Fig 1: Small EVs vs. whole serum nanoparticles. (A) Comparison of nanoparticle concentration by size range. (B) ApoB quantification on small EVs and whole plasma in all samples. (C) ApoB quantification on small EVs and whole plasma in Pre- and Post-surgery samples. (D) ApoA1 quantification on small EVs and whole plasma in all samples. (E) ApoA1 quantification on small EVs and whole plasma in Pre- and Post-surgery samples. *p < 0.05.
Fig 2: Changes from baseline in total APOA1 concentration and APOA1 concentration in high-density lipoprotein (HDL) subspecies containing APOC1, APOC3, APOE, a2-macroglobulin, complement C3, and plasminogen. Point estimates, 95% CIs, and P values extracted from a mixed-model framework. P values are for overall dose effect and comparison between the 5 mg daily dose group and placebo groups.
Fig 3: Experiments on ApoA1- and ApoE-immunodepleted CSF. A Protein aggregation assay performed using 0.7 mg/mL of recombinant a-syn in PBS with 40 µL of: CSF subjected to ApoA1 and ApoE IP (IP CSF), CSF subjected to IP without antibodies (IP CSF no Ab) and neat CSF belonging to different aliquots of the same NC pool. To remove the background fluorescence, the average fluorescence of three replicates containing the same reaction mix without a-syn was subtracted prior to the analysis. All ThT fluorescence traces are represented as average intensity over 3 replicates with error bars representing the SEM. B Average t2 fitted parameters with error bars representing the SEM. P-values were calculated by applying one-way ANOVA followed by Tukey post-hoc test
Fig 4: Different CSF fractions differently affect a-syn aggregation. A Scheme of the CSF fractionation procedure. From a starting aliquot of 4.5 mL of CSF in PBS 1x, we collected 6 aliquots containing compounds of different molecular weight and froze them in liquid nitrogen. After every filtration with centrifugal filters, the flow-through of the filtered fraction was passed to a filter with smaller cut-off. B The addition of CSF fractions, whole NC-CSF pool, and PBS (40 µL) was analysed by ThT protein aggregation assay to evaluate effects on a-syn spontaneous aggregation. Background signal was corrected by subtracting the average fluorescence of three replicates containing PBS, whole CSF and CSF fractions without a-syn. All ThT fluorescence traces are represented as average intensity over 3 replicates with error bars representing the SEM. C Mean fitted A2 parameters (fitting was not possible for samples with whole CSF and the > 100 kDa fraction) and maximum fluorescence values (Fmax) estimated from individual ThT traces. Two scales of fluorescence intensity were used to better compare the results. Represented values correspond to the average of three replicates with error bars reflecting the SEM. D Relative concentration (emPAI score multiplied by protein molecular weight) of the most abundant protein constituents measured by nLC-nESI HRMS/MS. Apolipoproteins scores were summed together, with ApoA1 and ApoE being the most abundant (~ 85% of the total). Scores for fractions 10–3 and < 3 kDa are not shown since the protein content of these fractions was negligible with respect to the others
Fig 5: Analysis of NPH CSF samples. A Protein aggregation assay performed using 0.7 mg/mL of recombinant a-syn in PBS with 40 µl of CSF from 2 NPH subjects (40 µL). The two ThT fluorescence traces are represented as average intensity over 3 replicates with error bars representing the SEM. B Portion of 1D 1H NMR spectra relative to the two NPH CSF samples. C Relative concentration (emPAI score multiplied by protein molecular weight) ratio of total protein and the three most abundant protein constituents measured by nLC-nESI HRMS/MS in neat NPH1 and NPH2 CSF samples. Approximately 200 and 400 different proteins were detected in NPH1 and NPH2, respectively. Albumin was the most abundant protein followed by apolipoproteins and complement proteins. Apolipoproteins scores were summed together, with ApoA1 and ApoE being the most abundant (~ 85% of the total). Complement C3 and C4 were found as the most abundant complement proteins (~ 65% of the total)
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