Novel Plexor SNP genotyping technology: comparisons with TaqMan and homogenous MassEXTEND MALDI-TOF mass spectrometry.

Analysis of SNPs for association, linkage, haplotype, and pharmacogenetic studies has led to a dramatic increase in the number and evolution of medium- to high-throughput genotyping technologies. This study introduces Plexor as a new method for medium-throughput (single SNP) genotyping. We compare this fluorescent-based chemistry for call rate, accuracy, affordability, throughput, and overall efficiency against two commonly used technologies. These include fluorescent-based TaqMan allelic discrimination for single SNP analysis (medium-throughput) and the homogenous MassEXTEND (hME) chemistry using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for multiple SNP analysis (high-throughput). Analysis of 11 SNPs, including all six possible nucleotide substitutions, showed Plexor to be highly comparable for both call rate (94.7%) and accuracy (99.2%) to the TaqMan (94.6% and 99.8%, respectively) and hME (91.9% and 98.1%, respectively) chemistries. We demonstrate that this novel method is an efficient, cost-effective alternative to TaqMan genotyping commonly used in diagnostic settings.

Visualizing early frog development with motion-sensitive 3-D optical coherence microscopy.

A motion-sensitive en-face-scanning 3-D optical coherence microscope (OCM) has been designed and constructed to study critical events in the early development of plants and animals. We describe the OCM instrument and present time-lapse movies of frog gastrulation, an early developmental event in which three distinct tissue layers are established that later give rise to all major organ systems. OCM images constructed with fringe-amplitude data show the mesendoderm migrating up along the blastocoel roof, thus forming the inner two tissue layers. Motion-sigma data, measuring the random motion of scatterers, is used to construct complementary images that indicate the presence of Brownian motion in the yolk cells of the endoderm. This random motion provides additional intrinsic contrast that helps to distinguish different tissue types. Depth penetration at 850 nm is sufficient for studies of the outer ectoderm layer, but is not quite adequate for detailed study of the blastocoel floor, about 500 to 800 mum deep into the embryo. However, we measure the optical attenuation of these embryos to be about 35% less at 1310 nm. 2-D OCT images at 1310 nm are presented that promise sufficient depth penetration to test current models of cell movement near the blastocoel floor during gastrulation.

Optical coherence microscopy for the evaluation of a tissue-engineered artificial cornea.

A transparent artificial cornea derived from biological material is the ultimate goal of corneal research. Attempts at artificial corneal constructs produced from synthetic polymers have proved unsuccessful due to lack of biocompatibility and ability to integrate into the tissue. We have designed a corneal model derived from collagenous biological materials that has several advantages: it has low antigenicity and therefore small chance of eliciting an immune reaction, it can be broken down by the body's own cells and gradually replaced over time by natural materials, and it may contain signaling information for native cells, thereby inducing normal phenotype and behavior. In addition, a transparent corneal model has the potential to be used for testing of novel ophthalmic drugs or gene therapy approaches, eliminating the need for animal testing. We have used an optical coherence microscope (OCM) to evaluate both the structure of our tissue constructs over time in culture and the optical properties of the tissue itself. This imaging technique promises to be an important diagnostic tool in our efforts to understand the influence of mechanical forces, cell phenotype, and soluble factors on the transparency of corneal tissue.

Novel mutations identified using a comprehensive CCR5-denaturing gradient gel electrophoresis assay.

BACKGROUND: Most mutations detected for the gene for CC chemokine receptor 5 (CCR5) are either relatively specific to different population groups or rarely observed in Africans. OBJECTIVES: To develop a comprehensive mutation detection assay for the entire coding region of CCR5 and to identify novel mutations that may play a role in genetic susceptibility to HIV-1 infection, within the diverse South African population. DESIGN: The study cohort consisted of 103 HIV-seropositive patients and 146 HIV-seronegative controls of predominantly African descent. METHODS: A mutation detection assay for the entire coding region of CCR5 was designed; this included amplification of part of the coding region of CCR2. The assay was based on denaturing gradient gel electrophoresis (DGGE) and allowed the complete analysis of samples from 10 individuals per denaturing gel. RESULTS: The use of the CCR5-DGGE assay led to the identification of seven novel and six previously reported mutations. All novel mutations, including a common polymorphism at codon 35, occurred exclusively in non-Caucasians, indicating possible African origin. CONCLUSION: A comprehensive DGGE mutation detection assay has been developed for the entire coding region of CCR5. Application of this assay resulted in the identification of novel CCR5 mutations, which may have a significant effect on the normal functioning of CCR5 and thus contribute to host variability and susceptibility to HIV-1 infection and/or progression to AIDS within this population.

Optical coherence microscopy. A technology for rapid, in vivo, non-destructive visualization of plants and plant cells.

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 microm in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 x 5 x 10 microm) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a three-dimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.

Water permeation through the lipid bilayer membrane. Test of the liquid hydrocarbon model.

According to the liquid hydrocarbon model, the lipid bilayer is viewed simply as a thin slice of bulk hydrocarbon liquid. This allows the water permeability of the bilayer to be calculated from bulk properties. In this paper the prediction of the liquid hydrocarbon model is compared with the known water permeability coefficient of the glycerol monoolein/n-hexadecane bilayer (Fettiplace, R. (1978) Biochim. Biophys. Acta 513, 1-10). As the alkyl chain of glycerol monoolein is equivalent to 8-heptadecene, the water permeability coefficient of 8-heptadecene/n-hexadecane mixtures was measured for temperatures between 20 and 35 degrees C. The mole fraction of n-hexadecane in the bulk liquid was chosen at each temperature to match the known mole fraction of n-hexadecane in the bilayer (White, S. (1976) Nature 262, 421-422). The predicted water permeability coefficient agrees with the measured value at 32 degrees C but is 40% above the measured value at 20 degrees C. The apparent activation energy predicted by the liquid hydrocarbon model is 9.0 +/- 0.3 kcal/mol, while the measured value is 14.2 +/- 1.0 kcal/mol. The failure of the liquid hydrocarbon model probably results from a different molecular organization of the hydrocarbon chains in the bilayer and in the bulk liquid.

Formation of planar bilayer membranes from lipid monolayers. A critique.

The formation of planar bilayer membranes from lipid monolayers as described by Montal and Mueller (Proc. Natl. Acad. Sci. 1972. 69:3561) is analyzed. Bilayers absolutely free of alkane solvents or other nonpolar hydrocarbons can be formed on polytetrafluoroethylene (PTFE) (e.g. Teflon) septa only if certain boundary conditions are satisfied. Measurements have been made of the contact angles between monolayer-coated water and PTFE in the presence and absence of alkane solvents. The measurement suggest that the boundary conditions for formation of stable bilayers can be satisfied only when a nonpolar solvent is present. We conclude that the bilayer must be surrounded by a torus of alkane solvent, petroleum jelly, or silicone grease depending upon the details of technique used to form the bilayer. The non-polar solvent used in the formation of the bilayer may or may not be present in the bilayer depending upon the water solubility and size of the solvent molecule relative to the size of the alkyl chain of the lipid. Detailed sketches describing the formation of bilayers from monolayers are presented.

The electric dipole moment of rhodopsin solubilized in Triton X-100.

The electric dipole moment of solubilized rhodopsin was determined with dielectric dispersion measurements. Rhodopsin was extracted from disc membranes of cattle rod outer segments with the nonionic detergent Triton X-100. The dipole moment of rhodopsin at its isoionic point in the detergent micelle is 720 D (150 charge-A). This value is comparable to dipole moments of nonmembrane proteins, especially those which tend to aggregate or polymerize. Flash irradiation of the rhodopsin results in an increase in the dipole moment of about 25 D (5 charge-A). The light-induced increase in dipole moment appears to be composed of two parts--a faster component related to a change in the number of protons bound by rhodopsin and a slower component apparently independent of the change in proton binding.