αE-catenin-dependent mechanotransduction is essential for proper convergent extension in zebrafish

Cadherin complexes mediate cell-cell adhesion and are crucial for embryonic development. Besides their structural function, cadherin complexes also transduce tension across the junction-actomyosin axis into proportional biochemical responses. Central to this mechanotransduction is the stretching of the cadherin-F-actin-linker α-catenin, which opens its central domain for binding to effectors such as vinculin. Mechanical unfolding of α-catenin leads to force-dependent reinforcement of cadherin-based junctions as studied in cell culture. The importance of cadherin mechanotransduction for embryonic development has not been studied yet. Here we used TALEN-mediated gene disruption to perturb endogenous αE-catenin in zebrafish development. Zygotic α-catenin mutants fail to maintain their epithelial barrier, resulting in tissue rupturing. We then specifically disrupted mechanotransduction, while maintaining cadherin adhesion, by expressing an αE-catenin construct in which the mechanosensitive domain was perturbed. Expression of either wild-type or mechano-defective α-catenin fully rescues barrier function in α-catenin mutants; however, expression of mechano-defective α-catenin also induces convergence and extension defects. Specifically, the polarization of cadherin-dependent, lamellipodia-driven cell migration of the lateral mesoderm was lost. These results indicate that cadherin mechanotransduction is crucial for proper zebrafish morphogenesis, and uncover one of the essential processes affected by its perturbatio

Effect of oxygen on the expression of hypoxia-inducible factors in human fetal lung explants

Background: Fetal lung development requires proper coordination between lung epithelial and vascular morphogenesis. A major determinant in lung vascular development is vascular endothelial growth factor (VEGF), which is regulated by hypoxia-inducible factors (HIFs). VEGF is expressed in the airway epithelium, while its receptors (VEGFRs) are expressed in the pulmonary mesenchyme. The hypoxic environment in utero is beneficial for fetal organogenesis, especially vascular development. However, little is known about the expression of HIFs and VEGFR-2 in the human fetal lung in vitro. Objectives: The purpose of this study was to investigate the effects of hypoxia on fetal lung morphology and mRNA expression of VEGF, VEGFR-2, HIF-2α, and HIF-3α. Methods: An explant culture technique was used to study the effects of normoxic and hypoxic conditions on human fetal lung. Results: The morphology remained largely unchanged in explants cultured under hypoxic or normoxic conditions. Quantitative RT-PCR showed that the mRNA expression of VEGF-A, but not VEGFR-2 is upregulated in explants cultured at 1.5% compared with 21% oxygen. We observed a nonsignificant increase in HIF-2α and HIF-3α mRNA expression in explants cultured at 1.5% oxygen. These data suggest that the mRNA expression of VEGF, and possibly HIF-2α and HIF-3α, is regulated by hypoxia in the developing human lung. Conclusion: This lung explant culture model appears to be a valuable model to unravel the molecular mechanisms of human lung development

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the β2-adrenergic receptor

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1–GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the Gαs-coupled β2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway

Call-duration and triage decisions in out of hours cooperatives with and without the use of an expert system

<p>Abstract</p> <p>Background</p> <p>Cooperatives delivering out of hours care in the Netherlands are hesitant about the use of expert systems during triage. Apart from the extra costs, cooperatives are not sure that quality of triage is sufficiently enhanced by these systems and believe that call duration will be prolonged drastically. No figures about the influence of the use of an expert system during triage on call duration and triage decisions in out of hours care in the Netherlands are available.</p> <p>Methods</p> <p>Electronically registered data concerning call duration and triage decisions were collected in two cooperatives. One in Tilburg, a cooperative in a Southern city of the Netherlands using an expert system, and one in Groningen, a cooperative in a Northern city not using an expert system. Some other relevant information about the care process was collected additionally. Data about call duration was compared using an independent sample t-test. Data about call decisions was compared using Chi Square.</p> <p>Results</p> <p>The mean call time in the cooperative using the TAS expert system is 4.6 minutes, in the cooperative not using the expert system 3.9 minutes. A significant difference of 0.7 minutes (0.4 – 1.0, 95% CI) minutes. In the cooperative with an expert system a larger percentage of patients is handled by the assistant, patients are less often referred to a telephone consultation with the GP and are less likely to be offered a visit by the GP.</p> <p>A quick interpretation of the impact of the difference in triage decisions, show that these may be large enough to support the hypothesis that longer call duration is compensated for by less contacts with the GP (by telephone or face-to-face). There is no proof, however, that these differences are caused by the use of the triage system. The larger amount of calls handled by the assistant may be partly caused by the fact that the assistants in the cooperative with an expert system more often consult the GP during triage. And it is not likely that the larger amount of home visits in Groningen can be attributed to the absence of an expert system. The expert system only offers advice whether a GP should be seen, not in which way (by consultation in the office or by home visit).</p> <p>Conclusion</p> <p>The differences in call times between a cooperative using an expert system and a cooperative not using an expert system are small; 0.4 – 1.0 min. Differences in triage decisions were found, but it is not proven that these can be contributed to the use of an expert system.</p

Airway responsiveness after a single dose of salmeterol and during four months of treatment in children with asthma

Background: Inhalation of a single dose of the long-acting β2- adrenoceptor agonist salmeterol protects against methacholine-induced airway obstruction and other bronchoconstricting stimuli for at least 12 hours. Hypothetically, twice daily dosing of salmeterol may result in continuous protection. Objective: this study was designed to investigate the protective effect of a single dose of salmeterol and of continuous twice daily treatment on airway responsiveness to methacholine. Methods: In a double-blind, parallel study, salmeterol 50 μg twice daily was compared with salbutamol 200 μg twice daily. Thirty children with mild asthma, who had little or no bronchial obstruction and were hyperresponsive to methacholine (PD20 ≤ 150 μg) were allocated to receive either salmeterol or salbutamol. Airway responsiveness was measured before study entry, 12 hours after a single dose of drug was given, and monthly during 4 months of daily treatment. Measurements were always performed at the same time of the day, 12 hours after the last dose of medication was administered. Results: No significant differences in FEV1 were found between treatments at any time point. PD20 significantly increased after the first dose of salmeterol was given (geometric mean, 100 μg). Geometric mean PD20 values were significantly better during salmeterol treatment than during salbutamol treatment, 52 and 25 μg, respectively (p = 0.005). Conclusion: The protection provided by salmeterol during maintenance treatment was less than that provided after the first dose (p < 0.001). However, protection did not diminish during the 4- month treatment period and remained significant compared with baseline (p = 0.003)

Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner

Vinculin localizes to tension-bearing cell–cell junctions to help transmit signals from E-cadherin to the actin cytoskeleton in response to mechanical stress

Serum Potassium and Risk of Death or Kidney Replacement Therapy in Older People With CKD Stages 4-5: Eight-Year Follow-up

Rationale &amp; objective: Hypokalemia may accelerate kidney function decline. Both hypo- and hyperkalemia can cause sudden cardiac death. However, little is known about the relationship between serum potassium and death or the occurrence of kidney failure requiring replacement therapy (KRT). We investigated this relationship in older people with chronic kidney disease (CKD) stage 4-5. Study design: Prospective observational cohort study. Setting &amp; participants: We followed 1,714 patients (≥65 years old) from the European Quality (EQUAL) study for 8 years from their first estimated glomerular filtration rate (eGFR)&lt;20mL/min/1.73m2 measurement. Exposure: Serum potassium was measured every 3 to 6 months and categorized as≤3.5,&gt;3.5-≤4.0,&gt;4.0-≤4.5,&gt;4.5-≤5.0 (reference),&gt;5.0-≤5.5, &gt;5.5-≤6.0, and&gt;6.0mmol/L. Outcome: The combined outcome death before KRT or start of KRT. Analytical approach: The association between categorical and continuous time-varying potassium and death or KRT start was examined using Cox proportional hazards and restricted cubic spline analyses, adjusted for age, sex, diabetes, cardiovascular disease, renin-angiotensin-aldosterone system (RAAS) inhibition, eGFR, and subjective global assessment (SGA). Results: At baseline, 66% of participants were men, 42% had diabetes, 47% cardiovascular disease, and 54% used RAAS inhibitors. Their mean age was 76±7 (SD) years, mean eGFR was 17±5 (SD) mL/min/1.73m2, and mean SGA was 6.0±1.0 (SD). Over 8 years, 414 (24%) died before starting KRT, and 595 (35%) started KRT. Adjusted hazard ratios for death or KRT according to the potassium categories were 1.6 (95% CI, 1.1-2.3), 1.4 (95% CI, 1.1-1.7), 1.1 (95% CI, 1.0-1.4), 1 (reference), 1.1 (95% CI, 0.9-1.4), 1.8 (95% CI, 1.4-2.3), and 2.2 (95% CI, 1.5-3.3). Hazard ratios were lowest at a potassium of about 4.9mmol/L. Limitations: Shorter intervals between potassium measurements would have allowed for more precise estimations. Conclusions: We observed a U-shaped relationship between serum potassium and death or KRT start among patients with incident CKD&nbsp;4-5, with a nadir risk at a potassium level of&nbsp;4.9mmol/L. These findings underscore the&nbsp;potential importance of preventing both high and low potassium in patients with CKD 4-5. Plain-language summary: Abnormal potassium blood levels may increase the risk of death or kidney function decline, especially in older people with chronic kidney disease (CKD). We studied 1,714 patients aged≥65 years with advanced CKD from the European Quality (EQUAL) study and followed them for 8 years. We found that both low and high levels of potassium were associated with an increased risk of death or start of kidney replacement therapy, with the lowest risk observed at a potassium level of 4.9 mmol/L. In patients with CKD, the focus is often on preventing high blood potassium. However, this relatively high optimum potassium level stresses the potential importance of also preventing low potassium levels in older patients with advanced CKD

VASP, zyxin and TES are tension-dependent members of Focal Adherens Junctions independent of the α-catenin-vinculin module

Mechanical forces are integrated at cadherin-based adhesion complexes to regulate morphology and strength of cell-cell junctions and organization of associated F-actin. A central mechanosensor at the cadherin complex is α-catenin, whose stretching recruits vinculin to regulate adhesion strength. The identity of the F-actin regulating signals that are also activated by mechanical forces at cadherin-based junctions has remained elusive. Here we identify the actin-regulators VASP, zyxin and TES as members of punctate, tensile cadherin-based junctions called Focal Adherens Junctions (FAJ) and show that they display mechanosensitive recruitment similar to that of vinculin. However, this recruitment is not altered by destroying or over-activating the α-catenin/vinculin module. Structured Illumination Microscopy (SIM) indicates that these tension sensitive proteins concentrate at locations within FAJs that are distinct from the core cadherin complex proteins. Furthermore, localization studies using mutated versions of VASP and zyxin indicate that these two proteins require binding to each other in order to localize to the FAJs. We conclude that there are multiple force sensitive modules present at the FAJ that are activated at distinct locations along the cadherin-F-actin axis and regulate specific aspects of junction dynamics