Sirtuin 6 attenuates periapical lesion propagation by modulating hypoxia-induced chemokine (C-C motif) ligand 2 production in osteoblasts.

AIM: To investigate the attenuating effect of sirtuin 6 (SIRT6) on hypoxia-induced production of chemokine (C-C motif) ligand 2 (CCL2) by osteoblasts and the relevance of this action on the pathogenesis of periapical lesions. METHODOLOGY: Sirtuin 6 was overexpressed in MC3T3-E1 murine osteoblasts by lentivirus-mediated gene transfer. The relationship between the antiglycolytic/antioxidative activities of SIRT6 and its effect on hypoxia-induced CCL2 production were examined. Pathogenetic relevance of the actions of SIRT6 was assessed in a rat model of induced apical periodontitis. The data were analysed statistically using Student's t-test or one-way analysis of variance (anova) and then a Tukey's multiple comparison test. RESULTS: In cultured murine osteoblasts, 24-h hypoxic treatment significantly enhanced the generation of reactive oxygen species (P = 0.003), expression of lactate dehydrogenase A (LDHA) and production of lactate (P = 0.007). A reciprocal effect between hypoxia-induced redox imbalance and hypoxia-enhanced glycolysis was noted which in turn augmented the secretion of CCL2. Through its antiglycolytic and antioxidative effects, SIRT6 blocked the vicious cycle to suppress CCL2 production. In normal periapical tissues of rats, strong expression of SIRT6 and low levels of LDHA and 8-OHdG (a marker of oxidative DNA damage) were found in osteoblasts. In induced apical periodontitis, osteoblastic expression of SIRT6 was significantly suppressed (P = 0.001) which was associated with significantly elevated levels of LDHA (P = 0.003) and 8-OHdG (P = 0.004) and significantly enhanced recruitment of macrophages (P = 0.004). CONCLUSIONS: Sirtuin 6 has a therapeutic effect on periapical lesions through suppression of CCL2 synthesis. The anti-inflammatory action of SIRT6 is closely related to its regulatory activities in cellular metabolism and redox homoeostasis.

Health impacts of Facebook usage and mobile texting among undergraduate dental students: it's time to understand the difference between usage and an excessive use.

BACKGROUND: Facebook and mobile texting are prevalent in the lives of almost every student. However, little is known about the relationship between Facebook usage or mobile texting and their impacts on health amongst undergraduate dental students. In this study, excessive Facebook use and excessive mobile texting were studied as they relate to impacts on health. MATERIALS AND METHODS: A cross-sectional study was conducted at a private university in Malaysia. A total of 188 undergraduate dental students were interviewed using a pre-tested and self-rated questionnaire. Data collected from participants were analysed using SPSS version 18.0. Chi-square test, Fisher's exact test and multiple logistic regression analyses were applied to study the relationship between explanatory variables and excessive Facebook use and excessive mobile texting. RESULTS: The prevalence of excessive Facebook use and excessive mobile texting amongst undergraduate dental students was found to be 33.2% and 33.0%, respectively. According to a multivariate analysis, texting habits, such as the presence of daytime sleepiness after texting late at night (aOR = 2.682, 95% CI = 1.142-6.301) and the presence of anxious feelings if students failed to receive a timely response (aOR = 3.819, 95% CI = 1.580-9.230), were determined to be significant predictors of excessive mobile texting. Excessive Facebook use was found to be significantly related to three variables as follows: fewer numbers of close friends (aOR = 2.275, 95% CI = 1.057-4.898), the checking of updates on the Facebook walls of their friends (aOR = 2.582, 95% CI = 1.189-5.605) and the absence of active and vigorous feelings during Facebook use (aOR = 3.401, 95% CI = 1.233-9.434). CONCLUSIONS: Approximately one-third of undergraduate dental students in this study experienced excessive Facebook use and/or excessive mobile texting. Health education and promotion should be instituted to create awareness, whilst students should be advised to practise self-control with respect to both mobile texting and Facebook usage.

Evaluation of developmentally hypomineralised enamel after surface pretreatment with Papacarie Duo gel and different etching modes: an in vitro SEM and AFM study.

PURPOSE: This study aimed at investigating the surface morphology and nanotopography of normal enamel (NE) and developmentally hypomineralised enamel (HE) when subjected to various pretreatment protocols under scanning electron microscopy (SEM) and atomic force microscopy (AFM). METHODS: Sixteen NE, 16 creamy/white (CW) HE and 16 yellow/brown (YB) HE specimens sectioned from extracted hypomineralised first permanent molars (FPMs) were included in this study. They were randomly distributed into 12 experimental groups (n = 4). Each group involved the following: (1) deproteinisation with Papacarie Duo® gel or no deproteinisation, and (2) the use of Scotchbond™ Universal Adhesive (Scotchbond) in self-etch (SE) mode or 37% phosphoric acid etchant. Subsequently, the surface morphology and nanotopography of pretreated enamel specimens were evaluated under SEM and AFM, respectively. RESULTS: SEM observation showed that deproteinisation with Papacarie Duo® gel before phosphoric acid etching led to favourable etching patterns. This was consistent across all groups irrespective of the type of enamel specimen and the severity of hypomineralisation. In contrast, AFM results identified three factors that influenced surface parameters: (1) type of enamel specimen, (2) severity of hypomineralisation and (3) etching mode. YB HE recorded higher surface roughness values than CW HE and NE when subjected to the same pretreatment protocol. Deproteinisation and the application of Scotchbond in SE mode led to minimal topographic changes; however, acid etching was associated with an increase in surface roughness. CONCLUSION: Deproteinisation with Papacarie Duo® gel followed by acid etching contributed to improved etching patterns on HE.

Effects of EDTA on the hydration mechanism of mineral trioxide aggregate.

Ethylenediaminetetraacetic acid (EDTA) is commonly used during the preparation of obstructed root canals that face a high risk of root perforation. Such perforations may be repaired with mineral trioxide aggregate (MTA). Due to EDTA's ability to chelate calcium ions, we hypothesized that EDTA may disrupt the hydration of MTA. Using scanning electron microscopy and energy-dispersive x-ray spectroscopy, we found that MTA specimens stored in an EDTA solution had no crystalline structure and a Ca/Si molar ratio considerably lower than those obtained for specimens stored in distilled water and normal saline. Poor cell adhesion in EDTA-treated MTA was also noted. X-ray diffraction indicated that the peak corresponding to portlandite, which is normally present in hydrated MTA, was not shown in the EDTA group. The microhardness of EDTA-treated specimens was also significantly reduced (p < 0.0001). These findings suggest that EDTA interferes with the hydration of MTA, resulting in decreased hardness and poor biocompatibility.

Demonstration of different modes of cell death upon herpes simplex virus 1 infection in different types of oral cells.

The effects of Herpes simplex virus 1 (HSV-1) infection on five different types of oral cancerous cells (neck metastasis of gingival carcinoma (GNM) cells and tongue squamous cells of carcinoma (TSCCa) and non-cancerous cells (buccal mucosal fibroblasts (BF), gingival fibroblasts (GF), oral submucosal fibrosis cells (OSF)) and one type of non-oral cancerous cells (KB cells) were investigated. In HSV-1-infected cells the cell viability, CPE, viral antigens accumulation, caspase-3 activity, annexin V binding and DNA fragmentation were estimated. Three different forms or pathways of cell death were considered: apoptosis (the presence or rise of caspase-3 activity, DNA fragmentation and annexin V binding), slow cell death (the presence or rise of DNA fragmentation, the absence or decline of caspase-3 activity and annexin V binding), and necrosis (the absence of decline of caspase-3 activity, DNA fragmentation and annexin V binding). The viability of all cell types, except for KB cells, was reduced by the infection. CPE and viral antigens data demonstrated that all six types of cells could be infected with HSV-1. Upon HSV-1 infection there occurred (i) a classical apoptosis in GF cells, (ii) apoptosis in the early phase of infection and necrosis in the late phase of infection in GNM and TSCCa cells, (iii) slow cell death followed by necrosis in BF and OSF cells (however, these cells showed a different type of CPE), (iv) a classical slow cell death in KB cells. It is hypothesized that HSV-1 infection has a potential to induce several distinct pathways leading to cell death or several forms of cell death. Moreover, more than one pathway may be involved in the death of particular cell type. As HSV-1 was demonstrated to infect different oral and non-oral cells and cause different pathways or forms of cell death, the safety of using HSV-1 as a vector for gene therapy should be re-considered.

Localization of fibronectin in gingival connective tissue of the beagle dog. Ultrastructural detection with ferritin and peroxidase-conjugated antibodies.

Ferritin and peroxidase-conjugated antibodies were used in an indirect antibody method to localize fibronectin in gingival connective tissues. Fibronectin was found in the basal lamina beneath the epithelium and endothelium. Collagen fibrils associated with the basement membranes were also heavily coated by fibronectin. Amorphous patches of fibronectin were found adjacent to the plasma membrane of epithelial cells as well as free in the interepithelial spaces. Fibronectin was present throughout the connective tissue in close association with individual collagen fibrils, apparently serving as an interfibrillar cementing substance. Patches of fibronectin were located at the cell surface of fibroblasts, plasma cells, lymphocytes, endothelial cells, smooth muscle cells, and neutrophils. These amorphous patches were observed to connect adjacent cells across narrow spaces and to connect cells to collagen fibrils. The heavy labeling for fibronectin visualized by fluorescent microscopy around gingival blood vessels (Cho et al., 1985) can be accounted for by a heavy coating of fibronectin on the collagen fibrils and basal laminas associated with endothelial cells, as well as by the presence of abundant deposits of fibronectin along the cell membranes of endothelial cells and in the intercellular spaces of the vessel wall.

Localization of fibronectin in gingival connective tissue of the beagle dog. Immunofluorescent light microscopic findings.

Affinity purified antibodies to plasma fibronectin were used to localize fibronectin in the connective tissues of inflamed and noninflamed beagle gingiva. In noninfiltrated gingival connective tissue, fibronectin was demonstrated in the basement membrane beneath gingival epithelium and around blood vessels as a uniform and intensely stained band about 3 to 10 micron thick. Fibronectin was also distributed throughout the connective tissue in association with collagen fibrils as a more diffuse, less intensely stained pattern. The inflamed gingiva included in this study was characterized by proliferation of epithelial pegs, heavy infiltration of plasma cells and loss of collagen within the subepithelial connective tissue. In these sites, fibronectin was present as an intensely stained band around blood vessels and at the crest of connective tissue papillae nearest the sulcular space. The fibronectin in the basement membrane beneath the epithelium appeared diminished and less uniformly distributed. A delicate network of fibronectin was present around plasma cells and the remaining collagen fibers.

Dentin-pulp complex responses to carious lesions.

To understand the molecular events underlying the dentin-pulp complex responses to carious progression, we systematically analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries. Dentin matrix proteins analyzed included collagen type I, phosphophoryn (PP) and dentin sialoprotein (DSP), all of which play decisive roles in the dentin mineralization process. Human non-carious and carious third molar teeth were freshly collected, demineralized, and processed for hematoxylin and eosin staining. The ABC-peroxidase method was used for immunohistochemical staining of collagen type I, PP and DSP proteins using specific antibodies. In situ hybridization was also performed. In contrast to elongated odontoblasts in non-carious teeth, odontoblasts subjacent to dentin caries were cuboidal and fewer in number. The predentin zone was also dramatically reduced in teeth with dentin caries. The staining intensity for collagen type I, PP and DSP in the dentin-pulp complex increased progressively from non-carious teeth, to teeth with enamel and dentin caries. In situ hybridization studies showed DSP-PP mRNA expression in odontoblasts and dental pulp that was consistent with our immunohistochemical results. These results suggest that carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type I, PP and DSP proteins. This response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation.

Ammonium ions induce inactivation of Kir2.1 potassium channels expressed in Xenopus oocytes.

1. The decay of inward currents was studied using the giant patch-clamp technique and a cloned inward rectifier K(+) channel, Kir2.1, expressed in Xenopus oocytes. 2. In inside-out patches, inward currents carried by NH4(+) or Tl(+) decayed over time. When the voltage was more negative, the degree and rate of decay were greater. The rate of NH4(+)-induced decay saturated at a symmetrical [NH4(+)] of approximately 100 mM. The decay rate was slow (2.6 x 10(3) M(-1) s(-1)) at -140 mV with 10 mM [NH4(+)]. 3. Upon a 10 degrees C increase in temperature, the single-channel NH4(+) current amplitude increased by a factor of 1.57, whereas the NH4(+)-induced decay rate increased by a factor of 2.76. In the R148Y Kir2.1 mutant (tyrosine 148 is at the external pore mouth), NH4(+)-induced inactivation was no longer observed. 4. NH4(+) single-channel currents revealed one open and one closed state. The entry rate into the closed state was voltage dependent whereas the exit rate from the closed state was not. An increase of internal [NH4(+)] not only decreased the entry rate into but also elevated the exit rate from the closed state, consistent with the occupancy model modified from the foot-in-the-door model of gating. 5. These results suggest that the decay of NH4(+) current is unlikely to be due to a simple bimolecular reaction leading to channel block. We propose that NH4(+) binding to Kir2.1 channels induces a conformational change followed by channel closure. 6. The decay induced by permeant ions other than K(+) may serve as a secondary selectivity filter, such that K(+) is the preferred permeant ion for Kir2.1 channels.

Radioautographic demonstration of receptors for epidermal growth factor in various cells of the oral cavity.

Mouse iodinated epidermal growth factor (EGF) was localized by light and electron microscopic radioautography in basal cells of oral epithelium, papillary cells of the enamel organ, periodontal ligament fibroblasts, preodontoblast precursor cells, and preosteoblasts of the alveolar bone of 13-day-old Sprague-Dawley rats. The specificity of binding in these cells was suggested by an observed reduction of about 90% in the labeling when excess unlabeled EGF was injected along with the 125I-EGF. In contrast, fully differentiated cells, such as ameloblasts, odontoblasts, and osteoblasts, were only poorly labeled. Quantitative analysis of the light microscopic radioautographs revealed that the papillary cells had the highest level of labeling (5.5 grains per 100 micron 2 of cell area). The significance of the rather high labeling of the preosteoblasts of the alveolar bone and the fibroblasts of the periodontal ligament is unknown. However, the well-known effect of EGF in producing precocious eruption of teeth may be a consequence of an effect on these two cell types.

Astrocyte culture on nitrocellulose membranes and plastic: detection of cytoskeletal proteins and mRNAs by immunocytochemistry and in situ hybridization.

Neonatal rat brain astrocyte secondary cultures were established on nitrocellulose membrane filters (13-mm diameter Millipore disk) and on plastic coverslips in serum-supplemented medium. On these substrata, cultured astrocytes changed their shape from flat and polygonal to stellate in the absence of hormones or growth factor supplements. Cultures became confluent after 4 days, and astrocytes on nitrocellulose filters continued to differentiate morphologically and biochemically, as evidenced by extensive cytoplasmic process formation and glial fibrillary acid protein (GFAP) accumulation. Cultures were immunostained for GFAP and vimentin. mRNAs to GFAP, vimentin, alpha and beta tubulin, and actin also were detected by in situ hybridization with biotinylated cDNA probes. The astrocyte culture method on nitrocellulose provides a simple, versatile means of comparing undifferentiated, morphologically mature, reactive, and neoplastic astrocytes in vitro.

Formation of protoberberine-type alkaloids by the tubers of somatic embryo-derived plants of Corydalis yanhusuo.

The effects of 0.5 - 5 mg/l abscisic acid [ABA], 0.5 - 10 mg/l (2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol [paclobutrazol] and 0.5 - 2 mg/l alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidinemethanol [ancymidol], 0.5 - 5 mg/l gibberellic acid [GA(3)] and 15 - 100 mg/l polyethylene glycol [PEG] 4000 supplemented in half-strength Murashige and Skoog's (MS) medium on the production of the two major protoberberine-type alkaloids (D,L-tetrahydropalmatine and D-corydaline) by the tubers of somatic embryo-derived plants of Corydalis yanhusuo were examined. Somatic embryo derived plants were also maintained for 6 months on half-strength MS medium containing 0.1 mg/l GA(3) or 0.5 mg/l paclobutrazol. The alkaloid contents were determined by high performance liquid chromatography (HPLC). The analysis revealed that the contents of D,L-tetrahydropalmatine and D-corydaline in the tubers of somatic embryo-derived plants were higher than the marketed crude drug and varied with growth regulator/PEG-4000 treatment and the age of the plant.