Publications by authors named "Ryohei Nakamura"

16 Publications

  • Page 1 of 1

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals.

Nat Commun 2020 11 24;11(1):5972. Epub 2020 Nov 24.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
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http://dx.doi.org/10.1038/s41467-020-19603-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686362PMC
November 2020

A Nearly Complete Genome of Ciona intestinalis Type A (C. robusta) Reveals the Contribution of Inversion to Chromosomal Evolution in the Genus Ciona.

Genome Biol Evol 2019 11;11(11):3144-3157

Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan.

Since its initial publication in 2002, the genome of Ciona intestinalis type A (Ciona robusta), the first genome sequence of an invertebrate chordate, has provided a valuable resource for a wide range of biological studies, including developmental biology, evolutionary biology, and neuroscience. The genome assembly was updated in 2008, and it included 68% of the sequence information in 14 pairs of chromosomes. However, a more contiguous genome is required for analyses of higher order genomic structure and of chromosomal evolution. Here, we provide a new genome assembly for an inbred line of this animal, constructed with short and long sequencing reads and Hi-C data. In this latest assembly, over 95% of the 123 Mb of sequence data was included in the chromosomes. Short sequencing reads predicted a genome size of 114-120 Mb; therefore, it is likely that the current assembly contains almost the entire genome, although this estimate of genome size was smaller than previous estimates. Remapping of the Hi-C data onto the new assembly revealed a large inversion in the genome of the inbred line. Moreover, a comparison of this genome assembly with that of Ciona savignyi, a different species in the same genus, revealed many chromosomal inversions between these two Ciona species, suggesting that such inversions have occurred frequently and have contributed to chromosomal evolution of Ciona species. Thus, the present assembly greatly improves an essential resource for genome-wide studies of ascidians.
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http://dx.doi.org/10.1093/gbe/evz228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836712PMC
November 2019

Targeted in vivo epigenome editing of H3K27me3.

Epigenetics Chromatin 2019 03 13;12(1):17. Epub 2019 Mar 13.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0033, Japan.

Background: Epigenetic modifications have a central role in transcriptional regulation. While several studies using next-generation sequencing have revealed genome-wide associations between epigenetic modifications and transcriptional states, a direct causal relationship at specific genomic loci has not been fully demonstrated, due to a lack of technology for targeted manipulation of epigenetic modifications. Recently, epigenome editing techniques based on the CRISPR-Cas9 system have been reported to directly manipulate specific modifications at precise genomic regions. However, the number of editable modifications as well as studies applying these techniques in vivo is still limited.

Results: Here, we report direct modification of the epigenome in medaka (Japanese killifish, Oryzias latipes) embryos. Specifically, we developed a method to ectopically induce the repressive histone modification, H3K27me3 in a locus-specific manner, using a fusion construct of Oryzias latipes H3K27 methyltransferase Ezh2 (olEzh2) and dCas9 (dCas9-olEzh2). Co-injection of dCas9-olEzh2 mRNA with single guide RNAs (sgRNAs) into one-cell-stage embryos induced specific H3K27me3 accumulation at the targeted loci and induced downregulation of gene expression.

Conclusion: In this study, we established the in vivo epigenome editing of H3K27me3 using medaka embryos. The locus-specific manipulation of the epigenome in living organisms will lead to a previously inaccessible understanding of the role of epigenetic modifications in development and disease.
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http://dx.doi.org/10.1186/s13072-019-0263-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419334PMC
March 2019

Unlinking the methylome pattern from nucleotide sequence, revealed by large-scale in vivo genome engineering and methylome editing in medaka fish.

PLoS Genet 2017 12 21;13(12):e1007123. Epub 2017 Dec 21.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

The heavily methylated vertebrate genomes are punctuated by stretches of poorly methylated DNA sequences that usually mark gene regulatory regions. It is known that the methylation state of these regions confers transcriptional control over their associated genes. Given its governance on the transcriptome, cellular functions and identity, genome-wide DNA methylation pattern is tightly regulated and evidently predefined. However, how is the methylation pattern determined in vivo remains enigmatic. Based on in silico and in vitro evidence, recent studies proposed that the regional hypomethylated state is primarily determined by local DNA sequence, e.g., high CpG density and presence of specific transcription factor binding sites. Nonetheless, the dependency of DNA methylation on nucleotide sequence has not been carefully validated in vertebrates in vivo. Herein, with the use of medaka (Oryzias latipes) as a model, the sequence dependency of DNA methylation was intensively tested in vivo. Our statistical modeling confirmed the strong statistical association between nucleotide sequence pattern and methylation state in the medaka genome. However, by manipulating the methylation state of a number of genomic sequences and reintegrating them into medaka embryos, we demonstrated that artificially conferred DNA methylation states were predominantly and robustly maintained in vivo, regardless of their sequences and endogenous states. This feature was also observed in the medaka transgene that had passed across generations. Thus, despite the observed statistical association, nucleotide sequence was unable to autonomously determine its own methylation state in medaka in vivo. Our results apparently argue against the notion of the governance on the DNA methylation by nucleotide sequence, but instead suggest the involvement of other epigenetic factors in defining and maintaining the DNA methylation landscape. Further investigation in other vertebrate models in vivo will be needed for the generalization of our observations made in medaka.
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http://dx.doi.org/10.1371/journal.pgen.1007123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755920PMC
December 2017

Centromere evolution and CpG methylation during vertebrate speciation.

Nat Commun 2017 11 28;8(1):1833. Epub 2017 Nov 28.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba, 277-8583, Japan.

Centromeres and large-scale structural variants evolve and contribute to genome diversity during vertebrate speciation. Here, we perform de novo long-read genome assembly of three inbred medaka strains that are derived from geographically isolated subpopulations and undergo speciation. Using single-molecule real-time (SMRT) sequencing, we obtain three chromosome-mapped genomes of length ~734, ~678, and ~744Mbp with a resource of twenty-two centromeric regions of length 20-345kbp. Centromeres are positionally conserved among the three strains and even between four pairs of chromosomes that were duplicated by the teleost-specific whole-genome duplication 320-350 million years ago. The centromeres do not all evolve at a similar pace; rather, centromeric monomers in non-acrocentric chromosomes evolve significantly faster than those in acrocentric chromosomes. Using methylation sensitive SMRT reads, we uncover centromeres are mostly hypermethylated but have hypomethylated sub-regions that acquire unique sequence compositions independently. These findings reveal the potential of non-acrocentric centromere evolution to contribute to speciation.
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http://dx.doi.org/10.1038/s41467-017-01982-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705604PMC
November 2017

Hypomethylated domain-enriched DNA motifs prepattern the accessible nucleosome organization in teleosts.

Epigenetics Chromatin 2017 09 20;10(1):44. Epub 2017 Sep 20.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Background: Gene promoters in vertebrate genomes show distinct chromatin features such as stably positioned nucleosome array and DNA hypomethylation. The nucleosomes are known to have certain sequence preferences, and the prediction of nucleosome positioning from DNA sequence has been successful in some organisms such as yeast. However, at gene promoters where nucleosomes are much more stably positioned than in other regions, the sequence-based model has failed to work well, and sequence-independent mechanisms have been proposed.

Results: Using DNase I-seq in medaka embryos, we demonstrated that hypomethylated domains (HMDs) specifically possess accessible nucleosome organization with longer linkers, and we reassessed the DNA sequence preference for nucleosome positioning in these specific regions. Remarkably, we found with a supervised machine learning algorithm, k-mer SVM, that nucleosome positioning in HMDs is accurately predictable from DNA sequence alone. Specific short sequences (6-mers) that contribute to the prediction are specifically enriched in HMDs and distribute periodically with approximately 200-bp intervals which prepattern the position of accessible linkers. Surprisingly, the sequence preference of the nucleosome and linker in HMDs is opposite from that reported previously. Furthermore, the periodicity of specific motifs at hypomethylated promoters was conserved in zebrafish.

Conclusion: This study reveals strong link between nucleosome positioning and DNA sequence at vertebrate promoters, and we propose hypomethylated DNA-specific regulation of nucleosome positioning.
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http://dx.doi.org/10.1186/s13072-017-0152-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607494PMC
September 2017

Comparative Analysis of Genome and Epigenome in Closely Related Medaka Species Identifies Conserved Sequence Preferences for DNA Hypomethylated Domains.

Zoolog Sci 2016 Aug;33(4):358-65

1 Department of Biological Sciences, Graduate School of Science,The University of Tokyo, Tokyo 113-0033, Japan.

The genomes of vertebrates are globally methylated, but a small portion of genomic regions are known to be hypomethylated. Although hypomethylated domains (HMDs) have been implicated in transcriptional regulation in various ways, how a HMD is determined in a particular genomic region remains elusive. To search for DNA motifs essential for the formation of HMDs, we performed the genome-wide comparative analysis of genome and DNA methylation patterns of the two medaka inbred lines, Hd-rRII1 and HNI-II, which are derived from northern and southern subpopulations of Japan and exhibit high levels of genetic variations (SNP, ∼ 3%). We successfully mapped > 70% of HMDs in both genomes and found that the majority of those mapped HMDs are conserved between the two lines (common HMDs). Unexpectedly, the average genetic variations are similar in the common HMD and other genome regions. However, we identified short well-conserved motifs that are specifically enriched in HMDs, suggesting that they may play roles in the establishment of HMDs in the medaka genome.
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http://dx.doi.org/10.2108/zs160030DOI Listing
August 2016

Assessing Cell-to-Cell DNA Methylation Variability on Individual Long Reads.

Sci Rep 2016 Feb 18;6:21317. Epub 2016 Feb 18.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwa, Japan.

Understanding cell-to-cell variability in cytosine methylation is essential for understanding cellular perturbation and its molecular machinery. However, conventional methylation studies have focused on the differences in the average levels between cell types while overlooking methylation heterogeneity within cell types. Little information has been uncovered using recent single-cell methods because of either technical limitations or the great labor required to process many single cells. Here, we report the highly efficient detection of cell-to-cell DNA methylation variability in liver tissue, based on comparing the methylation status of adjacent CpG sites on long sequencing reads. This method provides abundant methylation linkage information and enables genome-wide estimation of cell-to-cell variability. We observed repressed methylation variability in hypomethylated regions compared with the variability in hypomethylated regions across the genome, which we confirmed using public human sperm data. A gradual change in methylation status at the boundaries of hypomethylated regions was observed for the first time. This approach allows the concise, comprehensive assessment of cell-to-cell DNA methylation variability.
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http://dx.doi.org/10.1038/srep21317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758075PMC
February 2016

Associations between nucleosome phasing, sequence asymmetry, and tissue-specific expression in a set of inbred Medaka species.

BMC Genomics 2015 Nov 19;16:978. Epub 2015 Nov 19.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, 277-0882, Japan.

Background: Transcription start sites (TSSs) with pronounced and phased nucleosome arrays downstream and nucleosome-depleted regions upstream of TSSs are observed in various species.

Results: We have characterized sequence variation and expression properties of this set of TSSs (which we call "Nucleocyclic TSSs") using germline and somatic cells of three medaka (Oryzias latipes) inbred isolates from different locations. We found nucleocyclic TSSs in medaka to be associated with higher gene expression and characterized by a clear boundary in sequence composition with potentially-nucleosome-destabilizing A/T-enrichment upstream (p < 10(-60)) and nucleosome- accommodating C/G-enrichment downstream (p < 10(-40)) that was highly conserved from an ancestor. A substantial genetic distance between the strains facilitated the in-depth analysis of patterns of fixed mutations, revealing a localization-specific equilibrium between the rates of distinct mutation categories that would serve to maintain the conserved sequence anisotropy around TSSs. Downstream of nucleocyclic TSSs, C to T, T to C, and other mutation rates on the sense strand increased around first nucleosome dyads and decreased around first linkers, which contrasted with genomewide mutational patterns around nucleosomes (p < 5 %). C to T rates are higher than G to A rates around nucleosome associated with germline nucleocyclic TSS sites (p < 5 %), potentially due to the asymmetric effect of transcription-coupled repair.

Conclusions: Our results demonstrate an atypical evolutionary process surrounding nucleocyclic TSSs.
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http://dx.doi.org/10.1186/s12864-015-2198-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653950PMC
November 2015

Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates.

Development 2014 Jul 12;141(13):2568-80. Epub 2014 Jun 12.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan

DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, we generated epigenomic profiles that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish and compared the profiles with those of human ES cells. We found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. We propose that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells.
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http://dx.doi.org/10.1242/dev.108548DOI Listing
July 2014

[Multivariate quantification of acupuncture and moxibustion treatment in combination with questionnaire and salivary stress markers].

Yakugaku Zasshi 2014 ;134(5):655-63

Faculty of Pharmacy, Kinki University.

Pain and stress alleviation after acupuncture treatment was assessed in this study. Patients responded to a questionnaire designed to determine the amount of stress they were experiencing, and data were obtained for patient salivary amylase, cortisol, secretary IgA (s-IgA), and leptin receptor (OBRb). As a part of this study on acute pain, 6 factors were extracted from the questionnaire. The second factor (pain removal) was well correlated with salivary amylase activity in patients with cervico-omo-brachial syndrome. An evaluation of cumulative acupuncture treatments showed that salivary cortisol increased and s-IgA decreased. In addition, a decreased s-IgA level significantly correlated with chronic pain removal. The questionnaire correlated well with measurements of salivary markers suggesting that they can be taken as indices of therapeutic efficacy in acupuncture treatment.
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http://dx.doi.org/10.1248/yakushi.13-00240DOI Listing
December 2014

The effects of frequent electroejaculation on the semen characteristics of a captive Siberian tiger (Panthera tigris altaica).

J Reprod Dev 2013 Oct 16;59(5):491-5. Epub 2013 Jun 16.

Asahikawa Municipal Asahiyama Zoological Park & Wildlife Conservation Center, Asahikawa 078-8205, Japan.

Artificial insemination (AI) can help to avoid inbreeding and genetic degeneration for sustaining genetically healthy populations of endangered species in captivity. Collection of a sufficient quantity of viable sperm is an essential first step in the AI process. In the present study, we examined the effects of frequent electroejaculation on semen characteristics in a Siberian tiger. We collected semen in all 17 trials during 6 breeding seasons (6 years). The mean number of sperm and the percentage of motile sperm were 294.3 ± 250.2 × 10⁶/ejaculate and 82.4 ± 11.4%, respectively. The number of motile sperm tended to increase during frequent electroejaculation in the same breeding season. Semen collection by electroejaculation can be performed effectively up to the fourth sequential ejaculate, which contained the most sperm in the study. In conclusion, frequent collection of sperm by electroejaculation from tigers may be effective for collection of a large number of motile sperm.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934116PMC
http://dx.doi.org/10.1262/jrd.2013-016DOI Listing
October 2013

Timing of puberty and its relationship with body growth and season in male raccoons (Procyon lotor) in Hokkaido.

J Reprod Dev 2013 20;59(4):361-7. Epub 2013 Apr 20.

Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido 060-0818, Japan.

The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944363PMC
http://dx.doi.org/10.1262/jrd.2012-198DOI Listing
April 2014

The medaka zic1/zic4 mutant provides molecular insights into teleost caudal fin evolution.

Curr Biol 2012 Apr 1;22(7):601-7. Epub 2012 Mar 1.

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan.

Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.
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http://dx.doi.org/10.1016/j.cub.2012.01.063DOI Listing
April 2012

Seasonal changes in spermatogenesis and peripheral testosterone concentration in raccoons (Procyon lotor) in Hokkaido.

J Vet Med Sci 2012 Jun 10;74(6):727-32. Epub 2012 Jan 10.

Laboratory of Wildlife Biology and Medicine, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan.

Feral raccoons (Procyon lotor) have been increasing in number since 1979 and are currently subject to pest control in Hokkaido. One of the reasons for the increase in numbers is thought to be the high reproductive potential of raccoons, but little is known about their reproduction. The main aim of this study was to clarify seasonal changes in spermatogenesis and peripheral testosterone concentration of raccoons in Hokkaido. In the present study, external characteristics and histology of the testis and epididymis and the plasma testosterone concentration were investigated in 68 feral, male raccoons culled for pest control and once a month in one live, captive male. The feral males exhibited seasonal changes in spermatogenesis, showing active spermatogenesis in autumn, winter and spring (October-June) with noted spermatogenesis and inactive spermatogenesis in summer (July-September) with lower mean levels of spermatozoa in the cauda epididymis. Even in the inactive period, spermatozoa were observed in about half of the individuals (14/26); therefore, individuals producing spermatozoa existed every month throughout the year. Testosterone concentrations were significantly high in the winter mating season. In the captive male, the testosterone concentrations were low from June to August, and spermatozoa could not be observed from July to September. These results suggest that raccoons exhibit seasonality of reproduction, but the time and duration of spermatogenetic decline varies widely among individuals. This individual variation in the inactive period is a feature of male raccoon reproduction and is unique among seasonally breeding mammals.
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http://dx.doi.org/10.1292/jvms.11-0351DOI Listing
June 2012