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Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors., Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors., Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

Cell signaling is mostly studied in in vitro 2D-cell culture models that lack the complex in vivo environment provided by neighboring cells, soluble secreted factors and non-cellular matrix components. Given that many environmental factors control cell signaling, it comes as no surprise that in vitro observations often poorly correlate with in vivo observations. Recent developments in intravital imaging techniques have made it possible to visualize and study cell signaling in individual cells within living animals. Here, we review intravital imaging techniques based on fluorescence microscopy and give examples of how these techniques are being used to study cell signaling., Cell signaling is mostly studied in in vitro 2D-cell culture models that lack the complex in vivo environment provided by neighboring cells, soluble secreted factors and non-cellular matrix components. Given that many environmental factors control cell signaling, it comes as no surprise that in vitro observations often poorly correlate with in vivo observations. Recent developments in intravital imaging techniques have made it possible to visualize and study cell signaling in individual cells within living animals. Here, we review intravital imaging techniques based on fluorescence microscopy and give examples of how these techniques are being used to study cell signaling.

Cell signaling is mostly studied in in vitro 2D-cell culture models that lack the complex in vivo environment provided by neighboring cells, soluble secreted factors and non-cellular matrix components. Given that many environmental factors control cell signaling, it comes as no surprise that in vitro observations often poorly correlate with in vivo observations. Recent developments in intravital imaging techniques have made it possible to visualize and study cell signaling in individual cells within living animals. Here, we review intravital imaging techniques based on fluorescence microscopy and give examples of how these techniques are being used to study cell signaling., Cell signaling is mostly studied in in vitro 2D-cell culture models that lack the complex in vivo environment provided by neighboring cells, soluble secreted factors and non-cellular matrix components. Given that many environmental factors control cell signaling, it comes as no surprise that in vitro observations often poorly correlate with in vivo observations. Recent developments in intravital imaging techniques have made it possible to visualize and study cell signaling in individual cells within living animals. Here, we review intravital imaging techniques based on fluorescence microscopy and give examples of how these techniques are being used to study cell signaling.

Acute cough is one of the most common reasons for patients to visit a general practitioner. In this revised guideline acute cough is defined as cough lasting less than 3 weeks at presentation. The guideline covers the diagnosis, treatment, and education of patients with cough, pneumonia, bronchiolitis, croup, whooping cough, and Q-fever. It is important to distinguish an uncomplicated respiratory tract infection from a complicated respiratory tract infection that requires antibiotic treatment. In most cases, cough is caused by an uncomplicated respiratory tract infection (viral or bacterial) A patient with an uncomplicated respiratory tract infection has no risk factors for complications (age > 3 months and < 75 years, no relevant comorbidity), is not very ill, doesn't have signs of a complicated respiratory tract infection and has a fever < 7 days. The symptoms (cough) can last up to 4 weeks. There is no effective therapy. There are two groups of patients with a complicated respiratory tract infection. 1 Patients with a pneumonia (severely ill [tachypnea, tachycardia, hypotension or confusion] OR moderately ill and one-sided auscultatory findings, CRP > 100 mg/l [a CRP of 20-100 mg/l doesn't exclude a pneumonia, [management depends on presentation and risk-factors], infiltrate on chest X-ray or sick > 7 days with fever and a cough). These patients are prescribed an antibiotic. 2 Patients with other risk factors for complications (age < 3 months or > 75 years and/or relevant comorbidity [in children cardial and pulmonary disease not being astma, in adults congestive heart failure, severe chronic obstructive pulmonary disease, diabetes mellitus, neurological disorders, severe renal failure, compromised immunity]). In these patients, the decision to prescribe antibiotics is based on the presentation, supported, if necessary, by measurement of CRP. Specific management recommendations are made for croup, bronchiolitis and whooping cough. In cases

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting., Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting., Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.