Kawakami Lab

Kawakami Lab

"Ten to 20 percent of the population of industrialized countries suffers from some form of allergies. There is a huge need to understand this disease and to find therapeutic interventions." — Toshiaki Kawakami, M.D., Ph.D. // Professor
Division of Cellular Biology

Overview

Toshiaki Kawakami, M.D., Ph.D., and his team study signal transduction in the immune and hematopoietic systems. The laboratory has been studying mainly mast cells, an important cell type found in mucosal as well as connective tissues that is responsible for the allergic reactions that trigger itching, wheezing, and sneezing. These symptoms of allergic reactions occur after mast cells are activated.

When exposed to allergens (substances, such as pollen, that cause allergies), IgE-bound mast cells are activated and secrete numerous chemical, lipid, nucleotide, peptide, and protein mediators. These mediators, together with other immune cells, orchestrate complex cellular and molecular interactions to induce allergic reactions. Increasing numbers of signaling molecules are shown to be important for mast cell activation. Dr. Kawakami and his colleagues focus on dissecting the complex network of signaling molecules, particularly those involved in the early phase of mast cell activation.
Conventional belief was that IgE-mediated mast cell activation requires multivalent allergen or antigen to induce mast cell activation. However, in 2001 Dr. Kawakami and his colleagues found that IgE can induce mast activation without the involvement of allergen or antigen. They later showed a vast heterogeneity in IgE molecules in their ability to induce this IgE effect (so-called “monomeric IgE effect”): some IgEs can induce all kinds of activation events, but others can do so very inefficiently. In addition to this form of heterogeneity, they found that 20-30% of IgE molecules directly bind to histamine-releasing factor (HRF), a cytokine-like molecule secreted during allergic reactions. Recently, they developed inhibitors that block the function of HRF and demonstrated the role of HRF in enhancing allergic inflammation in asthma and other allergic diseases. Currently, they are trying to understand how these fundamental observations can be translated in human allergic diseases.

Principles of signaling mechanisms in mast cells are shared not only by other immune and hematopoietic cells but also more distantly related cells. Abnormalities in signaling networks can lead to many types of disorders including cancer, autoimmune diseases, and immunodeficiencies. Microbes often hijack such intricate networks for their advantage. Therefore, Dr. Kawakami’s team sometimes ends up studying cancer and virus infection, even when their original interest is in allergy research. An interesting extension of their study is an establishment of a mouse model of atopic dermatitis and a subsequent use of that model to study eczema vaccinatum and eczema herpeticum. Eczema vaccinatum is a serious infection with vaccinia virus often experienced when patients with atopic dermatitis are vaccinated against smallpox and eczema herpeticum is a rare, but severe infection with herpes simplex virus. Another exciting finding is a recent identification of a novel tumor suppressor that was originally supposed to play an important role in mast cell activation. They hope their study of signaling molecules may lead to an increased understanding of these diseases and contribute to the development of new preventive measures, diagnostics, and treatments.

From The Lab

Jan 9, 2014

La Jolla Institute scientist identifies pivotal cellular protein underlying eczema

Kawakami Lab

Publications

Journal of Cell Biology

Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation

2015-08
Carroll-Portillo A, Cannon JL, Te Riet J, Holmes A, Kawakami Y, Kawakami T, Cambi A, Lidke DS
Frontiers in Immunology

Hypothetical atopic dermatitis-myeloproliferative neoplasm syndrome

2015-08
Kawakami T, Ando T, Kawakami Y
Journal of Investigative Dermatology

Mast cells are required for full expression of allergen/SEB-induced skin inflammation

2015-03
Ando T, Matsumoto K, Namiranian S, Yamashita H, Glatthorn H, Kimura M, Dolan BR, Lee JJ, Galli SJ, Kawakami Y, Jamora…
Cell Reports

Critical role for mast cell stat5 activity in skin inflammation

2014-01
Ando T, Xiao W, Gao P, Namiranian S, Matsumoto K, Tomimori Y, Hong H, Yamashita H, Kimura M, Kashiwakura JI, Hata TR,…
Allergy Asthma & Immunology Research

Histimine-releasing factor and immunoglobulins in asthma and allergy

2014-01
Kawakami T, Kashiwakura J, Kawakami Y
Journal of Investigative Dermatology

Mast cells are required for full expression of allergen/SEB-induced skin inflammation

2013-12
Ando T, Matsumoto K, Namiranian S, Yamashita H, Glatthorn H, Kimura M, Dolan BR, Lee JJ, Galli SJ, Kawakami Y, Jamora…
Immunity

Salmonella's masterful skill in mast cell suppression

2013-12
Kawakami T, Ando T
Advances in Biological Regulation

Phospholipase C-β in immune cells

2013-09
Kawakami T, Xiao W
Immunologic Research

Immune regulation by phospholipase C-β isoforms

2013-05
Xiao W, Kawakami Y, Kawakami T
Allergy Asthma & Immunology Research

Most highly cytokinergic IgEs have polyreactivity to autoantigens

2012-11
Kasjiwakura JI, Okayama Y, Furue M, Kabashima K, Shimada S, Ra C, Siraganian RP, Kawakami Y, Kawakami T
Biochemical Journal

A novel role of the transcription factor HIF-1α in the formation of mast cell extracellular traps

2012-08
Branitzki-Heinemann K, Okumura CY, Völlger L, Kawakami Y, Kawakami T, Naim HY, Nizet V, von Köckritz-Blickwede M
Journal of Immunology

The FcRβ- and γ-ITAMs play crucial but distinct roles in the full activation of mast cells induced by IgEκ and protein L

2012-04
Nunomura S, Kawakami Y, Kawakami T, Ra C
Blood

Human FcyRIIA at center stage

2012-03
Kawakami T
Annals of Allergy Asthma & Immunology

Omalizumab inhibits acceleration of FceRI-mediated responsiveness of immature human mast cells by immunoglobulin EOmalizumab inhibits acceleration of FceRI-mediated responsiveness of immature human mast cells by immunoglobulin EOmalizumab inhibits acceleration of FceRI-mediated responsiveness of immature human mast cells by immunoglobulin EOmalizumab inhibits acceleration of FceRI-mediated responsiveness of immature human mast cells by immunoglobulin E

2012-03
Okayama Y, Kashiwakura J, Sasaki-Sakamoto T, Matsumoto K, Hashimoto N, Ohmori K, Kawakami T, Saito H, Ra C
Journal of Clinical Investigation

Histamine-releasing factor has a proinflammatory role in mouse models of asthma and allergy

2012-01
Kashiwakura JC, Ando T, Matsumoto K, Kimura M, Kitaura J, Matho MH, Zajonc DM, Ozeki T, Ra C, MacDonald SM, Siraganian…
Advances in Biological Regulation

Regulation of proliferation, survival, differentiation, and activation by the Signaling Platform for SHP-1 phosphatase

2012-01
Kawakami T, Xiao W, Yasudo H, Kawakami Y
Journal of Clinical Investigation

IgE stimulates human and mouse arterial cell apoptosis and cytokine expression and promotes atherogenesis in Apoe-/- mice

2011-09
Wang J, Cheng X, Xiang MX, Alanne-Kinnunen M, Wang JA, Chen H, He A, Sun X, Lin Y, Tang TT, Tu X, Sjoberg S, Sukhova…
Immunity

Phospholipase C-β3 regulates FcεRI-mediated mast cell activation by recruiting the proteinphosphatase SHP-1

2011-06
Xiao W, Kashiwakura J, Hong H, Yasudo H, Ando T, Maeda-Yamamoto M, Wu D, Kawakami Y, Kawakami T
Journal of Immunology

SHIP represses Th2 skewing by inhibiting IL-4 production from basophils

2011-01
Kuroda E, Antinano F, Ho VW, Hughes MR, Ruschmann J, Lam V, Kawakami T, Kerr WG, McNagny KM, Sly LM, Krystal G
Methods in Molecular Biology

A mouse model of atopic dermatitis

2016-07
Kawakami Y, Kawakami T
Methods in Molecular Biology

Basic techniques to study FcεRI signaling in mast cells

2016-07
Kawakami Y, Kawakami T
Open Allergy Journal

Editorial: Histamine-releasing factor and TCTP

2016-07
Kawakami T
Open Allergy Journal

HRF-interacting molecules

2016-07
Kawakami T, Ando T, Kawakami Y
Antiviral Therapy

Protective murine and human monoclonal antibodies against eczema vaccinatum

2016-07
Tomimori Y, Kawakami Y, McCausland MM, Ando T, Koriazova L, Kato S, Kawakami T, Crotty S
Advances in Experimental Medicine and Biology

Monomeric IgE and mast cell development, survival and function

2016-07
Kashiwakura J, Otani IM, Kawakami T
PLoS One

Short stat5-interacting peptide derived from phospholipase C-β3 inhits hematopoietic cell proliferation and myeloid differentiation

2016-07
Yasudo H, Ando T, Xiao W, Kawakami Y, Kawakami T

Principal Investigator

kawakami

Toshiaki Kawakami, M.D., Ph.D.

Professor

Dr. Kawakami has been with LIAI since 1990, joining originally as an Assistant and then Associate Professor in the Biochemistry Section of the Division of Immunobiology. Dr. Kawakami then became an Associate Professor in the Allergy Division in 1996 and in 2000 he became a full Professor. He is also Team leader of the Laboratory for Allergic Disease at RIKEN Center for the Integrative Medical Sciences, Yokohama, Japan.
Dr. Kawakami received both M.D. and Ph.D degrees from the University of Tokyo. While working towards his Ph.D., Dr. Kawakami was hired as an Assistant Professor in the department of Physiological Chemistry and Nutrition at the University of Tokyo, but in 1984 he began to work as a visiting fellow at the National Cancer Institute in Bethesda, Maryland. In 1987, Dr. Kawakami spent a year in the Cell Development and Oncology lab at the National Institute of Dental Research, also in Bethesda, before returning to Kyoto in 1988 where he spent two years as an Assistant Professor in the Department of Medical Chemistry, Kyoto University, Faculty of Medicine.

Dr. Kawakami has been giving talks at a Major Symposium at an AAI meeting, America Academy of Allergy, Asthma, and Immunology (AAAA&I), FASEB and Aegean conferences as well as platform presentations at other national and international meetings. He is currently co-organizing a FASEB Conference “IgE and Allergy, 50 Years and Onward” to commemorate the 50th anniversary of the discovery of IgE by Kimishige Ishizaka (Founding Director of La Jolla Institute for Allergy and Immunology) in 2016.

He served as Associate Editor and Section Editor for the Journal of Immunology and he is currently a board member of several research journals. He is an elected member of Collegium Internationale Allergologicum. In addition to several NIH Study Sections and other NIH review panels, he has been serving as a reviewer of grants for Agency for Science, Technology and Research’s Biomedical Research Council (Singapore), Arthritis National Research Foundation, Asthma UK, British Lung Foundation, German-Israeli Foundation for Scientific Research and Development, JAPAN Health Sciences Foundation, The Israel Science Foundation, The Wellcome Trust, Deutsche Forschungsgemeinschaft, and de l’Agence Nationale de la Recherche (ANR). He also serves as a Consultant for The Japan Prize, The Science and Technology Foundation of Japan and as a reviewer of research programs at the Campus for Research Excellence and Technological Enterprise (CREATE) for the National Research Foundation of Singapore.

Lab Members

Minato Baba

Postdoctoral Fellow

Natsuhiko Inoue

Visiting Scientist

YukoKawakami

Yuko Kawakami

Scientific Associate

Biosketch:
I obtained M.D. from the University of Tokyo Medical School. After several years of clinical work, I went to NIH with my husband and worked as a postdoc. We went back to Kyoto, Japan, 4 years later and I resumed clinical work again while writing thesis and obtained Ph.D. from the University of Tokyo. We came here in 1990 and I started to work as a postdoc, and eventually became a scientific associate.

Research Focus:
We have been working on the pathogenesis of allergic diseases using a variety of experimental techniques from molecular biology to animal models. Our research has been especially focused on the mechanism of mast cell activation through IgE and antigen stimulation. We also use animal models of allergic diseases including asthma, atopic dermatitis and food allergy.

Career Goals:
As a physician scientist, I want to find the pathogenesis that helps to develop novel diagnostic, preventative and treatment means of the diseases. Now we are very close to connecting our findings to diagnostics and treatment of a type of allergic disease.

Yu Kawakami

Visiting Scientist

Yuko Kawakami

Scientific Associate

TheaLu

Thea Lu

Postdoctoral Fellow

Thea Lu is currently a postdoctoral fellow in the Kawakami Lab at the La Jolla Institute for Allergy and Immunology. She received her Ph.D. in Microbiology, Molecular Biology, and Biochemistry from the University of Idaho in 2012 and her undergraduate studies from the California Institute of Technology. She was also a postdoctoral fellow at Rocky Mountain Laboratories in the National Institute of Allergy and Infectious Diseases before joining LJI.

Her recent research interests lie in understanding mast cell activation via the high-affinity IgE receptor (FcεRI) and its relationship to antiviral signaling pathways. Her focus includes characterizing the signaling molecules involved in these pathways since FcεRI-mediated mast cell activation is a central event for causing allergic reactions and allergic diseases.

Ryohei Shibata

Visiting Scientist

Jing Song

Sayo Suzuki

Visiting Scientist

Ikuo

Ikuo Takazawa

Postdoctoral Researcher

Biosketch:
I graduated from The University of Tokyo, School of Medicine, JAPAN in 2012 with a MD degree in medicine. After finishing my internship at the University of Tokyo Hospital, I worked as a doctor in the division of respiratory medicine at the National Center for Global Health and Medicine, JAPAN. I began working as a postdoc in the Kawakami laboratory at the La Jolla Institute for Allergy and Immunology in October 2015.

Research Focus:
My research projects are focused on the roles of histamine releasing factor in the allergic disease and phospholipase in the inflammatory bowel disease. I am interested in understanding how histamine releasing factor reactive Immunoglobulin works in the allergic disease and in defining new drug targets for allergic diseases by inhibiting histamine releasing factor function.

Career Goals:
I plan to pursue a career in scientific research focused on immunoglobulin and histamine releasing factor in human allergic disease.

Kawakami Lab

Research Projects

Dr. Kawakami has been interested in immunology and cancer from the beginning of his scientific career. After molecular cloning of immunoglobulin m gene during the course of the graduate school at The University of Tokyo (the mentor, Tasuku Honjo), he discovered a novel Src family protein-tyrosine kinase (PTK), Fyn, during his postdoctoral stint (the mentor, Stuart A. Aaronson) at the National Institutes of Health. These findings led him to study the normal function of PTKs and their networks in immunological settings.

Bruton’s tyrosine kinase (Btk) in mast cell activation

Dr. Kawakami has been using the high-affinity IgE receptor (FceRI) signaling in mast cells as a model system. Following his co-discovery of two Tec family PTKs, Btk and Itk, in mast cells, his group revealed crucial roles of these kinases in FceRI-mediated activation and apoptosis. By introducing retroviral transduction into the field of mast cell research, they established a standard methodology to investigate the FceRI signaling system.

Selected References

Yamada, N., Kawakami, Y., Kimura, H., Fukamachi, H., Baier, G., Altman, A., Kato, T., Inagaki, Y., and Kawakami, T. Structure and expression of novel protein-tyrosine kinases, Emb and Emt, in hematopoietic cells. Biochem. Biophys. Res. Comm. 192:231-240, 1993.

Kawakami, Y., Yao, L., Miura, T., Tsukada, S., Witte, O. N., and Kawakami, T. Tyrosine
phosphorylation and activation of Bruton tyrosine kinase (Btk) upon FceRI cross-linking. Mol. Cell. Biol. 14:5108-5113, 1994.

Kawakami, Y., Miura, T., Bissonnette, R., Hata, D., Khan, W. N., Kitamura, T., Maeda-Yamamoto, M., Hartman, S. E., Yao, L., Alt, F. W., and Kawakami, T. Bruton’s tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity. Proc. Natl. Acad. Sci. USA 94:3938-3942, 1997.

Hata, D., Kawakami, Y., Inagaki, N., Lantz, C. S., Kitamura, T., Khan, W. N., Tashiro, M., Maeda-
Yamamoto, M., Miura, T., Han, W., Hartman, S. E., Yao, L., Nagai, H., Goldfeld, A. E., Alt, F. W., Galli, S. J., Witte, O. N., and Kawakami, T. Involvement of Bruton’s tyrosine kinase in FceRI-dependent mast cell degranulation and cytokine production. J. Exp. Med. 187:1235-1247, 1998.

Btk-mediated regulation of cytokine production

During the course of the above-mentioned study, they found interactions between Btk and protein kinase C (PKC)-b and terreic acid as a novel Btk inhibitor that blocked Btk-PKC-b interactions. Their study also showed that PKC-b and Akt are essential for degranulation and cytokine production via activation of several transcription factors. These studies established how cytokine production is regulated by FceRI-stimulated mast cells.

Selected References

Yao, L., Kawakami, Y., and Kawakami, T. The pleckstrin homology domain of Btk tyrosine kinase interacts with protein kinase C. Proc. Natl. Acad. Sci. USA 91:9175-9179, 1994

Kitaura, J., Asai, K., Maeda-Yamamoto, M., Kawakami, Y., Kikkawa, U., and Kawakami, T. Akt-dependent cytokine production in mast cells. J. Exp. Med. 192:729-739, 2000.

Kawakami, Y., Kitaura, J., Hartman, S. E., Lowell, C. A., Siraganian, R. P., and Kawakami, T.
Regulation of protein kinase CbI by two protein-tyrosine kinases, Btk and Syk. Proc. Natl. Acad. Sci. USA. 97:7423-7428, 2000.

Kawakami, Y., Kitaura, J., Yao, L., McHenry, R. W., Kawakami, Y., Newton, A. C., Kang, S., Kato, R. M., Leitges, M., Rawlings, D. J., and Kawakami, T. A Ras activation pathway dependent on Syk
phosphorylation of protein kinase C. Proc. Natl. Acad. Sci. USA. 100:9470-9475, 2003.

Mouse model of atopic dermatitis (AD)

As our knowledge of the FceRI-mediated signal transduction has grown for the last two decades, the need to study in vivo roles of this pathway in disease settings encouraged the Kawakami group to develop novel in vivo models of allergic diseases. They devised a novel procedure to induce AD-like skin lesions in mice using a house dust mite extract and staphylococcal enterotoxin B. This model allowed them to show that mast cells and T cells, but not B cells or eosinophils, are required for the full expression of dermatitis. Its application to develop a model to study the susceptibility of AD patients to vaccinia virus revealed the weakened ability of NK cells to control this virus in AD-bearing mice, which recapitulates eczema vaccinatum, a complication sometimes seen in severe cases of human AD. This model was also useful to develop or confirm the efficacy of certain compounds and monoclonal anti-viral antibodies to fight against eczema vaccinatum and eczema herpeticum.

Selected References

Kawakami, Y., Yumoto, K., and Kawakami, T. An improved mouse model of atopic dermatitis and suppression of skin lesions by an inhibitor of Tec family kinases. Allergol. International 56: 403-409, 2007.

Kawakami, Y., Tomimori, Y., Yumoto, K., Hasegawa, S., Ando, T., Tagaya, Y., Crotty, S., and
Kawakami, T. Inhibition of NK cell activity by IL-17 allows vaccinia virus to induce severe skin lesions in a mouse model of eczema vaccinatum. J. Exp. Med. 206:1219-1225, 2009. PMCID: PMC2715052

Tomimori, Y., Kawakami, Y., McCausland, M.M., Ando, T., Koriazova, L., Kato, S., Kawakami, T., and Crotty, S. Protective murine and human monoclonal antibodies against eczema vaccinatum. Antivir. Ther. 16:67-75, 2011. PMCID: PMC3046123

Phospholipase C (PLC)-b3-mediated regulation of Stat5 activity

Given their previous studies on crosstalks between PTKs-dependent and G protein-coupled receptor-dependent signals in FceRI-stimulated mast cells, they have been characterizing PLC-b isoforms. In addition to its role in an early FceRI signal transduction, they found that deficiency in PLC-b3 results in myeloproliferative neoplasm (MPN) and AD. They demonstrated that a novel multi-molecular complex, SPS complex, that contains SHP-1 (SH2 domain-containing protein phosphatase 1), PLC-b3 and Stat5, regulates Stat5 activity. Increased Stat5 activity due to the loss of PLC-b3 was crucial for the leukemogenesis of MPN as well as the development of AD. This mechanism seems important in the pathogenesis of Burkitt’s lymphoma, chronic lymphocytic leukemia, and human AD.

Selected References

Xiao, W., Hong, H., Kawakami, Y., Kato, Y., Wu, D., Yasudo, H., Kimura, A., Kubagawa, H., Bertoli, L. F., Davis, R. S., Chau, L. A., Madrenas. J., Hsia, C. C., Xenocostas. A., Kipps, T. J., Hennighausen, L., Iwama, A., Nakauchi, H., and Kawakami, T. Tumor suppression by phospholipase C-b3 via SHP-1-mediated dephosphorylation of Stat5. Cancer Cell 16: 161-171, 2009. PMCID: PMC2744338

Xiao, W., Ando, T., Wang, H., Kawakami, Y., and Kawakami, T. Lyn- and PLC-b3-dependent regulation of SHP-1 phosphorylation controls Stat5 activity and myelomonocytic leukemia-like disease. Blood 116:6003-6013, 2010. PMCID: PMC3031387

Xiao, W., Kashiwakura, J., Hong, H., Yasudo, H., Ando, T., Maeda-Yamamoto, M., Wu, D., Kawakami, Y., and Kawakami, T. Phospholipase C-b3 regulates FceRI-mediated mast cell activation by recruiting the protein phosphatase SHP-1. Immunity 34:893-904, 2011. PMCID: PMC3124618

Ando, T., Xiao, W., Gao, P., Namiranian, S., Matsumoto, K., Tomimori, Y., Hong, H., Yamashita, H., Kimura, M., Kashiwakura, J., Hata, T.R., Izuhara, K., Gurish, M.F., Roers, A., Rafaels, N.M., Barnes, K.C., Jamora, C., Kawakami, Y., and Kawakami, T. Critical role for mast-cell Stat5 activity in skin inflammation. Cell Reports 6:366-376, 2014. PMCID: PMC4329986

IgE heterogeneity

Since their finding of ‘monomeric IgE effects’ on mast cell survival in 2001, Dr. Kawakami and associates have shown that IgEs exhibit a tremendous heterogeneity in their ability to induce mast cell activation, with highly cytokinergic (HC) IgEs at one end of the spectrum and poorly cytokinergic (PC) IgEs at the other: HC IgEs can induce strong survival promotion, degranulation, cytokine production, and migration very efficiently, whereas PC IgEs do so inefficiently. Intrigued by a similar dichotomy for the requirement of a certain type of IgE to prime basophils and mast cells in response to HRF, they recently found that a subset (~25%) of IgE and IgG molecules directly interact with HRF. HRF-reactive IgE together with HRF could activate mast cells in vitro. By mapping the binding sites on both HRF and IgE/IgG molecules, they developed competitive inhibitors of HRF-IgE (or IgG) interactions, which inhibited IgE+HRF-induced mast cell activation. Using these inhibitors, they could show that HRF promotes allergic inflammation in in vivo mouse models of passive cutaneous anaphylaxis and asthma. Thus, their study discovered the long sought receptors for HRF and enabled them to investigate the role of HRF in allergic diseases and potential utility of HRF inhibitors as therapeutics.

Selected References

Asai, K., Kitaura, J., Kawakami, Y., Yamagata, N, Tsai, M., Carbone, D. P., Liu, F. –T., Galli, S. J., and Kawakami, T. Regulation of mast cell survival by IgE. Immunity 14:791-800, 2001.

Kitaura, J., Song, J., Tsai, M., Asai, K., Maeda-Yamamoto, M., Mocsai, A., Kawakami, Y., Liu, F. -T., Lowell, C. A., Barisas, B. G., Galli, S. J., and Kawakami, T. Highly or poorly cytokinergic IgE molecules mediate a spectrum of effects on mast cell survival and activation. Proc. Natl. Acad. Sci. USA 100:12911-12916, 2003.

Kitaura, J., Kinoshita, T., Matsumoto, M., Chung, S., Kawakami, Y., Leitges, M., Wu, D., Lowell, C. L., and Kawakami, T. IgE- and IgE+Ag-mediated mast cell migration in an autocrine/paracrine fashion. Blood 105: 3222-3229, 2005.

Kashiwakura, J.*, Ando, T.*, Matsumoto, K., Kimura, M., Zajonc, D.M., Ozeki, T., Siraganian, R.P., Broide, D., Kawakami, Y., and Kawakami, T. Histamine-releasing factor has a proinflammatory role in mouse models of asthma and allergy. J. Clin. Invest. 122:218-228, 2012. (*equal contributions). PMCID: PMC3248297