Publications by authors named "Makoto Nakanishi"

124 Publications

PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage.

Cancer Sci 2021 May 3. Epub 2021 May 3.

Department of Biochemistry, Joint Faculty of Veterinary Science, Yamaguchi University, Yamaguchi, Japan.

DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3-Thr phosphorylation (H3-pThr ) at E2F1 target gene promoters. Dephosphorylation of H3-pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ-mediated H3-pThr by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ-mediated dephosphorylation of H3-pThr both in vivo and in vitro. By generating NIPP1-depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1-Ser residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.
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http://dx.doi.org/10.1111/cas.14924DOI Listing
May 2021

HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation.

Nucleic Acids Res 2021 Apr 19. Epub 2021 Apr 19.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.
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http://dx.doi.org/10.1093/nar/gkab269DOI Listing
April 2021

TP53/p53-FBXO22-TFEB controls basal autophagy to govern hormesis.

Autophagy 2021 Mar 11:1-18. Epub 2021 Mar 11.

Division of Cancer Cell Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy. Mild stress-activated TP53 transcriptionally induced FBXO22, which in turn ubiquitinated KDM4B (lysine-specific demethylase 4B) complexed with MYC-NCOR1 suppressors for degradation, leading to transcriptional induction of TFEB. Upregulation of autophagy-related genes by increased TFEB dramatically enhanced autophagic activity and cell survival upon following a severe stressor. Mitogen-induced AKT1 activation counteracted this process through the phosphorylation of KDM4B, which inhibited FBXO22-mediated ubiquitination. Additionally, mice died within 10 h of birth, and their mouse embryonic fibroblasts (MEFs) showed a lowered basal autophagy, whereas FBXO22-overexpressing mice were resistant to chemotherapy. Taken together, these results suggest that TP53 upregulates basal autophagy through the FBXO22-TFEB axis, which governs the hormetic effect in chemotherapy.: BBC3/PUMA: BCL2 binding component 3; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; ChIP-seq: chromatin immunoprecipitation followed by sequencing; DDB2: damage specific DNA binding protein 2; DRAM: DNA damage regulated autophagy modulator; ESR/ER: estrogen receptor 1; FMD: fasting mimicking diet; HCQ: hydroxychloroquine; KDM4B: lysine-specific demethylase 4B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; NCOR1: nuclear receptor corepressor 1; SCF: SKP1-CUL-F-box protein; SQSTM1: sequestosome 1; TFEB: transcription factor EB.
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http://dx.doi.org/10.1080/15548627.2021.1897961DOI Listing
March 2021

Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders.

Science 2021 01;371(6526):265-270

Division of Cancer Cell Biology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Removal of senescent cells (senolysis) has been proposed to be beneficial for improving age-associated pathologies, but the molecular pathways for such senolytic activity have not yet emerged. Here, we identified glutaminase 1 () as an essential gene for the survival of human senescent cells. The intracellular pH in senescent cells was lowered by lysosomal membrane damage, and this lowered pH induced kidney-type glutaminase (KGA) expression. The resulting enhanced glutaminolysis induced ammonia production, which neutralized the lower pH and improved survival of the senescent cells. Inhibition of KGA-dependent glutaminolysis in aged mice eliminated senescent cells specifically and ameliorated age-associated organ dysfunction. Our results suggest that senescent cells rely on glutaminolysis, and its inhibition offers a promising strategy for inducing senolysis in vivo.
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http://dx.doi.org/10.1126/science.abb5916DOI Listing
January 2021

Development of an indirect ELISA based on soluble antigen produced from virus-infected cells for detection of porcine hemagglutinating encephalomyelitis virus.

J Virol Methods 2021 03 5;289:114016. Epub 2020 Dec 5.

Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo, 183-8509, Japan; Cooperative Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo, 183-8509, Japan. Electronic address:

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus Betacoronavirus and is the etiologic agent of encephalomyelitis or vomiting and wasting disease in neonatal pigs. Although there are only a few epidemiological studies that document the seroprevalence of PHEV infection, there are reports of sporadic outbreaks, including recent documentation of an influenza-like respiratory disease associated with PHEV in the United States. To address this issue, we have developed a new indirect enzyme linked immunosorbent assay (ELISA) for use in sero-epidemiological research of PHEV infection. One hundred and fifty porcine serum samples that were determined as antibody-positive or antibody-negative in virus neutralization (VN) tests were used in conjunction with PHEV-specific antigen extracted from virus-infected FS-L3 cells using RBS buffer containing 0.2 % NP-40 to develop this assay. The ELISA showed a high sensitivity (95.35 %) and specificity (96.88 %) by receiver operating characteristic (ROC) analysis, with an area under the curve (AUC) of 0.996 attesting to its accuracy. Our results revealed a strong correlation between the results of the indirect ELISA and VN test (R = 0.850, P < 0.05), with near-perfect agreement (kappa value = 0.932). These results indicate that this new indirect ELISA might be useful for diagnosis and sero-epidemiological tracking of PHEV infection.
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http://dx.doi.org/10.1016/j.jviromet.2020.114016DOI Listing
March 2021

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 3D tissue model-on-a-chip for studying the effects of human senescent fibroblasts on blood vessels.

Biomater Sci 2021 Jan;9(1):199-211

Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

All human tissues experience aging that eventually causes organ dysfunction and disease. Cellular senescence was discovered in fibroblasts cultured in vitro. In adults, it is a primary defense mechanism against cancer, but also a major contributor to lifespan limits and disorders associated with aging. To assess how human blood vessels change in an aged environment, we developed an elementary tissue model-on-a-chip that comprises an in vitro three-dimensional model of a blood vessel embedded in a collagen gel with young or senescent skin fibroblasts. We found that senescent fibroblasts mechanically altered the surrounding extracellular matrix by exerting excessive traction stress. We then found that senescent fibroblasts induced sprouting angiogenesis of a microvessel via their senescence-associated secretory phenotype (SASP). Finally, we gathered evidence that the mechanical changes of the microenvironment play a role in sustaining SASP-induced angiogenesis. The model proved useful in monitoring morphological changes in blood vessels induced by senescent fibroblasts while controlling the proportion of senescent cells, and enabled the study of SASP inhibitors, a class of drugs useful in aging and cancer research.
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http://dx.doi.org/10.1039/d0bm01297aDOI Listing
January 2021

Preparation and Characterization of Additional Metallic Element-Containing Tubular Iron Oxides of Bacterial Origin.

ACS Omega 2020 Oct 16;5(42):27287-27294. Epub 2020 Oct 16.

Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.

Biogenic microtubular iron oxides (BIOXs) derived from spp. are known as promising multifunctional materials for industrial applications such as ceramic pigments and catalyst carriers. Here, we report unprecedented BIOX products with additive depositions of various metallic elements prepared by a newly devised "two-step" method using an artificial culture system of strain OUMS1; the method comprises a biotic formation of immature organic sheaths and subsequent abiotic deposition of Fe and intended elements on the sheaths. Chemical composition ratios of the additional elements Al, Zr, and Ti in the respective BIOXs were arbitrarily controllable depending on initial concentrations of metallic salts added to reaction solutions. Raman spectroscopy exemplified an existence of Fe-O-Al linkage in the Al-containing BIOX matrices. Time-course analyses revealed the underlying physiological mechanism for the BIOX formation. These results indicate that our advanced method can contribute greatly to creations of innovative bioderived materials with improved functionalities.
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http://dx.doi.org/10.1021/acsomega.0c03574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594126PMC
October 2020

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 11 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Two mouse models carrying truncating mutations in Magel2 show distinct phenotypes.

PLoS One 2020 17;15(8):e0237814. Epub 2020 Aug 17.

Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

Schaaf-Yang syndrome (SYS) is a neurodevelopmental disorder caused by truncating variants in the paternal allele of MAGEL2, located in the Prader-Willi critical region, 15q11-q13. Although the phenotypes of SYS overlap those of Prader-Willi syndrome (PWS), including neonatal hypotonia, feeding problems, and developmental delay/intellectual disability, SYS patients show autism spectrum disorder and joint contractures, which are atypical phenotypes for PWS. Therefore, we hypothesized that the truncated Magel2 protein could potentially produce gain-of-function toxic effects. To test the hypothesis, we generated two engineered mouse models; one, an overexpression model that expressed the N-terminal region of Magel2 that was FLAG tagged with a strong ubiquitous promoter, and another, a genome-edited model that carried a truncating variant in Magel2 generated using the CRISPR/Cas9 system. In the overexpression model, all transgenic mice died in the fetal or neonatal period indicating embryonic or neonatal lethality of the transgene. Therefore, overexpression of the truncated Magel2 could show toxic effects. In the genome-edited model, we generated a mouse model carrying a frameshift variant (c.1690_1924del; p(Glu564Serfs*130)) in Magel2. Model mice carrying the frameshift variant in the paternal or maternal allele of Magel2 were termed Magel2P:fs and Magel2M:fs, respectively. The imprinted expression and spatial distribution of truncating Magel2 transcripts in the brain were maintained. Although neonatal Magel2P:fs mice were lighter than wildtype littermates, Magel2P:fs males and females weighed the same as their wildtype littermates by eight and four weeks of age, respectively. Collectively, the overexpression mouse model may recapitulate fetal or neonatal death, which are the severest phenotypes for SYS. In contrast, the genome-edited mouse model maintains genomic imprinting and distribution of truncated Magel2 transcripts in the brain, but only partially recapitulates SYS phenotypes. Therefore, our results imply that simple gain-of-function toxic effects may not explain the patho-mechanism of SYS, but rather suggest a range of effects due to Magel2 variants as in human SYS patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237814PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430741PMC
October 2020

FBXO22, an epigenetic multiplayer coordinating senescence, hormone signaling, and metastasis.

Cancer Sci 2020 Aug 17;111(8):2718-2725. Epub 2020 Jul 17.

Division of Cancer Cell Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Japan.

Ubiquitin-dependent protein degradation has been implicated in the control of various cellular processes such as cell cycle control, transcriptional regulation, DNA damage repair, and apoptosis, many of which are involved in the initiation, progression, metastasis, and drug resistance of cancers. E3 ubiquitin ligases are known to be the second most prevalent cancer-related functional gene family next to protein kinases. Of these, FBXO22, an F-box receptor subunit of SCF E3 ligase, has recently been proposed to play a critical role in multiple aspects related to cancer development and therapy response. Firstly, FBXO22 is a key regulator of senescence induction through ubiquitylation of p53 for degradation. FBXO22 also acts as a molecular switch for the antagonistic and agonistic actions of selective estrogen receptor modulators (SERM) and determines the sensitivity of breast cancer to SERM by ubiquitylating KDM4B complexed with unliganded or SERMs-bound estrogen receptor (ER). Furthermore, FBXO22 binds to Bach1, a pro-metastatic transcription factor, suppressing Bach1-driven metastasis of lung adenocarcinoma, and loss of FBXO22 facilitates metastasis. These findings, as well as other reports, unveiled strikingly important roles of FBXO22 in cancer development and therapeutic strategy. In this review, we summarize recent findings of how FBXO22 regulates major cancer suppression pathways.
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http://dx.doi.org/10.1111/cas.14534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419058PMC
August 2020

Validity of the Wide-range Assessment of Vision-related Essential Skills in Japanese Children with Learning Problems.

Optom Vis Sci 2020 04;97(4):275-285

Department of Pediatrics, Osaka Medical College, Takatsuki, Japan.

Significance: Our study revealed that the validity of a new Japanese visual-perceptual test was acceptable. Visual-perceptual abilities are important to activities of daily living; thus, accurate assessment of visual perception is especially important for children with neurodevelopmental disorders and particularly so for those with learning problems.

Purpose: Visual perception refers to the process by which one receives visual information through sensory impulses and then translates those impulses into meaning based on a previously developed view of the environment. A problem in Japan is the paucity of visual perception tests that use normative data from children who are native Japanese. The Wide-range Assessment of Vision-related Essential Skills (WAVES), which measures visual perception and eye-hand coordination skills and is based on Japanese normative data, was recently published in Japan. The validity of this test has not been comprehensively established.

Methods: To investigate the validity of the WAVES, we used the Pearson correlation coefficient to calculate the degrees of association among WAVES scaled and index scores compared with Developmental Test of Visual Perception, Third Edition, scores from 108 elementary school children with symptoms of learning problems. Participants were recruited at Osaka Medical College Learning Disability Center and Sakai Seikeikai Learning Disability Center.

Results: The concurrent validity of the WAVES was supported by moderate correlation (r = 0.67, P < .01) between the total scores for visual perception and eye-hand coordination index from the WAVES and general visual perception index from the Developmental Test of Visual Perception, Third Edition, even though a correlation analysis of subtests found differences between the two tests.

Conclusions: Our results showed that the indices from the two tests measured nearly the same underlying visual-perceptual constructs and indicated that the WAVES had acceptable levels of concurrent validity.
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http://dx.doi.org/10.1097/OPX.0000000000001504DOI Listing
April 2020

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Nat Commun 2020 03 6;11(1):1222. Epub 2020 Mar 6.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
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http://dx.doi.org/10.1038/s41467-020-15006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060239PMC
March 2020

Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

Genes Cells 2020 Jan 3;25(1):22-32. Epub 2019 Dec 3.

Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Japan.

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.
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http://dx.doi.org/10.1111/gtc.12732DOI Listing
January 2020

[Cellular senescence and age-related diseases].

Authors:
Makoto Nakanishi

Rinsho Ketsueki 2019 ;60(9):1027-1032

Division of Cancer Cell Biology, Institute of Medical Science, The University of Tokyo.

Cellular senescence is a state of durable cell cycle arrest after a defined number of cell divisions. The number of population doublings of normal cells in culture depends on the species but not on the types of cells used to establish the culture, showing a positive correlation with the life span of the animals. Therefore, the results suggest a physiological link between a limited proliferative capacity in cell culture and the processes observed in organismal aging. The pivotal role of senescence in organismal aging and the onset of age-related disorders, such as atherosclerosis, type II diabetes, and Alzheimer's disease, is supported by the observation that the clearance of p16-positive senescent cells delays various age-associated disorders and extends healthy lifespan. In this review, I provide an overview of recent advances in understanding the mechanisms underlying the induction of senescence and maintenance of the specific phenotypes, such as senescence associated secretory phenotypes (SASP).
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http://dx.doi.org/10.11406/rinketsu.60.1027DOI Listing
October 2019

Intermittent parathyroid hormone 1-34 induces oxidation and deterioration of mineral and collagen quality in newly formed mandibular bone.

Sci Rep 2019 05 29;9(1):8041. Epub 2019 May 29.

Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, Japan.

Intermittent parathyroid hormone (PTH) administration is known to promote bone healing after surgical procedures. However, the mechanism and influence of PTH on the mineral and collagen quality of the jaw are not well understood. Most studies have focused on analyzing the bone density and microstructure of the mandible, and have insufficiently investigated its mineral and collagen quality. Oxidative stress activates osteoclasts, produces advanced glycation end products, and worsens mineral and collagen quality. We hypothesized that PTH induces oxidation and affects the mineral and collagen quality of newly formed mandibular bone. To test this, we examined the mineral and collagen quality of newly formed mandibular bone in rats administered PTH, and analyzed serum after intermittent PTH administration to examine the degree of oxidation. PTH administration reduced mineralization and worsened mineral and collagen quality in newly formed bone. In addition, total anti-oxidant capacity in serum was significantly decreased and the oxidative-INDEX was increased among PTH-treated compared to vehicle-treated rats, indicating serum oxidation. In conclusion, intermittent administration of PTH reduced mineral and collagen quality in newly formed mandibular bone. This effect may have been induced by oxidation.
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http://dx.doi.org/10.1038/s41598-019-44389-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541641PMC
May 2019

Cdk1-mediated DIAPH1 phosphorylation maintains metaphase cortical tension and inactivates the spindle assembly checkpoint at anaphase.

Nat Commun 2019 02 28;10(1):981. Epub 2019 Feb 28.

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan.

Animal cells undergo rapid rounding during mitosis, ensuring proper chromosome segregation, during which an outward rounding force abruptly increases upon prometaphase entry and is maintained at a constant level during metaphase. Initial cortical tension is generated by the actomyosin system to which both myosin motors and actin network architecture contribute. However, how cortical tension is maintained and its physiological significance remain unknown. We demonstrate here that Cdk1-mediated phosphorylation of DIAPH1 stably maintains cortical tension after rounding and inactivates the spindle assembly checkpoint (SAC). Cdk1 phosphorylates DIAPH1, preventing profilin1 binding to maintain cortical tension. Mutation of DIAPH1 phosphorylation sites promotes cortical F-actin accumulation, increases cortical tension, and delays anaphase onset due to SAC activation. Measurement of the intra-kinetochore length suggests that Cdk1-mediated cortex relaxation is indispensable for kinetochore stretching. We thus uncovered a previously unknown mechanism by which Cdk1 coordinates cortical tension maintenance and SAC inactivation at anaphase onset.
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http://dx.doi.org/10.1038/s41467-019-08957-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395754PMC
February 2019

Fbxo22-mediated KDM4B degradation determines selective estrogen receptor modulator activity in breast cancer.

J Clin Invest 2018 12 12;128(12):5603-5619. Epub 2018 Nov 12.

Division of Cancer Cell Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

The agonistic/antagonistic biocharacter of selective estrogen receptor modulators (SERMs) can have therapeutic advantages, particularly in the case of premenopausal breast cancers. Although the contradictory effects of these modulators have been studied in terms of crosstalk between the estrogen receptor α (ER) and coactivator dynamics and growth factor signaling, the molecular basis of these mechanisms is still obscure. We identify a series of regulatory mechanisms controlling cofactor dynamics on ER and SERM function, whose activities require F-box protein 22 (Fbxo22). Skp1, Cullin1, F-box-containing complex (SCFFbxo22) ubiquitylated lysine demethylase 4B (KDM4B) complexed with tamoxifen-bound (TAM-bound) ER, whose degradation released steroid receptor coactivator (SRC) from ER. Depletion of Fbxo22 resulted in ER-dependent transcriptional activation via transactivation function 1 (AF1) function, even in the presence of SERMs. In living cells, TAM released SRC and KDM4B from ER in a Fbxo22-dependent manner. SRC release by TAM required Fbxo22 on almost all ER-SRC-bound enhancers and promoters. TAM failed to prevent the growth of Fbxo22-depleted, ER-positive breast cancers both in vitro and in vivo. Clinically, a low level of Fbxo22 in tumor tissues predicted a poorer outcome in ER-positive/human epidermal growth factor receptor type 2-negative (HER2-negative) breast cancers with high hazard ratios, independently of other markers such as Ki-67 and node status. We propose that the level of Fbxo22 in tumor tissues defines a new subclass of ER-positive breast cancers for which SCFFbxo22-mediated KDM4B degradation in patients can be a therapeutic target for the next generation of SERMs.
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http://dx.doi.org/10.1172/JCI121679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264734PMC
December 2018

Isozyme-Specific Role of SAD-A in Neuronal Migration During Development of Cerebral Cortex.

Cereb Cortex 2019 08;29(9):3738-3751

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.
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http://dx.doi.org/10.1093/cercor/bhy253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335017PMC
August 2019

Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance.

Mol Cell 2017 Oct;68(2):350-360.e7

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Electronic address:

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.
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http://dx.doi.org/10.1016/j.molcel.2017.09.037DOI Listing
October 2017

Exon 7 splicing variant of estrogen receptor α is associated with pathological invasiveness in smoking-independent lung adenocarcinoma.

Oncol Lett 2017 Jul 19;14(1):891-898. Epub 2017 May 19.

Department of Oncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, Mizuho, Nagoya 467-8601, Japan.

Patients with smoking-independent lung cancer mainly consist of females, yet the molecular background of this epidemiological feature, other than epidermal growth factor receptor (EGFR) mutation, remains unclear. Several studies have revealed the association between female hormone-associated factors and the prognosis of lung cancer, however the data remain inconsistent. The present study focused on the expression of estrogen receptor (ER)α in order to elucidate this association in smoking-independent lung cancer. Immunohistochemistry staining (IHC) of aromatase, ERα and ERβ was performed against formalin-treated tissues from 38 patients who had never-smoked who underwent complete surgical resection between 2012 and 2013. Among them, adequate RNA of the tumor and adjacent normal lung cancer was extracted from 31 matching deep frozen samples. Considering the IHC results, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to measure the expression level of 2 different exons of ERα, exon 6 and exon 7, which are part of the ligand binding domain of ERα, using the Taqman gene expression assay. Extra-nuclear expression of ERα using IHC demonstrated a statistically significant association with pathological invasiveness. RT-qPCR results exhibited a decreased expression of ERα exon 7 in invasive tumor tissues, compared with their adjacent normal tissues. This is consistent with the findings of previous studies indicating that extra-nuclear ERα were exon 7 splicing variants. No difference was observed in ERα exon 7 expression between normal and tumor tissues in non-invasive lung cancer tissues. When considering the EGFR mutation status, EGFR wild-type lung cancers exhibited decreased ERα exon 7 expression levels compared with EGFR mutated lung cancers. Extra-nuclear expression of ERα, which may represent exon 7 splicing variants of ERα, showed statistical association with pathological invasiveness in smoking-independent lung cancer. The post-translational splicing mechanism of ERα may be involved in the acquired invasiveness of smoking independent lung cancer.
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http://dx.doi.org/10.3892/ol.2017.6216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494717PMC
July 2017

Biosorption of metal elements by exopolymer nanofibrils excreted from Leptothrix cells.

Water Res 2017 10 4;122:139-147. Epub 2017 May 4.

Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), Okayama 700-8530, Japan; Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan. Electronic address:

Leptothrix species, aquatic Fe-oxidizing bacteria, excrete nano-scaled exopolymer fibrils. Once excreted, the fibrils weave together and coalesce to form extracellular, microtubular, immature sheaths encasing catenulate cells of Leptothrix. The immature sheaths, composed of aggregated nanofibrils with a homogeneous-looking matrix, attract and bind aqueous-phase inorganics, especially Fe, P, and Si, to form seemingly solid, mature sheaths of a hybrid organic-inorganic nature. To verify our assumption that the organic skeleton of the sheaths might sorb a broad range of other metallic and nonmetallic elements, we examined the sorption potential of chemically and enzymatically prepared protein-free organic sheath remnants for 47 available elements. The sheath remnants were found by XRF to sorb each of the 47 elements, although their sorption degree varied among the elements: >35% atomic percentages for Ti, Y, Zr, Ru, Rh, Ag, and Au. Electron microscopy, energy dispersive x-ray spectroscopy, electron and x-ray diffractions, and Fourier transform infrared spectroscopy analyses of sheath remnants that had sorbed Ag, Cu, and Pt revealed that (i) the sheath remnants comprised a 5-10 nm thick aggregation of fibrils, (ii) the test elements were distributed almost homogeneously throughout the fibrillar aggregate, (iii) the nanofibril matrix sorbing the elements was nearly amorphous, and (iv) these elements plausibly were bound to the matrix by ionic binding, especially via OH. The present results show that the constitutive protein-free exopolymer nanofibrils of the sheaths can contribute to creating novel filtering materials for recovering and recycling useful and/or hazardous elements from the environment.
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http://dx.doi.org/10.1016/j.watres.2017.05.003DOI Listing
October 2017

The G2 checkpoint inhibitor CBP-93872 increases the sensitivity of colorectal and pancreatic cancer cells to chemotherapy.

PLoS One 2017 30;12(5):e0178221. Epub 2017 May 30.

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Aichi, Japan.

CBP-93872 suppresses maintenance of DNA double-stranded break-induced G2 checkpoint, by inhibiting the pathway between ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) activation. To examine the potential use of CBP-93872 for clinical applications, we analyzed the synergistic effects of platinum-containing drugs, oxaliplatin and cisplatin, pyrimidine antimetabolites, gemcitabine and 5-fluorouracil (5-FU), in combination with CBP-93872, on cell lethality in colorectal and pancreatic cancer cell lines. Treatment with CBP-93872 significantly increased cancer cell sensitivities to various chemotherapeutic agents tested through suppression of checkpoint activation. Our results thus reveal that combination treatment of CBP-93872 with known chemotherapeutic agents inhibits phosphorylation of ATR and Chk1, and induces cell death.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178221PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448762PMC
September 2017

Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation.

Sci Rep 2017 03 3;7(1):55. Epub 2017 Mar 3.

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1, Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan.

Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.
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http://dx.doi.org/10.1038/s41598-017-00136-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427934PMC
March 2017

A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly.

BMC Med Genet 2017 Jan 13;18(1). Epub 2017 Jan 13.

Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.

Background: Constitutive activation of the PI3K-AKT-mTOR pathway (mTOR pathway) underlies megalencephaly in many patients. Yet, prevalence of the involvement of the PI3K-AKT-mTOR pathway in patients with megalencephaly remains to be elucidated, and molecular diagnosis is challenging. Here, we have successfully established a combination of genetic and biochemical methods for diagnosis of mTOR pathway-associated megalencephaly, and have attempted to delineate the clinical characteristics of the disorder.

Methods: Thirteen patients with an increased head circumference and neurological symptoms participated in the study. To evaluate the activation of the mTOR pathway, we performed western blot analysis to determine the expression levels of phosphorylated S6 ribosomal protein (phospho-S6 protein) in lymphoblastoid cell lines from 12 patients. Multiplex targeted sequencing analysis for 15 genes involved in the mTOR pathway was performed on 12 patients, and whole-exome sequencing was performed on one additional patient. Clinical features and MRI findings were also investigated.

Results: We identified pathogenic mutations in six (AKT3, 1 patient; PIK3R2, 2 patients; PTEN, 3 patients) of the 13 patients. Increased expression of phospho-S6 protein was demonstrated in all five mutation-positive patients in whom western blotting was performed, as well as in three mutation-negative patients. Developmental delay, dysmorphic facial features were observed in almost all patients. Syndactyly/polydactyly and capillary malformations were not observed, even in patients with AKT3 or PIK3R2 mutations. There were no common phenotypes or MRI findings among these patients.

Conclusions: A combination of genetic and biochemical methods successfully identified mTOR pathway involvement in nine of 13 (approximately 70%) patients with megalencephaly, indicating a major contribution of the pathway to the pathogenesis of megalencephaly. Our combined approach could be useful to identify patients who are suitable for future clinical trials using an mTOR inhibitor.
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http://dx.doi.org/10.1186/s12881-016-0363-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5237172PMC
January 2017

HP1 regulates the localization of FANCJ at sites of DNA double-strand breaks.

Cancer Sci 2016 Oct 1;107(10):1406-1415. Epub 2016 Sep 1.

Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.

The breast and ovarian cancer predisposition protein BRCA1 forms three mutually exclusive complexes with Fanconi anemia group J protein (FANCJ, also called BACH1 or BRIP1), CtIP, and Abraxas/RAP80 through its BRCA1 C terminus (BRCT) domains, while its RING domain binds to BRCA1-associated RING domain 1 (BARD1). We recently found that the interaction between heterochromatin protein 1 (HP1) and BARD1 is required for the accumulation of BRCA1 and CtIP at sites of DNA double-strand breaks. Here, we investigated the importance of HP1 and BARD1-HP1 interaction in the localization of FANCJ together with the other BRCA1-BRCT binding proteins to clarify the separate role of the HP1-mediated pathway from the RNF8/RNF168-induced ubiquitin-mediated pathway for BRCA1 function. FANCJ interacts with HP1γ in a BARD1-dependent manner, and this interaction was enhanced by ionizing radiation or irinotecan hydrochloride treatment. Simultaneous depletion of all three HP1 isoforms with shRNAs disrupts the accumulation of FANCJ and CtIP, but not RAP80, at double-strand break sites. Replacement of endogenous BARD1 with a mutant BARD1 that is incapable of binding to HP1 also disrupts the accumulation of FANCJ and CtIP, but not RAP80. In contrast, RNF168 depletion disrupts the accumulation of only RAP80, but not FANCJ or CtIP. Consequently, the accumulation of conjugated ubiquitin was only inhibited by RNF168 depletion, whereas the accumulation of RAD51 and sister chromatid exchange were only inhibited by HP1 depletion or disruption of the BARD1-HP1 interaction. Taken together, the results suggest that the BRCA1-FANCJ and BRCA1-CtIP complexes are not downstream of the RNF8/RNF168/ubiquitin pathway, but are instead regulated by the HP1 pathway that precedes homologous recombination DNA repair.
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http://dx.doi.org/10.1111/cas.13008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5084677PMC
October 2016

Multiple facets of p53 in senescence induction and maintenance.

Cancer Sci 2016 Nov 4;107(11):1550-1555. Epub 2016 Nov 4.

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.

Cellular senescence is a state of durable cell cycle arrest with metabolic activities distinct from those of the proliferative state. Since senescence was originally reported to be induced by various genotoxic stressors, such as telomere erosion and oncogenic signaling, it has been proposed to play a pivotal role in aging-related changes and as an antitumorigenic barrier in vivo. However, the mechanisms underlying its induction and maintenance remain entirely elusive. We have recently found that abrupt activation of p53 at G results in a cell skipping mitosis and subsequently undergoing senescence. Surprisingly, we have also found that downregulation of p53 by SCF is crucial for the induction of a senescence-associated phenotype. In this review, we provide an overview of recent advances in understanding the mechanisms underlying the timing and magnitude of activation of p53 during senescence.
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http://dx.doi.org/10.1111/cas.13060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132285PMC
November 2016

Aurora B twists on histones for activation.

Cell Cycle 2016 12 25;15(24):3321-3322. Epub 2016 Aug 25.

b Division of Cancer Cell Biology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo , Tokyo , Japan.

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http://dx.doi.org/10.1080/15384101.2016.1224758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224459PMC
December 2016

Defective DNA repair increases susceptibility to senescence through extension of Chk1-mediated G2 checkpoint activation.

Sci Rep 2016 08 10;6:31194. Epub 2016 Aug 10.

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.

Susceptibility to senescence caused by defective DNA repair is a major hallmark of progeroid syndrome patients, but molecular mechanisms of how defective DNA repair predisposes to senescence are largely unknown. We demonstrate here that suppression of DNA repair pathways extends the duration of Chk1-dependent G2 checkpoint activation and sensitizes cells to senescence through enhancement of mitosis skipping. Extension of G2 checkpoint activation by introduction of the TopBP1 activation domain and the nondegradable mutant of Claspin sensitizes cells to senescence. In contrast, a shortening of G2 checkpoint activation by expression of SIRT6 or depletion of OTUB2 reduces susceptibility to senescence. Fibroblasts from progeroid syndromes tested shows a correlation between an extension of G2 checkpoint activation and an increase in the susceptibility to senescence. These results suggest that extension of G2 checkpoint activation caused by defective DNA repair is critical for senescence predisposition in progeroid syndrome patients.
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http://dx.doi.org/10.1038/srep31194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979019PMC
August 2016