Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated. However, it is unclear whether functional aggregation...

Reactive Crystallization Kinetics of K2SO4 from Picromerite-Based MgSO4 and KCl

In this work, the kinetic parameters, the degrees of initial supersaturation (S0) and the profiles of supersaturation (S) were determined for the reactive crystallization of K2SO4 from picromerite (K2SO4.MgSO4.6H2O) and KCl. Different reaction temperatures between 5 and 45 °C were considered, and several process analytical techniques were applied. Along with the solution temperature, the crystal chord length distribution (CLD) was continuously followed by an FBRM probe, images of nucleation and growth events as well as the crystal morphology were captured, and the absorbance of the solution was measured via ATR-FTIR spectroscopy. In addition, the ion concentrations were analyzed. It was found that S0 is inversely proportional to the reactive crystallization temperature in the K+, Mg2+/Cl−, SO42−//H2O system at 25 °C, where S0 promotes nucleation and crystal growth of K2SO4 leading to a bimodal CLD. The CLD was converted to square-weighted chord lengths for each S0 to determine the secondary nucleation rate (B), crystal growth rate (G), and suspension density (MT). By correlation, from primary nucleation rate (Bb) and G with S0, the empirical parameters b = 3.61 and g = 4.61 were obtained as the order of primary nucleation and growth, respectively. B versus G and MT were correlated to the reaction temperature providing the rate constants of B and respective activation energy, E = 69.83 kJ∙mol−1. Finally, a general Equation was derived that describes B with parameters KR = 13,810.8, i = 0.75 and j = 0.71. The K2SO4 crystals produced were of high purity, containing maximal 0.51 wt% Mg impurity, and were received with ~73% yield at 5 °C.

A Chemical Chaperone That Prevents Insulin Fibrillation

Insulin, a peptide hormone, is susceptible to amyloid formation upon exposure to aberrant physiological conditions, result-ing in a loss of its bioactivity. For mitigating insulin aggregation, we report a molecule called PAD-S, which completely inhibit-ed insulin fibril formation, and preserved insulin in its soluble form. Circular Dichroism spectroscopy showed that PAD-S was able to maintain the native structure of insulin, thus acting as a chemical chaperone. Seeded aggregation kinetics suggest that PAD-S inhibited primary nucleation events during aggregation. This is consistent with molecular docking results which suggest that PAD-S binds strongly to native insulin monomers/dimers. Through a competitive binding experiment with ‘LVEALYL’ peptide, we conclude that PAD-S likely binds to the amyloid prone B11-B17 residues of insulin thereby prevent-ing its aggregation. PAD-S was also effective in disaggregating preformed insulin fibrils to non-toxic species. PAD-S treated insulin was functional as indicated by its ability to phosphorylate Akt. PAD-S was also highly effective in preventing the ag-gregation of insulin biosimilars. The low cellular cytotoxicity of PAD-S, and amelioration of aggregation-induced toxicity by PAD-S treated insulin further highlights its potential as an effective chemical chaperone.

Dependence of Crystallization Behavior of Interacting Telechelic Poly(butylene succinate) Oligomer on Molecular Weight

A large spherulite structure deteriorates the mechanical properties of crystalline polymers, and therefore various methods have been explored to increase primary nucleation density. Recently, chain-end modification has been proposed as an effective approach for regulating polymer crystal nucleation. However, the relevant nucleation mechanism still requires investigation. Therefore, in this work, 2-ureido-4[1H]-pyrimidinone (UPy) units, which can form stacks via quadruple hydrogen bonds with each other, are introduced as end groups for the preparation of interacting telechelic poly(butylene succinate) (PBS-UPy) oligomers with different molecular weights (Mns). The crystallization, especially the nucleation behavior of PBS-UPy, is studied in detail by comparing with the corresponding pre-polymer, the hydroxyl-terminal PBS (PBS-OH). The thermal properties of PBS-UPy exhibit similar Mn-dependent tendency to those of PBS-OH, but with weaker total crystallization rate. The spherulite growth rate is significantly reduced, whereas the primary nucleation density is highly promoted, after introducing UPy groups. Further investigation reveals that the mechanism of UPy stacks’ influence on nucleation ability changes from inhibition to promotion with respect to Mn. Even under an inhibition of nucleation ability, the final nucleation density is obviously increased because of a significant decline of the growth rate. In addition, the change in the impact of UPy stacks on nucleation ability is speculated to originate from the memory expression feasibility of ordered conformation in the melt during crystallization.

Fundamental studies on shear-induced nucleation and beta-phase formation in the isotactic polypropylene—effect of the temperature

The complex and incompletely understood phenomenon of shear-induced crystallization of polymers may be nowadays analysed via the in situ POM-shear stage methodology. In this research, the two main issues were investigated with the use of the Linkam CCS450 shear stage connected with POM microscope. It was found that the secondary nucleation in the tree well-known temperature regimes plays the greater role in the overall crystallization kinetics than the shear induced primary nucleation. Furthermore, it was found that the tendency towards β-phase formation in shear conditions is dependent on the temperature value during shear treatment. It may be concluded that the temperature is the key parameter in the primary and secondary nucleation process and beta-phase formation in the iPP melts.

Spontaneous nucleation and fast aggregate-dependent proliferation of α-synuclein aggregates within liquid condensates at physiological pH

It is well-established that α-synuclein aggregation may proceed through an initial lipid-dependent aggregate formation and, if at acidic pH, a subsequent aggregate-dependent proliferation. It has also been recently reported that the aggregation of α-synuclein may also take place through an alternative pathway, which takes place within dense liquid condensates produced through liquid-liquid phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we developed a fluorescence-based assay to perform a kinetic analysis of the aggregation process of α-synuclein within liquid condensates, and applied it to determine the corresponding mechanism of aggregation. Our analysis shows that at pH 7.4 the aggregation process of α-synuclein within dense condensates starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Taken together, these results reveal a highly efficient pathway for the appearance and proliferation of α-synuclein aggregates at physiological pH.

Hot Deformation Behavior and Microstructure Evolution of Fe–5Mn–3Al–0.1C High-Strength Lightweight Steel for Automobiles

The hot deformation behavior of a newly designed Fe–5Mn–3Al–0.1C (wt.%) medium manganese steel was investigated using hot compression tests in the temperature range of 900 to 1150 °C, at constant strain rates of 0.1, 1, 2.5, 5, 10, and 20 s−1. A detailed analysis of the hot deformation parameters, focusing on the flow behavior, hot processing map, dynamic recrystallization (DRX) critical stress, and nucleation mechanism, was undertaken to understand the hot rolling process of the newly designed steel. The flow behavior is sensitive to deformation parameters, and the Zener–Hollomon parameter was coupled with the temperature and strain rate. Three-dimensional processing maps were developed considering the effect of strain and were used to determine safe and unsafe deformation conditions in association with the microstructural evolution. In the deformation condition, the microstructure of the steel consisted of δ-ferrite and austenite; in addition, there was a formation of DRX grains within the δ-ferrite grains and austenite grains during the hot compression test. The microstructure evolution and two types of DRX nucleation mechanisms were identified; it was observed that discontinuous dynamic recrystallization (DDRX) is the primary nucleation mechanism of austenite, while continuous dynamic recrystallization (CDRX) is the primary nucleation mechanism of δ-ferrite. The steel possesses unfavorable toughness at the deformation temperature of 900 °C, which is mainly due to the presence of coarse κ-carbides along grain boundaries, as well as the lower strengthening effect of grain boundaries. This study identified a relatively ideal hot processing region for the steel. Further exploration of hot roll tests will follow in the future.

Secondary Nucleation and the Conservation of Structural Characteristics of Amyloid Fibril Strains

Amyloid fibrils are ordered protein aggregates and a hallmark of many severe neurodegenerative diseases. Amyloid fibrils form through primary nucleation from monomeric protein, grow through monomer addition and proliferate through fragmentation or through the nucleation of new fibrils on the surface of existing fibrils (secondary nucleation). It is currently still unclear how amyloid fibrils initially form in the brain of affected individuals and how they are amplified. A given amyloid protein can sometimes form fibrils of different structure under different solution conditions in vitro, but often fibrils found in patients are highly homogeneous. These findings suggest that the processes that amplify amyloid fibrils in vivo can in some cases preserve the structural characteristics of the initial seed fibrils. It has been known for many years that fibril growth by monomer addition maintains the structure of the seed fibril, as the latter acts as a template that imposes its fold on the newly added monomer. However, for fibrils that are formed through secondary nucleation it was, until recently, not clear whether the structure of the seed fibril is preserved. Here we review the experimental evidence on this question that has emerged over the last years. The overall picture is that the fibril strain that forms through secondary nucleation is mostly defined by the solution conditions and intrinsic structural preferences, and not by the seed fibril strain.

Tau AD fragment aggregates proliferate through autocatalytic secondary nucleation

The self-assembly of the protein tau into neurofibrillary tangles is one of the hallmarks of Alzheimer’s disease and related tauopathies. Still, the molecular mechanism of tau aggregation is largely unknown. This problem may be addressed by systematically obtaining reproducible in vitro kinetic measurements under quiescent conditions in the absence of triggering substances. Here, we implement this strategy by developing protocols for obtaining an ultra-pure tau fragment (residues 304-380 of tau441, tau AD fragment) and for performing spontaneous aggregation assays with reproducible kinetics under quiescent conditions. We are thus able to identify the mechanism of fibril formation of the tau AD fragment at physiological pH using fluorescence spectroscopy and mass spectrometry. We find that primary nucleation is slow, and that secondary processes dominate the aggregation process once the initial aggregates are formed. Moreover, our results further show that secondary nucleation of monomers on fibril surfaces dominate over fragmentation of fibrils. Using separate isotopes in monomers and fibrils, through mass spectroscopy measurements, we verify the isotope composition of the intermediate oligomeric species, which reveals that these small aggregates are generated from monomer through secondary nucleation. Our results provide a framework for understanding the processes leading to tau aggregation in disease, and for selecting possible tau forms as targets in the development of therapeutic interventions in Alzheimer’s disease.