Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling.

G protein-coupled receptors (GPCRs) relay extracellular stimuli into specific cellular functions. Cells express many different GPCRs, but all these GPCRs signal to only a few second messengers such as cAMP. It is largely unknown how cells distinguish between signals triggered by different GPCRs to orchestrate their complex functions. Here, we demonstrate that individual GPCRs signal via receptor-associated independent cAMP nanodomains (RAINs) that constitute self-sufficient, independent cell signaling units. Low concentrations of glucagon-like peptide 1 (GLP-1) and isoproterenol exclusively generate highly localized cAMP pools around GLP-1- and ß2-adrenergic receptors, respectively, which are protected from cAMP originating from other receptors and cell compartments. Mapping local cAMP concentrations with engineered GPCR nanorulers reveals gradients over only tens of nanometers that define the size of individual RAINs. The coexistence of many such RAINs allows a single cell to operate thousands of independent cellular signals simultaneously, rather than function as a simple "on/off" switch.

Long-term performance of the bovine pericardium patch in conventional carotid endarterectomy.

OBJECTIVE: The aim of the study was to analyze long-term results of carotid endarterectomy (CEA) using bovine pericardium patch. PATIENTS AND METHODS: This study is a retrospective analysis of 274 consecutive cases (173 in CEA group and 101 patients in an internal control group of eversion endarterectomy [EEA]) operated between January 2005 and May 2007. Operations were performed according to standard surgical technique. Primary endpoints of the study were 30-day mortality, ipsilateral neurologic event rate, and high-grade restenosis in the long-term follow-up. RESULTS: No statistically significance between both groups was found in terms of gender, age, risk factors, medication (statine, platelet inhibition), and incidence of symptomatic stenoses (50.9 vs. 50.5%, n.s.). Early mortality was 0% for EEA and 1% for CEA (nonsignificant [n.s.]) and neurologic event rate (transitory ischemic attack [TIA] + stroke) was 4 versus 4% (n.s.), respectively. After 5 years of follow-up (mean 81 months for CEA and 73 months for EEA), the rate of severe (> 70%) restenosis was 2.9% for EEA and 2.7% for CEA (p =0.729). CONCLUSION: Bovine pericardium patch yielded promising results with regard to mortality, perioperative neurologic event rate (TIA, stroke), and occurrence of severe restenosis after 5 years of follow-up.

The challenge of making the right choice: patient avatars in the era of cancer immunotherapies.

Immunotherapies are a key therapeutic strategy to fight cancer. Diverse approaches are used to activate tumor-directed immunity and to overcome tumor immune escape. The dynamic interplay between tumor cells and their tumor(immune)microenvironment (T(I)ME) poses a major challenge to create appropriate model systems. However, those model systems are needed to gain novel insights into tumor (immune) biology and a prerequisite to accurately develop and test immunotherapeutic approaches which can be successfully translated into clinical application. Several model systems have been established and advanced into so-called patient avatars to mimic the patient´s tumor biology. All models have their advantages but also disadvantages underscoring the necessity to pay attention in defining the rationale and requirements for which the patient avatar will be used. Here, we briefly outline the current state of tumor model systems used for tumor (immune)biological analysis as well as evaluation of immunotherapeutic agents. Finally, we provide a recommendation for further development to make patient avatars a complementary tool for testing and predicting immunotherapeutic strategies for personalization of tumor therapies.

Spatiotemporal GPCR signaling illuminated by genetically encoded fluorescent biosensors.

G protein-coupled receptors (GPCRs) are ligand-activated cell membrane proteins and represent the most important class of drug targets. GPCRs adopt several active conformations that stimulate different intracellular G proteins (and other transducers) and thereby modulate second messenger levels, eventually resulting in receptor-specific cell responses. It is increasingly accepted that not only the type of active signaling protein but also the duration of its stimulation and the subcellular location from where receptors signal distinctly contribute to the overall cell response. However, the molecular principles governing such spatiotemporal GPCR signaling and their role in disease are incompletely understood. Genetically encoded, fluorescent biosensors-in particular for the GPCR/cAMP signaling axis-have been pivotal to the discovery and molecular understanding of novel concepts in spatiotemporal GPCR signaling. These include GPCR priming, location bias, and receptor-associated independent cAMP nanodomains. Here, we review such technologies that we believe will illuminate the spatiotemporal organization of other GPCR signaling pathways that define the complex signaling architecture of the cell.

Real-Time Measurements of Intracellular cAMP Gradients Using FRET-Based cAMP Nanorulers.

3',5'-cyclic adenosine monophosphate (cAMP) is one of the most important and ubiquitous second messengers in cells downstream of G protein-coupled receptors (GPCRs). In a single cell, cAMP can exert innumerous specific cell functions in response to more than one hundred different GPCRs. Cells achieve this extraordinary functional specificity of cAMP signaling by limiting the spread of these signals in space and time. To do so, cells establish nanometer-size cAMP gradients by immobilizing cAMP via cAMP binding proteins and via targeted activity of cAMP-degrading phosphodiesterases (PDEs). As cAMP gradients appear to be essential for cell function, new technologies are needed to accurately measure cAMP gradients in intact cells with nanometer-resolution. Here we describe FRET-based cAMP nanorulers to measure local, nanometer-size cAMP gradients in intact cells in the direct vicinity of PDEs.