Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells

Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function

Clinical validation of cutoff target ranges in newborn screening of metabolic disorders by tandem mass spectrometry: A worldwide collaborative project.

Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass 215 spectrometry through a worldwide collaboration. Methods: Cumulative percentiles of amino 216 acids and acylcarnitines in dried blood spots of approximately 30 million normal newborns and 217 10,615 true positive cases are compared to assign clinical significance, which is achieved when 218 the median of a disease range is either >99%ile or <1%ile of the normal population. The cutoff 219 target ranges of analytes and ratios are then defined as the interval between the limits of the two 220 populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity 221 taking in consideration all available factors. Results: As of December 1, 2010, 129 sites in 45 222 countries have uploaded to the project website a total of 23,970 percentile data points, 558,168 223 analyte results of 10,615 true positive cases with 64 conditions, and 5,088 cutoff values. The 224 average rate of submission of true positive cases between December 1, 2008 and December 1, 225 2010 was 4.7 cases per day (3,418 cases). This cumulative evidence generated 91 and 23 high 226 and low target cutoff ranges, respectively. The overall proportion of cutoff values within the 227 respective target range was 43% (2,176/5,088). Conclusions: An unprecedented level of 228 cooperation and collaboration has allowed the objective definition of cutoff target ranges for 114 229 markers applied to newborn screening of rare metabolic disorders. This set of data could be used 230 as baseline for monitoring of future performance