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

Journal: Journal of Neuroinflammation

Article Title: Validation of semaphorin 7A and ala-β-his-dipeptidase as biomarkers associated with the conversion from clinically isolated syndrome to multiple sclerosis

doi: 10.1186/s12974-014-0181-8

Figure Lengend Snippet: Clinical information and CSF characteristics of CIS patients included in the study

Article Snippet: Levels of plasminogen were measured with the AssayMax Human Plasminogen ELISA Kit (EP1200-1; Assaypro, St Charles, Massachusetts, United States) following 1:100 and 1:20000 dilutions in CSF and serum samples respectively.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Validation of semaphorin 7A and ala-β-his-dipeptidase as biomarkers associated with the conversion from clinically isolated syndrome to multiple sclerosis

doi: 10.1186/s12974-014-0181-8

Figure Lengend Snippet: Clinical information and CSF characteristics of validation cohorts of CIS patients

Article Snippet: Levels of plasminogen were measured with the AssayMax Human Plasminogen ELISA Kit (EP1200-1; Assaypro, St Charles, Massachusetts, United States) following 1:100 and 1:20000 dilutions in CSF and serum samples respectively.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Validation of semaphorin 7A and ala-β-his-dipeptidase as biomarkers associated with the conversion from clinically isolated syndrome to multiple sclerosis

doi: 10.1186/s12974-014-0181-8

Figure Lengend Snippet: Serum levels of apolipoprotein AI, apolipoprotein AIV, vitronectin, and plasminogen. Boxplots showing serum protein levels determined by ELISA in CIS patients who converted to CDMS and CIS who remained with CIS. Outliers are represented with asterisks. Statistically significant p-values are shown in bold. CIS → CIS: CIS patients who did not convert to clinically definite MS during the follow-up period. CIS → CDMS: CIS patients who converted to clinically definite MS. C: controls with other neurological disorders. Numbers in parentheses indicate individuals available for analysis. CDMS, clinically definite multiple sclerosis; CIS, clinically isolated syndromes, CSF, cerebrospinal fluid.

Article Snippet: Levels of plasminogen were measured with the AssayMax Human Plasminogen ELISA Kit (EP1200-1; Assaypro, St Charles, Massachusetts, United States) following 1:100 and 1:20000 dilutions in CSF and serum samples respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation

Journal: Journal of Neuroinflammation

Article Title: Validation of semaphorin 7A and ala-β-his-dipeptidase as biomarkers associated with the conversion from clinically isolated syndrome to multiple sclerosis

doi: 10.1186/s12974-014-0181-8

Figure Lengend Snippet: Validation of apolipoprotein AI, semaphorin 7A, and ala-beta-his-dipeptidase as biomarkers associated with conversion to CDMS in a second and totally independent cohort of CIS patients. Boxplots depicting CSF levels of apolipoprotein AI determined by ELISA, and CSF abundance of CNDP1 and semaphorin 7A determined by selected reaction monitoring in MS converters and non-converters to MS. Statistically significant P values are shown in bold. *Indicates that significant differences were also observed between CIS → CDMS and controls ( P = 2.9 × 10 −3 ). CIS → CIS: CIS patients who did not convert to clinically definite MS during the follow-up period. CIS → CDMS: CIS patients who converted to clinically definite MS. C: controls with other neurological disorders. Numbers in parentheses indicate individuals available for analysis. CDMS, clinically definite multiple sclerosis; CDNP1, ala-beta-his-dipeptidase; CIS, clinically isolated syndromes, CSF, cerebrospinal fluid.

Article Snippet: Levels of plasminogen were measured with the AssayMax Human Plasminogen ELISA Kit (EP1200-1; Assaypro, St Charles, Massachusetts, United States) following 1:100 and 1:20000 dilutions in CSF and serum samples respectively.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Targeted Proteomics, Isolation