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Volumn 96, Issue 7, 2009, Pages 2935-2948

Absolute quantitation of bacterial biofilm adhesion and viscoelasticity by microbead force spectroscopy

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL CELL; ARTICLE; ATOMIC FORCE MICROSCOPY; BACTERIAL BIOFILM; BACTERIAL CELL; BACTERIAL GROWTH; BACTERIAL STRAIN; BACTERIUM ADHERENCE; BIOFILM; CELL ADHESION; ECOSYSTEM REGENERATION; ENVIRONMENTAL STRESS; GRAM NEGATIVE BACTERIUM; MATHEMATICAL PARAMETERS; MICROBEAD FORCE SPECTROSCOPY; MICROBIAL COMMUNITY; NONHUMAN; PSEUDOMONAS AERUGINOSA; QUANTITATIVE STUDY; SPECTROSCOPY; VISCOELASTICITY; BIOMECHANICS; COST; CYTOLOGY; ELASTICITY; GENE EXPRESSION REGULATION; GENETICS; METABOLISM; MUTATION; PHYSIOLOGY; SENSITIVITY AND SPECIFICITY; SURFACE PROPERTY; TIME; VISCOSITY;

EID: 67649337216     PISSN: 00063495     EISSN: 15420086     Source Type: Journal    
DOI: 10.1016/j.bpj.2008.12.3943     Document Type: Article
Times cited : (137)

References (52)
  • 1
    • 0033591467 scopus 로고    scopus 로고
    • Bacterial biofilms: A common cause of persistent infections
    • Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms: a common cause of persistent infections. Science. 284:1318-1322.
    • (1999) Science , vol.284 , pp. 1318-1322
    • Costerton, J.W.1    Stewart, P.S.2    Greenberg, E.P.3
  • 2
    • 0037027377 scopus 로고    scopus 로고
    • Viscoelastic fluid description of bacterial biofilm material properties
    • Klapper, I., C. J. Rupp, R. Cargo, B. Purvedorj, and P. Stoodley. 2002. Viscoelastic fluid description of bacterial biofilm material properties. Biotechnol. Bioeng. 80:289-296.
    • (2002) Biotechnol. Bioeng , vol.80 , pp. 289-296
    • Klapper, I.1    Rupp, C.J.2    Cargo, R.3    Purvedorj, B.4    Stoodley, P.5
  • 5
    • 34447648722 scopus 로고    scopus 로고
    • Influence of culture conditions on Escherichia coli O157:H7 biofilm formation by atomic force microscopy
    • Oh, Y. J., W. Jo, Y. Yang, and S. Park. 2007. Influence of culture conditions on Escherichia coli O157:H7 biofilm formation by atomic force microscopy. Ultramicroscopy. 107:869-874.
    • (2007) Ultramicroscopy , vol.107 , pp. 869-874
    • Oh, Y.J.1    Jo, W.2    Yang, Y.3    Park, S.4
  • 6
    • 0033968812 scopus 로고    scopus 로고
    • Quantification of bacterial adhesion forces using atomic force microscopy (AFM)
    • Fang, H. H., K. Y. Chan, and L. C. Xu. 2000. Quantification of bacterial adhesion forces using atomic force microscopy (AFM). J. Microbiol. Methods. 40:89-97.
    • (2000) J. Microbiol. Methods , vol.40 , pp. 89-97
    • Fang, H.H.1    Chan, K.Y.2    Xu, L.C.3
  • 7
    • 0037142776 scopus 로고    scopus 로고
    • The measurement of Bacillus mycoides spore adhesion using atomic force microscopy, simple counting methods, and a spinning disk technique
    • Bowen, W. R., A. S. Fenton, R. W. Lovitt, and C. J. Wright. 2002. The measurement of Bacillus mycoides spore adhesion using atomic force microscopy, simple counting methods, and a spinning disk technique. Biotechnol. Bioeng. 79:170-179.
    • (2002) Biotechnol. Bioeng , vol.79 , pp. 170-179
    • Bowen, W.R.1    Fenton, A.S.2    Lovitt, R.W.3    Wright, C.J.4
  • 8
    • 8144222102 scopus 로고    scopus 로고
    • Nanoscale investigation of pathogenic microbial adhesion to a biomaterial
    • Emerson, R. J. T., and T. A. Camesano. 2004. Nanoscale investigation of pathogenic microbial adhesion to a biomaterial. Appl. Environ. Microbiol. 70:6012-6022.
    • (2004) Appl. Environ. Microbiol , vol.70 , pp. 6012-6022
    • Emerson, R.J.T.1    Camesano, T.A.2
  • 9
    • 33646565383 scopus 로고    scopus 로고
    • Nanoscale investigation on adhesion of E. coli to surface modified silicone using atomic force microscopy
    • Cao, T., H. Tang, X. Liang, A. Wang, G. W. Auner, et al. 2006. Nanoscale investigation on adhesion of E. coli to surface modified silicone using atomic force microscopy. Biotechnol. Bioeng. 94:167-176.
    • (2006) Biotechnol. Bioeng , vol.94 , pp. 167-176
    • Cao, T.1    Tang, H.2    Liang, X.3    Wang, A.4    Auner, G.W.5
  • 10
    • 30744471193 scopus 로고    scopus 로고
    • Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy
    • Touhami, A., M. H. Jericho, J. M. Boyd, and T. J. Beveridge. 2006. Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy. J. Bacteriol. 188:370-377.
    • (2006) J. Bacteriol , vol.188 , pp. 370-377
    • Touhami, A.1    Jericho, M.H.2    Boyd, J.M.3    Beveridge, T.J.4
  • 11
    • 34247542875 scopus 로고    scopus 로고
    • Force measurements of bacterial adhesion on metals using a cell probe atomic force microscope
    • Sheng, X., Y. P. Ting, and S. O. Pehkonen. 2007. Force measurements of bacterial adhesion on metals using a cell probe atomic force microscope. J. Colloid Interface Sci. 310:661-669.
    • (2007) J. Colloid Interface Sci , vol.310 , pp. 661-669
    • Sheng, X.1    Ting, Y.P.2    Pehkonen, S.O.3
  • 12
    • 36649013657 scopus 로고    scopus 로고
    • Direct measurement of hydrophobic forces on cell surfaces using AFM
    • Alsteens, D., E. Dague, P. G. Rouxhet, A. R. Baulard, and Y. F. Dufrene. 2007. Direct measurement of hydrophobic forces on cell surfaces using AFM. Langmuir. 23:11977-11979.
    • (2007) Langmuir , vol.23 , pp. 11977-11979
    • Alsteens, D.1    Dague, E.2    Rouxhet, P.G.3    Baulard, A.R.4    Dufrene, Y.F.5
  • 13
    • 34247873024 scopus 로고    scopus 로고
    • Intermolecular forces and enthalpies in the adhesion of Streptococcus mutans and an antigen I/II-deficient mutant to laminin films
    • Busscher, H. J., B. van de Belt-Gritter, R. J. B. Dijkstra, W. Norde, F. C. Petersen, et al. 2007. Intermolecular forces and enthalpies in the adhesion of Streptococcus mutans and an antigen I/II-deficient mutant to laminin films. J. Bacteriol. 189:2988-2995.
    • (2007) J. Bacteriol , vol.189 , pp. 2988-2995
    • Busscher, H.J.1    van de Belt-Gritter, B.2    Dijkstra, R.J.B.3    Norde, W.4    Petersen, F.C.5
  • 14
    • 36849078685 scopus 로고    scopus 로고
    • Quantifying the adhesion and interaction forces between Pseudomonas aeruginosa and natural organic matter
    • Abu-Lail, L. I., Y. Liu, A. Atabek, and T. A. Camesano. 2007. Quantifying the adhesion and interaction forces between Pseudomonas aeruginosa and natural organic matter. Environ. Sci. Technol. 41:8031-8037.
    • (2007) Environ. Sci. Technol , vol.41 , pp. 8031-8037
    • Abu-Lail, L.I.1    Liu, Y.2    Atabek, A.3    Camesano, T.A.4
  • 16
    • 0033527329 scopus 로고    scopus 로고
    • Structural deformation of bacterial biofilms caused by shortterm fluctuations in fluid shear: An in situ investigation of biofilm rheology
    • Stoodley, P., Z. Lewandowski, J. D. Boyle, and H. M. Lappin-Scott. 1999. Structural deformation of bacterial biofilms caused by shortterm fluctuations in fluid shear: an in situ investigation of biofilm rheology. Biotechnol. Bioeng. 65:83-92.
    • (1999) Biotechnol. Bioeng , vol.65 , pp. 83-92
    • Stoodley, P.1    Lewandowski, Z.2    Boyle, J.D.3    Lappin-Scott, H.M.4
  • 17
    • 0036955558 scopus 로고    scopus 로고
    • Biofilm material properties as related to shear-induced deformation and detachment phenomena
    • Stoodley, P., R. Cargo, C. J. Rupp, S. Wilson, and I. Klapper. 2002. Biofilm material properties as related to shear-induced deformation and detachment phenomena. J. Ind. Microbiol. Biotechnol. 29:361-367.
    • (2002) J. Ind. Microbiol. Biotechnol , vol.29 , pp. 361-367
    • Stoodley, P.1    Cargo, R.2    Rupp, C.J.3    Wilson, S.4    Klapper, I.5
  • 18
    • 0036945238 scopus 로고    scopus 로고
    • The influence of fluid shear on the structure and material properties of sulphate-reducing bacterial biofilms
    • Dunsmore, B. C., A. Jacobsen, L. Hall-Stoodley, C. J. Bass, H. M. Lappin-Scott, et al. 2002. The influence of fluid shear on the structure and material properties of sulphate-reducing bacterial biofilms. J. Ind. Microbiol. Biotechnol. 29:347-353.
    • (2002) J. Ind. Microbiol. Biotechnol , vol.29 , pp. 347-353
    • Dunsmore, B.C.1    Jacobsen, A.2    Hall-Stoodley, L.3    Bass, C.J.4    Lappin-Scott, H.M.5
  • 19
    • 17444410016 scopus 로고    scopus 로고
    • Viscoelasticity of Staphylococcus aureus biofilms in response to fluid shear allows resistance to detachment and facilitates rolling migration
    • Rupp, C. J., C. A. Fux, and P. Stoodley. 2005. Viscoelasticity of Staphylococcus aureus biofilms in response to fluid shear allows resistance to detachment and facilitates rolling migration. Appl. Environ. Microbiol. 71:2175-2178.
    • (2005) Appl. Environ. Microbiol , vol.71 , pp. 2175-2178
    • Rupp, C.J.1    Fux, C.A.2    Stoodley, P.3
  • 20
    • 0242424331 scopus 로고    scopus 로고
    • Viscoelastic properties of a mixed culture biofilm from rheometer creep analysis
    • Towler, B. W., C. J. Rupp, A. B. Cunningham, and P. Stoodley. 2003. Viscoelastic properties of a mixed culture biofilm from rheometer creep analysis. Biofouling. 19:279-285.
    • (2003) Biofouling , vol.19 , pp. 279-285
    • Towler, B.W.1    Rupp, C.J.2    Cunningham, A.B.3    Stoodley, P.4
  • 21
    • 4944228515 scopus 로고    scopus 로고
    • Rheology of biofilms formed from the dental plaque pathogen Streptococcus mutans
    • Vinogradov, A. M., M. Winston, C. J. Rupp, and P. Stoodley. 2004. Rheology of biofilms formed from the dental plaque pathogen Streptococcus mutans. Biofilms. 1:49-56.
    • (2004) Biofilms , vol.1 , pp. 49-56
    • Vinogradov, A.M.1    Winston, M.2    Rupp, C.J.3    Stoodley, P.4
  • 23
    • 34247617672 scopus 로고    scopus 로고
    • Does water activity rule P. mirabilis periodic swarming? II. Viscoelasticity and water balance during swarming
    • Lahaye, E., T. Aubry, V. Fleury, and O. Sire. 2007. Does water activity rule P. mirabilis periodic swarming? II. Viscoelasticity and water balance during swarming. Biomacromolecules. 8:1228-1235.
    • (2007) Biomacromolecules , vol.8 , pp. 1228-1235
    • Lahaye, E.1    Aubry, T.2    Fleury, V.3    Sire, O.4
  • 24
    • 41949124185 scopus 로고    scopus 로고
    • Rheology of biofilms formed at the surface of NF membranes in a drinking water production unit
    • Houari, A., J. Picard, H. Habarou, L. Galas, H. Vaudry, et al. 2008. Rheology of biofilms formed at the surface of NF membranes in a drinking water production unit. Biofouling. 24:235-240.
    • (2008) Biofouling , vol.24 , pp. 235-240
    • Houari, A.1    Picard, J.2    Habarou, H.3    Galas, L.4    Vaudry, H.5
  • 25
    • 0034972313 scopus 로고    scopus 로고
    • Uniaxial compression measurement device for investigation of the mechanical stability of biofilms
    • Korstgens, V., H. C. Flemming, J. Wingender, and W. Borchard. 2001. Uniaxial compression measurement device for investigation of the mechanical stability of biofilms. J. Microbiol. Methods. 46:9-17.
    • (2001) J. Microbiol. Methods , vol.46 , pp. 9-17
    • Korstgens, V.1    Flemming, H.C.2    Wingender, J.3    Borchard, W.4
  • 26
    • 33750347282 scopus 로고    scopus 로고
    • Mechanical properties and failure of Streptococcus mutans biofilms, studied using a microindentation device
    • Cense, A. W., E. A. Peeters, B. Gottenbos, F. P. Baaijens, A. M. Nuijs, et al. 2006. Mechanical properties and failure of Streptococcus mutans biofilms, studied using a microindentation device. J. Microbiol. Methods. 67:463-472.
    • (2006) J. Microbiol. Methods , vol.67 , pp. 463-472
    • Cense, A.W.1    Peeters, E.A.2    Gottenbos, B.3    Baaijens, F.P.4    Nuijs, A.M.5
  • 27
    • 0347930300 scopus 로고    scopus 로고
    • Measurement of creep compliance of solid polymers by nanoindentation
    • Lu, H., B. Wang, J. Ma, G. Huang, and H. Viswanathan. 2003. Measurement of creep compliance of solid polymers by nanoindentation. Mech. Time-Depend. Mater. 7:189-207.
    • (2003) Mech. Time-Depend. Mater , vol.7 , pp. 189-207
    • Lu, H.1    Wang, B.2    Ma, J.3    Huang, G.4    Viswanathan, H.5
  • 28
    • 7244225073 scopus 로고    scopus 로고
    • A simple phenomenological approach to nanoindentation creep
    • Fischer-Cripps, A. C. 2004. A simple phenomenological approach to nanoindentation creep. Mater. Sci. Eng. 385:74-82.
    • (2004) Mater. Sci. Eng , vol.385 , pp. 74-82
    • Fischer-Cripps, A.C.1
  • 30
    • 44949238738 scopus 로고    scopus 로고
    • Surface viscoelasticity of individual gram-negative bacterial cells measured using atomic force microscopy
    • Vadillo-Rodriguez, V., T. J. Beveridge, and J. R. Dutcher. 2008. Surface viscoelasticity of individual gram-negative bacterial cells measured using atomic force microscopy. J. Bacteriol. 190:4225-4232.
    • (2008) J. Bacteriol , vol.190 , pp. 4225-4232
    • Vadillo-Rodriguez, V.1    Beveridge, T.J.2    Dutcher, J.R.3
  • 31
    • 0032518146 scopus 로고    scopus 로고
    • Direct measurement of interactions between adsorbed protein layers using an atomic force microscope
    • Bowen, W. R., N. Hilal, R. W. Lovitt, and C. J. Wright. 1998. Direct measurement of interactions between adsorbed protein layers using an atomic force microscope. J. Colloid Interface Sci. 197:348-352.
    • (1998) J. Colloid Interface Sci , vol.197 , pp. 348-352
    • Bowen, W.R.1    Hilal, N.2    Lovitt, R.W.3    Wright, C.J.4
  • 32
    • 0036353152 scopus 로고    scopus 로고
    • Direct surface force measurement in water using a nanosize colloidal probe technique
    • Cho, J. M., and W. M. Sigmund. 2002. Direct surface force measurement in water using a nanosize colloidal probe technique. J. Colloid Interface Sci. 245:405-407.
    • (2002) J. Colloid Interface Sci , vol.245 , pp. 405-407
    • Cho, J.M.1    Sigmund, W.M.2
  • 33
    • 1642565241 scopus 로고    scopus 로고
    • Lubricating properties of the initial salivary pellicle-an AFM study
    • Berg, I. C., M. W. Rutland, and T. Arnebrant. 2003. Lubricating properties of the initial salivary pellicle-an AFM study. Biofouling. 19:365-369.
    • (2003) Biofouling , vol.19 , pp. 365-369
    • Berg, I.C.1    Rutland, M.W.2    Arnebrant, T.3
  • 34
    • 27744587245 scopus 로고    scopus 로고
    • Force measurements with the atomic force microscope: Technique, interpretation and applications
    • Butt, H. J., B. Cappella, and M. Kappl. 2005. Force measurements with the atomic force microscope: technique, interpretation and applications. Surf. Sci. Rep. 59:1-152.
    • (2005) Surf. Sci. Rep , vol.59 , pp. 1-152
    • Butt, H.J.1    Cappella, B.2    Kappl, M.3
  • 35
    • 0018613990 scopus 로고
    • Outer membrane of Pseudomonas aeruginosa: Heat- 2-mercaptoethanol-modifiable proteins
    • Hancock, R. E., and A. M. Carey. 1979. Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins. J. Bacteriol. 140:902-910.
    • (1979) J. Bacteriol , vol.140 , pp. 902-910
    • Hancock, R.E.1    Carey, A.M.2
  • 36
    • 40449113870 scopus 로고    scopus 로고
    • Functional characterization of MigA and WapR: Putative rhamnosyltransferases involved in outer core oligosaccharide biosynthesis of Pseudomonas aeruginosa
    • Poon, K. K., E. L. Westman, E. Vinogradov, S. Jin, and J. S. Lam. 2008. Functional characterization of MigA and WapR: putative rhamnosyltransferases involved in outer core oligosaccharide biosynthesis of Pseudomonas aeruginosa. J. Bacteriol. 190:1857-1865.
    • (2008) J. Bacteriol , vol.190 , pp. 1857-1865
    • Poon, K.K.1    Westman, E.L.2    Vinogradov, E.3    Jin, S.4    Lam, J.S.5
  • 37
    • 36449007442 scopus 로고
    • Calibration of atomic-force microscope tips
    • Hutter, J. L., and J. Bechhoefer. 1993. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64:1868-1873.
    • (1993) Rev. Sci. Instrum , vol.64 , pp. 1868-1873
    • Hutter, J.L.1    Bechhoefer, J.2
  • 38
    • 0032661215 scopus 로고    scopus 로고
    • Organic and inorganic contamination on commercial AFM cantilevers
    • Lo, Y. S., N. D. Huefner, W. S. Chan, P. Dryden, B. Hagenhoff, et al. 1999. Organic and inorganic contamination on commercial AFM cantilevers. Langmuir. 15:6522-6526.
    • (1999) Langmuir , vol.15 , pp. 6522-6526
    • Lo, Y.S.1    Huefner, N.D.2    Chan, W.S.3    Dryden, P.4    Hagenhoff, B.5
  • 41
    • 0012489320 scopus 로고    scopus 로고
    • Nanoindentation
    • F. F. Ling and W. H. Hart, editors. Springer-Verlag, New York
    • Fischer-Cripps, A. C. 2004. Nanoindentation. In Mechanical Engineering Series. F. F. Ling and W. H. Hart, editors. Springer-Verlag, New York. 39-68.
    • (2004) Mechanical Engineering Series , pp. 39-68
    • Fischer-Cripps, A.C.1
  • 43
    • 40549131623 scopus 로고    scopus 로고
    • Correlation between Enterococcus faecalis biofilms development stage and quantitative surface roughness using atomic force microscopy
    • Santos, R. P., T. T. Arruda, C. B. Carvalho, V. A. Carneiro, L. Q. Braga, et al. 2008. Correlation between Enterococcus faecalis biofilms development stage and quantitative surface roughness using atomic force microscopy. Microsc. Microanal. 14:150-158.
    • (2008) Microsc. Microanal , vol.14 , pp. 150-158
    • Santos, R.P.1    Arruda, T.T.2    Carvalho, C.B.3    Carneiro, V.A.4    Braga, L.Q.5
  • 44
    • 0035047821 scopus 로고    scopus 로고
    • Effects of cell surface damage on surface properties and adhesion of Pseudomonas aeruginosa
    • Bruinsma, G. M., M. Rustema-Abbing, H. C. van der Mei, and H. J. Busscher. 2001. Effects of cell surface damage on surface properties and adhesion of Pseudomonas aeruginosa. J. Microbiol. Methods. 45:95-101.
    • (2001) J. Microbiol. Methods , vol.45 , pp. 95-101
    • Bruinsma, G.M.1    Rustema-Abbing, M.2    van der Mei, H.C.3    Busscher, H.J.4
  • 45
    • 0037453672 scopus 로고    scopus 로고
    • Macroscopic and nanoscale measurements of the adhesion of bacteria with varying outer layer surface composition
    • Burks, G. A., S. B. Velegol, E. Paramonova, B. E. Lindenmuth, J. D. Feick, et al. 2003. Macroscopic and nanoscale measurements of the adhesion of bacteria with varying outer layer surface composition. Langmuir. 19:2366-2371.
    • (2003) Langmuir , vol.19 , pp. 2366-2371
    • Burks, G.A.1    Velegol, S.B.2    Paramonova, E.3    Lindenmuth, B.E.4    Feick, J.D.5
  • 46
    • 36749070884 scopus 로고    scopus 로고
    • Atomic force microscopy study of the effect of lipopolysaccharides and extracellular polymers on adhesion of Pseudomonas aeruginosa
    • Atabek, A., and T. A. Camesano. 2007. Atomic force microscopy study of the effect of lipopolysaccharides and extracellular polymers on adhesion of Pseudomonas aeruginosa. J. Bacteriol. 189:8503-8509.
    • (2007) J. Bacteriol , vol.189 , pp. 8503-8509
    • Atabek, A.1    Camesano, T.A.2
  • 47
    • 34948864413 scopus 로고    scopus 로고
    • Nanomechanical properties of glucans and associated cell-surface adhesion of Streptococcus mutans probed by atomic force microscopy under in situ conditions
    • Cross, S. E., J. Kreth, L. Zhu, R. Sullivan, W. Shi, et al. 2007. Nanomechanical properties of glucans and associated cell-surface adhesion of Streptococcus mutans probed by atomic force microscopy under in situ conditions. Microbiology. 153:3124-3132.
    • (2007) Microbiology , vol.153 , pp. 3124-3132
    • Cross, S.E.1    Kreth, J.2    Zhu, L.3    Sullivan, R.4    Shi, W.5
  • 48
    • 33646905042 scopus 로고    scopus 로고
    • Deformation and nano-rheology of red blood cells: An AFM investigation
    • Bremmell, K. E., A. Evans, and C. A. Prestidge. 2006. Deformation and nano-rheology of red blood cells: an AFM investigation. Colloids Surf. B Biointerfaces. 50:43-48.
    • (2006) Colloids Surf. B Biointerfaces , vol.50 , pp. 43-48
    • Bremmell, K.E.1    Evans, A.2    Prestidge, C.A.3
  • 49
    • 2042493009 scopus 로고
    • Fundamental mechanisms of interfacial friction. 1. relation between adhesion and friction
    • Yoshizawa, H., Y. L. Chen, and J. Israelachvili. 1993. Fundamental mechanisms of interfacial friction. 1. relation between adhesion and friction. J. Phys. Chem. 97:4128-4140.
    • (1993) J. Phys. Chem , vol.97 , pp. 4128-4140
    • Yoshizawa, H.1    Chen, Y.L.2    Israelachvili, J.3
  • 51
    • 3042785070 scopus 로고    scopus 로고
    • The role of the biofilm matrix in structural development
    • Cogan, N. G., and J. P. Keener. 2004. The role of the biofilm matrix in structural development. Math. Med. Biol. 21:147-166.
    • (2004) Math. Med. Biol , vol.21 , pp. 147-166
    • Cogan, N.G.1    Keener, J.P.2


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