ABSTRACT
Creutzfeldt-Jakob disease (CJD) is a fatal neurological illness for which accurate diagnosis is paramount. Real-time quaking-induced conversion (RT-QuIC) is a prion-specific assay with high sensitivity and specificity for CJD. The Canadian endpoint quaking-induced conversion (EP-QuIC) test is similar, but unlike RT-QuIC there is little data regarding its diagnostic utility in clinical practice. In this exploratory predictive value analysis of EP-QuIC in CJD, the negative predictive value (NPV) and positive predictive value (PPV) was 100% and 83%, respectively, with one false-positive result identified. Re-testing this sample with an optimized EP-QuIC protocol eliminated this false-positive result, leading to a PPV of 100%.
La valeur prédictive de la méthode diagnostique dite de conversion provoquée par tremblement au point final dans le cas de la maladie de Creutzfeldt-Jakob. La maladie de Creutzfeldt-Jakob (MCJ) est une maladie neurologique qui entraîne à terme un décès et pour laquelle l'établissement d'un diagnostic précis est primordial. La conversion provoquée par tremblement en temps réel (RT-QuIC en anglais) est une méthode diagnostique qui repose sur la détection de la protéine prion. Dans le cas de la MCJ, cette méthode est réputée posséder une sensibilité et une spécificité élevées. La méthode canadienne dite de conversion provoquée par tremblement au point final (EP-QuIC en anglais) se veut similaire mais, à la différence de la RT-QuIC, il existe peu de données en ce qui concerne son utilité diagnostique dans le cadre d'une pratique clinique. Dans cette analyse prédictive exploratoire de la EP-QuIC en lien avec la MCJ, la valeur prédictive négative (VPN) et la valeur prédictive positive (VPP) ont été respectivement de 100 % et de 83 %, un seul résultat faux-positif ayant été identifié. Le fait de soumettre notre échantillon à un nouveau test effectué à l'aide d'un protocole d'EP-QuIC optimisé a permis d'éliminer ce résultat faux-positif, ce qui a débouché sur une VPP de 100 %.
Subject(s)
Creutzfeldt-Jakob Syndrome/diagnosis , PrPSc Proteins/analysis , Prion Proteins/analysis , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Predictive Value of TestsSubject(s)
Amnesia/chemically induced , Analgesics, Opioid/adverse effects , Chronic Pain/drug therapy , Fentanyl/adverse effects , Opioid-Related Disorders/diagnosis , Administration, Cutaneous , Amnesia/diagnosis , Analgesics, Opioid/administration & dosage , Female , Fentanyl/administration & dosage , Humans , Middle Aged , Opioid-Related Disorders/etiology , Syndrome , Transdermal PatchSubject(s)
Alzheimer Disease/complications , Alzheimer Disease/genetics , Cognition Disorders/etiology , Leukoencephalopathies/genetics , Mutation/genetics , Receptor, Macrophage Colony-Stimulating Factor/genetics , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/pathology , Cognition Disorders/diagnostic imaging , Disease Progression , Female , Humans , Leukoencephalopathies/diagnostic imaging , Leukoencephalopathies/physiopathology , Magnetic Resonance Imaging , Mental Disorders/diagnostic imaging , Mental Disorders/etiology , Microglia/pathology , Middle Aged , Neuropsychological Tests , SiblingsABSTRACT
Quantitative measurement of enzyme activity is a valuable approach to study how cells function. We present a method to measure the activity of the enzyme Cdk1/cyclin B. This enzyme is required by all eukaryotic cells to enter mitosis. Therefore, a biochemical assay to measure Cdk1/cyclin B activity can be used to identify cell populations that are in mitosis or to detect inhibitors of Cdk1/Cyclin B in vitro. A key distinction of the method presented here, compared to others, is that it uses a recombinant protein, a specific antibody, and a western blot apparatus, which makes the technique available to cell and molecular biology laboratories who do not wish to use radioisotopes, which are commonly required for other protein kinase assays.
Subject(s)
Antibody Specificity , Blotting, Western/methods , CDC2 Protein Kinase/immunology , CDC2 Protein Kinase/metabolism , Cyclin B/immunology , Cyclin B/metabolism , Enzyme Assays/methods , CDC2 Protein Kinase/antagonists & inhibitors , Cell Cycle Checkpoints/drug effects , Electrophoresis, Polyacrylamide Gel , Humans , Mitosis/drug effects , Protein Kinase Inhibitors/pharmacologyABSTRACT
We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.5 µM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity.
