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1. Clinical use of duplicate complete dentures: A narrative review

Author

Nobuhiro Yoda, Masumi Abe, Hirofumi Yamaguchi, Naomi Tanoue, and Tetsuo Yamamori

Subjects
Duplicate denture, 3D printing, CAD-CAM, Dentistry, RK1-715
Abstract

Most reports on duplicate dentures are introduction to fabrication methods or clinical case reports. Only a few studies have verified their clinical effectiveness; hence, evidence to construct useful clinical guidelines for duplicate denture use is lacking. This review aimed to comprehensively investigate reports on duplicate dentures to accumulate evidences that will contribute to the formulation of clinical practice guidelines. Duplicate dentures are effectively used for impression making and bite registration when fabricating new dentures, thereby reducing the number of clinic visits and treatment time. Duplicate denture can also be used as temporary or new dentures. Older people in whom various adaptive abilities have declined, may find it difficult to adjust to new dentures and experience stress, even if the shape is appropriate. Duplicate dentures, which reproduces the shape of old dentures that they are used to, have the advantage of being more familiar to older people and less stressful. When manufacturing duplicate dentures, digital methods such as milling and three-dimensional printing are superior to conventional methods regarding working time and cost. A notable advantage of the digital method is that the denture shape can be saved as digital data, and the denture can be easily duplicated if lost.

Published
2024
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2. Activities of the dorsolateral and medial prefrontal cortices during oral function training with cognitive training elements: a NIRS study

Author

Masumi Abe, Rui Nouchi, Toru Ogawa, Naru Shiraishi, Hiroki Hihara, Keiichi Sasaki, and Nobuhiro Yoda

Subjects
Oral function training, Cognitive training, PFC, Oral health, Tongue, Lip, Dentistry, RK1-715
Abstract

Abstract Background Cognitive function plays a crucial role in human life, and its maintenance and improvement are essential in both young and older adults. Since cognitive decline can be associated with oral function decline, preventing the decline in both cognitive and oral functions is an urgent social issue. Several training methods to improve each function have been proposed. Previous studies have indicated that greater brain activity during training is associated with increased benefits for cognitive function. Although adding cognitive function elements to oral function training may promote the activation of brain activity during oral function training, the effects have not been validated. The main purpose of this study is to develop a novel training program that combines oral function training with cognitive training, which is expected to activate key brain regions involved in oral and cognitive functions, such as the left dorsolateral prefrontal cortex (DLPFC) and right medial prefrontal cortex (mPFC). Methods Four types of training programs combining oral and cognitive training: PaTaKaRa × calculation, lip exercise × N-back, tongue exercise × inhibition, and tongue exercise × memory, were developed. Each program had seven levels of difficulty [level 0 (no cognitive load) and level 6 (maximum difficulty)]. Twelve healthy young adults participated in the study and were instructed to perform all four programs. Brain activity in the left DLPFC and right mPFC were measured during each training session using two-channel near-infrared spectroscopy (NIRS). Results No significant brain activity was observed during training at level 0. Brain activity in the left DLPFC was significantly increased at levels 1 and 2 and in the left DLPFC and right mPFC at level 6 during PaTaKaRa × calculation training. Brain activity in the left DLPFC was significantly increased at level 6 during tongue exercise × inhibition training. Brain activity in the left DLPFC and right mPFC was significantly increased at level 6 during lip exercise × N-back training. Conclusion Oral function training did not significantly increase brain activity; nevertheless, oral function with cognitive training stimulated brain activity in the prefrontal cortex. Trial registration : UMIN-CTR. ID: UMIN000039678. date: 06/03/2020.

Published
2024
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3. iPS cell generation-associated point mutations include many C > T substitutions via different cytosine modification mechanisms

Author

Ryoko Araki, Tomo Suga, Yuko Hoki, Kaori Imadome, Misato Sunayama, Satoshi Kamimura, Mayumi Fujita, and Masumi Abe

Subjects
Science
Abstract

Abstract Genomic aberrations are a critical impediment for the safe medical use of iPSCs and their origin and developmental mechanisms remain unknown. Here we find through WGS analysis of human and mouse iPSC lines that genomic mutations are de novo events and that, in addition to unmodified cytosine base prone to deamination, the DNA methylation sequence CpG represents a significant mutation-prone site. CGI and TSS regions show increased mutations in iPSCs and elevated mutations are observed in retrotransposons, especially in the AluY subfamily. Furthermore, increased cytosine to thymine mutations are observed in differentially methylated regions. These results indicate that in addition to deamination of cytosine, demethylation of methylated cytosine, which plays a central role in genome reprogramming, may act mutagenically during iPSC generation.

Published
2024
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4. Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency

Author

Ryoko Araki, Yuko Hoki, Tomo Suga, Chizuka Obara, Misato Sunayama, Kaori Imadome, Mayumi Fujita, Satoshi Kamimura, Miki Nakamura, Sayaka Wakayama, Andras Nagy, Teruhiko Wakayama, and Masumi Abe

Subjects
Science
Abstract

Point mutations have been found in induced pluripotent stem cells (iPSCs) but when they arise is unclear. Here, the authors show that a G1/S cell cycle checkpoint deficiency transiently occurs early in genome reprogramming, suggesting a common developmental pathway between iPSC and tumorigenesis, and generate genetic burden-free human iPSCs.

Published
2020
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5. In situ delivery of iPSC-derived dendritic cells with local radiotherapy generates systemic antitumor immunity and potentiates PD-L1 blockade in preclinical poorly immunogenic tumor models

Author

Kunle Odunsi, Takaaki Oba, Hans Minderman, Kenichi Makino, Ryutaro Kajihara, Toshihiro Yokoi, Ryoko Araki, Masumi Abe, and Alfred E Chang

Subjects
Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

Background Dendritic cells (DCs) are a promising therapeutic target in cancer immunotherapy given their ability to prime antigen-specific T cells, and initiate antitumor immune response. A major obstacle for DC-based immunotherapy is the difficulty to obtain a sufficient number of functional DCs. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs); however, therapeutic strategies to engage iPSC-derived DCs (iPSC-DCs) into cancer immunotherapy remain to be elucidated. Accumulating evidence showing that induction of tumor-residing DCs enhances immunomodulatory effect of radiotherapy (RT) prompted us to investigate antitumor efficacy of combining intratumoral administration of iPSC-DCs with local RT.Methods Mouse iPSCs were differentiated to iPSC-DCs on OP9 stromal cells expressing the notch ligand delta-like 1 in the presence of granulocyte macrophage colony-stimulating factor. Phenotype and the capacities of iPSC-DCs to traffic tumor-draining lymph nodes (TdLNs) and prime antigen-specific T cells were evaluated by flow cytometry and imaging flow cytometry. Antitumor efficacy of intratumoral injection of iPSC-DCs and RT was tested in syngeneic orthotopic mouse tumor models resistant to anti-PD-1 ligand 1 (PD-L1) therapy.Results Mouse iPSC-DCs phenotypically resembled conventional type 2 DCs, and had a capacity to promote activation, proliferation and effector differentiation of antigen-specific CD8+ T cells in the presence of the cognate antigen in vitro. Combination of in situ administration of iPSC-DCs and RT facilitated the priming of tumor-specific CD8+ T cells, and synergistically delayed the growth of not only the treated tumor but also the distant non-irradiated tumors. Mechanistically, RT enhanced trafficking of intratumorally injected iPSC-DCs to the TdLN, upregulated CD40 expression, and increased the frequency of DC/CD8+ T cell aggregates. Phenotypic analysis of tumor-infiltrating CD8+ T cells and myeloid cells revealed an increase of stem-like Slamf6+ TIM3− CD8+ T cells and PD-L1 expression in tumor-associated macrophages and DCs. Consequently, combined therapy rendered poorly immunogenic tumors responsive to anti-PD-L1 therapy along with the development of tumor-specific immunological memory.Conclusions Our findings illustrate the translational potential of iPSC-DCs, and identify the therapeutic efficacy of a combinatorial platform to engage them for overcoming resistance to anti-PD-L1 therapy in poorly immunogenic tumors.

Published
2021
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6. Hotspots of De Novo Point Mutations in Induced Pluripotent Stem Cells

Author

Masahito Yoshihara, Ryoko Araki, Yasuji Kasama, Misato Sunayama, Masumi Abe, Kohji Nishida, Hideya Kawaji, Yoshihide Hayashizaki, and Yasuhiro Murakawa

Subjects
Biology (General), QH301-705.5
Abstract

Summary: Induced pluripotent stem cells (iPSCs) are generated by direct reprogramming of somatic cells and hold great promise for novel therapies. However, several studies have reported genetic variations in iPSC genomes. Here, we investigated point mutations identified by whole-genome sequencing in mouse and human iPSCs in the context of epigenetic status. In contrast to disease-causing single-nucleotide polymorphisms, de novo point mutations introduced during reprogramming were underrepresented in protein-coding genes and in open chromatin regions, including transcription factor binding sites. Instead, these mutations occurred preferentially in structurally condensed lamina-associated heterochromatic domains, suggesting that chromatin organization is a factor that can bias the regional mutation rate in iPSC genomes. Mutation signature analysis implicated oxidative stress associated with reprogramming as a likely cause of point mutations. Altogether, our study provides deeper understanding of the mutational landscape of iPSC genomes, paving an important way toward the translation of iPSC-based cell therapy. : Yoshihara et al. show that de novo point mutations introduced during iPSC reprogramming preferentially occur in structurally condensed lamina-associated heterochromatic domains and exhibit an oxidative stress-induced DNA damage mutation signature. This study provides better characterization of iPSC mutations at the whole-genome level and accelerates the translation of iPSC-based cell therapies. Keywords: iPSCs, genomics, point mutation, epigenetics, heterochromatin, lamina-associated domains, iPSC-based cell therapy

Published
2017
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7. Supplementary Figure S3 from Generation of Mouse Pluripotent Stem Cell–Derived Proliferating Myeloid Cells as an Unlimited Source of Functional Antigen-Presenting Cells

Author

Yasushi Uemura, Kiyotaka Kuzushima, Yasuharu Nishimura, Yoshiaki Sonoda, Yasushi Sakamoto, Yoshiki Akatsuka, Masumi Abe, Ryoko Araki, Hayao Nakanishi, Shinobu Tsuzuki, Yutaka Sasaki, Yoshikazu Matsuoka, Ryusuke Nakatsuka, Hiroyuki Maki, Norihiro Ueda, Minako Tatsumi, Motoharu Suzuki, Narumi Hirosawa, Miwa Haruta, Satoru Senju, Tian-Yi Liu, and Rong Zhang

Abstract

Protection against subcutaneously inoculated OVA-expressing melanoma by OVA-peptide loaded iPS-pMC-DCs.

Published
2023
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8. Data from Generation of Mouse Pluripotent Stem Cell–Derived Proliferating Myeloid Cells as an Unlimited Source of Functional Antigen-Presenting Cells

Author

Yasushi Uemura, Kiyotaka Kuzushima, Yasuharu Nishimura, Yoshiaki Sonoda, Yasushi Sakamoto, Yoshiki Akatsuka, Masumi Abe, Ryoko Araki, Hayao Nakanishi, Shinobu Tsuzuki, Yutaka Sasaki, Yoshikazu Matsuoka, Ryusuke Nakatsuka, Hiroyuki Maki, Norihiro Ueda, Minako Tatsumi, Motoharu Suzuki, Narumi Hirosawa, Miwa Haruta, Satoru Senju, Tian-Yi Liu, and Rong Zhang

Abstract

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow–derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2Kb-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs. Cancer Immunol Res; 3(6); 668–77. ©2015 AACR.

Published
2023
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9. Supplementary Figure Legends from Generation of Mouse Pluripotent Stem Cell–Derived Proliferating Myeloid Cells as an Unlimited Source of Functional Antigen-Presenting Cells

Author

Yasushi Uemura, Kiyotaka Kuzushima, Yasuharu Nishimura, Yoshiaki Sonoda, Yasushi Sakamoto, Yoshiki Akatsuka, Masumi Abe, Ryoko Araki, Hayao Nakanishi, Shinobu Tsuzuki, Yutaka Sasaki, Yoshikazu Matsuoka, Ryusuke Nakatsuka, Hiroyuki Maki, Norihiro Ueda, Minako Tatsumi, Motoharu Suzuki, Narumi Hirosawa, Miwa Haruta, Satoru Senju, Tian-Yi Liu, and Rong Zhang

Abstract

Supplementary Figure Legends

Published
2023
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10. Supplementary Materials and Methods from Generation of Mouse Pluripotent Stem Cell–Derived Proliferating Myeloid Cells as an Unlimited Source of Functional Antigen-Presenting Cells

Author

Yasushi Uemura, Kiyotaka Kuzushima, Yasuharu Nishimura, Yoshiaki Sonoda, Yasushi Sakamoto, Yoshiki Akatsuka, Masumi Abe, Ryoko Araki, Hayao Nakanishi, Shinobu Tsuzuki, Yutaka Sasaki, Yoshikazu Matsuoka, Ryusuke Nakatsuka, Hiroyuki Maki, Norihiro Ueda, Minako Tatsumi, Motoharu Suzuki, Narumi Hirosawa, Miwa Haruta, Satoru Senju, Tian-Yi Liu, and Rong Zhang

Abstract

Supplementary Materials and Methods

Published
2023
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12. Empagliflozin in Patients with Chronic Kidney Disease

Author

Herrington, William G, Staplin, Natalie, Wanner, Christoph, Green, Jennifer B, Hauske, Sibylle J, Emberson, Jonathan R, Preiss, David, Judge, Parminder, Mayne, Kaitlin J, Ng, Sarah Y A, Sammons, Emily, Zhu, Doreen, Hill, Michael, Stevens, Will, Wallendszus, Karl, Brenner, Susanne, Cheung, Alfred K, Liu, Zhi-Hong, Li, Jing, Hooi, Lai Seong, Liu, Wen, Kadowaki, Takashi, Nangaku, Masaomi, Levin, Adeera, Cherney, David, Maggioni, Aldo P, Pontremoli, Roberto, Deo, Rajat, Goto, Shinya, Rossello, Xavier, Tuttle, Katherine R, Steubl, Dominik, Petrini, Michaela, Massey, Dan, Eilbracht, Jens, Brueckmann, Martina, Landray, Martin J, Baigent, Colin, Haynes, Richard, The EMPA-KIDNEY Collaborative Group: Colin Baigent, Martin J Landray, Christoph Wanner, William G Herrington, Richard Haynes, Jennifer B Green, Sibylle J Hauske, Martina Brueckmann, Mark Hopley, Maximillian von-Eynatten, Jyothis George, Susanne Brenner, Alfred K Cheung, David Preiss, Zhi-Hong Liu, Jing Li, Laiseong Hooi, Wen Liu, Takashi Kadowaki, Masaomi Nangaku, Adeera Levin, David Cherney, Roberto Pontremoli, Aldo P Maggioni, Natalie Staplin, Jonathan Emberson, Stefan Hantel, Shinya Goto, Rajat Deo, Katherine R Tuttle, Michael Hill, Parminder Judge, Kaitlin J Mayne, Sarah Y A Ng, Xavier Rossello, Emily Sammons, Doreen Zhu, Peter Sandercock, Rudolf Bilous, Charles Herzog, Paul Whelton, Janet Wittes, Derrick Bennett, Patricia Achiri, Chrissie Ambrose, Cristina Badin, Jill Barton, Richard Brown, Andy Burke, Sebastian Butler, Rejive Dayanandan, Pia Donaldson, Robert Dykas, Lucy Fletcher, Kate Frederick, Hannah Kingston, Mo Gray, Emily Harding, Akiko Hashimoto, Lyn Howie, Susan Hurley, Ryonfa Lee, Nik Luker, Kevin Murphy, Mariko Nakahara, John Nolan, Michelle Nunn, Sorcha Mulligan, Akiko Omata, Sandra Pickworth, YanRu Qiao, Shraddha Shah, Karen Taylor, Alison Timadjer, Monique Willett, Liz Wincott, Qin Yan, Hui Yu, Louise Bowman, Fang Chen, Robert Clarke, Michelle Goonasekera, Waseem Karsan, Marion Mafham, Christina Reith, Mohammed Zayed, Ritva Ellison, Rowan Moys, Will Stevens, Kevin Verdel, Karl Wallendszus, Chris Bowler, Anna Brewer, Andy Measor, Guanguo Cui, Charles Daniels, Angela Field, Bob Goodenough, Ashley Lawson, Youcef Mostefai, Dheeptha Radhakrishnan, Samee Syed, Shuang Xia, Ruth Adewuyi-Dalton, Thomas Arnold, Anne-Marie Beneat, Anoushka Bhatt, Chloe Bird, Andrew Breach, Laura Brown, Mark Caple, Tatyana Chavagnon, Karen Chung, Sarah Clark, Luminita Condurache, Katarzyna Eichstadt, Marta Espino Obrero, Scarlett Forest, Helen French, Nick Goodwin, Andrew Gordon, Joanne Gordon, Cat Guest, Tina Harding, Michal Hozak, Matthew Lacey, David MacLean, Louise Messinger, Stewart Moffat, Martin Radley, Claire Shenton, Sarah Tipper, Jon Tyler, Lesley Weaving, James Wheeler, Elissa Williams, Tim Williams, Hamish Woodhouse, Angela Chamberlain, Jo Chambers, Joanne Davies, Denise Donaldson, Pati Faria-Shayler, Denise Fleming-Brown, Jennifer Ingell, Carol Knott, Anna Liew, Helen Lochhead, Juliette Meek, Isabel Rodriguez-Bachiller, Andrea Wilson, Patrick Zettergren, Rach AitSadi, Ian Barton, Alex Baxter, Yonghong Bu, Lukasz Danel, Sonja Grotjahn, Rijo Kurien, Michael Lay, Archie Maskill, Aleksandra Murawska, Rachel Raff, Allen Young, Rebecca Sardell, Vladimir Cejka, Marcela Fajardo-Moser, Christian Hartner, Doris Poehler, Janina Renner, Franziska Scheidemantel, Miya Bryant, Anita Hepditch, Cassandra Johnson, Erin Latore, Yolanda Miller, Lauren Price, Merilee Whalen, Ashleigh Wheeler, Jenny Ingell, Yu An, Yinghua Chen, Peiling Chen, Hao Dai, Hong Du, Fang Feng, Qing Guo, Libo Hou, Wuhanbilige Hundei, Binbin Jin, Yan Li, Jiamin Liu, Xia Song, Yanping Wang, Yanwu Yu, Ning Zhang, Lingshan Zhao, Hui Zhong, Cheng Beng Goh, Ye Mun Low, Soon Yi Sor, Farah Hanis Zulkipli, Sarojini Sivanandam, Natsuki Arai, Ai Fukasawa, Mizue Furukawa, Keisuke Habuki, Shoko Hayashi, Wakako Isari, Saki Kanegae, Maria Kawai, Reiki Kobayashi, Takako Kuramae, Chika Kuribayashi, Sawako Maeno, Satoshi Masumoto, Tomoko Morisaki, Minoru Oda, Kazue Sawada, Kenta Sugamori, Ayana Tatsuzawa, Aiko Tomita, Kazuyuki Yuasa, Hiroko Inazawa, Amanda Axler, Kerri Gallo, Ester Baldini, Barbara Bartolomei Mecatti, Francesca Bianchini, Martina Ceseri, Laura Cipressa, Gianna Fabbri, Andrea Lorimer, Donata Lucci, Sharang Ghavampour, Anja Knoppe, Hans Schmidt-Gurtler, Hubert Dumann, Sybille Merscher, Margret Patecki, Georg Rainer Schlieper, Anke Torp, Bianca Weber, Maja Zietz, Bernd Hohenstein, Urs Benck, Diliana Draganova, Thomas Weinreich, Lothar Wolf, Jasmine Gaidu, Hanna Reiner, Mandy Visnjic, Daniel Steffl, Marie Breitenfeldt, Annette Kraemer-Guth, Christine Braun, Simone Hagge, Michael Schomig, Stephan Matthias, Dominik Stoffler, Beate Schumacher, Thomas Sitter, Louise Fuessl, Julia Krappe, Jerome Loutan, Volker Vielhauer, Luciano Andriaccio, Magdalena Maurer, Bernhard Winkelmann, Martin Dursch, Linda Seifert, Linda Tenbusch, Julia Weinmann-Menke, Simone Boedecker, Wiebke Kaluza-Schilling, Daniel Kraus, Carina Krieger, Margit Schmude, Anne Schreiber, Ewelina Eckrich, Diethelm Tschope, Abdulwahab Arbi, Young Lee-Barkey, Bernd Stratmann, Natalie Prib, Sina Rolfsmeier, Irina Schneider, Lars Rump, Johannes Stegbauer, Christine Pötz, Mara Schemmelmann, Claudia Schmidt, Michael Koch, Sendogan Aker, Annika Küpper, Manuela Martin, Thiemo Pfab, Christian Albert, Michael Haase, Barbara Zander, Claudia Schneider-Danwitz, Wolfgang Seeger, Wolf-Adam Seeger, Britta Zemann, Christoph Stellbrink, Kristin Marx, Ekaterina Stellbrink, Britta Brettschneider, Stephanie Watson, Marion Iselt, Gerhard Klausmann, Inga-Nadine Kummer, Auguste Kutschat, Simone Streitenberger, Matthias Girndt, Silke Markau, Ina Girakossyan, Claudia Hanf, Joachim Beige, Ralph Wendt, Ulrike Schmidt, Andreas Schneider, Roland Veelken, Claudia Donhauser, Luis Becker, Nexhat Miftari, Ricarda Wolfling, Sarah Morlok, Christian Hugo, Alexander Paliege, Jens Passauer, Julian Stumpf, Annegret Fleischer, Kerstin Haaser, Bernhard Kraemer, Jan Jochims, Bernd Kruger, Claudia Foellinger, Anastassiya Reisler, Frank Strutz, Stefan Haack, Ursula Hohenstatt, Martin Busch, Konstantin Herfurth, Gunter Wolf, Rainer Paul, Hermann Haller, Jessica Kaufeld, Jan Menne, Elisabeth Bahlmann-Kroll, Angela Bergner, Horst Weihprecht, Aydin Er, Florian Sonntag, Elif Turan, Michael Wittmann, Franziska Klauser, Eva Voigt, Volker Schettler, Egbert Schulz, Madlen Rohnstock, Elke Schettler, Bernd Schroppel, Rene van Erp, Martin Kachele, Ulla Ludwig, Lena Schulte-Kemna, Waltraud Kmietschak, Elke Preiss, Martina Ruocco, Gunnar Heine, Martin Brzoska, Sebastian Gabel, Christina Büttner, Asma Sabarai, Bernhard Banas, Tobias Bergler, Yvonne Ehrl, Franz Putz, Antonia Schuster, Stefanie Kuhn, Torsten Schramm, Stefan Degenhardt, Gerhard Schmidt, Lea Weiland, Ulrike Giebeln-Hudnell, Jan Kielstein, Gabriele Eden, Brigitte Fuchs, Gina Morig, Manuela Winkler, Harald Darius, Charalampos Kriatselis, Carl-Philipp Roesch, Astrid Maselli, Dominik Alscher, Markus Ketteler, Moritz Schanz, Severin Schricker, Bianka Rettenmaier, Andrea Schwab, Pablo Pergola, Irene Leal, Melissa Cagle, Anna Romo, Anthony Torres, Sucharit Joshi, Kulli Barrett, Alexis Africano, Vicki Dodds, Dorleena Gowen, Ashlee Morris, Juan Fernandez, Guillermo Jimenez, Ricardo Viera, Kendaling Bruce, Ryan Barrios, Maylin Garcia, Kerelyn Garcia, Iradis Leal, David Tietjen, David Bains, Carlo Castillo, Genielle Brewer, Justin Davis, Natalie Freking, Brittany Golson, Sally Ham, Jesslyn Roesch, Pusadee Suchinda, Shameem Beigh, Usah Lilavivat, Joyce Bilton, Kim Bocchicchia, Jeffrey Turner, Neera Dahl, Aldo Peixoto, Yasemin Kavak, Lauren Liberti, Hari Nair, Nicolas Page, Stephanie Rosenberg, Kathryn Simmons, Tamara Isakova, Rebecca Frazier, Rupal Mehta, Anand Srivastava, Patrick Fox, Jonathan Hecktman, Alexander Hodakowski, Carlos Martinez, Rachel Phillips, Alexis Stevenson, Reem Mustafa, Kyle Jansson, Cassandra Kimber, Jason Stubbs, Ahmad Tuffaha, Sri Yarlagadda, Debbie Griffin, Elisabeth Laundy, Zhuo Tang, Radica Alicic, Ann Cooper, Lisa Davis, Ashwini Gore, Rebecca Goldfaden, Leslie Harvill, Lisa Hichkad, Barry Johns, Thomas Jones, Kayla Merritt, Jennifer Sheldon, Jennifer Stanfield, Lindsay Alexander, Kaitlyn Preston, Lindsey Wood, Rajesh Pradhan, Roger DeRaad, Kelli McIntosh, Louis Raymond, Michael Shepperd, Susan McLaughlin, Mary Seifert, Andrew Shepherd, Joseph Aiello, William Durham, Laurie Loudermilk, John Manley, Sabrina Burnette, Stephanie Evans, Tara Johnson, Lance Sloan, Judy Ann Acosta, Stacy Gillham, Katia Sloan, SueAnn Squyres, Michael Rocco, Amret Hawfield, Ben Bagwell, Lauren Richmond, Joseph Soufer, Subha Clarke, Amanda Aliu, Kristine Calabrese, Amanda Davis, Veronica Poma, Tracy Spinola, James Magee, Ricardo Silva, Rushab Choksi, Lorraine Dajani, John Evans, Anil George, Prasanth Krish, Gerard Martins, Mae Sheikh-Ali, David Sutton, Freda Driver, Abraham Hanburry, Laura Hume, Amber Hurst, Matthew Taddeo, Marla Turner, Veronica Yousif, Srinivasan Beddhu, Laith Al-Rabadi, Nikita Abraham, Amalia Caamano, Judy Carle, Victoria Gonce, Kaitlyn Staylor, Na Zhou, Shweta Bansal, Manoj Bhattarai, Kumar Sharma, Subrata Debnath, Aliseiya Garza, Chakradhar Velagapudi, Sergio Rovner, Javier Almeida, Pablo Casares, Verlaine Stewart-Ray, Rene Almaraz, Renata Dayrell, Ana Moncada, Ricardo Pulido, Roxana Rodriquez, Wasim Deeb, Kathryn DeGoursey, Rodel Gloria, Trevor Greene, Robert Miller, Edward Pereira, Miguel Roura, Debbie Domingo, Sasha Dorestin, William Hodge, Cathy Jackson, Deborah Lund, Katrina Taylor, Kenneth Boren, Brittany Cleveland, Sandra Gaiser, Mandeep Sahani, Logan Aldrich, Exodus Edmerson, Edmond Limon, Cole Valletta, Patricia Vasquez, Christopher Provenzano, Navkiranjot Brar, Heather Henderson, Bellovich Keith, Qur Khai, Quresh Khairullah, Gail Makos, Joel Topf, Sherry Gasko, Rosemarie Henschel, Kaitlin Knapp, Teresa Kozlowski, Paula LaFleur, Ashwathy Varughese, Hui Xue, Patricia Wu, Olga Arechiga, Shan Darbeau, Michael Fechter, Stephanie Martinez, Lenita Hanson, Nyla Cooper, Arelis Madera, Jay Cadorna, Rita Sheridan, Helen Sparks, Bradley Eilerman, Susanne Bodine, Wael Eid, Rebecca Flora, Amber Avery, Cashmere Hardy, Mihaela Biscoveanu, Steven Nagelberg, Tracey Cummins, Frederic Rahbari-Oskoui, Anju Oommen, Zohreh Forghani, Stacie Hitchcock, Darya Hosein, Diane Watkins, Minesh Patel, Anthony Lambert, Elizabeth Newman, Autumn Wood, Tammy Ross, Stephany Topping, Jeffrey Mulhern, Lorna Murphy, Ann Vasseur, Gregory Greenwood, Alexander Hadley, Denise Laurienti, Christopher Marshall, Nicholas McLean, Scott Satko, Brandy Caudill, Jacob Maris, Janice Rogers, Cindy Vanhoy, George Thomas, Georges Nakhoul, John O'Toole, Jonathan Taliercio, Leslie Cooperman, Marina Markovic, Barbara Tucky, Devasmita Dev, Alia Hasan, Hima Yalamanchili, Namita Jain, Lesley McNeil, Eric Wines, Jean Park, Adline Ghazi, Mia Hamm, Tejas Patel, Amy Mottl, Emily Chang, Vimal Derebail, Emmie Cole, Anne Froment, Sara Kelley, Jordan Osmond Foster, Vahid Mahabadi, Golriz Jafari, Anita Kamarzarian, Wendy Arriaga, Daisy Arteaga, Rosario Machicado, Genesis Naverrete, Prashant Kumar, Imran Nazeer, Karina Urquia, Tammi Glider, Vickie Jones, Savannah Rucker, Jennifer Wiley, Rahul Pandey, Jesus Arroyo, Harish Pariani, Mohammad Ahmad, Shahin Mozaffari, Erika Perez, Matthew Budoff, Sion Roy, Divya Birudaraju, Ahmed Ghanem, Sajad Hamal, Stephen Aronoff, Elisa Joye Petr, Richard Sachson, Jaime Wiebel, Sana Akram, Laurie Jones, Curtis Knight, Maurie Tarlac, Shahbaz Ahmed, Harold Szerlip, Akinwande Akinfolarin, Ankit Mehta, Shana Camp, Cindy Castro, Zanaida Cooper, Jessica Terry, Ahmed Awad, Bhavya Kothapalli, Ryan Lustig, Serine Alfaress, Hyder Jasim, Mary Parrigon, Dennis Karounos, Sadiq Ahmed, Maggie Berry, Ruth Oremus, Carlos Hernandez-Cassis, Elias Ugwu, Nazia Junejo, Nancy Suazo, Mark Segal, Amir Kazory, Sherry Brown, Tristan Daniels, Sofia Dayi, Renee Hogan, Kathy McCray, Jennifer Stickley, Mahboob Rahman, Mirela Dobre, Lavinia Negrea, Aparna Padiyar, Nishigandha Pradhan, Arash Rashidi, Nagaraju Sarabu, Vicki Donley, Tricia Young, Godson Oguchi, Judepatricks Onyema, Kahla Damianik, Jack Dienes, Judith Plummer-Morgan, Marilyn Roman, Mauver Skipper, Stacey-Ann Villaruel, Krystle Williams, Danny Sugimoto, Jeffrey Dugas, Ismeal Ahmed, Jamie Bhairoo, Dolores Rijos, Huzaifa Salim, Madita Gavrila, Kathryn Lafferty, Ria Rabara, Sally Ruse, Maria Weetman, James Bushnell, Albert Power, Alison Jenkins, Stefanie Jones, Amanda Scott, Cath Byrne, Mark Jesky, Alison Cowley, Emma McHaffie, Holly Waterfall, Jo Taylor, Laura Bough, Thomas Phillips, Barbara Winter-Goodwin, Sui Phin Kon, Iain MacDougall, Eirini Lioudaki, Sapna Shah, Claire Sharpe, Francisco Aguilar, Abegail Hernandez Pena, Conception Pugay, Amelia Te, Hugh Finn, Wasim Hanif, Samiul Mostafa, Alice Aitken, Katharine Draxlbauer, Evelina Grobovaite, Jennifer Kearney, Theresa McCarthy, Giorgio Gentile, Duncan Browne, Palanichamy Chellamuthu, Tabinda 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Cipressa, Laura, Fabbri, Gianna, Lorimer, Andrea, Lucci, Donata, Ghavampour, Sharang, Knoppe, Anja, Schmidt-Gurtler, Han, Dumann, Hubert, Merscher, Sybille, Patecki, Margret, Rainer Schlieper, Georg, Torp, Anke, Weber, Bianca, Zietz, Maja, Hohenstein, Bernd, Benck, Ur, Draganova, Diliana, Weinreich, Thoma, Wolf, Lothar, Gaidu, Jasmine, Reiner, Hanna, Visnjic, Mandy, Steffl, Daniel, Breitenfeldt, Marie, Kraemer-Guth, Annette, Braun, Christine, Hagge, Simone, Schomig, Michael, Matthias, Stephan, Stoffler, Dominik, Schumacher, Beate, Sitter, Thoma, Fuessl, Louise, Krappe, Julia, Loutan, Jerome, Vielhauer, Volker, Andriaccio, Luciano, Maurer, Magdalena, Winkelmann, Bernhard, Dursch, Martin, Seifert, Linda, Tenbusch, Linda, Weinmann-Menke, Julia, Boedecker, Simone, Kaluza-Schilling, Wiebke, Kraus, Daniel, Krieger, Carina, Schmude, Margit, Schreiber, Anne, Eckrich, Ewelina, Tschope, Diethelm, Arbi, Abdulwahab, Lee-Barkey, Young, Stratmann, Bernd, Prib, Natalie, Rolfsmeier, Sina, Schneider, 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Herfurth, Konstantin, Wolf, Gunter, Paul, Rainer, Haller, Hermann, Kaufeld, Jessica, Menne, Jan, Bahlmann-Kroll, Elisabeth, Bergner, Angela, Weihprecht, Horst, Er, Aydin, Sonntag, Florian, Turan, Elif, Wittmann, Michael, Klauser, Franziska, Voigt, Eva, Schettler, Volker, Schulz, Egbert, Rohnstock, Madlen, Schettler, Elke, Schroppel, Bernd, van Erp, Rene, Kachele, Martin, Ludwig, Ulla, Schulte-Kemna, Lena, Kmietschak, Waltraud, Preiss, Elke, Ruocco, Martina, Heine, Gunnar, Brzoska, Martin, Gabel, Sebastian, Büttner, Christina, Sabarai, Asma, Banas, Bernhard, Bergler, Tobia, Ehrl, Yvonne, Putz, Franz, Schuster, Antonia, Kuhn, Stefanie, Schramm, Torsten, Degenhardt, Stefan, Schmidt, Gerhard, Weiland, Lea, Giebeln-Hudnell, Ulrike, Kielstein, Jan, Eden, Gabriele, Fuchs, Brigitte, Morig, Gina, Winkler, Manuela, Darius, Harald, Kriatselis, Charalampo, Roesch, Carl-Philipp, Maselli, Astrid, Alscher, Dominik, Ketteler, Marku, Schanz, Moritz, Schricker, Severin, Rettenmaier, Bianka, Schwab, Andrea, Pergola, Pablo, Leal, Irene, Cagle, Melissa, Romo, Anna, Torres, Anthony, Joshi, Sucharit, Barrett, Kulli, Africano, Alexi, Dodds, Vicki, Gowen, Dorleena, Morris, Ashlee, Fernandez, Juan, Jimenez, Guillermo, Viera, Ricardo, Bruce, Kendaling, Barrios, Ryan, Garcia, Maylin, Garcia, Kerelyn, Leal, Iradi, Tietjen, David, Bains, David, Castillo, Carlo, Brewer, Genielle, Davis, Justin, Freking, Natalie, Golson, Brittany, Ham, Sally, Roesch, Jesslyn, Suchinda, Pusadee, Beigh, Shameem, Lilavivat, Usah, Bilton, Joyce, Bocchicchia, Kim, Turner, Jeffrey, Dahl, Neera, Peixoto, Aldo, Kavak, Yasemin, Liberti, Lauren, Nair, Hari, Page, Nicola, Rosenberg, Stephanie, Simmons, Kathryn, Isakova, Tamara, Frazier, Rebecca, Mehta, Rupal, Srivastava, Anand, Fox, Patrick, Hecktman, Jonathan, Hodakowski, Alexander, Martinez, Carlo, Phillips, Rachel, Stevenson, Alexi, Mustafa, Reem, Jansson, Kyle, Kimber, Cassandra, Stubbs, Jason, Tuffaha, Ahmad, Yarlagadda, Sri, Griffin, Debbie, Laundy, Elisabeth, Tang, Zhuo, Alicic, Radica, Cooper, Ann, Davis, Lisa, Gore, Ashwini, Goldfaden, Rebecca, Harvill, Leslie, Hichkad, Lisa, Johns, Barry, Jones, Thoma, Merritt, Kayla, Sheldon, Jennifer, Stanfield, Jennifer, Alexander, Lindsay, Preston, Kaitlyn, Wood, Lindsey, Pradhan, Rajesh, Deraad, Roger, Mcintosh, Kelli, Raymond, Loui, Shepperd, Michael, Mclaughlin, Susan, Seifert, Mary, Shepherd, Andrew, Aiello, Joseph, Durham, William, Loudermilk, Laurie, Manley, John, Burnette, Sabrina, Evans, Stephanie, Johnson, Tara, Sloan, Lance, Ann Acosta, Judy, Gillham, Stacy, Sloan, Katia, Squyres, Sueann, Rocco, Michael, Hawfield, Amret, Bagwell, Ben, Richmond, Lauren, Soufer, Joseph, Clarke, Subha, Aliu, Amanda, Calabrese, Kristine, Davis, Amanda, Poma, Veronica, Spinola, Tracy, Magee, Jame, Silva, Ricardo, Choksi, Rushab, Dajani, Lorraine, Evans, John, George, Anil, Krish, Prasanth, Martins, Gerard, Sheikh-Ali, Mae, Sutton, David, Driver, Freda, Hanburry, Abraham, Hume, Laura, Hurst, Amber, Taddeo, Matthew, Turner, Marla, Yousif, Veronica, Beddhu, Srinivasan, Al-Rabadi, Laith, Abraham, Nikita, Caamano, Amalia, Carle, Judy, Gonce, Victoria, Staylor, Kaitlyn, Zhou, Na, Bansal, Shweta, Bhattarai, Manoj, Sharma, Kumar, Debnath, Subrata, Garza, Aliseiya, Velagapudi, Chakradhar, Rovner, Sergio, Almeida, Javier, Casares, Pablo, Stewart-Ray, Verlaine, Almaraz, Rene, Dayrell, Renata, Moncada, Ana, Pulido, Ricardo, Rodriquez, Roxana, Deeb, Wasim, Degoursey, Kathryn, Gloria, Rodel, Greene, Trevor, Miller, Robert, Pereira, Edward, Roura, Miguel, Domingo, Debbie, Dorestin, Sasha, Hodge, William, Jackson, Cathy, Lund, Deborah, Taylor, Katrina, Boren, Kenneth, Cleveland, Brittany, Gaiser, Sandra, Sahani, Mandeep, Aldrich, Logan, Edmerson, Exodu, Limon, Edmond, Valletta, Cole, Vasquez, Patricia, Provenzano, Christopher, Brar, Navkiranjot, Henderson, Heather, Keith, Bellovich, Khai, Qur, Khairullah, Quresh, Makos, Gail, Topf, Joel, Gasko, Sherry, Henschel, Rosemarie, Knapp, Kaitlin, Kozlowski, Teresa, Lafleur, Paula, Varughese, Ashwathy, Xue, Hui, Wu, Patricia, Arechiga, Olga, Darbeau, Shan, Fechter, Michael, Martinez, Stephanie, Hanson, Lenita, Cooper, Nyla, Madera, Areli, Cadorna, Jay, Sheridan, Rita, Sparks, Helen, Eilerman, Bradley, Bodine, Susanne, Eid, Wael, Flora, Rebecca, Avery, Amber, Hardy, Cashmere, Biscoveanu, Mihaela, Nagelberg, Steven, Cummins, Tracey, Rahbari-Oskoui, Frederic, Oommen, Anju, Forghani, Zohreh, Hitchcock, Stacie, Hosein, Darya, Watkins, Diane, Patel, Minesh, Lambert, Anthony, Newman, Elizabeth, Wood, Autumn, Ross, Tammy, Topping, Stephany, Mulhern, Jeffrey, Murphy, Lorna, Vasseur, Ann, Greenwood, Gregory, Hadley, Alexander, Laurienti, Denise, Marshall, Christopher, Mclean, Nichola, Satko, Scott, Caudill, Brandy, Maris, Jacob, Rogers, Janice, Vanhoy, Cindy, Thomas, George, Nakhoul, George, O'Toole, John, Taliercio, Jonathan, Cooperman, Leslie, Markovic, Marina, Tucky, Barbara, Dev, Devasmita, Hasan, Alia, Yalamanchili, Hima, Jain, Namita, Mcneil, Lesley, Wines, Eric, Park, Jean, Ghazi, Adline, Hamm, Mia, Patel, Teja, Mottl, Amy, Chang, Emily, Derebail, Vimal, Cole, Emmie, Froment, Anne, Kelley, Sara, Osmond Foster, Jordan, Mahabadi, Vahid, Jafari, Golriz, Kamarzarian, Anita, Arriaga, Wendy, Arteaga, Daisy, Machicado, Rosario, Naverrete, Genesi, Kumar, Prashant, Nazeer, Imran, Urquia, Karina, Glider, Tammi, Jones, Vickie, Rucker, Savannah, Wiley, Jennifer, Pandey, Rahul, Arroyo, Jesu, Pariani, Harish, Ahmad, Mohammad, Mozaffari, Shahin, Perez, Erika, Budoff, Matthew, Roy, Sion, Birudaraju, Divya, Ghanem, Ahmed, Hamal, Sajad, Aronoff, Stephen, Joye Petr, Elisa, Sachson, Richard, Wiebel, Jaime, Akram, Sana, Jones, Laurie, Knight, Curti, Tarlac, Maurie, Ahmed, Shahbaz, Szerlip, Harold, Akinfolarin, Akinwande, Mehta, Ankit, Camp, Shana, Castro, Cindy, Cooper, Zanaida, Terry, Jessica, Awad, Ahmed, Kothapalli, Bhavya, Lustig, Ryan, Alfaress, Serine, Jasim, Hyder, Parrigon, Mary, Karounos, Denni, Ahmed, Sadiq, Berry, Maggie, Oremus, Ruth, Hernandez-Cassis, Carlo, Ugwu, Elia, Junejo, Nazia, Suazo, Nancy, Segal, Mark, Kazory, Amir, Brown, Sherry, Daniels, Tristan, Dayi, Sofia, Hogan, Renee, Mccray, Kathy, Stickley, Jennifer, Rahman, Mahboob, Dobre, Mirela, Negrea, Lavinia, Padiyar, Aparna, Pradhan, Nishigandha, Rashidi, Arash, Sarabu, Nagaraju, Donley, Vicki, Young, Tricia, Oguchi, Godson, Onyema, Judepatrick, Damianik, Kahla, Dienes, Jack, Plummer-Morgan, Judith, Roman, Marilyn, Skipper, Mauver, Villaruel, Stacey-Ann, Williams, Krystle, Sugimoto, Danny, Dugas, Jeffrey, Ahmed, Ismeal, Bhairoo, Jamie, Rijos, Dolore, Salim, Huzaifa, Gavrila, Madita, Lafferty, Kathryn, Rabara, Ria, Ruse, Sally, Weetman, Maria, Bushnell, Jame, Power, Albert, Jenkins, Alison, Jones, Stefanie, Scott, Amanda, Byrne, Cath, Jesky, Mark, Cowley, Alison, Mchaffie, Emma, Waterfall, Holly, Taylor, Jo, Bough, Laura, Phillips, Thoma, Winter-Goodwin, Barbara, Phin Kon, Sui, Macdougall, Iain, Lioudaki, Eirini, Shah, Sapna, Sharpe, Claire, Aguilar, Francisco, Hernandez Pena, Abegail, Pugay, Conception, Te, Amelia, Finn, Hugh, Hanif, Wasim, Mostafa, Samiul, Aitken, Alice, Draxlbauer, Katharine, Grobovaite, Evelina, Kearney, Jennifer, Mccarthy, Theresa, Gentile, Giorgio, Browne, Duncan, Chellamuthu, Palanichamy, Dugal, Tabinda, Chant, Terri, Jones, Laura, Laity, Emily, Miners, Megan, Muir, Jame, Swanson, Elizabeth, Frankel, Andrew, Tomlinson, Jame, Alegata, Marlon, Almasarwah, Rashid, Apostolidi, Anthoula, Vourvou, Maria, Walters, Thoma, Taal, Maarten, Dukka, Hari, Kolhe, Nitin, Mcdonald, Carly, White, Kelly, Ugni, Shiva, Gunda, Smita, Oluyombo, Rotimi, Brindle, Vicki, Coutts, Ping, Fuller, Tracy, Nadar, Evelyn, Ramadoss, Suresh, Motherwell, Nichola, Pajak, Susannah, Tonks, Louise, Bhandari, Sunil, Bodington, Richard, Hazara, Adil, Fellowes, Dominic, Wong, Christopher, Goldsmith, Christopher, Barnes, Sherald, Bennett, Ann, Burston, Claire, Hope, Samantha, Hunt, Nicola, Kurian, Lini, Fish, Richard, Farrugia, Daniela, Lee, Judy, Sadler, Emma, Turner, Hannah, Hill, Christopher, Brown, Henry, Masengu, Agne, Maxwell, Peter, Bleakley, Nina, Murtagh, Hugh, Petchey, William, Yiu, Vivian, Kellett, Joanne, Williams, Angharad, Clarke, Helen, Carnall, Victoria, Benyon, Sarah, Blake, Caroline, Estcourt, Stephanie, Piper, Jane, Morgan, Neal, Hutchinson, Carolyn, Mckinley, Teresa, Woodman, Alastair, Graham, Judi, Leonard, Niall, Smyth, John, Adell, Vicki, Hagan, Samantha, Caplin, Ben, Oomatia, Amin, Damian, Eleanor, Sobande, Toluleyi, Doulton, Tim, Delaney, Michael, Montasser, Mahmoud, Hansen, Jenny, Loader, David, Moon, Angela, Morris, France, Sinha, Smeeta, Chukwu, Chukwuma, Hudson, Amy, Campbell, Diane, Kershaw, Melanie, Whittaker, Stephanie, Irtiza-Ali, Ayesha, Ghalli, Farid, Nosseir, Heba, Leslie, Allison, Trivedi, Kate, Fraser, Donald, Alhadj Ali, Mohammad, Griffin, Sian, Latif, Farah, Witczak, Justyna, Wonnacott, Alexa, Jeffers, Lynda, Webley, Yvette, Phelan, Paul, Miller-Hodges, Eve, Geddes, Ailsa, Glenwright, Margaret, Hunter, Amy, Pickett, Thoma, Moriarty, Jim, Hill, Linda, Tyler, Amanda, Ayub, Waqar, Evans, Gail, Hewins, Sue, Hewitt, Davina, Read, Kerry, Bell, Samira, Cosgrove, Leanne, Craik, Rachel, Murray, Shona, Bhandary, Nitin, Coles, Holly, Easow, Rashmi, Joseph, Maya, Khwaja, Arif, Jackson, Yvonne, Mbuyisa, Angeline, Sellars, Rachel, Chitalia, Nihil, Mohandas, Cynthia, Gherman, Anca, Kamundi, Charlotte, Olufuwa, Olumide, Mccafferty, Kieran, Adeleke, Adedolapo, Healy, Cara, Jeyarajah, Damini, Kinsella-Perks, Edward, Smith, Richard, Camilleri, Brian, Buckman, Carol, Finch, Jenny, Rivers, Vanessa, Connor, Andrew, Carr, Sheila, Shainberg, Lisa, Lewington, Andrew, Baker, Richard, Dorey, Suzannah, Tobin, Kay, Wheatley, Rosalyn, Banerjee, Debasish, Hull, Richard, Abat, Sharirose, Paul, Riny, Karim, Mahzuz, Htet, Zay, Tufail, Saad, Varma, Ravi, Convery, Karen, Fottrell-Gould, Deirdre, Hudig, Lisa, Tropman, Emily, Abdul-Samad, Thahir, Grace, Anne, Phipps, Marie, Suckling, Rebecca, Somalanka, Subash, Sood, Bhrigu, Swift, Pauline, Acheampong, Sarah, Ansu, Kwame, Augustin, Martia, Sampson, Anna, Vinall, Lynn, Wren, Kim, Wanninayake, Shamila, Wooding, Nichola, Edwards, Heather, Owen, Lydia, Bolton, Stephanie, Carson, Marion, Matthews, Michael, Brunskill, Nigel, Jesus-Silva, Jorge, Howson, Alex, Quashie-Akponeware, Mary, Tindall, Hilary, Nethaji, Chidambaram, Eldon, Helen, Patel, Rajan, Mark, Patrick, Rankin, Alastair, Sullivan, Michael, Forsyth, Kirsty, Mcdougall, Rowan, Dasgupta, Tanaji, Davies, Louisa, Ryder, Maggie, Grimmer, Philip, Macdonald, Clare, Webster, Mary, Newcastle, Newcastle, Ellam, Timothy, Wong, Edwin, Meshykhi, Christine, Webster, Andrea, Wilson, Peter, Vilar, Enric, Berdeprado, Jocelyn, Doctolero, Eunice, Wilkinson, Lily, Mccarroll, Frank, Ammar, Hesham, Kuan, Ying, Moran, Conor, Shivashankar, Girish, Campbell, Ryan, Glowski, Deborah, Mcdermott, Paula, Ali, Amar, Patel, Zuber, Bond, Christine, Whalley, Gillian, Zhang, Haitao, Yang, Liu, Zhang, Lihua, Kan, Tingting, Zhu, Ling, Zhao, Jinghong, Hou, Weiping, Wu, Jing, Cheng, Hong, Bian, Weijing, Zhao, Zhirui, Shao, Fengmin, Cao, Huixia, Jiao, Xiaojing, Niu, Peiyuan, Niu, Jianying, Chen, Yu, Zhang, Lihong, Zhu, Shenglang, Lin, Haiyan, Yao, Shaopeng, Chen, Jiehui, Jiang, Ying, Hu, Ying, Xiao, Huaying, Yang, Fuye, Zhang, Xinzhou, Guo, Baochun, Jin, Qiu, Liu, Lixia, Xiao, Xiangcheng, Xie, Yanyun, Meng, Ting, Xu, Chuanwen, Huang, Jie, Xu, Yanmei, Kong, Weixin, Wang, Xiaoliang, Liu, Qianpan, Wang, Xueying, Gao, Ming, Hu, Xiumei, Lu, Ying, Wang, Li, Peng, Kun, Wang, Wei, Gong, Qiuhong, Cai, Jianfang, Li, Xiaojue, Liu, Xuejiao, Zhou, Shuhan, Liu, Hong, Weng, Yao, Tang, Shuai, Yao, Yao, Zhao, Shi, Cheng, Chen, Wei, Wei, Li, Na, Aqashiah Mazlan, Sadanah, Zubaidah Bahtar, Alia, Katiman, Elliyyin, Othman, Noraini, Mushahar, Lily, Mazlan, Nurdiana, Sharafina Safiee, Nur, Ramasamy, Sarasa, Seng Wong, Hin, Ahmad Rosdi, Hajar, Zhao Zhi Tan, Esther, Fan Tay, Ju, Seng Teng, Kok, Yahaya, Hasnah, Jiun Liu, Wen, Wee Ee, Lik, Kay Leong Khoo, Kenneth, Mohd Yusoff, Yuana, Safhan Mohamad Nor, Fariz, Kamil Ahmad, Mohd, Ramli Seman, Mohd, Hui Hong Tan, Clare, Lui Sian Ngu, Laura, Yoke May Chan, Jaime, Peji, Javelin, Loong Loh, Chek, Yan Lee, Yee, Ramanaidu, Sridhar, Mean Thong, Kah, Hong Wong, Yik, Junus, Suria, Hua Ching, Chen, Faisal Asmee, Mohammad, Ruziana Ku Md Razi, Ku, Leong Low, Chun, Sze Bing Sim, Christopher, Duan Tham, Zhang, Kamila Abdullah, Noor, Meng Chen, Tai, Chieh Chan, Yong, Chang, Eason, Yean Kang, Huan, Quan Lee, Kai, Ann Lee, Sue, Kheng Lee, Aik, Vinathan, Jeevika, Abdul Cader, Rizna, Mustafar, Ruslinda, Kamaruzaman, Lydia, Mohd, Rozita, Ismail, Rahimah, Men Leong, Chong, Koon Low, Chee, Wei Wong, Liang, Adnan, Norlezah, Ibrahim, Sabariah, Zaimi Abdul Wahab, Mohamad, Bavanandan, Sunita, Shen Lim, Yik, Hazlina Wan Mohamad, Wan, Munirah Jaafar, Siti, Ashykeen Mohd Fauzi, Nur, Sudin, Aziee, Kun Lim, Soo, Chung Gan, Chye, Hing, Albert, Ahmad Faizal Alaidin Razali, Wan, Fong Liew, Yew, Bao Tyng Chan, Chelsia, Chih Cheng, Mei, Chen Ong, Yu, Meng Ong, Loke, Amalina Mohamed Affandi, Farah, Rahmat, Korina, Chai Peng, Ban, Amat, Masayu, Hadafi Ahmad, Nuzaimin, Yee Mah, Doo, Loon Tye, Yi, Azhari, Zaid, Nabilah Mohamad Zaini, Siti, Aidil Musa, Mohd, Ahmad Miswan, Norazinizah, Ramli, Rafizanur, Aziah Ahmad, Nor, Leong Goh, Bak, Izah Ahmad, Nurul, Huda Ibrahim, Fairol, Jian Ng, Tze, Shanmuganathan, Malini, Lian Tay, Li, Harun, Zaiha, Ramli, Salmi, 'Ain Yusof, Nurul, Abd Rahman, Rossenizal, Iqbal Abdul Hafidz, Muhammad, Hidayati Mohd Sharif, Nur, Yasmoon Awang, Irda, Nakashima, Eitaro, Imamine, Rui, Minatoguchi, Makiko, Miura, Yukari, Nakaoka, Miduki, Suzuki, Yoshiki, Yoshikawa, Hitomi, Shin, Koki, Fujita, Kanae, Iwasa, Misuzu, Sasajima, Haruka, Sato, Airi, Hamamoto, Yoshiyuki, Fujita, Yuki, Haraguchi, Takuya, Hyo, Takanori, Izumi, Kiyohiro, Komiya, Toshiyuki, Kubota, Sodai, Kurose, Takeshi, Kuwata, Hitoshi, Nakatani, Susumu, Oishi, Kaori, Okamoto, Saki, Okamura, Kaori, Takeoka, Jun, Tanaka, Nagaaki, Tanigaki, Katsuya, Toda, Naohiro, Watanabe, Koin, Komori, Hiromi, Kumuji, Rika, Takesada, Asako, Tanaka, Aya, Maruyama, Shoichi, Hasegawa, Tomonori, Ishiguro, Akiko, Ishimoto, Takuji, Ito, Kazuhiro, Kamimura, Yutaka, Kato, Noritoshi, Kato, Sawako, Kojima, Hiroshi, Kosugi, Tomoki, Maeda, Kayaho, Mizuno, Masasi, Saito, Shoji, Sato, Hitomi, Sato, Yuka, Suzuki, Yasuhiro, Tanaka, Akihito, Yasuda, Yoshinari, Hasegawa, Fujiko, Hayashi, Maiko, Higashi, Shizuka, Shimamura, Kaho, Sumi, Momoko, Tajima, Kazuki, Unekawa, Chimaki, Wakayama, Kana, Wakita, Yukiko, Otani, Takatoshi, Imai, Ayako, Kawashima, Sayaka, Kogure, Eri, Sato, Tomoe, Takezawa, Misato, Yoshida, Shinya, Araki, Hideo, Katsuda, Yuko, Konishi, Masahiro, Matsunaga, Takahiro, Oe, Masashi, Ogane, Kunihiro, Sakai, Masato, Takahashi, Tomoko, Yamano, Takahiro, Yokoyama, Takuya, Ito, Hitomi, Katayama, Masayo, Kuroda, Emi, Ikeda, Toru, Kojo, Takuma, Yoshidome, Etsuo, Mizumachi, Rieko, Yamamoto, Akane, Yamasaki, Narihisa, Yamasaki, Yoshihiko, Wada, Jun, Eguchi, Jun, Higuchi, Chigusa, Katayama, Akihiro, Kinomura, Masaru, Kitagawa, Masashi, Kitamura, Shinji, Miyamoto, Satoshi, Morinaga, Hiroshi, Nakatsuka, Atsuko, Nojima, Ichiro, Shikata, Kenichi, Sugiyama, Hitoshi, Tanabe, Katsuyuki, Tsuji, Kenji, Uchida, Haruhito, Watanabe, Mayu, Hashimoto, Chie, Kato, Takahiro, Yamamoto, Sayaka, Wada, Takehiko, Fukagawa, Masafumi, Hamano, Naoto, Koizumi, Masahiro, Komaba, Hirotaka, Nakagawa, Yosuke, Iwamoto, Michiyo, Masutani, Kosuke, Katanosaka, Akane, Kiyota, Mayu, Uchi, Hikari, Ueda, Yuka, Yamamoto, Sonoka, Nagasu, Hajime, Itano, Seiji, Iwakura, Tsukasa, Kadoya, Hiroyuki, Kanda, Eiichiro, Kashihara, Naoki, Kidokoro, Kengo, Kondo, Megumi, Sasaki, Tamaki, Satoh, Minoru, Tokuyama, Atsuyuki, Umeno, Reina, Wada, Yoshihisa, Yamamoto, Toshiya, Yamanouchi, Yu, Abe, Masumi, Inukai, Yoko, Ogawa, Wataru, Asahara, Shunichiro, Fujii, Hideki, Goto, Shunsuke, Hirota, Yushi, Hosooka, Tetsuya, Kono, Keiji, Nishi, Shinichi, Okada, Yuko, Sakaguchi, Kazuhiko, Sugawara, Kenji, Takahashi, Michiko, Takai, Tomoko, Tamori, Yoshikazu, Watanabe, Kentaro, Kitajima, Miyu, Nishi, Misaki, Wada, Junko, Ito, Yasuhiko, Kamiya, Hideki, Asai, Akimasa, Asai, Nao, Asano, Saeko, Banno, Shogo, Ejima, Yohei, Hase, Hanako, Hayami, Tomohide, Himeno, Tatsuhito, Ishikawa, Takahiro, Ito, Mayumi, Iwagaitsu, Shiho, Kasagi, Rina, Kato, Yoshiro, Kato, Makoto, Kato, Koichi, Katsuno, Takayuki, Kawai, Miyuka, Kinashi, Hiroshi, Kondo, Masaki, Koshino, Masako, Matsuoka, Naoya, Morishita, Yoshiaki, Motegi, Mikio, Nakamura, Jiro, Shimoda, Hiromi, Sugiyama, Hirokazu, Tsunekawa, Shin, Yamaguchi, Makoto, Takahashi, Kazuyo, Watada, Hirotaka, Funayama, Takashi, Furukawa, Yasuhiko, Gohda, Tomohito, Goto, Hiromasa, Kaga, Hideyoshi, Kanaguchi, Yasuhiko, Kanazawa, Akio, Kaneko, Kayo, Kano, Toshiki, Kihara, Masao, Kimura, Shogo, Kobayashi, Takashi, Maiguma, Masayuki, Makita, Yuko, Mano, Satoshi, Mita, Tomoya, Miyatsuka, Takeshi, Murakoshi, Maki, Muto, Masahiro, Nakata, Masami, Nakata, Junichiro, Nishida, Yuya, Nohara, Nao, Ogihara, Takeshi, Sato, Daisuke, Sato, Junko, Sato, Hiroaki, Suzuki, Yusuke, Suzuki, Ruka, Suzuki, Hitoshi, Takagi, Miyuki, Tamura, Yoshifumi, Uchida, Toyoyoshi, Ueda, Seiji, Asawa, Miki, Miyaji, Minako, Nagashima, Eri, Shibata, Yoshie, Yanagisawa, Eri, Yamauchi, Toshimasa, Hirakawa, Yosuke, Nishi, Hiroshi, Shojima, Nobuhiro, Horikawa, Satoko, Nakayama, Yukiko, Yamada, Naoko, Omori, Yuki, Yano, Shintaro, Ioka, Miyabi, Kuwabara, Nahoko, Nagano, Remi, Nozawa, Megumi, Osawa, Yumi, Maegawa, Hiroshi, Kume, Shinji, Araki, Shinichi, Miyazawa, Itsuko, Morino, Katsutaro, Kawai, Ikuko, Sobata, Masumi, Takaoka, Motoko, Iwaita, Yasushi, Udagawa, Takashi, Inamori, Ami, Kawase, Aya, Yamanaka, Aya, Shimano, Hitoshi, Fujita, Akiko, Iwasaki, Hitoshi, Kai, Hirayasu, Osaki, Yoshinori, Saito, Chie, Sekiya, Motohiro, Tsunoda, Ryoya, Yamagata, Kunihiro, Nakamura, Rikako, Yamada, Aiko, Ohsugi, Mitsuru, Awazawa, Motoharu, Bouchi, Ryotaro, Hashimoto, Shota, Hashimoto, Makiko, Hisatake, Tomoko, Ihana, Noriko, Ishizuka, Koko, Izumi, Kazuo, Kajio, Hiroshi, Kobayashi, Michi, Kodani, Noriko, Maruyama, Koji, Matsumoto, Michihiro, Matsushita, Maya, Nakamura, Tomoka, Sugiyama, Takehiro, Tanabe, Akiyo, Terakawa, Aiko, Ueki, Kojiro, Orimo, Yuko, Ozawa, Takako, Takahira, Eriko, Yamasaki, Yoshimitsu, Haneda, Masakazu, Tomita, Tadahiro, Akimoto, Saori, Fujimoto, Akihiro, Ishihara, Kenji, Murakami, Chiho, Nishiyama, Akiyo, Toyonaga, Yukiko, Uozumi, Kana, Yamaji, Yukihiro, Shigehara, Tetsuya, Okajyo, Jun, Shimizu, Yukihiro, Iwasaki, Shingo, Fukao, Yuki, Furusho, Megumi, Nunokawa, Shintaro, Katagiri, Hideki, Izumi, Tomohito, Kaneko, Keizo, Kodama, Shinjiro, Miyazaki, Mariko, Munakata, Yuichiro, Nagasawa, Tasuku, Oe, Yuji, Sugawara, Hiroto, Takahashi, Kei, Hirata, Kazushige, Inomata, Keiko, Otomo, Shoko, Uchida, Taeko, Yamashita, Chigusa, Kiyosue, Arihiro, Tamura, Ryota, Dube, Francoi, Bolduc, Marilene, Talbot, Marie-Christine, Cham, Leslie, Lai, Vesta, Tse, Josephine, Jolly, Shivinder, Duck, Tabbatha, Lyle, Scott, Epp, Rachel, Galloway, Camille, Haskett, Susan, Matvienko, Elizabeta, Paulsen, Liam, Moist, Louise, Lozon, Zabrina, Ramsey, Tina, Whitmore, Brittany, Al-Zeer, Bader, Macleod, Paula, O'Sullivan, Aoife, Sheriff, Zainab, Tholl, Sam, Pandey, Amritanshu, Armstrong, Samantha, Gebeyehu, Bethelihem, Toth, Patrick, Goldenberg, Ronald, Jahangiriesmaili, Mahsa, Sanguila, Shariff, Suresh, Neethi, Talsania, Tanvi, Zalunardo, Nadia, Agharazii, Mohsen, Roussel, Marie-Pier, Saillant, Annie, Samson, France, Bajaj, Harpreet, Bhavsar, Miken, Dhall, Parul, Dhillon, Gagandeep, Grewal, Bhupinder, Nimbkar, Taniya, Madore, Francoi, Marcotte, Guylaine, Steen, Oren, Bullen, Mathura, Raguwaran, Shayani, Valleteau, Andre, Langlois, Marie-France, Brown, Christine, Steele, Andrew, Garrity, Melissa, Ghate, Taneera, Robinson, Holly, Tolibas, Michael, Tailor, Chetna, Elliott, Lauren, McClary-Wright, Christine, Boreky, Fadia, Fikry, Sameh, Ali, Ayesha, Barot, Chintankumar, Basily, Wagdy, Saram, Thisun, Varad, Vinay, Khandwala, Hasnain, Aguilera, Alex, Alvarez, Patricia, Gill, Balwinder, Huda, Nazihah, Navivala, Aamir, Pinto, Daniel, Bevilacqua, Micheli, Fung, Elaine, Hernandez, Geraldine, Mann, Puneet, Saini, Jaskiran, Rabasa-Lhoret, Remi, Bovan, Danijela, Devaux, Marie, Barnini, Cecilia, Leoncini, Giovanna, Manco, Luca, Nobili, Giulia, Piemontese, Matteo, Aucella, Filippo, Grifa, Rachele, Totaro, Francesco, La Manna, Gaetano, Capelli, Irene, Cianciolo, Giuseppe, Lerario, Sarah, Zappulo, Fulvia, Rosati, Alberto, Fani, Filippo, Spatoliatore, Giuseppe, Gesualdo, Loreto, Pesce, Francesco, Russo, Maria, Zippo, Maria, Cafiero, Cesira, Motta, Daria, Bianco, Simona, Bilucaglia, Donatella, Messa, Piergiorgio, Pavone, Laura, Tripodi, Federica, Vettoretti, Simone, Fioretto, Paola, Carraro, Gianni, Farnia, Filippo, Postal, Anna, D'Amelio, Alessandro, Cardone, Antonio, Piccinni, Giovanni, Aloisi, Annalisa, Scolari, Francesco, Alberici, Federico, Guerini, Alice, Saccà, Chiara, Salviani, Chiara, Zani, Roberta, DE NICOLA, Luca, Garofalo, Carlo, Elena Liberti, Maria, Minutolo, Roberto, Pennino, Luigi, Polese, Lucio, Mené, Paolo, Barberi, Simona, Falcone, Clorinda, Russo, Francesco, Caroppo, Maurizio, Santorelli, Gennaro, Rivera, Rodolfo, Santoro, Domenico, Giuffrida, Alfio, Zirino, Fortunata, Calvi, Cristina, Estienne, Luca, Gambaro, Giovanni, Gangemi, Concetta, Ortalda, Vittorio, Pessolano, Giuseppina, Grandaliano, Giuseppe, Baccaro, Rocco, Ferraro, Pietro, Mangiacapra, Roberto, Melandri, Marco, Foligno, Nadia, Quartagno, Rita, Vezzoli, Giuseppe, Brioni, Elena, and Group, EMPA-KIDNEY Collaborative

Subjects
chronic renal disease, empagliflozin, empa-kidney, General Medicine
Abstract

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m2 of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m2 with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; PConclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo. (Funded by Boehringer Ingelheim and others; EMPA-KIDNEY ClinicalTrials.gov number, NCT03594110. opens in new tab; EudraCT number, 2017-002971-24. opens in new tab.)

Published
2023

14. Insertion/deletion and microsatellite alteration profiles in induced pluripotent stem cells

Author

Kaori Imadome, Yuko Hoki, Satoshi Kamimura, Mayumi Fujita, Ryoko Araki, Misato Sunayama, Miki Nakamura, Masumi Abe, and Tomo Suga

Subjects
genome reprogramming, Somatic cell, Cell, Induced Pluripotent Stem Cells, cord blood erythroblasts, Biology, Biochemistry, Genome, Article, Mice, microsatellite alterations, sister clones set, INDEL Mutation, Genetics, medicine, Insertion deletion, Animals, Humans, Cellular Reprogramming Techniques, Indel, Induced pluripotent stem cell, Cells, Cultured, Whole genome sequencing, InDels, mouse iPSCs, Whole Genome Sequencing, Cell Biology, Genetic Profile, mouse ntESCs, Cellular Reprogramming, Cell biology, Mice, Inbred C57BL, hotspots of microsatellite alteration, medicine.anatomical_structure, whole-genome sequencing, Microsatellite, human iPSCs, Developmental Biology, Microsatellite Repeats
Abstract

Summary We here demonstrate that microsatellite (MS) alterations are elevated in both mouse and human induced pluripotent stem cells (iPSCs), but importantly we have now identified a type of human iPSC in which these alterations are considerably reduced. We aimed in our present analyses to profile the InDels in iPSC/ntESC genomes, especially in MS regions. To detect somatic de novo mutations in particular, we generated 13 independent reprogramed stem cell lines (11 iPSC and 2 ntESC lines) from an identical parent somatic cell fraction of a C57BL/6 mouse. By using this cell set with an identical genetic background, we could comprehensively detect clone-specific alterations and, importantly, experimentally validate them. The effectiveness of employing sister clones for detecting somatic de novo mutations was thereby demonstrated. We then successfully applied this approach to human iPSCs. Our results require further careful genomic analysis but make an important inroad into solving the issue of genome abnormalities in iPSCs., Graphical abstract, Highlights • InDels and microsatellite alterations are elevated in iPSCs • These alterations are reduced in human iPSCs derived from cord blood erythroblasts • Employing sister clones is an effective way to detect somatic de novo mutations, In this article, Abe and colleagues conclusively demonstrate that InDels and microsatellite alterations are elevated in reprogrammed pluripotent stem cells, both mouse and human, by employing sister clones and conducting large-scale validation experiments. Furthermore, they show that these alterations are considerably reduced in human iPSCs derived from cord blood erythroblasts.

Published
2021

15. Insertion/deletion and microsatellite alteration profiles in induced pluripotent stem cells

Author

Kamimura, Satoshi, Suga, Tomo, Hoki, Yuko, Sunayama, Misato, Imadome, Kaori, Fujita, Mayumi, Nakamura, Miki, Araki, Ryoko, Abe, Masumi, Satoshi, Kamimura, Tomo, Suga, Yuko, Fujimori, Misato, Sunayama, Kaori, Imadome, Mayumi, Fujita, Miki, Nakamura, Ryoko, Araki, and Masumi, Abe

Abstract

We here demonstrate that microsatellite (MS) alterations are elevated in both mouse and human induced pluripotent stem cells (iPSCs), but importantly we have now identified a type of human iPSC in which these alterations are considerably reduced. We aimed in our present analyses to profile the InDels in iPSC/ntESC genomes, especially in MS regions. To detect somatic de novo mutations in particular, we generated 13 independent reprogramed stem cell lines (11 iPSC and 2 ntESC lines) from an identical parent somatic cell fraction of a C57BL/6 mouse. By using this cell set with an identical genetic background, we could comprehensively detect clone-specific alterations and, importantly, experimentally validate them. The effectiveness of employing sister clones for detecting somatic de novo mutations was thereby demonstrated. We then successfully applied this approach to human iPSCs. Our results require further careful genomic analysis but make an important inroad into solving the issue of genome abnormalities in iPSCs.

Published
2021

16. Induced Pluripotent Stem Cell Generation-Associated Point Mutations Arise during the Initial Stages of the Conversion of These Cells

Author

Mayumi Sugiura, Yasuji Kasama, Ryoko Araki, Yuko Hoki, Misato Sunayama, Masahiro Uda, Miki Nakamura, Shunsuke Ando, and Masumi Abe

Subjects
Medicine (General), R5-920, Biology (General), QH301-705.5
Abstract

A large number of point mutations have been identified in induced pluripotent stem cell (iPSC) genomes to date. Whether these mutations are associated with iPSC generation is an important and controversial issue. In this study, we approached this critical issue in different ways, including an assessment of iPSCs versus embryonic stem cells (ESCs), and an investigation of variant allele frequencies and the heterogeneity of point mutations within a single iPSC clone. Through these analyses, we obtained strong evidence that iPSC-generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation. Further, our results suggest a possible approach for establishing iPSCs with fewer point mutations.

Published
2014
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17. Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station

Author

Yasuyuki Kikuchi, Masumi Abe, Ryoko Araki, Aiko Nagamatsu, Akira Higashibata, Daiyu Ito, Rei Inoue, Ikuko Osada, Hiroaki Nagatomo, Yuko Kamada, Teruhiko Wakayama, Masatoshi Ooga, Sayaka Wakayama, Hiromi Sano, Satoshi Kamimura, Hiromi Suzuki, Takashi Kohda, Tomomi Suzuki, Li Yang, Erika Hayashi, Sachiko Yano, Toru Shimazu, Satoshi Kishigami, Takahiro Ishikawa, Ren Watanabe, Kousuke Kazama, Rina Emura, Haruo Kasahara, Naoki Hirose, Tohru Yamamori, Masumi Umehara, and Motoki N. Tada

Subjects
0301 basic medicine, endocrine system, Multidisciplinary, Offspring, urogenital system, media_common.quotation_subject, SciAdv r-articles, Fertility, NASA Deep Space Network, Biology, Sperm, Cell biology, Mammalian reproduction, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Deep space exploration, 030220 oncology & carcinogenesis, International Space Station, Genetics, Health and Medicine, Normality, Research Articles, media_common, Research Article
Abstract

The effect of space radiation on offspring was evaluated using mouse freeze-dried spermatozoa preserved on the Space Station., Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

Published
2021

18. In situ delivery of iPSC-derived dendritic cells with local radiotherapy generates systemic antitumor immunity and potentiates PD-L1 blockade in preclinical poorly immunogenic tumor models

Author

Fumito Ito, Kenichi Makino, Takaaki Oba, Ryutaro Kajihara, Alfred E. Chang, Kunle Odunsi, Hans Minderman, Toshihiro Yokoi, Masumi Abe, and Ryoko Araki

Subjects
0301 basic medicine, Cancer Research, Skin Neoplasms, medicine.medical_treatment, Melanoma, Experimental, Priming (immunology), CD8-Positive T-Lymphocytes, Immunotherapy, Adoptive, B7-H1 Antigen, 0302 clinical medicine, Cancer immunotherapy, Tumor Microenvironment, Immunology and Allergy, Immune Checkpoint Inhibitors, RC254-282, biology, Chemistry, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, adaptive immunity, Acquired immune system, Tumor Burden, Phenotype, medicine.anatomical_structure, Oncology, 030220 oncology & carcinogenesis, Molecular Medicine, Signal Transduction, T cell, Induced Pluripotent Stem Cells, Immunology, Mice, Transgenic, programmed cell death 1 receptor, 03 medical and health sciences, Lymphocytes, Tumor-Infiltrating, Immune system, Antigen, Cell Line, Tumor, medicine, Animals, dendritic cells, Pharmacology, CD40, Immunotherapy, vaccination, Coculture Techniques, Mice, Inbred C57BL, Oncolytic and Local Immunotherapy, 030104 developmental biology, radioimmunotherapy, Cancer research, biology.protein, Radiotherapy, Adjuvant
Abstract

BackgroundDendritic cells (DCs) are a promising therapeutic target in cancer immunotherapy given their ability to prime antigen-specific T cells, and initiate antitumor immune response. A major obstacle for DC-based immunotherapy is the difficulty to obtain a sufficient number of functional DCs. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs); however, therapeutic strategies to engage iPSC-derived DCs (iPSC-DCs) into cancer immunotherapy remain to be elucidated. Accumulating evidence showing that induction of tumor-residing DCs enhances immunomodulatory effect of radiotherapy (RT) prompted us to investigate antitumor efficacy of combining intratumoral administration of iPSC-DCs with local RT.MethodsMouse iPSCs were differentiated to iPSC-DCs on OP9 stromal cells expressing the notch ligand delta-like 1 in the presence of granulocyte macrophage colony-stimulating factor. Phenotype and the capacities of iPSC-DCs to traffic tumor-draining lymph nodes (TdLNs) and prime antigen-specific T cells were evaluated by flow cytometry and imaging flow cytometry. Antitumor efficacy of intratumoral injection of iPSC-DCs and RT was tested in syngeneic orthotopic mouse tumor models resistant to anti-PD-1 ligand 1 (PD-L1) therapy.ResultsMouse iPSC-DCs phenotypically resembled conventional type 2 DCs, and had a capacity to promote activation, proliferation and effector differentiation of antigen-specific CD8+ T cells in the presence of the cognate antigen in vitro. Combination of in situ administration of iPSC-DCs and RT facilitated the priming of tumor-specific CD8+ T cells, and synergistically delayed the growth of not only the treated tumor but also the distant non-irradiated tumors. Mechanistically, RT enhanced trafficking of intratumorally injected iPSC-DCs to the TdLN, upregulated CD40 expression, and increased the frequency of DC/CD8+ T cell aggregates. Phenotypic analysis of tumor-infiltrating CD8+ T cells and myeloid cells revealed an increase of stem-like Slamf6+ TIM3− CD8+ T cells and PD-L1 expression in tumor-associated macrophages and DCs. Consequently, combined therapy rendered poorly immunogenic tumors responsive to anti-PD-L1 therapy along with the development of tumor-specific immunological memory.ConclusionsOur findings illustrate the translational potential of iPSC-DCs, and identify the therapeutic efficacy of a combinatorial platform to engage them for overcoming resistance to anti-PD-L1 therapy in poorly immunogenic tumors.

Published
2021

19. In situ delivery of iPSC-derived dendritic cells with local radiotherapy generates systemic antitumor immunity and potentiates PD-L1 blockade in preclinical poorly immunogenic tumor models.

Author

Oba, Takaaki, Makino, Kenichi, Kajihara, Ryutaro, Yokoi, Toshihiro, Araki, Ryoko, Abe, Masumi, Minderman, Hans, E Chang, Alfred, Odunsi, Kunle, Ito, Fumito, Ryoko, Araki, Masumi, Abe, Oba, Takaaki, Makino, Kenichi, Kajihara, Ryutaro, Yokoi, Toshihiro, Araki, Ryoko, Abe, Masumi, Minderman, Hans, E Chang, Alfred, Odunsi, Kunle, Ito, Fumito, Ryoko, Araki, and Masumi, Abe

Abstract

Dendritic cells (DCs) are a promising therapeutic target in cancer immunotherapy given their ability to prime antigen-specific T cells, and initiate antitumor immune response. A major obstacle for DC-based immunotherapy is the difficulty to obtain a sufficient number of functional DCs. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs); however, therapeutic strategies to engage iPSC-derived DCs (iPSC-DCs) into cancer immunotherapy remain to be elucidated. Accumulating evidence showing that induction of tumor-residing DCs enhances immunomodulatory effect of radiotherapy (RT) prompted us to investigate antitumor efficacy of combining intratumoral administration of iPSC-DCs with local RT.

Published
2021

20. Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station.

Author

Wakayama, Sayaka, Ito, Daiyu, Kamada, Yuko, Shimazu, Toru, Suzuki, Tomomi, Nagamatsu, Aiko, Araki, Ryoko, Ishikawa, Takahiro, Kamimura, Satoshi, Hirose, Naoki, Kazama, Kousuke, Yang, Li, Inoue, Rei, Kikuchi, Yasuyuki, Hayashi, Erika, Emura, Rina, Watanabe, Ren, Nagatomo, Hiroaki, Suzuki, Hiromi, Yamamori, Tohru, N Tada, Motoki, Osada, Ikuko, Umehara, Masumi, Sano, Hiromi, Kasahara, Haruo, Higashibata, Akira, Yano, Sachiko, Abe, Masumi, Kishigami, Satoshi, Kohda, Takashi, Ooga, Masatoshi, Wakayama, Teruhiko, Ryoko, Araki, Takahiro, Ishikawa, Satoshi, Kamimura, Masumi, Abe, Wakayama, Sayaka, Ito, Daiyu, Kamada, Yuko, Shimazu, Toru, Suzuki, Tomomi, Nagamatsu, Aiko, Araki, Ryoko, Ishikawa, Takahiro, Kamimura, Satoshi, Hirose, Naoki, Kazama, Kousuke, Yang, Li, Inoue, Rei, Kikuchi, Yasuyuki, Hayashi, Erika, Emura, Rina, Watanabe, Ren, Nagatomo, Hiroaki, Suzuki, Hiromi, Yamamori, Tohru, N Tada, Motoki, Osada, Ikuko, Umehara, Masumi, Sano, Hiromi, Kasahara, Haruo, Higashibata, Akira, Yano, Sachiko, Abe, Masumi, Kishigami, Satoshi, Kohda, Takashi, Ooga, Masatoshi, Wakayama, Teruhiko, Ryoko, Araki, Takahiro, Ishikawa, Satoshi, Kamimura, and Masumi, Abe

Abstract

Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

Published
2021

21. Cloning and characterization of the antiviral activity of feline Tetherin/BST-2.

Author

Aiko Fukuma, Masumi Abe, Yuko Morikawa, Takayuki Miyazawa, and Jiro Yasuda

Subjects
Medicine, Science
Abstract

Human Tetherin/BST-2 has recently been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. In this study, we cloned a cDNA fragment encoding a feline homolog of Tetherin/BST-2 and characterized the protein product. The degree of amino acid sequence identity between human Tetherin/BST-2 and the feline homolog was 44.4%. Similar to human Tetherin/BST-2, the expression of feline Tetherin/BST-2 mRNA was inducible by type I interferon (IFN). Exogenous expression of feline Tetherin/BST-2 efficiently inhibited the release of feline endogenous retrovirus RD-114. The extracellular domain of feline Tetherin/BST-2 has two putative N-linked glycosylation sites, N79 and N119. Complete loss of N-linked glycosylation by introduction of mutations into both sites resulted in almost complete abolition of its antiviral activity. In addition, feline Tetherin/BST-2 was insensitive to antagonism by HIV-1 Vpu, although the antiviral activity of human Tetherin/BST-2 was antagonized by HIV-1 Vpu. Our data suggest that feline Tetherin/BST-2 functions as a part of IFN-induced innate immunity against virus infection and that the induction of feline Tetherin/BST-2 in vivo may be effective as a novel antiviral strategy for viral infection.

Published
2011
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23. Restoration of ATM Expression in DNA-PKcs–Deficient Cells Inhibits Signal End Joining

Author

Yao Xu, Jessica A. Neal, Eric A. Hendrickson, Katheryn Meek, and Masumi Abe

Subjects
0301 basic medicine, Genetics, Immunology, HEK 293 cells, Cell, V(D)J recombination, Biology, Embryonic stem cell, Cell biology, enzymes and coenzymes (carbohydrates), 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, medicine.anatomical_structure, Cell culture, Gene expression, medicine, Immunology and Allergy, Ectopic expression, biological phenomena, cell phenomena, and immunity, DNA-PKcs, 030215 immunology
Abstract

Unlike most DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)–deficient mouse cell strains, we show in the present study that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficency results in spontaneous reduction of ATM expression in many cultured cell lines (including those examined in this study) and in DNA-PKcs–deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs–deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs–deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the classical nonhomologous end-joining pathway.

Published
2016
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24. Abstract 2484: Development of a gene expression database of pancreatic ductal adenocarcinoma cases by NGS-combined HiCEP to identify tumor markers

Author

Mikiya Takao, Junji Yamamoto, Akiyoshi Nakayama, Yoji Kishi, Kazuki Maehara, Ryoko Araki, Masumi Abe, Yusuke Kawamura, Mayumi Hoshikawa, Yosuke Kitamura, Keiichi Ito, Hirotaka Matsuo, Makoto Kawaguchi, Nariyoshi Shinomiya, and Seiko Shimizu

Subjects
Cancer Research, Pancreatic ductal adenocarcinoma, Oncology, Cancer research, Biology, Gene
Abstract

Objectives: High coverage expression profiling (HiCEP) is an AFLP-based comprehensive gene expression analysis invented in Japan. There are two advantages of HiCEP compared with existing methods, such as DNA microarrays and RNA sequencing. First, it can efficiently detect an especially low amount of mRNA with high sensitivity and reliability, and second, it enables us to analyze mRNA expression much more quantitatively and reproducibly. On the other hand, it requires complicated processes, including TA cloning of isolated transcripts, to obtain the sequence information of the detected peaks. In order to solve this problem, we established the gene expression database of human cancers by combining the next-generation sequencing (NGS) with HiCEP method. Materials and methods: We applied the NGS-combined HiCEP method to analyze pancreatic ductal adenocarcinoma(PDAC) cases in this study, and tried to establish the gene expression database of PDAC to identify effective tumor markers. We collected samples of both the cancerous and macroscopically non-cancerous tissues from 49 patients diagnosed with PDAC who underwent surgical resection at our institute. Among them, four cases were analyzed by HiCEP. Total RNA was extracted from the cancerous and normal tissues of the four PDAC cases, and transcribed to cDNA. The cDNA was synthesized and subjected to digestion with the restriction enzymes, MspI and MseI, followed by adapter ligation. Selective PCR by 256 kinds of primer pairs was used to amplify the HiCEP fragments, and products with fluorescently-labeled primer were then analyzed by capillary electrophoresis. HiCEP fragments were sequenced by the next-generation sequencer (ion PGM, Thermo Fisher Scientific). Furthermore, we compared the expression levels of HiCEP peaks in cancerous tissues with those in normal tissues. Results: We detected multiple HiCEP peaks that showed higher expression in cancerous tissues than in normal tissues in all four cases, and also found out different peaks showing higher expression in cancerous tissues of cases which had a recurrence of cancer after surgery than in cancerous tissues of cases without recurrences. We determined the sequences of the HiCEP fragments by NGS, and developed the first ever HiCEP fragment database for PDAC. Conclusion: We successfully established a PDAC gene expression database by NGS-combined HiCEP method. We are now performing replication analyses with the other PDAC cases, and further analyses of blood samples from the same PDAC cases, aiming to identify diagnostic and prognostic markers of PDAC. Citation Format: Mikiya Takao, Hirotaka Matsuo, Ryoko Araki, Seiko Shimizu, Makoto Kawaguchi, Akiyoshi Nakayama, Yosuke Kitamura, Yusuke Kawamura, Kazuki Maehara, Masumi Abe, Keiichi Ito, Mayumi Hoshikawa, Junji Yamamoto, Yoji Kishi, Nariyoshi Shinomiya. Development of a gene expression database of pancreatic ductal adenocarcinoma cases by NGS-combined HiCEP to identify tumor markers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2484.

Published
2020
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27. Generation of Mouse Pluripotent Stem Cell–Derived Proliferating Myeloid Cells as an Unlimited Source of Functional Antigen-Presenting Cells

Author

Satoru Senju, Miwa Haruta, Norihiro Ueda, Yoshikazu Matsuoka, Ryusuke Nakatsuka, Yoshiki Akatsuka, Motoharu Suzuki, Yasuharu Nishimura, Yutaka Sasaki, Hayao Nakanishi, Tian Yi Liu, Masumi Abe, Minako Tatsumi, Yoshiaki Sonoda, Ryoko Araki, Hiroyuki Maki, Rong Zhang, Yasushi Uemura, Shinobu Tsuzuki, Kiyotaka Kuzushima, Yasushi Sakamoto, and Narumi Hirosawa

Subjects
Pluripotent Stem Cells, Cancer Research, Adoptive cell transfer, Cellular differentiation, Immunology, Antigen presentation, Antigen-Presenting Cells, Biology, Immunophenotyping, Mice, Antigens, Neoplasm, Neoplasms, Animals, Cytotoxic T cell, Myeloid Cells, Induced pluripotent stem cell, Antigen-presenting cell, Melanoma, Cells, Cultured, Cell Proliferation, Antigen Presentation, CD40, Cell Differentiation, Dendritic Cells, Adoptive Transfer, Embryonic stem cell, Cell biology, Phenotype, Antigens, Surface, biology.protein, Cytokines, Female, Peptides, T-Lymphocytes, Cytotoxic
Abstract

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow–derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2Kb-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs. Cancer Immunol Res; 3(6); 668–77. ©2015 AACR.

Published
2015
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29. Suppression of production of baboon endogenous virus by dominant negative mutants of cellular factors involved in multivesicular body sorting pathway

Author

Jiro Yasuda, Akira Hashimoto, Rie Nakaoka Miyaho, Takayuki Miyazawa, Masumi Abe, and Rokusuke Yoshikawa

Subjects
Cancer Research, Endosome, viruses, Amino Acid Motifs, Molecular Sequence Data, Mutant, Gene Products, gag, Genome, Viral, Virus Replication, ESCRT, Cell Line, Virology, biology.animal, Chlorocebus aethiops, Animals, Humans, Protein Interaction Domains and Motifs, Amino Acid Sequence, Vero Cells, Virus Release, Gammaretrovirus, Budding, Endosomal Sorting Complexes Required for Transport, biology, Multivesicular body sorting pathway, Multivesicular Bodies, biology.organism_classification, Infectious Diseases, Host-Pathogen Interactions, Mutation, Vero cell, RNA, Viral, Baboon
Abstract

Baboon endogenous virus (BaEV) is an infectious endogenous gammaretrovirus isolated from a baboon placenta. BaEV-related sequences have been identified in both Old World monkeys and African apes, but not in humans or Asian apes. Recently, it was reported that BaEV-like particles were produced from Vero cells derived from African green monkeys by chemical induction, and thus BaEV-like particles may contaminate biological products manufactured using Vero cells. In this study, we constructed an infectious molecular clone of BaEV strain M7. We found two putative L-domain motifs, PPPY and PSAP, in the pp15 region of Gag. To examine the function of the L-domain motifs, we conducted virus budding assay using L-domain motif mutants. We revealed that the PPPY motif, but not the PSAP motif, plays a major role as the L-domain in BaEV budding. We also demonstrated that Vps4A/B are involved in BaEV budding. These data suggest that BaEV Gag recruits the cellular endosomal sorting complex required for transport (ESCRT) machinery through the interaction of the PPPY L-domain with cellular factors. These data will be useful for controlling contamination of BaEV-like particles in biological products in the future.

Published
2015
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30. Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving

Author

Jingyi Nie, Sophia Moore, Ryuichi Okayasu, Nakako Izumi Nakajima, Mayumi Sugiura, Akira Fujimori, Jac A. Nickoloff, Neelam Sharma, Christopher P. Allen, Ryoko Araki, Masumi Abe, Yuko Hoki, and Hirokazu Hirakawa

Subjects
0301 basic medicine, Genome instability, DNA Repair, DNA repair, Biophysics, Somatic hypermutation, Biology, medicine.disease_cause, Article, Ionizing radiation, 03 medical and health sciences, Chromosome instability, Cell Line, Tumor, medicine, Humans, Radiology, Nuclear Medicine and imaging, Linear Energy Transfer, Homologous Recombination, Mutation, Radiation, Dose-Response Relationship, Radiation, Radiotherapy Dosage, Neoplasms, Experimental, Molecular biology, 030104 developmental biology, Cancer cell, Homologous recombination
Abstract

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

Published
2017

31. Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency

Author

Araki, Ryoko, Hoki, Yuko, Suga, Tomo, Obara, Chizuka, Sunayama, Misato, Imadome, Kaori, Fujita, Mayumi, Kamimura, Satoshi, Nakamura, Miki, Wakayama, Sayaka, Nagy, Andras, Wakayama, Teruhiko, Abe, Masumi, Ryoko, Araki, Yuko, Fujimori, Tomo, Suga, Chizuka, Obara, Misato, Sunayama, Kaori, Imadome, Mayumi, Fujita, Satoshi, Kamimura, and Masumi, Abe

Abstract

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

Published
2020

32. Inhibition of budding/release of porcine endogenous retrovirus

Author

Aiko Fukuma, Rokusuke Yoshikawa, Takayuki Miyazawa, Jiro Yasuda, and Masumi Abe

Subjects
Budding, Xenotransplantation, medicine.medical_treatment, Immunology, Mutant, Biology, biology.organism_classification, Microbiology, Virology, law.invention, Retrovirus, Cell culture, law, Tetherin, medicine, Recombinant DNA, TSG101
Abstract

PERV is integrated into the genome of all pigs. PERV-A and PERV-B are polytropic and can productively infect human cell lines, whereas PERV-C is ecotropic. Recombinant PERV-A/C can infect human cells and exhibits high titer replication. Therefore, use of pigs for human xenotransplantation raises concerns about the risks of transfer of this infectious agent from donors to xenotransplantation recipients. To establish strategies to inhibit PERV production from cells, in the present study, we investigated the mechanism of PERV budding and anti-PERV activity of Tetherin/BST-2. The results showed that DN mutants of WWP-2, Tsg101, and Vps4A/B markedly reduced PERV production in human and porcine cell lines, suggesting that PERV budding uses these cellular factors and the cellular MVB sorting pathway as well as many other retroviruses. Moreover, PERV production was also reduced by human and porcine Tetherin/BST-2. These data are useful for developing strategies to inhibit PERV production and may reduce the risk of PERV infection in xenotransplantation.

Published
2014
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33. Abstract 5237: Development of a gene expression database of renal cell carcinoma cases by NGS-combined HiCEP to identify tumor markers

Author

Makoto Kawaguchi, Hirotaka Matsuo, Ryoko Araki, Seiko Shimizu, Mikiya Takao, Akiyoshi Nakayama, Yosuke Kitamura, Masumi Abe, Keiichi Ito, and Nariyoshi Shinomiya

Subjects
Cancer Research, Oncology
Abstract

Objectives: High coverage expression profiling (HiCEP) is an AFLP-based comprehensive gene expression analysis technique invented in Japan. HiCEP has two unique characteristics. First, it can detect low amounts of mRNA with high sensitivity and reliability. Second, HiCEP enables highly quantitative and reproducible mRNA expression analyses. However, it requires complicated processes, including TA cloning of isolated transcripts, to obtain sequence information on detected peaks. We performed next-generation sequencing (NGS)-combined HiCEP and tried to establish a gene expression database of renal cell carcinoma (RCC) cases to identify effective tumor markers. Materials and methods: We collected cancerous and macroscopically non-cancerous regions from 83 RCC cases. Of these, six cases with clear cell RCC were analyzed by HiCEP. Total RNA was extracted from the cancerous and non-cancerous tissues of six clear cell RCC cases, and transcribed to cDNA. The cDNA was synthesized and subjected to digestion with the restriction enzymes MspI or MseI, followed by adapter ligation. Selective PCR by 256 kinds of primer pairs was used to amplify the HiCEP fragments, and products with fluorescently-labeled primer were then analyzed by capillary electrophoresis. HiCEP fragments were sequenced using a next-generation sequencer (ion PGM, Thermo Fisher Scientific). We compared the expression levels of HiCEP peaks in cancerous tissues with those in non-cancerous tissues. Results: We detected several HiCEP peaks in cancerous tissues that showed five times higher expression than in normal tissues. We determined the sequences of the HiCEP fragments by NGS, and developed the first ever cancerous tissue HiCEP fragment database. Conclusion: We successfully established an RCC gene expression database by NGS-combined HiCEP. We are now performing replication analyses and further analyses of blood samples from the same RCC cases to be able to identify diagnostic and prognostic markers of RCC. Citation Format: Makoto Kawaguchi, Hirotaka Matsuo, Ryoko Araki, Seiko Shimizu, Mikiya Takao, Akiyoshi Nakayama, Yosuke Kitamura, Masumi Abe, Keiichi Ito, Nariyoshi Shinomiya. Development of a gene expression database of renal cell carcinoma cases by NGS-combined HiCEP to identify tumor markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5237.

Published
2019
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34. Induced Pluripotent Stem Cell Generation-Associated Point Mutations Arise during the Initial Stages of the Conversion of These Cells

Author

Miki Nakamura, Masahiro Uda, Ryoko Araki, Yasuji Kasama, Shunsuke Ando, Yuko Hoki, Mayumi Sugiura, Misato Sunayama, and Masumi Abe

Subjects
Induced Pluripotent Stem Cells, Clone (cell biology), Biology, medicine.disease_cause, Biochemistry, Article, Cell Line, Genetic Heterogeneity, Mice, Gene Frequency, Genetics, medicine, Animals, Point Mutation, Induced pluripotent stem cell, Allele frequency, lcsh:QH301-705.5, Embryonic Stem Cells, Mutation, lcsh:R5-920, Genome, Genetic heterogeneity, Point mutation, Chromosome Mapping, High-Throughput Nucleotide Sequencing, Cell Biology, Sequence Analysis, DNA, Embryonic stem cell, Mice, Inbred C57BL, lcsh:Biology (General), Cell culture, lcsh:Medicine (General), Developmental Biology
Abstract

Summary A large number of point mutations have been identified in induced pluripotent stem cell (iPSC) genomes to date. Whether these mutations are associated with iPSC generation is an important and controversial issue. In this study, we approached this critical issue in different ways, including an assessment of iPSCs versus embryonic stem cells (ESCs), and an investigation of variant allele frequencies and the heterogeneity of point mutations within a single iPSC clone. Through these analyses, we obtained strong evidence that iPSC-generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation. Further, our results suggest a possible approach for establishing iPSCs with fewer point mutations., Graphical Abstract, Highlights • iPSCs versus ESCs—an obvious difference in the frequency and mode of point mutations • A large number of iPSC generation-associated, not preexisting, point mutations • Heterogeneity of point mutation profiles in an iPSC clone • No common point mutations among iPSC clones, Sugiura et al. have obtained strong evidence that iPSC generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation.

Published
2014

35. Investigation of transcriptional responses of juvenile mouse bone marrow to power frequency magnetic fields

Author

Sylwia Kabacik, Masumi Abe, Kevin J. Whitehill, Claudine Raffy, Kevin M. Brindle, Margaret Coster, Simon Bouffler, Zenon Sienkiewicz, Christophe Badie, and Heide L. Kirschenlohr

Subjects
Male, Microarray, Health, Toxicology and Mutagenesis, Pilot Projects, Biology, Real-Time Polymerase Chain Reaction, PICALM, Mice, Bone Marrow, In vivo, Transcription (biology), Genetics, medicine, Animals, Molecular Biology, Oligonucleotide Array Sequence Analysis, Phosphatidylinositol binding, Molecular biology, Mice, Inbred C57BL, Gene expression profiling, Magnetic Fields, medicine.anatomical_structure, Gene Expression Regulation, Monomeric Clathrin Assembly Proteins, Bone marrow, DNA microarray
Abstract

To seek alterations in gene transcription in bone marrow cells following in vivo exposure of juvenile mice to power frequency magnetic fields, young (21–24-day old) C57BL/6 mice were exposed to a 100 μT 50 Hz magnetic field for 2 h. Transcription was analysed by three methods, High Coverage Expression Profiling (HiCEP), Illumina microarrays and quantitative real-time polymerase chain reaction (QRT-PCR). A pilot HiCEP experiment with 6 exposed (E) and 6 non-exposed (NE) mice identified four candidate responsive transcripts (two unknown transcripts (AK152075 and F10-NED), phosphatidylinositol binding clathrin assembly protein (Picalm) and exportin 7 (Xpo7)). A larger experiment compared 19 E and 15 NE mice using two independent QRT-PCR assays and repeated microarray assays. No significant field-dependent changes were seen, although Picalm showed a trend to significance in one QRT-PCR assay (E/NE = 0.91; P = 0.06). However, the study was underpowered to detect an effect of this magnitude (52% power at P = 0.05). These data indicate the current experimental constraints in detecting small changes in transcription that may occur in response to magnetic fields. These constraints result from technical limitations in the accuracy of assays and biological variation, which together were sufficient to account statistically for the number of differentially expressed transcripts identified in the pilot experiment.

Published
2013
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36. Two-step differentiation of mast cells from induced pluripotent stem cells

Author

Satoshi Tanaka, Waka Ishida, Katsuhisa Tashiro, Ryoko Araki, Ken Fukuda, Tomoko Yamaguchi, Atsuki Fukushima, Sumie Katayama, Masumi Abe, Hiroyuki Mizuguchi, and Kenji Kawabata

Subjects
Cell Degranulation, Cellular differentiation, Induced Pluripotent Stem Cells, Embryoid body, Histidine Decarboxylase, Immunoglobulin E, Cell Line, Tetraspanin 28, Mice, Vancomycin, Animals, Mast Cells, RNA, Messenger, Induced pluripotent stem cell, Interleukin 5, Connective Tissue Cells, biology, Passive Cutaneous Anaphylaxis, Degranulation, Cell Differentiation, Cell Biology, Hematology, 3T3 Cells, Cell biology, Interleukin 33, Mice, Inbred C57BL, biology.protein, Phenazines, Developmental Biology
Abstract

Mast cells play important roles in the pathogenesis of allergic diseases. They are generally classified into 2 phenotypically distinct populations: connective tissue-type mast cells (CTMCs) and mucosal-type mast cells (MMCs). The number of mast cells that can be obtained from tissues is limited, making it difficult to study the function of mast cells. Here, we report the generation and characterization of CTMC-like mast cells derived from mouse induced pluripotent stem (iPS) cells. iPS cell-derived mast cells (iPSMCs) were generated by the OP9 coculture method or embryoid body formation method. The number of Safranin O-positive cells, expression levels of CD81 protein and histidine decarboxylase mRNA, and protease activities were elevated in the iPSMCs differentiated by both methods as compared with those in bone marrow-derived mast cells (BMMCs). Electron microscopic analysis revealed that iPSMCs contained more granules than BMMCs. Degranulation was induced in iPSMCs after stimulation with cationic secretagogues or vancomycin. In addition, iPSMCs had the ability to respond to stimulation with the IgE/antigen complex in vitro and in vivo. Moreover, when iPSMCs generated on OP9 cells were cocultured with Swiss 3T3 fibroblasts, protease activities as maturation index were more elevated, demonstrating that mature mast cells were differentiated from iPS cells. iPSMCs can be used as an in vitro model of CTMCs to investigate their functions.

Published
2013

37. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells

Author

Masahiro Uda, Masumi Abe, Ryoko Araki, Shunsuke Ando, Akira Nifuji, Misato Sunayama, Akemi Shimada, Mayumi Sugiura, Miki Nakamura, Yuko Hoki, and Hisashi Ideno

Subjects
Male, Cellular differentiation, Induced Pluripotent Stem Cells, Bone Marrow Cells, Cell Cycle Proteins, Biology, Mice, Immune system, Bone Marrow, medicine, Animals, Induced pluripotent stem cell, Embryonic Stem Cells, Bone Marrow Transplantation, Skin, Multidisciplinary, Gene Expression Profiling, Immunogenicity, Teratoma, Membrane Proteins, Cell Differentiation, Skin Transplantation, Embryonic stem cell, Mice, Inbred C57BL, Transplantation, medicine.anatomical_structure, Immunology, Cancer research, Bone marrow, Reprogramming
Abstract

Immune rejection may limit the therapeutic use of induced pluripotent stem cells (iPSCs); here, terminally differentiated mouse iPSCs are shown to generate negligible immune rejection in their host. Induced pluripotent stem cells (iPSCs) derived from a patient's own somatic cells could have great therapeutic potential. The hope is that iPSC-derived differentiated cells would avoid any immunogenic responses. In this study, Masumi Abe and colleagues assess the immunogenicity of skin and bone marrow tissues derived from a large set of isogenic mouse embryonic stem cell and iPSC lines. Their results are consistent with negligible immune rejection by the host. The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues1,2. However, partial reprogramming and genetic instabilities in iPSCs3,4,5,6 could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues7,8,9,10 and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas11 and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings12. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

Published
2013
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38. Comprehensive gene expression analyses in pluripotent stem cells of a planarian, Dugesia japonica

Author

Tetsutaro Hayashi, Nobuko Suzuki, Fuyan Son, Syozo Sano, Ryoko Araki, Norito Shibata, Ryutaro Fukumura, Junsuke Fujii, Osamu Nishimura, Masumi Abe, Kiyokazu Agata, and Tomomi Kudome-Takamatsu

Subjects
Pluripotent Stem Cells, Embryology, Population, Real-Time Polymerase Chain Reaction, Transcriptome, Animals, Regeneration, RNA, Messenger, Induced pluripotent stem cell, education, Gene, In Situ Hybridization, Oligonucleotide Array Sequence Analysis, Genetics, Regulation of gene expression, education.field_of_study, biology, Gene Expression Profiling, Gene Expression Regulation, Developmental, Helminth Proteins, Planarians, biology.organism_classification, Cell biology, Gene expression profiling, Planarian, Biomarkers, Developmental Biology, Adult stem cell
Abstract

The neoblasts are the only somatic stem cells in planarians possessing pluripotency, and can give rise to all types of cells, including germline cells. Recently, accumulated knowledge about the transcriptome and expression dynamics of various pluripotent somatic stem cells has provided important opportunities to understand not only fundamental mechanisms of pluripotency, but also stemness across species at the molecular level. The neoblasts can easily be eliminated by radiation. Also, by using fluorescence activated cell sorting (FACS), we can purify and collect many neoblasts, enabling identification of neoblast-related genes by comparison of the gene expression level among intact and X-ray-irradiated animals, and purified neoblasts. In order to find such genes, here we employed the high coverage expression profiling (HiCEP) method, which enables us to observe and compare genome-wide gene expression levels between different samples without advance sequence information, in the planarian D. japonica as a model organism of pluripotent stem cell research. We compared expression levels of ~17,000 peaks corresponding to independent genes among different samples, and obtained 102 peaks as candidates. Expression analysis of genes identified from those peaks by in situ hybridization revealed that at least 42 genes were expressed in the neoblasts and in neoblast-related cells that had a different distribution pattern in the body than neoblasts. Also, single-cell PCR analysis of those genes revealed heterogeneous expression of some genes in the neoblast population. Thus, using multidimensional gene expression analyses, we were able to obtain a valuable data set of neoblast-related genes and their expression patterns.

Published
2012
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39. ERK signaling controls blastema cell differentiation during planarian regeneration

Author

Yoshimichi Tabata, Nobuko Suzuki, Fuyan Son, Yoshihiko Umesono, Junichi Tasaki, Ryoko Araki, Norito Shibata, Osamu Nishimura, Masumi Abe, Kazu Itomi, and Kiyokazu Agata

Subjects
MAPK/ERK pathway, education.field_of_study, biology, MAP Kinase Signaling System, Stem Cells, Regeneration (biology), Cellular differentiation, fungi, Population, Cell Differentiation, Planarians, biology.organism_classification, Cell biology, body regions, Planarian, Animals, Regeneration, Dugesia japonica, Extracellular Signal-Regulated MAP Kinases, education, Molecular Biology, Blastema, Developmental Biology, Adult stem cell
Abstract

The robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Here, using the planarian Dugesia japonica we found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, was specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic [RNA interference (RNAi)] approaches provided evidence that ERK activity was required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induced an increased level of ERK activity and rescued the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration.

Published
2011
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40. Suppressive effects of electrolyzed reduced water on alloxan-induced apoptosis and type 1 diabetes mellitus

Author

Shigeru Kabayama, Taichi Kashiwagi, Takeki Hamasaki, Munenori Kawamura, Noboru Nakamichi, Sanetaka Shirahata, Yupin Li, Hanxu Yan, Kiichiro Teruya, Tomoya Kinjo, Jun Ye, Takaaki Komatsu, and Masumi Abe

Subjects
chemistry.chemical_classification, Type 1 diabetes, medicine.medical_specialty, Reactive oxygen species, endocrine system diseases, Pancreatic islets, Clinical Biochemistry, Biomedical Engineering, Bioengineering, Cell Biology, medicine.disease, medicine.disease_cause, chemistry.chemical_compound, Endocrinology, medicine.anatomical_structure, chemistry, Apoptosis, Internal medicine, Diabetes mellitus, Alloxan, medicine, Beta cell, Oxidative stress, Original Research, Biotechnology
Abstract

Electrolyzed reduced water, which is capable of scavenging reactive oxygen species, is attracting recent attention because it has shown improved efficacy against several types of diseases including diabetes mellitus. Alloxan produces reactive oxygen species and causes type 1 diabetes mellitus in experimental animals by irreversible oxidative damage to insulin-producing β-cells. Here, we showed that electrolyzed reduced water prevented alloxan-induced DNA fragmentation and the production of cells in sub-G1 phase in HIT-T15 pancreatic β-cells. Blood glucose levels in alloxan-induced type 1 diabetes model mice were also significantly suppressed by feeding the mice with electrolyzed reduced water. These results suggest that electrolyzed reduced water can prevent apoptosis of pancreatic β-cells and the development of symptoms in type 1 diabetes model mice by alleviating the alloxan-derived generation of reactive oxygen species.

Published
2010
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41. Generation of Genome Integration-free Induced Pluripotent Stem Cells from Fibroblasts of C57BL/6 Mice without c-Myc Transduction

Author

Shunsuke Ando, Masumi Abe, Yasuji Kasama, Yuko Hoki, Miki Nakamura, Yuko Jincho, Chihiro Tamura, and Ryoko Araki

Subjects
Induced Pluripotent Stem Cells, Cell Culture Techniques, Cell Biology, Fibroblasts, Biology, Biochemistry, Molecular biology, Embryonic stem cell, Genome, Clone Cells, Cell biology, Proto-Oncogene Proteins c-myc, Mice, Transduction (genetics), Species Specificity, Transduction, Genetic, Cell culture, Methods, Animals, Stem cell, Induced pluripotent stem cell, Molecular Biology, Gene, Reprogramming, Embryonic Stem Cells
Abstract

Although the induction of genome integration-free induced pluripotent stem cells (iPSCs) has been reported, c-Myc was still required for the efficient generation of these cells. Herein, we report mouse strain-dependent differences in the c-Myc dependence for iPSC generation and the successful generation of genome integration-free iPSCs without c-Myc transduction using C57BL/6 mouse embryonic fibroblasts. We performed 49 independent experiments and obtained a total of 24 iPSC clones, including 18 genome integration-free iPSC clones. These iPSCs were indistinguishable from embryonic stem cells and from iPSCs generated using other methods. Furthermore, the generation of three-factor iPSCs free of virus vectors revealed the contribution of c-Myc to the genomic integration of external genes. C57BL/6 is an inbred mouse strain with substantial advantages for use in genetic and molecular biological studies due to its use in the whole mouse genome sequencing project. Thus, the present series of C57BL/6 iPSCs generated by various procedures will serve as a valuable resource for future genetic studies of iPSC generation.

Published
2010
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42. Conversion of Ancestral Fibroblasts to Induced Pluripotent Stem Cells

Author

Yasuji Kasama, Yuko Hoki, Yuko Jincho, Shunsuke Ando, Ryoko Araki, Masumi Abe, Chihiro Tamura, and Miki Nakamura

Subjects
Genetics, Induced stem cells, Reverse Transcriptase Polymerase Chain Reaction, Somatic cell, Induced Pluripotent Stem Cells, Cell Biology, Embryoid body, Fibroblasts, Biology, Embryonic stem cell, Cell biology, Mice, Animals, Molecular Medicine, Stem cell, Induced pluripotent stem cell, Reprogramming, Cells, Cultured, Developmental Biology, Adult stem cell
Abstract

The emergence of induced pluripotent stem cells (iPSCs) from an ancestral somatic cell is one of the most important processes underlying their generation, but the mechanism has yet to be identified. This is principally because these cells emerge at a low frequency, about 0.1% in the case of fibroblasts, and in a stochastic manner. In our current study, we succeeded in identifying ancestral fibroblasts and the subsequent processes leading to their conversion to iPSCs. The ancestral fibroblasts were found to divide several times in a morphologically symmetric manner, maintaining a fibroblastic shape, and then gradually transform into embryonic stem-like cells. Interestingly, this conversion occurred within 48 hours after gene introduction in most iPSC generations. This is the first report to directly observe a cell lineage conversion of somatic cells to stem cells and provides a critical new insight into the “black box” of iPSCs, that is, the first three days of their generation.

Published
2009
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43. High-fidelity fractionation of ssDNA fragments differing in size by one-base on a spiral-channel electrophoretic chip

Author

Hiroaki Misawa, Masumi Abe, Nobuko Suzuki, Sumihare Noji, Zheyu Li, Saulius Juodkazis, Kosei Ueno, Kai Sun, and Ryoko Araki

Subjects
Chromatography, Base pair, Gene Expression Profiling, Clinical Biochemistry, Extraction (chemistry), Analytical chemistry, DNA, Single-Stranded, Electrophoresis, Capillary, Fractionation, Biology, Chip, Biochemistry, DNA sequencing, Analytical Chemistry, chemistry.chemical_compound, Electrophoresis, chemistry, DNA, Spiral
Abstract

For the fractionation of fragments of interest from selective PCR products generated by high coverage gene expression profiling (HiCEP) analysis, high-resolution with the ability to discriminate and fractionate fragments differing by one base (base pair) in size is highly required. We report here on a new 4-inch diameter spiral-channel chip device for automatic high-fidelity fractionation. Overlapping DNA fragments of 180, 181 and 182 bases, with only one-base difference in size, were successfully fractionated. The collected fragments were PCR amplified, and then evaluated by size checking analysis, DNA sequencing, and homolog search. The high-resolution fractionation has been achieved because of the combined contributions of (i) the high-resolution separation using a 30 cm long spiral channel, (ii) a blocking technique to avoid contamination from unselected fragments during CE, and (iii) precise micro-scale target extraction. Contaminations due to unselected fractions have been greatly decreased to a negligible level by optimization of the extraction position and extraction time corresponding to the targeted segment only. This technique can be adapted to a wide range of applications, such as protein or cell collections where requirements for the high purity are more important than the amount of recovered fractionated material.

Published
2009

44. Identification of cDNAs Encoding the WD Repeat Protein and Auxin-regulated Protein in Nicotiana Glutinosa Leaves Induced in Response to Tobacco Mosaic Virus-infection at 25 ℃

Author

Kazumasa Hada, Makoto Kimura, Masumi Abe, and Takeshi Hayashi

Subjects
chemistry.chemical_classification, WD-Repeat Protein, chemistry, Auxin, Complementary DNA, Tobacco mosaic virus, Nicotiana glutinosa, Biology, Agronomy and Crop Science, Molecular biology, Biotechnology
Published
2008
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45. Electrophoretic chip for fractionation of selective DNA fragment

Author

Kai Sun, Ryoko Araki, Kosei Ueno, Saulius Juodkazis, Hiroaki Misawa, Sumihare Noji, Zheyu Li, Masumi Abe, and Nobuko Suzuki

Subjects
Acrylamides, Bioanalysis, Microchannel, Chromatography, Clinical Biochemistry, Extraction (chemistry), Polyacrylamide, Analytical chemistry, DNA, Single-Stranded, DNA, Fractionation, Chemical Fractionation, Blocking (statistics), Chip, Biochemistry, Analytical Chemistry, Electrophoresis, Microchip, Electrophoresis, chemistry.chemical_compound, chemistry
Abstract

A microchannel chip has been used to fractionate selected segments from an electrophoretic flow of separated fragments. A sample, which covers the size from 35 to 670 bp, was initially separated using an 8.8-cm-long channel at the electric field strength of 100 V/cm. The target fragment of 318 bp was selected and extracted from the separation channel. High-resolution fractionation was achieved by introducing new procedures for blocking, extraction, and segment transfer. Fractionation quality with and without blocking were compared using a 310 Genetic Analyzer (Applied Biosystems). The results show that no contamination was found in the sample, which was fractionated with blocking; however, a contamination by short segments was found in the sample, which was fractionated without blocking. Furthermore, fractionation by the chip was found to be of higher fidelity than that by the polyacrylamide slab gel, which displayed a small overlapped peak after the target peak. Compared with the traditional method, our chips enable faster and high-fidelity fractionation, thus providing a new tool for bioanalysis and other applications.

Published
2008
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46. More then 40,000 transcripts including novel and noncoding transcripts in mouse embryonic stem cells

Author

Yasuji Kasama, Yoshimichi Tabata, Naokazu Sasaki, Ryoko Araki, Hirokazu Takahashi, Ryutaro Fukumura, Nobuko Suzuki, Masumi Abe, and Toshiyuki Saito

Subjects
Homeobox protein NANOG, RNA, Untranslated, Transcription, Genetic, Biology, Cell Line, Transcriptome, Mice, Gene expression, Databases, Genetic, Animals, Cell Lineage, RNA, Messenger, Gene, Embryonic Stem Cells, Regulation of gene expression, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling, Gene Expression Regulation, Developmental, Reproducibility of Results, Cell Differentiation, Cell Biology, Embryonic stem cell, Molecular biology, Gene expression profiling, Cell culture, Molecular Medicine, Developmental Biology
Abstract

To study the transcriptome of embryonic stem cells, we used a new gene expression profiling method that can measure the expression levels of unknown and rarely expressed transcripts precisely. We detected a total of 33,136 signal peaks representing transcripts in mouse embryonic stem cells, E14. Subsequent random cloning of the peaks suggests that mouse embryonic stem cells express at least 40,000 transcripts, of which about 2,000 are still unknown. In addition, we identified 1,022 noncoding transcripts, several of which change depending on differentiation in gene expression. Our database provides a high-resolution expression profile of E14 cells and is applicable to other mouse embryonic stem cell analyses. It includes most transcription regulation factor-encoding genes and a significant number of unknown and noncoding transcripts.

Published
2006

47. Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts

Author

Nguyen Van Thuan, Eiji Mizutani, Masako Suzuki, Shin-Ichi Nishikawa, Satoshi Tanaka, Teruhiko Wakayama, Sayaka Wakayama, Mitsuhiro Okada, Yuko Sakaide, Martin L. Jakt, Masashi Miyake, Takafusa Hikichi, Sho Senda, Kunio Shiota, Masumi Abe, Hiroshi Ohta, Satoshi Kishigami, and Ryoko Araki

Subjects
Pluripotent Stem Cells, KOSR, Somatic cell, Cellular differentiation, Mice, Transgenic, Embryoid body, Biology, Stem cell marker, Mice, Animals, Research Embryo Creation, reproductive and urinary physiology, Cell Nucleus, Mice, Inbred BALB C, urogenital system, Gene Expression Profiling, Cell Differentiation, Cell Biology, DNA Methylation, Embryo, Mammalian, Microarray Analysis, Embryonic stem cell, Molecular biology, Blastocyst, Fertilization, Karyotyping, embryonic structures, Molecular Medicine, Cattle, Female, Stem cell, biological phenomena, cell phenomena, and immunity, Developmental Biology, Adult stem cell
Abstract

Therapeutic cloning, whereby nuclear transfer (NT) is used to generate embryonic stem cells (ESCs) from blastocysts, has been demonstrated successfully in mice and cattle. However, if NT-ESCs have abnormalities, such as those associated with the offspring produced by reproductive cloning, their scientific and medical utilities might prove limited. To evaluate the characteristics of NT-ESCs, we established more than 150 NT-ESC lines from adult somatic cells of several mouse strains. Here, we show that these NT-ESCs were able to differentiate into all functional embryonic tissues in vivo. Moreover, they were identical to blastocyst-derived ESCs in terms of their expression of pluripotency markers in the presence of tissue-dependent differentially DNA methylated regions, in DNA microarray profiles, and in high-coverage gene expression profiling. Importantly, the NT procedure did not cause irreversible damage to the nuclei. These similarities of NT-ESCs and ESCs indicate that murine therapeutic cloning by somatic cell NT can provide a reliable model for preclinical stem cell research.

Published
2006

48. Comprehensive Analysis of Wound-inducible Genes from the Nicotiana glutinosa Leaves Using a Full-length cDNA Microarray

Author

Kosuke Tashiro, Masumi Abe, Yukiko Shimada, Satoru Kuhara, Makoto Kimura, Takeshi Hayashi, Yoshiaki Kouzuma, Keigo Uehashi, Kazumasa Hada, and Yusuke Tsutsumi

Subjects
Genetics, biology, Microarray, business.industry, Hypothetical protein, Biotechnology, Heat shock protein, Gene expression, biology.protein, Nicotiana glutinosa, business, Agronomy and Crop Science, Gene, Transcription factor, Peroxidase
Abstract

Wound-inducible gene expression in the the Nicotiana glutinosa leaves was studied by using a microarray with 9600 full-length CDNAS. As a result, 86 genes were identified as wound-inducible genes in the N. glutinosa leaves, including those encoding defense related proteins, such as heat shock proteins, glutathione S-transferase, ascorbate peroxidase and nonspecffic lipid-transfer proteins. Among 86 genes, 15 genes including 1 1 hypothetical protein genes and I unknown protein genes encode unknown functional proteins. Although the translational products of these genes have not been characterized, they are potential candidates for defense-related proteins toward wounding. The cluster analysis classified the genes into 6 groups on the basis of their expression patterns. It is likely that genes clustered in the same groups may be co-regulated by common transcriptional factors and also translational products belonging to the same clusters may share common functions in defense response to wounding.

Published
2005
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49. High-coverage profiling analysis of genes expressed during rice seed development, using an improved amplified fragment length polymorphism technique

Author

Atsushi Hattori, Shinichi Kitamura, Takehiro Masumura, Kiyoshi Suzuki, Seiko Tanaka, and Masumi Abe

Subjects
Genetics, Candidate gene, Oryza sativa, Gene Expression Profiling, Molecular Sequence Data, food and beverages, Oryza, General Medicine, Biology, Transcriptome, Complementary DNA, Seeds, Gene expression, biology.protein, Amplified fragment length polymorphism, Starch synthase, Nucleic Acid Amplification Techniques, Gene
Abstract

A novel method, based on quantitative PCR and amplified fragment length polymorphism was applied to the analysis of high-coverage gene expression profiles during the development of rice seeds. This represents the first report of the application of this method to plants, which permitted the detection and analysis of approximately 70% of all the genes that are expressed in rice. The method was used to compare gene expression at different developmental stages, subspecies or cultivars, and phyletic lines to identify genes of interest through differences in their level of expression. Using this approach, even novel anonymous genes could be detected. Examples of these include the soluble starch synthase (SS) II-I and the rice branching enzyme 4 (rbe4) genes in the starch synthesis pathway. A profiling database was compiled and the results compared with public data on full-length cDNA sequences of rice. The method enables candidate novel genes to be immediately identified among the large numbers of genes that are expressed during the development of rice seeds. Our results will contribute to a better understanding of comparative transcriptomics in all plant species.

Published
2004
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50. Growth retardation and skin abnormalities of the Recql4-deficient mouse

Author

Haruhiko Koseki, Ryutaro Fukumura, Yuko Noda, Akira Fujimori, Ryoko Araki, Hirokazu Takahashi, Seiji Kito, Yuko Hoki, Tatsuya Ohhata, Masumi Abe, and Miki Nakamura

Subjects
Time Factors, Ultraviolet Rays, Ratón, RecQ helicase, Mice, Exon, Radiation, Ionizing, Genetics, medicine, Animals, Humans, Molecular Biology, Rothmund–Thomson syndrome, Gene, Cells, Cultured, Germ-Line Mutation, Genetics (clinical), Adenosine Triphosphatases, RecQ Helicases, biology, X-Rays, Body Weight, DNA Helicases, Rothmund-Thomson Syndrome, Helicase, General Medicine, Fibroblasts, Embryo, Mammalian, medicine.disease, Embryonic stem cell, Molecular biology, Mice, Inbred C57BL, Animals, Newborn, Gene Targeting, Knockout mouse, Skin Abnormalities, biology.protein
Abstract

Mutations in the Recql4 gene are very likely responsible for a subset of Rothmund-Thomson syndrome (RTS) cases, but until now there has been no animal model to confirm this. Knockout mice in which the Recql4 gene is disrupted at exons 5-8 exhibit embryonic lethality at embryonic day 3.5-6.5. We generated a helicase activity-inhibited mouse by deleting exon 13 of Recql4, which is one of the coding exons of the consensus RecQ-helicase domain. This domain is the primary site of mutations that have been identified in RTS patients. The exon 13-deleted Recql4-deficient mice are viable, but exhibit severe growth retardation and abnormalities in several tissues, and embryonic fibroblasts show a defect in cell proliferation. Abnormalities in the Recql4-deficient mice are similar to those in RTS patients, suggesting that defects in the Recql4 gene may indeed be responsible for RTS. We speculate that the loss of Recql4 helicase activity results in the prematurely aged appearance observed in some RecQ helicase diseases.

Published
2003
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