of 80 nm and a length in the micrometer range. It is possible that these thick fibrils are formed by the inter-twisting of protofilaments [12]. After incubation for 3 d, there were no obvious transitions in the cross-sectional dimensions of these structures (Fig. 1). The proto-filaments developed into a more extended form with a width of 50 nm and a height of 2 nm. The filaments inter-connected with each other into network-like structures. The co-localization of protofilaments (2 nm in height) and mature fibrils (5 nm in height) supports our supposition that the fibrillar species are initiated from protofilaments. To identify the origin of massive aggregates, S100A9 was titrated with various concentrations of Ab1–40 and incubated at 370C for 3 d (Fig. 1). As the Ab1–40 concentration was reduced, the quantity of aggregates (fibrils and oligomers) decreased dramatically. Using AFM cross-section measurements, we found that these fibrillar structures shared comparable dimensions (2 nm in height and 30 nm in width). Short proto-filaments carrying a similar cross-sectional dimension were observed for concentrations of Ab1–40 as low as 0.02 mM. This similarity suggests that these fibrillar aggregates originated from Ab1–40. In conclusion, compared with the amyloid development in Ab1–40 control samples (Fig. S1: It took 3 d for Ab1–40 to develop small quantities of amyloid fibrils with a height of 2 nm), the presence of S100A9 promotes the rate of formation and the quantity of Ab1–40 amyloid species produced. Kinetics of amyloid formation The kinetics of S100A9/Ab1–40 amyloid formation was monitored using the ThT-binding assay (Fig. 2). The specific interaction of ThT with cross-b-sheet-containing amyloids, leads to an increase in fluorescence emission. The time dependence of amyloid formation depended primarily on the concentrations of Ab1–40. In contrast to the Ab1–40 (0.2 mM) control samples, a sharp increase in ThT intensity was observed with the addition of S100A9 (0.02 mM). After 2 d to 3 d of incubation, the ThT Inflammation and Alzheimer’s Disease (AD) intensity reached its maximum value. A plateau was attained following a decline in the ThT intensity after 7 d to 8 d of incubation. It is possible that the decline in the relative ThT value was induced by the formation of massive aggregates, which is consistent with our AFM observations (Fig. 1). When the concentration of Ab was decreased to 0.1 mM, the relative ThT intensity dropped from 2.0 to 1.6 in spite of the increasing S100A9 concentration (from 0.02 mM to 0.2 mM). The variation in amyloid formation kinetics is consistent with the hypothesis that amyloid formation is largely induced by Ab1–40 rather than by S100A9. Transitions in S100A9/Ab1–40 secondary structures The transition in secondary structures during the development of S100A9/Ab1–40 aggregates was monitored using far UV circular dichorism (CD). The CD spectrum of the control sample containing only Ab1–40 (0.2 mM) was dominated by random coil structure and characterized by a pronounced negative peak centered at the wavelength below 200 nm after 1 d of incubation at 370C (Fig. 2) [13]. With the addition of trace amount of S100A9 (0.02 mM) under the same conditions, spectra typical of b-sheet structures (centered at 217 nm) were observed. As there was no obvious secondary-structure transition for the samples containing only S100A9 or Ab1–40, we conclude that this evolution is caused by the interaction between these two proteins. To explore the effect of Ab peptide on the secondary structure of S100A9, S100A9 protein was incubated with various concen- trations of Ab1–40 for 3 d and 7 d. The CD spectra were characterized by two negative peaks centered at 208 nm and 222 nm (Fig. S2). There was little change in the spectra following the addition of Ab1–40 and incubation at 370C for 3 d and 7 d. The occurrence of S100A9 amyloid formation was excluded by a comparison with S100A9 samples exposed to extreme conditions (pH 3.0 and continuous shaking at 800 rpm). Under these conditions, S100A9 lost its native structure (a -helix) and took the form of b-sheet after 3d of incubation (Fig. S4). This transition in the secondary structures of S100A9 is consistent with the downward shift of monomer and dimer band positions in SDS PAGE. The transition in the secondary structures was exaggerated, when the spectra of S100A9 alone were subtracted from the spectra of the mixtures (Fig. 3). Following incubation for 3 d, the difference spectra of the mixtures showed a negative peak with a minimum below 200nm, which is thought to represent random- coil structure. It is worth noting that the absolute values of the Figure 1. AFM images of freshly dissolved Ab1–40 peptide (0.2 mM) and S100A9 protein (0.02 mM) mixture, incubated at pH 7.4 and 370C for (a) 1 d; and (b) 3 d. S100A9 (0.2 mM) with (c) Ab1– 40 (0.1 mM) ; (d) Ab1–40 (0.02 mM) incubated at pH 7.4 and 370C for 3 d. The scale bars denote 1000 nm for Fig. (a), 1200 nm for (b), 600 nm for (c) and 600 nm for (d). doi:10.1371/journal.pone.0032953.g001 Figure 2. ThT and CD spectra of S100A9/Ab1–40 (0.2 mM) complex. (a) Kinetics of S100A9/Ab1–40 mixture amyloid formation at pH 7.4 and 370C monitored by the thioflavin-T assay. Rhombuses correspond to 0.2 mM Ab1–40. Squares correspond to 0.2 mM S100A9 and 0.1 mM Ab1–40. Circles correspond to 0.02 mM S100A9 and 0.2 mM Ab1–40 (b) CD spectra of Ab1–40 peptide (0.2 mM), S100A9 (0.02 mM) and their mixture, incubated at pH 7.4 and 370C for 1 d. doi:10.1371/journal.pone.0032953.g002 PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32953