Figure 1.
Imaging of Aβ42 aggregation using a QDAβ nanoprobe.
(A) QDAβ was prepared by crosslinking CysAβ40 and amino (PEG) Qdot655 according to our recent study [12] (left). QDAβ coaggregated with unlabeled Aβ42, and the Aβ42 fibrils that formed could be visualized under fluorescence microscopy (right). (B) 30 nM QDAβ and 30 µM unlabeled Aβ42 was incubated at 37 °C for 24 h in a 1536-well plate, and was observed using an inverted fluorescence microscope using a 4x objective. Left and right panels show before and after incubation, respectively. (C) Magnified image of the aggregates observed using a 10× objective.
Figure 2.
Effect of EtOH on Aβ42 aggregation.
30 nM QDAβ and 30 µM Aβ42 were mixed in 1xPBS, 3% DMSO containing 0, 2.5, 5, 10, 20, or 40% EtOH, each sample was incubated at 37 °C for 24 h in a 1536-well plate. The wells were observed using an inverted fluorescence microscope using a 4x objective. The images show 200 × 200 pixels in the center of each well.
Figure 3.
Correlation between Aβ aggregation and variations of fluorescence intensity.
(A) Magnified images of center region (100 × 100 pixel) in fluorescence micrographs of QDAβ- Aβ42 coaggregates before (left) and after (right) incubation (Figure 1B). (B) Schematic illustrations of the distribution of QDAβ (red) and Aβ42 (gray) molecules before (left) and after (right) incubation of samples. QDAβ molecules are diffused in the sample solution before incubation (left), and QDAβ molecules are inserted in Aβ42 fibrils after incubation (right). (C) The histograms of fluorescence intensities of 10,000 pixels (100 × 100 pixel) before (left) and after (right) incubation of samples.
Figure 4.
Concentration-dependent Aβ aggregation.
(A) Various concentrations of Aβ42 and 30 nM QDAβ were incubated in a 1536-well plate at 37 °C for 24 h. Each well was observed using an inverted fluorescence microscope using a 4x objective. (B) Variations of fluorescence intensities of 10,000 pixels (100 × 100 pixel) in the center region of micrographs were estimated as SD values, the mean values were plotted against the concentrations of added Aβ42. A linear equation and R2 in B were determined using the data of less than 30 µM of Aβ concentrations. Error bars represent ±SDs of the mean values of fluorescence intensities (n=3 separate experiments).
Figure 5.
Time-dependent Aβ aggregation.
(A) 30 nM QDAβ and 30 µM Aβ42 were incubated in a 1536-well plate at 37 °C, and observed over time by an inverted fluorescence microscope using a 4x objective. All images show the same field of a well. (B) Variations of fluorescence intensities of 10,000 pixels (100 × 100 pixel) in the center region of micrographs were estimated as SD values, the mean values were plotted against incubation time periods. Error bars represent ±SDs of the mean values of fluorescence intensities (n=3 separate experiments).
Figure 6.
A flow diagram of the microliter-scale high-throughput screening system.
Various concentrations of known inhibitors or spice extracts were incubated with 30 µM Aβ42 and 30 nM QDAβ in a 1536-well plate, and incubated to induce the aggregation of Aβ42. Aggregates of Aβ42 and QDAβ were imaged by fluorescence microscopy, and EC50 of the inhibitory activities were estimated from the fluorescence micrograph data A more detailed method is mentioned in the experimental section.
Figure 7.
Estimation of EC50 by the microliter-scale high-throughput screening system.
(A) A schematic illustration of a microliter-scale high-throughput screening system for three samples. (B) The concept of estimation of EC50 values from inhibition curves that are a plotted percentage of SD versus concentrations of inhibitors. The EC50 of sample A is higher than that of sample C, and sample B did not inhibit Aβ aggregation. (C and D) Estimations of EC50 of well-known inhibitors, curcumin (Cur), rosmarinic acid (RA), tannic acid (TA), and myricetin (Myr). 30 nM QDAβ and 30 µM Aβ42 was incubated with various concentrations of the four inhibitors at 37 °C for 24 h (C). The SD values from the fluorescence images plotted against several concentrations of inhibitors (D). Error bars represent ±SDs of the mean values from fluorescence intensities (n=3 separate experiments). EC50 values of Cur, RA, TA, and Myr were 31 ± 16, 11 ± 2, 1.8 ± 1.5, and 1.0 ± 0.3 µM, respectively.
Figure 8.
Estimation of EC50 values of EtOH extracts from 52 spices using the microliter-scale high-throughput screening system.
Screening was applied to examine the EtOH extracts of dried spices. The ‘black’, ‘gray’, and ‘white’ cells indicate ‘not inhibited (SD ≥ 80%)’, ‘partially inhibited (80% > SD > 20%)’, and ‘completely inhibited (20% ≥ SD)’ wells, respectively. The percentage of SD was defined as SD values before and after incubation of control samples (0% and 100%, respectively). EC50 values were estimated from dose-dependent inhibition curves (n=3 separate experiments). Spices were aligned using the Angiosperm Phylogeny Group classification (APG III) [25].
Figure 9.
Isolation and identification of active compound from EtOH extract of summer savory.
(A) A flow diagram of isolation steps. (B) Inhibition curves of isolated RA from summer savory (squares) and standard RA (triangles) were determined by the microliter-scale high-throughput screening (MHS) system (B, top) and the ThT assay (B, bottom). Vertical axes of the MHS system and the ThT assay are the percentage of average SD values and the percentage of average fluorescence intensity (FI) values, respectively. The EC50 values of isolated RA and standard RA determined by the MHS system were 9.6 ± 0.1 and 11 ± 2 µM, respectively. In contrast to that, the EC50 values of isolated RA and standard RA determined by ThT assay were 8.6 ± 0.8 and 6.3 ± 1.5 µM, respectively. Error bars represent ±SDs (n=3 separate experiments).