Journal: MethodsX

Article Title: Determination of organic pollutants in Anguilla anguilla by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)

doi: 10.1016/j.mex.2021.101342

Figure Lengend Snippet: Validation of the selected method in terms of sensitivity (LODs, LOQs), Intra- R and Inter- R .

Article Snippet: Chlorfenvinphos-d10 (diethyl D5), chlorpyrifos-d10 (diethyl D10), and vildagliptin-d3 were purchased from LGC Standards.

Techniques:

Journal: MethodsX

Article Title: Determination of organic pollutants in Anguilla anguilla by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)

doi: 10.1016/j.mex.2021.101342

Figure Lengend Snippet: Recoveries of the validated method in both eel liver and muscle matrix.

Article Snippet: Chlorfenvinphos-d10 (diethyl D5), chlorpyrifos-d10 (diethyl D10), and vildagliptin-d3 were purchased from LGC Standards.

Techniques:

Journal: bioRxiv

Article Title: DPP9 is an endogenous and direct inhibitor of the NLRP1 inflammasome that guards against human auto-inflammatory diseases

doi: 10.1101/260919

Figure Lengend Snippet: A. DPP8/9 inhibitors cause ASC-GFP speck formation in the presence of NLRP1. 293T-ASC-GFP cells were transfected NLRP1 expressing plasmids at a ratio of 1 μg/5x10 5 cells. Transfected cells were treated with Talabostat (0.2 μM) and 1G244 (10 μM) for 16 hours before GFP imaging. Scale bar=20 μm. B. DPP8/9 inhibition does not activate the NLRP3 inflammasome or enhance a NLRP1 pyrin-domain (PYD) mutant, p. M77T. 293T-ASC-GFP cells were transfected and treated as in A. C. Talabostat leads to ASC oligomerization independently of DPP4. 293T-ASC-GFP cells were transfected and treated as in A. Cell pellets were lysed in 1xTBS buffer with 1% NP-40. Insoluble pellets were subjected to crosslinking with 1 mM DSS for 15 mins at 37 °C and solubilized in 1xTBS with 1% SDS. D. DPP8/9 inhibition by Talabostat causes NLRP1 self-oligomerization. 293T cells were transfected with the respective constructs at a ratio of 2 μg/5x10 5 cells and treated with Talabostat (2 μM) two days after transfection for 16 hours. E. Alanine mutations in the DPP9 catalytic triad dominantly activate the NLRP1-ASC inflammasome. 293T-ASC-GFP cells were co-transfected with NLRP1 and the respective DPP9 constructs and imaged two days after transfection. F. Opposing roles of wild-type DPP9 and S759A mutant in mediating ASC-GFP oligomerization in the presence of NLRP1. DSS crosslinking was performed as in C. G. Validation of CRISPR/Cas9-mediated deletion of DPP9 and subsequent knockdown of DPP8. Cells were harvested and lysed in 1xTBS buffer with 1% NP-40 4 days after siRNA transfection. H. DPP9 deletion activates the NLRP1 inflammasome with partial compensation by DPP8. 293T-ASC-GFP cells were treated with control or siRNAs against DPP8 for 3 days before NLRP1 transfection. Related to

Article Snippet: The small molecule inhibitors used are vildagliptin (MedChemExpress), saxagliptin (MedChemExpress), TC-E 5007 (Tocris), butabindide oxalate (MedChemExpress), Talabostat and (MedChemExpress), 1G244 (Santa Cruz Biotechnology), LPS (Ultrapure, Escherichia coli O111:B4, SigmaAldrich) and nigericin 10 uM (Invivogen, #tlrl-nig).

Techniques: Transfection, Expressing, Imaging, Inhibition, Mutagenesis, Construct, CRISPR

Journal: bioRxiv

Article Title: DPP9 is an endogenous and direct inhibitor of the NLRP1 inflammasome that guards against human auto-inflammatory diseases

doi: 10.1101/260919

Figure Lengend Snippet: A. IL-1B secretion from primary human keratinocytes and PBMCs upon Talabostat (2 μM) treatment. B. Human keratinocytes secrete IL-1B upon DPP9 S759A expression. Human keratinocytes were transfected with the respective plasmids with a ratio of 0.5 μg/ 5 x10 5 cells. Conditioned media were harvested 24 hours post transfection. C. Cykokine/chemokine response of keratinocytes to Talabostat is highly similar to MSPC/FKLC patient-derived keratinocytes harboring gain-of-function NLRP1 mutations. Luminex array was performed on conditioned media of Talabostat-treated keratinocytes. Cytokines/chemokines that were also enriched in MSPC/FKLC patient-derived primary keratinocytes are shown in red. D. Cytokine/chemokine analysis of PBMCs treated with 2 μM Talabostat. Luminex array was performed on conditioned media of Talabostat-treated PBMCs isolated from three donors. P-values were calculated based on Student‘s t-tests after log transformation. E. DPP8/9 inhibition by Talabostat causes dose-dependent IL-1B processing. Cultured immortalized keratinocytes were treated with different 0.2 μM, 2 μM, 20 μM and 200 μM Talabostat for 24 hours. Conditioned media was concentrated 10 times for SDS-PAGE. F. DPP8/9 inhibition by S759A and S759P leads to IL-1B processing and secretion. Keratinocytes were transfected with the DPP9 expressing constructs. Conditioned media was harvested 24 hours post-transfection. G. DPP8/9 inhibition by Talabostat causes endogenous ASC oligomerization. DSS crosslinking was performed as in . H. DPP8/9 inhibition by Talabostat leads to IL-1B processing and secretion in PBMCs. Conditioned media from PBMC (Donor 3) was used for SDS-PAGE without prior concentration. I. NLRP1 and ASC knockdown abrogates Talabostat-induced ASC oligomerization and IL-1B processing. Immortalized keratinocytes were treated with 2 μM Talabostat for 24 hours three days after siRNA incubation. Conditioned media was concentrated 10 times before SDS-PAGE. J. Genetic requirement of NLRP1 , ASC and CASP1 , but not NLRP3 in the effect of DPP8/9 inhibition. Immortalized keratinocytes treated with siRNAs and DPP8/9 inhibitors as in J. Conditioned media was diluted 1:5 before IL-1B ELISA. K. CRISPR/Cas9-mediated deletion of NLRP1 and ASC blocks Talabostat-induced pyroptosis. L. Dissection of the genetic requirement for inflammasome activation upon DPP8/9 inhibition in mouse bone marrow derived macrophages (BMDMs). Murine BMDMs from the indicated genotypes were primed with LPS (200 ng/ml), then treated with the indicated concentrations of Talabostat for 24 hours. Related to

Article Snippet: The small molecule inhibitors used are vildagliptin (MedChemExpress), saxagliptin (MedChemExpress), TC-E 5007 (Tocris), butabindide oxalate (MedChemExpress), Talabostat and (MedChemExpress), 1G244 (Santa Cruz Biotechnology), LPS (Ultrapure, Escherichia coli O111:B4, SigmaAldrich) and nigericin 10 uM (Invivogen, #tlrl-nig).

Techniques: Expressing, Transfection, Derivative Assay, Luminex, Isolation, Transformation Assay, Inhibition, Cell Culture, SDS Page, Construct, Concentration Assay, Incubation, Enzyme-linked Immunosorbent Assay, CRISPR, Dissection, Activation Assay

Journal: bioRxiv

Article Title: DPP9 is an endogenous and direct inhibitor of the NLRP1 inflammasome that guards against human auto-inflammatory diseases

doi: 10.1101/260919

Figure Lengend Snippet: A. Talabostat induces IL-1B secretion either alone or in cooperation with LPSpriming in primary human PBMCs. B. Overlap between Talabostat dependent cytokine/chemokine signature and MSPC/FKLC patient-derived keratinocytes. C. Luminex analysis of Talabostat-treated LPS-prestimulated PBMCs. D. List of cytokines/chemokines that are induced by Talabostat in PBMCs. E. Lytic cell death of Talabostat-treated keratinocytes. F. Talabostat induces ASC speck formation in immortalized human keratinocytes. G. Talabostat causes significant leukocyte cell death. H. The effect on IL-1B secretion by a panel of peptidase/protease inhibitors on immortalized keratinocytes. Compounds with known IC50 for DPP9<100 nM are colored red. I. Lytic death in keratinocytes upon Talabostat exposure requires NLRP1 and ASC, but not NLRP3. J. Caspase-1 knockdown abrogates the effect of Talabostat on IL-1B processing.

Article Snippet: The small molecule inhibitors used are vildagliptin (MedChemExpress), saxagliptin (MedChemExpress), TC-E 5007 (Tocris), butabindide oxalate (MedChemExpress), Talabostat and (MedChemExpress), 1G244 (Santa Cruz Biotechnology), LPS (Ultrapure, Escherichia coli O111:B4, SigmaAldrich) and nigericin 10 uM (Invivogen, #tlrl-nig).

Techniques: Derivative Assay, Luminex

Journal: Journal of clinical medicine

Article Title: Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation.

doi: 10.3390/jcm12144632

Figure Lengend Snippet: Figure 2. Effects of vildagliptin, sitagliptin or 1G244 on cell viability. (a) Quantification was performed on cultures of MSC treated with different concentrations of these chemicals for 48 h. (b) Viability quantification in cultures of MSC not differentiated or induced to differentiate into adipocytes (A) or osteoblasts (O) for seven days, non-treated or treated with 10 µM of vildagliptin, sitagliptin or 1G244. V: vildagliptin; S: sitagliptin. * p < 0.05; *** p < 0.001 vs. control (untreated).

Article Snippet: MSC cultures not differentiated or induced to differentiate into adipocytes or osteoblasts were treated with 10 μM of vildagliptin from Selleckchem (Houston, TX, USA), sitagliptin or 1G244 (both from Sigma-Aldrich).

Techniques: Control

Journal: Journal of clinical medicine

Article Title: Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation.

doi: 10.3390/jcm12144632

Figure Lengend Snippet: Figure 3. Effects of vildagliptin, sitagliptin or 1G244 on apoptosis. (a) Representative images of caspase activation and Hoechst nuclei staining in cultures of MSC, not differentiated or differen- tiated into osteoblasts or adipocytes, treated with 10 µM of vildagliptin, sitagliptin or 1G244 for seven days. (b) Apoptosis quantification of treatments described in (a). AU: arbitrary units of caspase staining/Hoechst staining. *** p < 0.001 vs. control (untreated).

Article Snippet: MSC cultures not differentiated or induced to differentiate into adipocytes or osteoblasts were treated with 10 μM of vildagliptin from Selleckchem (Houston, TX, USA), sitagliptin or 1G244 (both from Sigma-Aldrich).

Techniques: Activation Assay, Staining, Control

Journal: Journal of clinical medicine

Article Title: Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation.

doi: 10.3390/jcm12144632

Figure Lengend Snippet: Figure 4. Effects of vildagliptin, sitagliptin or 1G244 in osteoblastic differentiation. Cultures of MSC were induced to differentiate into osteoblasts. (a) Osteoblast mineralization. Representative images of cultures stained with alizarin red S, on day 21 of osteoblastic differentiation, in cultures treated with these chemicals. The graph represents dye elution quantification by spectrophotometry. (b) Gene expression of osteoblastic marker genes (RUNX2, SP7, IBSP and COL1A1) on days 7 and 14 after onset of osteoblastic differentiation, in presence or absence of vildagliptin, sitagliptin or 1G244 chemicals. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control (untreated).

Article Snippet: MSC cultures not differentiated or induced to differentiate into adipocytes or osteoblasts were treated with 10 μM of vildagliptin from Selleckchem (Houston, TX, USA), sitagliptin or 1G244 (both from Sigma-Aldrich).

Techniques: Staining, Spectrophotometry, Gene Expression, Marker, Control

Journal: Journal of clinical medicine

Article Title: Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation.

doi: 10.3390/jcm12144632

Figure Lengend Snippet: Figure 5. Effects of vildagliptin, sitagliptin or 1G244 in β-catenin, DKK1, LRP5 and LRP6 gene expression in MSC cultures induced to differentiate into osteoblasts. (a) Expression of CTNNB1, DKK1, LRP5 and LRP6 genes on day 14 of osteogenic differentiation. (b) Western blot for β-catenin protein of cultures treated as indicated before. Results of the quantification of Western blot bands are shown. O: osteoblasts; V: vildagliptin; S: sitagliptin. * p < 0.05; ** p < 0.01; vs. control (untreated).

Article Snippet: MSC cultures not differentiated or induced to differentiate into adipocytes or osteoblasts were treated with 10 μM of vildagliptin from Selleckchem (Houston, TX, USA), sitagliptin or 1G244 (both from Sigma-Aldrich).

Techniques: Gene Expression, Expressing, Western Blot, Control

Journal: Journal of clinical medicine

Article Title: Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation.

doi: 10.3390/jcm12144632

Figure Lengend Snippet: Figure 6. Effects of vildagliptin, sitagliptin or 1G244 on adipogenic differentiation. MSC cultures were induced to differentiate into adipocytes. (a) Representative light-microscopy pictures (200X) of oil red O staining of lipid droplets on day 14 of adipocyte differentiation in presence of 10 µM of these chemicals. Results of spectrophotometric quantification of dye elutions are shown in the plot. (b) Gene expression of adipocyte marker genes (PPARG2, ATGL and LPL) on days 7 and 14 after starting adipocyte differentiation. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control (untreated).

Article Snippet: MSC cultures not differentiated or induced to differentiate into adipocytes or osteoblasts were treated with 10 μM of vildagliptin from Selleckchem (Houston, TX, USA), sitagliptin or 1G244 (both from Sigma-Aldrich).

Techniques: Light Microscopy, Staining, Gene Expression, Marker, Control

Journal: Journal of clinical medicine

Article Title: Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation.

doi: 10.3390/jcm12144632

Figure Lengend Snippet: Figure 7. Effects of vildagliptin, sitagliptin or 1G244 in β-catenin, DKK1, LRP5 and LRP6 expression in MSC cultures induced to differentiate into adipocytes. (a) Gene expression of CTNNB1, DKK1, LRP5 and LRP6 genes on day 14 of osteogenic differentiation. (b) Western blot for β-catenin protein of cultures treated with these chemicals on day 14 of adipogenic differentiation. Results of the quantification of Western blot bands are shown. A: adipocytes; V: vildagliptin; S: sitagliptin. * p < 0.05; ** p < 0.01 vs. control (untreated).

Article Snippet: MSC cultures not differentiated or induced to differentiate into adipocytes or osteoblasts were treated with 10 μM of vildagliptin from Selleckchem (Houston, TX, USA), sitagliptin or 1G244 (both from Sigma-Aldrich).

Techniques: Expressing, Gene Expression, Western Blot, Control

Journal: Respiratory Research

Article Title: Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition

doi: 10.1186/s12931-017-0660-4

Figure Lengend Snippet: Vildagliptin restored normal pulmonary structure in a mouse model of post-ALI pulmonary fibrosis. a Flow cytometry (FCM) analyses revealed that the number of CD26-expressing pulmonary vascular endothelial cells (PVECs: CD31 + /CD45 − cells) isolated from mice was significantly increased 14 days after LPS administration. This effect was significantly inhibited by systemic vildagliptin administration (* P < 0.05, N = 5). b Representative FCM panels with CD26 + -gated PVECs. c Immunohistochemistry also revealed that the number of CD26-expressing pulmonary vascular endothelial cells (PVECs: CD31 + CD45 − cells) significantly increased 14 days after LPS administration, and this increase could be significantly inhibited by systemic vildagliptin administration. CD31, green; CD26, blue; CD45, red. Scale bars, 100 μm. d Effect of bleomycin on lung architecture in vehicle- or vildagliptin-treated mice as shown by Masson’s trichrome staining of lung tissue sections 28 days after LPS administration. e The Ashcroft fibrosis score was used to compare the degrees of pulmonary fibrosis. Pulmonary fibrosis induced by LPS was significantly attenuated by vildagliptin treatment (* P < 0.05, N = 5). Scale bars, 100 μm.

Article Snippet: For treatment in vivo with vildagliptin, mice were given once daily doses of either 10 mg/kg vildagliptin (Santa Cruz Biotechnology, Dallas, TX) or saline vehicle delivered by intraperitoneal injection for 14 consecutive days from 1 day before the first administration of LPS.

Techniques: Flow Cytometry, Expressing, Isolation, Immunohistochemistry, Staining

Journal: Respiratory Research

Article Title: Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition

doi: 10.1186/s12931-017-0660-4

Figure Lengend Snippet: Antifibrotic effects of vildagliptin were associated with EndMT inhibition in septic lungs. a Flow cytometry (FCM) analyses revealed that the percentage of α-SMA + -gated PVECs significantly increased in LPS-induced pulmonary fibrosis, and this increase was attenuated by systemic administration of vildagliptin (* P < 0.05, N = 5). b Representative FCM panels with α-SMA + -gated PVECs. c FCM analyses revealed that the percentage of S100A4 + -gated PVECs significantly increased in LPS-induced pulmonary fibrosis, and the increase was attenuated by systemic administration of vildagliptin (* P < 0.05, N = 5). d Representative FCM panels with S100A4 + -gated PVECs. e Gene expression of mesenchymal-specific markers ( Col1a1 , Col1a2 and S100a4 ) in isolated PVECs significantly increased 28 days after LPS challenge, whereas gene expression of endothelial specific markers in isolated PVECs ( Pecam1 and Cdh5 ) significantly decreased. Moreover, expression of Twist2 , a transcription factor related to EndMT in PVECs was significantly increased 28 days after LPS challenge. All of these changes were significantly attenuated by systemic administration of vildagliptin (* P < 0.05, N = 5). Values are means ± SEM

Article Snippet: For treatment in vivo with vildagliptin, mice were given once daily doses of either 10 mg/kg vildagliptin (Santa Cruz Biotechnology, Dallas, TX) or saline vehicle delivered by intraperitoneal injection for 14 consecutive days from 1 day before the first administration of LPS.

Techniques: Inhibition, Flow Cytometry, Gene Expression, Isolation, Expressing

Journal: Respiratory Research

Article Title: Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition

doi: 10.1186/s12931-017-0660-4

Figure Lengend Snippet: Vildagliptin suppressed EndMT in a mouse model of post-ALI pulmonary fibrosis. a Immunohistochemistry revealed that the number of CD31 + /α-SMA + -cells (EndMT-cells) isolated from mice increased 28 days after LPS administration, and the increase could be significantly inhibited by systemic vildagliptin administration. CD31, green; α-SMA, red; Hoechst, blue. Scale bars, 100 μm. b Immunohistochemistry revealed that the number of CD31 + /S100A4 + -cells (EndMT-cells) was increased 28 days after LPS administration, and the increase could be significantly inhibited by systemic vildagliptin administration. CD31, green; S100A4, red; Hoechst, blue. Scale bars, 100 μm

Article Snippet: For treatment in vivo with vildagliptin, mice were given once daily doses of either 10 mg/kg vildagliptin (Santa Cruz Biotechnology, Dallas, TX) or saline vehicle delivered by intraperitoneal injection for 14 consecutive days from 1 day before the first administration of LPS.

Techniques: Immunohistochemistry, Isolation

Journal: Respiratory Research

Article Title: Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition

doi: 10.1186/s12931-017-0660-4

Figure Lengend Snippet: Vildagliptin attenuated ROS production in PVECs. a After LPS injection, intracellular ROS significantly rose in PVECs, then peaked after 7 days, and eventually returned to the base line on day 14 (* P < 0.05; n = 6). Intracellular ROS measured in PVECs significantly decreased in LPS-PVECs treated with vildagliptin (Vilda). PVECs ROS production was determined by FCM using DCFDA. Values are means ± SEM. b Fluorescence intensity of oxidized DCFDA in viable HMVEC-Ls (PI − /CD31 + /CD45 − cells) from control- and LPS-treated HMVEC-Ls with or without vildagliptin (Vilda) or Linagliptin (Lina) are shown. The fluorescence intensity increased within 2 h of LPS challenge, which was before the increase in EndMT-HMVEC-Ls. These phenotypic changes were suppressed by vildagliptin or linagliptin (* P < 0.05; n = 4). Values are means ± SEM

Article Snippet: For treatment in vivo with vildagliptin, mice were given once daily doses of either 10 mg/kg vildagliptin (Santa Cruz Biotechnology, Dallas, TX) or saline vehicle delivered by intraperitoneal injection for 14 consecutive days from 1 day before the first administration of LPS.

Techniques: Injection, Fluorescence, Control

Journal: Respiratory Research

Article Title: Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition

doi: 10.1186/s12931-017-0660-4

Figure Lengend Snippet: Vildagliptin inhibited LPS-induced EndMT in the absence of immune cells. a Phase-contrast micrographs of PVECs (CD31 + /CD45 − cells) isolated from mice in the absence or presence of LPS (10 μg/ml for 144 h) and vildagliptin (10 nM)/ linagliptin (100 nM) treatment. The morphology of PVECs exposed to LPS changed to a spindle shape and vildagliptin or linagliptin treatment preserved the original morphology. Scale bars, 50 μm. b FCM analyses also revealed that the percentage of α-SMA + -PVECs significantly increased 144 h after LPS challenge and treatment by vildagliptin or linagliptin significantly suppressed this change (* P < 0.05, N = 5). Values are means ± SEM. c Immunocytochemistry revealed an increase in α-SMA + -PVECs 144 h after LPS challenge, and this increase was suppressed by vildagliptin or linagliptin. CD31, green; α-SMA, red; Hoechst, blue. Scale bars, 50 μm. Vilda; Vildagliptin, Lina; Linagliptin

Article Snippet: For treatment in vivo with vildagliptin, mice were given once daily doses of either 10 mg/kg vildagliptin (Santa Cruz Biotechnology, Dallas, TX) or saline vehicle delivered by intraperitoneal injection for 14 consecutive days from 1 day before the first administration of LPS.

Techniques: Isolation, Immunocytochemistry