohol drinkers. The exclusion criteria were failing to complete the questionnaire in full. To assess the associations between sleep bruxism, sleep quality, and SHS exposure, we used chi-squared test and structural equation modeling. Results We analyzed a total of 1,781 Japanese young adults. Females who had been exposed to SHS had worse sleep quality (p = 0.019) than those who had not. Females with worse sleep quality showed a higher prevalence of sleep bruxism (p = 0.034) than those with better sleep quality. Using structural equation modelling, direct associations were identified between SHS exposure and poor sleep quality (standardized coefficients, 0.153; p = 0.008) and between sleep bruxism and poor sleep quality (standardized coefficients, 0.187; p = 0.022) in females. However, no association was found among males. Conclusions These results suggest that SHS exposure is associated with poor sleep quality and sleep bruxism in Japanese young adult females.

Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid–osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin, which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II, and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1. In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and enhances both osteoblast and osteoclast activity by regulating specific transcription factors.

Endochondral ossification, including bone growth and other metab olic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detect ed mRNA expression of the genes which encode the melatonin-synthesizing enzymes AA NAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric anal ysis of primary rat and chick chondrocytes. Addition of melatonin to primary BALB/c mouse chondrocytes caused enhanced cell growth and increased expression of Col2a1, Aggrecan and Sox9, but inhibited Col10a1 expression. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes p roduce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of Aanat, Mt1, Mt2 and Pthrp expression, followed by Sox9 and Ihh. Furthermore, expression of the clock gene Bmal1 was induced, while that of Per1 was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of Aanat and modified the cyclic rhythm of Bmal1, Mt1 and Mt2. In contrast, Mt1 and Mt2 showed different rhythms from Bmal1 and Aanat, indicating the existence of different regulatory genes. Our results indicate that exogeno us and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic ex pression to the central suprachiasmatic nucleus clock. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate whether a Keap1-dependent oxidative stress detector-luciferase (OKD-LUC) mouse model would be useful for the visualization of oxidative stress induced by experimental periodontitis. A ligature was placed around the mandibular first molars for seven days to induce periodontitis. Luciferase activity was measured with an intraperitoneal injection of D-luciferin on days 0, 1, and 7. The luciferase activity in the periodontitis group was significantly greater than that in the control group at seven days. The expressions of heme oxygenase-1 (HO-1) and malondialdehyde in periodontal tissue were significantly higher in the periodontitis group than in the control group. Immunofluorescent analysis confirmed that the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) occurred more frequently in the periodontitis group than in the control group. This study found that under oxidative stress induced by experimental periodontitis, the Nrf2/antioxidant defense pathway was activated and could be visualized from the luciferase activity in the OKD-LUC model. Thus, the OKD-LUC mouse model may be useful for exploring the mechanism underlying the relationship between the Nrf2/antioxidant defense pathway and periodontitis by enabling the visualization of oxidative stress over time. [ABSTRACT FROM AUTHOR]