Screening for noncovalent ligand-receptor interactions by electrospray ionization mass spectrometry-based diffusion measurements

The application of a novel method for the identification of low-molecular-weight noncovalent ligands to a macromolecular target is reported. This technique is based on the measurement of analyte diffusion coefficients by electrospray mass spectrometry (ESI-MS) (Clark et al., Rapid Commun. Mass Spectrom. 2002, 16, 1454-1462). Potential ligands have large diffusion coefficients as long as they are free in solution. Binding to a macromolecular target, however, drastically reduces the diffusional mobility of any ligand species. Mixtures containing six different saccharides [ribose, rhamnose, glucose, maltose, maltotriose, and N,N',N'-triacetylchitotriose (NAG(3))] were screened for noncovalent binding to lysozyme. Of these six compounds, only NAG(3) is known to bind to the protein. In "direct" binding tests, NAG(3) shows a significantly reduced diffusion coefficient in the presence of the protein. No changes were observed for any of the other saccharides. In a second set of experiments, the use of a "competition" screening method was explored in which mixtures of candidate saccharides were tested for their ability to displace a reference ligand from the target. The addition of NAG(3)-containing mixtures significantly increased the diffusion coefficient of the reference ligand NAG(4) (N,N',N',N'-tetraacetylchitotetrose), whereas mixtures that did not contain NAG(3) had no effect. These data clearly indicate the potential of ESI-MS-based diffusion measurements as a novel tool to screen compound libraries for binding to proteins and other macromolecular targets. In contrast to conventional ESI-MS-based ligand-receptor binding studies, this method does not rely on the preservation of noncovalent interactions in the gas phase.     

Determination of ligand-protein dissociation constants by electrospray mass spectrometry-based diffusion measurements

A novel approach for the quantification of ligand-protein interactions is presented. Electrospray ionization mass spectrometry (ESI-MS) is used to monitor the diffusion behavior of noncovalent ligands in the presence of their protein receptors. These data allow the fraction of free ligand in solution to be determined, such that the corresponding dissociation constants can be calculated. A set of conditions is developed that provides an "allowable range" of concentrations for this type of assay. The method is tested by applying it to two different inhibitor-enzyme systems. The dissociation constants measured for benzamidine-trypsin and for N,N',N' '-triacetylchitotriose-lysozyme are (50 +/- 10) and (6 +/- 1) mM, respectively. Both of these results are in good agreement with previous data from the literature. In contrast to traditional ESI-MS-based methods, the approach used in this work does not rely on the preservation of specific solution-type noncovalent interactions in the gas phase. It is shown that this method allows an accurate determination of dissociation constants, even in cases in which the ion abundance ratio of free to ligand-bound protein in ESI-MS does not reflect the corresponding concentration ratio in solution.     

Reliability of scanning electron microscopy information

The reliability of information obtained with the scanning electron microscope is influenced by human and instrumental factors. The human factor enters, for example, when a microstructure is described as being typical of the material when in fact it is only a minor component. Some instrumental factors include charging of the specimen, problems differentiating between positive and negative relief and distortion of the shape of the sample in micrographs. The determination of the amount of a phase present in a composite is a complex problem involving both preparation of representative surfaces and appropriate methods of evaluation. A number of experimental results are provided. Where smearing of surfaces is not a problem, point counting evaluation on sawn surfaces is shown to provide reliable results.     

High-resolution field desorption/ionization fourier transform ion cyclotron resonance mass analysis of nonpolar molecules

We report the first field desorption ionization broadband high-resolution (m/Deltam(50%) approximately 65 000) mass spectra. We have interfaced a field ionization/field desorption source to a home-built 9.4-T FT-ICR mass spectrometer. The instrumental configuration employs convenient sample introduction (in-source liquid injection) and external ion accumulation. We demonstrate the utility of this configuration by generating high-resolution positive-ion mass spectra of C(60) and a midboiling crude oil distillate. The latter contains species not accessible by common soft-ionization methods, for example, low-voltage electron ionization, electrospray ionization, and matrix-assisted laser desorption/ionization. The present work demonstrates significant advantages of FI/FD FT-ICR MS for analysis of nonpolar molecules in complex mixtures.     

Evolving controllers for a homogeneous system of physical robots: structured cooperation with minimal sensors

We report on recent work in which we employed artificial evolution to design neural network controllers for small, homogeneous teams of mobile autonomous robots. The robots were evolved to perform a formation-movement task from random starting positions, equipped only with infrared sensors. The dual constraints of homogeneity and minimal sensors make this a non-trivial task. We describe the behaviour of a successful system in which robots adopt and maintain functionally distinct roles in order to achieve the task. We believe this to be the first example of the use of artificial evolution to design coordinated, cooperative behaviour for real robots.     

Researching haptics in higher education: The complexity of developing haptics virtual learning systems and evaluating its impact on students’ learning

hapTEL, an interdisciplinary project funded by two UK research councils from 2007 to 2011, involves a large interdisciplinary team (with undergraduate and post-graduate student participants) which has been developing and evaluating a virtual learning system within an HE healthcare education setting, working on three overlapping strands. Strand 1 involves the technical development and evaluation of the hapTEL workstation which simulates clinical conditions for dental training including haptics (sense of touch). Strand 2 involves examining the traditional undergraduate curriculum and how this could benefit from the use of haptics. Strand 3 is concerned with the educational evaluation of the impact of the work carried out within Strands 1 and 2. Two theoretical frameworks (Entwistle, (1987) and Webb and Cox (2004)) have been used to identify as many factors as possible which could affect the impact of Technology Enhanced Learning (TEL) on the quality of the learning achieved. These frameworks have formed a foundation for measuring the impact of TEL on curriculum change, teachers’ pedagogical practices, students’ learning and on institutional practices. A range of quantitative and qualitative methods were designed, piloted and evaluated in order to measure the impact of TEL on teaching and learning; and to have a rich and robust data set which also addresses the variables in the frameworks. The results from using these frameworks show that institutional and departmental factors should be considered when evaluating the impact of TEL in higher education and that these had a major influence on the design and curriculum integration of the hapTEL systems. We have also shown that by involving the end users from the beginning enabled not only an enhancement of the students’ learning experiences but also a modification to the traditional curriculum itself and the successful integration of TEL within a very traditional undergraduate higher education dental curriculum. The conclusions from this paper confirm earlier reviews of researching TEL that technology integration is extremely complex and the related research requires a comprehensive approach of both quantitative and qualitative methods if one is to take account of the range of variables identified by theoretical frameworks. Finally, repeating the range of empirical investigations for a second year enables researchers to validate the effectiveness of the methods used in the initial year and thereby maximise the reliability and generalisability of the research outcomes.     

An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection

A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 10(7) organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.     

Engineering in Genomics [variable number tandem repeats as agents of functional regulation in the genome]

The explosion of genetic data availability in the last decade has opened up many new avenues for the application of genetics to the improvement of human health, particularly, the common and complex disorders, which previously largely defied researchers seeking to understand their etiology. However, the utilization of this data in healthcare and by the pharmaceutical industry is still in its infancy, with the current focus being almost solely on two forms of genetic variation single nucleotide polymorphisms (SNPs), involving the substitution of one base in DNA for another and copy number variations, involving the deletion and multiplication of regions > 1,000 base pairs (bp). However, there is currently a relative paucity of funding and general interest in a third form of variation called microsatellites and mini- satellites.