Journal: Saudi Pharmaceutical Journal : SPJ

Article Title: Design, synthesis, molecular docking, and in vitro studies of 2-mercaptoquinazolin-4(3 H )-ones as potential anti-breast cancer agents

doi: 10.1016/j.jsps.2024.101971

Figure Lengend Snippet: Inhibition assay of the synthesized quinazolinone compounds (3a, 3b, 3e, 3 g, and 3 h) against KSP (% inhibition at 2 µM and IC 50 ), using Ispinesib as positive control, and against PI3Kδ using Idelalisib as positive control.

Article Snippet: For IC 50 value measurement, a microtubule-activated ATPase kinetic assay kit (Cytoskeleton, Cat. # BK060) was used as per the manufacturer’s instructions ( ).

Techniques: Inhibition, Synthesized, Positive Control, Control, Activity Assay

Journal: Scientific Reports

Article Title: Rapid development of neutralizing and diagnostic SARS-COV-2 mouse monoclonal antibodies

doi: 10.1038/s41598-021-88809-0

Figure Lengend Snippet: SARS-CoV-2 Spike protein subunit vaccine strategy and humoral immune response in mice. ( a ) Recombinant spike subunit 1 protein (His-S1, residues 1–681) or S1 Receptor Binding Domain (mouse Fc-RBD, residues 319–541, ACE2 contact residues in purple; PDBID: 6vxx) antigens. ( b ) VLP display of Fc-tagged antigens using the PP7 particle bearing 120 ZZ-domains; a 1:1 mass ratio of mFc-RBD and VLP provides a Fc:ZZ molar ratio of approximately 0.8. R h = hydrodynamic radius measured by dynamic light scattering in phosphate buffer. ( c ) Vaccine schedule and strategy. Six-week old female BALB/c mice (n = 3 per group) were immunized with primary antigen and adjuvant on day 0 followed by boosts on days 14 and 27. Blood was collected for ELISA on days 0, 14, 21 and 30. ( d ) ELISA responses for serum dilutions against plated His-S1 protein from the sacrificed mice at day 30. ( e ) Titer values from ELISA analysis as in panel ( d ), against plated His-S1 or spike ectodomain protein. Immunization series defined in panel ( c ); ( a ) and ( b ) designate different mice within that series. Experimental error represents standard deviation.

Article Snippet: His2 biosensors were hydrated for 10 min in kinetics buffer, then loaded for 300 s with 2.5 µg/mL ACE2-His (residues 1–740) (Sino Biological) or truncated ACE2 (described above for the mix-and-read assay) in kinetics buffer.

Techniques: Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Rapid development of neutralizing and diagnostic SARS-COV-2 mouse monoclonal antibodies

doi: 10.1038/s41598-021-88809-0

Figure Lengend Snippet: Functional utility of SARS-CoV-2 mAbs. ( a ) Assessment of inhibition of S1 RBD binding to human ACE2 receptor as measured by BLI, performed at a single antibody concentration. Both full-length ACE2 (residues #1-740, pink bars) and truncated ACE2 (residues #1-614, black bars) were included. Data represents percent of signal change normalized to response of ACE2/RBD binding with no mAb present, error bars represent standard deviation of technical replicates. Neutralizing antibodies in both assays are denoted by asterisks; adjacent mAbs in italics are identical. ( b ) Representative (mAb 3G7) dose-dependent antibody-mediated inhibition of ecto-Spike binding to full-length human ACE2 receptor; readout is the wavelength shift in nm in the BLI interferometric response , . ( c ) In vitro mAb neutralization of SARS-CoV-2 USA/WA1/2020 virus (blue bars) or of GFP-expressing SARS-CoV-2 (green dots). Asterisks mark particularly effective entries. ( d ) Binding of antibodies to biotinylated peptide (amino acids 486–501 with C488S substitution; containing four out of five reported RBD-hACE2 contacts) plated on streptavidin-coated plates as measured by ELISA. Dashed line represents the average plus twice the standard deviation of the values obtained for replicates involving three identical mAbs (3A2, 3A7, 3F5); error bars = standard deviation. ( e ) Immunohistochemical staining of lung tissue from an early confirmed SARS-CoV-2 fatal case by (top) known SARS polyclonal nucleocapsid antibody , (middle) mAbs 3D2, and (bottom) 3D7 (1:100 mAb dilution, antigen detection in red). ( f ) Specificity of mAbs determined by split nanoluciferase assay including either SARS1 or SARS2 RBD incubated with mAb followed by ACE2, normalized to ACE2/RBD signal without antibody. Negative values denote inhibition, positive indicate binding enhancement observed only with truncated ACE2. ( g ) ELISA detection of mAb binding to gamma-inactivated SARS-COV-2 (1 µg/mL TCID50 units plated); dotted line denotes background signal. ( h ) Dose-dependent gamma-inactivated SARS-COV-2 binding of select mAbs (5 µg/mL mAb) measured by ELISA, sigmoidal dose–response curve fitted by GraphPad Prism8. ( i ) Limit of detection quantification of mAbs in panel g. LOD calculated as the minimum virus concentration giving rise to a signal equivalent to the mean background signal plus 3 experimental standard deviations (GraphPad Prism 8).

Article Snippet: His2 biosensors were hydrated for 10 min in kinetics buffer, then loaded for 300 s with 2.5 µg/mL ACE2-His (residues 1–740) (Sino Biological) or truncated ACE2 (described above for the mix-and-read assay) in kinetics buffer.

Techniques: Functional Assay, Inhibition, Binding Assay, Concentration Assay, Standard Deviation, In Vitro, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Incubation

Journal: Journal of Neuroinflammation

Article Title: Early effects of LPS-induced neuroinflammation on the rat hippocampal glycolytic pathway

doi: 10.1186/s12974-022-02612-w

Figure Lengend Snippet: LPS activates neuroinflammatory pathways in the hippocampus. Pro-inflammatory immunocontent of cytokines (IL-1β and TNF-α) was measured by ELISA. Gene expressions of cytokines and receptors were evaluated by RT-PCR. Phospho-NFκB was evaluated in total homogenate and nucleus fraction by Western blot. LPS increases IL-1β at both time points, and at 6 h TNF-α was decreased ( A ). IL-1b mRNA increases only at 24 h after LPS injection, with no changes in its receptor (ILR1), and in TNF-α or its receptor, TNFR1 (B). Representative image of Western blot data ( C ). The translocation to the nucleus of phospho-NF-κB increases at both time points ( D ). No changes in mRNA of receptors (TLR2, TLR4, RAGE) involved in neuroinflammatory signaling. NLRP3 mRNA was increased only at 6 h ( E ). Values are expressed as means ± standard error. LPS 6 h (euthanized at 6 h after LPS administration), LPS 24 h (euthanized at 24 h after LPS administration). Data were analyzed by ANOVA, followed by the Tukey test, assuming P < 0.05. * means significant increase, when compared to sham group (* P < 0.05, ** P < 0.01), # means significant decrease, when compared to sham group

Article Snippet: The messenger RNAs (mRNAs) encoding TLR2 (#Rn02133647_s1), TLR4 (#Rn00569848_m1), RAGE (#Rn01525753_g1), NLRP3 (#Rn04244620_m1), IL-1β (#Rn00580432_m1), IL1R1 (#Rn00565482_m1), TNF-α (#Rn99999017_m1), TNFR1 (#Rn01492348_m1), G6PD (#Rn01529640_g1), and AMPK (#Rn00576935_m1) were quantified using the TaqMan real-time RT-PCR system, employing inventory primers and probes purchased from Applied Biosystems.

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Injection, Translocation Assay

Journal: Journal of Neuroinflammation

Article Title: Early effects of LPS-induced neuroinflammation on the rat hippocampal glycolytic pathway

doi: 10.1186/s12974-022-02612-w

Figure Lengend Snippet: Inhibitors of neuroinflammation signaling reverse the changes in the glycolytic pathway induced by LPS in acute hippocampal slices. Glucose uptake was measured by a radioactivity assay. Lactate medium levels were evaluated by a spectrophotometric method. PFK1 activity was analyzed by kinetic assay. Downregulation of neuroinflammation reverses the glucose uptake effects. The effects of arundic acid (AA), an inhibitor of reactive astrocytes ( A ), minocycline (mino), an inhibitor of microglia cells ( C ), and MCC950 (MCC), an inhibitor of NLRP3 assembly ( E ), on glucose uptake were evaluated. The high lactate extracellular levels were reversed by AA ( B ) and mino ( D ). The specific inhibition of NLRP3 reverses the activity of PFK1 ( G ). Values are expressed as means ± standard error. Data were analyzed by ANOVA, followed by the Tukey test, assuming P < 0.05. * means significant increase when compared to sham group

Article Snippet: The messenger RNAs (mRNAs) encoding TLR2 (#Rn02133647_s1), TLR4 (#Rn00569848_m1), RAGE (#Rn01525753_g1), NLRP3 (#Rn04244620_m1), IL-1β (#Rn00580432_m1), IL1R1 (#Rn00565482_m1), TNF-α (#Rn99999017_m1), TNFR1 (#Rn01492348_m1), G6PD (#Rn01529640_g1), and AMPK (#Rn00576935_m1) were quantified using the TaqMan real-time RT-PCR system, employing inventory primers and probes purchased from Applied Biosystems.

Techniques: Radioactivity, Activity Assay, Kinetic Assay, Inhibition

Journal: Journal of Neuroinflammation

Article Title: Early effects of LPS-induced neuroinflammation on the rat hippocampal glycolytic pathway

doi: 10.1186/s12974-022-02612-w

Figure Lengend Snippet: Inhibitors of neuroinflammation signaling reverse the effects of LPS on IL-1β and S100B in acute hippocampal slices. Pro-inflammatory immunocontents of IL-1β and the S100B protein were measured by ELISA. Astrocyte inhibition by arundic acid (AA) reversed the elevations in IL-1β and S100B ( A - C ). Microglia inhibition by minocycline (mino) only reversed the increase in IL-1β ( D - F ). NLRP3 inhibition by MCC950 (MCC) reversed the IL-1β and S100B secretion that was induced by LPS ( H , I ) and, when compared to LPS group, the intracellular immunocontent of IL-1β ( G ). Values are expressed as means ± standard error. Data were analyzed by ANOVA, followed by the Tukey test, assuming P < 0.05. * means significant increase, when compared to sham group

Article Snippet: The messenger RNAs (mRNAs) encoding TLR2 (#Rn02133647_s1), TLR4 (#Rn00569848_m1), RAGE (#Rn01525753_g1), NLRP3 (#Rn04244620_m1), IL-1β (#Rn00580432_m1), IL1R1 (#Rn00565482_m1), TNF-α (#Rn99999017_m1), TNFR1 (#Rn01492348_m1), G6PD (#Rn01529640_g1), and AMPK (#Rn00576935_m1) were quantified using the TaqMan real-time RT-PCR system, employing inventory primers and probes purchased from Applied Biosystems.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A GMR enzymatic assay for quantifying nuclease and peptidase activity

doi: 10.3389/fbioe.2024.1363186

Figure Lengend Snippet: Human Neutrophil Elastase Assay. (A) Illustration of the neutrophil elastase assay. The peptide substrate is covalently attached to the sensor surface and then bound to streptavidin-MNPs via a biotin group on the peptide. This results in an increase in magnetoresistance. The addition of human neutrophil elastase cleaves the substrate, decreasing the signal. (B) An example of how the signal is loaded and then reduced by adding human neutrophil elastase. The signal is displayed as a percentage of the loading signal after the magnetic nanoparticle binding has saturated. (C) Human neutrophil elastase assays with inhibition by sivelestat sodium. All three assays have 20 μg/mL of neutrophil elastase, but the inhibitor concentration increases from 0 to 20 μg/mL. (D) Human neutrophil elastase titration serial diluting from 125 to 3.9 nM by a factor of 2 in PBS containing 0.01% Tween-20. The magnetic assay results are shown as the maximum velocity.

Article Snippet: The assay is initiated by adding 100 μL of 677 nM (20 μg/mL) human neutrophil elastase (Athens Research & Technology #16-14–051200) in PBS containing 0.01% Tween-20.

Techniques: Binding Assay, Inhibition, Concentration Assay, Titration

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A GMR enzymatic assay for quantifying nuclease and peptidase activity

doi: 10.3389/fbioe.2024.1363186

Figure Lengend Snippet: Validation of Magnetic Neutrophil Elastase Assay in Buffer and Sputum. (A) Validation of the magnetic human neutrophil elastase assay by comparison to a traditional AMC assay readout with a spectrometer. Each point represents a different concentration of human neutrophil elastase in buffer readout by the GMR readout station (shown in the y-axis) and the spectrometer (shown in the x-axis). The concentration ranges from 3.9 to 125 nM of human neutrophil elastase. (B) Validation of the magnetic human neutrophil elastase assay compared to a traditional amc assay readout with a spectrometer. Each point represents a different human sputum sample of patients with cystic fibrosis measured by the GMR station (shown in the y-axis) and the spectrometer (shown in the x-axis).

Article Snippet: The assay is initiated by adding 100 μL of 677 nM (20 μg/mL) human neutrophil elastase (Athens Research & Technology #16-14–051200) in PBS containing 0.01% Tween-20.

Techniques: Biomarker Discovery, Comparison, Ub-AMC Assay, Concentration Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A GMR enzymatic assay for quantifying nuclease and peptidase activity

doi: 10.3389/fbioe.2024.1363186

Figure Lengend Snippet: Potential applications of hydrolase activity assays.

Article Snippet: The assay is initiated by adding 100 μL of 677 nM (20 μg/mL) human neutrophil elastase (Athens Research & Technology #16-14–051200) in PBS containing 0.01% Tween-20.

Techniques: Activity Assay, Kinetic Assay, Mass Spectrometry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Nucleic Acid Electrophoresis, Infection, MicroChIP Assay, Electrophoresis, Colorimetric Assay, Fluorescence, Drug discovery, Reflux

Journal: BioMed Research International

Article Title: Characterization of the Modulatory Effect of Hydroxychloroquine on ACE2 Activity: New Insights in relation to COVID-19

doi: 10.1155/2021/6614000

Figure Lengend Snippet: Inhibition of ACE2 activity by different concentrations on MLN-4760 or HCQ. Graph shows % of inhibition for ACE2 activity in an inhibitory activity in vitro assay using different concentrations of the specific ACE2 blocker MLN-4760 (black circles) and the aminoquinoline HCQ (diamonds). n = 3‐5.

Article Snippet: Recombinant Human ACE2 protein and Recombinant Human Coronavirus SARS-CoV-2 Spike Glycoprotein RBD (Active) (ab151852 and ab273065, respectively) were purchased from ABCAM; MLN-4760 (Cat. No.: HY-19414-10) was purchased from https://www.medchemexpress.com/ , and SensoLyte®390 ACE2 Activity Assay Kit (AS-72086) was purchased from https://www.anaspec.com ; HCQ was purchased from a local pharmaceutical provider.

Techniques: Inhibition, Activity Assay, In Vitro

Journal: BioMed Research International

Article Title: Characterization of the Modulatory Effect of Hydroxychloroquine on ACE2 Activity: New Insights in relation to COVID-19

doi: 10.1155/2021/6614000

Figure Lengend Snippet: Modulation of ACE2 activity by SARS-CoV-2 spike glycoprotein and its interaction with the inhibitors MLN-4760 and HCQ. Enzyme activity was assayed in the presence of SARS-CoV-2 spike protein (SP), ACE2 inhibitor MLN4760 (MLN), aminoquinoline hydroxychloroquine (HCQ), or their combinations. (a) ACE2 activity in presence of HCQ, MLN4760, or its combination was measured in a kinetic assay using the SensoLyte®390 kit. Fluorescence level and linear regression are represented for each reaction. (b) Kinetic assay showing the effect of SP on HCQ inhibition. (c) Quantification of the effect of SP, MLN, and HCQ on ACE2 activity. SP effect on the inhibition of ACE2 produced by (d) MLN and (e) HCQ. (f) Combined effect HCQ and MLN on ACE2 activity. Relevant comparisons are shown within brackets. ∗ P < 0.05. n = 3‐5.

Article Snippet: Recombinant Human ACE2 protein and Recombinant Human Coronavirus SARS-CoV-2 Spike Glycoprotein RBD (Active) (ab151852 and ab273065, respectively) were purchased from ABCAM; MLN-4760 (Cat. No.: HY-19414-10) was purchased from https://www.medchemexpress.com/ , and SensoLyte®390 ACE2 Activity Assay Kit (AS-72086) was purchased from https://www.anaspec.com ; HCQ was purchased from a local pharmaceutical provider.

Techniques: Activity Assay, Kinetic Assay, Fluorescence, Inhibition, Produced

Journal: International journal of molecular sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

doi: 10.3390/ijms23169311

Figure Lengend Snippet: Figure 1. CNTF inhibits mineralization in cementoblasts. (A,B) Representative micrographs demon- strated that CNTF significantly inhibited mineralization, measured by solubilized Alizarin Red staining at day 7. Mineralization nodules are stained in brown and quantified by further dilution with cetylpiridiumchlorid. Results are shown as an optical density of 490 nm and are normalized to the control group. (C) Exogenous CNTF effects are immediate, and inhibition of OPG secretion is detected within 90 min. Results are shown as pg/mL. (D) A total of 1.5 h after CNTF administration to OCCM-30 cell culture that had been differentiated for 3 days, RUNX-2, OCN, BMP-7, and BSP mRNA expression was significantly reduced, while SPON-2 expression was not significantly altered. Gene expression levels were normalized to PPIB using the 2−∆∆Cq method. Data are indicated as mean ± standard deviation (SD) of three independent experiments (triplicate wells). ns, no significant difference; # p < 0.05; ## p < 0.005; ### p < 0.0005; #### p < 0.0005 vs. unstimulated control.

Article Snippet: OCCM-30 cells were kinetically stimulated using recombinant mouse CNTF protein (Novus Biologicals, Wiesbaden, Germany).

Techniques: Staining, Control, Inhibition, Cell Culture, Expressing, Gene Expression, Standard Deviation

Journal: International journal of molecular sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

doi: 10.3390/ijms23169311

Figure Lengend Snippet: Figure 2. CNTF phosphorylates STAT3 and triggers COX-2, SHP-2, and cPLAβ expression in cementoblasts. (A,B) The protein expression of STAT3 and its phosphorylated form were determined by WB. Results were calculated relative to loading control β-actin and reported in the line chart. (C–F) WB showed protein expression of the COX-2, SHP-2, cPLAα, and cPLAβ in OCCM-30 cells induced by CNTF protein (400 ng/mL) for different periods. Internal β-actin served as the loading control. Data are indicated as mean ± standard deviation (SD) of three independent experiments (triplicate wells). ns, no significant difference; # p < 0.05; ## p < 0.005 vs. unstimulated control.

Article Snippet: OCCM-30 cells were kinetically stimulated using recombinant mouse CNTF protein (Novus Biologicals, Wiesbaden, Germany).

Techniques: Expressing, Control, Standard Deviation

Journal: International journal of molecular sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

doi: 10.3390/ijms23169311

Figure Lengend Snippet: Figure 3. CNTF activates ERK1/2, P38, and JNK MAPK signaling. (A,C,E) Representative im- munoblot of p-ERK1/2, p-P38, and p-JNK protein expression in the presence of CNTF (400 ng/mL) at different time points. Internal β-actin served as the loading control. (B,D,F) Densitometric analysis of the bands indicated a significant upregulation of p-ERK1/2, p-P38, and p-JNK expression relative to the non-stimulated control group. Data are indicated as mean ± standard deviation (SD) of three independent experiments (triplicate wells). ns, no significant difference; # p < 0.05; ## p < 0.005; ### p < 0.0005 vs. unstimulated control.

Article Snippet: OCCM-30 cells were kinetically stimulated using recombinant mouse CNTF protein (Novus Biologicals, Wiesbaden, Germany).

Techniques: Expressing, Control, Standard Deviation

Journal: International journal of molecular sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

doi: 10.3390/ijms23169311

Figure Lengend Snippet: Figure 4. CNTF functions as an autophagy inducer. (A) Representative images from Cyto-ID Green staining of autophagic vesicles were captured using a fluorescence microscope. Individual and merged images of blue florescence (cell nuclei) and green fluorescence (red arrow, autophagosome) staining are shown (image magnification: 60×). Flow cytometric analysis of autophagosomes in cementoblasts stained with the Cyto-ID@ dye is shown. (B) mRNA expression analysis of autophagy related genes LC3A, Beclin-1, and Atg-5 was assessed using RT-qPCR in cementoblasts after stimulation of exogenous CNTF. (C,D) Time-dependent response of OCCM-30 cells to CNTF was evaluated by Western blots. (E) Densitometrical analysis of the bands showed an upregulation of LC3A/B, Beclin-1, and Atg-5. Data are indicated as mean ± standard deviation (SD) of three independent experiments (triplicate wells). ns, no significant difference; # p < 0.05; ## p < 0.005 vs. unstimulated control.

Article Snippet: OCCM-30 cells were kinetically stimulated using recombinant mouse CNTF protein (Novus Biologicals, Wiesbaden, Germany).

Techniques: Staining, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Control

Journal: International journal of molecular sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

doi: 10.3390/ijms23169311

Figure Lengend Snippet: Figure 5. The STAT3/ERK signal is involved in the regulation of the mineralization of cementoblasts and autophagy. (A,B) Graphics show the mineralization assay of cells exposed to the ERK1/2 inhibitor (FR180204) or the STAT3 inhibitor (sc-202818) and co-stimulation with CNTF (400 ng/mL). Solubilized Alizarin Red staining at day 7 and day 14 was measured at a wavelength of 490 nm by spectrophotometer. (C) OPG secretion after STAT3 and ERK1/2 antagonists (sc-202818 and FR180204) stimulation is detected by ELISA assay at day 14. Results are shown as pg/mL. n = 3 independent biological replicates. (D,E) Flow cytometric analysis of autophagosomes in cementoblasts stained with the Cyto-ID@ dye. DMSO-only treated cells served as negative control. The STAT3 and ERK1/2 antagonists were added 4 h prior to staining. Data are indicated as mean ± standard deviation (SD) of three independent experiments (triplicate wells). ns, no significant difference; # p < 0.05; ## p < 0.005; ### p < 0.0005 vs. unstimulated control.

Article Snippet: OCCM-30 cells were kinetically stimulated using recombinant mouse CNTF protein (Novus Biologicals, Wiesbaden, Germany).

Techniques: Mineralization Assay, Staining, Spectrophotometry, Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation, Control

Journal: International journal of molecular sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

doi: 10.3390/ijms23169311

Figure Lengend Snippet: Scheme 1. The scheme summarizes the possible mechanisms of CNTF on the autophagy and cementogenesis regulation of cementoblasts. CNTF impaired cementogenesis along with STAT3, COX-2, SHP-2, cPLAα, and MAPK signalling networks are involved. CNTF induced autophago- some formation through LC3A/B, Beclin-1, and Atg-5 activation. Furthermore, the autophagy and cementogenesis were partially regulated by STAT3 or ERK1/2.

Article Snippet: OCCM-30 cells were kinetically stimulated using recombinant mouse CNTF protein (Novus Biologicals, Wiesbaden, Germany).

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Natural Potent NAAA Inhibitor Atractylodin Counteracts LPS-Induced Microglial Activation

doi: 10.3389/fphar.2020.577319

Figure Lengend Snippet: Screening model of NAAA inhibitors based on fluorescence. (A) Illustrator of the hydrolysis of the PAMCA substrate into the fluorescent compound AMC by NAAA. (B) Kinetic assay of NAAA with PAMCA. (C) Concentration-dependent inhibition of NAAA by AM9053 and F96. The data are expressed as mean ± SEM.

Article Snippet: TNF-α, IL-1β, and IL-6 Valukine ELISA kits and recombinant human NAAA protein were purchased from R&D Systems (Shanghai, China).

Techniques: Fluorescence, Kinetic Assay, Concentration Assay, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Natural Potent NAAA Inhibitor Atractylodin Counteracts LPS-Induced Microglial Activation

doi: 10.3389/fphar.2020.577319

Figure Lengend Snippet: Atractylodin screened from a small natural compound library as potent NAAA inhibitor. (A) Five compounds screened from 465 compounds showing NAAA inhibition > 50% at 50 μM. (B) Concentration-dependent inhibition of NAAA activity by compounds 1–5, and (C) . Chemical structure of natural NAAA inhibitors. 1. Atractylodin, 2. Sesamin, 3. D-Tetrandrine, 4. Amentoflavone, 5. Anwuligan.

Article Snippet: TNF-α, IL-1β, and IL-6 Valukine ELISA kits and recombinant human NAAA protein were purchased from R&D Systems (Shanghai, China).

Techniques: Drug discovery, Inhibition, Concentration Assay, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Natural Potent NAAA Inhibitor Atractylodin Counteracts LPS-Induced Microglial Activation

doi: 10.3389/fphar.2020.577319

Figure Lengend Snippet: Characterization of the NAAA inhibitor atractylodin. (A) Lineweaver-Burk plot indicates competitive inhibition of NAAA by atractylodin (Atr, 3 µM). (B) Rapid, (C) Dilution assay indicates Atr, a reversible NAAA inhibitor. Data are shown as the mean ± SEM. ***P < 0.001 compared with the control group (V).

Article Snippet: TNF-α, IL-1β, and IL-6 Valukine ELISA kits and recombinant human NAAA protein were purchased from R&D Systems (Shanghai, China).

Techniques: Inhibition, Dilution Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Natural Potent NAAA Inhibitor Atractylodin Counteracts LPS-Induced Microglial Activation

doi: 10.3389/fphar.2020.577319

Figure Lengend Snippet: Interaction of NAAA with atractylodin. (A) Docking detail of the substrate-binding site of human NAAA (PDB ID: 6DXX) bound to the inhibitor atractylodin (light pink carbons). (B) The 2D Schematic diagram of the interaction. (C) The RMSD fluctuation of NAAA Cα atoms, and the RMSD fluctuation of Atractylodin along the 100 ns MD simulations.

Article Snippet: TNF-α, IL-1β, and IL-6 Valukine ELISA kits and recombinant human NAAA protein were purchased from R&D Systems (Shanghai, China).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Natural Potent NAAA Inhibitor Atractylodin Counteracts LPS-Induced Microglial Activation

doi: 10.3389/fphar.2020.577319

Figure Lengend Snippet: Effects of NAAA inhibitors on cell viability and nitrite expression in BV-2 cells. (A) Cell viability and (B) The inhibitory effect of compounds on nitrite production. Numbers represent Sesamin (1), Atractylodin (2), D-tetrandrine (3), amentoflavone (4), and anwuligan (5). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group (V).

Article Snippet: TNF-α, IL-1β, and IL-6 Valukine ELISA kits and recombinant human NAAA protein were purchased from R&D Systems (Shanghai, China).

Techniques: Expressing, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Helicobacter pylori –Induced Rev-erbα Fosters Gastric Bacteria Colonization by Impairing Host Innate and Adaptive Defense

doi: 10.1016/j.jcmgh.2021.02.013

Figure Lengend Snippet: Antibodies and Other Reagents

Article Snippet: Real-time PCR Master Mix (QPK-201) , Toyobo.

Techniques: Flow Cytometry, Immunohistochemical staining, Staining, Immunofluorescence, Blocking Assay, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Protein-Protein interactions, Inhibition, Phospho-proteomics, Luciferase, Reporter Assay, Plasmid Preparation, Purification, Cell Isolation, Pore Size, Protein Extraction, Transfection, Real-time Polymerase Chain Reaction, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes

doi: 10.1074/jbc.m116.770560

Figure Lengend Snippet: FIGURE 4. Effect of the P1 residue (Met versus Arg) on the inhibition of elastases and chymotrypsins by SGPI-2 variants E1 and E8 containing a P1 Leu. The graph shows pairwise comparisons of relative KD values of inhib- itor binding to the indicated proteinases, normalized to the E1 value.

Article Snippet: Porcine CELA1 (catalog no. E0127) used for phage selection was purchased from Sigma, and the enzyme used for kinetic and inhibition assays (code ESFF, catalog no. LS006363) was from Worthington.

Techniques: Residue, Inhibition, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes

doi: 10.1074/jbc.m116.770560

Figure Lengend Snippet: FIGURE 5. Effect of the P2 residue on the inhibition of elastases and chy- motrypsins by SGPI-2 variants. A, inhibition by variants E2 and E7 contain- ingaP1IleandaP2LeuversusGlu.PairwisecomparisonsofrelativeKDvalues of inhibitor binding to the indicated proteinases are shown, normalized to the E2 value. ND, no inhibition was detected. B, inhibition by variants E1 and E9 containing a P1 Leu and a P2 Leu versus Tyr. Pairwise comparisons of relative KD values normalized to the E1 value are shown. C, inhibition by vari- ants E10 and E11 containing a P1 Leu, a P4 Ala, and a P2 Leu versus Tyr. Pairwise comparisons of relative KD values normalized to the E10 value are shown.

Article Snippet: Porcine CELA1 (catalog no. E0127) used for phage selection was purchased from Sigma, and the enzyme used for kinetic and inhibition assays (code ESFF, catalog no. LS006363) was from Worthington.

Techniques: Residue, Inhibition, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes

doi: 10.1074/jbc.m116.770560

Figure Lengend Snippet: FIGURE 6. Effect of the P4 residue (His versus Ala) on the inhibition of elastases and chymotrypsins by SGPI-2 variants E1 and E12 containing a P1 Leu. The graph shows pairwise comparisons of relative KD values of inhib- itor binding to the indicated proteinases, normalized to the E1 value.

Article Snippet: Porcine CELA1 (catalog no. E0127) used for phage selection was purchased from Sigma, and the enzyme used for kinetic and inhibition assays (code ESFF, catalog no. LS006363) was from Worthington.

Techniques: Residue, Inhibition, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes

doi: 10.1074/jbc.m116.770560

Figure Lengend Snippet: FIGURE 7. Effect of the P4 residue on the inhibition of elastases and chy- motrypsins by SGPI-2 variants. A, inhibition by variants E1 and E10 contain- ing a P1 Leu, a P2 Leu, and a P4 Tyr versus Ala. Pairwise comparisons of relative KD values of inhibitor binding to the indicated proteinases are shown, normalized to the E1 value. B, inhibition by variants E9 and E11 containing a P1 Leu, a P2 Tyr, and a P4 Tyr versus Ala. Pairwise comparisons of relative KD values normalized to the E9 value are shown.

Article Snippet: Porcine CELA1 (catalog no. E0127) used for phage selection was purchased from Sigma, and the enzyme used for kinetic and inhibition assays (code ESFF, catalog no. LS006363) was from Worthington.

Techniques: Residue, Inhibition, Binding Assay