Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.     

Endcaps for stabilizing short DNA duplexes

The syntheses of endcaps for covalently linking the 3' and 5' hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by Tm experiments on the self-complementary octamer.     

Medical monitoring during the NASA Artificial Gravity-Bed Rest Pilot Study

The NASA artificial gravity-bed rest pilot study (AGPS) was designed to investigate the efficacy of daily exposure to a +Gz acceleration gradient for counteracting the physiologic decrements induced by prolonged bed rest. Test subjects were continuously monitored by a physician for signs and symptoms of pre-syncope, motion sickness, and arrhythmias while on the centrifuge. In this article, we have summarized the medical monitoring observations that were made during the AGPS and included an assessment of the relative usefulness of the information provided by the various monitoring tools in making a decision to terminate a centrifuge spin.     

Observers exploit stochastic models of sensory change to help judge the passage of time

Sensory stimulation can systematically bias the perceived passage of time, but why and how this happens is mysterious. In this report, we provide evidence that such biases may ultimately derive from an innate and adaptive use of stochastically evolving dynamic stimuli to help refine estimates derived from internal timekeeping mechanisms. A simplified statistical model based on probabilistic expectations of stimulus change derived from the second-order temporal statistics of the natural environment makes three predictions. First, random noise-like stimuli whose statistics violate natural expectations should induce timing bias. Second, a previously unexplored obverse of this effect is that similar noise stimuli with natural statistics should reduce the variability of timing estimates. Finally, this reduction in variability should scale with the interval being timed, so as to preserve the overall Weber law of interval timing. All three predictions are borne out experimentally. Thus, in the context of our novel theoretical framework, these results suggest that observers routinely rely on sensory input to augment their sense of the passage of time, through a process of Bayesian inference based on expectations of change in the natural environment.     

Responses of brain and non-brain endothelial cells to meningitis-causing Escherichia coli K1

Bacterial interaction with specific host tissue may contribute to its propensity to cause an infection in a particular site. In this study, we examined whether meningitis-causing Escherichia coli K1 interaction with human brain microvascular endothelial cells, which constitute the blood-brain barrier, differed from its interaction with non-brain endothelial cells derived from skin and umbilical cord. We showed that E. coli K1 association was significantly greater with human brain microvascular endothelial cells than with non-brain endothelial cells. In addition, human brain microvascular endothelial cells maintained their morphology and intercellular junctional resistance in response to E. coli K1. In contrast, non-brain endothelial cells exhibited decreased transendothelial electrical resistance and detachment from the matrix upon exposure to E. coli K1. These different responses of brain and non-brain endothelial cells to E. coli K1 may form the basis of E. coli K1's propensity to cause meningitis.     

Coiled coils at the edge of configurational heterogeneity. Structural analyses of parallel and antiparallel homotetrameric coiled coils reveal configurational sensitivity to a single solvent-exposed amino acid substitution

A detailed understanding of the mechanisms by which particular amino acid sequences can give rise to more than one folded structure, such as for proteins that undergo large conformational changes or misfolding, is a long-standing objective of protein chemistry. Here, we describe the crystal structures of a single coiled-coil peptide in distinct parallel and antiparallel tetrameric configurations and further describe the parallel or antiparallel crystal structures of several related peptide sequences; the antiparallel tetrameric assemblies represent the first crystal structures of GCN4-derived peptides exhibiting such a configuration. Intriguingly, substitution of a single solvent-exposed residue enabled the parallel coiled-coil tetramer GCN4-pLI to populate the antiparallel configuration, suggesting that the two configurations are close enough in energy for subtle sequence changes to have important structural consequences. We present a structural analysis of the small changes to the helix register and side-chain conformations that accommodate the two configurations and have supplemented these results using solution studies and a molecular dynamics energetic analysis using a replica exchange methodology. Considering the previous examples of structural nonspecificity in coiled-coil peptides, the findings reported here not only emphasize the predisposition of the coiled-coil motif to adopt multiple configurations but also call attention to the associated risk that observed crytstal structures may not represent the only (or even the major) species present in solution.     

Scaffold hybridization in generation of indenoindolones as anticancer agents that induce apoptosis with cell cycle arrest at G2/M phase

Scaffold hybridization of several natural and synthetic anticancer leads led to the consideration of indenoindolones as potential novel anticancer agents. A series of these compounds were prepared by a diversity-feasible synthetic method. They were found to possess anticancer activities with higher potency compared to etoposide and 5-fluorouracil in kidney cancer cells (HEK 293) and low toxicity to corresponding normal cells (Vero). They exerted apoptotic effect with blocking of cell cycle at G2/M phase.