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.     

Damage Modeling of Notched Graphite/Epoxy Sandwich Panels in Compression

Open-hole honeycomb sandwich panels with woven graphite/epoxy facesheets and Nomex cores were tested uniaxially in compression to characterize their damage tolerance. A plain weave T-300 graphite fiber fabric was used for the facesheets in two stacking sequences: [45/0₂] and [0₃]. Observations of macroscopic sub-critical damage behavior were different in the two material systems. Linear damage zones (LDZ), consisting of fiber micro-buckles and extensive delamination, were typically observed in the [0₃] material. The [45/0₂] material exhibited a delamination/bulge zone (DBZ), which consisted of an out-of-plane curved deformation of the outer 45° ply accompanied by a delamination from the interior 0° plies. Modeling of these apparently distinct failure modes, and comparison to experimental data, revealed that the only mode representative of damage tolerant behavior is linear damage zone formation and propagation for both material systems, and that the delamination/bulge behavior is a secondary phenomenon.     

Iterated Phantom Induction: A Knowledge-Based Approach to Learning Control

We advance a knowledge-based learning method that allows prior domain knowledge to be effectively utilized by machine learning systems. The domain knowledge is incorporated not into the learning algorithm itself but instead affects only the training data. The domain knowledge is used to explain and then transform the actual training examples into a more informative set of imaginary, or phantom examples. These phantom examples are added to the training set; the experienced examples are discarded. A new control policy is induced from the phantom training set. This policy is then exercised, yielding additional training points, and the process repeats.We investigate the performance of this method in a stylized air-hockey domain which demands a difficult nonlinear control policy. Our experiments show that, surprisingly, an accurate policy can be learned even if the domain theory is only imprecise and approximate. We advance an interpretation which indicates that the information available from a plausible qualitative domain theory is sufficient for robust successful learning. This interpretation is used to make a number of predictions which are tested in subsequent experiments. The outcomes confirm the interpretation and the robustness of the approach.     

Distributed Control for 3D Metamorphosis

In this paper, we define Proteo as a class of three-dimensional (3D) metamorphic robotic system capable of approximating arbitrary 3D shapes by utilizing repeated modules. Each Proteo module contains embedded sensors, actuators and a controller, and each resides in a 3D grid space. A module can move itself to one of its open neighbor sites under certain motion constraints. Distributed control for the self-reconfiguration of such robots is an interesting and challenging problem. We present a class of distributed control algorithms for the reconfiguration of Proteo robots based on the goal-ordering mechanism. Performance results are shown for experiments of these algorithms in a simulation environment, and the properties of these algorithms are analyzed.