Search

Gammaretroviral vectors are widely used in cellular and gene therapy products because of the availability of stable vector producer cells. Accurately assessing vector copy number (VCN) is critical for selecting appropriate clones to avoid the risks of homologous recombination and complications in mutation detection. Traditional methods such as quantitative polymerase chain reaction (PCR) and Southern blotting have limitations in accuracy and throughput. This study presents a triplex droplet digital PCR (ddPCR) method for analyzing the VCN in gammaretroviral vector producer cells. We designed a universal primer- probe set targeting the packaging signal sequence common to murine leukemia virus- and murine stem cell virus- based gammaretroviral vectors. Two reference genes were selected after karyotyping the PG13 gammaretroviral vector packaging cell line to identify stable chromosomes. The triplex ddPCR assay was optimized and verified using PG13 cells transduced with constructs of different transgene and vector backbones. The assay showed high concordance with Southern blot. Using multiple reference genes ensures accurate and robust VCN assessment, especially in cell lines with chromosomal instability. This method improves the clone selection process for gammaretroviral vector producer cells, accelerates the development of novel cellular and gene therapy products, and ensures their rapid delivery to patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are employees of Takeda Pharmaceutical Company Limited, and have no commercial, proprietary, or financial interests in the products or companies described in this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Leptin receptor-positive (LepR + ) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR + cells., Design: Bone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers., Results: Bone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly., Conclusion: LRP1 in LepR + cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Biosensors with two-dimensional materials have gained wide interest due to their high sensitivity. Among them, single-layer MoS 2 has become a new class of biosensing platform owing to its semiconducting property. Immobilization of bioprobes directly onto the MoS 2 surface with chemical bonding or random physisorption has been widely studied. However, these approaches potentially cause a reduction of conductivity and sensitivity of the biosensor. In this work, we designed peptides that spontaneously align into monomolecular-thick nanostructures on electrochemical MoS 2 transistors in a non-covalent fashion and act as a biomolecular scaffold for efficient biosensing. These peptides consist of repeated domains of glycine and alanine in the sequence and form self-assembled structures with sixfold symmetry templated by the lattice of MoS 2 . We investigated electronic interactions of self-assembled peptides with MoS 2 by designing their amino acid sequence with charged amino acids at both ends. Charged amino acids in the sequence showed a correlation with the electrical properties of single-layer MoS 2 , where negatively charged peptides caused a shift of threshold voltage in MoS 2 transistors and neutral and positively charged peptides had no significant effect on the threshold voltage. The transconductance of transistors had no decrease due to the self-assembled peptides, indicating that aligned peptides can act as a biomolecular scaffold without degrading the intrinsic electronic properties for biosensing. We also investigated the impact of peptides on the photoluminescence (PL) of single-layer MoS 2 and found that the PL intensity changed sensitively depending on the amino acid sequence of peptides. Finally, we demonstrated a femtomolar-level sensitivity of biosensing using biotinylated peptides to detect streptavidin.

Here we demonstrate that short peptides, de novo designed from first principles, self-assemble on the surface of graphite to produce a highly robust and catalytic nanoarchitecture, which promotes peroxidation reactions with activities that rival those of natural enzymes in both single and multi-substrate reactions. These designable peptides recapitulate the symmetry of the underlying graphite surface and act as molecular scaffolds to immobilize hemin molecules on the electrode in a hierarchical self-assembly manner. The highly ordered and uniform hybrid graphite-peptide-hemin nanoarchitecture shows the highest faradaic efficiency of any hybrid electrode reported. Given the explosive growth of the types of chemical reactions promoted by self-assembled peptide materials, this new approach to creating complex electrocatalytic assemblies will yield highly efficient and practically applicable electrocatalysts.

Dipeptide repeat proteins (DRPs) are considered a significant cause of amyotrophic lateral sclerosis (ALS), and their liquid-liquid phase separation (LLPS) formation with other biological molecules has been studied both in vitro and in vivo. The immobilization and wetting of the LLPS droplets on glass surfaces are technically crucial for the measurement with optical microscopy. In this work, we characterized the surface diffusion of LLPS droplets of the DRPs with different lengths to investigate the multivalent effect on the interactions of their LLPS droplets with the glass surface. Using fluorescence microscopy and the single-particle tracking method, we observed that the large multivalency drastically changed the surface behavior of the droplets. The coalescence and wetting of the droplets were accelerated by increasing the multivalency of peptides in the LLPS system. Our findings on the effect of multivalency on interactions between droplets and glass surfaces could provide a new insight to enhance the understanding of LLPS formation and biophysical properties related to the solid/liquid interface., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Introduction: Orthodontic anchoring screws (OASs) have been placed around midpalatal sutures in patients of various ages. Our previous study found that OAS placement more than 1.5 mm from midpalatal suture was more successful than placement directly at the suture. This study aimed to investigate the relationship between age and midpalatal suture maturation, considering factors affecting the failure of OASs using cone-beam computed tomography., Methods: In total, 150 patients who underwent cone-beam computed tomography were selected. The total depth and sutured depth of the midpalatal suture corresponding area to anterior (interpremolar zone) and posterior region (mesial and distal borders of the first molar) were measured, and the ratio of sutured depth to total depth (sutured ratio) was calculated., Results: The mean sutured ratios at interpremolar zone and mesial and distal borders of the first molar according to age were 40%, 60%, and 63% in the younger group (≤17 years), 46%, 76%, and 76% in the middle group (18-25 years), and 47%, 74%, and 76% in the older group (≥26 years), respectively. The sutured ratio of the anterior region was significantly lower than that of the posterior region (P <0.01). Each mean sutured ratio of the middle and older group was significantly higher than that of the younger group on both sides (P <0.01). According to the cervical vertebral maturation, the mean sutured ratio of cervical vertebral stages 5-6 was significantly higher than cervical vertebral stages 1-3 on the distal side (P <0.05)., Conclusions: Incomplete closure of the midpalatal suture was observed frequently, even in the older group. This might be caused by insufficient calcification of the midpalatal suture, including in elder patients. To prevent OAS placement to the unsutured area, the midpalatal suture should be avoided regardless of age., (Copyright © 2022 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The detailed mechanism of the process during bone healing of drill-hole injury has been elucidated, but a crucial factor in regulating drill-hole healing has not been identified. The transcription factor p53 suppresses osteoblast differentiation through inhibition of osterix expression. In present study, we demonstrate the effects of p53 deficiency on the capacity of MSCs and osteoblasts during drill-hole healing., Materials and Methods: Mesenchymal stromal cells (MSCs) and osteoblasts were collected from bone marrow and calvaria of p53 knockout (KO) mice, respectively. The activities of cell mobility, cell proliferation, osteoblast differentiation, and wound healing of MSCs and/or osteoblasts were determined by in vitro experiments. In addition, bone healing of drill-hole injury in KO mice was examined by micro-CT and immunohistological analysis using anti-osterix, Runx2, and sclerostin antibodies., Results: KO MSCs stimulated cell mobility, cell proliferation, and osteoblast differentiation. Likewise, KO osteoblasts enhanced cell proliferation and wound healing. KO MSCs and osteoblasts showed high potency in the inflammation and callus formation phases compared to those from wild-type (WT) mice. In addition, increased expression of osterix and Runx2 was observed in KO MSCs and osteoblasts that migrated in the drill-hole. Conversely, sclerostin expression was inhibited in KO mice. Eventually, KO mice exhibited high repairability of drill-hole injury, suggesting a novel role of p53 in MSCs and osteoblasts in improving bone healing., Conclusion: p53 Deficiency promotes bone healing of drill-hole injury by enhancing the bone-regenerative ability of MSCs and osteoblasts., (© 2022. The Japanese Society Bone and Mineral Research.)

Self-disclosure of life experiences from the viewpoint of integrity is considered beneficial to the psychological health of older adults. It has been shown that people tend to self-disclose more to people they like. Compared to a consistent invariant reward, an improvement in the rewarding behavior of a person has been shown to have a greater positive impact on an individual's liking for the person. Based on these previous studies, we explored the psychological impact of self-disclosure of integrated life experiences on the elderly and the effect of the change in the robot's listening attitude on the elderly's self-disclosure. We conducted an experiment in which 38 elderly participants were asked to self-disclose their life experiences to a robot for approximately 20 min. The participants interacted with either a robot with a consistently positive listening attitude or a robot that initially had a neutral listening attitude that changed to a positive listening attitude. The results showed that self-disclosure of integrated life experiences to the robot had a psychological impact on improving self-esteem. In addition, changes in the robot's listening attitude were found to promote self-disclosure and enhance its impact on self-esteem., Competing Interests: Conflict of interestThe authors declare no conflicts of interest associated with this manuscript., (© The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

The liquid-liquid phase separation (LLPS) of proteins and RNA molecules has emerged in recent years as an important physicochemical process to explain the organization of membrane-less organelles in living cells and cellular functions and even some fatal neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) due to the spontaneous condensation and growth of LLPS droplets. In general, the characterization of LLPS droplets has been performed by optical microscopy, where we need transparent substrates. By virtue of the liquid and wetting properties of LLPS droplets on a glass surface, there have been some technical protocols recommended to immobilize droplets on the surfaces. However, interactions between LLPS droplets and glass surfaces still remain unclear. Here, we investigated the surface diffusion of LLPS droplets on the glass surface to understand the interactions of droplets in a dynamic manner, and employed chemically modified glass surface with charges to investigate their Coulombic interaction with the surface. Using the single-particle tracking method, we first analyzed the diffusion of droplets on an untreated glass surface. Then, we compared the diffusion modes of LLPS droplets on each substrate and found that there were two major states of droplets on a solid surface: fix and diffusion mode for the LLPS droplet diffusion. While untreated glass showed a diffusion of droplets mainly, chemically modified glass with positive charges exhibited droplets fixed on the surface. It could arise from the Coulombic interaction between droplets and solid surface, where LLPS droplets have a negative ζ-potential. Our findings on the dynamics of LLPS at the solid/liquid interface could provide a novel insight to advance fundamental studies for understanding the LLPS formation.

During orthodontic treatment, plaque tends to form around fixed orthodontic appliances, which increases the risk of dental caries. It has been reported that ultarafine bubble with a diameter <1 μm water (UFBW) effectively removes organic matter. In addition, UFBW is harmless and stable for at least one month with refrigeration. The aim of this study was to examine the plaque-removal effect of ultrafine bubble water (UFBW) to establish a new method to prevent dental caries in patients during orthodontic treatment procedures. The in vitro study examined different concentrations of UFBW and compared the cleaning effect to that of existing mouthwashes. High-concentration UFBW (HUFBW) was most effective in cleaning. In the subsequent clinical study, HUFBW showed a significantly higher plaque-removal effect compared to distilled water (p<0.01). Thus, supplementary use of HUFBW could decrease the incidence of dental caries during orthodontic treatment.

M-doped WO 3 (M = Sn or In) films were prepared from aqueous coating solutions via evaporation-driven deposition during low-speed dip coating. Sn- and In-doping were easily achieved by controlling the chemical composition of simple coating solutions containing only metal salts and water. The crystallinity of the WO 3 , Sn-doped WO 3 , and In-doped WO 3 films varied with heating temperature, where amorphous and crystalline films were obtained by heating at 200 and 500 °C, respectively. All the amorphous and crystalline films showed an electrochromic response, but good photoelectrochemical stability was observed only for the crystalline samples heated at 500 °C. The crystalline In-WO 3 films exhibited a faster electrochromic color change than the WO 3 or Sn-WO 3 films, and good cycle stability for the electrochromic response in the visible wavelength region., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Purpose: The aim of this study was to investigate the stability of orthodontic anchor screws (OASs) in the mid-palatal area according to pre-drilling diameter., Methods: The success rate of 161 OASs (83 patients, φ2.0 mm, 6.0 mm in length) placed in a corresponding area to the mesial and distal borders of the first molar (mesial zone and distal zone) was assessed according to placement location and pre-drilling diameter (1.2 and 1.5 mm). Placement torque values from 73 OASs with a pre-drilling diameter of 1.2 mm were compared between success and failure groups., Results: The success rates of OASs pre-drilled with φ1.2 and 1.5 mm were 94.5% and 83.0%, respectively (P < 0.05); corresponding rates in the mesial zone were 100.0% and 77.3% (P < 0.005), and those in the distal zone were 89.2% and 88.6%, respectively. Placement torques of OASs predrilled with φ1.2 mm in the success and failure groups were 25.9 and 19.2 N·cm, respectively (P < 0.05)., Conclusion: A smaller pre-drilling diameter was associated with a higher success rate of OASs in the mid-palatal area, especially in the mesial zone. When pre-drilling diameter of 1.2 mm was used for φ2.0 mm OAS, greater placement torque was indicative of greater OAS stability.

Bone destructive diseases such as periodontitis are common worldwide and are caused by excessive osteoclast formation and activation. Receptor activator of nuclear factor-κB ligand (RANKL) is essential factor for osteoclastogenesis. This triggers reactive oxygen species (ROS), which has a key role in intracellular signaling as well exerting cytotoxicity. Cells have protective mechanisms against ROS, such as nuclear factor E2-related factor 2 (Nrf2), which controls the expression of many antioxidant enzyme genes. Conversely, BTB and CNC homology 1 (Bach1), a competitor for Nrf2, transcriptionally represses the expression of anti-oxidant enzymes. Previously, we demonstrated that RANKL induces Bach1 nuclear import and attenuates the expression of Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular ROS signaling and osteoclastogenesis. However, it remains unknown if Bach1 inhibitors attenuate osteoclastogenesis. In this study, we hypothesized that Bach1 inhibition would exert an anti-osteoclastogenic effects via diminishing of intracellular ROS signaling through augmented antioxidation. We used RAW 264.7 cells as osteoclast progenitor cells. Using flow cytometry, we found that Bach1 inhibitors attenuated RANKL-mediated ROS generation, which resulted in the inhibition of osteoclastogenesis. Local injection of a Bach1 inhibitor into the calvaria of male BALB/c mice blocked bone destruction induced by lipopolysaccharide. In conclusion, we demonstrate that Bach1 inhibitor attenuates RANKL-mediated osteoclastogenesis and bone destruction in mice by inducing the expression of Nrf2-regulated antioxidant enzymes that consequently decrease intracellular ROS levels. Bach1 inhibitors have potential in inhibiting bone destructive diseases such as periodontitis, rheumatoid arthritis and osteoporosis., (Copyright © 2020 Wada, Kanzaki, Katsumata, Yamaguchi, Narimiya, Attucks, Nakamura and Tomonari.)

Occlusal disharmony leads to morphological changes in the hippocampus and osteopenia of the lumbar vertebra and long bones in mice, and causes stress. Various types of stress are associated with increased incidence of cardiovascular disease, but the relationship between occlusal disharmony and cardiovascular disease remain poorly understood. Therefore, in this work, we examined the effects of occlusal disharmony on cardiac homeostasis in bite-opening (BO) mice, in which a 0.7 mm space was introduced by cementing a suitable applicance onto the mandibular incisior. We first examined the effects of BO on the level of serum corticosterone, a key biomarker for stress, and on heart rate variability at 14 days after BO treatment, compared with baseline. BO treatment increased serum corticosterone levels by approximately 3.6-fold and the low frequency/high frequency ratio, an index of sympathetic nervous activity, was significantly increased by approximately 4-fold by the BO treatment. We then examined the effects of BO treatment on cardiac homeostasis in mice treated or not treated with the non-selective β-blocker propranolol for 2 weeks. Cardiac function was significantly decreased in the BO group compared to the control group, but propranolol ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage were significantly increased in the BO group, but propranolol blocked these changes. The BO-induced cardiac dysfunction was associated with increased phospholamban phosphorylation at threonine-17 and serine-16, as well as inhibition of Akt/mTOR signaling and autophagic flux. These data suggest that occlusal disharmony might affect cardiac homeostasis via alteration of the autonomic nervous system., Competing Interests: SO received funding from Pfizer Japan. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLoS One policies on sharing data and meterials.

Introduction: Because mechanical stimulation of the periodontal ligament by low-intensity pulsed ultrasound (LIPUS) has been shown to increase the speed of bone remodeling, this study aimed to examine the effects of LIPUS stimulation on the rate of tooth movement and bone remodeling during lateral tooth movement., Methods: Twelve-week-old Wistar rats were divided into 2 groups. The LIPUS group received experimental tooth movement with LIPUS stimulation, and the tooth movement (TM) group were provided experimental tooth movement without LIPUS. For each group, the upper right first molars were moved buccally with fixed appliances. LIPUS exposure was placed in the region corresponding to the right maxillary first molar. Three days after tooth movement, tartrate-resistant acid phosphatase was examined. Fourteen days after tooth movement, the intermolar width, bone mineral content, and bone volume fraction were analyzed by micro-computed tomography, and newly formed bone was measured histomorphometrically., Results: The number of TRAP-positive cells in the compressed region was higher in the LIPUS group. The intermolar width was significantly higher in the LIPUS group than in the TM group. The alveolar bone around the maxillary first molar showed no differences in bone mineral content and bone volume fraction between the LIPUS and TM groups. The LIPUS group exhibited a more significant amount of newly formed alveolar bone than the TM group., Conclusions: The present study provides evidence of the beneficial effects of LIPUS on the lateral tooth movement., (Copyright © 2019 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Mastication is closely related to brain function. Animal experiments have revealed that tooth loss has a negative influence on brain function. Clinical studies also suggest that normal occlusion is an essential factor for favorable brain function. Mandibular prognathism (MP) usually results in occlusal dysfunction. However, the relationship between MP and brain function remains unclear. In the present study, we examined the relationship between MP and brain function by measuring brain blood flow (BBF). Seventeen subjects with normal occlusion (NORM) and 25 patients with MP participated in this study. The number of occlusal contacts were counted. Electromyography of the masseter muscles during clenching was also recorded. BBF was measured with non-invasive functional near-infrared spectroscopy during calculation task and chewing task. The number of the occlusal contacts and masseter muscle activity were lower in MP compared with NORM. The calculation task increased BBF in both groups. The chewing task also increased BBF in the inferior frontal gyrus in both groups, although the increase in MP was smaller than in NORM. We discovered that patients with MP exhibited a smaller increase in BBF at the inferior frontal gyrus during chewing as compared with NORM. As such, MP would negatively affect brain function.

In this study, we examined 239 outpatients receiving chemotherapy for breast cancer for a period of 6 months from July 2016 to December 2016. Using a questionnaire, we investigated the patients' symptom score and uneasiness. A symptom score of 2 and over was found in 24.7%(59)of the cases. Twenty-seven of the 59 cases experienced adverse effects of chemotherapy. Peripheral neuropathy was observed in 20 cases, of which only 2 cases improved after providing palliative care. Palliative care was effective against nausea, constipation, malaise, and sleeping disorders. Thirty-two cases(13.4%)had 5 or more painful feeling score. Among these, 10 cases resulted from the adverse effects of treatment, 10 cases from the aggravation of existing cancer, and 6 cases showed anxiety for the illness, family, and future. In 15 of the 32 cases, the pain score improved by providing palliative care, conversation with the nursing staff, reduction in the quantity of drug intake, etc.

We investigated the potency of TAK-828F, a RORγt inverse agonist, in murine experimental autoimmune encephalomyelitis (EAE) model. TAK-828F inhibited the differentiation of Th17 and Th1/17 cells in inguinal lymph node. Increase of these cells in central nervous system (CNS) was also inhibited by TAK-828F. Prophylactic and therapeutic treatments of TAK-828F were efficacious in the model. Plasma concentration of TAK-828F was higher than that in CNS. These results indicate that TAK-828F mainly acts at peripheral and results in the reduction of Th17- and Th1/17-dependent inflammation in CNS. Blocking RORγt may be a promising strategy for treatment of multiple sclerosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective and Design: To evaluate the potency of RORγt blockade for treatment of Inflammatory Bowel Disease (IBD), the efficacy of TAK-828F, a novel RORγt inverse agonist, in anti-TNF-α mAb non-responsive mouse colitis model and effect of TAK-828F on IL-17 production in peripheral mononuclear blood cells (PBMCs) of anti-TNF-α naive and treatment-failure patients of IBD was investigated., Methods and Results: The colitis model showed Th17-dependent pathogenicity and response to anti-IL-12/23p40 monoclonal antibody (mAb), but no response to anti-TNF-α mAb. In the model, TAK-828F, at oral dosages of 1 and 3 mg/kg, inhibited progression of colitis and reduced the immune reaction that characterize Th17 cells. Anti-IL-17A mAb showed neither efficacy nor change in the T cell population and colonic gene expression in the model. In the normal mouse, a 4-week treatment of TAK-828F at 30 mg/kg did not severely reduce lymphocyte cell counts in peripheral and intestinal mucosa, which was observed in RORγ -/- mice. TAK-828F strongly inhibited IL-17 gene expression with IC 50 values from 21.4 to 34.4 nmol/L in PBMCs from anti-TNF mAb naive and treatment-failure patients of IBD., Conclusions: These results indicate that RORγt blockade would provide an effective approach for treating refractory patients with IBD by blocking IL-23/Th17 pathway.

The aim of the present study was to evaluate albumin adsorption to stainless steel (SUS), diamond-like carbon (DLC) and fluorinedoped DLC (F-DLC) films using the quartz crystal microbalance (QCM) method. Each sensor was characterized using atomic force microscopy, surface roughness and surface wettability measurements and surface free energy calculations. Adsorbed amounts of bovine serum albumin on DLC and F-DLC were significantly lower than that on SUS (p<0.05). The apparent first-order reaction rate, k obs , of F-DLC was significantly larger than those of SUS and DLC (p<0.05). Moreover, significantly lower total surface free energies of DLC and F-DLC influenced the albumin absorbed amounts and k obs . Furthermore, a clear correlation was found between the albumin absorbed amounts and the hydrogen bond component of the total surface free energy. Thus, DLC or F-DLC coating is effective for preventing protein adsorption on orthodontic appliances.

In skeletal muscle, the major isoform of β-adrenergic receptor (β-AR) is β2-AR and the minor isoform is β1-AR, which is opposite to the situation in cardiac muscle. Despite extensive studies in cardiac muscle, the physiological roles of the β-AR subtypes in skeletal muscle are not fully understood. Therefore, in this work, we compared the effects of chronic β1- or β2-AR activation with a specific β1-AR agonist, dobutamine (DOB), or a specific β2-AR agonist, clenbuterol (CB), on masseter and cardiac muscles in mice. In cardiac muscle, chronic β1-AR stimulation induced cardiac hypertrophy, fibrosis and myocyte apoptosis, whereas chronic β2-AR stimulation induced cardiac hypertrophy without histological abnormalities. In masseter muscle, however, chronic β1-AR stimulation did not induce muscle hypertrophy, but did induce fibrosis and apoptosis concomitantly with increased levels of p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), calmodulin kinase II (Thr-286) and mammalian target of rapamycin (mTOR) (Ser-2481) phosphorylation. On the other hand, chronic β2-AR stimulation in masseter muscle induced muscle hypertrophy without histological abnormalities, as in the case of cardiac muscle, concomitantly with phosphorylation of Akt (Ser-473) and mTOR (Ser-2448) and increased expression of microtubule-associated protein light chain 3-II, an autophagosome marker. These results suggest that the β1-AR pathway is deleterious and the β2-AR is protective in masseter muscle. These data should be helpful in developing pharmacological approaches for the treatment of skeletal muscle wasting and weakness., Competing Interests: The authors have declared that no competing interests exist.

Background: Osteoclasts play a critical role in bone resorption due to orthodontic tooth movement (OTM). In OTM, a force is exerted on the tooth, creating compression of the periodontal ligament (PDL) on one side of the tooth, and tension on the other side. In response to these mechanical stresses, the balance of receptor activator of nuclear-factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) shifts to stimulate osteoclastogenesis. However, the mechanism of OPG expression in PDL cells under different mechanical stresses remains unclear. We hypothesized that compression and tension induce different microRNA (miRNA) expression profiles, which account for the difference in OPG expression in PDL cells. To study miRNA expression profiles resulting from OTM, compression force (2 g/cm 2 ) or tension force (15% elongation) was applied to immortalized human PDL (HPL) cells for 24 h, and miRNA extracted. The miRNA expression in each sample was analyzed using a human miRNA microarray, and the changes of miRNA expression were confirmed by real-time RT-PCR. In addition, miR-3198 mimic and inhibitor were transfected into HPL cells, and OPG expression and production assessed., Results: We found that certain miRNAs were expressed differentially under compression and tension. For instance, we observed that miR-572, - 663, - 575, - 3679-5p, UL70-3p, and - 3198 were upregulated only by compression. Real-time RT-PCR confirmed that compression induced miR-3198 expression, but tension reduced it, in HPL cells. Consistent with previous reports, OPG expression was reduced by compression and induced by tension, though RANKL was induced by both compression and tension. OPG expression was upregulated by miR-3198 inhibitor, and was reduced by miR-3198 mimic, in HPL cells. We observed that miR-3198 inhibitor rescued the compression-mediated downregulation of OPG. On the other hand, miR-3198 mimic reduced OPG expression under tension. However, RANKL expression was not affected by miR-3198 inhibitor or mimic., Conclusions: We conclude that miR-3198 is upregulated by compression and is downregulated by tension, suggesting that miR-3198 downregulates OPG expression in response to mechanical stress.

Introduction: The purpose of this study was to examine growth-promoting effects of myo-inositol nutritional supplementation on the mandible in experimental animals., Methods: Mice were fed on diets that contained various concentration of myo-inositol for 3 to 12 weeks. The length of the mandible, maxilla, and femur were measured on μCT images. The mandible and tibia were examined histologically and immunohistochemically. The effects of myo-inositol on cell proliferation and chondrocytic differentiation were examined using ATDC5 cells., Results: Myo-inositol supplementation had no effects on body weight, length, and maxilla and femur lengths. However, the length of mandible and the thickness of the mandibular condylar cartilage (MCC) were increased by myo-inositol supplement. Microarray analysis revealed that Pik3cd was highly expressed in MCC as compared to that in the cartilage of the tibial growth plate, which was confirmed by real-time RT-PCR and immunohistochemistry. ATDC5 cells also highly expressed Pik3CD. Myoinositol induced increases in cell proliferation and chondrocytic differentiation in ATDC5 cells. The addition of a PIK3CD inhibitor blocked the induction of cell proliferation by myo-inositol in ATDC5 cells., Conclusions: Nutritional supplementation with myo-inositol in growing mice augmented mandibular endochondral growth without any systemic effects. The specific promotion of mandibular growth by myoinositol is primarily dependent on the specific intensive expression of PIK3CD in the MCC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective and Design: Protease activity of MALT lymphoma-translocation protein 1 (Malt1) plays an important role in the development of colitis, but the detailed mechanism has not been fully elucidated., Method: Effects of Malt1 protease on the activation of T cells and the development of experimental colitis was investigated using Malt1 protease-deficient (PD) mouse., Results: IL-2 production from CD4 + T cells of Malt1 PD mice was decreased compared with that of wild-type (WT) mice. Intraperitoneal injection of anti-CD3 antibody into Malt1 PD mouse induced less productions of IL-17 in the plasma, as well as the colonic gene expression of IL-17A, compared with WT mice, whereas IFN-γ production was not impaired. In naïve T-cell transfer colitis model, Malt1 PD T cells induced less disease severity than WT T cells. Then, reduction in the populations of Th17 and Th1/17 cells was observed in the mesenteric lymph nodes of the recipient mice transferred with Malt1 PD T cells, whereas those of Th1 cells were not impaired. IL-17A expression in the colon was also decreased in the mouse receiving Malt1 PD T cells., Conclusions: Inactivation of Malt1 protease activity abrogates Th17 and Th1/17 cell activation, resulting in the amelioration of experimental colitis.

IL-17-producing Th17 cells and IFN-γ and IL-17 double-producing Th1/17 cells have been identified as the pathogenic cells in inflammatory bowel disease (IBD). Retinoic acid-related orphan receptor γt (RORγt) is a master regulator for the differentiation and activation of Th17 and Th1/17 cells. We discovered a novel orally available TAK-828F, a strong and selective RORγt inverse agonist. To assess the potential of RORγt blockade in the therapy for IBD, the efficacy of TAK-828F in activated T cell transfer mouse colitis model was investigated. This model was highly sensitive to the prophylactic treatment of anti-TNF-α monoclonal antibody but partially susceptible to sulfasalazine, tacrolimus, and prednisolone. Oral administration of TAK-828F, at doses of 1 and 3 mg/kg, b.i.d, strongly protected the progression of colitis. TAK-828F decreased the population of Th17 and Th1/17 cells in a dose-dependent manner in the mesenteric lymph node. Moreover, expression of mRNA that are characteristic of the Th17 signature, such as IL-17A and IL-17F in the colon, were inhibited by TAK-828F, while the expression of IL-10, an anti-inflammatory cytokine, was increased. In the therapeutic treatment, TAK-828F lessened disease severity compared to the vehicle control mice. Interestingly, gene expression of zonula occludens-1 (ZO-1) and mucin 2 (Muc2), which play an important role in barrier function of the intestinal mucosa, was recovered by TAK-828F. These results indicate that blocking RORγt has promising pharmacological profile in the colitis model. RORγt blockade may provide a novel therapeutic paradigm for treatment of IBD with unique mechanism by which improves imbalance of the immune system.

Osteoclastic bone resorption enables orthodontic tooth movement (OTM) in orthodontic treatment. Previously, we demonstrated that local epigallocatechin gallate (EGCG) injection successfully slowed the rate of OTM; however, repeat injections were required. In the present study, we produced a liquid form of EGCG-modified gelatin (EGCG-GL) and examined the properties of EGCG-GL with respect to prolonging EGCG release, NF-E2-related factor 2 (Nrf2) activation, osteoclastogenesis inhibition, bone destruction, and OTM. We found EGCG-GL both prolonged the release of EGCG and induced the expression of antioxidant enzyme genes, such as heme oxygenase 1 (Hmox1) and glutamate-cysteine ligase (Gclc), in the mouse macrophage cell line, RAW264.7. EGCG-GL attenuated intracellular reactive oxygen species (ROS) levels were induced by the receptor activator of nuclear factor-kB ligand (RANKL) and inhibited RANKL-mediated osteoclastogenesis in vitro. An animal model of bone destruction, induced by repeat Lipopolysaccharide (LPS)-injections into the calvaria of male BALB/c mice, revealed that a single injection of EGCG-GL on day-1 could successfully inhibit LPS-mediated bone destruction. Additionally, experimental OTM of maxillary first molars in male mice was attenuated by a single EGCG-GL injection on day-1. In conclusion, EGCG-GL prolongs the release of EGCG and inhibits osteoclastogenesis via the attenuation of intracellular ROS signaling through the increased expression of antioxidant enzymes. These results indicate EGCG-GL would be a beneficial therapeutic approach both in destructive bone disease and in controlling alveolar bone metabolism.

Acquired open bite mainly occurs after adolescence and is caused by a pathological or biological process. We herein report the case of an 18-year and 10-month-old Japanese woman. Her chief complaint was difficulty with mastication. Only distal cusps of first molars and second molars had contact. An angle class II molar relationship and extreme mesially inclined mandibular first molars were observed. The degree of overjet was +6.1mm and overbite was -2.0mm. There were no symptoms or abnormal findings in temporomandibular joints by radiography and magnetic resonance imaging. A skeletal class II jaw relationship was indicated. Therefore, the patient was diagnosed with a skeletal class II acquired open bite malocclusion accompanied with extreme mesial inclination of mandibular first molars. Orthodontic treatment with extraction of maxillary first premolars was performed. After stabilizing the maxillary dental arch, mandibular molars were uprighted and intruded by class III elastics. After treatment, overjet was +3.0mm and overbite was +2.3mm. Occlusal cusp-fossa relationships were established and canine relationships were class I. The lower occlusal plane and mandible were rotated counterclockwise, which helped to achieve proper overbite. After 2 years of retention, occlusion was well maintained., (Copyright © 2018 CEO. Published by Elsevier Masson SAS. All rights reserved.)

Purpose: Surgical orthodontic patients with facial asymmetry frequently show asymmetry of the lips, and this is often a major complaint of patients. This study investigated whether lip asymmetry associated with the maxilla and mandible was improved when 2-jaw surgery was performed in surgical orthodontic treatment., Materials and Methods: Inclusion criteria for this retrospective cohort study were 1) an anteroposterior maxillary relation defined as skeletal Class I; 2) menton (Me) tranverse deviation greater than 5.0 mm; 3) maxillary cant greater than 3.0°; and 4) 2-jaw surgery. Primary predictor variables in this study were skeletal morphologic measurements (Me deviation, maxillary cant, and maxillary distance ratio) before and after treatment. Outcome variables were lip morphology measurements (labial commissure distance, lip angle, and lip area). Additional variables included age and gender. Vertical distances, angles, and area of the upper and lower lips were measured and compared before and after treatment. Hard tissues were measured using posteroanterior cephalograms. Paired t test and correlation coefficients were calculated., Results: Fourteen patients (4 men [28.5%] and 10 women [71.5%]; mean age, 29 yr) were included. Meaningful changes were observed in distance and angle measurements of the lips from before to after treatment. In area measurement, ratios of the area on the deviated side to that on the contralateral side for the upper and lower lips changed markedly and were close to 1.0 compared with before treatment. A relevant correlation was found between change in Me deviation and change in ratio of the height of the labial commissure., Conclusion: In cases of facial asymmetry caused by deviation of the maxilla and mandible, lip asymmetry can be adequately corrected by leveling the canted occlusal plane and positioning the Me toward the midline with 2-jaw surgery., (Copyright © 2018 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5 -/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5 -/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5 -/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5 -/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5 -/- and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5 -/- mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

C-C chemokine receptor 9 (CCR9) is the homing receptor for C-C motif chemokine ligand 25 (CCL25), and contributes to the maintenance of mucosal immunity and pathogenesis of inflammatory bowel disease (IBD) through the recruitment of T cells into the gut mucosa. Recent reports suggest that the interaction of CCR9 and CCL25 in the large intestine correlate with disease severity of colonic IBD. MLN3126 is an orally available small molecular compound with potent and selective CCR9 antagonist activity. MLN3126 inhibited CCL25-induced calcium mobilization in human CCR9 transfected cells and CCL25-induced chemotaxis of mouse primary thymocytes in a dose-dependent manner. The potential effect of MLN3126 in an activated T cell transfer mouse colitis model was compared with that of an anti-tumor necrosis factor (TNF)-α antibody. CCL25 protein was detected in the colon of mucosal epithelial cells and CCR9 + CD4 + T cells were observed in the lamina propria of the colon of mice with colitis. Dietary administration of MLN3126 to the mice maintained sufficient concentration of the compound in the plasma and dose-dependently inhibited progression of colitis compared to the vehicle control group. Anti-TNF-α antibody, a surrogate for a standard of care for IBD treatment, was also efficacious in the colitis model. These results suggest that MLN3126 would be a promising orally available CCR9 antagonist to treat colonic IBD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Photocatalysts have multiple applications in air purifiers, paints, and self-cleaning coatings for medical devices such as catheters, as well as in the elimination of xenobiotics. In this study, a coating of a UV-responsive photocatalyst, titanium dioxide (TiO₂), was applied to an orthodontic resin. The antibacterial activity on oral bacteria as well as hydrophilic properties and mechanical properties of the TiO₂-coated resin were investigated. ultraviolet A (UVA) (352 nm) light was used as the light source. Antibacterial activity was examined with or without irradiation. Measurements of early colonizers and cariogenic bacterial count, i.e., colony forming units (CFU), were performed after irradiation for different time durations. Hydrophilic properties were evaluated by water contact angle measurements. While, for the assessment of mechanical properties, flexural strength was measured by the three-point bending test. In the coat(+)light(+) samples the CFU were markedly decreased compared to the control samples. Water contact angle of the coat(+)light(+) samples was decreased after irradiation. The flexural strength of the specimen irradiated for long time showed a higher value than the required standard value, indicating that the effect of irradiation was weak. We suggest that coating with the ultraviolet responsive photocatalyst TiO₂ is useful for the development of orthodontic resin with antimicrobial properties.

A series of tetrahydronaphthyridine derivatives as novel RORγt inverse agonists were designed and synthesized. We reduced the lipophilicity of tetrahydroisoquinoline compound 1 by replacement of the trimethylsilyl group and SBDD-guided scaffold exchange, which successfully afforded compound 7 with a lower log  D value and tolerable in vitro activity. Consideration of LLE values in the subsequent optimization of the carboxylate tether led to the discovery of [ cis-3-({(5 R)-5-[(7-fluoro-1,1-dimethyl-2,3-dihydro-1 H-inden-5-yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-naphthyridin-6(5 H)-yl}carbonyl)cyclobutyl]acetic acid, TAK-828F (10), which showed potent RORγt inverse agonistic activity, excellent selectivity against other ROR isoforms and nuclear receptors, and a good pharmacokinetic profile. In animal studies, oral administration of compound 10 exhibited robust and dose-dependent inhibition of IL-17A cytokine expression in a mouse IL23-induced gene expression assay. Furthermore, development of clinical symptoms in a mouse experimental autoimmune encephalomyelitis model was significantly reduced. Compound 10 was selected as a clinical compound for the treatment of Th17-driven autoimmune diseases.

Retinoic acid-related orphan receptor γt (RORγt) is a key master regulator of the differentiation and activation of IL-17 producing CD4 + Th17, CD8 + Tc17 and IL-17/IFN-γ co-producing cells (Th1/17 cells). These cells play critical roles in the pathogenesis of autoimmune diseases such as inflammatory bowel disease and multiple sclerosis. Thus, RORγt is an attractive target for the treatment of these diseases. We discovered TAK-828F, an orally available potent and selective RORγt inverse agonist. The inhibitory effect on the activation and differentiation of Th17 cells by TAK-828F was evaluated in mouse and human primary cells. TAK-828F inhibited IL-17 production from mouse splenocytes and human peripheral blood mononuclear cells dose-dependently at concentrations of 0.01-10 μM without affecting the production of IFN-γ. Additionally, TAK-828F strongly inhibited Th17, Tc17 and Th1/17 cells' differentiation from naive T cells and memory CD4 + T cells at 100 nM without affecting Th1 cells' differentiation. In addition, TAK-828F improved Th17/Treg cells' population ratio by inhibiting Th17 cells' differentiation and up-regulating Treg cells. Furthermore, TAK-828F, at 100 nM, reduced the production of Th17-related cytokines (IL-17, IL-17F and IL-22) without affecting IFN-γ production in whole blood. These results demonstrate that TAK-828F has the potent and selective inhibitory activity against RORγt both in mouse and human cells. Additionally, oral administration of TAK-828F showed promising efficacy in naive T cell transfer mouse colitis model. TAK-828F may provide a novel therapeutic option to treat immune diseases by inhibiting Th17 and Th1/17 cells' differentiation and improving imbalance between Th17 and Treg cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

MALT lymphoma-translocation protein 1 (Malt1) protease activity is triggered by stimulation of various immune receptors. Activation of Malt1 protease induces cleavage of negative regulators for immune responses, resulting in lymphocytes activation. Although Malt1 protease mediates the signaling process downstream of the T cell, B cell, and dectin receptors, its contribution in Fcγ receptor (FcγR) signaling has not been elucidated. In this study, we investigated the role of Malt1 protease activity in FcγR signaling using Malt1 protease-deficient (PD) mouse. In addition, role of Malt1 protease for the development of FcγR-mediated autoimmune disease was also investigated in vivo. Malt1 protease cleaves their substrates, such as RelB and cylindromatosis (CYLD). However, the Malt1 proteolytic activity was silenced in the Malt1 PD mice. Production of inflammatory cytokines via FcγR stimulation was decreased on dendritic cells prepared from Malt1 PD mice. In FcγR-dependent murine immune thrombocytopenia (ITP) model, gene expressions of the inflammatory cytokines in the spleen of Malt1 PD mice were lower than those of WT mice. Then, Malt1 PD mice protected the development of thrombocytopenia. These results clearly figured out that Malt1 protease activity plays an important role in the activation of innate immune cells via FcγR, and the development of FcγR-mediated autoimmune diseases. Therefore, Malt1 is an attractive target for the treatment of inflammatory diseases mediated by FcγR., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bone destructive diseases are common worldwide and are caused by dysregulation of osteoclast formation and activation. During osteoclastogenesis, reactive oxygen species (ROS) play a role in the intracellular signalling triggered by receptor activator of nuclear factor-κB ligand (RANKL) stimulation. Previously, we demonstrated that induction of antioxidant enzymes by Nrf2 activation using Nrf2-gene transfer, an ETGE-peptide or polyphenols, successfully ameliorated RANKL-dependent osteoclastogenesis. Dimethyl fumarate (DMF) has been shown to activate Nrf2 signalling and has been lately used in clinical trials for neurodegenerative diseases. In this study, we hypothesized that Nrf2 activation by DMF would inhibit osteoclastogenesis and bone destruction via attenuation of intracellular ROS signalling through antioxidant mechanisms. RAW 264.7 cells were used as osteoclast progenitor cells. We found that DMF induced Nrf2 translocation to the nucleus, augmented Nrf2 promoter-luciferase reporter activity and increased antioxidant enzyme expression. Using flow cytometry, we found that DMF attenuated RANKL-mediated intracellular ROS generation, which resulted in the inhibition of RANKL-mediated osteoclastogenesis. Local DMF injection into the calvaria of male BALB/c mice resulted in attenuated bone destruction in lipopolysaccharide-treated mice. In conclusion, we demonstrated in a preclinical setting that DMF inhibited RANKL-mediated osteoclastogenesis and bone destruction via induction of Nrf2-mediated transcription of antioxidant genes and consequent decrease in intracellular ROS levels. Our results suggest that DMF may be a promising inhibitor of bone destruction in diseases like periodontitis, rheumatoid arthritis and osteoporosis., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Introduction: The purpose of this study was to investigate whether a local unilateral IGF-1 injection into the mandibular condylar cavity can induce unilateral endochondral mandibular growth without any systemic adverse effects., Methods: Seventy-five 3-week-old male Jcl:ICR mice were used in this study. The mice were divided into 2 groups: control group (n = 22) and IGF-1 group (n = 53). In the IGF-1 group, human IGF-1 was injected into the right mandibular condylar cavity, and phosphate-buffered saline solution was injected into the left cavity, 3 times per week for 10 weeks., Results: There was no significant difference in body weight, serum human IGF-1 concentration, and soft tissue thickness of the cheeks including the masseter muscles between the 2 groups. Unilateral IGF-1 injection induced a lateral shift of the mandible to the contralateral side, and microcomputed tomogtraphy analysis showed that unilateral IGF-1 injection induced endochondral growth in the condyle. Col2, Ihh, and Runx2 were extensively upregulated by the local unilateral IGF-1 injection in real-time reverse transcription polymerase chain reaction analysis. Proliferation marker KI67, IGF-1 signaling molecule AKT1, and chondrogenic differentiation marker Col2 were strongly expressed in the IGF-1 injected condyle by immunohistochemistry. Vital labeling showed that the distance between the labels was increased in the IGF-1 injection group compared with that of the control group., Conclusions: The results verified in this study indicated that local unilateral IGF-1 injection into the mandibular condylar cavity successfully induced unilateral endochondral mandibular growth in mice without any systemic adverse effects. Thus, local unilateral IGF-1 injection into the mandibular condylar cavity could be a useful alternative for mandibular asymmetry therapy during the growth period. However, additional experimental and clinical studies will be necessary to prove the real effect of this new therapy., (Copyright © 2017 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of the present study was to examine the effect of extracellular heat shock protein (HSP) 72 on human periodontal ligament fibroblast cells (hPDLFs) in vitro., Design: hPDLFs were stimulated by recombinant human HSP72 (rhHSP72). TAK-242 was used to inhibit toll-like receptor 4 (TLR4) activity. Interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α mRNA levels were analyzed by real-time PCR and protein levels were analyzed by enzyme-linked immunosorbent assay. p65/RelA phosphorylation was analyzed by western blot., Results: IL-6, IL-8 and TNF-α mRNA and protein levels were significantly increased by rhHSP72 stimulation. These effects were inhibited by TAK-242 treatment. Additionally, p65/RelA phosphorylation was increased after 5-min rhHSP72 stimulation, which was inhibited by TAK-242 treatment., Conclusion: Extracellular HSP72 induces proinflammatory cytokines through TLR4/NF-κB in hPDLFs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Teeth develop through interactions between epithelial and mesenchymal tissues mediated by a signaling network comprised of growth factors and transcription factors. However, little is known about how epigenetic modifiers affect signaling pathways and thereby regulate tooth formation. We previously reported that the histone 3 lysine 9 (H3K9) methyltransferase (MTase) G9a is specifically enriched in the tooth mesenchyme during mouse development. In this study, we investigated the functions of G9a in tooth development using G9a conditional knockout (KO) mice. We used Sox9-Cre mice to delete G9a in the tooth mesenchyme because Sox9 is highly expressed in the mesenchyme derived from the cranial neural crest. Immunohistochemical analyses revealed that G9a expression was significantly decreased in the mesenchyme of Sox9-Cre;G9afl/fl (G9a cKO) mice compared with that in Sox9-Cre;G9a fl/+(control) mice. Protein levels of the G9a substrate H3K9me2 were also decreased in the tooth mesenchyme. G9a cKO mice showed smaller tooth germ after embryonic day (E) 16.5 and E17.5, but not at E15.5. The developing cusp tips, which were visible in control mice, were absent in G9a cKO mice at E17.5. At 3 weeks after birth, small first molars with smaller cusps and unseparated roots were formed. Organ culture of tooth germs derived from E15.5 cKO mouse embryos showed impaired tooth development, suggesting that tooth development per se is affected independently of skull development. BrdU labeling experiments revealed that the proliferation rates were decreased in the mesenchyme in G9a cKO mice at E17.5. In addition, the proliferation rates in the tooth inner enamel epithelium were also decreased. In situ hybridization revealed altered localization of genes associated with tooth development. In cKO mice, intensively localized expression of mRNAs encoding bone morphogenic protein (Bmp2 and Bmp4) was observed in the tooth mesenchyme at E17.5, similar to the expression patterns observed in control mice at E15.5. Localization of Shh and related signaling components, including Gli1, Ptch1, and Ptch2, in the tooth mesenchyme of cKO mice was generally similar to that at earlier stages in control mice. In addition, expression of Fgf3 and Fgf10 in the mesenchyme was decreased in G9a cKO mice at P0. Expression levels of Fgf9 and p21, both of which were expressed in the secondary enamel-knot, were also decreased. Thus, the expression of genes associated with tooth development was delayed in cKO mice. Our results suggest that H3K9MTase G9a regulates cell proliferation and timing of differentiation and that G9a expression in the tooth mesenchyme is required for proper tooth development., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Polyetheretherketone (PEEK) is now attracting attention as an alternative to metal alloys in the dental field. In the present study, we evaluated the load-deflection characteristics of PEEK wires in addition to their frictional properties. Three types of PEEK wires are used: two sizes of rectangular shape, 0.016 × 0.022 in² and 0.019 × 0.025 in² (19-25PEEK), and rounded shape, diameter 0.016 in (16PEEK). As a control, Ni-Ti orthodontic wire, diameter 0.016 in, was used. The three-point bending properties were evaluated in a modified three-point bending system for orthodontics. The static friction between the orthodontic wire and the bracket was also measured. The load-deflection curves were similar among Ni-Ti and PEEK wires, except for 16PEEK with slot-lid ligation. The bending force of 19-25PEEK wire was comparable with that of Ni-Ti wire. 19-25PEEK showed the highest load at the deflection of 1500 μm ( p < 0.05) in the case of slot-lid ligation. No significant differences were seen in the permanent deformation between Ni-Ti and all three PEEK wires ( p > 0.05). No significant difference was seen in static friction between all three PEEK wires and Ni-Ti wire ( p > 0.05). It is suggested that 19-25PEEK will be applicable for orthodontic treatment with the use of slot-lid ligation., Competing Interests: The authors declare no conflict of interest.

Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC. Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining. Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age. In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time.

Periodontitis, an inflammatory disease that affects the tissues surrounding the teeth, is a common disease worldwide. It is caused by a dysregulation of the host inflammatory response to bacterial infection, which leads to soft and hard tissue destruction. In particular, it is the excessive inflammation in response to bacterial plaque that leads to the release of reactive oxygen species (ROS) from neutrophils, which, then play a critical role in the destruction of periodontal tissue. Generally, ROS produced from immune cells exhibit an anti-bacterial effect and play a role in host defense and immune regulation. Excessive ROS, however, can exert cytotoxic effects, cause oxidative damage to proteins, and DNA, can interfere with cell growth and cell cycle progression, and induce apoptosis of gingival fibroblasts. Collectively, these effects enable ROS to directly induce periodontal tissue damage. Some ROS also act as intracellular signaling molecules during osteoclastogenesis, and can thus also play an indirect role in bone destruction. Cells have several protective mechanisms to manage such oxidative stress, most of which involve production of cytoprotective enzymes that scavenge ROS. These enzymes are transcriptionally regulated via NRF2, Sirtuin, and FOXO. Some reports indicate an association between periodontitis and these cytoprotective enzymes' regulatory axes, with superoxide dismutase (SOD) the most extensively investigated. In this review article, we discuss the role of oxidative stress in the tissue destruction manifest in periodontitis, and the mechanisms that protect against this oxidative stress.

During orthodontic tooth movement, the periodontal ligament (PDL) is exposed to continuous mechanical strain. However, many researchers have applied cyclic tensile strain, not continuous tensile strain, to PDL cells in vitro because there has been no adequate device to apply continuous tensile strain to cultured cells. In this study, we contrived a novel device designed to apply continuous tensile strain to cells in culture. The continuous tensile strain was applied to human immortalized periodontal ligament cell line (HPL cells) and the cytoskeletal structures of HPL cells were examined by immunohistochemistry. The expression of both inflammatory and osteogenic markers was also examined by real-time reverse transcription polymerase chain reaction. The osteogenic protein, Osteopontin (OPN), was also detected by western blot analysis. The actin filaments of HPL cells showed uniform arrangement under continuous tensile strain. The continuous tensile strain increased the expression of inflammatory genes such as IL-1β , IL-6 , COX-2 and TNF-α , and osteogenic genes such as RUNX2 and OPN in HPL cells. It also elevated the expression of OPN protein in HPL cells. These results suggest that our new simple device is useful for exploring the responses to continuous tensile strain applied to the cells., (© 2017. Published by The Company of Biologists Ltd.)

This case report describes the importance of combining morphological and functional examination with psychological examination in the establishment ofstable mandibular position in the treatment of maxillary protrusion with unstable mandibular position accompanied by unidentified complaints, which ensures safe orthodontic treatment.

Reactive oxygen species (ROS) play a role in intracellular signaling during osteoclastogenesis. We previously reported that transcriptional factor nuclear factor E2-related factor 2 (Nrf2) was exported from the nucleus to the cytoplasm by receptor activator of nuclear factor-κB ligand (RANKL), and that Nrf2 negatively regulated osteoclastogenesis via antioxidant enzyme up-regulation. Knockout mice of BTB and CNC homology 1 (Bach1)-the competitor for Nrf2 in transcriptional regulation-was known to attenuate RANKL-mediated osteoclastogenesis, although the mechanism remains unclear. Therefore, we hypothesized that RANKL could be involved in the nuclear translocation of Bach1, which would attenuate Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular ROS signaling in osteoclasts. RANKL induced Bach1 nuclear import and Nrf2 nuclear export. Induction of Bach1 nuclear export increased Nrf2 nuclear import, augmented antioxidant enzyme expression, and, thus, diminished RANKL-mediated osteoclastogenesis via attenuated intracellular ROS signaling. Finally, an in vivo mouse bone destruction model clearly demonstrated that induction of Bach1 nuclear export inhibited bone destruction. In this study, we report that RANKL favors osteoclastogenesis via attenuation of Nrf2-mediated antioxidant enzyme expression by competing with Bach1 nuclear accumulation. Of importance, induction of Bach1 nuclear export activates Nrf2-dependent antioxidant enzyme expression, thereby attenuating osteoclastogenesis. Bach1 nuclear export might be a therapeutic target for such bone destructive diseases as rheumatoid arthritis, osteoporosis, and periodontitis.-Kanzaki, H., Shinohara, F., Itohiya, K., Yamaguchi, Y., Katsumata, Y., Matsuzawa, M., Fukaya, S., Miyamoto, Y., Wada, S., Nakamura, Y. RANKL induces Bach1 nuclear import and attenuates Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular reactive oxygen species signaling and osteoclastogenesis in mice., (© FASEB.)

When considering camouflage orthodontic treatment of a malocclusion associated with significant facial asymmetry, it is important to define the location of the dental midline. The patient, a 19-year-old Japanese woman, had an anterior open bite and a dental midline discrepancy associated with facial asymmetry. A nonsurgical treatment plan was considered. The main treatment objective was to correct the anterior open bite and the dental midlines in both arches. The dental midline discrepancy was eliminated, and proper overjet and overbite were achieved. Although the facial asymmetry remained, oral esthetics dramatically improved and a favorable occlusion was obtained. The results suggest that appropriately defining the location of the dental midline is critical for successful camouflage treatment of facial asymmetry., (Copyright © 2016 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

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

It has been reported that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, take part in osteoclast differentiation as intra-cellular signaling molecules. The current assumed signaling cascade from RANK to ROS production is RANK, TRAF6, Rac1, and then Nox. The target molecules of ROS in RANKL signaling remain unclear; however, several reports support the theory that NF-κB signaling could be the crucial downstream signaling molecule of RANKL-mediated ROS signaling. Furthermore, ROS exert cytotoxic effects such as peroxidation of lipids and phospholipids and oxidative damage to proteins and DNA. Therefore, cells have several protective mechanisms against oxidative stressors that mainly induce cytoprotective enzymes and ROS scavenging. Three well-known mechanisms regulate cytoprotective enzymes including Nrf2-, FOXO-, and sirtuin-dependent mechanisms. Several reports have indicated a crosslink between FOXO- and sirtuin-dependent regulatory mechanisms. The agonists against the regulatory mechanisms are reported to induce these cytoprotective enzymes successfully. Some of them inhibit osteoclast differentiation and bone destruction via attenuation of intracellular ROS signaling. In this review article, we discuss the above topics and summarize the current information available on the relationship between cytoprotective enzymes and osteoclastogenesis., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)