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1.
J Vis Exp ; (175)2021 09 09.
Article in English | MEDLINE | ID: mdl-34570089

ABSTRACT

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved Förster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 µL), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.


Subject(s)
STAT3 Transcription Factor , Signal Transduction , Energy Transfer , Humans , Phosphorylation , Reproducibility of Results , STAT3 Transcription Factor/metabolism , Transducers
2.
Case Rep Gastrointest Med ; 2019: 3104187, 2019.
Article in English | MEDLINE | ID: mdl-30838142

ABSTRACT

A rare combination of severe volume depletion and electrolyte imbalance caused by a rectal villous adenoma is often referred to as the McKittrick-Wheelock syndrome. Patients usually seek medical care because of chronic hypersecretory diarrhea and display renal failure, metabolic acidosis, hyponatremia, and hypokalemia. We report the case of a 68-year-old woman who presented with this condition but showed unusual features such as severe hypokalemia and metabolic alkalosis, without diarrhea. She subsequently underwent transanal endoscopic microsurgery (TEMS), an innovative procedure in the management of large rectal adenomas. We also provide a narrative review of the literature on this rare entity.

3.
Prev Med Rep ; 12: 6-11, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30116704

ABSTRACT

BACKGROUND AND AIMS: FIT's value has been ascertained across Canada and worldwide, but still needs to be assessed within the province of Quebec. There also remains a gap between formal indications for FIT, and its actual use in clinical practice. This research aims to evaluate some aspects of FIT's effectiveness in our setting, and its application by prescribers. METHODS: We retrospectively identified and reviewed all the colonoscopies conducted for a positive FIT in 2014 at 2 hospitals located in Quebec City. RESULTS: Five hundred and fifty-nine (559) colonoscopies were reviewed. We obtained PPVs of 6.8% and 46.9% for the detection of CRC and AA, respectively. The PPV for the detection of SCL was higher in men compared to women (OR 1.56, 95%CI 1.11-2.20) and among justified FITs compared to unwarranted ones (OR 1.88, 95%CI 1.34-2.63). The PPV for CRC detection was 25.0% in the presence of unexplained iron deficiency anemia and 6.5% when anemia was absent (p = 0.0058). In 49.9% of cases, the prescription of a FIT was inappropriate. CONCLUSION: The FIT holds a better PPV for detecting SCL among men and when it is indicated. Anemia is associated with a higher CRC detection rate. Half of the FITs were not initially indicated.

4.
J Biomol Screen ; 17(1): 49-58, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21940714

ABSTRACT

Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z' factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.


Subject(s)
Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Lysine/metabolism , Biotinylation , Epigenesis, Genetic/drug effects , Histones/metabolism , Immunoassay/methods , Peptides/metabolism , Small Molecule Libraries
5.
Assay Drug Dev Technol ; 10(2): 179-86, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22192306

ABSTRACT

Histone methylation is a regulated feature of nucleosomes that can have an impact on gene expression. The methylation state of histone residues has also been found in recent years to be associated with various disorders. Tools for detecting methylation state changes are very useful for dissecting the function of these epigenetic marks. In this work, a sensitive homogeneous assay for histone demethylase activity at the H3K4 site has been developed in a time-resolved fluorescent resonance energy transfer assay format. The assay is based on the detection of the unmethylated H3 peptide by a fluorescent europium-chelate labeled monoclonal antibody binding specifically to the H3K4 site. The assay was validated for histone lysine-specific demethylase 1 and was demonstrated to be a suitable assay for inhibitor profiling and high-throughput screening.


Subject(s)
High-Throughput Screening Assays/methods , Histone Deacetylase Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Antibodies, Monoclonal/chemistry , Binding Sites , Chelating Agents/chemistry , Data Interpretation, Statistical , Europium , Fluorescence Resonance Energy Transfer , Histone Demethylases/chemistry , Horseradish Peroxidase/chemistry , Humans , Hydrogen Peroxide/chemistry , Indicators and Reagents , Substrate Specificity
6.
J Recept Signal Transduct Res ; 22(1-4): 533-41, 2002.
Article in English | MEDLINE | ID: mdl-12503639

ABSTRACT

In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.


Subject(s)
Arrestins/metabolism , Biosensing Techniques , GTP-Binding Proteins/metabolism , Luminescent Proteins/analysis , Receptors, Vasopressin/metabolism , Cell Line , Green Fluorescent Proteins , Humans , Luciferases/analysis , Luminescent Measurements , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sensitivity and Specificity , beta-Arrestin 2 , beta-Arrestins
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