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Number of incurable diseases associated with neurodegenerative syndromes like Alzheimer's, and Parkinson's, are owing to protein aggregation which leads to amyloid fibril formation. In vitro, such fibrillation depends on concentration, temperature, pH, ionic strength, organic solvents, agitation, and stirring, which play a crucial role in understanding the mechanism of fibrillation as well as to identify potential inhibitors for fibrillation. Although these parameters were considered, the precise repeatability of amyloid fibrillation kinetics between laboratories remains challenging. Herein, we have demonstrated that another important parameter such as diameter of the vial in which protein undergoes fibrillation play a key role in the amyloid fibrillation. The various biophysical analyses indicated that the lag time, elongation, and the amount of fibril formation was significantly reduced with decreasing the diameter of the reaction vial from 24 to 15mm. Further, the minimum amount of protein required for fibrillation was determined by the diameter of the vial. The observed fibrillation difference in different vials is most likely due to the variation in the interface area between hydrophobic (air) and hydrophilic (water) surfaces as the diameter of the vial changes. The current results have a major role in the design of drug screening assays for amyloid inhibition., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Proteins under stressful conditions can lead to the formation of an ordered self-assembled structure, referred to as amyloid fibrils, to which many neurodegenerative diseases such as Type II diabetes, Alzheimer's, Parkinson's, Huntington's, etc., are attributed. Inhibition of amyloid fibril formation using natural products is one of the main therapeutic strategies to prevent the progression of these diseases. Polyphenols are the mostly consumed as antioxidants in a human nutrition. Herein, we have studied the effect of a simple polyphenol, gallic acid (GA), one of the main components in plant tissues, especially in tea leaves, on the insulin amyloid fibril formation. Different biophysical characterizations such as turbidity, atomic force microscopy (AFM), Thioflavin T (ThT) assays, circular dichroism, and Fourier transform-infrared spectroscopy have been used to analyze the inhibition of amyloid fibril formation. The occurrence of fibrils in an AFM image and ThT fluorescence enhancement confirms the formation of insulin amyloid fibrils when incubated under acidic pH 2 at 65 °C. In the presence of GA, absence of fibrils in AFM image and no change in the intensity of ThT fluorescence confirms the inhibition of insulin amyloid fibrils by GA. Spectroscopic results reveal that GA inhibits the conformational transition of α-helix → β-sheet, which is generally induced during the insulin fibril formation. It was found that the inhibitory effect of GA is concentration dependent and non-linear. Based on the observed results, we propose that GA interacts with native insulin, preventing nuclei formation, which is essential for fibril growth, thereby inhibiting the amyloid fibril formation. The present results thus demonstrate that GA can effectively inhibit insulin amyloid fibril formation in vitro., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)