Evaluating Nuclei Concentration in Amyloid Fibrillation Reactions Using Back-Calculation Approach

Background: In spite of our extensive knowledge of the more than 20 proteins associated with different amyloid diseases, we do not know how amyloid toxicity occurs or how to block its action. Recent contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. An estimate of their temporal concentration may broaden understanding of the amyloid aggregation process. Methodology/Principal Findings: Assuming that conversion of folded protein to fibril is initiated by a nucleation event, we back-calculate the distribution of nuclei concentration. The temporal in vitro concentration of nuclei for the model hormone, recombinant human insulin, is estimated to be in the picomolar range. This is a conservative estimate since the back-calculation method is likely to overestimate the nuclei concentration because it does not take into consideration fibril fragmentation, which would lower the amount of nuclei Conclusions: Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in isolating and blocking oligomers or nuclei toxicity and the long onset time for amyloid diseases

Nuclei concentration.

<p>Calculated profiles of nuclei concentrations (pM) versus length (nm), or equivalently time scale, as a function of the bin size. 2 monomers/bin corresponds to 0.47 nm/bin, while 20 monomers/bin corresponds to 4.7 nm/bin.</p

Equations and variables.

<p>Set of equations used to estimate the total number of insulin nuclei, <i>N_n,t</i>, from the available fibril length distribution. The number of measured fibrils per i-th bin, <i>N_fi</i>, Eqs. (1) & (3), were calculated using the Weibull distribution (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020072#pone-0020072-g001" target="_blank"><b>Figure 1</b></a>). From the definition of the nucleus, the total number of fibrils, <i>N_f,t</i>, is equivalent to the total number of nuclei, <i>N_n,t</i>, Eq. (4). A description and the units are provided for each variable.</p

Fibril length distribution.

<p>The histogram of frequency versus fibril length summarizes AFM data for 495 insulin fibrils in 36.6 nm/bin for a total of 100 bins. The parameters of this distribution were estimated using distribution-fitting software, EasyFit (MathWave Technologies). The software fitted the data using 60 different distributions and ranked the results based on three different goodness-of-fit tests. The histogram shows the best fit (Kolmogorov-Smirnov statistic, <i>D</i> = 0.0187, Anderson-Darling, <i>A²</i> = 0.323, and Chi-Squared, <i>χ²</i> = 5.113) using the Weibull distribution (line). The probability density function is with values of the parameters: α = 1.7409 and β = 1248.5. (A) Example of a 2D AFM image of insulin fibrils, with measurements: A free-hand curve was drawn on the fibril and two cursors placed at each fibril end. Measurements are in nm. (B) Example of a 3D image, which assisted in detecting individual fibrils.</p

A Potent and Selective Quinoxalinone-Based STK33 Inhibitor Does Not Show Synthetic Lethality in KRAS-Dependent Cells

The KRAS oncogene is found in up to 30% of all human tumors. In 2009, RNAi experiments revealed that lowering mRNA levels of a transcript encoding the serine/threonine kinase STK33 was selectively toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors of STK33 might selectively target KRAS-dependent cancers. To test this hypothesis, we initiated a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). Several hits were identified, and one of these, a quinoxalinone derivative, was optimized. Extensive SAR studies were performed and led to the chemical probe ML281 that showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors that were reported in the course of these studies. Even at the highest concentration tested (10 μM), ML281 had no effect on the viability of KRAS-dependent cancer cells. These results are consistent with other recent reports using small-molecule STK33 inhibitors. Small molecules having different chemical structures and kinase-selectivity profiles are needed to fully understand the role of STK33 in KRAS-dependent cancers. In this regard, ML281 is a valuable addition to small-molecule probes of STK33

Epitope spreading toward wild-type melanocyte-lineage antigens rescues suboptimal immune checkpoint blockade responses

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8+ T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod