Advertisement

Amebiasis pp 331-349 | Cite as

Metabolomic Analysis of Entamoeba Biology

  • Ghulam Jeelani
  • Dan Sato
  • Tomoyoshi Nozaki
Chapter

Abstract

Whole genome or transcriptome information provides the annotation of genes and proteins and predicts metabolic pathways, but unequivocal demonstration of the functionalities of the enzymes and metabolic pathways remains challenging. Because nearly 56 % of the Entamoeba histolytica genes remain unannotated, correlative “omics” analyses of genomics, transcriptomics, proteomics, and biochemical metabolic profiling can be useful in uncovering new, or poorly understood, metabolisms and metabolic pathways. Current understanding of metabolic pathways constructed by genes and pathway predictions are based on homology search of the genome, transcriptome, and proteome databases and conventional biochemical demonstration of enzymatic activities. However, it is well known that there are large disparities between the pathways predicted in silico and the pathways actually operating in vivo. Thus, it is important to demonstrate the presence and kinetics (flow or flux) of the metabolites involved in the pathways. To this end, a variety of analytical methods and platforms for metabolomics and metabolite profiling has been developed, in which intracellular and extracellular metabolites can be selectively or globally analyzed. Global metabolomics analysis of Entamoeba histolytica under environmental stress conditions, in different life-cycle stages, and heterogenic (i.e., clinical) isolates, should potentially uncover unpredictable metabolic pathways, interaction and regulation of pathways, and also directly demonstrate the role of individual genes on metabolic pathways, and thus helps our understanding of the physiological and biological roles of metabolic pathways and a network of regulatory interactions between them. Metabolomics of Entamoeba is still in its infancy and only a handful of studies have been reported thus far. In this chapter, we summarize a few examples of the application of metabolomics, combined with transcriptomic analysis, to the analysis of global changes in metabolism in response to three representative physiological conditions: encystation, oxidative stress, and cysteine deprivation. We also discuss future applications of metabolomics to understand the biology and pathogenesis of E. histolytica. Furthermore, because major metabolic differences between the parasite and its host provide rational drug targets, which are either selectively present in pathogens or highly divergent from humans, multi-“omics” approaches, including metabolomics, should lead to important discoveries of unique exploitable metabolic networks crucial to develop new effective drugs against amebiasis.

Keywords

Metabolic Network Biogenic Amine Pentose Phosphate Pathway Metabolomic Analysis Entamoeba Histolytica 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Dixon RA, Strack D (2003) Phytochemistry meets genome analysis, and beyond. Phytochemistry 62:815–816PubMedCrossRefGoogle Scholar
  2. 2.
    Tweeddale H, Notley-McRobb L, Ferenci T (1998) Effect of slow growth on metabolism of Escherichia coli, as revealed by global metabolite pool (“metabolome”) analysis. J Bacteriol 180:5109–5116PubMedCentralPubMedGoogle Scholar
  3. 3.
    Feist AM, Herrgard MJ, Thiele I, Reed JL, Palsson BO (2009) Reconstruction of biochemical networks in microorganisms. Nat Rev Microbiol 7:129–143PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Durot M, Bourguignon PY, Schachter V (2009) Genome-scale models of bacterial metabolism: reconstruction and applications. FEMS Microbiol Rev 33:164–190PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Trauger SA, Kalisak E, Kalisiak J, Morita H, Weinberg MV, Menon AL, Poole FL II, Adams MW, Siuzdak G (2008) Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile. J Proteome Res 7:1027–1035PubMedCrossRefGoogle Scholar
  6. 6.
    Rabinowitz JD (2007) Cellular metabolomics of Escherichia coli. Expert Rev Proteomics 4:187–198PubMedCrossRefGoogle Scholar
  7. 7.
    Brauer MJ, Yuan J, Bennett BD, Lu W, Kimball E, Botstein D, Rabinowitz JD (2006) Conservation of the metabolomic response to starvation across two divergent microbes. Proc Natl Acad Sci USA 103:19302–19307PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Cho K, Shibato J, Agrawal GK, Jung YH, Kubo A, Jwa NS, Tamogami S, Satoh K, Kikuchi S, Higashi T et al (2008) Integrated transcriptomics, proteomics, and metabolomics analyses to survey ozone responses in the leaves of rice seedling. J Proteome Res 7:2980–2998PubMedCrossRefGoogle Scholar
  9. 9.
    Sun G, Yang K, Zhao Z, Guan S, Han X, Gross RW (2007) Shotgun metabolomics approach for the analysis of negatively charged water-soluble cellular metabolites from mouse heart tissue. Anal Chem 79:6629–6640PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Pedersen KS, Kristensen TN, Loeschcke V, Petersen BO, Duus JO, Nielsen NC, Malmendal A (2008) Metabolomic signatures of inbreeding at benign and stressful temperatures in Drosophila melanogaster. Genetics 180:1233–1243PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Khoo SH, Al-Rubeai M (2007) Metabolomics as a complementary tool in cell culture. Biotechnol Appl Biochem 47:71–84PubMedCrossRefGoogle Scholar
  12. 12.
    Roessner U, Bowne J (2009) What is metabolomics all about? Biotechniques 46:363–365PubMedCrossRefGoogle Scholar
  13. 13.
    Lei Z, Huhman DV, Sumner LW (2011) Mass spectrometry strategies in metabolomics. J Biol Chem 286:25435–25442PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Sauer U (2006) Metabolic networks in motion: 13C-based flux analysis. Mol Syst Biol 2:62PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Zamboni N, Fendt SM, Rühl M, Sauer U (2009) (13)C-based metabolic flux analysis. Nat Protoc 4:878–892PubMedCrossRefGoogle Scholar
  16. 16.
    Blank LM, Kuepfer L, Sauer U (2005) Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast. Genome Biol 6:R49PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Munger J, Bajad SU, Coller HA, Shenk T, Rabinowitz JD (2006) Dynamics of the cellular metabolome during human cytomegalovirus infection. PLoS Pathog 2:e132PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Kwon YK, Lu W, Melamud E, Khanam N, Bognar A, Rabinowitz JD (2008) A domino effect in antifolate drug action in Escherichia coli. Nat Chem Biol 4:602–608PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Kalisiak J, Trauger SA, Kalisiak E, Morita H, Fokin VV, Adams MW, Sharpless KB, Siuzdak G (2009) Identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach. J Am Chem Soc 131:378–386PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Loftus B, Anderson I, Davies R, Alsmark UC, Samuelson J, Amedeo P, Roncaglia P, Berriman M et al (2005) The genome of the protist parasite Entamoeba histolytica. Nature (Lond) 433:865–868CrossRefGoogle Scholar
  21. 21.
    Anderson IJ, Loftus BJ (2005) Entamoeba histolytica: observations on metabolism based on the genome sequence. Exp Parasitol 110:173–177PubMedCrossRefGoogle Scholar
  22. 22.
    Lorenzi HA, Puiu D, Miller JR, Brinkac LM, Amedeo P, Hall N, Caler EV (2010) New assembly, reannotation and analysis of the Entamoeba histolytica genome reveal new genomic features and protein content information. PLoS Negl Trop Dis 4:e716PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Weinbach EC, Diamond LS (1974) Entamoeba histolytica. I. Aerobic metabolism. Exp Parasitol 35:232–243PubMedCrossRefGoogle Scholar
  24. 24.
    Tovar J, Fischer A, Clark CG (1999) The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica. Mol Microbiol 32:1013–1021PubMedCrossRefGoogle Scholar
  25. 25.
    Reeves RE (1984) Metabolism of Entamoeba histolytica Schaudinn, 1903. Adv Parasitol 23:105–142PubMedCrossRefGoogle Scholar
  26. 26.
    Müller M (1998) Enzymes and compartmentation of core energy metabolism of anaerobic protists: a special case in eukaryotic evolution. In: Coombs GH, Vickerman K, Sleigh MA, Warren A (eds) Evolutionary relationships among protozoa. Kluwer Academic, Dordrecht, pp 109–132Google Scholar
  27. 27.
    Nozaki T, Ali V, Tokoro M (2005) Sulfur-containing amino acid metabolism in parasitic protozoa. Adv Parasitol 60:1–99PubMedCrossRefGoogle Scholar
  28. 28.
    Husain A, Sato D, Jeelani G, Mi-ichi F, Ali V, Suematsu M, Soga T, Nozaki T (2010) Metabolome analysis revealed increase in S-methylcysteine and phosphatidyl isopropanolamine synthesis upon l-cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica. J Biol Chem 285:39160–39170PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Jeelani G, Sato D, Husain A, Escueta-de Cadiz A, Sugimoto M, Soga T, Suematsu M, Nozaki T (2012) Metabolic profiling of the protozoan parasite Entamoeba invadens revealed activation of unpredicted pathway during encystation. PLoS One 7:e37740PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Husain A, Sato D, Jeelani G, Soga T, Nozaki T (2012) Dramatic increase in glycerol biosynthesis upon oxidative stress in the anaerobic protozoan parasite Entamoeba histolytica. PLoS Negl Trop Dis 6:e1831PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Bharti SK, Jaiswal V, Ghoshal U, Ghoshal UC, Baijal SS, Roy R, Khetrapal CL (2012) Metabolomic profiling of amoebic and pyogenic liver abscesses: an in vitro NMR study. Metabolomics 8:540–555CrossRefGoogle Scholar
  32. 32.
    Saavedra E, Encalada R, Pineda E, Jasso-Chavez R, Moreno-Sanchez R (2005) Glycolysis in Entamoeba histolytica. Biochemical characterization of recombinant glycolytic enzymes and flux control analysis. FEBS J 272:1767–1783PubMedCrossRefGoogle Scholar
  33. 33.
    Saavedra E, Marín-Hernández A, Encalada R, Olivos A, Mendoza-Hernández G, Moreno-Sánchez R (2007) Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica. FEBS J 274:4922–4940PubMedCrossRefGoogle Scholar
  34. 34.
    Moreno-Sánchez R, Encalada R, Marín-Hernández A, Saavedra E (2008) Experimental validation of metabolic pathway modeling. An illustration with glycolytic segments from Entamoeba histolytica. FEBS J 275:3454–3469PubMedCrossRefGoogle Scholar
  35. 35.
    Montalvo FE, Reeves RE, Warren LG (1971) Aerobic and anaerobic metabolism in Entamoeba histolytica. Exp Parasitol 30:249–256PubMedCrossRefGoogle Scholar
  36. 36.
    Bakker-Grunwald T, Martin JB, Klein G (1995) Characterization of glycogen and amino acid pool of Entamoeba histolytica by 13C-NMR spectroscopy. J Eukaryot Microbiol 42:346–349PubMedCrossRefGoogle Scholar
  37. 37.
    Zuo X, Coombs GH (1995) Amino acid consumption by the parasitic, amoeboid protists Entamoeba histolytica and E. invadens. FEMS Microbiol Lett 130:253–258PubMedCrossRefGoogle Scholar
  38. 38.
    McConnachie EW (1969) The morphology, formation and development of cysts of Entamoeba. Parasitology 59:41–53PubMedCrossRefGoogle Scholar
  39. 39.
    Arroyo-Begovich A, Carabez-Trejo A, Ruiz-Herrera J (1980) Identification of the structural component in the cyst wall of Entamoeba invadens. J Parasitol 66:735–741PubMedCrossRefGoogle Scholar
  40. 40.
    Arroyo-Begovich A, Carabez-Trejo A (1982) Location on chitin in the cyst wall of Entamoeba invadens with colloidal gold tracers. J Parasitol 68:253–258PubMedCrossRefGoogle Scholar
  41. 41.
    Spindler KD, Spindler-Barth M, Londershausen M (1990) Chitin metabolism: a target for drugs against parasites. Parasitol Res 76:283–288PubMedCrossRefGoogle Scholar
  42. 42.
    Muller M (1991) Energy metabolism of anaerobic parasitic protists. In: Coombs GH, North MJ (eds) Biochemical protozoology. Taylor & Francis, London, pp 80–91Google Scholar
  43. 43.
    Slocum RD, Kaur-Sawhney R, Galston AW (1984) The physiology and biochemistry of polyamines in plants. Arch Biochem Biophys 235:283–303PubMedCrossRefGoogle Scholar
  44. 44.
    Tabor CW, Tabor H (1985) Polyamines in microorganisms. Microbiol Rev 49:81–99PubMedCentralPubMedGoogle Scholar
  45. 45.
    Lounvaud-Funel A (2001) Biogenic amines in wines: role of lactic acid bacteria. FEMS Microbiol Lett 199:9–13CrossRefGoogle Scholar
  46. 46.
    Loukou Z, Zotou A (2003) Determination of biogenic amines as dansyl derivatives in alcoholic beverages by high-performance liquid chromatography with fluorimetric detection and characterization of the dansylated amines by liquid chromatography atmospheric pressure chemical ionization mass spectrometry. J Chromatogr A 996:103–113PubMedCrossRefGoogle Scholar
  47. 47.
    Bauza T, Blaise A, Teissedre PL, Cabanis JC, Kanny G et al. (1995) Les amines biogenes du vin: metabolisme et toxicite. Bull L’OIV: 42–67Google Scholar
  48. 48.
    Verma AK, Raizada MK, Murti CK (1974) Effect of bioamines on the cellular differentiation of Hartmannella culbertsoni. Biochem Pharmacol 23:57–63PubMedCrossRefGoogle Scholar
  49. 49.
    Morales-Vallarta M, Villarreal-Treviño L, Guerrero Medrano L, Ramírez-Bon E, Navarro-Marmolejo L, Said-Fernández S, Mata-Cárdenas BD (1997) Entamoeba invadens differentiation and E. histolytica cyst-like formation induced by CO2. Arch Med Res 28:150–151PubMedGoogle Scholar
  50. 50.
    Stanley SL Jr (2003) Amoebiasis. Lancet 361:1025–1034PubMedCrossRefGoogle Scholar
  51. 51.
    Bogdan C, Rollinghoff M, Diefenbach A (2000) Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 12:64–76PubMedCrossRefGoogle Scholar
  52. 52.
    Mehlotra RK (1996) Antioxidant defense mechanisms in parasitic protozoa. Crit Rev Microbiol 22:295–314PubMedCrossRefGoogle Scholar
  53. 53.
    Fahey RC, Newton GL, Arrick B, Overdank-Bogart T, Aley SB (1984) Entamoeba histolytica: a eukaryote without glutathione metabolism. Science 224:70–72PubMedCrossRefGoogle Scholar
  54. 54.
    Saraiva LM, Vicente JB, Teixeira M (2004) The role of the flavodiiron proteins in microbial nitric oxide detoxification. Adv Microb Physiol 49:77–129PubMedCrossRefGoogle Scholar
  55. 55.
    Sen A, Chatterjee NS, Akbar MA, Nandi N et al (2007) The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress. Eukaryot Cell 6:664–673PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Jackson JB (1991) The proton-translocating nicotinamide adenine dinucleotide transhydrogenase. J Bioenerg Biomembr 23:715–741PubMedCrossRefGoogle Scholar
  57. 57.
    Yousuf MA, Mi-ichi F, Nakada-Tsukui K, Nozaki T (2010) Localization and targeting of an unusual pyridine nucleotide transhydrogenase in Entamoeba histolytica. Eukaryot Cell 9:926–933PubMedCentralPubMedCrossRefGoogle Scholar
  58. 58.
    Vicente J, Ehrenkaufer G, Saraiva L, Teixeira M, Singh U (2008) Entamoeba histolytica modulates a complex repertoire of novel genes in response to oxidative and nitrosative stresses: implications for amebic pathogenesis. Cell Microbiol 11:51–69PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Ramos-Martínez E, Olivos-García A, Saavedra E, Nequiz M, Sánchez EC, Tello E, El-Hafidi M, Saralegui A, Pineda E, Delgado J, Montfort I, Pérez-Tamayo R (2009) Entamoeba histolytica: oxygen resistance and virulence. Int J Parasitol 39:693–702PubMedCrossRefGoogle Scholar
  60. 60.
    Ramos E, Olivos-García A, Nequiz M, Saavedra E, Tello E, Saralegui A, Montfort I, Pérez Tamayo R (2007) Entamoeba histolytica: apoptosis induced in vitro by nitric oxide species. Exp Parasitol 116:257–265PubMedCrossRefGoogle Scholar
  61. 61.
    Lo HS, Reeves RE (1978) Pyruvate-to-ethanol pathway in Entamoeba histolytica. Biochem J 171(1):225–230PubMedCentralPubMedGoogle Scholar
  62. 62.
    Ali V, Nozaki T (2006) Biochemical and functional characterization of phosphoserine aminotransferase from Entamoeba histolytica, which possesses both phosphorylated and non-phosphorylated serine metabolic pathways. Mol Biochem Parasitol 145:71–83PubMedCrossRefGoogle Scholar
  63. 63.
    Bragg PD, Reeves RE (1962) Pathways of glucose dissimilation in Laredo strain of Entamoeba histolytica. Exp Parasitol 12:393–400PubMedCrossRefGoogle Scholar
  64. 64.
    Krüger A, Grüning NM, Wamelink MM, Kerick M, Kirpy A, Parkhomchuk D, Bluemlein K, Schweiger MR, Soldatov A, Lehrach H, Jakobs C, Ralser M (2011) The pentose phosphate pathway is a metabolic redox sensor and regulates transcription during the antioxidant response. Antioxid Redox Signal 15:311–324PubMedCrossRefGoogle Scholar
  65. 65.
    Jeelani G, Husain A, Sato D, Soga T, Suematsu M, Nozaki T (2013) Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica. Biochimie 95:309–319PubMedCrossRefGoogle Scholar
  66. 66.
    Chapman A, Linstead DJ, Lloyd D, Williams J (1985) 13C-NMR reveals glycerol as an unexpected major metabolite of the protozoan parasite Trichomonas vaginalis. FEBS J 191:287–292CrossRefGoogle Scholar
  67. 67.
    Hammond DJ, Bowman IB (1980) Studies on glycerol kinase and its role in ATP synthesis in Trypanosoma brucei. Mol Biochem Parasitol 2:77–91PubMedCrossRefGoogle Scholar
  68. 68.
    Lian LY, Al-Helal M, Roslaini AM, Fisher N, Bray PG (2009) Glycerol: an unexpected major metabolite of energy metabolism by the human malaria parasite. Malar J 6:38CrossRefGoogle Scholar
  69. 69.
    Reeves RE, Lobelle-Rich P (1983) Absence of α-glycerophosphate dehydrogenase in axenically grown Entamoeba histolytica. Am J Trop Med Hyg 32:1177–1178PubMedGoogle Scholar
  70. 70.
    Gillin FD, Diamond LS (1980) Attachment of Entamoeba histolytica to glass in a defined maintenance medium: specific requirement for cysteine and ascorbic acid. J Protozool 27:474–478PubMedCrossRefGoogle Scholar
  71. 71.
    Gillin FD, Diamond LS (1981) Entamoeba histolytica and Giardia lamblia: effects of cysteine and oxygen tension on trophozoite attachment to glass and survival in culture media. Exp Parasitol 52:9–17PubMedCrossRefGoogle Scholar
  72. 72.
    Gillin FD, Diamond LS (1981) Entamoeba histolytica and Giardia lamblia: growth responses to reducing agents. Exp Parasitol 51:382–391PubMedCrossRefGoogle Scholar
  73. 73.
    Jeelani G, Husain A, Sato D, Ali V, Suematsu M, Soga T, Nozaki T (2010) Two atypical l-cysteine-regulated NADPH-dependent oxidoreductases involved in redox maintenance, l-cystine and iron reduction, and metronidazole activation in the enteric protozoan Entamoeba histolytica. J Biol Chem 285:26889–26899PubMedCentralPubMedCrossRefGoogle Scholar
  74. 74.
    Rébeillé F, Jabrin S, Bligny R, Loizeau K, Gambonnet B, Van Wilder V, Douce R, Ravanel S (2006) Methionine catabolism in Arabidopsis cells is initiated by a gamma-cleavage process and leads to S-methylcysteine and isoleucine syntheses. Proc Natl Acad Sci USA 103:15687–15692PubMedCentralPubMedCrossRefGoogle Scholar
  75. 75.
    Hussain S, Ali V, Jeelani G, Nozaki T (2009) Isoform-dependent feedback regulation of serine O-acetyltransferase isoenzymes involved in l-cysteine biosynthesis of Entamoeba histolytica. Mol Biochem Parasitol 163:39–47PubMedCrossRefGoogle Scholar
  76. 76.
    Husain A, Jeelani G, Sato D, Nozaki T (2011) Global analysis of gene expression in response to l-cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica. BMC Genomics 12:275PubMedCentralPubMedCrossRefGoogle Scholar
  77. 77.
    Ali V, Nozaki T (2007) Current therapeutics, their problems, and sulfur-containing-amino-acid metabolism as a novel target against infections by “amitochondriate” protozoan parasites. Clin Microbiol Rev 20:164–187PubMedCentralPubMedCrossRefGoogle Scholar
  78. 78.
    Chayen A, Mirelman D, Chayen R (1984) Polyamines in Entamoeba invadens. Cell Biochem Funct 2:115–118PubMedCrossRefGoogle Scholar
  79. 79.
    Mi-ichi F, Yousuf MA, Nakada-Tsukui K, Nozaki T (2009) Mitosomes in Entamoeba histolytica contain a sulfate activation pathway. Proc Natl Acad Sci USA 106:21731–21736PubMedCentralPubMedCrossRefGoogle Scholar
  80. 80.
    Mi-ichi F, Makiuchi T, Furukawa A, Sato D, Nozaki T (2011) Sulfate activation in mitosomes plays an important role in the proliferation of Entamoeba histolytica. PLoS Negl Trop Dis 5:e1263PubMedCentralPubMedCrossRefGoogle Scholar
  81. 81.
    Makiuchi T, Mi-Ichi F, Nakada-Tsukui K, Nozaki T (2013) Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport. Sci Rep 3:1129PubMedCentralPubMedCrossRefGoogle Scholar
  82. 82.
    Maralikova B, Ali V, Nakada-Tsukui K, Nozaki T, van der Giezen M, Henze K, Tovar J (2010) Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria. Cell Microbiol 12:331–342PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2015

Authors and Affiliations

  1. 1.Department of ParasitologyNational Institute of Infectious DiseasesTokyoJapan
  2. 2.Department of Biochemistry and Integrative Medical BiologySchool of Medicine, Keio UniversityTokyoJapan
  3. 3.Institute for Advanced BiosciencesKeio UniversityYamagataJapan
  4. 4.Graduate School of Life and Environmental SciencesUniversity of TsukubaIbarakiJapan

Personalised recommendations