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Ice-Binding Proteins - Not Only for Ice Growth Control
Wednesday, February 15, 2017
589a
Erythrocytes from non-ischemic patients presented higher stiffness than those from the other two groups. Nevertheless, a significantly higher cell penetration depth at the same applied force was observed for ischemic heart failure patients. Erythrocyte deformability (assessed as elongation index) results show that heart failure patients presented higher erythrocyte deformability than the control group at lower shear stresses, and lower deformability at higher shear stresses. This indicates that patients’ erythrocytes are more deformable than those from healthy donors in blood vessels with larger internal diameters; however, in smaller-diameter vessels the opposite trend exists. Finally, a 12-month clinical follow-up shows that CHF patients with higher fibrinogen-erythrocyte binding forces, probed by AFM at the beginning of the assessment, had a significantly higher probability of being hospitalized due to cardiovascular complications on the subsequent year. Our results show that AFM can be a promising tool for clinical prognosis, pinpointing those patients with increased risk for cardiovascular diseases. Guedes et al. (2016) Nature Nanotechnology, 11:687-92.
of IBPs to the basal plane differs from one type of IBP to another. In another work we studied the IBP from an Antarctic marine bacterium, Marinomonas primoryensis. We showed that this IBP has a different function than any other known IBP, which is as an adhesin that helps the bacterium stick to ice crystals. Antibodies raised against the ice-binding part of the protein abolished the ability of the bacteria to bind ice, while antibodies raised against other parts of the protein failed to do so. This principle of ‘‘knocking out’’ the adhesion function can theoretically be used to prevent formation of biofilms, thereby combating infections. This is currently the only example known of an adhesin that binds to ice. Funding: ERC, ISF, CHIR, NSERC. References: 1) Bar-Dolev M., Braslavsky I. and Davies P.L., Annl. Rev. Biochemistry 2015. 2) Bar-Dolev M. et al. R. Soc. Interface 2016. 3) Haleva L., Celik Y. and Bar-Dolev M. et al. Biophysical Journal 2016. 4) Lewis J. K., Bischof J. C., Braslavsky I., et al. Cryobiology 2016.
Biosurfaces
2899-Pos Board B506 Aquaporin Biomimetic Membrane for Energy Conservative Water Desalination Ahmed Fuwad1, Hyunil Ryu2, Tae-Joon Jeon2, Sun Min Kim1. 1 Mechanical Engineering, Inha University, Incheon, Korea, Republic of, 2 Biological Engineering, Inha University, Incheon, Korea, Republic of. One of the important issues affecting people around the globe is inadequate access to clean and safe water. Problems with water are expected to increase in coming decades at an alarming rate, causing water scarcity around the world even in water rich areas. To address the problems a novel approach is needed for water purification at low energy consumption, high purity and at the same time the chemical treatment and waste production should be minimized. Here we present a nature inspired biomimetic water filter containing aquaporin protein. Aquaporin is the most efficient water channel in nature due to its high water selectivity and permeability. In spite of its merits, the manufactural difficulties of aquaporin embedded membranes as well as their fragility preclude their industrial applications. To overcome these problems, by crosslinking lipid molecules, we propose a novel method that stabilizes aquaporin embedded membranes using electrostatic interactions and alternative coating methods. Furthermore, surface morphological analysis show that the proteoliposomes were successfully immobilized on the substrate and retain their structure. The performance of this aquaporin biomimetic membrane was tested for a forward osmosis (FO) based water purification system.
2897-Pos Board B504 Graphene-Oxide Gel as Biomimetic Antimicrobial Cloak Valentina Palmieri1,2, Massimiliano Papi1,2, Francesca Bugli3, Maurizio Sanguinetti4, Luca Angelani5, Marco De Spirito1, Claudio Conti5. 1 Physics Institute, Universita` Cattolica del Sacro Cuore, Roma, Italy, 2 Institute for Complex Systems, National Research Council (ISC-CNR), Rome, Italy, 3Universita` Cattolica del Sacro Cuore, Roma, Italy, 4 Microbiology Institute, Universita` Cattolica del Sacro Cuore, Roma, Italy, 5 Institute for Complex Systems, National Research Council (ISC-CNR), Roma, Italy. Antibacterial surfaces have an enormous economic and social impact on the worldwide technological fight against diseases. However, bacteria develop resistance and coatings are often not uniform and not stable in time. The challenge is finding an antibacterial coating that is biocompatible, costeffective, not toxic, and spreadable over large and irregular surfaces. Here we demonstrate an antibacterial cloak by laser printing of graphene oxide hydrogels mimicking the Cancer Pagurus carapace. We observe up to 90% reduction of bacteria cells. This cloak exploits natural surface patterns evolved to resist to microorganisms infection, and the antimicrobial efficacy of graphene oxide. Cell integrity analysis by scanning electron microscopy and nucleic acids release show bacteriostatic and bactericidal effect. Nucleic acids release demonstrates microorganism cutting, and microscopy reveals cells wrapped by the laser treated gel. A theoretical active matter model confirms our findings. The employment of biomimetic graphene oxide gels opens unique possibilities to decrease infections in biomedical applications and chirurgical equipment; our antibiotic-free approach, based on the geometric reduction of microbial adhesion and the mechanical action of Graphene Oxide sheets, less likely induces bacterial resistance. 2898-Pos Board B505 Ice-Binding Proteins - Not Only for Ice Growth Control Maya Bar-Dolev1, Shuaiqi Guo2, Lotem Haleva1, Yeliz Celik3, Peter L. Davies2, Ido Braslavsky1,3. 1 Institute of Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot, Israel, 2Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada, 3Physics and Astronomy, Ohio University, Athens, OH, USA. Ice-binding proteins (IBPs) evolved in organisms living in cold ecosystems as a protective measure against freezing stresses. Some IBPs prevent their hosts from freezing mainly by depression of the freezing point of the body fluids below the melting point. Others help the organism avoid the damage of freezing by inhibiting ice recrystallization. These functions make IBPs excellent candidates for ice growth control, such as in cryopreservation of food and tissues. Organ banking, still in its infancy, is becoming a priority field for funding in order to revolutionize transplantation medicine and embryo preservation. To allow the use of IBPs in these applications, more basic knowledge about their function is needed. Recently we developed a new tool, called the mini cold finger, which allows us to grow a single ice crystal in a microfluidic channel and inspect the interactions of various IBPs with it. By injecting solutions of fluorescently-labeled IBPs into the device with an ice crystal present, we studied the ability of various IBPs to bind the basal plane of ice. Basal plane affinity is a crucial factor that directly affects the specific activity of IBPs. We further showed that the dynamics of binding
2900-Pos Board B507 Assembling Functional Proteins on Gold-Glass Surfaces Timothy Robson1, Deepan Shah2, Luke Clifton3, Becky Welbourn3, Jeremy Lakey1. 1 Newcastle University, Newcastle, United Kingdom, 2Orla Protein Technologies, Newcastle, United Kingdom, 3ISIS Pulsed Neutron and Muon Source, Harwell, United Kingdom. The generation of functional surfaces using biological molecules is of great interest for cell culture and diagnostic applications. Control of protein orientation is difficult but advantageous in many cases to retain activity. The results from simple adsorption methods can be extremely variable and often result in denatured or inactive proteins. The industrial sponsor of this work, Orla Protein Technologies, utilise gold-thiol chemistry and the self-assembling nature of a beta-barrel outer membrane protein (OMP) from E. coli to create reproducible protein arrays on gold surfaces. The E. coli OMP is a robust protein scaffold that can be engineered to display a wide variety of functional motifs or domains such as, cell adhesion proteins, single chain antibodies or antigens. Sputter coating is often used to achieve thin and even gold surfaces suitable for most uses but the method requires expensive equipment and specialist training and the layers are poorly transparent, limiting their use in cell biology and microscopy. This has led our group to develop a simple, flexible method for depositing high densities of gold nanoparticles on to glass substrates. These are cheap to produce using inexpensive equipment and all steps are carried out at room temperature without harsh solvents. Moving to a gold-glass platform enables the more flexible use of microscopy and spectroscopic techniques in cell culture and biosensing applications. Characterisation of the new surfaces by electron microscopy, fluorescence microscopy and neutron reflection reveals wellordered fields of nanoparticles coated in a functional protein layer. Further analysis of functionalised nanoparticles in solution has been carried out using UV-Visible spectroscopy, dynamic light scattering, small angle neutron scattering and electron microscopy. This shows several proteins bound to the
589a
Erythrocytes from non-ischemic patients presented higher stiffness than those from the other two groups. Nevertheless, a significantly higher cell penetration depth at the same applied force was observed for ischemic heart failure patients. Erythrocyte deformability (assessed as elongation index) results show that heart failure patients presented higher erythrocyte deformability than the control group at lower shear stresses, and lower deformability at higher shear stresses. This indicates that patients’ erythrocytes are more deformable than those from healthy donors in blood vessels with larger internal diameters; however, in smaller-diameter vessels the opposite trend exists. Finally, a 12-month clinical follow-up shows that CHF patients with higher fibrinogen-erythrocyte binding forces, probed by AFM at the beginning of the assessment, had a significantly higher probability of being hospitalized due to cardiovascular complications on the subsequent year. Our results show that AFM can be a promising tool for clinical prognosis, pinpointing those patients with increased risk for cardiovascular diseases. Guedes et al. (2016) Nature Nanotechnology, 11:687-92.
of IBPs to the basal plane differs from one type of IBP to another. In another work we studied the IBP from an Antarctic marine bacterium, Marinomonas primoryensis. We showed that this IBP has a different function than any other known IBP, which is as an adhesin that helps the bacterium stick to ice crystals. Antibodies raised against the ice-binding part of the protein abolished the ability of the bacteria to bind ice, while antibodies raised against other parts of the protein failed to do so. This principle of ‘‘knocking out’’ the adhesion function can theoretically be used to prevent formation of biofilms, thereby combating infections. This is currently the only example known of an adhesin that binds to ice. Funding: ERC, ISF, CHIR, NSERC. References: 1) Bar-Dolev M., Braslavsky I. and Davies P.L., Annl. Rev. Biochemistry 2015. 2) Bar-Dolev M. et al. R. Soc. Interface 2016. 3) Haleva L., Celik Y. and Bar-Dolev M. et al. Biophysical Journal 2016. 4) Lewis J. K., Bischof J. C., Braslavsky I., et al. Cryobiology 2016.
Biosurfaces
2899-Pos Board B506 Aquaporin Biomimetic Membrane for Energy Conservative Water Desalination Ahmed Fuwad1, Hyunil Ryu2, Tae-Joon Jeon2, Sun Min Kim1. 1 Mechanical Engineering, Inha University, Incheon, Korea, Republic of, 2 Biological Engineering, Inha University, Incheon, Korea, Republic of. One of the important issues affecting people around the globe is inadequate access to clean and safe water. Problems with water are expected to increase in coming decades at an alarming rate, causing water scarcity around the world even in water rich areas. To address the problems a novel approach is needed for water purification at low energy consumption, high purity and at the same time the chemical treatment and waste production should be minimized. Here we present a nature inspired biomimetic water filter containing aquaporin protein. Aquaporin is the most efficient water channel in nature due to its high water selectivity and permeability. In spite of its merits, the manufactural difficulties of aquaporin embedded membranes as well as their fragility preclude their industrial applications. To overcome these problems, by crosslinking lipid molecules, we propose a novel method that stabilizes aquaporin embedded membranes using electrostatic interactions and alternative coating methods. Furthermore, surface morphological analysis show that the proteoliposomes were successfully immobilized on the substrate and retain their structure. The performance of this aquaporin biomimetic membrane was tested for a forward osmosis (FO) based water purification system.
2897-Pos Board B504 Graphene-Oxide Gel as Biomimetic Antimicrobial Cloak Valentina Palmieri1,2, Massimiliano Papi1,2, Francesca Bugli3, Maurizio Sanguinetti4, Luca Angelani5, Marco De Spirito1, Claudio Conti5. 1 Physics Institute, Universita` Cattolica del Sacro Cuore, Roma, Italy, 2 Institute for Complex Systems, National Research Council (ISC-CNR), Rome, Italy, 3Universita` Cattolica del Sacro Cuore, Roma, Italy, 4 Microbiology Institute, Universita` Cattolica del Sacro Cuore, Roma, Italy, 5 Institute for Complex Systems, National Research Council (ISC-CNR), Roma, Italy. Antibacterial surfaces have an enormous economic and social impact on the worldwide technological fight against diseases. However, bacteria develop resistance and coatings are often not uniform and not stable in time. The challenge is finding an antibacterial coating that is biocompatible, costeffective, not toxic, and spreadable over large and irregular surfaces. Here we demonstrate an antibacterial cloak by laser printing of graphene oxide hydrogels mimicking the Cancer Pagurus carapace. We observe up to 90% reduction of bacteria cells. This cloak exploits natural surface patterns evolved to resist to microorganisms infection, and the antimicrobial efficacy of graphene oxide. Cell integrity analysis by scanning electron microscopy and nucleic acids release show bacteriostatic and bactericidal effect. Nucleic acids release demonstrates microorganism cutting, and microscopy reveals cells wrapped by the laser treated gel. A theoretical active matter model confirms our findings. The employment of biomimetic graphene oxide gels opens unique possibilities to decrease infections in biomedical applications and chirurgical equipment; our antibiotic-free approach, based on the geometric reduction of microbial adhesion and the mechanical action of Graphene Oxide sheets, less likely induces bacterial resistance. 2898-Pos Board B505 Ice-Binding Proteins - Not Only for Ice Growth Control Maya Bar-Dolev1, Shuaiqi Guo2, Lotem Haleva1, Yeliz Celik3, Peter L. Davies2, Ido Braslavsky1,3. 1 Institute of Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot, Israel, 2Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada, 3Physics and Astronomy, Ohio University, Athens, OH, USA. Ice-binding proteins (IBPs) evolved in organisms living in cold ecosystems as a protective measure against freezing stresses. Some IBPs prevent their hosts from freezing mainly by depression of the freezing point of the body fluids below the melting point. Others help the organism avoid the damage of freezing by inhibiting ice recrystallization. These functions make IBPs excellent candidates for ice growth control, such as in cryopreservation of food and tissues. Organ banking, still in its infancy, is becoming a priority field for funding in order to revolutionize transplantation medicine and embryo preservation. To allow the use of IBPs in these applications, more basic knowledge about their function is needed. Recently we developed a new tool, called the mini cold finger, which allows us to grow a single ice crystal in a microfluidic channel and inspect the interactions of various IBPs with it. By injecting solutions of fluorescently-labeled IBPs into the device with an ice crystal present, we studied the ability of various IBPs to bind the basal plane of ice. Basal plane affinity is a crucial factor that directly affects the specific activity of IBPs. We further showed that the dynamics of binding
2900-Pos Board B507 Assembling Functional Proteins on Gold-Glass Surfaces Timothy Robson1, Deepan Shah2, Luke Clifton3, Becky Welbourn3, Jeremy Lakey1. 1 Newcastle University, Newcastle, United Kingdom, 2Orla Protein Technologies, Newcastle, United Kingdom, 3ISIS Pulsed Neutron and Muon Source, Harwell, United Kingdom. The generation of functional surfaces using biological molecules is of great interest for cell culture and diagnostic applications. Control of protein orientation is difficult but advantageous in many cases to retain activity. The results from simple adsorption methods can be extremely variable and often result in denatured or inactive proteins. The industrial sponsor of this work, Orla Protein Technologies, utilise gold-thiol chemistry and the self-assembling nature of a beta-barrel outer membrane protein (OMP) from E. coli to create reproducible protein arrays on gold surfaces. The E. coli OMP is a robust protein scaffold that can be engineered to display a wide variety of functional motifs or domains such as, cell adhesion proteins, single chain antibodies or antigens. Sputter coating is often used to achieve thin and even gold surfaces suitable for most uses but the method requires expensive equipment and specialist training and the layers are poorly transparent, limiting their use in cell biology and microscopy. This has led our group to develop a simple, flexible method for depositing high densities of gold nanoparticles on to glass substrates. These are cheap to produce using inexpensive equipment and all steps are carried out at room temperature without harsh solvents. Moving to a gold-glass platform enables the more flexible use of microscopy and spectroscopic techniques in cell culture and biosensing applications. Characterisation of the new surfaces by electron microscopy, fluorescence microscopy and neutron reflection reveals wellordered fields of nanoparticles coated in a functional protein layer. Further analysis of functionalised nanoparticles in solution has been carried out using UV-Visible spectroscopy, dynamic light scattering, small angle neutron scattering and electron microscopy. This shows several proteins bound to the