Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions.

Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior. mechanical modulator | 3,4-dichloroaniline | 4-hydroxyacetophenone | myosin II | pancreatic cancer C ell shape change processes include cell growth, division, motility, and the formation of complex structures like tissues and organs, all of which are governed by the intersection of biochemistry, genetics, and mechanics. These three modules are integral not just for normal function in healthy cells but also in disease states. Pharmacological manipulation of some of these modules has already led to treatment strategies for inflammation and cancer (e.g., paclitaxel) (1, 2). However, many presently available therapies, which address only one aspect of cell shape change, typically either fail to abolish the disease completely or lead to compensatory regulatory changes, and therefore to drug resistance. Targeting cell mechanics remains an underused approach for drug development. In cancer, altered cell mechanics are a hallmark of metastatic efficiency: cell stiffness decreases up to 70% in many metastatic cancer cells (3-5). It is rational then that one therapeutic approach is to increase cellular elasticity, which would, in turn, reduce metastatic potential and act downstream of cancer-inducing genetic alterations.Known mechanical modulators (e.g., latrunculin, blebbistatin) are often lethal, have numerous off-site targets, and act to generate a softer and metastatic-like mechanical phenotype (6, 7). However, the field's ability to increase cellular elasticity on acute time scales is highly restricted. In an effort to close this gap and find modulators that stiffen cells, we leveraged our molecular and analytical understanding of cytokinesis, an evolutionarily conserved and highly mechanical cell shape change event, to establish an in vivo, large-scale, high-throughput chemical screen for small-molecule modulators of cell shap...

Shear viscosities of supercritical nitrogen have been measured to 573 K and 7 GPa (70 kbars). Measurements were made in a diamond-anvil cell with a rolling-ball technique. Individual isotherms are well fitted by a modified Doolittle equation. The data demonstrate a close relation between viscosities and excess entropy; this relation is further explored for the systems argon, oxygen, carbon dioxide, sodium, cesium, and a Lennard-Jones fluid.