Analysis of Integrin αIIb Subunit Dynamics Reveals Long-Range Effects of Missense Mutations on Calf Domains

Integrin α_IIbβ₃, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin α_IIbβ₃ polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of α_IIb Calf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf-1 + Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.     

APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.     

Prolonged display or rapid internalization of the IgG-binding protein ZZ anchored to the surface of cells using the diphtheria toxin T domain

We have shown previously that the diphtheria toxin transmembranedomain (T) may function as a membrane anchor for soluble proteinsfused at its C-terminus. Binding to membranes is triggered byacidic pH. Here, we further characterized this anchoring device.Soluble proteins may be fused at the N-terminus of the T domainor at both extremities, without modifying its membrane bindingproperties. This allows one to choose the orientation of theprotein to be attached to the membrane. Maximum binding to thecell surface is reached within 1 h. Anchoring occurs on cellspreviously treated with proteinase K, suggesting that T interactswith the lipid phase of the membrane without the help of cellsurface proteins. Binding does not permeabilize cells or affectcell viability, despite the fact that it permeabilizes liposomesand alters their structure. When attached to L929 fibroblasts,the proteins are not internalized and remain displayed at theirsurface for more than 24 h. When bound to K562 myeloid cells,the molecules are internalized and degraded. Thus, dependingon the cell type, soluble proteins may be anchored to the surfaceof cells by the T domain for an extended time or directed towardsan internalization pathway.     

Synthesis of AlN nanopowder coated with a thin layer of C,using a thermal plasma reactor

High-purity nanocrystalline aluminum nitride powders were synthesized by using a 12?kW non-transferred arc plasma. The synthesis was conducted in a versatile, new designed, one-chamber thermal plasma reactor (TPR). The novel experimental assembly incorporated better working conditions like: high temperature gradient between the crucible and reactor's wall, and high super-saturation of the system by nitrogen and carbon. Thermodynamic modelling of the synthesis was conducted in order to achieve the best conditions for AlN formation. In this study, aluminum discs of Al 1100 were used as precursor material and pure nitrogen was the only gas used as reagent and plasmogenic gas.Nanopowders collected from reactor's wall were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-Ray diffraction (XRD). Synthesized h-AlN nano-powders were found to be free of oxides and aluminum metal. A thin carbon-layer around the particles was detected. TEM results indicated that the carbon-layer was around 5 and 10?nm. This outcome could make a significant difference with other synthesis reported in the literature since the occurrence of the carbon-layer, could delay AlN oxidation, prevent hydration, and could avoid the agglomeration of the particles.     

Electromagnetic modeling of waveguide amplifier based on Nd<Superscript>3+</Superscript> Si-rich SiO<Subscript>2</Subscript> layers by means of the ADE-FDTD method

By means of ADE-FDTD method, this paper investigates the electromagnetic modelling of a rib-loaded waveguide composed of a Nd³⁺ doped Silicon Rich Silicon Oxide active layer sandwiched between a SiO₂ bottom cladding and a SiO₂ rib. The Auxilliary Differential Equations are the rate equations which govern the levels populations. The Finite Difference Time Domain (FDTD) scheme is used to solve the space and time dependent Maxwell equations which describe the electromagnetic field in a copropagating scheme of both pumping (λ _pump = 488 nm) and signal (λ _signal = 1064 nm) waves. Such systems are characterized by extremely different specific times such as the period of electromagnetic field ~ 10^-15 s and the lifetimes of the electronic levels between ~ 10^-10s and ~ 10^-4 s. The time scaling method is used in addition to specific initial conditions in order to decrease the computational time. We show maps of the Poynting vector along the propagation direction as a function of the silicon nanograin (Si-ng) concentrations. A threshold value of 10²⁴ Si-ng m^-3 is extracted below which the pump wave can propagate so that a signal amplication is possible.     

Alumina foam catalyst supports for industrial steam reforming processes

The manufacture and the characterisation of alumina foams as alternative catalysts supports for industrial steam reforming processes are presented here. The possibility of use of alumina foams as catalysts supports in such processes is evaluated by studying their resistance toward mechanical and chemical stresses. The alumina foams produced are characterised owing to their processing parameters (slurry infiltration, sintering temperature, template pore size). Their ability to work in hydrothermal atmosphere is assessed by characterising the evolution of microstructures and mechanical strengths upon aging. Thermodynamic studies of the stability of alumina in industrial steam reforming working conditions are performed and correlated to the experiments to demonstrate the stability of such a system.     

Anticoagulant, Antioxidant and Antitumor Activities of Heterofucans from the Seaweed Dictyopteris delicatula

In the present study, six families of sulfated polysaccharides were obtained from seaweed Dictyopteris delicatula by proteolytic digestion, followed by acetone fractionation and molecular sieving on Sephadex G-100. Chemical analyses demonstrated that all polysaccharides contain heterofucans composed mainly of fucose, xylose, glucose, galactose, uronic acid, and sulfate. The fucans F0.5v and F0.7v at 1.0 mg/mL showed high ferric chelating activity (～45%), whereas fucans F1.3v (0.5 mg/mL) showed considerable reducing power, about 53.2% of the activity of vitamin C. The fucan F1.5v presented the most prominent anticoagulant activity. The best antiproliferative activity was found with fucans F1.3v and F0.7v. However, F1.3v activity was much higher than F0.7v inhibiting almost 100% of HeLa cell proliferation. These fucans have been selected for further studies on structural characterization as well as in vivo experiments, which are already in progress.     

Simulation of pH-controlled dissolution of aluminium based on a modified Scanning Electrochemical Microscope experiment to mimic localized trenching on aluminium alloys

Some constituent intermetallic (IMPs) particles at the surface of aluminium alloys are considered as preferential sites for the initiation of structural corrosion resulting in localized trenching around the particles and the surrounding Al matrix. In this work, a modified scanning electrochemical microscope (SECM) experiment was used to induce such phenomena via a local alcalinisation on 200 nm thick aluminium coatings promoting their local dissolution in an aerated 0.1 M NaCl electrolyte. The local alcalinisation was induced by the oxygen reduction reaction on the tip of a SECM which mimics the surface of an isolated IMP. From a phenomenological point of view, reproducible cylindrical damage develops in front of the platinum ultramicroelectrode (UME). Using a 2D finite element modelling to simulate the SECM experiments, the role of the local alcalinisation was validated and the calculated Al dissolution rate was found in agreement with the experimental evaluation.     

Multivariate Analysis of the Stability of Spray-Dried Eupenicillium javanicum Peptidases

Peptidases occupy a central position in the enzyme market because of their importance in many areas, such as for physiological processes, foods, and detergents, as well as in the pharmaceutical, leather, and biotechnology industries. Microbial production is among the major sources of peptidases because it presents many advantages when compared with other methods. In this study, the metallopeptidases produced by the fungus Eupenicillium javanicum under a solid-state fermentation bioprocess were spray-dried. The enzymatic extract was dried using drying adjuvants, and optimal conditions for preserving enzymatic activity were studied following a Box-Behnken experimental design. The spray process factors studied were the air-drying temperature, enzyme feed flow rate, and the proportion of enzyme/additive. The responses analyzed were the dry extract yield and enzymatic activity after spray drying. Additionally, the stability of the dry extracts was assessed during 180 days at 4°C and 25°C. The results revealed extract yields of up to 66.12% and good enzymatic activity for intermediate values of temperature and adjuvant proportions. Furthermore, the dried enzymatic extracts showed potential for future commercial applications because of their stability at 25°C for 180 days.