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Abstract Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis.

Congenital tooth agenesis is a common anomaly in humans. We investigated the etiology of human tooth agenesis by exome analysis in Japanese patients, and found a previously undescribed heterozygous deletion (NM_002448.3(MSX1_v001):c.433_449del) in the first exon of the MSX1 gene. The deletion leads to a frameshift and generates a premature termination codon. The truncated form of MSX1, namely, p.(Trp145Leufs*24) lacks the homeodomain, which is crucial for transcription factor function.

Abstract Congenital tooth agenesis is a common anomaly in human development. We performed exome sequence analysis of genomic DNA collected from Japanese patients with tooth agenesis and their relatives. We found a novel single-nucleotide insertion in the LRP6 gene, the product of which is involved in Wnt/β-catenin signaling as a coreceptor for Wnt ligands. The single-nucleotide insertion results in a premature stop codon in the extracellular region of the encoded protein.

Abstract Analysis of various genetically modified mice, with supernumerary teeth, has revealed the following two intrinsic molecular mechanisms that increase the number of teeth. One plausible explanation for supernumerary tooth formation is the rescue of tooth rudiments. Topical application of candidate molecules could lead to whole tooth formation under suitable conditions. Congenital tooth agenesis is caused by the cessation of tooth development due to the deletion of the causative gene and suppression of its function. The arrest of tooth development in Runx2 knockout mice, a mouse model of congenital tooth agenesis, is rescued in double knockout mice of Runx2 and Usag-1. The Usag-1 knockout mouse is a supernumerary model mouse. Targeted molecular therapy could be used to generate teeth in patients with congenital tooth agenesis by stimulating arrested tooth germs. The third dentition begins to develop when the second successional lamina is formed from the developing permanent tooth in humans and usually regresses apoptotically. Targeted molecular therapy, therefore, seems to be a suitable approach in whole-tooth regeneration by the stimulation of the third dentition. A second mechanism of supernumerary teeth formation involves the contribution of odontogenic epithelial stem cells in adults. Cebpb has been shown to be involved in maintaining the stemness of odontogenic epithelial stem cells and suppressing epithelial-mesenchymal transition. Odontogenic epithelial stem cells are differentiated from one of the tissue stem cells, enamel epithelial stem cells, and odontogenic mesenchymal cells are formed from odontogenic epithelial cells by epithelial-mesenchymal transition. Both odontogenic epithelial cells and odontogenic mesenchymal cells required to form teeth from enamel epithelial stem cells were directly induced to form excess teeth in adults. An approach for the development of targeted therapeutics has been the local application of monoclonal neutralizing antibody/siRNA with cationic gelatin for USAG-1 or small molecule for Cebpb.

In this study, the authors developed age-specific physical head phantoms simulating the physique of Japanese children for dose evaluation in paediatric head computed tomography (CT) examinations. Anatomical structures at 99 places in 0-, 0.5-, 1- and 3-y-old Japanese patients were measured using DICOM viewer software from CT images, and the head phantom of each age was designed. For trial manufacture, a 3-y-old head phantom consisting of acrylic resin and gypsum was produced by machine processing. Radiation doses for the head phantom were measured with radiophotoluminescence glass dosemeters and Si-pin photodiode dosemeters. To investigate whether the phantom shape was suitable for dose evaluation, organ doses in the same scan protocol were compared between the 3-y-old head and commercially available anthropomorphic phantoms having approximately the same head size. The doses of organs in both phantoms were equivalent. The authors’ designed paediatric head phantom will be useful for dose evaluation in paediatric head CT examinations. [ABSTRACT FROM AUTHOR]

Supernumerary teeth and tooth agenesis are common morphological anomalies in humans. We previously obtained evidence that supernumerary maxillary incisors form as a result of the successive development of the rudimentary maxillary incisor tooth germ in Usag-1 null mice. The development of tooth germs is arrested in Runx2 null mice, and such mice also exhibit lingual epithelial buds associated with the upper molars and incisors. The aim of this study is to investigate the potential crosstalk between Usag-1 and Runx2 during tooth development. In the present study, three interesting phenomena were observed in double null Usag-1-/-/Runx2-/- mice: the prevalence of supernumerary teeth was lower than in Usag-1 null mice; tooth development progressed further compared than in Runx2 null mice; and the frequency of molar lingual buds was lower than in Runx2 null mice. Therefore, we suggest that RUNX2 and USAG-1 act in an antagonistic manner. The lingual bud was completely filled with odontogenic epithelial Sox2-positive cells in the Usag-1+/+/Runx2-/- mice, whereas almost no odontogenic epithelial Sox2-positive cells contributed to supernumerary tooth formation in the rudimentary maxillary incisors of the Usag-1-/-/Runx2+/+ mice. Our findings suggest that RUNX2 directly or indirectly prevents the differentiation and/or proliferation of odontogenic epithelial Sox2-positive cells. We hypothesize that RUNX2 inhibits the bone morphogenetic protein (BMP) and/or Wnt signaling pathways regulated by USAG-1, whereas RUNX2 expression is induced by BMP signaling independently of USAG-1.

Bone morphogenetic proteins (BMPs) are highly conserved signaling molecules that are part of the transforming growth factor (TGF)-beta superfamily, and function in the patterning and morphogenesis of many organs including development of the dentition. The functions of the BMPs are controlled by certain classes of molecules that are recognized as BMP antagonists that inhibit BMP binding to their cognate receptors. In this study we tested the hypothesis that USAG-1 (uterine sensitization-associated gene-1) suppresses deciduous incisors by inhibition of BMP-7 function. We learned that USAG-1 and BMP-7 were expressed within odontogenic epithelium as well as mesenchyme during the late bud and early cap stages of tooth development. USAG-1 is a BMP antagonist, and also modulates Wnt signaling. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Using explant culture and subsequent subrenal capsule transplantation of E15 USAG-1 mutant maxillary incisor tooth primordia supplemented with BMP-7 demonstrated in USAG-1+/- as well as USAG-1-/- rescue and supernumerary tooth development. Based upon these results, we conclude that USAG-1 functions as an antagonist of BMP-7 in this model system. These results further suggest that the phenotypes of USAG-1 and BMP-7 mutant mice reported provide opportunities for regenerative medicine and dentistry.

BACKGROUND: Several studies have reported osteomyelitis of the jaw (OMJ) as a side effect of bisphosphonates (BPs), and the risk of oral BPs has been recently clarified. However, other systemic risk factors of OMJ remain unclear. Importantly, the possibility of risk classification based on the clinical characteristics of patients has not been explored. Here, we clarified risk factors of OMJ and evaluate the predictive accuracy of risk indices in osteoporosis patients. METHODS: We performed sub-analysis using a database developed for a retrospective cohort study in patients taking medications for osteoporosis at Kyoto University Hospital. Risk indices for OMJ were constructed using logistic regression analysis, and odds ratios (OR) for OMJ cases and 95% confidence intervals (CI) were estimated. Potential risk factors included in the statistical analysis were age; sex; diabetes; use of oral BPs, corticosteroids, cancer chemotherapy, antirheumatic drugs, and biologic agents; and their interactions. Risk indices were calculated by the sum of potential risk factors of an individual patient multiplied by the regression coefficients. The discriminatory power of the risk indices was assessed by receiver operating characteristic (ROC) analysis. RESULTS: In analysis of all patients, oral BPs (OR: 4.98, 95% CIs: 1.94-12.75), age (OR: 1.28, 95% CI: 1.06-1.60) and sex-chemotherapy interaction (OR: 11.70, 95% CI: 1.46-93.64) were significant risk factors of OMJ. Areas under the ROC curves of these risk indices provided moderate sensitivity or specificity regardless of group (0.683 to 0.718). CONCLUSIONS: Our data suggest that oral BP use, age, and sex-chemotherapy are predictors of OMJ in osteoporosis patients. The risk indices are moderately high, and allow the prediction of OMJ incidence.

BACKGROUND: Associations between mastication and insufficient nutrient intake, obesity, and glucose metabolism have been shown in previous studies. However, the association between mastication and diabetes has not been clarified. Our objective was to examine the association between mastication, namely masticatory performance or rate of eating, and diabetes in a population-based cohort. METHODS: We conducted a cross-sectional study of the association between mastication and diabetes in the Nagahama Prospective Cohort Study, an ongoing study which recruits citizens of Nagahama City in Shiga Prefecture, central Japan. 2,283 male and 4,544 female residents aged 40-74 years were enrolled from July 2009 to November 2010. Masticatory performance was evaluated by spectrophotometric measurement of color changes after masticating color-changeable chewing gum. Categorical rate of eating (fast, intermediate or slow) was self-assessed using a questionnaire. RESULTS: 177 males (7.7%) and 112 (2.4%) females were diagnosed with diabetes. We divided participants into four groups by quartile of masticatory performance, namely Q1 (lowest), 2, and 3 and 4 (highest). Compared to the lowest performance group, the multivariable adjusted odds ratio (OR) of diabetes was 0.91 (95% confidence interval (CI), 0.58-1.4) in Q2, 0.77 (95% CI, 0.48-1.2) in Q3, and 0.53 (95% CI, 0.31-0.90) in the highest group in males, and 1.2 (95% CI, 0.73-2.0), 0.95 (95% CI, 0.54-1.6) and 0.56 (95% CI, 0.30-1.0) in females. We also estimated ORs of diabetes by rate of eating. Compared to the fast eating group, ORs in males were 0.87 (95% CI, 0.61-1.2) in the intermediate group and 0.38 (95% CI, 0.16-0.91) in the slow group, and ORs in females were 0.92 (95% CI, 0.59-1.4) and 1.5 (95% CI, 0.73-3.0). CONCLUSIONS: These findings support the hypothesis that higher masticatory performance and slow eating prevent the occurrence of diabetes.

Background. The expression term of the gene transfected in cells needs to belong enough inorder to make a gene therapy clinically effective. The controlled release of the transfected gene can be utilized. The new biodegradable hydrogel material created by 20 w/w% aldehyded dextran and 10 w/w% ε-poly(L-lysine) (ald-dex/PLL) was developed. We examined whether it could be as a nonviral carrier of the gene transfer. Methods. A plasmid (Lac-Z) was mixed with ald-dex/PLL. An in vitro study was performed to assess the expression of Lac-Z with X-gal stain after gene transfer into the cultured 293 cells and bone marrow cells. As a control group, PLL was used as a cationic polymer. Results. We confirmed that the transfection efficiency of the ald-dex/PLL had a higher transfection efficiency than PLL in 293 cells (plasmid of 2 μg: ald-dex/PLL 1.1%, PLL 0.23%, plasmid of 16 μg: ald-dex/PLL 1.23%, PLL 0.48%). In bone marrow cells, we confirmed the expression of Lac-Z by changing the quantity of aldehyded dextran. In the groups using ald-dextran of the quantity of 1/4 and 1/12 of PLL, their transfection efficiency was 0.43% and 0.41%, respectively. Conclusions. This study suggested a potential of using ald-dex/PLL as a non-carrier for gene transfer.

OBJECTIVES: This study aimed to carry out a histological examination of the temporomandibular joint (TMJ) in ank mutant mice and to identify polymorphisms of the human ANKH gene in order to establish the relationship between the type of temporomandibular disorders (TMD) and ANKH polymorphisms. MATERIALS AND METHODS: Specimens from the TMJ of ank mutant and wild-type mice were inspected with a haematoxylin and eosin staining method. A sample of 55 TMD patients were selected. Each was examined with standard clinical procedures and genotyping techniques. RESULTS: The major histological finding in ank mutant mice was joint space narrowing. Within TMD patients, closed lock was more prevalent among ANKH-OR homozygotes (p = 0.011, OR = 7.7, 95% CI 1.6-36.5) and the elder (p = 0.005, OR = 2.4, 95% CI 1.3-4.3). CONCLUSIONS: Fibrous ankylosis was identified in the TMJ of ank mutant mice. In the human sample, ANKH-OR polymorphism was found to be a genetic marker associated with TMJ closed lock. Future investigations correlating genetic polymorphism to TMD are indicated.

Abstract Background Several lines of evidence have supported possible roles of the sigma receptors in the etiology of schizophrenia and mechanisms of antipsychotic efficacy. An association study provided genetic evidence that the sigma receptor type 1 gene (SIGMAR1) was a possible susceptibility factor for schizophrenia, however, it was not replicated by a subsequent study. It is necessary to evaluate further the possibility that the SIGMAR1 gene is associated with susceptibility to schizophrenia. Methods A case-control association study between two polymorphisms of the SIGMAR1 gene, G-241T/C-240T and Gln2Pro, and schizophrenia in Japanese population, and meta-analysis including present and previous studies. Results There was no significant association of any allele or genotype of the polymorphisms with schizophrenia. Neither significant association was observed with hebephrenic or paranoid subtype of schizophrenia. Furthermore, a meta-analysis including the present and previous studies comprising 779 controls and 636 schizophrenics also revealed no significant association between the SIGMAR1 gene and schizophrenia. Conclusion In view of this evidence, it is likely that the SIGMAR1 gene does not confer susceptibility to schizophrenia.

Antimicrobial peptides that act by disrupting bacterial membranes are attractive agents for treating drug-resistant bacteria. This study investigates a membrane-disrupting peptide mimic made of a cyclic oligosaccharide cyclodextrin scaffold that can be chemically polyfunctionalized. An antibacterial functional group on the peptide was simplified to an alkylamino group that combines cationic and hydrophobic moieties, the former to interact with the anionic bacterial membrane and the latter with the membrane interior. The cyclodextrins equipped with eight alkylamino groups on the molecules using a poly-click reaction exhibited antibacterial activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens such as carbapenem-resistant Enterobacteriaceae . Several lines of evidence showed that these agents disrupt bacterial membranes, leading to rapid bacterial cell death. The resulting membrane perturbation was directly visualized using high-speed atomic force microscopy imaging. In Gram-negative bacteria, the membrane-permeabilizing action of these derivatives allowed the entry of co-treated traditional antibiotics, which were then active against these bacteria., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

A membrane-active antimicrobial peptide gramicidin S-like amphiphilic structure was prepared from cyclodextrin. The mimic was a cyclic oligomer composed of 6-amino-modified glucose 2,3-di- O -propanoates and it exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria, together with no resistance development and low haemolytic activity against red blood cells., (This journal is © The Royal Society of Chemistry 2019.)

We analyzed the structure-activity relationship of porphyrins with the photoinactivation of membrane function in bacteria and erythrocytes. The porphyrins tested were protoporphyrin (PP), mesoporphyrin (MP), deuteroporphyrin (DP), hematoporphyrin (HP), coproporphyrin (CP) and uroporphyrin (UP), along with hematoporphyrin derivative (HPD) and photofrin (PF). These porphyrins dissipated membrane potential of Staphylococcus aureus cells depending on the degrees of respiratory inhibition and K+ leakage. The dysfunction of bacterial membrane was caused within minutes and in the order of PP ∼ MP > DP > HPD ≫ HP > PF > CP ∼ UP. For bovine erythrocytes, these porphyrins induced leakage of K+ and inhibition of the enzyme acetylcholinesterase, which is located on the outer layer of the erythrocyte membrane, in the same order as that observed in bacteria. At high concentrations of PP, MP, DP and HPD, hemolysis (the lysis of erythrocytes with liberation of hemoglobin) was also induced. We found that the degree of photoinactivation of membrane function was closely associated with porphyrin-induced morphological changes in bovine erythrocytes, forming a crenated form from the normal discoid, which is the index of the amount of porphyrins in the outer layer of the cytoplasmic membrane. Furthermore, the degree of morphological changes was related with the octanol/water partition coefficients of porphyrins. These results strongly supported that porphyrins located in the outer layer of cytoplasmic membrane inactivated the cell membrane function by photo-irradiation, and the strength of photoinactivation by porphyrins depended on their affinity to the cell membrane.