Suppression of Liquid-Liquid Phase Separation and Aggregation of Antibodies by Modest Pressure Application

The high colloidal stability of antibody (immunoglobulin) solutions is important for pharmaceutical applications. Inert cosolutes, excipients, are generally used in therapeutic protein formulations to minimize physical instabilities, such as liquid–liquid phase separation (LLPS), aggregation and precipitation, which are often encountered during manufacturing and storage. Despite their widespread use, a detailed understanding of how excipients modulate the specific protein-protein interactions responsible for these instabilities is still lacking. In this work, we demonstrate the high sensitivity to pressure of globulin condensates as a suitable means to suppress LLPS and subsequent aggregation of concentrated antibody solutions. The addition of excipients has only a minor effect. The high pressure sensitivity observed is due to the fact that these flexible Y-shaped molecules create a considerable amount of void volume in the condensed phase, leading to an overall decrease in the volume of the system upon dissociation of the droplet phase by pressure already at a few tens of to hundred bar. Moreover, we show that immunoglobulin molecules themselves are highly resistant to unfolding under pressure, and can even sustain pressures up to about 6 kbar without conformational changes. This implies that immunoglobulins are resistant to the pressure treatment of foods, such as milk, in high-pressure food-processing technologies, thereby preserving their immunological activity.     

Critical clusters in interdependent economic sectors

In this paper we develop a data-driven hierarchical clustering methodology to group the economic sectors of a country in order to highlight strongly coupled groups that are weakly coupled with other groups. Specifically, we consider an input-output representation of the coupling among the sectors and we interpret the relation among sectors as a directed graph; then we recursively apply the spectral clustering methodology over the graph, without a priori information on the number of groups that have to be obtained. In order to do this, we resort to the eigengap criterion, where a suitable number of groups is selected automatically based on the intensity and structure of the coupling among the sectors. We validate the proposed methodology considering a case study for Italy, inspecting how the coupling among clusters and sectors changes from the year 1995 to 2011, showing that in the years the Italian structure underwent deep changes, becoming more and more interdependent, i.e., a large part of the economy has become tightly coupled.     

Catalytic Effect of Electric Fields on the Kemp Elimination Reactions with Neutral Bases

The effect of solvent reaction fields and oriented electric fields on the Kemp elimination reaction between methylamine or imidazole and 5-nitrobenzisoxazole has been theoretically studied. The Kemp reaction is the most widely used for the design of new enzymes. Our results, using the SMD continuous model for solvents, are in quite good agreement with the experimental fact that the rate of the analogous reaction with butylamine is one order of magnitude smaller in water than in acetonitrile. In the case of external electric fields, our results show that they can increase or decrease the energy barrier depending on the magnitude and orientation of the field. A duly oriented electric field may have a notable catalytic effect on the reaction. So, external electric fields and reaction fields due to the medium can contribute to the design of new enzymes. Several factors that must be taken into account to increase the catalytic effect are discussed.     

Numerical analysis of conservative unstructured discretisations for low Mach flows

Unstructured meshes allow easily representing complex geometries and to refine in regions of interest without adding control volumes in unnecessary regions. However, numerical schemes used on unstructured grids have to be properly defined in order to minimise numerical errors. An assessment of a low Mach algorithm for laminar and turbulent flows on unstructured meshes using collocated and staggered formulations is presented. For staggered formulations using cell‐centred velocity reconstructions, the standard first‐order method is shown to be inaccurate in low Mach flows on unstructured grids. A recently proposed least squares procedure for incompressible flows is extended to the low Mach regime and shown to significantly improve the behaviour of the algorithm. Regarding collocated discretisations, the odd–even pressure decoupling is handled through a kinetic energy conserving flux interpolation scheme. This approach is shown to efficiently handle variable‐density flows. Besides, different face interpolations schemes for unstructured meshes are analysed. A kinetic energy‐preserving scheme is applied to the momentum equations, namely, the symmetry‐preserving scheme. Furthermore, a new approach to define the far‐neighbouring nodes of the quadratic upstream interpolation for convective kinematics scheme is presented and analysed. The method is suitable for both structured and unstructured grids, either uniform or not. The proposed algorithm and the spatial schemes are assessed against a function reconstruction, a differentially heated cavity and a turbulent self‐igniting diffusion flame. It is shown that the proposed algorithm accurately represents unsteady variable‐density flows. Furthermore, the quadratic upstream interpolation for convective kinematics scheme shows close to second‐order behaviour on unstructured meshes, and the symmetry‐preserving is reliably used in all computations. Copyright © 2016 John Wiley & Sons, Ltd.     

Dendronized Anionic Gold Nanoparticles: Synthesis,Characterization, and Antiviral Activity

Anionic carbosilane dendrons decorated with sulfonate functions and one thiol moiety at the focal point have been used to synthesize water‐soluble gold nanoparticles (AuNPs) through the direct reaction of dendrons, gold precursor, and reducing agent in water, and also through a place‐exchange reaction. These nanoparticles have been characterized by NMR spectroscopy, TEM, thermogravimetric analysis, X‐ray photoelectron spectroscopy (XPS), UV/Vis spectroscopy, elemental analysis, and zeta‐potential measurements. The interacting ability of the anionic sulfonate functions was investigated by EPR spectroscopy with copper(II) as a probe. Different structures and conformations of the AuNPs modulate the availability of sulfonate and thiol groups for complexation by copper(II). Toxicity assays of AuNPs showed that those produced through direct reaction were less toxic than those obtained by ligand exchange. Inhibition of HIV‐1 infection was higher in the case of dendronized AuNPs than in dendrons.     

Electrochemical studies and square wave adsorptive stripping voltammetry of the antidepressant fluoxetine

The electrochemical reduction of the antidepressant drug fluoxetine was investigated by cyclic, linear sweep, differential pulse and square wave voltammetry using a hanging mercury drop electrode in alkaline buffer solution in water and in a water/acetonitrile mixed solvent. Cyclic voltammograms in aqueous solution showed very strong adsorption of fluoxetine on the electrode with formation of a compact film. The effect of addition of different percentages of acetonitrile on the voltammetric response was evaluated. It is shown that acetonitrile protects the electrode surface, thus preventing the adsorption of fluoxetine as a compact film, although reduction occurs at more negative potentials. Adsorption was used to accumulate the drug onto the electrode surface. The adsorbed species were measured voltammetrically by reduction at -1.3 V in an aqueous 0.05 M Ringer buffer, pH 12, 20% acetonitrile v/v. Linear calibration graphs were obtained in the range 0.52-5.2 M. The quantification of fluoxetine in pharmacological formulations existing in the market was performed using adsorptive square wave cathodic stripping voltammetry. and compared with data from UV spectrophotometry. The method is simple and not time-consuming. A comparative high performance liquid chromatography assay with UV detection was performed. Recovery data for both methods are reported.