Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Deciphering complement receptor type 1 interactions with recognition proteins of the lectin complement pathway.

doi: 10.4049/jimmunol.1202451

Figure Lengend Snippet: FIGURE 1. Characterization of the recom- binant CR1 CCP 22–30_His fragment. (A) SDS-PAGE analysis of 4 mg CR1 CCP22-30 under reducing (R) and nonreducing (NR) conditions. The positions of the m.w. mark- ers are indicated. (B) CD spectroscopy of CR1 CCP22–30. A spectrum was recorded in the far-UV (200–260 nm) and collected six times. The mean values for each wavelength were calculated. The maximal ellipticity is indicated by an arrowhead. (C) Electron microscopy analysis of CR1 CCP22–30 (375000) after negative staining with 2% sodium silicotungstate.

Article Snippet: Recombinant soluble human CR1 was purchased from R&D Systems Europe (Lille, France).

Techniques: SDS Page, Circular Dichroism, Electron Microscopy, Negative Staining

Journal: Journal of inorganic biochemistry

Article Title: Impact of the dynamics of the catalytic arginine on nitrite and chlorite binding by dimeric chlorite dismutase.

doi: 10.1016/j.jinorgbio.2021.111689

Figure Lengend Snippet: Fig. 1. Formation of nitrite complexes of wild-type chlorite dismutase from Cyanothece sp. PCC7425 and the variants Q74V, Q74E and R127A at pH 5.0, 7.0 and 9.0. (A) Dissociation constants (KD) calculated from thermodynamic titrations followed by UV–vis spectroscopy. (B) Spectral transitions during high-spin (HS) to low-spin (LS) transition of wild-type CCld at pH 5, 7 and 9 (left to right). For Q74V and Q74E see Fig. S2. The insets depict the wavelength range between 450 and 750 nm for better visualization of the Q-bands and CT-bands. Blue spectra represent ferric HS spectra or partially hydroxo-LS spectra, whereas red spectra represent the LS nitrite complexes at the end of titration. A 100- to 3000-fold excess of nitrite was used to obtain the final spectra, for pH 5 and pH 9 respectively. Note that the background absorbance in the UV-region at pH 9.0 derives from the high nitrite concentration that is necessary for formation of a stable nitrite-complex. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Sigma plot (Version 13.0, Systat Software, San Jose, CA, USA) was used to fit the resulting curves to the following hyperbola equation with b corresponding to KD, x representing the nitrite concentration, a corresponding to the maximal absorption of the Soret band in saturation of the ligand and y being the absorption at the respective wavelength: I. Serra et al. Journal of Inorganic Biochemistry 227 (2022) 111689 y = ax b + x For electronic circular dichroism spectroscopy, Chirascan from Applied Photophysics (Leatherhead, UK) was used.

Techniques: UV-Vis Spectroscopy, Titration, Concentration Assay

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a Schematic representation of the different domains of p53. The DBD (residues 102–292) contains an aggregation-nucleating subdomain (residues 251–258) that is necessary and sufficient to drive p53 aggregation , , . Another segment of interest comprises residues 213–217, which is the antigen recognized by the PAb 240 antibody that binds to partially unfolded p53. Also highlighted in the DBD is R248, one of the most common mutation hotspots in p53 (IARC TP53 database; https://p53.iarc.fr ) . b Structure of p53 DBD. Highlighted are the aggregation-nucleating subdomain (green) and the epitope recognized by PAb 240 (red). Both segments are buried in the fully folded p53 structure. The 3D image was generated using PyMOL 2.3.5 (Schrödinger, New York, NY). c Primary sequences of the studied WT and mutant R248W p53 DBD-derived peptides, denoted pWT and pR248W, respectively, which span residues 248–273. The peptides include the aggregation-prone 252–258 sequence, as well as R248 and another of the most common mutation hotspots in p53 and R273 (IARC TP53 database; https://p53.iarc.fr ) . d Chemical structures of the oligopyridylamides ADH-1 and ADH-6. e , f Effects of the oligopyridylamides on pR248W amyloid formation. Kinetic profiles (left panel) and representative transmission electron microscopy (TEM) images (right panel) for aggregation of 25 μM pR248W in the absence or presence of an equimolar amount of ADH-1 or ADH-6 co-mixed at the start of the reaction ( e ) or added during the growth phase (i.e. 5 h after the start of the reaction) ( f ). Kinetic aggregation profiles were acquired by measuring the fluorescence of the thioflavin T (ThT) reporter ( λ ex/em = 440/480 nm) at 5-min intervals at 37 °C ( n = 4). TEM images were acquired at 10 h after the start of the aggregation reaction. Scale bar = 100 nm. g Characterization of the binding interaction of the oligopyridylamides and pR248W measured using steady-state intrinsic tryptophan fluorescence quenching. A 5 µM solution of pR248W was titrated with increasing concentrations of ADH-1 (left panel) or ADH-6 (right panel) and the tryptophan fluorescence after each addition was normalized to account for the dilution (total dilution during the titration was <1%) and plotted against the ligand concentration. The equilibrium dissociation constants ( K d ) were then determined using a one-site-specific binding equation (Eq. ). h Effects of the oligopyridylamides on pR248W oligomerization monitored using the dot blot assay. Samples of 10 μM pR248W were incubated with or without an equimolar amount of ADH-1 or ADH-6 for 0–24 h, and the presence of oligomers was detected using an amyloid oligomer-specific polyclonal antibody (A11) . i Effects of the oligopyridylamides on the self-assembly driven structural transition of pR248W. Time-dependent circular dichroism (CD) spectra of 10 µM pR248W alone (left panel) or in the presence of an equimolar amount of ADH-1 (middle panel) or ADH-6 (right panel).

Article Snippet: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

Techniques: Mutagenesis, Generated, Derivative Assay, Sequencing, Transmission Assay, Electron Microscopy, Fluorescence, Binding Assay, Titration, Concentration Assay, Dot Blot, Incubation, Circular Dichroism

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a , b Overlay of 15 N- 1 H HSQC maps of 19 μM WT ( a ) and 24 μM R248W ( b ) p53 DBD in H O/D O (96/4) with 16.7 mM DTT, without (green contours) or with (red contours) ADH-6 addition (protein:ligand 1:11 in a and 1:15 in b ). The assignments are reported only outside the rightmost regions. These regions are crowded because of the presence of partially unfolded species that also interact with ADH-6 as highlighted by the boxed peak in each panel. c HSQC contour maps overlay of mutant R248W p53 DBD at different protein:ADH-6 ratios (1:0 green, 1:8 cyan, and 1:15 red) showing the increment of cumulated chemical shift perturbation (CSP) with ligand concentration (Eq. ). d The five clusters of the two p53 DBD variants (WT and mutant R248W) that show high (>0.025) or medium (>0.015) CSP values . Cluster 1 (highlighted in blue) includes residues T118, Y126, E271, C275, and G279; cluster 2 (highlighted in magenta) includes residues R196, E198, G199, L201, Y220, and E221; cluster 3 (green) includes T102, Y103, Q104, G105, L257, L264, and R267; cluster 4 (orange) includes E171, R174, H179, R209, and G244; and cluster 5 (cyan) includes S94, A161, I162, L206, and S215. Clusters 1 and 2 are at the front in the cartoon on the left; clusters 3–5 are at the front in the cartoon on the right. The 3D image was generated using PyMOL 2.3.5 (Schrödinger, New York, NY).

Article Snippet: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

Techniques: Mutagenesis, Concentration Assay, Generated

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a Confocal fluorescence microscopy images showing thioflavin S (ThS) staining of mutant p53 (R248W) aggregates in MIA PaCa-2 cells treated with vehicle (0.02% DMSO) or ADH-6 (5 µM) for 0.5 or 6 h. Imaging experiments were performed in quadruplicate and representative images are shown. b Quantification of ThS-positive MIA PaCa-2 cells after treatment with vehicle or ADH-6. The number of positively stained cells in 3–5 different fields of view are expressed as % of the total number of cells ( n = 4 biologically independent samples). Data presented are mean ± SD. Statistical analysis was performed using two-tailed unpaired t -test. P < 0.0001 for ADH-6 vs vehicle at 6 h. c Confocal fluorescence microscopy images of ThS and PAb 240 antibody staining of R248W aggregates in MIA PaCa-2 cells treated with vehicle or 5 µM ADH-6 for 0.5 or 6 h. Images shown are representative of four independent experiments. d – f Quantification of PAb 240-positive MIA PaCa-2 cells after treatment with the indicated concentrations of ADH-1, ReACp53, or ADH-6 for 0.5 or 6 h relative to controls (vehicle-treated cells). The number of positively stained cells in 3–5 different fields of view are expressed as % of the total number of cells (mean ± SD; n = 4). Statistical analysis was performed using repeated measures two-way ANOVA followed by Holm-Sidak’s post hoc test. P < 0.0001 for ReACp53 (2.5–10 µM) vs vehicle at 6 h ( e ); P < 0.0001 for ADH-6 (2.5–10 µM) vs vehicle at 6 h ( f ). g Colocalization of FITC-labeled ADH-6 (ADH-6 FITC ) with PAb 240-stained R248W aggregates following incubation with the oligopyridylamide (5 µM) for 0.5 or 6 h. Colocalization was quantified using directional Pearson correlation coefficient, r , which measures pixel-by-pixel covariance in the signal level of two images . Scale bar = 5 µm. h , i Cellular thermal shift assay (CETSA) analysis of intracellular target engagement. Melting curves for p53 mutants R248W ( h ) and R175H ( i ) in MIA PaCa-2 and SK-BR-3 cells, respectively, in the absence or presence of the oligopyridylamides (mean ± SD; n = 3). *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls.

Article Snippet: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

Techniques: Fluorescence, Microscopy, Staining, Mutagenesis, Imaging, Two Tailed Test, Labeling, Incubation, Thermal Shift Assay, Drug discovery

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a – c Effects of ADH-6 on viability of cancer cells bearing WT or mutant p53. MIA PaCa-2 (mutant R248W p53) ( a ), MCF-7 (WT p53) ( b ), and SK-BR-3 (mutant R175H p53) ( c ), cells treated with increasing oligopyridylamide concentrations for 24 or 48 h. ( d – f ) p53 null Saos-2 cells before ( d ) and after transfection with p53 mutants, R248W ( e ) or R175H ( f ), treated with increasing concentrations of ADH-6 for 24 or 48 h. Cell viability in a – f was assessed using the MTS assay, with the % viability determined form the ratio of the absorbance of the treated cells to the control cells ( n = 3 biologically independent samples). Data presented are mean ± SD. Statistical analysis in a – f was performed using two-tailed unpaired t -test. P < 0.0001 for ADH-6 vs ADH-1 at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( a , c ); P < 0.0001 for ADH-6 treatment of Saos-2/R248W compared with untransfected cells (data shown in d ) at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( e ); P < 0.0001 for ADH-6 treatment of Saos-2/R175H compared with untransfected cells (data shown in d ) at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( f ). g , h Flow cytometry analysis of annexin V/propidium iodide (PI) staining of MIA PaCa-2 cells that were treated with vehicle (control), or 5 µM ADH-1, ReACp53, or ADH-6, for 24 h. The bottom left quadrant (annexin V−/PI−) represents live cells; bottom right (annexin V+/PI−), early apoptotic cells; top right (annexin V+/PI+), late apoptotic cells; and top left (annexin V−/PI+), necrotic cells ( g ). A summary of the incidence of early/late apoptosis and necrosis in the different treatment groups determined from the flow cytometry analysis of annexin V/PI staining (mean ± SD; n = 4) ( h ). Statistical analysis in h was performed using one-way ANOVA followed by Dunnett’s post hoc test. P < 0.0001 for ReACp53 vs vehicle (live and early apoptosis); P < 0.0001 for ADH-6 vs vehicle (live, early apoptosis, and late apoptosis). i Cell cycle distribution of MIA PaCa-2 cells treated w i th vehicle (control), or 5 µM ADH-1, ReACp53 or ADH-6, for 6 h as determined by measurement of DNA content using flow cytometry (mean ± SD; n = 4). Two-tailed unpaired t -test: P = 0.0071 and 0.0037 for ReACp53 vs vehicle (G0/G1 and G2/M, respectively); P < 0.0001 and P = 0.0004 for ADH-6 vs vehicle (G0/G1 and G2/M, respectively). ** P < 0.01, *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls.

Article Snippet: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

Techniques: Mutagenesis, Transfection, MTS Assay, Control, Two Tailed Test, Concentration Assay, Incubation, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a , b In vivo pharmacokinetics of ADH-6. Concentration of ADH-6 in plasma ( a ) and in MIA PaCa-2 xenografts ( b ) of mice ( n = 5–6 per group), after an intraperitoneal injection of the oligopyridylamide (15 mg kg -1 ), was quantified using LC-MS/MS . Shown are the circulation half-life ( T 1/2 ) ( a ) as well as the maximum (or peak) concentration ( C max ) in tumors and the time to achieve C max ( T max ) ( b ). Data presented are mean ± SD. c , d Design of the tumor reduction studies. A representative mouse bearing both MIA PaCa-2 (mutant R248W p53) and MCF-7 (WT p53) xenografts ( c ) and treatment schedule for the dual xenograft model ( d ). Once the tumor volume reached ~25 mm , the mice were randomized into the different treatment groups ( n = 8 per group), which were injected intraperitoneally with vehicle (0.02% DMSO), ReACp53 (716.4 µM), or ADH-6 (716.4 µM). Injections were done every 2 days for a total of 12 doses, with the first day of treatment defined as day 0. e Body weight changes of the tumor-bearing mice in the different treatment groups monitored for the duration of the experiment (mean ± SD; n = 8). f , g Tumor volume growth curves for the MIA PaCa-2 ( f ) and MCF-7 ( g ) xenografts in the different treatment groups over the duration of the experiment (mean ± SD; n = 8). Tumor volume was calculated using Eq. . Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 ( f ). h , i Tumor mass analysis for the different treatment groups. After 25 days of treatment, four mice per treatment group were sacrificed and the tumor tissues were isolated and imaged ( h ) and subsequently weighed to determine the tumor mass ( i ). Data presented are mean ± SD, and statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 (MIA PaCa-2 xenografts; i ). j Hematoxylin and eosin (H&E)-stained xenograft sections from the different treatment groups following 25 days of treatment. Images on the right are magnified views of the boxed regions in the images on the left. Scale bar = 20 μm (50 μm for the magnified views). k – m Immunohistochemistry (IHC) analysis of the residual xenografts. Images of sections of MIA PaCa-2 and MCF-7 xenografts stained using the anti-p53 PAb 240 and DO-7 antibodies, respectively, from the different treatment groups ( k ). Images on the right are magnified views of the boxed regions in the images on the left. Scale bar = 20 μm (50 μm for the magnified views). Quantification of PAb 240 ( l ) and DO-7 ( m ) positive cells in 3–5 different fields of view expressed as % of the total number of cells (mean ± SD; n = 4). One-way ANOVA followed by Tukey’s post hoc test: P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 ( l ). n Survival curves for the vehicle, ReACp53 and ADH-6 treatment groups over 30 days ( n = 4 per group). Statistical analysis was performed using log-rank (Mantel-Cox) test. P = 0.0062 for ADH-6 vs vehicle. ** P < 0.01, *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls and between the different treatment groups.

Article Snippet: Subsequently, the cells were incubated with 5 μg/ml p53 antibody (PAb 240, sc-99; Santa Cruz Biotechnology, Dallas, TX), overnight at 4 °C, which was followed by incubation with Alexa 488- or Alexa 594-labeled anti-mouse IgG (ab150117 or ab150120, respectively; Abcam) secondary antibody (1:800 dilution) for 1 h at room temperature.

Techniques: In Vivo, Drug discovery, Concentration Assay, Clinical Proteomics, Injection, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Isolation, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: Self-assembling dipeptide antibacterial nanostructures with membrane disrupting activity

doi: 10.1038/s41467-017-01447-x

Figure Lengend Snippet: The diphenylalanine nanostructures hinder E. coli growth and viability and severely damage bacterial morphology. a Morphology of the nanostructures formed by the studied peptides. Micrographs were obtained via scanning electron microscopy. Scale bar is 10 µm. b Secondary structure of the studied peptides as obtained by circular dichroism (CD). CD spectra were obtained for both of the peptides diluted in ultra-pure water at 25 °C. c Kinetics of the inhibition of bacterial growth. The effect of the diphenylalanine nanostructures and controls on bacterial growth was evaluated by turbidity analysis via absorbance readings at 600 nm of bacteria treated overnight. d Effect of the diphenylalanine nanostructures on bacterial viability. Bacterial viability was evaluated using the Live/Dead backlight bacterial viability kit. Green fluorescence of the Syto9 probe indicates bacterial cells with an intact membrane, while red fluorescence of Propidium Iodide (PI) indicates dead bacterial cells. Scale bar is 50 µm. e Evaluation of the effect of the diphenylalanine nanostructures on bacterial morphology. Micrographs were obtained using a high-resolution scanning electron microscope. Scale bar is 1 µm

Article Snippet: The strain utilized in this study is E. coli ATCC 25922, a clinical isolate widely used for testing the susceptibility of bacteria to antibiotics and antimicrobial peptides.

Techniques: Electron Microscopy, Circular Dichroism, Inhibition, Bacteria, Fluorescence, Membrane, Microscopy

Journal: Nature Communications

Article Title: Self-assembling dipeptide antibacterial nanostructures with membrane disrupting activity

doi: 10.1038/s41467-017-01447-x

Figure Lengend Snippet: The diphenylalanine nanostructures cause outer and inner membrane perturbations in E. coli and interact with model membrane systems. a Outer membrane permeation by the diphenylalanine nanostructures. Outer membrane permeation was determined using the ANS sulfonic amine fluorescent probe that displays enhanced fluorescence upon exposure of hydrophobic regions. b Cytoplasmic membrane depolarization by the diphenylalanine nanostructures. The cytoplasmic membrane depolarization activities were determined using the membrane potential sensitive dye DiSC3(5). c , d The diphenylalanine nanostructures interact with model phospholipid membrane systems. Membrane interactions of each compound were determined via measurements of the color transitions induced upon interaction of the compounds with DMPE/DMPG/PDA (1:1:3) vesicles. c Kinetics of the initial membrane interaction of the diphenylalanine nanostructures. d Interaction of the diphenylalanine nanostructures with the model membrane systems after incubation for 24 h

Article Snippet: The strain utilized in this study is E. coli ATCC 25922, a clinical isolate widely used for testing the susceptibility of bacteria to antibiotics and antimicrobial peptides.

Techniques: Membrane, Fluorescence, Incubation

Journal: Nature Communications

Article Title: Self-assembling dipeptide antibacterial nanostructures with membrane disrupting activity

doi: 10.1038/s41467-017-01447-x

Figure Lengend Snippet: The effect of diphenylalanine nanostructure treatment on the bacterial stress response and their proposed mechanism of action. a Treatment with diphenylalanine nanostructures causes upregulation of cellular stress-related genes. E. coli bacteria were transformed with plasmids containing each of the six evaluated genes fused to mCherry and the level of fluorescent signal of mCherry, which correlates to the level of expression of the corresponding gene, was measured following the indicated treatments. b Model of the proposed mechanism of action of the diphenylalanine nanostructures on Gram-negative bacteria. The interaction of the diphenylalanine nanostructures with the bacterial membrane causes outer membrane permeation and inner membrane depolarization, resulting in severe changes to membrane morphology, such as the appearance of nicks and tears, leading to bacterial growth inhibition and cell death

Article Snippet: The strain utilized in this study is E. coli ATCC 25922, a clinical isolate widely used for testing the susceptibility of bacteria to antibiotics and antimicrobial peptides.

Techniques: Bacteria, Transformation Assay, Expressing, Membrane, Inhibition

Journal: Translational Psychiatry

Article Title: Impairment in stimulus-response learning as a cognitive biomarker in a model of synucleinopathy

doi: 10.1038/s41398-025-03795-5

Figure Lengend Snippet: A Schematic of α-synuclein pre-formed fibril production. α-synuclein type 1 pre-formed fibrils (PFF-1) were generated from monomers expressed in E.coli at room temperature overnight, while α-synuclein type 2 pre-formed fibrils (PFF-2) were generated from monomers expressed in E.coli grown at 37 °C for four hours. Following this, both fibril precursors underwent monomer purification and identical fibrilization protocols and were characterized in vitro. B SDS-PAGE of PFF-1 and PFF-2 after sedimentation revealed similar populations of insoluble (pellet) vs soluble (supernatant) materials, both consisting of predominantly insoluble species. C Transmission Electron Microscopy (TEM) of unsonicated PFF-1 and PFF-2 displayed similar architecture and range of fibril sizes, both appearing as elongated fibrils of mixed length. D Far UV circular dichroism (UV-CD) measurements identified a significant β-sheet presence in fibrils compared monomers with subtle differences between PFF-1 and PFF-2s. Thioflavin T (ThT) binding assay revealed a stronger binding capacity of PFF-1 (E) relative to PFF-2 (F) after fibrilization and during a seeding assay with additional monomer.

Article Snippet: Jacob A. McPhail generated and characterized the recombinant alpha-synuclein pre-formed fibrils while an employee of StressMarq Biosciences, and Ariel Louwrier is the founder of StressMarq Biosciences.

Techniques: Generated, Purification, In Vitro, SDS Page, Sedimentation, Transmission Assay, Electron Microscopy, Circular Dichroism, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Anti-amyloid Compounds Inhibit α-Synuclein Aggregation Induced by Protein Misfolding Cyclic Amplification (PMCA) *

doi: 10.1074/jbc.M113.542340

Figure Lengend Snippet: Characterization of α-synuclein PMCA-derived fibrils. A , circular dichroism of recombinant α-synuclein before and after 24-h PMCA. B , transmission electron microscopy of 24-h PMCA ( upper panels ) and incubation ( lower panels ) α-synuclein fibrils in carbon coated grids at two different magnifications. C , Coomassie Blue-stained ( left ) and anti-α-synuclein fluorescence-immunostained ( right ) native gels of recombinant α-synuclein subjected (+) or not (−) to 24-h PMCA. An increase in high molecular mass species is present following PMCA. D , Coomassie Blue-stained ( left ) or anti-α-synuclein immunostained ( right ) Bis-Tris gel before and after 24-h PMCA samples following digestion with different concentrations of PK. Whereas non-PMCA samples contain mainly monomeric protein that is completely degraded by PK, the PMCA-derived samples show specific bands following PK digestion. Asterisk indicates the position of the PK band in the Coomassie Blue-stained gel. E , α-synuclein 24-h PMCA, before (−) and after (+) PK digestion, epitope mapping using several anti-α-synuclein-specific antibodies. F , Coomassie Blue-stained SDS (Bis-Tris) gel of serial α-synuclein 24-h PMCA samples after 2.5 μg/ml PK digestion. No clear significant difference is observed in band pattern after PK digestion in samples from different PMCA passages.

Article Snippet: Three-μl aliquots of α-synuclein PMCA or non-PMCA control samples were either mixed with 1 μl of 4× loading buffer (NuPAGE LDS®; Invitrogen) and incubated at 100 °C for 10 min (for SDS gels) or mixed with 1 μl of 4× native loading buffer (NativePAGE®; Invitrogen), and 3.5 μl of the mixture was loaded into 4–12% SDS (Bis-Tris)- or native gels (Invitrogen).

Techniques: Derivative Assay, Circular Dichroism, Recombinant, Transmission Assay, Electron Microscopy, Incubation, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Anti-amyloid Compounds Inhibit α-Synuclein Aggregation Induced by Protein Misfolding Cyclic Amplification (PMCA) *

doi: 10.1074/jbc.M113.542340

Figure Lengend Snippet: Compound screening using α-synuclein PMCA. A , 16-h α-synuclein ( alpha-syn ) PMCA reaction, alone or in the presence of solvent (DMSO) and two concentrations of 10 different drugs added at the beginning of the reaction. ThT signal is measured as readout for α-synuclein fibril assembly. Data are normalized for α-synuclein signal (α-synuclein) without drugs and presented as mean ± S.D. ( error bars ) of triplicate samples in two independent experiments. B and C , Coomassie Blue staining of SDS (Bis-Tris) gel of the drug-PMCA samples before ( B ) and after ( C ) digestion with 2.5 μg/ml PK. The order of the drugs (as indicated by name abbreviation) corresponds to that in A . The lanes indicated as syn show α-synuclein without drugs. D , ThT signal of defibrillated α-synuclein ( alpha-syn ) PMCA samples following 30-min incubation with 50 μ m concentration of 10 different drugs added to preformed fibrils.

Article Snippet: Three-μl aliquots of α-synuclein PMCA or non-PMCA control samples were either mixed with 1 μl of 4× loading buffer (NuPAGE LDS®; Invitrogen) and incubated at 100 °C for 10 min (for SDS gels) or mixed with 1 μl of 4× native loading buffer (NativePAGE®; Invitrogen), and 3.5 μl of the mixture was loaded into 4–12% SDS (Bis-Tris)- or native gels (Invitrogen).

Techniques: Solvent, Staining, Incubation, Concentration Assay

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a Schematic representation of the different domains of p53. The DBD (residues 102–292) contains an aggregation-nucleating subdomain (residues 251–258) that is necessary and sufficient to drive p53 aggregation , , . Another segment of interest comprises residues 213–217, which is the antigen recognized by the PAb 240 antibody that binds to partially unfolded p53. Also highlighted in the DBD is R248, one of the most common mutation hotspots in p53 (IARC TP53 database; https://p53.iarc.fr ) . b Structure of p53 DBD. Highlighted are the aggregation-nucleating subdomain (green) and the epitope recognized by PAb 240 (red). Both segments are buried in the fully folded p53 structure. The 3D image was generated using PyMOL 2.3.5 (Schrödinger, New York, NY). c Primary sequences of the studied WT and mutant R248W p53 DBD-derived peptides, denoted pWT and pR248W, respectively, which span residues 248–273. The peptides include the aggregation-prone 252–258 sequence, as well as R248 and another of the most common mutation hotspots in p53 and R273 (IARC TP53 database; https://p53.iarc.fr ) . d Chemical structures of the oligopyridylamides ADH-1 and ADH-6. e , f Effects of the oligopyridylamides on pR248W amyloid formation. Kinetic profiles (left panel) and representative transmission electron microscopy (TEM) images (right panel) for aggregation of 25 μM pR248W in the absence or presence of an equimolar amount of ADH-1 or ADH-6 co-mixed at the start of the reaction ( e ) or added during the growth phase (i.e. 5 h after the start of the reaction) ( f ). Kinetic aggregation profiles were acquired by measuring the fluorescence of the thioflavin T (ThT) reporter ( λ ex/em = 440/480 nm) at 5-min intervals at 37 °C ( n = 4). TEM images were acquired at 10 h after the start of the aggregation reaction. Scale bar = 100 nm. g Characterization of the binding interaction of the oligopyridylamides and pR248W measured using steady-state intrinsic tryptophan fluorescence quenching. A 5 µM solution of pR248W was titrated with increasing concentrations of ADH-1 (left panel) or ADH-6 (right panel) and the tryptophan fluorescence after each addition was normalized to account for the dilution (total dilution during the titration was <1%) and plotted against the ligand concentration. The equilibrium dissociation constants ( K d ) were then determined using a one-site-specific binding equation (Eq. ). h Effects of the oligopyridylamides on pR248W oligomerization monitored using the dot blot assay. Samples of 10 μM pR248W were incubated with or without an equimolar amount of ADH-1 or ADH-6 for 0–24 h, and the presence of oligomers was detected using an amyloid oligomer-specific polyclonal antibody (A11) . i Effects of the oligopyridylamides on the self-assembly driven structural transition of pR248W. Time-dependent circular dichroism (CD) spectra of 10 µM pR248W alone (left panel) or in the presence of an equimolar amount of ADH-1 (middle panel) or ADH-6 (right panel).

Article Snippet: Human bone cancer Saos-2 and ovarian cancer SKOV-3 cells were plated at a density of 1 × 10 5 cells/well in 2 mL complete medium in six-well plates and cultured for 48 h. For each well of cells to be transfected, 2.5 μg pCMV-Neo-Bam p53 R175H or pCMV-Neo-Bam p53 R248W vectors (plasmids #16436 or #16437, respectively; Addgene) containing the amp r gene was diluted in 500 μL Opti-MEM Reduced Serum Medium in an Eppendorf tube, to which 10 μL Lipofectamine LTX Reagent was added, mixed gently, and incubated for 30 min at room temperature.

Techniques: Mutagenesis, Generated, Derivative Assay, Sequencing, Transmission Assay, Electron Microscopy, Fluorescence, Binding Assay, Titration, Concentration Assay, Dot Blot, Incubation, Circular Dichroism

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a , b Overlay of 15 N- 1 H HSQC maps of 19 μM WT ( a ) and 24 μM R248W ( b ) p53 DBD in H O/D O (96/4) with 16.7 mM DTT, without (green contours) or with (red contours) ADH-6 addition (protein:ligand 1:11 in a and 1:15 in b ). The assignments are reported only outside the rightmost regions. These regions are crowded because of the presence of partially unfolded species that also interact with ADH-6 as highlighted by the boxed peak in each panel. c HSQC contour maps overlay of mutant R248W p53 DBD at different protein:ADH-6 ratios (1:0 green, 1:8 cyan, and 1:15 red) showing the increment of cumulated chemical shift perturbation (CSP) with ligand concentration (Eq. ). d The five clusters of the two p53 DBD variants (WT and mutant R248W) that show high (>0.025) or medium (>0.015) CSP values . Cluster 1 (highlighted in blue) includes residues T118, Y126, E271, C275, and G279; cluster 2 (highlighted in magenta) includes residues R196, E198, G199, L201, Y220, and E221; cluster 3 (green) includes T102, Y103, Q104, G105, L257, L264, and R267; cluster 4 (orange) includes E171, R174, H179, R209, and G244; and cluster 5 (cyan) includes S94, A161, I162, L206, and S215. Clusters 1 and 2 are at the front in the cartoon on the left; clusters 3–5 are at the front in the cartoon on the right. The 3D image was generated using PyMOL 2.3.5 (Schrödinger, New York, NY).

Article Snippet: Human bone cancer Saos-2 and ovarian cancer SKOV-3 cells were plated at a density of 1 × 10 5 cells/well in 2 mL complete medium in six-well plates and cultured for 48 h. For each well of cells to be transfected, 2.5 μg pCMV-Neo-Bam p53 R175H or pCMV-Neo-Bam p53 R248W vectors (plasmids #16436 or #16437, respectively; Addgene) containing the amp r gene was diluted in 500 μL Opti-MEM Reduced Serum Medium in an Eppendorf tube, to which 10 μL Lipofectamine LTX Reagent was added, mixed gently, and incubated for 30 min at room temperature.

Techniques: Mutagenesis, Concentration Assay, Generated

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a Confocal fluorescence microscopy images showing thioflavin S (ThS) staining of mutant p53 (R248W) aggregates in MIA PaCa-2 cells treated with vehicle (0.02% DMSO) or ADH-6 (5 µM) for 0.5 or 6 h. Imaging experiments were performed in quadruplicate and representative images are shown. b Quantification of ThS-positive MIA PaCa-2 cells after treatment with vehicle or ADH-6. The number of positively stained cells in 3–5 different fields of view are expressed as % of the total number of cells ( n = 4 biologically independent samples). Data presented are mean ± SD. Statistical analysis was performed using two-tailed unpaired t -test. P < 0.0001 for ADH-6 vs vehicle at 6 h. c Confocal fluorescence microscopy images of ThS and PAb 240 antibody staining of R248W aggregates in MIA PaCa-2 cells treated with vehicle or 5 µM ADH-6 for 0.5 or 6 h. Images shown are representative of four independent experiments. d – f Quantification of PAb 240-positive MIA PaCa-2 cells after treatment with the indicated concentrations of ADH-1, ReACp53, or ADH-6 for 0.5 or 6 h relative to controls (vehicle-treated cells). The number of positively stained cells in 3–5 different fields of view are expressed as % of the total number of cells (mean ± SD; n = 4). Statistical analysis was performed using repeated measures two-way ANOVA followed by Holm-Sidak’s post hoc test. P < 0.0001 for ReACp53 (2.5–10 µM) vs vehicle at 6 h ( e ); P < 0.0001 for ADH-6 (2.5–10 µM) vs vehicle at 6 h ( f ). g Colocalization of FITC-labeled ADH-6 (ADH-6 FITC ) with PAb 240-stained R248W aggregates following incubation with the oligopyridylamide (5 µM) for 0.5 or 6 h. Colocalization was quantified using directional Pearson correlation coefficient, r , which measures pixel-by-pixel covariance in the signal level of two images . Scale bar = 5 µm. h , i Cellular thermal shift assay (CETSA) analysis of intracellular target engagement. Melting curves for p53 mutants R248W ( h ) and R175H ( i ) in MIA PaCa-2 and SK-BR-3 cells, respectively, in the absence or presence of the oligopyridylamides (mean ± SD; n = 3). *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls.

Article Snippet: Human bone cancer Saos-2 and ovarian cancer SKOV-3 cells were plated at a density of 1 × 10 5 cells/well in 2 mL complete medium in six-well plates and cultured for 48 h. For each well of cells to be transfected, 2.5 μg pCMV-Neo-Bam p53 R175H or pCMV-Neo-Bam p53 R248W vectors (plasmids #16436 or #16437, respectively; Addgene) containing the amp r gene was diluted in 500 μL Opti-MEM Reduced Serum Medium in an Eppendorf tube, to which 10 μL Lipofectamine LTX Reagent was added, mixed gently, and incubated for 30 min at room temperature.

Techniques: Fluorescence, Microscopy, Staining, Mutagenesis, Imaging, Two Tailed Test, Labeling, Incubation, Thermal Shift Assay, Drug discovery

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a – c Effects of ADH-6 on viability of cancer cells bearing WT or mutant p53. MIA PaCa-2 (mutant R248W p53) ( a ), MCF-7 (WT p53) ( b ), and SK-BR-3 (mutant R175H p53) ( c ), cells treated with increasing oligopyridylamide concentrations for 24 or 48 h. ( d – f ) p53 null Saos-2 cells before ( d ) and after transfection with p53 mutants, R248W ( e ) or R175H ( f ), treated with increasing concentrations of ADH-6 for 24 or 48 h. Cell viability in a – f was assessed using the MTS assay, with the % viability determined form the ratio of the absorbance of the treated cells to the control cells ( n = 3 biologically independent samples). Data presented are mean ± SD. Statistical analysis in a – f was performed using two-tailed unpaired t -test. P < 0.0001 for ADH-6 vs ADH-1 at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( a , c ); P < 0.0001 for ADH-6 treatment of Saos-2/R248W compared with untransfected cells (data shown in d ) at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( e ); P < 0.0001 for ADH-6 treatment of Saos-2/R175H compared with untransfected cells (data shown in d ) at the same compound concentration (2.5–10 µM) and incubation time (24 or 48 h) ( f ). g , h Flow cytometry analysis of annexin V/propidium iodide (PI) staining of MIA PaCa-2 cells that were treated with vehicle (control), or 5 µM ADH-1, ReACp53, or ADH-6, for 24 h. The bottom left quadrant (annexin V−/PI−) represents live cells; bottom right (annexin V+/PI−), early apoptotic cells; top right (annexin V+/PI+), late apoptotic cells; and top left (annexin V−/PI+), necrotic cells ( g ). A summary of the incidence of early/late apoptosis and necrosis in the different treatment groups determined from the flow cytometry analysis of annexin V/PI staining (mean ± SD; n = 4) ( h ). Statistical analysis in h was performed using one-way ANOVA followed by Dunnett’s post hoc test. P < 0.0001 for ReACp53 vs vehicle (live and early apoptosis); P < 0.0001 for ADH-6 vs vehicle (live, early apoptosis, and late apoptosis). i Cell cycle distribution of MIA PaCa-2 cells treated w i th vehicle (control), or 5 µM ADH-1, ReACp53 or ADH-6, for 6 h as determined by measurement of DNA content using flow cytometry (mean ± SD; n = 4). Two-tailed unpaired t -test: P = 0.0071 and 0.0037 for ReACp53 vs vehicle (G0/G1 and G2/M, respectively); P < 0.0001 and P = 0.0004 for ADH-6 vs vehicle (G0/G1 and G2/M, respectively). ** P < 0.01, *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls.

Article Snippet: Human bone cancer Saos-2 and ovarian cancer SKOV-3 cells were plated at a density of 1 × 10 5 cells/well in 2 mL complete medium in six-well plates and cultured for 48 h. For each well of cells to be transfected, 2.5 μg pCMV-Neo-Bam p53 R175H or pCMV-Neo-Bam p53 R248W vectors (plasmids #16436 or #16437, respectively; Addgene) containing the amp r gene was diluted in 500 μL Opti-MEM Reduced Serum Medium in an Eppendorf tube, to which 10 μL Lipofectamine LTX Reagent was added, mixed gently, and incubated for 30 min at room temperature.

Techniques: Mutagenesis, Transfection, MTS Assay, Control, Two Tailed Test, Concentration Assay, Incubation, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

doi: 10.1038/s41467-021-23985-1

Figure Lengend Snippet: a , b In vivo pharmacokinetics of ADH-6. Concentration of ADH-6 in plasma ( a ) and in MIA PaCa-2 xenografts ( b ) of mice ( n = 5–6 per group), after an intraperitoneal injection of the oligopyridylamide (15 mg kg -1 ), was quantified using LC-MS/MS . Shown are the circulation half-life ( T 1/2 ) ( a ) as well as the maximum (or peak) concentration ( C max ) in tumors and the time to achieve C max ( T max ) ( b ). Data presented are mean ± SD. c , d Design of the tumor reduction studies. A representative mouse bearing both MIA PaCa-2 (mutant R248W p53) and MCF-7 (WT p53) xenografts ( c ) and treatment schedule for the dual xenograft model ( d ). Once the tumor volume reached ~25 mm , the mice were randomized into the different treatment groups ( n = 8 per group), which were injected intraperitoneally with vehicle (0.02% DMSO), ReACp53 (716.4 µM), or ADH-6 (716.4 µM). Injections were done every 2 days for a total of 12 doses, with the first day of treatment defined as day 0. e Body weight changes of the tumor-bearing mice in the different treatment groups monitored for the duration of the experiment (mean ± SD; n = 8). f , g Tumor volume growth curves for the MIA PaCa-2 ( f ) and MCF-7 ( g ) xenografts in the different treatment groups over the duration of the experiment (mean ± SD; n = 8). Tumor volume was calculated using Eq. . Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 ( f ). h , i Tumor mass analysis for the different treatment groups. After 25 days of treatment, four mice per treatment group were sacrificed and the tumor tissues were isolated and imaged ( h ) and subsequently weighed to determine the tumor mass ( i ). Data presented are mean ± SD, and statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 (MIA PaCa-2 xenografts; i ). j Hematoxylin and eosin (H&E)-stained xenograft sections from the different treatment groups following 25 days of treatment. Images on the right are magnified views of the boxed regions in the images on the left. Scale bar = 20 μm (50 μm for the magnified views). k – m Immunohistochemistry (IHC) analysis of the residual xenografts. Images of sections of MIA PaCa-2 and MCF-7 xenografts stained using the anti-p53 PAb 240 and DO-7 antibodies, respectively, from the different treatment groups ( k ). Images on the right are magnified views of the boxed regions in the images on the left. Scale bar = 20 μm (50 μm for the magnified views). Quantification of PAb 240 ( l ) and DO-7 ( m ) positive cells in 3–5 different fields of view expressed as % of the total number of cells (mean ± SD; n = 4). One-way ANOVA followed by Tukey’s post hoc test: P < 0.0001 for ADH-6 vs vehicle, ReACp53 vs vehicle and ADH-6 vs ReACp53 ( l ). n Survival curves for the vehicle, ReACp53 and ADH-6 treatment groups over 30 days ( n = 4 per group). Statistical analysis was performed using log-rank (Mantel-Cox) test. P = 0.0062 for ADH-6 vs vehicle. ** P < 0.01, *** P < 0.001 or non-significant (n.s., P > 0.05) for comparisons with vehicle-treated controls and between the different treatment groups.

Article Snippet: Human bone cancer Saos-2 and ovarian cancer SKOV-3 cells were plated at a density of 1 × 10 5 cells/well in 2 mL complete medium in six-well plates and cultured for 48 h. For each well of cells to be transfected, 2.5 μg pCMV-Neo-Bam p53 R175H or pCMV-Neo-Bam p53 R248W vectors (plasmids #16436 or #16437, respectively; Addgene) containing the amp r gene was diluted in 500 μL Opti-MEM Reduced Serum Medium in an Eppendorf tube, to which 10 μL Lipofectamine LTX Reagent was added, mixed gently, and incubated for 30 min at room temperature.

Techniques: In Vivo, Drug discovery, Concentration Assay, Clinical Proteomics, Injection, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Isolation, Staining, Immunohistochemistry