JNK inhibition enhances cell-cell adhesion impaired by desmoglein 3 gene disruption in keratinocytes.

c-Jun NH2-terminal protein kinase (JNK) and p38 are stress-activated mitogen-activated protein kinases (MAPK) that are phosphorylated by various stimuli. It has been reported that the loss of desmoglein (DSG) 3, a desmosomal transmembrane core molecule, in keratinocytes impairs cell-cell adhesion accompanied by p38 MAPK activation. To understand the biological role of DSG3 in desmosomes and its relationship with stress-activated MAPKs, we established DSG3 knockout keratinocytes (KO cells). Wild-type cells showed a linear localization of DSG1 to cell-cell contacts, whereas KO cells showed a remarkable reduction despite the increased protein levels of DSG1. Cell-cell adhesion in KO cells was impaired over time, as demonstrated by dispase-based dissociation assays. The linear localization of DSG1 to cell-cell contacts and the strength of cell-cell adhesion were promoted by the pharmacological inhibition of JNK. Conversely, pharmacological activation of JNK, but not p38 MAPK, in wild-type cells reduced the linear localization of DSG1 in cell-cell contacts. Our data indicate that DSG1 and DSG2 in KO cells cannot compensate for the attenuation of cell-cell adhesion strength caused by DSG3 deficiency and that JNK inhibition restores the strength of cell-cell adhesion by increasing the linear localization of DSG1 in cell-cell contacts in KO cells. Inhibition of JNK signaling may improve cell-cell adhesion in diseases in which DSG3 expression is impaired.

FANCD2 counteracts O6-methylguanine-induced mismatch repair-dependent apoptosis.

BACKGROUND: Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process. METHODS AND RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci. CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.

Providing a monolithic zirconia fixed partial denture with rigid and nonrigid connectors to overcome nonparallel abutment teeth.

This clinical report describes the fabrication of a monolithic zirconia fixed partial denture using rigid and nonrigid connectors to overcome nonparallel abutment teeth. A precise key and keyway in the ceramic material was designed with digital technology, reducing material costs, improving biocompatibility, and using esthetically superior nonmetallic materials.

The efficacy of a novel zinc-containing desensitizer CAREDYNE Shield for cervical dentin hypersensitivity: a pilot randomized controlled trial.

BACKGROUND: Recently, a novel zinc-containing desensitizer, CAREDYNE Shield, was developed. This new type of desensitizer induces chemical occlusion of dentinal tubules for desensitization and releases zinc ion for root caries prevention. Despite these features, its clinical effectiveness in the improvement of cervical dentine hypersensitivity remains to be elucidated. Thus, we aimed to evaluate the effectiveness of CAREDYNE Shield in patients with CDH. METHODS: Forty CDH teeth which matched the eligibility criteria were randomly allocated to two groups in a 1:1 ratio: the CAREDYNE Shield group (intervention group) and the Nanoseal group (control group). The pain intensity in response to air stimuli, gingival condition, and oral hygiene status of CDH teeth were assessed before and at 4 weeks after treatment. The primary outcome was the reduction of pain intensity in response to air stimuli from baseline to 4 weeks after intervention. RESULTS: From November 2019 to April 2021, 24 participants with 40 teeth were enrolled in this study and 33 teeth in 20 participants were assessed at 4 weeks after treatment. A significant reduction of pain in response to air stimuli was observed in both groups; however, no significant difference was observed between the groups. CONCLUSIONS: This study showed that CAREDYNE Shield is effective for CDH and its effectiveness is similar to Nanoseal. TRIAL REGISTRATION: UMIN Clinical Trials Registry (UMIN-CTR), UMIN000038072. Registered on 21st September 2019, https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000043331.

The reduced susceptibility of mouse keratinocytes to retinoic acid may be involved in the keratinization of oral and esophageal mucosal epithelium.

Keratinocytes take up serum-derived retinol (vitamin A) and metabolize it to all-trans-retinoic acid (atRA), which binds to the nuclear retinoic acid receptor (RAR). We previously reported that serum-affected keratinocyte differentiation and function; namely, it inhibited keratinization, decreased loricrin (LOR) and claudin (CLDN) 1 expression, increased keratin (K) 4 and CLDN4 levels, and reduced paracellular permeability in three-dimensional (3D) cultures of mouse keratinocytes (COCA). Contrarily, RAR inhibition reversed these changes. Here, we aimed to examine whether atRA exerted the same effects as serum, and whether it was involved in the differential oral mucosa keratinization among animal species. Porcine oral mucosal keratinocytes, which form non-keratinized epithelium in vivo, established keratinized epithelium in 3D cultures. Both mouse and porcine sera induced non-keratinized epithelium at 0.1% in COCA 3D cultures. Although atRA caused the same changes as serum, its effective concentration differed. atRA inhibited keratinization at 0.1 nM and 1 nM in porcine or human keratinocytes and COCA, respectively. Furthermore, atRA upregulated CLDN7 in the cytoplasm but not in cell-cell contacts. These atRA-induced changes were reverted by RAR inhibition. The results indicate that serum-induced changes are probably due to the effect of serum-derived atRA, and that mouse keratinocytes require higher atRA concentrations to suppress keratinization than porcine and human keratinocytes. We propose that the lower susceptibility of mouse keratinocytes to atRA, rather than a lower retinol concentration, is a possible reason for the keratinization of mouse oral mucosal epithelium.

Formation of keratinocyte multilayers on filters under airlifted or submerged culture conditions in medium containing calcium, ascorbic acid, and keratinocyte growth factor.

Three-dimensional (3D) cell culture is a powerful in vitro technique to study the stratification and differentiation of keratinocytes. However, culture conditions, including culture media, supplements, and scaffolds (e.g., collagen gels with or without fibroblasts), can vary considerably. Here, we evaluated the roles of calcium, L-ascorbic acid phosphate magnesium salt n-hydrate (APM), and keratinocyte growth factor (KGF) in a chemically defined medium, EpiLife, in 3D cultures of primary human epidermal keratinocytes directly plated on polycarbonate filter inserts under airlifted or submerged conditions. Eight culture media containing various combinations of these three supplements were examined. Calcium was necessary for the stratification and differentiation of keratinocytes based on the localization of keratins and involucrin. However, the localization patterns of keratins and integrin ß4 were partially disrupted and Ki67-positive basal cells almost disappeared 3 weeks after airlift. The addition of KGF, but not APM, prevented these changes. Further addition of APM markedly improved the tissue architecture, including basal cell morphology and the appearance of keratohyalin granules and localized involucrin in the upper suprabasal cells, even after 1 week. Although the submerged culture also formed cornified epithelium-like multilayers, involucrin was localized in the cornified layer, where nuclei were often found. Based on these results, it is most effective to culture keratinocytes at the air-liquid interface in EpiLife medium supplemented with calcium, APM, and KGF to form well-organized and orthokeratinized multilayers as skin analogues.

[A Case of Local Recurrence of Bile Duct Cancer Completely Responding to Chemoradiotherapy with S-1].

An 80-year-old man with common bile duct cancer was treated by pancreaticoduodenectomy with D2 lymph node dissection in October 2005. The patient presented with frequent episodes of bloody-mucous rectal discharge in July 2009. An abdominal CT demonstrated local recurrence at the hepatoduodenal ligament. We treated him with concurrent chemoradiotherapy (CRT) with single-dose S-1 chemotherapy. After 6 months, we diagnosed a complete response (CR) by follow-up CT. The patient was treated with S-1 for 3 years after the diagnosis of a CR. He is alive without disease 6 years after the diagnosis of the recurrence. Concurrent CRT with S-1 chemotherapy may be the therapy of choice for recurrence of bile duct cancer.

Characterization of the bone matrix and its contribution to tooth loss in human cadaveric mandibles.

OBJECTIVE: It is uncertain as to what extent the major bone matrix constituents, mineral and collagen, show inter-individual variation and dependence on age and sex in jawbones. The purpose of this study was to clarify this uncertainty using cadaveric mandibles and investigate the association of bone matrix with the number of existing teeth. MATERIALS AND METHODS: Cortical bone samples (1 × 1 cm) collected from the mental of 48 cadaveric mandibles (27 men and 21 women; age range = 56-93 years and 63-103 years, respectively) were used to quantify three bone matrix indices: mineral content, collagen content and extent of lysine hydroxylation of collagen. Associations with age and comparisons by sex were evaluated based on bone matrix indices and the numbers of existing teeth. The numbers of existing teeth were compared between the groups showing low and high bone matrix index values. RESULTS: A great amount of inter-individual variation was seen in all bone matrix indices. No bone matrix indices were associated with age, while the number of existing teeth was negatively associated with age. The bone matrix indices and number of existing teeth did not differ by sex. The number of existing teeth was nearly twice as high in the group showing high collagen content as in the low collagen group; however, an analysis of covariance showed a significant inter-group difference not from bone matrix indices, but rather from age. Interestingly, in comparison to femoral collagen, mandibular collagen showed lower lysine hydroxylation, which can represent an aspect of bone quality. CONCLUSIONS: Mandibular bone matrix shows great inter-individual variation and is independent of age and sex, but did not show as strong a relationship with tooth loss as age. Even so, mandibular collagen may represent a unique characteristic of bone matrix and deserves to be further investigated.

[A case report of residual gastric cancer 15 years after pancreatoduodenectomy with modified child's reconstruction].

A 75-year-old man underwent pancreatoduodenectomy for pancreatic cancer. He had presented with epigastralgia in June 2008. Gastrointestinal endoscopy revealed type 2 gastric cancer in the cardiac area. Enhanced abdominal CT scanning confirmed an enhanced mass in the cardiac area. Gastrectomy with Roux-en-Y reconstruction was performed for residual gastric cancer. Histopathological findings revealed, pT3(SS), pN0, pH0, pP0, pStageIIA. Single-agent TS-1 therapy was chosen as adjuvant chemotherapy but was changed to TS-1+CDDP because of CT-detected recurrence 3 months after the second operation. After a 6 month course of chemotherapy, complete reduction of the tumor was obtained.

[A case of portal hypertension after 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) chemotherapy].

A 46-year-old man with cancer of the sigmoid colon with hepatic metastasis underwent sigmoidectomy, partial hepatectomy, and cholecystectomy in May 2008. He subsequently received 10 cycles of a modified 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) regimen as adjuvant chemotherapy from June 2008 to December 2008, following which he developed thrombocytopenia and splenomegaly. In May 2011, upper gastrointestinal endoscopy was performed, which revealed esophageal and gastric varices. The varices were treated endoscopically with ligation and balloon-occluded retrograde transvenous obliteration. A liver biopsy was performed to determine the cause of the portal hypertension in the absence of severe hepatic dysfunction or liver cirrhosis. The biopsy revealed obliteration of the peripheral portal veins with sinusoidal dilatation without fibrosis or inflammatory cell infiltration in the hepatic lobules. Oxaliplatin-based chemotherapy has been associated with hepatovascular injury, such as sinusoidal dilatation and fibrosis, resulting in non-cirrhotic portal hypertension as seen in this case.

Effect of Particle Sizes and Contents of Surface Pre-Reacted Glass Ionomer Filler on Mechanical Properties of Auto-Polymerizing Resin.

Herein, the mechanical properties of an auto-polymerizing resin incorporated with a surface pre-reacted glass ionomer (S-PRG) filler were evaluated. For this, S-PRG fillers with particle sizes of 1 µm (S-PRG-1) and 3 µm (S-PRG-3) were mixed at 10, 20, 30, and 40 wt% to prepare experimental resin powders. The powders and a liquid (powder/liquid ratio = 1.0 g/0.5 mL) were kneaded and filled into a silicone mold to obtain rectangular specimens. The flexural strength and modulus (n = 12) were recorded via a three-point bending test. The flexural strengths of S-PRG-1 at 10 wt% (62.14 MPa) and S-PRG-3 at 10 and 20 wt% (68.68 and 62.70 MPa, respectively) were adequate (>60 MPa). The flexural modulus of the S-PRG-3-containing specimen was significantly higher than that of the S-PRG-1-containing specimen. Scanning electron microscopy observations of the specimen fracture surfaces after bending revealed that the S-PRG fillers were tightly embedded and scattered in the resin matrix. The Vickers hardness increased with an increasing filler content and size. The Vickers hardness of S-PRG-3 (14.86-15.48 HV) was higher than that of S-PRG-1 (13.48-14.97 HV). Thus, the particle size and content of the S-PRG filler affect the mechanical properties of the experimental auto-polymerizing resin.

[A case of unresectable multiple hepatic metastases from ascending colon cancer successfully treated with panitumumab and FOLFIRI(5-FU/LV/irinotecan)therapy].

A 68-year-old man had undergone right hemicolectomy of ascending colon cancer with multiple liver metastases. This case of k-ras status on the cancer tissue also showed wild type. Chemotherapy with panitumumab and 5-FU/LV/irinotecan (FOLFIRI)regimen was performed after the resection of the ascending colon cancer. After seven curses of treatment, metastatic liver tumors were reduced considerably(PR). Liver resection(left hepatic lobectomy and partial resection of S4 and S5)and radiofrequency ablation therapy were performed. Recently, chemotherapy has improved overall survival of initially unresectable patients by allowing tumor downstaging and complete resection. Combination chemotherapy using panitumumab, and FOLFIRI plus operation is a candidate as a standard treatment strategy for multiple liver metastases of colon cancer.

Fabrication of bioresorbable hydroxyapatite bone grafts through the setting reaction of calcium phosphate cement.

We prepared hydroxyapatite (HAp) bone grafts by the setting reaction of calcium phosphate cement, and investigated the effects of the porosity and crystallinity on the osteoconductivity and bioresorbability. We examined the effect of the water-mixing ratio, pressure, and post-heat treatment temperature during preparation on the crystallite size and porosity of the HAp blocks. The quantity of protein adsorption increased with increasing porosity and specific surface area (SSA) of the HAp blocks, whereas the initial cell attachment was similar despite the different porosities and crystallinities. In in vitro dissolution tests with a pH 5.5 buffer, which mimics an osteoclast-created Howship's lacuna, both the porosity and SSA of the HAp blocks affected the solubility; most likely due to the increased contact area with the buffer. Thus, HAp blocks prepared by the setting reaction of calcium phosphate cement could be applicable for bioresorbable HAp bone grafts because of the high porosity and SSA.

Sivelestat suppresses iNOS gene expression in proinflammatory cytokine-stimulated hepatocytes.

BACKGROUND: Recent evidence has indicated that sivelestat, a neutrophil elastase inhibitor, has liver-protective effects in a variety of liver injuries. Proinflammatory cytokines including interleukin (IL)-1ß stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression, leading to excess production of NO and resulting in liver damage. We hypothesized that inhibition of iNOS induction underlies the protective effects of sivelestat on the liver. The objective of this study was to investigate whether sivelestat directly influences iNOS induction in cultured hepatocytes, which is used as a simple in vitro injury model, and to determine the mechanism involved. METHODS: Primary cultured rat hepatocytes were treated with IL-1ß in the presence or absence of sivelestat. The induction of iNOS and its signaling pathway were analyzed. RESULTS: Sivelestat inhibited the induction of iNOS mRNA and its protein, followed by decreased production of NO. Transfection and iNOS gene antisense-transcript experiments revealed that sivelestat reduced the levels of iNOS mRNA at both the promoter activation and mRNA stabilization steps. However, sivelestat had no effects on the degradation of IκB and nuclear translocation of NF-κB subunit p65, although it moderately blocked the activation of NF-κB. In contrast, sivelestat blocked the upregulation of IL-1 receptor I through the inactivation of phosphatidylinositol 3-kinase/Akt. CONCLUSIONS: Delayed sivelestat addition experiments demonstrated that the destabilization of the iNOS mRNA contributed more significantly to the inhibitory effects of sivelestat than the reduction in iNOS mRNA synthesis. Sivelestat may provide useful therapeutic effects through the suppression of iNOS induction involved in liver injury.

Contact Angle and Cell Adhesion of Micro/Nano-Structured Poly(lactic-co-glycolic acid) Membranes for Dental Regenerative Therapy.

Biodegradable membranes are used in regenerative dentistry for guided tissue regeneration (GTR) and guided bone regeneration (GBR). In this study, patterned poly(lactic-co-glycolic acid) (PLGA) membranes with groove, pillar, and hole structures were successfully fabricated by thermal nanoimprinting. Their surfaces were evaluated for topography by scanning electron microscopy and laser microscopy, for hydrophobicity/hydrophilicity by contact angle analysis, and for MC3T3-E1 cell adhesion. The sizes of the patterns on the surfaces of the membranes were 0.5, 1.0, and 2.0 µm, respectively, with the height/depth being 1.0 µm. The pillared and holed PLGA membranes were significantly more hydrophobic than the non-patterned PLGA membranes (p < 0.05). However, the 0.5 µm- and 1.0 µm-grooved PLGA membranes were significantly more hydrophilic than the non-patterned PLGA membranes (p < 0.05). The 0.5 µm-grooved, pillared, and holed membranes exhibited significantly superior adhesion to the MC3T3-E1 cells than the non-patterned PLGA (p < 0.05). These results suggest that patterned PLGA membranes can be clinically used for GTR and GBR in the dental regeneration field.

Functional roles of fish collagen peptides on bone regeneration.

Fish collagen peptides (FCP) derived from the skin, bones and scales are commercially used as a functional food or dietary supplement for hypertension and diabetes. However, there is limited evidence on the effects of FCP on the osteoblast function in contrast to evidence of the effects on wound healing, diabetes and bone regeneration, which have been obtained from animal studies. In this narrative review, we expound on the availability of FCP by basic research using osteoblasts. Low-concentration FCP upregulates the expression of osteoblast proliferation, differentiation and collagen modifying enzyme-related genes. Furthermore, it could accelerate matrix mineralization. FCP may have potential utility as a biomaterial to improve collagen quality and promote mineralization through the mitogen-activated protein kinase and Smad cascades. However, there are few clinical studies on bone regeneration in human subjects. It is desirable to be applied clinically through clinical study as soon as possible, based on the results from basic research.

Long-term clinical and radiographic evaluation of the effectiveness of direct pulp capping materials: A meta-analysis.

The aim of this review is to evaluate the long-term effectiveness of calcium silicate-based cement (CS) and calcium hydroxide (CH) for direct pulp capping (DPC) to human pulp-exposed permanent teeth. An electronic search and manual search were performed on 21 June 2019. Long-term clinical and radiographic evaluations of the effectiveness of CS and CH for DPC to human pulp-exposed teeth were included, and data extraction, risk-of-bias assessment and meta-analyses were performed. From 645 identified articles, 7 articles met the eligibility criteria. The meta-analyses comparing CS with CH and Biodentine with mineral trioxide aggregates (MTA) on DPC success rate were performed, and significant difference was observed between CS and CH (risk ratio=1.20; p=0.005), whereas no significant difference was observed between Biodentine and MTA. CS seems to be a more effective and predictable DPC material than CH; however, these analyses are based on the studies judged at high risk of bias.

CT and MRI findings of human herpesvirus 6-associated encephalopathy: comparison with findings of herpes simplex virus encephalitis.

OBJECTIVE: It is important to differentiate human herpesvirus 6 (HHV-6)-associated encephalopathy from herpes simplex encephalitis (HSE). Although these conditions are similar with regard to involvement of the mesial temporal lobe, HSE is sensitive to acyclovir but HHV-6 encephalopathy is not. We compared the imaging findings of the two conditions. MATERIALS AND METHODS: We encountered eight cases of HHV-6 encephalopathy and nine cases of HSE. We divided an observation time into early, middle, and late periods defined as 0-2, 3-30, and more than 30 days from the onset of neurologic symptoms. Differences between HHV-6 encephalopathy and HSE on CT scans in the early period and in distribution and temporal changes in the affected regions on MR images in the three periods were analyzed. RESULTS: At MRI in the early and middle periods, all eight patients with HHV-6 encephalopathy had exclusive involvement of the mesial temporal lobes, and all nine patients with HSE had involvement of both the mesial temporal lobes and the extratemporal regions (p < 0.01). Among patients who underwent head MRI, six of six with HHV-6 encephalopathy but none of six with HSE had resolution of high signal intensity on T2-weighted and FLAIR images (p < 0.01). Among patients who underwent head CT in the early period, none of the four with HHV-6 encephalopathy and six of the seven with HSE had abnormal findings, including parenchymal swelling, decreased attenuation of affected regions, and abnormal gyral enhancement (p < 0.05). CONCLUSION: Serial MRI showed transient abnormal signal intensity in the mesial temporal lobes in patients with HHV-6 encephalopathy but persistent abnormal signal intensity in both the mesial temporal lobes and the extratemporal regions in patients with HSE. CT in the early period showed no abnormality in patients with HHV-6 encephalopathy but definite abnormal findings in patients with HSE. These differences may be useful in the differential diagnosis of the two conditions.

Unusual multiple gastric carcinoids with hypergastrinemia: report of a case.

We report the case of a patient demonstrating multiple gastric carcinoids with hypergastrinemia. A 50-year-old Japanese woman was admitted to our hospital for the further examination of multiple carcinoids of the stomach with hypergastrinemia, although she was asymptomatic. However, based on our clinical examination, this case seemed to be neither type I nor II carcinoid. We performed a total gastrectomy with D1 lymph node dissection. A pathological examination showed numerous endocrine micronests, hyperplasia of the parietal cells extending to the foveolar neck region, and numerous dilated oxyntic glands filled with eosinophilic secretions. Many parietal cells exhibited vacuolated cytoplasms and apical snouts. Furthermore, the dilated glands at the base of the mucosa had hyperchromatic nuclei and ciliated surfaces. The postoperative serum gastrin level was soon normalized to 47 pg/ml. This is only the third reported case of multiple gastric carcinoids with hypergastrinemia due to an intrinsic abnormality in the acid secretion of the parietal cells.

Proliferation of Saos-2 and Ca9-22 cells on grooved and pillared titanium surfaces.

BACKGROUND: Surface nanostructures in titanium (Ti) oral implants are critical for rapid osseointegration. OBJECTIVE: The purpose of this study was to evaluate the growth of osteoblast-like (Saos-2) and epithelial-like (Ca9-22) cells on nanopatterned Ti films. METHODS: Ti films with 500 nm grooves and pillars were fabricated by nanoimprinting, and seeded with Saos-2 and Ca9-22 cells. Cell viability and morphology were assessed by cell proliferation assay and scanning electron microscopy, respectively. RESULTS: As assessed after 1 hour, proliferation of Saos-2 cells was most robust on grooved films than on pillared and smooth films, in this order. These cells approximately doubled on grooved and pillared substrates in 24 hours and after 5 days, but not on smooth surfaces. In contrast, Ca9-22 cells favored smooth surfaces, followed by grooved and pillared films. Indeed, cells sparsely adhered to pillared films over 5 days of incubation (p < 0.05). CONCLUSIONS: The data show that Saos-2 and Ca9-22 cells respond differently to different nanostructures, and highlight the potential use of nanopatterns to promote bone regeneration or to prevent epithelial downgrowth at the implant-bone interface.