Publications by authors named "Hiroshi Masumoto"

131 Publications

Kinetochore stretching-mediated rapid silencing of the spindle-assembly checkpoint required for failsafe chromosome segregation.

Curr Biol 2021 Feb 22. Epub 2021 Feb 22.

Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan. Electronic address:

The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived force, which causes kinetochores to undergo repetitive cycles of deformation; this phenomenon is referred to as kinetochore stretching. The nature of the forces and the relevance relating this deformation are not well understood. Here, we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespective of microtubule poleward pulling force. An experimental method to conditionally interfere with the stretching allowed us to determine that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment after the establishment of end-on attachments and removal of the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that a lower frequency of kinetochore stretching largely correlates with a prolonged metaphase in cancer cell lines with chromosomal instability. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges, which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability and underscores the importance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.
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http://dx.doi.org/10.1016/j.cub.2021.01.062DOI Listing
February 2021

Analysis of Complex DNA Rearrangements during Early Stages of HAC Formation.

ACS Synth Biol 2020 12 8;9(12):3267-3287. Epub 2020 Dec 8.

Wellcome Trust Centre for Cell Biology, Edinburgh EH9 3BF, United Kingdom.

Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.
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http://dx.doi.org/10.1021/acssynbio.0c00326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754191PMC
December 2020

Deletion of the GAPDH gene contributes to genome stability in Saccharomyces cerevisiae.

Sci Rep 2020 12 3;10(1):21146. Epub 2020 Dec 3.

Biomedical Research Support Center (BRSC), Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki, Nagasaki, 852-8523, Japan.

Cellular metabolism is directly or indirectly associated with various cellular processes by producing a variety of metabolites. Metabolic alterations may cause adverse effects on cell viability. However, some alterations potentiate the rescue of the malfunction of the cell system. Here, we found that the alteration of glucose metabolism suppressed genome instability caused by the impairment of chromatin structure. Deletion of the TDH2 gene, which encodes glyceraldehyde 3-phospho dehydrogenase and is essential for glycolysis/gluconeogenesis, partially suppressed DNA damage sensitivity due to chromatin structure, which was persistently acetylated histone H3 on lysine 56 in cells with deletions of both HST3 and HST4, encoding NAD-dependent deacetylases. tdh2 deletion also restored the short replicative lifespan of cells with deletion of sir2, another NAD-dependent deacetylase, by suppressing intrachromosomal recombination in rDNA repeats increased by the unacetylated histone H4 on lysine 16. tdh2 deletion also suppressed recombination between direct repeats in hst3∆ hst4∆ cells by suppressing the replication fork instability that leads to both DNA deletions among repeats. We focused on quinolinic acid (QUIN), a metabolic intermediate in the de novo nicotinamide adenine dinucleotide (NAD) synthesis pathway, which accumulated in the tdh2 deletion cells and was a candidate metabolite to suppress DNA replication fork instability. Deletion of QPT1, quinolinate phosphoribosyl transferase, elevated intracellular QUIN levels and partially suppressed the DNA damage sensitivity of hst3∆ hst4∆ cells as well as tdh2∆ cells. qpt1 deletion restored the short replicative lifespan of sir2∆ cells by suppressing intrachromosomal recombination among rDNA repeats. In addition, qpt1 deletion could suppress replication fork slippage between direct repeats. These findings suggest a connection between glucose metabolism and genomic stability.
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http://dx.doi.org/10.1038/s41598-020-78302-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713361PMC
December 2020

Successful treatment of recurrent small cell carcinoma of urinary bladder with pembrolizumab.

IJU Case Rep 2020 Nov 5;3(6):252-256. Epub 2020 Aug 5.

Department of Urology National Hospital Organization Fukuyama Medical Center Hiroshima Japan.

Introduction: Small cell carcinoma of urinary bladder is rare and has an aggressive malignant behavior and poor prognosis. Advanced bladder cancers are treated with immune checkpoint inhibitors, however, its efficacy for small cell carcinoma of urinary bladder is unclear.

Case Presentation: A 54-year-old female, diagnosed with clinical stage T2N0M0 small cell carcinoma of urinary bladder, underwent radical cystectomy after three cycles of etoposide-cisplatin neoadjuvant chemotherapy. Despite the fact that pathological examination revealed no residual carcinoma in bladder in her cystectomy specimen, local recurrence of a 60-mm mass detected in the follow-up investigation 7.5 months later. This was completely treated by pembrolizumab without any adverse effects. Immunohistochemical staining revealed that the tumor had no programmed death ligand 1 expression but it showed CD8-positive T-lymphocyte infiltration into the tumor.

Conclusion: Immune checkpoint inhibitors might have curative potentials for treatment of small cell carcinoma of urinary bladder.
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http://dx.doi.org/10.1002/iju5.12208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609171PMC
November 2020

Exploration of Pericyte-Derived Factors Implicated in Lung Cancer Brain Metastasis Protection: A Pilot Messenger RNA Sequencing Using the Blood-Brain Barrier In Vitro Model.

Cell Mol Neurobiol 2020 Nov 2. Epub 2020 Nov 2.

Department of Neurosurgery, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.

Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood-brain barrier (BBB) implication is supposed to play a major role in brain metastasis. However, the role of pericytes remains to be elucidated in the brain metastasis. This pilot study described the expression profile of interactions between pericytes, endothelial cells, and cancer cells. We applied an in vitro BBB model with rat primary cultured BBB-related cells (endothelial cells and pericytes), and performed the gene expression analyses of pericytes under the lung cancer cells coculture conditions. Pericytes demonstrated inhibition of the cancer cell proliferation significantly (p < 0.05). RNA was extracted from the pericytes, complementary DNA library was prepared, and RNA-seq was performed. The sequence read data were analyzed on the Management and Analysis System for Enormous Reads and Tag Count Comparison-Graphical User Interface platforms. No statistically or biologically significant differentially expressed genes (DEGs) were detected in the explanatory analyses. Lot-specific DEG detection demonstrated significant decreases in the expression of two genes (Wwtr1 and Acin1), and enrichment analyses using Metascape software revealed the inhibition of apoptotic processes in fibroblasts. Our results suggest that the expression profiles of brain pericytes are partially implicated in the prevention of lung cancer metastasis to the brain. Pericytes exerted an anti-metastatic effect in the BBB model, and their neurohumoral factors remain to be elucidated.
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http://dx.doi.org/10.1007/s10571-020-00988-yDOI Listing
November 2020

Effect of surface modification of Ti-6Al-4V alloy by electron cyclotron resonance plasma oxidation.

Dent Mater J 2021 Jan 15;40(1):228-234. Epub 2020 Oct 15.

Division of Advanced Prosthetic Dentistry, Tohoku University Graduate School of Dentistry.

Ti-6Al-4V alloy is used as biomaterials for dental and orthopedic implants because of their excellent biocompatibilities and mechanical properties. However, it is unclear that electron cyclotron resonance (ECR) plasma oxidation can create the oxide films on Ti-6Al-4V alloy surface, and this technique improves the ability of its osseointegration. The purpose of this study was to investigate the characteristics and calcification ability of the oxide films. X-ray diffraction (XRD) peaks of rutile phase were intensified with increasing the temperature. Scanning electron microscopy (SEM) images showed a crater-like structure, and bonding strengths between the substrate and oxide film reached a maximum at 400°C. Calcium phosphate (CaP) compounds after calcification process were identified as octacalcium phosphate (OCP) and precipitation amount was maximized at 400°C. The results suggested that the altered surface of Ti-6Al-4V alloy by ECR plasma oxidation might have the potential of accelerating the ability of its osseointegration through enhancement of OCP.
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http://dx.doi.org/10.4012/dmj.2020-051DOI Listing
January 2021

Artificial chromosomes.

Exp Cell Res 2020 11 25;396(1):112302. Epub 2020 Sep 25.

Genome Structure and Function Group, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA. Electronic address:

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http://dx.doi.org/10.1016/j.yexcr.2020.112302DOI Listing
November 2020

Damaged Myofiber-Derived Metabolic Enzymes Act as Activators of Muscle Satellite Cells.

Stem Cell Reports 2020 10 3;15(4):926-940. Epub 2020 Sep 3.

Department of Muscle Development and Regeneration, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan; Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 1-7-1, Sakamoto 852-8588, Japan; Center for Metabolic Regulation of Healthy Aging, Kumamoto University Faculty of Life Sciences, 1-1-1 Honjo, Kumamoto 860-8556, Japan. Electronic address:

Muscle satellite cells are normally quiescent but are rapidly activated following muscle damage. Here, we investigated whether damaged myofibers influence the activation of satellite cells. Our findings revealed that satellite cells are directly activated by damaged-myofiber-derived factors (DMDFs). DMDFs induced satellite cells to enter the cell cycle; however, the cells stayed at the G1 phase and did not undergo S phase, and these cells were reversible to the quiescent-like state. Proteome analysis identified metabolic enzymes, including GAPDH, as DMDFs, whose recombinant proteins stimulated the activation of satellite cells. Satellite cells pre-exposed to the DMDFs demonstrated accelerated proliferation ex vivo. Treatment with recombinant GAPDH prior to muscle injury promoted expansion of the satellite cell population in vivo. Thus, our results indicate that DMDFs are not only a set of biomarkers for muscle damage, but also act as moonlighting proteins involved in satellite cell activation at the initial step of muscle regeneration.
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http://dx.doi.org/10.1016/j.stemcr.2020.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561495PMC
October 2020

An Artificial Conversion of Roots into Organs with Shoot Stem Characteristics by Inducing Two Transcription Factors.

iScience 2020 Jul 14;23(7):101332. Epub 2020 Jul 14.

Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan; The Kisarazu Laboratory, Graduate School of Life Sciences, Tohoku University, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan. Electronic address:

Somatic plant cells can regenerate shoots and/or roots or adventitious embryonic calluses, which may induce organ formation under certain conditions. Such regenerations occur via dedifferentiation of somatic cells, induction of organs, and their subsequent outgrowth. Despite recent advances in understanding of plant regeneration, many details of shoot induction remain unclear. Here, we artificially induced shoot stem-like green organs (SSOs) in Arabidopsis thaliana roots via simultaneous induction of two transcription factors (TFs), ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 25 (ATHB25, At5g65410) and the B3 family transcription factor REPRODUCTIVE MERISTEM 7 (REM7, At3g18960). The SSOs exhibited negative gravitropism and differentiated vascular bundle phenotypes. The ATHB25/REM7 induced the expression of genes controlling shoot stem characteristics by ectopic expression in roots. Intriguingly, the restoration of root growth was seen in the consecutive and adjacent parts of the SSOs under gene induction conditions. Our findings thus provide insights into the development and regeneration of plant shoot stems.
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http://dx.doi.org/10.1016/j.isci.2020.101332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385925PMC
July 2020

CENP-B creates alternative epigenetic chromatin states permissive for CENP-A or heterochromatin assembly.

J Cell Sci 2020 08 11;133(15). Epub 2020 Aug 11.

Laboratory of Chromosome Engineering, Department of Frontier Research and Development, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu 292-0818, Japan

CENP-B binds to CENP-B boxes on centromeric satellite DNAs (known as alphoid DNA in humans). CENP-B maintains kinetochore function through interactions with CENP-A nucleosomes and CENP-C. CENP-B binding to transfected alphoid DNA can induce CENP-A assembly, functional centromere and kinetochore formation, and subsequent human artificial chromosome (HAC) formation. Furthermore, CENP-B also facilitates H3K9 (histone H3 lysine 9) trimethylation on alphoid DNA, mediated by Suv39h1, at ectopic alphoid DNA integration sites. Excessive heterochromatin invasion into centromere chromatin suppresses CENP-A assembly. It is unclear how CENP-B controls such different chromatin states. Here, we show that the CENP-B acidic domain recruits histone chaperones and many chromatin modifiers, including the H3K36 methylase ASH1L, as well as the heterochromatin components Suv39h1 and HP1 (HP1α, β and γ, also known as CBX5, CBX1 and CBX3, respectively). ASH1L facilitates the formation of open chromatin competent for CENP-A assembly on alphoid DNA. These results indicate that CENP-B is a nexus for histone modifiers that alternatively promote or suppress CENP-A assembly by mutually exclusive mechanisms. Besides the DNA-binding domain, the CENP-B acidic domain also facilitates CENP-A assembly on transfected alphoid DNA. CENP-B therefore balances CENP-A assembly and heterochromatin formation on satellite DNA.
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http://dx.doi.org/10.1242/jcs.243303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438015PMC
August 2020

H3K9me3 maintenance on a human artificial chromosome is required for segregation but not centromere epigenetic memory.

J Cell Sci 2020 07 24;133(14). Epub 2020 Jul 24.

Wellcome Trust Centre for Cell Biology, Edinburgh, UK

Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoid human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.This article has an associated First Person interview with the first authors of the paper.
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http://dx.doi.org/10.1242/jcs.242610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390644PMC
July 2020

A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus.

EMBO J 2020 04 3;39(8):e104120. Epub 2020 Mar 3.

Department of Molecular and Cellular Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the β subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.
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http://dx.doi.org/10.15252/embj.2019104120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156963PMC
April 2020

Human artificial chromosome: Chromatin assembly mechanisms and CENP-B.

Exp Cell Res 2020 04 8;389(2):111900. Epub 2020 Feb 8.

Laboratory of Chromosome Engineering, Department of Frontier Research and Development, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, 292-0818, Japan. Electronic address:

The centromere is a specialized chromosomal locus required for accurate chromosome segregation. Heterochromatin also assembles around centromere chromatin and forms a base that supports sister chromatid cohesion until anaphase begins. Both centromere chromatin and heterochromatin assemble on a centromeric DNA sequence, a highly repetitive sequence called alphoid DNA (α-satellite DNA) in humans. Alphoid DNA can form a de novo centromere and subsequent human artificial chromosome (HAC) when introduced into the human culture cells HT1080. HAC is maintained stably as a single chromosome independent of other human chromosomes. For de novo centromere assembly and HAC formation, the centromere protein CENP-B and its binding sites, CENP-B boxes, are required in the repeating units of alphoid DNA. CENP-B has multiple roles in de novo centromere chromatin assembly and stabilization and in heterochromatin formation upon alphoid DNA introduction into the cells. Here we review recent progress in human artificial chromosome construction and centromere/heterochromatin assembly and maintenance, focusing on the involvement of human centromere DNA and CENP-B protein.
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http://dx.doi.org/10.1016/j.yexcr.2020.111900DOI Listing
April 2020

Deep sequence analysis of NS5A resistance-associated substitution changes in patients reinfected with the hepatitis C virus after liver transplantation.

J Viral Hepat 2020 05 15;27(5):552-555. Epub 2020 Jan 15.

Department of Gastroenterology and Hepatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

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http://dx.doi.org/10.1111/jvh.13256DOI Listing
May 2020

Histomorphometric assessments of peri-implant bone around Ti-Nb-Sn alloy implants with low Young's modulus.

Dent Mater J 2020 Jan 30;39(1):148-153. Epub 2019 Oct 30.

Division of Advanced Prosthetic Dentistry, Tohoku University Graduate School of Dentistry, Tohoku University.

Many β-Ti alloys have been developed for, and used in, medical devices because of the corrosion resistance, biocompatibility, and exceptionally low Young's modulus. The aim of the present study was to investigate the histomorphometric aspects of peri-implant bone around Ti-Nb-Sn alloy implants and compare them with those in the case of commercially pure Ti (Ti). Fluorescent morphological observations of ST-2 cells on the substrate were performed and bone morphogenesis around implants in rat femur was evaluated. There was no difference between the cell morphology on Ti and those on the Ti-Nb-Sn alloy during observation for 24 h. A comparison of the Ti-Nb-Sn alloy implant and the Ti implant showed no significant differences between the bone-to-implant contact ratios or the bone fractions. These results suggest that the biological adaptations with Ti-Nb-Sn implants during a healing period are similar to those with Ti. Ti-Nb-Sn is therefore suitable for use in dental implants.
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http://dx.doi.org/10.4012/dmj.2018-376DOI Listing
January 2020

CRISPR/Transposon gene integration (CRITGI) can manage gene expression in a retrotransposon-dependent manner.

Sci Rep 2019 10 25;9(1):15300. Epub 2019 Oct 25.

Biomedical Research Support Center (BRSC), Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki, Nagasaki, 852-8523, Japan.

The fine-tuning of gene expression contributes to both basic science and applications. Here, we develop a novel gene expression technology termed CRITGI (CRISPR/Transposon gene integration). CRITGI uses CRISPR/Cas9 to integrate multiple copies of the plasmid pTy1 into Ty1 loci, budding yeast retrotransposons. The pTy1 plasmid harbors a Ty1 consensus sequence for integration, a gene of interest with its own promoter and a selection marker gene. Interestingly, the expression of the pTy1 gene in Ty1 loci could be induced in synthetic complete amino acid depletion medium, which could activate the selection marker gene on pTy1. The induction or repression of the gene on pTy1 depended on Ty1 transcription. Activation of the selection marker gene on pTy1 triggered Ty1 transcription, which led to induction of the gene on pTy1. The gene on pTy1 was not transcribed with Ty1 mRNA; the transcription required its own promoter. Furthermore, the trimethylation of histone H3 on lysine 4, a landmark of transcriptionally active chromatin, accumulated at the 5' end of the gene on pTy1 following selection marker gene activation. Thus, CRITGI is a unique gene regulation system to induce the genes on pTy1 in amino acid depletion medium and utilizes Ty1 transcription to create a chromatin environment favorable for the transcription of the genes on pTy1.
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http://dx.doi.org/10.1038/s41598-019-51891-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814769PMC
October 2019

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission.

Genome Res 2019 10 12;29(10):1719-1732. Epub 2019 Sep 12.

Developmental Therapeutics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

One of the hallmarks of cancer is hromosome stability (CIN), which leads to aneuploidy, translocations, and other chromosome aberrations. However, in the vast majority of human tumors the molecular basis of CIN remains unknown, partly because not all genes controlling chromosome transmission have yet been identified. To address this question, we developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses a human artificial chromosome (HAC) expressing the transgene. When this assay was applied to screen an siRNA library of protein kinases, we identified , , , , and as potential novel genes whose knockdown induces various mitotic abnormalities and results in chromosome loss. The HAC-based assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, and transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.
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http://dx.doi.org/10.1101/gr.254276.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771407PMC
October 2019

Utilization of Natural Detergent Potassium Laurate for Decellularization in Lung Bioengineering.

Tissue Eng Part C Methods 2019 08;25(8):459-471

1Department of Surgical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Impact Statement: Recent advances in tissue engineering using decellularized organ scaffolds have expanded the possibilities for organ replacement therapy. However, detergent-based decellularization itself damages the extracellular matrix (ECM), which results in failure associated with the transplanted bioengineered organ. This study determined that potassium laurate (PL), a natural detergent, significantly reduces lung ECM damage during the decellularization process compared with protocols using sodium dodecyl sulfate. PL-decellularized lungs showed better microarchitecture preservation and low biological reactions after subcutaneous implantation. PL-decellularized scaffolds supported rat lung endothelial cell attachment/proliferation and the bioengineered lungs significantly reduced lung congestion after transplantation.
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http://dx.doi.org/10.1089/ten.TEC.2019.0016DOI Listing
August 2019

Bacteroides in colonic mucosa-associated microbiota affects the development of minimal hepatic encephalopathy in patients with cirrhosis.

Hepatol Int 2019 Jul 9;13(4):482-489. Epub 2019 Jul 9.

Department of Gastroenterology and Hepatology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, Nagasaki, 852-8501, Japan.

Background/purpose: Gut microbiota has been associated with liver cirrhosis and, possibly, hepatic encephalopathy. However, only a few studies have examined the link between mucosa-associated microbiota (MAM) and minimal hepatic encephalopathy (MHE). Our aim was to investigate this relationship.

Methods: Twenty-four patients with cirrhosis underwent colon biopsies at our institution, between January 2014 and April 2015. Patterns of microbial colonization were examined using 16S rRNA gene sequences. MHE was diagnosed using the Neuropsychological Test.

Results: Ten (41.7%) of the 24 patients were diagnosed as having MHE. There was no significant difference in the diversity of gut microbiota by sampling locations between those with and without MHE. However, the diversity of the gut microbiota and the proportion of the genus Bacteroides decreased as a function of declining liver function. We divided patients into those with the highest proportion of the genus Bacteroides (Bacteroides-dominant group; n = 9) and into a Bacteroides non-dominant group (n = 15). In the Bacteroides-dominant group, only 1 patient (11.1%) was diagnosed as having MHE, with the incidence rate of MHE being significantly lower in the Bacteroides-dominant group than in the non-dominant group (p = 0.019). The Child-Pugh score (p = 0.05) and use of proton-pump inhibitors (p = 0.015) were negatively correlated to the proportion of Bacteroides. Furthermore, the proportion of the family Clostridiaceae was significantly higher in the Bacteroides-dominant group than in the non-dominant group (p = 0.078).

Conclusions: The decrease in microbial diversity and genus Bacteroides in MAM is a risk factor for MHE in patients with liver cirrhosis.
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http://dx.doi.org/10.1007/s12072-019-09963-2DOI Listing
July 2019

Whole genome characterisation of G11P[25] and G9P[19] rotavirus A strains from adult patients with diarrhoea in Nepal.

Infect Genet Evol 2019 04 11;69:246-254. Epub 2019 Feb 11.

Sukraraj Tropical and Infectious Disease Hospital, Kathmandu, Nepal.

Rotavirus A (RVA) causes acute diarrhoea in children and less frequently in adults. However, the knowledge about the genotype distribution of RVA strains circulating in adults is limited particularly in developing countries. This study aimed to characterise the RVA strains detected from adult patients with diarrhoea in Nepal. A total of 47 RVA positive stool samples from adult patients with diarrhoea in Kathmandu, Nepal during 2007-2008 were examined for the G and P genotypes by sequencing. Nearly half (49%) of the samples were genotyped as G9P[8] (n = 23), G1P[8], G2P[4] (n = 5 each), G12P[8] (n = 4), G12P[6] (n = 3), G1P[6] (n = 2), G3P[8] and G9P[6] (n = 1 each). Interestingly, two G11P[25] and one G9P[19] strains detected were further subjected to Illumina MiSeq next generation sequencing to determine their whole genome sequences. The genotype constellations of RVA/Human-wt/NPL/TK2615/2008/G11P[25] and RVA/Human-wt/NPL/TK2620/2008/G11P[25] were I12-R1-C1-M1-A1-N1-T1-E1-H1, whereas that of RVA/Human-wt/NPL/TK1797/2007/G9P[19] was I5-R1-C1-M1-A8-N1-T1-E1-H1. The 11 genes of TK2615 and TK2620 were virtually identical, and they were either porcine-like or unique except the VP2 and NSP1 genes which were of human RVA origin. The two G11P[25] strains were also very similar to KTM368, another G11P[25] isolated from a child in Nepal in 2004. On the other hand, no gene of TK1797 was likely to be of human RVA origin. The observation that porcine-like RVAs were detected from adult patients justifies further studies to explore the role of adults in the interspecies transmission of animal RVA to humans.
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http://dx.doi.org/10.1016/j.meegid.2019.02.007DOI Listing
April 2019

De novo formation and epigenetic maintenance of centromere chromatin.

Curr Opin Cell Biol 2019 06 15;58:15-25. Epub 2019 Jan 15.

Laboratory of Chromosome Engineering, Department of Frontier Research and Development, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu 292-0818, Japan. Electronic address:

Accurate chromosome segregation is essential for cell proliferation. The centromere is a specialized chromosomal locus, on which the kinetochore structure is formed. The centromere/kinetochore is required for the equal separation of sister chromatids to daughter cells. Here, we review recent findings on centromere-specific chromatin, including its constitutive protein components, its de novo formation and maintenance mechanisms, and our progress in analyses with synthetic human artificial chromosomes (HACs).
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http://dx.doi.org/10.1016/j.ceb.2018.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626702PMC
June 2019

Systematic Analysis of Compounds Specifically Targeting Telomeres and Telomerase for Clinical Implications in Cancer Therapy.

Cancer Res 2018 11 30;78(21):6282-6296. Epub 2018 Aug 30.

Developmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD.

The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In this work, we designed a new protocol to screen for and rank the efficacy of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular human artificial chromosome (HAC, lacking telomeres) and a linear HAC (containing telomeres) marked with the transgene; compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. Our assay allowed quantification of chromosome loss by routine flow cytometry. We applied this dual-HAC assay to rank a set of known and newly developed compounds, including G-quadruplex (G4) ligands. Among the latter group, two compounds, Cu-ttpy and Pt-ttpy, induced a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges in late mitosis and cytokinesis as well as UFB (ultrafine bridges). Chromosome loss after Pt-ttpy or Cu-ttpy treatment correlated with the induction of telomere-associated DNA damage. Overall, this platform enables identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction and may expedite the development of new therapeutic strategies for cancer treatment. An assay provides a unique opportunity to screen thousands of chemical compounds for their ability to inactivate replication of telomeric ends in cancer cells and holds potential to lay the foundation for the discovery of new treatments for cancer. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214708PMC
November 2018

Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.

ACS Synth Biol 2018 09 16;7(9):1974-1989. Epub 2018 Aug 16.

Developmental Therapeutics Branch , National Cancer Institute, NIH , Bethesda , Maryland 20892 , United States.

Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in human cells. This HAC was termed the alphoid-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoid-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoid-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Importantly, the phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. Alphoid-HAC-based technology has also been used to develop new drug screening and assessment strategies to manipulate the CIN phenotype in cancer cells. In summary, the alphoid-HAC is proving to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies.
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http://dx.doi.org/10.1021/acssynbio.8b00230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154217PMC
September 2018

The combination of NAD+-dependent deacetylase gene deletion and the interruption of gluconeogenesis causes increased glucose metabolism in budding yeast.

PLoS One 2018 26;13(3):e0194942. Epub 2018 Mar 26.

Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, Japan.

Metabolic engineering focuses on rewriting the metabolism of cells to enhance native products or endow cells with the ability to produce new products. This engineering has the potential for wide-range application, including the production of fuels, chemicals, foods and pharmaceuticals. Glycolysis manages the levels of various secondary metabolites by controlling the supply of glycolytic metabolites. Metabolic reprogramming of glycolysis is expected to cause an increase in the secondary metabolites of interest. In this study, we constructed a budding yeast strain harboring the combination of triple sirtuin gene deletion (hst3∆ hst4∆ sir2∆) and interruption of gluconeogenesis by the deletion of the FBP1 gene encoding fructose-1,6-bisphosphatase (fbp1∆). hst3∆ hst4∆ sir2∆ fbp1∆ cells harbored active glycolysis with high glucose consumption and active ethanol productivity. Using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) analysis, hst3∆ hst4∆ sir2∆ fbp1∆ cells accumulated not only glycolytic metabolites but also secondary metabolites, including nucleotides that were synthesized throughout the pentose phosphate (PP) pathway, although various amino acids remained at low levels. Using the stable isotope labeling assay for metabolites, we confirmed that hst3∆ hst4∆ sir2∆ fbp1∆ cells directed the metabolic fluxes of glycolytic metabolites into the PP pathway. Thus, the deletion of three sirtuin genes (HST3, HST4 and SIR2) and the FBP1 gene can allow metabolic reprogramming to increase glycolytic metabolites and several secondary metabolites except for several amino acids.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194942PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868833PMC
July 2018

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies.

ACS Synth Biol 2018 04 29;7(4):1116-1130. Epub 2018 Mar 29.

Wellcome Trust Centre for Cell Biology, University of Edinburgh , Edinburgh EH9 3QR, United Kingdom.

It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoid-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoid-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.
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http://dx.doi.org/10.1021/acssynbio.8b00018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951608PMC
April 2018

[CUTANEOUS METASTASIS OF UROTHERIAL CARCINOMA FOR WHICH MOHS PASTE WAS USEFUL: A CASE REPORT].

Nihon Hinyokika Gakkai Zasshi 2017 ;108(1):41-44

Department of Urology, National Hospital Organization Higashi-Hiroshima Medical Center.

The patient was a 71-year-old man referred to our department because of asymptomatic gross hematuria. Detailed examination led to a diagnosis of bilateral ureteral cancer and prostate cancer. Total nephroureterectomy was performed for bilateral ureteral cancers on separate occasions, while prostate cancer was managed with hormone therapy. Because multiple bladder tumors were found by cystoscopy conducted after 3 months, total cystectomy was performed. Neither distant metastasis nor local recurrence had developed, 2 years later, when the patient visited our department again because of glans enlargement and received a diagnosis of penile cancer. Partial penectomy was performed, and a 1-cm indurated nodule was found in the skin of the hypogastric region during surgery and treated by simultaneous wedge-shaped excision. Histopathologically, the lesions were urothelial carcinomas. Multiple metastatic foci occurred in the skin postoperatively centering on the hypogastric region, growing rapidly and accompanied by bleeding and exudate causing a bad odor. Topical application of Mohs paste resulted in dramatic improvement of the pain and exudate, which thereby improved the patient's quality of life (QOL) to a level enabling discontinuation of narcotic drug therapy for cancer-related pain. Thereafter, hepatic metastasis was found and chemotherapy with gemcitabine and paclitaxel was administered. The patient did not respond to this chemotherapy and died of cancer 3 months later. Cutaneous metastasis of urothelial carcinoma, which is a very rare condition, is often managed with palliative therapy due to the lack of response to anticancer chemotherapy. In such cases, the use of Mohs paste appears to be of great value for maintaining the patient's QOL which otherwise might deteriorate markedly.
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http://dx.doi.org/10.5980/jpnjurol.108.41DOI Listing
February 2019

Rab3a-Bound CD63 Is Degraded and Rab3a-Free CD63 Is Incorporated into HIV-1 Particles.

Front Microbiol 2017 29;8:1653. Epub 2017 Aug 29.

Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki UniversityNagasaki, Japan.

CD63, a member of the tetraspanin family, is involved in virion production by human immunodeficiency virus type 1 (HIV-1), but its mechanism is unknown. In this study, we showed that a small GTP-binding protein, Rab3a, interacts with CD63. When Rab3a was exogenously expressed, the amounts of CD63 decreased in cells. The Rab3a-mediated reduction of CD63 was suppressed by lysosomal and proteasomal inhibitors. The amount of CD63 was increased by reducing the endogenous Rab3a level using a specific shRNA. These results indicate that Rab3a binds to CD63 to induce the degradation of CD63. Rab3a is thought to be involved in exocytosis, but we found that another function of Rab3a affects the fate of CD63 in lysosomes. CD63 interacted with Rab3a and was incorporated into HIV-1 particles. However, Rab3a was not detected in HIV-1 virions, thereby indicating that Rab3a-free CD63, but not Rab3a-bound CD63, is incorporated into HIV-1 particles. Overexpression or silencing of Rab3a moderately reduced HIV-1 virion formation. Overexpression of Rab3a decreased CD63 levels, but did not affect the incorporation of CD63 into HIV-1 particles. This study showed that Rab3a binds to CD63 to induce the degradation of CD63, and only Rab3a-free CD63 is incorporated into HIV-1 particles.
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http://dx.doi.org/10.3389/fmicb.2017.01653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581869PMC
August 2017

Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System.

ACS Synth Biol 2018 01 24;7(1):63-74. Epub 2017 Aug 24.

Developmental Therapeutics Branch, National Cancer Institute , Bethesda, Maryland 20892, United States.

The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoid-HAC). We have demonstrated that the IIS-alphoid-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoid-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoid-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoid-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.
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http://dx.doi.org/10.1021/acssynbio.7b00209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778389PMC
January 2018

Using human artificial chromosomes to study centromere assembly and function.

Chromosoma 2017 Oct 7;126(5):559-575. Epub 2017 Jul 7.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3QR, UK.

Centromeres are the site of assembly of the kinetochore, which directs chromosome segregation during cell division. Active centromeres are characterized by the presence of nucleosomes containing CENP-A and a specific chromatin environment that resembles that of active genes. Recent work using human artificial chromosomes (HAC) sheds light on the fine balance of different histone post-translational modifications and transcription that exists at centromeres for kinetochore assembly and maintenance. Here, we review the use of HAC technology to understand centromere assembly and function. We put particular emphasis on studies using the alphoid HAC, whose centromere can be specifically modified for epigenetic engineering studies.
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http://dx.doi.org/10.1007/s00412-017-0633-xDOI Listing
October 2017

Nucleosome-nucleosome interactions via histone tails and linker DNA regulate nuclear rigidity.

Mol Biol Cell 2017 Jun 20;28(11):1580-1589. Epub 2017 Apr 20.

Department of Genetics, School of Life Science, Sokendai (Graduate University for Advanced Studies), Mishima 411-8540, Japan

Cells, as well as the nuclei inside them, experience significant mechanical stress in diverse biological processes, including contraction, migration, and adhesion. The structural stability of nuclei must therefore be maintained in order to protect genome integrity. Despite extensive knowledge on nuclear architecture and components, however, the underlying physical and molecular mechanisms remain largely unknown. We address this by subjecting isolated human cell nuclei to microneedle-based quantitative micromanipulation with a series of biochemical perturbations of the chromatin. We find that the mechanical rigidity of nuclei depends on the continuity of the nucleosomal fiber and interactions between nucleosomes. Disrupting these chromatin features by varying cation concentration, acetylating histone tails, or digesting linker DNA results in loss of nuclear rigidity. In contrast, the levels of key chromatin assembly factors, including cohesin, condensin II, and CTCF, and a major nuclear envelope protein, lamin, are unaffected. Together with in situ evidence using living cells and a simple mechanical model, our findings reveal a chromatin-based regulation of the nuclear mechanical response and provide insight into the significance of local and global chromatin structures, such as those associated with interdigitated or melted nucleosomal fibers.
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http://dx.doi.org/10.1091/mbc.E16-11-0783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449155PMC
June 2017