本論文は、異種生体分子を酵素反応を用いて部位特異的にアセンブルする技術の基礎研究から応用に至る展開を発表したものである。意図する機能を発現するための分子設計指針から、特異な構造を有する生体分子の調製、得られたハイブリッド分子による高感度遺伝子検出試薬の実用化（JST育成研究成果）とセルロース系バイオマス分解触媒としての利用価値（JST-ALCA研究成果）について言及した。

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1021/acsinfecdis.2c00052

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Biointerfaces are regions where biomolecules, cells, and organic materials are exposed to environmental media or come in contact with other biomaterials, cells, and inorganic/organic materials. In this review article, six research topics on biointerfaces are described to show examples of state-of-art research approaches. First, biointerface design of nanoparticles for molecular detection is described. Functionalized gold nanoparticles can be used for sensitive detection of various target molecules, including chemical compounds and biomolecules, such as DNA, proteins, cells, and viruses. Second, the interaction between bacterial cell surfaces and material surfaces, including the introduction of advances in analytical methods and theoretical calculations, are explained as well as their applications to bioprocesses. Third, bioconjugation technologies for localizing functional proteins at biointerfaces are introduced, in particular, by focusing the potential of enzymes as a catalytic tool for designing different types of bioconjugates that function at biointerfaces. Forth topics is focusing on lipid–protein interaction in cell membranes as natural biointerfaces. Examples of membrane lipid engineering are introduced, and it is mentioned how their compositional profiles affect membrane protein functions. Fifth topic is the physical method for molecular delivery across the biointerface being developed currently, such as highly efficient nanoinjection, electroporation, and nanoneedle devices, in which the key is how to perforate the cell membrane. Final topic is the chemical design of lipid- or polymer-based RNA delivery carriers and their behavior on the cell interface, which are currently attracting attention as RNA vaccine technologies targeting COVID-19. Finally, future directions of biointerface studies are presented.

DOI： 10.1016/j.jbiosc.2021.12.004

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.bioconjchem.1c00235

Language：English   Publishing type：Research paper (scientific journal)  

Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7 ± 2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

DOI： 10.1016/j.pep.2020.105730

Language：English   Publishing type：Research paper (scientific journal)  

Liquid marbles (LMs) have recently shown a great promise as microbioreactors to construct self-supported aqueous compartments for chemical and biological reactions. However, the evaporation of the inner aqueous liquid core has limited their application, especially in studying cellular functions. Hydrogels are promising scaffolds that provide a spatial environment suitable for three-dimensional cell culture. Here, we describe the fabrication of redox-responsive hydrogel marbles (HMs) as a three-dimensional cell culture platform. The HMs are prepared by introducing an aqueous mixture of a tetra-thiolated polyethylene glycol (PEG) derivative, thiolated gelatin (Gela-SH), horseradish peroxidase, a small phenolic compound, and human hepatocellular carcinoma cells (HepG2) to the inner aqueous phase of LMs. Eventually, HepG2 cells are encapsulated in the HMs then immersed in culture media, where they proliferate and form cellular spheroids. Experimental results show that the Gela-SH concentration strongly influences the physicochemical and microstructure properties of the HMs. After 6 days in culture, the spheroids were recovered from the HMs by degrading the scaffold, and examination showed that they had reached up to about 180 μm in diameter depending on the Gela-SH concentration, compared with 60 μm in conventional HMs without Gela-SH. After long-term culture (over 12 days), the liver-specific functions (secretion of albumin and urea) and DNA contents of the spheroids cultured in the HMs were elevated compared with those cultured in LMs. These results suggest that the developed HMs can be useful in designing a variety of microbioreactors for tissue engineering applications.

DOI： 10.1016/j.jbiosc.2020.05.010

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1021/acsabm.0c00940

Language：English   Publishing type：Research paper (scientific journal)  

In this report, a strategy for constructing three-dimensional (3D) cellular architectures comprising viable cells is presented. The strategy uses a redox-responsive hydrogel that degrades under mild reductive conditions, and a confluent monolayer of cells (i.e., cell sheet) cultured on the hydrogel surface peels off and self-folds to wrap other cells. As a proof-of-concept, the self-folding of fibroblast cell sheet was triggered by immersion in aqueous cysteine, and this folding process was controlled by the cysteine concentration. Such folding enabled the wrapping of human hepatocellular carcinoma (HepG2) spheroids, human umbilical vein endothelial cells and collagen beads, and this process improved cell viability, the secretion of metabolites and the proliferation rate of the HepG2 cells when compared with a two-dimensional culture under the same conditions. A key concept of this study is the ability to interact with other neighbouring cells, providing a new, simple and fast method to generate higher-order cellular aggregates wherein different types of cellular components are added. We designated the method of using a cell sheet to wrap another cellular aggregate the ‘cellular Furoshiki’. The simple self-wrapping Furoshiki technique provides an alternative approach to co-culture cells by microplate-based systems, especially for constructing heterogeneous 3D cellular microstructures.

DOI： 10.1038/s41598-020-63362-4

Language：English   Publishing type：Research paper (scientific journal)  

Biosynthesis of natural lipidated proteins is linked to important signal pathways, and therefore analyzing protein lipidation is crucial for understanding cellular functions. Artificial lipidation of proteins has attracted attention in recent decades as it allows modulation of the amphiphilic nature of the protein of interest, and is used in the design of drug-delivery systems containing antibodies anchored on a lipid bilayer carrier. However, the intrinsic hydrophobicity of lipids makes the synthesis of lipid–protein conjugates challenging with respect to the yield and selectivity of the lipidation. In this Minireview, the development of chemical and enzymatic synthetic strategies for the preparation of a range of lipid–protein conjugates that do not compromise the functions of the proteins are discussed as well as applications of the conjugates.

DOI： 10.1002/chem.201904568

Language：English   Publishing type：Research paper (scientific journal)  

The structure of a protein complex needs to be controlled appropriately to maximize its functions. Herein, we report the linear polymerization of bacterial alkaline phosphatase (BAP) through the site-specific cross-linking reaction catalyzed by Trametes sp. laccase (TL). We introduced a peptide loop containing a tyrosine (Y-Loop) to BAP, and the Y-Looped BAP was treated with TL. The Y-Looped BAP formed linear polymers, whereas BAP fused with a C-terminal peptide containing a tyrosine (Y-tag) showed an irregular shape after TL treatment. The sterically confined structure of the Y-Loop could be responsible for the formation of linear BAP polymers. TL-catalyzed copolymerization of Y-Looped BAP and a Y-tagged chimeric antibody-binding protein, pG₂pA-Y, resulted in the formation of linear bifunctional protein copolymers that could be employed as protein probes in an enzyme-linked immunosorbent assay (ELISA). Copolymers comprising Y-Looped BAP and pG₂pA-Y at a molar ratio of 100:1 exhibited the highest signal in the ELISA with 26- and 20-fold higher than a genetically fused chimeric protein, BAP-pG₂pA-Y, and its polymeric form, respectively. This result revealed that the morphology of the copolymers was the most critical feature to improve the functionality of the protein polymers as detection probes, not only for immunoassays but also for other diagnostic applications.

DOI： 10.1021/acsomega.9b04163

Language：English   Publishing type：Research paper (scientific journal)  

Biofunctional hydrogels prepared by a peroxidase, especially horseradish peroxidase (HRP), serve as an excellent class of materials or platform for the development of cellular scaffolds because their biocompatibility and mild and tunable reaction conditions provide them with desirable properties. In this focus review, we summarize our decade of research into HRP-mediated fabrication of biofunctional hydrogels and their applications, in particular cell culture scaffolds. A brief overview of potential substrates employed in HRP and improvement of the HRP hydrogelation system from the initial step until the hydrogen peroxide removal stage in an effort to meet environmental standards is discussed. We highlight our system and describe its biocompatibility and ability to functionalize molecules to support biofabrication by increasing cellular adhesiveness, retaining growth factor affinity, and finally accelerating the formation of two- and three-dimensional multicellular architectures. In the last section, we outline the adoption of hydrogelation as a self-standing, compartmentalized reaction system, i.e., the use of hydrogel marble to conduct cell-free biosynthesis. We believe that this HRP-mediated hydrogel system offers great potential not only as a cell culture scaffold but also for various biomedical applications.

DOI： 10.1038/s41428-020-0344-7

Language：English   Publishing type：Research paper (scientific journal)  

Assembling proteins in close vicinity to each other provides an opportunity to gain unique function because collaborative and even synergistic functionalities can be expected in an assembled form. There have been a variety of strategies to synthesize functional protein assemblies but site-specific covalent assembly of monomeric protein units without impairing their intrinsic function remains challenging. Herein we report a powerful strategy to design protein assemblies by using microbial transglutaminase (MTG). A serendipitous discovery of self-crosslinking of enhanced green fluorescent protein (EGFP) fused with StrepTag I at the C-terminus revealed that EGFP was assembled through the crosslinking of the Lys (K) residue in the C-terminus of EGFP and the Gln (Q) residue in StrepTag I (AWRHPQFGG). Site-directed mutagenesis of the residues next to the K and Q yielded EGFP assemblies with higher molecular weights. An optimized peptide tag comprised of both K and Q residues (HKRWRHYQRGG) enabled the assembly of different types of proteins of interest (POI) when it was fused to either the N- or C-terminus. The peptide tag that enabled the self-polymerization of the functional POI without a scaffold was designated as a ‘PolyTag’.

DOI： 10.1016/j.ab.2020.113700

Language：English   Publishing type：Research paper (scientific journal)  

Biologically active artificial scaffolds for cell seeding are developed by mimicking extracellular matrices using synthetic materials. Here, we propose a feasible approach employing biocatalysis to integrate natural components, that is, gelatin and heparin, into a synthetic scaffold, namely a polyethylene glycol (PEG)-based hydrogel. Initiation of horseradish peroxidase-mediated redox reaction enabled both hydrogel formation of tetra-thiolated PEG via disulfide linkage and incorporation of chemically thiolated gelatin (Gela-SH) and heparin (Hepa-SH) into the polymeric network. We found that the compatibility of the type of gelatin with heparin was crucial for the hydrogelation process. Alkaline-treated gelatin exhibited superior performance over acid-treated gelatin to generate dual functionality in the resultant hydrogel originating from the two natural biopolymers. The Gela-SH/Hepa-SH dual functionalized PEG-based hydrogel supported both cellular attachment and binding of basic fibroblast growth factor (bFGF) under cell culture conditions, which increased the proliferation and phenotype transformation of NIH3T3 cells cultured on the hydrogel. Inclusion of bFGF and a commercial growth factor cocktail in hydrogel matrices effectively enhanced cell spreading and confluency of both NIH3T3 cells and HUVECs, respectively, suggesting a potential method to design artificial scaffolds containing active growth factors.

DOI： 10.1021/acsabm.9b00275

Language：English   Publishing type：Research paper (scientific journal)  

Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid substrates (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding 90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid–protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG.

DOI： 10.1002/chem.201900370

Language：English   Publishing type：Research paper (scientific journal)  

Cancer continues to pose health problems for people all over the world. Nanoparticles (NPs) have emerged as a promising platform for effective cancer chemotherapy. NPs formed by the assembly of proteins and chitosan (CH) through noncovalent interactions are attracting a great deal of interest. However, the poor water solubility of CH and low stability of this kind of NP limit its practical application. Herein, the formation of reduced bovine serum albumin (rBSA) and glycol chitosan (GC) nanoparticles (rBG-NPs) stabilized by hydrophobic interactions and disulfide bonds was demonstrated for paclitaxel (PTX) delivery. The effects of the rBSA:GC mass ratio and pH on the particle size, polydispersity index (PDI), number of particles, and surface charge were evaluated. The formation mechanism and stability of the NPs were determined by compositional analysis and dynamic light scattering. Hydrophobic and electrostatic interactions were the driving forces for the formation of the rBG-NPs, and the NPs were stable under physiological conditions. PTX was successfully encapsulated into rBG-NPs with a high encapsulation efficiency (90%). PTX-loaded rBG-NPs had a particle size of 400 nm with a low PDI (0.2) and positive charge. rBG-NPs could be internalized by HeLa cells, possibly via endocytosis. An in vitro cytotoxicity study revealed that PTX-loaded rBG-NPs had anticancer activity that was lower than that of a Taxol-like formulation at 24 h but had similar activity at 48 h, possibly because of the slow release of PTX into the cells. Our study suggests that rBG-NPs could be used as a potential nanocarrier for hydrophobic drugs.

DOI： 10.1021/acs.langmuir.8b02809

Language：English   Publishing type：Research paper (scientific journal)  

We herein designed bi-functional aromatic peptide amphiphiles both self-assembling to fibrous nanomaterials and working as a substrate of microbial transglutaminase, leading to peptidyl scaffolds with different morphologies that can be enzymatically post-functionalized with proteins.

DOI： 10.1039/C8CC08163H

Language：English   Publishing type：Research paper (scientific journal)  

Liquid marble (LM), a self-standing micro-scale aqueous droplet, emerges as a micro-bioreactor in biological applications. Herein, the potential of LM as media for cell-free synthesis and simultaneous immobilization of recombinant proteins is explored. Initially, formation of hydrogel marble (HM) by using an enzymatic disulfide-based hydrogelation technique is confirmed by incorporating three components, horseradish peroxidase (HRP), a tetra-thiolated poly(ethylene glycol) derivative, and glycyl-L-tyrosine, in LM. The compatibility of the enzymatic hydrogelation with cell-free protein synthesis in LM is then validated. Although the hydrogelation reduces the level of protein synthesis in LM when compared with that in a test tube, the biosynthesis of enhanced green fluorescent protein (EGFP) is achieved. Interestingly, EGFP synthesized in LM is entrapped in the HM, and the introduction of a cysteine residue to EGFP by genetic engineering further increases the amount of protein immobilization in the hydrogel matrices. These results suggest that the cell-free synthesis and HRP-catalyzed hydrogelation can be conducted in parallel in LM, and the eventual entrapment of the key components in HM is possible. Facile recovery of macromolecular products immobilized in HM by degrading the hydrogel network under reducing conditions should lead to the design of an easy-to-handle system to screen protein functions.

DOI： 10.1002/biot.201800085

Language：English   Publishing type：Research paper (scientific journal)  

Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid-G₃S-MRHKGS lipid (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding >90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid–protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG.

DOI： 10.1021/acsabm.8b00271

Language：English   Publishing type：Research paper (scientific journal)  

Phycocyanin (PC), a water-soluble protein-chromophore complex composed of hexameric (αβ)₆ subunits, has important biological functions in blue-green algae as well as pharmacological activities in biomedicine. We have previously developed a solid-in-oil (S/O) nanodispersion method to deliver biomacromolecules through the skin, although the transcutaneous mechanism has not yet been fully elucidated. To study the mechanism of transcutaneous protein delivery, we therefore enabled S/O nanodispersion by coating PC with hydrophobic surfactants and evaluated how the proteinaceous macromolecules formulated in an oil phase might permeate the skin. The extent of S/O nanodispersion of PC was dependent on the type of surfactant, suggesting that the selection of a suitable surfactant is crucial for encapsulating a large protein having a subunit structure. By measuring the intrinsic fluorescence of PC, we found that S/O nanodispersion facilitated the accumulation of PC in the stratum corneum (SC) of Yucatan micropig skin. Furthermore, after crossing the SC layer, the fluorescent recovery of PC was evident, indicating the release of the biologically active form of PC from the SC into the deeper skin layer.

DOI： 10.1016/j.ejpb.2018.01.020

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DOI： 10.1002/biot.201700624

Language：English   Publishing type：Research paper (scientific journal)  

Nanoparticles formed by the assembly of protein and polysaccharides are of great interest for the delivery of hydrophobic molecules. Herein, the formation of genipin-crosslinked nanoparticles from caseinate (CS) and chitosan (CH) is reported for the delivery of curcumin, a polyphenolic compound from turmeric, to cells. Genipin-crosslinked CS-CH nanoparticles (G-CCNPs) having a diameter of ∼250 nm and a low polydispersity index showed excellent stability over a wide pH range, as indicated by dynamic light scattering and transmission electron microscopic measurements. Cellular uptake of curcumin loaded into G-CCNPs by HeLa cells was improved, as measured by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell-sorting analysis. Cell proliferation assays indicated that G-CCNPs were nontoxic and that curcumin's anticancer activity in vitro was also improved by G-CCNPs. Stability of curcumin at neutral pH was enhanced by G-CCNPs. CLSM study revealed that G-CCNPs were poorly internalized by HeLa cells, possibly because of strong cell membrane interactions and a negative zeta potential. Overall, our results suggested that the enhanced curcumin cytotoxicity might be associated with the enhanced stability of curcumin by G-CCNPs and free curcumin released from G-CCNPs into the cell. These biocompatible NPs might be suitable carriers for enhancing curcumin's therapeutic potential.

DOI： 10.1016/j.colsurfb.2018.01.041

Language：English   Publishing type：Research paper (scientific journal)  

Conjugating enzymes into a large protein assembly often results in an enhancement of overall catalytic activity, especially when different types of enzymes that work cooperatively are assembled together. However, exploring the proper method to achieve protein assemblies with high stability and also to avoid loss of the function of each component for efficient enzyme clustering is remained challenging. Assembling proteins onto synthetic scaffolds through varied post-translational modification methods is particularly favored since the proteins can be site-specifically conjugated together with less activity loss. Here, a SpyCatcher polymer is prepared through catalytic reaction of horseradish peroxidase (HRP) and serves as a polymeric proteinaceous scaffold for construction of protein assemblies. Taking advantage of the favorable SpyCatcher–SpyTag interaction, SpyTagged proteins can be easily assembled onto the polymeric SpyCatcher scaffold with controllable binding ratio and site specificity. Firstly, the feasibility of construction of ratio-controllable binary artificial hemicellulosomes by assembling endoxylanase and arabinofuranosidase is explored. This construct achieves higher sugar conversion than that of the free enzymes when the proportion of arabinofuranosidase is high, because the close spatial proximity of the enzymes allows them to work in a synergistic manner. Another application for biosensing is developed by conjugating SpyTagged Nanoluc and protein G onto SpyCatcher polymer. Due to the protein clustering effect, an amplified luminescent intensity is achieved by the resulting conjugates than chimera protein of Nanoluc and protein G in ovalbumin detection in ELISA.

DOI： 10.1002/biot.201700195

Language：English   Publishing type：Research paper (scientific journal)  

DNA-protein conjugates are promising biomolecules for use in areas ranging from therapeutics to analysis because of the dual functionalities of DNA and protein. Conjugation requires site-specific and efficient covalent bond formation without impairing the activity of both biomolecules. Herein, we have focused on the use of a microbial transglutaminase (MTG) that catalyzes the cross-linking reaction between a glutamine residue and a primary amine. In a model bioconjugation, a highly MTG-reactive Gln (Q)-donor peptide (FYPLQMRG, FQ) was fused to enhanced green fluorescent protein (FQ-EGFP) and a primary amine-clustered DNA aptamer was enzymatically synthesized as a novel acyl-acceptor substrate of MTG, whose combination leads to efficient and convenient preparation of DNA-protein conjugates with high purity. Dual functionality of the obtained DNA-EGFP conjugate was evaluated by discrimination of cancer cells via c-Met receptor recognition ability of the DNA aptamer. The DNA aptamer-EGFP conjugate only showed fluorescence toward cells with c-Met overexpression, indicating the retention of the biochemical properties of the DNA and EGFP in the conjugated form.

DOI： 10.1021/acs.bioconjchem.7b00594

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DOI： 10.1002/biot.201600087

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DOI： 10.1021/acs.biomac.6b01141

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In biological systems, proteins can form well-organized, higher-order structures with unique functions that would be difficult to achieve with a single protein. These proteinaceous supramolecular structures form by self-assembly, and the spatial arrangement of the protein building blocks in them is very important. In the present study, an artificial system was developed using recombinant proteins as building blocks, which were assembled in a one-dimensional manner. The assembly of these building blocks was based on the avidin-biotin interaction. A tetrameric biotin ligand unit was designed so that the 1:4 stoichiometry of the avidin-biotin interaction was altered to a 1:2 directional interaction between the streptavidin and tetrabiotinylated protein units. In a proof-of-concept study, site-specifically tetrabiotin-labeled endoglucanase and cellulose-binding module units were prepared, then these components were self-assembled by mixing with streptavidin to mimic a natural cellulosome. The formation of one-dimensional assemblies of the protein units depended on the stoichiometry of the avidin-biotin interaction sites in the system. Interestingly, the saccharification efficiency improved when the component ratio of protein units in the assemblies was changed.

DOI： 10.1039/C6ME90005D

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DOI： 10.1016/j.jbiosc.2015.02.013

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DOI： 10.1016/j.biortech.2015.02.041

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DOI： 10.1039/c4ra12334d

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DOI： 10.1002/biot.201400512

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Horseradish peroxidase-mediated oxidative cross-linking of a thiolated poly(ethylene glycol) is promoted in the absence of exogenous hydrogen peroxide, by adding a small amount of phenolic compound under physiological conditions. The prepared hydrogel can encapsulate and release living mammalian cells.

DOI： 10.1039/c3cc49766f

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DOI： 10.1039/c3ra46384b

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生体内ではほとんどのタンパク質が何らかの翻訳後修飾を受けることに着目し、翻訳後修飾過程で働く酵素の基質特異性を利用すれば、狙った部位でタンパク質を修飾できると考えた。従来の有機化学的手法では、タンパク質の狙った部位を選択的に修飾するのは極めて困難であった。そこでラベル化したい有機分子内に基質となる部位（酵素の認識部位）を設計し、これを糊代として利用することで、糊代選択的な生体分子の連結・修飾法を確立した。
既往の方法ではDNAとタンパク質を１：１で連結することしかできなかったが、DNAを構成する分子を酵素の基質となるように設計し、DNA-(タンパク質)n型の新規ハイブリッド分子を高収率で得る技術を確立し、新たな遺伝子検出システムを提案した。

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DOI： 10.1016/j.febslet.2005.02.064

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DOI： 10.1021/bm0494895

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DOI： 10.1021/bm050193o

Language：English   Publisher：Chemical Communications  

The fusion protein of an engineered zymogen of microbial transglutaminase (EzMTG) with a protein G variant, EzMTG-pG, enabled the proximity-based, tag-free labeling of Lys65 in the heavy chain of a native IgG antibody (trastuzumab) with a Gln-donor peptidyl substrate functionalized with a fluorescent molecule.

DOI： 10.1039/d4cc01728e

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PubMed

Publisher：Waste and Biomass Valorization  

Purpose: This work evaluated the profiles of Industrial agar seaweed waste biomass (ISWB) and pretreated ISWB by using deep eutectic solvent (DESs) and NaOH. The profiles of nanocellulose obtained from pretreated ISWB (with various types of hydrogen bond donors of choline chloride (ChCl)-based hydrated DES) were also evaluated. Methods: ISWB underwent pre-treatment with either 100% wt. DES (1:1 molar ratio of ethylene glycol: citric acid) or 10% wt. NaOH at 80 °C to 90 °C. Subsequent treatment involved 10% wt. DES or ChCl-based hydrated DES with various HBDs (oxalic acid, citric acid, and urea) in a 1:2 molar ratio to produce nanocellulose. Results: Cellulose nanofibers isolated by the CE-DES approach exhibited a substantially higher yield compared with those obtained via the CE-NaOH method. CE-DES method induced swelling of ISWB and facilitate production of cellulose nanofibers. SEM images of samples indicates the presence of fibrous nanostructures across the majority of the specimens. Fourier-transform infrared (FTIR) spectroscopy demonstrated uniformity among all samples; they exhibited a consistent fingerprint pattern. X-ray diffraction (XRD) spectra indicated the presence of cellulose Type I in cellulose nanofibers produced by CE–DES, but not definitively in nanocellulose produced by the CE–NaOH method. Conclusion: Cellulose nanofibers can be isolated from ISWB by using various DES and exhibits substantial characteristics of cellulose nanofibers from different ISWB pretreatment protocols. Graphical Abstract: (Figure presented.)

DOI： 10.1007/s12649-024-02499-z

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Language：English   Publisher：Molecules  

Herein, we report a transdermal patch prepared using an ionic liquid-based solid in oil (IL-S/O) nanodispersion and a pressure-sensitive adhesive (PSA) to deliver the macromolecular antigenic protein, ovalbumin (OVA). The IL-S/O nanodispersion and a PSA were first mixed at an equal weight ratio, then coated onto a release liner, and covered with a support film. To evaluate the effect of the PSA, three types of PSAs, DURO-TAK 87-4098, DURO-TAK 87-4287, and DURO-TAK 87-235A, were used to obtain the corresponding IL-S/O patches SP-4098, SP-4287, and SP-235A, respectively. The prepared IL-S/O patches were characterized for surface morphology, viscoelasticity, and moisture content. In vitro skin penetration and in vivo immunization studies of the IL-S/O patches were performed using Yucatan micropig skin and the C57BL/6NJc1 mice model, respectively. The SP-4098 and SP-4287 delivered 5.49-fold and 5.47-fold higher amounts of drug compared with the aqueous formulation. Although both patches delivered a similar amount of drug, SP-4287 was not detached fully from the release liner after 30 days, indicating low stability. Mice immunized with the OVA-containing SP-4098 produced a 10-fold increase in anti-OVA IgG compared with those treated with an aqueous formulation. These findings suggested that the IL-S/O patch may be a good platform for the transdermal delivery of antigen molecules.

DOI： 10.3390/molecules29132995

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Bioscience and Bioengineering  

Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production.

DOI： 10.1016/j.jbiosc.2024.04.001

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Language：English   Publisher：(公社)日本生物工学会  

Publisher：Fisheries Science  

Ulva is an unutilized green seaweed that grows in the intertidal zone around the coast of Java Island, Indonesia. In this study, U. lactuca samples collected from three regions in south of Java Island (Cihara, Surade, and Tepus) were studied in terms of their chemical composition, physical properties and its bioactivity, to determine the best regions for establishing seaweed industries. The chemical characteristics differed significantly among different regions, where the seaweed from Tepus, Surade and Cihara had the highest content of protein (22.93%), carbohydrate (61.58%), and mineral (28.72%), respectively. The amino acids were dominated with L-aspartic acid and L-glutamic acid. All U. lactuca samples contained abundant pigments, such as chlorophylls and carotenoids, especially samples from Tepus. The highest content of crude ulvan was found in Surade seaweed (26.9%). Chemical and physical analyses showed the presence of S = O and C–O–S functional groups in ulvan, a sulfated polysaccharide unique to Ulva sp., with thermal degradation up to 220 °C. Crude ulvan from Surade and Tepus seaweed exhibited bioactivity to support proliferation of fibroblast cells at 100 and 1000 ppm, respectively. Based on the properties of U. lactuca, Tepus and Surade were identified as potential sites to establish aquaculture and/or processing industries.

DOI： 10.1007/s12562-024-01799-6

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Publisher：ASEAN Journal of Chemical Engineering  

Ulva lactuca is a green seaweed commonly called sea lettuce and contains sulphated polysaccharides that have biological activity. Despite its lack of ecological sustainability, strong acids (HCl) and strong bases (NaOH) are widely used as solvents in the conventional extraction process for ulvan. Deep eutectic solvent (DES) is an alternative to ulvan extraction solvent, which is more environmentally friendly and has low toxicity. This study aims to assess the efficacy of peracetic acid (PAA) pretreatment and post-treatment in enhancing the quality of extracted ulvan using a DES-based solvent in the extraction process. Ulvan extraction using DES with a temperature of 85-95°C for 1 hour and adding 2% PAA pretreatment, 0.1%, 0.5%, and 2% PAA post-treatment were conducted. PAA pre-and post-treatment was carried out with a 1:10 (w/v) ratio at room temperature for 30 minutes. The analysis included moisture content, yield, functional groups, sulphate content, and color. The test results revealed that ulvan treated with 2% PAA pretreatment had the highest moisture content and sulphate content, at 18.71% and 33.39%, respectively, while ulvan treated with 0.1% PAA post-treatment had the highest yield, at 41.96%. Adding peracetic acid concentration can increase the color quality of the ulvan. PAA pre-and post-treatment had a significant effect on all ulvan quality parameters.

DOI： 10.22146/ajche.12272

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical Communications  

Herein, we report ethosome (ET) formulations composed of a safe amount of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)-based ionic liquid with various concentrations of ethanol as a carrier for the transdermal delivery of a high molecular weight drug, insulin. The Insulin-loaded ET vesicles exhibited long-term stability compared to conventional DMPC ETs, showing significantly higher drug encapsulation efficiency and increased skin permeation ability.

DOI： 10.1039/d3cc06130b

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DOI： 10.1101/2024.03.04.583322

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Journal of Molecular Liquids  

Ionic liquid (IL)-based transdermal formulations have gained significant attention because of their high skin penetration efficiency and ease of design. However, many IL-based formulations exhibit low viscosity, which hinders direct administration and leads to less efficient drug delivery. Therefore, there is a pressing need for specialized formulations with improved viscosity and efficacy as a drug delivery platform. In this study, we report a ready-to-use transdermal patch system utilizing IL-in-oil (IL/O) microemulsions to deliver acyclovir, a sparingly soluble antiviral drug. To prepare the patch system, a mixture containing equal weights of IL/O microemulsions and adhesive (Durotak 87–4098) was homogenized, subsequently coated on a liner, and covered by a support film. These patches were produced with varying thicknesses (100 μm, 200 μm, 300 μm, and 500 μm) via a solvent evaporation technique. A 24-hour in vitro skin permeation study revealed that the thinnest patch (100 μm) delivered more than 45 % of the loaded drug. Drug delivery rate gradually reduced as the patch thickness increased, accounting for 22 % and 14 % drug permeation by 200 μm and 300 μm patches respectively. A 30-day stability study at room temperature demonstrated that neither the physical appearance nor the active ingredient content changed, validating the physical and chemical stability of the patches. Skin permeation mechanism by Fourier transform infrared spectroscopic analysis of the skin suggested that the IL/O patch possesses the capability to modify the lipid barrier in the stratum corneum thus facilitating drug diffusion. In vitro skin irritation studies on reconstructed human epidermal tissue and Yucatan micropig skin revealed that the patch exhibits no skin irritation. We believe that the adhesive nature of this newly developed IL-based patch holds significant promise as a transdermal drug delivery platform.

DOI： 10.1016/j.molliq.2024.124184

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Publishing type：Research paper (scientific journal)   Publisher：MYU K.K.  

Human orthogonal enzymes (HOEs) do not show the same activities as the endogenous enzymes of human cells and thus are useful as amplification enzymes to detect antigen proteins in biological samples. Here, we evaluate a new HOE from Escherichia coli, α-sulfoquinovosidase (α-SQase). We confirmed that the activity of α-SQase did not exist in examined human cell lines, and thus it was applicable to live-cell enzyme-linked immunosorbent assay (ELISA) in which the antigen membrane protein on cells was detected without inactivating endogenous enzymes, a pretreatment required for cell ELISA using conventional amplification enzymes. Here, we also developed a fluorescent substrate for α-SQase whose active residue is located at the end of the narrow, deep pocket of the substrate recognition site. The designed methylumbelliferyl substrate with a hydroxyl benzyl alcohol linker showed a similar reactivity to the p-nitrophenol substrate, a good substrate for α-SQase.

DOI： 10.18494/SAM4816

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Language：English   Publisher：(公社)日本生物工学会  

ペルオキシダーゼ触媒によるクロスリンク形成を介して生理的な条件下でゲル化を受けるアルブミン派生物を合成した。アルブミンをカルボジイミドを用いてフェノール性水酸基(Alb-Ph-OH)で修飾し、置換度がゲル化に及ぼす影響を解析した。Alb-Ph-OHヒドロゲルの多様な特性、すなわち、ゲル化時間、膨張率、孔径、貯蔵弾性率、および酵素的な分解性は置換度とポリマー濃度の調整により容易に調節可能であった。Alb-Ph-OHヒドロゲル中にカプセル化された細胞の生存率は高かった。

DOI： 10.1101/2023.10.09.561484

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Albumin is an attractive component for the development of biomaterials applied as biomedical implants, including drug carriers and tissue engineering scaffolds, because of its high biocompatibility and low immunogenicity. Additionally, albumin-based gelators facilitate cross-linking reactions under mild conditions, which maintains the high viability of encapsulated living cells. In this study, we synthesized albumin derivatives to undergo gelation under physiological conditions via the peroxidase-catalyzed formation of cross-links. Albumin was modified with phenolic hydroxyl groups (Alb–Ph–OH) using carbodiimide chemistry, and the effect of degree of substitution on gelation was investigated. Various properties of the Alb–Ph–OH hydrogels, namely the gelation time, swelling ratio, pore size, storage modulus, and enzymatic degradability, were easily controlled by adjusting the degree of substitution and the polymer concentration. Moreover, the viability of cells encapsulated within the Alb–Ph–OH hydrogel was high. These results demonstrate the potential applicability of Alb–Ph–OH hydrogels as cell-encapsulating materials for biomedical applications, including tissue engineering.

DOI： 10.1016/j.jbiosc.2023.09.007

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Language：English  

DOI： https://doi.org/10.1101/2023.10.09.561484

Repository Public URL： https://hdl.handle.net/2324/7236919

Language：Others   Publishing type：Research paper (scientific journal)   Publisher：ACS Applied Materials and Interfaces  

Nucleic acid drugs, including antisense oligonucleotides (ASOs), have received considerable attention as novel therapeutics for the treatment of intractable diseases. Despite their potential benefits, ASOs are currently administered via injection, which can negatively impact patient quality of life because of the prevalence of severe injection site reactions. Non-invasive transdermal administration of ASOs is desirable but highly challenging owing to the strong barrier imposed by the stratum corneum, which only permits the penetration of small molecules under 500 Da. For ASOs to exert their antisense effect, they must traverse the negatively charged cell membrane and reach the cytoplasm. In this study, we used the solid-in-oil (S/O) dispersion technology to facilitate the skin permeation of ASOs by coating the drug with a hydrophobic surfactant molecule, specifically lipid-based ionic liquid (IL) surfactants with high biocompatibility and transdermal penetration-enhancing properties. To induce the antisense effect, it was important to achieve simultaneous transdermal delivery and intracellular entrapment of ASOs. In vitro investigations indicated that the newly prepared IL-S/O enhanced the transdermal penetration and intracellular delivery of ASOs, thus inhibiting mRNA translation of the target TGF-β. In addition, in vivo investigations of tumor-bearing mice suggested that the anti-tumor effect of the IL-S/O was similar to that of injection. This study demonstrates the potential of non-invasive transdermal delivery carriers based on biocompatible ILs, which can be applied to a variety of nucleic acid drugs.

DOI： 10.1021/acsami.3c03900

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：The Chemical Society of Japan  

We developed a method to orthogonally immobilize multiple functional molecules on amyloid aggregates using different enzymes (sortase A and transglutaminase). Orthogonal binding was performed via the substrate amino acid sequence of each enzyme, and the amount of molecular immobilization could be changed according to the amount of substrate sequence. This method is expected to have applications such as improving the sensitivity of immunoassays and visualization of amyloid elongation reactions.

DOI： 10.1246/cl.230156

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Recently, functional nanowire production using amyloids as a scaffold for protein immobilization has attracted attention. However, protein immobilization on amyloid fibrils often caused protein inactivation. In this study, we investigated protein immobilization using enzymatic peptide ligation to suppress protein inactivation during immobilization. We attempted to immobilize functional molecules such as green fluorescent protein (GFP) and Nanoluc to a transthyretin (TTR) amyloid using microbial transglutaminase (MTG), which links the glutamine side chain to the primary amine. Linkage between amyloid fibrils and functional molecules was achieved through the MTG substrate sequence, and the functional molecules-loaded nanowires were successfully fabricated. We also found that the synthetic process from amyloidization to functional molecules immobilization could be achieved in a single-step procedure.All rights reserved.

DOI： 10.1016/j.jbiosc.2023.03.015

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

DOI： 10.34565/seibutsukogaku.101.3_117

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Journal of Pharmaceutical Sciences  

Malaria is a mosquito-borne infectious disease that is widespread in developing countries. Malaria vaccines are important in efforts to eradicate malaria; however, vaccines are usually administered by injection, which requires medical personnel and has a risk of causing infection. Transdermal vaccines can be administered without damaging the skin and thus are ideal for the prevention of malaria. However, the stratum corneum forms a "brick and mortar" like structure in which stratum corneum cells are embedded in a hydrophobic matrix composed of lipids, which strongly inhibits the permeation of hydrophilic substances. In the present study, we designed a transdermal vaccine against vivax malaria using a solid-in-oil (S/O) dispersion. The S/O dispersion of a transmission blocking vaccine candidate, Pvs25 from Plasmodium vivax, showed higher skin penetration than that of the aqueous solution. Mice immunized with the S/O dispersion generated antibodies at similar titers as the mice immunized by injection, over the mid- to long-term. These results provide information for the development of transdermally administered malaria vaccines toward the eradication of malaria.

DOI： 10.1016/j.xphs.2022.10.031

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical Communications  

This study investigated the localization of artificial peptide supramolecular fibers in liquid-liquid phase separation (LLPS). Hierarchical organization led to the localization of supramolecules in LLPS droplets. Moreover, proteins were recruited into confined droplets by the physical adsorption of proteins on the supramolecules, enabling an enhanced cascade reaction.

DOI： 10.1039/d2cc05910j

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Organic and Biomolecular Chemistry  

Cytoplasm contains high concentrations of biomacromolecules. Protein behavior under such crowded conditions is reportedly different from that in an aqueous buffer solution, mainly owing to the effect of volume exclusion caused by the presence of macromolecules. Using a crosslinking reaction catalyzed by microbial transglutaminase (MTG) as a model, we herein systematically determined how the substrate size affects enzymatic activity in both dilute and crowded solutions of dextran. We first observed a threefold reduction in MTG-mediated crosslinking of a pair of small peptide substrates in 15 wt% dextran solution. In contrast, when proteinaceous substrates were involved, the crosslinking rates in 15 wt% dextran solutions accelerated markedly to levels comparable with the level in the absence of dextran. Our results provide new insights into the action of enzymes with regard to macromolecular substrates under crowded conditions, of which the potential utility was demonstrated by the formation of highly crosslinked protein polymers.

DOI： 10.1039/d2ob01549h

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

DOI： 10.3390/ijms24010102

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Publisher：Journal of Molecular Liquids  

Luteolin (LUT), an important flavonoid, shows excellent antioxidant and antimicrobial properties. However, its low solubility and stability in water, as well as challenges related to its formulation, have limited its use in food preservation. Here, we develop a highly water-soluble LUT-loaded micellar formulation (LUT-MF) using a solid-in-water nanodispersion technique involving emulsification and lyophilization processes. LUT solubilized in an organic phase comprising acetone, methanol, and cyclohexane (5:10:85 v/v) was emulsified with an aqueous solution of a surface-active ionic liquid, choline oleate ([Cho][Ole]), and the oil in water emulsion was lyophilized to remove the organic phase and water and obtain a solid LUT-[Cho][Ole] complex. LUT-[Cho][Ole] complex was dispersed again in water to prepare the LUT-MF formulation with good stability. Dynamic light scattering and transmission electron microscopy confirmed that the LUT-MF formed spherical micelles with a mean particle size of approximately 73 nm. LUT-MF showed excellent encapsulation efficiency (94.3%) and enhanced aqueous solubility. An in vitro release study in PBS containing 30% ethanol demonstrated 3.94 times higher release of LUT from LUT-MF than from LUT suspension after 24 h. The developed formulation showed better antioxidant activity than both free LUT and ascorbic acid. LUT-MF demonstrated higher antibacterial activity than sodium benzoate. Food preservation studies revealed that treating strawberries with LUT-MF effectively preserved color and prevented shriveling and decay over 8 days storage at 10 °C, compared with sodium benzoate treatment. The results suggest that LUT-MF is a potential natural preservative alternative to conventional toxic chemical preservatives used in the food industry.

DOI： 10.1016/j.molliq.2022.120151

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Language：English   Publisher：(公社)日本生物工学会  

市販のアンフォテリシンB(AMB)製剤と表面電荷が異なるリポソームを組合せてハイブリッドリポソーム製剤を調製し、抗真菌活性の重要な要因を探索した。AMB含有リポソーム製剤(AMB-LF)の粒子サイズ分布とゼータ電位を含む特性評価では、陰イオン性と中性のAMB-LFはAMBを安定的にカプセル化できた。アニオン性または中性AMB-LFと未修飾LysMの組合せではAMBのMICが低下した。中性AMB-LFとPteris ryukyuensisキチナーゼ由来の人工パルミトイル化キチン結合ドメインの組合せではMICは更に減少し、中性AMB-LF単独と比較して最大15倍になった。以上より、脂質修飾性蛋白質結合剤と組合せたAMBの脂質基盤リポソーム製剤の真菌感染に対する有用であると考えられた。

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS Applied Bio Materials  

Transdermal drug delivery systems (TDDSs) may be useful for preventing various diseases including cancer. However, the stratum corneum (SC) inhibits the permeation of foreign particles into the skin. To obtain an effective TDDS, we developed a protein-containing nanocarrier (PCNC) comprising an antigenic protein (ovalbumin/OVA) stabilized by a combination of surfactants, i.e., a lipid-based surface-active ionic liquid and Tween-80. The PCNC was lyophilized to remove water and cyclohexane and then dispersed in isopropyl myristate. It is biocompatible both in vitro and in vivo, and is suitable for use in a therapeutic TDDS. The skin permeability of the PCNC was significantly (p < 0.0001) enhanced, and the transdermal distribution and transdermal flux of the OVA delivery system were 25 and 28 times greater, respectively, than those of its aqueous formulation. The PCNC disrupted the order of lipid orientation in the skin's SC and increased intercellular protein delivery. It demonstrated effective antitumor activity, drastically (p < 0.001) suppressed tumor growth, increased mouse survival rates, and significantly (p < 0.001) stimulated the OVA-specific tumor immune response. The PCNC also increased the number of cytotoxic T cells expressing CD8 antibodies on their surfaces (CD8 + T-cells) in the tumor microenvironment. These findings suggest that PCNCs may be promising biocompatible carriers for transdermal antigenic protein delivery in tumor immunotherapy.

DOI： 10.1021/acsabm.2c00061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS Infectious Diseases  

Combinations of antifungal drugs can have synergistic antifungal activity, achieving high therapeutic efficacy while minimizing the side effects. Amphotericin B (AMB) has been used as a standard antifungal drug for fungal infections; however, because of its high toxicity, new strategies to minimize the required dose are desirable. Chitinases have recently received attention as alternative safe antifungal agents. Herein, we report the combination of palmitoylated chitinase domains with AMB to enhance the antifungal activity. The chitin-binding domain (LysM) from Pteris ryukyuensis chitinase was site-specifically palmitoylated by conjugation reaction catalyzed by microbial transglutaminase. The palmitoylated LysM (LysM-Pal) exhibited strong antifungal activity against Trichoderma viride, inhibiting the growth completely at a concentration of 2 μM. This antifungal effect of LysM-Pal was mainly due to the effect of anchoring of palmitic acid motif to the plasma membrane of fungi. A combination of AMB with LysM-Pal resulted in synergistic enhancement of the antifungal activity. Intriguingly, LysM-Pal exhibited higher level of antifungal activity enhancement than palmitoylated catalytic domain (CatD) and fusion of LysM and CatD. Addition of 0.5 μM LysM-Pal to AMB reduced the minimal inhibition concentration of AMB to 0.31 μM (2.5 μM without LysM-Pal). The possible mechanism of the synergistic effect of AMB and LysM-Pal is destabilization of the plasma membrane by anchoring of palmitic acid and ergosterol extraction by AMB and destabilization of the chitin layer by LysM binding. The combination of LysM-Pal with AMB can drastically reduce the dose of AMB and may be a useful strategy to treat fungal infections.

DOI： 10.1021/acsinfecdis.2c00052

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Publisher：Journal of Molecular Liquids  

Ionic liquids (ILs) have gained great attention as potent solubilizing agents of poorly soluble bioactive compounds. The use of Luteolin (LUT) – a natural bioactive compound – is limited because of its poor aqueous solubility. To address this issue, this study reports potential ILs for effectively solubilizing LUT at ambient temperature. First, promising ILs were screened from a potential 180 ILs that were proposed by the combination of 20 standard amino acid ethyl esters cations and nine phenolic acids anions using the conductor-like screening model for real solvents (COSMO-RS); the activity coefficients of LUT at infinite dilution in ILs were used as the screening criteria. Four proline ethyl ester phenolate ILs (PEEP-ILs) composed of a biocompatible proline ethyl ester and bioactive phenolic acids (trans-ferulic, vanillic, p-coumaric, and 4-hydroxybenzoic acid) were selected and synthesized. The synthesized PEEP-ILs were characterized through 1H NMR, FT-IR, elemental analysis, TGA, and DSC. The experimental solubility of LUT in PEEP-ILs (60% IL in water) showed excellent solubility. Among the PEEP-ILs, proline ethyl ester ferulate (IL[ProEt][Fer]) exhibited the best LUT solubility, followed by ILs with p-coumarate (IL[ProEt][Cou]), vanillate (IL[ProEt][Van]), and 4-hydroxybenzoate (IL[ProEt][Ben]). 1H NMR assessment and COSMO-RS prediction confirmed that the ILs facilitated the solubilization of LUT through multiple hydrogen bonding, π − π, and cation − π interactions between the LUT and the ILs. The biocompatibility study revealed that PEEP-ILs were relatively harmless and low toxicity compared with conventional imidazolium, pyridinium, pyrrolidinium, piperidinium, and morpholinium-based ILs. The LUT dissolved in the newly-synthesized PEEP-ILs and demonstrated better organoleptic properties on red apple slices. These results indicate that PEEP-ILs can be potential green alternatives to conventional toxic solvents for dissolving poorly water-soluble bioactive natural preservatives.

DOI： 10.1016/j.molliq.2021.118103

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

The biomaterial-based immunoengineering has become one of the most attractive research fields in the last decade. In the present study, a solid-in-oil-in-water (S/O/W) emulsion encapsulating antigen in the oil phase of an oil-in-water (O/W) emulsion was prepared as a novel vaccine carrier consisting of similar materials to the emulsion adjuvant of which the safety, immunogenicity and vaccination efficacy have been already confirmed in human. Direct observation by high-resolution confocal laser scanning microscopy and small angle X-ray scattering analysis showed that the antigens were dispersed inside of the oil phase of the S/O/W emulsion as solid-state particles. The S/O/W emulsion robustly produced antigen-specific antibodies and enhanced the antitumor effects in a therapeutic cancer vaccination compared with free antigens or the O/W emulsion in vivo. This result is in good agreement with the activation effect of antigen-specific cytotoxic T lymphocytes and antigen presentation by the S/O/W emulsion, indicating that the S/O/W emulsion consisting of already approved materials is a promising vaccine carrier to produce both humoral and cellular immunity.

DOI： 10.1016/j.biomaterials.2022.121385

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Acta Biomaterialia  

Transcutaneous drug delivery is a promising method in terms of drug repositioning and reformulation because of its non-invasive and easy-to-use features. To overcome the skin barrier, which is the biggest challenge in transcutaneous drug delivery, a number of techniques, such as microemulsion, solid-in-oil dispersions and liposomes, have been studied extensively. However, the low viscosity of these formulations limits drug retention on the skin and reduces patient acceptability. Although viscosity can be increased by adding a thickening reagent, such an addition often alters formulation nanostructures and drug solubility, and importantly, decreases skin permeability. In this study, a gel-like lyotropic liquid crystal (LLC) was used as a tool to enhance skin permeability. In particular, we prepared 1-monolinolein (ML)-based LLCs with different water contents. All LLCs significantly enhanced skin permeation of a peptide drug, an epitope peptide of melanoma, despite their high viscoelasticity. Fourier transform infra-red spectroscopic analysis of the skin surface treated with the LLCs revealed that the gyroid geometry more strongly interacted with the lamellar structure inside the stratum corneum (SC) than the diamond geometry. Finally, as the result of the in vivo tumor challenge experiment using B16F10 melanoma-bearing mice, the LLC with the gyroid geometry showed stronger vaccine effect against tumor than a subcutaneous injection. Collectively, ML-based LLCs, especially with the gyroid geometry, are a promising strategy to deliver biomacromolecules into skin. Statement of significance: Transcutaneous drug delivery is a promising method for drug repositioning and reformulation because of its non-invasive and easy-to-use features. To overcome the skin barrier, which is the biggest challenge in transcutaneous drug delivery, we used a gel-like lyotropic liquid crystal (LLC) as a novel tool to enhance skin permeability. In this paper, we demonstrated that an LLC with a specific liquid crystalline structure has the highest skin permeation enhancement effect for a peptide antigen as a model drug. Moreover, the peptide antigen-loaded LLC showed a vaccine effect that was comparable to a subcutaneous injection in vivo. This study provides a basis for designing a transcutaneous delivery system of peptide drugs with LLC.

DOI： 10.1016/j.actbio.2021.11.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Self-sorting and co-assembly of aqueous supramolecular fibres were formed using peptide amphiphiles having immiscible hydrophobic tails.

DOI： 10.1039/d1cc05560g

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Here we proposed a-L-arabinofuranosidase from Clostridium thermocellum as an enzyme for signal amplification in bioanalysis whose activity does not exist in human cells. Combination of this enzyme and beta-galactosidase enabled simultaneous detection of two different antigens on live cells.

DOI： 10.1246/cl.210231

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DOI： 10.1016/j.aspen.2021.04.011

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The tumor necrosis factor α (TNFα) has been employed as a promising reagent in treating autoimmunity and cancer diseases. To meet the substantial requirement of TNFα proteins, we report in this study that mature types of recombinant human and murine TNFα proteins are successfully expressed in the baculovirus expression system using silkworm larvae as hosts. The biological activities of purified products were verified in culture murine L929 cells, showing better performance over a commercial Escherichia coli-derived murine TNFα. By comparing the activity of purified TNFα with or without the tag removal, it is also concluded that the overall activity of purified TNFα cytokines could be further improved by the complete removal of C-terminal fusion tags. Collectively, our current attempt demonstrates an alternative platform for supplying high-quality TNFα products with excellent activities for further pharmaceutical and clinical trials.

DOI： 10.3390/insects12060517

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DOI： 10.3390/ijms22073459

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DOI： https://doi.org/10.1101/2021.03.13.435221

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DOI： https://doi.org/10.2116/analsci.20SCR07

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DOI： 10.2116/analsci.20scp12

Language：English   Publishing type：Research paper (scientific journal)  

Supramolecular fibrous materials in biological systems play important structural and functional roles, and therefore, there is a growing interest in synthetic materials that mimic such fibrils, especially those bearing enzymatic reactivity. In this study, we investigated the self-assembly and enzymatic post-modification of short aromatic peptide amphiphiles (PAs), Fmoc-LnQG (n = 2 or 3), which contain an LQG recognition unit for microbial transglutaminase (MTG). These aromatic PAs self-assemble into fibrous structures via π-π stacking interactions between the Fmoc groups and hydrogen bonds between the peptides. The intermolecular interactions and morphologies of the assemblies were influenced by the solution pH because of the change in the ionization states of the C-terminal carboxy group of the peptides. Moreover, MTG-catalyzed post-modification of a small fluorescent molecule bearing an amine group also showed pH dependency, where the enzymatic reaction rate was increased at higher pH, which may be because of the higher nucleophilicity of the amine group and the electrostatic interaction between MTG and the self-assembled Fmoc-LnQG. Finally, the accumulation of the fluorescent molecule on these assembled materials was directly observed by confocal fluorescence images. Our study provides a method to accumulate functional molecules on supramolecular structures enzymatically with the morphology control.

DOI： 10.3390/ijms22073459

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.bioconjchem.1c00027

Language：English   Publishing type：Research paper (scientific journal)  

The objective of this study was to prepare a co-amorphous formulation of piroxicam (PIR), a non-steroidal anti-inflammatory drug, and citric acid (CA), and evaluate its skin permeation ability. A spray-drying method was employed to prepare the co-amorphous formulation and its physical properties were characterized. X-ray powder diffraction and thermal analysis confirmed a homogeneous amorphous state, and the infrared spectra revealed intermolecular interactions between PIR and CA, suggesting formation of a co-amorphous formulation of PIR and CA. The PIR-CA co-amorphous formulation exhibited no crystallization for 60 days at 4/25/40°C with silica gel. The PIR-CA co-amorphous formulation increased the solubility of PIR in polyethylene glycol 400 compared with that of the pure drug, and physical mixture (PM) of PIR and CA, confirming a supersaturated state in the formulation. The PIR-CA co-amorphous formulation demonstrated higher skin permeation than PIR alone or PM of PIR and CA, and the flux value was consistent with the degree of saturation. Thus, the increase in the skin permeation of PIR from the PIR-CA co-amorphous formulation directly depended on the increased thermodynamic activity by supersaturation in the absence of interactions between the drug and co-former in the vehicle.

DOI： 10.1016/j.ejps.2020.105667

Language：English   Publishing type：Research paper (scientific journal)  

Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7 ± 2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

DOI： 10.1016/j.pep.2020.105730

Language：English   Publishing type：Research paper (scientific journal)  

We report a new series of lipid-based biocompatible ionic liquids (LBILs) consisting of the long-chain phosphonium compound 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine as the cation and the long-chain fatty acids stearic acid, oleic acid, or linoleic acid as anions. These materials were found to be completely miscible with many polar and nonpolar organic solvents as well as dispersible in water. These LBILs also exhibited excellent biocompatibility with an artificial three-dimensional human epidermis model.

DOI： 10.1039/d0cc04491a

Language：English   Publishing type：Research paper (scientific journal)  

Ionic liquids (ILs) attract significant attention as novel solvents for drug delivery systems because of their ability to solubilize poorly soluble drugs and tune the physiological properties of active pharmaceutical ingredients. For the next generation of IL-based drug delivery systems, biocompatibility is a high priority. In the current study, choline-fatty acids ([Cho][FA]) were used as a biocompatible IL to mediate the dissolution of a water-soluble antigen peptide in an oil-based skin penetration enhancer. Among the candidate fatty acids (C8, C10, C12, C14, C16, C18:0, and C18:1), C18:1 was selected because of its low cytotoxicity and mediation of skin permeability for an antigen peptide. Using IL[Cho][C18:1] and an oil-based penetration enhancer, the flux of transdermal delivery of the peptide increased 28-fold compared with delivery using an aqueous vehicle. Furthermore, the IL-mediated transcutaneous vaccination succeeded in suppressing tumor growth in vivo compared to injection. The skin irritation produced by this formulation was tested using an in vitro 3D constructed skin tissue model and an in vivo histological study, which concluded that the formulation did not cause skin irritation. The results suggest that biocompatible IL-mediated dissolution in an oil-based skin penetration enhancer is a promising strategy for transdermal drug delivery.

DOI： 10.1021/acs.molpharmaceut.0c00598

Language：English   Publishing type：Research paper (scientific journal)  

Assembling proteins in close vicinity to each other provides an opportunity to gain unique function because collaborative and even synergistic functionalities can be expected in an assembled form. There have been a variety of strategies to synthesize functional protein assemblies but site-specific covalent assembly of monomeric protein units without impairing their intrinsic function remains challenging. Herein we report a powerful strategy to design protein assemblies by using microbial transglutaminase (MTG). A serendipitous discovery of self-crosslinking of enhanced green fluorescent protein (EGFP) fused with StrepTag I at the C-terminus revealed that EGFP was assembled through the crosslinking of the Lys (K) residue in the C-terminus of EGFP and the Gln (Q) residue in StrepTag I (AWRHPQFGG). Site-directed mutagenesis of the residues next to the K and Q yielded EGFP assemblies with higher molecular weights. An optimized peptide tag comprised of both K and Q residues (HKRWRHYQRGG) enabled the assembly of different types of proteins of interest (POI) when it was fused to either the N- or C-terminus. The peptide tag that enabled the self-polymerization of the functional POI without a scaffold was designated as a 'PolyTag'.

DOI： 10.1016/j.ab.2020.113700

Language：English   Publishing type：Research paper (scientific journal)  

Liquid marbles (LMs) have recently shown a great promise as microbioreactors to construct self-supported aqueous compartments for chemical and biological reactions. However, the evaporation of the inner aqueous liquid core has limited their application, especially in studying cellular functions. Hydrogels are promising scaffolds that provide a spatial environment suitable for three-dimensional cell culture. Here, we describe the fabrication of redox-responsive hydrogel marbles (HMs) as a three-dimensional cell culture platform. The HMs are prepared by introducing an aqueous mixture of a tetra-thiolated polyethylene glycol (PEG) derivative, thiolated gelatin (Gela-SH), horseradish peroxidase, a small phenolic compound, and human hepatocellular carcinoma cells (HepG2) to the inner aqueous phase of LMs. Eventually, HepG2 cells are encapsulated in the HMs then immersed in culture media, where they proliferate and form cellular spheroids. Experimental results show that the Gela-SH concentration strongly influences the physicochemical and microstructure properties of the HMs. After 6 days in culture, the spheroids were recovered from the HMs by degrading the scaffold, and examination showed that they had reached up to about 180 μm in diameter depending on the Gela-SH concentration, compared with 60 μm in conventional HMs without Gela-SH. After long-term culture (over 12 days), the liver-specific functions (secretion of albumin and urea) and DNA contents of the spheroids cultured in the HMs were elevated compared with those cultured in LMs. These results suggest that the developed HMs can be useful in designing a variety of microbioreactors for tissue engineering applications.

DOI： 10.1016/j.jbiosc.2020.05.010

Language：English   Publishing type：Research paper (scientific journal)  

Transdermal delivery of drugs is more challenging for drugs that are insoluble or sparingly soluble in water and most organic solvents. To overcome this problem, ionic liquid (IL)-mediated ternary systems have been suggested as potential drug carriers. Here, we report potent ternary (IL–EtOH–IPM) systems consisting of biocompatible ILs, ethanol (EtOH), and isopropyl myristate (IPM) that can dissolve a significant amount of the sparingly soluble drug acyclovir (ACV). The ternary systems were optically transparent and thermodynamically stable with a wide range of IL pertinence. An in vitro drug permeation study showed that the ILs in the ternary systems dramatically enhanced ACV permeation into and across the skin. Fourier Transform Infrared spectroscopy of the stratum corneum (sc) after treatment with ternary systems showed that the skin barrier function was reduced by disturbance of the regularly ordered arrangement of corneocytes and modification of the surface properties of the sc during permeation. Histological analysis, and skin irritation studies using a reconstructed human epidermis model showed the safety profile of the ternary system, and there were no significant changes in the structures of the sc, epidermis, and dermis. Therefore, ternary systems containing biocompatible ILs are promising for transdermal delivery of insoluble or sparingly soluble drugs.

DOI： 10.1016/j.ijpharm.2020.119335

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ijpharm.2020.119335

Language：English   Publishing type：Research paper (scientific journal)  

The transdermal delivery of sparingly soluble drugs is challenging due to of the need for a drug carrier. In the past few decades, ionic liquid (IL)-in-oil microemulsions (IL/O MEs) have been developed as potential carriers. By focusing on biocompatibility, we report on an IL/O ME that is designed to enhance the solubility and transdermal delivery of the sparingly soluble drug, acyclovir. The prepared MEs were composed of a hydrophilic IL (choline formate, choline lactate, or choline propionate) as the non-aqueous polar phase and a surface-active IL (choline oleate) as the surfactant in combination with sorbitan laurate in a continuous oil phase. The selected ILs were all biologically active ions. Optimized pseudo ternary phase diagrams indicated the MEs formed thermodynamically stable, spherically shaped, and nano-sized (<100 nm) droplets. An in vitro drug permeation study, using pig skin, showed the significantly enhanced permeation of acyclovir using the ME. A Fourier transform infrared spectroscopy study showed a reduction of the skin barrier function with the ME. Finally, a skin irritation study showed a high cell survival rate (>90%) with the ME compared with Dulbecco’s phosphate-buffered saline, indicates the biocompatibility of the ME. Therefore, we conclude that IL/O ME may be a promising nano-carrier for the transdermal delivery of sparingly soluble drugs.

DOI： 10.3390/pharmaceutics12040392

Language：Others  

Language：Others   Publishing type：Research paper (scientific journal)  

© 2019 Korean Society of Applied Entomology Interleukine-4 (IL-4) is a cytokine that plays an important role in the immune system and recognized as a biological medicine. Therefore, there is a demand for the production of IL-4 with high performance. The expression of a recombinant IL-4 protein in the prokaryotic system usually results in the formation of an inclusion body. To date, the solution to obtain those active products without the refolding process remains to be established. In this study, we tried to acquire a biologically active recombinant Mus musculus IL-4 (rMmIL-4) using a silkworm-baculovirus expression vector system (silkworm-BEVS). We constructed two recombinant baculoviruses coding rMmIL-4 with the distinct location of affinity purification tags and succeeded in the expression and purification of rMmIL-4 proteins directly without the refolding process. Both purified proteins displayed comparable biological activity to the commercial proteins produced by the E. coli expression system. Besides, we performed screening of silkworm strains to seek optimal hosts for the mass-production of rMmIL-4. Intriguingly, we found that some silkworm strains showed significantly higher secretion levels of rMmIL-4 in silkworm sera. Our study provides meaningful insights into the industrial-scale production of rMmIL-4 with high productivity for pharmaceutical applications in the future.

DOI： 10.1016/j.aspen.2019.12.014

Language：English   Publishing type：Research paper (scientific journal)  

The transdermal delivery of sparingly soluble drugs is challenging due to of the need for a drug carrier. In the past few decades, ionic liquid (IL)-in-oil microemulsions (IL/O MEs) have been developed as potential carriers. By focusing on biocompatibility, we report on an IL/O ME that is designed to enhance the solubility and transdermal delivery of the sparingly soluble drug, acyclovir. The prepared MEs were composed of a hydrophilic IL (choline formate, choline lactate, or choline propionate) as the non-aqueous polar phase and a surface-active IL (choline oleate) as the surfactant in combination with sorbitan laurate in a continuous oil phase. The selected ILs were all biologically active ions. Optimized pseudo ternary phase diagrams indicated the MEs formed thermodynamically stable, spherically shaped, and nano-sized (<100 nm) droplets. An in vitro drug permeation study, using pig skin, showed the significantly enhanced permeation of acyclovir using the ME. A Fourier transform infrared spectroscopy study showed a reduction of the skin barrier function with the ME. Finally, a skin irritation study showed a high cell survival rate (>90%) with the ME compared with Dulbecco's phosphate-buffered saline, indicates the biocompatibility of the ME. Therefore, we conclude that IL/O ME may be a promising nano-carrier for the transdermal delivery of sparingly soluble drugs.

DOI： 10.3390/pharmaceutics12040392

Language：English   Publishing type：Research paper (scientific journal)  

In this report, a strategy for constructing three-dimensional (3D) cellular architectures comprising viable cells is presented. The strategy uses a redox-responsive hydrogel that degrades under mild reductive conditions, and a confluent monolayer of cells (i.e., cell sheet) cultured on the hydrogel surface peels off and self-folds to wrap other cells. As a proof-of-concept, the self-folding of fibroblast cell sheet was triggered by immersion in aqueous cysteine, and this folding process was controlled by the cysteine concentration. Such folding enabled the wrapping of human hepatocellular carcinoma (HepG2) spheroids, human umbilical vein endothelial cells and collagen beads, and this process improved cell viability, the secretion of metabolites and the proliferation rate of the HepG2 cells when compared with a two-dimensional culture under the same conditions. A key concept of this study is the ability to interact with other neighbouring cells, providing a new, simple and fast method to generate higher-order cellular aggregates wherein different types of cellular components are added. We designated the method of using a cell sheet to wrap another cellular aggregate the 'cellular Furoshiki'. The simple self-wrapping Furoshiki technique provides an alternative approach to co-culture cells by microplate-based systems, especially for constructing heterogeneous 3D cellular microstructures.

DOI： 10.1038/s41598-020-63362-4

Language：English   Publishing type：Research paper (scientific journal)  

Interleukine-4 (IL-4) is a cytokine that plays an important role in the immune system and recognized as a biological medicine. Therefore, there is a demand for the production of IL-4 with high performance. The expression of a recombinant IL-4 protein in the prokaryotic system usually results in the formation of an inclusion body. To date, the solution to obtain those active products without the refolding process remains to be established. In this study, we tried to acquire a biologically active recombinant Mus musculus IL-4 (rMmIL-4) using a silkworm-baculovirus expression vector system (silkworm-BEVS). We constructed two recombinant baculoviruses coding rMmIL-4 with the distinct location of affinity purification tags and succeeded in the expression and purification of rMmIL-4 proteins directly without the refolding process. Both purified proteins displayed comparable biological activity to the commercial proteins produced by the E. coli expression system. Besides, we performed screening of silkworm strains to seek optimal hosts for the mass-production of rMmIL-4. Intriguingly, we found that some silkworm strains showed significantly higher secretion levels of rMmIL-4 in silkworm sera. Our study provides meaningful insights into the industrial-scale production of rMmIL-4 with high productivity for pharmaceutical applications in the future.

DOI： 10.1016/j.aspen.2019.12.014

Language：English   Publishing type：Research paper (scientific journal)  

The aim of this study was to prepare binary supercooled liquid (SCL) by intermolecular interaction and apply this formulation to transdermal drug delivery. Ketoprofen (KET) and ethenzamide (ETH) were selected as binary SCL component. Thermal analysis of physical mixtures of KET and ETH showed decreases in melting points and glass transition below room temperature, thereby indicating formation of KET–ETH SCL. Intermolecular interactions between KET and ETH in the SCL were evaluated from Fourier transform (FT)-IR spectra. KET–ETH SCL maintained SCL state at 25°C with silica gel over 31d and at 40°C/89% relative humidity (RH) over 7d. KET SCL and KET–ETH SCL showed similar permeability of KET for hairless mice skin, which was two-fold higher than that of KET aqueous suspension. Our findings suggest that the SCL state could enhance the skin permeation of drugs and the binary SCL formed by intermolecular interaction could also improve the stability of the SCL. The binary SCL system could become a new drug form for transdermal drug delivery.

DOI： 10.1248/bpb.b19-00642

Language：English   Publishing type：Research paper (scientific journal)  

Japanese cedar pollinosis (JCP) is a common affliction caused by an allergic reaction to cedar pollen and is considered a disease of national importance in Japan. Antigen-specific immunotherapy (AIT) is the only available curative treatment for JCP. However, low compliance and persistence have been reported among patients subcutaneously or sublingually administered AIT comprising a conventional antigen derived from a pollen extract. To address these issues, many research studies have focused on developing a safer, simpler, and more effective AIT for JCP. Here, we review the novel antigens that have been developed for JCP AIT, discuss their different administration routes, and present the effects of anti-allergy treatment. Then, we describe a new form of AIT called transcutaneous immunotherapy (TCIT) and its solid-in-oil (S/O) nanodispersion formulation, which is a promising antigen delivery system. Finally, we discuss the applications of S/O nanodispersions for JCP TCIT. In this context, we predict that TCIT delivery by using a S/O nanodispersion loaded with novel antigens may offer an easier, safer, and more effective treatment option for JCP patients.

DOI： 10.3390/pharmaceutics12030240

Language：English   Publishing type：Research paper (scientific journal)  

The structure of a protein complex needs to be controlled appropriately to maximize its functions. Herein, we report the linear polymerization of bacterial alkaline phosphatase (BAP) through the site-specific cross-linking reaction catalyzed by Trametes sp. laccase (TL). We introduced a peptide loop containing a tyrosine (Y-Loop) to BAP, and the Y-Looped BAP was treated with TL. The Y-Looped BAP formed linear polymers, whereas BAP fused with a C-terminal peptide containing a tyrosine (Y-tag) showed an irregular shape after TL treatment. The sterically confined structure of the Y-Loop could be responsible for the formation of linear BAP polymers. TL-catalyzed copolymerization of Y-Looped BAP and a Y-tagged chimeric antibody-binding protein, pG2pA-Y, resulted in the formation of linear bifunctional protein copolymers that could be employed as protein probes in an enzyme-linked immunosorbent assay (ELISA). Copolymers comprising Y-Looped BAP and pG2pA-Y at a molar ratio of 100:1 exhibited the highest signal in the ELISA with 26- and 20-fold higher than a genetically fused chimeric protein, BAP-pG2pA-Y, and its polymeric form, respectively. This result revealed that the morphology of the copolymers was the most critical feature to improve the functionality of the protein polymers as detection probes, not only for immunoassays but also for other diagnostic applications.

DOI： 10.1021/acsomega.9b04163

Language：English   Publishing type：Research paper (scientific journal)  

Japanese cedar pollinosis (JCP) is a common affliction caused by an allergic reaction to cedar pollen and is considered a disease of national importance in Japan. Antigen-specific immunotherapy (AIT) is the only available curative treatment for JCP. However, low compliance and persistence have been reported among patients subcutaneously or sublingually administered AIT comprising a conventional antigen derived from a pollen extract. To address these issues, many research studies have focused on developing a safer, simpler, and more effective AIT for JCP. Here, we review the novel antigens that have been developed for JCP AIT, discuss their different administration routes, and present the effects of anti-allergy treatment. Then, we describe a new form of AIT called transcutaneous immunotherapy (TCIT) and its solid-in-oil (S/O) nanodispersion formulation, which is a promising antigen delivery system. Finally, we discuss the applications of S/O nanodispersions for JCP TCIT. In this context, we predict that TCIT delivery by using a S/O nanodispersion loaded with novel antigens may offer an easier, safer, and more effective treatment option for JCP patients.

DOI： 10.3390/pharmaceutics12030240

Language：English   Publishing type：Research paper (scientific journal)  

An allergy to cow’s milk proteins is the most common food allergy in infants and toddlers. Conventional oral immunotherapy for cow’s milk allergies requires hospital admission due to the risk of severe allergic reactions, including anaphylaxis. Therefore, a simpler and safer immunotherapeutic method is desirable. We examined transcutaneous immunotherapy with a solid-in-oil (S/O) system. In the S/O system, nano-sized particles of proteins are dispersed in an oil-vehicle with the assistance of nonionic surfactants. In the present study, the S/O system enhanced the skin permeation of the allergen molecule β-lactoglobulin (BLG), as compared with a control PBS solution. The patches containing BLG in the S/O nanodispersion skewed the immune response in the allergy model mice toward T helper type 1 immunity, indicating the amelioration of allergic symptoms. This effect was more pronounced when the immunomodulator resiquimod (R-848) was included in the S/O system.

DOI： 10.3390/pharmaceutics12030205

Language：English   Publishing type：Research paper (scientific journal)  

The development of non-toxic ionic liquid-based active pharmaceutical ingredients (IL-APIs) for effective topical drug delivery is still challenging. The properties of IL-APIs can be boosted up by selecting potential biocompatible cations. Here, we introduced N-methyl-2-pyrrolidone (NMP) as a potent biocompatible counter ion to prepare ionic liquefied drugs for topical drug delivery. The cytotoxicity of NMP cation was investigated using mammalian cell lines (HepG2, NIH3T3 and L929 cells) and compared with conventional IL-forming cations. The synthesized NMP cation has lower toxicity than that of conventional IL-forming cations. The NMP cation showed at least 3.6, 15.2 and 58.9 times lower toxicity than that of conventional imidazolium, ammonium and phosphonium cations, respectively. The synthesized NMP-based ionic liquid (NMP-IL) was characterized using ¹H & ¹³C NMR, FT-IR, DSC and TGA. NMP-IL showed better physico-thermal stability, enhanced skin penetration, and enriched drug accumulation 2.6 times higher than that of IL [Cho][Ibu] in the target tissue. These results suggested that NMP cation based API-IL can be an effective biocompatible formulation for topical drug delivery by accumulating active drugs in the skin.

DOI： 10.1016/j.molliq.2019.112166

Language：English   Publishing type：Research paper (scientific journal)  

Skin dendritic cells (DCs) such as Langerhans cells and dermal dendritic cells have a pivotal role in inducing antigen-specific immunity; therefore, transcutaneous cancer vaccines are a promising strategy to prophylactically prevent the onset of a variety of diseases, including cancers. The largest obstacle to delivering antigen to these skin DC subsets is the barrier function of the stratum corneum. Although reverse micellar carriers are commonly used to enhance skin permeability to hydrophilic drugs, the transcutaneous delivery of antigen, proteins, or peptides has not been achieved to date because of the large molecular weight of drugs. To achieve effective antigen delivery to skin DCs, we developed a novel strategy using a surfactant as a skin permeation enhancer in a reverse micellar carrier. In this study, glyceryl monooleate (MO) was chosen as a skin permeation enhancer, and the MO-based reverse micellar carrier enabled the successful delivery of antigen to Langerhans cells and dermal dendritic cells. Moreover, transcutaneous vaccination with the MO-based reverse micellar carrier significantly inhibited tumor growth, indicating that it is a promising vaccine platform against tumors.

DOI： 10.1021/acs.molpharmaceut.9b01104

Language：English   Publishing type：Research paper (scientific journal)  

Skin dendritic cells (DCs) such as Langerhans cells and dermal dendritic cells have a pivotal role in inducing antigen-specific immunity; therefore, transcutaneous cancer vaccines are a promising strategy to prophylactically prevent the onset of a variety of diseases, including cancers. The largest obstacle to delivering antigen to these skin DC subsets is the barrier function of the stratum corneum. Although reverse micellar carriers are commonly used to enhance skin permeability to hydrophilic drugs, the transcutaneous delivery of antigen, proteins, or peptides has not been achieved to date because of the large molecular weight of drugs. To achieve effective antigen delivery to skin DCs, we developed a novel strategy using a surfactant as a skin permeation enhancer in a reverse micellar carrier. In this study, glyceryl monooleate (MO) was chosen as a skin permeation enhancer, and the MO-based reverse micellar carrier enabled the successful delivery of antigen to Langerhans cells and dermal dendritic cells. Moreover, transcutaneous vaccination with the MO-based reverse micellar carrier significantly inhibited tumor growth, indicating that it is a promising vaccine platform against tumors.

DOI： 10.1021/acs.molpharmaceut.9b01104

Language：English   Publishing type：Research paper (scientific journal)  

En masse vaccination is a promising strategy for combatting infectious diseases. Intranasal vaccination is a viable route of mass vaccination, and it could be performed easily via needle-free administration. However, it is not widely used because it tends not to evoke sufficient immunity. The aim of the present study was to improve the performance of intranasal vaccination by extending the amount of time that administered antigens remain in the nasal cavity, and enhancing immune responses via a nanocarrier-based adjuvant. A simple and safe solid-in-oil (S/O) system was investigated as a nanocarrier in intranasal vaccination. S/O nanodispersions are oil-based dispersions of antigens coated with surfactants. Because of the mucoadhesive capacities of surfactant and oil they have high potential to extend the amount of time that administered antigens remain in the nasal cavity, and can induce strong immune responses due to a nanocarrier-based adjuvant effect. In nasal absorption experiments antigens administered intranasally via S/O nanodispersions remained in the nasal cavity longer and induced strong mucosal and systemic immune responses. Histopathology analysis indicated that S/O nanodispersions did not modify the nasal epithelium or cilia, suggesting non-toxicity of the carrier. These results indicate the potential of intranasal vaccination using S/O nanodispersions for future vaccination.

DOI： 10.1016/j.ijpharm.2019.118777

Language：English   Publishing type：Research paper (scientific journal)  

Synthetic Strategies for Artificial Lipidation of Functional Proteins

DOI： 10.1002/chem.201904568

Language：Others  

Solid-in-oil nanodispersions for intranasal vaccination: Enhancement of mucosal and systemic immune responses.

DOI： 10.1016/j.ijpharm.2019.118777

Language：Others  

A novel binary supercooled liquid formulation for transdermal drug delivery.

DOI： 10.1248/bpb.b19-00642

Language：English   Publishing type：Research paper (scientific journal)  

Japanese cedar pollinosis is a type I allergic disease and has already become a major public health problem in Japan. Conventional subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) cannot meet patients’ needs owing to the side effects caused by both the use of conventional whole antigen molecules in the pollen extract and the administration routes. To address these issues, a surface-modified antigen and transcutaneous administration route are introduced in this research. First, the pollen extract (PE) was conjugated to galactomannan (PE-GM) to mask immunoglobulin E (IgE)-binding epitopes in the PE to avoid side effects. Second, as a safer alternative to SCIT and SLIT, transcutaneous immunotherapy (TCIT) with a solid-in-oil (S/O) nanodispersion system carrying PE-GM was proposed. Hydrophilic PE-GM was efficiently delivered through mouse skin using S/O nanodispersions, reducing the antibody secretion and modifying the type 1 T helper (Th1)/ type 2 T helper (Th2) balance in the mouse model, thereby demonstrating the potential to alleviate Japanese cedar pollinosis.

DOI： 10.3390/pharmaceutics11110563

Language：English   Publishing type：Research paper (scientific journal)  

The objective of this study was to prepare a supersaturated formulation based on formation of a co-amorphous system of a drug and a coformer in order to enhance skin permeation. Atenolol (ATE) and urea (URE) were used as the model drug and the coformer, respectively. Thermal analysis of physical mixtures of ATE and URE showed decreases in the melting points and the formation of a co-amorphous system which was in a supercooled liquid state because of a low glass transition temperature. Supersaturated solutions of ATE and URE at different molar ratios in polyethylene glycol 400 (PEG400) were prepared. The precipitations were observed under storage at 25 °C for all formulations except for ATE-URE at 1:8 molar ratio which remained in the supersaturated state for 2 months. ¹H NMR analysis confirmed the interactions between ATE and URE in PEG400. The ATE-URE supersaturated formulation showed higher permeability for mice skin than that of ATE saturated formulation, which was superior to the expected permeability from the degree of supersaturation. We concluded that co-amorphous based supersaturated formulation offers much promise for transdermal drug delivery.

DOI： 10.1016/j.ijpharm.2019.118582

Language：Others  

Transcutaneous Delivery of Immunomodulating Pollen Extract-Galactomannan Conjugate by Solid-in-Oil Nanodispersions for Pollinosis Immunotherapy.

DOI： 10.3390/pharmaceutics11110563

Language：English   Publishing type：Research paper (scientific journal)  

Ionic liquid (IL)surfactants have attracted great interest as promising substitutes for conventional surfactants owing to their exceptional and favorable physico-chemical properties. However, most IL surfactants are not eco-friendly and form unstable micelles, even when using a high concentration of the surfactant. In this study, we prepared a series of halogen-free and biocompatible choline–fatty-acid-based ILs with different chain lengths and degrees of saturation, and we then investigated their micellar properties in aqueous solutions. Characterization of the synthesized surface-active ILs (SAILs)was performed by ¹ H and ¹³ C nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and elemental analysis. The surface-active properties of the SAILs were investigated by tensiometry, conductometry, and dynamic light scattering measurements. The critical micelle concentration of the SAILs was found to be 2–4 times lower than those of conventional surfactants. The thermodynamic properties of micellization (ΔG ⁰ _m , ΔH ⁰ _m , and ΔS ⁰ _m )indicate that the micellization process of the SAILs is spontaneous, stable, and entropy-driven at room temperature. The cytotoxicity of the SAILs was evaluated using mammalian cell line NIH 3T3. Importantly, [Cho][Ole]shows lower toxicity than the analogous ILs with conventional surfactants. These results clearly suggest that these environmentally friendly SAILs can be used as a potential alternative to conventional ILs for various purposes, including biological applications.

DOI： 10.1016/j.jcis.2019.04.095

Language：English   Publishing type：Research paper (scientific journal)  

We developed a new oil-based delivery system for transdermal protein delivery, a gel-in-oil (G/O) nanosuspension, where gelatin-based hydrogel was coated with hydrophobic surfactants. The high entrapment efficiency of a model protein, phycocyanin (PC), into nano-sized gelatin hydrogel particles was achieved. Spectroscopic evaluation of PC suggested that the G/O nanosuspension could retain the functional form of PC in isopropyl myristate. In vitro skin permeation studies showed that the G/O nanosuspension facilitated the delivery of PC through the stratum corneum of Yucatan micropig skin.

DOI： 10.1016/j.ijpharm.2019.118495

Language：English   Publishing type：Research paper (scientific journal)  

A nano-sized gel-in-oil suspension for transcutaneous protein delivery
We developed a new oil-based delivery system for transdermal protein delivery, a gel-in-oil (G/O) nanosuspension, where gelatin-based hydrogel was coated with hydrophobic surfactants. The high entrapment efficiency of a model protein, phycocyanin (PC), into nano-sized gelatin hydrogel particles was achieved. Spectroscopic evaluation of PC suggested that the G/O nanosuspension could retain the functional form of PC in isopropyl myristate. In vitro skin permeation studies showed that the G/O nanosuspension facilitated the delivery of PC through the stratum corneum of Yucatan micropig skin.

DOI： 10.1016/j.ijpharm.2019.118495

Language：English   Publishing type：Research paper (scientific journal)  

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.

DOI： 10.1016/j.pep.2019.03.010

Language：Others  

Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system.

DOI： 10.1016/j.pep.2019.03.010

Language：English   Publishing type：Research paper (scientific journal)  

In order to prevent common hypersensitivity reactions to paclitaxel injections (Taxol), we previously reported an ionic liquid-mediated paclitaxel (IL-PTX)formulation with small particle size and narrow size distribution. The preliminary work showed high PTX solubility in the IL, and the formulation demonstrated similar antitumor activity to Taxol, while inducing a smaller hypersensitivity effect in in vitro cell experiments. In this study, the stability of the IL-PTX formulation was monitored by quantitative HPLC analysis, which showed that IL-PTX was more stable at 4 °C than at room temperature. The in vivo study showed that the IL-PTX formulation could be used in a therapeutic application as a biocompatible component of a drug delivery system. To assess the in-vivo biocompatibility, IL or IL-mediated formulations were administered intravenously by maintaining physiological buffered conditions (neutral pH and isotonic salt concentration). From in vivo pharmacokinetics data, the IL-PTX formulation was found to have a similar systemic circulation time and slower elimination rate compared to cremophor EL mediated paclitaxel (CrEL-PTX). Furthermore, in vivo antitumor and hypersensitivity experiments in C57BL/6 mice revealed that IL-PTX had similar antitumor activity to CrEL-PTX, but a significantly smaller hypersensitivity effect compared with CrEL-PTX. Therefore, the IL-mediated formulation has potential to be an effective and safe drug delivery system for PTX.

DOI： 10.1016/j.ijpharm.2019.05.020

Language：English   Publishing type：Research paper (scientific journal)  

Polymerization of Horseradish Peroxidase by a Laccase-Catalyzed Tyrosine Coupling Reaction.
The polymerization of proteins can create newly active and large bio-macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine-tag (Y-tag) through a flexible linker at the N- and/or C-termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y-tagged HRPs with molecular O2 to form a tyrosyl-free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP-catalyzed self-crosslinking reaction in the presence of H2 O2 . The addition of H2 O2 in the self-polymerization of Y-tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site-selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y-tagged HRPs and HRP-protein G (Y-HRP-pG) units catalyzed by TL shows a higher signal in enzyme-linked immunosorbent assay (ELISA) than the genetically pG-fused HRP, Y-HRP-pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.

DOI： 10.1002/biot.201800531

Language：Others   Publishing type：Research paper (scientific journal)  

© 2019 American Chemical Society. Biologically active artificial scaffolds for cell seeding are developed by mimicking extracellular matrices using synthetic materials. Here, we propose a feasible approach employing biocatalysis to integrate natural components, that is, gelatin and heparin, into a synthetic scaffold, namely a polyethylene glycol (PEG)-based hydrogel. Initiation of horseradish peroxidase-mediated redox reaction enabled both hydrogel formation of tetra-thiolated PEG via disulfide linkage and incorporation of chemically thiolated gelatin (Gela-SH) and heparin (Hepa-SH) into the polymeric network. We found that the compatibility of the type of gelatin with heparin was crucial for the hydrogelation process. Alkaline-treated gelatin exhibited superior performance over acid-treated gelatin to generate dual functionality in the resultant hydrogel originating from the two natural biopolymers. The Gela-SH/Hepa-SH dual functionalized PEG-based hydrogel supported both cellular attachment and binding of basic fibroblast growth factor (bFGF) under cell culture conditions, which increased the proliferation and phenotype transformation of NIH3T3 cells cultured on the hydrogel. Inclusion of bFGF and a commercial growth factor cocktail in hydrogel matrices effectively enhanced cell spreading and confluency of both NIH3T3 cells and HUVECs, respectively, suggesting a potential method to design artificial scaffolds containing active growth factors.

DOI： 10.1021/acsabm.9b00275

Language：Others  

Development of a novel ionic liquid-curcumin complex to enhance its solubility, stability, and activity

Language：Others  

Complementary interaction with peptide amphiphiles guides size-controlled assembly of small molecules for intracellular delivery

DOI： 10.1039/c9cc02473e

Language：English   Publishing type：Research paper (scientific journal)  

Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP)
₄
–Stav or Stav–(HRP)
₄
, respectively) using a baculovirus-silkworm expression system. Both (HRP)
₄
–Stav and Stav–(HRP)
₄
were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP)
₄
–Stav and Stav–(HRP)
₄
could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP)
₄
–Stav was twofold higher than that of Stav–(HRP)
₄
, and the sensitivity of (HRP)
₄
-Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP)
₄
–Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.

DOI： 10.1016/j.jbiotec.2019.03.007

Language：Others  

Synthesis and characterization of choline–fatty-acid-based ionic liquids: A new biocompatible surfactant

Language：English   Publishing type：Research paper (scientific journal)  

Molecular chaperone prefoldin-assisted biosynthesis of gold nanoparticles with improved size distribution and dispersion
Here we report a novel aspect of molecular chaperone prefoldin (PFD) as a biomaterial in the biocatalytic synthesis of gold nanoparticles (AuNPs) using glycerol dehydrogenase (GLD). We found that PFD could inhibit the aggregation of AuNPs during the biosynthesis, leading to the formation of AuNPs with controlled size distribution.

DOI： 10.1039/c8bm01026a

Language：Others  

In vivo biocompatibility, pharmacokinetics, antitumor efficacy, and hypersensitivity evaluation of ionic liquid-mediated paclitaxel formulations

Language：Others  

Functional horseradish peroxidase-streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes.

DOI： 10.1016/j.jbiotec.2019.03.007

Language：English   Publishing type：Research paper (scientific journal)  

© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid-G3S-MRHKGS lipid (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding >90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid–protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG.

DOI： 10.1002/chem.201900370

Language：English   Publishing type：Research paper (scientific journal)  

Semi-refined carrageenan (SRC) is one of the products from Kappaphycus alvarezii, which has the potential to be developed in Indonesia. However, unbleached seaweed will produce SRC with light brown colour due to natural seaweed pigments. Peracetic acid (PAA) is a strong oxidizing agent that has the potential to be used as a bleaching agent in the SRC production. This research aimed to study the effects of the PAA as a bleaching agent on the characteristics of SRC from K. alvarezii. Chopped seaweed was heated in 10% KOH at 80°C for 30 minutes. Bleaching was carried out at room temperature for 90 minutes using PAA at a concentration of 0.5%, 1.5%, and 2.5% (w/w) or using 1.5% (w/w) of sodium hypochlorite. Bleaching using PAA produced SRC whiter than without bleaching and bleaching using sodium hypochlorite. SRC brightness increased with increasing concentrations of PAA and while the yield reduced, as well as the viscosity, gel strength, sulfate content and ash content. The best concentration of PAA was 0.5%. At best, PAA concentration bleaching produced white SRC flour with a lightness value of 80.46±0.01; yield of 23.50%; viscosity 99.33 cP, gel strength 307.63 g/cm², sulfate 13.29% (w/w), ash 8.33% (w/w) and acid-insoluble ash 1.33% (w/w).

DOI： 10.1088/1755-1315/278/1/012077

Language：English   Publishing type：Research paper (scientific journal)  

Here we report a novel aspect of molecular chaperone prefoldin (PFD) as a biomaterial in the biocatalytic synthesis of gold nanoparticles (AuNPs) using glycerol dehydrogenase (GLD). We found that PFD could inhibit the aggregation of AuNPs during the biosynthesis, leading to the formation of AuNPs with controlled size distribution.

DOI： 10.1039/c8bm01026a

Language：Others  

Transcutaneous Codelivery of Tumor Antigen and Resiquimod in Solid-in-Oil Nanodispersions Promotes Antitumor Immunity

Language：Japanese  

昆虫工場を利用したウイルス様粒子ワクチン生産系の確立とその応用

Language：Others  

Self-Assembled Reduced Albumin and Glycol Chitosan Nanoparticles for Paclitaxel Delivery

DOI： 10.1021/acs.langmuir.8b02809

Language：English   Publishing type：Research paper (scientific journal)  

Macromolecular crowding modulates the conformational preference of a small molecular rotor, which is used as a molecular viscometer. The shift towards the planar conformation of the porphyrin rotor was observed in the presence of increasing concentrations and sizes of polyethylene glycols. This observation highlights the differences between the behaviour of this molecular viscometer in the crowding and non-crowding types of media.

DOI： 10.1016/j.jphotochem.2018.10.006

Language：Others  

Enhanced Potential of Therapeutic Applications of Curcumin Using Solid-in-Water Nanodispersion Technique

DOI： 10.1252/jcej.18we060

Language：Others  

Effect of macromolecular crowding on the conformational behaviour of a porphyrin rotor

DOI： 10.1016/j.jphotochem.2018.10.006

Language：English   Publishing type：Research paper (scientific journal)  

Cancer vaccines aim to prevent or inhibit tumor growth by inducing an immune response to tumor-associated antigens (TAAs) encoded by or present in the vaccine. Previous work has demonstrated that effective antitumor immunity can be induced using a codelivery system in which nonspecific immunostimulatory molecules are administered together with TAAs. In this study, we investigated the antitumor effects of a solid-in-oil (S/O) nanodispersion system containing a model TAA, ovalbumin (OVA), and resiquimod (R-848), a small molecular Toll-like receptor 7/8 ligand, which induces an antigen-nonspecific cellular immune response that is crucial for the efficacy of cancer vaccines. R-848 was contained in the outer oil phase of S/O nanodispersion. Analysis of OVA and R-848 permeation in mouse skin after application of an R-848 S/O nanodispersion indicated that R-848 rapidly permeated the skin and preactivated Langerhans cells, resulting in efficient uptake of OVA and migration of antigen-loaded Langerhans cells to the draining lymph nodes. Transcutaneous immunization of mice with an R-848 S/O nanodispersion inhibited the growth of E.G7-OVA tumors and prolonged mouse survival to a greater extent than did immunization with an S/O nanodispersion containing OVA alone. Consistent with this observation, antigen-specific secretion of the Th1 cytokine interferon-γand cytolytic activity were both high in splenocytes isolated from mice immunized with R-848 S/O. Our results thus demonstrate that codelivery of R-848 significantly amplified the antitumor immune response induced by antigen-containing S/O nanodispersions and further suggest that S/O nanodispersions may be effective formulations for codelivery of TAAs and R-848 in transcutaneous cancer vaccines.

DOI： 10.1021/acsbiomaterials.9b00260

Language：English   Publishing type：Research paper (scientific journal)  

We report a one-step emulsification and rapid freeze-drying process to develop a curcumin-ionic liquid (CCM-IL) complex that could be readily dispersed in water with a significantly enhanced solubility of ∼8 mg mL^-1 and half-life (t_1/2) of ∼260 min compared with free CCM (solubility ∼30 nM and t_1/2 ∼ 20 min). This process using an IL consisting of a long chain carbon backbone as a surfactant, may provide an alternative way of enhancing the solubility of poorly water-soluble drugs.

DOI： 10.1039/c9cc02812a

Language：English   Publishing type：Research paper (scientific journal)  

We introduced complementary interactions between peptide amphiphiles and a small fluorescence dye to develop a programmable multi-component supramolecular assembly, and intracellular delivery of the dye was controlled by the dimensions of the co-assembly, which was manipulated by the peptide design.

DOI： 10.1039/c9cc02473e

Language：English   Publishing type：Research paper (scientific journal)  

The polymerization of proteins can create newly active and large bio-macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine-tag (Y-tag) through a flexible linker at the N- and/or C-termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y-tagged HRPs with molecular O
₂
to form a tyrosyl-free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP-catalyzed self-crosslinking reaction in the presence of H
₂
O
₂
. The addition of H
₂
O
₂
in the self-polymerization of Y-tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site-selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y-tagged HRPs and HRP-protein G (Y-HRP-pG) units catalyzed by TL shows a higher signal in enzyme-linked immunosorbent assay (ELISA) than the genetically pG-fused HRP, Y-HRP-pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.

DOI： 10.1002/biot.201800531

Language：English   Publishing type：Research paper (scientific journal)  

Curcumin (Cur), a hydrophobic polyphenol compound, holds promising potential as an anticancer agent. However, the poor solubility in water and the low bioavailability of curcumin have limited its therapeutic applications. In this regard, we reported the formulation of curcumin using a solid-in-water (S/W) nanodispersion technique to enhance the water solubility and therapeutic activity of curcumin. This new aqueous formulation comprises simple preparation protocols: emulsification and freeze-drying for encapsulating hydrophobic biomolecules with a hydrophilic surfactant, followed by redispersion of the resultant solid complexes in an aqueous solution. Pluronics F68 and F127 were used here for the encapsulation of curcumin. Enhanced aqueous solubility of curcumin was achieved by encapsulating curcumin with a hydrophilic surfactant using the S/W nanodispersion technique. The resultant nanosized formulation had a narrow size distribution and high entrapment efficiency of curcumin. The highest loading capacity of curcumin in S/W nanodispersion was obtained with a weight ratio of curcumin to pluronic of 1: 10 for both surfactants. The release profile of the complexes was found to depend on the type of surfactant, suggesting that the selection of a proper surfactant is crucial for controlling curcumin delivery. The anticancer activity of the S/W formulation of curcumin was correlated with the drug release profiles and cellular uptake, which in turn was influenced by the hydrophobicity and chemical structure of the surfactant.

DOI： 10.1252/jcej.18we060

Language：Others   Publishing type：Research paper (scientific journal)  

Copyright © 2018 American Chemical Society. Lipid modification of proteins plays a significant role in regulating the cellular environment. Mimicking natural lipidated proteins is a key technique for assessing the function of proteins modified with lipids and also to render self-assembly of lipids to a target protein. Herein, we report a facile method of conjugating proteins with lipid-fused peptides under homogeneous physiological conditions by using the microbial transglutaminase (MTG) reaction. MTG catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and a lysine (K) in newly designed lipid-fused peptides. The water-soluble peptide substrates for lipid modification, C14-X-MRHKGS, were newly synthesized, where C14, X, and MRHKGS represent myristic acid, linker peptides composed of G, P, or S, and MTG-reactive K surrounded with basic amino acids, respectively. The MTG-mediated cross-linking reaction between a protein fused with LLQG at the C-terminus and C14-X-MRHKGS (5 molar eq) dissolved in a phosphate saline solution resulted in lipid-protein conjugates with yields of 70 to 100%. The anchoring ability of the obtained lipid-protein conjugates to cell membranes was dependent on the number of G residues in the GnS linker, suggesting that self-assembly and hydrophobicity of the GnS motif serves to enhance membrane anchoring of lipid-protein conjugates.

DOI： 10.1021/acsabm.8b00271

Language：English   Publishing type：Research paper (scientific journal)  

Liquid marble (LM), a self-standing micro-scale aqueous droplet, emerges as a micro-bioreactor in biological applications. Herein, the potential of LM as media for cell-free synthesis and simultaneous immobilization of recombinant proteins is explored. Initially, formation of hydrogel marble (HM) by using an enzymatic disulfide-based hydrogelation technique is confirmed by incorporating three components, horseradish peroxidase (HRP), a tetra-thiolated poly(ethylene glycol) derivative, and glycyl-L-tyrosine, in LM. The compatibility of the enzymatic hydrogelation with cell-free protein synthesis in LM is then validated. Although the hydrogelation reduces the level of protein synthesis in LM when compared with that in a test tube, the biosynthesis of enhanced green fluorescent protein (EGFP) is achieved. Interestingly, EGFP synthesized in LM is entrapped in the HM, and the introduction of a cysteine residue to EGFP by genetic engineering further increases the amount of protein immobilization in the hydrogel matrices. These results suggest that the cell-free synthesis and HRP-catalyzed hydrogelation can be conducted in parallel in LM, and the eventual entrapment of the key components in HM is possible. Facile recovery of macromolecular products immobilized in HM by degrading the hydrogel network under reducing conditions should lead to the design of an easy-to-handle system to screen protein functions.

DOI： 10.1002/biot.201800085

Language：Others  

Designer aromatic peptide amphiphiles for self-assembly and enzymatic display of proteins with morphology control

Language：English   Publishing type：Research paper (scientific journal)  

The site-specific cross-linking of functional proteins creates macromolecular assemblies that exhibit unique biochemical and/or physicochemical properties. Herein, we explored the potential of laccase as a biocatalyst for the site-specific cross-linking of tyrosine-tagged proteins. Trametes sp. laccase (TL) was selected as the cross-linking catalyst, and Escherichia coli alkaline phosphatase (BAP) and antibody-binding proteins (pG₂pAs) were employed as model proteins. The protein models were genetically fused to a peptide tag containing a tyrosine residue (Y-tag) at the N- and/or C-termini. Proteins without Y-tags were used as controls. The Y-tagged proteins could be recognized by TL as macromolecular substrates, leading to the oxidative formation of protein polymers, whereas no polymerization was observed with intact BAP or pG₂pA. The TL-catalyzed cross-linking of Y-tagged proteins proceeded at a relatively high pH in comparison with that of small phenolic substrates. Co-polymers of BAP and pG₂pA were able to be prepared by mixing the aqueous solution of each component in the presence of TL. A combination of bis-Y-tagged pG₂pA (Y-pG₂pA-Y) and Y-tagged BAP (BAP-Y) yielded functional co-polymers compatible with enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA of ovalbumin with anti-OVA IgG depended on the molar ratio of BAP-Y and Y-pG₂pA-Y in the TL-catalyzed cross-linking reaction. A high molar ratio of BAP-Y to Y-pG₂pA-Y (75:1) resulted in the highest absorbance in the ELISA. The results suggested that the formation of a bifunctional protein polymer with a high molar ratio of signaling unit to antibody-binding unit gave better performance in antigen detection than using lower ratios.

DOI： 10.1016/j.jbiosc.2018.05.013

Language：English   Publishing type：Research paper (scientific journal)  

The site-specific cross-linking of functional proteins creates macromolecular assemblies that exhibit unique biochemical and/or physicochemical properties. Herein, we explored the potential of laccase as a biocatalyst for the site-specific cross-linking of tyrosine-tagged proteins. Trametes sp. laccase (TL) was selected as the cross-linking catalyst, and Escherichia coli alkaline phosphatase (BAP) and antibody-binding proteins (pG2pAs) were employed as model proteins. The protein models were genetically fused to a peptide tag containing a tyrosine residue (Y-tag) at the N- and/or C-termini. Proteins without Y-tags were used as controls. The Y-tagged proteins could be recognized by TL as macromolecular substrates, leading to the oxidative formation of protein polymers, whereas no polymerization was observed with intact BAP or pG2pA. The TL-catalyzed cross-linking of Y-tagged proteins proceeded at a relatively high pH in comparison with that of small phenolic substrates. Co-polymers of BAP and pG2pA were able to be prepared by mixing the aqueous solution of each component in the presence of TL. A combination of bis-Y-tagged pG2pA (Y-pG2pA-Y) and Y-tagged BAP (BAP-Y) yielded functional co-polymers compatible with enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA of ovalbumin with anti-OVA IgG depended on the molar ratio of BAP-Y and Y-pG2pA-Y in the TL-catalyzed cross-linking reaction. A high molar ratio of BAP-Y to Y-pG2pA-Y (75:1) resulted in the highest absorbance in the ELISA. The results suggested that the formation of a bifunctional protein polymer with a high molar ratio of signaling unit to antibody-binding unit gave better performance in antigen detection than using lower ratios.

DOI： 10.1016/j.jbiosc.2018.05.013

Language：Others  

Design of lipid-protein conjugate with a self-assembling ability on a cell membrane by using microbial transglutaminase reaction

Language：English   Publishing type：Research paper (scientific journal)  

As a therapeutic treatment, recombinant human basic fibroblast growth factor (rhbFGF) is usually employed in tissue regeneration, and as an essential component in culture medium for maintaining the induced pluripotent stem (iPS) cell and embryonic stem (ES) cell in an undifferentiated state. Therefore, a large amount of biologically active rhbFGF is required. In this study, silkworm-baculovirus expression vector system (silkworm-BEVS) is employed to achieve a high productivity of recombinant rhbFGF with two small affinity tags (His-tag and STREP-tag) at the N or C-terminus. It is observed that rhbFGF with 30 K signal peptide of silkworm were successfully expressed but are not sufficiently secreted into the culture medium of cultured insect cells. Then we purified the N- or C-tagged intracellular rhbFGF protein and obtained a yield of about 0.7 mg/larva and 1.2 mg/larva, respectively. Although the final yield of the C-tagged rhbFGF is higher than that of the N-tagged, rhbFGF with N-tag demonstrated promising and comparable biological activity, which is evaluated through a mammalian cell proliferation assay. Taken together, these results indicate that silkworm-BEVS could contribute to the mass-production of the biologically active rhbFGF for medical uses.

DOI： 10.1016/j.aspen.2018.05.002

Language：Others   Publishing type：Research paper (scientific journal)  

© 2018 As a therapeutic treatment, recombinant human basic fibroblast growth factor (rhbFGF) is usually employed in tissue regeneration, and as an essential component in culture medium for maintaining the induced pluripotent stem (iPS) cell and embryonic stem (ES) cell in an undifferentiated state. Therefore, a large amount of biologically active rhbFGF is required. In this study, silkworm-baculovirus expression vector system (silkworm-BEVS) is employed to achieve a high productivity of recombinant rhbFGF with two small affinity tags (His-tag and STREP-tag) at the N or C-terminus. It is observed that rhbFGF with 30 K signal peptide of silkworm were successfully expressed but are not sufficiently secreted into the culture medium of cultured insect cells. Then we purified the N- or C-tagged intracellular rhbFGF protein and obtained a yield of about 0.7 mg/larva and 1.2 mg/larva, respectively. Although the final yield of the C-tagged rhbFGF is higher than that of the N-tagged, rhbFGF with N-tag demonstrated promising and comparable biological activity, which is evaluated through a mammalian cell proliferation assay. Taken together, these results indicate that silkworm-BEVS could contribute to the mass-production of the biologically active rhbFGF for medical uses.

DOI： 10.1016/j.aspen.2018.05.002

Language：English   Publishing type：Research paper (scientific journal)  

Phycocyanin (PC), a water-soluble protein-chromophore complex composed of hexameric (αβ)6 subunits, has important biological functions in blue-green algae as well as pharmacological activities in biomedicine. We have previously developed a solid-in-oil (S/O) nanodispersion method to deliver biomacromolecules through the skin, although the transcutaneous mechanism has not yet been fully elucidated. To study the mechanism of transcutaneous protein delivery, we therefore enabled S/O nanodispersion by coating PC with hydrophobic surfactants and evaluated how the proteinaceous macromolecules formulated in an oil phase might permeate the skin. The extent of S/O nanodispersion of PC was dependent on the type of surfactant, suggesting that the selection of a suitable surfactant is crucial for encapsulating a large protein having a subunit structure. By measuring the intrinsic fluorescence of PC, we found that S/O nanodispersion facilitated the accumulation of PC in the stratum corneum (SC) of Yucatan micropig skin. Furthermore, after crossing the SC layer, the fluorescent recovery of PC was evident, indicating the release of the biologically active form of PC from the SC into the deeper skin layer.

DOI： 10.1016/j.ejpb.2018.01.020

Language：English   Publishing type：Research paper (scientific journal)  

Paclitaxel (PTX) injection (i.e., Taxol) has been used as an effective chemotherapeutic treatment for various cancers. However, the current Taxol formulation contains Cremophor EL, which causes hypersensitivity reactions during intravenous administration and precipitation by aqueous dilution. This communication reports the preliminary results on the ionic liquid (IL)-based PTX formulations developed to address the aforementioned issues. The formulations were composed of PTX/cholinium amino acid ILs/ethanol/Tween-80/water. A significant enhancement in the solubility of PTX was observed with considerable correlation with the density and viscosity of the ILs, and with the side chain of the amino acids used as anions in the ILs. Moreover, the formulations were stable for up to 3 months. The driving force for the stability of the formulation was hypothesized to be the involvement of different types of interactions between the IL and PTX. In vitro cytotoxicity and antitumor activity of the IL-based formulations were evaluated on HeLa cells. The IL vehicles without PTX were found to be less cytotoxic than Taxol, while both the IL-based PTX formulation and Taxol exhibited similar antitumor activity. Finally, in vitro hypersensitivity reactions were evaluated on THP-1 cells and found to be significantly lower with the IL-based formulation than Taxol. This study demonstrated that specially designed ILs could provide a potentially safer alternative to Cremophor EL as an effective PTX formulation for cancer treatment giving fewer hypersensitivity reactions.

DOI： 10.1021/acs.molpharmaceut.8b00305

Language：English   Publishing type：Research paper (scientific journal)  

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.

DOI： 10.1002/biot.201700624

Language：English   Publishing type：Research paper (scientific journal)  

Paclitaxel (PTX) injection (i.e., Taxol) has been used as an effective chemotherapeutic treatment for various cancers. However, the current Taxol formulation contains Cremophor EL, which causes hypersensitivity reactions during intravenous administration and precipitation by aqueous dilution. This communication reports the preliminary results on the ionic liquid (IL)-based PTX formulations developed to address the aforementioned issues. The formulations were composed of PTX/cholinium amino acid ILs/ethanol/Tween-80/water. A significant enhancement in the solubility of PTX was observed with considerable correlation with the density and viscosity of the ILs, and with the side chain of the amino acids used as anions in the ILs. Moreover, the formulations were stable for up to 3 months. The driving force for the stability of the formulation was hypothesized to be the involvement of different types of interactions between the IL and PTX. In vitro cytotoxicity and antitumor activity of the IL-based formulations were evaluated on HeLa cells. The IL vehicles without PTX were found to be less cytotoxic than Taxol, while both the IL-based PTX formulation and Taxol exhibited similar antitumor activity. Finally, in vitro hypersensitivity reactions were evaluated on THP-1 cells and found to be significantly lower with the IL-based formulation than Taxol. This study demonstrated that specially designed ILs could provide a potentially safer alternative to Cremophor EL as an effective PTX formulation for cancer treatment giving fewer hypersensitivity reactions.

DOI： 10.1021/acs.molpharmaceut.8b00305

Language：English   Publishing type：Research paper (scientific journal)  

Nanoparticles formed by the assembly of protein and polysaccharides are of great interest for the delivery of hydrophobic molecules. Herein, the formation of genipin-crosslinked nanoparticles from caseinate (CS) and chitosan (CH) is reported for the delivery of curcumin, a polyphenolic compound from turmeric, to cells. Genipin-crosslinked CS-CH nanoparticles (G-CCNPs) having a diameter of ∼250 nm and a low polydispersity index showed excellent stability over a wide pH range, as indicated by dynamic light scattering and transmission electron microscopic measurements. Cellular uptake of curcumin loaded into G-CCNPs by HeLa cells was improved, as measured by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell-sorting analysis. Cell proliferation assays indicated that G-CCNPs were nontoxic and that curcumin's anticancer activity in vitro was also improved by G-CCNPs. Stability of curcumin at neutral pH was enhanced by G-CCNPs. CLSM study revealed that G-CCNPs were poorly internalized by HeLa cells, possibly because of strong cell membrane interactions and a negative zeta potential. Overall, our results suggested that the enhanced curcumin cytotoxicity might be associated with the enhanced stability of curcumin by G-CCNPs and free curcumin released from G-CCNPs into the cell. These biocompatible NPs might be suitable carriers for enhancing curcumin's therapeutic potential.

DOI： 10.1016/j.colsurfb.2018.01.041

Language：English   Publishing type：Research paper (scientific journal)  

Cellulosomal systems are known as highly efficient biocatalysts in the degradation of lignocellulosic biomass in nature, but they remain unsuitable for industrial applications. In seeking alternatives to natural cellulosomes, casein was chosen as a scaffold for cellulase clustering. Casein is recognized as an excellent substrate for microbial transglutaminase (MTG) because it contains naturally reactive glutamine and lysine residues. A substrate peptide containing an MTG-reactive lysine residue was inserted into the C-terminus of the endoglucanase Cel5A and Cel6A from Thermobifida fusca using genetic engineering. The engineered cellulases, EG(Cel5A) and EG(Cel6A), were conjugated onto casein in different ratios by an MTG-mediated site-specific protein crosslinking reaction. Overall, a more than two-fold increase was observed when EG(Cel5A) was conjugated onto N,N-dimethylcasein, but a small or no change was observed for EG(Cel6A).

DOI： 10.1016/j.procbio.2017.12.013

Language：English   Publishing type：Research paper (scientific journal)  

Cancer vaccines represent a prophylactic or therapeutic method of suppressing cancer by activating the adaptive immune system. The immune response is initiated by the delivery of tumor antigens to antigen presenting cells (APCs). The use of peptides as vaccine antigens is advantageous, especially in the availability and productivity of pure and defined antigens. However, their limited immunogenicity remains a major drawback, and therefore, the utilization of nanocarriers as a means of delivering antigens to target cells and/or the addition of immune stimulants have been investigated as an efficient peptide-based cancer vaccine. We have developed a solid-in-oil (S/O) nanodispersion as a transcutaneous nanocarrier for hydrophilic molecules. This system has attractive features as a peptide nanocarrier for cancer vaccines, including transcutaneous targeting of professional APCs in the skin, high encapsulation efficacy of hydrophilic molecules, and capacity for coloading with a variety of immune stimulants such as adjuvants. We therefore sought to utilize the developed S/O nanodispersion for the delivery of the tyrosine-related protein 2 peptide, TRP-2180-188, as a peptide antigen against melanoma. Transcutaneous vaccination of the S/O nanodispersion coloaded with adjuvant R-848 was associated with a significant inhibition of melanoma growth and suppression of lung metastasis in tumor-bearing mice. Our findings indicate the potential of S/O nanodispersions as an endogenous peptide carrier for cancer vaccines.

DOI： 10.1021/acs.molpharmaceut.7b00894

Language：English   Publishing type：Research paper (scientific journal)  

Cellulosomal systems are known as highly efficient biocatalysts in the degradation of lignocellulosic biomass in nature, but they remain unsuitable for industrial applications. In seeking alternatives to natural cellulosomes, casein was chosen as a scaffold for cellulase clustering. Casein is recognized as an excellent substrate for microbial transglutaminase (MTG) because it contains naturally reactive glutamine and lysine residues. A substrate peptide containing an MTG-reactive lysine residue was inserted into the C-terminus of the endoglucanase Cel5A and Cel6A from Thermobifida fusca using genetic engineering. The engineered cellulases, EG(Cel5A) and EG(Cel6A), were conjugated onto casein in different ratios by an MTG-mediated site-specific protein crosslinking reaction. Overall, a more than two-fold increase was observed when EG(Cel5A) was conjugated onto N,N-dimethylcasein, but a small or no change was observed for EG(Cel6A).

DOI： 10.1016/j.procbio.2017.12.013

Language：English   Publishing type：Research paper (scientific journal)  

Cancer vaccines represent a prophylactic or therapeutic method of suppressing cancer by activating the adaptive immune system. The immune response is initiated by the delivery of tumor antigens to antigen presenting cells (APCs). The use of peptides as vaccine antigens is advantageous, especially in the availability and productivity of pure and defined antigens. However, their limited immunogenicity remains a major drawback, and therefore, the utilization of nanocarriers as a means of delivering antigens to target cells and/or the addition of immune stimulants have been investigated as an efficient peptide-based cancer vaccine. We have developed a solid-in-oil (S/O) nanodispersion as a transcutaneous nanocarrier for hydrophilic molecules. This system has attractive features as a peptide nanocarrier for cancer vaccines, including transcutaneous targeting of professional APCs in the skin, high encapsulation efficacy of hydrophilic molecules, and capacity for coloading with a variety of immune stimulants such as adjuvants. We therefore sought to utilize the developed S/O nanodispersion for the delivery of the tyrosine-related protein 2 peptide, TRP-2_180-188, as a peptide antigen against melanoma. Transcutaneous vaccination of the S/O nanodispersion coloaded with adjuvant R-848 was associated with a significant inhibition of melanoma growth and suppression of lung metastasis in tumor-bearing mice. Our findings indicate the potential of S/O nanodispersions as an endogenous peptide carrier for cancer vaccines.

DOI： 10.1021/acs.molpharmaceut.7b00894

Language：English   Publishing type：Research paper (scientific journal)  

Curcumin holds promise as a therapeutic agent due to its capability of conferring several pharmacological activities. While curcumin shows efficacy in preclinical studies as an anti-cancer agent, its translation into the clinic as a drug has yet to be realized. One possible reason is its poor solubility and stability in water, which decreases the bioavailability. Here, we report the formation of biocompatible nanocomplexes (NCs) from caseinate (CS) and chitosan (CH) using an electrostatic interaction-based approach to stabilize and enhance water solubility of curcumin. The formation of CS-CH NCs (CCNCs) was studied as a function of CH concentration. We show that positively charged NCs, having size between approx. 250 nm with a narrow distribution was formed by adjusting CH concentration. CCNCs successfully entrapped curcumin with a high entrapment efficiency. Curcumin was possibly located in a hydrophobic region of CS as indicated by a blue-shift in the emission maxima of curcumin. Ultimately, the stability and water solubility of curcumin in CCNCs could be remarkably enhanced. These results suggest that CCNCs would be useful for increasing the potential of curcumin as a preventive or therapeutic agent.

DOI： 10.1252/jcej.17we293

Language：Others  

DOI： 10.1002/9783527803293.ch6

Language：English   Publishing type：Research paper (scientific journal)  

Virus-like particles (VLPs) are multi-protein complex, which mimic viral particles without viral genomes. Recently various applications of VLPs for medical and pharmaceutical fields are developed. Furthermore, surface modification of VLPs is expected to extend their functional versatility. As an enzymatic strategy for the modification, microbial transglutaminase (MTG) that catalyzes the covalent bond between a glutamine residue and a ly-sine residue or a primary amine is suitable for efficient protein ligation. In the previous study, we demonstrated the efficient production of immunogenic VLPs of porcine circovirus type 2 (PCV2) using silkworm-baculovirus expression vector system (silkworm-BEVS). Herein, we established the display system of exotic molecules to VLPs by enzymatic covalent cross-linking using MTG. For the target modification, Q-tag (YPLQMRG) that is MTG reactive sequence were introduced into the N-terminus, C-terminus, or loop BC of capsid protein of PCV2 and successfully expressed as VLPs in silkworm pupae. Of these, PCV2 VLPs with Q-tag at the loop BC and C-terminus were efficiently conjugated with FITC-cadaverine and K-tagged (MRHKGS) EGFP by MTG. These results showed that flexible surface modification of VLPs mediated by MTG could be used for development of several therapeutic tools such as multivalent vaccines.

DOI： 10.11416/jibs.87.2_053

Language：English   Publishing type：Research paper (scientific journal)  

Conjugating enzymes into a large protein assembly often results in an enhancement of overall catalytic activity, especially when different types of enzymes that work cooperatively are assembled together. However, exploring the proper method to achieve protein assemblies with high stability and also to avoid loss of the function of each component for efficient enzyme clustering is remained challenging. Assembling proteins onto synthetic scaffolds through varied post-translational modification methods is particularly favored since the proteins can be site-specifically conjugated together with less activity loss. Here, a SpyCatcher polymer is prepared through catalytic reaction of horseradish peroxidase (HRP) and serves as a polymeric proteinaceous scaffold for construction of protein assemblies. Taking advantage of the favorable SpyCatcher-SpyTag interaction, SpyTagged proteins can be easily assembled onto the polymeric SpyCatcher scaffold with controllable binding ratio and site specificity. Firstly, the feasibility of construction of ratio-controllable binary artificial hemicellulosomes by assembling endoxylanase and arabinofuranosidase is explored. This construct achieves higher sugar conversion than that of the free enzymes when the proportion of arabinofuranosidase is high, because the close spatial proximity of the enzymes allows them to work in a synergistic manner. Another application for biosensing is developed by conjugating SpyTagged Nanoluc and protein G onto SpyCatcher polymer. Due to the protein clustering effect, an amplified luminescent intensity is achieved by the resulting conjugates than chimera protein of Nanoluc and protein G in ovalbumin detection in ELISA.

DOI： 10.1002/biot.201700195

Language：English   Publishing type：Research paper (scientific journal)  

DNA-protein conjugates are promising biomolecules for use in areas ranging from therapeutics to analysis because of the dual functionalities of DNA and protein. Conjugation requires site-specific and efficient covalent bond formation without impairing the activity of both biomolecules. Herein, we have focused on the use of a microbial transglutaminase (MTG) that catalyzes the cross-linking reaction between a glutamine residue and a primary amine. In a model bioconjugation, a highly MTG-reactive Gln (Q)-donor peptide (FYPLQMRG, FQ) was fused to enhanced green fluorescent protein (FQEGFP) and a primary amine clustered DNA aptamer was enzymatically synthesized as a novel aryl-acceptor substrate of MTG, whose combination leads to efficient and convenient preparation of DNA-protein conjugates with high purity. Dual functionality of the obtained DNA-EGFP conjugate was evaluated by discrimination of cancer cells via c-Met receptor recognition ability of the DNA aptamer. The DNA aptamer-EGFP conjugate only showed fluorescence toward cells with c-Met overexpression, indicating the retention of the biochemical properties of the DNA and EGFP in the conjugated form.

DOI： 10.1021/acs.bioconjchem.7b00594

Language：English   Publishing type：Research paper (scientific journal)  

Pollinosis, a typical seasonal allergy, is a serious public health problem. Limited numbers of patients receive curative immunotherapy instead of symptomatic therapy; however, there are still some concerns about the inconvenience and side effects of subcutaneous injections and sublingual administration caused by immunotherapy. Here, we propose a simple and safe transcutaneous immunotherapy using solid-in-oil (S/O) nanodispersions loaded with vaccine T cell epitope peptides derived from pollen allergen. S/O nanodispersions are oil-based dispersions of antigens coated with hydrophobic surfactants. They have a high potential to deliver biomolecules including peptides or proteins to immune cells in the skin, and to induce an immune response. The result of quantitative and qualitative analysis by in vitro permeation experiments demonstrated the effective permeation of T cell epitope peptides into the skin. Furthermore, in vivo experiments using a pollinosis mouse model indicated that the S/O nanodispersions loaded with T cell epitopes suppressed serum antibody IgE and cytokine production, and alleviated allergic symptoms to a similar therapeutic level to that observed for subcutaneous injection. These results indicate the potential of transcutaneous immunotherapy using S/O nanodispersions for the future treatment of pollinosis.

DOI： 10.1016/j.ijpharm.2017.07.020

Language：English   Publishing type：Research paper (scientific journal)  

Pollinosis, a typical seasonal allergy, is a serious public health problem. Limited numbers of patients receive curative immunotherapy instead of symptomatic therapy; however, there are still some concerns about the inconvenience and side effects of subcutaneous injections and sublingual administration caused by immunotherapy. Here, we propose a simple and safe transcutaneous immunotherapy using solid-in-oil (S/O) nanodispersions loaded with vaccine T cell epitope peptides derived from pollen allergen. S/O nanodispersions are oil-based dispersions of antigens coated with hydrophobic surfactants. They have a high potential to deliver biomolecules including peptides or proteins to immune cells in the skin, and to induce an immune response. The result of quantitative and qualitative analysis by in vitro permeation experiments demonstrated the effective permeation of T cell epitope peptides into the skin. Furthermore, in vivo experiments using a pollinosis mouse model indicated that the S/O nanodispersions loaded with T cell epitopes suppressed serum antibody IgE and cytokine production, and alleviated allergic symptoms to a similar therapeutic level to that observed for subcutaneous injection. These results indicate the potential of transcutaneous immunotherapy using S/O nanodispersions for the future treatment of pollinosis. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ijpharm.2017.07.020

Language：English  

The recovery and separation of platinum group metals (PGMs) are of considerable significance for metal sustainability. To establish an effcient extraction process for PGMs, the novel extractant N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl] phenylalanine (D2EHAF), containing a phenylalanine moiety as the metal-affinity group, was synthesized. The extraction of PGMs from aqueous HCl and HNO3 solutions with D2EHAF, or our previously developed N-[ N, N-di(2-ethylhexyl) aminocarbonylmethyl]glycine (D2EHAG), in n-dodecane was investigated. Both D2EHAF and D2EHAG exhibited high extraction affinities for Os4+, Pt4+, and Pd2+ in aqueous HCl. In a HNO3 solution, D2EHAF extracted Pd2+ more efficiently than D2EHAG, and the extraction of Pd2+ from a 1 mol dm(-3) HNO3 solution with D2EHAF proceeded quantitatively. The extracted metal ions were stripped from the organic solution using a highly acidic solution, or thiourea.

DOI： 10.1252/jcej.16we335

Language：English   Publishing type：Research paper (scientific journal)  

Among the many types of lignocellulosic biomass pretreatment methods, the use of ionic liquids (ILs) is regarded as one of the most promising strategies. In this study, the effects of four kinds of ILs for pretreatment of lignocellulosic biomass such as bagasse, eucalyptus, and cedar were evaluated. In direct ethanol fermentation from biomass incorporated with ILs by cellulase-displaying yeast, 1-butyl-3-methylimidazolium acetate ([Bmim][OAc]) was the most effective IL. The ethanol production and yield from [Bmim][OAc]-pretreated bagasse reached 0.81 g/L and 73.4% of the theoretical yield after fermentation for 96 h. The results prove the initial concept, in which the direct fermentation from lignocellulosic biomass effectively promoted by the pretreatment with IL.

DOI： 10.1007/s12010-016-2322-2

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00726-016-2322-0

Language：English   Publishing type：Research paper (scientific journal)  

Transcutaneous pollinosis immunotherapy using a solid-in-oil nanodispersion system carrying T cell epitope peptide and R848.
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.

DOI： 10.1002/btm2.10048

Language：English  

Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.

DOI： 10.1007/s00726-016-2322-0

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.biomac.6b01538

Language：English   Publishing type：Research paper (scientific journal)  

Protein polymer conjugates have been developed in many fields. Most hybrids are composed of one protein attached to one or several polymer chains. The other form of hybrid involves the construction of multiple proteins on one polymer chain, thereby facilitating protein assemblies that provide multivalent effects. Unfortunately, synthetic methods for production of these types of hybrids are limited and challenging because precise control of the conjugation sites is needed. Herein, a novel synthetic polymer that can enzymatically assemble multiple proteins was developed. Polyacrylamide grafted with multiple microbial transglutaminase (MTG)-recognizable peptide derivatives was synthesized, and MTG-catalyzed site-specific conjugation of proteins with the polymer was achieved. The application for immunological biosensing was demonstrated using the assembly of a fusion protein composed of antibody-binding and enzyme moieties. This enzymatic method to synthesize a one-dimensional protein assembly on a synthetic polymer is versatile and can be expanded to a wide range of applications.

DOI： 10.1021/acs.biomac.6b01538

Language：English   Publishing type：Research paper (scientific journal)  

The recovery and separation of platinum group metals (PGMs) are of considerable significance for metal sustainability. To establish an efficient extraction process for PGMs, the novel extractant N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl] phenylalanine (D2EHAF), containing a phenylalanine moiety as the metal-affinity group, was synthesized. The extraction of PGMs from aqueous HCl and HNO₃ solutions with D2EHAF, or our previously developed N-[N,N-di(2-ethylhexyl) aminocarbonylmethyl]glycine (D2EHAG), in n-dodecane was investigated. Both D2EHAF and D2EHAG exhibited high extraction affinities for Os⁴⁺, Pt⁴⁺, and Pd²⁺ in aqueous HCl. In a HNO₃ solution, D2EHAF extracted Pd²⁺ more efficiently than D2EHAG, and the extraction of Pd²⁺ from a 1 mol dm^-3 HNO₃ solution with D2EHAF proceeded quantitatively. The extracted metal ions were stripped from the organic solution using a highly acidic solution, or thiourea.

DOI： 10.1252/jcej.16we335

Language：English   Publishing type：Research paper (scientific journal)  

Among the many types of lignocellulosic biomass pretreatment methods, the use of ionic liquids (ILs) is regarded as one of the most promising strategies. In this study, the effects of four kinds of ILs for pretreatment of lignocellulosic biomass such as bagasse, eucalyptus, and cedar were evaluated. In direct ethanol fermentation from biomass incorporated with ILs by cellulase-displaying yeast, 1-butyl-3-methylimidazolium acetate ([Bmim][OAc]) was the most effective IL. The ethanol production and yield from [Bmim][OAc]-pretreated bagasse reached 0.81 g/L and 73.4% of the theoretical yield after fermentation for 96 h. The results prove the initial concept, in which the direct fermentation from lignocellulosic biomass effectively promoted by the pretreatment with IL.

DOI： 10.1007/s12010-016-2322-2

Language：English   Publishing type：Research paper (scientific journal)  

Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.

DOI： 10.1007/s00726-016-2322-0

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/biot.201600081

Language：English   Publishing type：Research paper (scientific journal)  

Transdermal administration of drugs has advantages over conventional oral administration or administration using injection equipment. The route of administration reduces the opportunity for drug evacuation before systemic circulation, and enables long-lasting drug administration at a modest body concentration. In addition, the skin is an attractive route for vaccination, because there are many immune cells in the skin. Recently, solid-in-oil nanodisperison (S/O) technique has demonstrated to deliver cosmetic and pharmaceutical bioactives efficiently through the skin. S/O nanodispersions are nanosized drug carriers designed to overcome the skin barrier. This review discusses the rationale for preparation of efficient and stable S/O nanodispersions, as well as application examples in cosmetic and pharmaceutical materials including vaccines. Drug administration using a patch is user-friendly, and may improve patient compliance. The technique is a potent transcutaneous immunization method without needles.

DOI： 10.1002/biot.201600081

Language：English   Publishing type：Research paper (scientific journal)  

Cellular spheroids have been received much attention in the biological and biomedical fields, especially as a base material for drug assays, regenerative medicine, and tissue engineering. Hydrogels have potential for scalable preparation of spheroids because they provide a spatial environment suitable for three-dimensional cell cultivation. Herein, the potential use of a redox-responsive hydrogel as a scaffold for preparation and recovery of spheroids is reported. A hydrogel composed of poly(ethylene glycol) (PEG), which can be degraded using cysteine as a reducing agent under mild conditions, is prepared by mixing an octa-thiolated PEG derivative (8-arm PEG-SH), horseradish peroxidase and a small phenolic compound (Glycyl-L-tyrosine). Human hepatocellular carcinoma cells (HepG2) are encapsulated in the hydrogel and cellular spheroids formed by proliferation within the scaffolds. After seven days of cultivation, the size of the HepG2 spheroids reached a diameter between approximate to 40 and 60 mu m, depending on the 8-arm PEG-SH concentration. Liver-specific functions of the HepG2 spheroids such as albumin secretion and urea production are retained at higher levels than those of cells prepared from traditional two-dimensional monolayers. These results suggest that the system presented here has potential for preparation of cellular spheroids for tissue engineering applications.

DOI： 10.1002/biot.201600087

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/biot.201600650

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bej.2016.07.013

Language：English   Publishing type：Research paper (scientific journal)  

Effective degradation of hemicellulose is of utmost importance in a wide variety of applications in bioindustry. Five endoxylanases from different glycoside hydrolase families and microorganisms were tested with an arabinofuranosidase, Araf51A, for the hydrolysis of insoluble wheat arabinoxylan, which is a structural component of hemicellulose. The optimized combination was XynZ/Xyn11A/Araf51A with a loading ratio of 2:2:1, and the value of degree of synergy increased with the increase of Araf51A proportion in the enzyme mixture. Afterwards, selected enzymes were immobilized on commercial magnetic nanoparticles through covalent bonding. Both free and immobilized enzymes showed a similar conversion to reducing sugars after hydrolysis for 48 h. After 10 cycles, approximately 20% of the initial enzymatic activity of both the individual or mixture of immobilized enzymes was retained. A 5.5-fold increase in the production of sugars was obtained with a mixture of enzymes immobilized after 10 cycles in total compared with free enzymes. Importantly, a sustainable synergism between immobilized arabinofuranosidase and immobilized endoxylanases in the hydrolysis of arabinoxylan was demonstrated. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2016.07.013

Language：English   Publishing type：Research paper (scientific journal)  

A novel artificial cellulase was developed by conjugating a DNA aptamer to an endoglucanase catalytic domain, thereby substituting the natural carbohydrate-binding module. Circular dichroism spectroscopy and adsorption isotherm showed the binding motif of cellulose-binding DNA aptamer (CelApt) was G-quadruplex and stem-loop structures stabilized in the presence of salts, and CelApt binding preferred the amorphous region of the solid cellulose. By introducing the revealed salt-switchable cellulose binding nature of CelApt into a catalytic domain of a cellulase, we created CelApt-catalytic domain conjugate possessing both controllable adsorption on the solid substrates and equal enzymatic activity to the wild-type cellulase. Thus potential use of a responsive DNA aptamer for biocatalysis at a solid surface was demonstrated.

DOI： 10.1021/acs.biomac.6b01141

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bej.2016.06.005

Language：Others  

Diglycolic amic acid-modified E. coli as a biosorbent for the recovery of rare earth elements
© 2016 Elsevier B.V. Biosorption has recently attracted much attention as an alternative to conventional techniques for the recovery of rare earth elements (REEs). In this study, Escherichia coli (E. coli) was chemically modified to improve its performance as a biosorbent for REEs. The diglycolic amic acid group, which shows high affinity to REEs, was introduced by succinylation of the amine groups on the E. coli. Adsorption curves using the modified E. coli were characteristic of the diglycolic amic acid group. The adsorption performance for transition metal ions was not affected by the modification. These results suggest that modification of E. coli with a functional group with high affinity to REEs increases the effectiveness of adsorption. The maximum uptakes of REEs on the modified E. coli were doubled. Modification of E. coli is an effective method for enhancing the adsorption performance for REEs.

DOI： 10.1016/j.bej.2016.06.005

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/C6RA16926K

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.biortech.2016.04.121

Language：Others  

Powerful peracetic acid-ionic liquid pretreatment process for the efficient chemical hydrolysis of lignocellulosic biomass
© 2016 Elsevier Ltd. The aim of this work was to design a new method for the efficient saccharification of lignocellulosic biomass (LB) using a combination of peracetic acid (PAA) pretreatment with ionic liquid (IL)-HCl hydrolysis. The pretreatment of LBs with PAA disrupted the lignin fractions, enhanced the dissolution of LB and led to a significant increase in the initial rate of the IL-HCl hydrolysis. The pretreatment of Bagasse with PAA prior to its 1-buthyl-3-methylimidazolium chloride ([Bmim][Cl])-HCl hydrolysis, led to an improvement in the cellulose conversion from 20% to 70% in 1.5 h. Interestingly, the 1-buthyl-3-methylpyridium chloride ([Bmpy][Cl])-HCl hydrolysis of Bagasse gave a cellulose conversion greater than 80%, with or without the PAA pretreatment. For LB derived from seaweed waste, the cellulose conversion reached 98% in 1 h. The strong hydrolysis power of [Bmpy][Cl] was attributed to its ability to transform cellulose I to II, and lowering the degree of polymerization of cellulose.

DOI： 10.1016/j.biortech.2016.04.121

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/C6OB01227B

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.aspen.2016.03.014

Language：Others  

High-level expression and purification of biologically active human IL-2 using silkworm-baculovirus expression vector system

DOI： 10.1016/j.aspen.2016.03.014

Language：Others  

Enzymatic conjugation of multiple proteins on a DNA aptamer in a tail-specific manner
Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Conjugation of single-strand DNA aptamers and enzymes has been of great significance in bioanalytical and biomedical applications because of the unlimited functions provided by DNA aptamer direction. Therefore, we developed efficient tailing of a DNA aptamer, with end-specific conjugation of multiple enzymes, through enzymatic catalysis. Terminal deoxynucleotidyl transferase (TdT) added multiple Z-Gln-Gly (Z-QG) moieties to the 3′-end of a DNA aptamer via the addition of Z-QG-modified deoxyuridine triphosphate (Z-QG-dUTP) and deoxynucleoside triphosphates (dNTPs). The resultant (Z-QG)m-(dN)l-aptamer, whose Z-QGs with dN spacers served as stickers for microbial transglutaminase (MTG), were crosslinked between the Z-QGs on the DNA and a substrate peptide sequence containing lysine (K), fused to a recombinant enzyme (i.e. bacterial alkaline phosphatase; BAP) by MTG. The incorporation efficiency of Z-QG moieties on the aptamer tail and the subsequent conjugation efficiency with multiple enzyme molecules were dramatically altered by the presence of dNTPs, revealing that a combination of Z-QG-dUTP/dTTP comprised the best labeling efficiency and corresponding properties during analytical performance. Thus, a novel optimized platform for designing (BAP)n-(dT)l-DNA aptamers was demonstrated for the first time in this article, offering unique opportunities for tailoring new types of covalent protein-nucleic acid conjugates in a controllable way.

DOI： 10.1002/biot.201500560

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/biot.201500560

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Herein, we introduce an amino acid ester (AAE) as a potent biocompatible counter ion to formulate ionic liquefied active pharmaceutical ingredients. By equimolar pairing of a non-steroidal anti-inflammatory drug (NSAID) as the anion and the AAE as the cation, the liquid formulation was feasibly obtained. In vitro drug permeation results using pig skin revealed a significant enhancement of the transdermal delivery of the ionic liquefied NSAIDs. The AAE showed much lower cytotoxicity to mouse fibroblast L929 cells when compared with that of conventional counter cations, indicating the significant potential of the AAE in the development of new ionic liquid drug formulations.

DOI： 10.1039/c6ra16926k

Language：English  

Microbial transglutaminase (MTG), a robust enzyme developed initially for the manipulation of edible proteins in the food industry, has now been widely recognized as a practical protein-modifying reagent in the range of biotechnological applications. In this chapter, we introduce the potential use of MTG through our basic studies on the design of novel glutamine (Gln) donor substrates for lysine (Lys)-specific protein modification. Based on the core structure of a conventional transglutaminase substrate, benzyloxycarbonyl-L-glutaminylglycine (Z-QG), new Gln-donor substrates have been developed for the conjugation of recombinant proteins with different functionalities. The first target site for the substrate engineering was the C-terminal carboxylic group of Z-QG, which is feasibly labeled with functional moieties. For the preparation of protein-nucleic acid conjugates with novel molecular architecture, a new nucleotidyl substrate, Z-QG-(d)UTP, was created. We have also explored substitution of the N-terminal protecting group (Z) with fluorophores and biotin, and found that MTG accepts diverse functional groups at the N-terminus by inserting a short linker, leading to an increase in the utility of MTG in site-specific modification of functional proteins. Our results demonstrated how the design of (small) Gln-donor substrates of MTG can expand the scope of enzymatic manipulation in biomolecular engineering.

DOI： 10.1007/978-4-431-55825-5_17

Language：Others  

Self-assembly of Ni-NTA-modified β-annulus peptides into artificial viral capsids and encapsulation of His-tagged proteins
© The Royal Society of Chemistry 2016. β-Annulus peptides bearing Cys at the N-terminal from tomato bushy stunt virus were synthesised using a standard Fmoc-protected solid-phase method, and the peptide was modified with Ni-NTA at the N-terminal. The Ni-NTA-modified β-annulus peptide self-assembled into virus-like nanocapsules of approximately 40 nm in diameter. The critical aggregation concentration of these nanocapsules in 10 mM Tris-HCl buffer (pH 7.3) at 25 °C was 0.053 μM, which is 470 times lower than that of unmodified β-annulus peptides. Moreover, size exclusion chromatography of the peptide assembly indicated encapsulation of His-tagged green fluorescent protein in the Ni-NTA-modified artificial viral capsid.

DOI： 10.1039/c6ob01227b

Language：Others  

Selective extraction of scandium from transition metals by synergistic extraction with 2-thenoyltrifluoroacetone and tri-n-octylphosphine oxide
There has been an increasing demand for scandium (Sc). However the supply is insufficient because the separation and recovery of Sc as a byproduct from ores is difficult. In this study, extraction and separation of Sc3+from a sulfuric acid solution containing transition metals such as Al3+, Fe3+, Mn2+, Co2+, Ni2+and Zn2+was investigated using a β-diketone, HTTA and a neutral extractant, TOPO. Due to the synergistic effect with the two extractants, Sc3+was effectively and selectively separated even from Fe3+, the separation of which is difficult with HTTA alone. The extraction mechanism was examined and it was revealed that Sc3+is extracted with three HTTA and one TOPO molecules. Scandium was readily stripped from the extracting phase by 1 M sulfuric acid. The potential use of the combination of the β-diketone with TOPO for the recovery of Sc3+in a refining process was demonstrated.

DOI： 10.15261/serdj.23.137

Language：Others  

Mutual separation of indium, gallium, and zinc with the amic acid-type extractant D2EHAG containing glycine and amide moieties
Solvent extraction of indium (In3+), gallium (Ga3+), and zinc (Zn2+) was investigated using N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]glycine (D2EHAG) and N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]-sarcosine (D2EHAS) which we have developed recently. Indium and gallium were selectively extracted from zinc under high acid conditions (0 > pH ≥ 2.0), and easily stripped using an acidic solution such as 2 mol dm-3 HNO3. The extraction behavior was compared to that of N,N-dioctyldiglycol amic acid (DODGAA), which has a similar molecular structure to D2EHAG, a commercial alkyl monocarboxylic acid extractant, neodecanoic acid (Versatic 10), or an organophosphorus extractant, di(2-ethylhexyl) phosphoric acid (D2EHPA). Based on the results, D2EHAG and D2EHAS were found to be useful for the separation of In3+ and Ga3+ from Zn2+. The extraction mechanisms of these ions with D2EHAG were investigated through slope analysis and loading tests.

DOI： 10.15261/serdj.23.9

Language：Others  

Biocatalytic formation of gold nanoparticles decorated with functional proteins inside recombinant Escherichia coli cells
© The Japan Society for Analytical Chemistry. A novel strategy for the preparation of protein-decorated gold nanoparticles (Au NPs) was developed inside Escherichia coli cells, where an artificial oxidoreductase, composed of antibody-binding protein (pG), Bacillus stearothermophilus glycerol dehydrogenase (BsGLD) and a peptide tag with gold-binding affinity (H6C), was overexpressed in the cytoplasm. In situ formation of Au NPs was promoted by a natural electron-donating cofactor, nicotinamide adenine dinucleotide (NAD), which was regenerated to the reduced form of NADH by the catalytic activity of the fusion protein (pG-BsGLDH6C) overexpressed in the cytoplasm of E. coli, with the concomitant addition of exogenous glycerol to the reaction system. The fusion protein was self-immobilized on Au NPs inside the E. coli cells, which was confirmed by SDS-PAGE and western blotting analyses of the resultant Au NPs. Finally, the IgG binding ability of the pG moiety displayed on Au NPs was evaluated by an enzyme-linked immunosorbent assay. 2016

DOI： 10.2116/analsci.32.295

Language：Others  

BODIPY-labeled fluorescent aptamer sensors for turn-on sensing of interferon-gamma and adenine compounds on cells
© The Japan Society for Analytical Chemistry. An on-cell aptamer sensor has the potential to reveal cell-cell communications by signalling molecules. We attempted to design new fluorescent aptamer sensors for the sensing of IFN-? and adenine compounds on cells. BODIPY-labeled external quencher-free aptamer sensors have allowed a turn-on detection of the target molecule with improved off/on efficiency.

DOI： 10.2116/analsci.32.543

Language：English  

Microbial transglutaminase (MTG), a robust enzyme developed initially for the manipulation of edible proteins in the food industry, has now been widely recognized as a practical protein-modifying reagent in the range of biotechnological applications. In this chapter, we introduce the potential use of MTG through our basic studies on the design of novel glutamine (Gln) donor substrates for lysine (Lys)-specific protein modification. Based on the core structure of a conventional transglutaminase substrate, benzyloxycarbonyl-L-glutaminylglycine (Z-QG), new Gln-donor substrates have been developed for the conjugation of recombinant proteins with different functionalities. The first target site for the substrate engineering was the C-terminal carboxylic group of Z-QG, which is feasibly labeled with functional moieties. For the preparation of protein-nucleic acid conjugates with novel molecular architecture, a new nucleotidyl substrate, Z-QG-(d)UTP, was created. We have also explored substitution of the N-terminal protecting group (Z) with fluorophores and biotin, and found that MTG accepts diverse functional groups at the N-terminus by inserting a short linker, leading to an increase in the utility of MTG in site-specific modification of functional proteins. Our results demonstrated how the design of (small) Gln-donor substrates of MTG can expand the scope of enzymatic manipulation in biomolecular engineering.

DOI： 10.1007/978-4-431-55825-5_17

Language：English   Publishing type：Research paper (scientific journal)  

β-Annulus peptides bearing Cys at the N-terminal from tomato bushy stunt virus were synthesised using a standard Fmoc-protected solid-phase method, and the peptide was modified with Ni-NTA at the N-terminal. The Ni-NTA-modified β-annulus peptide self-assembled into virus-like nanocapsules of approximately 40 nm in diameter. The critical aggregation concentration of these nanocapsules in 10 mM Tris-HCl buffer (pH 7.3) at 25 °C was 0.053 μM, which is 470 times lower than that of unmodified β-annulus peptides. Moreover, size exclusion chromatography of the peptide assembly indicated encapsulation of His-tagged green fluorescent protein in the Ni-NTA-modified artificial viral capsid.

DOI： 10.1039/c6ob01227b

Language：English   Publishing type：Research paper (scientific journal)  

An on-cell aptamer sensor has the potential to reveal cell-cell communications by signalling molecules. We attempted to design new fluorescent aptamer sensors for the sensing of IFN-? and adenine compounds on cells. BODIPY-labeled external quencher-free aptamer sensors have allowed a turn-on detection of the target molecule with improved off/on efficiency.

DOI： 10.2116/analsci.32.543

Language：English   Publishing type：Research paper (scientific journal)  

There has been an increasing demand for scandium (Sc). However the supply is insufficient because the separation and recovery of Sc as a byproduct from ores is difficult. In this study, extraction and separation of Sc³⁺ from a sulfuric acid solution containing transition metals such as Al³⁺, Fe³⁺, Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ was investigated using a β-diketone, HTTA and a neutral extractant, TOPO. Due to the synergistic effect with the two extractants, Sc³⁺ was effectively and selectively separated even from Fe³⁺, the separation of which is difficult with HTTA alone. The extraction mechanism was examined and it was revealed that Sc³⁺ is extracted with three HTTA and one TOPO molecules. Scandium was readily stripped from the extracting phase by 1 M sulfuric acid. The potential use of the combination of the β-diketone with TOPO for the recovery of Sc³⁺ in a refining process was demonstrated.

DOI： 10.15261/serdj.23.137

Language：English   Publishing type：Research paper (scientific journal)  

In biological systems, proteins can form well-organized, higher-order structures with unique functions that would be difficult to achieve with a single protein. These proteinaceous supramolecular structures form by self-assembly, and the spatial arrangement of the protein building blocks in them is very important. In the present study, an artificial system was developed using recombinant proteins as building blocks, which were assembled in a one-dimensional manner. The assembly of these building blocks was based on the avidin-biotin interaction. A tetrameric biotin ligand unit was designed so that the 1:4 stoichiometry of the avidin-biotin interaction was altered to a 1:2 directional interaction between the streptavidin and tetrabiotinylated protein units. In a proof-of-concept study, site-specifically tetrabiotin-labeled endoglucanase and cellulose-binding module units were prepared, and then these components were self-assembled by mixing with streptavidin to mimic a natural cellulosome. The formation of one-dimensional assemblies of the protein units depended on the stoichiometry of the avidin-biotin interaction sites in the system. Interestingly, the saccharification efficiency improved when the component ratio of protein units in the assemblies was changed. The presence of the optimal ratio of the building blocks implies the modularity of the present protein assembly system.

DOI： 10.1039/c5me00011d

Language：English   Publishing type：Research paper (scientific journal)  

Solvent extraction of indium (In³⁺), gallium (Ga³⁺), and zinc (Zn²⁺) was investigated using N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]glycine (D2EHAG) and N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]-sarcosine (D2EHAS) which we have developed recently. Indium and gallium were selectively extracted from zinc under high acid conditions (0 > pH ≥ 2.0), and easily stripped using an acidic solution such as 2 mol dm^-3 HNO₃. The extraction behavior was compared to that of N,N-dioctyldiglycol amic acid (DODGAA), which has a similar molecular structure to D2EHAG, a commercial alkyl monocarboxylic acid extractant, neodecanoic acid (Versatic 10), or an organophosphorus extractant, di(2-ethylhexyl) phosphoric acid (D2EHPA). Based on the results, D2EHAG and D2EHAS were found to be useful for the separation of In³⁺ and Ga³⁺ from Zn²⁺. The extraction mechanisms of these ions with D2EHAG were investigated through slope analysis and loading tests.

DOI： 10.15261/serdj.23.9

Language：English   Publishing type：Research paper (scientific journal)  

A novel strategy for the preparation of protein-decorated gold nanoparticles (Au NPs) was developed inside Escherichia coli cells, where an artificial oxidoreductase, composed of antibody-binding protein (pG), Bacillus stearothermophilus glycerol dehydrogenase (BsGLD) and a peptide tag with gold-binding affinity (H ₆ C), was overexpressed in the cytoplasm. In situ formation of Au NPs was promoted by a natural electron-donating cofactor, nicotinamide adenine dinucleotide (NAD), which was regenerated to the reduced form of NADH by the catalytic activity of the fusion protein (pG-BsGLDH ₆ C) overexpressed in the cytoplasm of E. coli, with the concomitant addition of exogenous glycerol to the reaction system. The fusion protein was self-immobilized on Au NPs inside the E. coli cells, which was confirmed by SDS-PAGE and western blotting analyses of the resultant Au NPs. Finally, the IgG binding ability of the pG moiety displayed on Au NPs was evaluated by an enzyme-linked immunosorbent assay. 2016

DOI： 10.2116/analsci.32.295

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1208/s12249-015-0333-x

Language：English  

Transcutaneous Peptide Immunotherapy of Japanese Cedar Pollinosis Using Solid-in-Oil Nanodispersion Technology
© 2015, American Association of Pharmaceutical Scientists. Peptide immunotherapy is an attractive approach to relieve allergic symptoms such as rhinitis and asthma. Treatment of Japanse cedar pollinosis (Cryptomeria japonica; Cj), from which over one quarter of Japanese population suffer, is becoming a great concern. Recently, oral feeding of a peptide (7crp) consisting of seven immunodominant human T cell epitopes derived from two enzymes present in Cj pollen was demonstrated to have a benefit in treating Cj pollinosis. In this work, we aimed to apply a novel transcutaneous administration system as a simple and easy peptide delivery for an immunotherapy using a T cell epitope peptide. A modified 7crp peptide (7crpR) which contained triarginine linkers between each epitopes was designed to increase water solubility and was encapsulated in a unique solid-in-oil (S/O) nanodispersion. The S/O nanodispersion consists of a nano-sized peptide-surfactant complex dispersed in an oil vehicle. The S/O nanopartilces having an average diameter of 230 nm facilitated the permeation of the peptide 7crpR into the skin and suppressed serum total IgE and antigen-specific IgE levels in a Cj pollinosis mouse model. Transcutaneous administration of the T cell epitope peptide using the S/O nanodispersion system has potential for future simple and easy immunotherapy of Cj pollinosis.

DOI： 10.1208/s12249-015-0333-x

Language：English   Publishing type：Research paper (scientific journal)  

Interferon gamma (IFN-gamma) is a cytokine that is mainly secreted from immune cells. It plays an important role in regulating neighboring cellular activities through interactions with cell membrane proteins. Because of its biological importance, there has been increasing demand for a biosensor that can respond to IFN-gamma. Here, we report a fluorescent aptamer sensor that targets IFN-gamma. The 25-nt IFN-gamma aptamer was converted into a turn-on fluorescent sensor by labeling the 3'-end with fluorescein. The designed sensor achieved fluorescence sensing of IFN-gamma with nanomolar affinity.

DOI： 10.1246/cl.150794

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.biombioe.2015.05.023

Language：Others  

Heme precursor injection is effective for Arthromyces ramosus peroxidase fusion protein production by a silkworm expression system

DOI： 10.1016/j.jbiosc.2015.02.013

Language：Others  

Great potency of seaweed waste biomass from the carrageenan industry for bioethanol production by peracetic acid-ionic liquid pretreatment
© 2015 Published by Elsevier Ltd. Seaweed waste biomass from the carrageenan industry (SWBC) is a potential biomass feedstock for producing sustainable biofuel because it increases the product value and reduces the pollutant risk. Peracetic acid (PAA) followed by ionic liquid (IL) pretreatment has been used to increase the enzymatic saccharification of pretreated SWBC. The SWBC cellulose content was comparable with that of terrestrial biomasses. PAA+1-hexylpyridinium chloride ([Hpy][Cl]), and PAA+1-ethyl-3-methylimidazolium diethylphosphate ([Emim][DEP]) pretreatments produced saccharide and unknown oligosaccharide fractions in regenerated water (~4-6% SWBC cellulose content). For 48h of saccharification, the untreated SWBC and the SWBC pretreated using PAA followed by [Hpy][Cl], [Emim][DEP] or 1-ethyl-3-methylimidazole acetate ([Emim][OAc]) produced cellulose conversions of 77, 91, 84 and 62%, respectively. The untreated SWBC had a high cellulose conversion, which may be caused by the low lignin and hemicellulose contents of the SWBC. PAA+IL pretreatment could yield pretreated SWBCs with more amorphous cellulose structures, which lead to an almost-complete cellulose conversion.

DOI： 10.1016/j.biombioe.2015.05.023

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：Others  

Transcutaneous immunization against cancer using solid-in-oil nanodispersions
© The Royal Society of Chemistry. Transcutaneous immunization is a novel, non-invasive alternative to conventional immunization by injection. Skin immunocompetence composed of abundant antigen-presenting cells in the epidermal and dermal layers of the skin can provide an effective tool for transcutaneous immunization, whereas the outermost, hydrophobic layer of skin, the stratum corneum, hinders the penetration of antigens into the skin. To realize effective transcutaneous delivery of antigens, we have developed a solid-in-oil (S/O) technique that produces an oil dispersion of hydrophilic biomolecules. In this study, we applied the S/O nanodispersions in transcutaneous immunization to induce cancer immunity. The topical application of S/O nanodispersions bearing ovalbumin (OVA) as a model cancer-antigen allowed the penetration of OVA into the deep dermis region of the skin by intercellular and transfollicular pathways. Inhibition of OVA-bearing tumor growth and production of cytokines responsible for cellular immunity were achieved, demonstrating the applicability of S/O nanodispersions to the induction of cancer immunity.

DOI： 10.1039/c5md00168d

Language：English   Publishing type：Research paper (scientific journal)  

Transcutaneous immunization is a novel, non-invasive alternative to conventional immunization by injection. Skin immunocompetence composed of abundant antigen-presenting cells in the epidermal and dermal layers of the skin can provide an effective tool for transcutaneous immunization, whereas the outermost, hydrophobic layer of skin, the stratum corneum, hinders the penetration of antigens into the skin. To realize effective transcutaneous delivery of antigens, we have developed a solid-in-oil (S/O) technique that produces an oil dispersion of hydrophilic biomolecules. In this study, we applied the S/O nanodispersions in transcutaneous immunization to induce cancer immunity. The topical application of S/O nanodispersions bearing ovalbumin (OVA) as a model cancer-antigen allowed the penetration of OVA into the deep dermis region of the skin by intercellular and transfollicular pathways. Inhibition of OVA-bearing tumor growth and production of cytokines responsible for cellular immunity were achieved, demonstrating the applicability of S/O nanodispersions to the induction of cancer immunity.

DOI： 10.1039/c5md00168d

Language：English   Publishing type：Research paper (scientific journal)  

Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse

DOI： 10.1016/j.biortech.2015.02.041

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.enzmictec.2015.01.007

Language：Others  

Synergistic effect and application of xylanases as accessory enzymes to enhance the hydrolysis of pretreated bagasse.

DOI： 10.1016/j.enzmictec.2015.01.007

Language：English   Publishing type：Research paper (scientific journal)  

Purpose: Simple and noninvasive vaccine administration alternatives to injections are desired. A solid-in-oil (S/O) nanodispersion system was able to overcome skin barriers and induce an immune response; however, antibody levels remained low. We applied an immune potentiator, CpG oligodeoxynucleotide (ODN), to enhance the immune response by controlling the T helper 1 (Th1)/T helper 2 (Th2) balance. Methods: S/O nanodispersions containing ovalbumin (OVA) and CpG ODN (CpG-A or CpG-B) were characterized by size distribution analysis and a protein release test. The skin permeation of fluorescence-labeled OVA was observed by fluorescence microscopy. Antigen-specific IgG, IgG1, and IgG2a responses were measured by enzyme-linked immunosorbent assay. Results: Co-encapsulation of CpG ODNs in S/O nanodispersions enhanced induction of OVA-specific IgG. S/O nanodispersion containing OVA and CpG-A had a smaller mean particle size and permeated the skin more efficiently. In contrast, CpG-B showed the highest protein release and induction of OVA-specific IgG. IgG subclass analysis revealed that OVA induced a Th2-dominant immune response, while the S/O nanodispersion containing CpG-A skewed the immune response toward a Th1-bias. Conclusions: In combination with CpG ODN, the S/O nanodispersion system efficiently induced an antigen-specific antibody response. The Th1/Th2 immune balance could be controlled by the selection of CpG ODN type.

DOI： 10.1007/s11095-014-1554-5

Language：English   Publishing type：Research paper (scientific journal)  

To detect a target protein in biological samples, a fusion protein was designed composed of a peroxidase from Arthromyces ramosus (ARP) and parts of the antibody-binding domains of Staphylococcus aureus protein A and Streptococcus protein G (PG). The ARP-PG fusion protein was successfully expressed by a heterologous protein expression system in Brevibacillus choshinensis. The fusion protein was secreted as an active form in culture media. The production of ARP-PG with higher peroxidase activity was observed by the addition of 5-aminolevulinic acid to the culture media. The performance of purified ARP-PG was validated by dot blotting for the detection of transferrin as a model target protein. A comparable performance in the dot blot analysis was attained using a culture supernatant containing crude but active ARP-PG, indicating the practicality of the Brevibacillus protein secretion system.

DOI： 10.1252/kakoronbunshu.41.157

Language：English   Publishing type：Research paper (scientific journal)  

A novel polymeric ionic liquid (PIL) gel was synthesized via radical polymerization of the ionic liquid monomers, 1-vinyl- 3-ethylimidazoliumbis(trifluorometyl-sulfonyl) imide ([veim][Tf2N]). The gel was made porous by removing the water phase dispersed by polyoxyethylene (20) sorbitan monolaurate (Tween-20). The porous structure of the synthesized gel was observed by SEM imaging. In comparison to the nonporous PIL gel synthesized without Tween-20, the adsorption capacity of Au(III) onto porous PIL gel was enhanced. The adsorption behavior of the synthesized gel was studied in acidic chloride solutions containing various metal ions, and it was found that the gel selectively adsorbed Au(III) over other metal ions. Maximum adsorption capacity of Au(III) was more than 140 mg/g; whereas, those for Pt(IV) and Pd(II) were lower than 20 mg/g. The adsorbed Au(III) onto the gel can easily be desorbed using an acidified thiourea aqueous solution. The feasibility of utilizing the polymeric ionic liquid gel for the separation of Au(III) was thereby demonstrated.

DOI： 10.1252/jcej.14we162

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English  

In the most commonly used in situ hybridization (ISH) procedure, a hapten-labeled antisense nucleic acid (e.g., RNA) probe is employed to hybridize a target mRNA in tissue sections. The hapten-labeled RNA is then detected by a highly specific hapten-antibody interaction
however, it requires laborious immunostaining steps for spatial coloring on tissue sections. To simplify ISH-based mRNA detection systems, we created a new RNA-(enzyme) n conjugate for sensitive detection of mRNA in tissue sections. In the present simple, antibody-free ISH protocol, an antisense RNA probe of interest is first modified with a specific dipeptide substrate of microbial transglutaminase (MTG) by in vitro transcription. Alkaline phosphatase from a hyperthermophile is then covalently linked to the substrate-labeled RNA by MTG-catalyzed sitespecific conjugation. A robust, multi-enzyme-labeled RNA probe enables the direct labeling of a target mRNA in tissue sections with signaling enzymes under harsh hybridization conditions, leading to one-step signal amplification after hybridization. The application of the new trans glutaminase-mediated ISH (TransISH) strategy to mRNA detection in mammalian tissues was demonstrated.

DOI： 10.1007/978-1-4939-2303-8_29

Language：English  

In the most commonly used in situ hybridization (ISH) procedure, a hapten-labeled antisense nucleic acid (e.g., RNA) probe is employed to hybridize a target mRNA in tissue sections. The hapten-labeled RNA is then detected by a highly specific hapten-antibody interaction; however, it requires laborious immunostaining steps for spatial coloring on tissue sections. To simplify ISH-based mRNA detection systems, we created a new RNA-(enzyme) _n conjugate for sensitive detection of mRNA in tissue sections. In the present simple, antibody-free ISH protocol, an antisense RNA probe of interest is first modified with a specific dipeptide substrate of microbial transglutaminase (MTG) by in vitro transcription. Alkaline phosphatase from a hyperthermophile is then covalently linked to the substrate-labeled RNA by MTG-catalyzed sitespecific conjugation. A robust, multi-enzyme-labeled RNA probe enables the direct labeling of a target mRNA in tissue sections with signaling enzymes under harsh hybridization conditions, leading to one-step signal amplification after hybridization. The application of the new trans glutaminase-mediated ISH (TransISH) strategy to mRNA detection in mammalian tissues was demonstrated.

DOI： 10.1007/978-1-4939-2303-8_29

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：Others  

Transdermal immunization using solid-in-oil nanodispersion with CpG oligodeoxynucleotide adjuvants
© 2014 Springer Science+Business Media New York. Purpose: Simple and noninvasive vaccine administration alternatives to injections are desired. A solid-in-oil (S/O) nanodispersion system was able to overcome skin barriers and induce an immune response; however, antibody levels remained low. We applied an immune potentiator, CpG oligodeoxynucleotide (ODN), to enhance the immune response by controlling the T helper 1 (Th1)/T helper 2 (Th2) balance. Methods: S/O nanodispersions containing ovalbumin (OVA) and CpG ODN (CpG-A or CpG-B) were characterized by size distribution analysis and a protein release test. The skin permeation of fluorescence-labeled OVA was observed by fluorescence microscopy. Antigen-specific IgG, IgG1, and IgG2a responses were measured by enzyme-linked immunosorbent assay. Results: Co-encapsulation of CpG ODNs in S/O nanodispersions enhanced induction of OVA-specific IgG. S/O nanodispersion containing OVA and CpG-A had a smaller mean particle size and permeated the skin more efficiently. In contrast, CpG-B showed the highest protein release and induction of OVA-specific IgG. IgG subclass analysis revealed that OVA induced a Th2-dominant immune response, while the S/O nanodispersion containing CpG-A skewed the immune response toward a Th1-bias. Conclusions: In combination with CpG ODN, the S/O nanodispersion system efficiently induced an antigen-specific antibody response. The Th1/Th2 immune balance could be controlled by the selection of CpG ODN type.

DOI： 10.1007/s11095-014-1554-5

Language：Others  

Site-specific conjugation of an antibody-binding protein catalyzed by horseradish peroxidase creates a multivalent protein conjugate with high affinity to IgG
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Cross-linking proteins offers an approach to enhance the distinct function of proteins due to the multivalent effect. In this study, we demonstrated the preparation of a multivalent antibody-binding protein possessing high affinity to IgG by conjugating a number of antibody-binding proteins using the horseradish peroxidase (HRP)-mediated protein conjugation method. By introducing a peptide tag containing a tyrosine (Y-tag) to the C-terminus of the model protein, a chimera protein of protein G and protein A (pG2pA), the Tyr residue in the Y-tag was efficiently recognized by HRP and cross-linked with each other to yield a pG2pA conjugate, composed of mainly two to three units of pG2pA. The cross-linking occurred site specifically at the Tyr residue in the Y-tag and introduction of the Y-tag showed no effect on the function of pG2pA. The affinity of the Y-tagged pG2pA conjugate against IgG clearly increased because of the multivalent effect, demonstrating the benefit of this protein cross-linking reaction, which yields functional protein oligomers. Such multivalent protein conjugates created by this reaction should have potential to be used in ELISA and Western blotting applications in which highly sensitive detection of target molecules is desired.

DOI： 10.1002/biot.201400512

Language：Others  

Separation of gold(III) in acidic chloride solution using porous polymeric ionic liquid gel
© 2015 The Society of Chemical Engineers, Japan. A novel polymeric ionic liquid (PIL) gel was synthesized via radical polymerization of the ionic liquid monomers, 1-vinyl- 3-ethylimidazoliumbis(trifluorometyl-sulfonyl) imide ([veim][Tf2N]). The gel was made porous by removing the water phase dispersed by polyoxyethylene (20) sorbitan monolaurate (Tween-20). The porous structure of the synthesized gel was observed by SEM imaging. In comparison to the nonporous PIL gel synthesized without Tween-20, the adsorption capacity of Au(III) onto porous PIL gel was enhanced. The adsorption behavior of the synthesized gel was studied in acidic chloride solutions containing various metal ions, and it was found that the gel selectively adsorbed Au(III) over other metal ions. Maximum adsorption capacity of Au(III) was more than 140 mg/g; whereas, those for Pt(IV) and Pd(II) were lower than 20 mg/g. The adsorbed Au(III) onto the gel can easily be desorbed using an acidified thiourea aqueous solution. The feasibility of utilizing the polymeric ionic liquid gel for the separation of Au(III) was thereby demonstrated.

DOI： 10.1252/jcej.14we162

Language：Others  

Ionic liquid-mediated transcutaneous protein delivery with solid-in-oil nanodispersions
© 2015 The Royal Society of Chemistry. As a potentially safe and non-invasive vaccination method, transcutaneous immunization represents an attractive alternative to conventional vaccine delivery by injection. However, the development of transcutaneous immunization has remained a challenge for a large number of hydrophilic macromolecules including protein and peptide antigens. We report a novel ionic liquid (IL)-mediated transcutaneous vaccine formulation consisting of a solid-in-oil (S/O) nanodispersion of antigen coated with pharmaceutically accepted surfactants dispersed in IL-containing oil. The introduction of the IL [C12mim][Tf2N] (1-dodecyl-3-methyl imidazolium bis(trifluoromethyl sulfonyl) amide) as a penetration enhancer in the formulation significantly enhanced the skin permeability of ovalbumin (OVA), a model antigen. It was also found that the IL-mediated S/O nanodispersion obtained high levels of OVA-specific serum IgG compared with both S/O nanodispersions without IL and PBS control. These findings clearly indicate that ILs-which are potentially attractive "green" and "designer" solvents-could serve as potential skin penetration enhancers in transcutaneous vaccination for hydrophilic macromolecules.

DOI： 10.1039/c5md00378d

Language：Others  

Enzyme-mediated preparation of hydrogels composed of poly(ethylene glycol) and gelatin as cell culture platforms
© The Royal Society of Chemistry 2015. A redox-responsive hydrogel composed of poly(ethylene glycol) and gelatin was prepared via an enzymatic oxidative reaction. Fibroblast cells adhered on the hydrogel showed proliferation and they could be recovered as a cell sheet by degradation of the scaffold under mild reductive conditions, thus providing a novel platform for cell culture.

DOI： 10.1039/c4ra12334d

Language：Others  

Activity and stability of enzyme immobilized with ionic liquid based polymer materials
© School of Engineering, Taylor’s University. Enzyme immobilization methods are continuously being developed for a wide range of applications including biocatalysis and biotransformation. Compared to other immobilization methods, physical entrapment into a matrix is relatively simple and inexpensive, and causes a relatively small perturbation to the native enzyme structure and function. However, significant enzyme leaching from such biocatalytic polymers is the main constraint in using them for industrial levels. This study reports an ionic liquid (IL) polymer materials incorporating enzymes that can be used as active, stable and reusable biocatalysts to overcome the limitations. Lipase was microencapsulated in surfactant aggregates formed in an IL monomer or the solution of an IL monomer/IL and then incorporated into polymer frameworks through the free radical polymerization of an IL (1-vinyl-3-ethylimidazolium bis(trifluoromethylsulfonyl) amide ([veim][Tf2N]). The activity, stability and reusability of IL polymer materials containing lipase were evaluated using lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-PNB) as a model reaction. It was found that lipase IL polymer materials exhibited excellent stability in aqueous solutions. More importantly, these biopolymer materials retained most of their activity after six reaction cycles. We firmly believe that enzyme containing IL polymeric materials developed in this study will provide several advantages over conventional polymer based methods for diverse enzymatic reactions.

Language：English   Publishing type：Research paper (scientific journal)  

In the most commonly used in situ hybridization (ISH) procedure, a hapten-labeled antisense nucleic acid (e.g., RNA) probe is employed to hybridize a target mRNA in tissue sections. The hapten-labeled RNA is then detected by a highly specific hapten–antibody interaction; however, it requires laborious immunostaining steps for spatial coloring on tissue sections. To simplify ISH-based mRNA detection systems, we created a new RNA–(enzyme) n conjugate for sensitive detection of mRNA in tissue sections. In the present simple, antibody-free ISH protocol, an antisense RNA probe of interest is first modified with a specific dipeptide substrate of microbial transglutaminase (MTG) by in vitro transcription. Alkaline phosphatase from a hyperthermophile is then covalently linked to the substrate-labeled RNA by MTG-catalyzed sitespecific conjugation. A robust, multi-enzyme-labeled RNA probe enables the direct labeling of a target mRNA in tissue sections with signaling enzymes under harsh hybridization conditions, leading to one-step signal amplification after hybridization. The application of the new trans glutaminase-mediated ISH (TransISH) strategy to mRNA detection in mammalian tissues was demonstrated.

DOI： 10.1007/978-1-4939-2303-8_29

Language：English   Publishing type：Research paper (scientific journal)  

New fusion proteins for immunoassay were developed as an alternative to the conventional chemical linked enzyme-antibody complex. Human chimeric alkaline phosphatase (IPP) was used as the labeling enzyme, and partial domains of Staphylococcus aureus Protein A and Streptococcus Protein G were used as antibody binding protein (PG). The fusion protein composed of IPP and PG was produced using human cell lines because IPP is derived from human enzymes. The expression system was optimized by changing the plasmid vector, reducing the GC content of the DNA sequence, and employing different cell lines including adherent and suspension cells. As result, a 2,600-fold increase in the protein yield per unit of growth medium was achieved. The resultant IPP-PG fusion protein was successfully utilized for immunoassay applications such as western blotting and immunohistochemistry.

DOI： 10.1252/kakoronbunshu.41.38

Language：English   Publishing type：Research paper (scientific journal)  

Enzyme immobilization methods are continuously being developed for a wide range of applications including biocatalysis and biotransformation. Compared to other immobilization methods, physical entrapment into a matrix is relatively simple and inexpensive, and causes a relatively small perturbation to the native enzyme structure and function. However, significant enzyme leaching from such biocatalytic polymers is the main constraint in using them for industrial levels. This study reports an ionic liquid (IL) polymer materials incorporating enzymes that can be used as active, stable and reusable biocatalysts to overcome the limitations. Lipase was microencapsulated in surfactant aggregates formed in an IL monomer or the solution of an IL monomer/IL and then incorporated into polymer frameworks through the free radical polymerization of an IL (1-vinyl-3-ethylimidazolium bis(trifluoromethylsulfonyl) amide ([veim][Tf2N]). The activity, stability and reusability of IL polymer materials containing lipase were evaluated using lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-PNB) as a model reaction. It was found that lipase IL polymer materials exhibited excellent stability in aqueous solutions. More importantly, these biopolymer materials retained most of their activity after six reaction cycles. We firmly believe that enzyme containing IL polymeric materials developed in this study will provide several advantages over conventional polymer based methods for diverse enzymatic reactions.

Language：English   Publishing type：Research paper (scientific journal)  

Interferon gamma (IFN-γ) is a cytokine that is mainly secreted from immune cells. It plays an important role in regulating neighboring cellular activities through interactions with cell membrane proteins. Because of its biological importance, there has been increasing demand for a biosensor that can respond to IFN-γ. Here, we report a fluorescent aptamer sensor that targets IFN-γ. The 25-nt IFN-γ aptamer was converted into a turn-on fluorescent sensor by labeling the 3'-end with fluorescein. The designed sensor achieved fluorescence sensing of IFN-γ with nanomolar affinity.

DOI： 10.1246/cl.150794

Language：English   Publishing type：Research paper (scientific journal)  

As a potentially safe and non-invasive vaccination method, transcutaneous immunization represents an attractive alternative to conventional vaccine delivery by injection. However, the development of transcutaneous immunization has remained a challenge for a large number of hydrophilic macromolecules including protein and peptide antigens. We report a novel ionic liquid (IL)-mediated transcutaneous vaccine formulation consisting of a solid-in-oil (S/O) nanodispersion of antigen coated with pharmaceutically accepted surfactants dispersed in IL-containing oil. The introduction of the IL [C₁₂mim][Tf₂N] (1-dodecyl-3-methyl imidazolium bis(trifluoromethyl sulfonyl) amide) as a penetration enhancer in the formulation significantly enhanced the skin permeability of ovalbumin (OVA), a model antigen. It was also found that the IL-mediated S/O nanodispersion obtained high levels of OVA-specific serum IgG compared with both S/O nanodispersions without IL and PBS control. These findings clearly indicate that ILs-which are potentially attractive "green" and "designer" solvents-could serve as potential skin penetration enhancers in transcutaneous vaccination for hydrophilic macromolecules.

DOI： 10.1039/c5md00378d

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1186/s40508-014-0019-9

Language：English   Publishing type：Research paper (scientific journal)  

In the present study, we developed a novel surface-coated nanocarrier (SCN) for efficient and stable encapsulation of a poorly water-soluble anticancer agent, camptothecin (CPT). Using emulsification and freeze-drying processes in the presence of a hydrophilic surfactant, Pluronic F-68 or F-127, CPT-Pluronic complexes with crystalline features were synthesised. Investigation of the in vitro anticancer activities and cellular internalisation of the complexes revealed that the SCN efficiently delivered CPT to cells while maintaining anticancer activity.

DOI： 10.1039/c4md00179f

Language：Others  

Application of cellulose acetate to the selective adsorption and recovery of Au(III)
Cellulose acetyl derivatives were examined for the selective recovery of Au(III) from acidic chloride solutions as an adsorbent, and cellulose acetate fibers (CAF) were found to be effective for the separation of Au(III) from other metal ions, including the precious metal ions Pt(IV) and Pd(II). The amount of Au(III) adsorbed by the fibers increased with an increase in the hydrochloric acid concentration, but decreased with an increase in the ionic strength of the solution. The adsorption of Au(III) onto CAF took place quickly and an adsorption equilibrium was reached within 1 h. The maximum adsorption capacity of Au(III) was determined to be 110 mg/g at 2 M hydrochloric acid. The loaded Au(III) was readily recovered by incineration. © 2014 Elsevier Ltd.

DOI： 10.1016/j.carbpol.2014.05.003

Language：Others  

A novel surface-coated nanocarrier for efficient encapsulation and delivery of camptothecin to cells
© 2014 the Partner Organisations. In the present study, we developed a novel surface-coated nanocarrier (SCN) for efficient and stable encapsulation of a poorly water-soluble anticancer agent, camptothecin (CPT). Using emulsification and freeze-drying processes in the presence of a hydrophilic surfactant, Pluronic F-68 or F-127, CPT-Pluronic complexes with crystalline features were synthesised. Investigation of the in vitro anticancer activities and cellular internalisation of the complexes revealed that the SCN efficiently delivered CPT to cells while maintaining anticancer activity.

DOI： 10.1039/c4md00179f

Language：English   Publishing type：Research paper (scientific journal)  

Cellulose acetyl derivatives were examined for the selective recovery of Au(III) from acidic chloride solutions as an adsorbent, and cellulose acetate fibers (CAF) were found to be effective for the separation of Au(III) from other metal ions, including the precious metal ions Pt(IV) and Pd(II). The amount of Au(III) adsorbed by the fibers increased with an increase in the hydrochloric acid concentration, but decreased with an increase in the ionic strength of the solution. The adsorption of Au(III) onto CAF took place quickly and an adsorption equilibrium was reached within 1 h. The maximum adsorption capacity of Au(III) was determined to be 110 mg/g at 2 M hydrochloric acid. The loaded Au(III) was readily recovered by incineration.

DOI： 10.1016/j.carbpol.2014.05.003

Language：English   Publishing type：Research paper (scientific journal)  

Enzymatic hydrogelation has received much attention due to the high biocompatibility and the ease of control of reaction kinetics under physiological conditions. In particular, horseradish peroxidase (HRP)-mediated phenol coupling reaction has great potential for developing in situ hydrogelation systems. Herein, we report the HRP-catalyzed preparation and characterization of hydrogels composed of a terminally bis-phenolated linear poly(ethylene glycol) (PEG-Ph-OH) with different molecular weights (M_ws 3100, 8800, 11,000, 20,000g/mol). The gelation time of polymer solution can be controlled in the range from few second to few minute, suggestion that the PEG-Ph-OH has a potential as a in situ forming hydrogel. In addition, the physicochemical properties of the hydrogels, such as swelling ratio, mesh size and mechanical property, were controlled by the molecular weight of the PEG-Ph-OH. The results could be attributed to the alteration in the cross-linking density by the variation of molecular weight of the gel precursor. Furthermore, the viability of mammalian cells encapsulated in the PEG-Ph-OH hydrogels was approximately 90%. These results indicate that PEG-Ph-OH has potential for biomedical applications including tissue engineering.

DOI： 10.1016/j.bej.2014.09.003

Language：Others  

Characterization of enzymatically gellable, phenolated linear poly(ethylene glycol) with different molecular weights for encapsulating living cells
© 2014 Elsevier B.V. Enzymatic hydrogelation has received much attention due to the high biocompatibility and the ease of control of reaction kinetics under physiological conditions. In particular, horseradish peroxidase (HRP)-mediated phenol coupling reaction has great potential for developing in situ hydrogelation systems. Herein, we report the HRP-catalyzed preparation and characterization of hydrogels composed of a terminally bis-phenolated linear poly(ethylene glycol) (PEG-Ph-OH) with different molecular weights (Mws 3100, 8800, 11,000, 20,000g/mol). The gelation time of polymer solution can be controlled in the range from few second to few minute, suggestion that the PEG-Ph-OH has a potential as a in situ forming hydrogel. In addition, the physicochemical properties of the hydrogels, such as swelling ratio, mesh size and mechanical property, were controlled by the molecular weight of the PEG-Ph-OH. The results could be attributed to the alteration in the cross-linking density by the variation of molecular weight of the gel precursor. Furthermore, the viability of mammalian cells encapsulated in the PEG-Ph-OH hydrogels was approximately 90%. These results indicate that PEG-Ph-OH has potential for biomedical applications including tissue engineering.

DOI： 10.1016/j.bej.2014.09.003

Language：English  

The synergistic extraction of rare-earth metals by a beta-diketone and an organophosphine oxide is described. A combination of 2-thenoyltri'uor oacetone (HTTA) and tri-n-octyl phosphine oxide (TOPO) was found to be e degrees ective in synergistically extracting rare-earth metals into an ionic liquid (IL). The synergistic e degrees ect was strongly in(similar to)uenc ed by the diluent in which the extractants were dissolved. Extraction e ciencies for all the rare-earth metal ions were enhanced by synergy when a conventional organic solvent was used (n-dodecane). However, the synergistic e degrees ect in the IL preferentially improved the scandium extraction, leading to higher selectivity for Sc than for other rare-earth metals. Slope analysis was used to assess each extraction system, and the IL itself was found to be directly involved in the complex formation of the metal extraction system.

DOI： 10.1252/jcej.14we360

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/ie403524a

Language：Others  

The self-assembly and secondary structure of peptide amphiphiles determine the membrane permeation activity
Membrane fusogenic peptides have attracted increasing attention because of their unique biofunctions in membrane translocation and viral infection. Here, we designed GALA-related peptides with palmitoyl tails. Our study indicated that the self-assembling propensity and the secondary structure of these peptide amphiphiles greatly influenced the membrane permeability. This journal is © the Partner Organisations 2014.

DOI： 10.1039/c4ra02901a

Language：Others  

Sucrose laurate enhanced transcutaneous immunization with a solid-in-oil nanodispersion

DOI： 10.1039/c3md00164d

Language：Others  

Separation of precious metals by using undiluted ionic liquids
The ionic liquid (IL), 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Bmim][Tf2N]), was evaluated as an extractant without dilution for the separation and recovery of precious metals. Au(III) was efficiently extracted from the aqueous phase to the IL phase, and a concentrated Au(III) solution almost 8 times higher than the initial feed solutions could be produced. In contrast, Pd(II) was rarely extracted and Pt(IV) was partly extracted under the present experimental conditions. Furthermore, the effect of the IL anions on the extraction ability for the metal ions was examined, and it was found that an IL having an anion component slightly more hydrophilic compared to [Tf2N], such as 1-butyl-3-methylimidazolium hexafluorophosphate [Bmim][PF6], showed a higher extraction ability than [Bmim][Tf2N]. It was suggested that the extraction proceeded via an anion exchange mechanism and it was shown that ILs could be effective extractants for the separation of precious metals.

DOI： 10.15261/serdj.21.89

Language：Others  

One step effective separation of platinum and palladium in an acidic chloride solution by using undiluted ionic liquids
Imidazolium-based ionic liquids (ILs) with Tf2N- as anions were studied as extractants without dilution for the extractive separation of platinum and palladium in an acidic chloride solution. The effect of the alkyl chain length on the extraction performance was evaluated and, based on the performance, the IL, 1-methyl-3-octylimidazolium bis(trifluoromethylsulfonyl) imide ([Omim][Tf2N]), was selected to be studied in detail. The extraction of Pt(IV) underwent little change over the HCl concentration studied, while that of Pd(II) decreased with increasing HCl concentration. The separation of Pt(IV) and Pd(II) was highly effective, and the highest separation factor between Pt(IV) and Pd(II) reached 312 at the optimum conditions in a binary component system composed of Pt(IV) and Pd(II). The effect of the initial Pt(IV) concentration on loading capacity and extraction efficiency of Pt(IV) was also investigated.

DOI： 10.15261/serdj.21.129

Language：Others  

Development of novel extractants with amino acid structure for efficient separation of nickel and cobalt from manganese ions
The solvent extraction separation of nickel and cobalt from manganese is an important issue to be resolved. Novel amic acid type extractants with the glycine or sarcosine moiety N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]glycine (D2EHAG) and N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]sarcosine (D2EHAS) were synthesized to achieve this separation. These extractants were prepared by a simple two-step reaction and were readily soluble in aliphatic organic solvents. The extraction behavior of Ni2+, Co2+, and Mn 2+ with D2EHAG and D2EHAS in n-dodecane was investigated compared with that of conventional extractants. The novel extractants extracted Ni 2+ and Co2+ preferentially with a high selectivity to Mn2+. The metal ions could be back-extracted quantitatively using 1 mol dm-3 sulfuric acid. The extraction mechanism of the three metal ions with D2EHAG and D2EHAS was investigated, and results suggest that one divalent metal ion is extracted with two extractant molecules. © 2013 American Chemical Society.

DOI： 10.1021/ie403524a

Language：English   Publishing type：Research paper (scientific journal)  

The liquid-liquid extraction of rare earth metal ions (scandium (Sc³⁺), yttrium (Y³⁺) and the lanthanides (La³⁺, Nd³⁺, Eu³⁺ and Dy³⁺)) was investigated using N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]glycine (D2EHAG). Scandium was extracted selectively from lanthanides under highly acidic conditions (0 < pH ≤ 1.5), and stripped easily using a mild acidic solution such as 1 mol dm^-3 H₂SO₄. By comparing the extraction behavior with N,N-dioctyldiglycol amic acid, which has a similar molecular structure to D2EHAG, or the commercial alkyl monocarboxylic acid extractant, Versatic 10, it was concluded that the affinity of D2EHAG to scandium was caused by a chelating effect and the size recognition ability of D2EHAG. The extraction mechanism was examined, and it was proven that the trivalent scandium ion is extracted by forming a stable metal complex with four D2EHAG molecules. This journal is

DOI： 10.1039/c4ra08897b

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c3ra45668d

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c3md00164d

Language：English   Publishing type：Research paper (scientific journal)  

Imidazolium-based ionic liquids (ILs) with Tf²N- as anions were studied as extractants without dilution for the extractive separation of platinum and palladium in an acidic chloride solution. The effect of the alkyl chain length on the extraction performance was evaluated and, based on the performance, the IL, 1-methyl-3-octylimidazolium bis(trifluoromethylsulfonyl) imide ([Omim][Tf²N]), was selected to be studied in detail. The extraction of Pt(IV) underwent little change over the HCl concentration studied, while that of Pd(II) decreased with increasing HCl concentration. The separation of Pt(IV) and Pd(II) was highly effective, and the highest separation factor between Pt(IV) and Pd(II) reached 312 at the optimum conditions in a binary component system composed of Pt(IV) and Pd(II). The effect of the initial Pt(IV) concentration on loading capacity and extraction efficiency of Pt(IV) was also investigated.

DOI： 10.15261/serdj.21.129

Language：English   Publishing type：Research paper (scientific journal)  

Membrane fusogenic peptides have attracted increasing attention because of their unique biofunctions in membrane translocation and viral infection. Here, we designed GALA-related peptides with palmitoyl tails. Our study indicated that the self-assembling propensity and the secondary structure of these peptide amphiphiles greatly influenced the membrane permeability. This journal is

DOI： 10.1039/c4ra02901a

Language：English   Publishing type：Research paper (scientific journal)  

The synergistic extraction of rare-earth metals by a β-diketone and an organophosphine oxide is described. A combination of 2-thenoyltrifluoroacetone (HTTA) and tri-n-octyl phosphine oxide (TOPO) was found to be effective in synergistically extracting rare-earth metals into an ionic liquid (IL). The synergistic effect was strongly influenced by the diluent in which the extractants were dissolved. Extraction efficiencies for all the rare-earth metal ions were enhanced by synergy when a conventional organic solvent was used (n-dodecane). However, the synergistic effect in the IL preferentially improved the scandium extraction, leading to higher selectivity for Sc than for other rare-earth metals. Slope analysis was used to assess each extraction system, and the IL itself was found to be directly involved in the complex formation of the metal extraction system.

DOI： 10.1252/jcej.14we360

Language：English   Publishing type：Research paper (scientific journal)  

The ionic liquid (IL), 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Bmim][Tf₂N]), was evaluated as an extractant without dilution for the separation and recovery of precious metals. Au(III) was efficiently extracted from the aqueous phase to the IL phase, and a concentrated Au(III) solution almost 8 times higher than the initial feed solutions could be produced. In contrast, Pd(II) was rarely extracted and Pt(IV) was partly extracted under the present experimental conditions. Furthermore, the effect of the IL anions on the extraction ability for the metal ions was examined, and it was found that an IL having an anion component slightly more hydrophilic compared to [Tf₂N], such as 1-butyl-3-methylimidazolium hexafluorophosphate [Bmim][PF₆], showed a higher extraction ability than [Bmim][Tf₂N]. It was suggested that the extraction proceeded via an anion exchange mechanism and it was shown that ILs could be effective extractants for the separation of precious metals.

DOI： 10.15261/serdj.21.89

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2013.05.025

Language：Others  

Tailing DNA aptamers with a functional protein by two-step enzymatic reaction
An efficient, quantitative synthetic strategy for aptamer-enzyme conjugates was developed by using a two-step enzymatic reaction. Terminal deoxynucleotidyl transferase (TdT) was used to first incorporate a Z-Gln-Gly (QG) modified nucleotide which can act as a glutamine donor for a subsequent enzymatic reaction, to the 3&#039;-OH of a DNA aptamer. Microbial transglutaminase (MTG) then catalyzed the cross-linking between the Z-QG modified aptamers and an enzyme tagged with an MTG-reactive lysine containing peptide. The use of a Z-QG modified dideoxynucleotide (Z-QG-ddUTP) or a deoxyuridine triphosphate (Z-QG-dUTP) in the TdT reaction enables the controlled introduction of a single or multiple MTG reactive residues. This leads to the preparation of enzyme-aptamer and (enzyme)n-aptamer conjugates with different detection limits of thrombin, a model analyte, in a sandwich enzyme-linked aptamer assay (ELAA). Since the combination of two enzymatic reactions yields high site-specificity and requires only short peptide substrates, the methodology should be useful for the labeling of DNA/RNA aptamers with proteins. © 2013 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2013.05.025

Language：Others  

Needle-free immunization using a solid-in-oil nanodispersion enhanced by skin-permeable oligoarginine peptide

DOI： 10.1016/j.ijpharm.2013.10.006

Language：Others  

Enzymatic fabrication of protein-decorated gold nanoparticles by the aid of artificial peptides with gold-binding affinity
Here, we report a new approach for the biofabrication of protein-immobilized gold nanoparticles (Au NPs), using oxidoreductase with gold-binding peptide-tagged recombinant proteins. The reduction of Au ions to Au0 was achieved using a natural electron-donating cofactor, nicotinamide adenine dinucleotide, which was regenerated by the glycerol dehydrogenase (GLD) enzyme. First, we selected the A3 peptide (AYSSGAPPMPPF) as a gold binding moiety. The A3 peptide was introduced to the C-terminus of fusion proteins of immunoglobulin G (IgG)-binding domains of protein G and protein A. In the presence of the recombinant protein, the GLD-catalyzed cofactor reduction resulted in the efficient in situ fabrication of Au NPs immobilized with the fusion protein. Moreover, the protein-immobilized Au NPs were shown to have IgG binding activity. Although the A3 peptide had the ability to stabilize Au NPs, the results suggested that its binding affinity for Au NPs was unexpectedly weaker than that of His-tag. A cysteine residue was thus introduced to a recombinant protein adjacent to the A3 peptide. Finally, an artificial peptide, comprising A3 sequence with the C-terminal single cysteine residue, enabled the stable display of a fusion protein while maintaining its IgG binding activity through the Au-S bond. This enzyme-assisted one-pot methodology for protein-Au NPs conjugation offers one potent route for the facile fabrication of biomolecule-decorated metal NPs. © 2013 American Chemical Society.

DOI： 10.1021/la401327h

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ijpharm.2013.10.006

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/la401327h

Language：English   Publishing type：Research paper (scientific journal)  

Ionic liquid-in-oil (IL/o) microemulsion (ME) techniques have been employed to increase the solubility of sparingly soluble drugs and enhance the extent of their topical and transdermal delivery. Methotrexate (MTX) has been used as a model drug in the current study. The size and size distributions of the ME droplets in the presence and in the absence of MTX were determined by dynamic light scattering analysis. The mean droplet diameter of the ME droplets containing 4 mg/mL of MTX was less than 20 nm, and smaller than the corresponding value observed in the absence of MTX. MTX was incorporated into IL droplets as well as other formulations and its permeation across Yucatan micropig (YMP) porcine skin evaluated. The use of IL/o ME has been shown to dramatically increase MTX administration. The results obtained in this study reveal the versatility of IL/o MEs as efficient drug delivery systems for sparingly soluble drugs.

DOI： 10.1252/jcej.13we009

Language：Others  

Ionic liquid-in-oil microemulsions as potential carriers for the transdermal delivery of Methotrexate
Ionic liquid-in-oil (IL/o) microemulsion (ME) techniques have been employed to increase the solubility of sparingly soluble drugs and enhance the extent of their topical and transdermal delivery. Methotrexate (MTX) has been used as a model drug in the current study. The size and size distributions of the ME droplets in the presence and in the absence of MTX were determined by dynamic light scattering analysis. The mean droplet diameter of the ME droplets containing 4 mg/mL of MTX was less than 20 nm, and smaller than the corresponding value observed in the absence of MTX. MTX was incorporated into IL droplets as well as other formulations and its permeation across Yucatan micropig (YMP) porcine skin evaluated. The use of IL/o ME has been shown to dramatically increase MTX administration. The results obtained in this study reveal the versatility of IL/o MEs as efficient drug delivery systems for sparingly soluble drugs. © 2013 The Society of Chemical Engineers, Japan.

DOI： 10.1252/jcej.13we009

Language：Others  

Aligning an endoglucanase Cel5A from Thermobifida fusca on a DNA scaffold: potent design of an artificial cellulosome

DOI： 10.1039/c3cc42614a

Language：Others  

Biosorption of rare earth elements by Escherichia coli
Biosorption, which takes place by passive metal-sequestering, has recently attracted attention as an alternative to conventional separation processes such as precipitation and solvent extraction. In this work, the adsorption behavior of rare earth elements (REEs) onto an Escherichia coli (E. coli) bacterial adsorbent was examined. The adsorption of REEs increased with increasing pH of the feed solution. Also, E. coli showed a higher selectivity for heavier REEs, especially for scandium. To elucidate the adsorption mechanism, the functional groups on the E. coli were chemically modified. By analyzing the adsorption behavior and FT-IR spectra of these E. coli samples before and after adsorption, it was confirmed that REEs are adsorbed at phosphate and carboxyl groups on the surface of E. coli. It is concluded that E. coli is a potential novel adsorbent for REEs. © 2013 The Society of Chemical Engineers, Japan.

DOI： 10.1252/jcej.13we031

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/09205063.2012.755600

Language：Others  

Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol)
Hybrid gels constructed from proteins and polymers have attracted a wide range of attention in the field of biomedicine and bioengineering. We report herein the enzymatic preparation of polymer-protein hybrid hydrogels composed of terminally bis-functionalized linear poly(ethylene glycol) (PEG) and streptavidin (SA). PEG was conjugated with tyramine to introduce terminal phenolic hydroxyl (Ph-OH) groups. A peptide tag containing a tyrosine residue (G4Y-tag) was genetically introduced at the C-terminus of SA. The Ph-OH-modified PEG and G4Y-tagged SA (SA-G4Y) were treated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2) to yield (PEG-Ph-OH)-(SA-G4Y) hybrid gels. Biotinylated enhanced green fluorescent protein (biotin-EGFP) was selectively captured in the obtained hybrid gels, indicating that SA-G4Y retained its biological function. The amount of biotin-EGFP immobilized in the hybrid gels depended on the concentration of SA-G4Y. In addition, biotinylated bacterial alkaline phosphatase (biotin-BAP) was immobilized in the hybrid gel. The immobilized biotin-BAP exhibited more than 95% of the initial activity after 5 rounds of recycling. The results suggest the facile functionalization of the hybrid gel with a variety of biotinylated functional molecules. © 2013 Elsevier B.V.

DOI： 10.1016/j.bej.2013.04.007

Language：English   Publishing type：Research paper (scientific journal)  

A novel multi-cellulase conjugate assembled on a double-stranded DNA scaffold, a DNA–(endoglucanase)_n conjugate, exhibited unique hydrolytic activity toward crystalline cellulose (Avicel) depending on the cellulase/DNA ratio on the DNA-based artificial cellulosome.

DOI： 10.1039/c3cc42614a

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bej.2013.04.007

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.biortech.2013.03.147

Language：Others  

Selective extraction and recovery of rare earth metals from phosphor powders in waste fluorescent lamps using an ionic liquid system
The recycling of rare earth metals from phosphor powders in waste fluorescent lamps by solvent extraction using ionic liquids was studied. Acid leaching of rare earth metals from the waste phosphor powder was examined first. Yttrium (Y) and europium (Eu) dissolved readily in the acid solution; however, the leaching of other rare earth metals required substantial energy input. Ionization of target rare earth metals from the waste phosphor powders into the leach solution was critical for their successful recovery. As a high temperature was required for the complete leaching of all rare earth metals, ionic liquids, for which vapor pressure is negligible, were used as an alternative extracting phase to the conventional organic diluent. An extractant, N, N-dioctyldiglycol amic acid (DODGAA), which was recently developed, showed a high affinity for rare earth metal ions in liquid-liquid extraction although a conventional commercial phosphonic extractant did not. An effective recovery of the rare earth metals, Y, Eu, La and Ce, from the metal impurities, Fe, Al and Zn, was achieved from the acidic leach solution of phosphor powders using an ionic liquid containing DODGAA as novel extractant system. © 2013 Elsevier B.V.

DOI： 10.1016/j.jhazmat.2013.03.026

Language：English   Publishing type：Research paper (scientific journal)  

Ionic liquids have attracted attention as potential pretreatment agents in cellulosic biomass processing. Here we report on a new magnetic ionic liquid that can dissolve crystalline cellulose and be collected by a magnet. © 2013 Elsevier B.V.

DOI： 10.1016/j.molliq.2013.03.012

Language：English   Publishing type：Research paper (scientific journal)  

To enhance enzymatic saccharification of pine biomass, the pretreatment reagents peracetic acid (PAA) and ionic liquid (IL) were validated in single reagent pretreatments or combination pretreatments with different sequences. In a 1 h saccharification, 5-25% cellulose conversion was obtained from the single pretreatment of PAA or IL In contrast, a marked enhancement in conversion rates was achieved by PAA-IL combination pretreatments (45-70%). The PAA followed by IL (PAA + IL) pretreatment sequence was the most effective for preparing an enzymatic digestible regenerated biomass with 250-fold higher glucose formation rates than untreated biomass and 2- to 12-fold higher than single pretreatments with PAA or IL alone. Structural analysis confirmed that this pretreatment resulted in biomass with highly porous structural fibers associated with the reduction of lignin content and acetyl groups. Using the PAA + IL sequence, biomass loading in the pretreatment step can be increased from 5% to 15% without significant decrease in cellulose conversion. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biortech.2013.03.147

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molliq.2013.03.012

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jhazmat.2013.03.026

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.biortech.2012.06.096

Language：Japanese  

Application of E. coli to adsorption and recovery of rare earth elements

Language：English   Publishing type：Research paper (scientific journal)  

The potential of 1-hexylpyridinium chloride ([Hpy][Cl]), to pretreat cellulosic feedstocks was investigated using microcrystalline cellulose (Avicel) and Bagasse at 80 degrees C or 100 degrees C. Short [Hpy][Cl] pretreatments, <30 min, at lower temperature accelerate subsequent enzymatic saccharification of Avicel. Over 95% conversion of pretreated Avicel to glucose was attained after 24 h enzymatic saccharification under optimal conditions, whereas regenerated Bagasse showed 1-3-fold higher conversion than untreated biomass. FT-IR analysis of both Avicel and Bagasse samples pretreated with [Hpy][Cl] or 1-ethyl-3-methyimidazolium acetate ([Emim][OAc]) revealed that these ionic liquids behaved differently during pretreatment. [Hpy][Cl] pretreatment for an extended duration (180 min) released mono- and disaccharides without using cellulase enzymes, suggesting [Hpy][Cl] has capability for direct saccharification of cellulosic feedstocks. On the basis of the results obtained, [Hpy][Cl] pretreatment enhanced initial reaction rates in enzymatic saccharification by either crystalline polymorphic alteration of cellulose or partial degradation of the crystalline cellulosic fraction in biomass. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biortech.2012.06.096

Language：Others  

Preparation of Multiple Emulsions to Depress the Rlease of Drugs and Enhanced Permeation Effect in Transdermal Delivery

DOI： 10.5360/membrane.38.92

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/bc300606b

Language：Others  

Split Spy0128 as a potent scaffold for protein cross-linking and immobilization
Site-specific cross-linking techniques between proteins and additional functional groups have become increasingly important for expanding the utility of proteins in biochemistry and biotechnology. In order to explore powerful techniques for practical bioconjugation applications, we have validated a technique mediated by a unique property of Streptcoccus pyogenes pilin subunit Spy0128, an autocatalytic intramolecular isopeptide formation in Spy0128. Recently, it has been revealed that Spy0128 can be split into two fragments (split-Spy0128 (residues 18-299 of Spy0128) and isopeptag (residues 293-308 of Spy0128)) that were capable of forming an intermolecular covalent complex. We focused on this unique reconstitution property and first studied the bioconjugation of blue and green fluorescent proteins, enabling the direct monitoring of cross-linking reactions by Förster resonance energy transfer (FRET). A fluorescence lifetime study shows that spatial control of two proteins on the Spy0128 scaffold is possible when one protein is fused to the N-terminus of split-Spy0128 and another one is tethered at the N- or C-terminus of the isopeptag. Furthermore, we demonstrated site-specific protein immobilization mediated by the reconstitution of split-Spy0128 and isopeptag. In this case, a split-Spy0128 mutant with a free N-terminal Cys residue was first immobilized onto beads chemically modified with a maleimide group through a Michael addition process. Then, an isopeptagged protein was successfully immobilized onto the split-Spy0128-immobilized beads. These results suggest that Spy0128 is a potent proteinaceous scaffold available for bioconjugation both in solution and at a solid surface. © 2013 American Chemical Society.

DOI： 10.1021/bc300606b

Language：Others  

Protein supramolecular complex formation by site-specific avidin-biotin interactions
The precise accumulation of protein functions on a nanoscale to fabricate advanced biomaterials has become possible by a bottom-up approach based on molecular self-assembly. The avidin-biotin interaction is widely employed in the design of functional protein self-assemblies. Herein we assessed how the spatial arrangement of the avidin-biotin interaction between protein building blocks affects the formation of a protein supramolecular complex (PSC). The enzymatic site-specific internal labeling of a symmetric protein scaffold, bacterial alkaline phosphatase (AP), with specifically designed biotinylation substrates revealed that the precise positioning of the biotinylation sites on AP and the linker flexibility of the substrate are critical factors for the growth of PSCs in the presence of streptavidin (SA). A potential diagnostic application of the PSCs comprised of AP and SA was demonstrated in an enzyme-linked immunosorbent assay. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c2ob26625c

Language：English   Publishing type：Research paper (scientific journal)  

The precise accumulation of protein functions on a nanoscale to fabricate advanced biomaterials has become possible by a bottom-up approach based on molecular self-assembly. The avidin-biotin interaction is widely employed in the design of functional protein self-assemblies. Herein we assessed how the spatial arrangement of the avidin-biotin interaction between protein building blocks affects the formation of a protein supramolecular complex (PSC). The enzymatic site-specific internal labeling of a symmetric protein scaffold, bacterial alkaline phosphatase (AP), with specifically designed biotinylation substrates revealed that the precise positioning of the biotinylation sites on AP and the linker flexibility of the substrate are critical factors for the growth of PSCs in the presence of streptavidin (SA). A potential diagnostic application of the PSCs comprised of AP and SA was demonstrated in an enzyme-linked immunosorbent assay.

DOI： 10.1039/c2ob26625c

Language：Japanese  

Development of Ionic Liquid Pharmaceuticals for Transdermal Drug Delivery

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bej.2012.10.002

Language：English   Publishing type：Research paper (scientific journal)  

S/O-nanodispersion electrospun nanofiber mesh effective for sustained release of healthy plasmid DNA with the structuraland functional integrity
Localized and sustained delivery of the therapeutic genes using a solid carrier matrix is a potential approach to develop highly curative treatments. Electrospun nanofiber mesh of biodegradable polymer has been applied extensively as a carrier for localized and sustained delivery of drugs, proteins, and DNA, but it remains difficult to release sufficient amounts of DNA while maintaining structural and functional integrity. To realize the stable sustained release of the healthy plasmid DNA (pDNA) from electrospun fiber mesh, a novel method was examined for loading pDNA into the fibers based on solid-in-oil (S/O) nanodispersion of pDNA in organic solvent for electrospinning polymer solution: S/O nanodispersion electrospinning. A prepared pDNA-loaded fiber mesh made of biodegradable polymer showed sustained release of pDNA without burst release. From luciferase activity-based in vitro transcription-translation assay, pDNA released from meshes of the S/O nanodispersion retained about 60% luciferase activity of control pDNA, whereas pDNA released from the meshes of simple mixing showed only about 5% activity, indicating that S/O nanodispersion electrospinning is effective for loading pDNA into electrospun fiber meshes while maintaining their healthy functions. Effectiveness of S/O nanodispersion electrospinning was verified for fabricating a sustained release carrier matrix for high molecular weight bioactives, including therapeutic genes.

DOI： 10.1080/09205063.2012.755600

Language：English   Publishing type：Research paper (scientific journal)  

In this paper, we designed and validated a new media for biotransformation aided by ionic liquefied amino acid substrates. Thermolysin-catalyzed synthesis of Z-aspartame (N-carbobenzoxy-l-aspartame) in which ionic liquids play dual roles as both substrate and reaction medium was conducted. If the protease can retain catalytic activity in this new liquefied amino acid substrate/media, the effective substrate concentration can be maximized, leading to theoretically high yields. In fact, we observed a 34-fold enhancement in the catalytic activity compared with that obtained in a previous report. The yield of Z-aspartame reached at approximately 660mM at 24h with 50mgmL-1 of the enzyme, thus submolar range productivity was achieved. The results suggest the successful construction of a high productivity reaction system for peptide synthesis. © 2012 Elsevier B.V.

DOI： 10.1016/j.bej.2012.10.002

Language：English   Publishing type：Research paper (scientific journal)  

A comparative study of ionic liquids (ILs) and a conventional organic solvent on the extraction of rare-earth ions from a nitrate-contained solution with TOPO has been investigated. A slope analysis was carried out to determine the extraction mechanism, and it was found that more TOPO molecules and the ionic constituents of the ILs have taken part in the extraction reaction in the ILs-based TOPO extraction systems. The numbers of TOPO molecules coordinated to the metal ion in the extracted complexes were 6 and 4 in the [C₄mim] and [C₈mim][Tf₂N] systems, although 3 TOPO molecules were needed in the n-dodecane system. Higher extraction efficiency was obtained in the extraction system in which the higher coordinating number of TOPO was shown. The extraction ability of [C₈mim][Tf₂N]-based TOPO depends significantly on the ionic constituents of the ILs in the extracting phase, but depends only slightly on the anionic nitrate ion from the aqueous phase, and [C₄mim][Tf₂N]-based TOPO is not dependent on the nitrate ion.

DOI： 10.15261/serdj.20.225

Language：English   Publishing type：Research paper (scientific journal)  

The extraction behavior of rare-earth ions with an 8-hydroxyquinoline derivative, HO8Q (5-octyloxymethyl-8-quinolinol), was studied using an ionic liquid, [C₈mim][Tf₂N] (1-octyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide), as the extracting phase. Compared with a conventional organic solvent, n-dodecane, the ionic liquid system showed a higher extraction ability for a heavy rare earth, Dy, and a better selectivity between Dy and Nd (separation factor β_Dy/Nd: 108). In the liquid-liquid extraction system, the color of the extracting phase changed from colorless to light yellow along with the extraction of rare-earth ions. Furthermore, the extraction efficiency was enhanced by the addition of TOPO (tri-n-octylphosphine oxide) as a co-extractant.

DOI： 10.15261/serdj.20.123

Language：English   Publishing type：Research paper (scientific journal)  

Biosorption, which takes place by passive metal-sequestering, has recently attracted attention as an alternative to conventional separation processes such as precipitation and solvent extraction. In this work, the adsorption behavior of rare earth elements (REEs) onto an Escherichia coli (E. coli) bacterial adsorbent was examined. The adsorption of REEs increased with increasing pH of the feed solution. Also, E. coli showed a higher selectivity for heavier REEs, especially for scandium. To elucidate the adsorption mechanism, the functional groups on the E. coli were chemically modified. By analyzing the adsorption behavior and FT-IR spectra of these E. coli samples before and after adsorption, it was confirmed that REEs are adsorbed at phosphate and carboxyl groups on the surface of E. coli. It is concluded that E. coli is a potential novel adsorbent for REEs.

DOI： 10.1252/jcej.13we031

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c2md20059g

Language：English   Publishing type：Research paper (scientific journal)  

A solid-in-oil nanodispersion that utilizes an oil-based vehicle of proteins was developed as a unique protein-delivery system into the skin. We enhanced the permeability of insulin and accomplished transdermal delivery of insulin by the collaborative effects of a S/O nanodispersion and arginine-rich peptides.

DOI： 10.1039/c2md20059g

Language：English   Publishing type：Research paper (scientific journal)  

Drug delivery systems using a solid-in-oil (S/O) dispersion as a core technology have advanced significantly over the past ten years. A novel, effective and practical preparation method for a S/O dispersion was originally established in 1997 as a tool for enzymatic catalysis in organic media. This oil-based dispersion containing proteins in non-aqueous media had great potential for applications to other research with one of the most successful being its adaptation as a drug delivery system. The history and features of novel processes for preparing S/O dispersions are presented in this article. In addition, recent research into the use of S/O dispersions for innovative oral and skin drug delivery systems is discussed.

DOI： 10.1016/j.ijpharm.2012.09.007

Language：English  

Drug delivery systems using a solid-in-oil (S/O) dispersion as a core technology have advanced significantly over the past ten years. A novel, effective and practical preparation method for a S/O dispersion was originally established in 1997 as a tool for enzymatic catalysis in organic media. This oil-based dispersion containing proteins in non-aqueous media had great potential for applications to other research with one of the most successful being its adaptation as a drug delivery system. The history and features of novel processes for preparing S/O dispersions are presented in this article. In addition, recent research into the use of S/O dispersions for innovative oral and skin drug delivery systems is discussed. (c) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ijpharm.2012.09.007

Language：English   Publishing type：Research paper (scientific journal)  

Treatment of a hyperthermophilic enzyme, alkaline phosphatase from Pyrococcus furiosus (PfuAP), with EDTA completely deactivated PfuAP, indicating that the presence of one or more divalent metal ions is essential for its catalytic activity. Subsequent addition of various divalent metal ions to the apoprotein recovered the enzymatic activity and, in particular, the addition of Co(II) resulted in an over 50-fold increase in activity compared with PfuAP before EDTA treatment. Intriguingly, PfuAP with Co(II) exhibited weaker stability toward heat treatment, suggesting that Co2+ destabilizes the tertiary structure of PfuAP at high temperature.

DOI： 10.1007/s10529-012-0998-0

Language：English   Publishing type：Research paper (scientific journal)  

Treatment of a hyperthermophilic enzyme, alkaline phosphatase from Pyrococcus furiosus (PfuAP), with EDTA completely deactivated PfuAP, indicating that the presence of one or more divalent metal ions is essential for its catalytic activity. Subsequent addition of various divalent metal ions to the apoprotein recovered the enzymatic activity and, in particular, the addition of Co(II) resulted in an over 50-fold increase in activity compared with PfuAP before EDTA treatment. Intriguingly, PfuAP with Co(II) exhibited weaker stability toward heat treatment, suggesting that Co²⁺ destabilizes the tertiary structure of PfuAP at high temperature.

DOI： 10.1007/s10529-012-0998-0

Language：English   Publishing type：Research paper (scientific journal)  

The aim of this study was to develop ionic liquid (IL) polymer materials incorporating enzymes that can be used as active, stable and reusable biocatalysts. To this goal, Candida rugosa lipase has been microencapsulated in surfactant aggregates formed in an IL monomer or the solution of an IL monomer/IL and then incorporated into polymer frameworks through the free radical polymerization of an IL (1-vinyl-3-ethylimidazolium bis(trifluoromethyl- sulfonyl) amide) ([veim][Tf₂N]). The activity, stability and reusability of such IL polymer materials containing lipase were evaluated using lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-PNB) as a model reaction. Lipase encapsulated within ionic liquid polymer materials remained active and exhibited excellent stability in aqueous solutions. More importantly, these biopolymer materials retained most of their activity after five reaction cycles, in which biopolymers were recovered from the reaction mixture simply by centrifugation. This study promulgates a direction toward the design of IL-an interesting class of tunable and designable solvents-based polymer materials containing biomolecules via a combination of polymer and supramolecular chemistry for diverse applications.

DOI： 10.1039/c2ob25529d

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ejpb.2012.05.016

Language：English   Publishing type：Research paper (scientific journal)  

Transdermal delivery of methotrexate (MIX) was investigated by using the solid-in-oil (S/O) technique. Because MTX was coated with nonionic surfactant molecules, the resulting complex was easy to dissolve in various organic solvents and provided a transparent solution in isopropyl myristate (IPM). The stability of MIX-surfactant complexes are enhanced by the addition of a basic amino acid such as L-Arginine (L-Arg) or L-Lysine (L-Lys). The average size of the dispersed complex of MIX and amino acid was reduced to below 100 nm and gave a uniform distribution. A transdermal delivery experiment was conducted using the S/O nanocarrier, and the permeation behavior of MIX through Yucatan micropig (YMP) skin was evaluated with a Franz diffusion cell. The permeation efficiency for the S/O nanocarrier (not urea addition) was two- to threefold increased compared to that of the control aqueous solution because the oil-based nanocarrier is effective for penetrating the stratum corneum. Furthermore, addition of urea has dramatically improved the release property of MIX from the S/O nanocarrier, and the S/O nanocarrier containing urea showed an optimal permeation efficiency of approximately 8.8-fold increased compared to that of the control aqueous solution after 24 h (p < 0.01). (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejpb.2012.05.016

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/bc300137s

Language：English   Publishing type：Research paper (scientific journal)  

Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced. to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross linking From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross linking toward negatively charged BAP, and the BAP SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.

DOI： 10.1021/bc300137s

Language：English   Publishing type：Research paper (scientific journal)  

A novel intracellular delivery method both for genes and proteins is one of the most coveted systems in the drug delivery field. In the present study, we developed a double-coating carrier loaded with gene and protein produced by solid-in-oil and solid-in-water nanodispersion technology. The double-coating carriers did not require electrostatic interactions during the preparation so were able to encapsulate plasmid DNA, ovalbumin (pI 4.5), horseradish peroxidase (pI 7.2), and cytochrome-c (pI 10.5) in a consistent manner. The carriers had practical encapsulation efficiencies and release profiles for genes and proteins. Furthermore, effective gene expression and cellular uptakes of both anionic and cationic proteins were achieved by modification of carriers with functional molecules. These findings indicate that the double-coating carrier has high potential for cellular delivery of various drugs and is a novel, superior method for both gene and protein delivery into cells. (c) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jconrel.2012.05.001

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jconrel.2012.05.001

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

In situハイブリダイゼーション法は遺伝子機能解明のための重要な基礎的技術となっているが、免疫染色技術を利用する一般的な方法では、煩雑で時間のかかる操作が必要とされる。本研究では、簡易な操作でターゲット核酸を検出するための新たなRNA―酵素複合体の調製方法を開発し、それを用いた組織切片上での高感度核酸検出を達成した。

DOI： 10.1021/ac2034198

Language：English   Publishing type：Research paper (scientific journal)  

Detection and localization of specific DNA or RNA sequences in cells and tissues are of great importance for biological research, diagnosis, and environmental monitoring. However, the most common procedure for in situ hybridization employs laborious immunostaining techniques. In the present study, we report proof-of-concept for a new RNA enzyme conjugated probe for the detection of mRNA on tissue sections with a simple procedure. An RNA probe modified with a specific dipeptide substrate of transglutaminase was prepared. Alkaline phosphatase was then covalently and site specifically combined to the dipeptide-labeled RNA using microbial transglutaminase. The new RNA probe labeled with alkaline phosphatase-was validated by in situ hybridization (ISH) and proved to be a sensitive and sequence specific probe for mRNA detection in tissues.. The new transglutaminase-mediated ISH (TransISH) strategy is free from antigen-antibody reaction, leads to one-step signal amplification after hybridization, and thus will be widely applicable for highly sensitive nucleic acid detection.

DOI： 10.1021/ac2034198

Language：English   Publishing type：Research paper (scientific journal)  

We report a one-pot biological approach to fabricate gold nanoparticle (AuNP)-ZZ domain conjugates using peptide-functionalized proteins that can simultaneously direct both biomineralization and surface modification of AuNPs. In addition, immuno-AuNPs are readily prepared through the specific binding of antibodies to the ZZ domain on the AuNPs.

DOI： 10.1039/c2an35172b

Language：English   Publishing type：Research paper (scientific journal)  

Protein molecules were precisely arrayed on a designable DNA scaffold close to each other using a DNA aptamer. By adding a chemical cross-linker, the neighboring protein molecules were effectively and covalently cross-linked to each other without losing their activities.

DOI： 10.1039/c2cc30618b

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese  

チロシンタグ導入タンパク質/フェノール性水酸基含有ヒドロゲル融合基材の開発

Language：Japanese  

チロシルラジカルの電荷指向型カップリング反応によるタンパク質架橋

Language：English   Publishing type：Research paper (scientific journal)  

We report a method to prepare a DNA-enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K-d was approximately 10(-6) (M-1) toward a His-tag present on a recombinant protein via the complexation of Ni2+. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni2+ complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni2+. complex. The DNA-enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2011.11.028

Language：English   Publishing type：Research paper (scientific journal)  

Oral administration of biomolecular drugs such as peptides, proteins, and DNA is an attractive delivery method because of the safety and convenience of delivery in contrast to injection administration. However, oral delivery of biomolecules has several potential barriers such as enzymatic degradation in the gastrointestinal tract and low permeability across an intestinal membrane. In this study, we proposed an intestinal patch system that included surfactant-coated insulin for oral delivery. The intestinal patches, which have mucoadhesive and drug-impermeable layers, induced sustained unidirectional insulin release toward intestinal mucosa and inhibition of insulin leakage from the patches. Moreover, the surfactant-coated insulin, which has high compatibility with cell membranes, enhanced insulin transport across the intestinal membrane. This study demonstrates that the intestinal patches might improve protein permeability in the intestinal mucosa, thereby offering an innovative therapeutic strategy. (C) 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.actbio.2011.09.023

Language：English   Publishing type：Research paper (scientific journal)  

We report a method to prepare a DNA-enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K_d was approximately 10^-6 (M^-1) toward a His-tag present on a recombinant protein via the complexation of Ni²⁺. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni²⁺ complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni ²⁺ complex. The DNA-enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized.