On the lag phase in amyloid fibril formation.

The formation of nanoscale amyloid fibrils from normally soluble peptides and proteins is a common form of self-assembly phenomenon that has fundamental connections with biological functions and human diseases. The kinetics of this process has been widely studied and exhibits on a macroscopic level three characteristic stages: a lag phase, a growth phase and a final plateau regime. The question of which molecular events take place during each one of these phases has been a central element in the quest for a mechanism of amyloid formation. In this review, we discuss the nature and molecular origin of the lag-phase in amyloid formation by making use of tools and concepts from physical chemistry, in particular from chemical reaction kinetics. We discuss how, in macroscopic samples, it has become apparent that the lag-phase is not a waiting time for nuclei to form. Rather, multiple parallel processes exist and typically millions of primary nuclei form during the lag phase from monomers in solution. Thus, the lag-time represents a time that is required for the nuclei that are formed early on in the reaction to grow and proliferate in order to reach an aggregate concentration that is readily detected in bulk assays. In many cases, this proliferation takes place through secondary nucleation, where fibrils may present a catalytic surface for the formation of new aggregates. Fibrils may also break (fragmentation) and thereby provide new ends for elongation. Thus, at least two - primary nucleation and elongation - and in many systems at least four - primary nucleation, elongation, secondary nucleation and fragmentation - microscopic processes occur during the lag phase. Moreover, these same processes occur during all three phases of the macroscopic aggregation process, albeit at different rates as governed by rate constants and by the concentration of reacting species at each point in time.This work was supported by the Swedish Research Council (SL) and its Linneus Centre Organizing Molecular Matter for CD spectrometer, plate readers (SL), the Alzheimer Foundation Sweden (SL), the Frances and Augustus Newman Foundation (TPJK), the BBSRC (TPJK), and the Marie Curie fellowship scheme for career development (PA).This is the final version of the article. It first appeared from RSC via http://dx.doi.org/10.1039/C4CP05563

Universality of filamentous aggregation phenomena.

We use perturbative renormalization group theory to study the kinetics of protein aggregation phenomena in a unified manner across multiple timescales. Using this approach, we find that, irrespective of the specific molecular details or experimental conditions, filamentous assembly systems display universal behavior in time. Moreover, we show that the universality classes for protein aggregation correspond to simple autocatalytic processes and that the diversity of behavior in these systems is determined solely by the reaction order for secondary nucleation with respect to the protein concentration, which labels all possible universality classes. We validate these predictions on experimental data for the aggregation of several different proteins at several different initial concentrations, which by appropriate coordinate transformations we are able to collapse onto universal kinetic growth curves. These results establish the power of the perturbative renormalization group in distilling the ultimately simple temporal behavior of complex protein aggregation systems, creating the possibility to study the kinetics of general self-assembly phenomena in a unified fashion

Mimicking Hierarchical Complexity of the Osteochondral Interface Using Electrospun Silk-Bioactive Glass Composites

The anatomical complexity and slow regeneration capacity of hyaline cartilage at the osteochondral interface pose a great challenge in the repair of osteochondral defects (OCD). In this study, we utilized the processing feasibility offered by the sol derived 70S bioactive glass and silk fibroin (mulberry Bombyx mori and endemic Indian non-mulberry Antheraea assama), in fabricating a well-integrated, biomimetic scaffolding matrix with a coherent interface. Differences in surface properties such as wettability and amorphousness between the two silk groups resulted in profound variations in cell attachment and extracellular matrix protein deposition. Mechanical assessment showed that the biphasic composites exhibited both an elastic region pertinent for cartilage tissue and a stiff compression resistant region simulating the bone phase. In vitro biological studies revealed that the biphasic mats presented spatial confinement for the growth and maturation of both osteoblasts and chondrocytes, marked by increased alkaline phosphatase (ALP) activity, osteopontin (OPN), sulfated glycosaminoglycan (sGAG) and collagen secretion in the cocultured mats. The non-mulberry silk based biphasic composite mats performed better than their mulberry counterpart, as evidenced by enhanced expression levels of key cartilage and bone specific marker genes. Therefore, the developed biphasic scaffold show great promise for improving the current clinical strategies for osteochondral tissue repair

Microstructural characterisation of metallic shot peened and laser shock peened Ti–6Al–4V

A detailed analysis has been conducted of Ti–6Al–4V processed by metallic shot peening and laser shock peening. Analysis by incremental hole drilling, electron backscattered diffraction microscopy, transmission electron microscopy and transmission Kikuchi diffraction microscopy is evaluated and discussed. The results of this analysis highlight the very different dislocation structures in surfaces processed by these two techniques. Transmission Kikuchi diffraction also has been used to evaluate sub-grains generated by laser shock peening. A notable feature of material processed by laser shock peening is the almost complete absence of deformation twinning, contrasting with the frequent observation of extensive deformation twinning observed in the material processed by metallic shot peening.This work was supported by the Rolls-Royce plc/EPSRC strategic partnership under EP/H022309/1

Crucial role of nonspecific interactions in amyloid nucleation.

Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiologically high peptide concentrations. At low peptide concentrations, which mimic the physiologically relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet-rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only observed in rare fluctuations, which is why such aggregates might be hard to capture experimentally. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the critical nucleus size increases with increases in both concentration and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.This is the accepted manuscript. The final published version is available from PNAS at http://www.pnas.org/content/111/50/17869.abstract

Atomic force microscopy for single molecule characterisation of protein aggregation.

The development of atomic force microscopy (AFM) has opened up a wide range of novel opportunities in nanoscience and new modalities of observation in complex biological systems. AFM imaging has been widely employed to resolve the complex and heterogeneous conformational states involved in protein aggregation at the single molecule scale and shed light onto the molecular basis of a variety of human pathologies, including neurodegenerative disorders. The study of individual macromolecules at nanoscale, however, remains challenging, especially when fully quantitative information is required. In this review, we first discuss the principles of AFM with a special emphasis on the fundamental factors defining its sensitivity and accuracy. We then review the fundamental parameters and approaches to work at the limit of AFM resolution in order to perform single molecule statistical analysis of biomolecules and nanoscale protein aggregates. This single molecule statistical approach has proved to be powerful to unravel the molecular and hierarchical assembly of the misfolded species present transiently during protein aggregation, to visualise their dynamics at the nanoscale, as well to study the structural properties of amyloid-inspired functional nanomaterials

Ultrafast Photoinduced Dynamics of 1,3-Cyclohexadiene Using XMS-CASPT2 Surface Hopping

A full-dimensional simulation of the photodissociation of 1,3-cyclohexadiene in the manifold of three electronic states was performed via nonadiabatic surface hopping dynamics using extended multistate complete active space second-order perturbation (XMS-CASPT2) electronic structure theory with fully analytic nonadiabatic couplings. With the 47 ± 8% product quantum yield calculated from the 136 trajectories, generally 400 fs-long, and an estimated excited lifetime of 89 ± 9 fs, our calculations provide a detailed description of the nonadiabatic deactivation mechanism, showing the existence of an extended conical intersection seam along the reaction coordinate. The nature of the preferred reaction pathways on the ground state is discussed and extensive comparison to the previously published full dimensional dynamics calculations is provided