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Understanding the Electrostatic Contribution to Gold Nanoparticle-Protein Binding
504a
Tuesday, March 1, 2016
2483-Pos Board B627 Controlling Gliding Trajectories of Microtubules by Altering Microtubule Flexural Rigidity Naoto Isozaki1, Scott Erickson2, Shintaku Hirofumi1, Hidetoshi Kotera1, Taviare L. Hawkins2, Jennifer L. Ross3, Ryuji Yokokawa1. 1 Micro Engineering, Kyoto University, Kyoto, Japan, 2Physics, University of Wisconsin La Crosse, La Crosse, WI, USA, 3Physics, University of Massachusetts Amherst, Amherst, MA, USA. A kinesin-microtubule system plays an important role in intracellular transport and has been studied for potential applications as in vitro nanoactuators. A challenge for an engineered nanoactuator is to control gliding microtubule trajectories in a motility assay where microtubules glide on a kinesin-coated substrate. Our group has been focusing on altering intrinsic characteristics of microtubules to control their trajectories under the influence of an electric field. Here, we propose a method to alter their flexural rigidity (EI). The primary components of this project were: 1) Effects of fluorescent dye, nucleotide, total length, and tau on EI. 2) Control of microtubule trajectories with different EIs. 1) We polymerized six kinds of microtubules in the presence of different fluorescent dye, nucleotide, or tau binding. They were partially immobilized on a substrate via biotin-avidin bindings while the other end thermally fluctuated. Since the fluctuating segment dominated the leading ends of their gliding, we could calculate microtubule EI. As a result of solving an equation relating microtubule bending and thermal energy, EI was influenced by microtubule length and nucleotides but not by fluorescent dye. We also found EI of GMPCPP-polymerized microtubule was constant with tau, although the concentration of tau would change the EI of GTP-polymerized microtubule. 2) GTP- and GMPCPP-polymerized microtubules were expected to show different trajectories from the measured EI. They were glided under an electric field, and their gliding trajectories could be divided into two groups of corresponding EIs. Therefore, our method alters microtubule EIs by changing polymerization conditions and shows an ability to control microtubule trajectories under a given electric field. This method can be applied as a molecular sorter by utilizing the EI-altered microtubule as a molecular transporter in a microfluidic device. 2484-Pos Board B628 A Microfluidic Device for Single Cell Imaging and Intracellular Components Counting David C. Duran, Juan Manuel Pedraza. Physics, Universidad de los Andes, Bogota, Colombia. Today, there exist two powerful microfluidics technologies for cell population analysis at the single-cell level. One of them is called ‘‘mother-machine. It allows the analysis of protein level expression for a population with single-cell resolution during time. The other technology is Microfluidic Assisted Cell Screening (MACS). This one allows counting of low-number intracellular components (e.g. mRNA) with single cell resolution as cells passes through the device. The disadvantage of MACS is that, once a group of cells passes through the device, it is lost so time-lapse experiments are not possible with this technology. However, a complete analysis of stochastic gene expression, would need a measurement of the distribution across a population of both, low number intracellular components and proteins, and their evolution through time. We designed and fabricated a two-level microfluidic device which combine both technologies afore mentioned. This device brings great advantages to cell population analysis at the single cell level through time, since it enables is capable of trapping cells and allowing measurement of protein levels and low-number intra cellular components counting at the single-cell level for populations of E. Coli through time. In each channel, thousands of cells are trapped and can be analyzed individually. We show the capability of this technology by measuring protein level and low-number intra cellular components for populations of E.Coli. We hope this technology will enable better understanding of gene expression and cell-fate decision making. 2485-Pos Board B629 Understanding the Electrostatic Contribution to Gold NanoparticleProtein Binding Ailin Wang, Randika Perera, Nicholas Fitzkee. Chemistry, Mississippi State University, Mississippi State, MS, USA. Upon exposure to a solution containing gold nanoparticles (AuNPs), proteins spontaneously bind to the nanoparticle surface, leading to the formation of a stable surface coating, or biocorona. The composition of this biocorona in biological fluids depends on the protein concentration, binding affinity, and other physical factors, including size and charge. Because of the potential applications of AuNPs as drug delivery vectors, it is crucially important to understand the physical basis of the protein-nanoparticle interaction. Here, we investigate
the contribution of electrostatic interactions to protein binding. We find that both GB3 and ubiquitin exhibit a pH-dependent binding behavior that closely tracks with the proton binding curve of ionizable residues using conventional pKA values. To investigate this behavior more closely, we developed a novel NMR-based approach to monitor the binding competition between GB3 and ubiquitin in situ. The ratio of GB3 versus ubiquitin bound to the surface can be tuned depending on the pH, suggesting a significant interaction between the proteins and the surface-bound citrate molecules used to stabilize AuNPs. Moreover, chemical methylation of lysine residues significantly alters protein binding, suggesting that these residues are important in driving adsorption. Using linkage analysis, we have developed a thermodynamic model for binding that can explain these observations in terms of pKA shifts localized to a single binding site. Ultimately, this work provides a better understanding of the dynamics and biocompatibility of the protein-AuNP biocorona in biological systems. 2486-Pos Board B630 Filovirus Mimics Deliver Effectively Praful R. Nair1, Kyle R. Spinler1, Mohammed R. Vakili2, Afsaneh Lavasanifar2, Dennis E. Discher1. 1 University of Pennsylvania, Philadelphia, PA, USA, 2University of Alberta, Edmonton, AB, Canada. Filoviruses such as Ebola are microns long but biophysical advantages for such encapsulating/enveloped viruses have remained obscure. Flexible ‘filomicelles’ have been made from amphiphilic block copolymers and demonstrate effective delivery of two very different hydrophobic compounds. Retinoic acid (RA) and other retinoids regulate RA receptor transcription factors that induce differentiation and arrest proliferation of many cell types, including cancer cells. Lamin-A is transcriptionally regulated by RA receptors, and as a structural protein surrounding chromatin, lamin-A can affect differentiation and karyokinesis as well as nuclear viscosity. Paclitaxel, on the other hand, stabilizes microtubules and induces aneuploidy by blocking mitosis at the metaphase-anaphase transition, which greatly increases cell death. When cancer cells are treated with either of the drugs alone over several periods of the normal cell cycle, cancer cell populations revert back to the original proliferative state, consistent with relapse commonly seen after conventional chemotherapy. On the other hand, combining RA with select chemotherapeutics has for several decades produced durable cures of select cancers, notably pro-myeloblastic leukemia (PML) where RA differentiates cells while chemotherapeutic kills the cancer stem cell. With carcinoma lines, we find dual treatment with RA plus Paclitaxel increases lamin-A levels, aneuploidy, and cell death beyond those achieved by either drug single-handedly, with effects appearing irreversible. Trends with the key cell cycle factor Cyclin-D1 and proliferation marker Ki-67 help clarify the basis for drug synergy. These effects are greatly enhanced by loading the drugs into filomicelles selfassembled from degradable di-block copolymers of Polyethylene glycolPolybenzyl caprolactone (PEG-PBCL). Preliminary tests in vivo demonstrate sustained delivery for days as well as efficacy in shrinking tumors. These results highlight the irreversible synergy of killing cancerous cells while driving differentiation. 2487-Pos Board B631 Wormpharm: A Microfluidic Platform for Pharmacogenetic Studies on C. Elegans Andrew Moore1, Jung Doh2, Irem Celen3, Michael Moore2, Chandran Sabanayagam2. 1 Department of Biological Sciences, University of Delaware, Newark, DE, USA, 2Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA, 3Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE, USA. Our lab has developed the WormPharm platform that utilizes microfluidics to allow automated culturing and imaging of C. elegans exposed to pharmaceuticals. Current problems with pharmaceutical experiments using C. elegans include, extensive plate maintenance and animal transfer, and additional drug metabolism by bacterial food sources. As a solution, we have modified a chemically synthetic axenic media for use with the WormPharm platform. System automation allows us to perform experiments that can last weeks to months. Alcohol, caffeine, and nicotine constitute the three most commonly consumed psychoactive drugs around the globe, making them prime compounds for pharmaceutical assays. To date, our platform has been used to study the individual and combinatorial effects of all three drugs. In our study, we found that the effects of these drugs when introduced in axenic media varied greatly both individually and in combination. We have examined locomotive behavior, pharyngeal pump rate, and morphological deformities upon drug dose. Our
Tuesday, March 1, 2016
2483-Pos Board B627 Controlling Gliding Trajectories of Microtubules by Altering Microtubule Flexural Rigidity Naoto Isozaki1, Scott Erickson2, Shintaku Hirofumi1, Hidetoshi Kotera1, Taviare L. Hawkins2, Jennifer L. Ross3, Ryuji Yokokawa1. 1 Micro Engineering, Kyoto University, Kyoto, Japan, 2Physics, University of Wisconsin La Crosse, La Crosse, WI, USA, 3Physics, University of Massachusetts Amherst, Amherst, MA, USA. A kinesin-microtubule system plays an important role in intracellular transport and has been studied for potential applications as in vitro nanoactuators. A challenge for an engineered nanoactuator is to control gliding microtubule trajectories in a motility assay where microtubules glide on a kinesin-coated substrate. Our group has been focusing on altering intrinsic characteristics of microtubules to control their trajectories under the influence of an electric field. Here, we propose a method to alter their flexural rigidity (EI). The primary components of this project were: 1) Effects of fluorescent dye, nucleotide, total length, and tau on EI. 2) Control of microtubule trajectories with different EIs. 1) We polymerized six kinds of microtubules in the presence of different fluorescent dye, nucleotide, or tau binding. They were partially immobilized on a substrate via biotin-avidin bindings while the other end thermally fluctuated. Since the fluctuating segment dominated the leading ends of their gliding, we could calculate microtubule EI. As a result of solving an equation relating microtubule bending and thermal energy, EI was influenced by microtubule length and nucleotides but not by fluorescent dye. We also found EI of GMPCPP-polymerized microtubule was constant with tau, although the concentration of tau would change the EI of GTP-polymerized microtubule. 2) GTP- and GMPCPP-polymerized microtubules were expected to show different trajectories from the measured EI. They were glided under an electric field, and their gliding trajectories could be divided into two groups of corresponding EIs. Therefore, our method alters microtubule EIs by changing polymerization conditions and shows an ability to control microtubule trajectories under a given electric field. This method can be applied as a molecular sorter by utilizing the EI-altered microtubule as a molecular transporter in a microfluidic device. 2484-Pos Board B628 A Microfluidic Device for Single Cell Imaging and Intracellular Components Counting David C. Duran, Juan Manuel Pedraza. Physics, Universidad de los Andes, Bogota, Colombia. Today, there exist two powerful microfluidics technologies for cell population analysis at the single-cell level. One of them is called ‘‘mother-machine. It allows the analysis of protein level expression for a population with single-cell resolution during time. The other technology is Microfluidic Assisted Cell Screening (MACS). This one allows counting of low-number intracellular components (e.g. mRNA) with single cell resolution as cells passes through the device. The disadvantage of MACS is that, once a group of cells passes through the device, it is lost so time-lapse experiments are not possible with this technology. However, a complete analysis of stochastic gene expression, would need a measurement of the distribution across a population of both, low number intracellular components and proteins, and their evolution through time. We designed and fabricated a two-level microfluidic device which combine both technologies afore mentioned. This device brings great advantages to cell population analysis at the single cell level through time, since it enables is capable of trapping cells and allowing measurement of protein levels and low-number intra cellular components counting at the single-cell level for populations of E. Coli through time. In each channel, thousands of cells are trapped and can be analyzed individually. We show the capability of this technology by measuring protein level and low-number intra cellular components for populations of E.Coli. We hope this technology will enable better understanding of gene expression and cell-fate decision making. 2485-Pos Board B629 Understanding the Electrostatic Contribution to Gold NanoparticleProtein Binding Ailin Wang, Randika Perera, Nicholas Fitzkee. Chemistry, Mississippi State University, Mississippi State, MS, USA. Upon exposure to a solution containing gold nanoparticles (AuNPs), proteins spontaneously bind to the nanoparticle surface, leading to the formation of a stable surface coating, or biocorona. The composition of this biocorona in biological fluids depends on the protein concentration, binding affinity, and other physical factors, including size and charge. Because of the potential applications of AuNPs as drug delivery vectors, it is crucially important to understand the physical basis of the protein-nanoparticle interaction. Here, we investigate
the contribution of electrostatic interactions to protein binding. We find that both GB3 and ubiquitin exhibit a pH-dependent binding behavior that closely tracks with the proton binding curve of ionizable residues using conventional pKA values. To investigate this behavior more closely, we developed a novel NMR-based approach to monitor the binding competition between GB3 and ubiquitin in situ. The ratio of GB3 versus ubiquitin bound to the surface can be tuned depending on the pH, suggesting a significant interaction between the proteins and the surface-bound citrate molecules used to stabilize AuNPs. Moreover, chemical methylation of lysine residues significantly alters protein binding, suggesting that these residues are important in driving adsorption. Using linkage analysis, we have developed a thermodynamic model for binding that can explain these observations in terms of pKA shifts localized to a single binding site. Ultimately, this work provides a better understanding of the dynamics and biocompatibility of the protein-AuNP biocorona in biological systems. 2486-Pos Board B630 Filovirus Mimics Deliver Effectively Praful R. Nair1, Kyle R. Spinler1, Mohammed R. Vakili2, Afsaneh Lavasanifar2, Dennis E. Discher1. 1 University of Pennsylvania, Philadelphia, PA, USA, 2University of Alberta, Edmonton, AB, Canada. Filoviruses such as Ebola are microns long but biophysical advantages for such encapsulating/enveloped viruses have remained obscure. Flexible ‘filomicelles’ have been made from amphiphilic block copolymers and demonstrate effective delivery of two very different hydrophobic compounds. Retinoic acid (RA) and other retinoids regulate RA receptor transcription factors that induce differentiation and arrest proliferation of many cell types, including cancer cells. Lamin-A is transcriptionally regulated by RA receptors, and as a structural protein surrounding chromatin, lamin-A can affect differentiation and karyokinesis as well as nuclear viscosity. Paclitaxel, on the other hand, stabilizes microtubules and induces aneuploidy by blocking mitosis at the metaphase-anaphase transition, which greatly increases cell death. When cancer cells are treated with either of the drugs alone over several periods of the normal cell cycle, cancer cell populations revert back to the original proliferative state, consistent with relapse commonly seen after conventional chemotherapy. On the other hand, combining RA with select chemotherapeutics has for several decades produced durable cures of select cancers, notably pro-myeloblastic leukemia (PML) where RA differentiates cells while chemotherapeutic kills the cancer stem cell. With carcinoma lines, we find dual treatment with RA plus Paclitaxel increases lamin-A levels, aneuploidy, and cell death beyond those achieved by either drug single-handedly, with effects appearing irreversible. Trends with the key cell cycle factor Cyclin-D1 and proliferation marker Ki-67 help clarify the basis for drug synergy. These effects are greatly enhanced by loading the drugs into filomicelles selfassembled from degradable di-block copolymers of Polyethylene glycolPolybenzyl caprolactone (PEG-PBCL). Preliminary tests in vivo demonstrate sustained delivery for days as well as efficacy in shrinking tumors. These results highlight the irreversible synergy of killing cancerous cells while driving differentiation. 2487-Pos Board B631 Wormpharm: A Microfluidic Platform for Pharmacogenetic Studies on C. Elegans Andrew Moore1, Jung Doh2, Irem Celen3, Michael Moore2, Chandran Sabanayagam2. 1 Department of Biological Sciences, University of Delaware, Newark, DE, USA, 2Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA, 3Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE, USA. Our lab has developed the WormPharm platform that utilizes microfluidics to allow automated culturing and imaging of C. elegans exposed to pharmaceuticals. Current problems with pharmaceutical experiments using C. elegans include, extensive plate maintenance and animal transfer, and additional drug metabolism by bacterial food sources. As a solution, we have modified a chemically synthetic axenic media for use with the WormPharm platform. System automation allows us to perform experiments that can last weeks to months. Alcohol, caffeine, and nicotine constitute the three most commonly consumed psychoactive drugs around the globe, making them prime compounds for pharmaceutical assays. To date, our platform has been used to study the individual and combinatorial effects of all three drugs. In our study, we found that the effects of these drugs when introduced in axenic media varied greatly both individually and in combination. We have examined locomotive behavior, pharyngeal pump rate, and morphological deformities upon drug dose. Our