Leucyl-tRNA Synthetase Contributes to Muscle Weakness through Mammalian Target of Rapamycin Complex 1 Activation and Autophagy Suppression in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by loss-of-function mutations in the dystrophin gene, results in progressive muscle weakness and early fatality. Impaired autophagy is one of the cellular hallmarks of DMD, contributing to the disease progression. Molecular mechanisms underlying the inhibition of autophagy in DMD are not well understood. In the current study, the DMD mouse model mdx was used for the investigation of signaling pathways leading to suppression of autophagy. Mammalian target of rapamycin complex 1 (mTORC1) was hyperactive in the DMD muscles, accompanying muscle weakness and autophagy impairment. Surprisingly, Akt, a well-known upstream regulator of mTORC1, was not responsible for mTORC1 activation or the dystrophic muscle phenotypes. Instead, leucyl-tRNA synthetase (LeuRS) was overexpressed in mdx muscles compared with the wild type. LeuRS activates mTORC1 in a noncanonical mechanism that involves interaction with RagD, an activator of mTORC1. Disrupting LeuRS interaction with RagD by the small-molecule inhibitor BC-LI-0186 reduced mTORC1 activity, restored autophagy, and ameliorated myofiber damage in the mdx muscles. Furthermore, inhibition of LeuRS by BC-LI-0186 improved dystrophic muscle strength in an autophagy-dependent manner. Taken together, our findings uncovered a noncanonical function of the housekeeping protein LeuRS as a potential therapeutic target in the treatment of DMD.

Compliant 3D frameworks instrumented with strain sensors for characterization of millimeter-scale engineered muscle tissues.

Tissue-on-chip systems represent promising platforms for monitoring and controlling tissue functions in vitro for various purposes in biomedical research. The two-dimensional (2D) layouts of these constructs constrain the types of interactions that can be studied and limit their relevance to three-dimensional (3D) tissues. The development of 3D electronic scaffolds and microphysiological devices with geometries and functions tailored to realistic 3D tissues has the potential to create important possibilities in advanced sensing and control. This study presents classes of compliant 3D frameworks that incorporate microscale strain sensors for high-sensitivity measurements of contractile forces of engineered optogenetic muscle tissue rings, supported by quantitative simulations. Compared with traditional approaches based on optical microscopy, these 3D mechanical frameworks and sensing systems can measure not only motions but also contractile forces with high accuracy and high temporal resolution. Results of active tension force measurements of engineered muscle rings under different stimulation conditions in long-term monitoring settings for over 5 wk and in response to various chemical and drug doses demonstrate the utility of such platforms in sensing and modulation of muscle and other tissues. Possibilities for applications range from drug screening and disease modeling to biohybrid robotic engineering.

Why locking plates for the proximal humerus do not fit well.

INTRODUCTION: We compared the angle of the humerus and plate and to assess compatibility of a plate to the proximal humerus using three-dimensional (3D) printed models. MATERIALS AND METHODS: A total of 120 cases were included, who underwent anteroposterior shoulder radiographs. From these, 30 cases with 3D shoulder computed tomography scans were randomly selected to print 3D model. The lateral angle between the lateral cortex of the humeral shaft and lateral border of the greater tuberosity (GT), neck-shaft angle, and height from the most proximal point of the GT to the angular point were measured. When the plates were applied on the 3D models, the gap from the most proximal point of the GT to the proximal rim of the plate was measured. RESULTS: The mean lateral angle in plain radiographs was 12.9 ± 2.2° and height from the most proximal point of the GT to the angular point was 44.4 ± 4.7 mm. The bending angles of the three plates were 8° and 10°. Height from the proximal rim of the plate to the bending point was 42.4, 42.0 and 43.8 mm. In 98% of cases, the lateral angle of the humerus was larger than all three plates. In 43% of cases, height of the GT was smaller than height of plates. When plates were applied to the 3D model, the mean gap from GT to plate was 4.8 ± 2.8 mm. CONCLUSIONS: There was large variation in the lateral angle of the proximal humerus, which was not correlated with the neck-shaft angle. The lateral angle of the humerus was larger than the plates and prone to varus reduction and medial collapse. LEVEL OF EVIDENCE OR CLINICAL RELEVANCE: Basic science study.

Deacetylasperulosidic Acid Ameliorates Pruritus, Immune Imbalance, and Skin Barrier Dysfunction in 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis NC/Nga Mice.

The prevalence of atopic dermatitis (AD), a disease characterized by severe pruritus, immune imbalance, and skin barrier dysfunction, is rapidly increasing worldwide. Deacetylasperulosidic acid (DAA) has anti-atopic activity in the three main cell types associated with AD: keratinocytes, mast cells, and eosinophils. Our study investigated the anti-atopic activity of DAA in 2,4-dinitrochlorobenzene-induced NC/Nga mice. DAA alleviated the symptoms of AD, including infiltration of inflammatory cells (mast cells and eosinophils), epidermal thickness, ear thickness, and scratching behavior. Furthermore, DAA reduced serum IgE, histamine, and IgG1/IgG2a ratio and modulated the levels of AD-related cytokines and chemokines, namely interleukin (IL)-1ß, IL-4, IL-6, IL-9, IL-10, IL-12, tumor necrosis factor-α, interferon-γ, thymic stromal lymphopoietin, thymus and activation-regulated chemokine, macrophage-derived chemokine, and regulated on activation the normal T cell expressed and secreted in the serum. DAA restored immune balance by regulating gene expression and secretion of Th1-, Th2-, Th9-, Th17-, and Th22-mediated inflammatory factors in the dorsal skin and splenocytes and restored skin barrier function by increasing the expression of the pro-filaggrin gene and barrier-related proteins filaggrin, involucrin, and loricrin. These results suggest DAA as a potential therapeutic agent that can alleviate the symptoms of AD by reducing pruritus, modulating immune imbalance, and restoring skin barrier function.

Prognosis of teeth in mandibular fracture lines.

BACKGROUND/AIMS: Teeth in a jaw fracture line, because of the presence of the periodontal ligament, may communicate with the oral cavity. There are no guidelines for the management of teeth in mandibular fracture lines. The aim of this study was to investigate the factors related to dental problems with teeth involved in mandibular fracture lines and to determine the best treatment option. MATERIAL AND METHODS: This retrospective study was based on the medical and radiographic records of patients with mandibular fractures. The relationships among the patient's age, gender, smoking history, amount of bony displacement, surgery, trauma-surgery period, apical involvement, tooth mobility, and periodontal status were investigated. Group comparisons were performed using the chi-squared test, Fisher's exact test, and Mann-Whitney U-test. RESULT: A total of 238 patients (247 fracture lines) with mandibular fractures including a tooth in the line of the fracture were examined. Post-operative dental complications occurred in 42 cases (17.0%). Extraction of related teeth occurred in 34 cases (80.9%) compared to eight cases (19.0%) related to root canal therapy. This study defined "dental problem" as "a case with a tooth extracted or endodontically treated after trauma." The variables associated with an increased risk of dental problems were the amount of bony displacement (p < .01), tooth mobility (p < .01), and pre-existing marginal alveolar bone loss (p = .027). CONCLUSION: The prognosis of teeth in mandibular fracture lines was related to tooth mobility, periodontal state, and the amount of bony displacement.

Connective tissue growth factor (CTGF) regulates the fusion of osteoclast precursors by inhibiting Bcl6 in periodontitis.

Connective tissue growth factor (CTGF), an extracellular matrix protein with various biological functions, is known to be upregulated in multiple chronic diseases such as liver fibrosis and congestive heart failure, but the mechanism it undertakes to cause alveolar bone loss in periodontitis remains elusive. The present study therefore investigates the pathways involving CTGF in chronic periodontitis. RNA sequencing revealed a notable increase in the expression of CTGF in chronic periodontitis tissues. Also, TRAP staining, TRAP activity and bone resorption assays showed that osteoclast formation and function is significantly facilitated in CTGF-treated bone marrow-derived macrophages (BMMs). Interestingly, western blotting and immunofluorescence staining results displayed that CTGF had little effect on the osteoclastogenic differentiation mediated by the positive regulators of osteoclastogenesis such as nuclear factor of activated T cells 1 (NFATc1). However, following results showed that both the mRNA and protein expressions of B cell lymphoma 6 (Bcl6), a transcriptional repressor of "osteoclastic" genes, were significantly downregulated by CTGF treatment. Moreover, CTGF upregulated the expressions of v-ATPase V0 subunit d2 (ATP6v0d2) and Dendritic cell-specific transmembrane protein (DC-STAMP) which are osteoclastic genes specifically required for osteoclast cell-cell fusion in pre-osteoclasts. Findings from this study suggest that CTGF promotes the fusion of pre-osteoclasts by downregulating Bcl6 and subsequently increasing the expression of DC-STAMP in periodontitis. Understanding this novel mechanism that leads to increased osteoclastogenesis in periodontitis may be employed for the development of new therapeutic targets for preventing periodontitis-associated alveolar bone resorption.

Letrozole Suppresses the Fusion of Osteoclast Precursors through Inhibition of p38-Mediated DC-STAMP Pathway.

Letrozole is a reversible nonsteroidal aromatase inhibitor that is widely used in postmenopausal breast cancer patients. It is well established that letrozole decreases bone density owing to estrogen depletion; however, few studies have reported its direct effect on bone cells in vitro. Therefore, we investigated the effect of letrozole on bone metabolism, focusing on osteoclastogenesis. Letrozole did not affect the viability, proliferation, or migration of bone marrow-derived macrophages (BMMs); however, it reduced the multinucleation of immature osteoclasts and subsequent bone resorption in vitro. Overall, letrozole inhibited the expression of dendritic cell-specific transmembrane protein (DC-STAMP), tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K. Among them, the reduced expression of DC-STAMP was the most prominent. However, this downregulation of DC-STAMP expression following letrozole treatment was not related to the inhibition of major osteoclastogenesis pathways, such as the nuclear factor-κB (NF-κB), c-Fos, and nuclear factor of activated T cell c1 (NFATc1) pathways, but was attributed to the inhibition of p38, which is known to reside upstream of DC-STAMP expression. Notably, the anti-osteoclastogenic effect of letrozole was abolished following treatment with the p38 activator anisomycin. Contrary to our expectations, these results strongly suggest a previously unknown anti-osteoclastogenic activity of letrozole, mediated by the downregulation of the p38/DC-STAMP pathway.

Oral Administration of Porphyromonas gingivalis, a Major Pathogen of Chronic Periodontitis, Promotes Resistance to Paclitaxel in Mouse Xenografts of Oral Squamous Cell Carcinoma.

Chemotherapy is not a first-line therapy for oral squamous cell carcinoma (OSCC), which is the most common type of oral cancer, because most OSCC shows resistance to chemotherapeutic reagents. Inflammatory signals are suggested to be associated with chemoresistance as well as carcinogenesis in many different cancers, and thus chronic periodontitis, the most common chronic inflammatory disease of the oral cavity, could modulate responsiveness to chemotherapeutic agents used against oral cancer. This study was performed to define the role of chronic periodontitis in oral cancer progression and to determine the responsiveness of oral cancer to a chemotherapeutic reagent. First, we quantified the tumor growth rate and changes in serum cytokine profiles of mice administered Porphyromonas gingivalis, a major pathogen of chronic periodontitis. Compared with uninfected mice, the mice that were chronically administered P. gingivalis showed increased resistance to paclitaxel and a decreased tumor growth rate. In addition, P. gingivalis-treated mice exhibited higher serum levels of interleukin-6 (IL-6) than uninfected mice. Furthermore, the sensitivity of tumor xenografts to paclitaxel in mice administered P. gingivalis was dramatically increased when the mice were administered ibuprofen, an anti-inflammatory drug which supports the modulatory effect of periodontal pathogen-induced inflammation in chemoresistance.

Serum Levels of Interleukin-6 and Titers of Antibodies Against Porphyromonas gingivalis Could Be Potential Biomarkers for the Diagnosis of Oral Squamous Cell Carcinoma.

It has been suggested that Porphyromonas gingivalis (P. gingivalis), a keystone pathogen in chronic periodontitis, is associated with a variety of cancers, including oral cancer. Recently, studies have shown the effects of persistent exposure to P. gingivalis on the promotion of tumorigenic properties of oral epithelial cells, suggesting that chronic P. gingivalis infection is a potential risk factor for oral cancer. On the other hand, Fusobacterium nucleatum (F. nucleatum), one of the major periodontal pathogens, has emerged as an important factor in the colon cancer progression. Here, we investigated the diagnostic potential of serum immunoglobulin G antibody against periodontal pathogens, P. gingivalis and F. nucleatum, and serum IL-6 for oral squamous cell carcinoma (OSCC). An enzyme-linked immunosorbent assay (ELISA) was used to determine and compare the serum levels of interleukin 6 (IL-6), F. nucleatum IgG, and P. gingivalis IgG in 62 OSCC patients with 46 healthy controls. The serum levels of P. gingivalis IgG and IL-6 were higher in OSCC patients than in non-OSCC controls, and the difference was statistically significant. In addition, a high serum level of IL-6 was associated with a worse prognosis in OSCC patients. Thus, P. gingivalis IgG and IL-6 could be utilized as potential serum biomarkers for the diagnosis of OSCC, and the serum level of IL-6 contributes to improved prognostic performance.

A novel combination treatment to stimulate bone healing and regeneration under hypoxic conditions: photobiomodulation and melatonin.

Melatonin has anabolic effects on the bone, even under hypoxia, and laser irradiation has been shown to improve osteoblastic differentiation. The aim of this study was to investigate whether laser irradiation and melatonin would have synergistic effects on osteoblastic differentiation and mineralization under hypoxic conditions. MC3T3-E1 cells were exposed to 1% oxygen tension for the hypoxia condition. The cells were divided into four groups: G1-osteoblast differentiation medium only (as the hypoxic condition), G2-treatment with 50 µM melatonin only, G3-laser irradiation (808 nm, 80 mW, GaAlAs diode) only, and G4-treatment with 50 µM melatonin and laser irradiation (808 nm, 80 mW, GaAlAs diode). Immunoblotting showed that osterix expression was markedly increased in the melatonin-treated and laser-irradiated cells at 48 and 72 h. In addition, alkaline phosphatase activity significantly increased and continued to rise throughout the experiment. Alizarin Red staining showed markedly increased mineralized nodules as compared with only melatonin-treated or laser-irradiated cells at day 7, which significantly increased by day 14. Moreover, when melatonin-treated cells were laser-irradiated, the differentiation and mineralization of cells were found to involve p38 MAPK and PRKD1 signaling mechanisms. However, the enhanced effects of laser irradiation with melatonin were markedly inhibited when the cells were treated with luzindole, a selective melatonin receptor antagonist. Therefore, we concluded that laser irradiation could promote the effect of melatonin on the differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions, and that this process is mediated through melatonin 1/2 receptors and PKRD/p38 signaling pathways.

Suppression of Osteoclastogenesis by Melatonin: A Melatonin Receptor-Independent Action.

In vertebrates, melatonin is primarily secreted from the pineal gland but it affects various biological processes including the sleep-wake cycle, vasomotor control, immune system and bone homeostasis. Melatonin has been known to promote osteoblast differentiation and bone maturation, but a direct role of melatonin on osteoclast differentiation is still elusive. The present study investigated the effect of melatonin on the differentiation of macrophages to osteoclasts. The presence of melatonin significantly reduced receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and the siRNA-mediated knockdown of the melatonin receptor failed to overcome the anti-osteoclastogenic effect of melatonin. Although melatonin treatment did not affect the phosphorylation of extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), it markedly inhibited the activation of NF-κB and subsequent induction of nuclear factor of activated T cell cytoplasmic 1(NFATc1). Thus, our results suggest that melatonin could suppress osteoclast differentiation through downregulation of NF-κB pathway with concomitant decrease in the NFATc1 transcription factor induction. Furthermore, melatonin seems to have an anti-osteoclastogenic effect independent of plasma membrane melatonin receptors. In addition to previously reported properties of melatonin, our study proposes another aspect of melatonin and bone homeostasis.

Effects of Remifentanil Preconditioning on Osteoblasts under Hypoxia-Reoxygenation Condition.

BACKGROUND: Ischemia-reperfusion of bone occurs in a variety of clinical conditions, such as orthopedic arthroplasty, plastic gnathoplasty, spinal surgery, and amputation. Usually, cellular models of hypoxia-reoxygenation reflect in vivo models of ischemia-reperfusion. With respect to hypoxia-reoxygenation conditions, the effects of remifentanil on osteogenesis have received little attention. Therefore, we investigated the effects of remifentanil on the proliferation and differentiation of osteoblasts during hypoxic-reoxygenation. METHODS: After remifentanil (0.1, 1 ng/mL) preconditioning for 2 hours, human osteoblasts were cultured under 1% oxygen tension for 24 hours. Thereafter, the cells were reoxygenated for 12 hours at 37 °C. The naloxone groups were treated with naloxone for 30 minutes before remifentanil treatment. We measured cell viability via MTT assay. Osteoblast maturation was determined by assay of bone nodular mineralization. Quantitative PCR and western blot methods were used to determine BMP-2, osteocalcin, Akt, type I collagen, osterix, TGF-ß1, HIF-1α, and RUNX2 expression levels. RESULTS: Osteoblast viability and bone nodular mineralization by osteoblasts is recovered by remifentanil preconditioning from hypoxia-reoxygenation insult. During hypoxic-reoxygenation condition, remifentanil preconditioning induced the expression of BMP-2, osteocalcin, Akt, type I collagen, osterix, TGF-ß1, HIF-1α, and RUNX2 in osteoblasts. CONCLUSIONS: Under hypoxia-reoxygenation conditions, remifentanil preconditioning enhanced the cell viability and maturation of osteoblasts, and stimulated the expression of proteins associated with osteoblast proliferation and differentiation of the osteoblast. Our results suggest that remifentanil may help in the treatment of bone stress injuries.

Effect of low-level laser therapy on oral keratinocytes exposed to bisphosphonate.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a side effect of bisphosphonate therapy. However, its pathophysiology is not yet fully elucidated, and effective treatment of BRONJ remains unclear. The aim of this study is to investigate the effects of alendronate on oral keratinocytes and of low-level laser therapy (LLLT) on alendronate-treated keratinocytes, specifically by evaluating their viability, apoptosis, and wound healing function after irradiation. Oral keratinocyte cells (HaCaT) were exposed to 25 µM alendronate. Then, laser irradiation was performed with a low-level Ga-Al-As laser (λ = 808 ± 3 nm, 80 mW, and 80 mA; NDLux, Seoul, Korea) using 1.2 J/cm(2) energy dose. Viability was analyzed using MTT assay. Apoptosis was measured by Hoechst staining, caspase assay. Changes in secretion of IL-8, VEGF, and collagen type I were studied by ELISA and immunofluorescence microscopy. Scratch wound assays were also performed to measure cellular migration. Our results show that alendronate inhibits keratinocyte viability, expression of IL-8, VEGF, and collagen type I which are intimately related to healing events and cell migration while promoting apoptosis. Our results serve to demonstrate the utility of LLLT in partially overcoming the inhibitory effects of this bisphosphonate. From these results, the authors believe that the present study will provide an experimental basis for a fuller explanation of the clinical effects of LLLT as a BRONJ treatment modality.

Melatonin promotes osteoblast differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions through activation of PKD/p38 pathways.

Osteoblastic differentiation and bone-forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3-E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time- and concentration-dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3-E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3-E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways.

Inspiring a convergent engineering approach to measure and model the tissue microenvironment.

Understanding the molecular and physical complexity of the tissue microenvironment (TiME) in the context of its spatiotemporal organization has remained an enduring challenge. Recent advances in engineering and data science are now promising the ability to study the structure, functions, and dynamics of the TiME in unprecedented detail; however, many advances still occur in silos that rarely integrate information to study the TiME in its full detail. This review provides an integrative overview of the engineering principles underlying chemical, optical, electrical, mechanical, and computational science to probe, sense, model, and fabricate the TiME. In individual sections, we first summarize the underlying principles, capabilities, and scope of emerging technologies, the breakthrough discoveries enabled by each technology and recent, promising innovations. We provide perspectives on the potential of these advances in answering critical questions about the TiME and its role in various disease and developmental processes. Finally, we present an integrative view that appreciates the major scientific and educational aspects in the study of the TiME.

Deposition of metal nanoparticles on phospholipid multilayer membranes modified by gramicidin.

A planar dipalmitoyl phosphatidylcholine (DPPC) multilayer phospholipid membrane was structurally modified by introducing a transmembrane protein, gramicidin (up to 25 mol %), to study its effect on the metal nanoparticles deposited on the membrane. Without gramicidin, when 3-nm-thick Ag, Sn, Al, and Au were deposited, the nanoparticles hardly nucleated on the DPPC membrane in rigid gel state (except for Au); however, the gramicidin addition dramatically enhanced the DPPC membrane surface's affinity for metal atoms so that a dense array of metal (Ag, Sn, and Au) or metal-oxide (Al-oxide) nanoparticles was produced on the membrane surface. The particle sizes ranged from 3 to 15 nm depending on the metal and gramicidin concentration, whereas the particle density was strongly dictated by the gramicidin concentration. The proposed method provides a convenient, generally applicable synthesis route for preparing different metal or metal-oxide nanoparticles on a relatively robust biocompatible membrane.

Coalescence and polygonization of Au nanoparticles embedded in liquid-crystalline lipid membrane.

Coarsening behavior of Au nanoparticles (AuNPs) embedded in a liquid crystalline lipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) membrane was investigated by heat treating the AuNP-embedded DOTAP membrane at 80 degrees C with 15% and 80% relative humidity (RH). The coarsening rate was (D) to approximately t0.6 regardless of the humidity; however, the spatial distribution and the coarsening mechanism differed depending on the humidity. In addition, extended treatment at 15% RH resulted in formation of large polygonized AuNPs from the lipid segregation.

Maxillary yaw as the primary predictor of maxillary dental midline deviation: 3D analysis using cone-beam computed tomography.

PURPOSE: The purpose of this study was to investigate which factors affect upper dental midline deviation in dentofacial deformity patients using cone-beam computed tomography analysis. PATIENTS AND METHODS: Twenty-eight patients were selected for this study. Subjects were divided into 2 groups according to the amount of upper incisor (U1) midline deviation from the clinical facial midline: group 1 (U1 deviation <2 mm) and group 2 (U1 deviation >2 mm). Linear measurements, angles, and reference planes on 3-dimensional (3D) computed tomograms were obtained. The predictor variables were maxillary yaw, palatal plane angle, differences of maxillary point to the coronal and sagittal planes, and maxillary canting. The outcome variable was U1 deviation. The variables between the 2 groups and 2 sides were analyzed with a t test. Pearson correlation coefficient and multiple regression analysis were also calculated within each group for each measurement against the U1 deviation to determine which variables affect U1 deviation. P < .05 was considered statistically significant. RESULTS: The patients were evenly distributed between each group (n = 14 in each group). There was significant deviation of U1 from the sagittal plane in group 2 compared with group 1 (0.99 mm in group 1 vs 1.73 mm in group 2, P < .05). When we compared yaw with the sagittal plane, group 2 was more rotated than group 1 (1.16° in group 1 vs 2.28° in group 2, P < .01). Through multiple regression analysis, the primary predictor variable for U1 deviation was maxillary yaw (P < .05). CONCLUSIONS: This study suggests that maxillary yaw is the primary contributing factor for upper dental midline deviation. The use of maxillary yaw should be considered when one is performing orthognathic surgery in patients with U1 deviation to achieve optimum esthetics.

Low-level laser therapy induces the expressions of BMP-2, osteocalcin, and TGF-ß1 in hypoxic-cultured human osteoblasts.

The aim of this study was to examine the effect of low-level laser therapy (LLLT) on the cell viability and the expression of hypoxia-inducible factor-1s (HIF-1s), bone morphogenic protein-2 (BMP-2), osteocalcin, type I collagen, transforming growth factor-ß1 (TGF-ß1), and Akt in hypoxic-cultured human osteoblasts. Human fetal osteoblast cells (cell line 1.19) were cultured under 1 % oxygen tension for 72 h. Cell cultures were divided into two groups. At the experimental side, low-level laser (808 nm, GaAlAs diode) was applied at 0, 24, and 48 h. After irradiation, each cell culture was incubated 24 h more under hypoxia. Total energy was 1.2, 2.4, and 3.6 J/cm(2), respectively. Non-irradiated cultures served as controls. Comparisons between the two groups were analyzed by t test; a p value <0.05 was considered statistically significant. Hypoxia resulted in a decrease in the expression of type I collagen, osteocalcin, and TGF-ß1 (p < 0.001, p < 0.001, and p < 0.01, respectively). Cell viability and BMP-2 expression were not decreased by hypoxic condition. On the other hand, LLLT on hypoxic-cultured osteoblast promoted the expression of BMP-2, osteocalcin, and TGF-ß1 (p < 0.05, p < 0.01, and p < 0.001, respectively). Cell proliferation was also increased time-dependently. However, hypoxia decreased in type I collagen expression (p < 0.001), and LLLT did not affect type I collagen expression in hypoxic-cultured osteoblasts. Furthermore, LLLT inhibited HIF-1 and Akt expression in hypoxic conditioned osteoblasts. We concluded that LLLT induces the expression of BMP-2, osteocalcin, and TGF- ß1 in 1 % hypoxic-cultured human osteoblasts.

RhoA/ROCK signalling activated by ARHGEF3 promotes muscle weakness via autophagy in dystrophic mdx mice.

BACKGROUND: Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular perturbations. Emerging evidence implicates RhoA/Rho-associated protein kinase (ROCK) signalling in DMD pathology, yet its direct role in DMD muscle function, and related mechanisms, are unknown. METHODS: Three-dimensionally engineered dystrophin-deficient mdx skeletal muscles and mdx mice were used to test the role of ROCK in DMD muscle function in vitro and in situ, respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generating Arhgef3 knockout mdx mice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild-type or GEF-inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine. RESULTS: Inhibition of ROCK with Y-27632 improved muscle force production in 3D-engineered mdx muscles (+25% from three independent experiments, P < 0.05) and in mice (+25%, P < 0.001). Unlike suggested by previous studies, this improvement was independent of muscle differentiation or quantity and instead related to increased muscle quality. We found that ARHGEF3 was elevated and responsible for RhoA/ROCK activation in mdx muscles, and that depleting ARHGEF3 in mdx mice restored muscle quality (up to +36%, P < 0.01) and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromised mdx muscle quality (-13% vs. empty vector control, P < 0.01) in GEF activity- and ROCK-dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles. CONCLUSIONS: Our findings uncover a new pathological mechanism of muscle weakness in DMD involving the ARHGEF3-ROCK-autophagy pathway and the therapeutic potential of targeting ARHGEF3 in DMD.