Skip to main content
SpringerLink
Log in

In Silico Characterization of the Binding Affinity of Dendrimers to Penicillin-Binding Proteins (PBPs): Can PBPs be Potential Targets for Antibacterial Dendrimers?

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

We have shown that novel silver salts of poly (propyl ether) imine (PETIM) dendron and dendrimers developed in our group exhibit preferential antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus. This led us to examine whether molecular modeling methods could be used to identify the key structural design principles for a bioactive lead molecule, explore the mechanism of binding with biological targets, and explain their preferential antibacterial activity. The current article reports the conformational landscape as well as mechanism of binding of generation 1 PETIM dendron and dendrimers to penicillin-binding proteins (PBPs) in order to understand the antibacterial activity profiles of their silver salts. Molecular dynamics at different simulation protocols and conformational analysis were performed to elaborate on the conformational features of the studied dendrimers, as well as to create the initial structure for further binding studies. The results showed that for all compounds, there were no significant conformational changes due to variation in simulation conditions. Molecular docking calculations were performed to investigate the binding theme between the studied dendrimers and PBPs. Interestingly, in significant accordance with the experimental data, dendron and dendrimer with aliphatic cores were found to show higher activity against S. aureus than the dendrimer with an aromatic core. The latter showed higher activity against MRSA. The findings from this computational and molecular modeling report together with the experimental results serve as a road map toward designing more potent antibacterial dendrimers against resistant bacterial strains.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13

Similar content being viewed by others

References

  1. Davies, J., & Davies, D. (2010). Origins and evolution of antibiotic resistance. Microbiology and Molecular Biology Reviews, 74, 417–433. doi:10.1128/MMBR.00016-10.

    Article  CAS  Google Scholar 

  2. http://www.wired.com/2014/12/oneill-rpt-amr/ - date accessed 6 March 2015. (n.d.).

  3. Diacon, A. H., Dawson, R., von Groote-Bidlingmaier, F., Symons, G., Venter, A., Donald, P. R., et al. (2015). Bactericidal activity of pyrazinamide and clofazimine alone and in combinations with pretomanid and bedaquiline. American Journal of Respiratory and Critical Care Medicine. doi:10.1164/rccm.201410-1801OC.

    Google Scholar 

  4. Muhindo Mavoko, H., Nabasumba, C., Tinto, H., D’Alessandro, U., Grobusch, M. P., Lutumba, P., et al. (2013). Impact of retreatment with an artemisinin-based combination on malaria incidence and its potential selection of resistant strains: study protocol for a randomized controlled clinical trial. Trials, 14, 307. doi:10.1186/1745-6215-14-307.

    Article  Google Scholar 

  5. Duse, A. G. (2011). The global antibiotic resistance partnership (GARP). South African Medical Journal, 101, 551. http://www.ncbi.nlm.nih.gov/pubmed/21936137 (accessed February 9, 2015).

    Google Scholar 

  6. Hosseinipour, M. C., Gupta, R. K., Van Zyl, G., Eron, J. J., & Nachega, J. B. (2013). Emergence of HIV drug resistance during first- and second-line antiretroviral therapy in resource-limited settings. The Journal of Infectious Diseases, 207(Suppl), S49–S56. doi:10.1093/infdis/jit107.

    Article  CAS  Google Scholar 

  7. White, N. J. (2004). Antimalarial drug resistance. The Journal of Clinical Investigation, 113, 1084–1092. doi:10.1172/JCI21682.

    Article  CAS  Google Scholar 

  8. http://www.nanowerk.com/spotlight/spotid=32188.php - date accessed 7 Jan 2015. (n.d.).

  9. Ferber, D. (2010). Infectious disease. From pigs to people: the emergence of a new superbug. Science, 329, 1010–1011. doi:10.1126/science.329.5995.1010.

    Article  CAS  Google Scholar 

  10. Kardas, P. (2002). Patient compliance with antibiotic treatment for respiratory tract infections. The Journal of Antimicrobial Chemotherapy, 49, 897–903. doi:10.1093/jac/dkf046.

    Article  CAS  Google Scholar 

  11. Huh, A. J., & Kwon, Y. J. (2011). “Nanoantibiotics”: a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era. Journal of Controlled Release, 156, 128–145. doi:10.1016/j.jconrel.2011.07.002.

    Article  CAS  Google Scholar 

  12. Hajipour, M. J., Fromm, K. M., Akbar Ashkarran, A., Jimenez de Aberasturi, D., De Larramendi, I. R., Rojo, T., et al. (2012). Antibacterial properties of nanoparticles. Trends in Biotechnology, 30, 499–511. doi:10.1016/j.tibtech.2012.06.004.

    Article  CAS  Google Scholar 

  13. Pelgrift, R. Y., & Friedman, A. J. (2013). Nanotechnology as a therapeutic tool to combat microbial resistance. Advanced Drug Delivery Reviews, 65, 1803–1815. doi:10.1016/j.addr.2013.07.011.

    Article  CAS  Google Scholar 

  14. Aruguete, D. M., Kim, B., Hochella, M. F., Ma, Y., Cheng, Y., Hoegh, A., et al. (2013). Antimicrobial nanotechnology: its potential for the effective management of microbial drug resistance and implications for research needs in microbial nanotoxicology. Environmental Science: Processes and Impacts, 15, 93. doi:10.1039/c2em30692a.

    CAS  Google Scholar 

  15. Kalhapure, R. S., Suleman, N., Mocktar, C., Seedat, N., & Govender, T. (2014). Nanoengineered drug delivery systems for enhancing antibiotic therapy. Journal of Pharmaceutical Sciences. doi:10.1002/jps.24298.

    Google Scholar 

  16. Lopez, A. I., Reins, R. Y., McDermott, A. M., Trautner, B. W., & Cai, C. (2009). Antibacterial activity and cytotoxicity of PEGylated poly(amidoamine) dendrimers. Molecular BioSystems, 5, 1148–1156. doi:10.1039/b904746h.

    Article  CAS  Google Scholar 

  17. Wang, B., Navath, R. S., Menjoge, A. R., Balakrishnan, B., Bellair, R., Dai, H., et al. (2010). Inhibition of bacterial growth and intramniotic infection in a guinea pig model of chorioamnionitis using PAMAM dendrimers. International Journal of Pharmaceutics, 395, 298–308. doi:10.1016/j.ijpharm.2010.05.030.

    Article  CAS  Google Scholar 

  18. Xue, X., Chen, X., Mao, X., Hou, Z., Zhou, Y., Bai, H., et al. (2013). Amino-terminated generation 2 poly(amidoamine) dendrimer as a potential broad-spectrum, nonresistance-inducing antibacterial agent. The AAPS Journal, 15, 132–142. doi:10.1208/s12248-012-9416-8.

    Article  CAS  Google Scholar 

  19. Vembu, S., Pazhamalai, S., & Gopalakrishnan, M. (2015). Potential antibacterial activity of triazine dendrimer: synthesis and controllable drug release properties. Bioorganic and Medicinal Chemistry, 23, 4561–4566. doi:10.1016/j.bmc.2015.06.009.

    Article  CAS  Google Scholar 

  20. Meyers, S. R., Juhn, F. S., Griset, A. P., Luman, N. R., Grinstaff, M. W. (2008). Anionic amphiphilic dendrimers as antibacterial agents dendritic structure enables the attachment of a multitude of drugs. 14444–14445.

  21. Wu, L., Ficker, M., Christensen, J. B., Trohopoulos, P., Moghimi, S. M. (2015). Dendrimers in medicine: therapeutic concepts and pharmaceutical challenges. Bioconjugate Chemistry. doi:10.1021/acs.bioconjchem.5b00031.

  22. Labieniec-Watala, M., & Watala, C. (2015). PAMAM dendrimers: destined for success or doomed to fail? Plain and modified PAMAM dendrimers in the context of biomedical applications. Journal of Pharmaceutical Sciences, 104, 2–14. doi:10.1002/jps.24222.

    Article  CAS  Google Scholar 

  23. Tolosa, J., Romero-Nieto, C., Díez-Barra, E., Sánchez-Verdú, P., & Rodríguez-López, J. (2007). Control of surface functionality in poly(phenylenevinylene) dendritic architectures. The Journal of Organic Chemistry, 72, 3847–3852. doi:10.1021/jo070210v.

    Article  CAS  Google Scholar 

  24. Boas, U., Christensen, J. B., & Heegaard, P. M. H. (2006). Dendrimers in medicine and biotechnology. Cambridge: Royal Society of Chemistry. doi:10.1039/9781847552679.

    Google Scholar 

  25. Chen, C. Z., & Cooper, S. L. (2002). Interactions between dendrimer biocides and bacterial membranes. Biomaterials, 23, 3359–3368. http://www.ncbi.nlm.nih.gov/pubmed/12099278 (accessed February 11, 2015).

    Article  CAS  Google Scholar 

  26. Chen, C. Z., Beck-Tan, N. C., Dhurjati, P., van Dyk, T. K., LaRossa, R. A., & Cooper, S. L. (2000). Quaternary ammonium functionalized poly(propylene imine) dendrimers as effective antimicrobials: structure-activity studies. Biomacromolecules, 1, 473–480. http://www.ncbi.nlm.nih.gov/pubmed/11710139 (accessed February 11, 2015).

    Article  CAS  Google Scholar 

  27. Nishikawa, K., Matsuoka, K., Kita, E., Okabe, N., Mizuguchi, M., Hino, K., et al. (2002). A therapeutic agent with oriented carbohydrates for treatment of infections by Shiga toxin-producing Escherichia coli O157:H7. Proceedings of the National Academy of Sciences of the United States of America, 99, 7669–7674. doi:10.1073/pnas.112058999.

    Article  CAS  Google Scholar 

  28. Ortega, P., Copa-Patiño, J. L., Muñoz-Fernandez, M. A., Soliveri, J., Gomez, R., & de la Mata, F. J. (2008). Amine and ammonium functionalization of chloromethylsilane-ended dendrimers. Antimicrobial activity studies. Organic and Biomolecular Chemistry, 6, 3264–3269. doi:10.1039/b809569h.

    Article  CAS  Google Scholar 

  29. Janiszewska, J., & Urbańczyk-Lipkowska, Z. (2006). Synthesis, antimicrobial activity and structural studies of low molecular mass lysine dendrimers. Acta Biochimica Polonica, 53, 77–82. http://www.ncbi.nlm.nih.gov/pubmed/16496039 (accessed December 19, 2015).

    CAS  Google Scholar 

  30. Tülü, M., Ertürk, A. S. (2012). A search for antibacterial agents. InTech. doi:10.5772/1085.

  31. Soto-Castro, D., Evangelista-Lara, A., & Guadarrama, P. (2006). Theoretical design of dendrimeric fractal patterns for the encapsulation of a family of drugs: salicylanilides. Tetrahedron, 62, 12116–12125. doi:10.1016/j.tet.2006.08.053.

    Article  CAS  Google Scholar 

  32. Bharatam, P. V., Sundriyal, S. Molecular electrostatic potentials in the design of dendrimers for the delivery of glitazones. Journal of Nanoscience and Nanotechnology 6:3277–82. http://www.ncbi.nlm.nih.gov/pubmed/17048547. Accessed 26 Feb 2015.

  33. Pricl, S. (2001). Molecular simulation of host–guest inclusion compounds: an approach to the lactodendrimers case. Carbohydrate Polymers, 45, 23–33. doi:10.1016/S0144-8617(00)00241-1.

    Article  CAS  Google Scholar 

  34. Abderrezak, A., Bourassa, P., Mandeville, J.-S., Sedaghat-Herati, R., & Tajmir-Riahi, H.-A. (2012). Dendrimers bind antioxidant polyphenols and cisplatin drug. PloS One, 7, e33102. doi:10.1371/journal.pone.0033102.

    Article  CAS  Google Scholar 

  35. Ouyang, D., Zhang, H., Parekh, H. S., & Smith, S. C. (2011). The effect of pH on PAMAM dendrimer-siRNA complexation: endosomal considerations as determined by molecular dynamics simulation. Biophysical Chemistry, 158, 126–133. doi:10.1016/j.bpc.2011.06.003.

    Article  CAS  Google Scholar 

  36. Metullio, L., Ferrone, M., Coslanich, A., Fuchs, S., Fermeglia, M., Paneni, M. S., et al. Polyamidoamine (Yet Not PAMAM) dendrimers as bioinspired materials for drug delivery: structure-activity relationships by molecular simulations. Biomacromolecules. 5:1371–1378. doi:10.1021/bm049858x.

  37. Kim, S. H., & Lamm, M. H. (2011). Reintroducing explicit solvent to a solvent-free coarse-grained model. Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics, 84, 025701. http://www.ncbi.nlm.nih.gov/pubmed/21929055 (accessed February 26, 2015).

    Article  Google Scholar 

  38. Suleman, N., Kalhapure, R. S., Mocktar, C., Rambharose, S., Singh, M., & Govender, T. (2015). Silver salts of carboxylic acid terminated generation 1 poly (propyl ether imine) (PETIM) dendron and dendrimers as antimicrobial agents against S. aureus and MRSA. RSC Advances, 5, 34967–34978. doi:10.1039/C5RA03179F.

    Article  CAS  Google Scholar 

  39. Georgopapadakou, N. H., Dix, B. A., & Mauriz, Y. R. (1986). Possible physiological functions of penicillin-binding proteins in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 29, 333–336. doi:10.1128/AAC.29.2.333.

    Article  CAS  Google Scholar 

  40. Macheboeuf, P., Contreras-Martel, C., Job, V., Dideberg, O., & Dessen, A. (2006). Penicillin binding proteins: key players in bacterial cell cycle and drug resistance processes. FEMS Microbiology Review, 30, 673–691. doi:10.1111/j.1574-6976.2006.00024.x.

    Article  CAS  Google Scholar 

  41. Scheffers, D.-J., & Pinho, M. G. (2005). Bacterial cell wall synthesis: new insights from localization studies. Microbiology and Molecular Biology Reviews, 69, 585–607. doi:10.1128/MMBR.69.4.585-607.2005.

    Article  CAS  Google Scholar 

  42. Chambers, H. F., & Sachdeva, M. (1990). Binding of beta-lactam antibiotics to penicillin-binding proteins in methicillin-resistant Staphylococcus aureus. The Journal of Infectious Diseases, 161, 1170–1176.

    Article  CAS  Google Scholar 

  43. Contreras-Martel, C., Amoroso, A., Woon, E. C. Y., Zervosen, A., Inglis, S., Martins, A., et al. (2011). Structure-guided design of cell wall biosynthesis inhibitors that overcome β-lactam resistance in Staphylococcus aureus (MRSA). ACS Chemical Biology, 6, 943–951. doi:10.1021/cb2001846.

    Article  CAS  Google Scholar 

  44. Navratna, V., Nadig, S., Sood, V., Prasad, K., Arakere, G., & Gopal, B. (2010). Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance. Journal of Bacteriology, 192, 134–144. doi:10.1128/JB.00822-09.

    Article  CAS  Google Scholar 

  45. Otero, L. H., Rojas-Altuve, A., Llarrull, L. I., Carrasco-Lopez, C., Kumarasiri, M., Lastochkin, E., et al. (2013). How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function. Proceedings of the National Academy of Sciences of the United States of America, 110, 16808–16813. doi:10.1073/pnas.1300118110.

    Article  Google Scholar 

  46. Yoshida, H., Kawai, F., Obayashi, E., Akashi, S., Roper, D. I., Tame, J. R. H., et al. (2012). Crystal structures of penicillin-binding protein 3 (PBP3) from methicillin-resistant staphylococcus aureus in the Apo and cefotaxime-bound forms. Journal of Molecular Biology, 423, 351–364. doi:10.1016/j.jmb.2012.07.012.

    Article  CAS  Google Scholar 

  47. Wada, A., & Watanabe, H. (1998). Penicillin-binding protein 1 of Staphylococcus aureus is essential for growth. Journal of Bacteriology, 180, 2759–2765.

    CAS  Google Scholar 

  48. Pinho, M. G., de Lencastre, H., & Tomasz, A. (2001). An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug-resistant staphylococci. Proceedings of the National Academy of Sciences of the United States of America, 98, 10886–10891. doi:10.1073/pnas.191260798.

    Article  CAS  Google Scholar 

  49. Henze, U. U., & Berger-Bächi, B. (1995). Staphylococcus aureus penicillin-binding protein 4 and intrinsic beta-lactam resistance. Antimicrobial Agents and Chemotherapy, 39, 2415–2422.

    Article  CAS  Google Scholar 

  50. Case, D. A., Iii, T. E. C., Darden, T. O. M., Gohlke, H., Jr, K. M. M., Onufriev, A., et al. (2007). The amber biomolecular simulation programs. Journal of Combinatorial Chemistry, 26, 1668–1688.

    Google Scholar 

  51. Pearlman, D. A., Case, D. A., Caldwell, J. W., Ross, W. S., Cheatham, T. E., Debolt, S., et al. (1995). AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules. Computer Physics Communications, 91, 1–41.

    Article  CAS  Google Scholar 

  52. Lindorff-larsen, K., Piana, S., Palmo, K., Maragakis, P., Klepeis, J. L., Dror, R. O., et al. (2010). Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins: Structure, Function and Bioinformatics, 78, 1950–1958. doi:10.1002/prot.22711.

    CAS  Google Scholar 

  53. Taylor, P., Iii, T. E. C., Cieplak, P., Kollman, P. A., Version, A. M., Thomas, F., et al. (2012). A modified version of the Cornell et al. . Force field with improved sugar pucker phases and helical repeat. Journal of Biomolecular Structure and Dynamics, 16, 37–41. doi:10.1080/07391102.1999.10508297.

    Google Scholar 

  54. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A., & Case, D. A. (2004). Development and testing of a general amber force field. Journal of Computational Chemistry, 25, 1157–1174.

    Article  CAS  Google Scholar 

  55. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., Klein, M. L., Jorgensen, W. L., et al. (1983). Comparison of simple potential functions for simulating liquid water comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics, 79, 926–935. doi:10.1063/1.445869.

    Article  CAS  Google Scholar 

  56. Berendsen, H. J. C., Grigera, J. R., & Straatsma, T. P. (1987). The missing term in effective pair potentials. Journal of Physical Chemistry, 91, 6269–6271.

    Article  CAS  Google Scholar 

  57. Harvey, M. J., & De Fabritiis, G. (2009). An implementation of the smooth particle mesh Ewald method on GPU hardware. Journal of Chemical Theory and Computation, 5, 2371–2377.

    Article  CAS  Google Scholar 

  58. Berendsen, H. J. C., Postma, J. P. M., Van Gunsteren, W. F., Dinola, A., Haak, J. R., Berendsen, H. J. C., et al. (1984). Molecular dynamics with coupling to an external bath. The Journal of Chemical Physics, 81, 3684–3690. doi:10.1063/1.448118.

    Article  CAS  Google Scholar 

  59. Schrödinger Release 2015–1: Maestro, version 10.1. Schrödinger, LLC, New York, NY, 2015., (n.d.).

  60. Schrödinger Release 2015–1: LigPrep, version 3.3. Schrödinger, LLC, New York, NY, 2015., (n.d.).

  61. Schrödinger Release 2015–1: Schrödinger Suite 2015–1 Protein Preparation Wizard; Epik version 3.1, Schrödinger, LLC, New York, NY, 2015; Impact version 6.6, Schrödinger, LLC, New York, NY, 2015; Prime version 3.9, Schrödinger, LLC, New York, NY, 2015., (n.d.).

  62. Dundas, J., Ouyang, Z., Tseng, J., Binkowski, A., Turpaz, Y., & Liang, J. (2006). CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Research, 34, 116–118. doi:10.1093/nar/gkl282.

    Article  Google Scholar 

  63. Gohlke, H., & Klebe, G. (2002). Approaches to the description and prediction of the binding affinity of small-molecule ligands to macromolecular receptors. Angewandte Chemie – International Edition, 41, 2644–2676. doi:10.1002/1521-3773(20020802)41:15<2644::AID-ANIE2644>3.0.CO;2-O.

    Article  CAS  Google Scholar 

  64. Nagy, P. (2014). Competing intramolecular vs. intermolecular hydrogen bonds in solution. doi:10.3390/ijms151119562.

  65. Sauvage, E., Derouaux, A., Fraipont, C., Joris, M., Herman, R., Rocaboy, M., et al. (2014). Crystal structure of penicillin-binding protein 3 (PBP3) from Escherichia coli. PloS One, 9, e98042. doi:10.1371/journal.pone.0098042.

    Article  Google Scholar 

  66. Hargis, J. C., Vankayala, S. L., White, J. K., & Woodcock, H. L. (2014). Identification and characterization of noncovalent interactions that drive binding and specificity in DD-peptidases and β-lactamases. Journal of Chemical Theory and Computation, 10, 855–864. doi:10.1021/ct400968v.

    Article  CAS  Google Scholar 

  67. Gallivan, J. P., & Dougherty, D. A. (1999). Cation-pi interactions in structural biology. Proceedings of the National Academy of Sciences of the United States of America, 96, 9459–9464. doi:10.1073/pnas.96.17.9459.

    Article  CAS  Google Scholar 

  68. Lavanya, P., Ramaiah, S., & Anbarasu, A. (2013). Cation-π interactions in β-lactamases: the role in structural stability. Cell Biochemistry and Biophysics, 66, 147–155. doi:10.1007/s12013-012-9463-x.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are thankful to the National Research Foundation of South Africa and University of KwaZulu-Natal for financial support. Ms Carrin Martin is acknowledged for proofreading the manuscript.

Author information

Authors and Affiliations

  1. Discipline of Pharmaceutical Sciences, School of Health Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000, KwaZulu-Natal, South Africa

    Shaimaa Ahmed, Suresh B. Vepuri, Muthusamy Ramesh, Rahul Kalhapure, Nadia Suleman & Thirumala Govender

Authors
  1. Shaimaa Ahmed
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suresh B. Vepuri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Muthusamy Ramesh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rahul Kalhapure
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nadia Suleman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thirumala Govender
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thirumala Govender.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 3974 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ahmed, S., Vepuri, S.B., Ramesh, M. et al. In Silico Characterization of the Binding Affinity of Dendrimers to Penicillin-Binding Proteins (PBPs): Can PBPs be Potential Targets for Antibacterial Dendrimers?. Appl Biochem Biotechnol 178, 1546–1566 (2016). https://doi.org/10.1007/s12010-015-1967-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1967-6

Keywords

Search

Navigation