Journal: The Journal of Physical Chemistry. B

Article Title: Conformation of Pyroglutamated Amyloid β (3–40) and (11–40) Fibrils – Extended or Hairpin?

doi: 10.1021/acs.jpcb.3c07285

Figure Lengend Snippet: Cis / trans -conformations of the azobenzene photoswitch (3-(3-aminomethyl)phenylazo)phenylacetic acid (AMPP) introduced into Aβ 1–40 as well as pE 3 -Aβ 3–40 and pE 11 -Aβ 11–40 peptides. Conformations can be switched upon irradiation with a wavelength of 365 nm ( cis ) or 430 nm ( trans ).

Article Snippet: In our current study, for all three mutants containing the AMPP photoswitch (AMPP-Aβ 1–40 , pE 3 -AMPP-Aβ 3–40 , and pE 11 -AMPP-Aβ 11–40 ), the comparison of the ThT fibrillation and the UV/vis absorption kinetics of the respective trans - and cis -AMPP-Aβ conformers indicated that in order to start fibrillation, a critical amount of trans -conformational peptide must be available.

Techniques: Irradiation

Journal: The Journal of Physical Chemistry. B

Article Title: Conformation of Pyroglutamated Amyloid β (3–40) and (11–40) Fibrils – Extended or Hairpin?

doi: 10.1021/acs.jpcb.3c07285

Figure Lengend Snippet: AMPP-Aβ 1–40 (left column), pE 3 -AMPP-Aβ 3–40 (middle column), pE 11 -AMPP-Aβ 11–40 (right column) ThT fluorescence (top row), CV fluorescence (middle row), and AMPP UV/vis absorption (bottom row) measurements to follow the fibrillation and conformation kinetics. Sample stock solutions in DMSO were irradiated for 30 min with either blue light (ca. 450 nm, obtaining trans -conformation) or UV-light (365 nm, obtaining cis -conformation) prior to fibrillation initiation by dilution with fibrillation buffer containing ThT and CV. Three independent experiments were performed in triplicate for each measurement. The curves for initial trans -AMPP-Aβ conformation are plotted in blue and for the cis -AMPP-Aβ conformation in violet. The curves show the average values; the error bars (gray) indicate one standard deviation.

Article Snippet: In our current study, for all three mutants containing the AMPP photoswitch (AMPP-Aβ 1–40 , pE 3 -AMPP-Aβ 3–40 , and pE 11 -AMPP-Aβ 11–40 ), the comparison of the ThT fibrillation and the UV/vis absorption kinetics of the respective trans - and cis -AMPP-Aβ conformers indicated that in order to start fibrillation, a critical amount of trans -conformational peptide must be available.

Techniques: Fluorescence, Irradiation, Standard Deviation

Journal: Molecular Brain

Article Title: Extracellular vesicle-mediated amyloid transfer to neural progenitor cells: implications for RAGE and HIV infection

doi: 10.1186/s13041-020-0562-0

Figure Lengend Snippet: Release of brain endothelial ECVs and their Aβ levels. HBMEC were exposed to 30 ng/ml HIV particles and/or 100 nM Aβ (1–40) for 48 h. Selected cultures were pretreated with 500 nM FPS-ZM1 (FPS) for 2 h followed by cotreatment with 30 ng/ml HIV particles and/or 100 nM Aβ (1–40) for 48 h. ECVs were isolated from the culture media. a Total ECV number as measured by nanoparticle tracking analysis (NTA). Values are mean ± SEM; n = 3–4. b ECV number according to their size distribution and measured as in A. Values are mean ± SEM; n = 3–4. c Size, surface area, and volume distribution of ECVs shed by a representative control and FPS-treated HBMEC. d & e ECV Aβ (1–40) levels as measured by ELISA and normalized either to ( d ) media volume or ( e ) ECV protein levels. Values are mean ± SEM; n = 5–7. f ECV protein levels as measured by the BCA assay. Values are mean ± SEM; n = 3–4. Statistically significant at * p < 0.05, ** p < 0.01, or *** p < 0.001

Article Snippet: Aβ (1–40) and Aβ (1–40) HiLyte were purchased from Anaspec (San Jose, CA) and Aβ (1–40) was dissolved in PBS.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, BIA-KA

Journal: Molecular Brain

Article Title: Extracellular vesicle-mediated amyloid transfer to neural progenitor cells: implications for RAGE and HIV infection

doi: 10.1186/s13041-020-0562-0

Figure Lengend Snippet: Uptake of ECVs by NPCs. HBMEC were treated with HIV and/or Aβ and ECVs were isolated as in Fig. ; however, Aβ (1–40) HiLyte was used instead of non-florescent Aβ (1–40). Then, human NPCs were exposed to HBMEC-derived ECVs for 24 h, with selected cultures pretreated with 500 nM FPS-ZM1 (FPS) for 2 h followed by cotreatment with the isolated ECVs. Images were performed by confocal microscopy z-stacking. a Transferred Aβ (1–40) HiLyte (green) from brain endothelial ECVs to NPCs. Representative images from three experiments. DRAQ5 staining (red) visualizes the NPC nuclei. Scale bar: 10 μm. b Quantification of total Aβ (1–40) HiLyte fluorescence in recipient NPCs from the confocal images. Values are mean ± SEM; n = 8–11. c Quantification of nuclear Aβ (1–40) HiLyte fluorescence in recipient NPCs from the confocal images. Values are mean ± SEM; n = 47–86 from randomly selected images. Statistically significant at * p < 0.05, ** p < 0.01, or **** p < 0.0001

Article Snippet: Aβ (1–40) and Aβ (1–40) HiLyte were purchased from Anaspec (San Jose, CA) and Aβ (1–40) was dissolved in PBS.

Techniques: Isolation, Derivative Assay, Confocal Microscopy, Staining, Fluorescence

Journal: Molecular Brain

Article Title: Extracellular vesicle-mediated amyloid transfer to neural progenitor cells: implications for RAGE and HIV infection

doi: 10.1186/s13041-020-0562-0

Figure Lengend Snippet: Aβ colocalization with inflammasome proteins in ECV-exposed NPCs. HBMEC were treated, and ECVs were isolated as in Figs. and , followed by exposure to NPCs for 24 h. a NLRP3 immunoreactivity (red) and transferred Aβ (1–40) HiLyte (green) in NPCs as visualized by confocal microscopy. Nuclei are stained with DAPI (blue). Scale bar: 10 μm. b Nuclear colocalization of NLRP3 with Aβ (1–40) HiLyte. The graphs below the representative nuclear images depict fluorescence intensity profiles for colocalization of NLRP3 and Aβ (1–40) HiLyte in nuclear areas. c Quantification of NLRP3 and Aβ (1–40) HiLyte colocalization from B. d ASC immunoreactivity (red) and transferred Aβ (1–40) HiLyte (green) in NPCs as visualized by confocal microscopy. Nuclei are stained with DAPI (blue). Scale bar: 10 μm. e Nuclear colocalization of ASC with Aβ (1–40) HiLyte. The graphs below the representative nuclear images depict fluorescence intensity profiles for colocalization of ASC and Aβ (1–40) HiLyte in nuclear areas. f Quantification of ASC and Aβ (1–40) HiLyte colocalization from E. a & d Representative images from three experiments. c , f Values are mean ± SEM, n = 45–60. Statistically significant as compared to control at *** p < 0.001 or **** p < 0.0001

Article Snippet: Aβ (1–40) and Aβ (1–40) HiLyte were purchased from Anaspec (San Jose, CA) and Aβ (1–40) was dissolved in PBS.

Techniques: Isolation, Confocal Microscopy, Staining, Fluorescence