Quantification of protein delivery in live cells using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a quantitative single-molecule method that measures the concentration and rate of diffusion of fluorophore-tagged molecules, both large and small, in vitro and within live cells, and even within discrete cellular compartments. FCS is exceptionally well-suited to directly quantify the efficiency of intracellular protein delivery-specifically, how well different "cell-penetrating" proteins and peptides guide proteinaceous materials into the cytosol and nuclei of live mammalian cells. This article provides an overview of the procedures necessary to execute robust FCS experiments and evaluate endosomal escape efficiencies: preparation of fluorophore-tagged proteins, incubation with mammalian cells and preparation of FCS samples, setup and execution of an FCS experiment, and a detailed discussion of and custom MATLAB® script for analyzing the resulting autocorrelation curves in the context of appropriate diffusion models.

Association of Patient Characteristics and Tumor Genomics With Clinical Outcomes Among Patients With Non-Small Cell Lung Cancer Using a Clinicogenomic Database.

Importance: Data sets linking comprehensive genomic profiling (CGP) to clinical outcomes may accelerate precision medicine.

Objective: To assess whether a database that combines EHR-derived clinical data with CGP can identify and extend associations in non-small cell lung cancer (NSCLC).

Design, Setting, And Participants: Clinical data from EHRs were linked with CGP results for 28 998 patients from 275 US oncology practices.

A functional role for intrinsic disorder in the tau-tubulin complex.

Tau is an intrinsically disordered protein with an important role in maintaining the dynamic instability of neuronal microtubules. Despite intensive study, a detailed understanding of the functional mechanism of tau is lacking. Here, we address this deficiency by using intramolecular single-molecule Förster Resonance Energy Transfer (smFRET) to characterize the conformational ensemble of tau bound to soluble tubulin heterodimers.