Reconstruction of conductivity and current density images using only one component of magnetic field measurements

Magnetic resonance current density imaging (MRCDI) is to provide current density images of a subject using a magnetic resonance imaging (MRI) scanner with a current injection apparatus. The injection current generates a magnetic field that we can measure from MR phase images. We obtain internal current density images from the measured magnetic flux densities via Ampere's law. However, we must rotate the subject to acquire all of the three components of the induced magnetic flux density. This subject rotation is impractical in clinical MRI scanners when the subject is a human body. In this paper, we propose a way to eliminate the requirement of subject rotation by careful mathematical analysis of the MRCDI problem. In our new MRCDI technique, we need to measure only one component of the induced magnetic flux density and reconstruct both cross-sectional conductivity and current density images without any subject rotation.     

Estimation of anomaly location and size using electrical impedance tomography

We developed a new algorithm that estimates locations and sizes of anomalies in electrically conducting medium based on electrical impedance tomography (EIT) technique. When only the boundary current and voltage measurements are available, it is not practically feasible to reconstruct accurate high-resolution cross-sectional conductivity or resistivity images of a subject. In this paper, we focus our attention on the estimation of locations and sizes of anomalies with different conductivity values compared with the background tissues. We showed the performance of the algorithm from experimental results using a 32-channel EIT system and saline phantom. With about 1.73% measurement error in boundary current-voltage data, we found that the minimal size (area) of the detectable anomaly is about 0.72% of the size (area) of the phantom. Potential applications include the monitoring of impedance related physiological events and bubble detection in two-phase flow. Since this new algorithm requires neither any forward solver nor time-consuming minimization process, it is fast enough for various real-time applications in medicine and nondestructive testing.     

DNA hybridization using surface plasmon-coupled emission

We describe a new approach to measuring DNA hybridization using surface plasmon-coupled emission (SPCE). Excited fluorophores are known to couple with surface oscillations of electrons in thin metal films, typically 50 nm thick silver on a glass prism. These surface plasmons then radiate into the glass at a sharply defined angle determined by the emission wavelength and the optical properties of the glass and metal. This radiation has the same spectral profile as the emission spectrum of the fluorophores. We studied the emission due to Cy3-labeled DNA oligomers bound to complementary unlabeled oligomers which were themselves bound to the metal surface. Hybridization resulted in SPCE due to Cy3-DNA into the prism. Directional SPCE was observed whether the sample was illuminated from the sample side or through the glass substrate at the surface plasmon angle for the excitation wavelength. A large fraction of the total potential emission is coupled to the surface plasmons resulting in improved sensitivity. When illuminated through the prism at the surface plasmon angle, the sensitivity is increased due to the enhanced intensity of the resonance evanescent field. It is known that SPCE depends on proximity to the silver surface. As a result, changes in emission intensity are observed due to fluorophore localization even if hybridization does not affect the quantum yield of the fluorophore. The use of SPCE resulted in suppression of interfering emission from a noncomplementary Cy5-DNA oligomers due to weaker coupling of the more distant fluorophores with the surface plasmons. We expect SPCE to have numerous applications to nucleic acid analysis and for the measurement of bioaffinity reactions.     

Identification of water-soluble selenium-containing proteins in selenized yeast by size-exclusion-reversed-phase HPLC/ICPMS followed by MALDI-TOF and electrospray Q-TOF mass spectrometry

An approach to speciation of selenium incorporated in yeast proteins was developed. The tryptic digest of a water-soluble protein fraction isolated by size-exclusion chromatography was analyzed by reversed-phase HPLC/ICPMS. The selenopeptides selected owing to the detector's elemental specificity were then analyzed by MALDI-TOFMS in order to select target ions for collision-induced dissociation MS. The latter, carried out with an electrospray Q-TOF spectrometer, enabled the sequencing of the selenopeptides detected by HPLC/ICPMS. The approach allowed for the first time the identification of a family of Se-containing proteins resulting from the replacement by selenomethionine of 2-9 methionine residues in a salt-stress-induced protein SIP18 (Mr 8874). The presence of these proteins was confirmed by MALDI-TOFMS of the original (nondigested) protein fraction. Another selenium protein identified was a heat-shock protein HSP12 (Mr 11693) in which the only methionine residue was replaced by selenomethionine. These two Se-containing proteins accounted for more than 95% of selenium in the water-soluble protein fraction.     

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.     

Reproducibility of quantitative proteomic analyses of complex biological mixtures by multidimensional protein identification technology

If quantitative proteomic technologies are to be of widespread use to the biological community, the reproducibility of each method must be investigated and determined. We have analyzed the reproducibility of complex quantitative proteomic analyses of metabolically labeled S. cerevisiae analyzed via multidimensional protein identification technology (MudPIT). Three independent cell growths of S. cerevisiae grown in rich and minimal media and independent MudPIT analyses of each were compared and contrasted. Quantitative MudPIT was found to be intra- and interexperimentally reproducible at both the peptide and protein levels. Proteins of potential low abundance were detected, identified, and quantified by identical peptides from three independent samples. In addition, when multiple peptides were matched to a protein, the relative abundance of each peptide was in agreement across the three samples. Despite the reproducibility, errors in the experimental determination of protein expression levels occurred, but the impact of the variation was minimized by replicate experiments. Last, quantitative MudPIT analyses will likely be improved by increasing the number of peptide hits per protein in a given analysis, which will provide for greater intraexperimental reproducibility.     

Pharmaceutical development of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum anticancer agent AP 5280

AP 5280 is a novel polymer-conjugated platinum anticancer agent showing promising in vitro and in vivo activity against solid tumors. The aim of this study was to develop a parenteral pharmaceutical dosage form for phase I clinical trials. AP 5280 drug substance was characterized by using a wide range of analytical techniques and showed excellent solubility in water. However, as aqueous solutions of AP 5280 proved to be labile upon sterilization by moist heat, it was decided to develop a lyophilized dosage form. Initially, glass vials were used as primary packaging, but this led to a high breakage rate, which could be completely prevented by the use of CZ® resin vials. Stability studies to date show that the lyophilized product in glass vials is stable for at least 12 months when stored at 2-8°C in the dark and the lyophilized product in CZ resin vials is stable for at least 6 months under these conditions. Photostability testing revealed photolability of AP 5280 drug substance and lyophilized product in both types of primary container, necessitating storage in the dark. The first clinical experiences indicate that the proposed formulation is fully applicable for use in the clinical setting.     

A single-element tuning fork piezoelectric linear actuator

This paper describes the design of a piezoelectric tuning-fork, dual-mode motor. The motor uses a single multilayer piezoelectric element in combination with tuning fork and shearing motion to form an actuator using a single drive signal. Finite-element analysis was used in the design of the motor, and the process is described along with the selection of the device's materials and its performance. Swaging was used to mount the multilayer piezoelectric element within the stator. Prototypes of the 25-mm long bidirectional actuator achieved a maximum linear no-load speed of 16.5 cm/s, a maximum linear force of 1.86 N, and maximum efficiency of 18.9%.     

Thermal analysis of poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels

The influence of water on the physical properties of a hydrogel is important for understanding natural tissues and in designing synthetic materials to replace them. In this study, poly (2-hydroxyethyl methacrylate) (pHEMA) was used as a model system to understand how water interacts with the polymer of a hydrogel. Thermal analysis methods (thermogravimetric analysis coupled to mass spectrometry and differential scanning calorimetry) were used to determine: (i) the total water content of pHEMA gels; (ii) how this water was lost during heating; (iii) the relationship between water content of the gel and its glass transition temperature; and (iv) the behavior of the water in the gel on cooling. Previous researchers have invoked various models to describe the organization of water in a hydrogel. In this study, the simplest model which could explain all of the results from the different thermal analysis techniques was one which consisted of three classes of water: (i) hydration water in close proximity to the polymer; (ii) interstitial water in regions or cavities surrounded by polymer chains; and (iii) bulk water.