tht  (Mimetics)

Journal: bioRxiv

Article Title: Evolutionarily Conserved Amyloid Aggregation in the PACAP Peptide Family Is Controlled by Heparin-Sensitive Lys/Arg Gatekeeper Residues

doi: 10.64898/2026.05.04.722589

Figure Lengend Snippet: (A) General overview of the experimental setup used to monitor amyloid formation. Peptides were applied at a final concentration of 0.35 mM. (B) CD spectra were recorded for the initial peptide states prior to incubation. The samples were then divided into two groups and incubated for 72 hours under Thioflavin-T fluorescence monitoring, either in the presence of enoxaparin sodium (2.125 mg mL⁻¹) or in the absence of heparin. The end states were subsequently analysed again by CD spectroscopy. For the rest of screening results see the supporting material. (PACAP-38 – S ; VIP – S ; PRP – S ; PHI – S ; GHRH – S ; SCT – S ) (C) The deconvolution of the collected CD curves yields 7 components: two slightly different unfolded components; one helical component and two-two typical and atypical β-sheeted components. For the in-depth review of the CD deconvolution see S . (D) Overview of the measured biophysical characteristics of the end states extended with the overall charge of the peptides across the examined pH range. ThT values represent the fold increase in fluorescence relative to the corresponding blank, whereas CD values indicate the relative proportions of unfolded, helical, and β-sheet components determined by spectral deconvolution. ThT values and component triplets are highlighted in bold in cases where amyloid fibrils were confirmed by AFM. Overall charges: heparin carries a negative charge under all investigated pH values. The sulfonate groups of polyanionic heparin remain fully deprotonated between pH 2 and 10. In contrast, its carboxyl groups are predominantly protonated at low pH, present in roughly equal protonated and deprotonated proportions near their pK a and become fully deprotonated above pH 7. Approximate net charges of the peptides were calculated using generalized pKₐ values. (E) AFM micrographs recorded from the end states of the incubated samples after 20-fold dilution reveal nanoscale amyloid filaments with varying morphologies ( S -6 /G shows additional micrographs ) , often accompanied by amorphous aggregates. PACAP (pH 6 – /C ) and GHRH (pH 4 – S /G ) display clear polymorphism within the same sample. In the case of SCT incubated at pH 5, the observed structures are not typical amyloid fibrils; however, they are too structured to be considered mere precipitation.

Article Snippet: Thioflavin T (ThT) stock solutions (Acros Organics, Thermo Fisher Scientific) were prepared in distilled water, with the pH adjusted between 2 and 10 in increments of one pH unit.

Techniques: Concentration Assay, Circular Dichroism, Incubation, Fluorescence

Journal: bioRxiv

Article Title: A new therapeutic approach for Parkinson’s disease: dual targeting of α-Synuclein aggregation and microglial function by the novel immunomodulator 3-Monothiopomalidomide

doi: 10.64898/2026.03.26.714051

Figure Lengend Snippet: ( A ) ThT fluorescence intensity monitored during α-Syn (100 μM) aggregation at 37 °C, in PBS at pH 7.4 and under orbital shaking (200 rpm). Kinetic traces for isolated α-Syn (black) and in co-incubation with POM (yellow) and 3MP (orange) are reported. All conditions were tested in triplicate. The intrinsic fluorescence of 3MP was found to be negligible compared to the intensity of ThT fluorescence. ( B ) t½ values of the kinetic curves. ( C ) Plateau values of the kinetic curves. One-way ANOVA followed by Dunnett’s post-hoc test was performed against protein alone (control). ns = not significant, * p < 0.05, *** p < 0.001. Shapiro–Wilk and Bartlett’s tests confirmed normality and homogeneity of variance. ( D ) Transmission electron microscopy (TEM) images of samples post 120h of incubations. The images were taken at both 100 nm and 200 nm magnifications. Samples were prepared using negative staining with 2% phosphotungstic acid (PTA) on carbon-coated 200 mesh copper grids. ( E ) Representative images of ThS staining on SH-SY5Y cells challenged with α-Syn pre-formed fibrils and treated with POM and 3MP (magnification 63x; scale bar: 20µm). ( F ) Quantification of ThS + signal expressed as ThS□ volume per cell (µm³/cell) in SH-SY5Y cells challenged with α-Syn pre-formed fibrils and treated with POM or 3MP. Data are shown as mean ± SEM from three independent biological replicates. One-way ANOVA followed Tuckey’s post-hoc test.

Article Snippet: To monitor the aggregation kinetics via ThT fluorescence, we employed a FLUOStar Omega (BMG Labtech) plate reader with excitation and emission wavelengths set at 448 nm and 482 nm, respectively.

Techniques: Fluorescence, Isolation, Incubation, Control, Transmission Assay, Electron Microscopy, Negative Staining, Staining