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From temperature analysis of polyacrylamide gel electrophoresis data for rigid-rod DNA analytes, it is proposed that an entropic force term is responsible for the discrepancy between Ogston--Morris--Rodbard--Chrambach model predictions and experimental results. This entropic force originates from reduction of the orientational freedom of anisotropic analytes in small pores of polyacrylamide gels. Time-dependent fluorescence anisotropy decay measurements confirm that, even in the absence of an external field, orientation of anisotropic analytes is restricted in polyacrylamide gels. A new comprehensive model is proposed that takes this effect into consideration. Predictions based on this model are found to compare favorably with experimental data for linear and three-arm asymmetrically branched rigid-rod DNA analytes covering a broad range of molecular aspect ratios and sizes. A new length scale is also proposed for describing the effect of analyte topology on electrophoretic mobility. This length scale reduces to the analyte radius of gyration in the limiting cases of spherically symmetric and linear rigid-rod species. Based on these results, a general approach is proposed for interpreting gel electrophoresis data of charged analytes possessing simple and complex topologies.

We describe a microfluidic mixer that is well-suited for kinetic studies of macromolecular conformational change under a broad range of experimental conditions. The mixer exploits hydrodynamic focusing to create a thin jet containing the macromolecules of interest. Kinetic reactions are triggered by molecular diffusion into the jet from adjacent flow layers. The ultimate time resolution of these devices can be restricted by premature contact between co-flowing solutions during the focusing process. Here, we describe the design and characterization of a mixer in which hydrodynamic focusing is decoupled from the diffusion of reactants, so that the focusing region is free from undesirable contact between the reactants. Uniform mixing on the microsecond time scale is demonstrated using a device fabricated by imprinting optical-grade plastic. Device characterization is carded out using fluorescence correlation spectroscopy (FCS) and two-photon microscopy to measure flow speeds and to quantify diffusive mixing by monitoring the collisional fluorescence quenching, respectively. Criteria for achieving microsecond time resolution are described and modeled.

Mycobacterium abscessus is an increasingly important cause of human disease; however, virulence determinants are largely uncharacterized. Previously, it was demonstrated that a rough, wild-type human clinical isolate (390R) causes persistent, invasive infection, while a smooth isogenic mutant (390S) has lost this capability. During serial passage of 390S, a spontaneous rough revertant was obtained, which was named 390V. This revertant regained the ability to cause persistent, invasive infection in human monocytes and the lungs of mice. Glycopeptidolipid (GPL), which plays a role in environmental colonization, was present in abundance in the cell wall of 390S, and was associated with sliding motility and biofilm formation. In contrast, a marked reduction in the amount of GPL in the cell wall of 390R and 390V was correlated with cord formation, a property associated with mycobacterial virulence. These results indicate that the ability to switch between smooth and rough morphologies may allow M. abscessus to transition between a colonizing phenotype and a more virulent, invasive form.