Crystal structure and substrate-induced activation of ADAMTS13

Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor (VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13. Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domain cleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacer domains reveals that the metalloprotease domain exhibits a latent conformation in which the active-site cleft is occluded supporting the requirement for an allosteric change to enable accommodation of the substrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate for ADAMTS13

Artificial MiRNA Knockdown of Platelet Glycoprotein lbα: A Tool for Platelet Gene Silencing.

In recent years, candidate genes and proteins implicated in platelet function have been identified by various genomic approaches. To elucidate their exact role, we aimed to develop a method to apply miRNA interference in platelet progenitor cells by using GPIbα as a proof-of-concept target protein. After in silico and in vitro screening of siRNAs targeting GPIbα (siGPIBAs), we developed artificial miRNAs (miGPIBAs), which were tested in CHO cells stably expressing GPIb-IX complex and megakaryoblastic DAMI cells. Introduction of siGPIBAs in CHO GPIb-IX cells resulted in 44 to 75% and up to 80% knockdown of GPIbα expression using single or combined siRNAs, respectively. Conversion of siGPIBAs to miGPIBAs resulted in reduced silencing efficiency, which could however be circumvented by tandem integration of two hairpins targeting different regions of GPIBA mRNA where 72% GPIbα knockdown was achieved. CHO GPIb-IX cells transfected with the miGPIBA construct displayed a significant decrease in their ability to aggregate characterized by lower aggregate numbers and size compared to control CHO GPIb-IX cells. More importantly, we successfully silenced GPIbα in differentiating megakaryoblastic DAMI cells that exhibited morphological changes associated with actin organization. In conclusion, we here report the successful use of miRNA technology to silence a platelet protein in megakaryoblastic cells and demonstrate its usefulness in functional assays. Hence, we believe that artificial miRNAs are suitable tools to unravel the role of a protein of interest in stem cells, megakaryocytes and platelets, thereby expanding their application to novel fields of basic and translational research

Relationship between spatial proximity and travel-to-work distance : the effect of the compact city

In this paper, an assessment is made of the relationship between selected aspects of spatial proximity (density, diversity, minimum commuting distance, jobs-housing balance and job accessibility) and reported commuting distances in Flanders (Belgium). Results show that correlations may depend on the considered trip end. For example, a high residential density, a high degree of spatial diversity and a high level of job accessibility are all associated with a short commute by residents, while a high job density is associated with a long commute by employees. A jobs-housing balance close to one is associated with a short commute, both by residents and by employees. In general, it appears that the alleged sustainability benefits of the compact city model are still valid in a context of continuously expanding commuting trip lengths

Brain Changes Induced by Electroconvulsive Therapy Are Broadly Distributed

© 2019 Society of Biological Psychiatry Background: Electroconvulsive therapy (ECT) is associated with volumetric enlargements of corticolimbic brain regions. However, the pattern of whole-brain structural alterations following ECT remains unresolved. Here, we examined the longitudinal effects of ECT on global and local variations in gray matter, white matter, and ventricle volumes in patients with major depressive disorder as well as predictors of ECT-related clinical response. Methods: Longitudinal magnetic resonance imaging and clinical data from the Global ECT-MRI Research Collaboration (GEMRIC) were used to investigate changes in white matter, gray matter, and ventricle volumes before and after ECT in 328 patients experiencing a major depressive episode. In addition, 95 nondepressed control subjects were scanned twice. We performed a mega-analysis of single subject data from 14 independent GEMRIC sites. Results: Volumetric increases occurred in 79 of 84 gray matter regions of interest. In total, the cortical volume increased by mean ± SD of 1.04 ± 1.03% (Cohen\u27s d = 1.01, p \u3c .001) and the subcortical gray matter volume increased by 1.47 ± 1.05% (d = 1.40, p \u3c .001) in patients. The subcortical gray matter increase was negatively associated with total ventricle volume (Spearman\u27s rank correlation ρ = −.44, p \u3c .001), while total white matter volume remained unchanged (d = −0.05, p = .41). The changes were modulated by number of ECTs and mode of electrode placements. However, the gray matter volumetric enlargements were not associated with clinical outcome. Conclusions: The findings suggest that ECT induces gray matter volumetric increases that are broadly distributed. However, gross volumetric increases of specific anatomically defined regions may not serve as feasible biomarkers of clinical response

CVD diamond coated silicon nitride self-mated systems : tribological behaviour under high loads

Friction and wear behaviour of self-mated chemical vapour deposited (CVD) diamond films coating silicon nitride ceramics (Si3N4) were investigated in ambient atmosphere. The tribological tests were conducted in a reciprocal motion ball-on-flat type tribometer under applied normal loads up to 80 N (~10 GPa). Several characterisation techniques - including scanning electron microscopy (SEM), atomic force microscopy (AFM) and micro-Raman studies - were used in order to assess the quality, stress state and wear resistance of the coatings. In addition, a novel method is presented to estimate the wear coefficient of the diamond coated flat specimens from AFM and optical microscopy (OM) observations of the wear tracks

A relocatable ocean model in support of environmental emergencies

During the Costa Concordia emergency case, regional, subregional, and relocatable ocean models have been used together with the oil spill model, MEDSLIK-II, to provide ocean currents forecasts, possible oil spill scenarios, and drifters trajectories simulations. The models results together with the evaluation of their performances are presented in this paper. In particular, we focused this work on the implementation of the Interactive Relocatable Nested Ocean Model (IRENOM), based on the Harvard Ocean Prediction System (HOPS), for the Costa Concordia emergency and on its validation using drifters released in the area of the accident. It is shown that thanks to the capability of improving easily and quickly its configuration, the IRENOM results are of greater accuracy than the results achieved using regional or subregional model products. The model topography, and to the initialization procedures, and the horizontal resolution are the key model settings to be configured. Furthermore, the IRENOM currents and the MEDSLIK-II simulated trajectories showed to be sensitive to the spatial resolution of the meteorological fields used, providing higher prediction skills with higher resolution wind forcing.MEDESS4MS Project; TESSA Project; MyOcean2 Projectinfo:eu-repo/semantics/publishedVersio

Simvastatin and purine analogs have a synergic effect on apoptosis of chronic lymphocytic leukemia cells

Despite many therapeutic regimens introduced recently, chronic lymphocytic leukemia (CLL) is still an incurable disorder. Thus, there is an urgent need to discover novel, less toxic and more effective drugs for CLL patients. In this study, we attempted to assess simvastatin, widely used as a cholesterol-lowering drug, both as a single agent and in combination with purine analogs—fludarabine and cladribine—in terms of its effect on apoptosis and DNA damage of CLL cells. The experiments were done in ex vivo short-term cell cultures of blood and bone marrow cells from newly diagnosed untreated patients. We analyzed expression of active caspase-3 and the BCL-2/BAX ratio as markers of apoptosis and the expression of phosphorylated histone H2AX (named γH2AX) and activated ATM kinase (ataxia telangiectasia mutated kinase), reporters of DNA damage. Results of our study revealed that simvastatin induced apoptosis of CLL cells concurrently with lowering of BCL-2/BAX ratio, and its pro-apoptotic effect is tumor-specific, not affecting normal lymphocytes. We observed that combinations of simvastatin+fludarabine and simvastatin+cladribine had a synergic effect in inducing apoptosis. Interestingly, the rate of apoptosis caused by simvastatin alone and in combination was independent of markers of disease progression like ZAP-70 and CD38 expression or clinical stage according to Rai classification. We have also seen an increase in γH2AX expression in parallel with activation of ATM in most of the analyzed samples. The results suggest that simvastatin can be used in the treatment of CLL patients as a single agent as well as in combination with purine analogs, being equally effective both in high-risk and good-prognosis patients. One of the mechanisms of simvastatin action is inducing DNA damage that ultimately leads to apoptosis