Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells.

The stability of housekeeping genes (HKGs) is critical when performing real-time quantitative PCR. To date, the stability of common HKGs has not been systematically compared in human airway epithelial cells (AEC) in normal and atopic subjects. Expression levels of 12 HKGs were measured in AECs from a cohort of 30 healthy atopic nonasthmatic or atopic asthmatic children. Gene expression stability was determined using three different Visual Basic for Applications applets (geNorm, NormFinder and BestKeeper). All 12 HKGs were expressed in AECs. However, the hypoxanthine ribosyltransferase and TATA-binding protein genes were excluded from further analysis due to low expression levels. The cyclophilin A gene was ranked the most stable by all three methods. The expression levels of the beta-actin and glyceraldehyde-3-phosphate dehydrogenase genes were significantly different between the three groups of patients, with atopic asthmatics showing the highest expression levels for both genes. The results suggest that the cyclophilin A gene is the most suitable housekeeping gene analysed for expression studies utilising uncultured bronchial airway epithelial cells from healthy and asthmatic children, and highlight the importance of validating housekeeping genes for each experimental model.

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Reconstructing potential energy functions from simulated force-induced unbinding processes.

One-dimensional stochastic models demonstrate that molecular dynamics simulations of a few nanoseconds can be used to reconstruct the essential features of the binding potential of macromolecules. This can be accomplished by inducing the unbinding with the help of external forces applied to the molecules, and discounting the irreversible work performed on the system by these forces. The fluctuation-dissipation theorem sets a fundamental limit on the precision with which the binding potential can be reconstructed by this method. The uncertainty in the resulting potential is linearly proportional to the irreversible component of work performed on the system during the simulation. These results provide an a priori estimate of the energy barriers observable in molecular dynamics simulations.

Molecular dynamics study of unbinding of the avidin-biotin complex.

We report molecular dynamics simulations that induce, over periods of 40-500 ps, the unbinding of biotin from avidin by means of external harmonic forces with force constants close to those of AFM cantilevers. The applied forces are sufficiently large to reduce the overall binding energy enough to yield unbinding within the measurement time. Our study complements earlier work on biotin-streptavidin that employed a much larger harmonic force constant. The simulations reveal a variety of unbinding pathways, the role of key residues contributing to adhesion as well as the spatial range over which avidin binds biotin. In contrast to the previous studies, the calculated rupture forces exceed by far those observed. We demonstrate, in the framework of models expressed in terms of one-dimensional Langevin equations with a schematic binding potential, the associated Smoluchowski equations, and the theory of first passage times, that picosecond to nanosecond simulation of ligand unbinding requires such strong forces that the resulting protein-ligand motion proceeds far from the thermally activated regime of millisecond AFM experiments, and that simulated unbinding cannot be readily extrapolated to the experimentally observed rupture.