Dipotassium glycyrrhizate and hinokitiol enhance macrophage efferocytosis by regulating recognition, uptake, and metabolism of apoptotic cells in vitro.

BACKGROUND AND OBJECTIVE: Efferocytosis is a process whereby macrophages remove apoptotic cells, such as neutrophils, that have accumulated in tissues, which is required for resolution of inflammation. Efferocytosis is impaired in individuals with increasing age and in those with various systemic diseases. Recently, efferocytosis has been reported to be related to the pathogenesis and progression of periodontitis, and enhancement of efferocytosis, especially in the subjects with impaired efferocytosis, was suggested to lead to periodontitis prevention and care. Various anti-inflammatory ingredients are used in oral care products, but their effect on efferocytosis is unclear. Here, we aimed to identify ingredients contained in oral care products that are effective for efferocytosis regulation. METHODS: The ability of dead cells to induce inflammation in human gingival fibroblast (HGF) cells were evaluated by measuring IL-6 secretion. Six ingredients in oral care products used as anti-inflammatory agents were evaluated for their effect on efferocytosis using flow cytometry. The expression of various efferocytosis-related molecules, such as MERTK and LRP1 involved in recognition, and LXRα and ABCA1 that function in metabolism, were measured in RAW264.7 cells with or without ingredient treatment. Rac1 activity, which is related to the uptake of dead cells, was measured using the G-LISA kit. RESULTS: Dead cells elicited IL-6 secretion in HGF cells. Among the six ingredients, GK2 and hinokitiol enhanced efferocytosis activity. GK2 and hinokitiol significantly increased the expression of MERTK and LRP1, and also enhanced LXRα and ABCA1 expression after efferocytosis. Furthermore, they increased Rac1 activity in the presence of dead cells. CONCLUSION: Among the six ingredients tested, GK2 and hinokitiol promoted efferocytosis by regulating apoptotic cell recognition, uptake, and metabolism-related molecules. Efferocytosis upregulation may be one of the mechanisms of GK2 and hinokitiol in the treatment of inflammatory diseases, such as periodontitis.

Integrated analysis of the oral and intestinal microbiome and metabolome of elderly people with more than 26 original teeth: a pilot study.

Elderly subjects with more than 20 natural teeth have a higher healthy life expectancy than those with few or no teeth. The oral microbiome and its metabolome are associated with oral health, and they are also associated with systemic health via the oral-gut axis. Here, we analyzed the oral and gut microbiome and metabolome profiles of elderly subjects with more than 26 natural teeth. Salivary samples collected as mouth-rinsed water and fecal samples were obtained from 22 healthy individuals, 10 elderly individuals with more than 26 natural teeth and 24 subjects with periodontal disease. The oral microbiome and metabolome profiles of elderly individuals resembled those of subjects with periodontal disease, with the metabolome showing a more substantial differential abundance of components. Despite the distinct oral metabolome profiles, there was no differential abundance of components in the gut microbiome and metabolomes, except for enrichment of short-chain fatty acids in elderly subjects. Finally, to investigate the relationship between the oral and gut microbiome and metabolome, we analyzed bacterial coexistence in the oral cavity and gut and analyzed the correlation of metabolite levels between the oral cavity and gut. However, there were few associations between oral and gut for bacteria and metabolites in either elderly or healthy subjects. Overall, these results indicate distinct oral microbiome and metabolome profiles, as well as the lack of an oral-gut axis in elderly subjects with a high number of natural teeth.

Dysbiosis of oral microbiome persists after dental treatment-induced remission of periodontal disease and dental caries.

IMPORTANCE: We characterized the oral conditions, salivary microbiome, and metabolome after dental treatment by investigating the state after treatment completion and transition to self-care. Dental treatment improved oral health conditions, resulting in oral disease remission; however, the imbalanced state of the salivary microbiome continued even after remission. Although the results of this study are preliminary, owing to the small number of participants in each group when compared to larger cohort studies, they indicate that the risk of disease may remain higher than that of healthy participants, thereby demonstrating the importance of removing dental plaque containing disease-related bacteria using appropriate care even after treatment completion. We also identified bacterial species with relative abundances that differed from those of healthy participants even after remission of symptoms, which may indicate that the maturation of certain bacterial species must be controlled to improve the oral microbiome and reduce the risk of disease recurrence.

Identification of gravity-responsive proteins in the femur of spaceflight mice using a quantitative proteomic approach.

Although the microgravity (µ-g) environment that astronauts encounter during spaceflight can cause severe acute bone loss, the molecular mechanism of this bone loss remains unclear. To investigate the gravity-response proteins involved in bone metabolism, it is important to comprehensively determine which proteins exhibit differential abundance associated with mechanical stimuli. However, comprehensive proteomic analysis using small bone samples is difficult because protein extraction in mineralized bone tissue is inefficient. Here, we established a high-sensitivity analysis system for mouse bone proteins using data-independent acquisition mass spectrometry. This system successfully detected 40 proteins in the femoral diaphysis showing differential abundance between mice raised in a µ-g environment, where the bone mass was reduced by gravity unloading, and mice raised in an artificial 1-gravity environment on the International Space Station. Additionally, 22 proteins, including noncollagenous bone matrix proteins, showed similar abundance between the two groups in the mandible, where bone mass was unaltered due to mastication stimuli, suggesting that these proteins are responsive to mechanical stimuli. One of these proteins, SPARCL1, is suggested to promote osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand. We expect these findings to lead to new insights into the mechanisms of bone metabolism induced by mechanical stimuli. SIGNIFICANCE: We aimed to investigate the gravity-response proteins involved in bone metabolism. To this end, we established a comprehensive analysis system for mouse bone proteins using data-independent acquisition mass spectrometry, which is particularly useful in comprehensively analyzing the bone proteome using small sample volumes. In addition, a comprehensive proteomic analysis of the femoral diaphysis and mandible, which exhibit different degrees of bone loss in mice raised on the International Space Station, identified proteins that respond to mechanical stimuli. SPARCL1, a mechanical stimulus-responsive protein, was consequently suggested to be involved in osteoclast differentiation associated with bone remodeling. Our findings represent an important step toward elucidating the molecular mechanism of bone metabolism induced by mechanical stimuli.

An in vitro study of the effects of Phellodendron bark extract and berberine chloride on periodontal pathogenic bacteria in the oral microbiome.

OBJECTIVES: Periodontal disease is triggered by oral microbiome dysbiosis. Thus, to prevent its onset, it is important to maintain relative abundance of periodontal pathogenic bacteria in the oral microbiome at a low level. While Phellodendron bark extract (PBE) and its active ingredient, berberine, exert antibacterial effects on periodontal pathogenic bacteria, such as Porphyromonas gingivalis, their effects on the oral microbiome as a whole remain unknown. Therefore, we aimed to clarify the potential of PBE and berberine chloride (BC) in regulating the relative abundance of periodontal pathogenic bacteria in the oral microbiome. METHODS: Saliva was collected from 20 participants. Each participant's saliva was combined separately with P. gingivalis suspension and either PBE or BC in a modified basal medium. The samples were then incubated under anaerobic conditions for 24 h. After cultivation, we determined the total bacterial concentration using quantitative polymerase chain reaction analysis and the bacterial composition using 16 S ribosomal RNA gene sequencing. RESULTS: The total bacterial concentration was reduced because of treatment with PBE and BC. Bacterial 16 S ribosomal RNA gene sequencing confirmed that treatment with PBE and BC significantly reduced the relative abundance of periodontal pathogenic bacteria, including red and orange complex bacteria. CONCLUSIONS: Our findings suggest that PBE and BC reduce the relative abundance of periodontal pathogenic bacteria in the oral microbiome. Thus, PBE and BC can aid in preventing periodontal disease, given their ability to regulate the oral microbiome composition and their anti-inflammatory effects.

Comparison of oral metabolome profiles of stimulated saliva, unstimulated saliva, and mouth-rinsed water.

Saliva includes a substantial amount of biological information, which has enabled us to understand the relationship between oral metabolites and various oral and systemic disorders. However, collecting saliva using a controlled protocol is time-consuming, making saliva an unsuitable analyte in large cohort studies. Mouth-rinsed water (MW), the water used to rinse the mouth, can be collected easily in less time with less difference between subjects than saliva and could be used as an alternative in oral metabolome analyses. In this study, we investigated the potential of MW collection as an efficient alternative to saliva sample collection for oral metabolome profiling. MW, stimulated saliva, and unstimulated saliva were collected from 10 systemically healthy participants. The samples were subjected to metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry, and the types and amounts of metabolites in the samples were compared. Qualitatively, MW contained the same metabolites as unstimulated and stimulated saliva. While the quantity of the metabolites did not drastically change between the sampling methods, all three reflected individual differences, and the features of MW were the same as those of the unstimulated saliva. Overall, these results suggest that MW may be an appropriate alternative to saliva in oral metabolome profile analysis.

A cross-sectional study of relationships between periodontal disease and general health: The Hitachi Oral Healthcare Survey.

BACKGROUND: This cross-sectional study performed to clarify the relationship between periodontal disease and non-communicable diseases (NCDs), such as obesity, diabetes mellitus, impaired glucose tolerance (IGT), chronic obstructive pulmonary disease (COPD), and atherosclerotic cardiovascular disease (ASCVD) by introducing dental examinations into the annual health examinations conducted by Japanese companies, and to highlights the importance of a medical system that connects dental and medical professionals. METHODS: A total of 1.022 Hitachi Ltd. employees were enrolled in this cross-sectional study. We examined correlations and odds ratios (ORs) between the dental and overall health of employees using stratification and multiple logistic regression analyses based on the periodontal health indicators, general health indicators, and occlusal force. RESULTS: The adjusted OR of PPD for obesity (OR, 1.42; 95% confidence interval [CI], 1.09-1.84; p = 0.009), IGT (OR, 1.48; 95% CI, 1.00-2.20; p = 0.049), and COPD (OR, 1.38; 95% CI, 1.02-1.88; p = 0.038) significantly differed. The adjusted OR of body mass index (OR, 1.28; 95% CI 1.15-1.42; p < 0.001), haemoglobin A1C (HbA1c) (OR, 4.34; 95% CI, 1.89-9.98; p < 0.001), fasting blood glucose (FBG) levels (OR, 1.08; 95% CI 1.04-1.11; p < 0.001), postbronchodilator forced expiratory volume in one second/forced vital capacity ratio (%FEV1) (OR, 0.95; 95% CI 0.91-1.00; p = 0.031) and smoking (OR, 2.32; 95% CI 1.62-3.33; p < 0.001) for severe periodontal disease also significantly differed. Occlusal force was significantly reduced in employees aged 50-59 years compared to those aged 40-49 years. Both PPD, HbA1c, FBG levels were significantly associated with occlusal force among employees with moderate/severe periodontitis. PPD was significantly associated with occlusal force among employees with and moderate COPD, and ASCVD. %FEV1 was significantly associated with occlusal force among employees with IGT. CONCLUSIONS: This cross-sectional study revealed mutual relationships among periodontal disease, NCDs, and occlusal force on Japanese corporate workers. We demonstrated that a comprehensive, regional healthcare system centred on annual integrated dental and physical health examinations in the workplace will benefit employees and positively impact corporate health insurance.

Effects of microgravity exposure and fructo-oligosaccharide ingestion on the proteome of soleus and extensor digitorum longus muscles in developing mice.

Short-chain fatty acids produced by the gut bacterial fermentation of non-digestible carbohydrates, e.g., fructo-oligosaccharide (FOS), contribute to the maintenance of skeletal muscle mass and oxidative metabolic capacity. We evaluated the effect of FOS ingestion on protein expression of soleus (Sol) and extensor digitorum longus muscles in mice exposed to microgravity (µ-g). Twelve 9-week-old male C57BL/6J mice were raised individually on the International Space Station under µ-g or artificial 1-g and fed a diet with or without FOS (n = 3/group). Regardless of FOS ingestion, the absolute wet weights of both muscles tended to decrease, and the fiber phenotype in Sol muscles shifted toward fast-twitch type following µ-g exposure. However, FOS ingestion tended to mitigate the µ-g-exposure-related decrease in oxidative metabolism and enhance glutathione redox detoxification in Sol muscles. These results indicate that FOS ingestion mildly suppresses metabolic changes and oxidative stress in antigravity Sol muscles during spaceflight.

Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice.

Investigating protein abundance profiles is important to understand the differences in the slow and fast skeletal muscle characteristics. The profiles in soleus (Sol) and extensor digitorum longus (EDL) muscles in mice exposed to 1 g or 3 g for 28 d were compared. The biological implications of the profiles revealed that hypergravity exposure activated a larger number of pathways involved in protein synthesis in Sol. In contrast, the inactivation of signalling pathways involved in oxidative phosphorylation were conspicuous in EDL. These results suggested that the reactivity of molecular pathways in Sol and EDL differed. Additionally, the levels of spermidine synthase and spermidine, an important polyamine for cell growth, increased in both muscles following hypergravity exposure, whereas the level of spermine oxidase (SMOX) increased in EDL alone. The SMOX level was negatively correlated with spermine content, which is involved in muscle atrophy, and was higher in EDL than Sol, even in the 1 g group. These results indicated that the contribution of SMOX to the regulation of spermidine and spermine contents in Sol and EDL differed. However, contrary to expectations, the difference in the SMOX level did not have a significant impact on the growth of these muscles following hypergravity exposure. SIGNIFICANCE: The skeletal muscle-specific protein abundance profiles result in differences in the characteristics of slow and fast skeletal muscles. We investigated differences in the profiles in mouse slow-twitch Sol and fast-twitch EDL muscles following 28-d of 1 g and 3 g exposure by LC-MS/MS analysis and label-free quantitation. A two-step solubilisation of the skeletal muscle proteins increased the coverage of proteins identified by LC-MS/MS analysis. Additionally, this method reduced the complexity of samples more easily than protein or peptide fractionation by SDS-PAGE and offline HPLC while maintaining the high operability of samples and was reproducible. A larger number of hypergravity-responsive proteins as well as a prominent increase in the wet weights was observed in Sol than EDL muscles. The biological implications of the difference in the protein abundance profiles in 1 g and 3 g groups revealed that the reactivity of each molecular pathway in Sol and EDL muscles to hypergravity exposure differed significantly. In addition, we found that the biosynthetic and interconversion pathway of polyamines, essential factors for cell growth and survival in mammals, was responsive to hypergravity exposure; spermidine and spermine contents in Sol and EDL muscles were regulated by different mechanisms even in the 1 g group. However, our results indicated that the difference in the mechanism regulating polyamine contents is unlikely to have a significant effect on the differences in Sol and EDL muscle growth following hypergravity exposure.

Experimental evidence for the thermophilicity of ancestral life.

Theoretical studies have focused on the environmental temperature of the universal common ancestor of life with conflicting conclusions. Here we provide experimental support for the existence of a thermophilic universal common ancestor. We present the thermal stabilities and catalytic efficiencies of nucleoside diphosphate kinases (NDK), designed using the information contained in predictive phylogenetic trees, that seem to represent the last common ancestors of Archaea and of Bacteria. These enzymes display extreme thermal stabilities, suggesting thermophilic ancestries for Archaea and Bacteria. The results are robust to the uncertainties associated with the sequence predictions and to the tree topologies used to infer the ancestral sequences. Moreover, mutagenesis experiments suggest that the universal ancestor also possessed a very thermostable NDK. Because, as we show, the stability of an NDK is directly related to the environmental temperature of its host organism, our results indicate that the last common ancestor of extant life was a thermophile that flourished at a very high temperature.

Construction of optically active CF3-containing quaternary carbon centers via stereospecific S(N)2' reaction.

[reaction: see text] Phosphates from 3-substituted 4,4,4-trifluorobut-2-en-1-ols were found to be effective for construction of CF3-containing quaternary carbon centers by way of Cu(I)-catalyzed Grignard reactions in the presence of catalytic amounts of CuCN and trimethylsilyl chloride (TMSCl) in an anti S(N)2' manner.