Measurements of the lengths of free fibrils obtained from solutions with 0 or 100 mM NaCl, were obtained by TEM analysis (see Fig. 4 and Section 2 for details). In the absence of salt (white columns), a wide distribution of lengths occurs with fibrils of up to ∼20 μm (image on the right in Fig. 4) and a mean value of ∼4 μm. However, in the presence of PD-1/PD-L1 tumor 100 mM NaCl (black columns), this decreases to ∼700 nm. A single amyloid fibril grows via nucleus formation and subsequent elongation [3]. Spherulite growth is believed to occur with an initial formation (via nonspecific aggregation) of a precursor species from which multiple fibrils nucleate and grow radially [26]. The

structure and composition of the precursor associated with spherulite formation is still unknown. However,

it is expected that the final number of spherulites will be equal to the number of spherulite precursors formed in solution. The data for size and number of spherulites MLN0128 clinical trial presented in Fig. 1 and Fig. 2 can be described intuitively in terms of three key factors: i) colloidal stability, ii) conformational stability, and iii) the amount of available protein which is able to participate in spherulite formation. Increases in temperature or salt concentration both have a destabilizing effect by reducing both colloidal and conformational stability and increasing the rates of aggregation. Changing the salt concentration and temperature will affect two key parameters: namely the number of precursors present in solution prior to fibril growth, and the nucleation time at which the growth of fibrils begins.

Increasing the temperature is expected to increase the number of spherulite precursors as shown in Fig. 1c (○). It is not known how the precursor arises. It could be a nucleation dependent process or a gradual coalescence and coarsening of smaller aggregates. Interestingly, light scattering measurements show a steadily increasing intensity in solution in the early stages of the process Ribonucleotide reductase (see inset in Fig. 2a), which is in agreement with previous studies [19] and [31]. Particles of a larger size scatter light more strongly, suggesting that some form of aggregation is gradually occurring well before fibril nucleation. One expects that decreases in conformational and colloidal stability would increase the rates of precursor formation. However, such factors will also affect the rate of fibril nucleation at the spherulite precursor surface. The exact number of spherulite precursors will therefore depend on the relative rates of precursor formation and the fibril nucleation time. Once nucleation occurs on the surface of a spherulite precursor the number of spherulites is expected to remain approximately constant with fibril growth (either spherulites or free fibrils) expected to be the dominant process.