Journal: International Journal of Molecular Sciences

Article Title: Small-Molecule Cyclophilin Inhibitors Potently Reduce Platelet Procoagulant Activity

doi: 10.3390/ijms24087163

Figure Lengend Snippet: Small-molecule cyclophilin inhibitors inhibit coagulation induced by procoagulant platelets. ( A ) Whole blood was incubated with 125 µM F759 or F83236, 4 µM CsA before perfusion over a Horm collagen-coated surface in the presence of 6.6 mM CaCl 2 and 3.08 mM MgCl 2 . Shown are representative images after 9 min of top row: DIC images of platelet aggregates; middle row: anti-GpIb-VhH Alexa647 indicating platelets and bottom row: tPA-Alexa488 binding to fibrin. ( B ) Quantification of Δ MFI anti-GpIb-VhH Alexa647 signal during 9 min. ( C ) MFI tPA-Alexa488 over time. ( D ) Quantification of Δ MFI tPA-Alexa488 signal during 9 min. Data represented as mean ± SD of 5–7 separate experiments. * p < 0.05 compared to vehicle by One-Way ANOVA. SMCypIs, small-molecule cyclophilin inhibitors; CsA, Cyclosporin A; tPA, tissue plasminogen activator.

Article Snippet: Images were analyzed for the formation of fibrin via Alexa488 fluorescent intensity by the Image Analysis module within the ZEN2 Pro software (ZEISS Microscopy, Jena, Thüringen, Germany).

Techniques: Coagulation, Incubation, Binding Assay

Journal: PLoS Pathogens

Article Title: EhC2B, a C2 domain-containing protein, promotes erythrophagocytosis in Entamoeba histolytica via actin nucleation

doi: 10.1371/journal.ppat.1008489

Figure Lengend Snippet: (A) pEhEx-GFP (vector control) and pEhEx-GFPEhC2B transfected amoebic trophozoites were grown at 20μg ml -1 G418, incubated in glass wells for 15mins at 37°C, fixed and visualised using confocal microscopy. (B) The fluorescence intensity of GFPEhC2B was analysed across the amoebic trophozoites. The line intensity plot shows the change in the fluorescence intensity with the distance. (C) Total cell lysates (400μg) from GFPEhC2B and GFP (vector control) transfected amoebic trophozoites were analysed on SDS-PAGE followed by immunoblotting using anti-GFP antibody and anti-EhCS1 antibody. (D) Pseudopodal localization of EhC2B. The red square represents the magnified view. (E) Relative fluorescence intensities of EhC2B were quantified in pseudopod and non-pseudopod regions of stably-expressing trophozoites. Data Points shown in the graph represent mean, and SEM (error bars) for N = 50 cells and statistical significance is shown by ***P≤0.001. (F) The localisation of GFPEhC2B in amoebic trophozoites in the presence of BAPTA-AM or DMSO. (G) Normalised peripheral fluorescence intensities are plotted for GFPEhC2B cells in the presence of BAPTA-AM or DMSO (N = 30 cells). Statistical significance is shown by **P≤ 0.01. (H) The localisation of GFPEhC2B in amoebic trophozoites in the wild-type EhC2B strain and its calcium (D70N and D72N) and lipid mutants (K76A and N77A) cell lines. (I) Amoebic trophozoites stably expressing GFPEhC2B were incubated with Cell Tracker Orange-labelled RBCs (1:50) for 10mins at 37°C. Trophozoites were then fixed and later studied by confocal microscopy. The yellow arrow indicates the absence of GFPEhC2B at phagosome. (J) Montage of live imaging of GFPEhC2B expressing trophozoite ingesting CHO cells by trogocytosis. The time 0 min represents the initiation of the trogocytic cup. All scale bar , 5μm. (K) FRAP study of GFP-EhC2B, localising on phagocytic cups. Pre-bleached fluorescence intensity of GFPEhC2B from the region of interest was measured and assigned as 100%. The region of interest, as shown by the yellow circle was photobleached, and the recovery of the fluorescence intensity was monitored as described in ‘Materials and Methods.’ The corresponding magnified view of the area of interest is shown by square below . (L) The recovery of fluorescence was fitted into an exponential model. Recovery curve shows the corresponding times for 50% recovery (τ 1/2 ), and values are shown by Mean and SEM (error bars) for 6 independent cells. (All scale bars , 5μm; DIC, Differential interference contrast).

Article Snippet: The reaction was transferred to the cuvette immediately after the preparation, and fluorescence intensity was measured at 365nm excitation and 407nm emission wavelength (Horiba, Scientific).

Techniques: Plasmid Preparation, Control, Transfection, Incubation, Confocal Microscopy, Fluorescence, SDS Page, Western Blot, Stable Transfection, Expressing, Imaging

Journal: PLoS Pathogens

Article Title: EhC2B, a C2 domain-containing protein, promotes erythrophagocytosis in Entamoeba histolytica via actin nucleation

doi: 10.1371/journal.ppat.1008489

Figure Lengend Snippet: Colocalization of GFPEhC2B with actin at (A) Phagocytic cup (B) Pseudopod. The GFPEhC2B overexpressing amoebic trophozoites were immunostained with anti-actin antibody followed by Alexa568 labelled phalloidin and visualised under a confocal microscope. (C) His pull-down assay for direct interaction between EhC2B and actin. One micromolar of MBP-EhC2B, His-EhC2B121, MBP or His-mDia1 was immobilized on Nickle-NTA beads and incubated with an equimolar concentration of monomeric rabbit muscles actin at 4°C. The binding was detected by immunoblotting using an anti-actin antibody. (D) Pyrene-actin nucleation assay. 0.25μM pyrene-labelled of rabbit muscles monomeric actin was mixed with 47 μl of G buffer (10 mM Tris pH 8.0, 0.2 mM ATP, 0.2 mM DTT,0.2 mM CaCl 2 ), 6μl of exchange buffer (10 mM EGTA, 1 mM MgCl 2 ), and incubated for two minutes at room temperature. Finally, 5μl of ion mixture (1 M KCl, 40 mM MgCl 2 , 10 mM ATP) was added to the reaction, and actin polymerization was initiated. The fluorescence intensity of polymerization was measured for 600 seconds in the presence of a different concentration of EhC2B, EhC2B121 (C2 domain), mDia1 and MBP at 365nm excitation and 407nm emission wavelength. (E) Real-time actin polymerization using TIRF microscopy. Assembly of actin monomer was initiated from 0.1μM of rabbit muscles actin by the combination of G-buffer (10 mM Tris (pH8.0) 0.2 mM ATP, 0.2 mM DTT, 0.2 mM CaCl 2 ), exchange buffer (10 mM EGTA, 1 mM MgCl 2 ), and ion mix (1 M KCl, 40 mM MgCl2, 10 mM ATP) as mentioned in ‘ Material and Methods' section. The actin filaments were stained with 30nM of phalloidin labelled with Alexa-568. The polymerization of actin monomer was monitored for 30 minutes in real time under TIRF microscope (Nikon T i 2) using 60X objective lens with 1.5X zoom ( – Movies). To detect the effect of EhC2B on actin dynamics, the actin nucleation reactions were set in the presence of 0.1 μM of (F) MBP-EhC2B (G) HisEhC2B121 (H) mDia1 (I) MBP (All s cale bars , 5 μm).

Article Snippet: The reaction was transferred to the cuvette immediately after the preparation, and fluorescence intensity was measured at 365nm excitation and 407nm emission wavelength (Horiba, Scientific).

Techniques: Microscopy, Pull Down Assay, Incubation, Concentration Assay, Muscles, Binding Assay, Western Blot, Fluorescence, Staining

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: Molecular model of mannose/ConA-A488 binding. Mannose with α-5-pentylphosphonic acid was covalently bound to the ACG slide surface. Fluorescence-tagged concanavalin A (ConA-A488) was then bound to mannose specifically. Con A is a lectin tetramer with subunit dimension of 42 × 40 × 39 Å 3 . Each subunit has a mannose binding site. Geometrically, only two binding sites per molecule are available for mannose binding. The most effective mannose/ConA-A488 binding should give the strongest fluorescence intensity, and this model system has been used to optimize the AAO surface for the glycan microarray.

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Binding Assay, Fluorescence, Glycoproteomics, Microarray

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: (A) Changes in electrical current for the fabrication of ACG slide no. 5. (B) Image of fluorescence intensities that resulted from the mannose/ConA-A488 binding of ACG slide no. 5. This image shows a 10 × 12 matrix with 10 repeated arrays, with each column of the mannose solution concentration varied (in consecutively 10 times dilution) from 100 mM to 1 pM. (C) Response surface of the modified quadratic model for AAO thickness transformed into a function of voltage and reaction time, Y AAO thickness = a × ( V ) 1/2 + b × (RT) 1/2 ; intercept ≠ 0 ( P < 0.0001). (D) Response surface fluorescence intensity with respect to 100 μM sugar concentration arrayed on the ACG slide surfaces. (E) Response surface of B max (1 μM to 100 mM) derived from model fitting, Y B max = a × V + b × V 2 (with significant P value) given in Table S5 . Both (D) and (E) show an optimal curvature of high fluorescence intensity within the range of this study.

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Fluorescence, Binding Assay, Concentration Assay, Modification, Transformation Assay, Derivative Assay

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: (A) On-time measurement of the AAO layer formation under various voltages and reaction times. The starting pure Al-coated glass slide was as smooth as glass. The AAO growth (thickness increasing) depends on the voltage at the beginning, and electropolishing (surface smoothing) and extended AAO growth occur later. (B) Fluorescence intensity differences in ACG slide no.5 of RSM (response surface measurement) study vs NHS glass slide at various sugar solution concentrations arrayed on the surface.

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Fluorescence

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: (A) GenePix scanning images (at PMT 380) of ConA-A488 bound to mannose (1 mM) on ACG slide (A) vs that on NHS glass slide (B). (C) ConA-A488/mannose (1 mM) binding on ACG slide vs NHS-activated glass slide. The fluorescence intensities of the averaged 20 spots for ACG slide vs NHS glass slide. (D) Spot analysis of ACG slides vs NHS-activated glass slides. (E) Confocal microscope (Leica SP8) images of ConA-A488/mannose binding on ACG slide (left) vs NHS-activated glass slide (right).

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Binding Assay, Fluorescence, Microscopy

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: Optimization Experiment—Factors, Voltage (volt), Reaction Time (s) and Responses of AAO Layer Thickness (nm), Electrical Current (mA), Fluorescence Intensity of 100 μM Mannose Solution Arrayed on Each Slide Surface, and B max Derived from Michaelis–Menten Equation Using GraphPad Prism7.0

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Fluorescence, Derivative Assay

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: Optimized Reaction Condition for Making the ACG Slide from an Al-Coated Glass Slide Based on the Fluorescence Intensity Resulting from Mannose/ConA-A488 Binding and B max Analysis

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Fluorescence, Binding Assay