Adding precise nanoliter volume capabilities to liquid-handling automation for compound screening experimentation

Miniaturizing experimental sample volumes to the nanoliter volume range is one of the most economical ways to perform mid- and high-throughput compound screening experiments. Existing automation platforms for nanoliter fluid handling can be bulky, expensive, and require periodic calibration to provide consistent liquid dispensing. In addition, even with frequent calibration, significant instrument-to-instrument variation in low-volume dispensing can occur between different instrument platforms. Many of these issues can be addressed by the use of PocketTips. PocketTips are tips with a defined internal pocket designed to hold specific nanoliter volumes of compound dissolved in dimethylsulfoxide. Although the overall liquid-handling process with PocketTips uses the aspirate/dispense features of the specific liquid-handling device being used, the dispensed nanoliter volume is solely based on the dimensions of the pocket of the PocketTip and thus, the liquid-handling device itself need not have nanoliter dispensing capabilities. In this report, we demonstrate the performance of PocketTips on different automation platforms. In addition, we used a cell-based ?-lactamase reporter assay system to demonstrate that compound delivery by PocketTips compares favorably with a standard compound addition technique.     

Molecular dynamics at the receptor level of immunodominant myelin basic protein epitope 87-99 implicated in multiple sclerosis and its antagonists altered peptide ligands: triggering of immune response

This work reports molecular dynamics studies at the receptor level of the immunodominant myelin basic protein (MBP) epitope 87-99 implicated in multiple sclerosis, and its antagonists altered peptide ligands (APLs), namely [Arg91, Ala96] MBP87-99 and [Ala91,96] MBP87-99. The interaction of each peptide ligand with the receptor human leukocyte antigen HLA-DR2b was studied, starting from X-ray structure with pdb code: 1ymm. This is the first such study of APL-HLA-DR2b complexes, and hence the first attempt to gain a better understanding of the molecular recognition mechanisms that underlie TCR antagonism by these APLs. The amino acids His88 and Phe89 serve as T-cell receptor (TCR) anchors in the formation of the trimolecular complex TCR-peptide-HLA-DR2b, where the TCR binds in a diagonal, off-centered mode to the peptide-HLA complex. The present findings indicate that these two amino acids have a different orientation in the APLs [Arg91, Ala96] MBP87-99 and [Ala91,96] MBP87-99: His88 and Phe89 remain buried in HLA grooves and are not available for interaction with the TCR. We propose that this different topology could provide a possible mechanism of action for TCR antagonism.     

Computational models of a nano probe tip for static behaviors

It is difficult to predict the measurement bias arising from the compliance of the atomic force microscope (AFM) probe. The issue becomes particularly important in this situation where nanometer uncertainties are sought for measurements with dimensional probes composed of flexible carbon nanotubes mounted on AFM cantilevers. We have developed a finite element model for simulating the mechanical behavior of AFM cantilevers with carbon nanotubes attached. Spring constants of both the nanotube and cantilever in two directions are calculated using the finite element method with known Young's moduli of both silicon and multiwall nanotube as input data. Compliance of the nanotube-attached AFM probe tip may be calculated from the set of spring constants. This paper presents static models that together provide a basis to estimate uncertainties in linewidth measurement using nanotubes. In particular, the interaction between a multiwall nanotube tip and a silicon sample is modeled using the Lennard-Jones theory. Snap-in and snap-out of the probe tip in a scanning mode are calculated by integrating the compliance of the probe and the sample-tip interacting force model. Cantilever and probe tip deflections and points of contact are derived for both horizontal scanning of a plateau and vertically scanning of a wall. The finite element method and the Lennard-Jones model provide a means to analyze the interaction of the probe and sample and measurement uncertainty, including actual deflection and the gap between the probe tip and the measured sample surface.     

Phenotypic correlations among quality traits of fresh and dry-cured hams

In this study, fresh and processing quality traits were collected on a total of 312 Country Hams. Phenotypic correlations between traits were estimated and numerous values were significantly different (P<0.05) from zero. Yield was significantly correlated with several fresh pork quality traits measured on the fresh hams, including subjective color (0.34) and lipid percentage (0.32). Some meat color traits were significantly correlated, including associations between the color scores taken on the fresh and cured hams. Correlations between fresh pork quality traits were also determined, with results showing significant correlations between ultimate pH and other pork quality traits. The results indicate that yield can be maximized if hams with good fresh pork quality parameters are used for curing. The correlations estimates are helpful for understanding of the biological mechanisms behind the production of dry-cured hams and can potentially be used to improve the efficiency of the dry-cured ham production chain.     

Approach to 3-d ultrasound high resolution imaging for mechanically moving large-aperture transducer based upon Fourier transform

A new three-dimensional (3-D) acoustic image formation technique is proposed that is based on the transmission of wide bandwidth pulse signals and the application of the 3-D fast Fourier transform. A solution to the Helmholtz wave equation has been obtained using the Born approximation. The solution contains analytical expressions for the spatial spectra of the transmit and receive radiation patterns for transducers of various geometries with lenses of fixed focal distances. It has been shown that the proposed algorithms allow for radiation patterns with constant widths at depths both behind and in front of the focal point, starting practically from the plane of the transducer. The theoretical and experimental investigations and computer simulation for both spherical and rectangular transducer shapes have been performed. The results were used to estimate the beamwidths and the side lobe levels. A variant of the linear array has been studied for a cylindrical lens of a fixed focal distance moving in a lateral direction. It has been shown that, in this case, a high resolution (of the order of a few wavelengths) can be achieved along all three Cartesian coordinates at a very high scanning velocity. The influence of the moving scatterers' velocity in inhomogeneous medium on the spatial radiation pattern characteristics has been estimated.     

Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors

GABA(A) receptors composed of alpha1beta3 gamma2 and alpha1beta3 subunits were expressed in insect Sf9 cells and solubilized in 1% Triton X100. In sucrose density gradients, [3H]-Ro15-1788 binding activity, in the case of alpha1beta3 gamma2, and [3H]-muscimol binding activity, in the case of alpha1beta3 containing receptors sedimented as a single sharp peak suggesting the formation of receptors containing a defined number of subunits. When alpha1beta3gamma2 -containing receptors were incubated with an alpha-subunit specific antibody (bd24), a single class of antibody receptor complex was formed irrespective of the receptor-antibody ratio. This is consistent with two alpha subunits cross-linked within the receptor by the antibody. Similar results were obtained using a beta-subunit specific antibody (bd17). Several classes of antibody-receptor complex were formed when receptors were pre-incubated with a gamma specific antibody (anti gamma(2) 1-15 Cys). This profile is consistent with the presence of a single gamma subunit in each complex. Experiments with alpha1beta3 subunit containing receptors and antibody bd24 produced a profile similar to that seen with alpha1beta3 gamma2 receptors, consistent with two alpha subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with three beta subunits per receptor. This method permits the rapid determination of subunit stoichiometries of homogeneous receptor populations     

Hydrophilic sponges based on 2-hydroxyethyl methacrylate Part VII: Modulation of sponge characteristics by changes in reactivity and hydrophilicity of crosslinking agents

Despite previous unsuccessful attempts to use hydrated poly(2-hydroxyethyl methacrylate) sponges as implantable biomaterials, recently these materials became important as peripheral components in an artificial cornea of the core-and-skirt design. The low mechanical strength of sponges prompted this study on possible improvement of tensile properties by the use of a variety of crosslinking agents. Three vinylic (dimethacrylates) and two allylic compounds were used at different concentrations (0.1 to 2% (mol)) as crosslinking agents in the production of sponges. Their influence on the mechanical properties, porous morphology and swelling behavior of resulting sponges was evaluated. The onset of phase separation during polymerization was also measured by visible spectrophotometry. The results suggested an inherent heterogeneity of sponges, i.e. pores of non-uniform size and structural inhomogeneities. While the effects of changes in the nature and concentration of crosslinking agents on the equilibrium water content of sponges were ambiguous, some of the mechanical properties, such as toughness and elasticity, were improved by crosslinking with allylic agents. Scanning electron microscopic examination suggested that the mechanical effect is related to the variation of size of the polymer particles constituting the sponge structure, which was proved to be dependent upon the onset of phase separation during polymerization. ©2000 Kluwer Academic Publishers     

Local haemodynamics of arterial bypass graft anastomoses

One of the main causes of failure of expanded polytetrafluoroethylene (PTFE) bypass grafts used in the lower limbs is the development of myointimal hyperplasia (MIH). Clinical studies show that higher patency rates can be obtained with the use of an autologous vein cuff (the Miller cuff) interposed between the graft and artery. The reasons for the improved performance are still unclear, but preliminary studies suggest that the change in local haemodynamics due to the cuff geometry may be the significant factor rather than the presence of autologous material. If this is the case, then PTFE grafts can be produced with an integral cuff, i.e. a precuffed graft, with similar haemodynamic patterns to that of the Miller cuff. In this paper, two different types of precuffed graft are presented and their flow patterns are compared with those recorded in the Miller cuff and the conventional end-to-side anastomosis. The haemodynamic studies were carried out using optically clear silicone rubber models under simulated in vivo pulsatile flow conditions. Flow structures similar to those observed in the Miller cuff were seen in the precuffed grafts.