Naoya Higuchi

Localization of Major, High Molecular Weight Proteins in Bacteroides forsythus

Bacteroides forsythus produces species‐specific major proteins with high molecular weights of 270 and 230‐kDa (270K and 230K). A specific antibody raised against 270K was used for Western blot analysis and immunoelectron microscopy. Western blot analysis showed that the 270K and 230K proteins were immunologically similar. Immunogold labeling of ultrathin‐sectioned bacterial cells and biochemical fractionation revealed that these proteins were localized at the outermost cell surface, not in the cytoplasm. These results suggest that major proteins ubiquitous to this species may form the S‐layer.

Aetiology, incidence and morphology of the C-shaped root canal system and its impact on clinical endodontics

The C-shaped root canal constitutes an unusual root morphology that can be found primarily in mandibular second permanent molars. Due to the complexity of their structure, C-shaped root canal systems may complicate endodontic interventions. A thorough understanding of root canal morphology is therefore imperative for proper diagnosis and successful treatment. This review aims to summarize current knowledge regarding C-shaped roots and root canals, from basic morphology to advanced endodontic procedures. To this end, a systematic search was conducted using the MEDLINE, BIOSIS, Cochrane Library, EMBASE, Google Scholar, Web of Science, PLoS and BioMed Central databases, and many rarely cited articles were included. Furthermore, four interactive 3D models of extracted teeth are introduced that will allow for a better understanding of the complex C-shaped root canal morphology. In addition, the present publication includes an embedded best-practice video showing an exemplary root canal procedure on a tooth with a pronounced C-shaped root canal. The survey of this unusual structure concludes with a number of suggestions concerning future research efforts.

A survey of filling methods, intracanal medications, and instrument breakage.

The present survey was conducted to obtain answers to some basic questions regarding the use of intracanal medications, root canal filling methods, and the intracanal breakage of instruments. A letter was sent to 300 endodontists listed in the 1995-1996 membership roster of the American Association of Endodontists. Eighty-five replies were received. Calcium hydroxide as an intracanal medication was used by 91.7% of the responding endodontists. With regard to the root canal filling technique, 52.9% used the lateral condensation method. When intracanal breakage occurred, 95.3% of the respondents informed the patient. The results of this survey provide useful information for the education of undergraduate dental students.

Increased Expression of Interleukin (IL)-35 and IL-17, But Not IL-27, in Gingival Tissues With Chronic Periodontitis

BACKGROUND Interleukin (IL)-35 plays an important role in immune regulation through the suppression of effector T-cell populations, including T-helper 17 (Th17) cells. Although Th17 cells and IL-17 are involved in the pathogenesis of periodontitis, the level of IL-35 in inflamed periodontal tissues is unclear. Here, IL-35, IL-17, and IL-27 production/expression in gingival crevicular fluid (GCF) and human gingival tissue were investigated. METHODS GCF samples were collected from buccal (mesial, center, and distal) sites of teeth from patients with chronic periodontitis (CP) and healthy controls and were analyzed by enzyme-linked immunosorbent assay for IL-35 (periodontitis, n = 36; healthy, n = 30) and IL-17 (periodontitis, n = 16; healthy, n = 13). Gingival tissue, including sulcus/pocket epithelium and underlying connective tissue, was collected from an additional 10 healthy participants and 10 patients with CP and were analyzed by quantitative polymerase chain reaction (qPCR) for Epstein Barr virus-induced gene 3 (EBI3), IL12A, and IL17A. IL27p28 was also tested by qPCR. RESULTS IL-35 and IL-17 were significantly higher in GCF from patients with periodontitis than healthy participants (P <0.01, P <0.05, respectively). In both healthy participants and those with periodontitis, positive correlations were found among IL-35 and probing depth and clinical attachment level (CAL) as well as between IL-17 and CAL. EBI3, IL12A (components of IL-35), and IL17A messenger RNA expression levels were significantly higher in inflamed gingival tissue than in healthy control tissues (P <0.05). IL27p28 was not detected in any sample, suggesting that IL-27 is not produced in large quantities in periodontal tissue. CONCLUSION IL-35 and IL-17, but not IL-27, may play important roles in the pathogenesis of periodontitis.

Experimental pain in the gingiva and its impact on prefrontal cortical hemodynamics: A functional near-infrared spectroscopy study

Evaluating alterations in brain activity in response to pain stimulus can help understand the mechanisms underlying pain perception. We measured oxygenated hemoglobin (oxy-Hb) levels using functional near-infrared spectroscopy (fNIRS) in order to assess prefrontal cortex activation after inducing a pain stimulus to the gingiva. Twenty-three right-handed, healthy male subjects (mean age: 29.3±3.6 years) were subjected to a mild pain stimulus to the tissue around the right maxillary central incisor. The periodontal pain stimulus (PPS) was elicited from a pocket probe, and a multi-channel fNIRS system with its accompanying 22-channel probes was used for measuring oxy-Hb levels. Mean oxy-Hb levels for each channel were calculated on the basis of values obtained at rest and during the PPS load, for 1min each. The change in oxy-Hb level was calculated by subtracting oxy-Hb at rest from oxy-Hb levels during PPS load. Oxy-Hb levels in each channel during both conditions were then compared using the paired t-test and Bonferroni correction. Pain stimulation caused oxy-Hb levels to decrease in virtually all areas of the prefrontal cortex, particularly, in the superior frontal gyrus, the middle frontal gyrus, and the orbital part of the superior, middle, and inferior frontal gyrus, on the brain side contralateral to the pain load. This measurement could prove beneficial as an index for objective pain evaluation.

A morphological and immunolabeling study of freeze-substituted Bacteroides forsythus

We used a rapid freezing and freeze-substitution technique without glutaraldehyde and OsO4 fixation for the electron microscopic immunocytochemical demonstration of the surface structure of Bacteroides forsythus, an anaerobic Gram-negative periodontopathogen. Cells were applied to pieces of filter paper and freeze-substituted by plunge-freezing in liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR White resin. The membrane ultrastructure of B. forsythus was preserved well, and the labeling density of the freeze-submitted cells was compared to a conventional processing method. Our results show the usefulness of the freeze-substitution method for immunohistochemical studies of B. forsythus.

Gelatin scaffold combined with bone morphogenetic protein-4 induces odontoblast-like cell differentiation involving integrin profile changes, autophagy-related gene 10, and Wnt5 sequentially in human induced pluripotent stem cells.

While human induced pluripotent stem (hiPS) cells have potential use in regenerative medicine, there are no reports on odontoblastic differentiation of hiPS cells. In the current study, to examine integrin profiles and explore the early signaling cascade of odontoblastic differentiation in hiPS cells, we investigated the regulation of autophagy-related gene (Atg) and wingless/int1 (Wnt) signaling in gelatin scaffold (GS) combined with bone morphogenetic protein (BMP)-4 (GS/BMP-4)-mediated odontoblastic differentiation. Following GS/BMP-4 treatment, there was a dramatic loss of α3 and α6 integrins, and reciprocal strong induction of α1 integrin expression in the differentiated cells. GS/BMP-4 increased the mRNA and protein levels of Atg10, Lrp5/Fzd9 (an Atg10 receptor), and Wnt5 together with the amount of autophagosomes and autophagic fluxes. Treatment with siRNAs against Atg10 and Wnt5a individually suppressed the GS/BMP-4-induced increase in odontoblastic differentiation. The odontoblastic phenotype was inhibited by chloroquine, but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have replicated our results from the rodent system in a novel human system. We have revealed a unique sequential cascade involving Atg10, Wnt5a, α1 integrin, and matrix metalloproteinase-3 in GS/BMP-4-induced differentiation of hiPS cells into odontoblast-like cells at a relatively early stage.

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3′-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.

Measurement of cerebral blood volume dynamics during volitional swallowing using functional near-infrared spectroscopy: An exploratory study

The aim of this study was to examine cerebral blood volume dynamics during volitional swallowing using multi-channel functional near-infrared spectroscopy (fNIRS) to understand the basic cortical activation patterns. Fifteen volunteers (age, 26.5±1.3 years, mean±SD) performed volitional swallowing of a 5-ml bolus of water as a task. A 52-channel fNIRS system was used for measuring oxy-Hb levels. We determined the oxy-Hb concentration changes in each channel by calculating the differences between rest and task oxy-Hb levels. Differences in rest and task data were assessed using a paired-t test (p<0.05). A significant increase in oxy-Hb was found in 21 channels. The cortical regions that exhibited increased oxy-Hb concentration included the bilateral precentral gyrus, postcentral gyrus, inferior frontal gyrus, superior temporal gyrus, middle temporal gyrus, and supramarginal gyrus. These data provide a description of cortical activation patterns during volitional swallowing using fNIRS, which will be useful for the evaluation of dysphasia and the effects of the rehabilitation [Corrected].

Energy dispersive spectroscopy-scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non-opsonized Tannerella forsythia

We investigated the association between human polymorphonuclear leukocytes (PMNs) and non‐opsonized Tannerella forsythia ATCC 43037 displaying a serum‐resistant surface layer (S‐layer). When PMNs were mixed with T. forsythia in suspension, the cells phagocytosed T. forsythia cells. Nitro blue tetrazolium (NBT) reduction, indicative of O2− production, was observed by light microscopy; cerium (Ce) perhydroxide deposition, indicative of H2O2 production, was observed by electron microscopy. We examined the relationship between high‐molecular‐weight proteins of the S‐layer and Ce reaction (for T. forsythia phagocytosis) using electron microscopic immunolabeling. Immunogold particles were localized within the PMNs and on cell surfaces, labelling at the same Ce‐reacted sites where the S‐layer was present. We then used energy dispersive spectroscopy (EDS)‐scanning transmission electron microscope (STEM) to perform Ce and nitrogen (N) (for S‐layer immunocytochemistry) elemental analysis on the phagocytosed cells. That is, the elemental mapping and analysis of N by EDS appeared to reflect the presence of the same moieties detected by the 3,3′‐diaminobenzidine‐tetrahydrochloride (DAB) reaction with horseradish peroxidase (HRP)‐conjugated secondary antibodies, instead of immunogold labeling. We focused on the use of EDS‐STEM to visualize the presence of N resulting from the DAB reaction. In a parallel set of experiments, we used EDS‐STEM to perform Ce and gold (Au; from immunogold labeling of the S‐layer) elemental analysis on the same phagocytosing cells.