How time consuming are general practitioners' home visits? Insights from a cross-sectional study in Switzerland.

BACKGROUND: Worldwide, the number of home visits has been decreasing over past decades. Lack of time and long journeys have been reported to hinder general practitioners (GPs) from conducting home visits. In Switzerland also, home visits have declined. Time constraints in a busy GP practice could be one reason. Therefore, the aim of this study was to analyse the time requirements of home visits in Switzerland. METHODS: A one-year cross-sectional study involving GPs from the Swiss Sentinel Surveillance System (Sentinella) was conducted in 2019. GPs provided basic information on all home visits performed throughout the year and additionally detailed reports of up to 20 consecutive home visits. Univariable and multivariable logistic regression analyses were run to identify factors affecting journey and consultation duration. RESULTS: In total, 95 GPs conducted 8489 home visits in Switzerland, 1139 of which have been characterised in detail. On average, GPs made 3.4 home visits per week. Average journey and consultation duration were 11.8 and 23.9 minutes, respectively. Prolonged consultations were provided by GPs working part-time (25.1 minutes), in group practice (24.9 minutes) or in urban regions (24.7 minutes). Rural environments and short journey to patient's home were both found to lower the odds of performing a long consultation compared to a short consultation (odds ratio [OR] 0.27, 95% confidence interval [CI] 0.16-0.44 and OR 0.60, 95% CI 0.46-0.77, respectively). Emergency visits (OR 2.20, 95% CI 1.21-4.01), out-of-hours appointments (OR 3.06, 95% CI 2.36-3.97) and day care involvement (OR 2.78, 95% CI 2.13-3.62) increased the odds of having a long consultation. Finally, patients in their 60s had markedly higher odds of receiving long consultations than patients in their 90s (OR 4.13, 95% CI 2.27-7.62), whereas lack of chronic conditions lowered the odds of a long consultation (OR 0.09, 95% CI 0.00-0.43). CONCLUSION: GPs perform rather few but long home visits, especially for multimorbid patients. GPs working part-time, in group practice or in urban regions devote more time to home visits.

Control of Cell Shape, Neurite Outgrowth, and Migration by a Nogo-A/HSPG Interaction.

Heparan sulfate proteoglycans (HSPGs) critically modulate adhesion-, growth-, and migration-related processes. Here, we show that the transmembrane protein, Nogo-A, inhibits neurite outgrowth and cell spreading in neurons and Nogo-A-responsive cell lines via HSPGs. The extracellular, active 180 amino acid Nogo-A region, named Nogo-A-Δ20, binds to heparin and brain-derived heparan sulfate glycosaminoglycans (GAGs) but not to the closely related chondroitin sulfate GAGs. HSPGs are required for Nogo-A-Δ20-induced inhibition of adhesion, cell spreading, and neurite outgrowth, as well as for RhoA activation. Surprisingly, we show that Nogo-A-Δ20 can act via HSPGs independently of its receptor, Sphingosine-1-Phosphate receptor 2 (S1PR2). We thereby identify the HSPG family members syndecan-3 and syndecan-4 as functional receptors for Nogo-A-Δ20. Finally, we show in explant cultures ex vivo that Nogo-A-Δ20 promotes the migration of neuroblasts via HSPGs but not S1PR2.

Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors.

Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for measuring spatio-temporal signaling dynamics in single living cells with subcellular resolution. There are quite a number of already existing sensors and this technology is increasingly used to obtain quantitative dynamic datasets. In this chapter, we describe the analysis of endogenous extracellular signal-regulated kinase (ERK) activity in living cells using the EKAR2G (ERK activity reporter second generation) probe. We focus on the generation of stable cell lines expressing the EKAR2G sensor as well as data acquisition and analysis.

The dynamics of spatio-temporal Rho GTPase signaling: formation of signaling patterns.

Rho GTPases are crucial signaling molecules that regulate a plethora of biological functions. Traditional biochemical, cell biological, and genetic approaches have founded the basis of Rho GTPase biology. The development of biosensors then allowed measuring Rho GTPase activity with unprecedented spatio-temporal resolution. This revealed that Rho GTPase activity fluctuates on time and length scales of tens of seconds and micrometers, respectively. In this review, we describe Rho GTPase activity patterns observed in different cell systems. We then discuss the growing body of evidence that upstream regulators such as guanine nucleotide exchange factors and GTPase-activating proteins shape these patterns by precisely controlling the spatio-temporal flux of Rho GTPase activity. Finally, we comment on additional mechanisms that might feed into the regulation of these signaling patterns and on novel technologies required to dissect this spatio-temporal complexity.

Spatio-temporal co-ordination of RhoA, Rac1 and Cdc42 activation during prototypical edge protrusion and retraction dynamics.

The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility.

SrGAP2-Dependent Integration of Membrane Geometry and Slit-Robo-Repulsive Cues Regulates Fibroblast Contact Inhibition of Locomotion.

Migrating fibroblasts undergo contact inhibition of locomotion (CIL), a process that was discovered five decades ago and still is not fully understood at the molecular level. We identify the Slit2-Robo4-srGAP2 signaling network as a key regulator of CIL in fibroblasts. CIL involves highly dynamic contact protrusions with a specialized actin cytoskeleton that stochastically explore cell-cell overlaps between colliding fibroblasts. A membrane curvature-sensing F-BAR domain pre-localizes srGAP2 to protruding edges and terminates their extension phase in response to cell collision. A FRET-based biosensor reveals that Rac1 activity is focused in a band at the tip of contact protrusions, in contrast to the broad activation gradient in contact-free protrusions. SrGAP2 specifically controls the duration of Rac1 activity in contact protrusions, but not in contact-free protrusions. We propose that srGAP2 integrates cell edge curvature and Slit-Robo-mediated repulsive cues to fine-tune Rac1 activation dynamics in contact protrusions to spatiotemporally coordinate CIL.

A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space.

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.

Quantitative chemical evaluation of dilute GaNAs using ADF STEM: avoiding surface strain induced artifacts.

The high angle annular dark field intensity (HAADF) in scanning transmission electron microscopy (STEM) can be used for a quantitative evaluation of the chemical composition in dilute GaNAs quantum wells by comparison with simulated intensities. As the scattered intensity is highly sensitive to surface strain fields originating from the quantum wells embedded in GaAs, the HAADF intensity is difficult to evaluate in a quantitative way as long as strain contrast cannot be distinguished from chemical contrast. We present a method to achieve full 2D HAADF STEM compositional mapping of GaNAs/GaAs quantum well systems by making use of information from two different camera lengths.

Strain measurement in semiconductor heterostructures by scanning transmission electron microscopy.

This article deals with the measurement of strain in semiconductor heterostructures from convergent beam electron diffraction patterns. In particular, three different algorithms in the field of (circular) pattern recognition are presented that are able to detect diffracted disc positions accurately, from which the strain in growth direction is calculated. Although the three approaches are very different as one is based on edge detection, one on rotational averages, and one on cross correlation with masks, it is found that identical strain profiles result for an In x Ga1-x N y As1-y /GaAs heterostructure consisting of five compressively and tensile strained layers. We achieve a precision of strain measurements of 7-9·10-4 and a spatial resolution of 0.5-0.7 nm over the whole width of the layer stack which was 350 nm. Being already very applicable to strain measurements in contemporary nanostructures, we additionally suggest future hardware and software designs optimized for fast and direct acquisition of strain distributions, motivated by the present studies.

Determination of the chemical composition of GaNAs using STEM HAADF imaging and STEM strain state analysis.

The nitrogen concentration of GaN(0.01≤x≤0.05)As(1-x) quantum wells was determined from high resolution scanning transmission electron microscopy (HRSTEM) images taken with a high-angle annular dark field (HAADF) detector. This was done by applying two independent methods: evaluation of the scattering intensity and strain state analysis. The HAADF scattering intensity was computed by multislice simulations taking into account the effect of static atomic displacements and thermal diffuse scattering. A comparison of the mean intensity per atom column on the experimental images with these simulations enabled us to generate composition maps with atomic scale resolution. STEM simulations of large supercells proved that local drops of the HAADF intensity observed close to embedded quantum wells are caused by surface strain relaxation. The same STEM images were evaluated by strain state analysis. We suggest a real space method which is not affected by fly-back errors in HRSTEM images. The results of both evaluation methods are in accordance with data obtained from X-ray diffraction measurements.

CNK1 and other scaffolds for Akt/FoxO signaling.

FoxO transcription factors mediate anti-proliferative and pro-apoptotic signals and act as tumor suppressors in cancer. Posttranslational modifications including phosphorylation and acetylation regulate FoxO activity by a cytoplasmic-nuclear shuttle mechanism. Scaffold proteins coordinating signaling pathways in time and space play a critical role in this process. CNK1 acts as a scaffold protein in several signaling pathways controlling the function of FoxO proteins. An understanding of CNK1 and other scaffolds in the FoxO signaling network will provide insights how to release the tumor suppressor function of FoxO as a possibility to block oncogenic pathways. This article is part of a Special Issue entitled: P13K-AKT-FoxO axis in cancer and aging.

CNK1 promotes invasion of cancer cells through NF-kappaB-dependent signaling.

Hallmarks of cancer cells are uncontrolled proliferation, evasion of apoptosis, angiogenesis, cell invasion, and metastasis, which are driven by oncogenic activation of signaling pathways. Herein, we identify the scaffold protein CNK1 as a mediator of oncogenic signaling that promotes invasion in human breast cancer and cervical cancer cells. Downregulation of CNK1 diminishes the invasiveness of cancer cells and correlates with reduced expression of matrix metalloproteinase 9 (MMP-9) and membrane-type 1 MMP (MT1-MMP). Ectopic expression of CNK1 elevates MT1-MMP promoter activity in a NF-kappaB-dependent manner. Moreover, CNK1 cooperates with the NF-kappaB pathway, but not with the extracellular signal-regulated protein kinase pathway, to promote cell invasion. Mechanistically, CNK1 regulates the alternative branch of the NF-kappaB pathway because knockdown of CNK1 interferes with processing of NF-kappaB2 p100 to p52 and its localization to the nucleus. In agreement with this, the invasion of CNK1-depleted cells is less sensitive to RelB downregulation compared with the invasion of control cells. Moreover, CNK1-dependent MT1-MMP promoter activation is blocked by RelB siRNA. Thus, CNK1 is an essential mediator of an oncogenic pathway involved in invasion of breast and cervical cancer cells and is therefore a putative target for cancer therapy.

Characterization of a putative phosphorylation switch: adaptation of SPOT synthesis to analyze PDZ domain regulation mechanisms.

Transient macromolecular complexes are often formed by protein-protein interaction domains (e.g., PDZ, SH2, SH3, WW), which are often regulated (positively or negatively) by phosphorylation. To address the in vitro analysis of PDZ domain regulation by such phosphorylation, we improved the inverted peptide method. This method is based on standard SPOT synthesis, followed by inversion of the peptide under acidic conditions to generate the free C termini necessary for PDZ domain ligand recognition. The benefit of the newly introduced acidic conditions is the preservation of the incorporated phosphate group during peptide synthesis. Furthermore, the improved method is more robust and shows an increased signal-to-noise ratio. As representative examples, we used the AF6, ERBIN, and SNA1 (alpha-1-syntrophin) PDZ domains to analyze the influence of ligand-position-dependent phosphorylation. We could clearly demonstrate severe down-regulation by phosphorylation of the PDZ ligand position -2 (<50 %) and slightly less at position -1 ( approximately 50 %). These results are specific and reproducible for all three PDZ domains. Finally, we confirmed the influence of negative regulation by using the protein kinase BCR as the AF6 PDZ domain ligand. For the first time, this approach allows the SPOT synthesis technique to be used to screen large libraries of phosphorylated peptides in vitro. This should ultimately help in the identification of phosphorylation-dependent regulation mechanisms in vivo.

The scaffold protein CNK1 interacts with the angiotensin II type 2 receptor.

The scaffold protein CNK1 mediates proliferative as well as antiproliferative responses including differentiation and apoptosis. The angiotensin II type 2 (AT2) receptor belongs to the class of G protein-coupled receptors and also promotes antiproliferative effects. Here we report that CNK1 binds through the sterile alpha motif (SAM) and the conserved region in CNK (CRIC) to the AT2 receptor. The exchange of a conserved leucine residue with arginine in the CRIC domain increases the binding affinity of CNK1 to the AT2 receptor. The insertion of a negatively charged amino acid stretch into the linker region between the N- and the C-terminal part of CNK1 strengthens the interaction between CNK1 and the AT2 receptor in a Ras-regulated manner. The biological significance of the interaction was supported by coprecipitation of CNK1 and the AT2 receptor in mouse heart extracts. Thus, CNK1 may play a role in the AT2 receptor-mediated signaling pathways.