Expert consensus on difficulty assessment of endodontic therapy.

Endodontic diseases are a kind of chronic infectious oral disease. Common endodontic treatment concepts are based on the removal of inflamed or necrotic pulp tissue and the replacement by gutta-percha. However, it is very essential for endodontic treatment to debride the root canal system and prevent the root canal system from bacterial reinfection after root canal therapy (RCT). Recent research, encompassing bacterial etiology and advanced imaging techniques, contributes to our understanding of the root canal system's anatomy intricacies and the technique sensitivity of RCT. Success in RCT hinges on factors like patients, infection severity, root canal anatomy, and treatment techniques. Therefore, improving disease management is a key issue to combat endodontic diseases and cure periapical lesions. The clinical difficulty assessment system of RCT is established based on patient conditions, tooth conditions, root canal configuration, and root canal needing retreatment, and emphasizes pre-treatment risk assessment for optimal outcomes. The findings suggest that the presence of risk factors may correlate with the challenge of achieving the high standard required for RCT. These insights contribute not only to improve education but also aid practitioners in treatment planning and referral decision-making within the field of endodontics.

Expert consensus on irrigation and intracanal medication in root canal therapy.

Chemical cleaning and disinfection are crucial steps for eliminating infection in root canal treatment. However, irrigant selection or irrigation procedures are far from clear. The vapor lock effect in the apical region has yet to be solved, impeding irrigation efficacy and resulting in residual infections and compromised treatment outcomes. Additionally, ambiguous clinical indications for root canal medication and non-standardized dressing protocols must be clarified. Inappropriate intracanal medication may present side effects and jeopardize the therapeutic outcomes. Indeed, clinicians have been aware of these concerns for years. Based on the current evidence of studies, this article reviews the properties of various irrigants and intracanal medicaments and elucidates their effectiveness and interactions. The evolution of different kinetic irrigation methods, their effects, limitations, the paradigm shift, current indications, and effective operational procedures regarding intracanal medication are also discussed. This expert consensus aims to establish the clinical operation guidelines for root canal irrigation and a position statement on intracanal medication, thus facilitating a better understanding of infection control, standardizing clinical practice, and ultimately improving the success of endodontic therapy.

Expert consensus on digital guided therapy for endodontic diseases.

Digital guided therapy (DGT) has been advocated as a contemporary computer-aided technique for treating endodontic diseases in recent decades. The concept of DGT for endodontic diseases is categorized into static guided endodontics (SGE), necessitating a meticulously designed template, and dynamic guided endodontics (DGE), which utilizes an optical triangulation tracking system. Based on cone-beam computed tomography (CBCT) images superimposed with or without oral scan (OS) data, a virtual template is crafted through software and subsequently translated into a 3-dimensional (3D) printing for SGE, while the system guides the drilling path with a real-time navigation in DGE. DGT was reported to resolve a series of challenging endodontic cases, including teeth with pulp obliteration, teeth with anatomical abnormalities, teeth requiring retreatment, posterior teeth needing endodontic microsurgery, and tooth autotransplantation. Case reports and basic researches all demonstrate that DGT stand as a precise, time-saving, and minimally invasive approach in contrast to conventional freehand method. This expert consensus mainly introduces the case selection, general workflow, evaluation, and impact factor of DGT, which could provide an alternative working strategy in endodontic treatment.

Experts consensus on the procedure of dental operative microscope in endodontics and operative dentistry.

The dental operative microscope has been widely employed in the field of dentistry, particularly in endodontics and operative dentistry, resulting in significant advancements in the effectiveness of root canal therapy, endodontic surgery, and dental restoration. However, the improper use of this microscope continues to be common in clinical settings, primarily due to operators' insufficient understanding and proficiency in both the features and established operating procedures of this equipment. In October 2019, Professor Jingping Liang, Vice Chairman of the Society of Cariology and Endodontology, Chinese Stomatological Association, organized a consensus meeting with Chinese experts in endodontics and operative dentistry. The objective of this meeting was to establish a standard operation procedure for the dental operative microscope. Subsequently, a consensus was reached and officially issued. Over the span of about four years, the content of this consensus has been further developed and improved through practical experience.

Influence of silver nanoparticles on the resin-dentin bond strength and antibacterial activity of a self-etch adhesive system.

STATEMENT OF PROBLEM: The influence of silver nanoparticles on adhesive properties of Single Bond Universal (3M ESPE) and the antibacterial activity of silver nanoparticles-modified adhesives against Streptococcus mutans is unclear. PURPOSE: The purpose of this in vitro study was to evaluate the effect of silver nanoparticles on the dentin bond strength of modified adhesives and the antibacterial activity against the cariogenic pathogen S. mutans. MATERIAL AND METHODS: Single Bond Universal adhesive was used as the control. Silver nanoparticles were added to adhesives at 0.05% and 0.1% (by weight) (experimental groups), and scanning electron microscopy was used to observe the uniformity of the modified adhesives. The Single Bond Universal adhesive and the modified adhesives were then used to prepare dentin-composite resin blocks. The microtensile bond strength and microleakage of the prepared dentin-composite resin blocks were determined with or without thermocycling. The colony-forming units (CFU) of S. mutans cultured with the adhesives were evaluated, and the microtensile bond strength and microleakage of each group were tested after treatment with S. mutans. The differences in the microtensile bond strength or CFU were analyzed by using the 2-way analysis of variance and independent sample t test. The differences in microleakage between the groups were evaluated by using the Mann-Whitney test (α=.05). RESULTS: Silver nanoparticle-modified adhesives exhibited uniform morphologies without agglomeration and exhibited a homogeneous adhesive layer in dentin-composite resin blocks. The microtensile bond strength and microleakage of the modified adhesives were similar to those of the control group, with or without thermocycling (P>.05). However, thermocycling reduced the bond strength significantly (P<.001). Self-etch adhesives incorporated with silver nanoparticles showed significant antibacterial activities after less than 6 months of aging treatment. The modified adhesives did not exhibit a decreased bond strength after S. mutans exposure (P>.05), while the control group exhibited a markedly decreased bond strength after S. mutans exposure (P<.05). CONCLUSIONS: Silver nanoparticle-modified adhesives showed excellent antibacterial activities against S. mutans and resisted the destruction of dentin bond strength caused by S. mutans while not compromising the bonding properties of Single Bond Universal self-etch adhesives.

Proteomic study of plaque fluid in high caries and caries free children.

BACKGROUND: The occurrence and development of caries is a complex process affected by multiple factor. OBJECTIVE: The present study was envisaged to evaluate the plaque fluid in caries free children and children with high caries, in order to establish a data set of bacterial secretion proteomics. A non-labeled quantitative technique based on two-dimensional liquid chromatography-series mass spectroscopy was employed to detect plaque fluid. Based on the proteomics data, the database search, data processing and pathway analysis illuminated the function of these proteins, and clarified the role of plaque microecology in caries occurrence and development. METHODS: The study enrolled 8 caries free (CF) children, whose decayed-missed-filled surface of teeth is 0 (dmfs = 0), and caries sensitive (CS) children, whose decayed-missed-filled surface of teeth is > 10(dmfs > 10) (3 ∼ 5 years old) for the smooth tooth plaque and the plaque in the high caries group. The plaque protein was extracted using the unlabeled quantitative technique like liquid chromatography-series mass spectrometry, using DeCyderTM MS Differential Analysis Software (version 1.0, GE Healthcare) that detected and compared the spectra, and quantified the full scanning before series mass spectroscopy. After obtaining all peptides with quantitative information, significantly differential polypeptide molecules were obtained (p< 0.05), and a metabolic pathway analysis was performed. RESULTS: We identified 1,804 peptides with quantitative information, including 39 in CF, 30 in CS, and 1,735 similarly expressing peptides. After statistical analysis, 603 statistically different expression peptide data sets were obtained, including 202 high-expressed peptides in Group CF, 33 greater than 1.5 fold peptides, 401 high-expressed in Group CS and 199 greater than 1.5 fold peptide (173 nonredundant proteins). CONCLUSION: Our study obtained the largest known dataset of the bacterial secretion protein in children with high caries, and screened the data set of high caries state. 603 peptides were statistically rich in 101 pathways, including glycolysis pyruvate acid metabolism, tricarboxylic acid cycle, pentyl phosphate pathway, fructose mannose metabolism, starch and sucrose metabolism, and ABC transporters.

Carbohydrate Metabolism Affects Macrophage-Mediated Killing of Enterococcus faecalis.

Enterococcus faecalis, an opportunistic pathogen that causes severe community-acquired and nosocomial infections, has been reported to resist phagocyte-mediated killing, which enables its long-term survival in the host. Metabolism, especially carbohydrate metabolism, plays a key role in the battle between pathogens and hosts. However, the function of carbohydrate metabolism in the long-term survival of E. faecalis in phagocytes has rarely been reported. In this study, we utilized transposon insertion sequencing (TIS) to investigate the function of carbohydrate metabolism during the survival of E. faecalis in RAW264.7 cells. The TIS results showed that the fitness of carbohydrate metabolism-related mutants, especially those associated with fructose and mannose metabolism, were significantly enhanced, suggesting that the attenuation of carbohydrate metabolism promotes the survival of E. faecalis in macrophages. The results of our investigation indicated that macrophages responded to carbohydrate metabolism of E. faecalis and polarized to M1 macrophages to increase nitric oxide (NO) production, leading to the enhancement of macrophage-mediated killing to E. faecalis. Meanwhile, E. faecalis automatically decreased carbohydrate metabolism to escape from the immune clearance of macrophages during intracellular survival. The shift of primary carbon resources for macrophages affected the ability to clear intracellular E. faecalis. In summary, the results of the present study demonstrated that carbohydrate metabolism affects the macrophage-mediated killing of E. faecalis. IMPORTANCE E. faecalis has become a major pathogen leading to a variety of infections around the world. The metabolic interaction between E. faecalis and its host is important during infection but is rarely investigated. We used transposon insertion sequencing coupled with transcriptome sequencing to explore the metabolic interaction between E. faecalis and macrophages and uncovered that the shift of carbohydrate metabolism dramatically affected the inflammatory response of macrophages. In addition, E. faecalis attenuated carbohydrate metabolism to avoid the activation of the immune response of macrophages. This study provides new insights for the reason why E. faecalis is capable of long-term survival in macrophages and may facilitate the development of novel strategies to treat infectious diseases.

[Research Advance of CXCR4 Inhibitors in the Treatment of Acute Myeloid Leukemia--Review].

CXCL12/CXCR4 axis composed of chemokine CXCL12 and its specific ligand CXCR4 can regulate and control the adhesion of leukemia cells to protective bone marrow niche, promote cell survival, and resist apoptosis induced by signal transduction inhibitors and chemotherapeutic drugs. Therefore, CXCL12 /CXCR4 axis has become a new target for the treatment of acute myeloid leukemia. At present, CXCR4 inhibitors that have been developed are in different clinical trials, showing good anti-leukemia effect. In this review, the research advance of CXCR4 inhibitors in the treatment of acute myeloid leukemia is summarized briefly.

Phosphate transport system mediates the resistance of Enterococcus faecalis to multidrug.

Enterococcus faecalis, a severe nosocomial and community opportunistic pathogen, is difficult to control due to its multidrug resistance. Through heredity and the recombination of intrinsic resistance genes and horizontally acquired resistance genes, E. faecalis can rapidly evolve drug resistance. Nisin, an important antimicrobial peptide, is extensively employed in the healthcare and food industries to inhibit Gram-positive bacteria and may induce the emergence of nisin-resistant bacteria worldwide. However, the mechanism governing nisin resistance in E. faecalis has not been fully elucidated. This study utilizes transposon insertion sequencing (TIS) to comprehensively explore novel genes related to nisin resistance. According to the analysis of TIS results, hundreds of genes appear to be essential for nisin resistance in E. faecalis. The phosphate transport system (OG1RF_10018-10021, named PTS), which is screened by TIS results, enhances the resistance of E. faecalis to nisin, the mechanism of which may be involved in potA and/or OG1RF_10526 (hypothetical gene). Meanwhile, PTS also strongly represses the biosynthesis of ribosomes to increase the sensitivity of E. faecalis to gentamycin. In addition, the overexpression of PTS increases the sensitivity of E. faecalis to daptomycin, the mechanism of which is independent of the LiaFSR system. This study first demonstrated that E. faecalis utilizes PTS to mediate the resistance to multidrug, which may help to elucidate the mechanism governing drug resistance and to establish guidelines for the treatment of infectious diseases caused by E. faecalis.

Enterococcus Faecalis activates NLRP3 inflammasomes leading to increased interleukin-1 beta secretion and pyroptosis of THP-1 macrophages.

OBJECTIVES: Enterococcus faecalis is the bacterial species closely related to persistent infection in root canals. Interleukin-1 beta (IL-1ß) is the most commonly detected proinflammatory cytokine in periapical granulation tissue and plays a critical role in host defenses against microbial infection. The synthesis and secretion of IL-1ß are mediated mainly by Toll-like receptors and inflammasome activation. The previous study found that the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes are positively expressed in periapical granulation tissue. The aim of this study was to investigate the pathogenicity of E. faecalis and the molecular mechanisms of IL-1ß secretion by THP-1 macrophages infected with E. faecalis. METHODS: The IL-1ß and lactate dehydrogenase (LDH) levels induced by E. faecalis were investigated with enzyme-linked immunosorbent assay (ELISA) kit and cytotoxicity assay kit, caspase-1 and inflammasome expression levels were investigated using real time PCR and Western blot analysis. Then the effect of caspase-1, NLRP3, adenosine triphosphate (ATP), and extracellular K+ on IL-1ß and LDH secretion, Gasdermin-D (GSDMD) cleavage induced by E. faecalis were analyzed. RESULTS: E. faecalis significantly increased IL-1ß and LDH release, caspase-1 and GSDMD cleavage, and NLRP3 inflammasome activation. It also showed that IL-1ß and LDH release, GSDMD cleavage required caspase-1 and NLRP3 activation. Furthermore, the expression and activation of caspase-1 and NLRP3 were blocked by oxidized ATP and extracellular K+. CONCLUSION: E. faecalis infection activated caspase-1 and the NLRP3 inflammasome to induce IL-1ß secretion and inflammatory cell death (pyroptosis). Furthermore, the activation and expression of NLRP3 induced by E. faecalis required P2X7R and K+ efflux. This study furthers our understanding of the inflammatory response mechanism induced by E. faecalis indicates that NLRP3 may be a potential target for treatment and prevention of persistent periodontitis caused by E. faecalis.

miR-200a contributes to the migration of BMSCs induced by the secretions of E. faecalis via FOXJ1/NFκB/MMPs axis.

BACKGROUND: Upon migrating to the injured sites, bone marrow mesenchymal stem cells (BMSCs) play critical roles in the repair of bone lesion caused by chronic apical periodontitis. Emerging evidences have shown that Enterococcus faecalis is always associated with apical periodontitis, especially refractory apical periodontitis. But the mechanism underlying how Enterococcus faecalis affects the migration of BMSCs remains unclear. METHODS: The effects of Enterococcus faecalis supernatants on the migration of BMSCs were determined by transwell migration assays. miRNA sequencing was performed to detect the significantly differentially expressed miRNAs of BMSCs. Proteomics analysis was used to detect the protein expression alterations of BMSCs. Luciferase report assays were deployed to verify the targets of miRNA. Western blot analysis was performed to examine the expressions of matrix metalloproteinases-3, matrix metalloproteinases-9, Forkhead Box Protein J1 (FOXJ1), and nuclear factor kappa B (NFκB). The activations of NFκB were detected by luciferase assays with NFκBluc reporter. RESULTS: We found that Enterococcus faecalis supernatants could promote the migration of BMSCs. The upregulation of miR-200a-3p in this process contributed to BMSC migration through downregulating its target Forkhead Box Protein J1. Moreover, FOXJ1/ NFκB axis was found to regulate matrix metalloproteinases (MMPs) in this process. CONCLUSIONS: These results above suggest that miR-200a contributes to the migration of BMSCs induced by the secretions of E. faecalis via FOXJ1/NFκB/MMPs axis.

[In vitro study of antimicrobial efficacy of different irrigations on Enterococcus faecalis biofilm formation in root canal].

PURPOSE: To compare the antimicrobial effect of different irrigations on Enterococcus faecalis biofilms in extracted teeth and evaluate the antimicrobial activity of irrigating solutions residual against E. faecalis biofilms formation, in order to provide a better strategy for clinician. METHODS: Extracted human premolar teeth with single root canal were clearly autoclaved. These teeth were contaminated with E. faecalis（ATCC33186） and incubated for 60 days. The samples were randomly assigned to 4 experimental groups. During biomechanical instrumentation, the root canal was irrigated with different irrigating agents. The bacteria samples were collected with sterile paper points before and after instrumentation to F2. Then, samples that had been instrumented and autoclaved again were randomly divided into 2 groups treated with normal saline and 1%NaOCl for 30 min. E. faecalis was used to contaminate these root canals. The bacteria samples were collected with sterile paper points after 2, 6, 24, 48 h. SPSS19.0 software package was used for statistical analysis. RESULTS: Group using 1% NaOCI with ultrasound devices was significantly more effective than NS alone groups. 1% NaOCI groups showed a better residual activity than NS group. CONCLUSIONS: NaOCl is still the most important irrigating solutions, and it could be a better choice after biomechanical instrumentation, because of its long time substantivity achieves residual antimicrobial activity. Ultrasound devices is recommended to coordinate with irrigation.

[The effect of Streptococcus mutans luxS gene on mixed-species biofilm communities].

PURPOSE: To evaluate the effect of S.mutans luxS gene on mixed-species biofilms communities. METHODS: Biofilms were formed by S. mutans (wild type strain, its luxS overexpression strain and luxS knockout strain) and Lactobacillus acidophilus (ATCC4356) with a ratio of 1:1 at 37℃ for 4 h, 14 h and 24 h. MTT assay was used to detect the quantification of the biofilms formed. The structures of biofilms were observed under confocal laser scanning microscopy after 24 h, and expression of biofilm-related genes (ftf, smu630, brpA, gbpB, gtfB, vicR, comDE and relA) was investigated by real-time PCR. Statistical analysis was performed with SPSS17.0 software package. RESULTS: The results showed that biofilm formed by S. mutans(wild type strain, its luxS overexpression strain and luxS knockout strain) and L.acidophilus after 14 h were 0.481±0.024, 0.591±0.023 and 0.279±0.019, respectively. The same findings were present after 24 h, the biofilm formed by S.mutans overexpression strain with L.acidophilus was higher than wild type strain, and the biofilm formed by knockout strain significantly decreased; but there was no significant difference at 4 h time points. CLSM images revealed that both S.mutans overexpression strain and its wild type strain tended to aggregate into distinct clusters and dense structures, whereas the luxS knockout strain appeared relatively sparse. Compared with wild type strain, all of the genes examined were upregulated in the biofilms formed by the overexpression strain, and were downregulated in the biofilms formed by the luxS mutant strain in mixed-species biofilm. CONCLUSIONS: S.mutans luxS gene can affect mixed-species biofilm formation with L.acidophilus, which provides evidences for further study.

Comparison of the shaping ability of different nickel-titanium instruments in simulated root canals in resin / 口腔疾病防治

Objective @#To compare the shaping ability of 3 different nickel (Ni)-titanium (Ti) systems in simulated root canals in resin and to provide a reference for clinicians.@*Methods@#Forty-eight resin blocks were prepared using the F360 (Komet, Brasseler GmbH & Co., Lemgo, Germany) (Group 1), F6 SkyTaper (20/06) (Komet, Brasseler GmbH & Co., Lemgo, Germany) (Group 2), F6 SkyTaper (25/06) (Komet, Brasseler GmbH & Co., Lemgo, Germany) (Group 3) and Reciproc R25 systems (VDW, Munich, Germany) (Group 4) (n=12 canals/group). The images taken before and after preparation were superimposed and analyzed by Adobe Photoshop v7.0. The amount of resin removed by each system was measured, and the centering ability was assessed. The data were statistically analyzed using SPSS 20.0.@*Results @#At the 1 mm point, the transportation in Group 4 [(0.10 ± 0.03) mm] was significantly greater than that in Groups 2 [(0.05 ± 0.03) mm] and 3 [(0.05 ± 0.03) mm] (P < 0.05). At the 8 mm and 9 mm points, the transportation values in Group 4 [(0.12 ± 0.06) mm and (0.13 ± 0.05) mm] were significantly higher than those in Groups 2 [(0.05 ± 0.05) mm and (0.05 ± 0.05) mm] and 3 [(0.05 ± 0.04) mm and (0.06 ± 0.05) mm] (P < 0.05). At the 10 mm point, the transportation was significantly greater in Group 4 [(0.13 ± 0.06) mm] than in Group 2 [(0.06 ± 0.06) mm].@*Conclusion@#F6 SkyTaper exhibits better centering ability than Reciproc.

A comparison of the effect of different nickel-titanium instruments combined with ultrasonic irrigation on root canal preparation / 口腔疾病防治

Objective @#To compare the removal efficiency and the amounts of apically extruded debris using Twisted File (TF), Twisted File Adaptive (TFA), ProTaper, and ProTaper Next combined with ultrasonic irrigation and to provide an experimental basis for the selection of root canal instrumentation in the clinic.@*Methods@#Forty mandibular premolars were randomly divided into 4 groups (n=10 teeth per group). The canals were cut using a Twisted File, Twisted File Adaptive, ProTaper, or ProTaper Next nickel-titanium instrument. The canals were irrigated with ultrasonic irrigation. The apically extruded debris were collected in preweighted Eppendorf tubes. The amount of dental tissue removed and extruded debris were assessed with an electronic balance.@*Results @#The amount of tooth tissue removed in groups A, B, C and D was 20.5 ± 2.0 mg, 17.8 ± 4.2 mg, 20.8 ± 3.9 mg and 16.5 ± 2.2 mg, respectively. Combined with ultrasonic irrigation, the Twisted File and ProTaper had a better removal efficiency than the ProTaper Next(P < 0.05). There was no significant difference in the amount of extruded debris (χ2=4.057, P=0.255) among four groups.@*Conclusion@#The Twisted File and ProTaper had a better removal efficiency than the ProTaper Next combined with ultrasonic irrigation. There was no significant difference in the amount of extruded debris using four Nickel-titanium instruments combined with ultrasonic irrigation.

Variations in oral microbiota associated with oral cancer.

Individual bacteria and shifts in microbiome composition are associated with human disease, including cancer. To unravel the connections underlying oral bacterial dysbiosis and oral squamous cell carcinoma (OSCC), cancer lesion samples and anatomically matched normal samples were obtained from the same patients. We then profiled the bacteria within OSCC lesion surface samples at the species level using next-generation sequencing to comprehensively investigate bacterial community composition and functional genes in these samples. Significantly greater bacterial diversity was observed in the cancer samples than in the normal samples. Compared with previous studies, we identified many more taxa demonstrating remarkably different distributions between the groups. In particular, a group of periodontitis-correlated taxa, including Fusobacterium, Dialister, Peptostreptococcus, Filifactor, Peptococcus, Catonella and Parvimonas, was significantly enriched in OSCC samples. Additionally, several operational taxonomic units (OTUs) associated with Fusobacterium were highly involved in OSCC and demonstrated good diagnostic power. Our study revealed drastic changes in surface bacterial communities of OSCC. The findings enrich knowledge of the association between oral bacterial communities and oral cancer.

Analysis of the expression of NLRP3 and AIM2 in periapical lesions with apical periodontitis and microbial analysis outside the apical segment of teeth.

OBJECTIVE: To detect the distribution and expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes in periapical lesions and to analyse the possible microbial stimuli outside of teeth. DESIGN: The distribution of NLRP3 and AIM2 inflammasomes in sixteen periapical lesions was investigated by immunohistochemistry. Meanwhile, the relative gene expression levels of NLRP3 and AIM2 in sixteen periapical lesions and three health periodontal tissue were quantified by real-time polymerase chain reaction (PCR). Moreover, forty-seven teeth without sinus tracts were obtained in the clinic and included in bacterial analysis using PCR. Then, the mRNA levels of apoptosis-associated speck-like protein (ASC), caspase-1, interleukin-1ß (IL-1ß), NLRP3 and AIM2 in THP-1-derived macrophages treated with lipopolysaccharides (LPS) of Porphyromonas were also quantified by real-time PCR, and the IL-1ß secretion level was investigated using enzyme-linked immunosorbent assay (ELISA). RESULTS: NLRP3 and AIM2 were positively expressed in periapical lesions and were mainly distributed in inflammatory cells. Most of the samples that demonstrated up-regulation of NLRP3 mRNA also demonstrated up-regulation of caspase-1 mRNA. Microbial analysis revealed that Porphyromonas endodontalis was the most commonly detected species and was detected in 27 of 47 cases (57.4%), followed by Fusobacterium nucleatum (20/47, 42.6%), Porphyromonas gingivalis (19/47, 40.4%), Tannerella forsythia (19/47, 40.4%), Actinomyces sp. (17/47, 36.17%), Treponema denticola (10/47,21.28%), Actinomyces israelii (9/47,19.15%), Prevotella intermedia (6/47, 12.77%), Enterococcus faecalis (1/47,2.13%) and Enterococcus faecium (0/47,0). Furthermore, we found that LPS of P. gingivalis induced THP-1 cells to produce IL-1ß and to activate NLRP3 and AIM2 inflammasomes. CONCLUSIONS: Our results suggest that the NLRP3 and AIM2 proteins play a part in the pathogenesis of periapical periodontitis. Anaerobes, such as P. endodontalis, P. gingivalis, F. nucleatum and T. forsythia, were the main detected microbial stimuli that might activate inflammasomes in periapical tissues.

Differences in the chemical composition of Enterococcus faecalis biofilm under conditions of starvation and alkalinity.

ABSTACT This study aimed to investigate the dynamic changes that occur in the chemical composition of an Enterococcus faecalis (E. faecalis) biofilm under conditions of starvation and in an alkaline environment and to explore the function of chemical composition changes in the resistance of the E. faecalis biofilm to an extreme environment. This study established an in vitro E. faecalis biofilm model under starvation and in an alkaline environment. During the formation of the biofilm, the pH value and nutritional condition of the culture medium were changed, and the changes in chemical composition were observed using biochemical measures. The results showed that, when the pH value of the culture medium was 11, the percentage of water-insoluble polysaccharides in the biofilm was significantly lower than under other conditions. In addition, the percentage of water-soluble polysaccharides in culture medium with pH values of 9 and 11 gradually decreased. The level of the water-soluble polysaccharides in each milligram of dry weight of biofilm at pH 11 increased compared to that under other conditions. The results from this study indicate that the chemical composition of E. faecalis biofilm changed in extreme environments. These changes served as a defensive mechanism for E. faecalis against environmental pressures.

Melatonin Receptor Agonists as the "Perioceutics" Agents for Periodontal Disease through Modulation of Porphyromonas gingivalis Virulence and Inflammatory Response.

AIM: "Perioceutics" including antimicrobial therapy and host modulatory therapy has emerged as a vital adjunctive treatment of periodontal disease. Melatonin level was significantly reduced in patients with periodontal diseases suggesting melatonin could be applied as a potential "perioceutics" treatment of periodontal diseases. This study aims to investigate the effects of melatonin receptor agonists (melatonin and ramelteon) on Porphyromonas gingivalis virulence and Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced inflammation. METHODS: Effects of melatonin receptor agonists on Porphyromonas gingivalis planktonic cultures were determined by microplate dilution assays. Formation, reduction, and viability of Porphyromonas gingivalis biofilms were detected by crystal violet staining and MTT assays, respectively. Meanwhile, biofilms formation was also observed by confocal laser scanning microscopy (CLSM). The effects on gingipains and hemolytic activities of Porphyromonas gingivalis were evaluated using chromogenic peptides and sheep erythrocytes. The mRNA expression of virulence and iron/heme utilization was assessed using RT-PCR. In addition, cell viability of melatonin receptor agonists on human gingival fibroblasts (HGFs) was evaluated by MTT assays. After pretreatment of melatonin receptor agonists, HGFs were stimulated with Pg-LPS and then release of cytokines (IL-6 and lL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Melatonin and ramelteon did exhibit antimicrobial effects against planktonic culture. Importantly, they inhibited biofilm formation, reduced the established biofilms, and decreased biofilm viability of Porphyromonas gingivalis. Furthermore, they at sub-minimum inhibitory concentration (sub-MIC) concentrations markedly inhibited the proteinase activities of gingipains and hemolysis in a dose-dependent manner. They at sub-MIC concentrations significantly inhibited the mRNA expression of virulence factors (kgp, rgpA, rgpB, hagA, and ragA), while increasing the mRNA expression of ferritin (ftn) or hemolysin (hem). They did not show obvious cytotoxicity toward HGFs. They inhibited Pg-LPS-induced IL-6 and IL-8 secretion, which was reversed by luzindole, the melatonin receptor antagonist. CONCLUSION: Melatonin receptor agonists can inhibit planktonic and biofilm growth of Porphyromonas gingivalis by affecting the virulent properties, as well as Pg-LPS-induced inflammatory response. Our study provides new evidence that melatonin receptor agonists might be useful as novel "perioceutics" agents to prevent and treat Porphyromonas gingivalis-associated periodontal diseases.

Bacterial Diversity and Community Structure of Supragingival Plaques in Adults with Dental Health or Caries Revealed by 16S Pyrosequencing.

Dental caries has a polymicrobial etiology within the complex oral microbial ecosystem. However, the overall diversity and structure of supragingival plaque microbiota in adult dental health and caries are not well understood. Here, 160 supragingival plaque samples from patients with dental health and different severities of dental caries were collected for bacterial genomic DNA extraction, pyrosequencing by amplification of the 16S rDNA V1-V3 hypervariable regions, and bioinformatic analysis. High-quality sequences (2,261,700) clustered into 10,365 operational taxonomic units (OTUs; 97% identity), representing 453 independent species belonging to 122 genera, 66 families, 34 orders, 21 classes, and 12 phyla. All groups shared 7522 OTUs, indicating the presence of a core plaque microbiome. α diversity analysis showed that the microbial diversity in healthy plaques exceeded that of dental caries, with the diversity decreasing gradually with the severity of caries. The dominant phyla of plaque microbiota included Bacteroidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria, and TM7. The dominant genera included Capnocytophaga, Prevotella, Actinomyces, Corynebacterium, Neisseria, Streptococcus, Rothia, and Leptotrichia. ß diversity analysis showed that the plaque microbial community structure was similar in all groups. Using LEfSe analysis, 25 differentially abundant taxa were identified as potential biomarkers. Key genera (27) that potentially contributed to the differential distributions of plaque microbiota between groups were identified by PLS-DA analysis. Finally, co-occurrence network analysis and function predictions were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed.