Unravelling the non-classical nucleation mechanism of an amyloid nanosheet through atomic force microscopy and an infrared probe technique.

Understanding the amyloid nucleation mechanism is fundamentally important for the development of diagnostics and therapeutics of amyloid-related diseases and for the design and application of amyloid-based materials. To this end, we here explore the use of atomic force microscopy (AFM) and a side-chain-based infrared (IR) probe technique to investigate the amyloid nanosheet formation mechanism of an Aß16-22 variant, KLVFXAK, where X is p-cyanophenylalanine with its side-chain cyano group being an infrared probe. Using AFM, we reveal that the formation of KLVFXAK amyloid nanosheets follows a two-step non-classical nucleation mechanism. The first step is the rapid formation of a metastable fibrillar intermediate and the second step is slow transformation to the final nanosheet. Using the side-chain-based IR probe technique, we obtain spectroscopic evidence for the proposed nucleation mechanism of the amyloid nanosheet as well as the structural details for the intermediate and amyloid nanosheet. By using the structural constraints set by the two techniques, we propose the structural models for both the fibrillar intermediate and the amyloid nanosheet. In addition, we further investigated the amyloid nanosheet formation mechanism of a similar Aß16-22 variant, KLVFXAE, and showed the impact of mutation on the amyloid nucleation mechanism. Our work also provides a nice example of how to use the combined approach of AFM and a side-chain-based IR probe technique to unravel the complex nucleation mechanism of amyloid formation.

New Insight into the Structural Nature of Diphenylalanine Nanotube through Comparison with Amyloid Assemblies.

Diphenylalanine (FF) nanotubes are a star material in the field of peptide self-assembly and have demonstrated numerous intriguing applications. Due to its resemblance to amyloid assembly, the FF nanotube is widely regarded as a simplified mimic of amyloids. Yet, whether FF nanotube truly possesses amyloid structure remains an open question. To better understand the structural nature of FF nanotube, we herein performed a comparative structural investigation between FF nanotube and typical amyloid systems by Aß1-40, Aß1-42, Aß16-22, Aß13-23, α-synuclein, and lysozyme using Fourier transform infrared spectroscopy. Through this comparative investigation, we obtained clear evidence to support that the FF nanotube does not possess a ß-sheet structure, a key structural characteristic of amyloid assembly, thus revealing the non-amyloid structural nature of the FF nanotube. At last, in light of our new finding, we further discussed the unique self-assembly behaviors of FF during nanotube formation and the implications of our work for FF nanotube related applications.

Tutorial on the instrumentation of sum frequency generation vibrational spectroscopy: Using a Ti:sapphire based system as an example.

Sum frequency generation vibrational spectroscopy (SFG-VS) is an intrinsically surface-selective vibrational spectroscopic technique based on the second-order nonlinear optical process. Since its birth in the 1980s, SFG-VS has been used to solve interfacial structure and dynamics in a variety of research fields including chemistry, physics, materials sciences, biological sciences, environmental sciences, etc. Better understanding of SFG-VS instrumentation is no doubt an essential step to master this sophisticated technique. To address this need, here we will present a Tutorial with respect to the classification, setup layout, construction, operation, and data processing about SFG-VS. We will focus on the steady state Ti:sapphire based broad bandwidth SFG-VS system and use it as an example. We hope this Tutorial is beneficial for newcomers to the SFG-VS field and for people who are interested in using SFG-VS technique in their research.

Amyloid-Based Injectable Hydrogel Derived from Hydrolyzed Hen Egg White Lysozyme.

Injectable hydrogels based on synthetic peptides have shown great promise in many biomedical applications. Yet, the high cost generally associated with synthetic peptides hinders the practical use of such peptide-based injectable hydrogel. To overcome this drawback, here, we propose to use the peptides from hydrolyzed low-cost natural protein as an economical and convenient peptide source to prepare an injectable hydrogel. We demonstrate the effectiveness of this alternative strategy using hen egg white lysozyme (HEWL) as an example. We used the peptide fragments from hydrolyzed HEWL as the gelator, and the magnesium ion as the performance enhancer to prepare the injectable hydrogel. We showed that the hydrogel is an amyloid gel as it was formed by a dense network of amyloid fibrils. We also showed that the hydrogel possesses a thixotropic property and displays a low cytotoxicity. The hydrolysis extent of HEWL was found to be a critical factor that influences the performance of the hydrogel. A fluorescence assay based on 8-anilinonaphthalene-1-sulfonic acid was proposed as a mean to precisely and conveniently control the hydrolysis extent of HEWL to enable the best injectability performance. At last, using doxorubicin as a model compound, we explored the potential of this amyloid-based hydrogel as an injectable drug carrier.

Tannic Acid-Induced Surface-Catalyzed Secondary Nucleation during the Amyloid Fibrillation of Hen Egg-White Lysozyme.

Amyloid fibrillation by hen egg white lysozyme under the influence of tannic acid was investigated by atomic force microscopy and fluorescence spectroscopy. Tannic acid was found to be able to induce the formation of amyloid fibrils with an interesting mixed morphology. Such morphology features with the existence of areas of thickening alternating with areas of normal height. This novel modulation effect of tannic acid on amyloid fibrillation was interpreted by the established surface-catalyzed secondary nucleation theory. We further performed a fluorescence quenching study to investigate the intermolecular interaction between tannic acid and lysozyme. The results support that lysozyme and tannic acid interact with each other mainly through hydrophobic interactions. We also discussed why hydrogen-bonding interaction is not a dominant factor in the interaction between tannic acid and lysozyme though tannic acid contains a significant amount of hydroxyl groups. Our work provides new insight into the effect of tannic acid, a well-known amyloid inhibitor, on amyloid fibrillation.

A structural model of the hierarchical assembly of an amyloid nanosheet by an infrared probe technique.

Rational design of amyloid-based materials requires structural insight into such materials. Here, we explore the use of a side-chain-based infrared (IR) probe technique combined with atomic force microscopy, Raman spectroscopy, UV-Vis spectroscopy, and thermogravimetric analysis coupled with mass spectrometry to elucidate the structural details of an amyloid nanosheet formed by an Aß(16-22) variant, KLVXFAK, where X is p-cyanophenylalanine with its side-chain cyano group being an IR probe. Through the structural constraints obtained with the combined tools, we are able to propose a novel structural model for the amyloid nanosheet. The nanosheet can be viewed as a stack of class 7-type steric-zipper-like amyloid structures with a unique sheared intersheet arrangement: the ß-sheets are stacked along a zippering axis with each individual ß-sheet sheared relative to its adjacent ß-sheets by two residues through the cyano-lysine intersheet hydrogen bond. With such a configuration, the side view of the nanosheet is similar to that of a stack of tilted-aligned roof tiles with each individual ß-sheet being each tile. In addition, this work provides a nice example of how to utilize the side-chain-based IR probe technique combined with other supplemental tools to build a hierarchical structural model for a complex amyloid assembly.

A Plasma Biochemical Analysis of Acute Lead Poisoning in a Rat Model by Chemometrics-Based Fourier Transform Infrared Spectroscopy: An Exploratory Study.

In this work, we explored to use chemometrics-based Fourier transform infrared (FTIR) spectroscopy to investigate the plasma biochemical changes due to acute lead poisoning (ALP) in a rat model. We first collected the FTIR spectra of the plasma samples from the rats with and without suffering from ALP. We then performed the chemometric analysis of these FTIR spectra using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). We found that the chemometrics-based FTIR spectroscopy can discriminate the rats with and without ALP. Further analysis on the PLS-DA regression coefficient revealed that the spectral changes, in particular, corresponding to the biochemical changes of proteins in the plasma may be used as potential spectral biomarkers for the diagnostics of lead poisoning. Our work demonstrates the potential of chemometrics-based FTIR spectroscopy as a promising tool for the biochemical analysis of plasma that could consequently enable an objective, convenient and non-destructive diagnostics of lead poisoning. To the best of our knowledge, this work is the first application of chemometrics-based FTIR spectroscopy in the diagnostics of lead poisoning.

Regioselective copper-catalyzed chlorination and bromination of arenes with O(2) as the oxidant.

Electron-rich aromatic C-H bonds undergo regioselective chlorination and bromination in the presence of CuX(2), LiX (X = Cl, Br) and molecular oxygen. Preliminary mechanistic insights suggest that the bromination and chlorination reactions proceed by different pathways.

Iron-catalysed propylene epoxidation by nitrous oxide: dramatic shift of allylic oxidation to epoxidation by the modification with alkali metal salts.

A dramatic shift of allylic oxidation to epoxidation has been observed during the oxidation of propylene by N(2)O when the FeO(x)/SBA-15 catalyst is modified with alkali metal salts, and the roles of alkali metal salts are to suppress the reactivity of lattice oxygen and to induce an iron coordination structure effective for epoxidation with N(2)O.