Journal: Scientific Reports

Article Title: Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

doi: 10.1038/srep20069

Figure Lengend Snippet: ( a ) Caco-2 cells were infected with an EGFP-expressing GAS NIH35 strain as green images at an MOI of 10 for 2 h in the presence or absence of 2 μM human PLG. Tricellulin was labeled with anti-tricellulin and Alexa Fluor 594-conjugated antibodies as red images, whereas ZO-1 was labeled with anti-ZO-1 and Alexa Fluor 647-conjugated antibodies as blue images. GAS-infected cells were analyzed using a confocal laser-microscope. White arrows indicate bacterial association with tricellulin. Data shown are representatives of at least three separate experiments. ( b ) Bacterial localization in tTJs was assessed as described in the Methods section. Data obtained from ten fields of view (x630) are presented as the mean ± S.D. * P < 0.01.

Article Snippet: Bound PLG was detected using an antibody against human PLG (mouse, mAb, R&D systems) and an HRP-conjugated antibody against mouse IgG, as described above.

Techniques: Infection, Expressing, Labeling, Microscopy

Journal: Scientific Reports

Article Title: Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

doi: 10.1038/srep20069

Figure Lengend Snippet: ( a ) Caco-2 cells were transfected with tricellulin-targeted siRNA. A scrambled siRNA was utilized as a negative control. At 72 h after transfection, whole cell lysates were subjected to Western blot analysis using anti-tricellulin antibody. β-actin served as a loading control. ( b ) Tricellulin knockdown or control cells were infected with EGFP-expressing GAS strain at an MOI of 10 for 2 h in the presence of 2 μM human PLG. ZO-1 and tricellulin were immunostained and are shown as blue and red images, respectively. GAS-infected cells were analyzed using a confocal laser-microscope. Data shown are representatives of at least three separate experiments. ( c ) Bacterial localization in tTJs, bTJs, or other regions was assessed as described in the Methods section. Data obtained from ten fields of view (x630) are presented as the mean ± S.D. * P < 0.01.

Article Snippet: Bound PLG was detected using an antibody against human PLG (mouse, mAb, R&D systems) and an HRP-conjugated antibody against mouse IgG, as described above.

Techniques: Transfection, Negative Control, Western Blot, Control, Knockdown, Infection, Expressing, Microscopy

Journal: Scientific Reports

Article Title: Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

doi: 10.1038/srep20069

Figure Lengend Snippet: TRIC proteins ( a ) or TRIC-EC1 variants ( b ) were immobilized on microtiter plates, then increasing amounts of SEN were added in the absence or presence of 1 μM human PLG. Bound SEN was detected using an anti-SEN antibody. All experiments were performed in sextuplet with three technical repeats. Data are shown as the mean ± S.D. of six wells from a representative experiment. * P < 0.01.

Article Snippet: Bound PLG was detected using an antibody against human PLG (mouse, mAb, R&D systems) and an HRP-conjugated antibody against mouse IgG, as described above.

Techniques:

Journal: Scientific Reports

Article Title: Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

doi: 10.1038/srep20069

Figure Lengend Snippet: (a) Schematic diagram of SEN protein. Internal and C-terminus PLG-binding motifs are shown as green and pink images, respectively. The amino acid sequences of the PLG-binding motif in each strain are described below. ( b ) NIH35 and isogenic sen mutants were grown in THY broth. The culture densities were measured at 37 °C. ( c ) The strains were grown to an OD 600 of 0.8 in THY broth, then each fraction was prepared, as described in Methods. ( d ) NIH35 and the isogenic sen mutants (OD 600 = 0.6) were bound to microtiter plates, and bound cells were incubated with 1 μM human PLG. Cell-bound PLG was detected by ELISA using an anti-PLG antibody. Data are shown as the mean ± S.D. of six samples from a representative experiment. * P < 0.05; ** P < 0.01.

Article Snippet: Bound PLG was detected using an antibody against human PLG (mouse, mAb, R&D systems) and an HRP-conjugated antibody against mouse IgG, as described above.

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

doi: 10.1038/srep20069

Figure Lengend Snippet: ( a ) Caco-2 cells were grown using a Millicell filter system, and then infected with the NIH35 strain or sen mutants at an MOI of 10 for 2 h. After removing non-adherent bacteria, the ability of the GAS strains to translocate across epithelial cells at 8 h after infection in the presence of bovine PLG was assessed by examining medium samples obtained from the lower chambers. Data are shown as the mean ± S.D. of six wells from a representative experiment. * P < 0.01. ( b ) Human and bovine PLG were immobilized on a nitrocellulose membrane after serial two-fold dilutions. After blocking of the membrane, 40 μg of SEN was overlaid and the binding signal was detected with an anti-SEN antibody. ( c ) TRIC-EC1 protein was immobilized on microtiter plates, and increasing amounts of SEN were reacted in the presence of 1 μM human PLG or 1 μM bovine PLG. Bound SEN was detected using an anti-SEN antibody. Data are shown as the mean ± S.D. from three independent experiments. * P < 0.01. ( d ) The effect of human PLG on bacterial translocation was analyzed as described in ( a ). Data are shown as the mean ± S.D. of six wells from a representative experiment. * P < 0.01.

Article Snippet: Bound PLG was detected using an antibody against human PLG (mouse, mAb, R&D systems) and an HRP-conjugated antibody against mouse IgG, as described above.

Techniques: Infection, Bacteria, Membrane, Blocking Assay, Binding Assay, Translocation Assay

Journal: Scientific Reports

Article Title: Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

doi: 10.1038/srep20069

Figure Lengend Snippet: (a) Effects of bovine or human PLG on reduction of TER in cells infected with GAS strains for 8 h. The TER value of the non-infected cells was set to 100%. All experiments were performed in sextuplet with three technical repeats. Data are shown as the mean ± S.D. of six wells from a representative experiment. * P < 0.01. (b) Caco-2 cells (left panel) or Detroit 562 cells (right panel) were infected with NIH35 or sen mutants at an MOI of 10 for 7 h in the presence of 1 μM human PLG. Cleavage of ZO-1, occludin, tricellulin, and E-cadherin was detected in whole cell lysates by Western blot analysis. β-actin served as a loading control.

Article Snippet: Bound PLG was detected using an antibody against human PLG (mouse, mAb, R&D systems) and an HRP-conjugated antibody against mouse IgG, as described above.

Techniques: Infection, Western Blot, Control

Journal: Journal of Thrombosis and Haemostasis

Article Title: Non‐severe COVID‐19 is associated with endothelial damage and hypercoagulability despite pharmacological thromboprophylaxis

doi: 10.1111/jth.15660

Figure Lengend Snippet: Plasma tissue plasminogen activator (t‐PA) levels are significantly elevated in coronavirus disease 2019 (COVID‐19). Plasma levels of t‐PA (A) were significantly elevated in hospitalized patients with non‐severe COVID‐19 ( n = 14) compared to a group of hospitalized patients ( n = 11) who had screened negative for severe acute respiratory syndrome coronavirus 2 by real‐time polymerase chain reaction. Levels of soluble thrombomodulin (sTM; B), vascular cell adhesion molecule‐1 (VCAM‐1; C), intercellular adhesion molecule‐1 (ICAM‐1; D), plasminogen activator inhibitor‐1 (PAI‐1; E) and E‐selectin (F) were similar in both groups. All samples were assayed in technical duplicate. Data are presented as the mean ± standard deviation. Statistical analysis was performed using a two‐tailed Student’s t test. * P < .05

Article Snippet: ELISAs for TFPI (DTFP10), tissue plasminogen activator (t‐PA; DTPA00), thrombomodulin (DTHBD0), vascular cell adhesion molecule‐1 (VCAM‐1; DVC00), intercellular adhesion molecule‐1 (ICAM‐1; DCIM00), and E‐selectin (DSLE00) were purchased from R&D Systems and a plasminogen activator inhibitor‐1 (PAI‐1; ab108891) ELISA was purchased from Abcam.

Techniques: Clinical Proteomics, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

Journal: CNS neuroscience & therapeutics

Article Title: Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator-Induced Brain Hemorrhagic Transformation After Ischemic Stroke.

doi: 10.1111/cns.70345

Figure Lengend Snippet: FIGURE 3 | AEP KO ameliorates tPA-associated hemorrhagic transformation in the stroke mouse model. (A, B) Representative images of hema- toma in brain slices and quantification of hemoglobin levels. Data are presented as mean ± SEM and statistical analyses are performed using Welch test followed by Dunnett T3 multiple comparisons test were applied since the P value of Levene test < 0.05. (C) Recorded brain water content to as- sess brain edema at 24 h after delayed tPA administration. Data are presented as mean ± SEM and statistical analysis is performed using one-way ANOVA test followed by Tukey's multiple comparisons test. (D) ELISA for tPA activity detection. Data are presented as mean ± SEM and statistical analyses are performed using Welch test followed by Dunnett T3 multiple comparisons test were applied since the P value of Levene test < 0.05. (E) Representative images of HE staining in the cortical hematoma area (scale bar = 50 μm). (A), (C) n = 6, (D) n = 4 per group. Normality and variance are assessed via Shapiro-Wilk test and Levene's test, respectively. *P < 0.05, **P < 0.01.

Article Snippet: A human Tissue- type Plasminogen Activator ELISA kit (E- EL- H2106, Elabscience) was used for detection.

Techniques: Transformation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Staining

Journal: Parasitology

Article Title: NumerousFasciolaplasminogen-binding proteins may underlie blood-brain barrier leakage and explain neurological disorder complexity and heterogeneity in the acute and chronic phases of human fascioliasis

doi: 10.1017/s0031182018001464

Figure Lengend Snippet: Fig. 1. Plasminogen binding to 1 µg of FhES extract measured over a range of plas- minogen amounts using a microtitre plate method: (■) Incubation with increasing amounts of plasminogen, 0–3 µg. (●) Competition assay with 50 mM ϵACA included during plasminogen incubation. (▲) Negative control consisted of wells coated only with BSA. Each point is the mean of three replicates ± S.D. The asterisk (*) designates significant (P < 0.05) differences.

Article Snippet: In each well, 2 μg of human plasminogen (Acris Antibodies) were incubated in PBS with 3 μg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 μg of FhES.

Techniques: Binding Assay, Incubation, Competitive Binding Assay, Negative Control

Journal: Parasitology

Article Title: NumerousFasciolaplasminogen-binding proteins may underlie blood-brain barrier leakage and explain neurological disorder complexity and heterogeneity in the acute and chronic phases of human fascioliasis

doi: 10.1017/s0031182018001464

Figure Lengend Snippet: Fig. 2. Plasminogen activation and plasmin generation by FhES extract of Fasciola hepatica: (□) 15 ng of t-PA was added to mixtures containing 2 µg of human plas- minogen, 3 µg of D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) and 1 µg of FhES extract (or BSA as negative control) in the presence or absence of 50 mM of εACA in a test volume of 100 µL. (■) No t-PA was added to reaction mixtures. Each point is the mean of three replicates ± S.D. The asterisk (*) designates significant (P < 0.05) differences.

Article Snippet: In each well, 2 μg of human plasminogen (Acris Antibodies) were incubated in PBS with 3 μg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 μg of FhES.

Techniques: Activation Assay, Negative Control

Journal: Parasitology

Article Title: NumerousFasciolaplasminogen-binding proteins may underlie blood-brain barrier leakage and explain neurological disorder complexity and heterogeneity in the acute and chronic phases of human fascioliasis

doi: 10.1017/s0031182018001464

Figure Lengend Snippet: Fig. 3. Two-dimensional electrophoresis of the FhES and ligand blotting with plasminogen: (A) Representative 2-DE of 60 µg of the FhES extract from adult F. hep- atica flukes. The gels were in the 3–10 pH range, 12% polyacrylamide and silver-stained. (B) Ligand blotting assay to determine which proteins of FhES extract bind plasminogen. The plasminogen-binding spots revealed are circled and numbered. Reference molecular masses are indicated on the left.

Article Snippet: In each well, 2 μg of human plasminogen (Acris Antibodies) were incubated in PBS with 3 μg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 μg of FhES.

Techniques: Electrophoresis, Staining, Binding Assay

Journal: Parasitology

Article Title: NumerousFasciolaplasminogen-binding proteins may underlie blood-brain barrier leakage and explain neurological disorder complexity and heterogeneity in the acute and chronic phases of human fascioliasis

doi: 10.1017/s0031182018001464

Figure Lengend Snippet: Fig. 4. Quantitative analyses of the FhES extract by Sypro Ruby florescent dye: Representative 2-DE of 60 µg of the FhES extract from adult F. hepatica flukes. The gels were in the 3–10 pH range, 12% polyacrylamide and Sypro Ruby-stained. Note density of proteins as the appearance of clear spots on a dark background, which is directly proportional to the amount of each protein into the gel. The plasminogen- binding spots revealed on the ligand blotting assay are circled and numbered.

Article Snippet: In each well, 2 μg of human plasminogen (Acris Antibodies) were incubated in PBS with 3 μg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 μg of FhES.

Techniques: Staining, Binding Assay

Journal: Parasitology

Article Title: NumerousFasciolaplasminogen-binding proteins may underlie blood-brain barrier leakage and explain neurological disorder complexity and heterogeneity in the acute and chronic phases of human fascioliasis

doi: 10.1017/s0031182018001464

Figure Lengend Snippet: Fig. 5. Schematic representation of the interaction between infecting Fasciola behaviour, fibrinolysis system alterations trig- gering subsequent blood-brain barrier leakages and contact system alterations inducing systemic vasculitis: (A) In the acute phase of the disease in cases of many simultaneously migrating, small-sized juvenile flukes after ingestion of numer- ous metacercariae. (B) In the chronic phase of the disease after the release of large amounts of accumulated excretory/ secretory products following the breakage of encapsulating for- mations triggered by single worm tracks at different times. (C) Blood-brain barrier leakages subsequently occurring due to fibrinolytic system-dependent mechanism involving plasmin- dependent generation of bradykinin and subsequent activation of bradykinin B2 receptors, according to different plasminogen- binding protein agglomeration waves. (D) Inflammation and dilation of blood vessels due to contact system-dependent gen- eration of the proinflammatory peptide bradykinin. Schema design and drawing by S. Mas-Coma.

Article Snippet: In each well, 2 μg of human plasminogen (Acris Antibodies) were incubated in PBS with 3 μg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 μg of FhES.

Techniques: Activation Assay, Binding Assay