Es amyloid (rPrPspon) was detected in control reactions containing normal brain

Es amyloid (rPrPspon) was detected in control reactions containing normal brain homogenate (NBH) 23388095 (Figure 1). In contrast, NBH controls gave rPrPspon when moPrPSen23?31 was used with higher NaCl concentrations ( 200 mM). When using moPrPC 90?31 as substrate, rPrPspon generation was observed within 20?0 h with all NaCl concentrations tested (130?400 mM; data not shown).RT-QuIC of additional mouse-adapted scrapie strains in wild-type miceTo gauge the strain-dependence of murine RT-QuIC analyses, we also analyzed the 22L and ME7 scrapie strains in brain homogenates from clinically affected WT C57BL/10 miceSeeding activity in mice with little Title Loaded From File PrPResTo determine if prion seeding activity can be detected in hosts with clinical TSE disease but little or no detectable PrPRes, we compared two scrapie strains in knock-in transgenic mice homozygous for P101L PrPC (101LL mice) [51]. Inoculation ofRT-QuIC and eQuIC with Mouse Scrapie StrainsFigure 1. RT-QuIC NaCl titration in mouse RML scrapie model using moPrPC23?31 as substrate. RT-QuIC reactions were seeded with 561026 dilutions of normal brain homogenates (NBH) and WT RML scrapie BH containing ,200 fg of PrPRes. Final concentrations of 130, 200, 300 and 400 mM NaCl were used. The vertical axis indicates the average ThT fluorescence from four replicate wells. doi:10.1371/journal.pone.0048969.gthe 263K scrapie strain causes TSE disease and high infectivity titers in the brain but little or no PrPRes in these mice as detected by immunoblotting and other assays [35]. In contrast, when these animals are inoculated with the 1527786 139A scrapie strain, they accumulate readily detectable amounts of PrPRes [37]. Indeed, our immunoblot-based comparisons indicated that brain synaptosome preparations from the 263K-inoculated mice contained ,81-fold less PrPRes than those from 139A-inoculated mice in the clinical phase of disease (Figure 6). Previous work has shown that a majority of the infectivity fractionates with synaptosomes, and that similar titers are found with these two strains ([35]; unpublished data). Despite the large difference in PrPRes levels with these strains, we measured similar levels of seeding activity by end-point dilution RT-QuIC (Figure 7 A, B, E). Moreover, the profile of PK-resistant bands in the RT-QuIC reaction products was also similar between the two strains (Figure 8, lanes 4 6). Altogether, the data indicated abundant seeding activity associated with both high-PrPRes and very low-PrPRes TSE strains. Next we tested the PK-resistance of the seeding activities associated with the 263K and 139A strains in the 101LL transgenic mice (Figure 7 C, D). Synaptosomes were permeabilized with Triton X-100 and treated with 100 mg/mL PK prior to end-point dilution RT-QuIC. Following PK treatment, little PrPRes was present in the 263K synaptosomes, and the expected size shift in banding pattern was observed in 139A synaptosomes (Figure 6). The PK treatment appeared to cause a modest (,4fold) decrease in the mean SD50/mg brain value for 263K synaptosomes from three separate experiments (Figure 7E, red bars), but this was of minimal statistical significance (p = 0.056). No effect of PK treatment on the mean SD50/mg brain was seen with 139A synaptosomes (Figure 7E, orange bars). Overall, the results suggested that the 263K seeding activity may be somewhat sensitive to PK digestion, but less so than the total synaptosomal PrP Comparisons), total protein with dexamethasone treatment (2-way ANOVA). Count data was content.eQuIC detection of prion seeding activity in mouse plasmaBecause bloo.Es amyloid (rPrPspon) was detected in control reactions containing normal brain homogenate (NBH) 23388095 (Figure 1). In contrast, NBH controls gave rPrPspon when moPrPSen23?31 was used with higher NaCl concentrations ( 200 mM). When using moPrPC 90?31 as substrate, rPrPspon generation was observed within 20?0 h with all NaCl concentrations tested (130?400 mM; data not shown).RT-QuIC of additional mouse-adapted scrapie strains in wild-type miceTo gauge the strain-dependence of murine RT-QuIC analyses, we also analyzed the 22L and ME7 scrapie strains in brain homogenates from clinically affected WT C57BL/10 miceSeeding activity in mice with little PrPResTo determine if prion seeding activity can be detected in hosts with clinical TSE disease but little or no detectable PrPRes, we compared two scrapie strains in knock-in transgenic mice homozygous for P101L PrPC (101LL mice) [51]. Inoculation ofRT-QuIC and eQuIC with Mouse Scrapie StrainsFigure 1. RT-QuIC NaCl titration in mouse RML scrapie model using moPrPC23?31 as substrate. RT-QuIC reactions were seeded with 561026 dilutions of normal brain homogenates (NBH) and WT RML scrapie BH containing ,200 fg of PrPRes. Final concentrations of 130, 200, 300 and 400 mM NaCl were used. The vertical axis indicates the average ThT fluorescence from four replicate wells. doi:10.1371/journal.pone.0048969.gthe 263K scrapie strain causes TSE disease and high infectivity titers in the brain but little or no PrPRes in these mice as detected by immunoblotting and other assays [35]. In contrast, when these animals are inoculated with the 1527786 139A scrapie strain, they accumulate readily detectable amounts of PrPRes [37]. Indeed, our immunoblot-based comparisons indicated that brain synaptosome preparations from the 263K-inoculated mice contained ,81-fold less PrPRes than those from 139A-inoculated mice in the clinical phase of disease (Figure 6). Previous work has shown that a majority of the infectivity fractionates with synaptosomes, and that similar titers are found with these two strains ([35]; unpublished data). Despite the large difference in PrPRes levels with these strains, we measured similar levels of seeding activity by end-point dilution RT-QuIC (Figure 7 A, B, E). Moreover, the profile of PK-resistant bands in the RT-QuIC reaction products was also similar between the two strains (Figure 8, lanes 4 6). Altogether, the data indicated abundant seeding activity associated with both high-PrPRes and very low-PrPRes TSE strains. Next we tested the PK-resistance of the seeding activities associated with the 263K and 139A strains in the 101LL transgenic mice (Figure 7 C, D). Synaptosomes were permeabilized with Triton X-100 and treated with 100 mg/mL PK prior to end-point dilution RT-QuIC. Following PK treatment, little PrPRes was present in the 263K synaptosomes, and the expected size shift in banding pattern was observed in 139A synaptosomes (Figure 6). The PK treatment appeared to cause a modest (,4fold) decrease in the mean SD50/mg brain value for 263K synaptosomes from three separate experiments (Figure 7E, red bars), but this was of minimal statistical significance (p = 0.056). No effect of PK treatment on the mean SD50/mg brain was seen with 139A synaptosomes (Figure 7E, orange bars). Overall, the results suggested that the 263K seeding activity may be somewhat sensitive to PK digestion, but less so than the total synaptosomal PrP content.eQuIC detection of prion seeding activity in mouse plasmaBecause bloo.