Emilios K. Dimitriadis

Determination of Elastic Moduli of Thin Layers of Soft Material Using the Atomic Force Microscope

We address three problems that limit the use of the atomic force microscope when measuring elastic moduli of soft materials at microscopic scales. The first concerns the use of sharp cantilever tips, which typically induce local strains that far exceed the linear material regime. We show that this problem can be alleviated by using microspheres as probes, and we establish the criteria for their use. The second relates to the common use of the Hertz contact mechanics model, which leads to significant errors when applied to thin samples. We develop novel, simple to use corrections to apply for such cases. Samples that are either bonded or not bonded to a rigid substrate are considered. The third problem concerns the difficulty in establishing when contact occurs on a soft material. We obtain error estimates for the elastic modulus resulting from such uncertainty and discuss the sensitivity of the estimation methods to error in contact point. The theoretical and experimental results are compared to macroscopic measurements on poly(vinyl-alcohol) gels.

Robust Strategies for Automated AFM Force Curve Analysis—I. Non-adhesive Indentation of Soft, Inhomogeneous Materials

The atomic force microscope (AFM) has found wide applicability as a nanoindentation tool to measure local elastic properties of soft materials. An automated approach to the processing of AFM indentation data, namely, the extraction of Youngs modulus, is essential to realizing the high-throughput potential of the instrument as an elasticity probe for typical soft materials that exhibit inhomogeneity at microscopic scales. This paper focuses on Hertzian analysis techniques, which are applicable to linear elastic indentation. We compiled a series of synergistic strategies into an algorithm that overcomes many of the complications that have previously impeded efforts to automate the fitting of contact mechanics models to indentation data. AFM raster data sets containing up to 1024 individual force-displacement curves and macroscopic compression data were obtained from testing polyvinyl alcohol gels of known composition. Local elastic properties of tissue-engineered cartilage were also measured by the AFM. All AFM data sets were processed using customized software based on the algorithm, and the extracted values of Youngs modulus were compared to those obtained by macroscopic testing. Accuracy of the technique was verified by the good agreement between values of Youngs modulus obtained by AFM and by direct compression of the synthetic gels. Validation of robustness was achieved by successfully fitting the vastly different types of force curves generated from the indentation of tissue-engineered cartilage. For AFM indentation data that are amenable to Hertzian analysis, the method presented here minimizes subjectivity in preprocessing and allows for improved consistency and minimized user intervention. Automated, large-scale analysis of indentation data holds tremendous potential in bioengineering applications, such as high-resolution elasticity mapping of natural and artificial tissues.

The influence of cochlear shape on low-frequency hearing

The conventional theory about the snail shell shape of the mammalian cochlea is that it evolved essentially and perhaps solely to conserve space inside the skull. Recently, a theory proposed that the spirals graded curvature enhances the cochleas mechanical response to low frequencies. This article provides a multispecies analysis of cochlear shape to test this theory and demonstrates that the ratio of the radii of curvature from the outermost and innermost turns of the cochlear spiral is a significant cochlear feature that correlates strongly with low-frequency hearing limits. The ratio, which is a measure of curvature gradient, is a reflection of the ability of cochlear curvature to focus acoustic energy at the outer wall of the cochlear canal as the wave propagates toward the apex of the cochlea.

Tetrameric organization of vertebrate centromeric nucleosomes

Mitosis ensures equal genome segregation in the eukaryotic lineage. This process is facilitated by microtubule attachment to each chromosome via its centromere. In centromeres, canonical histone H3 is replaced in nucleosomes by a centromere-specific histone H3 variant (CENH3), providing the unique epigenetic signature required for microtubule binding. Due to recent findings of alternative CENH3 nucleosomal forms in invertebrate centromeres, it has been debated whether the classical octameric nucleosomal arrangement of two copies of CENH3, H4, H2A, and H2B forms the basis of the vertebrate centromere. To address this question directly, we examined CENH3 [centromere protein A (CENP-A)] nucleosomal organization in human cells, using a combination of nucleosome component analysis, atomic force microscopy (AFM), and immunoelectron microscopy (immuno-EM). We report that native CENP-A nucleosomes contain centromeric alpha satellite DNA, have equimolar amounts of H2A, H2B, CENP-A, and H4, and bind kinetochore proteins. These nucleosomes, when measured by AFM, yield one-half the dimensions of canonical octameric nucleosomes. Using immuno-EM, we find that one copy of CENP-A, H2A, H2B, and H4 coexist in CENP-A nucleosomes, in which internal C-terminal domains are accessible. Our observations indicate that CENP-A nucleosomes are organized as asymmetric heterotypic tetramers, rather than canonical octamers. Such altered nucleosomes form a chromatin fiber with distinct folding characteristics, which we utilize to discriminate tetramers directly within bulk chromatin. We discuss implications of our observations in the context of universal epigenetic and mechanical requirements for functional centromeres.

Active control of noise transmission through rectangular plates using multiple piezoelectric or point force actuators

This paper analytically demonstrates the use of multiple piezoelectric actuators bonded to the surface and point force actuators applied directly to a plate to reduce sound transmission through the plate. A harmonic plane wave incident on a simply supported, thin rectangular plate mounted in an infinite baffle was considered as the primary source. Both multiple piezoelectric and point force actuators are separately used as secondary (control) sources to attenuate the sound transmission through the plate. An optimal process was applied to obtain the input voltages of the piezoelectric actuators and the magnitude of the point forces, so that the radiated acoustic power can be minimized. Results show that a reduction of sound transmission through the plate is successfully achieved, if the proper size, number, and position of the piezoelectric or point force actuators are selected. Additionally, a comparison showed that point force actuators provide more effective control of the sound transmission than piezoelectric actuators; however, piezoelectric patches have more practical implementation than point force shakers, because of their low cost and light weight.

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network

High-density fibrillar collagen matrix induces invadopodia formation in both fibroblasts and carcinoma cell lines through a kindlin2-dependent mechanism that drives local ECM remodeling.

Initial Stages of V(D)J Recombination: The Organization of RAG1/2 and RSS DNA in the Postcleavage Complex

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.

Right-handed DNA supercoiling by an octameric form of histone-like protein HU: modulation of cellular transcription.

In bacteria, the contribution of global nucleoid organization in determining cellular transcription programs is unclear. Using a mutant form of the most abundant nucleoid-associated protein HU, HUαE38K,V42L, we previously showed that nucleoid remodeling by the mutant protein re-organizes the global transcription pattern. Here, we demonstrate that, unlike the dimeric wild-type HU, HUαE38K,V42L is an octamer and wraps DNA around its surface. The formation of wrapped nucleoprotein complexes by HUαE38K,V42L leads to a high degree of DNA condensation. The DNA wrapping is right-handed, which restrains positive supercoils. In vivo, HUαE38K,V42L shows altered association and distribution patterns with the genetic loci whose transcription are differentially affected in the mutant strain.

Arterial Wall Properties and Womersley Flow in Fabry Disease

BackgroundFabry disease is an X-linked recessive lysosomal storage disease resulting in the cellular accumulation of globotriaosylceramide particularly globotriaosylceramide. The disease is characterized by a dilated vasculopathy with arterial ectasia in muscular arteries and arterioles. Previous venous plethysomographic studies suggest enhanced endothelium-dependent vasodilation in Fabry disease indicating a functional abnormality of resistance vessels.MethodsWe examined the mechanical properties of the radial artery in Fabry disease, a typical fibro-muscular artery. Eight control subjects and seven patients with Fabry disease had a right brachial arterial line placed allowing real time recording of intra-arterial blood pressure. Real time B-mode ultrasound recordings of the right radial artery were obtained simultaneously allowing calculation of the vessel wall internal and external diameter, the incremental Youngs modulus and arterial wall thickness. By simultaneously measurement of the distal index finger-pulse oximetry the pulse wave speed was calculated. From the wave speed and the internal radial artery diameter the volume flow was calculated by Womersley analysis following truncation of the late diastolic phase.ResultsNo significant difference was found between Fabry patients and controls for internal or external arterial diameters, the incremental Youngs modulus, the arterial wall thickness, the pulse wave speed and the basal radial artery blood flow. Further, no significant difference was found for the radial artery blood flow in response to intra-arterial acetylcholine or sodium nitroprusside. Both drugs however, elevated the mean arterial flow.ConclusionsThe current study suggests that no structural or mechanical abnormality exists in the vessel wall of fibro-muscular arteries in Fabry disease. This may indicate that a functional abnormality downstream to the conductance vessels is the dominant feature in development Fabry vasculopathy.

Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd</=0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.