研究者詳細 - 清水　一憲

論文 - 清水　一憲

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Microarray profiling of gene expression in C2C12 myotubes trained by electric pulse stimulation

Fujita, H; Horie, M; Shimizu, K; Nagamori, E

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 132 巻 ( 4 ) 頁： 417 - 422 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bioscience and Bioengineering

Electric pulse-stimulated C2C12 myotubes are gaining interest in the field of muscle physiology and biotechnology because electric pulse stimulation (EPS) enhances sarcomere structure development and active tension generation capability. Recently, we found that termination of EPS results in the rapid loss of active tension generation accompanied by disassembly of the sarcomere structure, which may represent an in vitro muscle atrophy model. To elucidate the molecular mechanism underlying this rapid loss of active tension generation and sarcomere structure disassembly after termination of EPS, we performed transcriptomic analysis using microarray. After termination of EPS, 74 genes were upregulated and 120 genes were downregulated after 30 min; however, atrophy-related genes were not found among these genes. To further assess the effect of EPS on gene expression, we re-applied EPS after its termination for 8 h and searched for genes whose expression was reversed. Four genes were upregulated by termination of EPS and downregulated by the re-application of EPS, whereas two genes were downregulated by termination of EPS and upregulated by the re-application of EPS. Although none of these genes were atrophy- or hypertrophy-related, the results presented in this study will contribute to the understanding of gene expression changes that mediate rapid loss of active tension generation and sarcomere structure disassembly following termination of EPS in C2C12 myotubes.

DOI： 10.1016/j.jbiosc.2021.06.016

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In Silico Screening of a Bile Acid Micelle Disruption Peptide for Oral Consumptions from Edible Peptide Database Open Access

Imai, K; Takeuchi, Y; Shimizu, K; Honda, H

FOODS 10 巻 ( 10 ) 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Foods

Recently, many bioactive peptides have been identified using bioinformatics tools. Previously, our group developed a method to screen dual-functional peptides that have direct intestinal delivery with porous silica gel and bile acid micelle disruption. However, newly designed peptides were not found in any storage protein. Therefore, in this study, in silico screening was performed using a 350,000 edible peptide library consisting of 4-to 7-mer independent peptides. As an initial screening, all edible peptides were applied to the random forest model to select predicted positive peptides. For a second screening, the peptides were assessed for the possibility of intestinal delivery using a 3D color map. From this approach, three novel dual-functional peptides, VYVFDE, WEFIDF, and VEEFYC were identified, and all of them were derived from storage proteins (legumin, myosin, and 11S globulin). In particular, VEEFYCS, in which a serine residue (S) is added to VEEFYC, was assumed to be released by thermolysin from the 11S-globulin derived from Ginkgo biloba by LC-MS/MS analysis. VEEFYCS was found to have suitable direct intestinal delivery and bile acid micelle disruption activity.

DOI： 10.3390/foods10102496

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions

Ishihara, M; Kikkawa, M; Shimizu, K; Suzuki, H; Honda, H

BIOTECHNOLOGY LETTERS 43 巻 ( 9 ) 頁： 1905 - 1911 2021年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biotechnology Letters

Objectives: To develop a simple pectin-degrading microorganism screening method. Results: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. Conclusions: The developed method is useful for identifying pectin-degrading microorganisms.

DOI： 10.1007/s10529-021-03162-6

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Machine learning screening of bile acid-binding peptides in a peptide database derived from food proteins Open Access

Imai, K; Shimizu, K; Honda, H

SCIENTIFIC REPORTS 11 巻 ( 1 ) 頁： 16123 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Bioactive peptides (BPs) are protein fragments that exhibit a wide variety of physicochemical properties, such as basic, acidic, hydrophobic, and hydrophilic properties; thus, they have the potential to interact with a variety of biomolecules, whereas neither carbohydrates nor fatty acids have such diverse properties. Therefore, BP is considered to be a new generation of biologically active regulators. Recently, some BPs that have shown positive benefits in humans have been screened from edible proteins. In the present study, a new BP screening method was developed using BIOPEP-UWM and machine learning. Training data were initially obtained using high-throughput techniques, and positive and negative datasets were generated. The predictive model was generated by calculating the explanatory variables of the peptides. To understand both site-specific and global characteristics, amino acid features (for site-specific characteristics) and peptide global features (for global characteristics) were generated. The constructed models were applied to the peptide database generated using BIOPEP-UWM, and bioactivity was predicted to explore candidate bile acid-binding peptides. Using this strategy, seven novel bile acid-binding peptides (VFWM, QRIFW, RVWVQ, LIRYTK, NGDEPL, PTFTRKL, and KISQRYQ) were identified. Our novel screening method can be easily applied to industrial applications using whole edible proteins. The proposed approach would be useful for identifying bile acid-binding peptides, as well as other BPs, as long as a large amount of training data can be obtained.

DOI： 10.1038/s41598-021-95461-1

Development of a human neuromuscular tissue-on-a-chip model on a 24-well-plate-format compartmentalized microfluidic device 国際誌 Open Access

Yamamoto, K; Yamaoka, N; Imaizumi, Y; Nagashima, T; Furutani, T; Ito, T; Okada, Y; Honda, H; Shimizu, K

LAB ON A CHIP 21 巻 ( 10 ) 頁： 1897 - 1907 2021年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Lab on a Chip

Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the physically separated structures of motor neurons (MNs) and skeletal muscle (SkM) fibers in the motor units in vivo. In this study, we aimed to develop microdevices for precisely compartmentalized coculturing of MNs and engineered SkM tissues. The developed microdevices, which fit a well of 24 well plates, had a chamber for MNs and chamber for SkM tissues. The two chambers were connected by microtunnels for axons, permissive to axons but not to cell bodies. Human iPSC (hiPSC)-derived MN spheroids in one chamber elongated their axons into microtunnels, which reached the tissue-engineered human SkM in the SkM chamber, and formed functional neuromuscular junctions with the muscle fibers. The cocultured SkM tissues with MNs on the device contracted spontaneously in response to spontaneous firing of MNs. The addition of a neurotransmitter, glutamate, into the MN chamber induced contraction of the cocultured SkM tissues. Selective addition of tetrodotoxin or vecuronium bromide into either chamber induced SkM tissue relaxation, which could be explained by the inhibitory mechanisms. We also demonstrated the application of chemical or mechanical stimuli to the middle of the axons of cocultured tissues on the device. Thus, compartmentalized neuromuscular tissue models fabricated on the device could be used for phenotypic screening to evaluate the cellular type specific efficacy of drug candidates and would be a useful tool in fundamental research and drug development for neuromuscular disorders.

DOI： 10.1039/d1lc00048a

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Electrospray propelled by ionic wind in a bipolar system for direct delivery of charge reduced nanoparticles 国際共著 Open Access

Dau, VT; Vu, TH; Tran, CD; Nguyen, TV; Nguyen, TK; Dinh, T; Phan, HP; Shimizu, K; Nguyen, NT; Dao, DV

APPLIED PHYSICS EXPRESS 14 巻 ( 5 ) 2021年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Applied Physics Express

We present a conceptual design to generate and deliver nanoparticles in one unique system based on electrohydrodynamic atomisation (EHDA) without the restriction of the collector. The present EHDA bipolar configuration consists of a capillary nozzle and a pin, both act as emitters and as the reference electrodes of each other. Under an applied voltage, the capillary nozzle sprays droplets while the pin generates ion wind via corona discharge. During spraying process, droplets' charge is significantly reduced by interacting with counter ions and propelled away from the electrodes by the momentum of ion winds accumulated from corona discharge. Thus, the present technique can yield promising applications in effective respiratory delivery of nanomedicine. © 2021 The Japan Society of Applied Physics.

DOI： 10.35848/1882-0786/abf36b

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Screening of a novel free fatty acid receptor 1 (FFAR1) agonist peptide by phage display and machine learning based-amino acid substitution 査読有り

Yoshioka K.

Biochemical and Biophysical Research Communications 550 巻頁： 177 - 183 2021年4月

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character 査読有り

Kozaki I.

Journal of Peptide Science 27 巻 ( 1 ) 2021年1月

Screening of FFAR1-Activating Peptides by Molecular Structural Analysis

Yoshioka, K; Yamashita, H; Fujitani, M; Kato, R; Shimizu, K; Honda, H

KAGAKU KOGAKU RONBUNSHU 47 巻 ( 3 ) 頁： 64 - 68 2021年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Kagaku Kogaku Ronbunshu

To find FFAR1-activating peptides consisting of natural amino acid residues, molecular structure analysis was performed by hierarchical clustering using extraction of physicochemical parameters. When 3 mer to 6 mer peptide libraries were prepared and the molecular structure of each was compared with 5 chemical agonists, 12 peptides with high correlation coefficients of physicochemical parameters were found from the 6 mer peptide library. these peptides were then synthesized by Fmoc solid phase synthesis, and their activity was assessed by TGFα shedding assay using FFAR1-expressing HEK293. As a result, two peptides, GCGGSS and GASGCC, were identified as peptide agonists with relatively high activity. Although these peptides showed approximately one fithh of the activity of chemical agonist GW9508, they are the first examples that we know of FFAR1-activating peptides consisting of natural amino acid residues.

DOI： 10.1252/kakoronbunshu.47.64

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Study on functional expression and evaluation of cultured skeletal muscle cells using microfabricated devices

Shimizu K

Seibutsu-kogaku Kaishi 99 巻 ( 3 ) 頁： 122 - 128 2021年