Characterization of the protein profile of donkey's milk whey fraction

Characterization of the protein profile of the whey fraction from a milk sample taken from an individual donkey belonging to the 'Ragusana' species of the East of Sicily is reported. Direct RP-HPLC/electrospray ionization (ESI)-MS analysis of the whey fraction allowed the detection of some unknown components, together with the identification of already known whey proteins. Matrix-assisted laser desorption/ionization (MALDI)-TOF/MS and RP-HPLC/ESI-MS/MS analysis of the enzymatic digests of the unknown components resulted the identification and characterization of (1) two beta-casein fragments; (2) the sequence of donkey's serum albumin; and (3) the oxidized methionine forms of lysozyme B and alpha-lactoalbumin. One of the two beta-casein fragments corresponds to the sequence Val(176)-Arg(189) of the horse's beta-casein. The second one corresponds the C-terminal sequence Tyr(199)-Val(226) of the horse's beta-casein, with four amino acid substitutions (Q --> R(203), L/I --> P(206), F --> L(210) and P --> A(219)). Both fragments, reasonably arising by endogenous proteases cleavage of the donkey's beta-casein, could be potential biologically active peptides. Direct mass spectrometric sequence characterization of the detected donkey's serum albumin reveals the presence of the amino acid substitution Val --> Ile at position 497 with respect to the cDNA deduced sequence. The oxidized forms of lysozyme B and alpha-lactoalbumin are selectively oxidized at methionine 79 and methionine 90, respectively.     

Modelling counter-current and dual counter-current chromatography using longitudinal mixing cell and eluting counter-current distribution models

Two well known approaches are considered to analyze the processes of counter-current and dual counter-current chromatography: the longitudinal mixing cell model and the Craig's counter-current distribution model. The cell model represents perfectly mixed, equally sized cells in series. The number of cells characterizes the rates of longitudinal mixing in the stationary and mobile phases. In the eluting counter-current distribution (CCD) model, the CCC process is considered as a continuous form of Craig's counter-current distribution. For a cascade of equilibrium stages theoretical elution profiles of the CCC process by using the CCD and cell model approaches have been compared. It is shown that in general, distribution functions of the CCD and cell models differ. It is established that the distribution of a solute between two solvent phases in the dual CCC process is determined by the extraction factor c, the total number of equilibrium stages n and the position of the sample inlet m by the equation Q(x)=(1-c(m))/(1-c(n+1)) with c=F(2)K(D)/F(1) (K(D), F(1), F(2) and Q(x) are the distribution constant, the phase flow-rates and the portion of solute eluted by the first phase, respectively).     

One-dimensional SAMs of (12-pyrrol-1-yl-dodecyl)-phosphonic acid templated by polyelectrolyte molecules

We present a novel method for the fabrication of one-dimensional (1-D) self-assembled monolayers and multilayers (SAMs) of (12-pyrrol-1-yl-dodecyl)-phosphonic acid (Py-DPA) on various polar surfaces using polyelectrolyte nanostructures as positive templates. Particularly, we demonstrate that (i) patterns of aligned 1-D polycation structures on a poly(dimethylsiloxane) stamp can be prepared by moving a droplet of polycation solution along the surface; (ii) these patterns can be used as templates for the ordered assembly of Py-DPA in water where Py-DPA carries a charge opposite to the charge of the template; and (iii) Py-DPA SAMs can then be transferred onto mica or silicon wafers by a printing process. These nanostructures with a polymerizable pyrrole headgroup might be useful for the creation of electrically conductive patterns of conjugated polymers.     

Free-standing films of fluorinated surfactants as 2D matrices for organizing detergent-solubilized membrane proteins

The possibility of organizing detergent-solubilized membrane proteins in a plane within the core of Newton black films (NBFs) formed from fluorinated surfactants has been investigated. Fluorinated surfactants have the interesting characteristics of being poorly miscible with detergents and highly surface-active. As a result, when a membrane protein-the transmembrane domain of OmpA (tOmpA)-solubilized by the nonionic detergent C8E4 (tetraethylene glycol monooctyl ether) was injected under a monolayer of fluorinated surfactant, C8E4 and tOmpA/C8E4 complexes remained confined to the subphase. Vertical, macroscopic NBFs were drawn, and their structure was investigated by means of X-ray reflectivity. Depending on experimental conditions, the protein was shown to organize into either one or two monolayers stabilized by two monolayers of fluorinated surfactant. Two different mechanisms of protein insertion were investigated: (i) attachment of polyhistidine-tagged tOmpA/C8E4 complexes to nickel-bearing polar groups born by a fluorinated surfactant and (ii) spontaneous diffusion into the surfactant films. Possible applications are discussed.     

Locally Farkas-Minkowski linear inequality systems

This paper introduces thelocally Farkas-Minkowski (LFM) linear inequality systems in a finite dimensional Euclidean space. These systems are those ones that satisfy that any consequence of the system that is active at some solution point is also a consequence of some finite subsystem. This class includes the Farkas-Minkowski systems and verifies most of the properties that these systems possess. Moreover, it contains the locally polyhedral systems, which are the natural external representation of quasi-polyhedral sets. TheLFM systems appear to be the natural external representation of closed convex sets. A characterization based on their properties under the union of systems is provided. In linear semi-infinite programming, theLFM property is the more general constraint qualification such that the Karush-Kuhn-Tucker condition characterizes the optimal points. Furthermore, the pair of Haar dual problems has no duality gap.     

Polyurethane Based Materials for the Production of Biomedical Materials

Polyurethanes (PUs) composed by hard and soft segments have been extensively used in the manufacturing of biocompatible prosthesis and medical devices. A broad variety of PUs can be obtained by modifying the balance between both segments. In the present work, different basically-flexible PUs have been prepared by employing different combinations of aliphatic hexamethylene diisocyanate, poly(ethylene glycol) (Mw 400 Da), poly(ϵ-caprolactone) diol (Mw 530 Da), and 1,4-butanediol. Thermal analysis of the synthesized PUs demonstrated high thermal stability and the assumption of glassy state well below room temperature, in agreement with their marked flexibility. Morphological characterization of PUs films indicated that films prepared by spin coating were smoother and more homogeneous than those obtained by casting. Biological assays performed by using 3T3/BALB-C mouse embryo fibroblast cell line confirmed the absence of toxicity and hence the biocompatibility of PU-films.     

Picomolar FKBP inhibitors enabled by a single water-displacing methyl group in bicyclic [4.3.1] aza-amides

Methyl groups can have profound effects in drug discovery but the underlying mechanisms are diverse and incompletely understood. Here we report the stereospecific effect of a single, solvent-exposed methyl group in bicyclic [4.3.1] aza-amides, robustly leading to a 2 to 10-fold increase in binding affinity for FK506-binding proteins (FKBPs). This resulted in the most potent and efficient FKBP ligands known to date. By a combination of co-crystal structures, isothermal titration calorimetry (ITC), density-functional theory (DFT), and 3D reference interaction site model (3D-RISM) calculations we elucidated the origin of the observed affinity boost, which was purely entropically driven and relied on the displacement of a water molecule at the protein–ligand–bulk solvent interface. The best compounds potently occupied FKBPs in cells and enhanced bone morphogenic protein (BMP) signaling. Our results show how subtle manipulation of the solvent network can be used to design atom-efficient ligands for difficult, solvent-exposed binding pockets.

Enhancement by displacement. A single methyl group displaces a water molecule from the binding site of FKBPs, resulting in the most potent binders known, outperforming the natural products FK506 and rapamycin in biochemical and cellular assays.     

An approach formulated in terms of conserved variables for the characterisation of propellant combustion in internal ballistics

A model formulated in terms of conserved variables is proposed for its use in the study of internal ballistic problems of pyrotechnical mixtures and propellants. It is a transient two‐phase flow model adapted from the non‐conservative Gough model. This conversion is mathematically attractive because of the wide range of numerical methods for this kind of systems that may be applied. We propose the use of the AUSM+, AUSM + up and Rusanov schemes as an efficient alternative for this type of two‐phase problem. A splitting technique is applied, which solves the system of equations in several steps. A second‐order approach based on Monotonic Upstream‐Centred Scheme for Conservation Laws (MUSCL) is also used. Some tests are used to validate the code, namely a shock wave test, a contact discontinuity problem and an internal ballistics problem. In this last case, one‐dimensional numerical results are compared with experimental data of 155‐mm gunshots. Copyright © 2015 John Wiley & Sons, Ltd.