The aggregation of amyloidogenic proteins is infamous for being highly chaotic, with small variations in conditions sometimes leading to large changes in aggregation rates. Using the amyloidogenic protein IAPP (islet amyloid polypeptide protein, also known as amylin) as an example, we show that a part of this phenomenon may be related to the formation of micellelike oligomers at specific critical concentrations and temperatures. We show that pyrene fluorescence can sensitively detect micellelike oligomer formation by IAPP and discriminate between micellelike oligomers from fibers and monomers, making pyrene one of the few chemical probes specific to a prefibrillar oligomer. We further show that oligomers of this type reversibly form at critical concentrations in the low micromolar range and at specific critical temperatures. Micellelike oligomer formation has several consequences for amyloid formation by IAPP. First, the kinetics of fiber formation increase substantially as the critical concentration is approached but are nearly independent of concentration below it, suggesting a direct role for the oligomers in fiber formation. Second, the critical concentration is strongly correlated with the propensity to form amyloid: higher critical concentrations are observed for both IAPP variants with lower amyloidogenicity and for native IAPP at acidic pH in which aggregation is greatly slowed. Furthermore, using the DEST NMR technique, we show that the pathway of amyloid formation switches as the critical point is approached, with self-interactions primarily near the N-terminus below the critical temperature and near the central region above the critical temperature, reconciling two apparently conflicting views of the initiation of IAPP aggregation.

Our knowledge of the molecular events underlying type 2 diabetes mellitus-a protein conformational disease characterized by the aggregation of islet amyloid polypeptide (IAPP) in pancreatic β cells-is limited. However, amyloid-mediated membrane damage is known to play a key role in IAPP cytotoxicity, and therefore the effects of lipid composition on modulating IAPP-membrane interactions have been the focus of intense research. In particular, membrane cholesterol content varies with aging and consequently with adverse environmental factors such as diet and lifestyle, but its role in the development of the disease is controversial. In this study, we employ a combination of experimental techniques and in silico molecular simulations to shed light on the role of cholesterol in IAPP aggregation and the related membrane disruption. We show that if anionic POPC/POPS vesicles are used as model membranes, cholesterol has a negligible effect on the kinetics of IAPP fibril growth on the surface of the bilayer. In addition, cholesterol inhibits membrane damage by amyloid-induced poration on membranes, but enhances leakage through fiber growth on the membrane surface. Conversely, if 1:2 DOPC/DPPC raft-like model membranes are used, cholesterol accelerates fiber growth. Next, it enhances pore formation and suppresses fiber growth on the membrane surface, leading to leakage. Our results highlight a twofold effect of cholesterol on the amyloidogenicity of IAPP and help explain its debated role in type 2 diabetes mellitus.

Laser synthesis emerges as a suitable technique to produce ligand-free nanoparticles, alloys and functionalized nanomaterials for catalysis, imaging, biomedicine, energy and environmental applications. In the last decade, laser ablation and nanoparticle generation in liquids has proven to be a unique and efficient technique to generate, excite, fragment and conjugate a large variety of nanostructures in a scalable and clean way. In this work, we give an overview on the fundamentals of pulsed laser synthesis of nanocolloids and new information about its scalability towards selected applications. Biomedicine, catalysis and sensing are the application areas mainly discussed in this review, highlighting advantages of laser-synthesized nanoparticles for these types of applications and, once partially resolved, the limitations to the technique for large-scale applications.

The self-assembling of the amyloid β (Aβ) peptide into neurotoxic aggregates is considered a central event in the pathogenesis of Alzheimer's disease (AD). Based on the "amyloid hypothesis", many efforts have been devoted to designing molecules able to halt disease progression by inhibiting Aβ self-assembly. Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS), and computational (all-atom molecular dynamics) techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aβ aggregation. Despite TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that silybin B interacts mainly with the C-terminal hydrophobic segment MVGGVV of Aβ40. Consequently to silybin B binding, the peptide conformation remains predominantly unstructured along all the simulations. By contrast, silybin A interacts preferentially with the segments LVFF and NKGAII of Aβ40 which shows a high tendency to form bend, turn, and β-sheet conformation in and around these two domains. Both 2,3-dehydrosilybin enantiomers bind preferentially the segment LVFF but lead to the formation of different small-sized, ThT-positive Aβ aggregates. Finally, in vivo studies in a transgenic Caenorhabditis elegans strain expressing human Aβ indicated that silybin B is the most effective of the four compounds in counteracting Aβ proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of silybins and points to silybin B as a promising lead compound for further development in anti-AD therapeutics.

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