Phage display

The sequence of events that are followed in phage display screening to identify polypeptides that bind with high affinity to desired target protein or DNA sequence.

Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages (viruses that infect bacteria) to connect proteins with the genetic information that encodes them.[1] In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. These displaying phages can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection.

The most common bacteriophages used in phage display are M13 and fd filamentous phage,[2][3] though T4,[4] T7, and λ phage have also been used.

History

Phage display was first described by George P. Smith in 1985, when he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest onto gene III of filamentous phage.[1] A patent by George Pieczenik claiming priority from 1985 also describes the generation of phage display libraries.[5] This technology was further developed and improved by groups at the Laboratory of Molecular Biology with Greg Winter and John McCafferty, The Scripps Research Institute with Lerner and Barbas and the German Cancer Research Center with Breitling and Dübel for display of proteins such as antibodies for therapeutic protein engineering.

Principle

Like the two-hybrid system, phage display is used for the high-throughput screening of protein interactions. In the case of M13 filamentous phage display, the DNA encoding the protein or peptide of interest is ligated into the pIII or pVIII gene, encoding either the minor or major coat protein, respectively. Multiple cloning sites are sometimes used to ensure that the fragments are inserted in all three possible reading frames so that the cDNA fragment is translated in the proper frame. The phage gene and insert DNA hybrid is then inserted (a process known as "transduction") into Escherichia coli (E. coli) bacterial cells such as TG1, SS320, ER2738, or XL1-Blue E. coli. If a "phagemid" vector is used (a simplified display construct vector) phage particles will not be released from the E. coli cells until they are infected with helper phage, which enables packaging of the phage DNA and assembly of the mature virions with the relevant protein fragment as part of their outer coat on either the minor (pIII) or major (pVIII) coat protein. By immobilizing a relevant DNA or protein target(s) to the surface of a microtiter plate well, a phage that displays a protein that binds to one of those targets on its surface will remain while others are removed by washing. Those that remain can be eluted, used to produce more phage (by bacterial infection with helper phage) and so produce a phage mixture that is enriched with relevant (i.e. binding) phage. The repeated cycling of these steps is referred to as 'panning', in reference to the enrichment of a sample of gold by removing undesirable materials. Phage eluted in the final step can be used to infect a suitable bacterial host, from which the phagemids can be collected and the relevant DNA sequence excised and sequenced to identify the relevant, interacting proteins or protein fragments.

The use of a helper phage can be eliminated by using 'bacterial packaging cell line' technology.[6]

Applications

Applications of phage display technology include determination of interaction partners of a protein (which would be used as the immobilised phage "bait" with a DNA library consisting of all coding sequences of a cell, tissue or organism) so that the function or the mechanism of the function of that protein may be determined.[7] Phage display is also a widely used method for in vitro protein evolution (also called protein engineering). As such, phage display is a useful tool in drug discovery. It is used for finding new ligands (enzyme inhibitors, receptor agonists and antagonists) to target proteins.[8][9][10] The technique is also used to determine tumour antigens (for use in diagnosis and therapeutic targeting)[11] and in searching for protein-DNA interactions[12] using specially-constructed DNA libraries with randomised segments.

Competing methods for in vitro protein evolution include yeast display, bacterial display, ribosome display, and mRNA display.

Antibody maturation in vitro

The invention of antibody phage display revolutionised antibody drug discovery. Initial work was done by laboratories at the MRC Laboratory of Molecular Biology (Greg Winter and John McCafferty), the Scripps Research Institute (Richard Lerner and Carlos F. Barbas) and the German Cancer Research Centre (Frank Breitling and Stefan Dübel).[13][14][15] In 1991, The Scripps group reported the first display and selection of human antibodies on phage.[16] This initial study described the rapid isolation of human antibody Fab that bound tetanus toxin and the method was then extended to rapidly clone human anti-HIV-1 antibodies for vaccine design and therapy.[17][18][19][20][21]

Phage display of antibody libraries has become a powerful method for both studying the immune response as well as a method to rapidly select and evolve human antibodies for therapy. Antibody phage display was later used by Carlos F. Barbas at The Scripps Research Institute to create synthetic human antibody libraries, a principle first patented in 1990 by Breitling and coworkers (Patent CA 2035384), thereby allowing human antibodies to be created in vitro from synthetic diversity elements.[22][23][24][25]

Antibody libraries displaying millions of different antibodies on phage are often used in the pharmaceutical industry to isolate highly specific therapeutic antibody leads, for development into antibody drugs primarily as anti-cancer or anti-inflammatory therapeutics. One of the most successful was HUMIRA (adalimumab), discovered by Cambridge Antibody Technology as D2E7 and developed and marketed by Abbott Laboratories. HUMIRA, an antibody to TNF alpha, was the world's first fully human antibody,[26] which achieved annual sales exceeding $1bn.[27]

General protocol

Below is the sequence of events that are followed in phage display screening to identify polypeptides that bind with high affinity to desired target protein or DNA sequence:

  1. Target proteins or DNA sequences are immobilised to the wells of a microtiter plate.
  2. Many genetic sequences are expressed in a bacteriophage library in the form of fusions with the bacteriophage coat protein, so that they are displayed on the surface of the viral particle. The protein displayed corresponds to the genetic sequence within the phage.
  3. This phage-display library is added to the dish and after allowing the phage time to bind, the dish is washed.
  4. Phage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away.
  5. Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant phage (i.e. non-binding) than were present in the initial mixture.
  6. Steps 3 to 6 are optionally repeated one or more times, further enriching the phage library in binding proteins.
  7. Following further bacterial-based amplification, the DNA within in the interacting phage is sequenced to identify the interacting proteins or protein fragments.

Selection of the coat protein

Filamentous phages

pIII

pIII is the protein that determines the infectivity of the virion. pIII is composed of three domains (N1, N2 and CT) connected by glycine-rich linkers.[28] The N2 domain binds to the F pilus during virion infection freeing the N1 domain which then interacts with a TolA protein on the surface of the bacterium.[28] Insertions within this protein are usually added in position 249 (within a linker region between CT and N2), position 198 (within the N2 domain) and at the N-terminus (inserted between the N-terminal secretion sequence and the N-terminus of pIII).[28] However, when using the BamHI site located at position 198 one must be careful of the unpaired Cysteine residue (C201) that could cause problems during phage display if one is using a non-truncated version of pIII.[28]

An advantage of using pIII rather than pVIII is that pIII allows for monovalent display when using a phagemid (Ff-phage derived plasmid) combined with a helper phage. Moreover, pIII allows for the insertion of larger protein sequences (>100 amino acids)[29] and is more tolerant to it than pVIII. However, using pIII as the fusion partner can lead to a decrease in phage infectivity leading to problems such as selection bias caused by difference in phage growth rate[30] or even worse, the phage's inability to infect its host.[28] Loss of phage infectivity can be avoided by using a phagemid plasmid and a helper phage so that the resultant phage contains both wild type and fusion pIII.[28]

cDNA has also been analyzed using pIII via a two complementary leucine zippers system,[31] Direct Interaction Rescue[32] or by adding an 8-10 amino acid linker between the cDNA and pIII at the C-terminus.[33]

pVIII

pVIII is the main coat protein of Ff phages. Peptides are usually fused to the N-terminus of pVIII.[28] Usually peptides that can be fused to pVIII are 6-8 amino acids long.[28] The size restriction seems to have less to do with structural impediment caused by the added section[34] and more to do with the size exclusion caused by pIV during coat protein export.[34] Since there are around 2700 copies of the protein on a typical phages, it is more likely that the protein of interest will be expressed polyvalently even if a phagemid is used.[28] This makes the use of this protein unfavorable for the discovery of high affinity binding partners.[28]

To overcome the size problem of pVIII, artificial coat proteins have been designed.[35] An example is Weiss and Sidhu's inverted artificial coat protein (ACP) which allows the display of large proteins at the C-terminus.[35] The ACP's could display a protein of 20kDa, however, only at low levels (mostly only monovalently).[35]

pVI

pVI has been widely used for the display of cDNA libraries.[28] The display of cDNA libraries via phage display is an attractive alternative to the yeast-2-hybrid method for the discovery of interacting proteins and peptides due to its high throughput capability.[28] pVI has been used preferentially to pVIII and pIII for the expression of cDNA libraries because one can add the protein of interest to the C-terminus of pVI without greatly affecting pVI's role in phage assembly. This means that the stop codon in the cDNA is no longer an issue.[36] However, phage display of cDNA is always limited by the inability of most prokaryotes in producing post-translational modifications present in eukaryotic cells or by the misfolding of multi-domain proteins.

While pVI has been useful for the analysis of cDNA libraries, pIII and pVIII remain the most utilized coat proteins for phage display.[28]

pVII and pIX

In an experiment in 1995, display of Glutathione S-transferase was attempted on both pVII and pIX and failed.[37] However, phage display of this protein was completed successfully after the addition of a periplasmic signal sequence (pelB or ompA) on the N-terminus.[38] In a recent study, it has been shown that AviTag, FLAG and His could be displayed on pVII without the need of a signal sequence. Then the expression of single chain Fv's (scFv), and single chain T cell receptors (scTCR) were expressed both with and without the signal sequence.[39]

PelB (an amino acid signal sequence that targets the protein to the periplasm where a signal peptidase then cleaves off PelB) improved the phage display level when compared to pVII and pIX fusions without the signal sequence. However, this led to the incorporation of more helper phage genomes rather than phagemid genomes. In all cases, phage display levels were lower than using pIII fusion. However, lower display might be more favorable for the selection of binders due to lower display being closer to true monovalent display. In five out of six occasions, pVII and pIX fusions without pelB was more efficient than pIII fusions in affinity selection assays. The paper even goes on to state that pVII and pIX display platforms may outperform pIII in the long run.[39]

The use of pVII and pIX instead of pIII might also be an advantage because virion rescue may be undertaken without breaking the virion-antigen bond if the pIII used is wild type. Instead, one could cleave in a section between the bead and the antigen to elute. Since the pIII is intact it does not matter whether the antigen remains bound to the phage.[39]

T7 phages

The issue of using Ff phages for phage display is that they require the protein of interest to be translocated across the bacterial inner membrane before they are assembled into the phage.[40] Some proteins cannot undergo this process and therefore cannot be displayed on the surface of Ff phages. In these cases, T7 phage display is used instead.[40] In T7 phage display, the protein to be displayed is attached to the C-terminus of the gene 10 capsid protein of T7.[40]

The disadvantage of using T7 is that the size of the protein that can be expressed on the surface is limited to shorter peptides because large changes to the T7 genome cannot be accommodated like it is in M13 where the phage just makes its coat longer to fit the larger genome within it. However, it can be useful for the production of a large protein library for scFV selection where the scFV is expressed on an M13 phage and the antigens are expressed on the surface of the T7 phage.[41]

Bioinformatics resources and tools

Databases and computational tools for mimotopes have been an important part of phage display study.[42] Databases,[43] programs and web servers[44] have been widely used to exclude target-unrelated peptides,[45] characterize small molecules-protein interactions and map protein-protein interactions. Users can use three dimensional structure of a protein and the peptides selected from phage display experiment to map conformational eptiopes. Some of the fast and efficient computational methods are available online (e.g EpiSearch [44] http://curie.utmb.edu/episearch.html ).

See also

Competing techniques:

References

  1. 1 2 Smith GP (June 1985). "Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface". Science 228 (4705): 1315–7. Bibcode:1985Sci...228.1315S. doi:10.1126/science.4001944. PMID 4001944.
  2. ↑ Smith GP, Petrenko VA (April 1997). "Phage Display". Chem. Rev. 97 (2): 391–410. doi:10.1021/cr960065d. PMID 11848876.
  3. ↑ Kehoe JW, Kay BK (November 2005). "Filamentous phage display in the new millennium". Chem. Rev. 105 (11): 4056–72. doi:10.1021/cr000261r. PMID 16277371.
  4. ↑ Malys N, Chang DY, Baumann RG, Xie D, Black LW (2002). "A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4 terminase (gp17) and late sigma factor (gp55) interaction". J Mol Biol 319 (2): 289–304. doi:10.1016/S0022-2836(02)00298-X. PMID 12051907.
  5. ↑ US patent 5866363, Pieczenik G, "Method and means for sorting and identifying biological information", published 1999-02-02
  6. ↑ Chasteen L, Ayriss J, Pavlik P, Bradbury AR (2006). "Eliminating helper phage from phage display". Nucleic Acids Res. 34 (21): e145. doi:10.1093/nar/gkl772. PMC 1693883. PMID 17088290.
  7. ↑ Explanation of "Protein interaction mapping" from The Wellcome Trust
  8. ↑ Lunder M, Bratkovic T, Doljak B, Kreft S, Urleb U, Strukelj B, Plazar N (November 2005). "Comparison of bacterial and phage display peptide libraries in search of target-binding motif". Appl. Biochem. Biotechnol. 127 (2): 125–31. doi:10.1385/ABAB:127:2:125. PMID 16258189.
  9. ↑ Bratkovic T, Lunder M, Popovic T, Kreft S, Turk B, Strukelj B, Urleb U (July 2005). "Affinity selection to papain yields potent peptide inhibitors of cathepsins L, B, H, and K". Biochem. Biophys. Res. Commun. 332 (3): 897–903. doi:10.1016/j.bbrc.2005.05.028. PMID 15913550.
  10. ↑ Lunder M, Bratkovic T, Kreft S, Strukelj B (July 2005). "Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies". J. Lipid Res. 46 (7): 1512–6. doi:10.1194/jlr.M500048-JLR200. PMID 15863836.
  11. ↑ Hufton SE, Moerkerk PT, Meulemans EV, de Bruïne A, Arends JW, Hoogenboom HR (December 1999). "Phage display of cDNA repertoires: the pVI display system and its applications for the selection of immunogenic ligands". J. Immunol. Methods 231 (1–2): 39–51. doi:10.1016/S0022-1759(99)00139-8. PMID 10648926.
  12. ↑ Gommans WM, Haisma HJ, Rots MG (December 2005). "Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command". J. Mol. Biol. 354 (3): 507–19. doi:10.1016/j.jmb.2005.06.082. PMID 16253273.
  13. ↑ McCafferty, J.; Griffiths, A.; Winter, G.; Chiswell, D. (1990). "Phage antibodies: filamentous phage displaying antibody variable domains". Nature 348 (6301): 552–554. Bibcode:1990Natur.348..552M. doi:10.1038/348552a0. PMID 2247164.
  14. ↑ Scott JS, Barbas CF III, Burton, DA (2001). Phage Display: A Laboratory Manual. Plainview, N.Y: Cold Spring Harbor Laboratory Press. ISBN 0-87969-740-7.
  15. ↑ Breitling, F.; Dübel, S.; Seehaus, T.; Klewinghaus, I.; Little, M. (1991). "A surface expression vector for antibody screening". Gene 104 (2): 147–53. doi:10.1016/0378-1119(91)90244-6. PMID 1916287.
  16. ↑ Barbas CF, Kang AS, Lerner RA, Benkovic SJ (September 1991). "Assembly of combinatorial antibody libraries on phage surfaces: the gene III site". Proc. Natl. Acad. Sci. U.S.A. 88 (18): 7978–82. Bibcode:1991PNAS...88.7978B. doi:10.1073/pnas.88.18.7978. PMC 52428. PMID 1896445.
  17. ↑ Burton DR, Barbas CF, Persson MA, Koenig S, Chanock RM, Lerner RA (November 1991). "A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals". Proc. Natl. Acad. Sci. U.S.A. 88 (22): 10134–7. Bibcode:1991PNAS...8810134B. doi:10.1073/pnas.88.22.10134. PMC 52882. PMID 1719545.
  18. ↑ Barbas CF, Björling E, Chiodi F, Dunlop N, Cababa D, Jones TM, Zebedee SL, Persson MA, Nara PL, Norrby E (October 1992). "Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro". Proc. Natl. Acad. Sci. U.S.A. 89 (19): 9339–43. Bibcode:1992PNAS...89.9339B. doi:10.1073/pnas.89.19.9339. PMC 50122. PMID 1384050.
  19. ↑ Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PW, Sawyer LS, Hendry RM, Dunlop N, Nara PL (November 1994). "Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody". Science 266 (5187): 1024–7. Bibcode:1994Sci...266.1024B. doi:10.1126/science.7973652. PMID 7973652.
  20. ↑ Yang WP, Green K, Pinz-Sweeney S, Briones AT, Burton DR, Barbas CF (December 1995). "CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range". J. Mol. Biol. 254 (3): 392–403. doi:10.1006/jmbi.1995.0626. PMID 7490758.
  21. ↑ Barbas CF, Hu D, Dunlop N, Sawyer L, Cababa D, Hendry RM, Nara PL, Burton DR (April 1994). "In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity". Proc. Natl. Acad. Sci. U.S.A. 91 (9): 3809–13. Bibcode:1994PNAS...91.3809B. doi:10.1073/pnas.91.9.3809. PMC 43671. PMID 8170992.
  22. ↑ Barbas CF, Bain JD, Hoekstra DM, Lerner RA (May 1992). "Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem". Proc. Natl. Acad. Sci. U.S.A. 89 (10): 4457–61. Bibcode:1992PNAS...89.4457B. doi:10.1073/pnas.89.10.4457. PMC 49101. PMID 1584777.
  23. ↑ Barbas CF, Languino LR, Smith JW (November 1993). "High-affinity self-reactive human antibodies by design and selection: targeting the integrin ligand binding site". Proc. Natl. Acad. Sci. U.S.A. 90 (21): 10003–7. Bibcode:1993PNAS...9010003B. doi:10.1073/pnas.90.21.10003. PMC 47701. PMID 7694276.
  24. ↑ Barbas CF, Wagner J (October 1995). "Synthetic Human Antibodies: Selecting and Evolving Functional Proteins". Methods 8 (2): 94–103. doi:10.1006/meth.1995.9997.
  25. ↑ Barbas CF (August 1995). "Synthetic human antibodies". Nat. Med. 1 (8): 837–9. doi:10.1038/nm0895-837. PMID 7585190.
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  27. ↑ Cambridge Antibody: Sales update | Company Announcements | Telegraph
  28. 1 2 3 4 5 6 7 8 9 10 11 12 13 Lowman HB, Clackson T (2004). "1.3". Phage display: a practical approach. Oxford [Oxfordshire]: Oxford University Press. pp. 10–11. ISBN 0-19-963873-X.
  29. ↑ Sidhu SS, Weiss GA, Wells JA (February 2000). "High copy display of large proteins on phage for functional selections". J. Mol. Biol. 296 (2): 487–95. doi:10.1006/jmbi.1999.3465. PMID 10669603.
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  31. ↑ Crameri R, Jaussi R, Menz G, Blaser K (November 1994). "Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of genes by specific gene-product/ligand interaction". Eur. J. Biochem. 226 (1): 53–8. doi:10.1111/j.1432-1033.1994.tb20025.x. PMID 7957259.
  32. ↑ Gramatikoff K, Georgiev O, Schaffner W (December 1994). "Direct interaction rescue, a novel filamentous phage technique to study protein-protein interactions". Nucleic Acids Res. 22 (25): 5761–2. doi:10.1093/nar/22.25.5761. PMC 310144. PMID 7838733.
  33. ↑ Fuh G, Sidhu SS (September 2000). "Efficient phage display of polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein". FEBS Lett. 480 (2-3): 231–4. doi:10.1016/s0014-5793(00)01946-3. PMID 11034335.
  34. 1 2 Malik P, Terry TD, Bellintani F, Perham RN (October 1998). "Factors limiting display of foreign peptides on the major coat protein of filamentous bacteriophage capsids and a potential role for leader peptidase". FEBS Lett. 436 (2): 263–6. doi:10.1016/s0014-5793(98)01140-5. PMID 9781692.
  35. 1 2 3 Weiss GA, Sidhu SS (June 2000). "Design and evolution of artificial M13 coat proteins". J. Mol. Biol. 300 (1): 213–9. doi:10.1006/jmbi.2000.3845. PMID 10864510.
  36. ↑ Jespers LS, Messens JH, De Keyser A, Eeckhout D, Van den Brande I, Gansemans YG, Lauwereys MJ, Vlasuk GP, Stanssens PE (April 1995). "Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI". Biotechnology (N.Y.) 13 (4): 378–82. doi:10.1038/nbt0495-378. PMID 9634780.
  37. ↑ Endemann H, Model P (July 1995). "Location of filamentous phage minor coat proteins in phage and in infected cells". J. Mol. Biol. 250 (4): 496–506. doi:10.1006/jmbi.1995.0393. PMID 7616570.
  38. ↑ Gao C, Mao S, Lo CH, Wirsching P, Lerner RA, Janda KD (May 1999). "Making artificial antibodies: a format for phage display of combinatorial heterodimeric arrays". Proc. Natl. Acad. Sci. U.S.A. 96 (11): 6025–30. Bibcode:1999PNAS...96.6025G. doi:10.1073/pnas.96.11.6025. PMC 26829. PMID 10339535.
  39. 1 2 3 Løset GÅ, Roos N, Bogen B, Sandlie I (2011). "Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage". PLoS ONE 6 (2): e17433. Bibcode:2011PLoSO...617433L. doi:10.1371/journal.pone.0017433. PMC 3044770. PMID 21390283.
  40. 1 2 3 Danner S, Belasco JG (November 2001). "T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries". Proc. Natl. Acad. Sci. U.S.A. 98 (23): 12954–9. Bibcode:2001PNAS...9812954D. doi:10.1073/pnas.211439598. PMC 60806. PMID 11606722.
  41. ↑ Castillo J, Goodson B, Winter J (November 2001). "T7 displayed peptides as targets for selecting peptide specific scFvs from M13 scFv display libraries". J. Immunol. Methods 257 (1-2): 117–22. doi:10.1016/s0022-1759(01)00454-9. PMID 11687245.
  42. ↑ Huang J, Ru B, Dai P (2011). "Bioinformatics resources and tools for phage display". Molecules 16 (1): 694–709. doi:10.3390/molecules16010694. PMID 21245805.
  43. ↑ Huang J, Ru B, Zhu P, Nie F, Yang J, Wang X, Dai P, Lin H, Guo FB, Rao N (January 2012). "MimoDB 2.0: a mimotope database and beyond". Nucleic Acids Res. 40 (Database issue): D271–7. doi:10.1093/nar/gkr922. PMC 3245166. PMID 22053087.
  44. 1 2 Negi SS, Braun W (2009). "Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences". Bioinform Biol Insights 3: 71–81. PMID 20140073.
  45. ↑ Huang J, Ru B, Li S, Lin H, Guo FB (2010). "SAROTUP: scanner and reporter of target-unrelated peptides". J. Biomed. Biotechnol. 2010: 101932. doi:10.1155/2010/101932. PMC 2842971. PMID 20339521.

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