New insights into the size and stoichiometry of the plasminogen activator inhibitor type-1·vitronectin complex

Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glyco-protein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M(r) ~ 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces

Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators

Intergenerational mobility of housework time in the United Kingdom

This paper analyzes the relationship between parents’ time devoted to housework and the time devoted to housework by their children. Using data from the Multinational Time Use Study for the UK, we find positive intergenerational correlations in housework for both parents, indicating that the more time parents devote to housework, the more time their children will devote to housework. Using data from the British Household Panel Survey, we find that a higher father–mother housework ratio is positively related to a higher child–mother housework ratio, even after allowing for individual fixed-effects. In order to address the potential exacerbation of errors-in-variables arising from the fixed-effects specification, we instrument the father–mother ratio of housework using father’s and mother’s lagged weekly working hours. The Instrumental-Variable estimates fully support the fixed-effects estimates, and suggest that the latter should be regarded as a lower bound. We also present evidence of the link between housework during adolescence and duringadulthood, which may indicate that housework time during adulthood depends on the housework time during childhood, which may also be affected by parents’ housework time. Our results contribute to the field of the intergenerational mobility of behaviors

Unveiling the mechanisms of solid-state dewetting in Solid Oxide Cells with novel 2D electrodes

During the operation of Solid Oxide Cell (SOC) fuel electrodes, the mobility of nickel can lead to significant changes in electrode morphology, with accompanying degradation in electrochemical performance. In this work, the dewetting of nickel films supported on yttriastabilized zirconia (YSZ), hereafter called 2D cells, is studied by coupling in-situ environmental scanning electron microscopy (E-SEM), image analysis, cellular automata simulation and electrochemical impedance spectroscopy (EIS). Analysis of experimental E-SEM images shows that Ni dewetting causes an increase in active triple phase boundary (aTPB) length up to a maximum, after which a sharp decrease in aTPB occurs due to Ni de-percolation. This microstructural evolution is consistent with the EIS response, which shows a minimum in polarization resistance followed by a rapid electrochemical degradation. These results reveal that neither evaporation-condensation nor surface diffusion of Ni are the main mechanisms of dewetting at 560-800 °C. Rather, the energy barrier for pore nucleation within the dense Ni film appears to be the most important factor. This sheds light on the relevant mechanisms and interfaces that must be controlled to reduce the electrochemical degradation of SOC electrodes induced by Ni dewetting

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation. In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of 125I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (Kd ~ 3.5 μM) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI- 1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1-vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization