Transcriptome and metabolome analyses of Streptococcus gordonii DL1 under acidic conditions.

OBJECTIVES: Streptococcus gordonii is associated with the formation of biofilms, especially those that comprise dental plaque. Notably, S. gordonii DL1 causes infective endocarditis (IE). Colonization of this bacterium requires a mechanism that can tolerate a drop in environmental pH by producing acid via its own sugar metabolism. The ability to survive acidic environmental conditions might allow the bacterium to establish vegetative colonization even in the endocardium due to inflammation-induced lowering of pH, increasing the risk of IE. At present, the mechanism by which S. gordonii DL1 survives under acidic conditions is not thoroughly elucidated. The present study was thus conducted to elucidate the mechanism(s) by which S. gordonii DL1 survives under acidic conditions. METHODS: We analyzed dynamic changes in gene transcription and intracellular metabolites in S. gordonii DL1 exposed to acidic conditions, using transcriptome and metabolome analyses. RESULTS: Transcriptome analysis revealed upregulation of genes involved in heat shock response and glycolysis, and down regulation of genes involved in phosphotransferase systems and biosynthesis of amino acids. The most upregulated genes were a beta-strand repeat protein of unknown function (SGO_RS06325), followed by copper-translocating P-type ATPase (SGO_RS09470) and malic enzyme (SGO_RS01850). The latter two of these contribute to cytoplasmic alkalinization. S. gordonii mutant strains lacking each of these genes showed significantly reduced survival under acidic conditions. Metabolome analysis revealed that cytoplasmic levels of several amino acids were reduced. CONCLUSIONS: S. gordonii survives the acidic conditions by recovering the acidic cytoplasm using the various activities, which are regulated at the transcriptional level.

Development of an in vitro biofilm formation model for screening anti-periodontal disease agents.

PURPOSE: To devise a method for artificial biofilm formation using Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Streptococcus gordonii, as well as a method for evaluating the effects of various ingredients on the artificial biofilm. METHODS: An artificial biofilm was developed using P. gingivalis, T. forsythia, T. denticola, and S. gordonii, which was then observed using scanning electron microscopy and evaluated by microflora analysis. The artificial biofilm was exposed to chlorhexidine gluconate and stained with a fluorescent dye. Then, the fluorescent-stained biofilm was observed using a confocal laser microscope and measured using a fluorescent microplate reader. RESULTS: The microflora analysis confirmed that the culture medium developed was capable of culturing four different bacterial species at the same time. The distribution of dead bacteria differed according to the difference in the concentration of exposed chlorhexidine gluconate. Moreover, the rate of attachment of viable cells decreased in a concentration-dependent manner. Many bacteria were detached from the biofilm in the group exposed to 0.09% chlorhexidine gluconate. Exposure to chlorhexidine gluconate induced a concentration-dependent decrease in living microorganisms and an increase in dead microorganisms in the biofilm. CLINICAL SIGNIFICANCE: The results of this study revealed that S. gordonii, P. gingivalis, T. forsythia, and T. denticola could be used to develop artificial biofilms. The effects of chlorhexidine gluconate on the biofilm showed that evaluating the change in the artificial biofilm caused by the component effect in the experiments was possible via exposure to chlorhexidine gluconate. This method can efficiently evaluate the component effect and has a high potential for use as an indicator. This study demonstrated that this simulation could help develop preventive measures.

Preclinical study for the treatment of diabetes mellitus using ß-like cells derived from human dental pulp stem cells.

Aim: The current study assessed whether insulin-producing cells obtained from dental pulp stem cells (DPSCs) can be a new therapeutic approach in a rat model of diabetes mellitus (DM). Materials & methods: Stem cells were differentiated into pancreatic ß cells under hydrogen sulfide exposure via 2D and 3D methods. Each ß-like cell was immunostained and transplanted into DM rats, after which the in vivo therapeutic effect was determined. Results: Immunostaining revealed the expression of various ß-cell markers in each ß-like cell differentiated using the 3D method. DPSC-derived ß-like cell differentiated via the 3D method promoted a sufficient therapeutic effect. Conclusion: The 3D method promoted islet differentiation, indicating that DPSC-derived ß-like cell transplantation could be a new approach for DM treatment.

Biofilm accumulation on additive manufactured Ti-6Al-4V alloy surfaces.

PURPOSE: This study investigated whether additive manufactured (AM) surfaces inhibit accumulation of bacterial biofilm on the surfaces of Ti-6Al-4V alloy dental implants. Bacterial biofilms are thought to cause peri-implant disease, which develops in mucosa surrounding titanium (Ti) and Ti alloy dental implants and can lead to bone loss and implant failure. METHODS: Accumulation of a Streptococcus mutans (ATCC 25175) biofilm on Ti-6Al-4V alloy was compared in relation to fabrication method, ie, AM using electron beam melting (EBM) or laser beam melting (LBM). Conventional lost-wax casting was used as positive control, and Teflon was used as negative control. Biofilm accumulation on the alloys and negative control (each n = 10) was conducted at 37°C under anaerobic conditions. After 4 h, the number of metabolically active S. mutans bacteria adhering to the alloy was determined with a bioluminescence assay. RESULTS: The quantitative roughness values of the specimens, before exposure to bacteria, ranked EBM > LBM > cast > Teflon. CONCLUSION: The amount of biofilm accumulation on the investigated AM metals and cast metal controls did not significantly differ.

Characterization of dental pulp stem cells isolated from a patient diagnosed with Crouzon syndrome.

Stem cells isolated from patients with rare diseases are important to elucidate their pathogeny and mechanisms to enable regenerative therapy. However, the mechanisms underlying tissue regeneration using patient-derived dental pulp stem cells (DPSCs) are unclear. In this study, we investigated the levels of mRNA and protein expression related to cellular differentiation of Crouzon syndrome patient-derived DPSCs (CS-DPSCs) with a Gly338Arg fibroblast growth factor receptor 2 mutation. Multipotency-related gene expression levels were equivalent in both healthy donor DPSCs and CS-DPSCs. CS-DPSCs showed higher osteocalcin (OCN) expression than healthy donor DPSCs. CS-DPSCs showed a lower increase in the rate of OCN expression among phorbol 12-myristate 13-acetate (PMA)-treated cells than healthy donor DPSCs compared with untreated control cells. CS-DPSCs showed a lower phosphorylation rate of p38 and p44/42 in PMA-treated cells than healthy donor DPSCs compared with untreated control cells. These results demonstrate that CS-DPSCs have higher OCN expression and lower PMA stimulation-responsiveness than healthy donor DPSCs.

Enzyme-digested Colla Corii Asini (E'jiao) prevents hydrogen peroxide-induced cell death and accelerates amyloid beta clearance in neuronal-like PC12 cells.

As an aging-associated degenerative disease, Alzheimer's disease is characterized by the deposition of amyloid beta (Aß), oxidative stress, inflammation, dysfunction and loss of cholinergic neurons. Colla Corii Asini (CCA) is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging. CCA might delay aging-induced degenerative changes in neurons. In the present study, we evaluated antioxidant activity, cytoprotective effects, and Aß removability of enzyme-digested Colla Corii Asini (CCAD). Oxygen radical absorbance capacity (ORAC) activity assay showed that, as compared to gelatins from the skin of porcine, bovine and cold water fish, CCA exhibited the highest ORAC activity. The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage. Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008, 2013, 2017 and gelatin from cold water fish skin, CCA manufactured in 2008 presented the smoothest surface structure. We further tested the protective effects of CCAD (manufactured in 2008) and enzyme-digested gelatin from cold water fish skin (FGD) on hydrogen peroxide (H2O2)-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells. Presto blue assay showed that both FGD and CCAD at 0.5 mg/mL increased cell viability in H2O2-treated neuronal-like PC12 cells. The protection of CCAD was significantly superior to that of FGD. Acetylcholinesterase (AchE) assay showed that both FGD and CCAD inhibited AchE activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1% and 74.5% of that in non-treated cells, respectively. The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine. Although CCAD inhibited AchE activity in neuronal-like PC12 cells, CCAD prevented H2O2-induced abnormal deterioration of AchE. ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aß1-42-treated neuronal-like PC12 cells, suggesting that CCAD can enhance Aß clearance. Our results suggest that CCA might be useful for preventing and treating Alzheimer's disease.

Autophagy Increases Zinc Bioavailability to Avoid Light-Mediated Reactive Oxygen Species Production under Zinc Deficiency.

Zinc (Zn) is an essential micronutrient for plant growth. Accordingly, Zn deficiency (-Zn) in agricultural fields is a serious problem, especially in developing regions. Autophagy, a major intracellular degradation system in eukaryotes, plays important roles in nutrient recycling under nitrogen and carbon starvation. However, the relationship between autophagy and deficiencies of other essential elements remains poorly understood, especially in plants. In this study, we focused on Zn due to the property that within cells most Zn is tightly bound to proteins, which can be targets of autophagy. We found that autophagy plays a critical role during -Zn in Arabidopsis (Arabidopsis thaliana). Autophagy-defective plants (atg mutants) failed to grow and developed accelerated chlorosis under -Zn. As expected, -Zn induced autophagy in wild-type plants, whereas in atg mutants, various organelle proteins accumulated to high levels. Additionally, the amount of free Zn2+ was lower in atg mutants than in control plants. Interestingly, -Zn symptoms in atg mutants recovered under low-light, iron-limited conditions. The levels of hydroxyl radicals in chloroplasts were elevated, and the levels of superoxide were reduced in -Zn atg mutants. These results imply that the photosynthesis-mediated Fenton-like reaction, which is responsible for the chlorotic symptom of -Zn, is accelerated in atg mutants. Together, our data indicate that autophagic degradation plays important functions in maintaining Zn pools to increase Zn bioavailability and maintain reactive oxygen species homeostasis under -Zn in plants.

Investigation of molecular biomarker candidates for diagnosis and prognosis of chronic periodontitis by bioinformatics analysis of pooled microarray gene expression datasets in Gene Expression Omnibus (GEO).

BACKGROUND: Chronic periodontitis (CP) is a multifactorial inflammatory disease. For the diagnosis of CP, it is necessary to investigate molecular biomarkers and the biological pathway of CP. Although analysis of mRNA expression profiling with microarray is useful to elucidate pathological mechanisms of multifactorial diseases, it is expensive. Therefore, we utilized pooled microarray gene expression data on the basis of data sharing to reduce hybridization costs and compensate for insufficient mRNA sampling. The aim of the present study was to identify molecular biomarker candidates and biological pathways of CP using pooled datasets in the Gene Expression Omnibus (GEO) database. METHODS: Three pooled transcriptomic datasets (GSE10334, GSE16134, and GSE23586) of gingival tissue with CP in the GEO database were analyzed for differentially expressed genes (DEGs) using GEO2R, functional analysis and biological pathways with the Database of Annotation Visualization and Integrated Discovery database, Protein-Protein Interaction (PPI) network and hub gene with the Search Tool for the Retrieval of Interaction Genes database, and biomarker candidates for diagnosis and prognosis and upstream regulators of dominant biomarker candidates with the Ingenuity Pathway Analysis database. RESULTS: We shared pooled microarray datasets in the GEO database. One hundred and twenty-three common DEGs were found in gingival tissue with CP, including 81 upregulated genes and 42 downregulated genes. Upregulated genes in Gene Ontology were significantly enriched in immune responses, and those in the Kyoto Encyclopedia of Genes and Genomes pathway were significantly enriched in the cytokine-cytokine receptor interaction pathway, cell adhesion molecules, and hematopoietic cell lineage. From the PPI network, the 12 nodes with the highest degree were screened as hub genes. Additionally, six biomarker candidates for CP diagnosis and prognosis were screened. CONCLUSIONS: We identified several potential biomarkers for CP diagnosis and prognosis (e.g., CSF3, CXCL12, IL1B, MS4A1, PECAM1, and TAGLN) and upstream regulators of biomarker candidates for CP diagnosis (TNF and TGF2). We also confirmed key genes of CP pathogenesis such as CD19, IL8, CD79A, FCGR3B, SELL, CSF3, IL1B, FCGR2B, CXCL12, C3, CD53, and IL10RA. To our knowledge, this is the first report to reveal associations of CD53, CD79A, MS4A1, PECAM1, and TAGLN with CP.

Zinc starvation induces autophagy in yeast.

Zinc is an essential nutrient for all forms of life. Within cells, most zinc is bound to protein. Because zinc serves as a catalytic or structural cofactor for many proteins, cells must maintain zinc homeostasis under severely zinc-deficient conditions. In yeast, the transcription factor Zap1 controls the expression of genes required for uptake and mobilization of zinc, but to date the fate of existing zinc-binding proteins under zinc starvation remains poorly understood. Autophagy is an evolutionarily conserved cellular degradation/recycling process in which cytoplasmic proteins and organelles are sequestered for degradation in the vacuole/lysosome. In this study, we investigated how autophagy functions under zinc starvation. Zinc depletion induced non-selective autophagy, which is important for zinc-limited growth. Induction of autophagy by zinc starvation was not directly related to transcriptional activation of Zap1. Instead, TORC1 inactivation directed zinc starvation-induced autophagy. Abundant zinc proteins, such as Adh1, Fba1, and ribosomal protein Rpl37, were degraded in an autophagy-dependent manner. But the targets of autophagy were not restricted to zinc-binding proteins. When cellular zinc is severely depleted, this non-selective autophagy plays a role in releasing zinc from the degraded proteins and recycling zinc for other essential purposes.

Recycling of iron via autophagy is critical for the transition from glycolytic to respiratory growth.

Autophagy is a bulk degradation process conserved from yeast to mammals. To examine the roles of autophagy in cellular metabolism, we generated autophagy-defective (atg) mutants in the X2180-1B strain background. We compared the growth of wild-type (WT) and atg cells in minimal (synthetic dextrose, SD) and rich (yeast extract/peptone/dextrose, YEPD) medium, and we found that mutations in the core autophagy machinery result in defects in the diauxic shift, the transition from fermentation to respiratory growth upon glucose depletion, specifically in SD. Furthermore, we confirmed that autophagy was induced prior to the diauxic shift, implying that it plays a role in this process. In YEPD, atg mutants grew normally, so we assumed that the insufficiency of certain nutrients in SD was responsible for the defects. We ultimately identified iron, which is a necessary cofactor for respiratory activity, as the nutrient required for the diauxic shift in atg mutants. Indeed, atg mutants exhibited defects in respiration, which was rescued by supplementation with iron. Based on these data, we hypothesized that autophagy is involved in iron recycling during the diauxic shift. smf3Δfet5Δ or smf3Δftr1Δ cells, which are unable to export iron from the vacuole, also exhibit defects in the diauxic shift, so iron released from the vacuole is important for the shift in SD medium. Finally, we observed that smf3Δfet5Δ cells accumulated nearly twice as much vacuolar iron as smf3Δfet5Δatg2Δ cells, suggesting that autophagy is involved in iron recycling by the vacuolar transport and degradation of iron-containing cargos.

Bulk RNA degradation by nitrogen starvation-induced autophagy in yeast.

Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is non-selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy-dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2-type ribonuclease, generating 3'-NMPs that were immediately converted to nucleosides by the vacuolar non-specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re-assimilated, but excreted from the cell. Bulk non-selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.

Oxidative stress in skeletal muscle causes severe disturbance of exercise activity without muscle atrophy.

The increase in reactive oxygen species (ROS) levels that occurs during intense exercise has been proposed to be one of the major causes of muscle fatigue. In addition, the accumulation of cellular damage due to ROS is widely regarded to be one of the factors triggering age-related pathological conditions in skeletal muscle. To investigate the pathological significance of oxidative stress in skeletal muscle, we generated skeletal muscle-specific manganese superoxide dismutase-deficient (muscle-Sod2(-/-)) mice. The mutant mice showed severe disturbances in exercise activity, but no atrophic changes in their skeletal muscles. In histological and histochemical analyses, the mutant mice showed centralized nuclei in their muscle fibers and selective loss of enzymatic activity in mitochondrial respiratory chain complexes. In addition, the mutant mice displayed increased oxidative damage and reduced ATP content in their muscle tissue. Furthermore, a single administration of the antioxidant EUK-8 significantly improved exercise activity and increased the cellular ATP level in skeletal muscle. These results imply that the superoxide anions generated in mitochondria play a pivotal role in the progression of exercise intolerance.

Phosphorylated Ssk1 prevents unphosphorylated Ssk1 from activating the Ssk2 mitogen-activated protein kinase kinase kinase in the yeast high-osmolarity glycerol osmoregulatory pathway.

In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1 approximately P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1 approximately P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.