Characteristics of the dengue viruses used in this study.

a<p>genome equivalents/mL.</p>b<p>C, C6/36 mosquito cell line; M, mosquito; S, suckling mouse.</p

Monitoring Native p38α:MK2/3 Complexes via Trans Delivery of an ATP Acyl Phosphate Probe

Here we describe a chemical proteomics strategy using ATP acyl phosphates to measure the formation of a protein:protein complex between p38α and mapkap kinases 2 and/or 3. Formation of the protein:protein complex results in a new probe labeling site on p38α that can be used to quantify the extent of interaction in cell lysates and the equilibrium binding constant for the interaction in vitro. We demonstrate through RNA interference that the labeling site is dependent on formation of the protein:protein complex in cells. Further, we identify that active-site-directed, small-molecule inhibitors of MK2/3 selectively inhibit the heterodimer-dependent probe labeling, whereas p38α inhibitors do not. These findings afford a new method to evaluate p38α and MK2/3 inhibitors within native biological systems and a new tool for improved understanding of p38α signaling pathways

ATP Acyl Phosphate Reactivity Reveals Native Conformations of Hsp90 Paralogs and Inhibitor Target Engagement

Hsp90 is an ATP-dependent chaperone of widespread interest as a drug target. Here, using an LC-MS/MS chemoproteomics platform based on a lysine-reactive ATP acyl phosphate probe, several Hsp90 inhibitors were profiled in native cell lysates. Inhibitor specificities for all four human paralogs of Hsp90 were simultaneously monitored at their endogenous relative abundances. Equipotent inhibition of probe labeling in each paralog occurred at sites both proximal to and distal from bound ATP observed in Hsp90 cocrystal structures, suggesting that the ATP probe is assaying a native conformation not predicted by available structures. Inhibitor profiling against a comprehensive panel of protein kinases and other ATP-binding proteins detected in native cell lysates identified PMS2, a member of the GHKL ATPase superfamily as an off-target of NVP-AUY922 and radicicol. Because of the endogenously high levels of Hsp90 paralogs in typical cell lysates, the measured potency of inhibitors was weaker than published IC₅₀ values. Significant inhibition of Hsp90 required inhibitor concentrations above a threshold where off-target activity was detectable. Direct on- and off-target engagement was measured by profiling lysates derived from cells treated with Hsp90 inhibitors. These studies also assessed the downstream cellular pathway effects of Hsp90 inhibition, including the down regulation of several known Hsp90 client proteins and some previously unknown client proteins. Overall, the ATP probe-based assay methodology enabled a broad characterization of Hsp90 inhibitor activity and specificity in native cell lysates

Demographical characteristics of Chikungunya cases, Dominica, 2014.

<p>Demographical characteristics of Chikungunya cases, Dominica, 2014.</p

Spatial dispersion of chikungunya cases across the island of Dominica.

<p>The maroon dots represent new cases, while the orange dots represent previously reported cases. Data for 2013 and 2014 epidemiologic weeks 51, 6, and 11 are presented.</p

(a) Location of the ten Parishes of Dominica.

<p>(b) Spatial clusters identified by SaTScan. The clusters are located in St. Andrew and St. George Parishes.</p

Epidemic curve by week of symptom onset; (a) case classification status and (b) sex.

<p>Reports start on epidemiologic week 51 of 2013 and end on week 11 of 2014.</p

Three statistically significant space-time clusters identified by SaTScan.

<p>The clusters are located in St. George and St. Andrew Parishes.</p