Publications by authors named "Hisato Kobayashi"

77 Publications

Urinary reabsorption in the rat kidney by anticholinergics.

Sci Rep 2021 Apr 28;11(1):9191. Epub 2021 Apr 28.

Department of Urology, Faculty of Medical Science, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui, 910-1193, Japan.

Anticholinergics, therapeutic agents for overactive bladder, are clinically suggested to reduce urine output. We investigated whether this effect is due to bladder or kidney urine reabsorption. Various solutions were injected into the bladder of urethane-anesthetized SD rats. The absorption rate for 2 h was examined following the intravenous administration of the anticholinergics imidafenacin (IM), atropine (AT), and tolterodine (TO). The bilateral ureter was then canulated and saline was administered to obtain a diuretic state. Anticholinergics or 1-deamino-[8-D-arginine]-vasopressin (dDAVP) were intravenously administered. After the IM and dDAVP administrations, the rat kidneys were immunostained with AQP2 antibody, and intracellular cAMP was measured. The absorption rate was ~ 10% of the saline injected into the bladder and constant even when anticholinergics were administered. The renal urine among peaked 2 h after the saline administration. Each of the anticholinergics significantly suppressed the urine production in a dose-dependent manner, as did dDAVP. IM and dDAVP increased the intracellular cAMP levels and caused the AQP2 molecule to localize to the collecting duct cells' luminal side. The urinary reabsorption mechanism through the bladder epithelium was not activated by anticholinergic administration. Thus, anticholinergics suppress urine production via an increase in urine reabsorption in the kidneys' collecting duct cells via AQP2.
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http://dx.doi.org/10.1038/s41598-021-88738-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080556PMC
April 2021

Impact of self-decision to stop cancer treatment on advanced genitourinary cancer patients.

Medicine (Baltimore) 2021 Apr;100(14):e25397

Division of Urology, Department of Surgery, Faculty of Medical Sciences, University of Fukui, Fukui.

Abstract: Decision-making to stop cancer treatment in patients with advanced cancer is stressful, and it significantly influences subsequent end-of-life palliative treatment. However, little is known about the extent to which the patient's self-decisions influenced the prognostic period. This study focused on the patient's self-decision and investigated the impact of the self-decision to stop cancer treatment on their post-cancer treatment survival period and place of death.We retrospectively analyzed 167 cases of advanced genitourinary cancer patients (kidney cancer: 42; bladder cancer: 68; prostate cancer: 57) treated at the University of Fukui Hospital (UFH), who later died because of cancer. Of these, 100 patients decided to stop cancer treatment by themselves (self-decision group), while the families of the remaining 67 patients (family's decision group) decided to stop treatment on their behalf because the patient's decision-making ability was already impaired. Differences in the post-cancer-treatment survival period and place of death between the 2 groups were examined. The association between place of death and survival period was also analyzed.The median survival period after terminating cancer treatment was approximately 6 times longer in the self-decision group (145.5 days in self-decision group vs 23.0 days in family's decision group, P < .001). Proportions for places of death were as follows: among the self-decision group, 42.0% of patients died at UFH, 45.0% at other medical institutions, and 13.0% at home; among the family's decision group, 62.7% died at UFH, 32.8% at other medical institutions, and 4.5% at home. The proportion of patients who died at UFH was significantly higher among the family's decision group (P = .011). The median survival period was significantly shorter for patients who died at UFH (UFH: 30.0 days; other institutions/home: 161.0 days; P < .001).Significantly longer post-cancer-treatment survival period and higher home death rate were observed among patients whose cancer treatment was terminated based on their self-decision. Our results provide clinical evidence, especially in terms of prognostic period and place of death that support the importance of discussing bad news, such as stopping cancer treatment with patients.
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http://dx.doi.org/10.1097/MD.0000000000025397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036094PMC
April 2021

Dynamics of transcription-mediated conversion from euchromatin to facultative heterochromatin at the Xist promoter by Tsix.

Cell Rep 2021 Mar;34(13):108912

Department of Molecular Biology, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan. Electronic address:

The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the -1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment.
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http://dx.doi.org/10.1016/j.celrep.2021.108912DOI Listing
March 2021

Proposal of selective wedge instillation of pulmonary surfactant for COVID-19 pneumonia based on computational fluid dynamics simulation.

BMC Pulm Med 2021 Feb 22;21(1):62. Epub 2021 Feb 22.

Department of Respiratory Medicine and Hematology, Hyogo College of Medicine, Nishinomiya, Japan.

Background: The most important target cell of SARS-CoV-2 is Type II pneumocyte which produces and secretes pulmonary surfactant (PS) that prevents alveolar collapse. PS instillation therapy is dramatically effective for infant respiratory distress syndrome but has been clinically ineffective for ARDS. Nowadays, ARDS is regarded as non-cardiogenic pulmonary edema with vascular hyper-permeability regardless of direct relation to PS dysfunction. However, there is a possibility that this ineffectiveness of PS instillation for ARDS is caused by insufficient delivery. Then, we performed PS instillation simulation with realistic human airway models by the use of computational fluid dynamics, and investigated how instilled PS would move in the liquid layer covering the airway wall and reach to alveolar regions.

Methods: Two types of 3D human airway models were prepared: one was from the trachea to the lobular bronchi and the other was from a subsegmental bronchus to respiratory bronchioles. The thickness of the liquid layer covering the airway was assigned as 14 % of the inner radius of the airway segment. The liquid layer was assumed to be replaced by an instilled PS. The flow rate of the instilled PS was assigned a constant value, which was determined by the total amount and instillation time in clinical use. The PS concentration of the liquid layer during instillation was computed by solving the advective-diffusion equation.

Results: The driving pressure from the trachea to respiratory bronchioles was calculated at 317 cmHO, which is about 20 times of a standard value in conventional PS instillation method where the driving pressure was given by difference between inspiratory and end-expiratory pressures of a ventilator. It means that almost all PS does not reach the alveolar regions but moves to and fro within the airway according to the change in ventilator pressure. The driving pressure from subsegmental bronchus was calculated at 273 cm HO, that is clinically possible by wedge instillation under bronchoscopic observation.

Conclusions: The simulation study has revealed that selective wedge instillation under bronchoscopic observation should be tried for COVID-19 pneumonia before the onset of ARDS. It will be also useful for preventing secondary lung fibrosis.
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http://dx.doi.org/10.1186/s12890-021-01435-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897887PMC
February 2021

Severe Liver Disorder Following Liver Transplantation in STING-Associated Vasculopathy with Onset in Infancy.

J Clin Immunol 2021 Feb 5. Epub 2021 Feb 5.

Division of Immunology, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan.

Purpose: STING-associated vasculopathy with onset in infancy (SAVI) is a type-I interferonopathy, characterized by systemic inflammation, peripheral vascular inflammation, and pulmonary manifestations. There are three reports of SAVI patients developing liver disease, but no report of a SAVI patient requiring liver transplantation. Therefore, the relevance of liver inflammation is unclear in SAVI. We report a SAVI patient who developed severe liver disorder following liver transplantation.

Methods: SAVI was diagnosed in a 4-year-old girl based on genetic analysis by whole-exome sequencing. We demonstrated clinical features, laboratory findings, and pathological examination of her original and transplanted livers.

Results: At 2 months of age, she developed bronchitis showing resistance to bronchodilators and antibiotics. At 10 months of age, she developed liver dysfunction with atypical cholangitis, which required liver transplantation at 1 year of age. At 2 years of age, multiple biliary cysts developed in the transplanted liver. At 3.9 years of age, SAVI was diagnosed by whole-exome sequencing. Inflammatory cells from the liver invaded the stomach wall directly, leading to fatal gastrointestinal bleeding unexpectedly at 4.6 years of age. In pathological findings, there were no typical findings of liver abscess, vasculitis, or graft rejection, but biliary cysts and infiltration of inflammatory cells, including plasmacytes around the bile duct area, in the transplanted liver were noted, which were findings similar to those of her original liver.

Conclusion: Although further studies to clarify the mechanisms of the various liver disorders described in SAVI patients are needed, inflammatory liver manifestations may be amplified in the context of SAVI.
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http://dx.doi.org/10.1007/s10875-021-00977-wDOI Listing
February 2021

Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF.

EMBO Rep 2021 Feb 5;22(2):e51524. Epub 2021 Jan 5.

Department of Developmental Neuroscience, Tohoku University Graduate School of Medicine, Sendai, Japan.

Advanced paternal age can have deleterious effects on various traits in the next generation. Here, we establish a paternal-aging model in mice to understand the molecular mechanisms of transgenerational epigenetics. Whole-genome target DNA methylome analyses of sperm from aged mice reveal more hypo-methylated genomic regions enriched in REST/NRSF binding motifs. Gene set enrichment analyses also reveal the upregulation of REST/NRSF target genes in the forebrain of embryos from aged fathers. Offspring derived from young mice administrated with a DNA de-methylation drug phenocopy the abnormal vocal communication of pups derived from aged fathers. In conclusion, hypo-methylation of sperm DNA can be a key molecular feature modulating neurodevelopmental programs in offspring by causing fluctuations in the expression of REST/NRSF target genes.
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http://dx.doi.org/10.15252/embr.202051524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857438PMC
February 2021

Maternal DNMT3A-dependent de novo methylation of the paternal genome inhibits gene expression in the early embryo.

Nat Commun 2020 10 27;11(1):5417. Epub 2020 Oct 27.

Department of Medical Genetics, University of British Columbia, Vancouver, Canada.

De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.
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http://dx.doi.org/10.1038/s41467-020-19279-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591512PMC
October 2020

Germline development in rat revealed by visualization and deletion of .

Development 2020 02 21;147(4). Epub 2020 Feb 21.

Section of Mammalian Transgenesis, Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, 444-8787 Aichi, Japan

Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying , a unique marker and an essential PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until the sex determination stage in fetal gonads using knock-in rats. We uncover that the crucial role of in PGC specification is conserved between rat and mice, by analyzing -deficient rat embryos. Notably, loss of completely abrogates the PGC program, as demonstrated by failure of the maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the post-implantation epiblast and all PGC stages in rat to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional timeline for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved versus species-specific features for germline development in mammals.
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http://dx.doi.org/10.1242/dev.183798DOI Listing
February 2020

Evolution of imprinting via lineage-specific insertion of retroviral promoters.

Nat Commun 2019 12 12;10(1):5674. Epub 2019 Dec 12.

Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

Imprinted genes are expressed from a single parental allele, with the other allele often silenced by DNA methylation (DNAme) established in the germline. While species-specific imprinted orthologues have been documented, the molecular mechanisms underlying the evolutionary switch from biallelic to imprinted expression are unknown. During mouse oogenesis, gametic differentially methylated regions (gDMRs) acquire DNAme in a transcription-guided manner. Here we show that oocyte transcription initiating in lineage-specific endogenous retroviruses (ERVs) is likely responsible for DNAme establishment at 4/6 mouse-specific and 17/110 human-specific imprinted gDMRs. The latter are divided into Catarrhini- or Hominoidea-specific gDMRs embedded within transcripts initiating in ERVs specific to these primate lineages. Strikingly, imprinting of the maternally methylated genes Impact and Slc38a4 was lost in the offspring of female mice harboring deletions of the relevant murine-specific ERVs upstream of these genes. Our work reveals an evolutionary mechanism whereby maternally silenced genes arise from biallelically expressed progenitors.
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http://dx.doi.org/10.1038/s41467-019-13662-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908575PMC
December 2019

Food-Derived Compounds Apigenin and Luteolin Modulate mRNA Splicing of Introns with Weak Splice Sites.

iScience 2019 Dec 20;22:336-352. Epub 2019 Nov 20.

Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. Electronic address:

Cancer cells often exhibit extreme sensitivity to splicing inhibitors. We identified food-derived flavonoids, apigenin and luteolin, as compounds that modulate mRNA splicing at the genome-wide level, followed by proliferation inhibition. They bind to mRNA splicing-related proteins to induce a widespread change of splicing patterns in treated cells. Their inhibitory activity on splicing is relatively moderate, and introns with weak splice sites tend to be sensitive to them. Such introns remain unspliced, and the resulting intron-containing mRNAs are retained in the nucleus, resulting in the nuclear accumulation of poly(A) RNAs in these flavonoid-treated cells. Tumorigenic cells are more susceptible to these flavonoids than nontumorigenic cells, both for the nuclear poly(A) RNA-accumulating phenotype and cell viability. This study illustrates the possible mechanism of these flavonoids to suppress tumor progression in vivo that were demonstrated by previous studies and provides the potential of daily intake of moderate splicing inhibitors to prevent cancer development.
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http://dx.doi.org/10.1016/j.isci.2019.11.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909097PMC
December 2019

Distinct cell proliferation, myogenic differentiation, and gene expression in skeletal muscle myoblasts of layer and broiler chickens.

Sci Rep 2019 11 11;9(1):16527. Epub 2019 Nov 11.

Department of Agriculture, Graduate School of Science and Technology, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano, 399-4598, Japan.

Myoblasts play a central role during skeletal muscle formation and growth. Precise understanding of myoblast properties is thus indispensable for meat production. Herein, we report the cellular characteristics and gene expression profiles of primary-cultured myoblasts of layer and broiler chickens. Broiler myoblasts actively proliferated and promptly differentiated into myotubes compared to layer myoblasts, which corresponds well with the muscle phenotype of broilers. Transcriptomes of layer and broiler myoblasts during differentiation were quantified by RNA sequencing. Ontology analyses of the differentially expressed genes (DEGs) provided a series of extracellular proteins as putative markers for characterization of chicken myogenic cells. Another ontology analyses demonstrated that broiler myogenic cells are rich in cell cycle factors and muscle components. Independent of these semantic studies, principal component analysis (PCA) statistically defined two gene sets: one governing myogenic differentiation and the other segregating layers and broilers. Thirteen candidate genes were identified with a combined study of the DEGs and PCA that potentially contribute to proliferation or differentiation of chicken myoblasts. We experimentally proved that one of the candidates, enkephalin, an opioid peptide, suppresses myoblast growth. Our results present a new perspective that the opioids present in feeds may influence muscle development of domestic animals.
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http://dx.doi.org/10.1038/s41598-019-52946-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848216PMC
November 2019

Fluid dynamics approach to airway obstruction.

Med Hypotheses 2019 Nov 30;132:109341. Epub 2019 Jul 30.

Critical Care Medicine, National Center for Child Health and Development, Japan.

Background: Fluid dynamics theory, which is a fundamental underlying concept applied to fluid management, has not been introduced to analyze the human respiratory system. We hypothesized that one of the potential mechanisms that promotes airflow limitation in patients with airway obstructive disease would be elucidated by using fluid dynamics theory.

Methods: We calculated the values of pressure loss and static pressure change under virtual tracheal stenotic conditions using the fluid dynamics approach.

Results: Under normal conditions, the absolute values of pressure loss and static pressure change are very low. However, once airway stenosis occurs, it is confirmed that they would be dramatically elevated.

Conclusions: The fluid dynamics approach to airway obstruction is very constructive. The treatment strategy for airway obstruction and the reasons for airflow limitation are well explained by using this approach.
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http://dx.doi.org/10.1016/j.mehy.2019.109341DOI Listing
November 2019

Communicating bronchopulmonary foregut malformation: Volume change of the affected lung after birth.

Pediatr Pulmonol 2019 06 2;54(6):669-671. Epub 2019 Apr 2.

Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan.

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http://dx.doi.org/10.1002/ppul.24296DOI Listing
June 2019

Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging.

Sci Rep 2019 03 11;9(1):4050. Epub 2019 Mar 11.

Institute of Advanced Technology, Kindai University, Wakayama, 642-0017, Japan.

The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.
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http://dx.doi.org/10.1038/s41598-019-40546-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411884PMC
March 2019

Global transcriptome analysis of pig induced pluripotent stem cells derived from six and four reprogramming factors.

Sci Data 2019 02 26;6:190034. Epub 2019 Feb 26.

Tsukuba Gene Technology Laboratories Inc., Tsuchiura, 6-320 Arakawaoki, 300-0873 Japan.

Pigs are important, both for agriculture and as animal models for human diseases. However, due to the lack of embryonic stem cells, the possibility of genetic modification is quite limited. To overcome this limitation, induced pluripotent stem (iPS) cells have been derived from pigs. Despite the public availability of a large number of expression datasets from mice, rats, and primates-derived iPS cells, the expression profile of pig-derived iPS cells is quite limited. Furthermore, there is no dataset focused on the profiling of pig-derived iPS cell with six reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc, Lin28, and Nanog). Here, we used Illumina RNA sequencing platform to characterize the mRNA expression of four-factor derived and six-factor derived pig iPS cells. We observed that the expression levels of whole genes in our established six factors derived iPS cells and parent fibroblast, and compared with that of iPS cells with four factors in public database. These data are valuable in understanding species difference in the reprogramming process of stem cells, and could help identify the key regulating genes involved in the process.
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http://dx.doi.org/10.1038/sdata.2019.34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390709PMC
February 2019

Tuning water-use efficiency and drought tolerance in wheat using abscisic acid receptors.

Nat Plants 2019 02 8;5(2):153-159. Epub 2019 Feb 8.

Arid Land Research Center, Tottori University, Tottori, Japan.

Water availability is a key determinant of terrestrial plant productivity. Many climate models predict that water stress will increasingly challenge agricultural yields and exacerbate projected food deficits. To ensure food security and increase agricultural efficiency, crop water productivity must be increased. Research over past decades has established that the phytohormone abscisic acid (ABA) is a central regulator of water use and directly regulates stomatal opening and transpiration. In this study, we investigated whether the water productivity of wheat could be improved by increasing its ABA sensitivity. We show that overexpression of a wheat ABA receptor increases wheat ABA sensitivity, which significantly lowers a plant's lifetime water consumption. Physiological analyses demonstrated that this water-saving trait is a consequence of reduced transpiration and a concomitant increase in photosynthetic activity, which together boost grain production per litre of water and protect productivity during water deficit. Our findings provide a general strategy for increasing water productivity that should be applicable to other crops because of the high conservation of the ABA signalling pathway.
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http://dx.doi.org/10.1038/s41477-019-0361-8DOI Listing
February 2019

Sex-specific histone modifications in mouse fetal and neonatal germ cells.

Epigenomics 2019 04 22;11(5):543-561. Epub 2019 Jan 22.

Department of Bioscience, Tokyo University of Agriculture, Setagaya, Tokyo, Japan.

Aims: Epigenetic signatures of germline cells are dynamically reprogrammed to induce appropriate differentiation, development and sex specification. We investigated sex-specific epigenetic changes in mouse fetal germ cells (FGCs) and neonatal germ cells.

Materials & Methods: Six histone marks in mouse E13.5 FGCs and P1 neonatal germ cells were analyzed by chromatin immunoprecipitation and sequencing. These datasets were compared with transposase-accessible chromatin sites, DNA methylation and transcriptome.

Results: Different patterns of each histone mark were detected in female and male FGCs, and H3K4me3/H3K27me3 bivalent marks were enriched in different chromosomal regions of female and male FGCs.

Conclusion: Our results suggest that histone modifications may affect FGC gene expression following DNA methylation erasure, contributing to the differentiation into female and male germ cells.
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http://dx.doi.org/10.2217/epi-2018-0193DOI Listing
April 2019

SETDB1 is essential for mouse primordial germ cell fate determination by ensuring BMP signaling.

Development 2018 12 3;145(23). Epub 2018 Dec 3.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi 980-8575, Japan

In mouse embryos, primordial germ cells (PGCs) are fate-determined from epiblast cells. Signaling pathways involved in PGC formation have been identified, but their epigenetic mechanisms remain poorly understood. Here, we show that the histone methyltransferase SETDB1 is an epigenetic regulator of PGC fate determination. -deficient embryos exhibit drastic reduction of nascent PGCs. , and are de-repressed whereas mesoderm development-related genes, including BMP4 signaling-related genes, are downregulated by knockdown during PGC-like cell (PGCLC) induction. In addition, binding of SETDB1 is observed at the flanking regions of , and in cell aggregates containing PGCLCs, and trimethylation of lysine 9 of histone H3 is reduced by knockdown at those regions. Furthermore, DPPA2, OTX2 and UTF1 binding is increased in genes encoding BMP4 signaling-related proteins, including SMAD1. Finally, overexpression of , and in cell aggregates containing PGCLCs results in the repression of BMP4 signaling-related genes and PGC determinant genes. We propose that the localization of SETDB1 to , and , and subsequent repression of their expression, are crucial for PGC determination by ensuring BMP4 signaling.
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http://dx.doi.org/10.1242/dev.164160DOI Listing
December 2018

DNMTs and SETDB1 function as co-repressors in MAX-mediated repression of germ cell-related genes in mouse embryonic stem cells.

PLoS One 2018 7;13(11):e0205969. Epub 2018 Nov 7.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan.

In embryonic stem cells (ESCs), the expression of development-related genes, including germ cell-related genes, is globally repressed. The transcription factor MAX represses germ cell-related gene expression in ESCs via PCGF6-polycomb repressive complex 1 (PRC1), which consists of several epigenetic factors. However, we predicted that MAX represses germ cell-related gene expression through several additional mechanisms because PCGF6-PRC1 regulates the expression of only a subset of genes repressed by MAX. Here, we report that MAX associated with DNA methyltransferases (DNMTs) and the histone methyltransferase SETDB1 cooperatively control germ cell-related gene expression in ESCs. Both DNA methylation and histone H3 lysine 9 tri-methylation of the promoter regions of several germ cell-related genes were not affected by knockout of the PRC1 components, indicating that the MAX-DNMT and MAX-SETDB1 pathways are independent of the PCGF6-PRC1 pathway. Our findings provide insights into our understanding of MAX-based repressive mechanisms of germ cell-related genes in ESCs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221296PMC
April 2019

Relationship between PIWIL4-Mediated H3K4me2 Demethylation and piRNA-Dependent DNA Methylation.

Cell Rep 2018 10;25(2):350-356

Department of Pathology, Medical School, Osaka University, Suita 565-0871, Osaka, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Osaka, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Saitama 332-0012, Japan. Electronic address:

Retrotransposon genes are silenced by DNA methylation because of potential harm due to insertional mutagenesis. DNA methylation of retrotransposon genes is erased and re-established during male germ cell development. Both piRNA-dependent and piRNA-independent mechanisms are active during the re-establishment process, with the piRNA-independent mechanism occurring first. In this study, we analyzed the role of PIWIL4/MIWI2 in the modification of histone H3 and subsequent piRNA-dependent DNA methylation. Dimethylation at H3K4 is highly enriched at piRNA-dependent methylated regions and anti-correlated with de novo DNA methylation during the phase of piRNA-independent DNA methylation. In addition, PIWIL4, which binds the H3K4 demethylases KDM1A and KDM5B, is required for removing H3K4me2 marks. These data show that PIWIL4 plays important roles in histone modification and piRNA-dependent DNA methylation.
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http://dx.doi.org/10.1016/j.celrep.2018.09.017DOI Listing
October 2018

Repression of Somatic Genes by Selective Recruitment of HDAC3 by BLIMP1 Is Essential for Mouse Primordial Germ Cell Fate Determination.

Cell Rep 2018 09;24(10):2682-2693.e6

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi 980-8575, Japan; Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8577, Japan; Center for Regulatory Epigenome and Diseases, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan. Electronic address:

Primordial germ cells (PGCs) are fate determined from pluripotent epiblasts. Signaling pathways and transcriptional regulators involved in PGC formation have been identified, but detailed molecular mechanisms of PGC fate determination remains poorly understood. Using RNAi screening, we identified histone deacetylase 3 (HDAC3) as a regulator of PGC formation. Hdac3 deficiency resulted in decreased nascent PGCs in vitro and in vivo, and somatic developmental genes were de-repressed by Hdac3 knockdown during PGC induction. We also demonstrated BLIMP1-dependent enrichment of HDAC3 and deacetylation of H3 and H4 histones in the somatic developmental genes in epiblast-like cells. In addition, the HDAC3/BLIMP1-targeted somatic gene products were enriched in PGC determinant genes; overexpression of these gene products in PGC-like cells in culture resulted in repression of PGC determinant genes. We propose that selective recruitment of HDAC3 to somatic genes by BLIMP1 and subsequent repression of these somatic genes are crucial for PGC fate determination.
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http://dx.doi.org/10.1016/j.celrep.2018.07.108DOI Listing
September 2018

LTR retrotransposons transcribed in oocytes drive species-specific and heritable changes in DNA methylation.

Nat Commun 2018 08 20;9(1):3331. Epub 2018 Aug 20.

Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

De novo DNA methylation (DNAme) during mouse oogenesis occurs within transcribed regions enriched for H3K36me3. As many oocyte transcripts originate in long terminal repeats (LTRs), which are heterogeneous even between closely related mammals, we examined whether species-specific LTR-initiated transcription units (LITs) shape the oocyte methylome. Here we identify thousands of syntenic regions in mouse, rat, and human that show divergent DNAme associated with private LITs, many of which initiate in lineage-specific LTR retrotransposons. Furthermore, CpG island (CGI) promoters methylated in mouse and/or rat, but not human oocytes, are embedded within rodent-specific LITs and vice versa. Notably, at a subset of such CGI promoters, DNAme persists on the maternal genome in fertilized and parthenogenetic mouse blastocysts or in human placenta, indicative of species-specific epigenetic inheritance. Polymorphic LITs are also responsible for disparate DNAme at promoter CGIs in distantly related mouse strains, revealing that LITs also promote intra-species divergence in CGI DNAme.
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http://dx.doi.org/10.1038/s41467-018-05841-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102241PMC
August 2018

Inactivated influenza vaccine effectiveness and an analysis of repeated vaccination for children during the 2016/17 season.

Vaccine 2018 09 6;36(37):5510-5518. Epub 2018 Aug 6.

Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan.

Objectives: We assessed the vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children 6 months to 15 years of age during the 2016/17 season. In addition, we estimated the impact of repeated vaccination in children on VE.

Methods: Our study for VEs in preventing influenza and admission due to influenza were conducted according to a test-negative case-control design (TNCC) based on influenza rapid diagnostic test results. We also analyzed the VE by vaccine status in the current and previous seasons for the impact of repeated vaccination.

Results: During the 2016/17 season, the quadrivalent IIV was used in Japan. The adjusted VE in preventing influenza illness was 38% (95% CI, 29-46) against influenza A and 39% (95% CI, 18-54) against influenza B. Infants showed no significant VE. The VE in preventing hospitalization was not demonstrated. For the analysis of repeated vaccination, the vaccine was effective only when immunization occurred in the current season. The children who were immunized in two consecutive seasons were more likely to develop influenza compared to those immunized in the current season only (odds ratio, 1.58 [95% CI, 1.05-2.38], adjusted odds ratio, 1.53 [95% CI, 0.99-2.35]). However, the odds ratio of repeated vaccination was not significant when the analysis excluded those who developed influenza in the previous season.

Conclusions: VE in children in the 2016/17 season was similar to values previously reported. Repeated vaccination interfered with the VE against any influenza infection in the 2016/17 season. The results of our study suggest that decreased VE by repeat vaccination phenomenon was associated with immunity by influenza infection in the previous season. However, the influenza vaccine should be recommended every season for children.
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http://dx.doi.org/10.1016/j.vaccine.2018.07.065DOI Listing
September 2018

Primary jejunal Burkitt lymphoma in a child: ultrasonic detection.

J Surg Case Rep 2018 May 14;2018(5):rjy090. Epub 2018 May 14.

Department of Radiology, Otsu Red Cross Hospital, Otsu, Japan.

Gastrointestinal Burkitt lymphoma (BL) is a highly aggressive malignancy in childhood, and early treatment is critical for its favorable prognosis. Ultrasonography is a widely accepted initial imaging workup; therefore, recognition of the sonographic features of BL should contribute to its early diagnosis and initiation of treatment. We present a 4-year-old boy with primary jejunal BL with intussusception mimicking presentation, in which initial abdominal US allowed sustainable detection and characterization of the intestinal lesion. Jejunotomy was performed and histopathological analysis revealed a 'starry sky' pattern and c-myc split signals characteristic of BL. The patient remains disease-free following chemotherapy.
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http://dx.doi.org/10.1093/jscr/rjy090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951082PMC
May 2018

Genome-wide methylation profiles in primary intracranial germ cell tumors indicate a primordial germ cell origin for germinomas.

Acta Neuropathol 2017 03 11;133(3):445-462. Epub 2017 Jan 11.

Division of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka, 540-0006, Japan.

Intracranial germ cell tumors (iGCTs) are the second most common brain tumors among children under 14 in Japan. The World Health Organization classification recognizes several subtypes of iGCTs, which are conventionally subclassified into pure germinoma or non-germinomatous GCTs. Recent exhaustive genomic studies showed that mutations of the genes involved in the MAPK and/or PI3K pathways are common in iGCTs; however, the mechanisms of how different subtypes develop, often as a mixed-GCT, are unknown. To elucidate the pathogenesis of iGCTs, we investigated 61 GCTs of various subtypes by genome-wide DNA methylation profiling. We showed that pure germinomas are characterized by global low DNA methylation, a unique epigenetic feature making them distinct from all other iGCTs subtypes. The patterns of methylation strongly resemble that of primordial germ cells (PGC) at the migration phase, possibly indicating the cell of origin for these tumors. Unlike PGC, however, hypomethylation extends to long interspersed nuclear element retrotransposons. Histologically and epigenetically distinct microdissected components of mixed-GCTs shared identical somatic mutations in the MAPK or PI3K pathways, indicating that they developed from a common ancestral cell.
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http://dx.doi.org/10.1007/s00401-017-1673-2DOI Listing
March 2017

Inducible Transposition of a Heat-Activated Retrotransposon in Tissue Culture.

Plant Cell Physiol 2017 02;58(2):375-384

Faculty of Science, Hokkaido University, Sappor, Japan.

A transposition of a heat-activated retrotransposon named ONSEN required compromise of a small RNA-mediated epigenetic regulation that includes RNA-directed DNA methylation (RdDM) machinery after heat treatment. In the current study, we analyzed the transcriptional and transpositional activation of ONSEN to better understand the underlying molecular mechanism involved in the maintenance and/or induction of transposon activation in plant tissue culture. We found the transposition of heat-primed ONSEN during tissue culture independently of RdDM mutation. The heat activation of ONSEN transcripts was not significantly up-regulated in tissue culture compared with that in heat-stressed seedlings, indicating that the transposition of ONSEN was regulated independently of the transcript level. RdDM-related genes were up-regulated by heat stress in both tissue culture and seedlings. The level of DNA methylation of ONSEN did not show any change in tissue culture, and the amount of ONSEN-derived small RNAs was not affected by heat stress. The results indicated that the transposition of ONSEN was regulated by an alternative mechanism in addition to the RdDM-mediated epigenetic regulation in tissue culture. We applied the tissue culture-induced transposition of ONSEN to Japanese radish, an important breeding species of the family Brassicaceae. Several new insertions were detected in a regenerated plant derived from heat-stressed tissues and its self-fertilized progeny, revealing the possibility of molecular breeding without genetic modification.
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http://dx.doi.org/10.1093/pcp/pcw202DOI Listing
February 2017

Conservative follow-up of fractured percutaneously inserted central venous catheter.

Pediatr Int 2016 Dec;58(12):1369-1370

Department of Pediatrics, Saiseikai Utsunomiya Hospital, Tochigi, Japan.

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http://dx.doi.org/10.1111/ped.13082DOI Listing
December 2016

Repetitive DNA methylome analysis by small-scale and single-cell shotgun bisulfite sequencing.

Genes Cells 2016 Nov 3;21(11):1209-1222. Epub 2016 Oct 3.

Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502, Japan.

Whole-genome shotgun bisulfite sequencing (WG-SBS) is currently the most powerful tool available for understanding genomewide cytosine methylation with single-base resolution; however, the high sequencing cost limits its widespread application, particularly for mammalian genomes. We mapped high- to low-coverage SBS short reads of mouse and human female developing germ cells to consensus sequences of repetitive elements that were multiplied in the respective host genome. This mapping strategy effectively identified active and evolutionarily young retrotransposon subfamilies and centromeric satellite repeats that were resistant to DNA demethylation during the investigated progressive stages of germ cell development. Notably, quantities of only tens of thousands of uniquely mapped reads provided sufficient sensitivity to allow for methylation analyses of multiple retrotransposons and satellite repeats in mice. Furthermore, we produced SBS results from single female murine germ cells by an improved multiplexing and amplification-free SBS method (scPBAT). The scPBAT results quantitatively provided ≥5× sequencing coverage for at least 30 repeats, and the individual methylation patterns detected were similar to the bulk cell-based results. Our single-cell methylome sequencing technique will allow researchers to investigate intergenic methylation characteristics from limited amounts of mammalian cells as well as cells from other organisms with genomic annotations.
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http://dx.doi.org/10.1111/gtc.12440DOI Listing
November 2016

Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice.

Reproduction 2016 11 5;152(5):417-30. Epub 2016 Aug 5.

Center for Regenerative MedicineNational Research Institute for Child Health and Development, Setagaya, Tokyo, Japan Department of Stem Cell ResearchFukushima Medical University, 1 Hikarigaoka, Fukushima City, Fukushima, Japan

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8-16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3-5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming.
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http://dx.doi.org/10.1530/REP-16-0277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064760PMC
November 2016

DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

Biol Reprod 2016 06 20;94(6):128. Epub 2016 Apr 20.

Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan

The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.
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http://dx.doi.org/10.1095/biolreprod.116.138677DOI Listing
June 2016