Compartmentation and dynamics of cAMP and PKA signaling are important determinants of specificity among cAMP's myriad cellular roles. Both cardiac inotropy and the progression of heart disease are affected by spatiotemporal variations in cAMP/PKA signaling, yet the dynamic patterns of PKA-mediated phosphorylation that influence differential responses to agonists have not been characterized. We performed live-cell imaging and systems modeling of PKA-mediated phosphorylation in neonatal cardiac myocytes in response to G-protein coupled receptor stimuli and UV photolysis of ''caged'' cAMP. cAMP accumulation was rate-limiting in PKA-mediated phosphorylation downstream of the ␤-adrenergic receptor. Prostaglandin E 1 stimulated higher PKA activity in the cytosol than at the sarcolemma, whereas isoproterenol triggered faster sarcolemmal responses than cytosolic, likely due to restricted cAMP diffusion from submembrane compartments. Localized UV photolysis of caged cAMP triggered gradients of PKA-mediated phosphorylation, enhanced by phosphodiesterase activity and PKA-mediated buffering of cAMP. These findings indicate that combining live-cell FRET imaging and mechanistic computational models can provide quantitative understanding of spatiotemporal signaling.fluorescence imaging ͉ models ͉ signal transduction I ntracellular signaling through cAMP and its cAMP-dependent protein kinase (PKA) mediates hundreds of distinct cellular functions. Compartmentation and dynamics of cAMP͞PKA signaling are gaining increasing acceptance as general mechanisms used to maintain signaling specificity in a contextdependent manner. In the heart, compartmentation appears to contribute to functional differences between ␤ 1 -and ␤ 2 -adrenergic signaling and other stimuli that increase cAMP and thus has important consequences for understanding the role of ␤-adrenergic signaling in the development and treatment of heart failure (1, 2). Short-term ␤-adrenergic signaling increases heart contractility (3), whereas prolonged exposure to ␤ 1 -adrenergic agonists induces apoptosis (4). Recent live-cell imaging and electrophysiologic approaches are now providing direct measurements of compartmentation (5, 6) and cAMP signaling dynamics (7-10) in intact cells, and our increasing molecular understanding provides numerous candidate molecular mechanisms for compartmentation including caveolae (2, 11), ␤-arrestins (12, 13), and A-kinase anchoring proteins (AKAPs) (14, 15). A future challenge will be to understand quantitatively how these molecular signaling mechanisms orchestrate such precise context-dependent signaling in the cell.Here, we integrate fluorescent reporters of PKA-mediated phosphorylation (16) and mechanistic computational models to characterize rate-limiting biochemical reactions in ␤-adrenergic signaling and identify signaling mechanisms contributing to asynchronous and spatially heterogeneous PKA-mediated phosphorylation. This combination of techniques reveals restricted diffusion, phosphodiesterase (PDE)-mediated cAMP degradation, and c...

We measure high resolution photoelectron angular distributions (PADs) for above-threshold ionization of xenon atoms in infrared laser fields. Based on the Ammosov-Delone-Krainov theory, we develop an intuitive quantum-trajectory Monte Carlo model encoded with Feynman's path-integral approach, in which the Coulomb effect on electron trajectories and interference patterns are fully considered. We achieve a good agreement with the measured PADs of atoms for above-threshold ionization. The quantum-trajectory Monte Carlo theory sheds light on the role of ionic potential on PADs along the longitudinal and transverse direction with respect to the laser polarization, allowing us to unravel the classical coordinates (i.e., tunneling phase and initial momentum) at the tunnel exit for all of the photoelectrons of the PADs. We study the classical-quantum correspondence and build a bridge between the above-threshold ionization and the tunneling theory.

The role of cytomegalovirus (CMV) reactivation in mediating adverse clinical outcomes in nonimmunosuppressed adults with critical illness is unknown.OBJECTIVE To determine whether ganciclovir prophylaxis reduces plasma interleukin 6 (IL-6) levels in CMV-seropositive adults who are critically ill. Double-blind, placebo-controlled, randomized clinical trial (conducted March 10, 2011-April 29, 2016 with a follow-up of 180 days (November 10, 2016) that included 160 CMV-seropositive adults with either sepsis or trauma and respiratory failure at 14 university intensive care units (ICUs) across the United States. DESIGN, SETTING, AND PARTICIPANTSINTERVENTIONS Patients were randomized (1:1) to receive either intravenous ganciclovir (5 mg/kg twice daily for 5 days), followed by either intravenous ganciclovir or oral valganciclovir once daily until hospital discharge (n = 84) or to receive matching placebo (n = 76). MAIN OUTCOMES AND MEASURESThe primary outcome was change in IL-6 level from day 1 to 14. Secondary outcomes were incidence of CMV reactivation in plasma, mechanical ventilation days, incidence of secondary bacteremia or fungemia, ICU length of stay, mortality, and ventilator-free days (VFDs) at 28 days. RESULTS Among 160 randomized patients (mean age, 57 years; women, 43%), 156 received 1 or more dose(s) of treatment, and 132 (85%) completed the study. The mean between-group change in IL-6 level was not significantly different. Among secondary outcomes, CMV reactivation in plasma was significantly lower in the ganciclovir group. The ganciclovir group had more VFDs in both the intention-to-treat population and in the prespecified sepsis subgroup. There were no significant between-group differences in other secondary outcomes.

To treat complex human diseases effectively, a systemslevel approach is needed to understand the interplay of environmental cues, intracellular signals, and cellular behaviors that underlie disease states. This approach requires high-throughput, multiplex techniques that measure quantitative temporal variations of multiple protein activities in the intracellular signaling network. Here, we describe a single microtiter-based format that simultaneously quantifies protein kinase activities in the phosphatidylinositol 3-kinase pathway (Akt), nuclear factor-B pathway (IKK), and three core mitogen-activated protein kinase pathways (ERK, JNK1, MK2). These parallel highthroughput assays are stringently linear, redundantly specific, reproducible, and sensitive compared with classical low-throughput techniques. When applied to a model of sepsis-induced colon epithelial apoptosis, this approach identified a late phase of Akt activity as a critical mediator of cell survival that quantitatively contributed to the efficacy of insulin as an anti-apoptotic cue. Thus, sampling parallel nodes in the intracellular signaling network identified part of the molecular mechanism underlying the efficacy of insulin in the treatment of human sepsis. Molecular & Cellular Proteomics 2:463-473, 2003.Complex patterns of signal transduction arise when cells are exposed to combinations of extracellular cues that vary in onset, duration, origin, and synchrony. Cells process these cues through an interconnected network of multifunctional, redundant molecules to elicit a set of phenotypic responses that subsequently impact function at the cell, tissue, and organ level. In order to develop a molecular understanding of the complex pathophysiology underlying human diseases and utilize this information for prognosis and therapy, a systemslevel, network-biology approach should be applied to the signaling networks governing the relevant cell responses (1). This approach will require frequent temporal sampling of protein activity at critical nodes within parallel signaling pathways inside the cell in a quantitative manner to characterize the flow of information accurately. Such functional measurements are likely to be as valuable, or more valuable, than measurements of simple protein abundance. By quantitatively exploring the functional response of the signaling network to distinct extracellular cues and correlating these molecular events with phenotypic responses, one can construct predictive models of cue-signal and signal-response relationships.Evolving proteomic approaches to network biology have largely focused on measuring abundances of many proteins at only a few time points or under a limited number of experimental conditions (2). Complementary information on functional protein characteristics, such as enzyme activity, has been lacking in these systematic analyses, in large part because there do not exist quantitatively robust, high-throughput techniques that simultaneously measure multiple protein activities in cells. Initially, this type of dat...