Development of an Artificial Intelligence Diagnostic System Using Linked Color Imaging for Barrett's Esophagus.

Background: Barrett's esophagus and esophageal adenocarcinoma cases are increasing as gastroesophageal reflux disease increases. Using artificial intelligence (AI) and linked color imaging (LCI), our aim was to establish a method of diagnosis for short-segment Barrett's esophagus (SSBE). Methods: We retrospectively selected 624 consecutive patients in total at our hospital, treated between May 2017 and March 2020, who experienced an esophagogastroduodenoscopy with white light imaging (WLI) and LCI. Images were randomly chosen as data for learning from WLI: 542 (SSBE+/- 348/194) of 696 (SSBE+/- 444/252); and LCI: 643 (SSBE+/- 446/197) of 805 (SSBE+/- 543/262). Using a Vision Transformer (Vit-B/16-384) to diagnose SSBE, we established two AI systems for WLI and LCI. Finally, 126 WLI (SSBE+/- 77/49) and 137 LCI (SSBE+/- 81/56) images were used for verification purposes. The accuracy of six endoscopists in making diagnoses was compared to that of AI. Results: Study participants were 68.2 ± 12.3 years, M/F 330/294, SSBE+/- 409/215. The accuracy/sensitivity/specificity (%) of AI were 84.1/89.6/75.5 for WLI and 90.5/90.1/91.1/for LCI, and those of experts and trainees were 88.6/88.7/88.4, 85.7/87.0/83.7 for WLI and 93.4/92.6/94.6, 84.7/88.1/79.8 for LCI, respectively. Conclusions: Using AI to diagnose SSBE was similar in accuracy to using a specialist. Our finding may aid the diagnosis of SSBE in the clinic.

Perfluorooctanoic acid-induced cell death via the dual roles of ROS-MAPK/ERK signaling in ameloblast-lineage cells.

Perfluorooctanoic acid (PFOA) is an artificial fluorinated organic compound that has generated increased public attention due to its potential health hazards. Unsafe levels of PFOA exposure can affect reproduction, growth and development. During tooth enamel development (amelogenesis), environmental factors including fluoride can cause enamel hypoplasia. However, the effects of PFOA on ameloblasts and tooth enamel formation remain largely unknown. In the present study we demonstrate several PFOA-mediated cell death pathways (necrosis/necroptosis, and apoptosis) and assess the roles of ROS-MAPK/ERK signaling in PFOA-mediated cell death in mouse ameloblast-lineage cells (ALC). ALC cells were treated with PFOA. Cell proliferation and viability were analyzed by MTT assays and colony formation assays, respectively. PFOA suppressed cell proliferation and viability in a dose dependent manner. PFOA induced both necrosis (PI-positive cells) and apoptosis (cleaved-caspase-3, γH2AX and TUNEL-positive cells). PFOA significantly increased ROS production and up-regulated phosphor-(p)-ERK. Addition of ROS inhibitor N-acetyl cysteine (NAC) suppressed p-ERK and decreased necrosis, and increased cell viability compared to PFOA alone, whereas NAC did not change apoptosis. This suggests that PFOA-mediated necrosis was induced by ROS-MAPK/ERK signaling, but apoptosis was not associated with ROS. Addition of MAPK/ERK inhibitor PD98059 suppressed necrosis and increased cell viability compared to PFOA alone. Intriguingly, PD98059 augmented PFOA-mediated apoptosis. This suggests that p-ERK promoted necrosis but suppressed apoptosis. Addition of the necroptosis inhibitor Necrostatin-1 restored cell viability compared to PFOA alone, while pan-caspase inhibitor Z-VAD did not mitigate PFOA-mediated cell death. These results suggest that 1) PFOA-mediated cell death was mainly caused by necrosis/necroptosis by ROS-MAPK/ERK signaling rather than apoptosis, 2) MAPK/ERK signaling plays the dual roles (promoting necrosis and suppressing apoptosis) under PFOA treatment. This is the initial report to indicate that PFOA could be considered as a possible causative factor for cryptogenic enamel malformation. Further studies are required to elucidate the mechanisms of PFOA-mediated adverse effects on amelogenesis.

Yersinia enterocolitica Enteritis Diagnosed with Erythema Nodosum.

We herein report a rare case of Yersinia enterocolitica enteritis with a fever and abdominal pain followed by erythema nodosum (EN) a few days later. The diagnosis was confirmed based on characteristic colonoscopy and computed tomography findings, pathology, and mucosal culture. Yersinia enteritis is a curable disease provided a proper diagnosis and treatment are performed. Although EN is a rare clinical course, it should still be considered as a differential diagnosis.

Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses

Abstract Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

Linked color imaging improves visibility of reflux esophagitis.

BACKGROUND: With more prevalent gastroesophageal reflux disease comes increased cases of Barrett's esophagus and esophageal adenocarcinoma. Image-enhanced endoscopy using linked-color imaging (LCI) differentiates between mucosal colors. We compared LCI, white light imaging (WLI), and blue LASER imaging (BLI) in diagnosing reflux esophagitis (RE). METHODS: Consecutive RE patients (modified Los Angeles [LA] classification system) who underwent esophagogastroduodenoscopy using WLI, LCI, and BLI between April 2017 and March 2019 were selected retrospectively. Ten endoscopists compared WLI with LCI or BLI using 142 images from 142 patients. Visibility changes were scored by endoscopists as follows: 5, improved; 4, somewhat improved; 3, equivalent; 2, somewhat decreased; and 1, decreased. For total scores, 40 points was considered improved visibility, 21-39 points was comparable to white light, and < 20 points equaled decreased visibility. Inter- and intra-rater reliabilities (Intra-class Correlation Coefficient [ICC]) were also evaluated. Images showing color differences (ΔE*) and L* a* b* color values in RE and adjacent esophageal mucosae were assessed using CIELAB, a color space system. RESULTS: The mean age of patients was 67.1 years (range: 27-89; 63 males, 79 females). RE LA grades observed included 52 M, 52 A, 24 B, 11 C, and 3 D. Compared with WLI, all RE cases showed improved visibility: 28.2% (40/142), LA grade M: 19.2% (10/52), LA grade A: 34.6% (18/52), LA grade B: 37.5% (9/24), LA grade C: 27.3% (3/11), and LA grade D: 0% (0/3) in LCI, and for all RE cases: 0% in BLI. LCI was not associated with decreased visibility. The LCI inter-rater reliability was "moderate" for LA grade M and "substantial" for erosive RE. The LCI intra-rater reliability was "moderate-substantial" for trainees and experts. Color differences were WLI: 12.3, LCI: 22.7 in LA grade M; and WLI: 18.2, LCI: 31.9 in erosive RE (P < 0.001 for WLI vs. LCI). CONCLUSION: LCI versus WLI and BLI led to improved visibility for RE after subjective and objective evaluations. Visibility and the ICC for minimal change esophagitis were lower than for erosive RE for LCI. With LCI, RE images contrasting better with the surrounding esophageal mucosa were more clearly viewed.

Unsaturated fatty acids enhance the fibrinolytic activity of subtilisin NAT (nattokinase).

Subtilisin NAT (STN), alternatively designated nattokinase, is a serine protease with potent fibrinolytic activity. In this study, we screened several foods to enhance the fibrinolytic potential of STN and identified unsaturated fatty acid-rich ones as candidates. We isolated linoleic acid as a major active compound from one of the most active foods, red pepper. Linoleic acid promoted the STN-mediated fibrin/fibrinogen degradation at >20 µg/ml. STN cleaved three of the fibrinogen polypeptide chains, among which linoleic acid accelerated Bß-chain and γ-chain degradations, but slightly suppressed the degradation of α-chain fragments. Linoleic acid failed to affect small synthetic peptide degradation, suggesting a conformational modulation of fibrin/fibrinogen for the linoleic acid promotion of STN activity. Of the various fatty acids tested, unsaturated ones were active but saturated ones were rather inhibitory to STN-mediated fibrinolysis. Thus, our data shed new light on the dietary promotion of STN activity. PRACTICAL APPLICATIONS: Subtilisin NAT (STN) is a serine protease abundantly contained in natto, a soybean food fermented with Bacillus subtilis var. natto. The use of STN as functional foods to improve blood circulation is getting attention because STN actively degrades fibrin. Our results demonstrate that widely occurring unsaturated fatty acids such as linoleic, eicosapentaenoic, and docosahexaenoic acids enhance the fibrinolytic activity of STN. Thus, the intake of natto or STN supplements in combination with unsaturated fatty acid-containing oil can be a novel way to gain cardiovascular benefits.

Histone acetyltransferase promotes fluoride toxicity in LS8 cells.

Previously we demonstrated that fluoride increased acetylated-p53 (Ac-p53) in LS8 cells that are derived from mouse enamel organ epithelia and in rodent ameloblasts. However, how p53 is acetylated by fluoride and how the p53 upstream molecular pathway responds to fluoride is not well characterized. Here we demonstrate that fluoride activates histone acetyltransferases (HATs) including CBP, p300, PCAF and Tip60 to acetylate p53. HAT activity is regulated by post-translational modifications such as acetylation and phosphorylation. HAT proteins and their post-translational modifications (p300, Acetyl-p300, CBP, Acetyl-CBP, Tip60 and phospho-Tip60) were analyzed by Western blots. p53-HAT binding was detected by co-immunoprecipitation (co-IP). Cell growth inhibition was analyzed by MTT assays. LS8 cells were treated with NaF with/without HAT inhibitors MG149 (Tip60 inhibitor) and Anacardic Acid (AA; inhibits p300/CBP and PCAF). MG149 or AA was added 1 h prior to NaF treatment. Co-IP results showed that NaF increased p53-CBP binding and p53-PCAF binding. NaF increased active Acetyl-p300, Acetyl-CBP and phospho-Tip60 levels, suggesting that fluoride activates these HATs. Fluoride-induced phospho-Tip60 was decreased by MG149. MG149 or AA treatment reversed fluoride-induced cell growth inhibition at 24 h. MG149 or AA treatment decreased fluoride-induced p53 acetylation to inhibit caspase-3 cleavage, DNA damage marker γH2AX expression and cytochrome-c release into the cytosol. These results suggest that acetylation of p53 by HATs contributes, at least in part, to fluoride-induced toxicity in LS8 cells via cell growth inhibition, apoptosis, DNA damage and mitochondrial damage. Modulation of HAT activity may, therefore, be a potential therapeutic target to mitigate fluoride toxicity in ameloblasts.

Linked Color Imaging and the Kyoto Classification of Gastritis: Evaluation of Visibility and Inter-Rater Reliability.

BACKGROUND/AIMS: To compare white light imaging (WLI) with linked color imaging (LCI) and blue LASER imaging (BLI) in endoscopic findings of Helicobacter pylori presently infected, previously infected, and uninfected gastric mucosae for visibility and inter-rater reliability. METHODS: WLI, LCI and BLI bright mode (BLI-bright) were used to obtain 1,092 endoscopic images from 261 patients according to the Kyoto Classification of Gastritis. Images were evaluated retrospectively by 10 experts and 10 trainee endoscopists and included diffuse redness, spotty redness, map-like redness, patchy redness, red streaks, intestinal metaplasia, and an atrophic border (52 cases for each finding, respectively). Physicians assessed visibility as follows: 5 (improved), 4 (somewhat improved), 3 (equivalent), 2 (somewhat decreased), and 1 (decreased). Visibility was assessed from totaled scores. The inter-rater reliability (intraclass correlation coefficient) was also evaluated. RESULTS: Compared with WLI, all endoscopists reported improved visibility with LCI: 55.8% for diffuse redness; LCI: 38.5% for spotty redness; LCI: 57.7% for map-like redness; LCI: 40.4% for patchy redness; LCI: 53.8% for red streaks; LCI: 42.3% and BLI-bright: 80.8% for intestinal metaplasia; LCI: 46.2% for an atrophic border. For all endoscopists, the inter-rater reliabilities of LCI compared to WLI were 0.73-0.87. CONCLUSION: The visibility of each endoscopic finding was improved by LCI while that of intestinal metaplasia was improved by BLI-bright.

ADAM10 is Expressed by Ameloblasts, Cleaves the RELT TNF Receptor Extracellular Domain and Facilitates Enamel Development.

MMP20 cleaves cadherins and may facilitate cell movement, however MMP20 is not known to cleave tight junction or desmosome proteins. Ameloblasts had not previously been screened for membrane anchored proteases that could contribute to cell movement. Here we performed a PCR screen for proteolyticlly active A Disintegrin And Metalloproteinase (ADAM) family members. These proteinases are termed sheddases because they have a transmembrane domain and their catalytic domain on the cell surface can function to release anchored proteins. Significantly, ADAMs can be targeted to specific substrates on the cell membrane through their interaction with tetraspanins. Six ADAMs (ADAM8, 9, 10, 15, 17, 19) were expressed in mouse enamel organs. We show that Adam10 expression begins in the apical loop, continues through the secretory stage and abruptly ends at the transition stage when ameloblast migration ceases. ADAM10 cleaves cadherins and tight junction plus desmosome proteins and is well characterized for its role in cell movement. ADAM10 facilitated LS8 cell migration/invasion through a Matrigel coated membrane and we demonstrate that ADAM10, but not ADAM17 cleaves the RELT extracellular domain. This striking result is significant because RELT mutations cause amelogenesis imperfecta (AI) and this directly links ADAM10 to an important role in enamel development.

MDM2-Mediated p21 Proteasomal Degradation Promotes Fluoride Toxicity in Ameloblasts.

Fluoride overexposure is an environmental health hazard and can cause enamel and skeletal fluorosis. Previously we demonstrated that fluoride increased acetylated-p53 and its downstream target p21 in ameloblast-derived LS8 cells. However, p21 function in fluoride toxicity is not well characterized. This study seeks to gain a better understanding of how p53 down-stream mediators, p21 and MDM2, respond to fluoride toxicity. LS8 cells were treated with NaF with/without MG-132 (proteasome inhibitor) or Nutlin-3a (MDM2 antagonist). NaF treatment for 2-6 h increased phospho-p21, which can inhibit apoptosis. However, phospho-p21 and p21 were decreased by NaF at 24 h, even though p21 mRNA was significantly increased at this time point. MG-132 reversed the fluoride-mediated p21 decrease, indicating that fluoride facilitates p21 proteasomal degradation. MG-132 suppressed fluoride-induced caspase-3 cleavage, suggesting that the proteasome plays a pro-apoptotic role in fluoride toxicity. NaF increased phospho-MDM2 in vitro and in mouse ameloblasts in vivo. Nutlin-3a suppressed NaF-mediated MDM2-p21 binding to reverse p21 degradation which increased phospho-p21. This suppressed apoptosis after 24 h NaF treatment. These results suggest that MDM2-mediated p21 proteasomal degradation with subsequent phospho-p21 attenuation contributes to fluoride-induced apoptosis. Inhibition of MDM2-mediated p21 degradation may be a potential therapeutic target to mitigate fluoride toxicity.

Glucose Management during Insulinoma Resection Using Real-Time Subcutaneous Continuous Glucose Monitoring.

Insulinoma is a rare neuroendocrine tumor that causes hypoglycemia due to unregulated insulin secretion. Blood glucose management during insulinoma resection is therefore challenging. We present a case in which real-time subcutaneous continuous glucose monitoring (SCGM) in combination with intermittent blood glucose measurement was used for glycemic control during surgery for insulinoma resection. The SCGM system showed the trends and peak of interstitial glucose in response to glucose loading and the change of interstitial glucose before and after insulinoma resection. These data were helpful for adjusting the glucose infusion; therefore, we think that an SCGM system as a supportive device for glucose monitoring may be useful for glucose management during surgery.

Improved Visibility of Barrett's Esophagus with Linked Color Imaging: Inter- and Intra-Rater Reliability and Quantitative Analysis.

BACKGROUND/AIMS: To evaluate the usefulness of linked color imaging (LCI) and blue LASER imaging (BLI) in Barrett's esophagus (BE) compared with white light imaging (WLI). METHODS: Five expert and trainee endoscopists compared WLI, LCI, and BLI images obtained from 63 patients with short-segment BE. Physicians assessed visibility as follows: 5 (improved), 4 (somewhat improved), 3 (equivalent), 2 (somewhat decreased), and one (decreased). Scores were evaluated to assess visibility. The inter- and intra-rater reliability (intra-class correlation coefficient) of image assessments were also evaluated. Images were objectively evaluated based on L* a* b* color values and color differences (ΔE*) in a CIELAB color space system. RESULTS: Improved visibility compared with WLI was achieved for LCI: 44.4%, BLI: 0% for all endoscopists; LCI: 55.6%, BLI: 1.6% for trainees; and LCI: 47.6%, BLI: 0% for experts. The visibility score of trainees compared with experts was significantly higher for LCI (p = 0.02). Intra- and inter-rater reliability ratings for LCI compared with WLI were "moderate" for trainees, and "moderate-substantial" for experts. The ΔE* revealed statistically significant differences between WLI and LCI. CONCLUSION: LCI improved the visibility of short-segment BE compared with WLI, especially for trainees, when evaluated both subjectively and objectively.

Sirt1 overexpression suppresses fluoride-induced p53 acetylation to alleviate fluoride toxicity in ameloblasts responsible for enamel formation.

Low-dose fluoride is an effective caries prophylactic, but high-dose fluoride is an environmental health hazard that causes skeletal and dental fluorosis. Treatments to prevent fluorosis and the molecular pathways responsive to fluoride exposure remain to be elucidated. Previously we showed that fluoride activates SIRT1 as an adaptive response to protect cells. Here, we demonstrate that fluoride induced p53 acetylation (Ac-p53) [Lys379], which is a SIRT1 deacetylation target, in ameloblast-derived LS8 cells in vitro and in enamel organ in vivo. Here we assessed SIRT1 function on fluoride-induced Ac-p53 formation using CRISPR/Cas9-mediated Sirt1 knockout (LS8Sirt/KO) cells or CRISPR/dCas9/SAM-mediated Sirt1 overexpressing (LS8Sirt1/over) cells. NaF (5 mM) induced Ac-p53 formation and increased cell cycle arrest via Cdkn1a/p21 expression in Wild-type (WT) cells. However, fluoride-induced Ac-p53 was suppressed by the SIRT1 activator resveratrol (50 µM). Without fluoride, Ac-p53 persisted in LS8Sirt/KO cells, whereas it decreased in LS8Sirt1/over. Fluoride-induced Ac-p53 formation was also suppressed in LS8Sirt1/over cells. Compared to WT cells, fluoride-induced Cdkn1a/p21 expression was elevated in LS8Sirt/KO and these cells were more susceptible to fluoride-induced growth inhibition. In contrast, LS8Sirt1/over cells were significantly more resistant. In addition, fluoride-induced cytochrome-c release and caspase-3 activation were suppressed in LS8Sirt1/over cells. Fluoride induced expression of the DNA double strand break marker γH2AX in WT cells and this was augmented in LS8Sirt1/KO cells, but was attenuated in LS8Sirt1/over cells. Our results suggest that SIRT1 deacetylates Ac-p53 to mitigate fluoride-induced cell growth inhibition, mitochondrial damage, DNA damage and apoptosis. This is the first report implicating Ac-p53 in fluoride toxicity.

Soluble RANKL Cleaved from Activated Lymphocytes by TNF-α-Converting Enzyme Contributes to Osteoclastogenesis in Periodontitis.

Host immune responses play a key role in promoting bone resorption in periodontitis via receptor activator of NF-κB ligand (RANKL)-dependent osteoclastogenesis. Both membrane-bound RANKL (mRANKL) expressed on lymphocytes and soluble RANKL (sRANKL) are found in periodontal lesions. However, the underlying mechanism and cellular source of sRANKL release and its biological role in periodontitis are unclear. TNF-α-converting enzyme (TACE) is reported to cleave the following: 1) precursor TNF-α with release of mature, soluble TNF-α and 2) mRANKL with release of sRANKL. Both soluble TNF-α and sRANKL are found in the periodontitis lesion, leading to the hypothesis that TACE expressed on lymphocytes is engaged in RANKL shedding and that the resulting sRANKL induces osteoclastogenesis. In the current study, upon stimulating PBLs with mitogens in vitro, RANKL expression, sRANKL secretion, and TACE expression were all upregulated. Among the four putative mRANKL sheddases examined in neutralization assays, TACE was the only functional sheddase able to cleave mRANKL expressed on PBL. Moreover, PBL culture supernatant stimulated with mitogens in the presence of anti-TACE Ab or anti-RANKL Ab showed a marked reduction of osteoclastogenesis from osteoclast precursors, indicating that TACE-mediated sRANKL may possess sufficient osteoclastogenic activity. According to double-color confocal microscopy, B cells expressed a more pronounced level of RANKL and TACE expression than T cells or monocytes in periodontally diseased gingiva. Conditioned medium of patients' gingival lymphocyte culture increased in vitro osteoclastogenic activity, which was suppressed by the addition of anti-TACE Ab and anti-RANKL Ab. Therefore, TACE-mediated cleavage of sRANKL from activated lymphocytes, especially B cells, can promote osteoclastogenesis in periodontitis.

A-Disintegrin and Metalloproteinase (ADAM) 17 Enzymatically Degrades Interferon-gamma.

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts anti-tumor and anti-osteoclastogenic effects. Although transcriptional and post-transcriptional regulation of IFN-γ is well understood, subsequent modifications of secreted IFN-γ are not fully elucidated. Previous research indicates that some cancer cells escape immune surveillance and metastasize into bone tissue by inducing osteoclastic bone resorption. Peptidases of the a-disintegrin and metalloproteinase (ADAM) family are implicated in cancer cell proliferation and tumor progression. We hypothesized that the ADAM enzymes expressed by cancer cells degrades IFN-γ and attenuates IFN-γ-mediated anti-tumorigenic and anti-osteoclastogenic effects. Recombinant ADAM17 degraded IFN-γ into small fragments. The addition of ADAM17 to the culture supernatant of stimulated mouse splenocytes decreased IFN-γ concentration. However, ADAM17 inhibition in the stimulated mouse T-cells prevented IFN-γ degradation. ADAM17-expressing human breast cancer cell lines MCF-7 and MDA-MB-453 also degraded recombinant IFN-γ, but this was attenuated by ADAM17 inhibition. Degraded IFN-γ lost the functionality including the inhibititory effect on osteoclastogenesis. This is the first study to demonstrate the extracellular proteolytic degradation of IFN-γ by ADAM17. These results suggest that ADAM17-mediated degradation of IFN-γ may block the anti-tumorigenic and anti-osteoclastogenic effects of IFN-γ. ADAM17 inhibition may be useful for the treatment of attenuated cancer immune surveillance and/or bone metastases.

Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling.

Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through c-Jun N-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125 ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0 ppm fluoride). These results suggest that fluoride-induced ROS generation causes mitochondrial damage and DNA damage, which may lead to impairment of ameloblast function. To counteract this impairment, SIRT1/autophagy is induced via JNK signaling to protect cells/ameloblasts from fluoride-induced oxidative damage that may cause dental fluorosis.

A Combination of Low-Intensity Pulsed Ultrasound and Nanohydroxyapatite Concordantly Enhances Osteogenesis of Adipose-Derived Stem Cells From Buccal Fat Pad.

The osteogenic induction of adipose-derived stem cells (ADSCs) has been regarded as an important step in bone tissue engineering. In the present study, we focused on the buccal fat pad (BFP) as a source of adipose tissue, since BFPs are encapsulated by adipose tissue and are often coextirpated during oral surgery. Low-intensity pulsed ultrasound (LIPUS) is effective in the treatment of fractures, and nanohydroxyapatite (NHA) is known as a bone substitute material. Here we investigated the synergistic effects of LIPUS and NHA in the osteogenesis of ADSCs. A combination of LIPUS irritation and NHA as a scaffold significantly increased the osteogenic differentiation of ADSCs in vitro, and in our in vivo study in which ADSCs were transplanted into calvarial bone defects of nude mice, the combinational effect greatly enhanced the new bone formation of the margin of the defects. These results demonstrate that synergistic effects of LIPUS and NHA are capable of effectively inducing the differentiation of ADSCs into osteoblasts, and they suggest a novel therapeutic strategy for bone regeneration by the autotransplantation of ADSCs.

Molecular pathogenesis of keratocystic odontogenic tumors developing in nevoid basal cell carcinoma syndrome.

OBJECTIVE: The aim of this study was to investigate the molecular pathogenesis of keratocystic odontogenic tumors (KCOTs) that developed in nevoid basal cell carcinoma syndrome (NBCCS) patients. STUDY DESIGN: We analyzed germline and somatic mutations of the PTCH1 and its related genes, SMO and SUFU in 10 KCOTs that developed in 8 unrelated NBCCS patients. Methylation status of the PTCH1 promoter was also investigated by bisulfite sequencing. RESULTS: Somatic mutations of PTCH1 were detected in 3 KCOTs. Two out of 3 somatic mutations were either identified as a polymorphism or located on the same allele as the germline mutation. Neither abnormal methylation of the PTCH1 promoter, loss of PTCH1, nor somatic mutation of SMO or SUFU was detected in any of the samples. CONCLUSIONS: Our results suggest that the tumorigenesis of most KCOTs associated with NBCCS cannot be explained by the classical 2-hit theory.

Frameshift mutation in the PTCH2 gene can cause nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1, encoding a receptor for the secreted protein, sonic hedgehog. Recently, a Chinese family with NBCCS carrying a missense mutation in PTCH2, a close homolog of PTCH1, was reported. However, the pathological significance of missense mutations should be discussed cautiously. Here, we report a 13-year-old girl diagnosed with NBCCS based on multiple keratocystic odontogenic tumors and rib anomalies carrying a frameshift mutation in the PTCH2 gene (c.1172_1173delCT). Considering the deleterious nature of the frameshift mutation, our study further confirmed a causative role for the PTCH2 mutation in NBCCS. The absence of typical phenotypes in this case such as palmar/plantar pits, macrocephaly, falx calcification, hypertelorism and coarse face, together with previously reported cases, suggested that individuals with NBCCS carrying a PTCH2 mutation may have a milder phenotype than those with a PTCH1 mutation.

Two cases of nevoid basal cell carcinoma syndrome associated with meningioma caused by a PTCH1 or SUFU germline mutation.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1. The development of meningioma in NBCCS patients is a rare event. Here, we report two cases of NBCCS in which meningiomas did develop. The first patient carried a germline mutation in one allele of PTCH1, c.290dupA (p.N97KfsX43). In addition, the meningioma sample carried a somatic mutation, c.307delG (p.Val103LeufsX15), in the other allele of the same gene, suggesting a second hit. This is the first case of NBCCS-associated meningioma explained by the standard two-hit hypothesis. The second patient had a germline nonsense mutation in the SUFU gene, c.550C>T (p.Q184X). SUFU is located downstream of PTCH1 in the sonic hedgehog signaling pathway. This is the second time a germline mutation in SUFU has been found to cause NBCCS. Together with the previous report describing three cases of non-NBCCS medulloblastoma carrying a germline mutation in this gene, individuals with a SUFU germline mutation are expected to have a markedly high risk of developing medulloblastoma and probably meningioma.