Journal: European Journal of Medicinal Chemistry

Article Title: Structure-activity relationships of potentiators of the antibiotic activity of clarithromycin against Escherichia coli

doi: 10.1016/j.ejmech.2019.05.075

Figure Lengend Snippet: Figure 3. Membrane permeabilization assays of 16 in E. coli ATCC 25922. Polymyxin B was included as a comparator membrane-targeting agent. Both 16 and polymyxin B were used at concentrations ranging from 1/32X to 1/2X the minimum inhibitory concentration (MIC), with the MIC values of 16 and polymyxin B being 100 and 1.56 µg/ml, respectively. The compound vehicle was DMSO. (A,B) Impact of E. coli treatment with 16 (A) or polymyxin B (B) on the intensity of the fluorophore N- phenyl-1-naphthylamine (NPN) at a concentration of 10 µM. (C,D) Impact of treatment with 16 (C) or polymyxin B (D) on the percentage of E. coli cells stained with the fluorophore propidium iodide (PI) at a concentration of 50 µM.

Article Snippet: M AN US CR IP T AC CE PT ED Graphical Abstract Compound 16 is associated with a 32-fold enhancement in the potency of clarithromycin against E. coli ATCC 25922 in vitro Graphical Abstract Compound 16 is associated with a 32-fold enhancement in the potency of clarithromycin against E. coli ATCC 25922 in vitro H2N H N NH2 16 H2N H N NH2 16 M AN US CR IP T AC CE PT ED -1- Structure-Activity Relationships of Potentiators of the Antibiotic Activity of Clarithromycin against Escherichia coli Gifty Blanksona, Ajit K. Parhia,c, Malvika Kaulb, Daniel S. Pilchb and Edmond J. LaVoiea aDepartment of Medicinal Chemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey, 08820, U.S.A. bDepartment of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, U.S.A cTAXIS Pharmaceuticals, Inc., Monmouth Junction, New Jersey 08552, U.S.A. 1.

Techniques: Membrane, Concentration Assay, Staining

Journal: European Journal of Medicinal Chemistry

Article Title: Structure-activity relationships of potentiators of the antibiotic activity of clarithromycin against Escherichia coli

doi: 10.1016/j.ejmech.2019.05.075

Figure Lengend Snippet: Figure 4. Assay for inhibition of the efflux of the fluorophore Hoechst 33342 (H33342) by 16 in E. coli ATCC 25922. When present, 16 was used at concentrations ranging from 1/32X to 1/2X MIC (3.13 to 50 µg/mL). All reactions were initiated by the addition of 50 mM glucose except where indicated. H33342 was used at a concentration of 10 µM. Compound vehicle was DMSO.

Article Snippet: M AN US CR IP T AC CE PT ED Graphical Abstract Compound 16 is associated with a 32-fold enhancement in the potency of clarithromycin against E. coli ATCC 25922 in vitro Graphical Abstract Compound 16 is associated with a 32-fold enhancement in the potency of clarithromycin against E. coli ATCC 25922 in vitro H2N H N NH2 16 H2N H N NH2 16 M AN US CR IP T AC CE PT ED -1- Structure-Activity Relationships of Potentiators of the Antibiotic Activity of Clarithromycin against Escherichia coli Gifty Blanksona, Ajit K. Parhia,c, Malvika Kaulb, Daniel S. Pilchb and Edmond J. LaVoiea aDepartment of Medicinal Chemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey, 08820, U.S.A. bDepartment of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, U.S.A cTAXIS Pharmaceuticals, Inc., Monmouth Junction, New Jersey 08552, U.S.A. 1.

Techniques: Inhibition, Concentration Assay

Journal: International Immunopharmacology

Article Title: Cyanorona-20: The first potent anti-SARS-CoV-2 agent

doi: 10.1016/j.intimp.2021.107831

Figure Lengend Snippet: Chemical structures of favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide) and the newly-designed target compound cyanorona-20 (( E )- N -(4-cyanobenzylidene)-6-fluoro-3-hydroxypyrazine-2-carboxamide).

Article Snippet: Surprisingly, cyanorona-20 (EC 50 = 0.45 μM) was found to be about 209 and 45 times more potent than favipiravir (EC 50 = 94.09 μM) and remdesivir (EC 50 = 20.17 μM), respectively, in anti-SARS-CoV-2 activity ( in vitro ).

Techniques:

Journal: International Immunopharmacology

Article Title: Cyanorona-20: The first potent anti-SARS-CoV-2 agent

doi: 10.1016/j.intimp.2021.107831

Figure Lengend Snippet: Schematic representation of the conventional and microwave-assisted synthetic pathways of cyanorona-20 from favipiravir.

Article Snippet: Surprisingly, cyanorona-20 (EC 50 = 0.45 μM) was found to be about 209 and 45 times more potent than favipiravir (EC 50 = 94.09 μM) and remdesivir (EC 50 = 20.17 μM), respectively, in anti-SARS-CoV-2 activity ( in vitro ).

Techniques:

Journal: International Immunopharmacology

Article Title: Cyanorona-20: The first potent anti-SARS-CoV-2 agent

doi: 10.1016/j.intimp.2021.107831

Figure Lengend Snippet: Anti-COVID-19/antiviral activities (along with human/mammalian cells toxicities) of cyanorona-20 and the four reference drugs (favipiravir, remdesivir, HCl-arbidol-H 2 O, and hydroxychloroquine sulfate) against SARS-CoV-2 in Vero E6 cells.

Article Snippet: Surprisingly, cyanorona-20 (EC 50 = 0.45 μM) was found to be about 209 and 45 times more potent than favipiravir (EC 50 = 94.09 μM) and remdesivir (EC 50 = 20.17 μM), respectively, in anti-SARS-CoV-2 activity ( in vitro ).

Techniques: Inhibition, In Vitro, Concentration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mast Cells Inhibit Stem Cell-driven Epithelial Repair in Inflammatory Bowel Disease via Suppressing Wnt/lrp6/β-Catenin Signaling Pathway

doi: 10.1016/j.jcmgh.2026.101741

Figure Lengend Snippet: Analysis results of bulk RNA-seq data from intestinal tissue biopsies of MSCCR cohort, focusing on the association between MCs and transcriptional levels of Wnt/Notch signaling pathway-related genes. ( A–D ) The correlations of MC activation on genes related to Wnt ( Wnt3a , Wnt3 , Ctnnb1 , Lrp5 , Lrp6 , Axin2 ) and Notch ( Notch1 , JAG1 , Dll1 , Dll4 ) signaling pathways. ( A and B ) show correlations in the ileum and colon of patients with CD; ( C–E ) show correlations in the cecum, colon, and rectum of patients with UC. ( E–J ) GSEA of Wnt signaling pathways in patients with IBD stratified by MC infiltration levels. ( E ) GSEA indicated “WNT pathway” enriched in MC-low infiltration group of CD ileum. ( F and G ) In CD colon, GSEA indicated “WNT signaling inhibition” enriched in MC high-infiltration group ( F ), and “WNT pathway” enriched in MC-low infiltration group ( G ). ( H and I ) In UC colon, GSEA indicated “WNT signaling inhibition” enriched in MC high infiltration group ( H ), and “WNT pathway” enriched in MC low infiltration group ( I ). ( J ) GSEA indicated “WNT signaling inhibition” enriched in MC high infiltration group of UC rectum. High, high infiltration group of activated MCs; Low, low infiltration group of activated MCs. Statistical significance: Nominal P values are indicated in each subpanel, and Nominal P < .05 indicated significant enrichment.

Article Snippet: To investigate whether MC-induced inhibitory and cytotoxic effects on organoids could be rescued by activating lrp6/β-catenin signaling, the potent Wnt/lrp6 pathway activator HLY78 (20μM, MCE, HY-122816) was applied.

Techniques: RNA Sequencing, Activation Assay, Protein-Protein interactions, Inhibition

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mast Cells Inhibit Stem Cell-driven Epithelial Repair in Inflammatory Bowel Disease via Suppressing Wnt/lrp6/β-Catenin Signaling Pathway

doi: 10.1016/j.jcmgh.2026.101741

Figure Lengend Snippet: Physiological baseline comparison between BN rat vs MC-deficient Ws/Ws rat. ( A ) Schematic of the experimental workflow. This experiment is conducted concurrently with the establishment of the DSS-induced colitis model. ( B ) Body weight changes (%): Percentage of body weight change relative to the initial weight (day 0). ( C ) DAI score: DAI score was evaluated daily. ( D ) Colon length. ( E and F ) H&E staining of colon tissue: ( E ) Representative H&E-stained. ( F ) Quantitative analysis of epithelial damage score and inflammatory cell infiltration. ( G and H ) IHC of MC tryptase: ( G ) Representative IHC images of colon sections stained with MC tryptase. ( H ) Quantitative analysis of MC number. ( I and J ). Representative IF staining images of total β-catenin ( I ) and active β-catenin ( J ) in colon (n = 3). ( K ) WB analysis of LRP6 and LGR5 proteins with β-actin as the loading control and quantitative analysis was performed. Statistical significance: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: To investigate whether MC-induced inhibitory and cytotoxic effects on organoids could be rescued by activating lrp6/β-catenin signaling, the potent Wnt/lrp6 pathway activator HLY78 (20μM, MCE, HY-122816) was applied.

Techniques: Comparison, Staining, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mast Cells Inhibit Stem Cell-driven Epithelial Repair in Inflammatory Bowel Disease via Suppressing Wnt/lrp6/β-Catenin Signaling Pathway

doi: 10.1016/j.jcmgh.2026.101741

Figure Lengend Snippet: Effect of MCs on ISC maintenance in DSS-induced acute colitis model in rat. ( A ) Schematic of the in vivo experimental workflow. ( B ) Body weight changes (%): Percentage of body weight change relative to the initial weight (day 0) was recorded daily for each group. ( C ) DAI score: DAI score was evaluated daily. ( D ) Colon length. ( E and F ) H&E staining of colon tissue: ( E ) Representative H&E-stained images showing colonic mucosal changes in each group. ( F ) Quantitative analysis of epithelial damage score and inflammatory cell infiltration. ( G and H ) IHC of MC tryptase: ( G ) Representative IHC images of colon sections stained with a primary antibody against MC tryptase. ( H ) Quantitative analysis of MC number. ( I and L ) Relative mRNA expression of MC mediators ( I ), ISC marker Lgr5 ( J ), Wnt signaling ( K ), and Notch signaling ( L ) pathways. ( M and N ) Representative IF staining images of total β-catenin ( M ) and active β-catenin ( N ) in colon (n = 3/group). ( O ) WB analysis of LRP6 and LGR5 proteins with β-actin as the loading control, and quantitative analysis was performed. ( P ) Relative mRNA expression of LTB4R1 and Cxcl2 in colon tissues. Note: For the DSS-treated groups (BN + DSS vs Ws/Ws + DSS), we normalize all gene mRNA levels to their respective genotype-matched controls (ie, BN + DSS values are normalized to BN + water, and Ws/Ws + DSS values to Ws/Ws + water) to eliminate potential genetic background differences. Statistical significance: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: To investigate whether MC-induced inhibitory and cytotoxic effects on organoids could be rescued by activating lrp6/β-catenin signaling, the potent Wnt/lrp6 pathway activator HLY78 (20μM, MCE, HY-122816) was applied.

Techniques: In Vivo, Staining, Expressing, Marker, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mast Cells Inhibit Stem Cell-driven Epithelial Repair in Inflammatory Bowel Disease via Suppressing Wnt/lrp6/β-Catenin Signaling Pathway

doi: 10.1016/j.jcmgh.2026.101741

Figure Lengend Snippet: In vivo validation of MCs’ effect on ISC-mediated epithelial regeneration using a DSS-induced colitis mouse model. ( A ) Schematic of the in vivo experimental workflow. ( B ) Body weight loss: Body weight was recorded daily for each group. ( C ) DAI score: DAI was evaluated daily based on a standard scoring system (combining stool consistency, bleeding, and weight loss) to assess the severity of colitis. ( D and E ) Colon length measurement: Representative images of colon tissues ( D ) and quantitative analysis of colon length ( E ) were used to reflect colonic atrophy. ( F and G ) H&E staining of colon tissue: H&E-stained sections were observed under a light microscope to visualize pathological changes ( F ), and histological analysis was performed to quantify epithelial injury score and mean crypt depth ( G ). ( H and I ) IHC of MC tryptase: Colon sections were stained with a primary antibody against MC tryptase to visualize MCs in colon ( H ); The number of tryptase-positive MCs were counted, reflecting MC infiltration levels in different groups ( I ). ( J ) Expression levels of MC mediator: Relative mRNA expression of MC-specific mediators ( HDC; CPA3 ) detected by qRT-PCR, and CPA3 content in colon detected by ELISA. ( K and L ) Expression levels of ISC-related ( K ) and stemness-related ( L ) genes: qRT-PCR was used to measure the mRNA expression of ISC markers ( Lgr5, Lrig1, Tert ) and stemness-associated genes ( Dek ). ( M and N ) Expression levels of genes related to Wnt signaling ( M ) and Notch signaling ( N ) pathways: qRT-PCR was performed to detect mRNA expression of key genes in Wnt ( Wnt3a, Ctnnb1, Lrp5, Lrp6 ) and Notch ( Notch1, Jagged1 ) pathways, reflecting the activity of these stem cell-regulating signaling pathways. ( O and P ) Representative images showing IF staining of total β-catenin ( O ) and active β-catenin ( P ) in colon (n = 3/group). ( Q ) WB analysis of LRP6 and LGR5 proteins: WB was performed with β-actin as the loading control, and quantification analysis was performed to compare the differences. Statistical significance: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: To investigate whether MC-induced inhibitory and cytotoxic effects on organoids could be rescued by activating lrp6/β-catenin signaling, the potent Wnt/lrp6 pathway activator HLY78 (20μM, MCE, HY-122816) was applied.

Techniques: In Vivo, Biomarker Discovery, Staining, Light Microscopy, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Protein-Protein interactions, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mast Cells Inhibit Stem Cell-driven Epithelial Repair in Inflammatory Bowel Disease via Suppressing Wnt/lrp6/β-Catenin Signaling Pathway

doi: 10.1016/j.jcmgh.2026.101741

Figure Lengend Snippet: In vitro analysis of activated MCs’ direct inhibitory/cytotoxic effects on mouse intestinal organoids, and reversal by Wnt agonists. ( A – I ) Activated BMMCs’ effects on organoids (BMMC− group: organoid cocultured without active BMMC; BMMC+ group: organoid cocultured with active BMMC) ( A ) Co-culture schematic: Activated BMMCs and organoids were co-cultured to assess direct interactions. ( B and C ) Organoid growth: ( B ) Bright-field images; ( C ) quantification of organoid size, bud number, and density. ( D–G ) qRT-PCR analysis: ( D ) ISC markers, ( E ) stemness genes, ( F ) apoptosis genes, ( G ) Wnt pathway genes. ( H and I ) Representative images showing IF staining: Staining for total β-catenin ( H ) and active β-catenin ( I ). (n = 6–10 organoids/group). ( J–Q ) Wnt agonists reverse activated BMMC-induced inhibition (Vehicle group: organoid with DMSO and Substance P; BMMC+DMSO group: organoid cocultured with activated BMMC and treated with DMSO; BMMC+HLY78 group: organoid cocultured with activated BMMC and treated with Wnt agonist HLY78), ( J and K ) Organoid growth recovery: ( J ) Bright-field images; ( K ) quantification of size, bud number, density. ( L–O ) qRT-PCR analysis: ( L ) ISC markers, ( M ) stemness genes, ( N ) Wnt pathway genes, ( O ) apoptosis genes. ( P and Q ) Representative images showing IF staining: Staining for total β-catenin ( P ) and active β-catenin ( Q ). (n = 4–6 organoids/group). Statistical significance: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: To investigate whether MC-induced inhibitory and cytotoxic effects on organoids could be rescued by activating lrp6/β-catenin signaling, the potent Wnt/lrp6 pathway activator HLY78 (20μM, MCE, HY-122816) was applied.

Techniques: In Vitro, Co-Culture Assay, Cell Culture, Quantitative RT-PCR, Staining, Inhibition

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mast Cells Inhibit Stem Cell-driven Epithelial Repair in Inflammatory Bowel Disease via Suppressing Wnt/lrp6/β-Catenin Signaling Pathway

doi: 10.1016/j.jcmgh.2026.101741

Figure Lengend Snippet: The effect of CPA inhibitor on ISCs-mediated epithelial regeneration in DSS-induced colitis. ( A ) Schematic of the in vivo experimental workflow. ( B ) Body weight loss: Body weight was recorded daily for each group. ( C ) DAI score: DAI was evaluated daily based on a standard scoring system (combining stool consistency, bleeding, and weight loss) to assess the severity of colitis. ( D and E ) Colon length measurement: Representative images of colon tissues ( D ) and quantitative analysis of colon length ( E ). ( F and G ) H&E staining of colon tissue: H&E-stained sections were observed under a light microscope to visualize pathological changes ( F ), and histological analysis was performed to quantify epithelial injury score ( G ). ( H and I ) Expression levels of ISC-related ( H ) and stemness-related ( I ) genes: qRT-PCR was used to measure the mRNA expression of ISC markers (Lgr5, Lrig1, Tert) and stemness-associated genes (Dek). ( J ) Expression levels of genes related to Wnt signaling pathways. ( K and L ) Representative images showing IF staining of total β-catenin ( K ) and active β-catenin ( L ) in colon (n = 3/group). ( M ) WB analysis of LRP6 and LGR5 proteins: WB was performed with β-actin as the loading control. and quantification analysis was performed to compare the differences. Statistical significance: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: To investigate whether MC-induced inhibitory and cytotoxic effects on organoids could be rescued by activating lrp6/β-catenin signaling, the potent Wnt/lrp6 pathway activator HLY78 (20μM, MCE, HY-122816) was applied.

Techniques: In Vivo, Staining, Light Microscopy, Expressing, Quantitative RT-PCR, Protein-Protein interactions, Control

Journal: Cell Proliferation

Article Title: T‐LAK cell‐originated protein kinase (TOPK) is a Novel Prognostic and Therapeutic Target in Chordoma

doi: 10.1111/cpr.12901

Figure Lengend Snippet: Effects of TOPK inhibition by OTS514 on chordoma cell lines. (A) Dose‐response curve of UCH2 treated by OTS514 for two, three and five days. The data represent mean ± SD of the independent triple experiment. (B) Dose‐response curve of CH22 treated by OTS514 for two, three and five days. The data represent mean ± SD of the independent triple experiment. (C) Microscopic images exhibit cell number reduction after TOPK inhibition with OTS514 for five days in both UCH2 and CH22 cell lines. (Scale bar; 100 μm). (D) Expression of proteins involved in the anti‐apoptotic activity of TOPK in UCH2 and CH22 chordoma cells after treated with OTS514 for three days, as examined by Western blot. * P < .001

Article Snippet: The highly selective and potent TOPK inhibitor OTS514, ((R)‐9‐(4‐(1‐aminopropan‐2‐yl)phenyl)‐8‐hydroxy‐6‐methylthieno(2,3‐c)quinoline‐4(5H)‐one, (Selleckchem), has been shown to inhibit the effect of TOPK in lung cancer, ovarian cancer, kidney cancer and haematologic malignancies both in vitro and in vivo., , , , OTS514 inhibited TOPK kinase activity at a half‐maximal inhibitory concentration (IC50) value of 2.6 nmol/L.

Techniques: Inhibition, Expressing, Activity Assay, Western Blot

Journal: Cell Proliferation

Article Title: T‐LAK cell‐originated protein kinase (TOPK) is a Novel Prognostic and Therapeutic Target in Chordoma

doi: 10.1111/cpr.12901

Figure Lengend Snippet: TOPK inhibition by OTS514 reduced chordoma cell clonogenicity and spheroid diameter of cells in a 3D cell culture. (A) Representative images of colony formation in UCH2 and CH22. The number of colonies and their sizes were markedly decreased in chordoma cells treated with OTS514. (B) Representative images of spheroid formation of UCH2 and CH22 chordoma cells cultured with 5 nmol/L of OTS514. Fluorescence images of spheroid formation were captured after 14 days of culturing. Spheroid formation of UCH2 and CH22 cultured with OTS514 was significantly smaller than untreated cells at all observation points. (Scale bar; 50 μm). (C) The curve of spheroid diameters of UCH2 cells treated with OTS514 showed significant decrease compared with untreated cells. The data represent mean ± SD of the independent triple experiment. (D) The curve of spheroid diameters of CH22 cells treated with OTS514 showed significant decrease compared with untreated cells. The data represent mean ± SD of the independent triple experiment. * P < .001

Article Snippet: The highly selective and potent TOPK inhibitor OTS514, ((R)‐9‐(4‐(1‐aminopropan‐2‐yl)phenyl)‐8‐hydroxy‐6‐methylthieno(2,3‐c)quinoline‐4(5H)‐one, (Selleckchem), has been shown to inhibit the effect of TOPK in lung cancer, ovarian cancer, kidney cancer and haematologic malignancies both in vitro and in vivo., , , , OTS514 inhibited TOPK kinase activity at a half‐maximal inhibitory concentration (IC50) value of 2.6 nmol/L.

Techniques: Inhibition, Cell Culture, Fluorescence

Journal: Microorganisms

Article Title: Antimicrobial Potential of Cannabinoids: A Scoping Review of the Past 5 Years

doi: 10.3390/microorganisms13020325

Figure Lengend Snippet: Characteristics of the included studies and the main results of the antibacterial activity of cannabinoids. (Abbreviations: CBD: cannabidiol, CBC: cannabichromene, CBG: cannabigerol, CBN: cannabinol, CBCA: cannabichromenic acid, CBDA: cannabidiolic acid, CBDV: cannabidivarin, CBGA: cannabigerolic acid, EPS: extracellular polysaccharide, H 2 CBD: dihydrocannabidiol, LPS: lipopolysaccharide, MIC: minimum inhibitory concentration, THCBD: tetrahydrocannabidiol, VRE: vancomycin-resistant E. faecium , MRSA: methicillin-resistant S. aureus , MSSA: methicillin-sensitive S. aureus , VISA: vancomycin-intermediate resistant S. aureus ).

Article Snippet: Cham et al. [ ] , In vitro study , To evaluate the antibacterial potential of a semisynthetic phytocannabinoid, tetrahydrocannabidiol (THCBD, 4) against sensitive and resistant strains of Staphylococcus aureus . , - CBD showed potent activity against S. aureus ATCC-29213 and E. coli ATCC-25922, with an MIC of 4 μg/mL. - THCBD, 4 showed antibacterial activity against those bacteria but at a lower MIC against S. aureus ATCC-29213 (0.25 μg/mL). - THCBD, 4 showed strong effectiveness against efflux pump-overexpressing strains as well as MRSA. - THCBD, 4 demonstrated additive effects with tetracycline, mupirocin, and penicillin G..

Techniques: Activity Assay, Concentration Assay, In Vitro, Bacteria, Membrane, Clone Assay, Disruption, Modification, Transmission Assay, Permeability, Expressing, Ex Vivo, In Vivo, Formulation, Clinical Proteomics, Lysis, Infection, Inhibition

Journal: PLoS ONE

Article Title: Effect of the Transient Pharmacological Inhibition of Mapk3/1 Pathway on Ovulation in Mice

doi: 10.1371/journal.pone.0119387

Figure Lengend Snippet: Granulosa cells collected by follicular puncture from mice treated with PD0325901 showed absence of phosphorylation of Mapk3/1 at Thr302/Tyr204 compared to vehicle treated mice (n = 3 mice/group/time point).

Article Snippet: A potent selective Map2k (MEK) inhibitor PD0325901 (Selleckchem) was initially dissolved in DMSO (Fisher Scientific) to prepare a stock solution of 100 μg/μl concentration.

Techniques: Phospho-proteomics

Journal: PLoS ONE

Article Title: Effect of the Transient Pharmacological Inhibition of Mapk3/1 Pathway on Ovulation in Mice

doi: 10.1371/journal.pone.0119387

Figure Lengend Snippet: Treatment of immature mice with single dose of 25μg/g of PD0325901 resulted in anovulation compared to vehicle treated mice (A) during superovulation (n = 5 mice/ group). This anovulation as evidenced by trapped GV stage oocytes with compact cumulus cells (C&D) inside the preovulatory follicles at 18h hCG (n = 5 mice/ group). Vehicle treated mice showed well-developed corpus luteum (B) evidencing follicular rupture, ovulation and luteinization. CL—corpus luteum; GV—germinal vesicle.

Article Snippet: A potent selective Map2k (MEK) inhibitor PD0325901 (Selleckchem) was initially dissolved in DMSO (Fisher Scientific) to prepare a stock solution of 100 μg/μl concentration.

Techniques:

Journal: PLoS ONE

Article Title: Effect of the Transient Pharmacological Inhibition of Mapk3/1 Pathway on Ovulation in Mice

doi: 10.1371/journal.pone.0119387

Figure Lengend Snippet: Treatment of immature mice with single dose of 25μg/g of PD0325901 did not alter relative mRNA abundance of Mapk1 and Mapk3 (A). Expression of known hCG regulated genes like Fshr and Nr5a2 (B) as well as hCG induced genes like Scarb1 and Pappa (C) were also not altered in PD0325901 treated mice (n = 3mice/group/time point).

Article Snippet: A potent selective Map2k (MEK) inhibitor PD0325901 (Selleckchem) was initially dissolved in DMSO (Fisher Scientific) to prepare a stock solution of 100 μg/μl concentration.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Effect of the Transient Pharmacological Inhibition of Mapk3/1 Pathway on Ovulation in Mice

doi: 10.1371/journal.pone.0119387

Figure Lengend Snippet: Treatment of immature mice with single dose of 25μg/g of PD0325901 inhibited the expression of hCG-induced genes involved in follicular rupture like Ptgs2 , Pgr , Adamts1 ; genes involved in cumulus expansion like Has2 , Ptx3 , Tnfaip6 ; gene involved in oocyte meiotic maturation like Areg and luteinization like Cebpb (n = 3mice/group/time point). Mapk3/1 regulated LH induced genes like Ptgs2 , Ptx3 and Tnfaip6 were not regulated by Cebpb.

Article Snippet: A potent selective Map2k (MEK) inhibitor PD0325901 (Selleckchem) was initially dissolved in DMSO (Fisher Scientific) to prepare a stock solution of 100 μg/μl concentration.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Effect of the Transient Pharmacological Inhibition of Mapk3/1 Pathway on Ovulation in Mice

doi: 10.1371/journal.pone.0119387

Figure Lengend Snippet: A) Treatment of immature mice with single dose of 25μg/g of PD0325901 inhibited the expression of hCG-induced expression of Egr1 1h and 4h hCG. B) Abundance of Egr1 protein in granulosa cells of the inhibitor and vehicle treated mice. Actb was used as loading control.

Article Snippet: A potent selective Map2k (MEK) inhibitor PD0325901 (Selleckchem) was initially dissolved in DMSO (Fisher Scientific) to prepare a stock solution of 100 μg/μl concentration.

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Effect of the Transient Pharmacological Inhibition of Mapk3/1 Pathway on Ovulation in Mice

doi: 10.1371/journal.pone.0119387

Figure Lengend Snippet: A) Treatment with PD0325901 decreased Fo+PMA induced increases in the abundance of Ptgs2 protein relative to vehicle treated primary granulosa cells. B) Pre-treatment with Egr1-siRNA inhibited Fo+PMA induced expression of Egr1 when compared to control siRNA treatment in primary granulosa cells in vitro. This was associated with reduced abundance of Pgts2 transcript in Egr1-siRNA treated cells. Fo—forskolin; PMA—phorbol-12-myristate.

Article Snippet: A potent selective Map2k (MEK) inhibitor PD0325901 (Selleckchem) was initially dissolved in DMSO (Fisher Scientific) to prepare a stock solution of 100 μg/μl concentration.

Techniques: Expressing, Control, In Vitro

Journal: PLoS ONE

Article Title: Effect of the Transient Pharmacological Inhibition of Mapk3/1 Pathway on Ovulation in Mice

doi: 10.1371/journal.pone.0119387

Figure Lengend Snippet: Preovulatory LH surge induced Mapk3/1 activity regulates transcription of ovulatory genes such as Ptgs2 trough transcription factor Egr1. Inhibition of this LH-induced Mapk3/1 activity by Map2k inhibitor PD0325901 results in anovulation.

Article Snippet: A potent selective Map2k (MEK) inhibitor PD0325901 (Selleckchem) was initially dissolved in DMSO (Fisher Scientific) to prepare a stock solution of 100 μg/μl concentration.

Techniques: Activity Assay, Inhibition

Journal: Journal of immunological methods

Article Title: Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25 + cells

doi: 10.1016/j.jim.2014.01.008

Figure Lengend Snippet: Binding of the human IL-2 fusion toxins to human CD25+ HUT 102/6TG cells. A) Histograms of the DT390-hIL-2 (middle panel), DT390-bi-hIL-2 (right panel) as well as positive control human IL-2 (left panel). Cells with only the secondary staining (PE-conjugated streptavidin) served as the negative control and PE-conjugated mouse anti-human CD25 mAb (clone# M-A251, BD Pharmingen, Cat# 555432) was used for the positive control. Biotin-labeled porcine CD3-εγ (Peraino et al., 2012b) was included as a negative control for background due to protein biotinylation. The data are representative of multiple individual experiments. B–C) Kd determination using flow cytometry and nonlinear least squares fit. MFI was plotted over a range of concentrations of biotinylated B) DT390-hIL-2 and C) DT390-bi-hIL-2. The accompanying least-squares fit and parameters are shown based on the hyperbolic equation y = m1 + m2 * m0 / (m3 + m0) where y = MFI at the given biotinylated human IL-2 fusion toxin concentration, m0 = biotinylated or human IL-2 fusion toxin concentration, m1 = MFI of zero biotinylated human IL-2 fusion toxin, m2 = MFI at saturation and m3 = Kd. The inset table in B) shows a fitted Kd of 15.9 nM for DT390-hIL-2. The inset table in C) shows a fitted Kd of 0.21 nM for DT390-bi-hIL-2.

Article Snippet: All of the binding and potency assay results in this study were confirmed using a second human CD25 + lymphoma cell line, SR (ATCC CRL-2262) ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Binding of the human IL-2 fusion toxins to human CD25 + HUT 102/6TG cells.

Techniques: Binding Assay, Positive Control, Staining, Negative Control, Labeling, Flow Cytometry, Concentration Assay

Journal: Journal of immunological methods

Article Title: Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25 + cells

doi: 10.1016/j.jim.2014.01.008

Figure Lengend Snippet: In vitro binding analysis of the bivalent human IL-2 fusion toxin to human PBMC. A) Human PBMC were stained with biotinylated bivalent human IL-2 fusion toxin and analyzed by flow cytometry. The concentration of the biotinylated bivalent human IL-2 fusion toxin is indicated on the top of each panel. The X axis indicates the FITC-conjugated anti-human CD4 mAb (clone# SK3, Biolegend, Cat# 344604) as the only stain. The Y axis indicates the biotinylated bivalent human IL-2 fusion toxin as the primary stain and the PE-conjugated streptavidin as the secondary stain. The first panel (labeled as “0 nM” of the biotinylated bivalent human IL-2 fusion toxin) is the negative control stained with the biotinylated porcine CD3 εγ (20 nM) (Peraino et al., 2012b) for any background as a result of protein biotinylation. Data are representative of two individual assays. B) Biotinylated bivalent human IL-2 fusion toxin bound to 91% of the human CD4+CD25+ PBMC. In the left panels, the X axis indicates the stain with FITC-conjugated anti-human CD4 mAb. The Y axis indicates the stain with PE-conjugated anti-human CD25 mAb (clone# M-A251, BD, Cat# 555432). Biotinylated bivalent human IL-2 fusion toxin was used as primary stain and the PE-conjugated streptavidin as the secondary stain (right panels). Data are representative of two individual assays.

Article Snippet: All of the binding and potency assay results in this study were confirmed using a second human CD25 + lymphoma cell line, SR (ATCC CRL-2262) ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Binding of the human IL-2 fusion toxins to human CD25 + HUT 102/6TG cells.

Techniques: In Vitro, Binding Assay, Staining, Flow Cytometry, Concentration Assay, Labeling, Negative Control

Journal: Journal of immunological methods

Article Title: Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25 + cells

doi: 10.1016/j.jim.2014.01.008

Figure Lengend Snippet: Binding specificity of the human IL-2 fusion toxins for the human IL-2 receptor on human CD25+ HUT 102/6TG cells. Unlabeled human IL-2 fusion toxins as well as the positive control human IL-2 were each incubated with HUT 102/6TG cells at a range of concentrations for 5 min at 4 °C in the dark. Subsequently, without washing the cells, biotin-labeled human IL-2 was added to each tube containing cells in the presence of the unlabeled fusion toxin or human IL-2. Binding specificity of the human IL-2 fusion toxin or human IL-2 to the IL-2 receptor on HUT 102/6TG cells was measured by a decrease in biotin-labeled human IL-2 staining in the presence of increasing concentrations of the unlabeled proteins. Biotin-labeled porcine CD3-εγ was included as a negative control for background due to protein biotinylation.

Article Snippet: All of the binding and potency assay results in this study were confirmed using a second human CD25 + lymphoma cell line, SR (ATCC CRL-2262) ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Binding of the human IL-2 fusion toxins to human CD25 + HUT 102/6TG cells.

Techniques: Binding Assay, Positive Control, Incubation, Labeling, Staining, Negative Control

Journal: Journal of immunological methods

Article Title: Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25 + cells

doi: 10.1016/j.jim.2014.01.008

Figure Lengend Snippet: A) Human IL-2 fusion toxin-mediated protein synthesis inhibition in human CD25+ HUT 102/6TG cells in vitro: 1) Ontak®-like monovalent human IL-2 fusion toxin (DT390-hIL-2, red line); 2) DT390-bi-hIL-2 (green line); 3) human IL-2 alone (blue line); 4) DT390 alone (black line). Y-axis: cpm value measuring the incorporation of tritiated leucine. X-axis: plated IL-2 fusion toxin concentration. Cycloheximide (1:8) was used as a positive control. The negative control contained cells without fusion toxin. Data are representative of multiple assays. B–C) Binding specificity analysis of the human IL-2 fusion toxins to the target human CD25+ HUT 102/6TG cells in this in vitro protein synthesis inhibition assay using human IL-2 as inhibitor: B) Monovalent human IL-2 fusion toxin (DT390-hIL-2) with (blue) and without (pink) human IL-2 inhibitor; C) bivalent human IL-2 fusion toxin (DT390-bi-hIL-2) with (green) and without (orange) human IL-2 inhibitor. Y-axis: cpm value measuring the incorporation of tritiated leucine. X-axis: plated human IL-2 fusion toxin concentration. Wells containing the human IL-2 inhibitor were incubated for 1 h at 37 °C before addition of fusion toxin. Cycloheximide (1:8) was used as a positive control. Cells without fusion toxin served as the negative control. Data are representative of multiple assays.

Article Snippet: All of the binding and potency assay results in this study were confirmed using a second human CD25 + lymphoma cell line, SR (ATCC CRL-2262) ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Binding of the human IL-2 fusion toxins to human CD25 + HUT 102/6TG cells.

Techniques: Inhibition, In Vitro, Concentration Assay, Positive Control, Negative Control, Binding Assay, Incubation

Journal: Journal of immunological methods

Article Title: Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25 + cells

doi: 10.1016/j.jim.2014.01.008

Figure Lengend Snippet: A) Human IL-2 fusion toxin-mediated cellular proliferation inhibition in human CD25+ HUT 102/6TG cells in vitro: 1) Ontak®-like monovalent human IL-2 fusion toxin (DT390-hIL-2, red line); 2) DT390-bi-hIL-2 (green line); 3) human IL-2 (blue line); 4) DT390 (black line). Y-axis: cpm value measuring incorporation of tritiated thymidine. X-axis: plated IL-2 fusion toxin concentration. Cycloheximide (1:8) was used as a positive control. The negative control contained cells without fusion toxin. Data are representative of multiple assays. B–C) Binding specificity analysis of the human IL-2 fusion toxins to the target human CD25+ HUT 102/6TG cells during the in vitro cellular proliferation inhibition assay using human IL-2 as inhibitor: B) Ontak®-like monovalent human IL-2 fusion (DT390-hIL-2) toxin with (blue) and without (pink) human IL-2 inhibitor; C) bivalent human IL-2 fusion toxin (DT390-bi-hIL-2) with (green) and without (orange) human IL-2 inhibitor. Y-axis: cpm value measuring cellular incorporation of tritiated thymidine. X-axis: plated human IL-2 fusion toxin concentration. Wells containing the human IL-2 inhibitor were incubated for 1 h at 37 °C before addition of the fusion toxin. Cycloheximide (1:8) was used as a positive control. Cells without fusion toxin served as the negative control. Data are representative of multiple assays.

Article Snippet: All of the binding and potency assay results in this study were confirmed using a second human CD25 + lymphoma cell line, SR (ATCC CRL-2262) ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Binding of the human IL-2 fusion toxins to human CD25 + HUT 102/6TG cells.

Techniques: Inhibition, In Vitro, Concentration Assay, Positive Control, Negative Control, Binding Assay, Incubation