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The angiotensin II (ANG II)-ANG II type 1 receptor (AT 1 R) axis is a key player in the pathophysiology of obesity. Angiotensin-converting enzyme 2 (ACE2) counteracts the ANG II/AT 1 R axis via converting ANG II to angiotensin 1-7 (Ang 1-7), which is known to have an anti-obesity effect. In this study, we hypothesized that ACE2 exerts a strong anti-obesity effect by increasing Ang 1-7 levels. We injected intraperitoneally recombinant human ACE2 (rhACE2, 2.0 mg·kg -1 ·day -1 ) for 28 days to high-fat diet (HFD)-induced obesity mice. rhACE2 treatment decreased body weight and improved glucose metabolism. Furthermore, rhACE2 increased oxygen consumption and upregulated thermogenesis in HFD-fed mice. In the rhACE2 treatment group, brown adipose tissue (BAT) mass increased, accompanied with ameliorated insulin signaling and increased protein levels of uncoupling protein-1 (UCP-1) and PRD1-BF1-RIZ1 homologous domain containing 16. Importantly, subcutaneous white adipose tissue (sWAT) mass decreased, concomitant with browning, which was established by the increase of UCP-1 expression. The browning is the result of increased H3K27 acetylation via the downregulation of histone deacetylase 3 and increased H3K9 acetylation via upregulation of GCN5 and P300/CBP-associated factor. These results suggest that rhACE2 exerts anti-obesity effects by stimulating BAT and inducing browning in sWAT. ACE2 and the Ang 1-7 axis represent a potential therapeutic approach to prevent the development of obesity.

To develop a remote control system of transgene expression through localized cellular heating of magnetic nanoparticles, a heat-inducible transgene expression system was introduced into mammalian cells. Cells were labeled with magnetic nanoparticles and exposed to an alternating magnetic field. The magnetically labeled cells expressed the transgene in a monolayer and multilayered cell sheets in which cells were heated around the magnetic nanoparticles without an apparent temperature increase in the culture medium. Magnetic cells were also generated by genetically engineering with a ferritin gene, and transgene expression could be induced by exposure to an alternating magnetic field. This approach may be applicable to the development of novel gene therapies in cell-based medicine., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Investigating neural differentiation of pluripotent stem cells, including induced pluripotent stem (iPS) cells, is of importance for studying early neural development and providing a potential source of cells for nerve regeneration. Stromal cell-derived inducing activity (SDIA) using PA6 stromal cells promotes neural differentiation of iPS cells. Thus, we hypothesized that cadherin gene-engineered PA6 feeder cells will enhance the performance of SDIA by facilitating cell-cell interactions. Consequently, we created cadherin gene-engineered PA6 cells. Efficiency of neural differentiation from mouse iPS cells on PA6 feeder cells overexpressing E-cadherin gene (46%) or N-cadherin gene (27%) was significantly higher compared with parental PA6 feeder cells (19%). In addition, efficiency of motor neuron differentiation from mouse iPS cells on cadherin-gene engineered feeder cells (E-cadherin, 7.4%; N-cadherin, 11%) was significantly higher compared with parental PA6 feeder cells (4.1%). Altogether, these results indicate that cadherin gene-engineered feeder cells are a potent tool for promoting neural differentiation of pluripotent stem cells., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Impact Statement: In this study, we fabricated innervated skeletal muscle tissue constructs comprising C2C12 myoblasts and PC12 neural cells using a magnetic force-based tissue engineering technique. We found that the C2C12/PC12 co-culture enhanced neural differentiation of PC12 cells and sarcomere formation of C2C12 myotubes, accompanying with neuromuscular junction formation. The innervated skeletal muscle tissue constructs generated significantly higher contractile forces compared with aneural (C2C12 monoculture) skeletal muscle tissue constructs. These innervated skeletal muscle tissue constructs can be a useful tool for drug testing and biological research for neuromuscular diseases.

Bombyxin-II, an insulin-like peptide of the silkmoth Bombyx mori , has been shown to reduce both the trehalose concentration in the hemolymph and the glycogen content in some tissues of B. mori larvae. However, little is known about how these storage carbohydrates are utilized. To address this question, the effects of bombyxin-II injection into Bombyx larvae on the tissue lipid level, respiration rate, and glycolytic activity of tissues were investigated. Bombyxin-II did not affect lipid accumulation in the hemolymph and fat body, while it increased the rate of oxygen consumption and increased the content of fructose 2, 6-bisphosphate, a potent activator of glycolysis, in the gonads, imaginal discs, and midgut. These results suggest that bombyxin facilitates cellular energy production thereby supporting the tissue growth of insects.

The unique electronic, optical, and catalytic properties of AuNPs caused by localized surface plasmon resonance (LSPR) have attracted many scientists, but the LSPR diminishes the captivating luster of bulk gold. An exciting challenge is the fabrication of golden-colored AuNPs, but a decisive factor for controlling the absorption/reflection of AuNPs remains elusive. We now propose a simple and versatile method for the fabrication of glowing AuNPs to restore the "lost golden color" of AuNPs in combination with the deposition of AuNPs on a cellulose filter or a PET/cotton fabric by the successive ionic layer adsorption and reaction (SILAR) method and simple pencil drawing. The obtained materials exhibited the glowing golden-color on the pencil-drawn surface and common red and blue colors on the other parts. Surprisingly, the golden-colored AuNPs still maintain a catalytic activity different from that of bulk gold and could be used as a catalyst for the reduction of p-nitrophenol, pendimethalin or 2,4-dinitrophenol in the presence of NaBH 4 . We believe that the re-endowment of such a property characteristic of bulk gold into gold nanomaterials would lead to further advancement in the arts and culture as well as electronics, optics, and catalysis.

The natural attenuation behavior of chlorinated solvents and their risks to human health at a contaminated groundwater site in Takahata, Japan, were investigated. It was found that volatile organic compound (VOC) concentrations gradually decreased via two attenuation mechanisms, namely dilution and biodegradation. It was estimated that the VOC concentrations will be below the Japanese limits within 30 years after stopping the active remediation in 2003, which suggests that there is a high possibility that monitored natural attenuation can be adopted as the clean-up method at this contaminated site. The risk levels of VOCs at the present time are much lower than those at the time when the contamination was discovered. Vinyl chloride still presents a risk in some wells, and there were occasional unexpected increases in the risk levels of tetrachloroethylene, trichloroethylene, and cis-1,2-dichloloethylene, which means that continuous monitoring of the groundwater is necessary for forecasting risk levels.

Erythropoietin (EPO) has been reported to exert a beneficial effect on glucose metabolism in obesity. However, the effect of EPO on lipid metabolism and non-alcoholic fatty liver disease (NAFLD) was unclear. Furthermore, the effect of long acting erythropoiesis stimulating agents (ESA) on metabolism has not been poorly understood. The objective of this study was to investigate the effect of EPO and long acting ESA on NAFLD and lipid metabolism. We administered EPO and darbepoetin alpha (DEPO), a long acting ESA, by intraperitoneally injection for 4 weeks to mice with high-fat-diet (HFD)-induced obesity. EPO and DEPO treatment reduced body weight, ameliorated glucose tolerance and insulin resistance, and prevented lipid accumulation in liver and white adipose tissue (WAT). Administration of EPO and DEPO suppressed lipid synthesis-related protein in liver, including sterol regulatory element-binding protein 1 (SREBP-1), acetyl-CoA carboxylase (ACC1) and fatty acid synthase (FAS). EPO and DEPO also increased lipolysis protein in visceral WAT, including hormone-sensitive lipase (HSL), atni-adipose triglyceride lipase (ATGL). EPO and DEPO increased phosphorylation signal transducer and activator of transcription 3 (STAT3) and STAT5, transcriptional factors with crucial roles of lipid metabolism. These data suggest that EPO and DEPO ameliorated NAFLD by improving lipid metabolism via EPO/EPOR-induced STAT3 and STAT5 activation. EPO and DEPO may be a therapeutic option for NAFLD., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The well-orchestrated multicellular life cycle of Dictyostelium discoideum has fascinated biologists for over a century. Self-organisation of its amoebas into aggregates, migrating slugs and fruiting structures by pulsatile cAMP signalling and their ability to follow separate differentiation pathways in well-regulated proportions continue to be topics under investigation. A striking aspect of D. discoideum development is the recurrent use of cAMP as chemoattractant, differentiation inducing signal and second messenger for other signals that control the developmental programme. D. discoideum is one of >150 species of Dictyostelia and aggregative life styles similar to those of Dictyostelia evolved many times in eukaryotes. Here we review experimental studies investigating how phenotypic complexity and cAMP signalling co-evolved in Dictyostelia. In addition, we summarize comparative genomic studies of multicellular Dictyostelia and unicellular Amoebozoa aimed to identify evolutionary conservation and change in all genes known to be essential for D. discoideum development.

Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. In order to neutralize the increased C5, an even higher dosage of the antibody would be required. This antigen accumulation can be suppressed by giving the antibody a pH-dependent C5-binding property so that C5 is released from the antibody in the acidic endosome and then trafficked to the lysosome for degradation, while the C5-free antibody returns back to plasma. We recently demonstrated that a pH-dependent C5-binding antibody, SKY59, exhibited long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration. Here we report the details of the antibody engineering involved in generating SKY59, from humanizing a rabbit antibody to improving the C5-binding property. Moreover, because the pH-dependent C5-binding antibodies that we first generated still accumulated C5, we hypothesized that the surface charges of the ICs partially contributed to a slow uptake rate of the C5-antibody ICs. This idea motivated us to engineer the surface charges of the antibody. Our surface-charge engineered antibody consequently exhibited a high capacity to sweep C5 and suppressed the C5 accumulation in vivo by accelerating the cycle of sweeping: uptake of ICs into cells, release of C5 from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, our engineered anti-C5 antibody, SKY59, is expected to provide significant benefits for patients with complement-mediated disorders., Competing Interests: All the authors are employees and/or shareholders of Chugai, which is conducting the clinical study of SKY59. The authors report the filing of the following patents relevant to the investigational drug SKY59. ZS and YR are the inventors of the patents, “Anti-C5 antibodies and methods of use” (WO/2016/098356) and “Anti-C5 antibodies and methods of use” (WO/2017/217524). ZS is the inventor of the patent, “Anti-C5 antibodies and methods of use” (WO/2017/104779). ZS, K. Haraya, and TF are listed as inventors of the patent, “A pharmaceutical composition for use in the treatment or prevention of a C5-related disease and a method for treating or preventing a C5-related disease” (WO/2018/143266). These do not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Mixed lineage leukemia [MLL; now known as lysine methyltransferase 2A (KMT2A)] rearrangement-positive acute myeloid leukemia (AML) and juvenile myelomonocytic leukemia (JMML) are distinct diseases, although age of susceptibility (infancy or early childhood) and abnormal monocytosis are common clinical features. Here, we report two cases of KMT2A-rearranged infantile AML masquerading as JMML at initial presentation. Both cases showed leukocytosis accompanied by atypical monocytosis. However, in both cases, leukemic blasts were absent at the initial examination. Thus, a diagnosis of JMML was suspected. However, initial cytogenetic analysis revealed that both cases had an 11q23 rearrangement, which is atypical in JMML. Eventually, due to the emergence of leukemic blasts and further cytogenetic studies, both cases were diagnosed with infantile AML with a KMT2A rearrangement. Although one patient remains in complete remission after the completion of AML appropriate chemotherapy, the other died of AML due to treatment failure. Our experience suggests that AML with KMT2A rearrangement should be considered for the differential diagnosis of infantile cases with atypical monocytosis suggestive of JMML. Cytogenetic studies, including fluorescence in situ hybridization analysis of KMT2A, may be helpful in distinguishing between AML with KMT2A rearrangement and JMML.

Essentials Emicizumab (Emi) affects the APTT-based assays of factor (F)VIII activity and inhibitor titer. A mixture of two anti-Emi monoclonal antibodies (mAb) effectively neutralized the Emi activity. Anti-Emi mAbs completely eliminated the influence of Emi on FVIII activity and inhibitor titer. The inclusion of anti-Emi mAbs in routine FVIII assays would be useful for Emi-treated patients., Summary: Background Emicizumab is an anti-factor (F)IXa/X bispecific monoclonal antibody (mAb), mimicking the factor (F)VIIIa cofactor activity. Emicizumab does not require activation by thrombin and its shortening effect on the activated partial prothrombin time (APTT) is more pronounced than that of factor (F)VIII. APTT-based FVIII activity (FVIII:C) and FVIII inhibiter titer measurements are influenced by the presence of emicizumab. Aim To establish a reliable APTT-based assay to measure FVIII in the presence of emicizumab. Methods Plasmas from hemophilia A (HA) patients without or with inhibitors were studied using one-stage FVIII:C and Bethesda inhibitor assays. Two recombinant anti-idiotype mAbs to emicizumab (anti-emicizumab mAbs) were prepared, rcAQ8 to anti-FIXa-Fab and rcAJ540 to anti-FX-Fab. Results The combined anti-idiotype mAbs (2000 nm each) eliminated the effects of emicizumab on APTTs of HA plasmas without or with inhibitor by competitive inhibition of antibody binding to FIX(a)/FX(a). Measurements of FVIII coagulation activity in HA plasmas without inhibitor were overestimated in the presence of emicizumab (1 μm = ~150 μg mL -1 ) at all reference levels of FVIII. The addition of anti-emicizumab mAbs to the assay mixtures completely neutralized the emicizumab and facilitated accurate determination of FVIII:C. Anti-FVIII inhibitor titers were undetectable in the presence of emicizumab in HA plasmas with inhibitor or normal plasmas mixed with anti-FVIII neutralizing antibodies. These effects of emicizumab were completely counteracted by the addition of the anti-idiotype mAbs, allowing accurate assessment of inhibitor titers. Conclusion The in vitro inclusion of anti-emicizumab mAbs in the standard one-stage coagulation assays prevented interference by emicizumab and enabled accurate measurements of FVIII:C and inhibitor titers., (© 2018 International Society on Thrombosis and Haemostasis.)

Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes are integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre-loxP system using minicircle DNA vectors as transgene donors. The targeted transgene integration efficiency is improved 2-3-fold (≈1.4%) using minicircle DNA vectors compared with conventional plasmid vectors. Moreover, clones with expected structures after transgene integration are obtained with a high frequency. When a transgene together with bacterial sequences derived from a plasmid vector is integrated into the hprt locus, the cell growth rate and antibody titer decrease. These results indicate that minicircle DNA vectors are more suitable than conventional plasmid vectors for transgene delivery in recombinant protein production using CHO cells., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Glycogen synthase kinase 3 (GSK3) regulates many cell fate decisions in animal development. In multicellular structures of the group 4 dictyostelid Dictyostelium discoideum , GSK3 promotes spore over stalk-like differentiation. We investigated whether, similar to other sporulation-inducing genes such as cAMP-dependent protein kinase (PKA), this role of GSK3 is derived from an ancestral role in encystation of unicellular amoebas., Results: We deleted GSK3 in Polysphondylium pallidum , a group 2 dictyostelid which has retained encystation as an alternative survival strategy. Loss of GSK3 inhibited cytokinesis of cells in suspension, as also occurs in D. discoideum , but did not affect spore or stalk differentiation in P. pallidum . However, gsk3 - amoebas entered into encystation under conditions that in wild type favour aggregation and fruiting body formation. The gsk3 - cells were hypersensitive to osmolytes, which are known to promote encystation, and to cyst-inducing factors that are secreted during starvation. GSK3 was not itself regulated by these factors, but inhibited their effects., Conclusions: Our data show that GSK3 has a deeply conserved role in controlling cytokinesis, but not spore differentiation in Dictyostelia. Instead, in P. pallidum , one of many Dictyostelia that like their solitary ancestors can still encyst to survive starvation, GSK3 promotes multicellular development into fruiting bodies over unicellular encystment.

Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The liver plays a major role in whole-body energy homeostasis by releasing secretory factors, termed hepatokines. To identify novel target genes associated with insulin resistance, we performed a comprehensive analysis of gene expression profiles using a DNA chip method in liver biopsy samples from humans with varying degrees of insulin resistance. Inhibin βE (INHBE) was identified as a novel putative hepatokine with hepatic gene expression that positively correlated with insulin resistance and body mass index in humans. Quantitative real time-PCR analysis also showed an increase in INHBE gene expression in independent liver samples from insulin-resistant human subjects. Additionally, Inhbe gene expression increased in the livers of db/db mice, a rodent model of type 2 diabetes. To preliminarily screen the role of Inhbe in vivo in whole-body energy metabolic status, hepatic mRNA was knocked down with siRNA for Inhbe (siINHBE) in db/db mice. Treatment with siINHBE suppressed body weight gain during the two-week experimental period, which was attributable to diminished fat rather than lean mass. Additionally, treatment with siINHBE decreased the respiratory quotient and increased plasma total ketone bodies compared with treatment with non-targeting siRNA, both of which suggest enhanced whole-body fat utilization. Our study suggests that INHBE functions as a possible hepatokine to alter the whole-body metabolic status under obese insulin-resistant conditions.

Emicizumab is a humanized bispecific antibody that binds simultaneously to factor (F) IXa and FX replacing the cofactor function of FVIIIa. Because emicizumab recognizes FIX/FIXa and FX/FXa, a question may arise whether emicizumab competes with antithrombin (AT) and/or tissue factor pathway inhibitor (TFPI), thereby enhancing overall hemostatic potential by blocking their antihemostatic effects. To address this question, we performed enzymatic assays using purified coagulation factors to confirm whether emicizumab interferes with the action of AT on FIXa or FXa, or with the action of TFPI on FXa. In those assays, we found no interference of emicizumab on the actions of AT and TFPI. We next assessed emicizumab's influences on the anticoagulation actions of AT or TFPI in thrombin generation assays triggered with FXIa or tissue factor (TF) in AT-depleted or TFPI-depleted plasma supplemented with AT or TFPI in vitro. In those assays, we employed anti-FIXa and anti-FX monospecific one-armed antibodies derived from emicizumab instead of emicizumab itself so as to prevent emicizumab's FVIIIa cofactor activity from boosting thrombin generation. Consequently, we found that neither anti-FIXa, anti-FX monospecific antibody, nor the mixture of the two interfered with the anticoagulation actions of AT or TFPI in plasma. Although emicizumab can bind to FIXa and FXa, our results showed no interference of emicizumab with the action of AT or TFPI on FIXa or FXa. This indicates that the presence of emicizumab is irrelevant to the action of AT and TFPI, and thus should not alter the coagulant/anticoagulant balance related to AT and TFPI.

Astrocytes are the most abundant glia cell type in the central nervous system (CNS), and are known to constitute heterogeneous populations that differ in their morphology, gene expression and function. Although glial fibrillary acidic protein (GFAP) is the cardinal cytological marker of CNS astrocytes, GFAP-negative astrocytes can easily be found in the adult CNS. Astrocytes are also allocated to spatially distinct regional domains during development. This regional heterogeneity suggests that they help to coordinate post-natal neural circuit formation and thereby to regulate eventual neuronal activity. Here, during lineage-tracing studies of cells expressing Olig2 using Olig2 CreER ; Rosa-CAG-LSL-eNpHR3.0-EYFP transgenic mice, we found Olig2-lineage mature astrocytes in the adult forebrain. Long-term administration of tamoxifen resulted in sufficient recombinant induction, and Olig2-lineage cells were found to be preferentially clustered in some adult brain nuclei. We then made distribution map of Olig2-lineage astrocytes in the adult mouse brain, and further compared the map with the distribution of GFAP-positive astrocytes visualized in GFAP Cre ; Rosa-CAG-LSL-eNpHR3.0-EYFP mice. Brain regions rich in Olig2-lineage astrocytes (e.g., basal forebrain, thalamic nuclei, and deep cerebellar nuclei) tended to lack GFAP-positive astrocytes, and vice versa. Even within a single brain nucleus, Olig2-lineage astrocytes and GFAP astrocytes frequently occupied mutually exclusive territories. These findings strongly suggest that there is a subpopulation of astrocytes (Olig2-lineage astrocytes) in the adult brain, and that it differs from GFAP-positive astrocytes in its distribution pattern and perhaps also in its function. Interestingly, the brain nuclei rich in Olig2-lineage astrocytes strongly expressed GABA-transporter 3 in astrocytes and vesicular GABA transporter in neurons, suggesting that Olig2-lineage astrocytes are involved in inhibitory neuronal transmission.

The renin-angiotensin system is a key regulator of metabolism with beneficial effects of the angiotensin 1-7 (Ang 1-7) peptide. We hypothesized that the antiobesity effect of Ang 1-7 was related to the stimulation of brown adipose tissue (BAT). We administered Ang 1-7 (0.54 mg kg -1 day -1 ) for 28 days via implanted micro-osmotic pumps to mice with high-fat diet (HFD)-induced obesity. Ang 1-7 treatment reduced body weight, upregulated thermogenesis, and ameliorated impaired glucose homeostasis without affecting food consumption. Furthermore, Ang 1-7 treatment enlarged BAT and the increased expression of UCP1, PRDM16, and prohibitin. Alterations in PRDM16 expression correlated with increased AMPK and phosphorylation of mTOR. Ang 1-7 treatment elevated thermogenesis in subcutaneous white adipose tissue without altering UCP1 expression. These changes occurred in the context of decreased lipid accumulation in BAT from HFD-fed mice, preserved insulin signaling concomitant with phosphorylation of hormone-sensitive lipase and decreased expression of perilipin. These data suggest that Ang 1-7 induces brown adipocyte differentiation leading to upregulation of thermogenesis and improved metabolic profile in diet-induced obesity. Enhancing Ang 1-7 action represents a promising therapy to increase BAT and to reduce the metabolic complications associated with diet-induced obesity.

Advances in the diagnosis and treatment of patients with maxillary sinus carcinoma have improved patient prognosis. This study investigated changes in demographic and clinical characteristics, treatment methods, and outcomes of patients with maxillary sinus carcinomas during three different 10-year periods spanning 60 years at our university hospital. Of the 233 patients with maxillary sinus carcinomas managed at Nagoya University Hospital, 135 were treated between 1951 and 1960 (first period), 35 between 1981 and 1990 (second period), and 63 between 2003 and 2012 (third period). Patient age, sex, TN classifications, treatment methods, and survival rates were compared among patients treated during these time periods. Of the 135, 35, and 63 patients with maxillary sinus carcinomas treated during these time periods, 86 (63.7%), 21 (51.4%), and 48 (76.2%), respectively, were men; 14 (10.4%), six (17.1%), and 14 (22.2%), respectively, were aged ≥70 years; and 135 (100%), 28 (80.0%), and 43 (68.3%), respectively, were treated surgically. The 5-year overall survival rates in patients treated during the first, second, and third periods were 29.7%, 44.3%, and 57.5%, respectively. These findings indicated that advances in the diagnosis and treatment of patients with maxillary sinus carcinoma, including computed tomography and craniofacial resection, have contributed to improvements in patient survival rates.

Scratching is an important factor exacerbating skin lesions through the so-called itch-scratch cycle in atopic dermatitis (AD). In mice, interleukin (IL)-31 and its receptor IL-31 receptor A (IL-31RA) are known to play a critical role in pruritus and the pathogenesis of AD; however, study of their precise roles in primates is hindered by the low sequence homologies between primates and mice and the lack of direct evidence of itch sensation by IL-31 in primates. We showed that administration of cynomolgus IL-31 induces transient scratching behaviour in cynomolgus monkeys and by that were able to establish a monkey model of scratching. We then showed that a single subcutaneous injection of 1 mg/kg nemolizumab, a humanized anti-human IL-31RA monoclonal antibody that also neutralizes cynomolgus IL-31 signalling and shows a good pharmacokinetic profile in cynomolgus monkeys, suppressed the IL-31-induced scratching for about 2 months. These results suggest that the IL-31 axis and IL-31RA axis play as critical a role in the induction of scratching in primates as in mice and that the blockade of IL-31 signalling by an anti-human IL-31RA antibody is a promising therapeutic approach for treatment of AD. Nemolizumab is currently under investigation in clinical trials., (© 2016 The Authors. Experimental Dermatology Published by John Wiley & Sons Ltd.)

New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Hepatoma cells are a candidate cell source for bio-artificial livers. However, they exhibit reduced liver functions compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells were created by transduction with vectors mediating inducible overexpression of eight liver-enriched transcription factors. Upon the induction of the liver-enriched transcription factors transduced, the cells expressed both phenotypic and genotypic liver functions at high levels. In the present study, we performed three-dimensional culture of these cells using macroporous gelatin beads. When immobilized on the macroporous gelatin beads, these cells exhibited further enhancement in liver functionality, including increased albumin secretion, ammonia removal and cytochrome P450 activity. The levels of these functions were significantly enhanced compared to monolayer culture. The method is simple and scalable, and provides highly functional cells that can be used in basic and applied fields of hepatic research.

We have previously developed an accumulative site-specific gene integration system (AGIS) using Cre-recombinase and mutated loxP sites. AGIS enables repeated transgene integration into a predetermined chromosomal site in mammalian cells. However, the process of establishing cells with multiple integrated copies of the transgene is still time-consuming. In the present study, we describe an improved version of AGIS that facilitates and accelerates the establishment of high-producer Chinese hamster ovary (CHO) cells. Two donor vectors were simultaneously introduced into the cells in a single transfection. Cells with successfully targeted transgene integration were screened based on a change in the color of the reporter fluorescent protein that they express. Repeated rounds of integration allowed the transgene copy number to be increased. As a model, an scFv-Fc antibody gene was integrated into the hprt locus of the CHO cell genome. After three rounds of integration, a high-producer CHO cell clone with six copies of the scFv-Fc gene was successfully established. scFv-Fc productivity was approximately four-fold greater than a control cell line harboring a single copy of the transgene. This newly designed AGIS procedure should facilitate the development of producer cells suitable for biopharmaceutical protein production., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Precise control of gene expression using an artificial gene circuit is a major challenge in the application of synthetic biology. Here, we designed a hypoxia-responsive transgene expression system by combining a hypoxia-inducible RTP801 promoter and a tetracycline-responsive transactivator fused with an oxygen-dependent degradation domain (TA-ODD). The reporter gene expression was highly induced by hypoxia when a transactivator-expression plasmid, pRTP801/TA-ODD, harboring a TA-ODD gene driven by the RTP801 promoter, was cotransfected with a reporter plasmid, pTRE/EGFP, harboring an EGFP gene controlled under the transactivator-responsive promoter. A stable cell line into which the expression units RTP801/TA-ODD and TRE/EGFP had been introduced responded to hypoxia and expressed the reporter gene in an oxygen-concentration-dependent manner. Moreover, the cells demonstrated potential as sensors to detect hypoxic conditions in a three-dimensional tissue culture in vitro. These results indicate that the hypoxia-responsive transgene expression system is useful for constructing cell-based hypoxia detection systems., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Emicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. However, details of the emicizumab-antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities (K D = 1.58, 1.52, 1.85, and 0.978 µM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens' epidermal growth factor (EGF)-like domains. We then performed K D -based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX-emicizumab-FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab's cofactor activity. The simulation also predicted that at 10.0-100 µg/ml of emicizumab-levels shown in a previous study to be clinically effective-the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The K D -based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab's cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions.

Narcolepsy-cataplexy is a debilitating disorder of sleep/wakefulness caused by a loss of orexin-producing neurons in the lateroposterior hypothalamus. Genetic or pharmacologic orexin replacement ameliorates symptoms in mouse models of narcolepsy-cataplexy. We have recently discovered a potent, nonpeptide OX2R-selective agonist, YNT-185. This study validates the pharmacological activity of this compound in OX2R-transfected cells and in OX2R-expressing neurons in brain slice preparations. Intraperitoneal, and intracerebroventricular, administration of YNT-185 suppressed cataplexy-like episodes in orexin knockout and orexin neuron-ablated mice, but not in orexin receptor-deficient mice. Peripherally administered YNT-185 also promotes wakefulness without affecting body temperature in wild-type mice. Further, there was no immediate rebound sleep after YNT-185 administration in active phase in wild-type and orexin-deficient mice. No desensitization was observed after repeated administration of YNT-185 with respect to the suppression of cataplexy-like episodes. These results provide a proof-of-concept for a mechanistic therapy of narcolepsy-cataplexy by OX2R agonists., Competing Interests: The authors declare no conflict of interest.

Although skeletal muscle tissue engineering has been extensively studied, the physical forces produced by tissue-engineered skeletal muscles remain to be improved for potential clinical utility. In this study, we examined the effects of mild heat stimulation and supplementation of a l-ascorbic acid derivative, l-ascorbic acid 2-phosphate (AscP), on myoblast differentiation and physical force generation of tissue-engineered skeletal muscles. Compared with control cultures at 37°C, mouse C2C12 myoblast cells cultured at 39°C enhanced myotube diameter (skeletal muscle hypertrophy), whereas mild heat stimulation did not promote myotube formation (differentiation rate). Conversely, AscP supplementation resulted in an increased differentiation rate but did not induce skeletal muscle hypertrophy. Following combined treatment with mild heat stimulation and AscP supplementation, both skeletal muscle hypertrophy and differentiation rate were enhanced. Moreover, the active tension produced by the tissue-engineered skeletal muscles was improved following combined treatment. These findings indicate that tissue culture using mild heat stimulation and AscP supplementation is a promising approach to enhance the function of tissue-engineered skeletal muscles. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.

Skeletal muscle tissue engineering holds great promise for pharmacological studies. Herein, we demonstrated an in vitro drug testing system using tissue-engineered skeletal muscle constructs. In response to epigenetic drugs, myotube differentiation of C2C12 myoblast cells was promoted in two-dimensional cell cultures, but the levels of contractile force generation of tissue-engineered skeletal muscle constructs prepared by three-dimensional cell cultures were not correlated with the levels of myotube differentiation in two-dimensional cell cultures. In contrast, sarcomere formation and contractile activity in two-dimensional cell cultures were highly correlated with contractile force generation of tissue-engineered skeletal muscle constructs. Among the epigenetic drugs tested, trichostatin A significantly improved contractile force generation of tissue-engineered skeletal muscle constructs. Follistatin expression was also enhanced by trichostatin A treatment, suggesting the importance of follistatin in sarcomere formation of muscular tissues. These observations indicate that contractility data are indispensable for in vitro drug screening.

Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Antigen-specific immunotherapy is the only curative approach for the treatment of allergic diseases such as Japanese cedar pollinosis. Immunotherapy using a T cell epitope vaccine in combination with the adjuvant R848 is of particular interest as a safe and effective approach to treat allergic diseases. Herein, we propose a simple and easy to handle vaccine administration method using the original solid-in-oil (S/O) nanodispersion system that permeates through the skin. The S/O nanodispersion system is composed of nanoparticles of hydrophilic molecules surrounded with hydrophobic surfactants that are dispersed in an oil vehicle. The system has potential to carry and deliver both hydrophilic and hydrophobic bioactives. Hydrophilic T cell epitope peptide was efficiently delivered through mouse skin using the S/O nanodispersion system and lowered antigen-specific IgE levels in pollinosis model mice. Addition of the hydrophobic adju1vant R848 significantly lowered the antibody secretion and shifted the Th1/Th2-balance toward Th1-type immunity in the model mice, showing the potential to alleviate Japanese cedar pollinosis.

Objective: We investigated clinical outcomes in patients with remitting seronegative symmetrical synovitis with pitting edema (RS3PE) syndrome., Methods: This is a retrospective multicenter study conducted in Nagasaki, Japan. We consecutively diagnosed a total of 41 patients with RS3PE syndrome between October 2003 and September 2012 and evaluated their outcomes from medical records from the first year of follow-up., Results: Although an excellent initial response to corticosteroids was noted in all 41 patients, 34 (82.9%) were still receiving corticosteroids and 13 (31.7%) showed elevated C-reactive protein (CRP) at one year. Multivariate analysis demonstrated that male gender and high CRP level at entry were independent variables associated with patients' one-year CRP level being ≥0.5 mg/dL. Odds ratios were 17.05 ([95% CI 2.41-370.12], p < 0.026) and 12.99 ([95% CI 1.78-269.62], p < 0.0096), respectively. Twenty-four patients (58.5%) were still receiving prednisolone (PSL) ≥ 5 mg/day at one year. Disease-modifying anti-rheumatic drugs including methotrexate were required in three patients (10.3%). Neoplasms were found in 14 patients (34.1%) and 1 of these had died due to lung cancer at one year., Conclusions: RS3PE syndrome initially responds well to corticosteroids with remission of symptoms. However, outcomes of RS3PE syndrome appear to be worse than expected, and may be influenced by gender and initial CRP level.

Myoepithelial cells (MECs) exist on the basal surface of acini in major exocrine glands, include myofilaments and various constructive proteins, and share characteristics with smooth muscle and epithelial cells. MECs project several ramified processes to invest acini, and possibly contract to compress acini to support the secretion by the glandular cells. However, the functional roles of MECs in salivary secretion are still unclear. We investigated morphological changes in immunostained MECs using the anti-α-smooth muscle actin (αSMA) antibody in operated or non-operated contralateral (NC) submandibular glands after partial or total resection. Furthermore, we investigated and discuss other salivary glands of rats. MECs in the parotid, sublingual and submandibular gland of adult rats exhibited different shapes and localizations. After surgery, in both operated and NC glands, the number of MECs and αSMA-immunopositive areas increased significantly. Three-dimensional analysis using a confocal laser-scanning microscope revealed that substantial and significant enhancement became evident in the number, length, and thickness of MEC-processes covering acini of the operated and NC submandibular glands. The preset findings indicate that MECs alter the morphology of their processes in operated and NC glands after surgery of the partial or total resection. It is suggested that MECs promote salivary secretion using elongated, thickened, and more ramified processes.

Parathyroid hormone (PTH) is essential for calcium homeostasis and its action is mediated by the PTH type 1 receptor (PTHR1), a class B G-protein-coupled receptor. Hypoparathyroidism and osteoporosis can be treated with PTH injections; however, no orally effective PTH analogue is available. Here we show that PCO371 is a novel, orally active small molecule that acts as a full agonist of PTHR1. PCO371 does not affect the PTH type 2 receptor (PTHR2), and analysis using PTHR1-PTHR2 chimeric receptors indicated that Proline 415 of PTHR1 is critical for PCO371-mediated PTHR1 activation. Oral administration of PCO371 to osteopenic rats provokes a significant increase in bone turnover with limited increase in bone mass. In hypocalcemic rats, PCO371 restores serum calcium levels without increasing urinary calcium, and with stronger and longer-lasting effects than PTH injections. These results strongly suggest that PCO371 can provide a new treatment option for PTH-related disorders, including hypoparathyroidism.

Hypoparathyroidism is a disease of chronic hypocalcemia and hyperphosphatemia due to a deficiency of parathyroid hormone (PTH). PTH and analogs of the hormone are of interest as potential therapies. Accordingly, we examined the pharmacological properties of a long-acting PTH analog, [Ala(1,3,12,18,22) , Gln(10) ,Arg(11) ,Trp(14) ,Lys(26) ]-PTH(1-14)/PTHrP(15-36) (LA-PTH) in thyroparathyroidectomized (TPTX) rats, a model of HP, as well as in normal monkeys. In TPTX rats, a single intravenous administration of LA-PTH at a dose of 0.9 nmol/kg increased serum calcium (sCa) and decreased serum phosphate (sPi) to near-normal levels for longer than 48 hours, whereas PTH(1-34) and PTH(1-84), each injected at a dose 80-fold higher than that used for LA-PTH, increased sCa and decreased sPi only modestly and transiently (<6 hours). LA-PTH also exhibited enhanced and prolonged efficacy versus PTH(1-34) and PTH(1-84) for elevating sCa when administered subcutaneously (s.c.) into monkeys. Daily s.c. administration of LA-PTH (1.8 nmol/kg) into TPTX rats for 28 days elevated sCa to near normal levels without causing hypercalciuria or increasing bone resorption markers, a desirable goal in the treatment of hypoparathyroidism. The results are supportive of further study of long-acting PTH analogs as potential therapies for patients with hypoparathyroidism. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Novel near-infrared phosphorescent iridium(III) complexes based on benzo-annulated N-linked corrole analogue (termed as benzonorrole) were synthesized. The structures of the complexes revealed octahedral coordination geometries involving an organometallic iridium-carbon bond with two external axial ligands. Interestingly, the iridium(III) complex exhibits near-infrared phosphorescence at room temperature at wavelengths beyond 900 nm. The significant redshift of the emission, as compared to the corrole congener, is originated from the ligand-centered triplet character. The fine-tuning of the photophysical properties of the complexes was achieved by introducing electron-donating and electron-withdrawing substituents on the axial pyridine ligands.

Objectives: Interferon-gamma release assays (IGRAs) can be positive in patients infected with Mycobacterium kansasii (M. kansasii), which carries some of Mycobacterium tuberculosis specific antigens adopted for IGRAs. Our aim is to evaluate positive rate and factors associated with positive IGRAs in patients with M. kansasii pulmonary infection., Methods: We retrospectively investigated 105 M. kansasii cases in which IGRAs were performed before or ≦14 days after treatment initiation. Clinical characteristics including a history of tuberculosis, radiographic features and laboratory data were collected from medical records., Results: Positive rate of each IGRA was 25.9% (15/58) in QuantiFERON TB-Gold (QFT-G), 31.8% (7/22) in QuantiFERON-TB Gold In Tube (QFT-GIT), and 33.3% (7/21) in T-SPOT. TB (T-SPOT). After excluding cases having a history of tuberculosis, positive rate of each IGRA decreased to 19% (8/42) in QFT-G, 20% (3/15) in QFT-GIT, and 18.8% (3/16) in T-SPOT. The multivariate analysis revealed that only previous tuberculosis was significantly associated with positive IGRAs (odds ratio, 4.758; 95% confidence interval, 1.73-13.05; p = 0.002)., Conclusions: Positive rates of IGRAs were low in patients with M. kansasii, especially in those without previous tuberculosis. M. kansasii pulmonary infection alone might induce less interferon-gamma production with the antigens., (Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Urate-lowering therapy is indispensable for the treatment of gout, but available drugs do not control serum urate levels tightly enough. Although the uricosurics benzbromarone and probenecid inhibit a urate reabsorption transporter known as renal urate transporter 1 (URAT1) and thus lower serum urate levels, they also inhibit other transporters responsible for secretion of urate into urine, which suggests that inhibiting URAT1 selectively would lower serum urate more effectively. We identified a novel potent and selective URAT1 inhibitor, UR-1102, and compared its efficacy with benzbromarone in vitro and in vivo. In human embryonic kidney (HEK)293 cells overexpressing URAT1, organic anion transporter 1 (OAT1), and OAT3, benzbromarone inhibited all transporters similarly, whereas UR-1102 inhibited URAT1 comparably to benzbromarone but inhibited OAT1 and OAT3 quite modestly. UR-1102 at 3-30 mg/kg or benzbromarone at 3-100 mg/kg was administered orally once a day for 3 consecutive days to tufted capuchin monkeys, whose low uricase activity causes a high plasma urate level. When compared with the same dosage of benzbromarone, UR-1102 showed a better pharmacokinetic profile, increased the fractional excretion of urinary uric acid, and reduced plasma uric acid more effectively. Moreover, the maximum efficacy of UR-1102 was twice that of benzbromarone, suggesting that selective inhibition of URAT1 is effective. Additionally UR-1102 showed lower in vitro potential for mechanisms causing the hepatotoxicity induced by benzbromarone. These results indicate that UR-1102 achieves strong uricosuric effects by selectively inhibiting URAT1 over OAT1 and OAT3 in monkeys, and could be a novel therapeutic option for patients with gout or hyperuricemia., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

An efficient and versatile synthetic method for labile polyphenols was established using 2-nitrobenzenesulfonate (Ns) as a protecting group for phenol. This methodology provides regio- and stereoselective access to a range of methylated catechins, such as methylated epigallocatechin gallates, that are not readily available from natural sources. In addition, biomimetic synthesis of theaflavins from catechins was accomplished using Ns protection to minimize undesired side reactions of electron-rich aromatic rings during oxidation, enabling construction of the complex benzotropolone core in a single-step oxidative coupling reaction. Availability of these compounds will aid detailed structure-biological activity relationship studies of catechins.

Obesity is a global health concern that promotes chronic low-grade inflammation, leading to insulin resistance, a key factor in many metabolic diseases. Angiotensin 1-7 (Ang 1-7), a component of the renin-angiotensin system (RAS), exhibits anti-inflammatory effects in obesity and related disorders, though its mechanisms remain unclear. In this study, we examined the effect of Ang 1-7 on inflammation of white adipose tissue (WAT) in dietary-induced obese mice. Monocyte chemoattractant protein-1 (MCP-1) produced by white adipocytes and tumour necrosis factor-α (TNF-α) produced by macrophages are pro-inflammatory cytokines and interact to form a pathogenic loop to exacerbate obesity-induced inflammation. We found that Ang 1-7 reduced MCP-1 and TNF-α gene expressions and the number of crown-like structures, which are histological hallmarks of the pro-inflammatory process, in visceral epididymal WAT (eWAT) and reduced circulating MCP-1 and TNF-α levels, accompanied by improvement in insulin resistance, in dietary-induced obese mice. Furthermore, Ang 1-7 reduced MCP-1 and TNF-α secretions in 3T3-L1 white adipocytes and RAW 264.7 macrophages, respectively, which are in vitro experimental models mimicking obesity condition. Our results suggest that Ang 1-7 directly acts on WAT to mitigate obesity-induced inflammation. Thus, this study provides novel insights into the underlying mechanism of anti-obesity effects of Ang 1-7.

Introduction: Tissue-engineered skeletal muscle constructs should be designed to generate contractile force with directional movement. Because electrical impulses from a somatic nervous system are crucial for in vivo skeletal muscle development, electrical pulse stimulation (EPS) culture as an artificial exercise is essential to fabricate functional skeletal muscle tissues in vitro . To further improve muscle functions, the activation of cell-signaling pathways from myogenic growth factors, such as insulin-like growth factor (IGF)-I, is also important. Because tissue-engineered skeletal muscle constructs should maintain a high cell-dense structure, the expression of an anti-apoptotic factor, such as B-cell lymphoma 2 (Bcl-2), could be effective in preventing cell death., Methods: In the present study, myoblasts were genetically modified with inducible expression units of IGF-I and Bcl-2 genes, and the tissue-engineered skeletal muscle constructs fabricated from the myoblasts were cultured under continuous EPS., Results: Overexpression of IGF-I gene induced muscular hypertrophy in the muscle tissue constructs, and Bcl-2-overexpressing myoblasts formed significantly cell-dense and viable muscle tissue constructs. Furthermore, the combination of IGF-I and Bcl-2 gene transfer with EPS culture highly improved the force generation of the tissue-engineered skeletal muscle constructs., Conclusions: This approach has the potential to yield functional skeletal muscle substitutes with high force generation ability.

It has been known that the phosphoSer/Thr-Pro-specific peptidyl prolyl cis/trans isomerase Pin1 regulates a variety of intracellular signaling pathways, including the response to the genotoxic drug doxorubicin. Pin1 binds phosphorylated p53 and stabilizes p53 to cause cell cycle arrest and apoptosis quickly in response to doxorubicin. Here we show another mechanism of Pin1 to maintain cell sensitivity to genotoxic stress, irrespective of whether p53 is present or not. In response to the genotoxic drug, Pin1 binds and decreases levels of the phosphorylated Foxo3, the positive transcription factor of P-glycoprotein (P-gp) gene. Through this mechanism of action, Pin1 decreases the level of P-gp and signals the cell to pump the genotoxic drugs out. This shows that Pin1 is implemented in maintaining the susceptibility to the genotoxic drugs by controlling P-gp level as well as p53-dependent apoptosis and cell cycle signaling pathways., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tsunami deposits accumulated on the Tohoku coastal area in Japan due to the impact of the Tohoku-oki earthquake. In the study reported in this paper, we applied principal component analysis (PCA) and cluster analysis (CA) to determine the concentrations of heavy metals in tsunami deposits that had been diluted with water or digested using 1 M HCl. The results suggest that the environmental risk is relatively low, evidenced by the following geometric mean concentrations: Pb, 16 mg kg(-1) and 0.003 ml L(-1); As, 1.8 mg kg(-1) and 0.004 ml L(-1); and Cd, 0.17 mg kg(-1) and 0.0001 ml L(-1). CA was performed after outliers were excluded using PCA. The analysis grouped the concentrations of heavy metals for leaching in water and acid. For the acid case, the first cluster contained Ni, Fe, Cd, Cu, Al, Cr, Zn, and Mn; while the second contained Pb, Sb, As, and Mo. For water, the first cluster contained Ni, Fe, Al, and Cr; and the second cluster contained Mo, Sb, As, Cu, Zn, Pb, and Mn. Statistical analysis revealed that the typical toxic elements, As, Pb, and Cd have steady correlations for acid leaching but are relatively sparse for water leaching. Pb and As from the tsunami deposits seemed to reveal a kind of redox elution mechanism using 1 M HCl., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although mitogen-activated protein kinase kinase (MEK) is a key signaling molecule and a negative regulator of insulin action, it is still uncertain whether MEK can be a therapeutic target for amelioration of insulin resistance (IR) in type 2 diabetes (T2D) in vivo. To clarify whether MEK inhibition improves T2D, we examined the effect of continuous MEK inhibition with two structurally different MEK inhibitors, RO5126766 and RO4987655, in mouse models of T2D. RO5126766 and RO4987655 were administered via dietary admixture. Both compounds decreased blood glucose and improved glucose tolerance in doses sufficient to sustain inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation downstream of MEK in insulin-responsive tissues in db/db mice. A hyperinsulinemic-euglycemic clamp test showed increased glucose infusion rate (GIR) in db/db mice treated with these compounds, and about 60% of the increase was attributed to the inhibition of endogenous glucose production, suggesting that the liver is responsible for the improvement of IR. By means of adenovirus-mediated Mek1 shRNA expression, we confirmed that blood glucose levels are reduced by suppression of MEK1 expression in the liver of db/db mice. Taken together, these results suggested that the MEK signaling pathway could be a novel therapeutic target for novel antidiabetic agents.

Introduction: Patients with rheumatoid arthritis (RA) treated with abatacept (ABT) are at increased risk for vaccine-preventable infections. The aim of the present study is to evaluate the humoral response to 23-valent pneumococcal polysaccharide (PPSV23) vaccination in RA patients receiving ABT., Methods: The immunogenicity study was nested within a randomized, double-blind placebo-controlled study, designed to evaluate the efficacy of the PPSV23. PPSV23 was given to 111 RA patients, who were classified into three groups: RA control (n = 35), methotrexate (MTX) alone (n = 55), and ABT (n = 21). Before and 4-6 weeks after vaccination, we measured the patients' concentrations of antibodies against pneumococcal serotypes 6B and 23F using an enzyme-linked immunosorbent assay and determined their antibody functionality using a multiplexed opsonophagocytic killing assay, reported as the opsonization index (OI)., Results: The pneumococcal serotype-specific IgG concentrations and OIs were both significantly increased in all treatment groups in response to PPSV23 vaccination. In the ABT group, the IgG responses for the 6B serotype were lower compared with those in the MTX alone or control groups, whereas the OI responses were similar to those in the other two groups. In a subgroup analysis, the pneumococcal serotype-specific IgG responses were significantly lower in both serotypes (6B and 23F) in the ABT/MTX group; however, the OI responses in the ABT group were not different from the control group. There was no association between the pneumococcal serotype-specific IgG and OI responses for the 6B serotype in patients receiving ABT in contrast to the control or MTX alone patients. No severe adverse effects were observed in any of the treatment groups., Conclusions: OI responses indicate antibody functionality rather than simply their amount, so the similarity of these measurements between all three groups suggests that RA patients receiving ABT still benefit from receiving the PPSV23 vaccination, even though they produce less IgG in response to it. The results suggest an influence of ABT on the humoral response to PPSV23 vaccination under MTX treatment; however, preserved opsonin responses are expected in RA patients treated with ABT plus MTX., Trial Registration: University Hospital Medical Information Network Clinical Trials Registry: UMIN000009566. Registered 12 December 2012.

Purpose: To examine the relation of work type with health-related quality of life (HRQoL) in healthy workers., Methods: We cross-sectionally examined 4427 (3605 men and 822 women) healthy workers in Japan, aged 19-69 years. We assessed HRQoL based on scores for five scales of the SF-36. Multiple regression was applied to examine the relation of work type (nighttime, shift, day to night, and daytime) with the five HRQoL norm-based scores, lower scores of which indicate poorer health status, adjusted for confounding factors, including sleeping duration., Results: Shiftwork was inversely related to role physical [regression estimate (β) = -2.12, 95 % confidence intervals (CI) -2.94, -1.30, P < 0.001], general health (β = -1.37, 95 % CI -2.01, -0.72, P < 0.001), role emotional (β = -1.24, 95% CI -1.98, -0.50, P < 0.001), and mental health (β = -1.31, 95% CI -2.01, -0.63, P < 0.001) independent of confounding factors, but not to vitality. Day-to-nighttime work was inversely related to all the five HRQoL subscales (Ps 0.012 to <0.001)., Conclusion: Shiftwork was significantly inversely related to four out of the five HRQoL, except for vitality, and day-to-nighttime work was significantly inversely related to all five HRQoL, independent of demographic and lifestyle factors.