Characterization of oral microbiota in 6-8-month-old small breed dogs.

BACKGROUND: Periodontitis is the most common oral disease in dogs, and its progression and severity are influenced by risk factors, such as age and body size. Recent studies have assessed the canine oral microbiota in relation to different stages of periodontitis and niches within the oral cavity. However, knowledge of the bacterial composition at different ages and body sizes, especially in puppies, is limited. This study aimed to characterize the oral microbiota in the healthy gingiva of small breed puppies using next-generation sequencing. Additionally, we assessed the impact of dental care practices and the presence of retained deciduous teeth on the oral microbiota. RESULTS: In this study, plaque samples were collected from the gingival margin of 20 small breed puppies (age, 6.9 ± 0.6 months). The plaque samples were subjected to next-generation sequencing targeting the V3-V4 region of the 16 S rRNA. The microbiota of the plaque samples was composed mostly of gram-negative bacteria, primarily Proteobacteria (54.12%), Bacteroidetes (28.79%), and Fusobacteria (5.11%). Moraxella sp. COT-017, Capnocytophaga cynodegmi COT-254, and Bergeyella zoohelcum COT-186 were abundant in the oral cavity of the puppies. In contrast, Neisseria animaloris were not detected. The high abundance of Pasteurellaceae suggests that this genus is characteristic of the oral microbiota in puppies. Dental care practices and the presence of retained deciduous teeth showed no effects on the oral microbiota. CONCLUSIONS: In this study, many bacterial species previously reported to be detected in the normal oral cavity of adult dogs were also detected in 6-8-month-old small breed dogs. On the other hand, some bacterial species were not detected at all, while others were detected in high abundance. These data indicate that the oral microbiota of 6-8-month-old small breed dogs is in the process of maturating in to the adult microbiota and may also have characteristics of the small dog oral microbiota.

Evaluation of a Novel Immunochromatographic Device for Detecting Porphyromonas gingivalis in Patients with Periodontal Disease.

Porphyromonas gingivalis is the most pathogenic periodontal bacterium in the world. Recently, P. gingivalis has been considered responsible for dysbiosis during the development of periodontitis. This study aimed to evaluate a novel immunochromatographic device using monoclonal antibodies against P. gingivalis in subgingival plaques. A total of 72 patients with chronic periodontitis and 53 periodontally healthy volunteers underwent clinical and microbiological examinations. Subgingival plaque samples were analyzed for the presence of P. gingivalis and compared using real-time polymerase chain reaction (PCR). In the periodontitis group, a significant positive correlation was observed between the test device scores and the real-time PCR results. The specificity, positive predictive value, negative predictive value, and accuracy of the test device for P. gingivalis, as determined by real-time PCR, were 98%, 94%, 89%, and 90%, respectively. There were significant differences in bacterial counts by real-time PCR among the groups with different ranges of device scores. Additionally, there was a significant positive correlation between the device scores for P. gingivalis and periodontal parameters. These results suggest that this novel immunochromatographic device can be effectively used for rapid detection and semi-quantification of P. gingivalis in subgingival plaques.

Ascorbic acid enhances chondrocyte differentiation of ATDC5 by accelerating insulin receptor signaling.

Chondrogenesis is strictly regulated by several factors, including cytokines, hormones, and extracellular matrix proteins. Mouse teratocarcinoma-derived lineage cells, differentiate into chondrocytes in the presence of insulin. Although ascorbic acid promotes chondrogenic differentiation, the detailed regulative mechanisms underlying its role in chondrogenesis remain unclear. Therefore, in this study, we evaluated the effects of ascorbic acid on insulin-induced chondrogenic differentiation of ATDC5 cells and the underlying intracellular signaling. The results revealed that insulin-stimulated collagen deposition, matrix formation, calcification, and expression of chondrogenic differentiation marker genes in ATDC5 cells. This enhancement by insulin was amplified with the addition of ascorbic acid. Molecular analysis revealed that the activation of insulin-induced phosphoinositide 3-kinase (PI3K)/Akt signaling was enhanced in the presence of ascorbic acid. In contrast, Wnt/ß-catenin signaling was suppressed during chondrocyte differentiation via upregulation of the Wnt agonist, secreted Frizzled-related protein 1 (sFRP-1) and 3 (sFRP-3). Notably, ascorbic acid upregulated the expression of insulin receptors and their substrates (IRS-1 and IRS-2). Furthermore, ascorbic acid reversed the suppression of IRS-1 and IRS-2 protein by insulin. These results indicate that ascorbic acid positively regulates the chondrogenic differentiation of ATDC5 cells via enhancement of insulin signaling. Our findings provide a substantial basis for further elucidation of the regulatory mechanisms of chondrocyte differentiation and the pathophysiology of OA, thus aiding in development of effective treatment strategies.

Evaluation of financial conflicts of interest and quality of evidence in Japanese gastroenterology clinical practice guidelines.

BACKGROUND: Clinical practice guidelines assist healthcare professionals in providing evidence-based care. However, pharmaceutical companies' financial interests often influence guideline content. This study aimed to elucidate the magnitude of financial ties among Japanese gastroenterology guideline authors and the pharmaceutical industry. METHODS: Using pharmaceutical company disclosed payment data, we evaluated financial conflicts of interest (COI) among Japanese Society of Gastroenterology guideline authors between 2016 and 2021. Additionally, we assessed the evidence quality supporting guideline recommendations and associations with financial COI. Finally, we evaluated author COI management during guideline development against global standards. RESULTS: Overall, 88.2% (231/262) of guideline authors received a median of $12 968 (interquartile range [IQR]: $1839-$70 374) in payments between 2016 and 2019 for lectures, writings, and consulting. Chairpersons received significantly higher payments (median: $86 444 [IQR: $15 455-$165 679]). Notably, 41 (15.6%) authors had undeclared payments exceeding declaration requirements. Low or very low-quality evidence supported 41.0% of recommendations. There was a negative association between the median 4-year payment per author and the proportion of recommendations based on low-quality evidence (odds ratio: 0.966 [95% confidence interval [95% CI]: 0.945-0.987], P = 0.002) and positive association with moderate-quality evidence (odds ratio: 1.018 [95% CI: 1.011-1.025], P < 0.001). Still, the Japanese Society of Gastroenterology guideline development process remains less transparent, with insufficient COI policies relative to global standards. CONCLUSION: There were extensive financial COI between pharmaceutical companies and guideline authors, and more than 40% of recommendations were based on low-quality evidence. More rigorous and transparent COI policies for guideline development adhering to global standards are warranted.

The Mechanism of Interleukin 33-Induced Stimulation of Interleukin 6 in MLO-Y4 Cells.

The differentiation and function of osteocytes are controlled by surrounding cells and mechanical stress; however, the detailed mechanisms are unknown. Recent findings suggest that IL-33 is highly expressed in periodontal tissues in orthodontic tooth movement. The present study aimed to elucidate the effect of IL-33 on the expression of regulatory factors for bone remodeling and their molecular mechanisms in the osteocyte-like cell line MLO-Y4. MLO-Y4 cells were treated with IL-33, and the activation of intracellular signaling molecules and transcriptional factors was determined using Western blot analysis and chromatin immunoprecipitation assay. IL-33 treatment enhanced the expression of IL-6 in MLO-Y4 cells, which was suppressed by the knockdown of the IL-33 receptor ST2L. Additionally, IL-33 treatment induced activation of NF-κB, JNK/AP-1, and p38 MAPK signaling pathways in MLO-Y4 cells. Moreover, pretreatment with specific inhibitors of NF-κB, p38 MAPK, and JNK/AP-1 attenuated the IL-33-induced expression of IL-6. Furthermore, chromatin immunoprecipitation indicated that IL-33 increased c-Jun recruitment to the IL-6 promoter. Overall, these results suggest that IL-33 induces IL-6 expression and regulates osteocyte function via activation of the NF-κB, JNK/AP-1, and p38 MAPK pathways through interaction with ST2L receptors on the plasma membrane.

Dectin-1-mediated suppression of RANKL-induced osteoclastogenesis by glucan from baker's yeast.

Immunoreceptors expressed on osteoclast precursor cells modify osteoclast differentiation and bone resorption activity. Dectin-1 is a lectin receptor of ß-glucan and is specifically expressed in osteoclast precursor cells. In this study, we evaluated the bioactivity of ß-glucan on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and observed that glucan from baker's yeast inhibited this process in mouse bone marrow cells and dectin-1-overexpressing RAW264.7 (d-RAW) cells. In conjunction, RANKL-induced nuclear factor of activated T cell c1 expression was suppressed, subsequently downregulating TRAP and Oc-stamp. Additionally, nuclear factor-kappa B activation and the expression of c-fos and Blimp1 were reduced in d-RAW cells. Furthermore, glucan from baker's yeast induced the degradation of Syk protein, essential factor for osteoclastogenesis. These results suggest that glucan from baker's yeast suppresses RANKL-induced osteoclastogenesis and can be applied as a new treatment strategy for bone-related diseases.

Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation.

Cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as an active stress response. How these phage fossils affect persister cell resuscitation has, however, not been explored. Persister cells form as a result of stresses such as starvation, antibiotics and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis and Lyme disease. Deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system resulting from inactivating the transcriptional regulator AlpA of the CP4-57 cryptic prophage. Deletion of alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Both phosphate regulators PhoP and PhoB stimulate resuscitation. This suggests a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.

Biological Effects of ß-Glucans on Osteoclastogenesis.

Although the anti-tumor and anti-infective properties of ß-glucans have been well-discussed, their role in bone metabolism has not been reviewed so far. This review discusses the biological effects of ß-glucans on bone metabolisms, especially on bone-resorbing osteoclasts, which are differentiated from hematopoietic precursors. Multiple immunoreceptors that can recognize ß-glucans were reported to be expressed in osteoclast precursors. Coordinated co-stimulatory signals mediated by these immunoreceptors are important for the regulation of osteoclastogenesis and bone remodeling. Curdlan from the bacterium Alcaligenes faecalis negatively regulates osteoclast differentiation in vitro by affecting both the osteoclast precursors and osteoclast-supporting cells. We also showed that laminarin, lichenan, and glucan from baker's yeast, as well as ß-1,3-glucan from Euglema gracilisas, inhibit the osteoclast formation in bone marrow cells. Consistent with these findings, systemic and local administration of ß-glucan derived from Aureobasidium pullulans and Saccharomyces cerevisiae suppressed bone resorption in vivo. However, zymosan derived from S. cerevisiae stimulated the bone resorption activity and is widely used to induce arthritis in animal models. Additional research concerning the relationship between the molecular structure of ß-glucan and its effect on osteoclastic bone resorption will be beneficial for the development of novel treatment strategies for bone-related diseases.

Rhamnolipids and surfactin inhibit the growth or formation of oral bacterial biofilm.

BACKGROUND: Bacteria survive in various environments by forming biofilms. Bacterial biofilms often cause significant problems to medical instruments and industrial processes. Techniques to inhibit biofilm formation are essential and have wide applications. In this study, we evaluated the ability of two types of biosurfactants (rhamnolipids and surfactin) to inhibit growth and biofilm formation ability of oral pathogenic bacteria such as Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Streptococcus sanguinis. RESULTS: Rhamnolipids inhibited the growth and biofilm formation ability of all examined oral bacteria. Surfactin showed effective inhibition against S. sanguinis ATCC10556, but lower effects toward A. actinomycetemcomitans Y4 and S. mutans UA159. To corroborate these results, biofilms were observed by scanning electron microscopy (SEM) and confocal microscopy. The observations were largely in concordance with the biofilm assay results. We also attempted to determine the step in the biofilm formation process that was inhibited by biosurfactants. The results clearly demonstrated that rhamnolipids inhibit biofilm formation after the initiation process, however, they do not affect attachment or maturation. CONCLUSIONS: Rhamnolipids inhibit oral bacterial growth and biofilm formation by A. actinomycetemcomitans Y4, and may serve as novel oral drug against localized invasive periodontitis.

σ54 -Dependent regulator DVU2956 switches Desulfovibrio vulgaris from biofilm formation to planktonic growth and regulates hydrogen sulfide production.

Microbiologically influenced corrosion causes $100 billion in damage per year, and biofilms formed by sulfate-reducing bacteria (SRB) are the major culprit. However, little is known about the regulation of SRB biofilm formation. Using Desulfovibrio vulgaris as a model SRB organism, we compared the transcriptomes of biofilm and planktonic cells and identified that the gene for σ54 -dependent regulator DVU2956 is repressed in biofilms. Utilizing a novel promoter that is primarily transcribed in biofilms (Pdvu0304 ), we found production of DVU2956 inhibits biofilm formation by 70%. Corroborating this result, deleting dvu2956 increased biofilm formation, and this biofilm phenotype could be complemented. By producing proteins in biofilms from genes controlled by DVU2956 (dvu2960 and dvu2962), biofilm formation was inhibited almost completely. A second round of RNA-seq for the production of DVU2956 revealed DVU2956 influences electron transport via an Hmc complex (high-molecular-weight cytochrome c encoded by dvu0531-dvu0536) and the Fe-only hydrogenase (encoded by dvu1769, hydA and dvu1770, hydB) to control H2 S production. Corroborating these results, producing DVU2956 in biofilms decreased H2 S production by half, deleting dvu2956 increased H2 S production by 131 ± 5%, and producing DVU2956 in the dvu2956 strain reduced H2 S production. Therefore, DVU2956 maintains SRB in the planktonic state and reduces H2 S formation.

Identification of a potent indigoid persister antimicrobial by screening dormant cells.

The subpopulation of bacterial cells that survive myriad stress conditions (e.g., nutrient deprivation and antimicrobials) by ceasing metabolism, revive by activating ribosomes. These resuscitated cells can reconstitute infections; hence, it is imperative to discover compounds which eradicate persister cells. By screening 10,000 compounds directly for persister cell killing, we identified 5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride (NPIMA) kills Escherichia coli persister cells more effectively than the best indigoid found to date, 5-iodoindole, and better than the DNA-crosslinker cisplatin. In addition, NPIMA eradicated Pseudomonas aeruginosa persister cells in a manner comparable to cisplatin. NPIMA also eradicated Staphylococcus aureus persister cells but was less effective than cisplatin. Critically, NPIMA kills Gram-positive and Gram-negative bacteria by damaging membranes and causing lysis as demonstrated by microscopy and release of extracellular DNA and protein. Furthermore, NPIMA was effective in reducing P. aeruginosa and S. aureus cell numbers in a wound model, and no resistance was found after 1 week. Hence, we identified a potent indigoid that kills persister cells by damaging their membranes.

Viable but non-culturable and persistence describe the same bacterial stress state.

Bacteria are often thought of as having two dormant phenotypes: the viable but non-culturable (VBNC) state and the persister state. Here we investigate the relatedness of the two stress-induced phenotypes at the single-cell level and examine cell morphology and quantify cell resuscitation. Using the classic starvation conditions to create VBNC cells, we found that the majority of the remaining Escherichia coli population are spherical, have empty cytosol and fail to resuscitate; however, some of the spherical cells resuscitate immediately (most probably those with dense cytosol). Critically, all the culturable cells in this starved population became persister cells within 14 days of starvation. We found that the persister cells initially are rod-like, have clear but limited membrane damage, can resuscitate immediately and gradually become spherical by aging. After 24 h, only rod-shaped persister cells survive, and all the spherical cells lyse. Both cell populations formed under the VBNC-inducing conditions and the persister conditions are metabolically inactive. Therefore, the bacterial population consists of dead cells and persister cells in the VBNC-inducing conditions; that is, the non-lysed particles that do not resuscitate are dead, and the dormant cells that resuscitate are persister cells. Hence, 'VBNC' and 'persister' describe the same dormant phenotype.

Single cell observations show persister cells wake based on ribosome content.

Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising after antibiotic treatment at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through eight sets of experiments), we used microscopy of single cells to determine that the resuscitation of dormant persisters is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found during the observation of persister cells when fresh nutrients were added: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.

Current state and perspectives in hydrogen production by Escherichia coli: roles of hydrogenases in glucose or glycerol metabolism.

Escherichia coli has been a robust host strain for much biological research, in particular, research in metabolic engineering, protein engineering, and heterologous gene expression. In this mini review, to understand bacterial hydrogen production by E. coli, the effect of glucose and glycerol metabolism on hydrogen production is compared, and the current approaches to enhance hydrogen production from glycerol as a substrate are reviewed. In addition, the argument from past to present on the functions of E. coli hydrogenases, hydrogenase 1, hydrogenase 2, hydrogenase 3, and hydrogenase 4 is summarized. Furthermore, based on the literature that the E. coli formate-hydrogen lyase is essential for bacterial hydrogen production via recombinant hydrogenases, research achievements from the past regarding heterologous production of hydrogenase are rethought.

Medicinal herbs, especially Hibiscus sabdariffa, inhibit oral pathogenic bacteria.

OBJECTIVES: Medicinal herbs are plants with potential medicinal and health benefits. In recent years, they are being increasingly used as a treatment alternative owing to their effectiveness against various diseases. In this study, we investigated the inhibitory effects of 15 medicinal herbs on causative bacteria for dental caries and periodontal disease. METHODS: This study evaluated the effects of the extracts of 15 medicinal herbs on growth and biofilm formation in five oral pathogenic bacterial strains. The herbs were processed into extracts, and bacterial strains were cultured. Then, bacterial growth and biofilm formation were assessed using various methods. Finally, the extract of the herb Hibiscus sabdariffa (hibiscus) was analyzed using high-performance liquid chromatography. RESULTS: Incubation of bacteria with the herbal extracts showed that hibiscus exerted a significant inhibitory effect on all the oral pathogenic bacterial strains evaluated in this study. In addition, the pigment delphinidin-3-sambubioside, which is found in hibiscus extract, was identified as a particularly important inhibitory component. CONCLUSIONS: These results lay the ground work for the potential development of novel therapeutic or preventive agents against dental caries and periodontal disease, two major oral diseases.

Enhancement of cryopreservation with intracellularly permeable zwitterionic polymers.

A novel copolymer containing zwitterionic and methylsulfinyl structures was developed, which enhanced cryoprotective efficacy by enabling intracellular cytoplasmic permeation without relying on mediated endocytosis and diffused out of the cells within approximately 30 min, making it more advantageous than polymeric nanoparticles for the transport of membrane-impermeable cryoprotectants such as trehalose.

Persister Cell Formation and Elevated lsrA and lsrC Gene Expression upon Hydrogen Peroxide Exposure in a Periodontal Pathogen Aggregatibacter actinomycetemcomitans.

The effect of hydrogen peroxide, an antiseptic dental treatment, on Aggregatibacter actinomycetemcomitans, the main causative agent of localized invasive periodontitis, was investigated. Hydrogen peroxide treatment (0.06%, 4× minimum inhibitory concentration) resulted in the persistence and survival of approximately 0.5% of the bacterial population. The surviving bacteria did not genetically acquire hydrogen peroxide resistance but exhibited a known persister behavior. Sterilization with mitomycin C significantly reduced the number of A. actinomycetemcomitans persister survivors. RNA sequencing of hydrogen peroxide-treated A. actinomycetemcomitans showed elevated expression of Lsr family members, suggesting a strong involvement of autoinducer uptake. In this study, we found a risk of A. actinomycetemcomitans persister residual from hydrogen peroxide treatment and hypothesized associated genetic mechanisms of persister from RNA sequencing.

Magnesium Hydroxide Nanoparticles Inhibit the Biofilm Formation of Cariogenic Microorganisms.

Although various caries-preventive agents have been developed, dental caries is still a leading global disease, mostly caused by biological factors such as mutans streptococci. Magnesium hydroxide nanoparticles have been reported to exhibit antibacterial effects; however, they are rarely used in oral care practical applications. In this study, we examined the inhibitory effect of magnesium hydroxide nanoparticles on biofilm formation by Streptococcus mutans and Streptococcus sobrinus-two typical caries-causing bacteria. Three different sizes of magnesium hydroxide nanoparticles (NM80, NM300, and NM700) were studied, all of which inhibited biofilm formation. The results showed that the nanoparticles were important for the inhibitory effect, which was not influenced by pH or the presence of magnesium ions. We also determined that the inhibition process was mainly contact inhibition and that medium (NM300) and large (NM700) sizes were particularly effective in this regard. The findings of our study demonstrate the potential applications of magnesium hydroxide nanoparticles as caries-preventive agents.

Fabrication of a Three-Dimensional Spheroid Culture System for Oral Squamous Cell Carcinomas Using a Microfabricated Device.

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24-48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

Mechanisms Underlying the Suppression of IL-1ß Expression by Magnesium Hydroxide Nanoparticles.

In recent years, magnesium hydroxide has been widely studied due to its bioactivity and biocompatibility. The bactericidal effects of magnesium hydroxide nanoparticles on oral bacteria have also been reported. Therefore, in this study, we investigated the biological effects of magnesium hydroxide nanoparticles on inflammatory responses induced by periodontopathic bacteria. Macrophage-like cells, namely J774.1 cells, were treated with LPS derived from Aggregatibacter actinomycetemcomitans and two different sizes of magnesium hydroxide nanoparticles (NM80/NM300) to evaluate their effects on the inflammatory response. Statistical analysis was performed using an unresponsive Student's t-test or one-way ANOVA followed by Tukey's post hoc test. NM80 and NM300 inhibited the expression and secretion of IL-1ß induced by LPS. Furthermore, IL-1ß inhibition by NM80 was dependent on the downregulation of PI3K/Akt-mediated NF-κB activation and the phosphorylation of MAPK molecules such as JNK, ERK1/2, and p38 MAPK. By contrast, only the deactivation of the ERK1/2-mediated signaling cascade is involved in IL-1ß suppression by NM300. Although the molecular mechanism involved varied with size, these results suggest that magnesium hydroxide nanoparticles have an anti-inflammatory effect against the etiologic factors of periodontopathic bacteria. These properties of magnesium hydroxide nanoparticles can be applied to dental materials.