Optical approach for determining strain anisotropy in quantum wells

Anisotropic in-plane strain arises in quantum-well systems by design or unintentionally. We propose two methods of measuring the in-plane strain anisotropy based on the optical polarization anisotropy that arises with anisotropic in-plane strain. One method uses purely optical means to determine the strain anisotropy in quantum wells under a compressive strain that is spatially varying. A second approach, applicable to quantum wells under tensile strain or with strain that does not vary with position, requires the application of a uniaxial in-plane stress. Although the second method is experimentally more difficult, it allows analysis of systems that would otherwise be inaccessible.     

Analysis and simulation of changes in EMG amplitude during high-level fatiguing contractions

Changes in surface electromyographic (EMG) amplitude during sustained, fatiguing contractions are commonly attributed to variations in muscle fiber conduction velocity (MFCV), motor unit firing rates, transmembrane action potentials and the synchronization or recruitment of motor units. However, the relative contribution of each factor remains unclear. Analytical relationships relating changes in MFCV and mean motor unit firing rates to the root mean square (RMS) and average rectified (AR) value of the surface EMG signal are derived. The relationships are then confirmed using model simulation. The simulations and analysis illustrate the different behaviors of the surface EMG RMS and AR value with changing MFCV and firing rate, as the level of motor unit superposition varies. Levels of firing rate modulation and short-term synchronization that, combined with variations in MFCV, could cause changes in EMG amplitude similar to those observed during sustained isometric contraction of the brachioradialis at 80% of maximum voluntary contraction were estimated. While it is not possible to draw conclusions about changes in neural control without further information about the underlying motor unit activation patterns, the examples presented illustrate how a combined analytical and simulation approach may provide insight into the manner in which different factors affect EMG amplitude during sustained isometric contractions.     

An improved reconstruction algorithm for 3-D diffraction tomography using spherical-wave sources

Diffraction tomography (DT) is an inversion technique that reconstructs the refractive index distribution of a weakly scattering object. In this paper, a novel reconstruction algorithm for three-dimensional diffraction tomography employing spherical-wave sources is mathematically developed and numerically implemented. Our algorithm is numerically robust and is much more computationally efficient than the conventional filtered backpropagation algorithm. Our previously developed algorithm for DT using plane-wave sources is contained as a special case.     

Study of residual particle concentrations generated by the ultrasonic nebulization of deionized water stored in different container types

A scanning mobility particle sizer has been used to quantify residual particle number and mass concentrations generated by ultrasonic nebulization of deionized (DI) water stored in a variety of bottles. High variability of residual particles was found not only between different bottle types but also between different bottles of the same type. Degradation of the water quality, quantified as increased residual mass and number concentrations as a function of time, occurred to varying degrees for water stored in different bottle types. Overall, glass bottles showed the highest residual particle concentrations and exhibited the poorest stability over time. After a storage period of 3 weeks, DI water stored in Pyrex bottles showed average increases in particle mass and number densities in the aerosol of over 250% and 60%, respectively. Total dissolved impurity levels in the water increased from 110 to 290 ng mL(-1) over the 3-week period. It is hypothesized that leaching from the bottle walls increases impurity levels in the water over time. Leaching was observed for both glass and polymer bottles. Contrary to this trend, residual particle concentrations from deionized water stored in Teflon bottles showed a net decrease during the measurement period. With respect to absolute residual particle concentrations and storage stability, a Teflon bottle yielded the best performance. Total residual particle mass and number densities for Teflon were less than a factor of 15% and 1%, respectively, as compared to residual particle levels observed for the Pyrex bottle. Absolute dissolved impurity levels in the water for the Teflon bottle decreased from 7.8 to 3.7 ng mL(-1) over the 4-week period.     

Chip-based analysis of protein-protein interactions by fluorescence detection and on-chip immunoprecipitation combined with microLC-MS/MS analysis

A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by microLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein-protein interactions.     

Use of a microchip device coupled with mass spectrometry for ligand screening of a multi-protein target

Nanoflow electrospray mass spectrometry has been applied previously to investigate noncovalent protein-protein and protein-ligand interactions. Here we evaluate a commercial microchip device for this application. We show that the microchip can be used to obtain mass spectra of the noncovalent tetramer transthyretin. The device showed a 10-fold increase in signal stability compared with a nanoflow capillary and a high level of nozzle-to-nozzle reproducibility. Binding of the natural ligand thyroxine was clearly observed, and a range of small molecules proposed as inhibitors of transthyretin amyloidosis were shown to be effective in stabilizing the tetramer. We propose that measuring the ability of small molecules to stabilize protein complexes using this automated microchip technology will enable high-throughput screening of multi-protein complexes by mass spectrometry.     

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.     

Factors affecting the static shear strength of the prosthetic stem–bone cement interface

Debonding of the stem–cement interface has been implicated in the initiation of failure of cemented femoral stems. The objective of this work was to examine some of the parameters which influence the interface static shear strength, including surface finish, cement type, pre-treatments and porosity. Surface finish was found to have the greatest effect on the interface strength. Increasing the surface roughness by a factor of 100 increases the interface shear strength by a factor of 20. However, increasing the surface roughness above a certain value was found to have no additional affect. This was due to failure in the cement itself rather than at the cement–stem interface. There were significant differences between some of the different cement types regarding the interface strength. Pre-heating the stem produced a six fold reduction in cement porosity at the stem–cement interface, however, resulting in only a minor influence on the static interface strength. Generally, no significant correlation was found between the cement porosity and the static interfacial shear strength.     

Prediction of the multimeric assembly of staphylococcal enterotoxin A with cell-surface protein receptors

Staphylococcal enterotoxin A (SEA) cross-links two class II major histocompatibility complex (MHC) molecules and forms a multimeric assembly with T-cell receptors (TcRs). The X-ray crystal structure of SEA has been solved, yet details describing molecular recognition and association remain unclear. We present a structural model for the interactions of SEA with cell-surface proteins. Molecular docking calculations predicting SEA association with the class II MHC molecule HLA-DR1 were performed by using a rigid-body docking method. Docked orientations were evaluated by a Poisson-Boltzmann model for the electrostatic free energy of binding and the hydrophobic effect calculated from molecular surface areas. We found that the best-scoring SEA conformers for the DR1alpha interface display a binding mode similar to that determined crystallographically for staphylococcal enterotoxin B bound to HLA-DR1. For the zinc-binding site of SEA, docking DR1beta yielded several orientations exhibiting tetrahedral-like coordination geometries. Combining the two interfaces, tetramers were modeled by docking an alphabeta TcR with trimolecular complexes DR1beta-SEA-DR1alpha and SEA-betaDR1alpha-SEA. Our results indicate that the complex DR1beta-SEA-DR1alpha provides a more favorable assembly for the engagement of TcRs, forming SEA molecular contacts that are in accord with reported mutagenesis studies. In contrast, the cooperative association of two SEA molecules on a single DR1 molecule sterically inhibits interactions with TcRs. We suggest that signal transduction stimulated by SEA through large-scale assembly is limited to four or five TcR-(DR1beta-SEA-DR1alpha) tetramers and requires the dimerization of class II MHC molecules, while TcR dimerization is unlikely.