Associated with neurodegenerative disorders such as Alzheimer, Parkinson, or prion diseases, the conversion of soluble proteins into amyloid fibrils remains poorly understood. Extensive “in vitro” measurements of protein aggregation kinetics have been reported, but no consensus mechanism has emerged until now. This contribution aims at overcoming this gap by proposing a theoretically consistent crystallization-like model (CLM) that is able to describe the classic types of amyloid fibrillization kinetics identified in our literature survey. Amyloid conversion represented as a function of time is shown to follow different curve shapes, ranging from sigmoidal to hyperbolic, according to the relative importance of the nucleation and growth steps. Using the CLM, apparently unrelated data are deconvoluted into generic mechanistic information integrating the combined influence of seeding, nucleation, growth, and fibril breakage events. It is notable that this complex assembly of interdependent events is ultimately reduced to a mathematically simple model, whose two parameters can be determined by little more than visual inspection. The good fitting results obtained for all cases confirm the CLM as a good approximation to the generalized underlying principle governing amyloid fibrillization. A perspective is presented on possible applications of the CLM during the development of new targets for amyloid disease therapeutics.

Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. While the pathogenic role of these fibrils is not totally established, increasing evidences suggest off-pathway aggregation (OPA) as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step ahead towards the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of OPA relatively to amyloid fibrillization; these include non-linear semi-log plots of amyloid progress curves, highly variable endpoint signals and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of Dynamic Light Scattering (DLS), Circular Dichroism (CD) and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery.

The aggregation of amyloid-β peptide (Aβ) has been linked to the formation of neuritic plaques, which are pathological hallmarks of Alzheimer's disease. We synthesized peptides containing fluorinated amino acids and studied their effect on the Aβ aggregation. The peptides were based on the sequence LVFFD, in which valine was substituted by either 4,4,4-trifluorovaline or 4-fluoroproline, or the phenylalanine at position 3 was replaced by 3,4,5-trifluorophenylalanine. Our results demonstrate that fluorination of the hydrophobic residue valine or phenylalanine is effective in preventing the Aβ aggregation. This study opens up the possibility of using new sequences based on fluorinated amino acids to inhibit the amyloid-fibril formation.

The aim of this work was to investigate the possibility of producing microparticles containing β-galactosidase, using a modified chitosan (water soluble) as encapsulating agent. β-galactosidase microparticles were prepared by a spray-drying method and were characterized in terms of particle size, surface morphology, zeta potential, and stability over a storage period of six months. Microparticles were also analyzed by FTIR spectroscopy and thermogravimetry techniques. Structural analysis of the surface of the particles was performed by scanning electron microscopy (SEM). SEM results show that the obtained microparticles have an average diameter smaller than 3.5 µm and a regular shape. The β-galactosidase activity decreases when microencapsulated. The parameters Km and Vmax of the Michaelis-Menten equation were calculated for the different experimental conditions. The optimal pH ranges from 6.4 to 7.2, approximately, depending on the enzyme concentration in the microcapsule. After six months of storage, the enzyme activity presents a small decrease, although no significant differences in the appearance, color, and particle size distribution were identified.