Bradley E. Layton

Design and microfabrication of a high-aspect-ratio PDMS microbeam array for parallel nanonewton force measurement and protein printing

Cell and protein mechanics has applications ranging from cellular development to tissue engineering. Techniques such as magnetic tweezers, optic tweezers and atomic force microscopy have been used to measure cell deformation forces of the order of piconewtons to nanonewtons. In this study, an array of polymeric polydimethylsiloxane (PDMS) microbeams with diameters of 10–40 µm and lengths of 118 µm was fabricated from Sylgard® with curing agent concentrations ranging from 5% to 20%. The resulting spring constants were 100–300 nN µm−1. The elastic modulus of PDMS was determined experimentally at different curing agent concentrations and found to be 346 kPa to 704 kPa in a millimeter-scale array and ~1 MPa in a microbeam array. Additionally, the microbeam array was used to print laminin for the purpose of cell adhesion. Linear and nonlinear finite element analyses are presented and compared to the closed-from solution. The highly compliant, transparent, biocompatible PDMS may offer a method for more rapid throughput in cell and protein mechanics force measurement experiments with sensitivities necessary for highly compliant structures such as axons.

Dehydration-induced expression of a 31-kDa dehydrin in Polypodium polypodioides (Polypodiaceae) may enable large, reversible deformation of cell walls.

Current and predicted climate changes caused by global warming compel greater understanding of the molecular mechanisms that plants use to survive drought. The desiccation-tolerant fern Polypodium polypodioides exhibits extensive cell wall folding when dried to less than 15% relative water content (RWC) and rapidly (within 24 h) rehydrates when exposed to water and high humidity. A 31-kDa putative dehydrin polypeptide expressed in partially and fully dry tissues detected via western blotting was present only during drying and rapidly dissipated (within 24 h) upon tissue rehydration. Immunostaining indicates the presence of dehydrin near the cell wall of partially and fully dried tissues. Atomic force microscopy of tracheal scalariform perforations indicates that dry vascular tissue does not undergo significant strain. Additionally, environmental scanning electron microscopy reveals differential hydrophilicity between the abaxial and adaxial leaf surfaces as well as large, reversible deformation. The ability to avoid cell wall damage in some desiccation-tolerant species may be partially attributed to cell wall localization of dehydrins enabling reversible, large cell-wall deformation. Thus, the de novo synthesis of dehydrin proteins and potential localization to the cell walls of these desiccation-tolerant species may play a role in avoiding mechanical failure during drought.

Nerve collagens from diabetic and nondiabetic Sprague–Dawley and biobreeding rats: an atomic force microscopy study

Alterations in rats nerve collagens due to diabetes may be related to the permanence of damage due to diabetic neuropathy. We (1) provide a methodology for determining the diameters of collagen fibers accounting for atomic force microscope (AFM) imaging artifacts, (2) present data on structural differences in sciatic nerve endoneurial, epineurial and tail tendon collagens of control and diabetic Sprague–Dawley and BioBreeding rats, and (3) compare results with literature values.

Molecular Modeling of the Axial and Circumferential Elastic Moduli of Tubulin

Microtubules play a number of important mechanical roles in almost all cell types in nearly all major phylogenetic trees. We have used a molecular mechanics approach to perform tensile tests on individual tubulin monomers and determined values for the axial and circumferential moduli for all currently known complete sequences. The axial elastic moduli, in vacuo, were found to be 1.25 GPa and 1.34 GPa for α- and β-bovine tubulin monomers. In the circumferential direction, these moduli were 378 MPa for α- and 460 MPa for β-structures. Using bovine tubulin as a template, 269 homologous tubulin structures were also subjected to simulated tensile loads yielding an average axial elastic modulus of 1.10 ± 0.14 GPa for α-tubulin structures and 1.39 ± 0.68 GPa for β-tubulin. Circumferentially the α- and β-moduli were 936 ± 216 MPa and 658 ± 134 MPa, respectively. Our primary finding is that that the axial elastic modulus of tubulin diminishes as the length of the monomer increases. However, in the circumferential direction, no correlation exists. These predicted anisotropies and scale dependencies may assist in interpreting the macroscale behavior of microtubules during mitosis or cell growth. Additionally, an intergenomic approach to investigating the mechanical properties of proteins may provide a way to elucidate the evolutionary mechanical constraints imposed by nature upon individual subcellular components.

Collagen’s Triglycine Repeat Number and Phylogeny Suggest an Interdomain Transfer Event from a Devonian or Silurian Organism into Trichodesmium erythraeum

Two competing effects at two vastly different scales may explain collagen’s current translation length. The necessity to have long molecules for maintaining mechanical integrity at the organism and supraorganism scales may be limited by the need to have small molecules capable of robust self-assembly at the nanoscale. The triglycine repeat regions of all 556 currently cataloged organisms with collagen-like genes were ranked by length. This revealed a sharp boundary in the GXY transcript number at 1032 amino acids (344 GXY repeats). An anomalous exception, however, is the intron-free Trichodesmium erythraeum collagen gene. Immunogold atomic force microscopy reveals, for the first time, the presence of a collagen-like protein in T. erythraeum. A phylogenetic protein sequence analysis which includes vertebrates, nonvertebrates, shrimp white spot syndrome virus, Streptococcus equi, and Bacillus cereus predicts that the collagen-like sequence may have emerged shortly after the divergence of fibrillar and nonfibrillar collagens. The presence of this anomalously long collagen gene within a prokaryote may represent an interdomain transfer from eukaryotes into prokaryotes that gives T. erythraeum the ability to form blooms that cover hundreds of square kilometers of ocean. We propose that the collagen gene entered the prokaryote intron-free only after it had been molded by years of mechanical selective pressure in larger organisms and only after large, dense food sources such as marine vertebrates became available. This anomalously long collagen-like sequence may explain T. erythraeum’s ability to aggregate and thus concentrate its toxin for food-source procurement.

In situ imaging of mitochondrial outer-membrane pores using atomic force microscopy.

Here we describe a technique for imaging of the outer contours of the mitochondrial membrane using atomic force microscopy, subsequent to or during a toxic or metabolic challenge. Pore formation in both glucose-challenged and 1,3-dinitrobenzene (DNB)-challenged mitochondria was observed using this technique. Our approach enables quantification of individual mitochondrial membrane pore formations. With this work, we have produced some of the highest resolution images of the outer contours of the in situ mitochondrial membrane published to date. These are potentially the first images of the component protein clusters at the time of formation of the mitochondrial membrane transition pore in situ. With the current work, we have extended the application of atomic force microscopy of mitochondrial membranes to fluid imaging. We have also begun to correlate 3-D surface features of mitochondria dotted with open membrane pores with features previously viewed with electron microscopy (EM) of fixed sections.

Recent Patents in Bionanotechnologies: Nanolithography,Bionanocomposites, Cell-Based Computing and Entropy Production

This article reviews recent disclosures of bio-inspired, bio-mimicked and bionanotechnologies. Among the patents discussed is a nanoscale porous structure for use in nanocomposites and nanoscale processing. Patents disclosing methods for printing biological materials using nanolithography techniques such as dip-pen technology are discussed, as are patents for optimizing drug design. The relevance of these technologies to disease prevention, disease treatment and disease resistance is discussed. The paper closes with a review of cell-based computing and a brief examination of how information technology has enabled the development of these technologies. Finally, a forecast of the how these technologies are likely to accelerate global entropization is discussed as well as a new classification of machine types.

A Comparison of Energy Densities of Prevalent Energy Sources in Units of Joules Per Cubic Meter

Typically, the energy densities of solids or liquids such as coal and oil are measured in dimensions of energy per unit volume or energy per unit mass, whereas solar, wind, and hydroelectric sources are rated in dimensions of power per unit area. This article provides a unifying framework for comparing several prevalent energy sources on an energy-per-unit volume basis for the purpose of unifying conventional metrics. The energy density of oil is 35 to 45 gigajoules (10,000 kWh) per cubic meter*. When measured using the methods presented, solar energy has a density of 1.5 microjoules per cubic meter, over twenty quadrillion times less than oil. Human energy density is approximately 1000 J/m3, while other inexhaustibles such as wind and tidal have energy densities of 0.5 to 50 J/m3. This article provides an educational engineering mathematics framework for calculating energy densities of prevalent energy sources. The goal is to provide a new perspective on how to compare energy sources on a more fundamental basis. Finally, the article provides a method of estimating the dollars-per-joule for natural resources versus human resources and concludes with commentary on how political decisions may be affected by energy densities and energy costs. *One gigajoule equals one billion joules, and there are 3,600,000 joules in a kWh. A cubic meter is about half the volume of a kitchen refrigerator.

Determination of the mechanical properties of DOPC:DOPS liposomes using an image procession algorithm and micropipette-aspiration techniques.

Quantification of the mechanical properties of liposomes is critical in helping to predict their behavior during various applications such as targeted drug delivery, response to mechanical characterization or their interactions with isolated cytoskeletal elements. A numerical implementation of the Evans aspiration technique, and an image processing algorithm for measuring deformation of spherical DOPC:DOPS liposomes is presented. Liposomes were aspirated to pressures of -10mmHg (∼-1300Pa). The area expansion and Youngs moduli of the liposomes were found to be 0.067Nm⁻¹ (67±4dyn/cm) and 15±1 MPa.

Cytoskeleton-membrane interactions in neuronal growth cones: a finite analysis study.

Revealing the molecular events of neuronal growth is critical to obtaining a deeper understanding of nervous system development, neural injury response, and neural tissue engineering. Central to this is the need to understand the mechanical interactions between the cytoskeleton and the cell membrane, and how these interactions affect the overall growth mechanics of neurons. Using finite element analysis, the stress in the membrane produced by an actin filament or a microtubule acting against a deformable membrane was modeled, and the deformation, stress, and strain were computed for the membrane. Parameters to represent the flexural rigidities of the well-studied actin and tubulin cytoskeletal proteins, as well as the mechanical properties of cell membranes, were used in the simulations. Our model predicts that a single actin filament is able to produce a normal contact stress on the cell membrane that is sufficient to cause membrane deformation but not growth. Our model also predicts that under clamped boundary conditions a filament with a buckling strength equal to or smaller than an actin filament would not cause the areal strain in the membrane to exceed 3%, and therefore the filament is incapable of causing membrane rupture or puncture to a safety factor of approximately 15-25. Decreasing the radius of the membrane upon which the normal contact stress is acting allows an increase in the amount of normal contact stress that the membrane can withstand before rupture. The model predicts that a 50 nm radius membrane can withstand approximately 4 MPa of normal contact stress before membrane rupture whereas a 250 nm radius membrane can withstand approximately 2.5 MPa. Understanding how the mechanical properties of cytoskeletal elements have coevolved with their respective cell membranes may yield insights into the events that gave rise to the sequences and superquaternary structures of the major cytoskeletal proteins. Additionally, numerical modeling of membranes can be used to analyze the forces and stresses generated by nanoscale biological probes during cellular injection.