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Although bone dehiscence may occur during orthodontic tooth movement into the narrow alveolar ridge, a non-invasive prevention method is yet to be fully established. We show for the first time prevention of bone dehiscence associated with orthodontic tooth movement by prophylactic injection of bone anabolic agents in mice. In this study, we established a bone dehiscence mouse model by applying force application and used the granular type of scaffold materials encapsulated with bone morphogenetic protein (BMP)-2 and OP3-4, the receptor activator of NF-κB ligand (RANKL)-binding peptide, for the prophylactic injection to the alveolar bone. In vivo micro-computed tomography revealed bone dehiscence with decreased buccal alveolar bone thickness and height after force application, whereas no bone dehiscence was observed with the prophylactic injection after force application, and alveolar bone thickness and height were kept at similar levels as those in the control group. Bone histomorphometry analyses revealed that both bone formation and resorption parameters were significantly higher in the injection with force application group than in the force application without the prophylactic injection group. These findings suggest that the prophylactic local delivery of bone anabolic reagents can prevent bone dehiscence with increased bone remodelling activity. [ABSTRACT FROM AUTHOR]

Bow-tie architecture is a layered network structure that has a narrow middle layer with multiple inputs and outputs. Such structures are widely seen in the molecular networks in cells, suggesting that a universal evolutionary mechanism underlies the emergence of bow-tie architecture. The previous theoretical studies have implemented evolutionary simulations of the feedforward network to satisfy a given input-output goal and proposed that the bow-tie architecture emerges when the ideal input-output relation is given as a rank-deficient matrix with mutations in network link intensities in a multiplicative manner. Here, we report that the bow-tie network inevitably appears when the link intensities representing molecular interactions are small at the initial condition of the evolutionary simulation, regardless of the rank of the goal matrix. Our dynamical system analysis clarifies the mechanisms underlying the emergence of the bow-tie structure. Further, we demonstrate that the increase in the input-output matrix reduces the width of the middle layer, resulting in the emergence of bow-tie architecture, even when evolution starts from large link intensities. Our data suggest that bow-tie architecture emerges as a side effect of evolution rather than as a result of evolutionary adaptation. [ABSTRACT FROM AUTHOR]

The extracellular signal-regulated kinase (ERK) is a serine/threonine kinase that is known to regulate cellular events such as cell proliferation and differentiation. The ERK signaling pathway is activated by fibroblast growth factors, and is considered to be indispensable for the differentiation of primitive endoderm cells, not only in mouse preimplantation embryos, but also in embryonic stem cell (ESC) culture. To monitor ERK activity in living undifferentiated and differentiating ESCs, we established EKAREV-NLS-EB5 ESC lines that stably express EKAREV-NLS, a biosensor based on the principle of fluorescence resonance energy transfer. Using EKAREV-NLS-EB5, we found that ERK activity exhibited pulsatile dynamics. ESCs were classified into two groups: active cells showing high-frequency ERK pulses, and inactive cells demonstrating no detectable ERK pulses during live imaging. Pharmacological inhibition of major components in the ERK signaling pathway revealed that Raf plays an important role in determining the pattern of ERK pulses. [ABSTRACT FROM AUTHOR]

Purpose: There are only a few epidemiological studies of dry eye syndrome (DES) in populations with a common academic background. in this study, the prevalence of DES and associated factors were evaluated separately in men and women physical education and sports science graduates. Study design: Cross-sectional survey. Methods: A questionnaire about the diagnosis of DES and associated factors was mailed to 9507 graduates of the Faculty of Health and Sports Science, Juntendo University. The questions covered subjective DES using Schaumberg et al.'s questionnaire and the prevalence of diagnosed DES. Associated factors, age, sex, smoking, alcohol consumption, body mass index, daily screen viewing time, and contact lens (CL) use were analyzed. Results: A total of 2048 valid responses were received. The prevalence of diagnosed DES was 2.9% in men and 9.3% in women. For subjective DES, the prevalence was 14.8% in men and 39.8% in women. The odds ratio for DES was high in men and women who used CLs and women whose daily screen viewing time was ≥ 4 h. Conclusion: Both diagnosed and subjective DES were highly prevalent in men and women of all ages, particularly among those in their 20 and 30s. CL use was associated with DES in both men and women. Measures to deal with the factors that can be corrected might have a positive effect on the ocular health and quality of life of physical education and sports science graduates. [ABSTRACT FROM AUTHOR]

Advances in mechanistic understanding of integrin-mediated adhesion highlight the importance of precise control of ligand presentation in directing cell migration. Top-down nanopatterning limited the spatial presentation to sub-micron placing restrictions on both fundamental study and biomedical applications. To break the constraint, here we propose a bottom-up nanofabrication strategy to enhance the spatial resolution to the molecular level using simple formulation that is applicable as treatment agent. Via self-assembly and co-assembly, precise control of ligand presentation is succeeded by varying the proportions of assembling ligand and nonfunctional peptide. Assembled nanofilaments fulfill multi-functions exerting enhancement to suppression effect on cell migration with tunable amplitudes. Self-assembled nanofilaments possessing by far the highest ligand density prevent integrin/actin disassembly at cell rear, which expands the perspective of ligand-density-dependent-modulation, revealing valuable inputs to therapeutic innovations in tumor metastasis. Engineering peptide assembly that controls integrin ligand presentation on the molecular level possesses by far the highest ligand density, expanding the perspective of ligand-density-dependent modulation. [ABSTRACT FROM AUTHOR]

Cell tracking is one of the most critical tools for time-lapse image analysis to observe cell behavior and cell lineages over a long period of time. However, the accompanying graphical user interfaces are often difficult to use and do not incorporate seamless manual correction, data analysis tools, or simple training set design tools if it is machine learning based. In this paper, we introduce our cell tracking software "LIM Tracker". This software has a conventional tracking function consisting of recognition processing and link processing, a sequential search-type tracking function based on pattern matching, and a manual tracking function. LIM Tracker enables the seamless use of these functions. In addition, the system incorporates a highly interactive and interlocking data visualization method, which displays analysis result in real time, making it possible to flexibly correct the data and reduce the burden of tracking work. Moreover, recognition functions with deep learning (DL) are also available, which can be used for a wide range of targets including stain-free images. LIM Tracker allows researchers to track living objects with good usability and high versatility for various targets. We present a tracking case study based on fluorescence microscopy images (NRK-52E/EKAREV-NLS cells or MCF-10A/H2B-iRFP-P2A-mScarlet-I-hGem-P2A-PIP-NLS-mNeonGreen cells) and phase contrast microscopy images (Glioblastoma-astrocytoma U373 cells). LIM Tracker is implemented as a plugin for ImageJ/Fiji. The software can be downloaded from https://github.com/LIMT34/LIM-Tracker. [ABSTRACT FROM AUTHOR]

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, subcellular dynamics of actomyosin contractility underlying such processes remains elusive. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility at the subcellular level. The system, named OptoMYPT, combines a protein phosphatase 1c (PP1c)-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination is sufficient to induce dephosphorylation of myosin regulatory light chains and a decrease in actomyosin contractile force in mammalian cells and Xenopus embryos. The OptoMYPT system is further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We find that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system provides opportunities to understand cellular and tissue mechanics. The subcellular dynamics of actomyosin contractility is currently hard to study. Here the authors report OptoMYPT, an optogenetic method to induce relaxation of actomyosin contractility by allowing light-dependent recruitment of endogenous protein phosphatase 1c (PP1c) to the plasma membrane. [ABSTRACT FROM AUTHOR]

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol–gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments. [ABSTRACT FROM AUTHOR]

Plasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand. [ABSTRACT FROM AUTHOR]

Collective cell migration is a fundamental process in embryonic development and tissue homeostasis. This is a macroscopic population-level phenomenon that emerges across hierarchy from microscopic cell-cell interactions; however, the underlying mechanism remains unclear. Here, we addressed this issue by focusing on epithelial collective cell migration, driven by the mechanical force regulated by chemical signals of traveling ERK activation waves, observed in wound healing. We propose a hierarchical mathematical framework for understanding how cells are orchestrated through mechanochemical cell-cell interaction. In this framework, we mathematically transformed a particle-based model at the cellular level into a continuum model at the tissue level. The continuum model described relationships between cell migration and mechanochemical variables, namely, ERK activity gradients, cell density, and velocity field, which could be compared with live-cell imaging data. Through numerical simulations, the continuum model recapitulated the ERK wave-induced collective cell migration in wound healing. We also numerically confirmed a consistency between these two models. Thus, our hierarchical approach offers a new theoretical platform to reveal a causality between macroscopic tissue-level and microscopic cellular-level phenomena. Furthermore, our model is also capable of deriving a theoretical insight on both of mechanical and chemical signals, in the causality of tissue and cellular dynamics. [ABSTRACT FROM AUTHOR]

This commentary summarizes the recent biophysical research conducted at the National Institute for Basic Biology, the National Institute for Physiological Sciences, and the Institute for Molecular Science in Okazaki, Japan. [ABSTRACT FROM AUTHOR]

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest. The secretion of blood coagulation factor V and factor VIII (FV and FVIII) are driven by secretion factors ERGIC-53 and MCDF2. Here, the authors report a 10 amino acid motif from FVIII that can enhance secretion of another glycoprotein erythropoietin (EPO). [ABSTRACT FROM AUTHOR]

Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis. Osteoclasts secrete small extracellular vesicles that stimulate osteoblasts, promoting bone formation via receptor activator of nuclear factor-kappa B ligand (RANKL), thereby linking bone formation and resorption. [ABSTRACT FROM AUTHOR]

To examine the environmental factors controlling the inshore recruitment dynamics of Anguilla japonica in the Oyodo River, Miyazaki Prefecture, Kyushu, glass eel samplings were carried out using fyke nets during winter (November-March) of 1994-2014. The peak CPUEs (catch per unit effort) were observed between November and February, but differed from year to year. The yearly CPUE was extremely high in 2002, when the sea surface temperature (SST) in the offshore area of the Oyodo River was the lowest in winter of all the sampling years. The negative SST anomaly of less than − 0.5 °C was sustained in the offshore area during the recruitment season in 2002, which was caused by two combined factors; low air temperature and the Kuroshio path. The oceanographic data showed that the dominant path of the Kuroshio was displaced eastward at 31°N in 2002, which was different from the average Kuroshio path. The eastward displacement of the Kuroshio induced a cyclonic mesoscale eddy in the offshore area of the Oyodo River, resulting in the entrainment of the cold seawater into coastal waters from deep water. The oceanographic condition in relation to the continuous low SST could be favorable for local recruitment of glass eels. [ABSTRACT FROM AUTHOR]

Composite analysis was conducted using high-frequency radar data obtained during 2006-2015 in order to gain a better understanding of the current field in the Ariake Sea. The seasonally averaged surface current in the Ariake Sea was directed southward in all seasons, except around river mouths during summer. Heavy rainfall enhanced the outflow along the eastern coast of the Shimabara Peninsula from Isahaya Bay to the southern area 2-5 days after heavy rainfall. Spring-neap differences were clearly seen in the southward current along the Shimabara Peninsula. Interannual variation in the M2 tidal current amplitude was synchronized with the lunar nodal cycle. [ABSTRACT FROM AUTHOR]

The response to the mechanical loading of bone tissue has been extensively investigated; however, precisely how much strain intensity is necessary to promote bone formation remains unclear. Combination studies utilizing histomorphometric and numerical analyses were performed using the established murine maxilla loading model to clarify the threshold of mechanical strain needed to accelerate bone formation activity. For 7 days, 191 kPa loading stimulation for 30 min/day was applied to C57BL/6J mice. Two regions of interest, the AWAY region (away from the loading site) and the NEAR region (near the loading site), were determined. The inflammatory score increased in the NEAR region, but not in the AWAY region. A strain intensity map obtained from $$\upmu \hbox {CT}$$ images was superimposed onto the images of the bone formation inhibitor, sclerostin-positive cell localization. The number of sclerostin-positive cells significantly decreased after mechanical loading of more than $$150\,{\upmu }{\upvarepsilon }$$ in the AWAY region, but not in the NEAR region. The mineral apposition rate, which shows the bone formation ability of osteoblasts, was accelerated at the site of surface strain intensity, namely around $$170\,{\upmu }{\upvarepsilon }$$ , but not at the site of lower surface strain intensity, which was around $$80\,{\upmu }{\upvarepsilon }$$ in the AWAY region, thus suggesting the existence of a strain intensity threshold for promoting bone formation. Taken together, our data suggest that a threshold of mechanical strain intensity for the direct activation of osteoblast function and the reduction of sclerostin exists in a murine maxilla loading model in the non-inflammatory region. [ABSTRACT FROM AUTHOR]

In both 2009 and 2010, massive Chattonella blooms occurred in Tachibana Bay. Observation results show that high cell densities of C hattonella were distributed in the central area of Tachibana Bay with low salinity water. Model results indicate that the low salinity water originated from the Ariake Sea and intruded into Tachibana Bay during the northerly or weak winds. It is suggested that low salinity water was mainly discharged from the northern area of the Ariake Sea. Northerly wind enhanced the horizontal advection of the low salinity water intruding into Tachibana Bay originating from the northern area of the Ariake Sea. [ABSTRACT FROM AUTHOR]

Mass mortality of Seriola quinqueradiata was caused by a Chattonella bloom in summer 2010 in the southern area of the Yatsushiro Sea. We analyzed the diurnal records of the mortality occurrence and cell density of C. marina to examine the short-term mortality dynamics. In addition, a hydrodynamic model and Lagrangian particle tracking method were used to examine interregional differences in the mortalities. Mortalities were concentrated along the southern coast of Shishi Island and western coast of Nagashima Island in early July. In late July, mortalities occurred along the southern coast of Shishi Island and eastern coast of Nagashima Island. Severe mortalities occurred where the bloom (>100 cells ml of Chattonella marina) appeared in both early and late July. Model results show that interregional difference of the bloom was controlled by the advection system in the Yatsushiro Sea. The consistent spatio-temporal pattern between fish mortalities and bloom dynamics indicates the potential benefits of real-time monitoring of harmful algae and assessing factors controlling hydrographic conditions to mitigate mortalities. [ABSTRACT FROM AUTHOR]

The relationship between harmful blooms of raphidophycean flagellate Chattonella spp. and preceding meteorological conditions was investigated in the Yatsushiro Sea, Japan, using data over a 25-year period from 1988 to 2012. The occurrence date of Chattonella blooms was significantly correlated with the average air temperature from February through April and with the onset date of Baiu, the Japanese name for the rainy season in early summer. These two meteorological factors showed significant differences between occurrence and non-occurrence years of Chattonella blooms in the Yatsushiro Sea. The Chattonella blooms tended to occur in years that were relatively warm from winter to spring and had a relatively late onset of Baiu. The duration of Chattonella blooms was significantly correlated with the interval between the closing date of Baiu and the occurrence date of the Chattonella blooms, suggesting an important role of Baiu as nutrient input affecting the magnitude of the bloom event. The East Asian monsoon, which controls local meteorological conditions, is likely involved in the population dynamics of Chattonella through its life cycle and physiological characteristics. [ABSTRACT FROM AUTHOR]

Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in α-MEM containing macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA levels of osteoclast-related genes, assayed by real-time RT-PCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factor-β1 (TGF-β1), SB431542, a TGF-β receptor inhibitor, and ELISA were used to determine the role of TGF-β1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-β1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-β1 involvement. The addition of exogenous TGF-β1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-β1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-β1. [ABSTRACT FROM AUTHOR]

To examine the properties of winter mixed layer (ML) variability in the shelf-slope waters facing the Kuroshio, we analyzed historical temperature records and the simulated results of a triply nested high-resolution numerical model. As a candidate of the shelf-slope waters, we focused on Tosa Bay, off the southern Japan. A time series of observed monthly mean ML temperatures and depths in the bay exhibits a remarkable seasonal variation. The period when the ML develops can be divided into two regimes: from September to November, when the sea surface cooling is gradually enhanced, the ML temperature and depth decreases and increases, respectively; from January to March, the ML temperature and depth are kept nearly constant, while the sea surface cooling in January reaches its annual maximum. In the latter regime, variance for the monthly mean ML depth is the largest of the year. To further study the ML properties in the latter regime corresponding to winter, we examined simulated results. It was found that the largest variance for ML depth is attributed to a dominant intramonthly variation. This is related to a submesoscale variation with typical spatial scales of 10-20 km, induced by the Kuroshio and its frontal disturbances. Simulated monthly mean heat balance within the ML showed that heat advection balances with heat flux at the sea surface and entrainment through the ML bottom. Moreover, the monthly mean heat advection is determined mainly by the intramonthly eddy heat advection, suggesting that the high-frequency intramonthly variation related to submesoscale variations contributes significantly to the low-frequency monthly variations of the ML in winter. [ABSTRACT FROM AUTHOR]

Aims: Nitrogen (N) management strategies for reducing the risk of groundwater contamination around agricultural fields require precise prediction of N leaching using a process-based model. We modified LEACHM model for use in Andosols, which are characterized by slow soil organic carbon (SOC) mineralization and nitrate adsorption. Methods: The modification was made with regard to the SOC mineralization of incoming plant-residue/manure and humus following the RothC model, as well as for nitrate adsorption. Empirical equations were employed to determine the parameters of the modified model. The ability of the modified LEACHM to predict N leaching was tested against existing data from a 4-year lysimeter study for cropped Andosol and sandy soils and compared with that of the original model. Results: The modified model improved the prediction of leached N concentrations and the loss of N from Andosol with relative improvements of 63.5 and 76.5 %, respectively, over the original model, while retaining model applicability in sandy soil. This effective modeling was achieved by using precise predictions of N mineralization in the humus pool along with SOC mineralization processes that were based on the RothC model. Conclusions: The modification extended the applicability of LEACHM and may provide better N management strategies for reducing leaching from cultivated Andosols. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF)-α exerts its biological function via TNF type 1 and type 2 receptors (TNFR1 and TNFR2). We have previously reported that bone resorption induced by lipopolysaccharide (LPS) in TNFR2-deficient mice is accelerated compared to that in wild-type (WT) mice. Although these results suggested that TNFR2 might have a protective role in bone resorption, we could not exclude the possibility that TNFR2 has no role in bone resorption. To clarify the role of TNFR2, we developed a TNF-α-induced bone resorption model using cholesterol-bearing pullulan nanogel as a TNF-α carrier to minimize the influence of inflammatory cytokines other than TNF-α. Injections of human TNF-α (hTNF), an agonist of mouse TNFR1, stimulated bone resorption lacunae on the calvariae in WT mice, but mouse TNF-α (mTNF), an agonist of both mouse TNFR1 and TNFR2, could not. To eliminate the possibility that the TNFR1 agonistic effects of hTNF were stronger than those of mTNF, we used the same model in TNFR2-deficient mice. Injection of mTNF resulted in clear bone resorption lacunae to the same extent observed after using hTNF in the TNFR2-deficient mice. Histomorphometric analysis of osteoclast number supported the observed changes in bone resorption lacunae. These data suggest that TNFR2 has a protective role in TNF-α-induced bone resorption. [ABSTRACT FROM AUTHOR]

Our goal in this study was to determine to what extent the physiologic consequences of ovariectomy (OVX) in bones are exacerbated by a lack of daily activity such as walking. We forced 14-week-old female rats to be inactive for 15 weeks with a unique experimental system that prevents standing and walking while allowing other movements. Tibiae, femora, and 4th lumbar vertebrae were analyzed by peripheral quantitative computed tomography (pQCT), microfocused X-ray computed tomography (micro-CT), histology, histomorphometry, Raman spectroscopy, and the three-point bending test. Contrary to our expectation, the exacerbation was very much limited to the cancellous bone parameters. Parameters of femur and tibia cortical bone were affected by the forced inactivity but not by OVX: (1) cross-sectional moment of inertia was significantly smaller in Sham-Inactive rat bones than that of their walking counterparts; (2) the number of sclerostin-positive osteocytes per unit cross-sectional area was larger in Sham-Inactive rat bones than in Sham-Walking rat bones; and (3) material properties such as ultimate stress of inactive rat tibia was lower than that of their walking counterparts. Of note, the additive effect of inactivity and OVX was seen only in a few parameters, such as the cancellous bone mineral density of the lumbar vertebrae and the structural parameters of cancellous bone in the lumbar vertebrae/tibiae. It is concluded that the lack of daily activity is detrimental to the strength and quality of cortical bone in the femur and tibia of rats, while lack of estrogen is not. Our inactive rat model, with the older rats, will aid the study of postmenopausal osteoporosis, the etiology of which may be both hormonal and mechanical. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-17-producing helper T (TH17) cells are a distinct T-cell subset characterized by its pathological role in autoimmune diseases. IL-6 and transforming growth factor-β (TGF-β) induce TH17 development, in which the orphan nuclear receptors, RORγt and RORα, have an indispensable role. However, in the absence of IL-6 and TGF-β, the ectopic expression of RORγt or RORα leads to only a modest IL-17 production. Here we identify a nuclear IκB family member, IκBζ (encoded by the Nfkbiz gene), as a transcription factor required for TH17 development in mice. The ectopic expression of IκBζ in naive CD4+ T cells together with RORγt or RORα potently induces TH17 development, even in the absence of IL-6 and TGF-β. Notably, Nfkbiz-/- mice have a defect in TH17 development and a resistance to experimental autoimmune encephalomyelitis (EAE). The T-cell-intrinsic function of IκBζ was clearly demonstrated by the resistance to EAE of the Rag2-/- mice into which Nfkbiz-/- CD4+ T cells were transferred. In cooperation with RORγt and RORα, IκBζ enhances Il17a expression by binding directly to the regulatory region of the Il17a gene. This study provides evidence for the transcriptional mechanisms underlying TH17 development and points to a molecular basis for a novel therapeutic strategy against autoimmune disease. [ABSTRACT FROM AUTHOR]

The classical NF-kappaB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-kappaB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-kappaB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-kappaB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p < 0.001) and depths (p < 0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-kappaB pathway plays a pivotal role in osteoclast bone-resorbing activity. [ABSTRACT FROM AUTHOR]

The release of tumor necrosis factor (TNF)-alpha from macrophages upon stimulation of lipopolysaccharide (LPS) is a major etiological factor of inflammatory bone disease and elicits the effects through TNF receptors type 1 and 2. Given the importance of TNF-alpha action on osteoclastic bone resorption, the role of TNF type 2 receptor (TNFR2) on bone resorption has not been elucidated well so far. The purpose of this study is to investigate the role of TNFR2 on LPS-induced inflammatory bone resorption in vivo. LPS at 10 mg/kg (Re 595) was injected subcutaneously on calvariae of wild-type (WT), TNF type 1 receptor (TNFR1)-deficient (KO), and TNFR2 KO mice, killed on day 5 after the LPS injection. The calvarial bone mineral density (BMD) was significantly decreased by LPS compared to the vehicle-injected control in WT mice, but not in TNFR1 KO mice. Interestingly, the decrease of calvarial BMD and the increase of the osteoclast number by LPS in TNFR2 KO mice seemed to be more than those in WT mice. Furthermore, the significant decrease by LPS on the BMD of tibiae, femurs, and lumber vertebrae were observed only in TNFR2 KO mice. Histomorphometric analysis of tibiae showed the significant increases of osteoclast number and surface in the LPS-injected TNFR2 KO mice, and the levels of urinary deoxypyridinoline reflected these increases of bone resorption parameters. The present data indicate that TNFR1 is critical for bone resorption at the site of LPS injection and that TNFR2 might have a protective role on the LPS-induced inflammatory bone resorption process. [ABSTRACT FROM AUTHOR]

Genetically encoded probes based on Förster resonance energy transfer (FRET) enable us to decipher spatiotemporal information encoded in complex tissues such as the brain. Firstly, this review focuses on FRET probes wherein both the donor and acceptor are fluorescence proteins and are incorporated into a single molecule, i.e. unimolecular probes. Advantages of these probes lie in their easy loading into cells, the simple acquisition of FRET images, and the clear evaluation of data. Next, we introduce our recent study which encompasses FRET imaging and in silico simulation. In nerve growth factor-induced neurite outgrowth in PC12 cells, we found positive and negative signaling feedback loops. We propose that these feedback loops determine neurite-budding sites. We would like to emphasize that it is now time to accelerate crossover research in neuroscience, optics, and computational biology. [ABSTRACT FROM AUTHOR]

Calcium (Ca2+) signaling is essential for a variety of cellular responses and higher biological functions. Ca2+/calmodulin-dependent kinases (CaMKs) and the phosphatase calcineurin activate distinct downstream pathways that are mediated by the transcription factors cAMP response element (CRE)-binding protein (CREB) and nuclear factor of activated T cells (NFAT), respectively. The importance of the calcineurin-NFAT pathway in bone metabolism has been demonstrated in osteoclasts, osteoblasts and chondrocytes. However, the contribution of the CaMK-CREB pathway is poorly understood, partly because of the difficulty of dissecting the functions of homologous family members. Here we show that the CaMKIV-CREB pathway is crucial for osteoclast differentiation and function. Pharmacological inhibition of CaMKs as well as the genetic ablation of Camk4 reduced CREB phosphorylation and downregulated the expression of c-Fos, which is required for the induction of NFATc1 (the master transcription factor for osteoclastogenesis) that is activated by receptor activator of NF-κB ligand (RANKL). Furthermore, CREB together with NFATc1 induced the expression of specific genes expressed by differentiated osteoclasts. Thus, the CaMK-CREB pathway biphasically functions to regulate the transcriptional program of osteoclastic bone resorption, by not only enhancing induction of NFATc1 but also facilitating NFATc1-dependent gene regulation once its expression is induced. This provides a molecular basis for a new therapeutic strategy for bone diseases. [ABSTRACT FROM AUTHOR]

Acute and chronic peripheral inflammation is known to induce the expression of cyclo-oxygenase (COX)-2 in spinal cord neurons and increase the synthesis and release of prostaglandins (PG). Although these PG are presumed to cause inflammatory pain or hyperalgesia, the relationship between PG-producing cells in the dorsal horn and substance P (SP)-containing, pain-transmittimg nerve fibers remains unknown. In the present study we investigated immunohistochemically changes in the number of COX-2-containing neurons using the avidin–biotinylated peroxidase complex method in dorsal horn superficial laminae in chicken lumbosacral enlargement (L4, L5) under inflammatory conditions induced by unilateral intraplantar injection of complete Freund's adjuvant. After 12–24 h, a significant increase in the number of small COX-2-containing neurons was observed in lamina II on the injected side compared with the contralateral side. Furthermore, using fluorescent double-labeling for COX-2 and SP, an increase in the number of small COX-2-containing neurons in contact with SP-containing elements was observed ipsilaterally (1.4–1.6-fold compared with the contralateral side) in lamina II. Fluorescence triple-labeling of COX-2, SP and calcitonin gene-related peptide (CGRP) confirmed that the majority of these SP-containing elements coexisted with CGRP, indicating that these elements originated from primary afferent neurons. Using electron microscopy, two types of SP-containing axon terminals were found to form synapses with COX-2-containing neurons in lamina II. These results indicate that the number of COX-2-containing neurons increases concomitantly with an increase in the number of contacts of these neurons with SP-containing primary afferent fibers and suggest that this phenomenon is associated with PG production and the persistence of inflammatory pain. [ABSTRACT FROM AUTHOR]

Many clinical reports suggest that deciduous teeth exhibit a greater susceptibility to resorption than permanent ones. To examine the difference between deciduous and permanent dentine in their susceptibility to osteoclastic resorption, osteoclast-like cells (OCLs) were cultured on deciduous and permanent dentine slices. The number, area, depth, and volume of resorption pits were then measured, using image-analyzing systems. We measured the level of degraded collagen (cross-linked N-telopeptide of type I collagen; NTx) in culture medium using an enzyme-linked immunosorbent assay (ELISA). The levels of cathepsin K, matrix metallo proteinase (MMP)-9, and MMP-13 mRNAs in the cells attached to dentine were also analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). Deciduous dentine slices exhibited a significant (twofold) increase in resorbed area compared with the permanent slices. Three-dimensional analysis revealed that the volume of pits in deciduous dentine differed significantly (fourfold) compared to that in the permanent dentine. The depth of pits also followed the same trend. However, there was no significant difference in the number of pits or osteoclasts on the dentine slices. The NTx level in deciduous media was significantly more than that in permanent media. The mRNA levels also followed the same trend. These results suggest that deciduous dentine is more susceptible to resorption than permanent dentine and signals from the substrate play an important role in physiological resorption. [ABSTRACT FROM AUTHOR]

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis and periodontitis. Inflammation-induced bone loss of this sort results from elevated numbers of bone-resorbing osteoclasts. Gene targeting studies have shown that the transcription factor nuclear factor-?B (NF-?B) has a crucial role in osteoclast differentiation, and blocking NF-?B is a potential strategy for preventing inflammatory bone resorption. We tested this approach using a cell-permeable peptide inhibitor of the I?B-kinase complex, a crucial component of signal transduction pathways to NF-?B. The peptide inhibited RANKL-stimulated NF-?B activation and osteoclastogenesis both in vitro and in vivo. In addition, this peptide significantly reduced the severity of collagen-induced arthritis in mice by reducing levels of tumor necrosis factor-a and interleukin-1ß, abrogating joint swelling and reducing destruction of bone and cartilage. Therefore, selective inhibition of NF-?B activation offers an effective therapeutic approach for inhibiting chronic inflammatory diseases involving bone resorption. [ABSTRACT FROM AUTHOR]

Diallyl phthalate(DAP) was co-cured with vinyl monomers having long-chain alkyl groups, including vinyl laurate(VL), dioctyl fumarate(DOF), lauryl methacrylate(LMA), and stearyl methacrylate(SMA) with the intention of the improvement in mechanical properties of commercially important DAP resins. Thus the elongation and fracture energy increased with an increase in the mole fraction of comonomers and their extents were enlarged in the order VL < DOF < LMA < SMA, although a reverse tendency was observed for the tensile strength; in particular, LMA and SMA as comonomers showed a remarkable effect. These results are discussed in detail by considering the copolymerizability of DAP with comonomers and, moreover, the formation of microgel in microheterogeneous copolymerization systems. [ABSTRACT FROM AUTHOR]

To examine the environmental factors controlling the inshore recruitment dynamics of Anguilla japonica in the Oyodo River, Miyazaki Prefecture, Kyushu, glass eel samplings were carried out using fyke nets during winter (November-March) of 1994-2014. [ABSTRACT FROM AUTHOR]

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of α-galactosidase A. Ocular findings, such as cornea verticillata, cataracts, and retinal vascular tortuosity, serve as important diagnostic markers. We aimed to evaluate ocular phenotypes in α-galactosidase A-deficient (Fabry) rats and hypothesized that these rats would manifest ocular signs similar to those observed in patients. Slit lamp biomicroscopy was used to evaluate the cornea and lens, and retinal vasculature was examined by fluorescein angiography in WT and Fabry rats. Mass spectrometry was used to characterize and quantify ocular glycosphingolipids, and histology and electron microscopy revealed the location of the glycosphingolipid storage. We found that Fabry rats developed corneal and lenticular opacities to a statistically greater degree than WT rats. Retinal vascular morphology did not appear grossly different, but there was vascular leakage in at least one Fabry rat. Fabry rat eyes accumulated substrates of α-galactosidase A, and these α-galactosyl glycoconjugates were found in corneal keratocytes, lens fibers, and retinal vascular endothelial cells. Electron-dense lamellar inclusions were observed in keratocytes. Because Fabry rats recapitulate many ocular phenotypes observed in patients, they can be used to study disease pathogenesis and determine whether ocular findings serve as noninvasive indicators of therapeutic efficacy. [ABSTRACT FROM AUTHOR]

The bone mineral density (BMD) of astronauts decreases specifically in the weight-bearing sites during spaceflight. It seems that osteoclasts would be affected by a change in gravity; however, the molecular mechanism involved remains unclear. Here, we show that the mineral density of the pharyngeal bone and teeth region of TRAP-GFP/Osterix-DsRed double transgenic medaka fish was decreased and that osteoclasts were activated when the fish were reared for 56 days at the international space station. In addition, electron microscopy observation revealed a low degree of roundness of mitochondria in osteoclasts. In the whole transcriptome analysis, fkbp5 and ddit4 genes were strongly up-regulated in the flight group. The fish were filmed for abnormal behavior; and, interestingly, the medaka tended to become motionless in the late stage of exposure. These results reveal impaired physiological function with a change in mechanical force under microgravity, which impairment was accompanied by osteoclast activation. [ABSTRACT FROM AUTHOR]

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. [ABSTRACT FROM AUTHOR]