Kanto Chemical Research Articles

B-370 Clinical Utility of microRNA Quantitative Analysis Using Double Quencher Probes and Methylation Analysis by Methylation Sensitive Restriction Enzyme Polymerase Chain Reaction for Bladder Cancer

Abstract Background Histone modifications, DNA methylation, and microRNA (miRNA)-mediated gene silence are examples of epigenetic changes that regulate gene expression without causing sequence changes. The early phases of neoplastic transformation are characterized by the occurrence of these epigenetic changes, which are believed to be crucial for the development and progression of cancer. To increase the sensitivity and practicality of miRNA analysis, we developed a measuring technique using a ZEN double quencher into hydrolysis probe in reverse transcription quantitative polymerase chain reaction (RT-qPCR). Additionally, we developed a method for DNA methylation analysis employing methylation-sensitive restriction enzymes (MSRE) and methylation-insensitive restriction enzymes (MIRE) that can analyze a number of samples quickly. We examined the clinical usefulness of using these systems to understand the level of miRNA expression and methylation in bladder cancer. Methods In this study, 64 biopsied tissues from transurethral bladder tumor resection (TURBT) were used. For miRNA extraction, Cica geniusTM RNA Prep Kit (For Tissue) II (KANTO CHEMICAL CO.,INC.) and a solution that improves miRNA extraction efficiency were used. Using stem-loop RT primers, complementary DNA was created from the isolated miRNA solution. Quantitative analysis was performed by RT-qPCR using a ZEN double quencher probe and miR-21,30a, 200c, and U6 were used as target. Genomic DNA (gDNA) was extracted from the TURBT sample using High Pure PCR Template Preparation Kit (Roche). Restriction enzyme digestion was performed using HapII (Takara Bio) as MSRE and MspI (Takara Bio) as MIRE; the latter has the same recognition site as HapII but is not affected by the presence or absence of methylation. Real-time PCR was performed on the LightCycler 96 (Roche) using Universal ProbeLibrary probes (Roche) targeting ESM1, SFMBT2, and SEPTIN9. From the obtained Cp value, each methylation rate was calculated. The Fisher’s exact test was used to statistically analyze the relationship among miRNA, methylation data and other clinicopathological factors. Results For miR-30a, we observed a higher expression of miRNA was linked to more advanced T stage (P = 0.047). Additionally, ESM1 methylation exhibited a significant correlation with greater grade (P = 0.004) and T stage progress (P = 0.018). Data on miRNA abundance and methylation rate were combined in order to uncover a link with clinical data that was not apparent from either set of data separately. Samples with higher values for both miR-21 and ESM1 methylation rates were shown to have high grade (P = 0.007) and progress to the T stage (P = 0.006). Furthermore, samples with higher values for both miR-200c and SEPTIN9 methylation rates did not progress to the T stage (P = 0.013). Conclusion The combined miRNA and methylation data revealed relationships with clinical results that could not be discovered with either data separately, highlighting the clinical usefulness of the current study’s findings.

Methodological considerations of the acetaminophen Detection Kit®: Involvement of molecular oxygen (O2) in an indophenol reaction

The Acetaminophen Detection Kit® (Kanto Chemical Company Co. Inc., Tokyo, Japan) is a colorimetric test based on an indophenol reaction. The test involves three reactions: deproteination of the sample, hydrolysis of acetaminophen to yield p-aminophenol, and coupling p-aminophenol with a derivative of phenol in alkali conditions to form a blue-colored indophenol dye. The kit was devised to accomplish these three reactions with only two reagents, allowing the prompt diagnosis of acetaminophen overdose in emergency medicine.In the user instructions included with the kit and in reports introducing the kit, the chemical composition of the two reagents was not disclosed. Details about the composition can be found in the Safety Data Sheet from the manufacturer; however, there is little explanation about the principle (mechanism) of the coupling reaction. This lack of information appears to have hampered the use of this kit in forensic medicine. In this report, we conducted the coupling reaction by successively adding the two reagents to a p-aminophenol (intermediate molecule) solution with the reaction vessel open to the air and under an anaerobic condition. Development of the blue color was inhibited in the absence of air but gradually developed when the reaction vessel was opened to air. Thus, the coupling reaction is an oxidation–reduction (redox) reaction that requires molecular oxygen (O2) dissolved from the air to act as an oxidant. This finding corroborates statements in previous reports and will hopefully facilitate the use of the kit for forensic purposes.

Phase Transition Behavior of LiFePO4 in Non-Aqueous and Aqueous Electrolytes

Lithium iron phosphate (LiFePO4) is known to undergo a two-phase coexistence reaction during charging and discharging, and this phase transition is reported to be the rate-determining step of the cell reaction (1). Such a phase transition involves not only the rearrangement of the LiFePO4/FePO4 border but also the charge transfer process at the electrode/electrolyte interface, but the detail has been so far unknown. If the rearrangement of the solid border is truly the rate-determining step, it is expected that the phase transition behavior is not affected by the type of electrolyte used for the cell. In this study, we compare the activation energy of the phase transition reaction of LiFePO4 using the potential step measurements in non-aqueous and aqueous electrolyte cells to clarify the effect of the charge transfer at the electrode/electrolyte interface. The LiFePO4 powder (Hosen) was mixed with carbon black (Denka) and polyvinylidene fluoride (Kureha) in a weight ratio of 85:10:5 with 1-methyl-2-pyrrolidone (KANTO CHEMICAL, 99%). The slurry was coated onto a titanium current collector and dried at 80°C. A non-aqueous-type three-electrode cell composed of the LiFePO4 as a working electrode, lithium foils as a counter and reference electrodes, and a 1 mol dm–3 solution of LiPF6 in ethylene carbonate and diethyl carbonate (1:1 v/v) as an electrolyte solution was constructed in an Ar-filled glove box. An aqueous-type three-electrode cell was composed of the LiFePO4 as a working electrode, a Ni wire as a counter electrode, Ag/AgCl/sat’d KClaq as a reference electrode, and a 0.5 mol dm–3 Li2SO4 aqueous solution as an electrolyte solution. The charging–discharging behavior was firstly measured to confirm the cell characteristics and then the potential step measurement was employed. The Avrami plots based on the potential step results (2) were used to obtain the reaction rate constant k. These electrochemical measurements were performed at the chosen temperature. Then we calculated the activation energy of kusing the Arrhenius plot. The electrochemical measurements were performed in a constant temperature oven.Figure 1 (a) shows the transient current behavior for the cell with the aqueous electrolyte measured during the potential step from open circuit potential to 0.4 V, where LiFePO4 is completely converted into FePO4. Figure 1 (b) shows the Avlami plot (2), where f corresponds to the volume fraction of the converted FePO4, calculated from the accumulated current values. The slope was nearly unity, indicating that the system follows the first-order reaction kinetics f = 1-exp(-kt), as well as the case for the non-aqueous electrolytes. Then, we calculated the activation energy using the Arrhenius plot in the aqueous electrolyte and the non-aqueous electrolyte. The activation energy of the aqueous electrolyte was smaller than that of the non-aqueous electrolyte, suggesting the effect of the electrode/electrolyte interface on the phase transition behavior.References(1) C. Fongy, A.-C. Gaillot, S. Jouanneau, D.Guyomard, and B. Lestriez, J. Electrochem. Soc., 157, A885-A891 (2010).(2) J. Allen, T.R. Jow, and J. Wolfenstine, Chem. Mater., 19, 2108-2011 (2007).(3) Y. Orikasa, T. Maeda, Y. Koyama, T. Minato, H. Murayama, K. Fukuda, H. Tanida, H. Arai, E. Matsubara, Y. Uchimoto, and Z. Ogumi, J. Electrochem. Soc. 160, A3061-A3065 (2013). Figure 1

Ni-Alloy Fuel Electrodes for Reversible Solid Oxide Cells

Introduction Reversible solid oxide cells (r-SOCs, or solid oxide reversible cells) are devices that can efficiently operate in both fuel cell and electrolysis operating modes. It is expected that they can serve as a technology for green, flexible, and efficient energy systems coupled with renewable energy (1). However, a fuel electrode of r-SOCs might be degraded due to redox cycles in switching operation modes. In our research group, we have fabricated redox-resistant anodes with Ni-Co alloy cermet (2). Here in this study, we apply such alloy-based cermet to the fuel electrode, and evaluate electrochemical performance, reverse cycling durability, and the effect of Co addition. Moreover, we compare the cell performance of Ni-Co alloy cermet with conventional Ni-ScSZ fuel electrode. Experimental In this study, scandia-stabilized zirconia (ScSZ, 10mol% Sc2O3-1mol% CeO2-89mol% ZrO2) was used as the solid electrolyte. The powder prepared by mixing Ni-Co-based oxide powder prepared by ammonia co-precipitation with Ce0.9Gd0.1O2 (GDC) in a weight ratio of 48.1:51.9, was used as the fuel electrode material. Ni-Co-GDC fuel electrode with x mol% of Co added to Ni, is denoted as Ni(100 – x)Cox -GDC fuel electrode. The fuel electrodes of x = 0, 5, 10, and 20 were fabricated. Two types of Ni-GDC cermet were prepared. One was fabricated by using NiO powder by ammonia co-precipitation, and the other was by using commercial NiO powder (Kanto Chemical, Japan). We prepared the Ni-Co alloy cermet by mixing powder and binder, preparing and printing electrode paste on the electrolyte plate, followed by heat treatment. (La0.6Sr0.4)(Co0.2Fe0.8)O3 (LSCF) was used as the air electrode material. The electrochemical characteristics and r-SOC reversible cycle durability of the fuel electrodes were evaluated under the condition at an operating temperature of 800℃, while 100 ml/min of 50%-humidified hydrogen fuel was supplied to the fuel electrode, and 150 ml/min of air was supplied to the air electrode. Electrochemical characteristics were evaluated by electrochemical impedance measurements (1255WB, Solartron, UK), which separate ohmic and non-ohmic resistances. The durability in reverse r-SOC operation was evaluated by repeatedly varying current density within ± 0.2 A cm-2 at a current sweep rate of 1.56 mA cm-2 s-1 up to 1,000 cycles. The degradation was evaluated by averaging the percentage change in fuel electrode potential at ± 0.2 A cm-2. Results and discussion Figure 1 shows the initial performance of the cells with each fuel electrode. Figure 2 shows the degradation of each fuel electrode within 1,000 cycles after the r-SOC reverse durability test. The initial performance of Ni-Co-GDC was superior to Ni-ScSZ, and it increased with decreasing Co content. Two types of Ni-GDC exhibited identical performance. Similarly, the r-SOC reverse durability of Ni-Co-GDC was superior to Ni-ScSZ, and it increased with decreasing Co content. Furthermore, two types of Ni-GDC exhibited almost the same durability. These results indicate that GDC contributes to the increased cell performance and durability rather than alloying or the feature of original NiO powder. It appears that Co is not essential for r-SOC fuel electrodes in terms of r-SOC durability. However, fuel electrode materials should be selected by comprehensively considering the performance and the durability at both SOFC and SOEC modes. As Ni-Co alloy shows higher redox cycle durability compared with Ni-GDC (3), Ni-Co alloy is still one of the promising materials for r-SOC fuel electrodes. Acknowledgements A part of this study was supported by “Research and Development Program for Promoting Innovative Clean Energy Technologies Through International Collaboration” of the New Energy and Industrial Technology Development Organization (NEDO). References N. Q. Minh and M. B. Mogensen, Electrochem. Soc. Interface, 22, 55 (2013).Y. Ishibashi, S. Futamura, Y. Tachikawa, J. Matsuda, Y. Shiratori, S. Taniguchi, and K. Sasaki, J. Electrochem. Soc., 167, 124517 (2020).K. Matsumoto, Y. Tachikawa, J. Matsuda, S. Taniguchi, and K. Sasaki, ECS Trans., 103, 1549 (2021). Figure 1

Impact of beta-lactamase detection reagent on rapid diagnosis of ESBL-producing pathogens using urine samples of patients with Gram-negative bacteriuria

The rapid increase of extended-spectrum beta-lactamase (ESBL)-producing pathogens makes it difficult to choose appropriate antimicrobials in patients with Gram-negative bacterial infection. The Cica-beta reagent (Kanto Chemical, Tokyo, Japan) is a chromogenic test that detects ESBLs from bacterial colonies. This study aimed to reveal whether Cica-beta reagent could detect ESBLs directly from urine samples to facilitate rapid diagnosis of antibiotic susceptibility. A prospective study was conducted from July 2019 to November 2019. Patients in whom urine culture tests were performed were eligible. Each urine sample was centrifuged, and the pellet was mixed with Cica-beta reagent. The test was considered positive when the enzymatic reaction turned from yellow to red or orange. In total, 350 urine samples were analysed. Urinary tract infection (UTI) was diagnosed in 214 patients. ESBL-producing Enterobacterales were isolated from 79 samples. The Cica-beta test showed sensitivity of 79.8% and specificity of 99.3% in patients with Gram-negative bacteriuria. Sensitivity and specificity improved to 90.2% and 100%, respectively, in patients with UTI. The Cica-beta test could be an efficient test for the detection of ESBL-producing pathogens in urine. By providing immediate information about ESBLs, it might be a useful point-of-care test to guide appropriate antimicrobial use in patients with UTI.

Highly Specific Monoclonal Antibodies Against SARS-CoV-2 Nucleocapsid Antigen for Accurate Diagnosis of COVID-19

The ongoing COVID-19 pandemic is a major global public health concern. Although rapid point of care testing for detecting viral antigen is important for management the outbreak, none of current antigen detection kits provide a reliable diagnosis due to the lack of a specific monoclonal antibody (mAb) against SARS-CoV-2. In our current study, we produced mAbs exclusively react with SARS-CoV-2 and exhibited no cross-reactivity with other human coronaviruses including SARS-CoV. Molecular modeling revealed that the mAbs bound to epitopes present on the exterior surface of the nucleocapsid, encompassing their scope for detecting SARS-CoV-2 in clinical samples. We further selected the optimal pair of anti-SARS-CoV-2 NP mAbs for designing ELISA and immunochromatographic assay. An integrated bioinformatics analysis revealed that our mAbs could comprehensively detect divergent strains of SARS-CoV-2. Due to the high redundancy and absence of cross-reactivity, our newly developed mAbs would be instrumental for developing reliable diagnosis kits for COVID-19.Funding: This work was supported in part by Japan Agency for Medical Research and Development (AMED, Grant number: JP19fk0108110 and JP20he0522001), and by Health Labour Sciences research grant from The Ministry of Health Labour and Welfare.Conflict of Interest: The authors have no conflicts of interest directly relevant to the content of this article. YY, SK, KS and DA is a current employee of Kanto Chemical Co., Inc.Ethical Approval: This study was approved by the Institutional Review Board of Yokohama City University (IRB No. B200800106), and the protocols used in the study were approved by the ethicscommittee.

Pembentukan Fasa Co3O4 Dengan Metoda Dekomposisi CoCO3 Menggunakan Sinar Diffraksi

Experiments have been carried out to obtain the decomposition of cobalt carbonate cobalt oxide to be used as raw material for the manufacture of calcium cobalt oxide thermoelectric materials. The experiments were performed by heating cobalt carbonate powder material (CoCO3) from Kanto Chemical with a temperature of 1000°C. Heating aims to obtain Co3O4 phase. Co3O4 phase is what will be used in the manufacture of thermoelectric materials based on the phase diagram of the system Ca-Co-O. The results of X-ray diffraction CoCO3 materials are heated to 1000°C showed that Co3O4 phase has been formed. Analysis quantitative diffraction pattern shows the diffraction peaks are the property throughout the Co3O4 phase. The crystal structure of Co3O4 is Face Centered Cubic (FCC) with space group F d -3 m. Lattice parameters of the diffraction pattern is the result of smoothing a = 8.0838 Å.

Sulfurized Polyethylene Glycol As Electrode Material for Li-S Battery

The battery of safe, low cost, durable and high capacity is strongly demanded for vehicles, mobile phones, personal computers, power backup systems and so on. Lithium-sulfur batteries gather attentions because sulfur as a cathode material has a high capacity of 1672 mAhg-1, is abandon, cheap and benign to environment. The sulfur cathode, however, has disadvantages such as high resistance and dissolution into electrolyte in the form of Li2Sx, which degrade the charge/discharge efficiency and capacity [1]. In order to mitigate the dissolution of sulfur species, several kinds of nano carbon materials such as graphene [2], carbon nanotube [3], and nano carbon architectures [4] have been investigated as the container for sulfur. Polymer and sulfur composites also have been examined. Among them, a material prepared through heat treatment of polyacrylonitrile and sulfur mixture showed good capacity and cyclability [5-7]. We found that heat-treated mixture of cheap polyethylene glycol and sulfur, sulfurized polyethylene glycol (SPEG) also showed good capacity and relatively good cycle stability as Li-S battery cathode. SPEG was synthesized by heat treatment 25.0 g of polyethylene glycol (PEG) (Kishida Chemical) and 51.6 g sulfur (Kanto Chemical) in an alumina tube (Nikkato), which was set in an electric furnace. The mixture was heated-up to 723 K under N2 flow then cooled. The reaction of SPEG production proceeded above 523 K with refluxing both of sulfur and PEG releasing H2S. This kind of production reaction for sulfur containing material is quite unique because not only sulfur but also PEG exist both liquid and vapor. The obtained crude SPEG was pulverized and heat-treated again at 573 K under N2 flow for more than 2h in order to take rid of residual molecular sulfur. Black powder was obtained by 8.9 g. The production of SPEG was easily scaled-up to 112 g in an experimental batch. SPEG was characterized using elemental analysis, XRD, and TG-DTA. For electrochemical measurement, cathode was prepared by mixing 50 wt% SPEG, 40 wt% acetylene black (AB), and 10 wt% polytetrafluoroethylene (PTFE), attached to aluminum mesh. The cathode was assembled into coin-type cells (CR2032), with the use of polypropylene sheet as separator, lithium foil as counter electrode, and lithium bis(trifluoromethanesulfonyl)amide (LiTFSA) : tetraglyme (1:1 molar ratio) as electrolyte. Galvanostatic charge-discharge cycle tests were performed at a current density of 25 mA g-1 (C/20 rate) in the potential range of 1.0 – 3.0 V vs. Li/Li+. From elemental analysis of SPEG, it consist of C: S: H: O = 35.3: 61.4: 0.4: 2.9 wt%. The content ratio of sulfur in SPEG is relatively higher than that of the other sulfur containing cathode materials. The XRD profiles of SPEG showed only a halo profile typical for such as low-crystalline or amorphous material without reflection of crystalline sulfur. TG-DTA result showed that there was only 17% weight-loss of SPEG until 773 K. This showed SPEG was a heat-resistive material and strongly held sulfur in it. The electrochemical properties of SPEG were investigated in a half cell. The capacity was calculated based on the mass of SPEG not sulfur. SPEG cathode showed large first discharge capacity of 910 mAh/g, and first charge one 550 mAh/g, therefore this material had irreversible capacity of 360 mAh/g. The cathode showed second discharge capacity 488 mAh/g, and kept 433 mAh/g at 10th cycle. The average discharge voltage of the cathode was 1.8 V vs. Li/Li+. Because relatively large capacity cathodes like one of Li-S battery changes its volume during charge/discharge, cathode structure is apt to be destroyed. A suitable binder would improve SPEG cathode cyclability. As a conclusion, the cheap law materials of PEG and sulfur were changed through new method to novel cathode material SPEG with good productivity up to 112 g. SPEG has high content ratio of sulfur with heat-resistive up to 773 K, and has large capacity with good cycle stability.

New High-Capacity Positive Electrode Materials on Li-Ni(II)-Nb(V) System

High-capacity positive electrode materials are needed to further increase energy density of Li-ion battery. Numerous materials have been studied as positive electrodes. Among them, Li2MnO3-based electrode materials are generally recognized as the most promising high-capacity positive electrode materials for rechargeable lithium batteries. Recently, our group has reported that Li3NbO4,[1] which has higher lithium contents than that of Li2MnO3, is potentially utilized as host structures for a new series of high-capacity electrode material. Although Li3NbO4 crystallizes as the lithium-excess rocksalt-type structure, Li3NbO4 is electrochemically inactive because of the absence of electrons in a conduction band (4d0 configuration for Nb5+). Transition metals substituted for Li+ and Nb5+ donate electrons in the conduction band. Mn3+-substituted Li3NbO4 delivers large reversible capacity (approximately 300 mAh g-1) with highly reversible solid-state redox reaction of oxide ions.[1] In this study, crystal structures and electrode performance of a binary system between Li3NbO4 and NiO (x Li3NbO4 – (1 – x) NiO) are systematically examined as potential new high-capacity electrode materials. Binary system of xLi3NbO4 - (1-x) NiO was prepared from Li2CO3 (98.5 %, Kanto Chemical CO., Inc.), Nb2O5 (99.9 %, Wako Pure Chemical Industries, Ltd.), and NiCO3 • 2Ni(OH)2 • 4H2O (Wako Pure Chemical Industries, Ltd.). Composite electrodes consisted of 80 wt% active materials, 10 wt% acetylene black, and 10 wt% poly(vinylidene fluoride), pasted on aluminum foil as a current collector. Metallic lithium was used as a negative electrode. The electrolyte solution used was 1.0 mol·dm−3 LiPF6 dissolved in ethylene carbonate and dimethyl carbonate (3:7) (Kishida Chemical CO., Ltd). A polyolefin membrane was used as a separator. Electrode performance of samples was evaluated in Li cells at a rate of 5 mA g- 1 at room temperature or 50 oC. Figure 1a shows X-ray diffraction patterns of different samples found in x Li3NbO4 – (1 – x) NiO binary system. All samples assigned to rocksalt-related structures. Samples of x = 0.33 and 0.36 are found to be isostructural with Li3Ni2NbO6, which has cation ordering of Nb and Li/Ni.[3] In addition, the sample of x = 0.44 is found to be cation-disordered rocksalt-type structure. Electrochemical properties of the samples in Li cells are compared in Figure 1b. Although the sample (x = 0.33) shows small capacity (100 mAh g-1), high discharge voltage based on Ni2+/Ni4+ is observed. In contrast, the sample (x = 0.44) shows high reversible capacity (200 mAh g-1), but huge polarization on charge/discharge is observed. Together with these results, we will further discuss the possibility of x Li3NbO4 – (1 – x) NiO binary system as positive electrode materials with high energy density for rechargeable lithium batteries.

Electrochemical Behavior of Tris(2,2'-bipyridine)Cobalt Complex in Some Ionic Liquids

Electrostatic interaction between a charged species and the ions composing ionic liquids is considered much stronger than that in conventional electrolyte solutions. Strong electrostatic interaction is expected to influence electrochemical reactions of the charged species. We have already reported the diffusion and charge transfer of tris(2,2'-bipyridine) and tris(1,10-phenanthroline) complexes of some transition metals are affected by the charge densities of redox species and the ions composing ionic liquids[1-6]. In the present study, the electrochemical behavior of tris(2,2'-bipyridine)cobalt complex ( [Co(bpy)3]3+ and [Co(bpy)3]2+) was investigated in 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)amide (BMPTFSA) and 1-ethyl-3-methylimidazolioum tetrafluoroborate (EMIBF4). BMPBr was prepared by the reaction of methylpyrrolidine and butyl bromide, purified by recrystallization and dried under vacuum. BMPTFSA was prepared by the metathesis of BMPBr and LiTFSA. EMIBF4 was purchased from Kanto Chemical. [Co(bpy)3](TFSA)2 was prepared by addition of LiTFSA into CoCl2 and bpy aqueous solution. [Co(bpy)3](TFSA)3 was prepared by oxidation of [Co(bpy)3]2+ with H2O2 under acidic condition and addition of LiTFSA. [Co(bpy)3](BF4)2 and [Co(bpy)3](BF4)2 were prepared in the same way using NaBF4 instead of LiTFSA. Electrochemical measurements were performed with an air-tight three electrode cell assembled in a glovebox of dry Ar atmosphere. Platinum was used as a working and counter electrode. Silver immersed in BMPTFSA containing 0.1 M AgCF3SO3was used as a reference electrode. The diffusion coefficients of [Co(bpy)3]3+ and [Co(bpy)3]2+ were determined by chronoamperometry at various temperatures. The diffusion coefficients of [Co(bpy)3]3+ and [Co(bpy)3]2+ were close to those of [M(bpy)3]3+ and [M(bpy)3]2+ (M = Fe and Ru) in each ionic liquid, verifying the diffusion of the charged species is related to the size and charge density of the diffusing species. The charge transfer rate of [Co(bpy)3]3+/2+ was slower than those of [M(bpy)3]3+/2+ (M = Fe and Ru), probably due to the difference in the electron configurations. Furthermore, the apparent reaction entropy change of [Co(bpy)3]3+/2+ was larger than those of [M(bpy)3]3+/2+ (M = Fe and Ru), suggesting the large contribution of the difference in the electron configurations of [Co(bpy)3]3+ and [Co(bpy)3]2+to the apparent reaction entropy change.

Characterization for Sulfurized Polyethylene Glycol As Electrode Material for Li-S Battery

Sulfur is one of the promising electrode materials because of a large theoretical capacity (1672 mAh g-1). However, both ionic and electronic conductivities of sulfur are poor and the lithium polysulfide (Li2Sx) dissolves into the electrolyte solution during charge/discharge process [1]. To improve the ionic and electronic conductivities, the composites with carbon, such as nano-carbon architectures [2, 3], graphene [4-6], and carbon nanotubes [7, 8], have been investigated as positive electrode materials. Recently, it was reported that the sulfurized carbon composite prepared by heat treatment of polyacrylonitrile with sulfur exhibits a high capacity and long cyclability [9-11]. In this study, we propose a novel sulfurized carbon composite (here after denoted as SPEG), which is synthesized by heat treatment of polyethylene glycol (PEG: a representative polymer) with sulfur. SPEG showed a second discharge capacity of 488 mAh g(SPEG)-1 and was maintained at 433 mAh g(SPEG)-1 even after the 10thcycle. Although SPEG has high capacity and long life-cycle, crystallographic structure of SPEG itself is not clear. In this paper, we investigate the local structure and chemical states of S in SPEG by X-ray absorption spectroscopy and examined the chemical state of SPEG by Raman spectroscopy. SPEG was prepared through refluxing 25.0 g of polyethylene glycol (PEG; Kishida Chemical, mean molecular weight: 200 g mol-1) and 51.6 g sulfur (Kanto Chemical) in an alumina tube (Nikkato) in an electric furnace. The mixture of PEG and S was heated up to 673 K, then cooled. The obtained crude product was heated again at 573 K under nitrogen flow (100 ml min-1) in order to remove the residual molecular sulfur. Composite obtained (8.9 g) was pulverized using mortar and pestle. The composite powder is a black powder and contained about 60 wt.% of S in the sample. The local structure and chemical states of S in SPEG were examined by S K-edge X-ray absorption fine structure (XAFS, BL-10 of the Synchrotron Radiation Center, Ritsumeikan University). The total electron yield method was used and the incident X-ray beam was monochromatized with a Ge(111) crystal (2d = 0.6532 nm) pair. The photon energy was calibrated with the strong resonance of K2SO4 appearing at 2481.7 eV dipole transition. The XANES spectrum of this composite shows three distinct peaks at 2469, 2472 and 2473 eV, which however do not correspond to profiles of pure S and Li2S. Therefore, this suggests that free S is absent in the SPEG. The chemical states of composites were investigated by Raman spectroscopy (ALMEGA XR, Thermo Fisher SCIENTIFIC) at room temperature. The excitation source is a 532 nm laser. The wavenumber of monochromator was calibrated by a silicon plate at 520 cm-1. The spectrum was recorded by accumulation of 12 scans (each of 5 s) in the range of 100 < ν/cm-1 < 3000. The highest Raman peak of SPEG appears at 1441 cm-1 between G (1600 cm-1) and D (1300 cm-1) bands of graphite. The other peaks appear at 1279, 1066, 846, and 772 cm-1. Those peaks are not assigned to various vibration modes of pure S and carbon. Thus, the new chemical states are formed between S and C in this composite. This result is consistent with result of XAFS. The chemical states in SPEG are related to the electrochemical properties. We will discuss a possible structural model of SPEG and also the correlation between the electrochemical properties and the electronic/local structural changes in SPEG.

Electrorefining of Na in Hydrophobic Ionic Liquids

Introduction The sodium-sulfur secondary battery (Na-S battery) has several advantages such as high energy density, low material cost, long lifetime, and maintenance free1-2) and commercial scale of the battery had sold since 2003. 3) Recently, the battery has been applied to load leveling of electric power, emergency power supply for interruption in factory, and storage device of power generation from solar or wind energy. Lifetime of the battery is about 15 years and large amount of the used batteries will be discarded in near future. In the used battery, metallic sodium and sodium polysulfide remain in inside of the battery. Since metallic sodium is an active metal, effective recycle of metallic sodium is required. We have proposed electrorefining process of metallic sodium from the used Na-S battery.4) We investigated propylene carbonate as an electrolytic melt in previous study. From the results, it was found that temperature of upper limit in propylene carbonate was 373 K. In this paper, to survey the electrolytic melt that can be used above 373 K, impedance measurement, voltammogram measurement, and constant current electrolysis were carried out in NaTFSI-TEATFSI ionic liquids and NaTFSI-TBATFSI ionic liquidsExperimental NaTFSI (sodium-bis(trifluoremethylsulfonyl)imide) was prepared by HTFSI (bis(trifluoremethylsulfonyl) imide, 99% pure, Kanto chemical) and NaOH(99.99% pure, Merk). TEATFSI (Tetraethylammonium bis (trifluoremethylsulfonyl) imide, 99% pure, Kishida chemical) and TBATFSI (Tetrabuthylammonium bis (trifluoremethylsulfonyl) imide, 97% pure, Iolitech) were used as received. Electrochemical cell was made of Pyrex glass cylinder. 10-30mol%NaTFSI-TEATFSI or 10-30mol%NaTFSI-TBATFSImixture melt of 30 ml were kept at 433 K. Glassy carbon (GC, Tokai carbon, GC-20) plates were used for working and counter electrode. Pt (99.99% pure, Tanaka Kikinzoku Kogyo) wire was used as a quasi reference electrode. Impedance measurement was carried out with amplitude of 15 mV and frequency from 1 Hz to 20 kHz. Electrical conductivity was calculated from the resistance value at a high frequency limit in impedance measurement. In voltammogram measurement, metallic sodium (Aldrich, 99.9% pure, calcium content : 500 ppm) was used as a counter electrode. The electrochemical measurement was carried out in a glove box under pure argon gas atmosphere. Electrolysis was done by constant current of 1-10 mAcm-2in 20 mol% NaTFSI-TEATFSI melt or 20 mol% NaTFSI-TBATFSI melt and the electrodeposit was analyzed by ICP atomic emission spectroscopy (Thermo i-CAP6000). Results and Discussion The conductivity of electrolytic melts were determined by AC impedance measurement in 10 - 30 mol% NaTFSI-TEATFSI. The maximum conductivity was attained to 36 mScm-1 at 20 mol% NaTFSI-TEATFSI. On the other hand, maximum conductivity was 16 mScm-1 at 20 mol% NaTFSI-TBATFSI. From the result, we selected 20 mol% NaTFSI melt was a suitable concentration and the following experiment was made in the melt. In voltammogram on GC electrode in 20 mol% NaTFSI-TEATFSI, a cathodic current was observed from -1.0 V and the current increased until -3.0 V. When the potential is reversed at -3.0 V, anodic current is observed from -2.2 to 0.5 V. It is considered that the cathodic and anodic currents correspond to electrodeposition and electrodissolution of sodium. From ICP analysis, the electrodeposit formed by constant current electrolysis was found to contain sodium of 99.99 % pure and 12 ppm calcium as impurity. The calcium content was seen to be decreased to about one fortieth by the electrorefining process. References1) J. L. Sudworth and A. R. Tilley, The Sodium Sulfur Battery, Chapman and Hall, (1985).2) R. Okuyama, H. Nakashima, T. Sano, and E. Nomura, J. Power Sources, 93, 50 (2001).3) T. Oshima, M. Kajita, A. Okuno, Int. J. Appl. Ceram. Technol., 1, 269(2004).4) M. Ueda, H. Hayashi, and T. Ohtsuka, J. Japan Inst. Metals, 73, 691(2009). Acknowledgements We thank New Energy and Industrial Technology Development Organization (NEDO) and Hokkaido Bureau of Economy, Trade and Industry for supporting this research.

COVALENT IMMOBILIZATION OF GLUCOSE OXIDASE ON WELL-DEFINED POLYMER-SILICON WAFER HYBRIDS VIA SURFACE-INITIATED RAFT-MEDIATED PROCESS

A surface modification technique was developed for the functionalization of silicon surface with glucose oxidase (GOD). The silicon surface was first graft copolymerized with glycidyl methacrylate (GMA) via surface-initiated reversible addition-fragmentation chain-transfer (RAFT)-mediated process. GOD was then covalently immobilized through the ring-opening reaction between the amine groups of the GOD and the epoxide groups of the grafted GMA polymer chains. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface-modified surface after each modification stage. Increasing the thickness of the polymer layer and the immobilization time could allow a great amount of GOD to be immobilized on the silicon surface. The GOD-functionalized silicon hybrids are promising candidates for the silicon-based glucose biosensors. 1-4 It is of great importance to improve the biocompatibility of the silicon microdevices and to develop silicon surfaces with controllable cell adhesion. Immobilization of biomolecules on silicon substrates can be achieved via many methods such as adsorption, entrapment, covalent binding, etc. Functionalization of silicon surface with organics is a key in the development of new silicon-based biomedical devices. Tethering of polymer brushes on the solid substrate is an effective method of modifying the surface of the various substrates. 5-6 The dense polymer brushes can alter the properties of the surface, supply excellent mechanical and chemical protection to the substrate, and indicate new pathways to the functionalization of silicon surfaces for molecular recognition and sensing. 7-10 USA). The silicon wafers were cut into square chips of about 1.2 cm × 1.2 cm in size. The monomer, GMA, was passed through inhibitor-removing columns and then stored under an argon atmosphere at -10 °C. The initiator a,a'-azobis(isobutyronitrile) (AIBN, 97%) was obtained from Kanto Chemical Co. (Tokyo, Japan) and was recrystallized in anhydrous ethanol. The silane coupling agent (3-(trimethoxysilyl) propyl methacrylate (TMSPM, 98%)) was obtained from Aldrich Chemical Co. (St. Louis, MO, USA). The chain transfer agent (CAT), 1-phenylethyl dithiobenzoate (PDB), was synthesized according to the published procedures.19 GOD (Type II, 15500 units g-1 from Aspergillus niger) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's phosphate buffer solution (PBS, pH=7.4) was freshly prepared. Bio-Rad dye reagent for protein assay (Catalog No. 500-0006) was obtained from BioRad (Hercules, CA, USA). The other reagents were of analytical grade and were used without further purification unless otherwise mentioned. Immobilization process The process of surface functionalization of silicon was shown in Fig. 1. Coupling of the RAFT initiator to the silicon wafer was first achieved by adding TMSPM (0.41 mmol), AIBN (0.23 mmol), and PDB (0.46 mmol) at a molar ratio of 1.8:1:2 into 5 mL of dry DMF in a Pyrex tube containing the silicon chip. After purging the mixture with argon for 30 min, the surface coupling reaction continued at 60 °C for 24 h under an argon atmosphere. After the reaction, the modified substrate was washed with CH 2 Cl 2 and then with an ethanol/water mixture.

Determination of Artepillin-C in Brazilian Propolis by HPLC with Photodiode Array Detector

A method for the determination of artepillin-C using HPLC coupled with photodiode array (PDA) detector was developed. Artepillin-C is abundantly present in Brazilian propolis of Baccharis species origin. The chromatographic separation was performed on a Mightysil RP-18 GP II column (150 mm × 4.6 mm, 5.0 ?m; Kanto Chemical Company Limited), and column temperature was maintained at 40°C. The mobile phase was a linear gradient elution of 0.5% aqueous acetic acid (A) and acetonitrile (B) at a flow rate of 1.0 ml/min. The chromatogram was monitored at 320 nm. The calibration range of artepillin-C was 0.75-2500 ?g/ml (r2 = 0.9991). The limit of detection (LOD) and limit of quantification (LOQ) were 0.50 ?g/ml and 0.75 ?g/ml, respectively. The intra-day and inter-day precision of the assay (RSD) were in the range 1.28-5.60% and 1.45-6.75%, respectively. The artepillin-C content in Brazilian propolis is an important factor in ensuring the quality of Brazilian propolisof Baccharis species origin. This method was used to determine artepillin-C content in Brazilian propolis.

Stability of Solvents against Superoxide Radical Species for the Electrolyte of Lithium-Air Battery

Candidate solvents which we examined were PC (Kishida chemical, >99.5%), dimethylsulfoxide (DMSO, Wako pure chemical, >99%), acetonitrile (ACN, Wako pure chemical, >99%), 3-methoxypropionitrile (MPN, Wako pure chemical, >99%), trimethyl phosphate (TMP, Wako pure chemical, >99%), sulfolane (SULF, Aldrich, >99%), tetramethylene sulfoxide (TMSO, Aldrich, >99%), tetraglyme (TGly, Aldrich, >99%) and ionic liquid, N-methyl-N-propylpiperidinium bis(trifluoromethanesulfonyl)imide (PP13-TFSI, Kanto Chemical), and these solvents were used as received without further purification. 0.2 g (2.8 mmol) of yellow KO2 powder (Strem chemicals, 96.5%) was slowly added into 30 mL sample bottle containing 10 mL of candidate solvent with/without 1 M of supporting electrolyte, lithium bis(trifluoromethanesulfonyl)imide (LiTFSI, Kanto Chemical, >99.7%), and the bottle was closed and placed 24 hours in room temperature. Precipitates were filtrated by separable syringe filter (1 μm pore) and washed in DMSO and chloroform three times, respectively. After drying in vacuum, the precipitates were analyzed by Raman and IR spectroscopy. The yields of the precipitates were in the range of 50‐130 wt% as compared with the weight of added KO2. All process and analysis were carried out under inert gas (Ar) atmosphere. Analytical method of Raman and IR were identical of our previous report. 13 Baseline calibration of

Rapid detection of multidrug resistant Gram-negative bacilli by Cica-Beta-Test strips

Aim of the study This study aimed to evaluate the sensitivity and specificity of a new system (Cica-Beta-Test, Kanto Chemical, Japan) for rapid detection of AmpC-derepressed, extended-spectrum β-lactamases (ESβL) and metallo-β-lactamases (MβL). Methods Two hundred Multi-Drug Resistant (MDR)-Gram-negative bacilli were studied: 170 Enterobacteriaceae and 30 Gram-negative non-fermentative bacteria. One hundred and eighteen strains produced an ESβL, seven MβL and 75 derepressed cephalosporinases. One drop of substrate was dispensed onto the filter pad of the Cica-Beta-Test strip. The bacterial colonies were spread on the filter pad of strip. The reading was performed after 2 to 15 min: turning chromogenic indicated the positive test. Three tests were used: Cica-Beta I for detection of MDR bacteria; Cica-Beta MβL for detection of MβL-producing bacteria and Cica-Beta CVA, which distinguish ESβL and AmpC-derepressed producers. Results were compared with molecular assays. Results Cica-Beta-Test I has detected 194 MDR (sensitivity 97%), Cica-Beta-Test MβL has shown the presence of six MβL tested (sensitivity 85.7%). Five strains were non-MβL false positive (specificity 97.3%). Cica-Beta-Test CVA allowed the differentiation of ESβL-producing strains (109/115) and AmpC-derepressed strains (56/67) (sensitivity 94.8%, specificity 83.8%). Conclusions Because of their epidemic nature, the MDR strains are screened and require strict hygienic measures patients. The simultaneous use of three strips can quickly determine the presence of MDR including ESβL and MβL. Rapid screening of MDR avoids transmission and limits the use of broad-spectrum antibiotics.

Reduction of Brain β-Amyloid (Aβ) by Fluvastatin, a Hydroxymethylglutaryl-CoA Reductase Inhibitor, through Increase in Degradation of Amyloid Precursor Protein C-terminal Fragments (APP-CTFs) and Aβ Clearance

Epidemiological studies suggest that statins (hydroxymethylglutaryl-CoA reductase inhibitors) could reduce the risk of Alzheimer disease. Although one possible explanation is through an effect on beta-amyloid (Abeta) metabolism, its effect remains to be elucidated. Here, we explored the molecular mechanisms of how statins influence Abeta metabolism. Fluvastatin at clinical doses significantly reduced Abeta and amyloid precursor protein C-terminal fragment (APP-CTF) levels among APP metabolites in the brain of C57BL/6 mice. Chronic intracerebroventricular infusion of lysosomal inhibitors blocked these effects, indicating that up-regulation of the lysosomal degradation of endogenous APP-CTFs is involved in reduced Abeta production. Biochemical analysis suggested that this was mediated by enhanced trafficking of APP-CTFs from endosomes to lysosomes, associated with marked changes of Rab proteins, which regulate endosomal function. In primary neurons, fluvastatin enhanced the degradation of APP-CTFs through an isoprenoid-dependent mechanism. Because our previous study suggests additive effects of fluvastatin on Abeta metabolism, we examined Abeta clearance rates by using the brain efflux index method and found its increased rates at high Abeta levels from brain. As LRP1 in brain microvessels was increased, up-regulation of LRP1-mediated Abeta clearance at the blood-brain barrier might be involved. In cultured brain microvessel endothelial cells, fluvastatin increased LRP1 and the uptake of Abeta, which was blocked by LRP1 antagonists, through an isoprenoid-dependent mechanism. Overall, the present study demonstrated that fluvastatin reduced Abeta level by an isoprenoid-dependent mechanism. These results have important implications for the development of disease-modifying therapy for Alzheimer disease as well as understanding of Abeta metabolism.

Identification of a Novel Selenium-containing Compound, Selenoneine, as the Predominant Chemical Form of Organic Selenium in the Blood of Bluefin Tuna

A novel selenium-containing compound having a selenium atom in the imidazole ring, 2-selenyl-N(alpha),N(alpha),N(alpha)-trimethyl-L-histidine, 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate, was identified from the blood and other tissues of the bluefin tuna, Thunnus orientalis. The selenium-containing compound was purified from the tuna blood in several chromatographic steps. High resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the exact mass of the [M+H](+) ion of the compound was 533.0562 and the molecular formula was C(18)H(29)N(6)O(4)Se(2). Its gross structure was assigned as the oxidized dimeric form of an ergothioneine selenium analog in which the sulfur of ergothioneine is replaced by selenium. Therefore, we named this novel selenium-containing compound "selenoneine." By speciation analysis of organic selenium compounds using liquid chromatography inductively coupled plasma mass spectrometry, selenoneine was found widely distributed in various tissues of the tuna, with the highest concentration in blood; mackerel blood contained similar levels. Selenoneine was measurable at 2-4 orders of magnitude lower concentration in a limited set of tissues from squid, tilapia, pig, and chicken. Quantitatively, selenoneine is the predominant form of organic selenium in tuna tissues.

Comparative Structural Biology of Eubacterial and Archaeal Oligosaccharyltransferases

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes N-glycosylation. We report the 2.8 A resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.

JBIR-52, a new antimycin-like compound, from Streptomyces sp. ML55

GRP78/Bip is a molecular chaperone in the endoplasmic reticulum (ER) induced by ER stress that promotes protein folding and has an important role as a survival factor in solid tumors by providing resistance to both chemotherapy and hypoglycemic stress.1 Thus, specific downregulators of GRP78 expression can reasonably be expected to become promising drugs in cancer chemotherapy.2 In the course of our screening program for downregulators of GRP78 expression, we have isolated versipelostatin A-F,3–11 prunustatin A,12,13 JBIR-04, -0514 and JBIR-06.15 Further screening resulted in the isolation of a new inhibitor designated as JBIR-52 (1, Figure 1) from culture of a JBIR-06 producer, Streptomyces sp. ML55.15,16 In this paper, we report the isolation, structure elucidation and brief biological activity of a new member of antimycin, 1. Streptomyces sp. ML55 was cultured on a rotary shaker (220 r.p.m.) at 27 1C for 5 days in 500-ml Erlenmeyer flasks containing 100 ml of a production medium consisting of 2% glycerol (Nacalai Tesque, Kyoto, Japan), 1% molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan), 0.5% casein (Kanto Chemical, Tokyo, Japan), 0.1% polypepton (Nihon Pharmaceutical, Tokyo, Japan), 0.4% CaCO3 (Kozaki Pharmaceutical, Tokyo, Japan) (pH 7.2 before sterilization). The mycelium from the culture broth (2 l) was extracted with Me2CO (400 ml). After concentration in vacuo, the residue was extracted twice with EtOAc. The organic layer was dried over anhydrous Na2SO4, and concentrated in vacuo. The dried residue (1.99 g) was applied to normal-phase MPLC (Purif-Pack SI-60, size:60, Moritex, Tokyo, Japan) and developed with a n-hexane–EtOAc linear gradient system (0–100% EtOAc), and peak detection was carried out by UV absorption at 254 nm. The 60–75% EtOAc eluate (470 mg) was further chromatographed on normal-phase MPLC (Purif-Pack SI-60, size:20, Moritex) with n-hexane–EtOAc (80:20). A portion (44.5 mg) of the fraction (388 mg) including both 1 and JBIR-06 was finally purified by preparative reversed-phase HPLC using an L-column2 ODS (20 i.d. 150 mm, Chemical Evaluation Research Institute, Tokyo, Japan) with 60% CH3CN–H2O containing 0.1% formic acid (flow rate, 9.5 ml min 1) to yield 1 (1.7 mg, retention time (Rt) 28.0 min) and JBIR-06 (2.3 mg, Rt 37.0 min). Compound 1 was obtained as a white powder ([a]D +40.0, c 0.07, 29 1C in MeOH, UV (MeOH) lmax (e) 225 (22 500), 336 nm (4050)). The IR spectrum of 1 revealed the characteristic absorptions of esters (nmax 1750, 1280 cm 1), amide (nmax 1645 cm 1), hydroxyl and/or amide NH (nmax 3400 cm 1) groups. The HR electron spray ionization MS spectrum of 1 gave the (M+H)+ ion at m/z 549.2429 (calcd. for C27H37N2O10, 549.2448) consistent with a molecular formula of C27H36N2O10. Direct connectivity between protons and carbons was established by the heteronuclear single quantum coherence spectrum and the 13C and 1H NMR spectral data for 1 are shown in Table 1. The observed double-quantum-filtered (DQF)-COSY and constant time heteronuclear multiple bond correlation (HMBC)17 spectra established four partial structures. The sequence from an oxymethine proton 2-H (dH 5.46) to 11-H (dH 1.64), which in turn coupled to two methyl protons 12-H (dH 0.95) and 13-H (dH 0.94), through 10-H (dH 1.84, 1.72) in the DQF-COSY spectrum established a 3-methyl-1-oxybutyl moiety. A doublet methyl proton 27-H (dH 1.33) and a low-field shifted methine proton 9-H (dH 3.32), which were spin–spin coupled to each other, were each long-range coupled to an ester carbonyl carbon C-8 (dC 170.2) and a ketone carbonyl carbon C-1 (dC 202.1), which in turn long-range coupled to 2-H and 10-H. These HMBC correlations indicated the successive connectivity of C-2, C-1, C-9 and C-8 as shown in Figure 1b. Thus, a 2,6-dimethyl-3-oxo-4-oxyheptanoic acid moiety was elucidated as a partial structure of 1 (Figure 1b). The proton sequence between the aromatic protons 22-H (dH 8.58), 23-H (dH 6.97) and 24-H (dH 7.36) indicated the presence of a 1,2, 3-trisubstituted benzene ring moiety. An amide proton 21-NH (dH 7.97) was coupled to an aldehyde proton 25-H (dH 8.52), which was