Advertisement

Molecular Neurobiology

, Volume 53, Issue 5, pp 3088–3101 | Cite as

GNE Myopathy and Cell Apoptosis: A Comparative Mutation Analysis

  • Reema Singh
  • Ranjana AryaEmail author
Article

Abstract

In a number of genetic disorders such as GNE myopathy, it is not clear how mutations in target genes result in disease phenotype. GNE myopathy is a progressive neuro-degenerative disorder associated with homozygous or compound heterozygous missense mutations in either epimerase or kinase domain of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). This bifunctional enzyme catalyses the rate limiting step in sialic acid biosynthesis. Many mechanisms have been suggested as possible cause of muscle degeneration. These include hyposialylation of critical proteins, defects in cytoskeletal network, sarcomere organization and apoptosis. In order to elucidate the role of GNE in cell apoptosis, we have used HEK cell-based model system overexpressing pathologically relevant GNE mutations. These cells display a reduction in the levels of sialic acid-bound glycoconjugates. These mutants GNE overexpressing cells have defect in cell proliferation as compared to vector or wild-type GNE (wtGNE) controls. Moreover, effect of different GNE mutations on cell apoptosis was also observed using staining with annexin V-FITC and TUNEL assay. The downstream apoptosis signalling pathway involving activation of caspases and increased PARP cleavage were observed in all GNE mutant cell lines. In addition, morpho-structural changes in mitochondria in cells overexpressing different GNE mutants were noticed by transmission electron microscopy, and mitochondrial transmembrane potential was found to be altered in absence of functional GNE. Our results clearly indicate role of GNE in mitochondria-dependent cell apoptosis and provide insights into the pathomechanism of GNE myopathy.

Keywords

Hereditary inclusion body myopathy (HIBM) GNE myopathy Cell apoptosis Sialic acid Mitochondria Proliferation 

Notes

Acknowledgments

We thank Prof. Alok Bhattacharya (School of Life Sciences, Jawaharlal Nehru University, and New Delhi) for thoughtful discussions and progressive comments during the project. This work was supported by grants from the Indian Council of Medical Research, India and Council of Scientific and Industrial Research, Govt. of India. We acknowledge Mr. Ashok and Mr. Prabhat Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, for technical assistance in confocal microscopy and live cell imaging. We acknowledge Dr. Anwar Alam and Mrs. Sarika (School of Life Sciences, Jawaharlal Nehru University, NewDelhi) for technical assistance in flow cytometry.

Supplementary material

12035_2015_9191_MOESM1_ESM.jpg (86 kb)
Figure S1 Determination of DNA damage/nuclear fragmentation by TUNEL assay after supplementation with 5 mM NANA: Representative image of wtGNE and GNE mutants obtained by confocal microscopy, depicting DNA damage/nuclear fragmentation by TUNEL assay. Arrows indicate TUNEL-positive cells (green).The images were viewed using Olympus Fluoview FV1000 laser scan at 60 X magnification. (JPEG 85 kb)
12035_2015_9191_MOESM2_ESM.jpg (111 kb)
Figure S2 Study of cell apoptosis by Annexin V-FITC/Propidium iodide staining after supplementation with 5 mM NANA: Annexin V-FITC and Propidium iodide staining of wtGNE and different GNE mutant cells were analyzed by flow cytometry after NANA supplementation. B.Graphical representation of fold changein apoptosis of various GNE mutant cells compared to wtGNE cell line. (JPEG 111 kb)
12035_2015_9191_MOESM3_ESM.jpg (145 kb)
Figure S3 Effect of GNE mutation on mitochondrial dysfunction after 5 mM NANA supplementation: A. Effect of GNE mutation on dissipation of mitochondrial membrane potential (as measured by JC-1) using confocal microscopy, magnification 60 X. B. Histogram shows the ratio of red to green fluorescence intensity observed in various cell lines characterizing Δψ (m). (JPEG 145 kb)

References

  1. 1.
    Severi E, Hood DW, Thomas GH (2007) Sialic acid utilization by bacterial pathogens. Microbiology 153(Pt 9):2817–2822. doi: 10.1099/mic.0.2007/009480-0 CrossRefPubMedGoogle Scholar
  2. 2.
    Frenzel R, Krohn K, Eszlinger M, Tonjes A, Paschke R (2005) Sialylation of human thyrotropin receptor improves and prolongs its cell-surface expression. Mol Pharmacol 68(4):1106–1113. doi: 10.1124/mol.105.012906 CrossRefPubMedGoogle Scholar
  3. 3.
    Edelman GM, Crossin KL (1991) Cell adhesion molecules: implications for a molecular histology. Annu Rev Biochem 60:155–190. doi: 10.1146/annurev.bi.60.070191.001103 CrossRefPubMedGoogle Scholar
  4. 4.
    Narayanan S (1994) Sialic acid as a tumor marker. Ann Clin Lab Sci 24(4):376–384PubMedGoogle Scholar
  5. 5.
    Wielgat P, Braszko JJ (2012) Significance of the cell adhesion molecules and sialic acid in neurodegeneration. Adv Med Sci 57(1):23–30. doi: 10.2478/v10039-012-0011-0 CrossRefPubMedGoogle Scholar
  6. 6.
    Prajna K, Kumar JA, Rai S, Shetty SK, Rai T, Shrinidhi, Begum M, Shashikala MD (2013) Predictive value of serum sialic Acid in type-2 diabetes mellitus and its complication (nephropathy). J Clin Diagn Res 7(11):2435–2437. doi: 10.7860/JCDR/2013/6210.3567 Google Scholar
  7. 7.
    Ito M, Sugihara K, Asaka T, Toyama T, Yoshihara T, Furuichi K, Wada T, Asano M (2012) Glycoprotein hyposialylation gives rise to a nephrotic-like syndrome that is prevented by sialic acid administration in GNE V572L point-mutant mice. PLoS One 7(1):e29873. doi: 10.1371/journal.pone.0029873 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Valles-Ayoub Y, Esfandiarifard S, Sinai P, Carbajo R, Khokher Z, No D, Pietruszka M, Darvish B et al (2012) Serum neural cell adhesion molecule is hyposialylated in hereditary inclusion body myopathy. Genet Test Mol Biomarkers 16(5):313–317. doi: 10.1089/gtmb.2011.0146 CrossRefPubMedGoogle Scholar
  9. 9.
    Huizing M, Krasnewich DM (2009) Hereditary inclusion body myopathy: a decade of progress. Biochim Biophys Acta 1792(9):881–887. doi: 10.1016/j.bbadis.2009.07.001 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hinderlich S, Weidemann W, Yardeni T, Horstkorte R, Huizing M (2013) UDP-GlcNAc 2-Epimerase/ManNAc Kinase (GNE): a Master Regulator of Sialic Acid Synthesis. Top Curr Chem. doi: 10.1007/128_2013_464 Google Scholar
  11. 11.
    Leroy JG, Seppala R, Huizing M, Dacremont G, De Simpel H, Van Coster RN, Orvisky E, Krasnewich DM et al (2001) Dominant inheritance of sialuria, an inborn error of feedback inhibition. Am J Hum Genet 68(6):1419–1427. doi: 10.1086/320598 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ghaderi D, Strauss HM, Reinke S, Cirak S, Reutter W, Lucka L, Hinderlich S (2007) Evidence for dynamic interplay of different oligomeric states of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase by biophysical methods. J Mol Biol 369(3):746–758. doi: 10.1016/j.jmb.2007.03.037 CrossRefPubMedGoogle Scholar
  13. 13.
    Tong Y, Tempel W, Nedyalkova L, Mackenzie F, Park HW (2009) Crystal structure of the N-acetylmannosamine kinase domain of GNE. PLoS One 4(10):e7165. doi: 10.1371/journal.pone.0007165 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Eisenberg I, Avidan N, Potikha T, Hochner H, Chen M, Olender T, Barash M, Shemesh M et al (2001) The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy. Nat Genet 29(1):83–87. doi: 10.1038/ng718 CrossRefPubMedGoogle Scholar
  15. 15.
    Seppala R, Tietze F, Krasnewich D, Weiss P, Ashwell G, Barsh G, Thomas GH, Packman S et al (1991) Sialic acid metabolism in sialuria fibroblasts. J Biol Chem 266(12):7456–7461PubMedGoogle Scholar
  16. 16.
    Mori-Yoshimura M, Monma K, Suzuki N, Aoki M, Kumamoto T, Tanaka K, Tomimitsu H, Nakano S et al (2012) Heterozygous UDP-GlcNAc 2-epimerase and N-acetylmannosamine kinase domain mutations in the GNE gene result in a less severe GNE myopathy phenotype compared to homozygous N-acetylmannosamine kinase domain mutations. J Neurol Sci 318(1-2):100–105. doi: 10.1016/j.jns.2012.03.016 CrossRefPubMedGoogle Scholar
  17. 17.
    Galeano B, Klootwijk R, Manoli I, Sun M, Ciccone C, Darvish D, Starost MF, Zerfas PM et al (2007) Mutation in the key enzyme of sialic acid biosynthesis causes severe glomerular proteinuria and is rescued by N-acetylmannosamine. J Clin Invest 117(6):1585–1594. doi: 10.1172/JCI30954 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Eisenberg I, Grabov-Nardini G, Hochner H, Korner M, Sadeh M, Bertorini T, Bushby K, Castellan C et al (2003) Mutations spectrum of GNE in hereditary inclusion body myopathy sparing the quadriceps. Hum Mutat 21(1):99. doi: 10.1002/humu.9100 CrossRefPubMedGoogle Scholar
  19. 19.
    Tanboon J, Rongsa K, Pithukpakorn M, Boonyapisit K, Limwongse C, Sangruchi T (2014) A Novel Mutation of the GNE Gene in Distal Myopathy with Rimmed Vacuoles: a Case with Inflammation. Case Rep Neurol 6(1):55–59. doi: 10.1159/000360730 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Nalini A, Gayathri N, Nishino I, Hayashi YK (2013) GNE myopathy in India. Neurol India 61(4):371–374. doi: 10.4103/0028-3886.117609 CrossRefPubMedGoogle Scholar
  21. 21.
    Huizing M, Carrillo-Carrasco N, Malicdan MC, Noguchi S, Gahl WA, Mitrani-Rosenbaum S, Argov Z, Nishino I (2014) GNE myopathy: new name and new mutation nomenclature. Neuromuscul Disord 24(5):387–389. doi: 10.1016/j.nmd.2014.03.004 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Park YE, Kim HS, Choi ES, Shin JH, Kim SY, Son EH, Lee CH, Kim DS (2012) Limb-girdle phenotype is frequent in patients with myopathy associated with GNE mutations. J Neurol Sci 321(1-2):77–81. doi: 10.1016/j.jns.2012.07.061 CrossRefPubMedGoogle Scholar
  23. 23.
    Celeste FV, Vilboux T, Ciccone C, de Dios JK, Malicdan MC, Leoyklang P, McKew JC, Gahl WA et al (2014) Mutation Update for GNE Gene Variants Associated with GNE Myopathy. Hum Mutat 35(8):915–926. doi: 10.1002/humu.22583 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Cho A, Hayashi YK, Monma K, Oya Y, Noguchi S, Nonaka I, Nishino I (2014) Mutation profile of the GNE gene in Japanese patients with distal myopathy with rimmed vacuoles (GNE myopathy). J Neurol Neurosurg Psychiatry 85(8):914–917. doi: 10.1136/jnnp-2013-305587 CrossRefPubMedGoogle Scholar
  25. 25.
    Grover S, Arya R (2014) Role of UDP-N-Acetylglucosamine2-Epimerase/N-Acetylmannosamine Kinase (GNE) in beta1-Integrin-Mediated Cell Adhesion. Mol Neurobiol. doi: 10.1007/s12035-013-8604-6 PubMedGoogle Scholar
  26. 26.
    Amsili S, Zer H, Hinderlich S, Krause S, Becker-Cohen M, MacArthur DG, North KN, Mitrani-Rosenbaum S (2008) UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) binds to alpha-actinin 1: novel pathways in skeletal muscle? PLoS One 3(6):e2477. doi: 10.1371/journal.pone.0002477 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Sela I, Milman Krentsis I, Shlomai Z, Sadeh M, Dabby R, Argov Z, Ben-Bassat H, Mitrani-Rosenbaum S (2011) The proteomic profile of hereditary inclusion body myopathy. PLoS One 6(1):e16334. doi: 10.1371/journal.pone.0016334 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Weidemann W, Stelzl U, Lisewski U, Bork K, Wanker EE, Hinderlich S, Horstkorte R (2006) The collapsin response mediator protein 1 (CRMP-1) and the promyelocytic leukemia zinc finger protein (PLZF) bind to UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. FEBS Lett 580(28-29):6649–6654. doi: 10.1016/j.febslet.2006.11.015 CrossRefPubMedGoogle Scholar
  29. 29.
    Weidemann W, Klukas C, Klein A, Simm A, Schreiber F, Horstkorte R (2010) Lessons from GNE-deficient embryonic stem cells: sialic acid biosynthesis is involved in proliferation and gene expression. Glycobiology 20(1):107–117. doi: 10.1093/glycob/cwp153 CrossRefPubMedGoogle Scholar
  30. 30.
    Wang Z, Sun Z, Li AV, Yarema KJ (2006) Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase outside of sialic acid biosynthesis: modulation of sialyltransferase and BiP expression, GM3 and GD3 biosynthesis, proliferation, and apoptosis, and ERK1/2 phosphorylation. J Biol Chem 281(37):27016–27028. doi: 10.1074/jbc.M604903200 CrossRefPubMedGoogle Scholar
  31. 31.
    Kemmner W, Kessel P, Sanchez-Ruderisch H, Moller H, Hinderlich S, Schlag PM, Detjen K (2012) Loss of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) induces apoptotic processes in pancreatic carcinoma cells. FASEB J 26(2):938–946. doi: 10.1096/fj.11-186700 CrossRefPubMedGoogle Scholar
  32. 32.
    Jiang X, Wang X (2004) Cytochrome C-mediated apoptosis. Annu Rev Biochem 73:87–106. doi: 10.1146/annurev.biochem.73.011303.073706 CrossRefPubMedGoogle Scholar
  33. 33.
    Cosentino K, Garcia-Saez AJ (2014) Mitochondrial alterations in apoptosis. Chem Phys Lipids 181:62–75. doi: 10.1016/j.chemphyslip.2014.04.001 CrossRefPubMedGoogle Scholar
  34. 34.
    Eisenberg I, Novershtern N, Itzhaki Z, Becker-Cohen M, Sadeh M, Willems PH, Friedman N, Koopman WJ et al (2008) Mitochondrial processes are impaired in hereditary inclusion body myopathy. Hum Mol Genet 17(23):3663–3674. doi: 10.1093/hmg/ddn261 CrossRefPubMedGoogle Scholar
  35. 35.
    Amsili S, Shlomai Z, Levitzki R, Krause S, Lochmuller H, Ben-Bassat H, Mitrani-Rosenbaum S (2007) Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events. Cell Death Differ 14(11):1916–1924. doi: 10.1038/sj.cdd.4402208 CrossRefPubMedGoogle Scholar
  36. 36.
    Arabkhari M, Bunda S, Wang Y, Wang A, Pshezhetsky AV, Hinek A (2010) Desialylation of insulin receptors and IGF-1 receptors by neuraminidase-1 controls the net proliferative response of L6 myoblasts to insulin. Glycobiology 20(5):603–616. doi: 10.1093/glycob/cwq010 CrossRefPubMedGoogle Scholar
  37. 37.
    Gu J, Zhou S, Ding R, Aizezi W, Jiang A, Chen J (2013) Necrotizing scleritis and peripheral ulcerative keratitis associated with Wegener's granulomatosis. Ophthalmol Ther 2(2):99–111. doi: 10.1007/s40123-013-0016-1 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Wittmann S, Bali P, Donapaty S, Nimmanapalli R, Guo F, Yamaguchi H, Huang M, Jove R et al (2003) Flavopiridol down-regulates antiapoptotic proteins and sensitizes human breast cancer cells to epothilone B-induced apoptosis. Cancer Res 63(1):93–99PubMedGoogle Scholar
  39. 39.
    Berger NA (1985) Poly(ADP-ribose) in the cellular response to DNA damage. Radiat Res 101(1):4–15CrossRefPubMedGoogle Scholar
  40. 40.
    Smith L, Wang Z, Smith JB (2003) Caspase processing activates atypical protein kinase C zeta by relieving autoinhibition and destabilizes the protein. Biochem J 375(Pt 3):663–671. doi: 10.1042/BJ20030926 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Broccolini A, Mirabella M (2014) Hereditary inclusion-body myopathies. Biochim Biophys Acta. doi: 10.1016/j.bbadis.2014.08.007 PubMedGoogle Scholar
  42. 42.
    Li H, Chen Q, Liu F, Zhang X, Li W, Liu S, Zhao Y, Gong Y et al (2013) Unfolded protein response and activated degradative pathways regulation in GNE myopathy. PLoS One 8(3):e58116. doi: 10.1371/journal.pone.0058116 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Fischer C, Kleinschnitz K, Wrede A, Muth I, Kruse N, Nishino I, Schmidt J (2013) Cell stress molecules in the skeletal muscle of GNE myopathy. BMC Neurol 13:24. doi: 10.1186/1471-2377-13-24 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Malicdan MC, Noguchi S, Nonaka I, Hayashi YK, Nishino I (2007) A Gne knockout mouse expressing human GNE D176V mutation develops features similar to distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy. Hum Mol Genet 16(22):2669–2682. doi: 10.1093/hmg/ddm220 CrossRefPubMedGoogle Scholar
  45. 45.
    Malicdan MC, Noguchi S, Nonaka I, Hayashi YK, Nishino I (2007) A Gne knockout mouse expressing human V572L mutation develops features similar to distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy. Hum Mol Genet 16(2):115–128. doi: 10.1093/hmg/ddl446 CrossRefPubMedGoogle Scholar
  46. 46.
    Malicdan MC, Noguchi S, Hayashi YK, Nonaka I, Nishino I (2009) Prophylactic treatment with sialic acid metabolites precludes the development of the myopathic phenotype in the DMRV-hIBM mouse model. Nat Med 15(6):690–695. doi: 10.1038/nm.1956 CrossRefPubMedGoogle Scholar
  47. 47.
    Schwarzkopf M, Knobeloch KP, Rohde E, Hinderlich S, Wiechens N, Lucka L, Horak I, Reutter W et al (2002) Sialylation is essential for early development in mice. Proc Natl Acad Sci U S A 99(8):5267–5270. doi: 10.1073/pnas.072066199 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Daya A, Vatine GD, Becker-Cohen M, Tal-Goldberg T, Friedmann A, Gothilf Y, Du SJ, Mitrani-Rosenbaum S (2014) Gne depletion during zebrafish development impairs skeletal muscle structure and function. Hum Mol Genet 23(13):3349–3361. doi: 10.1093/hmg/ddu045 CrossRefPubMedGoogle Scholar
  49. 49.
    Salama I, Hinderlich S, Shlomai Z, Eisenberg I, Krause S, Yarema K, Argov Z, Lochmuller H et al (2005) No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation. Biochem Biophys Res Commun 328(1):221–226. doi: 10.1016/j.bbrc.2004.12.157 CrossRefPubMedGoogle Scholar
  50. 50.
    Weidemann W, Reinhardt A, Thate A, Horstkorte R (2011) Biochemical characterization of the M712T-mutation of the UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosaminekinase in hereditary inclusion body myopathy. Neuromuscul Disord 21(12):824–831. doi: 10.1016/j.nmd.2011.06.004 CrossRefPubMedGoogle Scholar
  51. 51.
    Paccalet T, Coulombe Z, Tremblay JP (2010) Ganglioside GM3 levels are altered in a mouse model of HIBM: GM3 as a cellular marker of the disease. PLoS One 5(4):e10055. doi: 10.1371/journal.pone.0010055 CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Bork K, Reutter W, Weidemann W, Horstkorte R (2007) Enhanced sialylation of EPO by overexpression of UDP-GlcNAc 2-epimerase/ManAc kinase containing a sialuria mutation in CHO cells. FEBS Lett 581(22):4195–4198. doi: 10.1016/j.febslet.2007.07.060 CrossRefPubMedGoogle Scholar
  53. 53.
    Kontou M, Weidemann W, Bork K, Horstkorte R (2009) Beyond glycosylation: sialic acid precursors act as signaling molecules and are involved in cellular control of differentiation of PC12 cells. Biol Chem 390(7):575–579. doi: 10.1515/BC.2009.058 CrossRefPubMedGoogle Scholar
  54. 54.
    Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25(34):4798–4811. doi: 10.1038/sj.onc.1209608 CrossRefPubMedGoogle Scholar
  55. 55.
    Filosto M, Scarpelli M, Cotelli MS, Vielmi V, Todeschini A, Gregorelli V, Tonin P, Tomelleri G et al (2011) The role of mitochondria in neurodegenerative diseases. J Neurol 258(10):1763–1774. doi: 10.1007/s00415-011-6104-z CrossRefPubMedGoogle Scholar
  56. 56.
    Kang SU, Cho JH, Chang JW, Shin YS, Kim KI, Park JK, Yang SS, Lee JS et al (2014) Nonthermal plasma induces head and neck cancer cell death: the potential involvement of mitogen-activated protein kinase-dependent mitochondrial reactive oxygen species. Cell Death Dis 5:e1056. doi: 10.1038/cddis.2014.33 CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Adhihetty PJ, O'Leary MF, Hood DA (2008) Mitochondria in skeletal muscle: adaptable rheostats of apoptotic susceptibility. Exerc Sport Sci Rev 36(3):116–121. doi: 10.1097/JES.0b013e31817be7b7 CrossRefPubMedGoogle Scholar
  58. 58.
    Reissig JL, Storminger JL, Leloir LF (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217(2):959–966PubMedGoogle Scholar
  59. 59.
    Hamann S, Metrailler S, Schorderet DF, Cottet S (2013) Analysis of the cytoprotective role of alpha-crystallins in cell survival and implication of the alphaA-crystallin C-terminal extension domain in preventing Bax-induced apoptosis. PLoS One 8(2):e55372. doi: 10.1371/journal.pone.0055372 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.School of BiotechnologyJawaharlal Nehru UniversityNew DelhiIndia

Personalised recommendations