Schematic representation of APP processing and point mutations in Aβ.

<p>α-secretase-mediated proteolytic cleavage of APP occurs after residue 687 of APP, β-secretase-mediated cleavage occurs after residue 671, and γ-secretase cleavage at position 711 or 713. Successive cleavage by β-secretase and γ-secretase results in the release of an intact Aβ peptide. Several point mutations in APP and Aβ are indicated, including the Arctic, Italian, Iowa, and Flemish mutations, which were used in this study.</p

Representative <i>in situ</i> AFM images of Italian Aβ (1–40) aggregate formation on supported TBLE lipid bilayers.

<p>(A) Upon addition of a 20 µM solution of Italian Aβ (1–40) to the TBLE bilayer, oligomeric aggregates appeared within 2–4 hours. (B) After ∼10–12 hours of exposure to Italian Aβ (1–40), the bilayer developed large patches of increased bilayer roughness that often contained oligomeric aggregates. (B–D) Elongated fibrillar aggregates were observed within 8–10 hours of addition of Italian Aβ (1–40) to the lipid bilayer. These fibrillar aggregates displayed a variety of morphologies, predominantly displaying large curvature and branching.</p

Quantification of bilayer roughening.

<p>(A) Quantitative assessment of bilayer disruption by analysis of root mean square (RMS) roughness of images taken before and after exposure to various Aβ (1–40) peptides is shown. Control corresponds to RMS roughness measurements taken on supported TBLE lipid bilayers that had not been exposed to any Aβ (1–40) peptides. Exposure to Wild Type, Arctic, Italian, Iowa, or Flemish Aβ (1–40) induced significant (* indicates p<0.01) roughening of the supported TBLE lipid bilayer. RMS roughness measurements of disrupted areas were restricted to areas that displayed enhanced roughness to prevent biased based on the extent of disruption. (B) The percent area of TBLE bilayers containing increased roughness induced by exposure to Wild Type, Arctic, Italian, Iowa, or Flemish Aβ (1–40) was measured from images obtained 10–12 hours after the initial injection of Aβ (1–40) peptides. Exposure to Arctic Aβ (1–40) resulted in a significantly larger area of the bilayer being disrupted in comparison to Wild Type (* indicates p<0.01). The extent of bilayer disruption was significantly reduced when bilayers were exposed to Iowa or Flemish Aβ (1–40) in comparison to Wild Type (** indicates p<0.05).</p

Wild Type and mutant Aβ fibril morphologies.

<p>A series of <i>ex situ</i> AFM images demonstrating the fibrillar morphologies associated with (A) Wild Type, (B) Arctic (C) Italian, (D) Iowa, and (E) Flemish Aβ aggregates. For all examples, color lines in the AFM image correspond to the profile of the same color presented to the right of each image.</p

Representative <i>in situ</i> AFM images of Flemish Aβ (1–40) aggregate formation on supported TBLE lipid bilayers.

<p>(A) Within 3–5 hours after the injection of Flemish Aβ (1–40) into the fluid cell, oligomeric aggregates appeared on the bilayer surface. (B) Often small defects in the lipid bilayer that did not appear to be associated with aggregates would develop. There were also many large amorphous aggregates on the bilayer. (C) After ∼10–12 hours of exposure to Flemish Aβ (1–40), a few small patches of increased bilayer roughness associated with a high density of very short putative fibrillar aggregates developed. (D) Not all fibrils were associated with bilayer disruption.</p

Representative <i>in situ</i> AFM images of Arctic Aβ (1–40) aggregate formation on supported TBLE lipid bilayers.

<p>(A) When TBLE bilayers were exposed to freshly prepared aliquots of 20 µM solution of Arctic Aβ (1–40), small oligomeric aggregates were observed within 1–2 hours. (B) After ∼6–8 hours, large regions of increased bilayer roughness were observed that appeared to be co-localized with discrete oligomeric aggregates. (C–D) Elongated fibrillar aggregates were also observed within 6–8 hours. (C) Many of these fibrils displayed straight morphologies. (D) These fibrillar aggregates often displayed enhanced curvature, forming large circular structures with many branching points.</p

Formation of total brain lipid extract (TBLE) bilayer.

<p>A sequence of time-lapse <i>in situ</i> AFM images that demonstrate the formation of a supported TBLE bilayer on mica via vesicle fusion is shown. Initially, round bilayer patches appeared on mica as vesicles encountered the surface. With time, these patches gradually fused to form a defect-free bilayer patch, which provided an excellent model surface for studying the aggregation of Aβ and its mutant forms.</p

Quantification of morphological features of aggregates formed on TBLE bilayers by Wild Type or mutant forms of Aβ (1–40).

<p>(A) Histograms of height above the bilayer surface for oligomers formed by Wild Type, Arctic, Italian, Iowa, or Flemish Aβ (1–40) are shown. (B) When the average height above the bilayer surface of oligomers formed by Wild Type and the mutant forms were compared, oligomers were not significantly different as a function of mutation. (C) Based on corrected volume measurements and the molecular mass of Aβ (1–40), the numbers of peptides per oligomer and apparent mass of oligomers comprised of Wild Type, Arctic, Italian, Iowa, or Flemish Aβ (1–40) were calculated. The plots are color coded such that darker colors represent a greater abundance of oligomers composed of that number of molecules. Black arrows indicate where 10–15 peptides and 47–70 kDa oligomers would be observed. (D) The average height above the bilayer surface along the contour of elongated fibril aggregates comprised of Wild Type, Arctic, Italian, Iowa, or Flemish Aβ (1–40) are shown. Fibrils formed from Arctic (* indicates p<0.01) and Italian (** indicates p<0.05) Aβ (1–40) were significantly shorter compared to fibrils comprised of Wild Type Aβ (1–40). (E) Plots correlating the contour length to the end to end distance of fibrils formed from Wild Type, Arctic, or Italian Aβ (1–40) are shown. The dashed line represents the theoretical correlation for infinitely rigid rod-like structures.</p

Supplemental Tables - Comparative Analysis of the Views of Oncologic Subspecialists and Palliative/Supportive Care Physicians Regarding Advanced Care Planning and End-of-Life Care

<p>Supplemental Tables for Comparative Analysis of the Views of Oncologic Subspecialists and Palliative/Supportive Care Physicians Regarding Advanced Care Planning and End-of-Life Care by Phillip M. Pifer, Mark K. Farrugia, Malcolm D. Mattes in American Journal of Hospice and Palliative Medicine®</p

Representative <i>in situ</i> AFM images of Wild Type Aβ (1–40) aggregate formation on supported TBLE lipid bilayers.

<p>(A) 2–4 hours after injection of freshly prepared Wild Type Aβ (1–40), discrete oligomeric aggregates of Wild Type Aβ (1–40) appeared on the bilayer. Zoomed in examples of oligomers are shown. (B–D) After ∼9–12 hours, fibrils associated with vast regions of bilayer disruption were observed.</p