Associations between periodontal infection and cardiovascular disease have been documented. Porphyromonas gingivalis is a well-established periodontal pathogen, and tissue factor (TF) is a key initiator of the coagulation cascade. In this context, P. gingivalis has been reported to enhance TF expression in human endothelial cells. The present study investigated the underlying mechanisms of TF induction by P. gingivalis in human umbilical vein endothelial cells. P. gingivalis increased TF expression in a dose- and time-dependent manner. Not only live bacteria but also glutaraldehyde-fixed bacteria increased TF expression to the same extent. However, sonicates of P. gingivalis did not induce TF expression. Cytochalasin D and SMIFH2, which are inhibitors of actin polymerization and actin nucleation, respectively, inhibited the TF expression induced by P. gingivalis . Finally, TF production was decreased or increased in the presence of various signaling inhibitors, including mitogen-activated protein kinases. These results suggest that P. gingivalis induces endothelial TF expression by a bacterial internalization-dependent mechanism and through diverse signal transduction mechanisms.
The physicochemical properties of crystalline titanium dioxide nanoparticles (TiO2 NPs) were investigated by comparing amorphous (amTiO2), anatase (aTiO2), metaphase of anatase-rutile (arTiO2), and rutile (rTiO2) NPs, which were prepared at various calcination temperatures (100℃, 400℃, 600℃, and 900℃). X-ray diffraction (XRD) and scanning electron microscopy (SEM) analyses confirmed that the phase-transformed TiO2 had the characteristic features of crystallinity and average size. The surface chemical properties of the crystalline phases were different in the spectral analysis. As anatase transformed to the rutile phase, the band of the hydroxyl group at 3,600–3,100 cm–1 decreased gradually, as assessed using Fourier transform infrared spectroscopy (FT-IR). For ultraviolet-visible (UVVis) spectra, the maximum absorbance of anatase TiO2 NPs at 309 nm was blue-shifted to 290 nm at the rutile phase with reduced absorbance. Under the electric field of capillary electrophoresis (CE), TiO2 NPs in anatase migrated and detected as a broaden peak, whereas the rutile NPs did not. In addition, anatase showed the highest photocatalytic activity in an UV-irradiated dye degradation assay in the following order: aTiO2 > arTiO2 > rTiO2. Overall, the phases of TiO2 NPs showed characteristic physicochemical properties regarding size, surface chemical properties, UV absorbance, CE migration, and photocatalytic activity.
Periodontitis and periimplantitis are caused as a result of dental biofilm formation. This biofilm is composed of multiple species of pathogens. Therefore, controlling biofilm formation is critical for disease prevention. To inhibit biofilm formation, sugars can be used to interrupt lectin-involving interactions between bacteria or between bacteria and a host. In this study, we evaluated the effect of D-Arabinose on biofilm formation of putative periodontal pathogens as well as the quorum sensing activity and whole protein profiles of the pathogens. Crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy revealed that D-Arabinose inhibited biofilm formation of Porphyromonas gingivalis , Fusobacterium nucleatum , and Tannerella forsythia . D-Arabinose also significantly inhibited the activity of autoinducer 2 of F. nucleatum and the expression of representative bacterial virulence genes. Furthermore, D-Arabinose treatment altered the expression of some bacterial proteins. These results demonstrate that D-Arabinose can be used as an antibiofilm agent for the prevention of periodontal infections.
Activation of transient receptor potential vanilloid 1 (TRPV1), a calcium permeable channel expressed in primary sensory neurons, induces the release of glutamate from their central and peripheral afferents during normal acute and pathological pain. However, little information is available regarding the glutamate release mechanism associated with TRPV1 activation in primary sensory neurons. To address this issue, we investigated the expression of vesicular glutamate transporter (VGLUT) in TRPV1-immunopositive (+) neurons in the rat trigeminal ganglion (TG) under normal and complete Freund’s adjuvant (CFA)-induced inflammatory pain conditions using behavioral testing as well as double immunofluorescence staining with antisera against TRPV1 and VGLUT1 or VGLUT2. TRPV1 was primarily expressed in small and medium-sized TG neurons. TRPV1+ neurons constituted approximately 27% of all TG neurons. Among all TRPV1+ neurons, the proportion of TRPV1+ neurons coexpressing VGLUT1 (VGLUT1+/ TRPV1+ neurons) and VGLUT2 (VGLUT2+/TRPV1+ neurons) was 0.4% ± 0.2% and 22.4% ± 2.8%, respectively. The proportion of TRPV1+ and VGLUT2+ neurons was higher in the CFA group than in the control group (TRPV1+ neurons: 31.5% ± 2.5% vs. 26.5% ± 1.2%, VGLUT2+ neurons: 31.8% ± 1.1% vs. 24.6% ± 1.5%, p < 0.05), whereas the proportion of VGLUT1+, VGLUT1+/TRPV1+, and VGLUT2+/TRPV1+ neurons did not differ significantly between the CFA and control groups. These findings together suggest that VGLUT2, a major isoform of VGLUTs, is involved in TRPV1 activation-associated glutamate release during normal acute and inflammatory pain.
We previously showed that γ-glutamyltranspeptidase (GGT), an enzyme involved in glutathione metabolism, in Bacillus subtilis acts as a virulence factor for osteoclastogenesis via the RANKL-dependent pathway. Hence, it can be hypothesized that GGT of periodontopathic bacteria acts as a virulence factor in bone destruction. Because Fusobacterium nucleatum, which is a periodontopathic pathogen, has GGT with a primary structure similar to that of B. subtilis GGT (37.7% identify), the bone-resorbing activity of F. nucleatum GGT was examined here. Recombinant GGT (rGGT) of F. nucleatum was expressed in Escherichia coli and purified using the His tag of rGGT. F. nucleatum rGGT (Fn rGGT) was expressed as a precursor of GGT, and then processed to a heavy subunit and a light subunit, which is characteristic of general GGTs, including the human and B. subtilis enzymes. Osteoclastogenesis was achieved in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. Fn rGGT induced osteoclastogenesis to a level similar to that of B. subtilis rGGT; furthermore, osteoclastogenesis was induced in a dose-dependent manner. These results suggest that F. nucleatum GGT possesses a virulent bone-resorbing activity, which could play an important role in the pathogenesis of periodontitis.
Under physiological conditions, calcium (Ca2+) regulates essential functions of polarized secretory cells by the stimulation of specific Ca2+ signaling mechanisms, such as increases in intracellular Ca2+ concentration ([Ca2+]i) via the store-operated Ca2+ entry (SOCE) and the receptor-operated Ca2+ entry (ROCE). Homer proteins are scaffold proteins that interact with G protein-coupled receptors, inositol 1,4,5-triphosphate (IP3) receptors, Orai1-stromal interaction molecule 1, and transient receptor potential canonical (TRPC) channels. However, their role in the Ca2+ signaling in exocrine cells remains unknown. In this study, we investigated the role of Homer2 in the Ca2+ signaling and regulatory channels to mediate SOCE and ROCE in pancreatic acinar cells. Deletion of Homer2 (Homer2–/–) markedly increased the expression of TRPC3, TRPC6, and Orai1 in pancreatic acinar cells, whereas these expressions showed no difference in whole brains of wild-type and Homer2–/– mice. Furthermore, the response of Ca2+ entry by carbachol also showed significant changes to the patterns regulated by specific blockers of SOCE and ROCE in pancreatic acinar cells of Homer2–/– mice. Thus, these results suggest that Homer2 plays a critical role in the regulatory action of the [Ca2+]i via SOCE and ROCE in mouse pancreatic acinar cells.
This study aimed to develop Lautropia mirabilis -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis . The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/ RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.
Accurate identification of microbes facilitates the prediction, prevention, and treatment of human diseases. To increase the accuracy of microbiome data analysis, a long region of the 16S rRNA is commonly sequenced via paired-end sequencing. In paired-end sequencing, a sufficient length of overlapping region is required for effective joining of the reads, and high-quality sequencing reads are needed at the overlapping region. Trimming sequences at the reads distal to a point where sequencing quality drops below a specific threshold enhance the joining process. In this study, we examined the effect of trimming conditions on the number of reads that remained after quality control and chimera removal in the Illumina paired-end reads of the V3–V4 hypervariable region. We also examined the alpha diversity and taxa assigned by each trimming condition. Optimum quality trimming increased the number of good reads and assigned more number of operational taxonomy units. The pre-analysis trimming step has a great influence on further microbiome analysis, and optimized trimming conditions should be applied for Divisive Amplicon Denoising Algorithm 2 analysis in QIIME2 platform.