Publications by authors named "Yuki Shibata"

32 Publications

Quantification of image quality of intra-fractional cone-beam computed tomography for arc irradiation with various imaging condition.

Rep Pract Oncol Radiother 2021 9;26(3):495-501. Epub 2021 Jun 9.

Department of Radiology, University of Yamanashi, Yamanashi, Japan.

Background: 3-dimensional intra-cone beam computed tomography (intra-CBCT ) could be a potentially powerful tool for use with arc irradiation such as volumetric modulated arc therapy. The aim of the study was to evaluate the image quality of intra-cone beam computed tomography (intra-CBCT ) for arc irradiation with various imaging condition.

Materials And Methods: Two types of intra-CBCT imaging techniques were evaluated - intra-fractional CBCT with flattening filtered (FF) beam (intra-FF CBCT ) and that with flattening filter free (FFF) beam (intra-FFF CBCT ). For the intra-MV beams, four different field sizes (2 cm × 2 cm, 5 cm × 5 cm, 10 cm × 10 cm, and 20 cm × 20 cm) were used with dose rates of 500 MU/min and 1600 MU/min, for 6 MV FF and 6 MV FFF, respectively. For all image acquisitions, two rotation angles (full-arc and half-arc) were investigated. Thereafter, the linearity, contrast-to-noise ratio (CNR), and uniformity index (UI) of intra-CBCT image were compared with those of conventional CBCT image.

Results: All acquisition conditions had good linearity of the CT value (R > 0.99). For CNR, the change rates from conventional CBCT ranged from 0.6-33.7% for a 2 cm × 2 cm beam, whereas that for a 20 cm × 20 cm beam ranged from 62.7-82.3%. Similarly, the UI increased from 1.5% to 7.0% as the field size increased.

Conclusion: Quality of intra-CBCT image was affected by the field size and acquisition angle. Image quality of intra-CBCT was worse than that of conventional CBCT, but it was better under a smaller field and wider correction angle and would be acceptable for clinical use.
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http://dx.doi.org/10.5603/RPOR.a2021.0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8281903PMC
June 2021

KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma.

J Genet Genomics 2021 Mar 10. Epub 2021 Mar 10.

Laboratory of Comparative Pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan.

Epigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we find that lysine-specific demethylase 2b (KDM2B) is highly expressed in HSA cell lines compared with normal canine endothelial cells. Silencing of KDM2B in HSA cells results in increased cell death in vitro compared with the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced KDM2B silencing in tumor xenografts results in decreased tumor sizes compared with the control. Furthermore, KDM2B is also highly expressed in clinical cases of HSA. We hypothesize that pharmacological KDM2B inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treat HSA cells with GSK-J4, a histone demethylase inhibitor, and find that GSK-J4 treatment also induces apoptosis and cell death. In addition, GSK-J4 treatment decreases tumor size. Therefore, we demonstrate that KDM2B acts as an oncogene in HSA by enhancing the DNA damage response. Moreover, we show that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.
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http://dx.doi.org/10.1016/j.jgg.2021.02.005DOI Listing
March 2021

12-Hydroxyeicosapentaenoic acid inhibits foam cell formation and ameliorates high-fat diet-induced pathology of atherosclerosis in mice.

Sci Rep 2021 May 17;11(1):10426. Epub 2021 May 17.

Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Asagi Saito, Ibaraki, Osaka, 567-0085, Japan.

Atherosclerosis is a chronic inflammatory disease associated with macrophage aggregate and transformation into foam cells. In this study, we sought to investigate the impact of dietary intake of ω3 fatty acid on the development of atherosclerosis, and demonstrate the mechanism of action by identifying anti-inflammatory lipid metabolite. Mice were exposed to a high-fat diet (HFD) supplemented with either conventional soybean oil or α-linolenic acid-rich linseed oil. We found that as mice became obese they also showed increased pulsatility and resistive indexes in the common carotid artery. In sharp contrast, the addition of linseed oil to the HFD improved pulsatility and resistive indexes without affecting weight gain. Histological analysis revealed that dietary linseed oil inhibited foam cell formation in the aortic valve. Lipidomic analysis demonstrated a particularly marked increase in the eicosapentaenoic acid-derived metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) in the serum from mice fed with linseed oil. When we gave 12-HEPE to mice with HFD, the pulsatility and resistive indexes was improved. Indeed, 12-HEPE inhibited the foamy transformation of macrophages in a peroxisome proliferator-activated receptor (PPAR)γ-dependent manner. These results demonstrate that the 12-HEPE-PPARγ axis ameliorates the pathogenesis of atherosclerosis by inhibiting foam cell formation.
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http://dx.doi.org/10.1038/s41598-021-89707-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129127PMC
May 2021

Thyroid Hormone Receptor Is Essential for Larval Epithelial Apoptosis and Adult Epithelial Stem Cell Development but Not Adult Intestinal Morphogenesis during Metamorphosis.

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

Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Vertebrate postembryonic development is regulated by thyroid hormone (T3). Of particular interest is anuran metamorphosis, which offers several unique advantages for studying the role of T3 and its two nuclear receptor genes, α and , during postembryonic development. We have recently generated TR double knockout (TRDKO) animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. Here we show that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, T3 treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and the cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation, whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth-muscle development and epithelial folding.
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http://dx.doi.org/10.3390/cells10030536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000126PMC
March 2021

The development of adult intestinal stem cells: Insights from studies on thyroid hormone-dependent anuran metamorphosis.

Vitam Horm 2021 9;116:269-293. Epub 2021 Mar 9.

Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, United States.

Vertebrates organ development often takes place in two phases: initial formation and subsequent maturation into the adult form. This is exemplified by the intestine. In mouse, the intestine at birth has villus, where most differentiated epithelial cells are located, but lacks any crypts, where adult intestinal stem cells reside. The crypt is formed during the first 3 weeks after birth when plasma thyroid hormone (T3) levels are high. Similarly, in anurans, the intestine undergoes drastic remodeling into the adult form during metamorphosis in a process completely dependent on T3. Studies on Xenopus metamorphosis have revealed important clues on the formation of the adult intestine during metamorphosis. Here we will review our current understanding on how T3 induces the degeneration of larval epithelium and de novo formation of adult intestinal stem cells. We will also discuss the mechanistic conservations in intestinal development between anurans and mammals.
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http://dx.doi.org/10.1016/bs.vh.2021.02.010DOI Listing
March 2021

Transferrin-based radiolabeled probe predicts the sensitivity of human renal cancer cell lines to ferroptosis inducer erastin.

Biochem Biophys Rep 2021 Jul 24;26:100957. Epub 2021 Feb 24.

Department of Biomedical Imaging, Graduate School of Biomedical Science and Engineering, Hokkaido University, Sapporo, Hokkaido, 060-8638, Japan.

Ferroptosis induction has been recognized as a novel cancer therapeutic strategy. To effectively apply ferroptosis-targeting cancer therapy to individual patients, a diagnostic indicator for selecting this therapeutic strategy from a number of molecular targeting drugs is needed. However, to date, methods that can predict the efficacy of ferroptosis-targeting treatment have not been established yet. In this study, we focused on the iron metabolic pathway to develop a nuclear imaging technique for diagnosing the susceptibility of cancer cells to ferroptosis. As a nuclear probe, human transferrin (Tf) was labeled with Gallium-68 (Ga) using 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as a chelator (Ga-NOTA-Tf). Western blot assay and clonogenic survival assay with human renal cancer cell lines A498 and 786-O revealed that the protein expression level of transferrin receptor1 (TfR1) and sensitivity to a ferroptosis inducer, erastin, were correlated. A cellular uptake assay with Ga-NOTA-Tf revealed that the cancer cells sensitive to erastin highly internalized the Ga-NOTA-Tf. Furthermore, treatment with the TfR1 inhibitor ferristatin II reduced the cellular uptake of Ga-NOTA-Tf, indicating that the intracellular uptake of the probe was mediated by TfR1. These results suggest that Ga-NOTA-Tf can be useful in predicting the sensitivity of cancer cells to ferroptosis inducers.
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http://dx.doi.org/10.1016/j.bbrep.2021.100957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910409PMC
July 2021

Chitin-Based Double-Network Hydrogel as Potential Superficial Soft-Tissue-Repairing Materials.

Biomacromolecules 2020 10 27;21(10):4220-4230. Epub 2020 Sep 27.

Laboratory of Soft and Wet Matter, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.

Chitin is a biopolymer, which has been proven to be a biomedical material candidate, yet the weak mechanical properties seriously limit their potentials. In this work, a chitin-based double-network (DN) hydrogel has been designed as a potential superficial repairing material. The hydrogel was synthesized through a double-network (DN) strategy composing hybrid regenerated chitin nanofiber (RCN)-poly (ethylene glycol diglycidyl ether) (PEGDE) as the first network and polyacrylamide (PAAm) as the second network. The hybrid RCN-PEGDE/PAAm DN hydrogel was strong and tough, possessing Young's modulus (elasticity) 0.097 ± 0.020 MPa, fracture stress σ 0.449 ± 0.025 MPa, and work of fracture 5.75 ± 0.35 MJ·m. The obtained DN hydrogel was strong enough for surgical requirements in the usage of soft tissue scaffolds. In addition, chitin endowed the DN hydrogel with good bacterial resistance and accelerated fibroblast proliferation, which increased the NIH3T3 cell number by nearly five times within 3 days. Subcutaneous implantation studies showed that the DN hydrogel did not induce inflammation after 4 weeks, suggesting a good biosafety in vivo. These results indicated that the hybrid RCN-PEGDE/PAAm DN hydrogel had great prospect as a rapid soft-tissue-repairing material.
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http://dx.doi.org/10.1021/acs.biomac.0c01003DOI Listing
October 2020

Analysis of Thyroid Hormone Receptor α-Knockout Tadpoles Reveals That the Activation of Cell Cycle Program Is Involved in Thyroid Hormone-Induced Larval Epithelial Cell Death and Adult Intestinal Stem Cell Development During Metamorphosis.

Thyroid 2021 01 1;31(1):128-142. Epub 2020 Jul 1.

Section on Molecular Morphogenesis, Cell Regulation and Development Affinity Group, Division of Molecular and Cellular Biology, and Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland, USA.

There are two highly conserved thyroid hormone (triiodothyronine [T3]) receptor () genes, and , in all vertebrates, and the expression of but not is activated earlier than T3 synthesis during development. In human, high levels of T3 are present during the several months around birth, and T3 deficiency during this period causes severe developmental abnormalities including skeletal and intestinal defects. It is, however, difficult to study this period in mammals as the embryos and neonates depend on maternal supply of nutrients for survival. However, undergoes a T3-dependent metamorphosis, which drastically changes essentially every organ in a tadpole. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We have previously demonstrated that T3 signaling is essential for the formation of adult intestinal stem cells during metamorphosis. We studied the function of endogenous TRα in the tadpole intestine by using knockout animals and RNA-seq analysis. We observed that removing endogenous TRα caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild-type and -knockout tadpoles treated with or without T3 for one day, before any detectable T3-induced cell death and stem cell formation in the tadpole intestine, we identified more than 1500 genes, which were regulated by T3 treatment of the wild-type but not -knockout tadpoles. Gene Ontology and biological pathway analyses revealed that surprisingly, these TRα-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Our findings suggest that TRα-mediated T3 activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling.
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http://dx.doi.org/10.1089/thy.2020.0022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840310PMC
January 2021

Thyroid hormone receptor beta is critical for intestinal remodeling during metamorphosis.

Cell Biosci 2020 27;10:46. Epub 2020 Mar 27.

Section on Molecular Morphogenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 USA.

Background: Thyroid hormone (T3) is critical for development in all vertebrates. The mechanism underlying T3 effect has been difficult to study due to the uterus-enclosed nature of mammalian embryos. Anuran metamorphosis, which is dependent on T3 but independent of maternal influence, is an excellent model to study the roles of T3 and its receptors (TRs) during vertebrate development. We and others have reported various effects of knockout ( and ) during development. However, these studies were largely focused on external morphology.

Results: We have generated knockout animals containing an out-frame-mutation of 5 base deletion by using the CRISPR/Cas9 system and observed that knockout does not affect premetamorphic tadpole development. We have found that the basal expression of direct T3-inducible genes is increased but their upregulation by T3 is reduced in the intestine of premetamorphic homozygous knockout animals, accompanied by reduced target binding by TR. More importantly, we have observed reduced adult stem cell proliferation and larval epithelial apoptosis in the intestine during T3-induced metamorphosis.

Conclusions: Our data suggest that plays a critical role in intestinal remodeling during metamorphosis.
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http://dx.doi.org/10.1186/s13578-020-00411-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099810PMC
March 2020

Evaluation of the target dose coverage of stereotactic body radiotherapy for lung cancer using helical tomotherapy: A dynamic phantom study.

Rep Pract Oncol Radiother 2020 Mar-Apr;25(2):200-205. Epub 2020 Jan 14.

Department of Radiology, University of Yamanashi, Yamanashi, Japan.

Aim: To evaluate the target dose coverage for lung stereotactic body radiotherapy (SBRT) using helical tomotherapy (HT) with the internal tumor volume (ITV) margin settings adjusted according to the degree of tumor motion.

Background: Lung SBRT with HT may cause a dosimetric error when the target motion is large.

Materials And Methods: Two lung SBRT plans were created using a tomotherapy planning station. Using these original plans, five plans with different ITV margins (4.0-20.0 mm for superior-inferior [SI] dimension) were generated. To evaluate the effects of respiratory motion on HT, an original dynamic motion phantom was developed. The respiratory wave of a healthy volunteer was used for dynamic motion as the typical tumor respiratory motion. Five patterns of motion amplitude that corresponded to five ITV margin sizes and three breathing cycles of 7, 14, and 28 breaths per minute were used. We evaluated the target dose change between a static delivery and a dynamic delivery with each motion pattern.

Results: The target dose difference increased as the tumor size decreased and as the tumor motion increased. Although a target dose difference of <5 % was observed at ≤10 mm of tumor motion for each condition, a maximum difference of -9.94 % ± 7.10 % was observed in cases of small tumors with 20 mm of tumor motion under slow respiration.

Conclusions: Minimizing respiratory movement is recommended as much as possible for lung SBRT with HT, especially for cases involving small tumors.
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http://dx.doi.org/10.1016/j.rpor.2020.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994284PMC
January 2020

Preclinical investigation of potential use of thymidine phosphorylase-targeting tracer for diagnosis of nonalcoholic steatohepatitis.

Nucl Med Biol 2020 Mar - Apr;82-83:25-32. Epub 2019 Dec 16.

Central Institute of Isotope Science, Hokkaido University, Hokkaido 060-0815, Japan; Graduate School of Biomedical Science and Engineering, Hokkaido University, Hokkaido 060-0815, Japan.

Introduction: Although liver biopsy is the gold standard for the diagnosis of nonalcoholic steatohepatitis (NASH), it has several problems including high invasiveness and sampling errors. Therefore, the development of alternative methods to overcome these disadvantages is strongly required. In this study, we evaluated the potential use of our tracer targeting thymidine phosphorylase (TYMP), 5-[I]iodo-6-[(2-iminoimidazolidinyl)methyl]uracil ([I]IIMU) for the diagnosis of NASH.

Methods: The mice used as the NASH model (hereafter, NASH mice) were prepared by feeding a methionine- and choline-deficient diet for 4 weeks. A control group was similarly given a control diet. The expression levels of the TYMP gene and protein in the liver were examined by real-time reverse-transcription polymerase chain reaction and western blot analyses. The localizations of [I]IIMU and the TYMP protein in the liver were examined by autoradiography and immunohistochemical staining, respectively. Finally, the mice were injected with [I]IIMU and single-photon emission tomography (SPECT) imaging was conducted.

Results: The hepatic expression levels of TYMP were significantly lower in the NASH mice than in the control mice at both mRNA and protein levels, suggesting that a decrease in TYMP level could be an indicator of NASH. [I]IIMU was uniformly distributed in the liver of the control mice, whereas it showed a patchy distribution in that of the NASH mice. The localization of [I]IIMU was visually consistent with that of the TYMP protein in the liver of the control and NASH mice. SPECT analysis indicated that the hepatic accumulation of [I]IIMU in the NASH mice was significantly lower than that in the control mice [SUV (g/ml): 4.14 ± 0.87 (Control) vs 2.31 ± 0.29 (NASH)].

Conclusions: [I]IIMU may provide a noninvasive means for imaging TYMP expression in the liver and may be applicable to the diagnosis of NASH.
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http://dx.doi.org/10.1016/j.nucmedbio.2019.12.006DOI Listing
May 2021

Organ-Specific Requirements for Thyroid Hormone Receptor Ensure Temporal Coordination of Tissue-Specific Transformations and Completion of Metamorphosis.

Thyroid 2020 02 23;30(2):300-313. Epub 2020 Jan 23.

Section on Molecular Morphogenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.

Thyroid hormone (triiodothyronine [T3]) is essential for the development throughout vertebrates. Anuran metamorphosis mimics mammalian postembryonic development, a period around birth when plasma T3 level peaks and many organs/tissues mature into their adult forms. Compared with the uterus-enclosed mammalian embryos, tadpoles can be easily manipulated to study the roles of T3 and T3 receptors (TRs) in tissue remodeling and adult organ development. We and others have previously knocked out or in the diploid anuran and reported distinct effects of the two receptor knockouts on metamorphosis. However, animals lacking either TRα or TRβ can complete metamorphosis and develop into reproductive adults. We have generated and double knockout animals and carried out molecular and morphological analyses to determine if TR is required for development. We found that the double knockout tadpoles do not respond to T3, supporting the view that there are no other genes in and that TR is essential for mediating the effects of T3 . Surprisingly, the double knockout tadpoles are able to initiate metamorphosis and accomplish many metamorphic changes, such as limb development. However, all double knockout tadpoles stall and eventually die at stage 61, the climax of metamorphosis, before tail resorption takes place. Analyses of the knockout tadpoles at stage 61 revealed various developmental abnormalities, including precocious ossification and extra vertebrae. Our data indicate that TRs are not required for the initiation of metamorphosis but is essential for the completion of metamorphosis. Furthermore, the differential effects of knockout on different organs/tissues suggest tissue-specific roles for TR to control temporal coordination and progression of metamorphosis in various organs.
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http://dx.doi.org/10.1089/thy.2019.0366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047119PMC
February 2020

Erastin, a ferroptosis-inducing agent, sensitized cancer cells to X-ray irradiation via glutathione starvation in vitro and in vivo.

PLoS One 2019 4;14(12):e0225931. Epub 2019 Dec 4.

Department of Biomedical Imaging, Graduate School of Biomedical Science and Engineering, Hokkaido University, Sapporo, Hokkaido, Japan.

High concentrations of antioxidants in cancer cells are huge obstacle in cancer radiotherapy. Erastin was first discovered as an inducer of iron-dependent cell death called ferroptosis accompanied by antioxidant depletion caused by cystine glutamate antiporter inhibition. Therefore, treatment with erastin is expected to potentially enhance cellular radiosensitivity. In this study, we investigated the influence of treatment with erastin on the radiation efficiency against cancers. The clonogenic ability, glutathione peroxidase 4 (GPX4) expression, and glutathione concentration were evaluated using HeLa and NCI-H1975 adenocarcinoma cell lines treated with erastin and/or X-ray irradiation. For in vivo studies, NCI-H1975 cells were transplanted in the left shoulder of nude mice, and then radiosensitizing effect of erastin and glutathione concentration in the cancer were evaluated. Treatment with erastin induced ferroptosis and decreased the concentration of glutathione and GPX4 protein expression levels in the two tumor cell lines. Moreover, erastin enhanced X-ray irradiation-induced cell death in both human tumor cell lines. Furthermore, erastin treatment of a tumor-transplanted mouse model similarly demonstrated the radiosensitizing effect and decrease in intratumoral glutathione concentration in the in vitro study. In conclusion, our study demonstrated the radiosensitizing effect of erastin on two adenocarcinoma cell lines and the tumor xenograft model accompanied by glutathione depletion, indicating that ferroptosis inducers that reduce glutathione concentration could be applied as a novel cancer therapy in combination with radiotherapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225931PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892486PMC
March 2020

Knocking out histone methyltransferase PRMT1 leads to stalled tadpole development and lethality in Xenopus tropicalis.

Biochim Biophys Acta Gen Subj 2020 03 15;1864(3):129482. Epub 2019 Nov 15.

Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD 20892, USA. Electronic address:

Background: Asymmetric arginine dimethylation of histone H4R3 to H4R3me2a by protein arginine methyltransferase 1 (PRMT1) has been implicated to play a key role in gene activation throughout vertebrates. PRMT1 knockout in mouse leads to embryonic lethality. This and the uterus-enclosed nature of the mouse embryo make it difficult to determine the development role of PRMT1 in mammals.

Methods: We took advantage of the external development of the diploid anuran Xenopus tropicalis and adapted the TALEN genome editing technology to knock out PRMT1 in order to investigate how PRMT1 participates in vertebrate development.

Results: We observed that PRMT1 knockout had no apparent effect on embryogenesis because normally feeding tadpoles were formed, despite the reduced asymmetric H4R3 di-methylation (H4R3me2a) due to the knockout. However, PRMT1 knockout tadpoles had severely reduced growth even with normal growth hormone gene expression. These tadpoles were also stalled in development shortly after feeding began at stages 44/45 and died within 2 weeks, well before the onset of metamorphosis. In situ analyses revealed broad cessation or drastic reduction in cell proliferation in diverse organs including the eye, brain, spinal cord, liver, and intestine.

Conclusions: Our findings suggest that PRMT1 is not required for embryogenesis but is a key regulator for normal progression of vertebrate development and growth.

General Significance: The similarities and differences between PRMT1 knockout Xenopus tropicalis and mouse suggest that two distinct phases of vertebrate development: early embryogenesis and subsequent growth/organ maturation, have different but evolutionally conserved requirement for epigenetic modifications.
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http://dx.doi.org/10.1016/j.bbagen.2019.129482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980677PMC
March 2020

High drug efflux pump capacity and low DNA damage response induce doxorubicin resistance in canine hemangiosarcoma cell lines.

Res Vet Sci 2019 Dec 17;127:1-10. Epub 2019 Oct 17.

Laboratory of Comparative Pathology, Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan.

Canine hemangiosarcoma (HSA) is an aggressive malignant endothelial tumor in dogs and characterized by poor prognosis because of its high invasiveness, high metastatic potential, and poor responsiveness to anti-cancer drugs. Although doxorubicin-based chemotherapy is regularly conducted after surgical treatment, its effects on survival rates are limited. Acquisition of drug resistance is one of the causes of this problem, but the underlying mechanisms remain unclear. In the present study, we aimed to identify the drug-resistance mechanism in canine HSA by establishing doxorubicin-resistant (DR) HSA cell lines. HSA cell lines were exposed to doxorubicin at gradually increasing concentrations. When the cells were able to grow in the presence of a 16-fold higher doxorubicin concentration compared with the initial culture, they were designated DR-HSA cell lines. Characterization of these DR-HSA cell lines revealed higher drug efflux pump capacity compared with the parental cell lines. Furthermore, the DR-HSA cell lines did not show activation of the DNA damage response despite carrying high DNA damage burdens, meaning that apoptosis was not strongly induced. In conclusion, canine HSA cell lines acquired doxorubicin resistance by increasing their drug efflux pump capacity and decreasing the DNA damage response. This study provides useful findings to promote further research on the drug-resistance mechanisms in canine HSA.
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http://dx.doi.org/10.1016/j.rvsc.2019.09.011DOI Listing
December 2019

Evaluation of the robustness of 3-dimensional conformal technique with MLC position control into the planning target volume in stereotactic body radiotherapy for lung cancer.

Med Dosim 2020 Spring;45(1):e1-e5. Epub 2019 May 25.

Department of Radiology, University of Yamanashi, Yamanashi 409-3898, Japan.

The purpose of this study was to evaluate the robustness of 3-dimensional conformal technique with MLC position control into the planning target volume (PTV) in stereotactic body radiotherapy for lung cancer. Two techniques using fixed beams were compared; one technique involved setting the MLC position outside the PTV and was referred to as Plan "O." Another technique involved setting the MLC position inside the PTV and was referred to as Plan "I." Two tumor motions were simulated: (1) tumor motion on the internal target volume (ITV) boundary and (2) tumor motion on the PTV boundary. Ten-phase CT images that captured the tumor in respiratory motion were generated for 2 simulations. Then, 4-dimensional (4D) treatment planning was performed by using deformable image registration. The gross tumor volume (GTV) dose changes between the 4D accumulated dose and treatment planning dose were evaluated for Plan "O" and Plan "I," respectively. For the simulation of tumor motion on the ITV boundary, the changes in GTV D50% were -0.10 ± 0.31% and -0.22 ± 0.26% (p < 0.05) for Plan "O" and Plan "I," respectively. In the same manner, for the simulation of tumor motion on the PTV boundary, the changes in GTV D50% were -3.37 ± 2.16% and -3.68 ± 1.71% (p < 0.05). Our result suggested that the dose change would be negligible in a clinical situation where the tumor moves within the ITV margin for both techniques, while Plan "O" showed better robustness.
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http://dx.doi.org/10.1016/j.meddos.2019.04.007DOI Listing
December 2020

Dietary coconut oil ameliorates skin contact hypersensitivity through mead acid production in mice.

Allergy 2019 08 10;74(8):1522-1532. Epub 2019 Apr 10.

Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Ibaraki-city, Osaka, Japan.

Coconut oil is used as a dietary oil worldwide, and its healthy effects are recognized by the fact that coconut oil is easy to digest, helps in weight management, increases healthy cholesterol, and provides instant energy. Although topical application of coconut oil is known to reduce skin infection and inflammation, whether dietary coconut oil has any role in decreasing skin inflammation is unknown. In this study, we showed the impact of dietary coconut oil in allergic skin inflammation by using a mouse model of contact hypersensitivity (CHS). Mice maintained on coconut oil showed amelioration of skin inflammation and increased levels of cis-5, 8, 11-eicosatrienoic acid (mead acid) in serum. Intraperitoneal injection of mead acid inhibited CHS and reduced the number of neutrophils infiltrating to the skin. Detailed mechanistic studies unveiled that mead acid inhibited the directional migration of neutrophils by inhibiting the filamentous actin polymerization and leukotriene B production required for secondary recruitment of neutrophils. Our findings provide valuable insights into the preventive roles of coconut oil and mead acid against skin inflammation, thereby offering attractive therapeutic possibilities.
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http://dx.doi.org/10.1111/all.13762DOI Listing
August 2019

Isolation of halophilic lactic acid bacteria possessing aspartate decarboxylase and application to fish sauce fermentation starter.

Int J Food Microbiol 2019 Mar 19;292:137-143. Epub 2018 Dec 19.

BlessingFavour Co., Ltd., 1-1, Hanabata-cho, Chuo-ku, Kumamoto 860-0806, Japan.

The aims of this study were to isolate halophilic lactic acid bacteria possessing aspartate decarboxylase and elucidate the property of the isolates as starter cultures for fish sauce fermentation. Seventy-four strains were isolated from fermented fish foods on aspartate indicator broth containing bromocresol purple, and all isolates were identified as Tetragenococcus halophilus and confirmed to possess the aspartate decarboxylase gene (aspD) by PCR amplification. The isolates were classified into 14 groups based on their aspD-encoding plasmid construction. Strains selected from each group and a control strain incapable of aspartate decarboxylation were inoculated into fish sauce mash as starter cultures. Isolated strains possessing aspD converted aspartate into alanine almost completely in the fish sauce mash. In addition, the strains prevented the accumulation of biogenic amines, as did the control strain, whereas various amines were accumulated in fish sauce mash without starter cultures. Sensory evaluation tests indicated that converting the sour amino acid aspartate into the sweet amino acid alanine made the fish sauce taste milder. In conclusion, the use of T. halophilus possessing aspartate decarboxylase as a fish sauce fermentation starter causes the conversion of aspartate to alanine, accompanied by taste alteration, and prevents biogenic amine accumulation in fish sauce products.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2018.12.013DOI Listing
March 2019

Functional Studies of Transcriptional Cofactors via Microinjection-Mediated Gene Editing in Xenopus.

Methods Mol Biol 2019 ;1874:507-524

Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, USA.

The anuran Xenopus laevis has been studied for decades as a model for vertebrate cell and developmental biology. More recently, the highly related species Xenopus tropicalis has offered the opportunity to carry out genetic studies due to its diploid genome as compared to the pseudo-tetraploid Xenopus laevis. Amphibians undergo a biphasic development: embryogenesis to produce a free-living tadpoles and subsequent metamorphosis to transform the tadpole to a frog. This second phase mimics the so-called postembryonic development in mammals when many organs/tissues mature into their adult form in the presence of high levels of plasma thyroid hormone (T3). The total dependence of amphibian metamorphosis on T3 offers a unique opportunity to study postembryonic development in vertebrates, especially with the recent development gene editing technologies that function in amphibians. Here, we first review the basic molecular understanding of the regulation of Xenopus metamorphosis by T3 and T3 receptors (TRs), and then describe a detailed method to use CRISPR to knock out the TR-coactivator SRC3 (steroid receptor coactivator 3), a histone acetyltransferase, in order to study its involvement in gene regulation by T3 in vivo and Xenopus development.
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http://dx.doi.org/10.1007/978-1-4939-8831-0_29DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291954PMC
June 2019

Sulfasalazine, an inhibitor of the cystine-glutamate antiporter, reduces DNA damage repair and enhances radiosensitivity in murine B16F10 melanoma.

PLoS One 2018 12;13(4):e0195151. Epub 2018 Apr 12.

Laboratory of Biochemistry, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan.

The sodium-independent cystine-glutamate antiporter plays an important role in extracellular cystine uptake. It comprises the transmembrane protein, xCT and its chaperone, CD98. Because glutathione is only weakly cell membrane permeable, cellular uptake of its precursor, cystine, is known to be a key step in glutathione synthesis. Moreover, it has been reported that xCT expression affects the progression of tumors and their resistance to therapy. Sulfasalazine is an inhibitor of xCT that is known to increase cellular oxidative stress, giving it anti-tumor potential. Here, we describe a radio-sensitizing effect of sulfasalazine using a B16F10 melanoma model. Sulfasalazine decreased glutathione concentrations and resistance to H2O2 in B16F10 melanoma cells, but not in mouse embryonic fibroblasts. It synergistically enhanced the cyto-killing effect of X-irradiation in B16F10 cells. It inhibited cellular DNA damage repair and prolonged cell cycle arrest after X-irradiation. Furthermore, in an in vivo transplanted melanoma model, sulfasalazine decreased intratumoral glutathione content, leading to enhanced susceptibility to radiation therapy. These results suggest the possibility of using SAS to augment the treatment of radio-resistant cancers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195151PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896924PMC
July 2018

Comparison of MLC error sensitivity of various commercial devices for VMAT pre-treatment quality assurance.

J Appl Clin Med Phys 2018 May 3;19(3):87-93. Epub 2018 Mar 3.

Department of Radiology, University of Yamanashi, Yamanashi, Japan.

The purpose of this study was to compare the MLC error sensitivity of various measurement devices for VMAT pre-treatment quality assurance (QA). This study used four QA devices (Scandidos Delta4, PTW 2D-array, iRT systems IQM, and PTW Farmer chamber). Nine retrospective VMAT plans were used and nine MLC error plans were generated for all nine original VMAT plans. The IQM and Farmer chamber were evaluated using the cumulative signal difference between the baseline and error-induced measurements. In addition, to investigate the sensitivity of the Delta4 device and the 2D-array, global gamma analysis (1%/1, 2%/2, and 3%/3 mm), dose difference (1%, 2%, and 3%) were used between the baseline and error-induced measurements. Some deviations of the MLC error sensitivity for the evaluation metrics and MLC error ranges were observed. For the two ionization devices, the sensitivity of the IQM was significantly better than that of the Farmer chamber (P < 0.01) while both devices had good linearly correlation between the cumulative signal difference and the magnitude of MLC errors. The pass rates decreased as the magnitude of the MLC error increased for both Delta4 and 2D-array. However, the small MLC error for small aperture sizes, such as for lung SBRT, could not be detected using the loosest gamma criteria (3%/3 mm). Our results indicate that DD could be more useful than gamma analysis for daily MLC QA, and that a large-area ionization chamber has a greater advantage for detecting systematic MLC error because of the large sensitive volume, while the other devices could not detect this error for some cases with a small range of MLC error.
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http://dx.doi.org/10.1002/acm2.12288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978943PMC
May 2018

Technical Note: Evaluation of the latency and the beam characteristics of a respiratory gating system using an Elekta linear accelerator and a respiratory indicator device, Abches.

Med Phys 2018 Jan 3;45(1):74-80. Epub 2017 Dec 3.

Department of Radiology, University of Yamanashi, Yamanashi, Japan.

Purpose: To evaluate the basic performance of a respiratory gating system using an Elekta linac and an Abches respiratory-monitoring device.

Methods: The gating system was comprised of an Elekta Synergy linac equipped with a Response gating interface module and an Abches respiratory-monitoring device. The latencies from a reference respiratory signal to the resulting Abches gating output signal and the resulting monitor-ion-chamber output signal were measured. Then, the flatness and symmetry of the gated beams were measured using a two-dimensional ionization chamber array for fixed and arc beams, respectively. Furthermore, the beam quality, TPR , and the output of the fixed gated beams were also measured using a Farmer chamber. Each of the beam characteristics was compared with each of those for nongated irradiation.

Results: The full latencies at beam-on and beam-off for 6-MV gated beams were 336.4 ± 23.4 ms and 87.6 ± 7.1 ms, respectively. The differences in flatness between the gated and nongated beams were within 0.91% and 0.87% for the gun-target and left-right directions, respectively. In the same manner, the beam symmetries were within 0.68% and 0.82%, respectively. The percentage differences in beam quality and beam output were below 1% for a beam-on time range of 1.1-7 s.

Conclusion: The latency of the Elekta gating system combined with Abches was found to be acceptable using our measurement method. Furthermore, we demonstrated that the beam characteristics of the gating system using our respiratory indicator were comparable with the nongated beams for a single-arc gated beam delivery.
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http://dx.doi.org/10.1002/mp.12664DOI Listing
January 2018

Thyroid Hormone Receptor α Controls Developmental Timing and Regulates the Rate and Coordination of Tissue-Specific Metamorphosis in Xenopus tropicalis.

Endocrinology 2017 06;158(6):1985-1998

Section on Molecular Morphogenesis, Program on Cell Regulation and Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.

Thyroid hormone (T3) receptors (TRs) mediate the effects of T3 on organ metabolism and animal development. There are two TR genes, TRα and TRβ, in all vertebrates. During animal development, TRα expression is activated earlier than zygotic T3 synthesis and secretion into the plasma, implicating a developmental role of TRα both in the presence and absence of T3. Using T3-dependent amphibian metamorphosis as a model, we previously proposed a dual-function model for TRs, in particular TRα, during development. That is, unliganded TR represses the expression of T3-inducible genes during premetamorphosis to ensure proper animal growth and prevent premature metamorphosis, whereas during metamorphosis, liganded TR activates target gene transcription to promote the transformation of the tadpole into a frog. To determine if TRα has such a dual function, we generated homozygous TRα-knockout animal lines. We show that, indeed, TRα knockout affects both premetamorphic animal development and metamorphosis. Surprisingly, we observed that TRα is not essential for amphibian metamorphosis, given that homozygous knockout animals complete metamorphosis within a similar time period after fertilization as their wild-type siblings. On the other hand, the timing of metamorphosis for different organs is altered by the knockout; limb metamorphosis occurs earlier, whereas intestinal metamorphosis is completed later than in wild-type siblings. Thus, our studies have demonstrated a critical role of endogenous TRα, not only in regulating both the timing and rate of metamorphosis, but also in coordinating temporal metamorphosis of different organs.
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http://dx.doi.org/10.1210/en.2016-1953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460924PMC
June 2017

Molecular machinery for vasotocin-dependent transepithelial water movement in amphibians: aquaporins and evolution.

Biol Bull 2015 Aug;229(1):109-19

Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836, Ohya, Suruga-ward, Shizuoka-city, Shizuoka 422-8529, Japan.

Amphibians represent the first vertebrates to adapt to terrestrial environments, and are successfully distributed around the world. The ventral skin, kidney, and urinary bladder are important osmoregulatory organs for adult anuran amphibians. Water channel proteins, called aquaporins (AQPs), play key roles in transepithelial water absorption/reabsorption in these organs. At least 43 types of AQPs were identified in anurans; a recent phylogenetic analysis categorized anuran AQPs among 16 classes (AQP0-14, 16). Anuran-specific AQPa2 was assigned to AQP6, then was further subdivided into the ventral skin-type (AQP6vs; AQPa2S), whose expression is confined to the ventral skin, and the urinary bladder-type (AQP6ub; AQPa2U), which is basically expressed in the urinary bladder. For the osmoregulatory organs, AQP3 is constitutively located in the basolateral plasma membrane of tight-junctioned epithelial cells. AQP6vs, AQP2 and/or AQP6ub are also expressed in these epithelial cells and are translocated to the apical membrane in response to arginine vasotocin, thereby regulating water absorption/reabsorption. It was suggested recently that two subtypes of AQP6vs contribute to cutaneous water absorption in Ranid species. In addition, AQP5 (AQP5a) and AQP5L (AQP5b) were identified from Xenopus tropicalis Gray, 1864, and AQP5 was localized to the apical membrane of luminal epithelial cells of the urinary bladder in dehydrated Xenopus. This finding suggested that AQP5 may be involved in water reabsorption from this organ under dehydration. Based on the hitherto reported information, we propose models for the evolution of water-absorbing/reabsorbing mechanisms in anuran osmoregulatory organs in association with AQPs.
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http://dx.doi.org/10.1086/BBLv229n1p109DOI Listing
August 2015

Molecular and cellular characterization of urinary bladder-type aquaporin in Xenopus laevis.

Gen Comp Endocrinol 2015 Oct 16;222:11-9. Epub 2014 Sep 16.

Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan; Department of Biological Science, Graduate School of Science, Shizuoka University, Shizuoka 422-8529, Japan. Electronic address:

In contrast to many anuran amphibians, water is not reabsorbed from the urinary bladder in aquatic Xenopus, thereby helping to prevent excessive water influx. However, little is known about the molecular mechanisms for this process. In the present study, we have identified urinary bladder-type aquaporin, AQP-x2, in Xenopus laevis by cDNA cloning. The predicted amino acid sequence contained six putative transmembrane domains and the two conserved Asn-Pro-Ala motifs, characteristic of AQPs. The sequence also contained a putative N-glycosylation site and phosphorylation motifs for protein kinase A and protein kinase C. The oocyte swelling assay showed that AQP-x2 facilitated water permeability. Reverse transcription-PCR analysis indicated that AQP-x2 mRNA was expressed in the urinary bladder and lung, and faintly in the kidney. Immunomicroscopical study further localized AQP-x2 protein to the cytoplasm of granular cells in the luminal epithelium of the urinary bladder whilst AQP3 was observed along the basolateral side of these cells. In vitro stimulation of the urinary bladder with 10(-8)M vasotocin (AVT), 10(-8)M hydrin 1, or 10(-8)M hydrin 2 had no clear effect on the subcellular distribution of AQP-x2. When the AVT concentration was increased to 10(-6)M, however, AQP-x2 was partially transferred to the apical plasma membrane. The treatment with hydrin 1 or hydrin 2 at the same concentration failed to induce the translocation to the apical membrane. On the other hand, AQP3 remained along the basolateral side even after the treatment with vasotocin or hydrins. The results suggest that the poor responsiveness of AQP-x2 to neurohypophyseal peptides may be a main cause for the little water permeability of the urinary bladder of X. laevis.
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http://dx.doi.org/10.1016/j.ygcen.2014.09.001DOI Listing
October 2015

Psychometric properties of the parent and teacher forms of the Japanese version of the Strengths and Difficulties Questionnaire.

Brain Dev 2015 May 27;37(5):501-7. Epub 2014 Aug 27.

Center for Developmental Clinical Psychology and Psychiatry, Nagoya University, Japan.

Objective: This study examined the psychometric properties of the parent and teacher forms of the Japanese version of the Strengths and Difficulties Questionnaire (SDQ).

Method: Parents and teachers of 1487 elementary school children (759 boys and 728 girls aged 6-12 years) participated in this study.

Results: The results of confirmatory factor analyses of the parent and teacher versions of the SDQ supported the five-factor structure reported in previous studies. However, factor invariance across sex was not observed. The alpha coefficients for the subscales of the SDQs varied between 0.55 and 0.86, the same reliability measures that were also reported in previous studies. Moreover, analyses of variance showed significant differences on all of the subscales according to sex and teacher-parent ratings.

Conclusion: The factor structure of the SDQ was generally supported, but more gender-segregated investigations of the factor structures are needed. Parents tended to give higher ratings on the difficulties and strengths of children compared to the teachers. Boys were rated higher than girls were on difficulties, while girls were rated higher than boys were on strengths.
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http://dx.doi.org/10.1016/j.braindev.2014.08.001DOI Listing
May 2015

Gene expression and localization of two types of AQP5 in Xenopus tropicalis under hydration and dehydration.

Am J Physiol Regul Integr Comp Physiol 2014 Jul 9;307(1):R44-56. Epub 2014 Apr 9.

Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka, Japan; and Department of Biological Science, Graduate School of Science, Shizuoka University, Shizuoka, Japan; and

Two types of aquaporin 5 (AQP5) genes (aqp-xt5a and aqp-xt5b) were identified in the genome of Xenopus tropicalis by synteny comparison and molecular phylogenetic analysis. When the frogs were in water, AQP-xt5a mRNA was expressed in the skin and urinary bladder. The expression of AQP-xt5a mRNA was significantly increased in dehydrated frogs. AQP-xt5b mRNA was also detected in the skin and increased in response to dehydration. Additionally, AQP-xt5b mRNA began to be slightly expressed in the lung and stomach after dehydration. For the pelvic skin of hydrated frogs, immunofluorescence staining localized AQP-xt5a and AQP-xt5b to the cytoplasm of secretory cells of the granular glands and the apical plasma membrane of secretory cells of the small granular glands, respectively. After dehydration, the locations of both AQPs in their respective glands did not change, but AQP-xt5a was visualized in the cytoplasm of secretory cells of the small granular glands. For the urinary bladder, AQP-xt5a was observed in the apical plasma membrane and cytoplasm of a number of granular cells under normal hydration. After dehydration, AQP-xt5a was found in the apical membrane and cytoplasm of most granular cells. Injection of vasotocin into hydrated frogs did not induce these changes in the localization of AQP-xt5a in the small granular glands and urinary bladder, however. The results suggest that AQP-xt5a might be involved in water reabsorption from the urinary bladder during dehydration, whereas AQP-xt5b might play a role in water secretion from the small granular gland.
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http://dx.doi.org/10.1152/ajpregu.00186.2013DOI Listing
July 2014

Novel vasotocin-regulated aquaporins expressed in the ventral skin of semiaquatic anuran amphibians: evolution of cutaneous water-absorbing mechanisms.

Endocrinology 2014 Jun 21;155(6):2166-77. Epub 2014 Mar 21.

Department of Biology, Faculty of Science (Y.Sa., R.O., S.T., M.S.), and Integrated Bioscience Section, Graduate School of Science and Technology (Y.O., Y.Sh., R.O., S.T., M.S.), Shizuoka University, Shizuoka 422-8529, Japan.

Until now, it was believed that only one form of arginine vasotocin (AVT)-regulated aquaporin (AQP) existed to control water absorption from the ventral skin of semiaquatic anuran amphibians, eg, AQP-rj3(a) in Rana japonica. In the present study, we have identified a novel form of ventral skin-type AQP, AQP-rj3b, in R. japonica by cDNA cloning. The oocyte swelling assay confirmed that AQP-rj3b can facilitate water permeability. Both AQP-rj3a and AQP-rj3b were expressed abundantly in the ventral hindlimb skin and weakly in the ventral pelvic skin. For the hindlimb skin, water permeability was increased in response to AVT, although the hydroosmotic response was not statistically significant in the pelvic skin. Isoproterenol augmented water permeability of the hindlimb skin, and the response was inhibited by propranolol. These events were well correlated with the intracellular trafficking of the AQPs. Immunohistochemistry showed that both AQP-rj3 proteins were translocated from the cytoplasmic pool to the apical membrane of principal cells in the first-reacting cell layer of the hindlimb skin after stimulation with AVT and/or isoproterenol. The type-b AQP was also found in R. (Lithobates) catesbeiana and R. (Pelophylax) nigromaculata. Molecular phylogenetic analysis indicated that the type-a is closely related to ventral skin-type AQPs from aquatic Xenopus, whereas the type-b is closer to the AQPs from terrestrial Bufo and Hyla, suggesting that the AQPs from terrestrial species are not the orthologue of the AQPs from aquatic species. Based on these results, we propose a model for the evolution of cutaneous water-absorbing mechanisms in association with AQPs.
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http://dx.doi.org/10.1210/en.2013-1928DOI Listing
June 2014

A clinical study of attention-deficit/hyperactivity disorder in preschool children--prevalence and differential diagnoses.

Brain Dev 2014 Oct 2;36(9):778-85. Epub 2013 Dec 2.

Center for Developmental Clinical Psychology and Psychiatry, Nagoya University, Nagoya, Japan.

Objective: We aimed to examine (1) the prevalence and characteristics of ADHD in preschool children, and (2) differential diagnoses among children who display symptoms of inattention and hyperactivity-impulsivity in early childhood.

Methods: The participants were children living in Kanie-cho, in Japan's Aichi Prefecture, who underwent their age 5 exams at the municipal health center between April 2009 and March 2011. We first extracted children who were observed to be inattentive or hyperactive-impulsive during their age 5 exams and considered as possibly having ADHD. We conducted follow-ups with these children using post-examination consultations, visits to preschools, and group rehabilitation. The results of the age 5 exams were combined with behavior observations and interview content obtained during subsequent follow-ups. A child psychiatrist and several clinical psychologists discussed these cases and made a diagnosis in accordance with the DSM-IV-TR.

Results: 91 (15.6%) of the 583 children selected were considered as possibly having ADHD; we were able to conduct follow-ups with 83 of the 91 children. Follow-up results showed that 34 children (5.8% of all participants) remained eligible for a diagnosis of ADHD. Diagnoses for the remaining children included: pervasive developmental disorders (six children, or 6.6% of suspected ADHD children), intellectual comprehension problems (four children, or 4.4%), anxiety disorders (seven children, or 7.7%), problems related to abuse or neglect (four children, or 4.4%), a suspended diagnosis for one child (1.1%), and unclear diagnoses for 29 children (31.9%).

Conclusions: ADHD tendencies in preschool children vary with changing situations and development, and the present study provides prevalence estimates that should prove useful in establishing a diagnostic baseline.
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http://dx.doi.org/10.1016/j.braindev.2013.11.004DOI Listing
October 2014

Localization of water channels in the skin of two species of desert toads, Anaxyrus (Bufo) punctatus and Incilius (Bufo) alvarius.

Zoolog Sci 2011 Sep;28(9):664-70

Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan.

Anuran amphibians obtain water by osmosis across their ventral skin. A specialized region in the pelvic skin of semiterrestrial species, termed the seat patch, contains aquaporins (AQPs) that become inserted into the apical plasma membrane of the epidermis following stimulation by arginine vasotocin (AVT) to facilitate rehydration. Two AVT-stimulated AQPs, AQP-h2 and AQP-h3, have been identified in the epidermis of seat patch skin of the Japanese tree frog, Hyla japonica, and show a high degree of homology with those of bufonid species. We used antibodies raised against AQP-h2 and AQP-h3 to characterize the expression of homologous AQPs in the skin of two species of toads that inhabit arid desert regions of southwestern North America. Western blot analysis of proteins gave positive results for AQP-h2-like proteins in the pelvic skin and also the urinary bladder of Anaxyrus (Bufo) punctatus while AQP-h3-like proteins were found in extracts from the pelvic skin and the more anterior ventral skin, but not the urinary bladder. Immunohistochemical observations showed both AQP-h2- and AQP-h3-like proteins were present in the apical membrane of skin from the pelvic skin of hydrated and dehydrated A. punctatus. Further stimulation by AVT or isoproterenol treatment of living toads was not evident. In contrast, skin from hydrated Incilius (Bufo) alvarius showed very weak labeling of AQP-h2- and AQP-h3-like proteins and labeling turned intense following stimulation by AVT. These results are similar to those of tree frogs and toads that occupy mesic habitats and suggest this pattern of AQP expression is the result of phylogenetic factors shared by hylid and bufonid anurans.
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http://dx.doi.org/10.2108/zsj.28.664DOI Listing
September 2011
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