Utilizing Activated Carbon Nanopores for Electrochemical Oxidation of Perylene to Redox-Active 3,10-Perylenedione: Application in Electrochemical Capacitor Electrodes.

We demonstrate that nanopores of activated carbon (AC) function as nanoreactors that oxidize perylene (PER) to a redox-active organic compound, 3,10-perylenedione (PERD), without any metal catalysts or organic solvents. PER is first adsorbed on AC in the gas phase, and the PER-adsorbed AC is subjected to electrochemical oxidation in aqueous H2SO4 as the electrolyte. Because gas-phase adsorption is solvent-free, PER is completely adsorbed on AC as long as the amount of PER does not exceed the saturated adsorption capacity of the AC, which enables accurate control of the amount adsorbed. PER is electrochemically oxidized to PERD in the nanopores of AC at above 0.7 V vs Ag/AgCl. The hybridized PERD undergoes a rapid reversible two-electron redox reaction in the nanopores owing to the large contact interface between the conductive carbon pore surfaces and PERD. The resulting AC/PERD hybrids serve as electrodes for electrochemical capacitors, utilizing the rapid redox reaction of PERD. The hybridization method is advantageous for quantitatively optimizing electrochemical capacitor performance by adjusting the amount of adsorbed PER. Moreover, because PERD hybridization in the AC nanopores does not expand the electrode volume, the volumetric capacitance increases with increasing hybridized PERD content. In three-electrode cell measurements, the volumetric capacitance at 0.05 A g-1 reaches 299 F cm-3, and 61% of this capacitance is retained at 10 A g-1 when 5 mmol of PER is used per gram of AC. Meanwhile, pristine AC delivers 117 F cm-3 at 0.05 A g-1 with a capacitance retention of 46% at 10 A g-1. Two-electrode cell measurements reveal that self-discharge is significantly suppressed by the hybridized PERD when AC/PERD hybrids and AC are used as cathodes and anodes, respectively, compared to that of a symmetrical AC cell. Moreover, PERD does not undergo cross-diffusion in the asymmetrical cells during self-discharge tests for 24 h.

Intraspecific divergence in a coastal plant, Euphorbia jolkinii, at a major biogeographic boundary in East Asia.

PREMISE: Quaternary climatic fluctuations and long-distance seed dispersal across the sea are critical factors affecting the distribution of coastal plants, but the spatiotemporal nature of population expansion and distribution change of East Asian coastal plants during this period are rarely examined. To explore this process, we investigated the genome-wide phylogenetic patterns of Euphorbia jolkinii Boiss. (Euphorbiaceae), which grows widely on littoral areas of Japan, Korea, and Taiwan. METHODS: We used plastome sequences and genome-wide single nucleotide polymorphisms in samples across the species range to reveal phylogeographic patterns and spatiotemporal distributional changes. We conducted ecological niche modeling for the present and the last glacial maximum (LGM). RESULTS: Genetic differentiation was observed between the northern and southern populations of E. jolkinii, separated by the major biogeographic boundary, the Tokara Gap. These two groups of populations differentiated during the glacial period and subsequently intermingled in the intermorainic areas of the central Ryukyu Islands after the LGM. Ecological niche models suggested that the potential range of E. jolkinii was restricted to southern Kyushu; however, it was widespread in the southern Ryukyu Islands and Taiwan during the LGM. CONCLUSIONS: This study provides evidence of genetic differentiation among coastal plant populations separated by the prominent biogeographical boundary. Although coastal plants are typically expected to maintain population connectivity through sea-drifted seed dispersal, our findings suggest that genetic differences may arise because of a combination of limited gene flow and changes in climate during the glacial period.

AMP-activated protein kinase (AMPK) suppresses Ibaraki virus propagation.

The Ibaraki virus (IBAV) causes Ibaraki disease in cattle. Our previous studies have shown that IBAV uses macropinocytosis to enter the host cell and exit from the endosome to the cytosol in response to endosomal acidification. To further explore the mechanism of IBAV infection and replication, we examined the effect of inhibitors of mitochondrial oxidative phosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and antimycin A, on IBAV propagation. These inhibitors significantly suppressed IBAV propagation, with reduced cellular ATP levels resulting from suppression of ATP synthesis. Furthermore, we identified AMP-activated protein kinase (AMPK), which is activated by CCCP or antimycin A, as a key signaling molecule in IBAV suppression. We also observed that IBAV infection induces ATP depletion and increases AMPK activity. Our findings suggest that AMPK is a potential target in Ibaraki disease.

IgG and insulin enhance endocytosis in THP-1 cells via activation of phosphatidylinositol 3-kinase (PI3K).

Extracellular substances, including membrane-impermeable nutrients, are taken up by cells via endocytosis. Endocytosis is also an important pathway for antigen uptake by antigen-presenting cells such as monocytes, macrophages, dendritic cells, and B cells. In this study, we investigated the regulatory mechanism of endocytosis in THP-1 cells, a monocytic leukemia cell line. We analyzed the effect of IgG and insulin, which are abundant in the serum and play important roles in immunity and metabolism, respectively, on the endocytic activity in THP-1 cells. The results indicated that IgG and insulin enhance pinocytosis and phagocytosis via activation of phosphatidylinositol 3-kinase (PI3K). Our results suggest that IgG and insulin contribute to the maintenance of endocytic activity and are important for antigen presentation and nutrient uptake.

Cancer-specific apoptosis induction in canine lymphoma cell lines by the endocytosis inhibitor dynasore.

Canine lymphoma is the most common cancer in dogs and has a poor prognosis. We recently found that the endocytosis inhibitor dynasore suppresses the viability of human cancer cell lines, especially hematopoietic cancers, by inducing apoptosis. In the present study, we examined the anticancer effects of dynasore on five previously established canine lymphoma cell lines (CLBL-1, Ema, Nody-1, CLC, and GL-1). Dynasore suppressed cell viability in these canine lymphoma cell lines more effectively than in human cancer cell lines. It also induced apoptosis in CLBL-1 and Ema cells but not in peripheral blood mononuclear cells in healthy dogs or in Madin-Darby canine kidney (MDCK) cells, suggesting that the ability of dynasore to induce apoptosis is cancer-specific. Furthermore, dynasore induced a DNA damage response in CLBL-1 and Ema cells, suggesting that it acts as a genotoxic agent in canine lymphoma cell lines. These findings suggest that endocytosis inhibitors may provide a new anticancer treatment for canine lymphoma.

Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is involved in phagocytosis activation during THP-1 cell differentiation.

Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase that plays a major role in cell metabolism. Although mTORC1 inhibitors are known to exert immunosuppressive effects, their effects on immune cells are not fully understood. In the present study, we examined the involvement of mTORC1 in the differentiation and functions of macrophages using THP-1 cells, which are derived from human monocytic leukemia and differentiate into macrophage-like cells upon treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We also examined the effects of two mTOR inhibitors, Torin 1 and rapamycin, on TPA-stimulated THP-1 cells. Although mTORC1 activation was observed upon TPA stimulation, mTOR inhibitors did not affect TPA-induced morphological changes or expression of the general macrophage marker, CD11b. In contrast, phagocytosis and fluid endocytosis were significantly impaired by the mTOR inhibitors. Endocytosis suppression was observed when mTOR inhibitors were added during differentiation, but not before or after differentiation, suggesting that endocytosis suppression altered the direction of differentiation. Furthermore, mTOR inhibitors altered the expression of M1/M2 polarization markers. These results suggest that the immunosuppressive effects of mTOR inhibitors may involve the suppression of macrophage endocytosis caused by abnormal cell differentiation.

The endocytosis inhibitor dynasore induces a DNA damage response pathway that can be manipulated for enhanced apoptosis.

Endocytosis has been shown to play an important role in cancer proliferation and metastasis. Recent studies have accumulated evidence that endocytosis inhibitors suppress in vitro and in vivo proliferation and migration. In addition, endocytosis inhibition has been shown to induce apoptosis, but its mechanism remains largely unclear. In this study, we found that the endocytosis inhibitor dynasore causes a cell viability reduction in multiple cancer cell lines, especially in hematopoietic cancers. Dynasore induced massive apoptosis and an S-phase progression delay. In addition, dynasore activated the ATR-Chk1 DNA damage response, which suggests a single-stranded DNA exposure induced by DNA replication stress. Furthermore, an ATR inhibitor sensitized the dynasore-induced apoptosis. These findings suggest that endocytosis inhibitors may have an ability to suppress DNA replication, a common mechanism of genotoxic chemotherapies targeting cancer, and that the anti-cancer effects of endocytosis inhibitors may be sensitized by DNA damage response inhibitors.

Metal-Free Homocoupling of Pyrene inside the Pores of Mesoporous Carbons via Electrochemical Oxidation: Application for Electrochemical Capacitors.

A pyrene dimer (PYD) is synthesized by electrochemical oxidation via homocoupling of pyrene (PY) inside the pores of MgO-templated mesoporous carbons without any metal catalysts or organic solvents. The resulting MgO-C/PYD hybrids can be used as high-performance aqueous electrochemical capacitor electrodes due to the reversible redox property of PYD and large contact area between the hybridized PYD and conductive carbon surfaces, which enable rapid charge transfer at the large contact interface. In our previous study, PY was considered to polymerize through electrochemical oxidation, and activated carbon with the pore sizes of ∼4 nm was used as a porous carbon substrate. In this study, the MgO-templated carbons have the average pore sizes of 5, 10, and 30 nm, and their large mesopore volumes can accommodate a large amount of PYD for enhancing the capacitance. To develop high-performance electrochemical capacitors, the dependence of the capacitance enhancement and the capacitance retention on the amount of PY and the pore sizes of MgO-templated carbons are studied. It is found that mesopores are necessary for fast charging/discharging, but the capacitance retention and capacitance enhancement decrease with increasing the mesopore sizes and the amount of PY due to the decreased utilization ratio of PY.

Synthesis of Polynorbornadiene within the Pores of Activated Carbons: Effects on EDLC and Hydrogen Adsorption Performances.

Norbornadiene (NBD) is adsorbed on activated carbon (AC), and the adsorbed NBD is polymerized within the pores of AC. Two kinds of ACs─AC-2 with only micropores of ∼2 nm and AC-4 with not only micropores but also mesopores below 4 nm─are examined to study the effects of the hybridized polynorbornadiene (PNBD) on the electric double-layer capacitor and hydrogen adsorption performance. Various measurements are performed to determine the form of the hybridized PNBD inside the pores of AC. Scanning and transmittance electron microscopy observations of the AC/PNBD hybrids confirm that PNBD is hybridized inside the pores of AC, and there is little PNBD on the surface of AC particles. The nitrogen adsorption/desorption measurement for the hybrids of AC-4 reveals that PNBD is not hybridized preferentially inside micropores rather than mesopores irrespective of the amount of PNBD. In addition, both micropore and mesopore volumes decrease at a constant rate with increasing amounts of PNBD. These results suggest that PNBD is hybridized not as a layer but as an agglomerate for both ACs, and the agglomerate delocalizes over the whole AC pores, which is supported by the results of electrochemical measurements and hydrogen adsorption behavior of the hybrids.

Nuclear-targeted 4E-BP1 is dephosphorylated, induces nuclear translocation of eIF4E, and alters mRNA translation.

Mechanistic target of rapamycin complex 1 (mTORC1) phosphorylates and inhibits eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). This leads to the release of eIF4E from 4E-BP1 and the initiation of eIF4E-dependent mRNA translation. In this study, we examined the expression of a 4E-BP1-based reporter (mTORC1 activity reporter; TORCAR) with various localization signal tags to clarify the relationship between the localization of 4E-BP1 and its phosphorylation. Phosphorylation of 4E-BP1 at threonine 37/46 and serine 65 was efficient at lysosomes and the plasma membrane, whereas it was significantly decreased in the nucleus. In addition, the localization of endogenous eIF4E shifted from the cytoplasm to the nucleus only when nuclear-localized TORCAR was expressed. Nuclear-localized TORCAR decreased cyclin D1 protein levels and altered cell cycle distribution. These data provide an experimental tool to manipulate the localization of endogenous eIF4E without affecting mTORC1 and highlight the important role of nuclear-cytoplasmic shuttling of eIF4E.

The complete chloroplast genome of a coastal plant, Euphorbia jolkinii (Euphorbiaceae).

The complete chloroplast genome sequence of a coastal plant, Euphorbia jolkinii Boiss. (Euphorbiaceae), was determined. The chloroplast genome was 162,854 bp in length, consisting of a large single copy region (90,726 bp), a small single copy region (18,422 bp), and two inverted repeats (26,853 bp). The chloroplast genome contained 115 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA genes, four unique rRNA genes, and one pseudogene, rps16. GC content of the whole chloroplast genome was 35.6%. The phylogenetic analysis showed a close relationship between E. jolkinii and E. pekinensis Rupr. The sequence data would provide useful information to understand the evolutionary process of E. jolkinii.

Partial glycosylation of the Ibaraki virus NS3 protein is sufficient to support virus propagation.

Ibaraki virus (IBAV) causes Ibaraki disease. We have previously shown that IBAV NS3 protein is highly glycosylated and that tunicamycin, an inhibitor of N-linked glycosylation, suppressed NS3 glycosylation and viral propagation. Since tunicamycin is known to cause endoplasmic reticulum (ER) stress, we explored the effects of ER stress and NS3 glycosylation on IBAV infection using tunicamycin and thapsigargin. These reagents both induced ER stress and NS3 glycosylation inhibition in a concentration-dependent manner, and as in our previous report, high concentrations of tunicamycin and thapsigargin suppressed IBAV propagation. However, lower concentrations of these reagents produced limited differences in IBAV propagation, despite their ability to suppress NS3 glycosylation and induce ER stress. These findings suggest that a considerable degree of NS3 glycosylation inhibition and ER stress induction does not suppress IBAV propagation. Conversely, lower concentrations of thapsigargin enhanced IBAV propagation, suggesting that moderate ER stress could benefit IBAV.

Ibaraki virus enters host cells by macropinocytosis.

Ibaraki virus (IBAV) is the pathogen associated with Ibaraki disease. In a previous study, we suggested that IBAV enters hamster lung (HmLu-1) cells via endocytosis and subsequently escapes into the cytoplasm upon endosomal acidification. However, it is unclear which of the endocytic pathways IBAV utilizes. In this study, we aimed to further elucidate the pathway of IBAV entry into host cells. We found that IBAV replication was not suppressed by inhibitors of clathrin-mediated or caveolin-mediated endocytosis but was markedly suppressed by 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and cytochalasin D, both of which inhibit macropinocytosis. Monensin, which inhibits endosomal acidification, also suppressed IBAV replication. To assess the inhibitory effects of these reagents on endocytosis, dextran and transferrin were used as indicators of macropinocytosis and clathrin-mediated endocytic activity, respectively. Our data confirmed that EIPA and monensin inhibited dextran uptake, and cytochalasin D inhibited the uptake of both. Additionally, we confirmed that endosomal/lysosomal acidification was inhibited by monensin. These results suggest that the macropinocytosis pathway is the major route of IBAV entry and confirm that IBAV infection of HmLu-1 cells is dependent on endosomal acidification.

Thapsigargin suppresses alpha 1-acid glycoprotein secretion independently of N-glycosylation and ER stress.

Alpha-1 acid glycoprotein (AGP) is a major acute-phase protein that is involved in drug/ligand binding and regulation of immune response. In response to inflammation, AGP secretion from the liver increases, resulting in elevated concentration of plasma AGP. AGP exhibits multiple N-glycosylation sites, and thus, is highly glycosylated. Although AGP glycosylation is considered to affect its functions, the significance of AGP glycosylation for its secretion is unclear. In this study, we investigated the effects of AGP glycosylation using glycosylation-deficient mouse AGP mutants lacking one, four, or all five N-glycosylation sites. Furthermore, we examined the effects of endoplasmic reticulum (ER) stress-inducing reagents, including tunicamycin and thapsigargin, which induce ER stress in an N-glycosylation-dependent and -independent manner, respectively. Here, we found that glycosylation deficiency and ER stress induce a little or no effect on AGP secretion. Conversely, thapsigargin significantly suppressed AGP secretion in glycosylation-independent manner. These findings indicate that AGP secretion is regulated via thapsigargin-sensitive pathway that might be further controlled by the intracellular calcium concentrations.

α-Sens: The improved ARE-Nrf2-based sensitization screening assay using normalized transcriptional activity.

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2.

Enzalutamide versus flutamide for castration-resistant prostate cancer after combined androgen blockade therapy with bicalutamide: the OCUU-CRPC study.

BACKGROUND: Before the androgen target therapy era, flutamide was widely used for castration-resistant prostate cancer in Japan. Enzalutamide is currently the recommended treatment; however, the efficacy and safety of enzalutamide and flutamide after combined androgen blockade therapy with bicalutamide, has not been compared. METHODS: Patients with castration-resistant prostate cancer who received combined androgen blockade therapy with bicalutamide were randomly assigned to receive either enzalutamide or flutamide. The primary endpoint for efficacy was the 3-month prostate-specific antigen response rate. This trial is registered with ClinicalTrials.gov (NCT02346578) and the University hospital Medical Information Network (UMIN000016301). RESULTS: Overall, 103 patients were enrolled. The 3- (80.8% vs. 35.3%; p < 0.001) and 6-month (73.1% vs. 31.4%; p < 0.001) prostate-specific antigen response rates were higher in the enzalutamide than in the flutamide group. The 3-month disease progression rates (radiographic or prostate-specific antigen progression) were 6.4% and 38.8% in the enzalutamide and flutamide groups, respectively [hazard ratio (HR): 0.16; 95% confidence interval (CI): 0.05-0.47; p < 0.001]; the 6-month rates were 11.4% and 51.1%, respectively (HR 0.22; 95% CI 0.09-0.50; p < 0.001). Enzalutamide provided superior prostate-specific antigen progression-free survival compared with flutamide (HR 0.29; 95% CI 0.15-0.54; p < 0.001). Median time to prostate-specific antigen progression-free survival was not reached and was 6.6 months in the enzalutamide and flutamide groups, respectively. CONCLUSIONS: As an alternative anti-androgen therapy in patients with castration-resistant prostate cancer who fail bicalutamide-combined androgen blockade therapy, enzalutamide provides superior clinical outcomes compared with flutamide. Enzalutamide should be preferred over flutamide in these patients.

Amino acid starvation accelerates replication of Ibaraki virus.

Ibaraki virus (IBAV) is a strain of epizootic hemorrhagic disease virus 2 that belongs to the genus Orbivirus of the family Reoviridae. IBAV replication is suppressed by the inhibition of autophagy, and since mechanistic target of rapamycin complex 1 (mTORC1) is a key regulator of autophagy, we examined if mTORC1 inhibition by amino acid starvation or mTOR inhibitors (Torin 1 and rapamycin) affects IBAV replication. We found that IBAV replication is significantly enhanced after amino acid starvation of host cells, but not after treatment with mTOR inhibitors, during early stages of viral infection (0-1 hpi). Notably, inhibition of mTORC1 by amino acid starvation was reversible and thus restricted to 0-1 hpi, whereas mTOR inhibitors sustainably suppressed mTORC1 even after the 1-h treatment, suggesting that mTORC1 suppression itself does not affect IBAV replication. To investigate the mechanism of enhanced IBAV replication by amino acid starvation, we examined the endocytic pathway, since IBAV utilizes acidification of endosomes as a trigger for viral replication. Accordingly, we found that amino acid starvation, but not mTOR inhibitors, strongly induced acidification of endosomes/lysosomes and that inhibition of endosomal acidification by bafilomycin A1 effectively blocked enhancement of IBAV replication. Altogether, the inactivation of mTORC1 by amino acid starvation during early stages of infection enhances acidification of endosomes, which in turn enhances IBAV replication.

Study of the pore structure and size effects on the electrochemical capacitor behaviors of porous carbon/quinone derivative hybrids.

We demonstrate the hybridization of a redox-active quinone derivative, 2,5-dichloro-1,4-benzoquinone (DCBQ), and porous carbons with different pore structures for aqueous electrochemical capacitor electrodes. The hybridization is performed in the gas phase, which enables accurate porous carbon/DCBQ weight ratios. This method is advantageous over conventional liquid phase adsorption, in terms of facile optimization of the porous carbon/DCBQ weight ratio to obtain high-performance aqueous electrochemical capacitor electrodes, dependent on the kind of porous carbons; moreover, complete adsorption in the liquid phase cannot be achieved by the conventional liquid phase adsorption method. Their electrochemical capacitor performances are evaluated using an aqueous 1 M H2SO4 electrolyte, and the adsorbed DCBQ undergoes redox reactions inducing pseudocapacitance within the pores of porous carbons. To study the effect of the pore size on the electrochemical capacitor behavior, two kinds of activated carbon (AC) with different pore sizes are examined: the microporous AC and the AC with both micro- and mesopores. Additionally, we examine ordered microporous carbon with a uniform pore size of 1.2 nm and a three-dimensionally (3D) ordered and mutually connected pore structure. The results reveal that mesopores facilitate proton conduction inside the DCBQ-constrained carbon pores, whereas the 3D-ordered and mutually connected micropores balance high volumetric capacitance enhancement with excellent rate capability. Such high proton conduction inside such constrained spaces can be explained only by the Grotthuss mechanism.

Apoptosis induced by Ibaraki virus does not affect virus replication and cell death in hamster lung HmLu-1 cells.

Ibaraki virus (IBAV) is an arbovirus that is transmitted by biting midges and causes Ibaraki disease in cattle. IBAV induces apoptosis in several mammalian cell lines, and apoptosis in turn facilitates IBAV replication. In addition, virus-induced apoptosis may contribute to mammalian-specific pathogenicity considering that some arboviruses induce apoptosis in mammalian cells but not in insect cells. In this study, we found that when hamster lung cells (HmLu-1) are used as a virus host, IBAV causes severe cytopathic effects with little induction of apoptosis. Furthermore, pharmacological inhibition of apoptosis did not affect IBAV-induced cytotoxicity. These results indicate the existence of an apoptosis-independent pathway in which IBAV replicates and exerts cytotoxicity in mammalian cells.