UTHealth School of Dentistry Research Labs
Role of cellular metabolism during development and diseases
Cellular metabolic aberrations result in craniofacial deformities in humans and mice. Interestingly, mice with cholesterol synthesis deficiency have severe malformations specifically in the craniofacial region and the rest of their body is largely unaffected. The majority of cells in craniofacial region are derived from cranial neural crest (CNC) cells, which is a multi-potent cell population that gives rise to a variety of different cell types. These results suggest that CNC cells are more sensitive to cellular metabolic aberrations than are cells from other regions during embryogenesis. However, it is still unknown how molecules related to cellular metabolism are regulated during craniofacial development. In addition, the possible relationship between the cellular metabolism and craniofacial deformities remain unclear. The aim of our laboratory is to identify gene mutations and protein modifications related to craniofacial disorders and provide the basis for tests aimed at identifying higher-risk persons.
Role of autophagic machinery in development and diseases
Autophagy is an evolutionarily conserved bulk-protein degradation system, in which isolation membranes engulf cytoplasmic constituents and the resulting autophagosomes transport them to lysosomes, crucial for the removal and breakdown of cellular components such as damaged proteins and aged organelles. Autophagic activity is altered in various diseases and birth defects in humans and mice. An understanding of the manner in which autophagy is regulated is critical for understanding normal craniofacial development as well as congenital malformations. The aim of our laboratory is to identify the molecular regulatory mechanism of autophagic machinery related to developmental defects and diseases.
Craniofacial skeletal defects are some of the most prominent genetic disorders. However, the etiology of these defects remains unclear. We have recently reported that cholesterol metabolic defects result in altered ciliogenesis and cause bone anomalies (Bone Research, 2020).
Salivary Gland Study
Salivary Gland Study
The major and minor salivary glands (SGs) produce 1.5−4.5 gram of proteins in 0.5−1.5 liters of saliva daily, which is essential for oral health. Acinar cells, one of the major cell types within the SGs, are responsible for the production and secretion of prepackaged secretory granules that contain key functional salivary components such as amylase, mucins, and immunoglobulins. These salivary components are functionally important for the digestion and tasting of food, lubrication, buffering, and the prevention of dental caries, periodontitis, candidiasis, and halitosis (bad breath). This secretion process, called exocytosis, involves secretory vesicle trafficking, docking, priming, and membrane fusion, and a failure during any of these steps in the SGs results in altered secretion of salivary proteins. Salivary protein content is altered in patients with diabetes and obesity, who present a higher frequency of oral health issues, including xerostomia. In addition, deficient exocytosis, including reduced salivary protein secretion and altered saliva content, has been reported in patients with Sjögren’s syndrome (SjS) and in mouse models of SjS (e.g. NOD mice). However, the pathogenesis of SjS remains elusive, with various potential risk factors at play (e.g. environmental, genetic and hormonal factors). Either immune cells or exocrine gland cells are primarily damaged/disorganized, inducing inflammation in the SGs and lacrimal glands. To date, despite the important physiological functions of salivary proteins, we know very little about the regulatory mechanism(s) of exocytosis in the SGs. A detailed understanding of the mechanism(s) regulating exocytosis will provide new knowledge about its key function(s), not only in the SGs but also in other secretory organs, under physiological and pathological conditions (such as SjS). Ultimately, this approach will identify novel targets for therapeutics and contribute to the development of new diagnostic tools for identifying exocytosis defects and SjS in at-risk populations such as those with high cholesterol levels.
Adult regenerative myogenesis is vital for restoring normal tissue structure after muscle injury. Muscle repair is dependent on progenitor satellite cells, which proliferate in response to injury, and their progeny differentiate and undergo cell-cell fusion to form regenerating myofibers. Myogenic progenitor cells must be precisely regulated and positioned for proper cell fusion to occur. We previously reported that WNT signaling is regulated in a spatiotemporal specific manner during muscle development (Mol Cell Biol, 2015; Sci Rep, 2018). We are further investigating how this signaling pathway regulates muscle development and regeneration. Our long-term goal is to better understand the mechanisms of muscle development and repair, to develop therapeutic interventions for muscle injury and diseases/disorders, and to devise potential strategies to prevent muscle developmental defects and accelerate muscle regeneration.
Cleft Lip/ Cleft Palate Study
Cleft Lip/ Cleft Palate Study
The etiology of cleft lip and palate is complicated by a variety of genetic and environmental factors. This study will identify the distribution and contribution of non-coding RNAs (ncRNAs) in lip and palate development. We hypothesize that various environmental factors and metabolic aberrations that cause craniofacial anomalies affect the expression of ncRNAs that suppress the expression and activity of genes associated with craniofacial anomalies. For instance, mutations in genes involved in cholesterol synthesis (SC5D, DHCR7, and DHCR24) have been found in lathosterolosis, Smith-Lemli-Opitz Syndrome [SLOS], and desmosterolosis. Patients with these syndromes display craniofacial abnormalities (e.g. cleft palate) with a wide array of degree in severity. In addition, high-cholesterol diets during pregnancy are known to be a risk factor for birth defects, including various craniofacial abnormalities. Sterol-C5-desaturase (SC5D) catalyzes the dehydrogenation of a C-5(6) bond in a sterol intermediate during compound cholesterol biosynthesis. Mutations in SC5D result in cholesterol deficiency as well as in excessive lathosterol, a cholesterol precursor that causes lathosterolosis, which is characterized by craniofacial deformities such as cleft palate, dysmorphism and micrognathia, and limb anomalies. By contrast, mutations in DHCR7, which encodes 7-dehydrocholesterol reductase, lead to SLOS, which is characterized by less severe craniofacial deformities (e.g. high-arched palate, less frequent cleft palate, ptosis, a short nasal root, etc.) compared with lathosterolosis. These findings suggest that accumulated cholesterol precursors lathosterol and 7-dehydrocholesterol differently interfere with the process of craniofacial development. Thus, while proper regulation of cholesterol metabolism is crucial for craniofacial development, the exact mechanism of how cholesterol metabolism affects craniofacial development remains largely unknown. The results of this study will enhance our understanding of the role of ncRNAs in lip and palate development and will enable us to design future therapeutic approaches to diagnose and prevent cleft lip and palate.
Tooth Development Study
Tooth Development Study
We found that autophagy is crucial for ameloblast differentiation in tooth development and that loss of autophagy in teeth results in amelogenesis imperfecta. Using teeth from the mouse models we developed (Atg7 and Atg3 conditional knockout [cKO] mice), we are investigating how ameloblast differentiation is regulated through autophagy. The results of this study will serve as the foundation for tooth biology and tissue regeneration.
1: Suzuki A, Ogata K, Iwata J. Cell signaling regulation in salivary gland development. Cell Mol Life Sci. 2021 Jan 15. doi: 10.1007/s00018-020-03741-2. Epub ahead of print. PMID: 33449148.
2: Suzuki A, Ogata K, Yoshioka H, Shim J, Wassif CA, Porter FD, Iwata J. Disruption of Dhcr7 and Insig1/2 in cholesterol metabolism causes defects in bone formation and homeostasis through primary cilium formation. Bone Res. 2020 Jan 2;8(1):1. doi: 10.1038/s41413-019-0078-3. PMID: 33384405.
3: Suzuki A, Minamide M, Iwaya C, Ogata K, Iwata J. Role of Metabolism in Bone Development and Homeostasis. Int J Mol Sci. 2020 Nov 26;21(23):8992. doi: 10.3390/ijms21238992. PMID: 33256181; PMCID: PMC7729585.
4: Yan F, Jia P, Yoshioka H, Suzuki A, Iwata J, Zhao Z. A developmental stage- specific network approach for studying dynamic co-regulation of transcription factors and microRNAs during craniofacial development. Development. 2020 Dec 24;147(24):dev192948. doi: 10.1242/dev.192948. PMID: 33234712; PMCID: PMC7774895.
5: Yang Y, Suzuki A, Iwata J, Jun G. Secondary Genome-Wide Association Study Using Novel Analytical Strategies Disentangle Genetic Components of Cleft Lip and/or Cleft Palate in 1q32.2. Genes. 2020 Oct 29;11(11):1280. doi: 10.3390/genes11111280. PMID: 33137956; PMCID: PMC7693579.
6: Yan F, Dai Y, Iwata J, Zhao Z, Jia P. An integrative, genomic, transcriptomic and network-assisted study to identify genes associated with human cleft lip with or without cleft palate. BMC Med Genomics. 2020 Apr 3;13(Suppl 5):39. doi: 10.1186/s12920-020-0675-4. PMID: 32241273; PMCID: PMC7118807.
7: Suzuki A, Yoshioka H, Summakia D, Desai NG, Jun G, Jia P, Loose DS, Ogata K, Gajera MV, Zhao Z, Iwata J. MicroRNA-124-3p suppresses mouse lip mesenchymal cell proliferation through the regulation of genes associated with cleft lip in the mouse. BMC Genomics. 2019 Nov 14;20(1):852. doi: 10.1186/s12864-019-6238-4. PMID: 31727022; PMCID: PMC6854646.
9: Li A, Jia P, Mallik S, Fei R, Yoshioka H, Suzuki A, Iwata J, Zhao Z. Critical microRNAs and regulatory motifs in cleft palate identified by a conserved miRNA- TF-gene network approach in humans and mice. Brief Bioinform. 2020 Jul 15;21(4):1465-1478. doi:
10.1093/bib/bbz082. PMID: 31589286; PMCID: PMC7412957. 10: Suzuki A, Shim J, Ogata K, Yoshioka H, Iwata J. Cholesterol metabolism plays a crucial role in the regulation of autophagy for cell differentiation of granular convoluted tubules in male mouse submandibular glands. Development. 2019 Oct 17;146(20):dev178335. doi: 10.1242/dev.178335. PMID: 31558435; PMCID: PMC6826039.
11: Suzuki A, Li A, Gajera M, Abdallah N, Zhang M, Zhao Z, Iwata J. MicroRNA-374a, -4680, and -133b suppress cell proliferation through the regulation of genes associated with human cleft palate in cultured human palate cells. BMC Med Genomics. 2019 Jul 1;12(1):93. doi: 10.1186/s12920-019-0546-z. PMID: 31262291; PMCID: PMC6604454.
12: Gajera M, Desai N, Suzuki A, Li A, Zhang M, Jun G, Jia P, Zhao Z, Iwata J. MicroRNA-655-3p and microRNA-497-5p inhibit cell proliferation in cultured human lip cells through the regulation of genes related to human cleft lip. BMC Med Genomics. 2019 May 23;12(1):70. doi: 10.1186/s12920-019-0535-2. PMID: 31122291; PMCID: PMC6533741.
13: Li A, Qin G, Suzuki A, Gajera M, Iwata J, Jia P, Zhao Z. Network-based identification of critical regulators as putative drivers of human cleft lip. BMC Med Genomics. 2019 Jan 31;12(Suppl 1):16. doi: 10.1186/s12920-018-0458-3. PMID: 30704473; PMCID: PMC6357351.
14: Suzuki A, Minamide R, Iwata J. WNT/β-catenin signaling plays a crucial role in myoblast fusion through regulation of nephrin expression during development. Development. 2018 Nov 27;145(23):dev168351. doi: 10.1242/dev.168351. PMID: 30389854; PMCID: PMC6288386.
15: Suzuki A, Iwata J. Molecular Regulatory Mechanism of Exocytosis in the Salivary Glands. Int J Mol Sci. 2018 Oct 17;19(10):3208. doi: 10.3390/ijms19103208. PMID: 30336591; PMCID: PMC6214078.
16: Suzuki A, Minamide R, Iwata J. The role of acetyltransferases for the temporal-specific accessibility of β-catenin to the myogenic gene locus. Sci Rep. 2018 Oct 10;8(1):15057. doi: 10.1038/s41598-018-32888-z. PMID: 30305648; PMCID: PMC6180044.
17: Suzuki A, Jun G, Abdallah N, Gajera M, Iwata J. Gene datasets associated with mouse cleft palate. Data Brief. 2018 Mar 14;18:655-673. doi: 10.1016/j.dib.2018.03.010. PMID: 29896534; PMCID: PMC5996166.
19: Suzuki A, Abdallah N, Gajera M, Jun G, Jia P, Zhao Z, Iwata J. Genes and microRNAs associated with mouse cleft palate: A systematic review and bioinformatics analysis. Mech Dev. 2018 Apr;150:21-27. doi: 10.1016/j.mod.2018.02.003. Epub 2018 Feb 21. PMID: 29475039; PMCID: PMC5906164.
20: Suzuki A, Sangani DR, Ansari A, Iwata J. Molecular mechanisms of midfacial developmental defects. Dev Dyn. 2016 Mar;245(3):276-93. doi: 10.1002/dvdy.24368. Epub 2015 Dec 11. PMID: 26562615; PMCID: PMC4755841.
21: Suzuki A, Scruggs A, Iwata J. The temporal specific role of WNT/β-catenin signaling during myogenesis. J Nat Sci. 2015;1(8):e143. PMID: 26176019; PMCID: PMC4499510.
22: Sangani D, Suzuki A, VonVille H, Hixson JE, Iwata J. Gene Mutations Associated with Temporomandibular Joint Disorders: A Systematic Review. OAlib. 2015 Jun;2(6):e1583. doi: 10.4236/oalib.1101583. Epub 2015 Jun 3. PMID: 27695703; PMCID: PMC5045035.
23: Suzuki A, Pelikan RC, Iwata J. WNT/β-Catenin Signaling Regulates Multiple Steps of Myogenesis by Regulating Step-Specific Targets. Mol Cell Biol. 2015 May;35(10):1763-76. doi: 10.1128/MCB.01180-14. Epub 2015 Mar 9. PMID: 25755281; PMCID: PMC4405648.
24: Ho TV, Iwata J, Ho HA, Grimes WC, Park S, Sanchez-Lara PA, Chai Y. Integration of comprehensive 3D microCT and signaling analysis reveals differential regulatory mechanisms of craniofacial bone development. Dev Biol. 2015 Apr 15;400(2):180-90. doi: 10.1016/j.ydbio.2015.02.010. Epub 2015 Feb 23. PMID: 25722190; PMCID: PMC4385433.
26: Iwata J, Suzuki A, Yokota T, Ho TV, Pelikan R, Urata M, Sanchez-Lara PA, Chai Y. TGFβ regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate. Development. 2014 Feb;141(4):909-17. doi: 10.1242/dev.103093. PMID: 24496627; PMCID: PMC3912833.
27: Iwata J, Suzuki A, Pelikan RC, Ho TV, Sanchez-Lara PA, Chai Y. Modulation of lipid metabolic defects rescues cleft palate in Tgfbr2 mutant mice. Hum Mol Genet. 2014 Jan 1;23(1):182-93. doi: 10.1093/hmg/ddt410. Epub 2013 Aug 23. PMID: 23975680; PMCID: PMC3857953.
28: Iwata J, Suzuki A, Pelikan RC, Ho TV, Chai Y. Noncanonical transforming growth factor β (TGFβ) signaling in cranial neural crest cells causes tongue muscle developmental defects. J Biol Chem. 2013 Oct 11;288(41):29760-70. doi: 10.1074/jbc.M113.493551. Epub 2013 Aug 15. PMID: 23950180; PMCID: PMC3795273.
29: Parada C, Li J, Iwata J, Suzuki A, Chai Y. CTGF mediates Smad-dependent transforming growth factor β signaling to regulate mesenchymal cell proliferation during palate development. Mol Cell Biol. 2013 Sep;33(17):3482-93. doi: 10.1128/MCB.00615-13. Epub 2013 Jul 1. PMID: 23816882; PMCID: PMC3753855.
30: Koike M, Tanida I, Nanao T, Tada N, Iwata J, Ueno T, Kominami E, Uchiyama Y. Enrichment of GABARAP relative to LC3 in the axonal initial segments of neurons. PLoS One. 2013 May 9;8(5):e63568. doi: 10.1371/journal.pone.0063568. PMID: 23671684; PMCID: PMC3650058.
31: Song Z, Liu C, Iwata J, Gu S, Suzuki A, Sun C, He W, Shu R, Li L, Chai Y, Chen Y. Mice with Tak1 deficiency in neural crest lineage exhibit cleft palate associated with abnormal tongue development. J Biol Chem. 2013 Apr 12;288(15):10440-50. doi: 10.1074/jbc.M112.432286. Epub 2013 Mar 4. PMID: 23460641; PMCID: PMC3624426.
33: Iwata J, Suzuki A, Pelikan RC, Ho TV, Sanchez-Lara PA, Urata M, Dixon MJ, Chai Y. Smad4-Irf6 genetic interaction and TGFβ-mediated IRF6 signaling cascade are crucial for palatal fusion in mice. Development. 2013 Mar;140(6):1220-30. doi: 10.1242/dev.089615. Epub 2013 Feb 13. PMID: 23406900; PMCID: PMC3585659.
35: Pelikan RC, Iwata J, Suzuki A, Chai Y, Hacia JG. Identification of candidate downstream targets of TGFβ signaling during palate development by genome-wide transcript profiling. J Cell Biochem. 2013 Apr;114(4):796-807. doi: 10.1002/jcb.24417. PMID: 23060211; PMCID: PMC3777336.
36: Iwata J, Hacia JG, Suzuki A, Sanchez-Lara PA, Urata M, Chai Y. Modulation of noncanonical TGF-β signaling prevents cleft palate in Tgfbr2 mutant mice. J Clin Invest. 2012 Mar;122(3):873-85. doi: 10.1172/JCI61498. Epub 2012 Feb 13. PMID: 22326956; PMCID: PMC3287237.
37: Okamoto K, Okamoto Y, Kawakubo T, Iwata J, Yasuda Y, Tsukuba T, Yamamoto K. Role of the transcription factor Sp1 in regulating the expression of the murine cathepsin E gene. J Biochem. 2012 Mar;151(3):263-72. doi: 10.1093/jb/mvr135. Epub 2011 Nov 30. PMID: 22134960.
38: Iwata J, Tung L, Urata M, Hacia JG, Pelikan R, Suzuki A, Ramenzoni L, Chaudhry O, Parada C, Sanchez-Lara PA, Chai Y. Fibroblast growth factor 9 (FGF9)-pituitary homeobox 2 (PITX2) pathway mediates transforming growth factor β (TGFβ) signaling to regulate cell proliferation in palatal mesenchyme during mouse palatogenesis. J Biol Chem. 2012 Jan 20;287(4):2353-63. doi: 10.1074/jbc.M111.280974. Epub 2011 Nov 28. PMID: 22123828; PMCID: PMC3268397.
39: Ezaki J, Matsumoto N, Takeda-Ezaki M, Komatsu M, Takahashi K, Hiraoka Y, Taka H, Fujimura T, Takehana K, Yoshida M, Iwata J, Tanida I, Furuya N, Zheng DM, Tada N, Tanaka K, Kominami E, Ueno T. Liver autophagy contributes to the maintenance of blood glucose and amino acid levels. Autophagy. 2011 Jul;7(7):727-36. doi: 10.4161/auto.7.7.15371. Epub 2011 Jul 1. PMID: 21471734; PMCID: PMC3149698.
40: Hochheiser H, Aronow BJ, Artinger K, Beaty TH, Brinkley JF, Chai Y, Clouthier D, Cunningham ML, Dixon M, Donahue LR, Fraser SE, Hallgrimsson B, Iwata J, Klein O, Marazita ML, Murray JC, Murray S, de Villena FP, Postlethwait J, Potter S, Shapiro L, Spritz R, Visel A, Weinberg SM, Trainor PA. The FaceBase Consortium: a comprehensive program to facilitate craniofacial research. Dev Biol. 2011 Jul 15;355(2):175-82. doi: 10.1016/j.ydbio.2011.02.033. Epub 2011 Mar 31. PMID: 21458441; PMCID: PMC3440302.
41: Chung IH, Han J, Iwata J, Chai Y. Msx1 and Dlx5 function synergistically to regulate frontal bone development. Genesis. 2010 Nov;48(11):645-55. doi: 10.1002/dvg.20671. Epub 2010 Nov 2. PMID: 20824629; PMCID: PMC2995851.
42: Hosokawa R, Oka K, Yamaza T, Iwata J, Urata M, Xu X, Bringas P Jr, Nonaka K, Chai Y. TGF-beta mediated FGF10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis. Dev Biol. 2010 May 1;341(1):186-95. doi: 10.1016/j.ydbio.2010.02.030. Epub 2010 Feb 26. PMID: 20193675; PMCID: PMC3336866.
43: Iwata J, Hosokawa R, Sanchez-Lara PA, Urata M, Slavkin H, Chai Y. Transforming growth factor-beta regulates basal transcriptional regulatory machinery to control cell proliferation and differentiation in cranial neural crest-derived osteoprogenitor cells. J Biol Chem. 2010 Feb 12;285(7):4975-82. doi: 10.1074/jbc.M109.035105. Epub 2009 Dec 3. PMID: 19959467; PMCID: PMC2836101.
44: Sou YS, Waguri S, Iwata J, Ueno T, Fujimura T, Hara T, Sawada N, Yamada A, Mizushima N, Uchiyama Y, Kominami E, Tanaka K, Komatsu M. The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice. Mol Biol Cell. 2008 Nov;19(11):4762-75. doi: 10.1091/mbc.e08-03-0309. Epub 2008 Sep 3. PMID: 18768753; PMCID: PMC2575156.
45: Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, Mizushima N, Iwata J, Ezaki J, Murata S, Hamazaki J, Nishito Y, Iemura S, Natsume T, Yanagawa T, Uwayama J, Warabi E, Yoshida H, Ishii T, Kobayashi A, Yamamoto M, Yue Z, Uchiyama Y, Kominami E, Tanaka K. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell. 2007 Dec 14;131(6):1149-63. doi: 10.1016/j.cell.2007.10.035. PMID: 18083104.
46: Kawakubo T, Okamoto K, Iwata J, Shin M, Okamoto Y, Yasukochi A, Nakayama KI, Kadowaki T, Tsukuba T, Yamamoto K. Cathepsin E prevents tumor growth and metastasis by catalyzing the proteolytic release of soluble TRAIL from tumor cell surface. Cancer Res. 2007 Nov 15;67(22):10869-78. doi: 10.1158/0008-5472.CAN-07-2048. PMID: 18006832.
47: Shin M, Kadowaki T, Iwata J, Kawakubo T, Yamaguchi N, Takii R, Tsukuba T, Yamamoto K. Association of cathepsin E with tumor growth arrest through angiogenesis inhibition and enhanced immune responses. Biol Chem. 2007 Nov;388(11):1173-81. doi: 10.1515/BC.2007.154. PMID: 17976010.
48: Komatsu M, Wang QJ, Holstein GR, Friedrich VL Jr, Iwata J, Kominami E, Chait BT, Tanaka K, Yue Z. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14489-94. doi: 10.1073/pnas.0701311104. Epub 2007 Aug 28. PMID: 17726112; PMCID: PMC1964831.
49: Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y, Kominami E, Tanaka K. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature. 2006 Jun 15;441(7095):880-4. doi: 10.1038/nature04723. Epub 2006 Apr 19. PMID: 16625205.
50: Iwata J, Ezaki J, Komatsu M, Yokota S, Ueno T, Tanida I, Chiba T, Tanaka K, Kominami E. Excess peroxisomes are degraded by autophagic machinery in mammals. J Biol Chem. 2006 Feb 17;281(7):4035-41. doi: 10.1074/jbc.M512283200. Epub 2005 Dec 6. PMID: 16332691.
51: Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol. 2005 May 9;169(3):425-34. doi: 10.1083/jcb.200412022. Epub 2005 May 2. PMID: 15866887; PMCID: PMC2171928.
52: Iwata J, Yamamoto K. [Involvement of cathepsin D and cathepsin E in the inhibition of tumor growth and metastasis through the production of angiogenetic inhibitors]. Tanpakushitsu Kakusan Koso. 2003 Nov;48(14):1928-33. Japanese. PMID: 14619419.