High-throughput capillary electrophoresis for the identification and differentiation of seven species of Eimeria from chickens

A capillary electrophoretic approach has been evaluated for the identification of seven currently recognised species of Eimeria infecting chickens. The second internal transcribed spacer of ribosomal DNA is PCR-amplified from any of the seven species using a single set of oligonucleotide primers (one of which is fluorescently labelled). The amplicons are heat-denatured and subjected to capillary electrophoresis in a MegaBACE 1000 (Amersham). The chromatograms captured are stored electronically and then analysed using MegaBACE Fragment Profiler software. Using control DNA samples representing monospecific lines of Eimeria, specific peaks in the chromatograms were defined for the unequivocal identification of each of the seven species and their differentiation. Electrophoretic reading and analysis are carried out automatically, thus making it a time- and cost-effective method. This procedure should find applicability as a tool for the quality control of Eimeria vaccines, the monitoring of coccidiosis outbreaks and the high-throughput analysis of oocyst samples for epidemiological surveys.     

Capillary electrochromatography of peptides and proteins

This review surveys the accomplishments in the separation of peptides and proteins by capillary electrochromatography (CEC) over the last decade. A significant number of research articles have been published on this topic since the last review. Peptide and proteins separations have been carried out in all three formats of CEC, i.e., packed bed, continuous bed and open-tubular (OT) format. In addition to electrophoresis, different chromatographic modes have been successfully exploited with the most prevalent being reversed-phase mode followed by ion-exchange. Although many researchers continue to use model proteins and peptides primarily to evaluate the performance of novel stationary phases some researchers have also applied CEC to the analysis of real-life samples. The potential of CEC to yield complementary information and sometimes a superior separation with respect to established techniques, i.e., microbore HPLC and capillary electrophoresis has been demonstrated. Instrumental modifications in order to facilitate coupling of CEC to mass spectrometry have further upgraded the value of CEC for proteomic analysis. Capillaries are still the separation vehicle of choice for most researchers yet the microfluidic platform is gaining momentum, propelled particularly by its potential for multitasking, e.g., performing different chromatographic modes in series.     

Oxide ion diffusion in optimised LAMOX materials

A series of optimised materials in the family with parent compound La(2)Mo(2)O(9) have been studied using the isotope exchange depth profile technique and SIMS analysis. The data collected indicate that the oxide ion diffusion coefficients in both the parent compound and optimised materials are significantly higher than those reported for any of the fluorite structured electrolyte materials, with a peak value for the La(1.7)Gd(0.3)Mo(2)O(9) composition of 1.41 x 10(-6) cm(2) s(-1) at 800 degrees C. Further, it was found that the substitution of isotopic oxygen into the ceramic was limited in dry atmospheres but significantly enhanced in a H(2)(18)O/(16)O atmosphere, in common with recent reports on the diffusion/exchange behaviour of the BIMEVOX class of oxide ion conductors.     

Gamma radiation induced effects on silica and on silica-polymer interfacial interactions in filled polysiloxane rubber

We report in our studies to assess the impact of gamma radiation on silica and on the silica-polymer interface in filled polysiloxane rubber. Electron spin resonance (ESR) and solid-state nuclear magnetic resonance (NMR) studies have been performed on samples exposed to gamma radiation. In an effort to probe directly the effect of gamma radiation on the silica surface, we employed ¹H and ²⁹Si NMR. Our ESR studies show trapped paramagnetic species (positive holes and/or trapped electrons) within the host silica matrix for all samples exposed to gamma radiation. A sample of pure cab-o-sil irradiated to a dose of 50 kGy also shows an ESR signal. Our studies on real-time aged samples (derived from field trials) also show ESR signatures indicative of silica based trapped paramagnetic species. The growth of trapped paramagnetic species as a function of gamma dose was investigated. This shows that the build up of trapped species occurs rapidly at low gamma dose before reaching saturation at about 20-30 kGy. Radiation induced band gap excitation is the likely process leading to the creation of these paramagnetic species which may be trapped in regions of local charge deficit within the silica matrix. Species that are not trapped may take part in silica surface reactions leading to changes in filler-polymer interfacial interactions. NMR studies combined with ammonia modified swell studies have shown increased polymer segmental chain mobility (softening) at low gamma dose indicative of a possible reduction in filler-polymer interfacial interactions. For those samples exposed to high gamma dose, our ammonia modified swell studies suggest increased polymer-filler interactions presumably through silica-polymer crosslinking effects. Our ¹H and ²⁹Si NMR studies on irradiated silica suggest that the silica surface is sensitive to gamma radiation. Our observations are important as they highlight the need to better control the quality (size, purity, etc.) of the silica constituent in filled polymer components used in gamma radiation environments.     

1H-NMR relaxation times and water content of red blood cells from chronic alcoholic patients during withdrawal

Red blood cell proton nmr, T1 and T2 times, and water content were measured for normal control subjects and thirteen patients with chronic alcoholism during the withdrawal phase. T1 and T2 were significantly increased without significant alteration in cell water content. The relaxation times were more markedly affected in those with symptoms of delirium tremens. These findings suggest that the intracellular free:bound water states rather than water content is the more likely explanation for these changes. The results are discussed in relation to similar findings obtained from in vivo studies in man and in vitro studies in animals.     

How Ubiquitin Unfolds after Transfer into the Gas Phase

The structural evolution of ubiquitin after transfer into the gas phase was studied by electron capture dissociation. Site-specific fragment yields show that ubiquitin’s solution fold is overall unstable in the gas phase, but unfolding caused by loss of solvent is slowest in regions stabilized by salt bridges.     

Detection of transthyretin variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: A clinical application of mass spectrometry

In our continuing efforts to develop mass spectrometry-based methods for transthyretin (TTR) variant detection and characterization, we have sought to use matrix-assisted laser desorption/ionization (MALDI) bioreactive probes incorporating immobilized trypsin for screening purposes. These devices show good diagnostic potential as a clinical screening tool to detect amino acid substitutions in TTR. MALDI probes allow the on-probe generation of tryptic digests. The subsequent mass analysis of the on-probe digest yields the peptide map. The inherent advantages of this method include considerably reduced digestion times (minutes vs. hours), absence of autolysis products, minimized sample handling, and hence minimal sample loss. A further advantage is that the opportunity for loss of hydrophobic peptides is reduced because no sample transfer occurs. The method can be applied as a preliminary screen for TTR variants where TTR is isolated from patient serum through immunoprecipitation. This method should also be applicable to other proteins and suitable for automation.     

Design and application of robust rf pulses for toroid cavity NMR spectroscopy

We present robust radio frequency (rf) pulses that tolerate a factor of six inhomogeneity in the B? field, significantly enhancing the potential of toroid cavity resonators for NMR spectroscopic applications. Both point-to-point (PP) and unitary rotation (UR) pulses were optimized for excitation, inversion, and refocusing using the gradient ascent pulse engineering (GRAPE) algorithm based on optimal control theory. In addition, the optimized parameterization (OP) algorithm applied to the adiabatic BIR-4 UR pulse scheme enabled ultra-short (50 μs) pulses with acceptable performance compared to standard implementations. OP also discovered a new class of non-adiabatic pulse shapes with improved performance within the BIR-4 framework. However, none of the OP-BIR4 pulses are competitive with the more generally optimized UR pulses. The advantages of the new pulses are demonstrated in simulations and experiments. In particular, the DQF COSY result presented here represents the first implementation of 2D NMR spectroscopy using a toroid probe.