Influence of composition, bonding characteristics and microstructure on the electrochemical and optical stability of AlOxNy thin films

Thin films of AlOxNy were deposited by magnetron sputtering in a wide composition range. Different structures and morphologies were observed, depending on the composition and bonding states, which opened the possibility to tailor the properties of this oxynitride system between those of pure Al and those of nitride and oxide films. In a wide range of stoichiometries, one can report the formation of nanocomposite porous films, where Al nanoparticles are dispersed in an amorphous matrix of AlOxNy. The electrochemical behaviour of the films was studied in isotonic NaCl solution. It was observed that the pitting 2 potential characteristic of aluminium disappears with the incorporation of oxygen and nitrogen in the films, being replaced by a smooth current increase. Electrochemical impedance spectroscopy performed during 35 days showed that the corrosion resistance of the films steadily increases. The unusual optical reflectance profile of some films is maintained after immersion for several months.Fundação para a Ciência e a TecnologiaPrograma Pessoa 2010/2011, Cooperação Portugal/França, Proc.º 441.00, Project“COLOURCLUSTER”

RNA Aptamers Generated against Oligomeric Aβ40 Recognize Common Amyloid Aptatopes with Low Specificity but High Sensitivity

Aptamers are useful molecular recognition tools in research, diagnostics, and therapy. Despite promising results in other fields, aptamer use has remained scarce in amyloid research, including Alzheimer's disease (AD). AD is a progressive neurodegenerative disease believed to be caused by neurotoxic amyloid β-protein (Aβ) oligomers. Aβ oligomers therefore are an attractive target for development of diagnostic and therapeutic reagents. We used covalently-stabilized oligomers of the 40-residue form of Aβ (Aβ40) for aptamer selection. Despite gradually increasing the stringency of selection conditions, the selected aptamers did not recognize Aβ40 oligomers but reacted with fibrils of Aβ40, Aβ42, and several other amyloidogenic proteins. Aptamer reactivity with amyloid fibrils showed some degree of protein-sequence dependency. Significant fibril binding also was found for the naïve library and could not be eliminated by counter-selection using Aβ40 fibrils, suggesting that aptamer binding to amyloid fibrils was RNA-sequence-independent. Aptamer binding depended on fibrillogenesis and showed a lag phase. Interestingly, aptamers detected fibril formation with ≥15-fold higher sensitivity than thioflavin T (ThT), revealing substantial β-sheet and fibril formation undetected by ThT. The data suggest that under physiologic conditions, aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils. Nevertheless, the high sensitivity, whereby aptamers detect β-sheet formation, suggests that they can serve as superior amyloid recognition tools

Continuous Flow Reactor for the Production of Stable Amyloid Protein Oligomers

The predominant working hypothesis of Alzheimer's disease is that the proximate pathologic agents are oligomers of the amyloid β-protein (Aβ). "Oligomer" is an ill-defined term. Many different types of oligomers have been reported, and they often exist in rapid equilibrium with monomers and higher-order assemblies. This has made formal structure-activity determinations difficult. Recently, Ono et al. [Ono, K., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 14745-14750] used rapid, zero-length, in situ chemical cross-linking to stabilize the oligomer state, allowing the isolation and study of pure populations of oligomers of a specific order (number of Aβ monomers per assembly). This approach was successful but highly laborious and time-consuming, precluding general application of the method. To overcome these difficulties, we developed a "continuous flow reactor" with the ability to produce theoretically unlimited quantities of chemically stabilized Aβ oligomers. We show, in addition to its utility for Aβ, that this method can be applied to a wide range of other amyloid-forming proteins

Conformation and self-association of human recombinant transforming growth factor-beta3 in aqueous solutions

The transforming growth factors-beta (TGF-beta) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-beta isoforms TGF-beta3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-beta3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-beta3. In addition, the aggregation of TGF-beta3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-beta3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH > 3.8) the protein was not aggregated. At pH approximately 3.9, a monomer right arrow over left arrow dimer equilibrium could be detected that transformed into larger aggregates at pH pH > 9.8 with the aggregation maximum between pH 6.5 and 8. 5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-beta3 dissolved in solution and aggregated TGF-beta3. Aggregated TGF-beta3 showed a higher beta-sheet content and lower beta-turn and random coil contributions compared with monomeric TGF-beta3. Both the solution structure and the aggregate structure of TGF-beta3 were different from the crystal structure. This was in contrast to TGF-beta2, which showed very similar crystal and solution structures. Under alkaline conditions (pH pH > 11. 0 was reversible. Aggregation of TGF-beta3 was, furthermore, influenced by the presence of salt. For pH < 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, cNaCl = 164 mM) TGF-beta3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations