Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: Cleavage profile of the HMPV F: A fluorogenic peptide mimicking the cleavage site of HMPV F was incubated with the indicated proteases and cleavage was monitored by the increase of fluorescence at 390 nm. RFU = relative fluorescence units. StDev = Standard Deviation.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Incubation, Fluorescence

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: IC50 values of all protease/peptide combinations.: Fluorogenic peptides mimicking the cleavage sites of A/CA/04/09 H1N1, A/Japan/305/1957 H2N2 HA, A/Aichi/2/68 H3N2 HA, A/Vietnam/1203/2004 H5N1 LPAI HA, A/Vietnam/1204/2004 H5N1 HPAI HA, A/Taiwan/2/2013 H6N1 HA, A/Shanghai/2/2013 H7N9 HA, A/Hong Kong/2108/2003 H9N2 HA and HMPV F were incubated with the indicated proteases and different SPINT2 concentrations. Cleavage was monitored by the increase of fluorescence at 390 nm and the resulting Vmax values were used to calculate the IC50 values as described in the “Material and Methods” section. (A) IC50 values of influenza A fluorogenic cleavage site peptide mimics. Concentrations are in nanomolar. (B) IC50 values of the HMPV F cleavage site peptide mimic. Concentrations are in picomolar. NT = Not tested.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Incubation, Fluorescence

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 inhibits cleavage of HA protein expressed in 293T cells. Cells were transfected with plasmids encoding for the indicated HA and allowed to express the protein for ~18 h. The recombinant proteases were incubated for 15 min with the indicated SPINT2 concentrations and subsequently added to the cells for 10 min (trypsin) or 90 min (matriptase and KLK5). Western blots were performed and the HA 1 band was quantified using ImageJ. (A) Quantification of the HA1 band comparing the signal intensity of the 0 nM SPINT2 samples against 10 nM and 500 nM SPINT2 of the respective HA/protease combination. Three independent experiments were carried out and the western blots of each experiment were analyzed. Quantifications were conducted as described in the methods section. (B – D) Western blots showing the cleavage of (B) A/CA/04/09 H1N1 HA by matriptase and KLK5 at different SPINT2 concentration, (C) A/Aichi/2/68 H3N2 HA by KLK5 at different SPINT2 concentration and (D) A/Shanghai/2/2013 H7N9 HA by matriptase and KLK5 at different SPINT2 concentrations. Statistical analysis was performed using a non-paired student's t-test comparing the samples tested with 10 nM SPINT2 against the respective sample incubated with 500 nM SPINT2. Error bars indicate standard deviation. * indicates p = < 0.05.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Recombinant, Incubation, Western Blot, Concentration Assay, Standard Deviation

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: TMPRSS2, HAT, matriptase and KLK5 cleave HMPV F and SPINT2 is able to prevent cleavage by exogenous proteases . HMPV F was either expressed alone or co-transfected with protease and allowed to express for ~ 18 h. Cells were then metabolically starved of cysteine and methionine followed by radioactive S35 labeling of protein for 4 h in the presence of TPCK-trypsin or specified protease. SPINT2 treated proteases were incubated at room temperature for 10 min and placed onto cells for 4 h. Radioactive gels were quantified using ImageQuant software with percent cleavage equal to [ ( F 1 F 0 + F 1 ) x 100 ] . (A and B) Co-transfected proteases TMPRSS2, HAT and matriptase are able to cleave HMPV F (n = 4) while C and D) exogenous proteases KLK5 and matriptase but not KLK12 are able to cleave HMPV F (n = 5). (E and F) SPINT2 prevented cleavage of HMPV F by trypsin, KLK5 and matriptase at nm concentrations demonstrated by the loss of the F 1 cleavage product (n = 3). Statistical analysis was performed using a one-way ANOVA followed by a student's t-test with a bonferroni multiple comparisons test correction. P < 0.05 *, P < 0.005 **, P < 0.0005 ***, P < 0.0001 ****. N values represent independent replicates for each treatment group. Error bars represent SD.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Metabolic Labelling, Labeling, Incubation, Software

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 inhibits HA-mediated cell-cell fusion. VERO cells were transfected with plasmids encoding for (A) A/CA/04/09 H1N1 HA or (B) A/Shanghai/2/2013 H7N9 HA and allowed to express the protein for ~18 h. Recombinant matriptase and KLK5 were incubated with different SPINT2 concentrations for 15 min and then added to the HA-expressing cells for 3 h. After 3 h the cells were briefly treated with cell fusion buffer at pH5, washed, supplemented with growth medium and returned to the incubator for 1 h to allow fusion. HA protein was detected using HA-specific primary antibodies and a secondary fluorogenic Alexa488 antibody. Nuclei were stained using DAPI. Magnification 40x. (C) VERO cells expressed A/Vietnam/1204/2004 H5N1 HA that was cleaved during its maturation process in the cell. SPINT2 was added at 0 nM or 500 nM at the time of transfection. Magnification 25x.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Recombinant, Incubation, Expressing, Staining

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 reduces IAV growth in cell culture. MDCK cells were transfected with plasmids encoding for human matriptase or human TMPRSS2 and allowed to express the proteins for ~18 h. Cells expressing human matriptase (B) or human TMPRSS2 (C) were then infected with A/CA/04/09 H1N1 at a MOI of 0.1 and different SPINT2 concentration were added to each well. Non-transfected cells to which trypsin was added served as a control (A). (D) MDCK cells were infected with A/X31 H3N2 at an MOI of 0.1 and trypsin was added to assist viral propagation. Different SPINT2 concentration were added as indicated. After 48 h of infection the supernatants were collected and used for an immuno-plaque assay to determine the viral loads. Experiment was repeated three times and each dot represents the viral titer of a single experiment. Statistical analysis was performed using a non-paired student's t-test comparing the control (0 h) against the respective sample. Error bars indicate standard deviation. * indicates p = < 0.05. Extended horizontal line within the error bars represents mean value of the three independent replicates.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Cell Culture, Transfection, Expressing, Infection, Concentration Assay, Control, Plaque Assay, Standard Deviation

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: Viral titers measured in the infection studies: Table shows the average viral titers and standard deviation calculated from the 3 independent biological replicates depicted in Fig. 6 , Fig. 7 . (A) Titers and standard deviation from infection studies shown in Fig. 5 . (B) Titers and standard deviation shown in Fig. 7 .

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Infection, Standard Deviation

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: Specificity and potency of anti-PAR2 monoclonal antibody MEDI0618 . ( A ) Live staining of PAR2-expressing (1321N1-hPAR2.cl8) or non-expressing (1321N1 parental) cell lines with MEDI0618 directly conjugated to Alexa Fluor 647. Scale bar = 20 µm. ( B ) Flow cytometry of hPAR2 overexpressing cells (1321N1-hPAR2.cl8) or non-expressing cells (1321N1 parental cell line) or A549 cells endogenously expressing hPAR2 live labelled with MEDI0618. ( C and D ) Calcium imaging from 1321N1-hPAR2 cells pretreated with MEDI0618 hIgG or an isotype control protein. ( C ) Exemplar raw calcium trace from a single well pretreated with MEDI0618 or isotype control antibody both at 1 nM, followed by PAR2 agonist stimulation with matriptase (10 nM). ( D ) Antibody titration of the anti-PAR2 antibodies MEDI0618 or PAR650097 or an isotype control antibody. Data show the matriptase (10 nM)-mediated calcium signal after preincubation with antibody and normalized to the matriptase response in the absence of antibody treatment. ( E ) Calcium imaging from A549 cells pretreated with MEDI0618, isotype control protein or PAR1 inhibitors (ATAP2 + WEDE15 mAbs) followed by 10 nM thrombin (PAR1 agonist) stimulation. Data are presented as mean ± standard error of the mean, n = 4. The concentration of the inhibitor is shown on the x -axis. Data are normalized to the peak thrombin calcium response in the absence of inhibitor pretreatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Staining, Expressing, Flow Cytometry, Imaging, Control, Titration, Concentration Assay

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: PAR2 functional expression in human and mouse cells relevant to migraine . Whole well calcium imaging from primary human dural fibroblasts (HDuF), human dural microvascular endothelial (HDuMEC) and mouse brain endothelial (bEnd.3) cells. ( A , D and G ) PAR2 agonists concentration response curve in ( A ) HDuF, ( D ) HDuMEC and ( G ) bEnd.3 cells. ( B , E and F ) Effect of MEDI0618 and isotype control protein (IgG) on inhibition of matriptase-induced calcium signalling at 30 nM in ( B ) HDuF, ( E ) HDuMEC and ( H ) bEnd.3 cells. ( C , F and I ) Representative calcium imaging traces of 30 nM matriptase-evoked activity following MEDI0618 or isotype control protein preincubation in ( C ) HDuF, ( F ) HDuMEC and ( I ) bEnd.3 cells. ( J and K ) Single-cell calcium imaging from mouse trigeminal neuron cultures. ( J ) Pseudocolour images of fura-2 ratio intensity show a subset of trigeminal neurons activated by treatment with 10 µM LIGRLO in comparison to 20 mM KCl treatment. Scale bar = 20 µm. ( K ) Representation of fura-2 traces recorded from two individual neurons during acute LIGRLO (10 µM) or KCl (20 mM) treatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Functional Assay, Expressing, Imaging, Concentration Assay, Control, Inhibition, Activity Assay, Comparison

Journal: Scientific Reports

Article Title: Autophagy of Candida albicans cells after the action of earthworm Venetin-1 nanoparticle with protease inhibitor activity

doi: 10.1038/s41598-023-41281-4

Figure Lengend Snippet: Proteolytic activity of matriptase-1 ( A ) and matriptase-2 ( B ), trypsin ( C ), and chymotrypsin ( D ) after treatment with Venetin-1 versus the control sample without the inhibitor.

Article Snippet: Recombinant human matriptase-2 was obtained from Enzo Life Sciences (Switzerland).

Techniques: Activity Assay