Identification of GPI-anchored protein LYPD1 as an essential factor for odontoblast differentiation in tooth development.

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an ex vivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

Three-Dimensional Geometric Morphometry of Facial Soft Tissue Changes After Bilateral Sagittal Split Ramus Osteotomy.

ABSTRACT: This study aimed to evaluate the performance of geometric morphometry (GM) to assess the changes in facial soft tissue after orthognathic surgery. Subjects were 27 patients (skeletal class III) who underwent bilateral sagittal split ramus osteotomy and 27 volunteers as a control group. Computed tomography images of each patient were obtained before surgery (T0) and 6 months after surgery (T1). Computed tomography images of 27 volunteers (skeletal class I) were also obtained as a control group. Using a three-dimensional (3D) modeling software, 3D models were created and exported to a 3D surface analyzing software for geometric morphometry and principal component (PC) analysis. Significant differences in facial soft tissue were found in the first and second of 15 PC. The first PC represented variation in the lower facial height, and the second PC represented variation in the anterior-posterior position of the chin. Comparing the pre- and post-operative images, they illustrated that lower facial height was decreased, and the chin and lower lip moved posteriorly. Geometric morphometry showed to be a successful tool to isolate surgery-related changes from interindividual morphological variations.

Stomatognathic functional improvement in a patient with a canted occlusal plane and temporomandibular disorder treated with an edgewise appliance and miniscrews.

Generally, a canted occlusal plane results in esthetic problems, such as an asymmetric mandible with midline deviation, and functional problems, such as temporomandibular disorder (TMD). For many years, orthognathic surgery has been used to level a canted occlusal plane. However, similar effects might be achieved by intruding the posterior teeth using a miniscrew. This case report describes a patient with a canted occlusal plane, mandibular deviation, shifted dental midlines, and TMD treated with an edgewise appliance using miniscrews as anchorage. Vertical control of posterior teeth with miniscrews enabled flattening of the canted occlusal plane. Dental midlines were coincided with the midfacial line, thereby improving smile symmetry. During 4 years of retention, the patient maintained ideal occlusion. Furthermore, TMD symptoms disappeared, and significant improvements in stomatognathic functions were observed compared with those at pretreatment. These results suggest that miniscrews can be used to improve canted occlusal plane and stomatognathic malfunctions.

Quality of life in preadolescent orthodontic patients before and after secondary alveolar bone grafting.

INTRODUCTION: We evaluated the effects of secondary bone grafting (SBG) on oral health-related and generic health-related quality of life (OHRQOL and HRQOL, respectively) in preadolescent orthodontic patients with alveolar bone defects. METHODS: We divided 101 orthodontic patients aged 8-10 years into 3 groups: 39 general orthodontic patients, 18 patients with orofacial clefts who did not require SBG, and 44 patients with alveolar defects who required SBG using particulate cancellous bone and marrow obtained from the iliac crest. The participants completed the self-report Child Perceptions Questionnaire (CPQ) and Paediatric Quality of Life Inventory (version 4.0) for OHRQOL and HRQOL, respectively, and their scores were assessed. The quality of life (QOL) of patients who required SBG was examined before, 1 month, and 6 months after SBG. The relationships between OHRQOL or HRQOL and potential patient factors were also evaluated. RESULTS: Physical HRQOL subscale scores worsened 1 month after SBG, whereas the total OHRQOL and HRQOL scores before and after SBG showed no significant changes. OHRQOL and HRQOL showed no significant differences among the 3 groups before SBG. The presence of oronasal fistula was associated with poorer OHRQOL in patients with cleft lip and/or palate. CONCLUSIONS: SBG and orthodontic treatment had a relatively small impact on the QOL of the preadolescent children in this study. Understanding the influence of SBG and patient factors on QOL would enable better treatment and care for these patients.

Modulation of osteoclastogenesis through adrenomedullin receptors on osteoclast precursors: initiation of differentiation by asymmetric cell division.

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.

Persistent colonization of non-lymphoid tissue-resident macrophages by Stenotrophomonas maltophilia.

Accumulating evidence has revealed that lymphoid tissue-resident commensal bacteria (e.g. Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713-deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713.

The development, validation, and psychometric properties of the Japanese version of the Child Oral Health Impact Profile-Short Form 19 (COHIP-SF 19) for school-age children.

BACKGROUND: This study was performed to develop and validate a Japanese version of Child Oral Health Impact Profile-Short Form (COHIP-SF) 19 and to assess its psychometric properties in Japanese school-age children. METHODS: The original English COHIP-SF 19 was translated into Japanese (COHIP-SF 19 JP) using a standard forward and backward translation procedure. The psychometric properties of the COHIP-SF 19 JP were assessed in 379 public school students between 7 and 18 years of age in Fukuoka, Japan. Internal consistency (Cronbach's alpha) and test-retest reliability (intraclass correlation coefficient, ICC) were the metrics used for evaluation of this questionnaire. The discriminant validly was examined using the Wilcoxon rank sum test to identify significant differences in COHIP-SF 19 JP scores according to the results of dental examinations. The convergent validity was examined using the Spearman correlations to determine the relationships between COHIP-SF 19 JP scores and the self-perceived oral health ratings. Confirmatory factor analyses (CFA) were performed to verify the factor structure of the questionnaire. RESULTS: The COHIP-SF 19 JP revealed good internal consistency (Cronbach's alpha, 0.77) and test-retest reliability (ICC, 0.81). Discriminant validity indicated that children with dental caries or malocclusion had significantly lower COHIP-SF 19 JP scores (P <  0.05); convergent validity indicated that the self-perceived oral health rating was significantly correlated with the COHIP-SF 19 JP total score and subscores (rs = 0.352-0.567, P <  0.0001), indicating that the questionnaire had a sufficient construct validity. CFA suggested that the modified four-factor model had better model fit indices than the original three-factor model. CONCLUSION: The collected data showed that the COHIP-SF 19 JP possesses sufficient psychometric properties for use in Japanese school-age children.

The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development.

Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme. During tooth development, tooth cusps are regulated by precise control of proliferation of cell clusters, termed enamel knots, that are present among dental epithelial cells. The interaction of ectodysplasin-A (EDA) with its receptor, EDAR, plays a critical role in cusp formation by these enamel knots, and mutations of these genes is a cause of ectodermal dysplasia. It has also been reported that deficiency in Nkx2-3, encoding a member of the NK2 homeobox family of transcription factors, leads to cusp absence in affected teeth. However, the molecular role of NKX2-3 in tooth morphogenesis is not clearly understood. Using gene microarray analysis in mouse embryos, we found that Nkx2-3 is highly expressed during tooth development and increased during the tooth morphogenesis, especially during cusp formation. We also demonstrate that NKX2-3 is a target molecule of EDA and critical for expression of the cell cycle regulator p21 in the enamel knot. Moreover, NKX2-3 activated the bone morphogenetic protein (BMP) signaling pathway by up-regulating expression levels of Bmp2 and Bmpr2 in dental epithelium and decreased the expression of the dental epithelial stem cell marker SRY box 2 (SOX2). Together, our results indicate that EDA/NKX2-3 signaling is essential for enamel knot formation during tooth morphogenesis in mice.

Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene.

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

Enhancement of Self-Aggregation Properties of Linear Elastin-Derived Short Peptides by Simple Cyclization: Strong Self-Aggregation Properties of Cyclo[FPGVG] n, Consisting Only of Natural Amino Acids.

Elastin-like peptides (ELPs) consist of distinctive repetitive sequences, such as (VPGVG) n, exhibit temperature-dependent reversible self-assembly (coacervation), and have been considered to be useful for the development of thermoresponsive materials. Further fundamental studies evaluating coacervative properties of novel nonlinear ELPs could present design concepts for new thermoresponsive materials. In this study, we prepared novel ELPs, cyclic (FPGVG) n (cyclo[FPGVG] n, n = 1-5), and analyzed their self-assembly properties and structural characteristics. Cyclo[FPGVG] n ( n = 3-5) demonstrated stronger coacervation capacity than the corresponding linear peptides. The coacervate of cyclo[FPGVG]5 was able to retain water-soluble dye molecules at 40 °C, which implied that cyclo[FPGVG]5 could be employed as a base material of DDS (drug delivery system) matrices and other biomaterials. The results of molecular dynamics simulations and circular dichroism measurements suggested that a certain chain length was required for cyclo[FPGVG] n to demonstrate alterations in molecular structure that were critical to the exhibition of coacervation.

Intracytoplasmic eosinophilic inclusion bodies with embryonic folliculosebaceous-apocrine unit differentiation in syringocystadenoma papilliferum.

We describe a case of syringocystadenoma papilliferum (SCAP) with a unique histopathology. A 50-year-old Japanese woman presented with a pedunculated tumor in the pubic region. Histopathological examination showed that the tumor was composed of basaloid cell proliferation interconnecting from the epidermis to the dermis. Ductal structures in the tumor were lined by club-shaped columnar cells with apical snouts. Interestingly, numerous vacuolated cells with hyaline globule-like cytoplasmic inclusions were present among the columnar cells, the content of which was identified as sialomucin. Electron microscopy revealed that the vacuolated cytosol of luminal cells represented intracytoplasmic lumens with a structure similar to embryonic apocrine ducts. We assumed that this case represents a rare variant of SCAP that had differentiated toward the embryonic folliculosebaceous-apocrine unit.

Exploration of bacterial species associated with the salivary microbiome of individuals with a low susceptibility to dental caries.

OBJECTIVE: Dental caries is caused by acidogenic plaque microbiota formed on saliva-bathed tooth surfaces, in which multiple organisms act collectively to initiate and expand a cavity. We explored bacterial species associated with the salivary microbiome of individuals with low susceptibility to dental caries. MATERIALS AND METHODS: The bacterial composition of saliva from 19 young adults was analyzed using barcoded pyrosequencing of the 16S rRNA gene; we compared 10 caries-experienced (CE) and nine caries-free (CF) individuals. A quantitative PCR assay of saliva from 139 orally healthy adults aged 40-59 years was carried out to confirm the result obtained by pyrosequencing analysis. RESULTS: The microbiomes of CF individuals showed more diverse communities with a significantly greater proportion of the genus Porphyromonas. Among operational taxonomic units (OTUs) corresponding to the genus Porphyromonas, the OTU corresponding to P. pasteri was the most predominant and its relative abundance in CF individuals was significantly greater than in CE individuals (P < 0.001, Wilcoxon rank sum test). A quantitative PCR assay of saliva confirmed that the amounts of P. pasteri were significantly higher in individuals with lower caries experience (filled teeth <15, n = 67) than in those with higher caries experience (filled teeth ≥15, n = 72) (P < 0.001, Student's t test). CONCLUSION: These results revealed an association between a greater abundance of P. pasteri and lower susceptibility to dental caries. CLINICAL RELEVANCE: P. pasteri may be a bacterial species that could potentially be used as a marker for maintaining a healthy oral microbiome against dental caries.

A Longitudinal Study of the Presence of Dental Anomalies in the Primary and Permanent Dentitions of Cleft Lip and/or Palate Patients.

OBJECTIVE: The aim is to survey primary and permanent dental anomalies: hypodontia, microdontia, a supernumerary tooth, and fused teeth in patients with cleft lip and/or palate. DESIGN: Retrospective longitudinal study Subjects : The subjects were selected from all 1724 patients with cleft lip and/or palate who were registered at the orthodontic clinic of Kyushu University Hospital, Fukuoka, Japan, from 1970 to 2009. Finally, 994 subjects were evaluated for primary dentition, 1352 for permanent dentition, and 871 for the longitudinal changes from primary to permanent dentition. METHODS: The prevalence of dental anomalies was compared for each tooth type, among various cleft types, between males and females, and between the alveolar cleft area and the noncleft area. RESULTS: The prevalence of hypodontia was 16.2% for primary dentition and 52.7% for permanent dentition in the subjects with cleft lip and/or palate. Hypodontia increased with the severity of the cleft type. Multiple hypodontia was found more frequently in the subjects with bilateral cleft lip and palate and the subjects with unilateral cleft lip and palate. Microformed lateral incisors were found in 22.7% of permanent lateral incisors but not in primary dentition. Supernumerary teeth were found in 17.7% of the subjects with cleft lip and/or palate for primary maxillary dentition and in 5.7% for permanent maxillary dentition. CONCLUSION: The prevalence of hypodontia was greater in permanent dentition than in primary dentition; although, it was not much different between males and females or between the right and left sides. The prevalence of dental anomalies was significantly different among four groups by cleft type: cleft lip, cleft lip and alveolus, cleft lip and palate, and cleft palate.

Long-term oral administration of osteocalcin induces insulin resistance in male mice fed a high-fat, high-sucrose diet.

Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion, pancreatic ß-cell proliferation, and adiponectin expression in adipocytes. Previously, we showed that long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level, improved glucose tolerance, and increased the fasting serum insulin concentration as well as pancreatic ß-cell area in female mice fed a normal or high-fat, high-sucrose diet. We have now performed similar experiments with male mice and found that such GluOC administration induced glucose intolerance, insulin resistance, and adipocyte hypertrophy in those fed a high-fat, high-sucrose diet. In addition, GluOC increased the circulating concentration of testosterone and reduced that of adiponectin in such mice. These phenotypes were not observed in male mice fed a high-fat, high-sucrose diet after orchidectomy, but they were apparent in orchidectomized male mice or intact female mice that were fed such a diet and subjected to continuous testosterone supplementation. Our results thus reveal a sex difference in the effects of GluOC on glucose homeostasis. Given that oral administration of GluOC has been considered a potentially safe and convenient option for the treatment or prevention of metabolic disorders, this sex difference will need to be taken into account in further investigations.

Simian immunodeficiency virus SIVmac239 infection and simian human immunodeficiency virus SHIV89.6P infection result in progression to AIDS in cynomolgus macaques of Asian origin.

Simian immunodeficiency virus (SIV) infection models in cynomolgus macaques are important for analysis of the pathogenesis of immunodeficiency virus and for studies on the efficacy of new vaccine candidates. However, very little is known about the pathogenesis of SIV or simian human immunodeficiency virus (SHIV) in cynomolgus macaques from different Asian countries. In the present study, we analysed the infectivity and pathogenicity of CCR5-tropic SIVmac and those of dual-tropic SHIV89.6P inoculated into cynomolgus macaques in Indonesian, Malaysian or Philippine origin. The plasma viral loads in macaques infected with either SIVmac239 or SHIV89.6P were maintained at high levels. CD4+ T cell levels in macaques infected with SIVmac239 gradually decreased. All of the macaques infected with SHIV89.6P showed greatly reduced CD4+ T-cell numbers within 6 weeks of infection. Eight of the 11 macaques infected with SIVmac239 were killed due to AIDS symptoms after 2-4.5 years, while four of the five macaques infected with SHIV89.6P were killed due to AIDS symptoms after 1-3.5 years. We also analysed cynomolgus macaques infected intrarectally with repeated low, medium or high doses of SIVmac239, SIVmac251 or SHIV89.6P. Infection was confirmed by quantitative RT-PCR at more than 5000, 300 and 500 TCID50 for SIVmac239, SIVmac251 and SHIV89.6P, respectively. The present study indicates that cynomolgus macaques of Asian origin are highly susceptible to SIVmac and SHIV infection by both intravenous and mucosal routes. These models will be useful for studies on virus pathogenesis, vaccination and therapeutics against human immunodeficiency virus/AIDS.

Involvement of multiple taste receptors in umami taste: analysis of gustatory nerve responses in metabotropic glutamate receptor 4 knockout mice.

KEY POINTS: The taste receptor T1R1 + T1R3 heterodimer and metabotropic glutamate receptors (mGluR) may function as umami taste receptors. Here, we used mGluR4 knockout (mGluR4-KO) mice and examined the function of mGluR4 in peripheral taste responses of mice. The mGluR4-KO mice showed reduced responses to glutamate and L-AP4 (mGluR4 agonist) in the chorda tympani and glossopharyngeal nerves without affecting responses to other taste stimuli. Residual glutamate responses in mGluR4-KO mice were suppressed by gurmarin (T1R3 blocker) and AIDA (group I mGluR antagonist). The present study not only provided functional evidence for the involvement of mGluR4 in umami taste responses, but also suggested contributions of T1R1 + T1R3 and mGluR1 receptors in glutamate responses. ABSTRACT: Umami taste is elicited by L-glutamate and some other amino acids and is thought to be initiated by G-protein-coupled receptors. Proposed umami receptors include heterodimers of taste receptor type 1, members 1 and 3 (T1R1 + T1R3), and metabotropic glutamate receptors 1 and 4 (mGluR1 and mGluR4). Accumulated evidences support the involvement of T1R1 + T1R3 in umami responses in mice. However, little is known about the in vivo function of mGluR in umami taste. Here, we examined taste responses of the chorda tympani (CT) and the glossopharyngeal (GL) nerves in wild-type mice and mice genetically lacking mGluR4 (mGluR4-KO). Our results indicated that compared to wild-type mice, mGluR4-KO mice showed significantly smaller gustatory nerve responses to glutamate and L-(+)-2-amino-4-phosphonobutyrate (an agonist for group III mGluR) in both the CT and GL nerves without affecting responses to other taste stimuli. Residual glutamate responses in mGluR4-KO mice were not affected by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (an antagonist for group III mGluR), but were suppressed by gurmarin (a T1R3 blocker) in the CT and (RS)-1-aminoindan-1,5-dicarboxylic acid (an antagonist for group I mGluR) in the CT and GL nerve. In wild-type mice, both quisqualic acid (an agonist for group I mGluR) and L-(+)-2-amino-4-phosphonobutyrate elicited gustatory nerve responses and these responses were suppressed by addition of (RS)-1-aminoindan-1,5-dicarboxylic acid and (RS)-alpha-cyclopropyl-4-phosphonophenylglycine, respectively. Collectively, the present study provided functional evidences for the involvement of mGluR4 in umami taste responses in mice. The results also suggest that T1R1 + T1R3 and mGluR1 are involved in umami taste responses in mice. Thus, umami taste would be mediated by multiple receptors.

Involvement of PRIP (Phospholipase C-Related But Catalytically Inactive Protein) in BMP-Induced Smad Signaling in Osteoblast Differentiation.

Phospholipase C-related but catalytically inactive protein (PRIP) was first isolated as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient mice which exhibited the increased bone mineral density and trabecular bone volume, indicating that PRIP is implicated in the regulation of bone properties. In this study, we investigated the possible mechanisms by which PRIP plays a role in bone morphogenetic protein (BMP) signaling, by analyzing the culture of primary cells isolated from calvaria of two genotypes, the wild type and a mutant. In the mutant culture, enhanced osteoblast differentiation was observed by measuring alkaline phosphatase staining and activity. The promoter activity of Id1 gene, responding immediately to BMP, was also more increased. Smad1/5 phosphorylation in response to BMP showed an enhanced peak and was more persistent in mutant cells, but the dephosphorylation process was not different between the two genotypes. The luciferase assay using calvaria cells transfected with the Smad1 mutated as a constitutive active form showed increased transcriptional activity at similar levels between the genotypes. The expression of BMP receptors was not different between the genotypes. BMP-induced phosphorylation of Smad1/5 was robustly decreased in wild type cells, but not in mutant cells, by pretreatment with DB867, an inhibitor of methyltransferase of inhibitory Smad6. Furthermore, BMP-induced translocation of Smad6 from nucleus to cytosol was not much observed in PRIP-deficient cells. These results indicate that PRIP is implicated in BMP-induced osteoblast differentiation by the negative regulation of Smad phosphorylation, through the methylation of inhibitory Smad6.

Assessment of endovascular coil configuration for embolization of intracranial aneurysms using computational fluid dynamics.

Endovascular coil embolization of arterial aneurysms is often complicated by reduced blood flow to branching arteries. To determine the optimal coil configuration for safe embolization of endovascular aneurysms without compromising blood flow in branching arteries. A 3-dimensional voxel model, built based on an unruptured vertebral artery-posterior inferior cerebellar artery (VA-PICA) aneurysm, predicted to show impairment of flow in the PICA during endovascular coil embolization (Case 0). Six different models of final coil configuration were generated and applied to this aneurysm. Case 1 was a round coil mass. Case 2 was designed with a stent assist. Cases 3, and 4 were designed with a neck remnant and Cases 5 and 6 incorporated a balloon neck remodeling technique. Computational fluid dynamics was used to analyze the flow in the PICA in each model. The average outflow to the PICA was highest in Case 0 and lowest in Case 2 (in descending order, Case 0, 5, 4, 6, 1, 3, and 2). There was better preservation of outflow to the PICA in the balloon neck remodeling models than in the neck remnant models. In a model of endovascular coil embolization, we found considerable differences in outflow to the branching artery with small changes in coil configuration. Careful preoperative planning is important to minimize the risk of thromboembolic events during and after endovascular coil embolization.

Regulation of osteoclastogenesis through Tim-3: possible involvement of the Tim-3/galectin-9 system in the modulation of inflammatory bone destruction.

Galectins are a unique family of lectins bearing one or two carbohydrate recognition domains (CRDs) that have the ability to bind molecules with ß-galactoside-containing carbohydrates. It has been shown that galectins regulate not only cell growth and differentiation but also immune responses, as well as inflammation. Galectin-9, a tandem repeat type of galectin, was originally identified as a chemotactic factor for eosinophils, and is also involved in the regulatory process of inflammation. Here, we examined the involvement of galectin-9 and its receptor, T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3), in the control of osteoclastogenesis and inflammatory bone destruction. Expression of Tim-3 was detected in osteoclasts and its mononuclear precursors in vivo and in vitro. Galectin-9 markedly inhibited osteoclastogenesis as evaluated in osteoclast precursor cell line RAW-D cells and primary bone marrow cells of mice and rats. The inhibitory effects of galectin-9 on osteoclastogenesis was negated by the addition of ß-lactose, an antagonist for galectin binding, suggesting that the inhibitory effect of galectin-9 was mediated through CRD. When galectin-9 was injected into rats with adjuvant-induced arthritis, marked suppression of bone destruction was observed. Inflammatory bone destruction could be efficiently ameliorated by controlling the Tim-3/galectin-9 system in rheumatoid arthritis.

Cynomolgus monkey induced pluripotent stem cells established by using exogenous genes derived from the same monkey species.

Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.