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Postnatal cell fate has been postulated to be primarily determined by the local tissue microenvironment. Here, we found that Mediator 1 (Med1) dependent epigenetic mechanisms dictate tissue-specific lineage commitment and progression of dental epithelia. Deletion ofMed1, a key component of the Mediator complex linking enhancer activities to gene transcription, provokes a tissue extrinsic lineage shift, causing hair generation in the dental environment.Med1deficiency gives rise to unusual hair growth via primitive cellular aggregates on incisors. Mechanistically, we found that Med1 establishes super-enhancers that control enamel lineage transcription factors in dental stem cells and their progenies. However,Med1deficiency reshapes the enhancer landscapes and causes a switch from the dental epithelial transcriptional program towards hair and epidermis on incisorsin vivo, and in dental epithelial stem cellsin vitro. Med1loss also provokes an increase in the number and size of enhancers. Interestingly, control dental epithelia already exhibit enhancers for hair and epidermal key transcription factors; these expand in size and transform into active super-enhancers uponMed1loss suggesting that these epigenetic mechanisms cause the transcriptomic and phenotypic shift towards epidermal and hair lineages. Thus, we propose a role for Med1 in safeguarding lineage specific enhancers, highlight the central role of enhancer accessibility and usage in lineage reprogramming and provide new insights into ectodermal regeneration.

Root cause analysis (RCA) of failures is mandatory to obtain the reliability and productivity of LSIs. Although analyzing layout-induced defects is crucial to optimize design rules and to predict unknown defects, it is a challenging task due to the difficulty in explaining the relationship between defects and circuit layouts. We applied convolutional neural networks (CNNs) to classify LSI layout images to perform the RCA of layout-induced defects in a previous study. However, due to the low resolution of images, actual defect positions were not clearly distinguished. In the present study, we use image clips of different sizes and resolutions for the CNN classification. Experimental results indicate that the validity of the extracted layout features depends on the resolution of image clips. Using the visual explanation technique GradCAM++, the features of defective layouts can be accurately captured in local areas when CNN models are trained on smaller image clips with higher resolution. These layout features included a group of patterns with their surroundings. Conversely, utilizing smaller-size clips deteriorates the classification accuracy due to the incorporation of less information from the images. In the conducted experiments, even in the case of using smaller clips, acceptable performance (the detection rate of defect positions DTR $\cong ~90$ %, and the risk-image classification rate RCR $\cong ~10$ %) can be obtained by increasing the size of training datasets. Partial layouts extracted as features of defective layouts can then be used in RCA and in designing future products.

The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-β1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-β1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-β1 stimulation. These results suggested that TGF-β1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.

Recently, the development of dental materials has increased the availability of various hyperesthesia desensitizers. However, there are no studies on the duration of retreatment in terms of adherence rates. Thus, the adhesion rates of resin-based desensitizers were investigated. We used a conventional desensitizer and a recently developed desensitizer containing calcium salt of 4-methacryloxyethyl trimellitic acid (C-MET) and 10-methacryloyloxydecyl dihydrogen calcium phosphate (MDCP). These colored agents were applied to the surfaces of premolars and molars, and the area was measured from weekly oral photographs. Areas were statistically analyzed and mean values were calculated using 95% confidence intervals. A p-value of

To investigate the detailed ultrastructural patterns of dental abnormalities affected by Axenfeld-Rieger syndrome (ARS) with a heterozygous microdeletion involving paired-like homeodomain 2 (PITX2) and explored the underlying molecular mechanisms driving enamel defects.Sanger sequencing, genomic quantitative PCR analysis, and chromosomal microarray analysis (CMA) were used to screen the disease-causing mutation in one ARS proband. An exfoliated tooth from an ARS patient was analyzed with scanning electron microscopy and micro-computerized tomography. A stable Pitx2 knockdown cell line was generated to simulate PITX2 haploinsufficiency. Cell proliferation and ameloblast differentiation were analyzed, and the role of the Wnt/β-catenin pathway in proliferation of ameloblast precursor cells was investigated.An approximately 0.216 Mb novel deletion encompassing PITX2 was identified. The affected tooth displayed a thinner and broken layer of enamel and abnormal enamel biomineralization. PITX2 downregulation inhibited the proliferation and differentiation of inner enamel epithelial cells, and LiCl stifmulation partially reversed the proliferation ability after Pitx2 knockdown.Enamel formation is disturbed in some patients with ARS. Pitx2 knockdown can influence the proliferation and ameloblast differentiation of inner enamel epithelial cells, and PITX2 may regulate cell proliferation via Wnt/β-catenin signaling pathway.

Background Filamin-A (FLNA) is one of the adaptor proteins binding to both integrin beta chain and actin filament. Several kind of mutation in human FLNA gene were observed in Melnick-Needles syndrome (MNS). However, the role of FLNA in tooth formation is still unknown. Matrials and methods We analyzed the tissue expression of FLNA during tooth development using immunostaining. The role of FLNA in cell migration was analyzed using a scratch assay. In addition, we analyzed the phenotype of teeth in patients with Mernick-Needles syndrome caused by FLNA gene abnormalities. Results FLNA expressed in inner and outer enamel epithelium in mouse tooth germ and also expressed in cervical region of dental epithelium. In postnatal stage, FLNA expressed in ameloblast and odontoblast layers. In rat dental epithelium cell line SF2, FLNA is localized at the boundary between cells and co-localized with the terminal region of F-actin filament. shRNA-FLNA transfected cells showed delayed cell migration and scratch closure compared with control cells. Analysis of actin fiber movement by time-lapse confocal microscopy showed slower fiber movement in shRNA-FLNA over expressing SF2 cells. Novel mutation in FLNA gene was observed in MNS with tooth anormaly. This patient showed tooth agenesis and short root in both primary and permanent dentition with thin enamel. Conclusion Taken together, these results suggest that filamin-A is play a key role for dental epithelium migration and root formation.

Background Recently, various amelogenesis imperfecta such as molar incisor hypomineralization (MIH) have been reported in pediatric dentistry. The cause has been studied extensively but is not yet clear. Therefore, dentists focus on prevention to avoid becoming serious. In order to find a new approach, we also need to understand the mechanism of enamel formation. The immature dental epithelial cells exist on the posterior side of the mouse incisor, while differentiated cells such as ameloblasts occur on the anterior side, since mouse incisors grow continuously. All ameloblast differentiation can be observed on the mouse incisor. Thus, the dental epithelium of the mouse incisor was analyzed to identify molecules that affect ameloblast differentiation in enamel formation. Results Tgf superfamily genes, cell adhesion molecules, Wnt-related genes, and transcription factors such as homeobox genes were expressed in the dental epithelium of the mouse incisor. Furthermore, the expression levels of these molecules increased depending on the differentiation of the molar tooth germ. Site-specifically-expressed molecules were also observed in the frontal section of the incisor, concentrated at the enamel border. These may contribute to the morphology or play a role in crown-root determination. β1 integrin, fibronectin, Gdf5, and Sox2 were also analyzed to determine their function. These molecules were found to play important roles in ameloblast differentiation and tooth development. Conclusion Various molecules were intricately involved in tooth development and morphogenesis in this study. Mouse incisors are a useful model system for the analysis of not only ameloblast differentiation, but also that of all epithelial tissues.

Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein–coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop–derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.

Physical failure analysis (PFA) specifies layout designs that affect large-scale integration (LSI) failure. Because of their capability and cost-effectiveness, convolutional neural networks (CNNs) have been applied to LSI layout analysis. However, the information on failure for root cause analyses is generally limited. Moreover, information over a large area, which includes many geometries, is required to understand the effects of a layout on failure. These problems pose challenges in training the CNN models to achieve high accuracy for layout segment classification. In this study, we propose a scheme for layout segment classification that uses CNN to analyze the effects of layout on failure. Thus, multiple segment images of LSI layouts are used as inputs, and the outputs of CNN models are used to classify the input images as either risk or non-risk segments. First, we evaluated a simple 3-layer model, and then a 15-layer model fine-tuned using the transfer learning of the VGG16 model. The 15-layer model outperformed the 3-layer model for classifying the LSI layout segments. From a cross-validation of the 15-layer model, a true positive rate of >80% and a false positive rate of

Proteasome inhibitors exert an anabolic effect on bone formation with elevated levels of osteoblast markers. These findings suggest the important role of the proteasomal degradation of osteogenic regulators, while the underlying molecular mechanisms are not fully understood. Here, we report that the proteasome inhibitors bortezomib and ixazomib markedly increased protein levels of the osteoblastic key transcription factor osterix/Sp7 (Osx). Furthermore, we revealed that Osx was targeted by p38 and Fbw7 for proteasomal degradation. Mechanistically, p38-mediated Osx phosphorylation at S73/77 facilitated Fbw7 interaction to trigger subsequent Osx ubiquitination. Consistent with these findings, p38 knockdown or pharmacological p38 inhibition resulted in Osx protein stabilization. Treatment with p38 inhibitors following osteogenic stimulation efficiently induced osteoblast differentiation through Osx stabilization. Conversely, pretreatment of p38 inhibitor followed by osteogenic challenge impaired osteoblastogenesis via suppressing Osx expression, suggesting that p38 exerts dual but opposite effects in the regulation of Osx level to fine-tune its activity during osteoblast differentiation. Furthermore, Fbw7-depleted human mesenchymal stem cells and primary mouse calvarial cells resulted in increased osteogenic capacity. Together, our findings unveil the molecular mechanisms underlying the Osx protein stability control and suggest that targeting the Osx degradation pathway could help enhance efficient osteogenesis and bone matrix regeneration.

Space flight produces an extreme environment with unique stressors, but little is known about how our body responds to these stresses. While there are many intractable limitations for in-flight space research, some can be overcome by utilizing gene knockout-disease model mice. Here, we report how deletion of Nrf2, a master regulator of stress defense pathways, affects the health of mice transported for a stay in the International Space Station (ISS). After 31 days in the ISS, all flight mice returned safely to Earth. Transcriptome and metabolome analyses revealed that the stresses of space travel evoked ageing-like changes of plasma metabolites and activated the Nrf2 signaling pathway. Especially, Nrf2 was found to be important for maintaining homeostasis of white adipose tissues. This study opens approaches for future space research utilizing murine gene knockout-disease models, and provides insights into mitigating space-induced stresses that limit the further exploration of space by humans., Using Nrf2 knockout mice, Suzuki, Uruno, Yumoto et al. show that space travel activates Nrf2 signaling, which contributes to the weight gain of mice by regulating fat metabolism of white adipose tissues. This study provides insights into potential interventions to mitigate stresses that accompany space travels.

The electroslag welding process involves high heat inputs and high deposition rates. In this process, the weld region is surrounded by the base metal and/or devices; therefore, it is difficult to observe the phenomenon and understand the mechanism of weld formation. In this study, we directly observe the molten region formed during the process and construct a numerical model to discuss the phenomena especially the construction of the molten region, the flow field of the molten region, and the metal transfer. A device for direct observation was constructed, and the molten region was observed. The molten slag flows from the wire tip to the molten metal interface, and the direction of flow is changed to the side at the interface. The molten metal flows from the center to the side region at the interface of the slag and metal. The penetration depth is governed by the behavior of the molten metal. In addition, droplet transfer from the wire is observed, and this transfer occurs in the spray or rotating mode. The simulation results indicate a tendency similar to that deduced via direct observations. Thus, the mechanism of penetration can be elucidated more clearly using these tools.

A number of methods have been proposed for the response-time analysis (RTA) of controller area networks (CANs), which are the industry standard for in-vehicle network protocols. However, most of these methods are unsuitable for analyzing nonperiodically transmitted messages because they assume a constant transmission period for each message. Moreover, the calculated evaluation criteria are limited and comprise many over-pessimistic estimations, such as the worst-case response time (WCRT). To tackle these problems, this paper proposes an RTA method for CANs with randomly occurring messages, which replaces the constant transmission period with the average number of transmission instances per unit time. This approach finds the approximate probability distribution of the response time to any message in a CAN, providing a more general measure than the WCRT. To this end, a mathematical model based on M/G/1 queueing theory is developed. The model accurately emulates the behavior of nonperiodic CAN messages and enables RTA with minimal pessimistic estimations. When tested against the benchmarks, the proposed method accurately obtained response time distributions within several seconds to several tens of seconds.

Lipin-2 is a phosphatidate phosphatase with key roles in regulating lipid storage and energy homeostasis. LPIN2-genetic deficiency is associated with an autoinflammatory disorder, underscoring its critical role in innate immune signaling; however, the regulatory mechanisms underlying protein stability remain unknown. Here, we demonstrate that Lipin-2 interacts with β-TRCP, a substrate receptor subunit of the SCFβ-TRCP E3 ligase, and undergoes ubiquitination and proteasomal degradation. β-TRCP-knockout in RAW264.7 macrophages resulted in Lipin-2 accumulation, leading to the suppression of LPS-induced MAPK activation and subsequent proinflammatory gene expression. Consistent with this, treatment with MLN4924, a Cullin-neddylation inhibitor that suppresses SCF E3 activity, increased Lipin-2 protein and concomitantly decreased Il1b expression. These findings suggested that β-TRCP-mediated Lipin-2 degradation affects macrophage-elicited proinflammatory responses and could lead to new therapeutic approaches to treat inflammatory diseases.

Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction.

Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2+-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2+ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2+/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD.

Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca2+ channels. However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca2+ channel to promote osteogenesis.