Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner.
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The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy. (+info)
Direct measurement of force generation by actin filament polymerization using an optical trap.
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Actin filament polymerization generates force for protrusion of the leading edge in motile cells. In protrusive structures, multiple actin filaments are arranged in cross-linked webs (as in lamellipodia or pseudopodia) or parallel bundles (as in filopodia). We have used an optical trap to directly measure the forces generated by elongation of a few parallel-growing actin filaments brought into apposition with a rigid barrier, mimicking the geometry of filopodial protrusion. We find that the growth of approximately eight actin parallel-growing filaments can be stalled by relatively small applied load forces on the order of 1 pN, consistent with the theoretical load required to stall the elongation of a single filament under our conditions. Indeed, large length fluctuations during the stall phase indicate that only the longest actin filament in the bundle is in contact with the barrier at any given time. These results suggest that force generation by small actin bundles is limited by a dynamic instability of single actin filaments, and therefore living cells must use actin-associated factors to suppress this instability to generate substantial forces by elongation of parallel bundles of actin filaments. (+info)
Internal strain regulates the nucleotide binding site of the kinesin leading head.
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In the presence of ATP, kinesin proceeds along the protofilament of microtubule by alternated binding of two motor domains on the tubulin binding sites. Because the processivity of kinesin is much higher than other motor proteins, it has been speculated that there exists a mechanism for allosteric regulation between the two monomers. Recent experiments suggest that ATP binding to the leading head (L) domain in kinesin is regulated by the rearward strain built on the neck-linker. We test this hypothesis by explicitly modeling a Calpha-based kinesin structure whose motor domains are bound on the tubulin binding sites. The equilibrium structures of kinesin on the microtubule show disordered and ordered neck-linker configurations for the L and trailing head, respectively. The comparison of the structures between the two heads shows that several native contacts present at the nucleotide binding site in the L are less intact than those in the binding site of the rear head. The network of native contacts obtained from this comparison provides the internal tension propagation pathway, which leads to the disruption of the nucleotide binding site in the L. Also, using an argument based on polymer theory, we estimate the internal tension built on the neck-linker to be f approximately 12-15 pN. Both of these conclusions support the experimental hypothesis. (+info)
Mechanically-evoked C-fiber activity in painful alcohol and AIDS therapy neuropathy in the rat.
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While altered activities in sensory neurons were noticed in neuropathic pain, caused by highly diverse insults to the peripheral nervous system, such as diabetes, alcohol ingestion, cancer chemotherapy and drugs used to treat AIDS, other infections and autoimmune diseases, as well as trauma, our understanding of how these various peripheral neuropathies manifest as altered neuronal activity is still rudimentary. The recent development of models of several of those neuropathies has, however, now made it possible to address their impact on primary afferent nociceptor function. We compared changes in mechanically-evoked C-fiber activity, in models of painful peripheral neuropathy induced by drinking ethanol (alcohol) or administering 2',3'-dideoxycytidine (ddC), a nucleoside reverse transcriptase inhibitor for AIDS therapy, two co-morbid conditions in which pain is thought to be mediated by different second messenger signaling pathways. In C-fiber afferents, ddC decreased conduction velocity. In contrast, alcohol but not ddC caused enhanced response to mechanical stimulation (i.e., decrease in threshold and increase in response to sustained threshold and supra-threshold stimulation) and changes in pattern of evoked activity (interspike interval and action potential variability analyses). These marked differences in primary afferent nociceptor function, in two different forms of neuropathy that produce mechanical hyperalgesia of similar magnitude, suggest that optimal treatment of neuropathic pain may differ depending on the nature of the causative insult to the peripheral nervous system, and emphasize the value of studying co-morbid conditions that produce painful peripheral neuropathy by different mechanisms. (+info)
Preparation and properties of chitosan/calcium phosphate composites for bone repair.
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Chitosan/calcium phosphate (CaP) composites composed of bioactive calcium phosphate and flexible chitosan were made by a simple mixing-and-heating method. Phase composition, morphology, and mechanical properties--including in-air and in vitro fatigue behavior - were evaluated. Experimental results showed that the chitosan matrix did not affect the crystalline phase of CaP. However, the content of CaP additive affected the three-point bending strength of the composites. A CaP/ chitosan ratio of 5% by mass to volume in the composite achieved the significantly highest bending strength of 45.7 MPa. Stability of chitosan/CaP hybrid composites was apparently affected by in vitro cyclic loading. Nonetheless, when applied a loading stress of 11.4 MPa, the sample containing the optimal 5 mass/vol% CaP lasted 40 minutes in in vitro fatigue test until failure occurred. It was thus concluded that hybrid biocomposites with initial high strength might be a potential implant candidate for bone defect repair. (+info)
Extracting kinetics from single-molecule force spectroscopy: nanopore unzipping of DNA hairpins.
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Single-molecule force experiments provide powerful new tools to explore biomolecular interactions. Here, we describe a systematic procedure for extracting kinetic information from force-spectroscopy experiments, and apply it to nanopore unzipping of individual DNA hairpins. Two types of measurements are considered: unzipping at constant voltage, and unzipping at constant voltage-ramp speeds. We perform a global maximum-likelihood analysis of the experimental data at low-to-intermediate ramp speeds. To validate the theoretical models, we compare their predictions with two independent sets of data, collected at high ramp speeds and at constant voltage, by using a quantitative relation between the two types of measurements. Microscopic approaches based on Kramers theory of diffusive barrier crossing allow us to estimate not only intrinsic rates and transition state locations, as in the widely used phenomenological approach based on Bell's formula, but also free energies of activation. The problem of extracting unique and accurate kinetic parameters of a molecular transition is discussed in light of the apparent success of the microscopic theories in reproducing the experimental data. (+info)
Entropic elasticity controls nanomechanics of single tropocollagen molecules.
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We report molecular modeling of stretching single molecules of tropocollagen, the building block of collagen fibrils and fibers that provide mechanical support in connective tissues. For small deformation, we observe a dominance of entropic elasticity. At larger deformation, we find a transition to energetic elasticity, which is characterized by first stretching and breaking of hydrogen bonds, followed by deformation of covalent bonds in the protein backbone, eventually leading to molecular fracture. Our force-displacement curves at small forces show excellent quantitative agreement with optical tweezer experiments. Our model predicts a persistence length xi(p) approximately 16 nm, confirming experimental results suggesting that tropocollagen molecules are very flexible elastic entities. We demonstrate that assembly of single tropocollagen molecules into fibrils significantly decreases their bending flexibility, leading to decreased contributions of entropic effects during deformation. The molecular simulation results are used to develop a simple continuum model capable of describing an entire deformation range of tropocollagen molecules. Our molecular model is capable of describing different regimes of elastic and permanent deformation, without relying on empirical parameters, including a transition from entropic to energetic elasticity. (+info)
Finite machines, mental procedures, and modern physics.
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A Turing machine provides a mathematical definition of the natural process of calculating. It rests on trust that a procedure of reason can be reproduced mechanically. Turing's analysis of the concept of mechanical procedure in terms of a finite machine convinced Godel of the validity of the Church thesis. And yet, Godel's later concern was that, insofar as Turing's work shows that "mental procedure cannot go beyond mechanical procedures", it would imply the same kind of limitation on human mind. He therefore deems Turing's argument to be inconclusive. The question then arises as to which extent a computing machine operating by finite means could provide an adequate model of human intelligence. It is argued that a rigorous answer to this question can be given by developing Turing's considerations on the nature of mental processes. For Turing such processes are the consequence of physical processes and he seems to be led to the conclusion that quantum mechanics could help to find a more comprehensive explanation of them. (+info)