Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: 'ZP domain' of human zona pellucida glycoprotein-1 binds to human spermatozoa and induces acrosomal exocytosis

doi: 10.1186/1477-7827-8-110

Figure Lengend Snippet: Profile of lectins binding to baculovirus-expressed human ZP1 273-551aa in ELISA . Microtitration plates were coated with the baculovirus-expressed recombinant ZP1 273-551aa (open bar; 500 ng/well) and processed for evaluation of binding to 21 different biotinylated lectins in an ELISA as described in Methods . As an internal control, microtitration plates coated with same amount of E. coli -expressed recombinant human ZP1 273-551aa (grey bar; unpublished observations) were also used. Values are expressed as absorbance obtained with various lectins binding to the respective recombinant protein, after deducting the non-specific binding of the lectins to the uncoated wells. Each bar represents a mean of duplicate experiments and standard deviation of the absorbance values. The lectins tested were GSL-I: Griffon simplicifolia lectin I, SBA: Soybean agglutinin, DSL: Datura stramonium lectin, PSA: Pisum sativum agglutinin, ConA: Concanavalin A, ECL: Erythrina cristagalli lectin, LCA: Lens culinaris agglutinin, DBA: Dolichos biflorus agglutinin, LEL: Lycopersicon esculentum lectin, PHA-L: Phaseolus vulgaris leucoagglutinin, UEA-I: Ulex europaeus agglutinin I, STL: Solanum tuberosum lectin, PHA-E: Phaseolus vulgaris erythroagglutinin, RCA: Ricinus communis agglutinin, VVA: Vicia villosa agglutinin, SJA: Sophora japonica agglutinin, PNA: Peanut agglutinin, S- WGA: Succinylated Wheat germ agglutinin, Jacalin, WGA: Wheat Germ agglutinin and GSL II: Griffonia simplicifolia lectin II.

Article Snippet: The plate was blocked with 0.1% Tween-20 in PBS (PBST, 200 μl/well) for 90 min at 37°C followed by incubation with 21 biotinylated lectins (1 μg/ml; 100 μl/well) at 37°C for 1 h. The biotinylated lectins available in the Lectin kit-I, II and III (Vector Laboratories, Burlingame, CA, USA) were used in the lectin binding assay.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Immunity

Article Title: A vaccine-induced public antibody protects against SARS-CoV-2 and emerging variants

doi: 10.1016/j.immuni.2021.08.013

Figure Lengend Snippet:

Article Snippet: NEBNext Immune Sequencing Kit (Human) , New England Biolabs , Cat# E6320.

Techniques: Recombinant, Sequencing, Cell Isolation, Transfection, Infection, Variant Assay, Expressing, Software, Enzyme-linked Immunosorbent Assay