Publications by authors named "Hiroshi Ueda"

454 Publications

Surfactant protein D: a useful marker for differentiation of drug-induced pneumonia and bacterial pneumonia.

Pneumonia (Nathan) 2021 Jun 5;13(1):11. Epub 2021 Jun 5.

Third Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuoka Shimoaizuki, 910-1193, Eiheiji, Fukui, Japan.

Background: Drug-induced pneumonia (d-pneumonia) and bacterial pneumonia (b-pneumonia) are often difficult to differentiate; therefore, this study examined the possibility of differentiating them using serum biomarkers.

Methods: The study included 22 and 16 patients diagnosed with b- and d-pneumonia, respectively, at our institution or affiliated institutions. For d-pneumonia, the causative drug was minocycline hydrochloride in four patients, gefitinib in two patients, nivolumab in two patients, pembrolizumab in two patients, sulfasalazine in two patients, loxoprofen in one patient, Bouiougitou in one patient, edoxaban tosilate hydrate in one patient, and abemaciclib in one patient. White blood cell (WBC), C-reactive protein (CRP), Krebs von den Lungen-6 (KL-6), surfactant protein (SP)-D, and SP-A levels were measured in each patient and compared between the groups.

Results: Significant differences were noted in the WBC and SP-D levels between the two groups (P < 0.05, P < 0.001), but not in the CRP, KL-6, or SP-A levels.

Conclusion: The study results suggest that SP-D is a useful marker for differentiating b-pneumonia and d-pneumonia.
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http://dx.doi.org/10.1186/s41479-021-00087-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178843PMC
June 2021

An enhanced open sandwich immunoassay by molecular evolution for noncompetitive detection of Alternaria mycotoxin tenuazonic acid.

Food Chem 2021 May 13;361:130103. Epub 2021 May 13.

Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China. Electronic address:

Open sandwich enzyme-linked immunosorbent assay (OS-ELISA), a novel noncompetitive immunoassay format, has shown great potential in rapid detection for small molecules compared with traditional competitive format. Here, an enhanced OS-ELISA towards the mycotoxin tenuazonic acid (TeA) was developed for the first time based on heavy chain variable region (V) and light chain variable region (V) from the hybridoma cells (3F10) producing anti-TeA monoclonal antibody (mAb). The established OS-ELISA exhibited a limit of detection of 0.08 ng/mL, and was 13 times more sensitive than mAb-based indirect competitive ELISA (ic-ELISA). The proposed assay was also applied to detect TeA contents in juice, flour and tomato ketchup samples with satisfactory recoveries of 87.6%-111.3%. Finally, the great accuracy of the established OS-ELISA method was validated by the standard ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS).
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http://dx.doi.org/10.1016/j.foodchem.2021.130103DOI Listing
May 2021

Simple Fluorogenic Cellular Assay for Histone Deacetylase Inhibitors Based on Split-Yellow Fluorescent Protein and Intrabodies.

ACS Omega 2021 Apr 7;6(15):10039-10046. Epub 2021 Apr 7.

Laboratory for Chemistry and Life Science, and Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Nagatsuta-cho, Yokohama, Kanagawa 226-8503, Japan.

Histone deacetylase (HDAC) inhibitors that regulate the posttranslational modifications of histone tails are therapeutic drugs for many diseases such as cancers, neurodegenerative diseases, and asthma; however, convenient and sensitive methods to measure the effect of HDAC inhibitors in cultured mammalian cells remain limited. In this study, a fluorogenic assay was developed to detect the acetylation of lysine 9 on histone H3 (H3K9ac), which is involved in several cancers, Alzheimer's disease, and autism spectrum disorder. To monitor the changes in H3K9ac levels, an H3K9ac-specific intrabody fused with a small fragment FP11 of the split-yellow fluorescent protein (YFP) (scFv-FP11) was expressed in mammalian cells, together with a larger YFP fragment FP1-10 fused with a nuclear localization signal. When the intranuclear level of H3K9ac is increased, the scFv-FP11 is more enriched in the nucleus via passive diffusion through the nuclear pores from the cytoplasm, which increases the chance of forming a fluorescent complex with the nuclear YFP1-10. The results showed that the YFP fluorescence increased when the cells were treated with HDAC inhibitors. Moreover, the sensitivity of the split YFP reporter system to three HDAC inhibitors was higher than that of a conventional cell viability test. The assay system will be a simple and sensitive detection method to evaluate HDAC inhibitor activities at the levels of both single cells and cell populations.
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http://dx.doi.org/10.1021/acsomega.0c06281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153662PMC
April 2021

Development of a Single Fluorescent Protein-Based Green Glucose Indicator by Semirational Molecular Design and Molecular Evolution.

Methods Mol Biol 2021 ;2274:89-100

Cell Signaling Group, WASEDA Bioscience Research Institute in Singapore (WABIOS), Singapore, Singapore.

Advances in live-cell imaging have been accelerated by the development of various fluorescent indicators. However, indicators that are suitable for multicolor imaging remain a challenge to develop. Herein, we have developed a single fluorescent protein (FP)-based indicator using a semirational molecular design and a molecular evolution approach. We first inserted a ligand-binding domain into the vicinity of an FP chromophore to convert the conformational change induced by ligand binding into a change in fluorescence intensity. We then optimized the linker regions between the FP and the ligand-binding domain to greatly expand the dynamic range (F/F) of the indicator. Our design and optimization methods are highly versatile and can be used to develop any single FP-based indicators, which will further advance the utility of live-cell imaging.
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http://dx.doi.org/10.1007/978-1-0716-1258-3_9DOI Listing
January 2021

BRET Q-Body: A Ratiometric Quench-based Bioluminescent Immunosensor Made of Luciferase-Dye-Antibody Fusion with Enhanced Response.

Anal Chem 2021 Jun 20;93(21):7571-7578. Epub 2021 May 20.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Nagatsuta-cho, Yokohama, Kanagawa 226-8503, Japan.

A quenchbody (Q-body) is an immunosensor comprising an antibody fragment containing an antigen-binding site that is site-specifically labeled with a fluorescent dye. The fluorescent dye of a Q-body is quenched in the absence of an antigen; however, its fluorescence recovers in the presence of an antigen, offering simple and rapid systems for antigen detection. In this study, we fused luciferase NanoLuc to a Q-body to construct a new immunosensor termed the "BRET Q-body" that can detect antigens based on the bioluminescence resonance energy transfer (BRET) principle. The resulting BRET Q-bodies for an osteocalcin peptide that emit three different emission colors could detect an antigen without the requirement of an external light source, based on ratiometric detection and color change with two wavelengths for the luciferase and fluorophore. Furthermore, the BRET Q-body produced unexpectedly higher responses up to 12-fold because of the increased BRET efficiency, probably associated with antigen-dependent dye movement. Thus, the BRET Q-body is a useful biosensor as a core of point-of-care tests.
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http://dx.doi.org/10.1021/acs.analchem.0c05217DOI Listing
June 2021

Specific inhibition of oncogenic RAS using cell-permeable RAS-binding domains.

Cell Chem Biol 2021 May 4. Epub 2021 May 4.

United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan. Electronic address:

Oncogenic RAS proteins, common oncogenic drivers in many human cancers, have been refractory to conventional small-molecule and macromolecule inhibitors due to their intracellular localization and the lack of druggable pockets. Here, we present a feasible strategy for designing RAS inhibitors that involves intracellular delivery of RAS-binding domain (RBD), a nanomolar-affinity specific ligand of RAS. Screening of 51 different combinations of RBD and cell-permeable peptides has identified Pen-cRaf-v1 as a cell-permeable pan-RAS inhibitor capable of targeting both G12C and non-G12C RAS mutants. Pen-cRaf-v1 crosses the cell membrane via endocytosis, competitively inhibits RAS-effector interaction, and thereby exerts anticancer activity against several KRAS-mutant cancer cell lines. Moreover, Pen-cRaf-v1 exhibits excellent activity comparable with a leading pan-RAS inhibitor (BI-2852), as well as high target specificity in transcriptome analysis and alanine mutation analysis. These findings demonstrate that specific inhibition of oncogenic RAS, and possibly treatment of RAS-mutant cancer, is feasible by intracellular delivery of RBD.
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http://dx.doi.org/10.1016/j.chembiol.2021.04.013DOI Listing
May 2021

Numerical Modeling for Sensitive and Rapid Molecular Detection by Membrane-Based Immunosensors.

Anal Chem 2021 May 6;93(19):7210-7219. Epub 2021 May 6.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan.

Rapid, simple, and sensitive point-of-care testing (POCT) has attracted attention in recent years due to its excellent potential for early disease diagnosis and health monitoring. The flow-through biosensor design is a candidate for POCT that utilizes the small-sized pores of a porous membrane as a recognition space where it emits a signal comparable to that of a conventional enzyme-linked immunosorbent assay within 35 min of detection time. In this paper, we present a numerical model for this immunosensing technology to systematically design an improved recognition system. The model considers mass transfer into the pore (convection and diffusion), the kinetics between the immobilized receptor and the target molecule, and the flow conditions, successfully leading to a bottleneck step (capture of secondary antibody) in sandwich-type detection. Our simulation results also show that this problem can be solved by adopting both appropriate receptors and analytical conditions. Eventually, the requirements to achieve the sensitivity required for POCT were fulfilled, which will allow for further development of immunosensing devices for disease detection.
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http://dx.doi.org/10.1021/acs.analchem.1c00250DOI Listing
May 2021

Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions.

Cells 2021 Mar 5;10(3). Epub 2021 Mar 5.

Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan.

Prothymosin alpha (ProTα) and S100A13 are released from C6 glioma cells under serum-free conditions via membrane tethering mediated by Ca-dependent interactions between S100A13 and p40 synaptotagmin-1 (Syt-1), which is further associated with plasma membrane syntaxin-1 (Stx-1). The present study revealed that S100A13 interacted with annexin A2 (ANXA2) and this interaction was enhanced by Ca and p40 Syt-1. Amlexanox (Amx) inhibited the association between S100A13 and ANXA2 in C6 glioma cells cultured under serum-free conditions in the in situ proximity ligation assay. In the absence of Amx, however, the serum-free stress results in a flop-out of ANXA2 through the membrane, without the extracellular release. The intracellular delivery of anti-ANXA2 antibody blocked the serum-free stress-induced cellular loss of ProTα, S100A13, and Syt-1. The stress-induced externalization of ANXA2 was inhibited by pretreatment with siRNA for P4-ATPase, ATP8A2, under serum-free conditions, which ablates membrane lipid asymmetry. The stress-induced ProTα release via Stx-1A, ANXA2 and ATP8A2 was also evidenced by the knock-down strategy in the experiments using oxygen glucose deprivation-treated cultured neurons. These findings suggest that starvation stress-induced release of ProTα, S100A13, and p40 Syt-1 from C6 glioma cells is mediated by the ANXA2-flop-out via energy crisis-dependent recovery of membrane lipid asymmetry.
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http://dx.doi.org/10.3390/cells10030567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998613PMC
March 2021

Applicability of an Experimental Grade of Hydroxypropyl Methylcellulose Acetate Succinate as a Carrier for Formation of Solid Dispersion with Indomethacin.

Pharmaceutics 2021 Mar 8;13(3). Epub 2021 Mar 8.

Physical Chemistry, Laboratory for Medicinal Chemistry Research, Shionogi & Co., Ltd., Osaka 561-0825, Japan.

The transformation of a crystalline drug into an amorphous form is a promising way to enhance the oral bioavailability of poorly water-soluble drugs. Blending of a carrier, such as a hydrophilic polymer, with an amorphous drug is a widely used method to produce a solid dispersion and inhibit crystallization. This study investigates an experimental grade of hydroxypropyl methylcellulose acetate succinate, HPMCAS-MX (MX), as a solid dispersion carrier. Enhancement of thermal stability and reduction of the glass transition temperature () of MX compared with those of the conventional grade were evaluated through thermogravimetric analysis and differential scanning calorimetry (DSC). The formation of a homogeneous amorphous solid dispersion between MX and indomethacin was confirmed by X-ray powder diffraction analysis, DSC, and Raman mapping. It was observed that 10-30% MX did not act as an anti-plasticizer, but the utilization of >40% MX caused an increase in and reduction of molecular mobility. This could be explained by a change in intermolecular interactions, inferred from infrared spectroscopy combined with principal component analysis. HPMCAS-MX exhibited similar performance to that of conventional-grade, HPMCAS-MG. Although HPMCAS-MX has thermal properties different from those of conventional-grade HPMCAS-MG, it retains its ability as a solid dispersion carrier.
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http://dx.doi.org/10.3390/pharmaceutics13030353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001926PMC
March 2021

Masticatory Function Assessment of Adult Patients With Cleft Lip and Palate After Orthodontic Treatment.

Cleft Palate Craniofac J 2021 Feb 12:1055665621991733. Epub 2021 Feb 12.

Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.

Objective: To determine whether orthodontically treated patients with cleft lip and palate (CLP) possess a different masticatory function than those of untreated patients with normal occlusion.

Design: Occlusal contact area, occlusal force, as well as masseter and anterior temporal muscular activity were measured during maximum voluntary clenching (MVC) tests. Mandibular left and right lateral movements during mastication were also assessed. To further elucidate the nature of masticatory function, especially to determine the rate of abnormal jaw movement patterns, a parametric error index (EI) was set. Finally, masticatory efficiency was evaluated with a glucose sensitive measuring device.

Participants: Fifteen patients with CLP who had previously completed the orthodontic treatments required to achieve an acceptable and more harmonious occlusion accepted to volunteer in this study along with 21 untreated patients who already possessed a normal occlusion.

Results: Patients with CLP showed a significantly lower occlusal force, reduced occlusal contact area, and decreased masticatory efficiency as well as significantly higher EI value when compared with controls. However, there was no significant difference when analyzing muscle activity, although masticatory efficiency was significantly different between the 2 groups. Despite this result, the scores obtained by the patients with CLP in the masticatory efficiency tests were still in the normal range.

Conclusions: Orthodontic treatment for adult patients with CLP provides a satisfactory result for the patients' masticatory ability albeit significantly less ideal compared with untreated patients with normal occlusion.
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http://dx.doi.org/10.1177/1055665621991733DOI Listing
February 2021

Recent Advances in Quenchbody, a Fluorescent Immunosensor.

Sensors (Basel) 2021 Feb 9;21(4). Epub 2021 Feb 9.

Tokyo Tech World Research Hub Initiative (WRHI), Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8503, Japan.

The detection of viruses, disease biomarkers, physiologically active substances, drugs, and chemicals is of great significance in many areas of our lives. Immunodetection technology is based on the specificity and affinity of antigen-antibody reactions. Compared with other analytical methods such as liquid chromatography coupled with mass spectrometry, which requires a large and expensive instrument, immunodetection has the advantages of simplicity and good selectivity and is thus widely used in disease diagnosis and food/environmental monitoring. Quenchbody (Q-body), a new type of fluorescent immunosensor, is an antibody fragment labeled with fluorescent dyes. When the Q-body binds to its antigen, the fluorescence intensity increases. The detection of antigens by changes in fluorescence intensity is simple, easy to operate, and highly sensitive. This review comprehensively discusses the principle, construction, application, and current progress related to Q-bodies.
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http://dx.doi.org/10.3390/s21041223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916128PMC
February 2021

Chronic generalized pain disrupts whole brain functional connectivity in mice.

Brain Imaging Behav 2021 Jan 11. Epub 2021 Jan 11.

Douglas Hospital Research Center, Department of Psychiatry, School of Medicine, McGill University, Montreal, Quebec, Canada.

Fibromyalgia (FM) is a generalized chronic pain condition whose pathophysiology is poorly understood, and both basic and translational research are needed to advance the field. Here we used the Sluka model to test whether FM-like pain in mice would produce detectable brain modifications using resting-state (rs) functional Magnetic Resonance Imaging (fMRI). Mice received intramuscular acid saline treatment, images were acquired at 7 T 5 days post-treatment, and pain thresholds tested 3 weeks post-scanning. Data-driven Independent Component Analysis revealed significant reduction of functional connectivity (FC) across several component pairs, with major changes for the Retrosplenial cortex (RSP) central to the default mode network, and to a lesser extent the Periaqueductal gray (PAG), a key pain processing area. Seed-to-seed analysis focused on 14 pain-related areas showed strongest FC reduction for RSP with several cortical areas (somatosensory, prefrontal and insular), and for PAG with both cortical (somatosensory) and subcortical (habenula, thalamus, parabrachial nucleus) areas. RSP-PAG FC was also reduced, and this decreased FC tended to be positively correlated with pain levels at individual subject level. Finally, seed-voxelwise analysis focused on PAG confirmed seed-to-seed findings and, also detected reduced PAG FC with the anterior cingulate cortex, increasingly studied in aversive pain effects. In conclusion, FM-like pain triggers FC alterations in the mouse, which are detected by rs-fMRI and are reminiscent of some human findings. The study reveals the causal fingerprint of FM-like pain in rodents, and indicates that both RSP and PAG connectional patterns could be suitable biomarkers, with mechanistic and translational value, for further investigations.
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http://dx.doi.org/10.1007/s11682-020-00438-9DOI Listing
January 2021

Allodynia by Splenocytes From Mice With Acid-Induced Fibromyalgia-Like Generalized Pain and Its Sexual Dimorphic Regulation by Brain Microglia.

Front Neurosci 2020 23;14:600166. Epub 2020 Dec 23.

Department of Pharmacology and Therapeutic Innovation, Nagasaki University Institute of Biomedical Sciences, Nagasaki, Japan.

Fibromyalgia (FM), a disease of unknown etiology characterized by chronic generalized pain, is partly recapitulated in an animal model induced by repeated acid saline injections into the gastrocnemius muscle. Here, we attempted to investigate the sex difference in pain hypersensitivity (mechanical allodynia and hypersensitivity to electrical stimulation) in the repeated acid saline-induced FM-like generalized pain (AcGP) model. The first unilateral acid injection into gastrocnemius muscle at day 0/D0 and second injection at D5 (post day 0, P0) induced transient and long-lasting mechanical allodynia, respectively, on both sides of male and female mice. The pretreatment with gonadectomy did not affect the first injection-induced allodynia in both sexes, but gradually reversed the second injection-induced allodynia in male but not female mice. Moreover, the AcGP in male mice was abolished by intracerebroventricular minocycline treatments during D4-P4 or P5-P11, but not by early treatments during D0-D5 in male but not female mice, suggesting that brain microglia are required for AcGP in late-onset and sex-dependent manners. We also found that the intravenous treatments of splenocytes derived from male but not female mice treated with AcGP caused allodynia in naive mice. In addition, the purified CD4 T cells derived from splenocytes of acid-treated male mice retained the ability to cause allodynia in naive mice. These findings suggest that FM-like AcGP has multiple sexual dimorphic mechanisms.
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http://dx.doi.org/10.3389/fnins.2020.600166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785978PMC
December 2020

Temporally Coupled Coordination of Eye and Body Movements in Baseball Batting for a Wide Range of Ball Speeds.

Front Sports Act Living 2020 26;2:64. Epub 2020 Jun 26.

Department of Information and Communications Engineering, School of Engineering, Tokyo Institute of Technology, Tokyo, Japan.

We investigated the visuomotor strategies of baseball batting, in particular, the relationship between eye and body (head and hip) movements during batting for a wide range of ball speeds. Nine college baseball players participated in the experiment and hit balls projected by a pitching machine operating at four different ball speeds (80, 100, 120, 140 km/h). Eye movements were measured with a wearable eye tracker, and body movements were measured with an optical motion capture system. In the early period of the ball's flight, batters foveated the ball with overshooting head movements in the direction of the ball's flight while compensating for the overshooting head movements with eye movements for the two slower ball speeds (80 and 100 km/h) and only head rotations for the two faster ball speeds (120 and 140 km/h). After that, batters made a predictive saccade and a quick head rotation to the future ball position before the angular velocity of the ball drastically increased. We also found that regardless of the ball speed, the onsets of the predictive saccade and the quick head movement were temporally aligned with the bat-ball contact and rotation of the hip (swing motion), but were not correlated with the elapsed time from the ball's release or the ball's location. These results indicate that the gaze movements in baseball batting are not solely driven by external visual information (ball position or velocity) but are determined in relation to other body movements.
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http://dx.doi.org/10.3389/fspor.2020.00064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7739824PMC
June 2020

Eye and Head Movements of Elite Baseball Players in Real Batting.

Front Sports Act Living 2020 29;2. Epub 2020 Jan 29.

Department of Information and Communications Engineering, School of Engineering, Tokyo Institute of Technology, Tokyo, Japan.

In baseball, batters swing in response to a ball moving at high speed within a limited amount of time-about 0. 5 s. In order to make such movement possible, quick and accurate trajectory prediction followed by accurate swing motion with optimal body-eye coordination is considered essential, but the mechanisms involved are not clearly understood. The present study aims to clarify the strategies of eye and head movements adopted by elite baseball batters in actual game situations. In our experiment, six current professional baseball batters faced former professional baseball pitchers in a scenario close to a real game (i.e., without the batters informed about pitch type in advance). We measured eye movements with a wearable eye-tracker and head movements and bat trajectories with an optical motion capture system while the batters hit. In the eye movement measurements, contrary to previous studies, we found distinctive predictive saccades directed toward the predicted trajectory, of which the first saccades were initiated approximately 80-220 ms before impact for all participants. Predictive saccades were initiated significantly later when batters knew the types of pitch in advance compared to when they did not. We also found that the best three batters started predictive saccades significantly later and tended to have fewer gaze-ball errors than the other three batters. This result suggests that top batters spend slightly more time obtaining visual information by delaying the initiation of saccades. Furthermore, although all batters showed positive correlations between bat location and head direction at the time of impact, the better batters showed no correlation between bat location and gaze direction at that time. These results raise the possibility of differences in the coding process for the location of bat-ball contact; namely, that top batters might utilize head direction to encode impact locations.
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http://dx.doi.org/10.3389/fspor.2020.00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7739578PMC
January 2020

A new species closely related to Acartia sinjiensis (Copepoda: Calanoida), from river estuaries of northern Luzon, the Philippines.

Zootaxa 2020 Nov 20;4881(3):zootaxa.4881.3.6. Epub 2020 Nov 20.

Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan..

We describe a brackish-water calanoid copepod Acartia (Acanthacartia) cagayanensis sp. nov. collected from river estuaries in the northernmost Luzon, the Philippines. The new species has several characteristic features that are typical to the A. plumosa group (A. (A.) plumosa Scott T., 1894, A. (A.) sinjiensis Mori, 1940 and A. (A.) tropica Ueda Hiromi, 1987); specifically, a short apical spine on the long terminal segment of male left leg 5, which is unique to the group. The morphological features of A. cagayanensis sp. nov. different from those of the A. plumosa group are the barrel-shaped genital double somite and the cylindrical basal part of the terminal segment of female leg 5. Among the species in the group, A. cagayanensis sp. nov. is closest to A. sinjiensis in terms of the spinule patterns on the female antennule, the posterior corner of the prosome, and the male second urosomite. The genetic analysis using DNA sequences of mitochondrial gene cytochrome oxidase subunit I (COI) revealed that A. sinjiensis from Japan and A. cagayanensis sp. nov. differed by 16.5-16.9%, in contrast to a small variation (0.0-0.5%) within each population. We confirmed that previous records of A. sinjiensis from the Philippines were not A. cagayanensis sp. nov., and therefore, A. cagayanensis sp. nov. is the third species of the subgenus Acanthacartia Steuer, 1925, after A. sinjiensis and A. (A.) tsuensis Ito, 1956.
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http://dx.doi.org/10.11646/zootaxa.4881.3.6DOI Listing
November 2020

Co-amorphous formation of piroxicam-citric acid to generate supersaturation and improve skin permeation.

Eur J Pharm Sci 2021 Mar 6;158:105667. Epub 2020 Dec 6.

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan; Center for Transdermal Drug Delivery, Kyushu University, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan.

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.
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http://dx.doi.org/10.1016/j.ejps.2020.105667DOI Listing
March 2021

Pathogenic mechanisms of lipid mediator lysophosphatidic acid in chronic pain.

Authors:
Hiroshi Ueda

Prog Lipid Res 2021 01 28;81:101079. Epub 2020 Nov 28.

Department of Molecular Pharmacology, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto 606-8501, Japan. Electronic address:

A number of membrane lipid-derived mediators play pivotal roles in the initiation, maintenance, and regulation of various types of acute and chronic pain. Acute pain, comprising nociceptive and inflammatory pain warns us about the presence of damage or harmful stimuli. However, it can be efficiently reversed by opioid analgesics and anti-inflammatory drugs. Prostaglandin E and I, the representative lipid mediators, are well-known causes of acute pain. However, some lipid mediators such as lipoxins, resolvins or endocannabinoids suppress acute pain. Various types of peripheral and central neuropathic pain (NeuP) as well as fibromyalgia (FM) are representatives of chronic pain and refractory owing to abnormal pain processing distinct from acute pain. Accumulating evidence demonstrated that lipid mediators represented by lysophosphatidic acid (LPA) are involved in the initiation and maintenance of both NeuP and FM in experimental animal models. The LPAR-mediated peripheral mechanisms including dorsal root demyelination, Caα2δ1 expression in dorsal root ganglion, and LPAR-mediated amplification of central LPA production via glial cells are involved in the series of molecular mechanisms underlying NeuP. This review also discusses the involvement of lipid mediators in emerging research directives, including itch-sensing, sexual dimorphism, and the peripheral immune system.
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http://dx.doi.org/10.1016/j.plipres.2020.101079DOI Listing
January 2021

An Open Sandwich Immunochromatography for Non-competitive Detection of Small Antigens.

Anal Sci 2021 Mar 20;37(3):455-459. Epub 2020 Nov 20.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology.

Immunochromatography assay is an easy and rapid on-site detection method. However, conventional sandwich immunochromatographies using two antibodies can only detect target molecules above a threshold size. Small molecules below 1000 in molecular weight are usually detected using competitive immunoassay. However, competitive immunoassay is not suitable for visual detection of low concentration samples. Based on the principles of open sandwich immunoassay, which detects small molecules via interchain interaction of separated variable region fragments (V and V) from a single antibody, we developed non-competitive open sandwich immunochromatography. Bone Gla protein (BGP)-C7, a peptide containing the seven C-terminal amino acids of human osteocalcin, was selected as the target. By using V fragments fixed on a nitrocellulose membrane, and colored cellulose bead-labeled V fragments, we specifically detected 10 ng/mL of BGP-C7. This is the first report of open sandwich immunochromatography, which is an easy and rapid method for on-site, signal-on detection of small molecules.
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http://dx.doi.org/10.2116/analsci.20SCP06DOI Listing
March 2021

Expression dynamics of the genes for the hypothalamo-pituitary-gonadal axis in tiger puffer (Takifugu rubripes) at different reproductive stages.

Gen Comp Endocrinol 2021 Jan 13;301:113660. Epub 2020 Nov 13.

Marine Biological Station, Sado Island Center for Ecological Sustainability, Niigata University, Sado, Niigata 952-2135, Japan. Electronic address:

Tiger puffer, Takifugu rubripes, a commercially important long-distance migratory fish, return to specific spawning grounds for reproduction. To clarify reproductive neuroendocrine system of the tiger puffer, the changes in the expression levels of the genes encoding three gonadotropin-releasing hormones (GnRHs), gonadotropin-inhibitory hormone (GnIH), GnIH receptor (GnIH-R), kisspeptin and kisspeptin receptor in the brain and gonadotropin (GTH) subunits, growth hormone (GH) and prolactin (PRL) in the pituitary were examined in the tiger puffer captured in the wild at different reproductive stages, namely immature and mature fish of both sexes, and post-ovulatory females that were obtained by hormonal treatment. The amounts of three gnrh mRNAs, gnih, gnih-r, fshb and lhb were substantially increased in the mature fish compared to the immature fish, especially in the females, and these augmented expressions were drastically decreased in the post-ovulatory females. gh expression showed a slight increase in the mature males. In contrast, kiss2, kiss2r and prl did not show significant changes in the males but significantly decreased in the post-ovulatory females. The present results demonstrate the expression dynamics of the hypothalamo-pituitary-gonadal axis genes associated with the reproductive conditions and the possible involvement of the GnRH/GnIH/GTH system in the regulation of the sexual maturation and spawning in the wild tiger puffer.
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http://dx.doi.org/10.1016/j.ygcen.2020.113660DOI Listing
January 2021

Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging.

Sci Rep 2020 11 11;10(1):19562. Epub 2020 Nov 11.

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902, Japan.

Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can then produce lactate from pyruvate. Here, we developed single fluorescent protein (FP)-based lactate and pyruvate indicators with low EC for trace detection of metabolic molecules and live cell imaging and named them "Green Lindoblum" and "Green Pegassos," respectively. Green Lindoblum (EC of 30 µM for lactate) and Green Pegassos (EC of 70 µM for pyruvate) produced a 5.2- and 3.3-fold change in fluorescence intensity in response to lactate and pyruvate, respectively. Green Lindoblum measured lactate levels in mouse plasma, and Green Pegassos in combination with D-serine dehydratase successfully estimated D-serine levels released from mouse primary cultured neurons and astrocytes by measuring pyruvate level. Furthermore, live cell imaging analysis revealed their utility for dual-colour imaging, and the interplay between lactate, pyruvate, and Ca in human induced pluripotent stem cell-derived cardiomyocytes. Therefore, Green Lindoblum and Green Pegassos will be useful tools that detect specific molecules in clinical use and monitor the interplay of metabolites and other related molecules in diverse cell types.
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http://dx.doi.org/10.1038/s41598-020-76440-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659002PMC
November 2020

Creation of a Nanobody-Based Fluorescent Immunosensor Mini Q-body for Rapid Signal-On Detection of Small Hapten Methotrexate.

ACS Sens 2020 11 10;5(11):3457-3464. Epub 2020 Nov 10.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Nagatsuta-cho, Yokohama, Kanagawa 226-8503, Japan.

"Quenchbody (Q-body)" is a quench-based fluorescent biosensor labeled with a fluorescent dye near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules quickly by simply mixing with a sample. However, the development of Q-bodies using V-nanobodies derived from camelid heavy-chain antibodies has not been reported despite their favorable characteristics. Here, we report a "mini Q-body" that can detect the chemotherapy agent methotrexate (MTX) by using anti-MTX nanobody. Three kinds of constructs each encoding an N-terminal Cys-tag and anti-MTX VHH gene with a different length of linker (GGGS) ( = 0, 2, and 4) between them were prepared followed by the expression in and labeling with several dye maleimides. When the fluorescence intensities in the presence of varied MTX concentrations were measured, TAMRA-labeled nanobodies showed a higher response than ATTO520- or R6G-labeled ones. Especially, TAMRA C6-labeled mini Q-body with no linker showed the highest response of ∼6-fold and a low detection limit of 0.56 nM. When each Trp residue in the mini Q-body was mutated to address the quenching mechanism, the major role of Trp34 at CDR1 in quenching was revealed. Furthermore, the mini Q-body could detect MTX in 50% human serum with a low detection limit of 1.72 nM, showing its applicability to therapeutic drug monitoring. This study is expected to become the basis of the construction of highly responsive mini Q-bodies for sensitive detection of many molecules from small haptens to larger proteins, which will lead to broader applications such as point-of-care tests.
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http://dx.doi.org/10.1021/acssensors.0c01404DOI Listing
November 2020

Corner transfer matrix renormalization group analysis of the two-dimensional dodecahedron model.

Phys Rev E 2020 Sep;102(3-1):032130

Department of Physics, Graduate School of Science, Kobe University, Kobe 657-8501, Japan.

We investigate the phase transition of the dodecahedron model on the square lattice. The model is a discrete analog of the classical Heisenberg model, which has continuous O(3) symmetry. In order to treat the large on-site degree of freedom q=20, we develop a massively parallelized numerical algorithm for the corner transfer matrix renormalization group method, incorporating EigenExa, the high-performance parallelized eigensolver. The scaling analysis with respect to the cutoff dimension reveals that there is a second-order phase transition at T_{c}^{}=0.4398(8) with the critical exponents ν=2.88(8) and β=0.21(1). The central charge of the system is estimated as c=1.99(6).
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http://dx.doi.org/10.1103/PhysRevE.102.032130DOI Listing
September 2020

Involvement of SNARE Protein Interaction for Non-classical Release of DAMPs/Alarmins Proteins, Prothymosin Alpha and S100A13.

Cell Mol Neurobiol 2020 Aug 27. Epub 2020 Aug 27.

Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, 852-8521, Japan.

Prothymosin alpha (ProTα) is involved in multiple cellular processes. Upon serum-free stress, ProTα lacking a signal peptide sequence is non-classically released from C6 glioma cells as a complex with Ca-binding cargo protein S100A13. Thus, ProTα and S100A13 are conceived to be members of damage-associated molecular patterns (DAMPs)/alarmins. However, it remains to be determined whether stress-induced release of ProTα and S100A13 involves SNARE proteins in the mechanisms underlying membrane tethering of the multiprotein complex. In the present study, we used C6 glioma cells as a model of ProTα release. In pull-down assay, p40 synaptotagmin-1 (Syt-1), a vesicular SNARE, formed a hetero-oligomeric complex with homodimeric S100A13 in a Ca-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca-dependent in surface plasmon resonance (SPR). Immunoprecipitation using conditioned medium (CM) revealed that p40 Syt-1 was co-released with ProTα and S100A13 upon serum-free stress. In in situ proximity ligation assay (PLA), Syt-1 interacted with S100A13 upon serum-free stress in C6 glioma cells. The intracellular delivery of anti-Syt-1 IgG blocked serum free-induced release of ProTα and S100A13. Serum free-induced ProTα-EGFP release was significantly blocked by botulinum neurotoxin/C1 (BoNT/C1), which cleaves target SNARE syntaxin-1 (Stx-1). In immunocytochemistry, the cellular loss of ProTα-EGFP, S100A13, and Syt-1 was also blocked by BoNT/C1. Furthermore, the intracellular delivery of anti-Stx-1 IgG or Stx-1 siRNA treatment blocked Syt-1, S100A13 and ProTα release from C6 glioma cells. All these findings suggest that SNARE proteins play roles in stress-induced non-classical release of DAMPs/alarmins proteins, ProTα and S100A13 from C6 glioma cells.
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http://dx.doi.org/10.1007/s10571-020-00950-yDOI Listing
August 2020

Lysophosphatidic Acid Receptor 1- and 3-Mediated Hyperalgesia and Hypoalgesia in Diabetic Neuropathic Pain Models in Mice.

Cells 2020 08 16;9(8). Epub 2020 Aug 16.

Division of Genome Medicine, Institute of Advanced Medical Sciences, Tokushima University, Tokushim 770-8501, Japan.

Lysophosphatidic acid (LPA) signaling is known to play key roles in the initiation and maintenance of various chronic pain models. Here we examined whether LPA signaling is also involved in diabetes-induced abnormal pain behaviors. The high-fat diet (HFD) showing elevation of blood glucose levels and body weight caused thermal, mechanical hyperalgesia, hypersensitivity to 2000 or 250 Hz electrical-stimulation and hyposensitivity to 5 Hz stimulation to the paw in wild-type (WT) mice. These HFD-induced abnormal pain behaviors and body weight increase, but not elevated glucose levels were abolished in LPA and LPA mice. Repeated daily intrathecal (i.t.) treatments with LPA antagonist AM966 reversed these abnormal pain behaviors. Similar abnormal pain behaviors and their blockade by daily AM966 (i.t.) or twice daily Ki16425, another LPA antagonist was also observed in db/db mice which show high glucose levels and body weight. Furthermore, streptozotocin-induced similar abnormal pain behaviors, but not elevated glucose levels or body weight loss were abolished in LPA and LPA mice. These results suggest that LPA and LPA play key roles in the development of both type I and type II diabetic neuropathic pain.
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http://dx.doi.org/10.3390/cells9081906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465054PMC
August 2020

PM Q-probe: A fluorescent binding protein that converts many antibodies to a fluorescent biosensor.

Biosens Bioelectron 2020 Oct 3;165:112425. Epub 2020 Jul 3.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan; Tokyo Tech World Research Hub Initiative (WRHI), Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan. Electronic address:

Quenchbody (Q-body) is a fluorescent biosensor in which a fluorescent dye is tagged near the antigen binding site of an antibody. The fluorescence of the dye is quenched by the tryptophan residues present in the variable region of the antibody, and is recovered when the antigen binds. Q-bodies have been prepared using recombinant DNA technology by introducing one or more tag sequence(s) at either the N-terminal of the Fab or the single chain variable region fragment of the antibody, and labeling the tag with a fluorescent dye. However, preparation of recombinant antibody fragments is time-consuming and the performance of the Q-body is unpredictable. Here we report an antibody-binding quenching probe made from protein M from Mycoplasma genitalium that can transform the IgG antibody into an immunosensor. By using bacterially expressed and purified protein M and labeling the C-terminal cysteine-containing tag, we prepared a TAMRA-labeled PM Q-probe. When the Q-probe was incubated with Fab or IgG recognizing the bone Gla protein, the fluorescence of the probe was quenched and subsequently recovered by the adding of antigens in a dose-dependent manner. We also succeeded in detecting several small biomarkers with nanomolar sensitivity, including thyroxine extracted from human serum. The clone found to be suitable for the detection of cortisol was confirmed to work as a recombinant Q-body as well, which also worked in 50% human serum. The results suggest that the Q-probe can quickly convert an IgG to a biosensor, which will be useful in rapid diagnosis of small biomarkers.
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http://dx.doi.org/10.1016/j.bios.2020.112425DOI Listing
October 2020

Finite-m scaling analysis of Berezinskii-Kosterlitz-Thouless phase transitions and entanglement spectrum for the six-state clock model.

Phys Rev E 2020 Jun;101(6-1):062111

Department of Physics, Graduate School of Science, Kobe University, Kobe 657-8501, Japan.

We investigate the Berezinskii-Kosterlitz-Thouless transitions for the square-lattice six-state clock model with the corner-transfer matrix renormalization group (CTMRG). Scaling analyses for effective correlation length, magnetization, and entanglement entropy with respect to the cutoff dimension m at the fixed point of the CTMRG provide transition temperatures consistent with a variety of recent numerical studies. We also reveal that the fixed-point spectrum of the corner-transfer matrix in the critical intermediate phase of the six-state clock model is characterized by the scaling dimension consistent with the c=1 boundary conformal field theory associated with the effective Z_{6} dual sine-Gordon model.
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http://dx.doi.org/10.1103/PhysRevE.101.062111DOI Listing
June 2020

Formulation of co-amorphous systems from naproxen and naproxen sodium and in situ monitoring of physicochemical state changes during dissolution testing by Raman spectroscopy.

Int J Pharm 2020 Sep 16;587:119662. Epub 2020 Jul 16.

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Co-amorphous systems comprising low-molecular weight drugs and co-formers constitute an interesting approach to optimize pharmaceutical performance of drugs with low aqueous solubility. Within the different types of co-amorphous systems, the combination of a drug with its own salt may be an attractive formulation option due the absence of any inactive co-formers. The aim of this study was to investigate the possibility of forming a co-amorphous system from naproxen (NAP) and its sodium salt (NAP(Na)). Ball milling of NAP and NAP(Na) at equal molar ratio resulted in the formation of a co-amorphous system whilst NAP and NAP(Na) alone were crystalline following both, ball milling and melt quenching. Infrared spectroscopy and physical stability testing revealed that intermolecular interactions were able to maintain the ball milled NAP-NAP(Na) system amorphous for 2 months at 40 °C. Surprisingly, the dissolution rate of co-amorphous NAP-NAP(Na) was only intermediate between those of crystalline NAP and crystalline NAP(Na). In situ Raman spectroscopic measurements indicated an initial phase separation of the co-amorphous form to NAP and NAP(Na) followed by dissociation of sodium from NAP(Na) and crystallization to NAP. These findings contribute to the design of co-amorphous formulations with the combination of a drug and its own salt.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119662DOI Listing
September 2020

Mirtazapine, an 2 Antagonist-Type Antidepressant, Reverses Pain and Lack of Morphine Analgesia in Fibromyalgia-Like Mouse Models.

J Pharmacol Exp Ther 2020 10 14;375(1):1-9. Epub 2020 Jul 14.

Department of Pharmacology and Therapeutic Innovation, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

Treatment of fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly understood. In a series of studies, we have demonstrated that some pharmacological treatments reverse generalized chronic pain but do not affect the lack of morphine analgesia in the intermittent cold stress (ICS)-induced fibromyalgia-like pain model in mice. Here we report that repeated intraperitoneal treatments with mirtazapine, which is presumed to disinhibit 5-hydroxytriptamine (5-HT) release and activate 5-HT1 receptor through mechanisms of blocking presynaptic adrenergic 2 and postsynaptic 5-HT2 and 5-HT3 receptors, completely reversed the chronic pain for more than 4 to 5 days after the cessation of treatments. The repeated mirtazapine treatments also recovered the morphine analgesia after the return of nociceptive threshold to the normal level. The microinjection of small interfering RNA (siRNA) adrenergic 2a receptor (ADRA2A) into the habenula, which showed a selective upregulation of 2 receptor gene expression after ICS, reversed the hyperalgesia but did not recover the morphine analgesia. However, both reversal of hyperalgesia and recovery of morphine analgesia were observed when siRNA ADRA2A was administered intracerebroventricularly. As the habenular is reported to be involved in the emotion/reward-related pain and hypoalgesia, these results suggest that mirtazapine could attenuate pain and/or augment hypoalgesia by blocking the habenular 2 receptor after ICS. The recovery of morphine analgesia in the ICS model, on the other hand, seems to be mediated through a blockade of 2 receptor in unidentified brain regions. SIGNIFICANCE STATEMENT: This study reports possible mechanisms underlying the complete reversal of hyperalgesia and recovery of morphine analgesia by mirtazapine, a unique antidepressant with adrenergic 2 and serotonergic receptor antagonist properties, in a type of intermittently repeated stress (ICS)-induced fibromyalgia-like pain model. Habenula, a brain region which is related to the control of emotional pain, was found to play key roles in the antihyperalgesia, whereas other brain regions appeared to be involved in the recovery of morphine analgesia in the ICS model.
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http://dx.doi.org/10.1124/jpet.120.265942DOI Listing
October 2020

Molecular Design of Polyampholytes for Vitrification-Induced Preservation of Three-Dimensional Cell Constructs without Using Liquid Nitrogen.

Biomacromolecules 2020 08 27;21(8):3017-3025. Epub 2020 Jul 27.

The Joint Graduate School of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-8580, Japan.

Current slow-freezing methods are too inefficient for cryopreservation of three-dimensional (3D) tissue constructs. Additionally, conventional vitrification methods use liquid nitrogen, which is inconvenient and increases the chance of cross-contamination. Herein, we have developed polyampholytes with various degrees of hydrophobicity and showed that they could successfully vitrify cell constructs including spheroids and cell monolayers without using liquid nitrogen. The polyampholytes prevented ice crystallization during both cooling and warming, demonstrating their potential to prevent freezing-induced damage. Monolayers and spheroids vitrified in the presence of polyampholytes yielded high viabilities post-thawing with monolayers vitrified with PLL-DMGA exhibiting more than 90% viability. Moreover, spheroids vitrified in the presence of polyampholytes retained their fusibilities, thus revealing the propensity of these polyampholytes to stabilize 3D cell constructs. This study is expected to open new avenues for the development of off-the-shelf tissue engineering constructs that can be prepared and preserved until needed.
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http://dx.doi.org/10.1021/acs.biomac.0c00293DOI Listing
August 2020