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Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states, such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) is a crucial lipid that regulates PIP 3 dynamics. Investigations of how PIP 3 is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information concerning membrane dynamics upon PIP 3 production. In the present study, we applied an optogenetic approach using the CRY2-CIBN system. Using this system, we revealed that local PIP 3 generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP 3 -induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors, such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.

Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of pulmonary fibrosis. Nintedanib, a multi-kinase inhibitor that targets several tyrosine kinases, including PDGF receptor (PDGFR), was recently approved as an anti-fibrotic agent to reduce the deterioration of FVC in patients with idiopathic pulmonary fibrosis (IPF). However, the effects of PDGFR-α or -β on pulmonary fibrosis remain unclear. In an attempt to clarify their effects, we herein used blocking antibodies specific for PDGFR-α (APA5) and -β (APB5) in a bleomycin (BLM)-induced pulmonary fibrosis mouse model. The effects of these treatments on the growth of lung fibroblasts were examined using the 3H-thymidine incorporation assay in vitro. The anti-fibrotic effects of these antibodies were investigated with the Ashcroft score and collagen content of lungs treated with BLM. Their effects on inflammatory cells in the lungs were also analyzed using bronchoalveolar lavage fluid. We investigated damage to epithelial cells and the proliferation of fibroblasts in the lungs. APA5 and APB5 inhibited the phosphorylation of PDGFR-α and -β as well as the proliferation of lung fibroblasts induced by PDGF-AA and BB. The administration of APB5, but not APA5 effectively inhibited BLM-induced pulmonary fibrosis in mice. Apoptosis and the proliferation of epithelial cells and fibroblasts were significantly decreased by the treatment with APB5, but not by APA5. The late treatment with APB5 also ameliorated fibrosis in lungs treated with BLM. These results suggest that PDGFR-α and -β exert different effects on BLM-induced pulmonary fibrosis in mice. A specific approach using the blocking antibody for PDGFR-β may be useful for the treatment of pulmonary fibrosis., Competing Interests: The authors have declared that no competing interests exist.

Background: The combination of oral anticoagulant (OAC) and antiplatelet drug (APD) increases the bleeding risk in atrial fibrillation (AF). Non-vitamin K antagonist OAC (NOAC) have been increasingly used since 2011. We investigated current status, time trends and outcomes of AF patients using combination therapy in 2011-2017. Methods and Results: The Fushimi AF Registry is a community-based prospective survey of AF patients in Fushimi-ku, Kyoto, Japan. Of 2,378 patients with OAC at enrollment, 521 (22%) received combination therapy, while 1,857 (78%) received OAC alone. When compared with OAC alone, combination therapy patients had more comorbidities, but approximately 30% had no atherosclerotic disease. From 2011 to 2017, the prevalence of combination therapy decreased from 26% to 14%. The prevalence of NOAC increased in those on combination therapy. Off-label under-dosing of NOAC increased year by year, especially in combination therapy. During follow-up, the incidence of major bleeding (hazard ratio [HR], 1.42; 95% CI: 1.03-1.95) and stroke/systemic embolism (HR, 1.48; 95% CI: 1.09-2.00) was higher in the combination therapy than in the OAC alone group., Conclusions: In Japanese AF patients receiving OAC, the prevalence of combination therapy decreased, with the proportion of NOAC use increasing in 2011-2017. Many patients, however, received off-label NOAC under-dosing, especially in the combination therapy group. Patients with combination therapy had higher incidences of major bleeding as well as stroke/systemic embolism, compared with OAC monotherapy.

Sulindac sulfone is a metabolite of sulindac, a non-steroidal anti-inflammatory drug (NSAID), without anti-inflammatory ability. However, sulindac sulfone has been reported to significantly reduce polyps in patients with colorectal adenomatous polyposis in clinical trials. Thus, sulindac sulfone is expected to be useful for the chemoprevention of neoplasia with few side effects related to anti-inflammatory ability. To date, the molecular targets of sulindac sulfone have not yet fully investigated. Therefore, in order to newly identify sulindac sulfone-binding proteins, we generated sulindac sulfone-fixed FG beads and purified sulindac sulfone-binding proteins from human colon cancer HT-29 cells. we identified mitochondrial outer membrane proteins voltage-dependent anion channel (VDAC) 1 and VDAC2 as novel molecular targets of sulindac sulfone, and sulindac sulfone directly bound to both VDAC1 and VDAC2. Double knockdown of VDAC1 and VDAC2 by siRNA inhibited growth and arrested the cell cycle at G1 phase in HT-29 cells. Depletion of VDAC1 and VDAC2 also inhibited the mTORC1 pathway with a reduction in cyclin D1. Interestingly, these effects were consistent with those of sulindac sulfone against human colon cancer cells, suggesting that sulindac sulfone negatively regulates the function of VDAC1 and VDAC2. In the present study, our data suggested that VDAC1 and VDAC2 are direct targets of sulindac sulfone which suppresses the mTORC1 pathway and induces G1 arrest., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The nucleus accumbens contains delta-opioid receptors that may decrease inhibitory neurotransmission. As GABA B receptors inhibit dopamine release, decrease in activation of GABA B receptors may be a mediator of delta-opioid receptor-induced accumbal dopamine efflux. If so, accumbal dopamine efflux induced by delta-opioid receptor activation should be suppressed by stimulating GABA B receptors. As delta-opioid receptors are further subdivided into delta1- and delta2-opioid receptors, we analysed the effects of the GABA B receptor agonist baclofen on delta1- and delta2-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were applied intracerebrally through the dialysis probe. Doses of compounds show total amount administered (mol) during 25-50 min infusions. Baclofen (2.5 and 5.0 nmol), which did not alter basal dopamine levels, inhibited the delta1-opioid receptor agonist DPDPE (5.0 nmol)-induced dopamine efflux. Baclofen (2.5 and 5.0 nmol) also inhibited the delta2-opioid receptor agonist deltorphin II (25.0 nmol)-induced dopamine efflux. A low dose of the GABA B receptor antagonist 2-hydroxysaclofen (100.0 pmol), which failed to alter basal accumbal dopamine levels, counteracted the inhibitory effects of baclofen (5.0 nmol) on DPDPE (5.0 nmol)- and deltorphin II (25.0 nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABA B receptor-mediated inhibition of accumbal dopaminergic activity facilitates activation of delta1- and delta2-opioid receptor-induced increases in accumbal dopamine efflux. This study suggests that activation of delta1- and delta2-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABA B receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Purpose- Atrial fibrillation (AF) is a common arrhythmic disorder among the elderly and sometimes progresses from paroxysmal to sustained (persistent or permanent) types. Clinical outcomes of patients with progression of AF were unknown. This study assessed the characteristics of patients with AF progression and the impact of AF progression on various clinical events. Methods- The Fushimi AF Registry is a community-based prospective survey of the patients with AF in Fushimi-ku, Kyoto. Analyses were performed on 4045 patients, which included 1974 paroxysmal AF (PAF; 48.8%) and 2071 sustained (persistent or permanent) AF (SAF; 51.2%) at baseline. Results- During the median follow-up period of 1105 days, progression of AF occurred in 252 patients with PAF (4.22 per 100 person-years). Multivariate Cox regression analysis demonstrated that progression of AF was significantly associated with an increased risk of ischemic stroke or systemic embolism (adjusted hazard ratio [HR], 4.10; 95% CI, 1.95-8.24; P<0.001 [versus PAF without progression]; adjusted HR, 2.20; 95% CI, 1.11-4.00; P=0.025 [versus SAF]) during progression period from paroxysmal to sustained forms. The risk after the progression was equivalent to SAF (adjusted HR, 1.54; 95% CI, 0.78-2.75; P=0.201 [versus SAF]). AF progression was significantly associated with a higher risk of hospitalization for heart failure (adjusted HR, 2.70; 95% CI, 1.55-4.52; P<0.001 [versus PAF without progression]; adjusted HR, 1.81; 95% CI, 1.08-2.88; P=0.026 [versus SAF]). Conclusions- Progression of AF was associated with increased risk of clinical adverse events during arrhythmia progression period from PAF to SAF among Japanese patients with AF. The risk of adverse events was transiently elevated during progression period from PAF to SAF and declined to the level equivalent to SAF after the progression. Clinical Trial Registration- URL: http://www.umin.ac.jp/ctr/ . Unique identifier: UMIN000005834.

Cancer stem cells (CSCs) represent a minor population that have clonal tumor initiation and self-renewal capacity and are responsible for tumor initiation, metastasis, and therapeutic resistance. CSCs reside in niches, which are composed of diverse types of stromal cells and extracellular matrix components. These stromal cells regulate CSC-like properties by providing secreted factors or by physical contact. Fibrocytes are differentiated from bone marrow-derived CD14 + monocytes and have features of both macrophages and fibroblasts. Accumulating evidence has suggested that stromal fibrocytes might promote cancer progression. However, the role of fibrocytes in the CSC niches has not been revealed. We herein report that human fibrocytes enhanced the CSC-like properties of lung cancer cells through secreted factors, including osteopontin, CC-chemokine ligand 18, and plasminogen activator inhibitor-1. The PIK3K/AKT pathway was critical for fibrocytes to mediate the CSC-like functions of lung cancer cells. In human lung cancer specimens, the number of tumor-infiltrated fibrocytes was correlated with high expression of CSC-associated protein in cancer cells. These results suggest that fibrocytes may be a novel cell population that regulates the CSC-like properties of lung cancer cells in the CSC niches., (Copyright © 2018. Published by Elsevier B.V.)

Lactoferrin is a secretory protein with various physiological functions. Bovine lactoferrin has been demonstrated to alleviate psychological stresses in rats, but this effect in humans has not yet been assessed. The present study aimed to investigate the changes in psychological stress markers following a calculation task, with either lactoferrin or a placebo orally administered prior to the task. A total of 16 healthy female college students visited Juntendo University, Inzai, Japan following an overnight fast. Subjects were quietly seated for 15 min to stabilize the respiratory rate at 0.25 Hz (one breath every 4 sec). Then, subjects provided saliva, ingested either lactoferrin (800 mg of lactoferrin + soy milk) or a placebo (soy milk), remained seated for another 15 min with respiration rate at 0.25 Hz, and performed a calculation task. The task comprised two sets of 15-min calculations, with a 5-min interval between sets. Each calculation set consisted of various multiplications and divisions using pairs of three-digit numbers. Following the calculation task, saliva was collected again. Heart rate was also monitored to identify the frequency domain of heart-rate variability. The calculation task resulted in increased activity of salivary amylase, and decreased concentration of chromogranin A for both lactoferrin (P=0.028 and P<0.001, respectively) and placebo (P=0.003 and P<0.001, respectively) treatments. The degrees of changes in these salivary markers were similar between the two treatments. Heart rate variability exhibited an increase in the high-frequency (HF) component (P=0.022) and a decrease in low-frequency (LF)/HF and LF/(LF+HF) ratios (both P<0.001) following the calculation task under the placebo condition, demonstrating an upregulation of parasympathetic and a downregulation of sympathetic nervous activities. These changes in parasympathetic (HF) and sympathetic (LF/HF) activities, however, were alleviated by lactoferrin compared with the placebo (P=0.007 and P=0.026, respectively). Collectively these results suggest that oral lactoferrin may mitigate psychological stress in humans.

In advanced bladder cancer, cisplatin-based chemotherapy has been the standard treatment for many years, but there are many problems in terms of side-effects. Recently, a number of clinical trials using molecular-targeted agents have been conducted, and new therapies are expected that could replace conventional cytotoxic chemotherapy. We herein report that concurrent treatment with fibroblast growth factor receptor (FGFR) inhibitor BGJ398 and the novel histone deacetylase (HDAC) inhibitor OBP-801/YM753/spiruchostatin A synergistically inhibited cell growth and markedly induced apoptosis in high-grade bladder cancer cells. This combination activated caspase-3, -8 and -9, and the pan-caspase inhibitor zVAD-fmk significantly reduced the apoptotic response to the combined treatment. The combination upregulated the expression of Bim, one of the pro-apoptotic molecules. In the present study, Bim siRNA efficiently reduced apoptosis induced by the co-treatment of BGJ398 and OBP-801. Therefore, the apoptosis induced by the combination was shown to be at least partially dependent on Bim. Taken together, these results suggest that the combination of BGJ398 and OBP-801 is a novel high potential therapeutic strategy for muscle-invasive bladder cancer.

We analysed the roles of orexin receptors in the effects of orexin-A on KCl-induced increases in intracellular calcium ion levels ([Ca 2+ ] i ) in C-fiber-like small neurons of rats with inflammation induced by intraplantar injection of carrageenan into the hind paw. Controls were treated with saline. Paw withdrawal and threshold forces in response to tactile stimuli were determined using von Frey filaments. [Ca 2+ ] i in C-fiber-like neurons derived from dorsal root ganglia was visualised using a calcium fluorescence probe. Changes in neuronal [Ca 2+ ] i were assessed as relative fluorescence intensity (F/F 0 ). One day after carrageenan injection, the paw withdrawal response to tactile stimuli and the paw withdrawal threshold were increased and reduced, respectively. KCl loading of neurons from either carrageenan-treated or control rats increased F/F 0 to about 2.0. KCl-induced increases in F/F 0 of carrageenan-treated, but not control, rats were inhibited by orexin-A. The OX 1 and OX 2 receptor antagonist MK-4305, but not the OX 1 receptor antagonist SB334867, counteracted the effects of orexin-A on the KCl-induced increase in F/F 0 . These results suggest that OX 2 , but not OX 1 receptors mediate the inhibitory effect of orexin-A on KCl-induced increases in [Ca 2+ ] i in C-fiber-like neurons of rats with inflammation.

The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABA A receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABA A receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABA A receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25-50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABA A receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABA A receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABA A receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Nintedanib, a tyrosine kinase inhibitor that is specific for platelet-derived growth factor receptors (PDGFR), fibroblast growth factor receptors (FGFR), and vascular endothelial growth factor receptors (VEGFR), has recently been approved for idiopathic pulmonary fibrosis. Fibrocytes are bone marrow-derived progenitor cells that produce growth factors and contribute to fibrogenesis in the lungs. However, the effects of nintedanib on the functions of fibrocytes remain unclear., Methods: Human monocytes were isolated from the peripheral blood of healthy volunteers. The expression of growth factors and their receptors in fibrocytes was analyzed using ELISA and Western blotting. The effects of nintedanib on the ability of fibrocytes to stimulate lung fibroblasts were examined in terms of their proliferation. The direct effects of nintedanib on the differentiation and migration of fibrocytes were also assessed. We investigated whether nintedanib affected the accumulation of fibrocytes in mouse lungs treated with bleomycin., Results: Human fibrocytes produced PDGF, FGF2, and VEGF-A. Nintedanib and specific inhibitors for each growth factor receptor significantly inhibited the proliferation of lung fibroblasts stimulated by the supernatant of fibrocytes. Nintedanib inhibited the migration and differentiation of fibrocytes induced by growth factors in vitro. The number of fibrocytes in the bleomycin-induced lung fibrosis model was reduced by the administration of nintedanib, and this was associated with anti-fibrotic effects., Conclusions: These results support the role of fibrocytes as producers of and responders to growth factors, and suggest that the anti-fibrotic effects of nintedanib are at least partly mediated by suppression of fibrocyte function.

Quality assurance (QA) of clinical electron beams is essential for performing accurate and safe radiation therapy. However, with advances in radiation therapy, QA has become increasingly labor-intensive and time-consuming. In this paper, we propose a tissue-equivalent plastic scintillator for quick and easy QA of clinical electron beams. The proposed tool comprises a plastic scintillator plate and a charge-coupled device camera that enable the scintillation light by electron beams to be recorded with high sensitivity and high spatial resolution. Further, the Cerenkov image is directly subtracted from the scintillation image to discriminate Cerenkov emissions and accurately measure the dose profiles of electron beams with high spatial resolution. Compared with conventional methods, discrepancies in the depth profile improved from 7% to 2% in the buildup region via subtractive corrections. Further, the output brightness showed good linearity with dose, good reproducibility (deviations below 1%), and dose rate independence (within 0.5%). The depth of 50% dose measured with the tool, an index of electron beam quality, was within  ±0.5 mm of that obtained with an ionization chamber. Lateral brightness profiles agreed with the lateral dose profiles to within 4% and no significant improvement was obtained using Cerenkov corrections. Field size agreed to within 0.5 mm with those obtained with ionization chamber. For clinical QA of electron boost treatment, a disk scintillator that mimics the shape of a patient's breast is applied. The brightness distribution and dose, calculated using a treatment planning system, was generally acceptable for clinical use, except in limited zones. Overall, the proposed plastic scintillator plate tool efficiently performs QA for electron beam therapy and enables simultaneous verification of output constancy, beam quality, depth, and lateral dose profiles during monthly QAs at lower doses of irradiation (small monitor units, MUs).

The nucleus accumbens is a terminal area of the mesolimbic dopaminergic system that arises in the ventral tegmental area. Opioids are thought to enhance dopaminergic activity in the nucleus accumbens by activating δ- and μ-opioid receptors in the ventral tegmental area. However, δ- and μ-opioid receptor agonists increase extracellular levels of accumbal dopamine when infused directly into the nucleus accumbens of rats. Therefore, the roles of δ- and μ-opioid receptors in regulation of accumbal dopaminergic neural activity have been analyzed by using δ- and μ-opioid receptor ligands. This review describes the mechanisms underlying the stimulatory effects on accumbal dopamine efflux, which are induced by local administration of δ- and μ-opioid receptor agonists into the nucleus accumbens of freely moving rats. The focus of this article is neurochemical studies that use in vivo microdialysis techniques. Taken together, the in vivo neurochemical evidence from these studies indicates that δ- and μ-opioid receptor agonists increase accumbal dopamine efflux by activating naloxone-sensitive opioid receptors, and by mechanisms independent of naloxone-sensitive opioid receptors, in the nucleus accumbens.

Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H + -ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.

Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gα q and Gα s ). By utilizing this strategy, we demonstrate successful regulation of Ca 2+ and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.

Autophagy is a self-protection process against reactive oxygen species (ROS). The intracellular level of ROS increased when cells were cultured under nutrient starvation. Antioxidants such as glutathione and ascorbic acid play an important role in ROS removal. However, the cellular redox state in the autophagic pathway is still unclear. Herein, we developed a new redox-sensitive probe with a disulfide-linked silica scaffold to enable the sensing of the reduction environment in cell organelles. This redox-responsive silica nanoprobe (ReSiN) could penetrate the plant cell wall and release fluorescent molecules in response to redox states. By applying the ReSiN to tobacco BY-2 cells and tracing the distribution of fluorescence, we found a higher reducing potential in the central vacuole than in the autolysosomes. Upon cysteine protease inhibitor (E64-c) treatment in sucrose-free medium, the disulfide-silica structures of the ReSiNs were broken down in the vacuoles but were not degraded and were accumulated in the autolysosomes. These results reveal the feasibility of our nanoprobe for monitoring the endocytic and macroautophagic pathways. These pathways merge upstream of the central vacuole, which is the final destination of both pathways. In addition, different redox potentials were observed in the autophagic pathway. Finally, the expression of the autophagy-related protein (Atg8) fused with green fluorescence protein confirmed that the ReSiN treatment itself did not induce the autophagic pathway under normal physiological conditions, indicating the versatility of this nanoprobe in studying stimuli-triggered autophagy-related trafficking.

Cholinergic neurons in the nucleus accumbens express delta- and mu-opioid receptors that are thought to inhibit neural activity. Delta- and mu-opioid receptors are divided into delta1- and delta2-opioid receptors and mu1- and mu2-opioid receptors, respectively. We analysed the roles of delta- and mu-opioid receptor subtypes in regulating accumbal acetylcholine efflux of freely moving rats using in vivo microdialysis. Other than naloxonazine, given intraperitoneally, delta- and mu-opioid receptor ligands were administered intracerebrally through the dialysis probe. Doses of these compounds indicate total amount (mol) over an infusion time of 30-60min. To monitor basal acetylcholine, a low concentration of physostigmine (50nM) was added to the perfusate. The delta1-opioid receptor agonist DPDPE (3 and 300pmol) and delta2-opioid receptor agonist deltorphin II (3 and 30pmol) decreased accumbal acetylcholine in a dose-related manner. DPDPE (300pmol)- and deltorphin II (3pmol)-induced reductions in acetylcholine were each inhibited by the delta1-opioid receptor antagonist BNTX (0.3pmol) and delta2-opioid receptor antagonist naltriben (15pmol), respectively. The mu-opioid receptor agonists endomorphin-1 and endomorphin-2 (6 and 30nmol) decreased acetylcholine in a dose-related manner. Endomorphin-1- and endomorphin-2 (30nmol)-induced reductions in acetylcholine were prevented by the mu-opioid receptor antagonist CTOP (3nmol). The mu1-opioid receptor antagonist naloxonazine (15mg/kg ip), which inhibits endomorphin-1 (15nmol)-induced accumbal dopamine efflux, did not alter endomorphin-1- or endomorphin-2 (30nmol)-induced reductions in acetylcholine efflux. This study provides in vivo evidence for delta1-, delta2- and mu2-opioid receptors, but not mu1-opioid receptors, that inhibit accumbal cholinergic neural activity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The prognosis of muscle-invasive bladder cancer with metastasis is poor. There have been no therapeutic improvements for many years, and an innovative therapy for muscle-invasive bladder cancer has been awaited to replace the conventional cytotoxic chemotherapy. Here, we show a candidate method for the treatment of bladder cancer. The combined treatment with a novel histone deacetylase (HDAC) inhibitor, OBP-801, and celecoxib synergistically inhibited cell growth and markedly induced apoptosis through the caspase-dependent pathway in high-grade bladder cancer cells. Furthermore, the combined treatment induced expression of death receptor 5 (DR5). We identified that knockdown of DR5 by small interfering RNA (siRNA) significantly suppressed apoptosis by the combined treatment. Therefore, we conjectured that the apoptosis induced by OBP-801 and celecoxib is at least partially dependent on DR5. However, it was interesting that the combined treatment drastically suppressed expression of DR5 ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). These data suggest that there is no involvement of TRAIL in the induction of apoptosis by the combination, regardless of the dependence of DR5. Moreover, xenograft studies using human bladder cancer cells showed that the combined therapy suppressed tumor growth by upregulating expressions of DR5 and Bim. The inhibition of tumor growth was significantly more potent than that of each agent alone, without significant weight loss. This combination therapy provided a greater benefit than monotherapy in vitro and in vivo These data show that the combination therapy with OBP-801 and celecoxib is a potential novel therapeutic strategy for patients with muscle-invasive bladder cancer. Mol Cancer Ther; 15(9); 2066-75. ©2016 AACR., (©2016 American Association for Cancer Research.)

Cyanobacteriochromes (CBCRs) are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB) and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV). The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1&#95;C0023g2) from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near-infrared fluorescence from mammalian cells harboring PCB-binding AM1&#95;C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.

Lake and river ice seasonality (dates of ice freeze and breakup) responds sensitively to climatic change and variability. We analyzed climate-related changes using direct human observations of ice freeze dates (1443-2014) for Lake Suwa, Japan, and of ice breakup dates (1693-2013) for Torne River, Finland. We found a rich array of changes in ice seasonality of two inland waters from geographically distant regions: namely a shift towards later ice formation for Suwa and earlier spring melt for Torne, increasing frequencies of years with warm extremes, changing inter-annual variability, waning of dominant inter-decadal quasi-periodic dynamics, and stronger correlations of ice seasonality with atmospheric CO2 concentration and air temperature after the start of the Industrial Revolution. Although local factors, including human population growth, land use change, and water management influence Suwa and Torne, the general patterns of ice seasonality are similar for both systems, suggesting that global processes including climate change and variability are driving the long-term changes in ice seasonality.

Despite evidence from a number of Earth systems that abrupt temporal changes known as regime shifts are important, their nature, scale and mechanisms remain poorly documented and understood. Applying principal component analysis, change-point analysis and a sequential t-test analysis of regime shifts to 72 time series, we confirm that the 1980s regime shift represented a major change in the Earth's biophysical systems from the upper atmosphere to the depths of the ocean and from the Arctic to the Antarctic, and occurred at slightly different times around the world. Using historical climate model simulations from the Coupled Model Intercomparison Project Phase 5 (CMIP5) and statistical modelling of historical temperatures, we then demonstrate that this event was triggered by rapid global warming from anthropogenic plus natural forcing, the latter associated with the recovery from the El Chichón volcanic eruption. The shift in temperature that occurred at this time is hypothesized as the main forcing for a cascade of abrupt environmental changes. Within the context of the last century or more, the 1980s event was unique in terms of its global scope and scale; our observed consequences imply that if unavoidable natural events such as major volcanic eruptions interact with anthropogenic warming unforeseen multiplier effects may occur., (© 2015 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

Bevacizumab exerts anti-angiogenic effects in cancer patients by inhibiting vascular endothelial growth factor (VEGF). However, its use is still limited due to the development of resistance to the treatment. Such resistance can be regulated by various factors, although the underlying mechanisms remain incompletely understood. Here we show that bone marrow-derived fibrocyte-like cells, defined as alpha-1 type I collagen-positive and CXCR4-positive cells, contribute to the acquired resistance to bevacizumab. In mouse models of malignant pleural mesothelioma and lung cancer, fibrocyte-like cells mediate the resistance to bevacizumab as the main producer of fibroblast growth factor 2. In clinical specimens of lung cancer, the number of fibrocyte-like cells is significantly increased in bevacizumab-treated tumours, and correlates with the number of treatment cycles, as well as CD31-positive vessels. Our results identify fibrocyte-like cells as a promising cell biomarker and a potential therapeutic target to overcome resistance to anti-VEGF therapy.

This study used in vivo microdialysis to examine the effects of intragingival application of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg-LPS) on gingival tumour necrosis factor (TNF)-α and interleukin (IL)-6 levels in rats. A microdialysis probe with an injection needle attached to the surface of the dialysis membrane was implanted into the gingiva of the upper incisor. For comparison, the effects of LPS derived from Escherichia coli (Ec-LPS) on IL-6 and TNF-α levels were also analysed. Pg-LPS (1 μg/1 μL) or Ec-LPS (1 or 6 μg/1 μL) was applied by microsyringe, with gingival dialysates collected every hour. Enzyme-linked immunosorbent assay (ELISA) revealed that gingival dialysates contained approximately 389 pg·mL⁻¹ of IL-6 basally; basal TNF-α levels were lower than the detection limit of the ELISA. Pg-LPS failed to alter IL-6 levels but markedly increased TNF-α levels, which remained elevated for 2 h after treatment. Neither IL-6 nor TNF-α were affected by Ec-LPS. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the gingiva expresses Toll-like receptor (TLR) 2 and TLR4 mRNA. Immunohistochemical examination showed that TLR2 and TLR4 are expressed by gingival epithelial cells. The present study provides in vivo evidence that locally applied Pg-LPS, but not Ec-LPS, into the gingiva transiently increases gingival TNF-α without affecting IL-6. The present results suggest that TLR2 but not TLR4 expressed on gingival epithelial cells may mediate the Pg-LPS-induced increase in gingival TNF-α in rats.

Two-dimensional (2D) coordination polymers consisting of Mn(III) Schiff-base complexes and dicarboxylic acids, [{Mn(salen)}4(L1)](PF6)2·(CH3OH)2 (C4; H2L1 = adipid acid) and [{Mn(salen)}4(L2)](PF6)2·(CH3OH)4 (C4'; H2L2 = E,E-1,3-butadiene-1,4-dicarboxylic acid) (salen(2-) = N,N'-(ethylene)bis(salicylideneiminato), were synthesized by using a one-pot reaction and characterized by using single-crystal X-ray crystallographic analysis. One-dimensional (1D) chains composed of Mn(salen) dimers, [Mn2], bridged by carboxylato ligands (-[Mn2]-OCO--[Mn2]-), were linked by dicarboxylato ligands with n-butyl (-C4H8-) (C4) and butadienyl aliphatic groups (-C4H4-) (C4'). From static magnetic measurements on both C4 and C4', there were ferromagnetic interactions between the Mn(III) ions through the phenoxo oxygen atoms of the salen(2-), and antiferromagnetic interactions between the Mn(III) ions through carboxylato ligands (-OCO-). As a result, weak ferromagnetism occurred because of the zigzag-shaped chain structure of C4 and C4', and magnetic anisotropy for Mn(salen). In the magnetization curves for C4', weak interchain interactions (Jlinker) occurred through the π-conjugated butadienyl linkers in C4', which C4 did not have. In other words, changing from saturated to unsaturated aliphatic groups in the dicarboxylic acid linkers resulted in weak interactions between 1D-magnetic chain moieties. Therefore, in the case of only C4', antiferromagnetic phase transition appeared at 2.3 K. Both coordination polymers exhibited slow relaxation of the magnetizations, which originated from SCM moieties, because C4 and C4' showed magnetic correlations. It is noteworthy that alternating current (ac) susceptibilities for C4' are frequency-dependent around the Néel temperature. From analysis of the ac susceptibilities for C4, α (dispersion coefficient of the relaxation of magnetization) varied linearly with 1/T. This signifies that C4 behaved as an SCM with a single relaxation process. On the other hand, in α versus 1/T plots for C4', an inflection point was observed at the Néel temperature, indicating that Jlinkers had an effect on the distribution of the relaxation times. Moreover, the inflection point for C4' disappeared when a dc magnetic field was applied. This is the first report showing a direct correlation between an antiferromagnetic phase transition and slow magnetic relaxation.

Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB) protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3β (GSK-3β) induces the proteasomal degradation of cyclin D1. The addition of GSK-3β inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3β.

The changes in March mean temperatures in Edo (Tokyo), Japan, since the seventeenth century, were reconstructed using phenological data for the cherry blossoms of Prunus jamasakura deduced from old diaries and chronicles. The observations of the time of full blossoming and of cherry blossom viewing parties were acquired and used to construct a full-blossoming phenological data series for P. jamasakura. Phenological data from 207 of the years from 1601 to 1905 were used for this study. The reconstructed temperatures suggested the existence of two cold periods (the second half of the seventeenth century and the beginning of the nineteenth century), during which times the estimated March mean temperatures were about 4 °C and 5 °C, respectively. These two cold periods at Edo coincided with those reconstructed at Kyoto in previous studies. These cold periods coincided with two less extreme periods, the Maunder and Dalton minima, in the long-term solar variation cycle.

Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

Intra-accumbal infusion of the α1-adrenergic agonist methoxamine, which has comparable affinity for α1A-, α1B- and α1D-adrenoceptor subtypes, fails to alter noradrenaline efflux but reduces dopamine efflux in the nucleus accumbens of rats. In-vivo microdialysis experiments were carried out to analyse the putative contribution of α1A-, α1B- and α1D-adrenoceptor subtypes to the methoxamine-induced decrease in accumbal dopamine efflux in freely moving rats. The drugs used were dissolved in the infusion medium and administered locally through a dialysis membrane. Intra-accumbal infusions of the α1A-adrenoceptor antagonist 5-methylurapidil (6 pmol), the α1B-adrenoceptor antagonist cyclazosin (0.6 and 6 pmol) and the α1D-adrenoceptor antagonist BMY 7378 (0.6 pmol) did not alter accumbal efflux of noradrenaline or dopamine: pretreatment with each of these α1-adrenoceptor subtype-selective antagonists counteracted the methoxamine (24 pmol)-induced decrease in accumbal dopamine efflux. Doses indicated are the total amount of drug administered over a 60-min infusion period. These results clearly suggest that the α1A-, α1B- and α1D-adrenoceptor subtypes in the nucleus accumbens mediate the α1-adrenergic agonist methoxamine-induced decrease in accumbal dopamine efflux. The present study also provides in-vivo neurochemical evidence indicating that concomitant, but not separate, activation of the α1A-, α1B- and α1D-adrenoceptors in the nucleus accumbens is required for α1-adrenergic inhibition of accumbal dopaminergic activity.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors in cyanobacteria that absorb visible and near-ultraviolet light. CBCRs are divided into two types based on the type of chromophore they contain: phycocyanobilin (PCB) or phycoviolobilin (PVB). PCB-binding CBCRs reversibly photoconvert at relatively long wavelengths, i.e., the blue-to-red region, whereas PVB-binding CBCRs reversibly photoconvert at shorter wavelengths, i.e., the near-ultraviolet to green region. Notably, prior to this report, CBCRs containing biliverdin (BV), which absorbs at longer wavelengths than do PCB and PVB, have not been found. Herein, we report that the typical red/green CBCR AM1&#95;1557 from the chlorophyll d-bearing cyanobacterium Acaryochloris marina can bind BV almost comparable to PCB. This BV-bound holoprotein reversibly photoconverts between a far red light-absorbing form (Pfr, λmax = 697 nm) and an orange light-absorbing form (Po, λmax = 622 nm). At room temperature, Pfr fluoresces with a maximum at 730 nm. These spectral features are red-shifted by 48~77 nm compared with those of the PCB-bound domain. Because the absorbance of chlorophyll d is red-shifted compared with that of chlorophyll a, the BV-bound AM1&#95;1557 may be a physiologically relevant feature of A. marina and is potentially useful as an optogenetic switch and/or fluorescence imager.

In orthodontics, a reduction in static friction between the brackets and wire is important to enable easy tooth movement. The aim of this study was to examine the effects of a homogeneous diamond-like carbon (DLC) coating on the whole surfaces of slots in stainless steel orthodontic brackets on reducing the static friction between the brackets and the wire. The DLC coating was characterized using Raman spectroscopy, surface roughness and contact angle measurements, and SEM observations. Rectangular stainless steel and titanium-molybdenum alloy wires with two different sizes were employed, and the static friction between the brackets and wire was measured under dry and wet conditions. The DLC coating had a thickness of approximately 1.0 μm and an amorphous structure was identified. The results indicated that the DLC coating always led to a reduction in static friction.

Circulating fibrocytes have been reported to migrate into the injured lungs, and contribute to fibrogenesis via CXCL12-CXCR4 axis. In contrast, we report that imatinib mesylate prevented bleomycin (BLM)-induced pulmonary fibrosis in mice by inhibiting platelet-derived growth factor receptor (PDGFR), even when it was administered only in the early phase. The goal of this study was to test the hypothesis that platelet-derived growth factor (PDGF) might directly contribute to the migration of fibrocytes to the injured lungs. PDGFR expression in fibrocytes was examined by flow cytometry and RT-PCR. The migration of fibrocytes was evaluated by using a chemotaxis assay for human fibrocytes isolated from peripheral blood. The numbers of fibrocytes triple-stained for CD45, collagen-1, and CXCR4 were also examined in lung digests of BLM-treated mice. PDGFR mRNA levels in fibrocytes isolated from patients with idiopathic pulmonary fibrosis were investigated by real-time PCR. Fibrocytes expressed both PDGFR-α and -β, and migrated in response to PDGFs. PDGFR inhibitors (imatinib, PDGFR-blocking antibodies) suppressed fibrocyte migration in vitro, and reduced the number of fibrocytes in the lungs of BLM-treated mice. PDGF-BB was a stronger chemoattractant than the other PDGFs in vitro, and anti-PDGFR-β-blocking antibody decreased the numbers of fibrocytes in the lungs compared with anti-PDGFR-α antibody in vivo. Marked expression of PDGFR-β was observed in fibrocytes from patients with idiopathic pulmonary fibrosis compared with healthy subjects. These results suggest that PDGF directly functions as a strong chemoattractant for fibrocytes. In particular, the PDGF-BB-PDGFR-β biological axis might play a critical role in fibrocyte migration into the fibrotic lungs.

Background: Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease., Methods: C57BL/6 wild-type (WT) and SP-D-/- mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D., Results: Dp-challenged SP-D-/- mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D-/- mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D-/- mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D-/- mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D-/- mice., Conclusion: These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.

Objective: Thymidine phosphorylase (TP) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is induced by tumor necrosis factor α (TNFα) and other cytokines that have been reported to be major inflammation mediators in RA. We previously demonstrated that TP plays an important role in angiogenesis and tumor growth, invasion, and metastasis. The aim of this study was to investigate whether the role of TP in the pathogenesis of RA is similar to its role in tumors., Methods: In FLS obtained from 2 patients with RA, the expression of TP, interferon-γ (IFNγ)-inducible protein 10 (CXCL10), and other cytokines was measured by quantitative real-time polymerase chain reaction, immunoblotting, and enzyme-linked immunosorbent assays. Microarray analysis was performed using FLS transfected with TYMP complementary DNA and treated with a TP inhibitor., Results: The expression of TP in FLS was up-regulated by TNFα, interleukin-1β (IL-1β), IL-17, IFNγ, and lipopolysaccharide. Microarray analysis of FLS overexpressing TP identified CXCL10 as a thymidine phosphorylase-related gene. The expression of CXCL10 was induced by TNFα, and this induction was suppressed by TYMP small interfering RNA and TP inhibitor. Furthermore, the combination of TNFα and IFNγ synergistically augmented the expression of TP and CXCL10. TP-induced CXCL10 expression was suppressed by the antioxidant EUK-8. In the synovial tissue of patients with RA, TP levels were significantly correlated with CXCL10 expression., Conclusion: The combination of TNFα and IFNγ strongly induced the expression of thymidine phosphorylase in RA FLS. The induction of thymidine phosphorylase enhanced the expression of CXCL10, which may contribute to the Th1 phenotype and bone destruction observed in RA., (Copyright © 2014 by the American College of Rheumatology.)

Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2). The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3 × 10(3) s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in various biological functions, including cell survival, proliferation, migration, and adhesion. FAK is an essential factor for transforming growth factor β to induce myofibroblast differentiation. In the present study, we investigated whether the targeted inhibition of FAK by using a specific inhibitor, TAE226, has the potential to regulate pulmonary fibrosis. TAE226 showed inhibitory activity of autophosphorylation of FAK at tyrosine 397 in lung fibroblasts. The addition of TAE226 inhibited the proliferation of lung fibroblasts in response to various growth factors, including platelet-derived growth factor and insulin-like growth factor I, in vitro. TAE226 strongly suppressed the production of type I collagen by lung fibroblasts. Furthermore, treatment of fibroblasts with TAE226 reduced the expression of α-smooth muscle actin induced by transforming growth factor β, indicating the inhibition of differentiation of fibroblasts to myofibroblasts. Administration of TAE226 ameliorated the pulmonary fibrosis induced by bleomycin in mice even when used late in the treatment. The number of proliferating mesenchymal cells was reduced in the lungs of TAE226-treated mice. These data suggest that FAK signal plays a significant role in the progression of pulmonary fibrosis and that it can become a promising target for therapeutic approaches to pulmonary fibrosis.

The effects of intra-accumbal infusion of selective agonists for the β-adrenoceptor subtypes on the noradrenaline and dopamine efflux in the nucleus accumbens of freely moving rats were investigated, using in vivo microdialysis. Neither β1-(dobutamine: 0.06 and 0.12 pmol) nor β2-adrenoceptor agonist (salbutamol: 0.36 and 3.6 pmol) altered the basal noradrenaline and dopamine efflux in the nucleus accumbens. Co-administration of 0.06 pmol of dobutamine with salbutamol (3.6 pmol) did not affect the noradrenaline levels, but it increased the dopamine efflux to approximately 120%. Co-administration of 0.12 pmol of dobutamine with salbutamol (0.36 or 3.6pmol) also increased DA efflux to approximately 120% without affecting noradrenaline levels. The non-selective β-adrenoceptor antagonist l-propranolol (1200 pmol) that did not alter the basal noradrenaline and dopamine levels, suppressed the dopamine efflux, induced by co-administration of dobutamine (0.12 pmol) and salbutamol (3.6 pmol). The doses mentioned are the total amount of drug over the 60-min infusion period. The present results support our previously reported conclusion that stimulation of accumbal β-adrenoceptors which are suggested to be postsynaptically located on accumbal dopaminergic terminals, can enhance the dopamine efflux in the nucleus accumbens. The present study also provides in vivo neurochemical evidence that concomitant, but not separate, activation of accumbal β1- and β2-adrenoceptors synergistically increases the accumbal dopamine efflux., (© 2013 Elsevier B.V. All rights reserved.)

Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

Surfactant protein A (SP-A) is a large multimeric protein found in the lungs. In addition to its immunoregulatory function in infectious respiratory diseases, SP-A is also used as a marker of lung adenocarcinoma. Despite the finding that SP-A expression levels in cancer cells has a relationship with patient prognosis, the function of SP-A in lung cancer progression is unknown. We investigated the role of SP-A in lung cancer progression by introducing the SP-A gene into human lung adenocarcinoma cell lines. SP-A gene transduction suppressed the progression of tumor in subcutaneous xenograft or lung metastasis mouse models. Immunohistochemical analysis showed that the number of M1 antitumor tumor-associated macrophages (TAMs) was increased and the number of M2 tumor-promoting TAMs was not changed in the tumor tissue produced by SP-A-expressing cells. In addition, natural killer (NK) cells were also increased and activated in the SP-A-expressing tumor. Moreover, SP-A did not inhibit tumor progression in mice depleted of NK cells. Taking into account that SP-A did not directly activate NK cells, these results suggest that SP-A inhibited lung cancer progression by recruiting and activating NK cells via controlling the polarization of TAMs., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Circulating fibrocytes had been reported to migrate into the injured lungs, and contribute to fibrogenesis via chemokine-chemokine receptor systems including CXCL12-CXCR4 axis. Here we hypothesized that blockade of CXCR4 might inhibit the migration of fibrocytes to the injured lungs and the subsequent pulmonary fibrosis. To explore the antifibrotic effects of blockade of CXCR4, we used a specific antagonist for CXCR4, AMD3100, in bleomycin-induced pulmonary fibrosis model in mice. Administration of AMD3100 significantly improved the loss of body weight of mice treated with bleomycin, and inhibited the fibrotic lesion in subpleural areas of the lungs. The quantitative analysis demonstrated that treatment with AMD3100 reduced the collagen content and fibrotic score (Aschcroft score) in the lungs. Although AMD3100 did not affect cell classification in bronchoalveolar lavage fluid on day 7, the percentage of lymphocytes was reduced by AMD3100 on day 14. AMD3100 directly inhibited the migration of human fibrocytes in response to CXCL12 in vitro, and reduced the trafficking of fibrocytes into the lungs treated with bleocmycin in vivo. These results suggest that the blockade of CXCR4 might be useful strategy for therapy of patients with pulmonary fibrosis via inhibiting the migration of circulating fibrocytes.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the proliferation of fibroblasts and deposition of extracellular matrix. Since the prognosis of IPF is still poor, novel therapeutic modalities are strongly required. For this reason, to find molecular target for therapy of IPF is of much importance. The recent understanding of pathogenesis in IPF indicates the critical role of alveolar epithelial type II cells (AECII) and fibroblasts. Although the detailed mechanisms involved in IPF is still unclear, various profibrotic mediators which are produced by the injured AECII are thought to play a role in the progression of pulmonary fibrosis via stimulating fibroblasts. Among them, platelet-derived growth factor (PDGF) is one of critical growth factors by stimulating the proliferation and migration of fibroblasts. In this review, we discuss the role of PDGF in pulmonary fibrosis and the possibility as a therapeutic target for IPF.

In vivo microdialysis was used to analyse the role of the α(1)- and α(2)-adrenoceptor subtypes in the regulation of noradrenaline and dopamine efflux in the nucleus accumbens of freely moving rats. Intra-accumbal infusion of α(1)-adrenoceptor agonist methoxamine (24pmol) failed to alter the noradrenaline efflux, but decreased the dopamine efflux. The intra-accumbal infusion of α(1)-adrenoceptor antagonist prazosin (6, 600 and 6000pmol) produced a dose-related increase and decrease of the noradrenaline and dopamine efflux, respectively. An ineffective dose of prazosin (6pmol) counteracted the methoxamine (24pmol)-induced decrease of dopamine efflux. The prazosin (6000pmol)-induced increase of noradrenaline efflux, but not the decrease of dopamine efflux, was suppressed by the co-administration of an ineffective dose of methoxamine (0.024pmol). Neither the α(2)-adrenoceptor agonist clonidine (300pmol) and UK 14,304 (300pmol) nor the α(2)-adrenoceptor antagonist RX 821002 (0.6, 3, 600 and 6000pmol) significantly affected the accumbal noradrenaline and dopamine efflux. The doses mentioned are the total amount of drug over the 60-min infusion period. The present results show that (1) accumbal α(1)-adrenoceptors which are presynaptically located on noradrenergic nerve terminals inhibit the accumbal noradrenaline efflux, increasing thereby the accumbal dopamine efflux, (2) accumbal α(1)-adrenoceptors which are postsynaptically located on dopaminergic nerve terminals inhibit the accumbal dopamine efflux, and (3) accumbal α(2)-adrenoceptors play no major role in the regulation of accumbal efflux of noradrenaline and dopamine., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Rationale: Surfactant protein (SP)-D and SP-A have been implicated in immunomodulation in the lung. It has been reported that patients with idiopathic pulmonary fibrosis (IPF) often have elevated serum levels of SP-A and SP-D, although their role in the disease is not known., Objectives: The goal of this study was to test the hypothesis that SP-D plays an important role in lung fibrosis using a mouse model of fibrosis induced by bleomycin (BLM)., Methods: Triple transgenic inducible SP-D mice (iSP-D mice), in which rat SP-D is expressed in response to doxycycline (Dox) treatment, were administered BLM (100 U/kg) or saline subcutaneously using miniosmotic pumps., Measurements and Main Results: BLM-treated iSP-D mice off Dox (SP-D off) had increased lung fibrosis compared with mice on Dox (SP-D on). SP-D deficiency also increased macrophage-dominant cell infiltration and the expression of profibrotic cytokines (transforming growth factor [TGF]-β1, platelet-derived growth factor-AA). Alveolar macrophages isolated from BLM-treated iSP-D mice off Dox (SP-D off) secreted more TGF-β1. Fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, were increased to a greater extent in the lungs of the BLM-treated iSP-D mice off Dox (SP-D off). Fibrocytes isolated from BLM-treated iSP-D mice off Dox (SP-D off) expressed more of the profibrotic cytokine TGF-β1 and more CXCR4, a chemokine receptor that is important in fibrocyte migration into the lungs. Exogenous SP-D administered intratracheally attenuated BLM-induced lung fibrosis in SP-D(-/-) mice., Conclusions: These data suggest that alveolar SP-D regulates numbers of macrophages and fibrocytes in the lungs, profibrotic cytokine expression, and fibrotic lung remodeling in response to BLM injury.

GABA released from accumbal GABAergic interneurons plays an inhibitory role in the regulation of dopamine efflux through GABA(B) and GABA(A) receptors located on accumbal dopaminergic nerve endings. The cytosolic newly synthesised GABA alters vesicular GABA levels and, accordingly, the amount of GABA released from the neuron. Therefore, we hypothesised that glutamic acid decarboxylase (GAD) which generates GABA in accumbal GABAergic neurons, at least partly determines the GABA receptor subtype-mediated GABAergic tonus. To (in)validate this hypothesis, in vivo microdialysis was used to study the effects of an intra-accumbal infusion of the GAD inhibitor l-allylglycine (allylglycine) on the accumbal dopamine efflux of freely moving rats. The intra-accumbal infusion of allylglycine (50.0, 250.0 and 500.0 nmol) dose-dependently increased the accumbal dopamine levels. The co-administration of tetrodotoxin (720 pmol) suppressed the allylglycine (500.0 nmol)-induced dopamine efflux. The intra-accumbal infusion of GABA(B) receptor agonist baclofen (2.5 and 5.0 nmol) inhibited the allylglycine (500.0 nmol)-induced dopamine efflux. The baclofen's effects were counteracted by GABA(B) receptor antagonist saclofen (10.0 nmol). Neither GABA(A) receptor agonist (muscimol: 25.0 and 250.0 pmol) nor antagonist (bicuculline: 50.0 pmol) altered the allylglycine (250.0 and 500.0 nmol)-induced dopamine efflux. The present study provides in vivo neurochemical evidence that newly synthesised GABA that exerts an inhibitory tonus on the accumbal dopaminergic activity, acts at the level of GABA(B) receptors, but not GABA(A) receptors. The present study also shows that there is an allylglycine-insensitive GABA pool that release GABA exerting an inhibitory control of the accumbal dopaminergic activity, at the level of GABA(A) receptors. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'., (Copyright © 2011 Elsevier Ltd. All rights reserved.)