Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation

The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.     

Automatic verification of concurrent stochastic systems

Formal Methods in System Design - Automated verification techniques for stochastic games allow formal reasoning about systems that feature competitive or collaborative behaviour among rational...     

Preparation of grids for simulations of groundwater flow in fractured porous media

This work presents an extension of grid generation techniques for finite-volume discretizations of density-driven flow in fractured porous media, in which fractures are considered as low-dimensional manifolds and are resolved by sides of grid elements. The proposed technique introduces additional degrees of freedom for the unknowns assigned to the fractures and thus allows to reconstruct jumps of the solution over a fracture. Through the concept of degenerated elements, the proposed technique can be used for arbitrary junctions of fractures but is sufficiently simple regarding the implementation and allows for the application of conventional numerical solvers. Numerical experiments presented at the end of the paper demonstrate the applicability of this technique in two and three dimensions for complicated fracture networks.     

Fabry Disease and the Heart: A Comprehensive Review

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations of the GLA gene that result in a deficiency of the enzymatic activity of α-galactosidase A and consequent accumulation of glycosphingolipids in body fluids and lysosomes of the cells throughout the body. GB3 accumulation occurs in virtually all cardiac cells (cardiomyocytes, conduction system cells, fibroblasts, and endothelial and smooth muscle vascular cells), ultimately leading to ventricular hypertrophy and fibrosis, heart failure, valve disease, angina, dysrhythmias, cardiac conduction abnormalities, and sudden death. Despite available therapies and supportive treatment, cardiac involvement carries a major prognostic impact, representing the main cause of death in FD. In the last years, knowledge has substantially evolved on the pathophysiological mechanisms leading to cardiac damage, the natural history of cardiac manifestations, the late-onset phenotypes with predominant cardiac involvement, the early markers of cardiac damage, the role of multimodality cardiac imaging on the diagnosis, management and follow-up of Fabry patients, and the cardiac efficacy of available therapies. Herein, we provide a comprehensive and integrated review on the cardiac involvement of FD, at the pathophysiological, anatomopathological, laboratory, imaging, and clinical levels, as well as on the diagnosis and management of cardiac manifestations, their supportive treatment, and the cardiac efficacy of specific therapies, such as enzyme replacement therapy and migalastat.     

Detection of water ingress in composite sandwich structures: a magnetic resonance approach

Water ingress inside honeycomb sandwich panels during service has been linked to in-flight failure in some aircraft. There is an ongoing effort to develop nondestructive testing methods to detect the presence of water within the panels. Magnetic resonance (MR) represents an attractive approach in that it is sensitive to moisture. Using a unilateral MR sensor, testing can be applied directly to the surface of the panel. The viability of MR is demonstrated through laboratory imaging of both water within sandwich panels, as well as the adhesive itself. The detection of water using a one-sided handheld MR sensor is presented. It is shown that simple detection, as well as spatial localization of water within sandwich panels is possible.     

Carbonization reactions of inertinite macerals in Australian coals

Widespread disagreement about the degree of reactivity of the inertinite group of macerais is related to variations in experimental conditions of assessment and failure to appreciate technological modifications imposed on similar macerals by dissimilar source materials and depositional conditions. This has resulted in the constant under-estimation of the coking potential of post-Carboniferous inertinite-rich coals by predictive methods developed for vitrinite-rich Carboniferous coals. Coking tests up to 1000 °C have been carried out on 20 coals of different rank in such a manner that coked portions of the samples could be correlated with their uncoked equivalents. It has been found that an inverse relationship exists between the level of precarbonization reflectance (PCR) of inertinite and the reflectance and bireflectance of its coke. The increase in the latter parameter is non-linear and involves a sudden jump which is taken as the boundary between reactive (high bireflectance) and non-reactive (low bireflectance) inertinite. In relation to coal rank a reactivity field for inertinite has been delineated which can be subdivided into two areas of high and moderate reactivity, respectively. On the whole, the proportion of reactive inertinite is larger than allowed for in most petrography-based coke stability calculations.     

Comparison of a contactor catalytic membrane reactor with a conventional reactor: example of wet air oxidation

A wet air oxidation reaction was carried out in a gas/liquid catalytic membrane reactor of the contactor type. The oxidation of formic acid was used as a model reaction. The mesoporous top-layer of a ceramic tubular membrane was used as catalyst (Pt) support, and was placed at the interface of the gas (air) and liquid (HCOOH solution) phases.

A similar reaction was carried out in a conventional batch reactor, using a steering rate high enough to avoid gas-diffusion limitations, and exactly identical conditions than for the CMR (amount of catalyst, pressure, etc.). At room temperature, the CMR showed an initial activity three to six times higher than the conventional reactor. This activity increase was attributed to an easier oxygen access to the catalytic sites. Nevertheless, the catalytic membrane gradually deactivated after a few hours of operation. Different deactivation mechanisms are presented.