Kinetic rates obtained for scFv-IC16 binding to different immobilized Aβ(1–42) assembly states.

<p>Association rate constants (<i>k</i>_a) were plotted against dissociation rate constants (<i>k</i>_d). The dissociation constant (<i>K</i>_D) can be extracted from the diagonal lines. Circles, squares and triangles correspond to data from interactions with monomers, oligomers and fibrils, respectively, whereas filled symbols represent data for the second binding site. All data was determined with the heterogeneous fitting model. The grey circle represents monomer data obtained with a 1∶1 binding model.</p

Interaction of scFv-IC16 with different immobilized Aβ(1–42) assembly states.

<p>SPR sensorgrams were recorded separately with single-cycle kinetics. Experimentally obtained, double-referenced binding data (black traces) were superimposed with the corresponding fit (red traces). Monomer data was fit to a 1∶1 Langmuir binding model, and oligomer and fibril data were fit to a heterogeneous ligand binding model. ΔRU: delta of the response units. t/s: time in seconds.</p

Overview of kinetic rates for scFv-IC16 binding to different Aβ(1–42) assembly states obtained within the single-cycle kinetic SPR experiments.

<p>The hash (^#) denotes that kinetic rates were determined with a heterogeneous binding model. Standard deviation with number of experiments is given in brackets.</p>#<p>fit to a heterogeneous binding model. Units are: [a] Ms⁻¹, [b] s⁻¹, [c] M, and [d] RU.</p

On the Examination of the Strong Discontinuity Analysis for a Kinking Discontinuous Surface

The purpose of this paper is to examine the accuracy of the analysis of the strong discontinuity with a kinking discontinuous surface. We first examine the path independent J-integral and the E-integral formula for a damage model with a kinking cohesive region. As a result, we find that the strong discontinuity analysis based on the E-integral has a high accuracy in view of the energy release rate even when the discontinuous surface kinks

Surface preparation with different Aβ(1–42) assembly states for surface plasmon resonance (SPR) analysis.

<p>Size exclusion chromatography (monomers and oligomers) and density gradient centrifugation (fibrils) ensure highly pure samples for immobilization on sensor surfaces and subsequent SPR measurements.</p

Single phage ELISA—immobilization control.

<p>The immobilization efficiency of different Aβ_1–42 species was analyzed by the binding affinity of Aβ_1–42 specific antibody 6E10 to immobilized Aβ_1–42 on each plate used for single phage ELISA. The Aβ_1–42 specific antibody 6E10 was added to wells coated with 150 ng Aβ_1–42 monomers (red) or 150 ng Aβ_1–42 oligomers and fibrils (1:1; green) or to wells only coated with streptavidin (blue). Transformation of substrate by the secondary antibody-conjugated HRP was measured at 450 nm.</p

Quantitative concentration determination (RP-HPLC) of Aβ_1–42 distribution.

<p>Concentrations of Aβ_1–42 in each DGC fraction were determined quantitatively via RP-HPLC. Samples were loaded to a Zorbax 300SB-C8 column connected to a 1260 Infinity HPLC system. Separation of the samples was achieved by elevated column temperature (80°C) and an isocratic mobile phase of 30% acetonitrile/0.1% TFA in water. The averaged concentration of Aβ_1–42 from three independent experiments (with standard deviation) is plotted against the obtained fractions F1 to F15 of different incubation approaches of Aβ_1–42 without or with Mosd1. Shown in red are the concentrations of fractions from 80 μM Aβ_1–42 incubated without Mosd1. The following columns represent the Aβ_1–42 concentrations in the fractions from 80 μM Aβ_1–42 samples coincubated with increasing concentrations of Mosd1 (10 μM = light blue; 20 μM = dark blue; 40 μM = light green; 80 μM = dark green).</p

Effect of Mosd1 on Neuro-2a cells.

<p>Overview and detailed pictures of wild type Neuro-2a cells are shown. Neuro-2a cells were treated with 0, 10 and 100 μM of Mosd1, respectively. The left panel shows untreated wild type Neuro-2a cells. In the middle and right panel, incubation of wild type Neuro-2a cells with 10 μM Mosd1 and 100 μM Mosd1 are shown. Cell viability and morphology were analyzed with a LSM 710 laser scanning microscope. Scale bars equate 50 μm.</p

Transmission electron microscopy.

<p>After incubation of 10 μM pretreated Aβ_1–42 without (left picture) and with 10 μM Mosd1 (right picture) for 24 hours at room temperature, samples were spotted onto a formvar/carbon coated copper grid and stained with 1% aqueous uranyl acetate. Samples were analyzed with a Libra 120 TEM operating at 120 kV. Scale bar presents 0.25 μm.</p

Reduction of seeded Aβ_1–42 growth and fibrillar Aβ_1–42 content.

<p>Seedless Aβ_1–42 was incubated alone (black) or together with fibrillary Aβ_1–42 seeds previously incubated with (green) or without (red) a fivefold molar excess of Mosd1. ThT (20 μM) was added to each sample in order to measure fibrillar content. The data were fitted with an asymmetric five parameter fit. The (A) amplitude of relative fluorescence (RFU) of fibrillated seedless Aβ_1–42 served as 100% to which the other values were normalized. The (B) half-life (t_1/2), displaying the point in time, when half of the maximum ThT signal (i.e. fibrillary content) was reached. Statistical significance was determined by one-way ANOVA. Error bars display SEM. ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.</p