An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system.

Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called "eTev", which contains a recognition sequence for Tobacco Etch Virus (TEV) protease. In the crystal structure of 2H5-eTev complex, the long eTev peptide assumes compact α-helical conformation in the binding groove, exposing both ends to the solution. This architecture allowed us to connect eTev with another peptide tag called PA tag via linker sequence, ensuring the simultaneous access of two anti-tag antibodies. When this tandem double tag was attached at one end of various proteins, it enabled highly sensitive and protein-independent detection by sandwich ELISA. Utilizing this system during a rapid cell line screening, we succeeded in isolating stable cell clones expressing high level of mouse Wise protein.

Maxillary Hypoplasia With Congenital Oligodontia Treated by Maxillary Distraction Osteogenesis.

It is known that congenitally missing teeth can often cause differences in craniofacial morphology; however, there are few reported cases of orthognathic surgical treatment for these patients. Herein, the authors report a rare case of maxillary hypoplasia with congenital oligodontia treated by maxillary distraction osteogenesis with internal device. A 17-year-old male presenting with multiple tooth agenesis and maxillary recession was referred to our hospital for orthognathic surgical treatment. Preoperative simulation surgery was performed using Full-Color 3-dimensional salt model. After surgery, improvement in maxillary recession and occlusal stability was observed. This report demonstrates the advantages of the method used herein, which includes reduction in operating time with increase in the safety of the procedure.

The Relationship Between Cephalogram Analysis and Oxygen Desaturation Index During Sleep in Patients Submitted for Mandibular Setback Surgery.

OBJECTIVES: The aim of this study was to examine the relationship between morphologic factors of mandibular protrusion patients and clinical indices of obstructive sleep apnea (OSA). METHODS: Fifty-two Japanese patients divided into 2 groups: 1 jaw surgery group (30 patients) and 2 jaw surgery group (22 patients). Morphologic changes were studied using cephalograms taken before surgery and 1 year after surgery. Functional changes studied using impulse oscillometry and pulse oximetry during sleep, both of which are clinically useful measures in assessing OSA, taken before surgery and 1 year after surgery. RESULT: Lower face cage area significantly decreased in 1 jaw group than in 2 jaw group patients. Positive significant correlation was found between changes in 3% oxygen desaturation index (ODI) and changes of tongue area and vertical position of the hyoid bone in 1 jaw surgery group. Multiple regression analysis indicates that tongue area and airway area were independently significant predictors of 3% ODI in 1 jaw group patients. CONCLUSION: In 2 jaw surgery, maxillary surgery compensated for the effect of mandibular setback surgery. Mandibular setback surgery to mandibular protrusion patients was performed within the range of adequate movement distance, but precautions for risk of postoperative obstructive sleep apnea syndrome should be considered.

Patient With Cleft Maxillary Hypoplasia Who Underwent Distraction Osteogenesis and Conservative Therapies to Postoperative Velopharyngeal Insufficiency.

The authors performed distraction osteogenesis using The Maxillary Distractor System (SYNTHES) to maxillary hypoplasia patient with cleft lip palate, and consequently improved the aesthetic complexion of the patient. Velopharyngeal insufficiency developed after bone elongation; the authors improved the insufficiency with conservative therapies such as articulatory training using the bulb attached palatal lift prosthesis. The authors were successful and accepted postoperative speech outcome.

Effects of Usag-1 and Bmp7 deficiencies on murine tooth morphogenesis.

BACKGROUND: Wnt5a and Mrfzb1 genes are involved in the regulation of tooth size, and their expression levels are similar to that of Bmp7 during morphogenesis, including during the cap and early bell stages of tooth formation. We previously reported that Usag-1-deficient mice form supernumerary maxillary incisors. Thus, we hypothesized that BMP7 and USAG-1 signaling molecules may play important roles in tooth morphogenesis. In this study, we established double genetically modified mice to examine the in vivo inter-relationships between Bmp7 and Usag-1. RESULTS: We measured the volume and cross-sectional areas of the mandibular incisors using micro-computed tomography (micro-CT) in adult Bmp7- and Usag-1-LacZ knock-in mice and their F2 generation upon interbreeding. The mandibular incisors of adult Bmp7+/- mice were significantly larger than those of wild-type (WT) mice. The mandibular incisors of adult Usag-1-/- mice were the largest of all genotypes examined. In the F2 generation, the effects of these genes were additive; Bmp7+/- was most strongly associated with the increase in tooth size using generalized linear models, and the total area of mandibular supernumerary incisors of Usag-1-/-Bmp7+/- mice was significantly larger than that of Usag-1-/-Bmp7 +/+ mice. At embryonic day 15 (E15), BrdU assays demonstrated that the labeling index of Bmp7+/- embryos was significantly higher than that of WT embryos in the cervical loop. Additionally, the labeling index of Usag-1-/- embryos was significantly the highest of all genotypes examined in dental papilla. CONCLUSIONS: Bmp7 heterozygous mice exhibited significantly increased tooth sizes, suggesting that tooth size was controlled by specific gene expression. Our findings may be useful in applications of regenerative medicine and dentistry.

Significant association of periodontal disease with anti-citrullinated peptide antibody in a Japanese healthy population - The Nagahama study.

Anti-citrullinated peptide antibody (ACPA) is a highly specific autoantibody to rheumatoid arthritis (RA). Recent studies have revealed that periodontal disease (PD) is closely associated with RA and production of ACPA in RA. Analyses of associations between PD and ACPA production in a healthy population may deepen our understandings. Here, we analyzed a total of 9554 adult healthy subjects. ACPA and IgM-rheumatoid factor (RF) was quantified and PD status was evaluated using the number of missing teeth (MT), the Community Periodontal Index (CPI) and Loss of Attachment (LA) for these subjects. PD status was analyzed for its association with the positivity and categorical levels of ACPA and RF conditioned for covariates which were shown to be associated with PD, ACPA or RF. As a result, all of MT, CPI and LA showed suggestive or significant associations with positivity (p = 0.024, 0.0042 and 0.037, respectively) and levels of ACPA (p ≤ 0.00031), but none of the PD parameters were associated with those of RF. These association patterns were also observed when we analyzed 6206 non-smokers of the participants. The significant associations between PD parameters and positivity and levels of ACPA in healthy population support the fundamental involvement of PD with ACPA production.

Influence of non-orthodontic intervention on digit sucking and consequent anterior open bite: a preliminary study.

OBJECTIVES: This study aimed to assess behavioural and occlusal outcomes of non-orthodontic intervention (NOI) in a sample of children, 4-12 years of age, in Australia, in order to establish clinical relevance. MATERIALS AND METHODS: Data from 91 patient records of 4- to 12-year-old children reporting a habit of digit sucking, from two clinics in north-eastern Australia, were de-identified and used. Each patient had been examined at two visits, separated by an interval of 4 months, using standard clinical procedures. RESULTS: Of the 77 children who received a 4-month NOI, 69 (89.6%) had ceased their digit sucking habit by the end of the NOI period [χ2=67.0, degrees of freedom (d.f.)=1, P<0.001]. Of the 72 subjects who had front teeth, the number with anterior open bite decreased from 37 (51.4%) to 12 (16.7%) upon completion of NOI (χ2=21.3, d.f.=1, P<0.001). Among the 32 patients with a measurable overjet, the mean overjet was found to decrease from 4.2±2.4 mm to 3.1±1.9 mm after implementation of NOI (t=5.8, d.f.=31, P<0.001). Children who received NOI were more likely to quit the digit sucking habit in the 4-month period (P<0.001, OR=51.8, 95% CI: 9.8-273.9) and were more likely to appear without anterior open bite at a 4-month recall (P<0.001, OR=30.0, 95% CI: 5.9-151.6). CONCLUSIONS: This study demonstrated clinical relevance of NOI on the cessation of a digit sucking habit, closure of anterior open bite and reduction of overjet. Further investigations are indicated.

Prospective signs of cleidocranial dysplasia in Cebpb deficiency.

BACKGROUND: Although runt-related transcription factor 2 (RUNX2) has been considered a determinant of cleidocranial dysplasia (CCD), some CCD patients were free of RUNX2 mutations. CCAAT/enhancer-binding protein beta (Cebpb) is a key factor of Runx2 expression and our previous study has reported two CCD signs including hyperdontia and elongated coronoid process of the mandible in Cebpb deficient mice. Following that, this work aimed to conduct a case-control study of thoracic, zygomatic and masticatory muscular morphology to propose an association between musculoskeletal phenotypes and deficiency of Cebpb, using a sample of Cebpb-/-, Cebpb+/- and Cebpb+/+ adult mice. Somatic skeletons and skulls of mice were inspected with soft x-rays and micro-computed tomography (µCT), respectively. Zygomatic inclination was assessed using methods of coordinate geometry and trigonometric function on anatomic landmarks identified with µCT. Masseter and temporal muscles were collected and weighed. Expression of Cebpb was examined with a reverse transcriptase polymerase chain reaction (RT-PCR) technique. RESULTS: Cebpb-/- mice displayed hypoplastic clavicles, a narrow thoracic cage, and a downward tilted zygomatic arch (p < 0.001). Although Cebpb+/- mice did not show the phenotypes above (p = 0.357), a larger mass percentage of temporal muscles over masseter muscles was seen in Cebpb+/- littermates (p = 0.012). The mRNA expression of Cebpb was detected in the clavicle, the zygoma, the temporal muscle and the masseter muscle, respectively. CONCLUSIONS: Prospective signs of CCD were identified in mice with Cebpb deficiency. These could provide an additional aetiological factor of CCD. Succeeding investigation into interactions among Cebpb, Runx2 and musculoskeletal development is indicated.

ITGA3 and ITGB4 expression biomarkers estimate the risks of locoregional and hematogenous dissemination of oral squamous cell carcinoma.

BACKGROUND: Molecular biomarkers are essential for monitoring treatment effects, predicting prognosis, and improving survival rate in oral squamous cell carcinoma. This study sought to verify the effectiveness of two integrin gene expression ratios as biomarkers. METHODS: Gene expression analyses of integrin α3 (ITGA3), integrin ß4 (ITGB4), CD9 antigen (CD9), and plakoglobin (JUP) by quantitative real-time PCR were conducted on total RNA from 270 OSCC cases. The logrank test, Cox proportional hazards model, and Kaplan-Meier estimates were performed on the gene expression ratios of ITGA3/CD9 and ITGB4/JUP and on the clinicopathological parameters for major clinical events. RESULTS: A high rate (around 80%) of lymph node metastasis was found in cases with a high ITGA3/CD9 ratio (high-ITGA3/CD9) and invasive histopathology (YK4). Primary site recurrence (PSR) was associated with high-ITGA3/CD9, T3-4 (TNM class), and positive margin, indicating that PSR is synergistically influenced by treatment failure and biological malignancy. A high ITGB4/JUP ratio (high-ITGB4/JUP) was revealed to be a primary contributor to distant metastasis without the involvement of clinicopathological factors, suggesting intervention of a critical step dependent on the function of the integrin ß4 subunit. Kaplan-Meier curves revealed positive margin as a lethal treatment consequence in high-ITGA3/CD9 and YK4 double-positive cases. CONCLUSION: Two types of metastatic trait were found in OSCC: locoregional dissemination, which was reflected by high-ITGA3/CD9, and distant metastasis through hematogenous dissemination, uniquely distinguished by high-ITGB4/JUP. The clinical significance of the integrin biomarkers implies that biological mechanisms such as cancer cell motility and anchorage-independent survival are vital for OSCC recurrence and metastasis.

Association Between Tooth Loss and Longitudinal Changes in B-Type Natriuretic Peptide Over 5 Years in Postmenopausal Women: The Nagahama Study.

There is disparity between the sexes in cardiovascular diseases including heart failure (HF). This study aimed to investigate the effect of periodontal disease (PD) on plasma B-type natriuretic peptide (BNP) concentration across sex, age, and menopausal status, as well as the interaction effect of PD and diabetes mellitus (DM) on BNP. This large-scale prospective cohort study enrolled 7539 individuals with no myocardial infarctions or angina pectoris at baseline from the general Japanese population. The association between baseline number of missing teeth (MT) and the longitudinal changes in BNP over 5 years (ΔBNP) was evaluated according to sex and menopausal status. Among 7539 participants, 3190 were postmenopausal women with a mean age ± standard deviation of 61.1 ± 7.6 at baseline. Multivariate analysis revealed a positive association between MT and ΔBNP among postmenopausal women even after adjusting for covariates, including traditional HF risk factors (coefficient, 0.210; 95% confidence interval [CI], 0.107 to 0.312; P < 0.001), but not in men aged > 50. Including an interaction term (MT × DM) in the multivariate model revealed a positive interaction between MT and DM in ΔBNP among postmenopausal women (coefficient for interaction, 1.365; 95% CI, 0.902 to 1.827; P for interaction < 0.001). In conclusion, our study showed a positive association between MT and ΔBNP, as well as a positive effect of the interactive association between MT and DM, among postmenopausal women. Our results suggest a sex difference of an adverse effect of PD on initial myocardial wall stress in the ventricles.

Impact of different surgery modalities to correct class III jaw deformities on the pharyngeal airway space.

OBJECTIVE: The objective of the study was to compare the outcome of different modalities of orthognathic surgery to correct class III jaw deformities concerning the pharyngeal airway space, especially in patients with other predisposing factors for the development of obstructive sleep apnea. METHODS: Lateral cephalograms of 30 Japanese patients (12 males and 18 females, 24.4 [SD, 6.8] years), who underwent surgical-orthodontic treatment for class III jaw deformities, were obtained. Patients were divided into 3 groups: Group A included patients who underwent bilateral sagittal split ramus osteotomy; group B patients underwent bimaxillary surgery, and group C patients underwent intraoral vertical ramus osteotomy. Lateral cephalograms were assessed before surgery and around 3 months and 1 year after surgery. The paired t-test was used to compare the groups, and P < 0.05 was considered significant. RESULTS: In groups A and C who underwent sagittal split ramus osteotomy and intraoral vertical ramus osteotomy, respectively, the pharyngeal airway was constricted significantly at the 3 levels of the pharyngeal airway space on short- and long-term follow-up, whereas in group B, who underwent bimaxillary surgery, no significant changes were noted on long-term follow-up. CONCLUSIONS: Bimaxillary surgery rather than only mandibular setback surgery is preferable to correct class III jaw deformity to prevent narrowing of the pharyngeal airway, which might be a predisposing factor in the development of obstructive sleep apnea syndrome.

Social nicotine dependence in Australian dental undergraduate students.

OBJECTIVES: This study aimed to investigate dental undergraduate students' tobacco usage and social nicotine dependence in Australia. A special interest was to identify the role of factors such as age, gender, year of dental education and cohabitants' smoking status for social nicotine dependence. MATERIALS AND METHODS: A sample of 252, first-to-fifth year undergraduate students in an Australian dental school was used. Each completed a self-administered questionnaire. RESULTS: The smoking rate was 4.8%. Current smokers displayed higher social nicotine dependence than those that had never smoked (t=3.1, df=244, P=0.002). Dental undergraduate students that showed higher social nicotine dependence (P=0.001, OR=1.3, 95% CI: 1.1-1.6), or that had smoking cohabitants (P=0.016, OR=4.8, 95% CI: 1.3-17.0), were more likely to smoke. Students' social nicotine dependence increased with year of dental study (P=0.043, ß=0.4, t=2.0). Social nicotine dependence enhanced tobacco usage among Year-1-to-4 students (P=0.005, OR=1.4, 95% CI: 1.1-1.7) but not Year-5 undergraduates (P=0.432). CONCLUSIONS: Social nicotine dependence has become a developing issue in dental education. Tobacco control should be highlighted in the dental curriculum. Future investigations into the effects of dental education on social nocotine dependence and tobacco usage are indicated.

Novel MSX1 frameshift mutation in a Japanese family with nonsyndromic oligodontia.

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.

A novel LRP6 variant in a Japanese family with oligodontia.

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/ß-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.

Local application of Usag-1 siRNA can promote tooth regeneration in Runx2-deficient mice.

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.

Posttraumatic temporomandibular joint ankylosis in adults: is it mandatory to perform interposition arthroplasty?

Ankylosis of the temporomandibular joint (TMJ) is a severe disorder that leads to jaw function impairment and restricted mouth opening. Management of TMJ ankylosis poses a challenge to surgeons concerned because of the high rate of recurrence. The surgical approach to TMJ ankylosis can be performed according to different techniques and modalities. This report presents a case of a bilateral posttraumatic TMJ ankylosis that showed limited mouth opening (27 mm) along with dental occlusion abnormalities, and both TMJs showed severe fibro-osseous ankylosis. Bilateral gap arthroplasty was performed, and passive interincisal mouth opening of at least 47 mm was achieved after 15 days of physiotherapy. Eight months later, bilateral split ramus osteotomy was performed to correct the residual retrognathia and malocclusion. The outcome of the patient's treatment was satisfactory. Wide gap arthroplasty if followed by vigorous physiotherapy may be sufficient in treating TMJ ankylosis and prevent recurrence in adults.

Development of tooth regenerative medicine strategies by controlling the number of teeth using targeted molecular therapy.

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.

Uterine sensitization-associated gene-1 (USAG-1), a novel BMP antagonist expressed in the kidney, accelerates tubular injury.

Dialysis dependency is one of the leading causes of morbidity and mortality in the world, and once end-stage renal disease develops, it cannot be reversed by currently available therapy. Although administration of large doses of bone morphogenetic protein-7 (BMP-7) has been shown to repair established renal injury and improve renal function, the pathophysiological role of endogenous BMP-7 and regulatory mechanism of its activities remain elusive. Here we show that the product of uterine sensitization-associated gene-1 (USAG1), a novel BMP antagonist abundantly expressed in the kidney, is the central negative regulator of BMP function in the kidney and that mice lacking USAG-1 (USAG1 mice) are resistant to renal injury. USAG1 mice exhibited prolonged survival and preserved renal function in acute and chronic renal injury models. Renal BMP signaling, assessed by phosphorylation of Smad proteins, was significantly enhanced in USAG1 mice with renal injury, indicating that the preservation of renal function is attributable to enhancement of endogenous BMP signaling. Furthermore, the administration of neutralizing antibody against BMP-7 abolished renoprotection in USAG1 mice, indicating that USAG-1 plays a critical role in the modulation of renoprotective action of BMP and that inhibition of USAG-1 is a promising means of development of novel treatment for renal diseases.

Enhanced BMP signaling results in supernumerary tooth formation in USAG-1 deficient mouse.

Uterine sensitization associated gene-1 (USAG-1) is a BMP antagonist, and also modulates Wnt signaling. We previously reported that USAG-1 deficient mice have supernumerary teeth. The supernumerary maxillary incisor appears to form as a result of the successive development of the rudimentary upper incisor. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. We confirmed that BMPs were expressed in both the epithelium and mesenchyme of the rudimentary incisor at E14 and E15. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Wnt signaling as demonstrated by the nuclear localization of beta-catenin was also up-regulated. Inhibition of BMP signaling rescues supernumerary tooth formation in E15 incisor explant culture. Based upon these results, we conclude that enhanced BMP signaling results in supernumerary teeth and BMP signaling was modulated by Wnt signaling in the USAG-1 deficient mouse model.

Gender differences in morphological and functional outcomes after mandibular setback surgery.

PURPOSE: The aim of this study was to examine and compare morphological and functional outcomes after either isolated mandibular setback or bimaxillary surgery in males and females. MATERIALS AND METHODS: A retrospective study was done on 52 patients, in whom surgical correction for mandibular prognathism was performed either by isolated mandibular setback (30 cases) or by bimaxillary surgery (22 cases). Morphological changes were studied using cephalograms and functional changes studied using impulse oscillometry (IOS) taken before surgery (T0), 3 months (T1) and 1 year after surgery (T2). Also 3% oxygen desaturation index (ODI) was measured at T0 and T2. RESULT: Posterior airway space decreased significantly in both groups and both sexes but more so in males after mandibular setback surgery and in females after bimaxillary surgery. Changes in supine R20 (central airway resistance at 20 Hz) and supine R5 (total airway resistance at 5 Hz) in IOS statistically significantly increased in the period T0-T1 in males compared with females after mandibular setback surgery (p < 0.05). CONCLUSION: Gender dimorphism is present according to morphological and functional outcomes, with males at a higher risk for obstructive sleep apnea (OSA) after mandibular setback surgery and females after bimaxillary surgery; however, compensatory changes act as a barrier against this.