Journal: Pharmacology Research & Perspectives

Article Title: Inhibition of adenosine deaminase (ADA)-mediated metabolism of cordycepin by natural substances

doi: 10.1002/prp2.121

Figure Lengend Snippet: Secondary structures of ADA substrates and inhibitors. Secondary structure of ADA substrates (cordycepin, adenosine, 2′-deoxyadenosine) and inhibitors (EHNA, pentostatin, and flavonoids) are shown.

Article Snippet: Pentostatin, 1-deazaadenosine, and EHNA were obtained from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: Pharmacology Research & Perspectives

Article Title: Inhibition of adenosine deaminase (ADA)-mediated metabolism of cordycepin by natural substances

doi: 10.1002/prp2.121

Figure Lengend Snippet: Inhibition constants of pentostatin, EHNA, and naringin for deamination of cordycepin, adenosine, and 2′-deoxyadenosine in mouse and human erythrocytes

Article Snippet: Pentostatin, 1-deazaadenosine, and EHNA were obtained from Tocris Bioscience (Bristol, UK).

Techniques: Inhibition

Journal: Pharmacology Research & Perspectives

Article Title: Inhibition of adenosine deaminase (ADA)-mediated metabolism of cordycepin by natural substances

doi: 10.1002/prp2.121

Figure Lengend Snippet: ADA1 expression and activity in Jurkat cells. The total cell homogenates (2 μ g protein) of the ADA1-expressing HEK293 cells, Mock cells, and Jurkat cells were subjected to Western blot analysis with anti-ADA1 or antiactin antibodies (A). Cordycepin deamination activities were determined using the cell homogenates in the absence or presence of pentostatin (P), EHNA (E), and naringin (N) (B). Concentrations of substrate and inhibitors were 100 μ mol/L and 50 μ mol/L, respectively. * P < 0.05.

Article Snippet: Pentostatin, 1-deazaadenosine, and EHNA were obtained from Tocris Bioscience (Bristol, UK).

Techniques: Expressing, Activity Assay, Western Blot

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Biochemical characterization of adenosine deaminase (CD26; EC 3.5.4.4) activity in human lymphocyte-rich peripheral blood mononuclear cells

doi: 10.1590/1414-431X2020e10850

Figure Lengend Snippet: Adenosine deaminase (ADA) inhibition in human lymphocyte-rich peripheral blood mononuclear cells by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) (n=4).

Article Snippet: EHNA hydrochloride (0.1 mM) was used as an inhibitor (1261 - TOCRIS Bioscience, USA).

Techniques: Inhibition, Activity Assay, Control

Journal: medRxiv

Article Title: Nav1.7 and Nav1.8 form supramolecular active clusters with TRKB in mouse and human DRG neurons during development of neuropathic pain

doi: 10.1101/2022.11.05.22281929

Figure Lengend Snippet: (A-B) Quantitative analysis of the immunostained L4 DRG sections with antibodies against SCN9A (red), SCN10A (green) and NeuN (blue) showed that the percentages of both SCN9A + and SCN9A + /SCN10A + neurons in L4 DRG of SNI mice significantly increased at 2 WPO when compared to that in Sham mice, and the percentage of SCN9A + /SCN10A + neurons in L4 DRG of SNI mice significantly decreased at 6 WPO when compared to that in Sham mice (B). (C, D) The percentage of SCN9A + /SCN10A + neurons in L5 DRG of SNI mice significantly decreased at 2 and 6 WPO when compared to that in Sham mice, and that the percentage of SCN9A + neurons in L5 DRG of SNI mice significantly decreased at 2 WPO when compared to that in Sham mice. (E) The proportions of SCN9A + /SCN10A + neurons among total SCN9A + neurons did not statistical significantly change in L4 DRG of SNI mice either at 2 WPO or 6 WPO when compared to that in Sham mice, but it’s decreased significantly in L5 DRG of SNI mice at 6 WPO when compared to that in Sham mice. (F) The proportion of SCN9A + /SCN10A + neurons among total SCN10A + neurons statistical significantly increased only in L4 DRG of SNI mice at 2 WPO, not at 6 WPO when compared to that in Sham mice. Scale bar=100 μm, * p<0.05, ** p<0.01, unpaired Student’s T-test, n=4-6.

Article Snippet: The following primary antibodies were used: mouse antibodies against SCN10A (NeuroMab, 75-166, 1:200) and NeuN (Merck, MAB377, 1:100), rabbit antibodies against SCN9A (Proteintech Group, 20257-1-AP, 1:200) and NeuN (Cell Signaling Technology, 24307S, 1:200), and goat antibody against TrkB (R&D system, AF1494, 1:400).

Techniques:

Journal: medRxiv

Article Title: Nav1.7 and Nav1.8 form supramolecular active clusters with TRKB in mouse and human DRG neurons during development of neuropathic pain

doi: 10.1101/2022.11.05.22281929

Figure Lengend Snippet: (A) Immunostaining of SCN9A (red), SCN10A (green) and TrkB (blue) on L4-L5 DRG sections from Sham (a-e) and SNI (f-j) mice at 2 WPO, as well as from Sham (k-o) and SNI (p-t) mice at 6 WPO showed that cluster of SCN9A/SCN10A/TrkB formed in DRG from SNI mice at 2 WPO (f-j), and developed into supramolecular cluster by 6 WPO (p-t), e’, j’, o’ and t’ are the high magnification view of inset in e, j, o, t, respectively. Scale bar=100 μm. (B) The numbers of cells containing cluster of SCN9A/SCN10A were increased in L4-5 DRG of SNI mice at both 2 and 6 WPO when compared to that in Sham mice. (C) The numbers of cells contained cluster of SCN9A/SCN10A/TrkB were increased in L4-5 DRG of SNI mice at both 2 and 6 WPO when compared to that in Sham mice. (D) SNI mice had more TrkB - cells contained cluster of SCN9A/SCN10A than Sham mice. (E) The diameters of SCN9A particles in cluster of DRG neurons were significantly bigger in SNI mice than that in Sham mice at 6 WPO. (F) The diameters of SCN10A particles in cluster of DRG neurons were significantly bigger in SNI mice than that in Sham mice at both 2 and 6 WPO. (G) The diameters of TrkB particles in cluster of DRG neurons were significantly bigger in SNI mice than that in Sham mice at both 2 and 6 WPO. * p<0.05, ** p<0.01, unpaired Student’s T-test, n=3-4.

Article Snippet: The following primary antibodies were used: mouse antibodies against SCN10A (NeuroMab, 75-166, 1:200) and NeuN (Merck, MAB377, 1:100), rabbit antibodies against SCN9A (Proteintech Group, 20257-1-AP, 1:200) and NeuN (Cell Signaling Technology, 24307S, 1:200), and goat antibody against TrkB (R&D system, AF1494, 1:400).

Techniques: Immunostaining

Journal: medRxiv

Article Title: Nav1.7 and Nav1.8 form supramolecular active clusters with TRKB in mouse and human DRG neurons during development of neuropathic pain

doi: 10.1101/2022.11.05.22281929

Figure Lengend Snippet: (A-H) Immunostaining of SCN9A (blue), SCN10A (red) and TRKB (green) showed that compared to normal DRG from 29-weeks abortive embryos (A-D), pathological DRG of BPA patients had more TRKB/SCN9A neurons expressing high level of SCN10A (E-H), a-h are high magnification view of inset in A-H, Scale bar=500 μm.

Article Snippet: The following primary antibodies were used: mouse antibodies against SCN10A (NeuroMab, 75-166, 1:200) and NeuN (Merck, MAB377, 1:100), rabbit antibodies against SCN9A (Proteintech Group, 20257-1-AP, 1:200) and NeuN (Cell Signaling Technology, 24307S, 1:200), and goat antibody against TrkB (R&D system, AF1494, 1:400).

Techniques: Immunostaining, Expressing

Journal: medRxiv

Article Title: Nav1.7 and Nav1.8 form supramolecular active clusters with TRKB in mouse and human DRG neurons during development of neuropathic pain

doi: 10.1101/2022.11.05.22281929

Figure Lengend Snippet: (A-C) Immunostaining of SCN9A/TRKB/NEUN (A), or SCN10A/TRKB/NEUN (B), or SCN9A/SCN10A/TRKB (C) on sections of normal DRG from 29-weeks abortive embryos (upper panels in A-C) and of pathological DRG from BPA patients (lower panels in A-C). (D) The proportion of high (bar in red) and low (bar in blue) expression of SCN9A, SCN10A and TRKB neurons in normal DRG and pathological DRG, # represented the comparison of low expression in two groups, * represented the comparison of high expression in two groups. (E) The proportion of SCN9A/TRKB, SCN10A/TRKB and SCN9A/SCN10A/TRKB neurons in normal DRG and pathological DRG. (F) The percentage of SCN9A, SCN10A and SCN9A/SCN10A within TRKB neuronal population. (G) The proportion of neurons expressing high level of SCN9A (SCN9A High+ ) or SCN10A (SCN10A High+ ) within TRKB neuronal population. (H) The proportion of neurons expressing high level of SCN10A (SCN10A High+ ) within TRKB/SCN9A sub neuronal population. Scale bar=100 μm. * p<0.05, *** p<0.001, **** p<0.0001, ### p<0.001, #### p<0.0001, unpaired Student’s T-test, n=3-4.

Article Snippet: The following primary antibodies were used: mouse antibodies against SCN10A (NeuroMab, 75-166, 1:200) and NeuN (Merck, MAB377, 1:100), rabbit antibodies against SCN9A (Proteintech Group, 20257-1-AP, 1:200) and NeuN (Cell Signaling Technology, 24307S, 1:200), and goat antibody against TrkB (R&D system, AF1494, 1:400).

Techniques: Immunostaining, Expressing

Journal: medRxiv

Article Title: Nav1.7 and Nav1.8 form supramolecular active clusters with TRKB in mouse and human DRG neurons during development of neuropathic pain

doi: 10.1101/2022.11.05.22281929

Figure Lengend Snippet: (A-B) Immunostaining for SCN9A/SCN10A (a-d, m-p) or SCN9A/TRKB (e-h, q-t) or SCN10A/TRKB (i-l, u-x) on sections from normal DRG (a-l) of 29-weeks abortive embryos and pathological DRG (m-x) of BPA patients showed that supramolecular clusters of SCN9A/SCN10A/TRKB on and along cytoplasm membrane of DRG neurons were only detected in pathological DRG neurons (B), not in normal DRG (A). (C) Three-dimension view of SCN9A/SCN10A/TRKB supramolecular cluster in pathological DRG neuron. (D) The diameters of the particles of TRKB, SCN9A and SCN10A in single neuron from patients with BPA. (E) Immunostaining of SCN9A (green) and SCN10A (red) showed SCN9A/SCN10A clusters in pathological DRG nerve from BPA patients. Arrows pointed to small particles containing only SCN10A or only TrkB. Scale bar=100 μm.

Article Snippet: The following primary antibodies were used: mouse antibodies against SCN10A (NeuroMab, 75-166, 1:200) and NeuN (Merck, MAB377, 1:100), rabbit antibodies against SCN9A (Proteintech Group, 20257-1-AP, 1:200) and NeuN (Cell Signaling Technology, 24307S, 1:200), and goat antibody against TrkB (R&D system, AF1494, 1:400).

Techniques: Immunostaining

Journal: medRxiv

Article Title: Nav1.7 and Nav1.8 form supramolecular active clusters with TRKB in mouse and human DRG neurons during development of neuropathic pain

doi: 10.1101/2022.11.05.22281929

Figure Lengend Snippet: Western-blot results demonstrated that the expression of SCN9A, SCN10A and TRKB significantly increased in DRGs from BPA patients when compared to normal DRGs from 29-weeks abortive embryos. * p<0.05, unpaired Student’s T-test, n=3.

Article Snippet: The following primary antibodies were used: mouse antibodies against SCN10A (NeuroMab, 75-166, 1:200) and NeuN (Merck, MAB377, 1:100), rabbit antibodies against SCN9A (Proteintech Group, 20257-1-AP, 1:200) and NeuN (Cell Signaling Technology, 24307S, 1:200), and goat antibody against TrkB (R&D system, AF1494, 1:400).

Techniques: Western Blot, Expressing

Journal: medRxiv

Article Title: Nav1.7 and Nav1.8 form supramolecular active clusters with TRKB in mouse and human DRG neurons during development of neuropathic pain

doi: 10.1101/2022.11.05.22281929

Figure Lengend Snippet: (A) A schematic view of the generation of chronic diabetic neuropathy mouse model. (B) Fasting blood glucose concentration of the mice were tested before STZ treatment and 2 weeks after last injection of STZ. (C) The mechanical threshold of mice along the experimental course. (D) Immunostaining of SCN9A (green) and SCN10A (red) showed that supramolecular clusters of SCN9A/SCN10A were detected in DRG neurons from mice with 10-weeks diabetic neuropathy (d-f), not in DRG neurons of naïve mice (a-c), Scale bar=20 μm.

Article Snippet: The following primary antibodies were used: mouse antibodies against SCN10A (NeuroMab, 75-166, 1:200) and NeuN (Merck, MAB377, 1:100), rabbit antibodies against SCN9A (Proteintech Group, 20257-1-AP, 1:200) and NeuN (Cell Signaling Technology, 24307S, 1:200), and goat antibody against TrkB (R&D system, AF1494, 1:400).

Techniques: Concentration Assay, Injection, Immunostaining

Journal: Circulation

Article Title: The Polyphenols Resveratrol and S17834 prevent the Structural and Functional Sequelae of Diet-Induced Metabolic Heart Disease in Mice

doi: 10.1161/CIRCULATIONAHA.111.067801

Figure Lengend Snippet: Effects of HFHS diet and S17834 (S) on myocardial nitrotyrosine and 4-hydroxy-2-nonenal (HNE) staining, and HNE-adducts of LKB. Shown are representative photomicrographs of myocardial nitrotyrosine staining (Panels A, B) and HNE staining (Panels C, D). Panel E shows representative immnoprecipitation (IP) and immunoblot (IB) data for HNE-adducts of LKB. Panel F shows group densitometry analysis. The bar indicates 25 μm. Values are means ± SEM; n=3–4. *P<0.05 vs. normal diet-fed mice. †P<0.05 vs. HFHS diet-fed mice.

Article Snippet: Briefly, LV tissue sections (4 μm) were blocked with 10% goat serum in phosphate-buffered saline, incubated with rabbit anti-3-nitrotyrosine polyclonal antibody or mouse anti-HNE monoclonal antibody, and incubated with goat biotin-conjugated anti-rabbit IgG or goat biotin-conjugated anti-mouse IgG (Vector Laboratory, Burlingame, CA).

Techniques: Staining, Western Blot

Journal: Circulation

Article Title: The Polyphenols Resveratrol and S17834 prevent the Structural and Functional Sequelae of Diet-Induced Metabolic Heart Disease in Mice

doi: 10.1161/CIRCULATIONAHA.111.067801

Figure Lengend Snippet: A schema that summarizes the observed mechanisms by which polyphenols may exert beneficial effects in the HFHS-fed mouse. HFHS feeding is associated with oxidative stress and oxidant-mediated protein modifications as evidenced by generalized immunohistochemical staining for nitrotyrosine and 4-hydroxy-2-nonenal (HNE) staining, and specific HNE-adducts of LKB that are associated with decreased enzyme activity and increased hypertrophic signaling. Modifications of other proteins (e.g., calcium handling or sarcomeric proteins) may contribute to impaired relaxation. HFHS feeding is also associated with insulin resistance and hyper-insulinemia, which may promote hypertrophic growth. Finally, oxidative stress can lead to interstitial fibrosis. Together, these and possibly other mechanisms may contribute to diastolic dysfunction, which is common in metabolic heart disease. By decreasing oxidative stress and inflammation, treatment with S17834 a) may decrease the generation of oxidative protein modifications, b) interstitial fibrosis, and c) insulin resistance/hyper-insulinemia. S17834 also increases plasma adiponectin, which may inhibit hypertrophic signaling.

Article Snippet: Briefly, LV tissue sections (4 μm) were blocked with 10% goat serum in phosphate-buffered saline, incubated with rabbit anti-3-nitrotyrosine polyclonal antibody or mouse anti-HNE monoclonal antibody, and incubated with goat biotin-conjugated anti-rabbit IgG or goat biotin-conjugated anti-mouse IgG (Vector Laboratory, Burlingame, CA).

Techniques: Immunohistochemical staining, Staining, Activity Assay

Journal: Journal of Functional Biomaterials

Article Title: Physicochemical and Antioxidant Alterations of Modified and Free Epigallocatechin Gallate Under Thermal Treatment in Air and Vacuum

doi: 10.3390/jfb17010018

Figure Lengend Snippet: Characterization and antioxidant activity of samples. ( A ) Three types of sponges: vacuum-heated (150 °C, 16 h) gelatin sponges containing β-tricalcium phosphate (β-TCP) granules (vh-GSβ), vacuum-heated chemically synthesized gelatin sponges containing β-TCP (vhc-GSβ), and vacuum-heated chemically synthesized gelatin sponges containing EGCG and β-TCP (vhEc-GSβ). Macroscopic appearances ( left ; scale bar = 5 mm) and field-emission scanning electron microscopic appearances ( middle ; scale bar = 500 µm, right ; scale bar = 200 µm). ( B ) Quantitative analysis of pore size based on the average of the maximum Feret diameter. n = 6; one-way ANOVA with Tukey’s multiple comparisons test; ns: not significant; * p < 0.05. ( C ) X-ray powder diffraction patterns. Arrow heads: the characteristic diffraction peaks of β-TCP. ( D ) ATR-FTIR spectra of the samples. Arrows: the characteristic peaks of amide bands. ( E ) DPPH assay. ( F ) Quantitative analysis of DPPH scavenging activity using Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test. n = 4, ns: not significant, **** p < 0.0001. ( G ) Immunofluorescence staining for 4-Hydroxynonenal (4-HNE) in rat calvarial bone defects implanted with or without samples. Scale bars: top = 50 µm; bottom = 500 µm. ( H ) Quantitative analysis of the ratio of 4-HNE to 4′,6-diamidino-2-phenylindole (DAPI) fluorescence area. n = 4; Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test; ns: not significant; ** p < 0.01.

Article Snippet: Frozen sections of animal tissues were blocked and permeabilized in phosphate-buffered saline (164-28713; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 5% goat serum (S-1000, Vector Laboratories, Inc., Newark, CA, USA) and 0.3% Triton X-100 (Code 12967-32, Nacalai, Kyoto, Japan) for 30 min. Immunostaining was performed using an anti-4-hydroxynonenal (4-HNE) polyclonal antibody (bs-6313R-Cy5, Bioss Antibodies, Woburn, MA, USA; 1:100) in the dark at 4 °C overnight.

Techniques: Antioxidant Activity Assay, Synthesized, Pore Size, DPPH Assay, Activity Assay, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in cellular and infection microbiology

Article Title: Serine protease inhibitor derived from Trichinella spiralis (TsSERP) inhibits neutrophil elastase and impairs human neutrophil functions.

doi: 10.3389/fcimb.2022.919835

Figure Lengend Snippet: FIGURE 1 The inhibitory activity of rTsSERP1 against human neutrophil serine proteases (hNSPs). The inhibition of rTsSERP1 against NSPs including human neutrophil elastase (hNE) (A), human proteinase 3 (hPR3) (B), or human cathepsin G (hCG) (C) was assessed by enzyme inhibitory assays using specific peptide substrates. Each enzyme and rTsSERP1 was incubated at the molar ratios (enzyme: inhibitor) of 1:0, 1:1, 1:2, 1:4 and 1:10. Alpha-1 proteinase inhibitor (a1PI) was used as a positive control. The Dixon plot showed the inhibition constant (Ki) of TsSERP1 for hNE (D). The Ki was calculated from the inhibition of hNE by rTsSERP1 (0, 20, 40 and 80 nM) against the fluorogenic MeOSuc-AAPV-AMC substrate at three different concentrations (50, 100 and 200 µM).

Article Snippet: The inhibitory activity of rTsSERP1 against human neutrophil serine proteases (hNSPs) including neutrophil elastase (hNE) (Fitzgerald Industries International, Acton, MA), proteinase 3 (hPR3) (Diarect AG, Freiburg, Germany), and cathepsin G (hCG) (Calbiochem, Darmstadt, Germany) were evaluated by hydrolysis of specific substrates, fluorogenic (FRET) or colorimetric substrates, according to previous publications with some modifications (Korkmaz et al., 2008; Tausch et al., 2009; Perera et al., 2013; Anderson et al., 2019).

Techniques: Activity Assay, Inhibition, Incubation, Positive Control

Journal: Frontiers in cellular and infection microbiology

Article Title: Serine protease inhibitor derived from Trichinella spiralis (TsSERP) inhibits neutrophil elastase and impairs human neutrophil functions.

doi: 10.3389/fcimb.2022.919835

Figure Lengend Snippet: FIGURE 2 The neutrophil-secreted elastase activity inhibited by rTsSERP1. Neutrophils were induced with 100 nM of fMLP prior to treatment with 40 µM of rTsSERP1or an irrelevant control (rmDHFR) followed by detecting NE activity using a neutrophil elastase substrate for 1 h (A) and the percentage of the NE activity was calculated at 1h by setting neutrophils treated with fMLP alone as 100% activity (B). Media alone was used as a negative control and 0.1 mM of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) was used as a positive control for serine protease inhibition. The bar charts show the data of three different donors, which are presented as the mean ± SD. The experiments were performed in triplicate with three independent experiments. One-way ANOVA followed by a Bonferroni multiple comparison test were used for statistical analysis: **p < 0.01 and ***p < 0.001. The treatment conditions were included media alone (Mock), only fMLP induction (fMLP), fMLP induction and AEBSF treatment (AEBSF+fMLP), fMLP induction and rTsSERP1 treatment (rTsSERP1+fMLP), fMLP induction and irrelevant control (rmDHFR) treatment (rmDHFR+fMLP).

Article Snippet: The inhibitory activity of rTsSERP1 against human neutrophil serine proteases (hNSPs) including neutrophil elastase (hNE) (Fitzgerald Industries International, Acton, MA), proteinase 3 (hPR3) (Diarect AG, Freiburg, Germany), and cathepsin G (hCG) (Calbiochem, Darmstadt, Germany) were evaluated by hydrolysis of specific substrates, fluorogenic (FRET) or colorimetric substrates, according to previous publications with some modifications (Korkmaz et al., 2008; Tausch et al., 2009; Perera et al., 2013; Anderson et al., 2019).

Techniques: Activity Assay, Control, Negative Control, Positive Control, Inhibition, Comparison

Journal: Frontiers in cellular and infection microbiology

Article Title: Serine protease inhibitor derived from Trichinella spiralis (TsSERP) inhibits neutrophil elastase and impairs human neutrophil functions.

doi: 10.3389/fcimb.2022.919835

Figure Lengend Snippet: FIGURE 3 rTsSERP1 impairs neutrophil phagocytosis. The phagocytosis of neutrophils was induced by 100 nM of fMLP prior to treatment with 10 µg/ml of rTsSERP1 or an irrelevant control (rmDHFR), followed by incubation with the fluorescent E. coli BioParticle. After incubation, the fluorescent signal was measured and subsequently calculated as the percentage of phagocytosis (A). Neutrophils treated with fMLP alone were set as 100% phagocytotic effect. The bar charts show the data of three different donors, which are presented as the mean ± SD. The experiments were performed in triplicate with three independent experiments. One-way ANOVA followed by a Bonferroni multiple comparison test were used for statistical analysis: **p < 0.01 and ***p < 0.001. Fluorescent images (B) were captured to confirm the above result. The percentage of phagocytosis positive out of the total number of alive neutrophils was indicated in the merge of picture. The E. coli BioParticle was labeled with FITC (green) and the nucleus was counterstained with Hoechst 33342 (blue). Examples of non-phagocytotic neutrophils are indicated by white arrows. The treatment conditions were included media alone (Mock), only fMLP induction (fMLP), fMLP induction and rTsSERP1 treatment (rTsSERP1+fMLP), fMLP induction and irrelevant control (rmDHFR) treatment (rmDHFR+fMLP).

Article Snippet: The inhibitory activity of rTsSERP1 against human neutrophil serine proteases (hNSPs) including neutrophil elastase (hNE) (Fitzgerald Industries International, Acton, MA), proteinase 3 (hPR3) (Diarect AG, Freiburg, Germany), and cathepsin G (hCG) (Calbiochem, Darmstadt, Germany) were evaluated by hydrolysis of specific substrates, fluorogenic (FRET) or colorimetric substrates, according to previous publications with some modifications (Korkmaz et al., 2008; Tausch et al., 2009; Perera et al., 2013; Anderson et al., 2019).

Techniques: Control, Incubation, Comparison, Labeling

Journal: Frontiers in cellular and infection microbiology

Article Title: Serine protease inhibitor derived from Trichinella spiralis (TsSERP) inhibits neutrophil elastase and impairs human neutrophil functions.

doi: 10.3389/fcimb.2022.919835

Figure Lengend Snippet: FIGURE 4 rTsSERP1 inhibits neutrophil extracellular traps (NETosis). Neutrophils initially stained with Sytox green (5 µM) were treated with rTsSERP1 or rmDHFR (10 µg/ml) for 30 min followed by induction with PMA (50 nM) for 4 h. NETosis was analyzed by the quantitative fluorescence intensity (A) and fluorescent images (B) with different treatment conditions including media alone (Mock) (a), PMA induction alone (PMA) (b), rTsSERP1 treatment before PMA induction (rTsSERP1+PMA) (c), and treatment with an irrelevant control (rmDHFR) before PMA induction (rmDHFR+PMA) (d). The bar charts show the data of three different donors, which are presented as the mean ± SD. The experiments were performed in triplicate with three independent experiments. One-way ANOVA followed by a Bonferroni multiple comparison test were used for statistical analysis: **p < 0.01 and ***p < 0.001.

Article Snippet: The inhibitory activity of rTsSERP1 against human neutrophil serine proteases (hNSPs) including neutrophil elastase (hNE) (Fitzgerald Industries International, Acton, MA), proteinase 3 (hPR3) (Diarect AG, Freiburg, Germany), and cathepsin G (hCG) (Calbiochem, Darmstadt, Germany) were evaluated by hydrolysis of specific substrates, fluorogenic (FRET) or colorimetric substrates, according to previous publications with some modifications (Korkmaz et al., 2008; Tausch et al., 2009; Perera et al., 2013; Anderson et al., 2019).

Techniques: Staining, Control, Comparison

Journal: Frontiers in cellular and infection microbiology

Article Title: Serine protease inhibitor derived from Trichinella spiralis (TsSERP) inhibits neutrophil elastase and impairs human neutrophil functions.

doi: 10.3389/fcimb.2022.919835

Figure Lengend Snippet: FIGURE 5 rTsSERP1 suppresses the transcription levels of cytokine and chemokine mRNAs in human neutrophils. Neutrophils were treated with 10 µg/ml of rTsSERP1 or an irrelevant control (rmDHFR) for 1 h prior and then stimulated with 100 nM of fMLP for 4 h. Thereafter, the cells were collected for RNA isolation and qRT-PCR analysis for IL-1b (A), IL-6 (B), IFN-g (C), TNF-a (D), IL-8 (E), and CCL3 (F) transcripts. The transcription level of each target was normalized to its own housekeeping gene level [glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] and then compared with the untreated control (media only) to calculate the relative fold change. The bar charts show the data of three different donors. The results are presented as mean ± SD. The experiments were performed in triplicate with three independent experiments. One-way ANOVA followed by a Bonferroni multiple comparison test were used for statistical analysis: *p < 0.05, **p < 0.01, and ***p < 0.001. The treatment conditions were included media alone (Mock), only fMLP induction (fMLP), fMLP induction and AEBSF treatment (AEBSF+fMLP), fMLP induction and rTsSERP1 treatment (rTsSERP1+fMLP), fMLP induction and irrelevant control (rmDHFR) treatment (rmDHFR+fMLP).

Article Snippet: The inhibitory activity of rTsSERP1 against human neutrophil serine proteases (hNSPs) including neutrophil elastase (hNE) (Fitzgerald Industries International, Acton, MA), proteinase 3 (hPR3) (Diarect AG, Freiburg, Germany), and cathepsin G (hCG) (Calbiochem, Darmstadt, Germany) were evaluated by hydrolysis of specific substrates, fluorogenic (FRET) or colorimetric substrates, according to previous publications with some modifications (Korkmaz et al., 2008; Tausch et al., 2009; Perera et al., 2013; Anderson et al., 2019).

Techniques: Control, Isolation, Quantitative RT-PCR, Comparison

Journal: Frontiers in cellular and infection microbiology

Article Title: Serine protease inhibitor derived from Trichinella spiralis (TsSERP) inhibits neutrophil elastase and impairs human neutrophil functions.

doi: 10.3389/fcimb.2022.919835

Figure Lengend Snippet: FIGURE 6 Inhibitory effect of rTsSERP1 on the production of proinflammatory cytokines released by human neutrophils. In addition to determining the transcription levels of cytokine and chemokine mRNAs in neutrophils, culture media were also collected for ELISA to analyze IL-1b (A) and IFN-g (B) protein levels. ELISA results are reported as pg/ml. The bar charts show the data of three different donors, which are presented as the mean ± SD. The experiments were performed in triplicate with three independent experiments. One-way ANOVA followed by a Bonferroni multiple comparison test were used for statistical analysis: ***p < 0.001. The treatment conditions were included media alone (Mock), only fMLP induction (fMLP), fMLP induction and AEBSF treatment (AEBSF+fMLP), fMLP induction and rTsSERP1 treatment (rTsSERP1+fMLP), fMLP induction and irrelevant control (rmDHFR) treatment (rmDHFR+fMLP).

Article Snippet: The inhibitory activity of rTsSERP1 against human neutrophil serine proteases (hNSPs) including neutrophil elastase (hNE) (Fitzgerald Industries International, Acton, MA), proteinase 3 (hPR3) (Diarect AG, Freiburg, Germany), and cathepsin G (hCG) (Calbiochem, Darmstadt, Germany) were evaluated by hydrolysis of specific substrates, fluorogenic (FRET) or colorimetric substrates, according to previous publications with some modifications (Korkmaz et al., 2008; Tausch et al., 2009; Perera et al., 2013; Anderson et al., 2019).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Frontiers in cellular and infection microbiology

Article Title: Serine protease inhibitor derived from Trichinella spiralis (TsSERP) inhibits neutrophil elastase and impairs human neutrophil functions.

doi: 10.3389/fcimb.2022.919835

Figure Lengend Snippet: FIGURE 7 A diagram summarizing the main findings of this study. TsSERP1, presented at different developmental stages of T. spiralis and its excretory- secretory (ES) products, inhibited the hNE activity of human neutrophils. The inhibitory effect of TsSERP1 on hNE influences neutrophil functions by impairing phagocytosis, NETosis, and the production of proinflammatory cytokines and chemokines.

Article Snippet: The inhibitory activity of rTsSERP1 against human neutrophil serine proteases (hNSPs) including neutrophil elastase (hNE) (Fitzgerald Industries International, Acton, MA), proteinase 3 (hPR3) (Diarect AG, Freiburg, Germany), and cathepsin G (hCG) (Calbiochem, Darmstadt, Germany) were evaluated by hydrolysis of specific substrates, fluorogenic (FRET) or colorimetric substrates, according to previous publications with some modifications (Korkmaz et al., 2008; Tausch et al., 2009; Perera et al., 2013; Anderson et al., 2019).

Techniques: Activity Assay