Markus J. Buehler Massachusetts Institute of Technology
David Kaplan Tufts University
Chwee Teck Lim National University of Singapore
Joachim Spatz University of Heidelberg
QQ1: Cell Mechanics and Cell-Material Interactions I
Lim Chwee Teck
Tuesday PM, April 06, 2010
Room 3024 (Moscone West)
9:30 AM - **QQ1.1
Shaping Cells by Force and Rigidity Through Protein Stretching.
Michael Sheetz 1 2 Show Abstract
1 Biological Sciences, Columbia University, New York, New York, United States, 2 Biological Sciences, National University of Singapore, Singapore Singapore
Control of cell morphology involves the integration of mechanical sensing and different types of cell motility to produce the desired shape of the organism 1. Nanometer level analyses of cell behavior have revealed only a limited number of types of motility involving complex mechanochemical steps 2. For example, cell spreading on matrix-coated surfaces have revealed three different types of motility, an initial blebbing, continuous spreading, and periodic contraction motility. Overall, cell traction forces are primarily dependent upon myosin II 3. Long term matrix forces appear to be sensed by protein stretching and we have defined two different cytoplasmic mechanisms. One example is the activation of protein phosphorylation by stretching4. Secondly, the stretching of proteins can unveil binding sites such as the stretching of talin causing the increased binding of vinculin5. Because these different motility types are robust and occur in many different cell types, we suggest that most cell mechanical functions are accomplished by various combinations of these different motility types. Thus, it is important to define each type of motility at the nanometer level with the new nanotools that are available. Such a mechanistic understanding of cell function will open new ways to target specific cell functions that are critical for disease and wound repair. 1. Vogel, V. and Sheetz, M., Local force and geometry sensing regulate cell functions. Nat Rev Mol Cell Biol 7 (4), 265 (2006).2. Döbereiner, H.G. et al., Dynamic Phase Transitions in Cell Spreading. Phys Rev Letters 93 (10), 108105 (2004).3. Cai, Y. et al., Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow. Biophys J 91 (10), 3907 (2006).4. Sawada, Y. et al., Force Sensing by Mechanical Extension of the Src Family Kinase Substrate p130Cas. Cell 127 (5), 1015 (2006).5. del Rio, A. et al., Stretching single talin rod molecules activates vinculin binding. Science 323 (5914), 638 (2009).
10:15 AM - QQ1.3
Use of Self-assembling Patterned Scaffolds for Directed Cell Growth in Three Dimensions.
Mustapha Jamal 1 , Noy Bassik 1 2 , George Stern 1 , David Gracias 1 3 Show Abstract
1 Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 2 School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States, 3 Chemistry, Johns Hopkins University, Baltimore, Maryland, United States
Many models of disease in culture permit cells to grow on flat two dimensional substrates. In order to study the behavior of cells in physiologically relevant conditions, there is a need to permit growth on scaffolds that have anatomically relevant geometries. Cell morphology, behavior, and disease processes may differ in 3D which warrants complex scaffolds for in vivo culture. Cell placement in such a specific 3D fashion can only be achieved by deterministic construction of a scaffold with specified 3D geometry. We used conventional microfabrication to construct 3D micropatterned cell scaffolds featuring several biologically fundamental geometries. Shapes that were constructed, such as cylinders, spirals, and bi-directional sheets, are otherwise difficult to manufacture at this microscale with specific sizes and radii. Using thin film manufacturing, lithographic processing was used to create flat sheets with a variety of polymeric and metallic materials connected in different patterns. Thin film stress was localized to hinges, and rigid segments allowed for locally flat areas. The resulting 2D sheets assembled into 3D scaffolds when lifted off from the substrate. Fibroblasts were cultured on the scaffolds. Cell viability was confirmed for several weeks, and serial imaging showed the cells growing in a directed fashion over the scaffolds. The scaffolds, with cells adhered, were fixed and analyzed using scanning electron microscopy. These experiments demonstrate a methodology that allows for the design of complex 3D patterns as a technique to investigate cell behavior as a function of 3D growth.
10:30 AM - QQ1.4
The Influence of Topographic Features Produced by Laser Micromachining and Heat Treatment Techniques on the Adhesion of Bone Cells to Stainless Steel.
Jorge Sobral 1 , Athina Markaki 2 , William O'Neill 2 , Trevor Clyne 1 Show Abstract
1 Department of Materials Science and Metallurgy, University of Cambridge, Cambridge United Kingdom, 2 Department of Engineering, University of Cambridge, Cambridge United Kingdom
Metals like stainless steels are broadly used for different types of orthopedic implants. Despite their good mechanical characteristics, getting a bioactive surface is still a challenge. In the past few years there has been evidence that fine scale topographic features affect the adhesion and growth of cells. Therefore the aim of the present study was to develop several topographies by laser processing and heat treatment techniques on stainless steel specimens in order to evaluate their influence on bone cell behavior, in vitro. The materials used in this study were AISI 316L austenitic stainless steel. The topographic features were produced using laser micromachining and heat treatment techniques. Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and X-Ray Photoelectron Spectroscopy (XPS) techniques were used to characterize the morphology and chemistry of the surface. Human fetal osteoblasts (hFOB) were seeded on such specimens and AlamarBlue assay, SEM imaging and Alkaline phosphatase assays were used to access their proliferation, morphology and differentiation behavior respectively. A centrifuge technique was also used to measure the strength of adhesion of bone cells to the different topographies. The topographic features created varied in terms of morphology, size and distribution depending on the applied technique. Heat treatments produced nano features with a random distribution while laser micromachining produced micro features with different sizes and shapes depending on the laser parameters selected. The biological experiments showed there is a direct correlation between the surface topography and the proliferation, adhesion and differentiation of bone cells. In conclusion, a variety of controlled topographic features were successfully created using heat treatment and laser micromachining techniques and fully characterized in terms of surface morphology and chemistry. Using these techniques it is possible to create a wide range of topographies at micro and nano scale and this might be a feasible way of increasing the potential of metals in terms of biological response for biomedical applications.
10:45 AM - QQ1.5
Protein-based Biomaterials that Dynamically Respond to Disease-regulated Proteases.
Nicole Romano 1 , Karin Straley 2 , Sarah Heilshorn 1 Show Abstract
1 Materials Science & Engineering, Stanford University, Stanford, California, United States, 2 Chemical Engineering, Stanford University, Stanford, California, United States
Many diseases are associated with the altered regulation of proteases, enzymes that catalyze the degradation of peptide bonds leading to tissue remodeling. These proteases are common targets as diagnostic markers for disease identification and disease progression. We describe an engineered biomaterial system that dynamically responds to these patterns of protease secretion by undergoing site-specific degradation with tunable Michaelis-Menten kinetics. This degradation activity may be used to release multiple therapeutics and/or imaging agents with distinct spatial and temporal resolution, thereby coupling therapeutic dosing with specific events that occur during disease progression.Several proteases are known to be upregulated by neurodegenerative diseases such as Alzheimer’s and Parkinson’s, as well as certain brain cancers. The proteases urokinase plasminogen activor (uPA) and tissue plasminogen activator (tPA) are of particular interest because they are secreted selectively at the growth cone, the active process at the tip of an extending neurite. Using a modular, protein-engineering design strategy, we synthesized a family of biomaterials that enables tuning of the density of cell-adhesion ligands and the scaffold elastic modulus. In order to harness the upregulation of proteolytic factors in a neurodegenerative environment, these elastin-like proteins also incorporate proteolytic degradation sites that are targets for uPA and tPA enzymatic activity. The proteins are recombinantly expressed in E. coli and purified by temperature cycling, with a final yield of 100 mg/L. The engineered proteins are covalently crosslinked through lysine residues to form hydrogels with tailored initial elastic moduli ranging from 50 Pa to 50 kPa. We confirm that the hydrogel environment maintains high cellular viability of neuronal-like PC-12 cells and rat neural stem cells. We demonstrate that minor alterations in the primary amino acid sequence (97% identical sequence homology) can tailor target-site recognition by the uPA and tPA enzymes, resulting in a 200-fold change in proteolysis rate. By arranging biopolymers with widely differing and controllably tuned degradation rates within a single, composite hydrogel, patterns can be triggered to emerge over time in response to disease-regulated proteases. Because the polymeric degradation fragments are designed to be smaller than the hydrogel pore size, these three-dimensional patterns include internal void structures that emerge within a well-sealed hydrogel. To demonstrate that the material released during pattern formation can serve as a delivery vehicle for pharmaceuticals and/or imaging contrast agents, we demonstrate the release of two fluorophores from a single hydrogel with distinct spatial and temporal delivery profiles. These biomaterials may be suitable for the design of disease-responsive treatment strategies that harness the degradative action of disease-regulated proteases.
11:30 AM - **QQ1.6
Neuronal Robustness Under Extreme Mechanical Conditions.
Scott Seichen 2 , Shengyuan Yang 3 , Alireza Tofangchi 1 , Jagannathan Rajagopalan 1 , Akira Chiba 4 , Taher Saif 1 Show Abstract
2 , Parkland College, Champaign, Illinois, United States, 3 , University of Maimi, Maimi, Florida, United States, 1 Mechanical Science and Engineering, University of Illinois, Urbana, Illinois, United States, 4 , Florida Institute of Technology, Melbourne, Florida, United States
Neurons play a central role in memory and learning in animals. Most neurons have a long arm, called axon, that contacts other neurons or muscles to form synapse. Synaptic vesicles containing neurotransmitters are clustered at the synapse. In response to an action potential, neurotransmitters are released to the post synaptic terminal by exocytosis. The more a synapse is used, higher is the vesicle clustering, and higher is the neurotransmission efficiency, i.e., the synapse “remembers” its usage in the near past, and modifies its neurotransmission efficiency accordingly. Vesicle clustering has been believed to result from biochemical signaling processes that require the connectivity of the presynaptic terminal with the cell body, the central nervous system and the postsynaptic cell. Here we show, through in vivo experiments on embryonic Drosophila nervous system, that vesicle clustering at the neuromuscular presynaptic terminal requires mechanical tension in the axons. Without tension, clustering disappears, but reappears with application of tension. In vivo axons maintain a rest tension. Mechanical increase in tension results in an increase in clustering. The axons are mechanically robust against very large (more that 100%) stretches. Under sudden stretches, the axonal force increases momentarily, but the force relaxes over time if the stretch is held fixed. If the stretched axon is suddenly released, it shows an elastic recovery followed by an active shrinking when it builds the rest tension again. This restoration is maintained even after large stretches. Preliminary observation suggests that axons may grow under large stretches, and undo the growth upon release of stretch, to regain the rest tension. The synaptic junction is also mechanically robust. It maintains integrity even under mechanical forces that exceed the in vivo rest tension by an order of magnitude. The study reveals the link between axonal tension and neural functionality, and the robustness of both the axon and the synapse against mechanical injuries.
12:00 PM - QQ1.7
In situ Crosslinking Hydrogel for Traumatic Brain Injury Repair.
Xiaowei Li 1 , Xuejun Wen 1 , Xiaoyan Liu 1 Show Abstract
1 Bioengineering, Clemson University, Charleston, South Carolina, United States
Traumatic brain injury (TBI) evokes a cascade of secondary biochemical and molecular changes that result in delayed tissue damage and cell death. Current approaches to traumatic brain injury have been focused on managing the primary injury using hypothermia or neuroprotection with pharmacological agents, all with limited success. Treatments for TBI that attempt to promote functional replacement present a significant challenge because of the poor regenerative capabilities of the brain. Tissue engineering in the post-injury brain represents a promising option for cellular replacement and rescue, providing a cell scaffold for either transplanted or resident cells. Hydrogels are attractive scaffolds for improving tissue regeneration and CNS repair, owing to their tissue-like mechanical abilities, porous structure, potential to attach adhesion and growth-promoting molecules and capacity for drug and gene incorporation and delivery. This study evaluated an in situ crosslinking hydrogel following experimental traumatic brain injury. In vitro study showed growth factors incorporated into these hydrogels released over 6 weeks. Hydrogels also showed excellent neuron biocompatibility and supported mouse neural stem cells (mNSCs) three-dimensionally culture. In the in vivo studies, unilateral injury of the sensorimotor cortex was produced by controlled cortical impact. mNSCs loaded into hydrogels were injected to the lesions 3 days post-injury. The complete vascular network at the injury site was reconstructed 4 weeks after transplantation. mNSCs transplanted survived and differentiated into neurons and astrocytes in the lesions. Behavior tests, such as tape test, were used to confirm that mNSCs with hydrogels injection resulting in an enhanced behavioral recovery.
12:15 PM - QQ1.8
Novel Electrospraying Process for Tissue Regeneration.
Jong Kyu Hong 1 , Sundararajan Madihally 1 Show Abstract
1 Chemical Engineering, Oklahoma State University, Stillwater, Oklahoma, United States
Electrospinning has recently emerged as a novel technique for tissue regeneration because it allows fabricating nano and micro sizes of fibers which is very similar to the characteristic of natural extracellular membrane. A major problem in electrospinning technology is, however, the lack of generating structural features necessary for building 3D tissues. Manmade structures have tiny pores compared to human cells, and do not allow cells to infiltrate into the layers below the surface. Hence, cell growth is restricted to the surface only. In this study, we introduce a novel electrospraying process with the novel collector plate which allows thin layer of fibers and its application for tissue regeneration. Polycaprolactone was used to fabricate electrosprayed fibers. The setup of electrospraying consists of a syringe pump, syringe, needle tip, high voltage power supply, earth grounding, and the novel collector or a conventional one. The physical properties of fibers made by novel and conventional collectors were compared using SEM, CCD camera, and Sigma Scan Pro software. Load and extension curve was also confirmed using INSTRON 5542 and Merlin software. Cell culture study of human fibroblast was carried out in single and multiple layers using layer-by-layer assembly technique. The cells were cultured in serum media, immobilized, and stained with Alexa phalloidin and DAPI. Then, cell morphology was confirmed using fluorescent inverted microscopy, fluorescent confocal microscopy, and SEM. Three pairs of fibers (Fiber A and B, C and D, and E and F) were fabricated under the same conditions except for exchanging novel (A, C, and E) and conventional (B, D, and F) collectors. Physical properties of each pair of fibers were very similar except pore sizes. The pore sizes were 61.75 and 9.95 μm for A and B, 104.83 and 6.35 μm for C and D, 9.14 and 3.21 μm for E and F. The diameters of fibers were 3.18 and 3.37 μm for A and B, 1.80 and 2.97 μm for C and D, and 100 and 350 nm for E and F. The shape factors were 0.525 and 0.571 for A and B, 0.515 and 0.590 for C and D, and 0.355 and 0.585 for E and F. After cell culture using Fiber C, images of cells in the thin layer were collected. The images showed that cells in single and multiple layers of fibers were growing well in space. The innovation of this study is the design of the collector plate which allows for the formation of thin layers of electrosprayed fibers with large pore size. This collector is versatile because it is easy for the novel or a modified collector to be associated with existing techniques of electrospinning. Furthermore, 3D scaffold with layer-by-layer technique will expand its implications on other cells, fibers, tissues and organs.
12:30 PM - QQ1.9
Molecular Mechanistic Insights into the Adhesion of Malaria Infected Erythrocytes With Endothelium.
Chwee Teck Lim 1 2 , Ang Li 2 , Kevin Tan 3 Show Abstract
1 Division of Bioengineering, National University of Singapore, Singapore Singapore, 2 , Singapore-MIT Alliance, Singapore Singapore, 3 Department of Microbiology, National University of Singapore, Singapore Singapore
Plasmodium falciparum is the most deadly species of malaria parasites. When the parasite invades and matures within an erythrocyte, parasite induced proteins are secreted and embedded in the cell membrane. This gives rise to cytoadherence where infected red blood cells (IRBCs) are found to adhere to endothelial cells that line the blood vessel walls. Often, this phenomenon causes severe consequences such as blood clogging. Here, we quantify the molecular interactions between TSP and CD36 (receptor proteins on endothelial cell surface) and that of the parasite exported proteins PfEMP1 secreted to the surface of the IRBCs using an atomic force microscope (AFM). Although the binding kinetics of CD36 and TSP molecules were previously studied using flow based assay, they were performed on a population of cells and there is no quantification of binding forces. Also, there was uncertainty in the number of bonds formed in cell-cell or cell-molecule contact in these assays. Our study seek to help better understand quantitatively the molecular interactions involved in malaria parasitology and suggest strategies in quantitatively evaluating the effectiveness of drugs developed to inhibit cytoadherence.
QQ2: Cell Mechanics and Cell-Material Interactions II
Lim Chwee Teck
Tuesday PM, April 06, 2010
Room 3024 (Moscone West)
2:30 PM - **QQ2.1
Feeling for Cells With Light: Illuminating the Role of Biomechanics for Tumor Metastasis.
Josef Kas 1 Show Abstract
1 Faculty of Physics and Geosciences, University of Leipzig, Leipzig, Saxony, Germany
Light has been used to observe cells since Leeuwenhoek’s × however, we use the forces caused by light described by Maxwell’s surface tensor to feel for the cellular cytoskeleton. The cytoskeleton a compound of highly dynamic polymers and active nano-elements inside biological cells is responsible for a cell’s stability and organization. It mechanically senses a cell’s environment and generates cellular forces sufficiently strong to push rigid AFM-cantilevers out of the way. The active cytoskeleton is described by a new type of polymer physics since nano-sized molecular motors and active polymerization overcome the inherently slow, often glass-like brownian polymer dynamics. The optical stretcher exploits the nonlinear, thus amplified response of a cell’s mechanical strength to small changes between different cytoskeletal proteomic compositions as a high precision cell marker that uniquely characterizes different cell types. Consequentially, the optical stretcher detects tumors and their stages with accuracy unparalleled by molecular biology. As implied by developmental biology the compartmentalization of cells and the epithelial-mesenchymal transition that allows cells to overcome compartmental boundaries strongly depend on cell stiffness and adhesiveness. Consequentially, biomechanical changes are key when metastatic cells become able to leave the boundaries of the primary tumor.
3:00 PM - QQ2.2
Modeling and Simulation of Soft Contact and Adhesion of Stem Cells.
Shaofan Li 1 Show Abstract
1 Department of Civil and Environmental Engineering, University of California-Berkeley, Berkeley, California, California, United States
Recently, experimental results have shown that the rigidity and micro-structures of the substrate have significant influences on the differentiation of stem cellsor the fate of stem cells. To explain such bio-physical or physiology phenomenon has been one of the current focus of stem cell research as well as the cell mechanics and bio-physics research.In this presentation, we shall discuss our recent work on multiscale modeling and simulations of soft elasticity and focal adhesion of stem cells. In particular, we are interested in modeling and simulation of contact andadhesion of stem cells on substrates with different rigidities and micro-structures.In order to understand the precise mechanical influences oncell contact, adhesion, sensing process and to explainthe possible mechanotransduction mechanism, we have developed a three-dimensional soft-matter cell models that use liquid-crystal gel or liquid-crystal elastomer gel to model the overall constitutive relations of the cell and simulated their responses to extra-cellular stimulus andinteractions.To further advance bio-physics modeling of stem cells, in this work, we have systematically build a three-dimensional cell model by treating cells or stem cells as a special soft matter. A multi-component three-dimensional cell model with a coarse grain adhesion body force and various surface tension expressions have been formulated and constructed. In our model, the outerlayer of the cell is a layer of nematic or smetic-A liquid crystal, and the inner cell is modeled as a hyperelastic core with or without liquid crystal elastomer contents. The extra-cellular matrix is modeled as a substrate of hyperelastic block and liquid-crystal polymer layer.To accurately model the contact and adhesion of between the substrate and stem cells, the state-of-art numerical modeling techniques are adopted in our modeling and simulation procedures such as meshfree methods, the level-set method, the phase-field method, etc. Our simulations have shown some remarkable features of cell contact and adhesion that have never been observed before, such as(1) cell spreading induced by combination of adhesive force and surface tension and its sensitivity to substrate rigidities; (2) cell shape evolution and the evolution ofthe mesogen director orientation distribution, an indicator of overall myosin structure of the stem cell; (3) evolutions of the traction force between the cell and substrate and the the overall contractile force inside the cell.In this presentation, we shall focus on discussions of the following topics:(1) how to model the overall myosin responses at the early stage of differentiation process of the stem cell, (2) the effects of both the adhesive force due to ligand-receptor interaction or focal adhesion and the surface tension, and (3) possible cell structure or microstructure changestriggered by substrate's rigidity, stress and strain states, or microstructure.
3:15 PM - QQ2.3
Micromechanical Properties and Structure of the Pericellular Coat of Living Cells.
Heike Boehm 1 2 , Tabea Mundinger 1 2 , Valentin Hagel 1 2 , Uwe Rauch 3 , Jennifer Curtis 4 , Joachim Spatz 1 2 Show Abstract
1 New Materials and Biosystems, Max Planck Institute for Metals Research, Stuttgart, 0, Germany, 2 Biophysical Chemistry, University of Heidelberg, Heidelberg Germany, 3 Department of Experimental Medical Science, Lund University, Lund Sweden, 4 School of Physics, Georgia Instiute of Technology, Atlanta, Georgia, United States
Most mammalian cells are enveloped by a coat of polysaccharides and proteins. This pericellular coat (PCC) plays a vital role in biological processes such as adhesion, proliferation, motility and embryogenesis. Due to its invisibility to most microscopy techniques, little more than their size and molecular composition is currently known. Its backbone is composed of hyaluronan (HA), a highly hydrated polysaccharide that anchors the coat to the cell membrane. The molecular interaction of hyaluronan with different hyaluronan binding proteins determines the architecture of the PCC. Their mesoscopic arrangement influences not only the cell‘s perception of its environment but also its ability to withstand compression. This is especially important for our cells of interest: chondrocytes living and maintaing the load-bearing cartilage. As the PCC envelopes chondrocytes on all non-aderend sides, we are especially interested how this several micrometer thick ‘barrier’ influences the cell’s interaction with its environment. Therefore we employ a toolbox of different biophysical techniques, including confocal microscopy, particle tracking microrheology and adhesive nanostructured surfaces. Thus we were able to obtain a micromechanical map of the PCC, which could be correlated to its hyaluronan distribution profile  and are able to study the dynamic modulation of the PCC in response to different extracellular signals.  H. Boehm, T. A. Mundinger, C. H. J. Boehm, V. Hagel, U. Rauch, J. P. Spatz, J. E. Curtis, Soft-Matter, DOI: 10.1039/B905574F, 2009, 4331-4337.
3:30 PM - QQ2.4
A Coarse-grain Membrane Model for Healthy and Defective Erythrocytes.
George Lykotrafitis 1 , He Li 1 Show Abstract
1 Mechanical Engineering, University of Conneticut, Storrs, Connecticut, United States
We present a novel two-component Red Blood Cell (RBC) membrane model - based on Coarse Grain Molecular Dynamics (CGMD) - that will be then used to understand, at the sub-cellular and cellular levels, the mechanisms leading to morphological and mechanical properties of defective erythrocytes. The model for the lipid bilayer is solvent-free and the inter-grain interaction potential is anisotropic. The model also allows free diffusion of membrane agents. By simultaneously invoking these three characteristics, the proposed method facilitates simulations that span much larger length-scales (~ µm) and time-scales (~ ms) than currently possible with other methods based on classical molecular dynamics models or other coarse-grain approaches. The spectrin cortex is represented by a six-fold symmetric network whose elements follow the Worm-Like Chain model with adjustable connectivity. This model naturally facilitates comprehensive simulations of a wide spectrum of biophysical responses of human red blood cells that strongly influence a variety of human disease states. The model will be applied in the study of spherocyte and elliptocyte formation. Critical quantitative relationships between spectrin density, RBC membrane skeleton integrity, and Hereditary Spherocytosis (HS) and Hereditary Elliptocytosis (HE) will be established. In addition, the two predominant hypotheses regarding the mechanism of membrane loss in HS will be tested.
3:45 PM - QQ2.5
Structural Damage in Erythrocytes Exposed to Nanomaterials.
Asha Rani 1 2 , Prakash Hande 2 , Suresh Valiyaveettil 3 Show Abstract
1 NUS Nanoscience and nanotechnology initiative, National University of Singapore, Singapore Singapore, 2 Department of Physiology, National University of Singapore, Singapore Singapore, 3 Department of Chemistry, National University of Singapore, Singapore Singapore
Erythrocytes (RBC) are major cell populations in the blood which transport oxygen to all tissues. Structural or functional damage to erythrocytes thus could result in serious medical conditions. We have investigated the structural damage in human erythrocytes after exposure to gold, silver and platinum nanoparticles. Exposure to silver nanoparticle resulted in haemolysis, agglutination and pit formation on the RBC cytoskeleton. Additionally, silver nanoparticles appeared to cross link RBCs resulting in haemagglutination and subsequent lysis through membrane damage. An indirect effect of haemolysis was identified by exposing the haemolysed fractions of blood (after removal of nanoparticles) to human nucleated cells, which resulted in an increased DNA damage. Gold and platinum nanoparticle exposed RBCs did not show significant damage. Exposure of RBCs to corresponding metal ions and reactants used in nanoparticle synthesis demonstrated no damage implying that the observed effects are solely due to the nanoparticles. Based on the evidences, we propose that exposure to nanoparticles should be minimized and proper guidelines should be introduced to prevent heavy commercialization of such nanoparticles.
4:30 PM - **QQ2.6
Mechanics of Hierarchical Protein Materials; the Case of Intermediate Filaments.
Laurent Kreplak 1 , Harald Bar 2 , Douglas Staple 1 , Hans-Juergen Kreuzer 1 , Harald Herrmann 2 Show Abstract
1 Physics and Atmospheric Science, Dalhousie University, Halifax, Nova Scotia, Canada, 2 , DKFZ, Heidelberg Germany
Protein materials such as collagen fibers, fibrin clots or hair have in common the hierarchical nature of their building plan that spans three orders of magnitude from the nanoscale to the microscale. Understanding the mechanics of such materials remains a challenge that can only be tackled by combining bottom-up and top-down approaches. I will illustrate both approaches in the study of intermediate filament (IF) based materials such as hair, the stratum corneum of the skin and the slime threads produced by hagfishes. Using a combination of small and wide angle X-ray diffraction on a single hair under stretching, we have been able to characterize the multiscale deformation of keratin IFs inside hair. These experiments revealed the existence of both a structural transition from alpha-helices to beta-sheets of the keratin chains and a sliding of the keratin chains past each-others within each IF. However, keratin IFs in hair are embedded within a cross-linked protein matrix. Hence it is virtually impossible to extract single keratin IFs mechanical properties from the top-down analysis of hair mechanics. Instead, we developped a bottom-up approach using atomic force microscopy (AFM) to manipulate in liquid single IFs on various substrates. We were able to both bend and stretch a single IF thus estimating its Young’s modulus, shear modulus, persistence length and extensibility. By combining theses structural and mechanical data, we developped a nanomechanical model of IFs that was applied to the study of severe myopathy mutations.
5:00 PM - QQ2.7
Near Infrared (NIR) Bioimaging for Cancer Diagnosis and Therapy.
Kohei Soga 1 2 3 , Hiroshi Hyodo 1 2 3 , Kimikazu Tokuzen 1 , Kosuke Tsuji 1 , Hidehiro Kishimoto 3 4 , Tamotsu Zako 5 Show Abstract
1 Department of Materials Science and Technology, Tokyo University of Science, Chiba Japan, 2 PTRC, Tokyo University of Science, Chiba Japan, 3 CTC, Tokyo University of Science, Chiba Japan, 4 , Institute of Bio. Sci., Chiba Japan, 5 , Riken, Saitama Japan
Fluorescence bioimaging (FBI) is one of the most important tools for detecting cancer. For biomedical imaging, especially for the medical diagnosis and therapy, optical loss is the most serious problem compared to other imaging as X-ray CT and MRI, while the FBI has the advantage to provide multicolor and real time image. The optical loss is mainly caused by scattering, which the more is in the shorter wavelength, and IR absorption tail coming from the longer wavelength. A valley is located in near infrared region, between 1000 and 2000 nm, as so-called "biological window." Rare-earth doped ceramic materials are known as efficient fluorescence materials with near infrared (NIR) excitation. Nd:YAG laser, to emit 1064-nm light with 800 nm-excitation, and Er-doped optical fiber amplifier, to emit 1550-nm light with 980-nm excitation, are good examples. The authors have studied rare-earth doped ceramic nanophosphors (RED-CNP) to be applied for the NIR-FIB, which uses both NIR excitation and emission for the imaging in the "biological window." Important issues for using the RED-CNP in a physiological condition is dispersion, prohibition of the nonspecific interaction to the non-targeting part of the body and specific targeting to the object, of the RED-CNP. We have cleared those requirements by particle size control, introduction of a certain polymer and several kinds of ligands to the polymer. As well, NIR bioimaging systems were developed. Some of the demonstrative works toward cancer diagnosis and therapy will be presented.
5:15 PM - QQ2.8
Dynamics of Redox in a Porcine Model for Cancer.
Chunchen Lin 1 , Vladimir Kolossov 1 , Gene Tsvid 1 , Jerrod Henderson 1 , Paul Kenis 1 , Rex Gaskins 1 , Gregory Timp 1 Show Abstract
1 Institute for Genomic Biology, UIUC, Urbana, Illinois, United States
Drug discovery is expensive, and testing the efficacy of drugs is the most expensive part. New assays based on cells are currently being developed to screen candidates under physiological conditions inside living cells. In particular, cancer metabolism is recognized as a target for chemotherapy, but it has been difficult to establish causal relationships between changes in redox state and development of cancer till now. Current pharmacological methods to alter intracellular redox state in cultured cells are significantly limited by (i) their inability to precisely regulate intracellular electron transfer independent of global biochemical alterations and cellular toxicity, and (ii) their inherent design, which requires media replacement that perturbs intracellular homeostasis. To circumvent by these limitations, we created a novel genetic construct that enables real-time and extended assessment of alterations in intracellular redox without cellular disruption. The approach is based on FRET.We have tested the efficiency of a FRET-based redox sensor (CY-RL7) in an experimental setting directly related to cancer biology. We suppose that tumorigenesis in pigs to be similar to that in humans, even at a molecular level. Thus, we are motivated to test 161-T cells, which are primary porcine cells that were genetically engineered to be tumorigenic by expression of proteins known to perturb pathways commonly corrupted in human cancer.1 We performed challenge and recovery experiments with 161-T cells transiently transfected with CY-RL7 and cultured in a microfluidic. The microfluidic is used to precisely control the microenvironment of the cells. We evaluated the effect of an anti-tumor drug commonly known as Carmustine (BCNU).2 After in situ decomposition, BCNU inhibits cellular glutathione reductase (GR), which results in depletion of reduced glutathione (GSH) and accumulation of oxidized glutathione(GSSG). We quantified the effect of BSO and BCNU on redox states of cancer cells by (i) inhibition of GSH biosynthesis by BSO, and (ii) restriction of GSH restoration with the treatment of BCNU. The main outcome from these experiments is a comparison of the dynamics of reduced glutathione GSH depletion and recovery in single cells in real-time. The main difference in biosensor responsiveness was observed when diamide, an oxidant, was removed from the flow chamber, as BCNU-treated 161-T cells either did not or took an extraordinarily longer time to recover from the oxidative challenge than untreated 161-T cells. 1. S.J. Adam, et al., Oncogene, 26(7): 1038 (2007).2. R.H. Schirmer, Angewandte Chemie, 32(4): 13 (1995).
5:30 PM - QQ2.9
Nanoconfinement-induced Viability, Dormancy, and Drug Resistance of Cancer Cells.
Eric Carnes 1 , Carlee Ashley 1 , Walker Wharton 2 , C. Jeffrey Brinker 1 3 4 , Robert Castillo 1 , Theresa Lee 1 Show Abstract
1 Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico, United States, 2 School of Medicine - Pathology, University of New Mexico, Albuquerque, New Mexico, United States, 3 Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, New Mexico, United States, 4 Self-Assembled Materials Dept., Sandia National Laboratories, Albuquerque, New Mexico, United States
Recent attention has focused on the use of cell-influenced materials and architectures to understand environmental influences on cellular behavior, in particular, dormancy, drug resistance, and metastasis - enabling the development of new targeting and drug delivery strategies designed to selectively attack and kill these cells, thereby reducing a significant reservoir of human disease. Our research is based on our discovery that living cells can re-direct inorganic self-assembly to form a unique bio-nano interface, where cells are encapsulated within a fluid, phospholipid membrane that interfaces coherently with a surrounding lipid-templated, self-assembled silica nanostructure (Baca et al. Science 2006). Remarkably, this synergistic living material, which cannot be fabricated by any other known method, maintains cell viability for months to years under harsh desiccating conditions (including evacuation), yet allows accessibility of small molecules, antibodies, and even plasmids to the cell surface. Very recently we extended this cell directed assembly (CDA) approach to the development of a unique metabolically and optically controlled lithographic process that allows patterned integration of live cells into nominally solid-state materials. Equally fascinating are the behaviors induced by cellular integration. We have shown that through chemically and mechanically communicated cell signaling, integration synchronizes the cells and can direct their genetic re-programming. Depending on chemical and mechanical characteristics of the host matrix, we can induce unified populations of cells arrested in various states of the cell cycle and additionally develop new behaviors with no known natural counterparts. This finding is important both to medicine and combating terrorism, as dormant states characteristic of metastatic cancer, TB, and anthrax are resistant to standard drugs developed in culture. By reliably directing cell cycle in an integrated platform (as opposed to heterogeneous culture) we expect to develop improved delivery strategies to target and kill these cells. Freezing cell cycle in populations of desiccation-resistant cells is also needed to develop improved means of stabilizing vaccines for use in remote, undeveloped regions.
5:45 PM - QQ2.10
Blood Factor XII Activation by Mixed-Thiol Surfaces.
James Bauer 2 , Erwin Vogler 3 4 , Christopher Siedlecki 1 2 Show Abstract
2 Bioengineering, Penn State University, Hershey, Pennsylvania, United States, 3 Materials Science and Engineering, Penn State University, University Park, Pennsylvania, United States, 4 Bioengineering, Penn State University, University Park, Pennsylvania, United States, 1 Surgery, Penn State University, Hershey, Pennsylvania, United States
Coagulation resulting from contact activation in blood-material interactions remains a challenge for use of blood-contacting biomaterials. The widespread view of contact activation imparts coagulation factor XII (FXII) activating properties to anionic surfaces. However, previous studies from our group showed nearly equal levels of surface-mediated autoactivation of FXII at both hydrophilic and hydrophobic surfaces, and more recently have shown decreased levels of activation for surfaces with mid-level wetting (∼40-60° contact angle). Further, we have found that FXIIa generation in plasma is attenuated at hydrophobic surfaces rather than accentuated at hydrophilic surfaces. In this study, one- and two-component thiol-modified surfaces having different levels of wettability were utilized as procoagulants for in vitro coagulation assays and FXII activation in neat buffer solution.Mixed carboxyl/methyl- and hydroxyl/methyl modified surfaces were prepared by immersing gold-coated coverslips in 1mM thiol in ethanol solutions for 24 hours. Dodecanethiol, 11-mercaptoundecanoic acid, and 11-mercapto-1-undecanol were used. Prior to thiol deposition, gold-coated coverslips were immersed in 0.125% butyltrichlorosilane (BTS) in chloroform for 15 min to block any glass exposed due to scratches/edges. XPS was used to verify sample composition and estimate proportion of individual components. Samples were used for in vitro coagulation assays using a 50% mixture of recalcified plasma in PBS. Alternatively, samples were placed into purified FXII solution for 30 minutes, with or without an HSA displacement step after 25 min. [FXIIa]eq produced was calculated using methods developed in our lab. Briefly, an FXIIa titration curve was prepared from the same lot of plasma. From this curve, parameters describing activation and propogation of the cascade were derived, allowing for calculation of FXIIa produced by surface contact.The [FXIIa]eq produced by mixed hydroxyl/methyl-surfaces in plasma coagulation assays showed a decrease for materials having adhesion tensions in the range of 40-50 dyne/cm. For mixed carboxyl/methyl surfaces there was a transition region between 45-60 dyne/cm, with samples having adhesion tension <45 dyne/cm showing near 10-fold larger [FXIIa]eq than their mixed hydroxyl analogs. Results for FXII activation in neat buffer solution showed that both carboxyl/methyl- and hydroxyl/methyl surfaces having adhesion tensions near 45 dyne/cm have decreased activation, and also FXII activation by pure hydroxyl- and carboxyl-terminated surfaces were statistically indistinguishable.Plasma coagulation studies support a role for anionic surfaces in contact activation; however, results of FXII activation in neat buffer solution indicate activation is related to surface wettability. A minimum in FXII activation was seen for materials with adhesion tensions of 45-60 dyne/cm, and there was no statistical difference between pure COOH and OH surfaces.
Markus J. Buehler Massachusetts Institute of Technology
David Kaplan Tufts University
Chwee Teck Lim National University of Singapore
Joachim Spatz University of Heidelberg
QQ3: Cell Mechanics and Human Disease
Wednesday AM, April 07, 2010
Room 3024 (Moscone West)
9:30 AM - **QQ3.1
Mechanical Properties of Human Desmin Disease Mutants.
Harald Herrmann 1 Show Abstract
1 , DKFZ, Heidelberg Germany
The extra-sarcomeric cytoskeleton of muscle cells represents a complex organized structural element that gives mechanical support for the sarcomeric working units. In addition, it connects costameres to Z bands, nuclei and organelles such as mitochondria and thereby integrates them into the functional myocyte architecture. Mutations in the human muscle-specific intermediate filament (IF) protein desmin have been identified to cause myofibrillar myopathy (desminopathy). Up to now, mutations in the alpha-helical central “rod” domain are predominant, although a growing number of missense mutations in the non-alpha-helical "head" and “tail” domain leading to disease have been described recently. We have now investigated the biophysical behavior of desmin disease mutants of all three domains that are still able to form filaments in more detail. We have performed high frequency oscillatory squeeze flow measurements to determine the bending stiffness of the filaments, i.e. the persistence length lp, and large amplitude oscillatory shear experiments in order to characterize the mesh size of the filament networks and their strain stiffening properties. All investigated mutations exhibit a significant reduction in strain-stiffening and seem to promote non-affine network deformation. A tail-truncated desmin variant forms extended, regular filaments, but no strain stiffening is observed indicating that indeed the “tail” domain is responsible for this filament-filament interaction.We propose that these alterations of filament and network mechanics will influence the capacity of the extrasarcomeric desmin network to act in cellular mechano-sensing as well as in intracellular mechano-transduction pathways in patient muscle.
10:00 AM - QQ3.2
Resolving Cadherin Interactions at the Single Molecule Level.
Sanjeevi Sivasankar 1 2 , Yunxiang Zhang 3 , W. James Nelson 3 , Steven Chu 4 Show Abstract
1 Physics and Astronomy, Iowa State University, Ames, Iowa, United States, 2 , Ames Laboratory, Ames, Iowa, United States, 3 , Stanford University, Stanford, California, United States, 4 , Department of Energy, Washington, District of Columbia, United States
The cadherin family of calcium dependent cell adhesion proteins are essential for tissue formation and for maintaining tissue integrity. Disruption of cadherin adhesion is common in metastatic cancers. Adhesion is initiated by the binding of cadherins on opposing cell surfaces and is enhanced by the clustering of cadherins at the sites of cell-cell contact. However, the molecular interactions that mediate cadherin adhesion are controversial. Resolving these interactions are critical for understanding how cells adhere, dynamically modulate adhesion and transduce mechanical forces into intracellular signals. The generally accepted model is that cadherins adhere in three stages. First, the functional unit of cadherin adhesion is a cis-dimer formed by the binding of the extracellular region of two cadherins on the same cell surface. Second, formation of low affinity trans-interactions between cadherin cis-dimers on opposing cell surfaces initiates cell-cell adhesion, and third, lateral clustering of cadherins cooperatively strengthens adhesion. Direct molecular proof of these cadherin binding states during adhesion is however contradictory and evidence for cooperativity is lacking. We used single molecule structural (Fluorescence Resonance Energy Transfer) and functional (Atomic Force Microscopy) assays to demonstrate directly that cadherin monomers interact via their outermost domain to form trans-adhesive complexes. We could not detect the formation of cadherin cis-dimers but found that increasing the density of cadherin monomers cooperatively increased the probability of trans-adhesive binding. We also resolved the role of key amino acids in cadherin trans-binding. These results resolve conflicting data on trans- and cis-cadherin binding states and provide quantitative evidence for cooperativity in trans-cadherin adhesion.
10:15 AM - QQ3.3
Tetherless Microgrippers Actuated by Thermal, Chemical, and Biological Signals.
Noy Bassik 1 2 , Timothy Leong 1 , Alla Brafman 1 , Christina Randall 3 , Bryan Benson 1 , David Gracias 1 4 Show Abstract
1 Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 2 School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States, 3 Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 4 Chemistry, Johns Hopkins University, Baltimore, Maryland, United States
Miniaturization of medical devices is a requirement for enabling the development of autonomous microsurgeons. Micromedical tools that work in obscured anatomical areas must sense a disease state and carry out an action on command, either triggered by an observer or due to sensitivity to a specific bioenvironment. The engineering of micromachines to respond to specific physiological stimuli can lead to the development of “smarter” diagnostic and treatment options. We demonstrate miniaturized medical devices in the form of tetherless microgrippers. The grippers have a sensitive polymer layer that allows for actuation in response to specific changes in temperature, solvent, or biochemical environment. The grippers are produced in parallel via lithographic processing and can be actuated in large numbers simultaneously. Magnetic manipulation permits tasks such as entry into enclosed spaces or retrieval of beads from surfaces. We demonstrate a thermally controlled biopsy of a cell mass (an in vitro biopsy) and the subsequent culture of these cells to show their viability. In more recent work, we engineer these tools to respond to specific disease markers or biochemical commands. Many diseases, such as tumors, manifest with specific biochemical markers. We utilized chemically modified biomolecules by lithographically integrating them as the sensitive components into the multilayer microgrippers. This permitted parallel multistep fabrication of a biochemically sensitive hybrid device. When these components sense particular disease markers they actuate, closing the gripper and executing a specific function. We demonstrate the use of several biochemical families to show sensitivity and specificity of action as desired, triggering either due to a disease marker or by manual chemical intervention in a physiological setting. We believe this method of design will allow integration of specific action into other machines that can manipulate tissue and support mechanical loads.Reference: Tetherless thermobiochemically actuated microgrippers, Leong, Randall, Benson, Bassik, Stern, & Gracias, PNAS 106, 703-708 (2009).
10:30 AM - **QQ3.4
Cell Mechanobiology and Human Disease Pathology.
Subra Suresh 1 , Ming Dao 1 Show Abstract
1 School of Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
How do the changes in the mechanobiological characteristics of human cells lead to the onset and progression of disease states? How do the molecular structural changes in the membrane and cytoskeleton of living cells, precipitated by biological, chemical and genetic factors, lead to changes in mechanobiology? We address these questions through a variety of experimental and/or three-dimensional computational studies that probe single-cell deformation, dynamic flow of cells through the microvasculature, cell cytoadherence to the endothelium, as well as cell membrane fluctuations in physiologically relevant conditions. Examples considered here will include Plasmodium falciparum malaria, different types of human cancer as well as hereditary hemolytic disorders. On the basis of these studies, some general observations will be formulated to link cell mechanobiology to human disease diagnostics, therapeutics and drug efficacy assays.
11:30 AM - **QQ3.5
Multi-scale Changes in Nuclear Structure and Mechanics in Hutchison-Gilford Progeria Syndrome.
Kris Dahl 1 2 3 Show Abstract
1 Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States, 2 Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States, 3 Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States
The structural proteins of the nucleus primarily reside at the nuclear envelope and are responsible for maintaining nuclear integrity. Nuclear intermediate filament lamins A, B and C form the nuclear lamina network stabilized by many lamin binding proteins. Both mechanical stability and mechano-sensitive gene expression are altered in cells with mutations in lamin proteins, but the underlying nature of the mechanical properties of the lamina is poorly understood. We study the mechanical defects in these “laminopathies” at many length scales - molecular, multi-molecular, network, nuclear, cellular and multi-cellular - using biophysical and computational approaches. Hutchison-Gilford progeria syndrome (HGPS) is a premature aging syndrome causing systemic defects including cardiovascular disease. A mutant form of lamin A, Δ50 lamin A, causes lamin accumulation at the nuclear membrane resulting in mechanical anomalies. It is unknown if this membrane accumulation is primarily caused by the deletion of a 50 amino acid exon or the retention of a post-translational farnesylation on the mutant. We use purified protein fragments of the lamin A tail domain to quantify changes in molecular protein stability and intermolecular protein-membrane interactions in the cell and with synthetic membrane models.We use micromanipulation to quantify the viscoelastic properties of isolated nuclei from human fibroblasts from (HGPS) patients and in model cell systems which exogenously overexpress the Δ50 lamin A protein. Nuclei from HGPS patients show significant quantitative reduction in the ability to rearrange, and the HGPS nuclei collapse along major axes, suggesting catastrophic failure to distribute applied forces across the entire lamina. The HGPS lamin network appears to have locally-ordered micro-domains, which can explain most of the mechanical differences seen here between HGPS and normal cells and may have functional consequences in disease.In multi-cellular systems exposed to shear stress, we have shown that nuclei expressing Δ50 lamin A are unable to respond to force as control nuclei do: by global reorientation in response to flow and by subnuclear force-mediated reorganization possibly related to mechanotransduction. In a monolayer of cells in which only a fraction expresses the Δ50 lamin A, all cells show reductions in flow-induced reorganization. Thus, we see how changes in redistribution of proteins can lead to mechanical changes in the nucleus and further alter cellular response to flow in a multi-cellular system.
12:00 PM - **QQ3.6
Effect of Specific Lamin A/C Mutations on Nuclear Structure and Mechanics.
Diana Jaalouk 1 , Maria Lucia Lombardi 1 , Philipp Isermann 1 , Jan Lammerding 1 Show Abstract
1 Medicine, Brigham and Women's Hospital/Harvard Medical School, Cambridge, Massachusetts, United States
Mutations in the LMNA gene that encodes the nuclear envelope proteins lamin A and C cause a plethora of human diseases (laminopathies) that include Emery-Dreifuss muscular dystrophy (EDMD), familial partial lipodystrophy, dilated cardiomyopathy (DCM), and Hutchinson-Gilford progeria syndrome. At the cellular level, many of these mutations lead to defects in nuclear shape and morphology, but it remains unclear if the changes in nuclear structure directly contribute to the disease mechanism, and there is no clear correlation between nuclear shape abnormalities caused by specific mutations and the associated disease.Based on our previous findings that lamins A and C are the main contributors to nuclear stiffness, we hypothesized that specific lamin mutations associated with different diseases could differentially affect the structural properties of the lamin network, resulting in altered nuclear mechanics. To compare the effect of specific lamin A/C mutations associated with different laminopathies, we measured the nuclear mechanical properties in fibroblasts derived from patients with diverse laminopathies, from mouse models of these diseases, and using a retroviral expression system to introduce specific lamin A mutations into lamin A/C–null fibroblasts. We found that mutations in the LMNA gene associated with muscular dystrophies (i.e., EDMD and DCM) result in decreased nuclear stiffness, potentially rendering cells more susceptible to physical damage in mechanically stressed tissues such as muscles. In contrast, cells from familial partial lipodystrophy patients had normal nuclear stiffness, and cells from patients with Hutchinson-Gilford progeria syndrome had increased nuclear stiffness. In addition to altered nuclear mechanics, functional loss of lamins A/C resulted in impaired nuclear-cytoskeletal coupling, characterized by defective force transmission from the cytoskeleton to the nucleus in a microneedle manipulation assay.In conclusion, lamin mutations associated with muscular laminopathies can cause impaired nuclear mechanics and more fragile nuclei and, at least in part, defective nuclear-cytoskeletal coupling; taken together, these defects may lead to increased cellular sensitivity to mechanical stress and contribute to the muscle phenotype in these diseases.
12:30 PM - QQ3.7
Single-molecule Nanomechanical Studies of an Engineered Protein Modular Polymer having Exceptional Mechanical Properties.
Zhibin Guan 1 , Dora Guzman 1 , Arlo Randall 2 , Pierre Baldi 2 Show Abstract
1 Chemistry, University of California, Irvine, California, United States, 2 2School of Information and Computer Sciences, University of California, Irvine, California, United States
Modular multi-domain architecture is commonly used in biopolymers as a molecular strategy to combine apparently orthogonal properties such as strength, toughness, and elasticity. Inspired by nature, one research thrust in my group is to design biomimetic polymers having modular multi-domain architecture for advanced mechanical properties. For this purpose, resolving molecular determinants of mechanical stability of proteins is crucial in the rational design of advanced biomaterials for use in biomedical and nanotechnological applications. Here, we present an interdisciplinary study combining bioinformatics screening, steered molecular dynamics (SMD) simulations, protein engineering, and single-molecule force microscopy (SMFS) that explores the mechanical properties of a macro domain protein with mixed alpha + beta topology. The unique architecture is defined by a single 7-stranded beta-sheet in the core of the protein flanked by five alpha-helices. Unlike mechanically stable proteins studied thus far, the macro domain provides the distinct advantage of having the key load-bearing hydrogen bonds (H-bonds) buried in the hydrophobic core protected from water attacks. This feature allows direct measurement of the force required to break apart the load-bearing H-bonds under locally hydrophobic conditions. SMD simulations predicted extremely high mechanical stability of the macro domain using constant velocity and constant force methods. SMFS experiments confirm the exceptional mechanical strength of the macro domain, measuring a rupture force as high as 570 pN. Furthermore, through selective deletion of shielding peptide segments, we examined the same key H-bonds under hydrophilic environments in which the beta-strands are exposed to solvent, and verify that the high mechanical stability of the macro domain results from excellent shielding of the load-bearing H-bonds from competing water. Our study reveals that shielding water accessibility to the load-bearing strands is a critical molecular determinant for enhancing the mechanical stability of proteins. In current study, we are creating a protein-based modular polymer through an interdisciplinary approach combining computation, protein engineering, and single-molecule studies. In this presentation, we will discuss our interdisciplinary approach in bioinformatics search, computation, protein engineering, and single molecule studies for designing biomimetic multimodular protein polymers with exceptional mechanical properties.
12:45 PM - QQ3.8
Investigating the Mechanical Properties of Multiphoton Fabricated Protein Hydrogels Using Atomic Force Microscopy.
Constantine Khripin 1 , C. Jeffrey Brinker 1 2 , Bryan Kaehr 2 Show Abstract
1 Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico, United States, 2 Advanced Materials Laboratory, Sandia National Laboratory, Albuquerque, New Mexico, United States
Recent work has demonstrated the feasibility of employing 3D protein hydrogels, fabricated using multiphoton-induced photochemistry, for use as chemically responsive microactuators in lab on a chip devices. In addition, these materials show great promise as cell capture/incubation devices, allowing single bacterial cells to reproduce into multicellular constructs with “user-defined” 3D geometries. However to date, the mechanical properties of these materials have not been well characterized. Here, we fabricated protein microcantilevers using a wide range of protein building blocks (e.g., albumin, lysozyme, avidin) and probed their mechanical properties using atomic force microscopy (AFM). The length dependence of the spring constant displayed by protein cantilevers followed the predicted cantilever model, yielding the Young’s modulus of the material. We show the modulus of protein cantilevers could be tuned over 3 orders of magnitude (from 0.1 to 100 MPa) by varying either the laser dwell time or protein concentration in the fabrication solution. Further, the modulus was shown to vary strongly over a range of pH values (pH 2-12). Distinct profiles of pH vs. modulus for albumin, lysozyme and avidin cantilevers were observed; the highest modulus values occurring at a pH near the isoelectric point of the incorporated protein. Modification of protein cantilevers by silica condensation (avidin and lysozyme) and ligand binding (biotin to avidin), as well as fabrication of composite albumin matrices (e.g., incorporation of carbon nanotubes) resulted increased cantilever stiffness. Finally, using the calculated modulus of the albumin hydrogel, we determined the pressure generated by a replicating bacterial colony entrapped in an albumin microchamber to be 1-10 kPa. This work will enable the development of a robust platform to investigate cell/microenvironmental interactions with high spatial resolution, in three dimensions, using mechanically tunable biological materials.
QQ4: Ectopic Materials in the Context of Prion and Amyloid Diseases
Wednesday PM, April 07, 2010
Room 3024 (Moscone West)
2:30 PM - **QQ4.1
Self-assembly of Short Aromatic Peptides: From Amyloid Nano-fibrils to Nanotechnology.
Ehud Gazit 1 Show Abstract
1 , Tel Aviv University, Tel Aviv Israel
The formation of ordered amyloid fibrils is the hallmark of several diseases of unrelated origin. In spite of its grave clinical consequence, the mechanism of amyloid formation is not fully understood. We have suggested, based on experimental and bioinformatic analysis, that aromatic interactions may provide energetic contribution as well as order and directionality in the molecular-recognition and self-association processes that lead to the formation of these assemblies. This is in line with the well-known central role of aromatic-stacking interactions in self-assembly processes. Following this notion, we demonstrated that the diphenylalanine recognition motif of the Alzheimer’s β-amyloid polypeptide self-assembles into ordered peptide nanotubes with a remarkable persistence length. Our model recently gained directed support from high-resolution X-ray and electron diffraction and solid-state NMR structures of amyloid fibrils as well as parameter-free models and molecular dynamics studies. We are currently using this notion, as well as a novel β-breakage strategy that was developed in our lab, for the development of novel inhibitors of the process of amyloid formation by utilizing hetero-aromatic interactions. Our lead compound is a novel chemical entity that inhibits the formation of β-amyloid oligomers in vitro and protects cultured cell and isolated cortical neurons from cytotoxic effect of β-amyloid aggregates. Chronic administration of the compound was shown safe and significantly effective in preventing memory impairment in this animal model as assayed by Morris Water Maze experiments. Taken together, our hypothesis provides a new approach to understand the self-assembly mechanism that governs amyloid formation and indicates possible ways to control this process.Relevant recent publication:Frydman-Marom, A., Rechter, M., Shefler, I, Bram, Y., Shalev, D.E., & Gazit E (2009) Cognitive-Performance Recovery of Alzheimer's Disease Model Mice by Modulation of Early Soluble Amyloidal Assemblies. Angew. Chem. Int. Ed Engl. 48, 1981-1986.
3:00 PM - **QQ4.2
Seeded Growth of Beta-Amyloid Fibrils from Alzheimer’s Brain–derived Fibrils Produces a Distinct Fibril Structure.
Anant Paravastu 1 , Isam Qahwash 4 , Wei-Ming Yau 2 , Richard Leapman 3 , Stephen Meredith 4 , Robert Tycko 2 Show Abstract
1 Chemical and Biomedical Engineering, Florida State University and Florida A&M University, Tallahassee, Florida, United States, 4 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, United States, 2 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States, 3 , National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, United States
Recent solid state NMR structural investigations of amyloid fibrils of the Alzheimer’s beta-amyloid peptide (Abeta) have revealed structures that are highly sensitive to environmental conditions during self-assembly (Paravastu, A.K., Leampan, R.D., Tycko, R., et. al. PNAS 105, 18349 (2008).). These results have raised questions on amyloid fibril structures formed in physiological environments. Using amyloid fibrils purified from the brains of deceased Alzheimer’s patients, we have demonstrated that brain amyloid can seed the growth of synthetic Abeta amyloid fibrils (Paravastu, A.K., Meredith, S.C., Tycko, R., et. al. PNAS 106, 7443 (2009).). Resultant fibrils most likely represent disease-relevant amyloid fibril structures. Solid state NMR revealed identical structures from fibrils seeded from the brains of two different Alzheimer’s patients. Furthermore, the predominant brain seeded fibrils differ structurally from those of well characterized synthetic Abeta fibrils. These results suggest the existence of a unique pathological microenvironment and demonstrate the feasibility of structural analysis of in vivo amyloid fibril deposits.
3:30 PM - QQ4.3
Investigating Spherulite Formation in Vitro.
Danielle Cannon 1 , Athene Donald 1 Show Abstract
1 Cavendish Laboratory , University of Cambridge, Cambridge United Kingdom
Protein aggregation is a hugely important area of research, as it is linked to many pathological disorders, such as Alzheimer’s disease, Parkinson’s disease and type II diabetes. It has been suggested that the formation of amyloid fibrils  the aggregates implicated in neurodegenerative disorders, may be a generic ability of all proteins in denaturing conditions. It is known that higher ordered protein assemblies termed spherulites with a distinct Maltese cross can also form. In these structures, fibrils grow outwards from an inner core, which is thought to be either amorphous  or composed of compressed fibrils that have lost their orientation while the spherulite continues to grow .Previous studies of insulin and β-lactoglobulin (BLG) spherulites in the bulk show similarities in their mechanism of growth; however it is not clear as to how the core region forms. Using Raman Spectroscopy, spectra were recorded from various positions across spherulites in insulin and amyloid beta (Aβ). The secondary structure was mainly beta sheet, the proportion of which decreases within the core. Gel Electrophoresis was used to investigate the strong correlation between the formation of spherulites and the degradation of proteins. The degree of degradation increases with prolonged incubation time as does the growth of spherulites. To determine if this was the key to spherulite formation, tests were employed at pH values known not to form spherulites. The protein still degraded into fragments but no spherulites were formed, proving that degradation alone is not responsible for the formation of spherulites.Insulin and β-lactoglobulin are useful model systems to study protein aggregation; however they are not directly related to any pathological disease. From in vivo studies, it is apparent that the deposition of the peptides Aβ (1-40) and (1-42) as amyloid is associated with Alzheimer’s disease. A logical next step has been to move to in vitro studies using the Aβ peptides. Concentrations exceeding the normal concentration for fibrillization studies, 150 μM – 450 μM were employed. Under physiological conditions and at these concentrations, Aβ (1-40) forms spherulites with diameters ranging from 30 to 100 microns, comparable to spherulites formed from insulin and β-lactoglobulin. The peptide Aβ (1-42) seems to lack the ability to form spherulites but does however form fine stranded aggregates and smaller aggregates termed amylospheroids . References: (1). M Sunde, L. C Serpell, M Bartlam, P. E. Fraser, M. B. Pepsy Mol. Biol. 1997 273: 729-739 (2). M. R. H. Krebs, C. E Macphee, A. F. Miller, I. E. Dunlop, C. M. Dobson and A. M. Donald PNAS 2004 101: 14420-14422 (3). Domike Kristin, A.M.D., 2007 Biomarcomolecules. 12: p. 3930-3937. (4). M. Hoshi, M. Sato, S. Matsumoto, A. Noguchi, K. Yasutake PNAS 100: 6370-6375
3:45 PM - QQ4.4
Prion Protein Misfolding and Aggregation Across Ten Animal Species.
Alexander Scouras 1 , Valerie Daggett 1 2 Show Abstract
1 Biochemistry, University of Washington, Seattle, Washington, United States, 2 Bioengineering, University of Washington, Seattle, Washington, United States
The prion diseases are one of a number of amyloid diseases characterized by the misfolding and aggregation of a native host protein into a toxic form, causing neurodegeneration. The toxic particles are believed to be soluble oligomers polymerized via intermolecular β-sheets, but much about the misfolded monomer and the oligomers, including their structures and the basis of toxicity, remains unknown. Beyond their interest as disease vectors, amyloid fibrils may have remarkable structural properties, including high resistance to pH and temperature in the case of prion particles. We use molecular dynamics simulations of the prion protein (PrP) under misfolding conditions to study the molecular cause of and early structures in the oligomerization pathway. We have investigated the behavior of the prion protein (PrP) from 10 species of animals at pH 7, 5, and 3. Five 50ns simulations were performed at each pH regime. We find that in most species, at lower pH, β-strands can form in the natively unstructured N-terminus and dock onto the native sheet; we believe these structures are the early stages of disease-related misfolding. Experimental data on the level of structure in the X-Loop (residues 164-171) across species was confirmed and we have also found differential X-Loop structure at different pH’s, which may have implications for disease transmission. We have further identified other misfolding related properties, including Helix A positioning, solvent exposure of key residues, and important polar and charged contacts.Previously, we have shown that using our proposed misfolded structures, the new β-structure can dock to an amyloidogenic region on an adjacent monomer. By repetition, we created a protofibril of P31 symmetry, the “spiral model”, which was in line with a variety of experimental results. Spiral models from different species each had a characteristic handedness, suggesting a basis for the species barrier. More recent investigation finds that a well-structured X-Loop is necessary in human and bovine protofibrils, but not in those of hamster.
4:30 PM - **QQ4.5
Material Properties of Amyloid Fibrils.
Tuomas Knowles 1 , Mark Welland 2 , Christopher Dobson 1 Show Abstract
1 Chemistry, University of Cambridge, Cambridge United Kingdom, 2 Nanoscience Centre, University of Cambridge, Cambridge United Kingdom
The proliferation of proteinaceous nanostructures such as amyloid fibrils is implicated in a range of pathological conditions which include Alzheimer's disease, Parkinson's disease and late onset diabetes. This talk describes measurements of the material properties of these structures and explores the connections between the mechanical properties of such fibrils and the kinetics of their growth.
5:00 PM - QQ4.6
Mechanisms of Degradation of Beta Sheet Structures.
Keiji Numata 1 , Peggy Cebe 2 , David Kaplan 1 Show Abstract
1 Biomedical Engineering, Tufts University, Medford, Massachusetts, United States, 2 Physics, Tufts University, Medford, Massachusetts, United States
The anti-parallel beta pleated sheet is a fundamental secondary structure in proteins with a key role to stabilize these proteins via physical cross-links. Importantly, these beta-sheets are fully degradable and nontoxic structures in biology, in contrast for example to beta-amyloid structures formed in disease states. Yet mechanisms of enzymatic degradation of these structures are not well understood, and such insight would be instructive as a route to elucidating differences among these stable yet different structural features in biological systems. We will report on the mechanism of enzymatic degradation of anti-parallel beta pleated sheets, leading to fibrils and subsequently to nanofilaments (2 nm thickness and 160 nm length). These nanofilaments play a role as nucleators of the crystalline regions, a novel and important feature of the system that can be exploited to design silk-based biomaterials with predictable biodegradability and mechanical properties. The degradation mechanisms of beta-sheet silk crystals provide additional insight into the significant differences in biological impact between the anti-parallel beta-sheet silk biomaterials vs. amyloid structures in disease states, adding to prior descriptions of chemical and structural differences that are more extensively documented.
5:15 PM - QQ4.7
Imaging Disease-related Protein Aggregates Inside Human Cells Using a Selenium Label.
Eva McGuire 1 , Michael Motskin 2 , Tuomas Knowles 3 , Chris Dobson 3 , David McComb 1 , Alexandra Porter 1 Show Abstract
1 Department of Materials, Imperial College London, London United Kingdom, 2 Department of Anatomy, University of Cambridge, Cambridge United Kingdom, 3 Department of Chemistry, University of Cambridge, Cambridge United Kingdom
The aberrant folding and subsequent aggregation of proteins into insoluble plaques known as amyloid fibrils is the underlying mechanism behind a number of diseases, including Alzheimer’s and Parkinson’s diseases. The exact role that these aggregates play in the disease process is not yet well understood. Part of the reason for this lack of understanding is that it is extremely challenging to visualise the interactions between the protein aggregates and human cells or tissue due to the difficulties that arise when attempting to identify the carbon-rich aggregates within the carbonaceous cellular environment. Traditional strategies to overcome this lack of contrast have involved the use of stains or tags that can potentially be either unreliable or intrusive.In this work we have taken a fragment of the Alzheimer’s-related Aβ peptide and replaced the naturally occurring sulfur that is present in the methionine amino acid with a selenium atom. Human phagocytic cells were exposed to different aggregate species formed from the selenium-labelled Aβ fragment and its selenium-free analogue to examine the toxicity, uptake and distribution of the aggregates. Cells exposed to the selenium-labelled aggregates were imaged using HAADF-STEM, an electron microscopy technique that is highly sensitive to local variations in atomic number. This allows the selenium-labelled aggregates to be identified reliably within the cellular environment without the use of any other stains.In separate experiments it was found that the fully aggregated mature fibrils do not show high levels of toxicity whereas the pre-fibrillar early aggregates are highly cytotoxic, in agreement with previous studies of such species. Both the non-toxic mature fibrils and the highly toxic early aggregates were observed in cell lysosomes, associated with the cell membrane, and in the extracellular space in what appears to be a degraded form. Notably, however, the early aggregates were also localised in the cell cytoplasm and the cell nucleus in an undegraded form. Uptake to these sites implies they may interact with intracellular proteins, organelles and DNA, which would greatly enhance their toxic potential.
QQ5: Poster Session: Biological Materials and Structures in Physiologically Extreme Conditions and Disease
Lim Chwee Teck
Thursday AM, April 08, 2010
Salon Level (Marriott)
9:00 PM - QQ5.1
Controlling the Physical Properties of Random Network Based Shape Memory Polymer Foams.
Pooja Singhal 1 , Ya-Jen Yu 1 , Thomas Wilson 2 , Duncan Maitland 1 2 Show Abstract
1 Biomedical Engineering, Texas A&M University, College Station, Texas, United States, 2 , Lawrence Livermore National Laboratory, Livermore, California, United States
Shape memory polymers (SMPs) are materials that can be deformed into a stable secondary shape, and then controllably actuated to recover their primary shape. There is an increasing interest in these materials due to the possibility of their use in minimally invasive medical devices. Particularly, low density SMP foams are being considered for treatment of aneurysms, a condition in which a localized dilatation occurs in a weakened area of artery wall. This dilated section can eventually rupture and cause death of the patient. Of approximately 700,000 stroke victims per year, 5-15% constitute secondary or ruptured saccular aneurysms. For use of these shape memory polymer based thermally stimulated devices in treatment of aneurysms in humans, it is desirable to be able to closely control the actuation temperature and maintain the breadth of transition within a small range. We are studying the control of glass transition of the shape memory foams based on hexamethylene diisocyanate (HDI), triethanol amine (TEA) and hydroxyl propyl ethanol diamine (HPED). Shape memory foams were synthesized at varying compositions of the constituent chemicals using the blowing technique and the effect that the change in composition has on other physical properties like shape memory behavior and modulus of the material was investigated. For these network based shape memory polymers, this study is used to predict the effect of variation in the content of 3-point and 4-point attachments in the physical properties of the polymer, and in the foaming process of the polymer due to the changes in the pre-polymer rheology based on the composition variation. Glass transition values ranging from 40 °C to 80 °C were achieved with high shape fixity and recovery. These materials also demonstrated water based actuation due to plasticization effects and characterization of their shape memory behavior and modulus was also done under these conditions.
9:00 PM - QQ5.10
Long-term Stability of Fully Integrated Wireless Neural Interface Based on Utah Slant Electrode Array (USEA).
Asha Sharma 1 , Loren Rieth 1 , Prashant Tathireddy 1 , Reid Harrison 1 2 , Richard Normann 2 , Gregory Clark 2 , Florian Solzbacher 1 2 Show Abstract
1 Department of Electrical and Computer Engineering, University of Utah, Salt Lake City, Utah, United States, 2 Department of Bioengineering, University of Utah, Salt Lake City, Utah, United States
Implantable biomedical devices such as microelectrode arrays for neural recording/stimulation are being developed as viable treatments for sensory and movement disorder. One example is 100 electrode neural interface based on Utah Slant Electrode Array (USEA), which is designed to record and stimulate from peripheral nerves. One of the most significant challenges for implanted neural interfaces is the percutaneous connector, which are likely to cause infections during chronic use. To eliminate the use of wired connections, large and non-biocompatible batteries, we are investigating fully integrated wireless neural interfaces. Packaging and encapsulation that can protect the electronic circuitry of neural interface from the harsh in-vivo environment, is a major challenge. In this context, we demonstrate in-vitro, and in-vivo wireless recording of neural signals from an anesthetized cat by using a fully integrated and encapsulated USEA/INI-R5 (integrated neural interface-recording version 5) system. The USEA/INI-R5 was powered and configured wirelessly through 2.765 MHz inductive link, and the neural data was also transmitted wirelessly via RF (900 MHz ISM band) telemetry link. The long term in-vitro stability of USEA/INI-R5 was investigated in saline (to simulate physiological environment) at room temperature. The USEA/INI-R5 employed a biocompatible polymer Parylene C as an encapsulation layer and was immersed in saline for over a period of 3 months (device is still in saline and is being monitored continuously). The device was powered and configured wirelessly, and the transmitted FSK modulated RF signal was recorded as a function of soak time. The USEA/INI-R5 was fully functional (RF power and command transmission) even after 3 months soaking without significant change in the transmitted RF signal strength. Further, in-vitro recording of the artificial neural signals was performed in agarose from time to time by introducing the signals from Grass SD-9 stimulator into the agarose, and recording the signals wirelessly. Our current in-vitro stability (>3 months) of the USEA/INI-R5 provides a measure of the encapsulation reliability, and the functional stability in a fully integrated wireless neural interface that has not been reported previously in wireless implants. These results could potentially evaluate the usefulness of wireless neural interface for future chronic implants.
9:00 PM - QQ5.11
Enhanced Organophosphate Hydrolysis With Enzyme-chaperone Chimeras and Organic/Inorganic Hybrids.
Patrick Dennis 1 2 , Matthew Dickerson 1 , Wendy Crookes-Goodson 1 , Caitlin Knight 1 2 , Arnon Heyman 3 , Oded Shoseyov 3 , Nils Kroger 4 , Kenneth Sandhage 5 , Rajesh Naik 1 Show Abstract
1 Air Force Research Laboratory, Materials and Manufacturing Directorate, Wright-Patterson AFB, Ohio, United States, 2 , UES, Dayton, Ohio, United States, 3 Robert H. Smith Institute of Plant Science and Genetics, Hebrew University, Rehovot Israel, 4 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, United States, 5 School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
Organophosphate hydrolase (OPH) is a bacterial protein that holds promise as an enzymatic agent for the destruction of chemical agents. The use of OPH for decontamination applications would be greatly enhanced in a molecular context that increases enzymatic stability. One potential way to increase enzymatic stability would be to combine the enzyme with a protein chaperone to create an enzyme-chaperone hybrid. The plant chaperone protein, Stable Protein 1 (SP1) is expressed as a monomeric protein of 12.4 kDa, but forms a higher ordered, dodecameric structure through homo-oligomerization. Remarkably, the dodecameric structure of SP1 is resistant to denaturization by heat, organic solvents and ionic detergents, thus preserving its chaperone activity under denaturing conditions for the re-folding of protein substrates. The stabilizing activity of purified, wild-type SP1 has been tested against OPH as a chimera where SP1 was covalently attached to OPH in a single reading frame. Both native OPH and chimeric OPH-SP1 were purified to near homogeneity using a combination of cation exchange and affinity chromatographies. Compared to monomeric OPH, the OPH-SP1 chimera demonstrated significant stabilization when assayed at elevated temperatures. Also, the chimeric OPH-SP1 demonstrated higher temperature stability compared to that of monomeric OPH in the presence of free SP1, even at higher OPH:SP1 mass ratios. As with OPH, previous studies have found that chemically modified titania diatom microshells are able to induce rapid hydrolysis of organophosphates. Based on these studies, the heat stable OPH-SP1 chimera has been used to coat these titania-based diatoms in an effort to enhance the hydrolytic activity of both reagents toward organophosphates. The ability of SP1 to form higher-ordered structures and stabilize covalently associated proteins will be critical in the creation of multi-enzyme complexes as well as organic/inorganic hybrids that are able to catalyze complex reactions under adverse environmental conditions.
9:00 PM - QQ5.12
Miniature Electronic Biosensor for Glycan Biomarker Detection.
Srivatsa Aithal 1 , Vinay Nagaraj 2 , Seron Eaton 2 , Manish Bothara 3 , Peter Wiktor 2 , Shalini Prasad 1 Show Abstract
1 Center for Solid State Electronics Research, Arizona State University, Tempe, Arizona, United States, 2 Center for Bioelectronics and Biosensors, The Biodesign Institute at Arizona State University, Tempe, Arizona, United States, 3 Department of Electrical and Computer Engineering, Portland State University, Portland, Oregon, United States
Glycans (oligosaccharide chains attached to proteins) hold great promise as a new class of biomarkers for the early diagnosis of cancer and other diseases. To realize the potential of glycan biomarkers and to overcome the inherent limitations of current laboratory analytical techniques, our aim is to develop a novel, label free, ultrasensitive diagnostic platform to enable rapid label-free glycosylation analysis from human samples in a clinical setting. The operation of the NanoMonitor is based on the principles of electrochemical impedance spectroscopy (EIS). The device consists of a silicon chip with an array of gold electrodes forming multiple sensor sites. Each sensor site is overlaid with a nanoporous alumina membrane that forms a high density of nanowells. Lectins, proteins that bind to and recognize specific glycan structures, are conjugated to the surface of the electrode. A perturbation to the electrical double-layer is produced when specific glycoproteins from a test sample bind to lectins at the base of each nanowell. This perturbation results in a change in the impedance of the double layer. Unlike conventional laboratory methods our device allows for detection in 15min, requires lower sample volume of 10ul, five orders of magnitude more sensitive, highly selective over a broad range of concentrations and can detect multiple glycan biomarkers on the same platform allowing for multiple screening. We have used this sensor to analyze glycoform variants of the fetuin and cancer cell extracts. Results from biosensor compare very well with lectin-based ELISA.
9:00 PM - QQ5.13
Crystallization of Synthetic Haemozoin (Beta-Haematin) Nucleated at the Surface of Lipid Particles.
Anh Hoang 1 2 Show Abstract
1 Material Science, Vanderbilt University, Nashville, Tennessee, United States, 2 Chemistry, Vanderbilt University, Nashville, Tennessee, United States
The mechanism of formation of haemozoin, a detoxification by-product of several blood-feeding organisms including malaria parasites, has been a subject of debate; however, recent studies suggest that neutral lipids may serve as a catalyst. In this study, a model system consisting of an emulsion of neutral lipid particles was employed to investigate the formation of beta-haematin, the synthetic counterpart of haemozoin, at the lipid-water interface. A solution of monoglyceride, either monostearoylglycerol (MSG) or monopalmitoylglycerol (MPG), dissolved in acetone and methanol was introduced to an aqueous surface. Fluorescence, confocal and transmission electron microscopic (TEM) imaging and dynamic light scattering analysis of samples obtained from beneath the surface confirmed the presence of homogeneous lipid particles existing in two major populations: one in the low micrometer size range and the other in the hundred nanometre range. The introduction of haem (Fe(III)PPIX) to this lipid particle system under biomimetic conditions (37C, pH 4.8) produced beta-haematin with apparent first order kinetics and an average half life of 0.5 min. TEM of monoglycerides (MSG or MPG) extruded through a 200 nm filter with haem produced beta-haematin crystals aligned and parallel to the lipid/water interface. These TEM data, together with a model system replacing the lipid with an aqueous organic solvent interface using either methyl laurate or docosane demonstrated that the OH and C=O groups are apparently necessary for efficient nucleation. This suggests that beta-haematin crystallizes via epitaxial nucleation at the lipid-water interface through interaction of Fe(III)PPIX with the polar head group. Once nucleated, the crystal grows parallel to the interface until growth is terminated by the curvature of the lipid particle. The hydrophobic nature of the mature crystal favours an interior transport resulting in crystals aligned parallel to the lipid-water interface and each other, strikingly similar to that seen in malaria parasites.
9:00 PM - QQ5.15
PEGDMA Microstructures Attenuate Fibrotic Phenotype and Extracellular Matrix Deposition.
Perla Ayala 1 , Jose Lopez 2 , Tejal Desai 3 Show Abstract
1 Joint Graduate Group in Bioengineering, UCSF/UCB , San Francisco, California, United States, 2 Department of Surgery, University of California, San Francisco , San Francisco, California, United States, 3 Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, United States
Chemical signals modulate cell migration, proliferation, differentiation, and ultimately death. Similarly, physical signals, and mechanical stresses can be converted into intracellular responses that regulate cell behavior and fate. Knowledge of how these physical cues affect cell function in three dimensions is critical for successful development of novel regenerative therapies. In this work, the role of discrete micromechanical cues on cell proliferation and ECM synthesis is investigated in a three dimensional (3D) system. PEGDMA hydrogel microrods were fabricated using photolithography and suspended in Matrigel to create a 3D culture with microscale cues of defined mechanical properties in the physiological range (2 kPa-50 kPa). These microrods had a significant differential effect on fibroblast proliferation and gene expression. Cultures with stiff microrods reduced fibroblast proliferation and down-regulated expression of ECM proteins. In addition, the contractility marker alpha smooth muscle actin and adhesion molecule integrin alpha 3 were also significantly down-regulated. Cultures with soft microrods had no significant difference on fibroblast proliferation and expression of collagen VI, alpha-SMA, and integrin alpha 3 compared to cultures with no microrods. Here, we present a new platform of injectable microrods with tunable elasticity; in addition we show that cell proliferation and gene expression are influenced by discrete physical cues in 3D. In this work, engineered microrods are developed as a new therapeutic approach for tissue regeneration.
9:00 PM - QQ5.16
Multi-scale Mechanical Property Evaluation of Soft Materials.
Jonathan Henderson 1 , Chad Watson 2 , William Hughes 2 , William Knowlton 2 3 Show Abstract
1 Mechanical Engineering, Boise State University, Boise, Idaho, United States, 2 Material Science and Engineering, Boise State University, Boise, Idaho, United States, 3 Electrical Engineering, Boise State University, Boise, Idaho, United States
Comprehensive evaluation of the biomechanical properties associated with the initiation of joint-related diseases, such as osteoarthritis, is needed to promote a more thorough understanding of cartilage degeneration mechanisms. For instance, opportunities to probe the biomechanical properties, from a multi-scale perspective (cartilage matrix to the collagen fibrils), could provide insights into the failure modes of degenerative diseases. However, the complexity of cartilage (i.e., composite structure) presents a challenge for isolating the contribution of its individual components to local mechanical property variations. Thus, a multi-scale approach enabling high resolution quantitative mechanical property maps is needed to develop cartilage structure-property relationships. For the techniques used in this study, the quantitative mechanical property measurement capabilities with associated spatial resolution were inversely related. The techniques include vertical nanoindentation with loads ranging in the 1000s µN, atomic force microscopy (AFM) cantilevered nanoindentation with loads ranging in the 10s µN, and AFM-based torsional harmonics imaging (HMX) with loads ranging in the 0.01s µN were used. Prior to probing biomaterials, the accuracy of the three systems was evaluated using material systems, polycarbonate (PC) and polymethyl methacrylate (PMMA). The polymers were selected for multiple reasons: (i) to avoid substrate effects, (ii) to minimize the influence of heterogeneities inherent to biomaterial composites and (iii) because of their similar material properties (elastic modulus and viscoelasticity). With the use of the polymer material systems, it was determined that the AFM and HMX measurements are within 15% of the expected elastic modulus of the polymer standards as measured with vertical indentation. The predominant source of the variation is most likely from inaccurate tip area calculations, viscoelastic effects and cantilever spring constant calculations. With the variation between the three systems established, AFM and HMX-based mechanical property measurements were made on collagen, a component of cartilage with extensive mechanical property data available for comparison. The results for collagen were within the range of published values of 1- 3 GPa. In addition, HMX coupled with the AFM and vertical indentation techniques enable high spatial resolution mechanical property maps that can potentially be used to study the initiation of degenerative diseases.
9:00 PM - QQ5.17
Reduced Molecular Flexibility in the Large Arteries of Diabetic Rats.
Riaz Akhtar 1 , Natalie Gardiner 1 , Kennedy Cruickshank 1 , Brian Derby 1 , Michael Sherratt 1 Show Abstract
1 , The University of Manchester, Manchester United Kingdom
In Type 1 and 2 diabetes tissue stiffening is evident from measurements of the gross mechanical properties of the vasculature. In general, pathological glycosylation of extracellular matrix proteins may play an important role in increasing stiffness in diabetic patients. However, the effects of diabetes on individual elastic fibre components remain poorly defined.Fibrillin microfibrils, a key elastic fibre component, have a ‘beads-on-a-string’ structure with a periodicity of approximately 56 nm. We tested for possible disruption due to diabetes in fibrillin microfibrils isolated from rat aorta. Diabetes was induced in 250g adult Wistar rats by streptozotocin (STZ) injection (55mg/kg) and were sacrificed 8 weeks later along with age-matched controls. At sacrifice, the STZ-treated rats had severe hyperglycaemia (+/- 28 mmol/l). Fibrillin microfibrils were isolated and purified by well-established bacterial collagenase digestion and size-exclusion chromatography prior to imaging with atomic force microscopy.Initial experiments show that fibrillin microfibril periodicity is reduced following STZ treatment ; 52.0 +/- 0.4 nm (STZ) vs 56.9 +/- 0.4 nm (Control), n = 600 periodicity measurements, 2 animals per group), (p<0.01). In young, healthy tissues the structure of fibrillin microfibrils is stabilised by both intra- and inter-chain disulphide bonds and by transglutaminase cross-links which permit reversible extension in vivo. These observations suggest that the formation of pathological cross-links may limit microfibril elasticity and hence play an important role in increasing the stiffness of the diabetic vasculature.
9:00 PM - QQ5.2
Correlation Between Nanomechanical Properties and Antibiotic Resistance in S. Aureus.
David Carroll 1 Show Abstract
1 , Wake Forest University, Winston-Salem, North Carolina, United States
Bacterial slime is widely believed to play a significant role in adhesion to surfaces and assist in antimicrobial resistance, thereby increasing rates and severity of bacterial infections. Unfortunately, little is known about the mechanism through which slime contributes to resistance. In this investigation, we used in situ atomic force microscopy to compare strains of Staphylococcus aureus expressing a permeable slime capsule with non-permeable slime encapsulated S. aureus, as well as non-capsulated S. aureus, in order to understand how the capsule affects adhesiveness and behaves as a barrier. Detailed analysis of capsular response under local force application was completed to understand the relationship between mechanical properties and protective capabilities of the slime. Importantly, we find that the nano-scale mechanical properties of slime acting as a barrier to antimicrobials were found to be distinct from slime without barrier capability. Specifically, the barrier slime is more spatially uniform and the capsular polysaccharides composing the slime barrier had a stronger non-specific adhesive interaction than non-barrier slime. The stronger adhesive interaction may retard movement of antimicrobials through the extracellular layer and possibly explain treatment failures associated with infections caused by strains of S. aureus which typically are susceptible to antibiotics in vitro.
9:00 PM - QQ5.3
On-media Axon Branching and Adhesion Investigation of Neurons as Stimulated by Modulated Potentials on Micro-patterned Gold Substrate.
Tanunya Visessonchok 1 , Naruphan Pukalanant 1 , Anan Srikiatkhachorn 2 , Nipan Israsena 3 , Min Medhisuwakul 4 , Nirun Witit-anun 5 , Saknan Bongsebandhu-phubhakdi 2 , Boonrat Lohwongwatana 6 1 Show Abstract
1 Nanoengineering program, International School of Engineering, Chulalongkorn University, Bangkok Thailand, 2 Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok Thailand, 3 Department of Pharmacology, Faculty of Medicine, Chulalongkorn University, Bangkok Thailand, 4 Department of Physics, Faculty of Science, Chiang Mai University, Chiang Mai Thailand, 5 Department of Physics, Faculty of Science, Burapha University, Chonburi Thailand, 6 Department of Metallurgical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok Thailand
Gold is one of the increasingly used materials in biotechnology, this research will investigate its compatibility as a patterned substrate in relations with the adhesion and proliferation of neuron cells using electrical potentials to induce magnetic field. Gold is chosen due to its facile methods of synthesis, high degree of control over shape and size, long-term stability in a wide variety of solvents and pH conditions, and most importantly in the study regarding neurons, its conductive nature toward surface-molecule interaction and modification. Currently, gold is being tested and extensively developed as biomaterials in dental and drug delivery applications as well as compounds in treatments of cancer. Recent researches showed that gold and gold metallic glass could easily replicate 3D patterns in the 100-nm length-scale. Ordinary metals, such as copper, could be deposited as thin films on Polystyrene cell culture substrate, therefore it is speculated that the same results could be made using pure crystalline gold and amorphous gold alloy. Using various thin film synthesis techniques including Cathode Arc Vapor Deposition, Sputtering, Magnetron Sputtering, and flash evaporation, distributions of the thin films will then be analyzed. Different patterning techniques, such as Stencil Patterning and Microcontact Printing will be investigated. The 3D patterns will be induced by electrical potentials to generate electric field and magnetic field near neurons. Overall shapes of the magnetic fields are speculated to have various effects on neural behaviors. Two types of cells will be experimented in our protocols; namely Osteoblasts, T3T, and Neuro2A. Thus, cell-substrate adhesion interactions, manipulation of neuronal growth and proliferation using electrical potentials will be explored on pure gold substrates in this research. Specifically, the ambition of this research is to contribute to the development of neuron circuits that will allow more efficient procedures for nerve repair. This research’s greatest hope is not only to provide current developments with extensive data for further improvements, but also to comprehend better the constraints restraining the breakthroughs of novel technologies.
9:00 PM - QQ5.5
AFM Characterization of Type I Collagen Fibril Morphology in Healthy and Diseased Bones.
Ming Fang 1 , Joseph Wallace 1 , Blake Erikson 5 , Clifford Les 4 , Bradford Orr 2 3 5 , Mark Banaszak Holl 1 2 5 Show Abstract
1 chemistry, university of michigan, Ann Arbor, Michigan, United States, 5 Biophysics, University of Michigan, Ann Arbor, Michigan, United States, 4 Bone and Joint Center, Henry Ford Hospital, Detroit, Michigan, United States, 2 Applied Physics, University of Michigan, Ann Arbor, Michigan, United States, 3 physics, University of Michigan, Ann Arbor, Michigan, United States
Collagen fibrils play important roles in bone tissue, where they not only serve as structure scaffolding, but also provide resistance against fracture. Once excreted from osteoblastic cells, collagen molecules self-assemble into fibrils with overlap/gap spacing, conventionally known as the 67 nm D-spacing. In this study, Atomic Force Microscope (AFM) was used to quantitatively study surface morphology of collagen fibrils. AFM has proven its excellence in probing biological material due to its non-destructive nature and ambient working conditions. We hypothesize that AFM can be used to quantitatively measure fibril D-spacings in their native morphology. Previous studies have shown that collagen fibrils in different tissue (bone, teeth and tendon) all possess a distribution of D-spacings versus a single value 67 nm. By comparing the distribution of D-spacings in healthy and diseased bone, such as osteopenic bone, we hope to gain new insights into the effects of diseases on bone’s collagen phase. Current diagnostic methods for osteoporosis focus exclusively on abnormalities in bone quantity and mineral component, and disregard the role collagen plays in bone health. In that aspect, our study could have significant implications to understanding disease mechanism, and could further provide an alternative diagnostic technique which may lead to earlier disease detection.
9:00 PM - QQ5.6
3D Nano-tomography and Density Quantification of Mineralized Tissue in Mouse Bone Trabeculae Exposed to Radiation and to Hind Limb Unloading.
Florian Meirer 1 6 , Eduardo Almeida 2 , Josh Alwood 2 , Cathy Lee 3 , Marjolein C. van der Meulen 4 , Jie Chen 5 , Joy Andrews 6 , Piero Pianetta 6 Show Abstract
1 Institute for Atomic and Subatomic Physics, TU Vienna, Vienna , Vienna, Austria, 6 Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California, United States, 2 Ames Research Center, NASA , Moffett Field, California, United States, 3 Department of Biological Science, San Jose State University, San Jose, California, United States, 4 Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York, United States, 5 National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei China
The full-field Xradia transmission X-ray microscope (TXM) installed at SSRL beam line 6-2 is optimized to operate from 5-14 keV in absorption contrast and at 5 and 8keV in Zernike phase contrast in both modes providing resolution as high as 30 nm. The microscope offers a single flat field of view of 30 µm2 but further allows tiling together multiple fields of view (mosaic mode) for larger samples. Recently software has been developed within MATLAB for quantitative reconstruction of tomographic scans using single field of view or mosaic mode. Providing not only the distribution (qualitative data) but also the real values of the absorption coefficient enables a) calculation of the density values at each point when the attenuation coefficient is known or b) identification of chemical elements (with known density) by their absorption coefficient. We will present TXM images taken at 5.4 keV in Zernike phase contrast, as well as density quantification results from 3D tomography in absorption contrast, of trabeculae from mice exposed to radiation and to hind limb unloading (a NASA-developed method to simulate micro gravity). We hypothesize that in space travel exposure to weightlessness and radiation can affect mineral density in bone. High resolution details of osteocytic, lacunar and canalicular organization and localized changes in density near lacunae may yield insight into the nano-structural effects of space flight on bone. Quantification of bone density was performed by calibration of the X-ray attenuation with crystalline chlorapatite, a structure close to the mineral phase responsible for the x-ray attenuation of bone tissue.
9:00 PM - QQ5.7
Agarose-based Nerve Guidance Scaffolds for Nerve Lesions Spanning 1cm and Beyond.
Daniel Lynam 1 Show Abstract
1 , Michigan State University, East Lansing, Michigan, United States
Nerve damage is a common condition following a traumatic incident, such as an automobile accident or a serious fall. The result of this injury is usually devastating, and can lead to long-term paralysis. However, recent work has provided ways of encouraging nerve growth and reconnection, indicating the possibility of repair. For nerve repair over distances of human clinical relevance (one centimeter or longer), a scaffolding structure is required to guide nerves and establish reconnection with the non-damaged site. Certain requirements are necessary for such a nerve growth scaffold (NGS) to be viable. Firstly, the NGS must be compatible with the body to reduce immune responses, be mechanically stable, and properly guide nerve axons across lesion sites. Secondly, incorporation and gradual release of nerve growth proteins from the NGS is desired to promote nerve growth. Lastly, controlled degradation of the NGS is necessary to mitigate full nerve regeneration and remove