研究者詳細 - 清水　一憲

論文 - 清水　一憲

分割表示 131 件中 1 - 20 件目／全件表示 >>

Identification of barley-derived peptides with angiotensin converting enzyme inhibitory activity

Endo, K; Akiyama, H; Kano, K; Mishiro-Sato, E; Karashima, T; Kajiwara, Y; Takashita, H; Shimizu, K; Honda, H

INTERNATIONAL JOURNAL OF FOOD ENGINEERING 21 巻 ( 2 ) 頁： 87 - 95 2025年3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：International Journal of Food Engineering

This study aimed to obtain angiotensin converting enzyme (ACE) inhibitory peptides from barley hordein, a protein rich in proline and glutamine, and to identify novel inhibitors. Hordein was hydrolyzed using four enzymes: pepsin, trypsin, papain, and orientase, an enzyme from Aspergillus oryzae known to cleave the C-terminal side of proline residues. The hydrolysate from orientase showed strong ACE inhibitory activity. LC-MS/MS analysis identified 35 peptides, including 23 derived from hordein and 9 known ACE inhibitors. After synthesizing 15 peptides, 7 showed significant ACE inhibition, all containing proline at the C-terminus. Notably, QQP, a new peptide, exhibited notable activity (IC50 40 μM). The results suggest that barley hordein is a valuable source of ACE inhibitory peptides, with QQP as a promising candidate.

DOI： 10.1515/ijfe-2024-0255

Quercetin induces a slow myofiber phenotype in engineered human skeletal muscle tissues Open Access

Nagai, A; Kaneda, Y; Izumo, T; Nakao, Y; Honda, H; Shimizu, K

FASEB JOURNAL 38 巻 ( 16 ) 頁： e70009 2024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：FASEB Journal

Skeletal muscle comprises slow and fast myofibers, with slow myofibers excelling in aerobic metabolism and endurance. Quercetin, a polyphenol, is reported to induce slow myofibers in rodent skeletal muscle both in vitro and in vivo. However, its effect on human myofiber types remains unexplored. In this study, we evaluated quercetin's impact on slow myofiber induction using human skeletal muscle satellite cells. In a two-dimensional culture, quercetin enhanced gene expression, contributing to muscle differentiation, and significantly expanded the area of slow-type myosin heavy chain positive cells. It also elevated the gene expression of Pgc1α, an inducer of slow myofibers. Conversely, quercetin did not affect mitochondrial abundance, fission, or fusion, but it did increase the gene expression of Cox7A2L, which aids in promoting mitochondrial supercomplexity and endurance, and Mb, which contributes to oxidative phosphorylation. In a three-dimensional culture, quercetin significantly extended the time to peak tension and half relaxation time of the engineered human skeletal muscle tissues constructed on microdevices. Moreover, quercetin enhanced the muscle endurance of the tissues and curbed the rise in lactate secretion from the exercised tissues. These findings suggest that quercetin may induce slow myofibers in human skeletal muscle.

DOI： 10.1096/fj.202400914RR

Discovery of fibroblast growth factor 2-derived peptides for enhancing mice skeletal muscle satellite cell proliferation

Fujii, I; Kinoshita, R; Akiyama, H; Nakamura, A; Iwamori, K; Fukada, S; Honda, H; Shimizu, K

BIOTECHNOLOGY JOURNAL 19 巻 ( 8 ) 2024年8月

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12

Ding, R; Xi, Y; Ito, A; Shimizu, K; Nagamori, E; Fujita, H; Kawamoto, T; Horie, M

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 137 巻 ( 6 ) 頁： 480 - 486 2024年6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

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

DOI： 10.1016/j.jbiosc.2024.03.005

Separation and enrichment of multiple bile acid micelle-disrupting peptides by adsorption/desorption process with heat-treated porous silica gels

Iriyama, M; Hagawa, H; Shimizu, S; Akiyama, H; Shimizu, K; Honda, H

BIOCHEMICAL ENGINEERING JOURNAL 205 巻 2024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：Biochemical Engineering Journal

Bile acid (BA) micelle-disrupting peptides are bioactive peptides (BPs) that suppress intestinal cholesterol adsorption and ameliorate blood cholesterol levels. To facilitate their application in the food industry, an effective and straightforward technology for BP separation and enrichment of BPs is required. For this purpose, we assessed the potential of heat-treated (HT) porous silica gels with an enhanced capacity for peptide adsorption/desorption (AD). Given the key characteristics of these peptides involving basic amino acid (AA) residues, we applied casein hydrolysate to HT silica gels for adsorption at neutral pH, where the surface yielded anionic silanolates through deprotonation. Subsequently, desorption was conducted at pH 2, at which point the surface returned to its neutral state. We confirmed a 335-fold increase in the BA micelle-disrupting activity of the resulting hydrolysates. LC-MS/MS analysis identified 783 peptides, confirming that the peptides containing cationic AA residues were selectively enriched, as expected. Moreover, a dose-response of randomly selected 27 peptides with various enrichment levels revealed the presence of multiple active peptides in the enriched groups. These results indicate that our method is effective for the separation and enrichment of BA micelle-disrupting peptides and holds promise for BP production.

DOI： 10.1016/j.bej.2024.109283

Electrical pulse stimulation-induced tetanic exercise simulation increases the secretion of extracellular vesicles from C2C12 myotubes

Murata, A; Akiyama, H; Honda, H; Shimizu, K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 672 巻頁： 177 - 184 2023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：Biochemical and Biophysical Research Communications

Extracellular vesicles (EVs) released into the blood during exercise mediate its whole-body health effects. The differentiation of EVs released by skeletal muscle cells in vivo from those released by other cells is challenging, therefore, it is unclear whether exercise increases the number of EVs secreted by skeletal muscle cells. In this study, we investigated whether exercise affects the quantity of EVs released from skeletal muscle cells using in vitro exercise models. C2C12 myotubes were cultured on a gel layer with 1 or 30 Hz electrical pulse stimulation (EPS) to induce contractions as an artificial simulating exercise. We found that tetanic contraction induced by 30 Hz EPS increased the number of secreted EVs. MicroRNA (miRNA)-seq analysis revealed that 30 Hz EPS altered the miRNA in the secreted EVs. Furthermore, expression analysis of genes related to the biogenesis and transport of EVs revealed that the expression of ALG-2 interacting protein X (Alix) was increased in response to 30 Hz EPS, and the peak value of intracellular Ca2+ in myotubes at 30 Hz EPS was higher than that at 1 Hz, indicating that the increase in intracellular Ca2+ concentration may be related to the increased secretion of EVs in response to 30 Hz EPS.

DOI： 10.1016/j.bbrc.2023.06.054

l-Anserine Increases Muscle Differentiation and Muscle Contractility in Human Skeletal Muscle Cells

Nagai, A; Ida, M; Izumo, T; Nakai, M; Honda, H; Shimizu, K

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 71 巻 ( 23 ) 頁： 8952 - 8958 2023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：Journal of Agricultural and Food Chemistry

l-Anserine, an imidazole peptide, has a variety of physiological activities, but its effects on skeletal muscle differentiation and muscle contractile force remain unknown. Thus, in this study, we investigated the effect of l-anserine on muscle differentiation and muscle contractile force in human skeletal muscle cells. In two-dimensional culture, 1 μM l-anserine significantly increased the myotube diameters (26.5 ± 1.71, 27.7 ± 1.08, and 28.8 ± 0.85 μm with 0, 0.1, and 1 μM l-anserine, respectively) and the expression levels of genes involved in muscle differentiation and the sarcomere structure. In three-dimensional culture, 1 μM l-anserine significantly increased the contractile force of engineered human skeletal muscle tissues cultured on a microdevice (1.99 ± 0.30, 2.17 ± 0.62, 2.66 ± 0.39, and 3.28 ± 0.85 μN with 0, 0.1, 0.5, and 1 μM l-anserine, respectively). l-Anserine also increased the myotube diameters and the proportion of myotubes with sarcomere structures in the cultured tissues. Furthermore, the histamine receptor 1 (H1R) antagonist attenuated the l-anserine-induced increase in the contractile force, suggesting the involvement of H1R in the mechanism of action of l-anserine. This study showed for the first time that l-anserine enhances muscle differentiation and muscle contractility via H1R.

DOI： 10.1021/acs.jafc.3c01685

Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling Open Access

Rashid, MI; Ito, T; Miya, F; Shimojo, D; Arimoto, K; Onodera, K; Okada, R; Nagashima, T; Yamamoto, K; Khatun, Z; Shimul, RI; Niwa, J; Katsuno, M; Sobue, G; Okano, H; Sakurai, H; Shimizu, K; Doyu, M; Okada, Y

SCIENTIFIC REPORTS 13 巻 ( 1 ) 頁： 8146 2023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：Scientific Reports

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.

DOI： 10.1038/s41598-023-34445-9

Drug-preloadable methacrylated gelatin microspheres fabricated using an aqueous two-phase system

Mizukami, Y; Yamaguchi, T; Shiono, M; Takahashi, Y; Shimizu, K; Konishi, S; Takakura, Y; Nishikawa, M

EUROPEAN POLYMER JOURNAL 181 巻 2022年12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：European Polymer Journal

Gelatin microspheres (GMS) can retain biomolecules and be incorporated into artificial tissues and organs for tissue regeneration. However, the inactivation of biomolecules during the conventional fabrication process of GMS requires postloading of biomolecules to prefabricated GMS. Herein, a novel method for fabricating drug-preloaded GMS using a water-in-water (w/w) emulsification technique, is reported. A highly concentrated gelatin methacrylate (Gm) solution containing fibroblast growth factor 2 (FGF2), a model drug, was emulsified in a highly concentrated polyethylene glycol solution, and the mixture was irradiated with ultraviolet light. The obtained water-in-water gelatin methacrylate microspheres (w/w GmMS) were superior to conventional GMS in terms of stability, FGF2-loading efficiency, and sustained release of FGF2. The FGF2-loaded w/w GmMS efficiently activated the proliferation of adipose tissue-derived mesenchymal stem cells (ASCs). In addition, FGF2-loaded w/w GmMS incorporated into multicellular spheroids of ASCs increased the viability of ASCs in the spheroids. The therapeutic efficacy of ASC spheroids in a wound healing mouse model was significantly improved by the incorporation of FGF2-loaded w/w GmMS. These results indicate that drug-preloadable w/w GmMS can be widely applied in tissue engineering and regenerative medicine strategies.

DOI： 10.1016/j.eurpolymj.2022.111671

Quality evaluation of cell spheroids for transplantation by monitoring oxygen consumption using an on-chip electrochemical device. Open Access

Tsujimura M, Kusamori K, Takamura K, Ito T, Kaya T, Shimizu K, Konishi S, Nishikawa M

Biotechnology reports (Amsterdam, Netherlands) 36 巻頁： e00766 2022年12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 Reports

Three-dimensional cell spheroids are superior cell-administration form for cell-based therapy which generally exhibit superior functionality and long-term survival after transplantation. Here, we nondestructively measured the oxygen consumption rate of cell spheroids using an on-chip electrochemical device (OECD) and examined whether this rate can be used as a marker to estimate the quality of cell spheroids. Cell spheroids containing NanoLuc luciferase-expressing mouse mesenchymal stem cell line C3H10T1/2 (C3H10T1/2/Nluc) were prepared. Spheroids of high or low quality were prepared by altering the medium change frequency. After transplantation into mice, the high-quality C3H10T1/2/Nluc spheroids exhibited a higher survival rate than the low-quality ones. The oxygen consumption rate of the high-quality C3H10T1/2/Nluc spheroids was maintained at high levels, whereas that of the low-quality spheroids decreased with time. These results indicate that OECD-based measurement of the oxygen consumption rate can be used to estimate the quality of cell spheroids without destructive analysis of the spheroids.

DOI： 10.1016/j.btre.2022.e00766

Alignment of Skeletal Muscle Cells Facilitates Acetylcholine Receptor Clustering and Neuromuscular Junction Formation with Co-Cultured Human iPSC-Derived Motor Neurons Open Access

Shimizu, K; Kassai, H; Kamei, Y; Yamamoto, K; Nagashima, T; Maekawa, T; Akiyama, H; Honda, H

CELLS 11 巻 ( 23 ) 2022年12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：Cells

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models.

DOI： 10.3390/cells11233760

Analysis of plasmodesmata permeability using cultured tobacco BY-2 cells entrapped in microfluidic chips

Kurotani, K; Kawakatsu, Y; Kikkawa, M; Tabata, R; Kurihara, D; Honda, H; Shimizu, K; Notaguchi, M

JOURNAL OF PLANT RESEARCH 135 巻 ( 5 ) 頁： 693 - 701 2022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：Journal of Plant Research

Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as regulatory factors for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, a system was constructed to monitor the movement of substances by plasmodesmata using tobacco BY-2 cells, which are linearly organized cells, and a microfluidic device that traps them in place and facilitates observation. After targeting one cell for photobleaching, recovery of the lost H2B-GFP protein was detected within 200 min. No recovery was detected in that time frame by photobleaching the entire cell filaments. This suggested that the recovery of H2B-GFP protein was not due to de novo protein synthesis, but rather to translocation from neighboring cells. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, using the microfluidic device and BY-2 cells, it was confirmed that the behavior of plasmodesmata could be observed in real time under controllable conditions.

DOI： 10.1007/s10265-022-01406-8

Screening of anti-atrophic peptides by using photo-cleavable peptide array and 96-well scale contractile human skeletal muscle atrophy models

Yamamoto, K; Ohsumi, S; Nagashima, T; Akiyama, H; Honda, H; Shimizu, K

BIOTECHNOLOGY AND BIOENGINEERING 119 巻 ( 8 ) 頁： 2196 - 2205 2022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：Biotechnology and Bioengineering

Skeletal muscle atrophy is characterized by decreases in protein content, myofiber diameter, and contractile force generation. As muscle atrophy worsens the quality of life, the development of anti-atrophic substances is desirable. In this study, we aimed to demonstrate a screening process for anti-atrophic peptides using photo-cleavable peptide array technology and human contractile atrophic muscle models. We developed a 96-well system and established a screening process with less variability. Dexamethasone-induced human atrophic tissue was constructed in the system. Eight peptides were selected from the literature and used for the screening of peptides for preventing the decrease of the contractile forces of tissues. The peptide QIGFIW, which showed preventive activity, was selected as the seed sequence. As a result of amino acid substitution, we obtained QIGFIQ as a peptide with higher anti-atrophic activity. These results indicate that the combinatorial use of the photo-cleavable peptide array technology and 96-well screening system could comprise a powerful approach to obtaining anti-atrophic peptides, and suggest that the 96-well screening system and atrophic model represent a practical and powerful tool for the development of drugs/functional food ingredients.

DOI： 10.1002/bit.28125

Mechanical Regulation Of Provisional Matrix Assembly In 3D Fibrous Microtissues 国際共著

Eyckmans, J; Das, S; Shimizu, K; Chen, C

WOUND REPAIR AND REGENERATION 30 巻 ( 2 ) 頁： A20 - A21 2022年3月

Selective concentration of antimicrobial peptides to heat-treated porous silica gel using adsorption/desorption Open Access

Hagawa, H; Imai, K; Gao, ZW; Taniguchi, M; Shimizu, K; Honda, H

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 133 巻 ( 2 ) 頁： 161 - 167 2022年2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

Heat-treated porous silica gel (HT silica gel) previously developed by our group has selectively adsorbed cationic peptides at a pH of 7. Therefore, we focused on the use of antimicrobial peptides (AMPs) as bioactive peptides (BPs). First, 32 AMPs and 32 randomly designed peptides were generated using Fmoc solid synthesis, and their adsorption ratio to HT-silica gel was investigated. Thirty two AMPs showed a relatively higher adsorption ratio of 58.8% compared to that of randomly designed peptides, which was 35.3%. Desorption conditions were investigated using Amyl-1-18 antimicrobial peptides. Next, pepsin hydrolysate from rice endosperm protein (REP) powder was prepared by ourselves. The REP hydrolysate containing dry matter (7.5 mg) was applied to the adsorption/desorption (AD) procedure using HT silica gel to obtain 1.6 mg of AD hydrolysate. When the two hydrolysates were subjected to mass spectrometry, 305 concentrated peptides were obtained. In total, 26 peptides with high content and high enrichment ratios were listed and synthesized. When the antimicrobial activity of these 26 peptides was evaluated using Cutibacterium acnes, five peptides consisting of 12–27 amino acids were identified as novel AMPs. Two of these peptides, which were derived from rice glutelin, showed antimicrobial activity against all four microbes, including Porphyromonas gingivalis, Escherichia coli, and Streptococcus mutans. In the present study, we showed that AMPs could be easily enriched from protein hydrolysate using HT silica gel. The adsorption/desorption procedure using HT silica gel was confirmed to be a useful tool for convenient BP separation.

DOI： 10.1016/j.jbiosc.2021.11.002

Intravenous injection of mesenchymal stem cell spheroids improves the pulmonary delivery and prolongs in vivo survival

Shimazawa, Y; Kusamori, K; Tsujimura, M; Shimomura, A; Takasaki, R; Takayama, Y; Shimizu, K; Konishi, S; Nishikawa, M

BIOTECHNOLOGY JOURNAL 17 巻 ( 1 ) 頁： e2100137 2022年1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 Journal

Background: Because of the excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. Methods and Results: The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170 μm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. Conclusions: These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.

DOI： 10.1002/biot.202100137

Development of microfluidic chip for entrapping tobacco BY-2 cells Open Access

Shimizu, K; Kawakatsu, Y; Kurotani, KI; Kikkawa, M; Tabata, R; Kurihara, D; Honda, H; Notaguchi, M

PLOS ONE 17 巻 ( 4 ) 頁： e0266982 2022年

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

The tobacco BY-2 cell line has been used widely as a model system in plant cell biology. BY-2 cells are nearly transparent, which facilitates cell imaging using fluorescent markers. As cultured cells are drifted in the medium, therefore, it was difficult to observe them for a long period. Hence, we developed a microfluidic device that traps BY-2 cells and fixes their positions to allow monitoring the physiological activity of cells. The device contains 112 trap zones, with parallel slots connected in series at three levels in the flow channel. BY-2 cells were cultured for 7 days and filtered using a sieve and a cell strainer before use to isolate short cell filaments consisting of only a few cells. The isolated cells were introduced into the flow channel, resulting in entrapment of cell filaments at 25 out of 112 trap zones (22.3%). The cell numbers increased through cell division from 1 to 4 days after trapping with a peak of mitotic index on day 2. Recovery experiments of fluorescent proteins after photobleaching confirmed cell survival and permeability of plasmodesmata. Thus, this microfluidic device and one-dimensional plant cell samples allowed us to observe cell activity in real time under controllable conditions.

DOI： 10.1371/journal.pone.0266982

Detection of protease digestion site using fluorescence labeled peptide array and construction of machine-learning prediction model

Mizutani R., Mori Y., Ogawa S., Tazoe K., Akiyama H., Shimizu K., Honda H.

Seibutsu-kogaku Kaishi 100 巻 ( 10 ) 頁： 528 - 540 2022年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Seibutsu-kogaku Kaishi

For the prediction of protease digestion site, 1990 kinds of tetramer peptide were designed as a library, of which N terminal end was fluorescence labelled. Decrease of fluorescence intensity of each peptide was quantitatively determined and used as a training data for Random Forest (RF) modeling. All of dipeptides bond as a protease substrate were included in 1990 tetramer peptides. As an explanatory variable for model construction, 532 parameters were prepared and those were included not only appearance of amino acid residue or dipeptides but also positioning parameters (PP) or global parameters (GP) to explain the peptide property. Trypsin for biochemistry grade was used for hydrolysis. After digesting at pH 8, the histogram of digestion ratio was appeared that tetramers with arginine residue (R) and lysine residue (K) could be expectedly digested at higher ratio compared with tetramers without R or K. Constructed pH 8-RF model showed 77 % of prediction accuracy. The explanatory parameters with top 10 higher importance in pH 8-RF model were both of appearance of K and 9 GPs. GPs were including 4 isoelectric parameters and 1 polarity parameter, of which K or R residue have relatively large value. Digestion site of α-lactalbumin was determined using pH 8-RF model and the protein was actually digested by trypsin. When the hydrolysate was analyzed by LC-MS/MS, 42 peptides were identified. Fourteen sites among 19 predicted digestion sites were coincided with each other. Many peptide fragments digested at 69Y-70G site or 50F-51H site was detected and it was strongly suggested to be digested by chymotrypsin as a contaminated protease. These sites were fairly predicted by constructed RF model. In addition, trypsin digestion at pH 5 was carried out to investigate the effect of pH decrease on trypsin digestion. The prediction accuracy of pH 5-RF model was only 59 %. The parameters with top 10 higher importance in pH 5-RF model were including 4 GPs which was the same with 4 isoelectric parameter in pH 8 model. Twenty digestion sites of α-lactalbumin were predicted from pH 5-RF model. α-lactalbumin was digested at pH 5 by trypsin, and 64 peptides were identified. The predicted sites were compared with identified fragments at the region from 48T to 77K in which a lot of fragments were detected. It was concluded that constructed model could fairly predict some of newly identified sites and could roughly grasp slight modification of digestion site by pH change. The proposed methodology, which contained 1990 kinds of tetramer peptides as a library, 532 explanatory parameters and RF model, was effective for prediction of protease digestion site.

DOI： 10.34565/seibutsukogaku.100.10_528

Agonist/Antagonist Activity of Oxytocin Variants Obtained from Free Cyclic Peptide Libraries Generated via Amino Acid Substitution Open Access

Kinoshita, R; Kozaki, I; Shimizu, K; Shibata, T; Ochiai, A; Honda, H

ACS OMEGA 6 巻 ( 46 ) 頁： 31244 - 31252 2021年11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：ACS Omega

We established a method for synthesizing a free cyclic peptide library via peptide array synthesis to demonstrate the sequence activity of cyclic peptides. Variants of the cyclic nonapeptide oxytocin (OXT) were synthesized via residue substitution. Natural amino acids (AAs) were classified into eight groups based on their physical properties and the size of their side chains, and a representative AA from each group was selected for residue substitution. All OXT variants were systematically evaluated for agonist/antagonist activity. Consequently, no improvement in agonist activity was observed, although substitution of the P4 and P8 residues resulted in decreased activity due to AA substitution. A few OXT variants exhibited antagonistic activity. In particular, the variants with P2 Leu residue substitution (Y2L) and Phe substitutions at residues 4 (Q4F), 5 (N5F), and 7 (P7F) showed high antagonistic activity. Variant Y2W was found to have the highest inhibitory effect, with a dissociation constant of 44 nM, which was comparable to that of the commercial antagonist atosiban (21 nM). Therefore, a free cyclic peptide library constructed via substitution with a natural AA residue was confirmed to be a powerful tool for bioactive peptide screening.

DOI： 10.1021/acsomega.1c04982

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply Open Access

Mizukami, Y; Takahashi, Y; Shimizu, K; Konishi, S; Takakura, Y; Nishikawa, M

BIOLOGICAL & PHARMACEUTICAL BULLETIN 44 巻 ( 10 ) 頁： 1458 - 1464 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：Biological and Pharmaceutical Bulletin

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16–BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

DOI： 10.1248/bpb.b21-00269

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