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OBJECTIVE To assess the diagnostic performance of a benchtop fluorescent enzyme immunoassay analyzer (AIA-360; Tosoh Bioscience Inc) for the measurement of serum cortisol concentration as a screening test for hypoadrenocorticism in dogs. ANIMALS 173 client-owned dogs (20 with hypoadrenocorticism and 153 with nonadrenal illness). PROCEDURES Medical records of all dogs that underwent an ACTH stimulation test between June 2015 and October 2019 were reviewed retrospectively. Dogs were excluded if the ACTH stimulation test was performed on the basis of a suspicion of hypercortisolism, serum cortisol concentrations were measured using an analyzer other than the one assessed in the present study, or dogs had received medication known to affect the pituitary-adrenal axis in the 4 weeks1,2 preceding ACTH stimulation testing. The diagnostic performance of the benchtop analyzer was evaluated by calculating sensitivity, specificity, and likelihood ratios at various cutoff points. RESULTS Serum resting cortisol cutoff point concentrations of 0.8 μg/dL (22 nmol/L), 1 μg/dL (28 nmol/L), and 2 μg/dL (55 nmol/L) had a sensitivity of 100%. An optimal serum resting cortisol cutoff point of 0.58 μg/dL (16 nmol/L) had a sensitivity, specificity, and positive and negative likelihood ratios of 100%, 97%, and 30.6 and 0.0, respectively. CLINICAL RELEVANCE Findings indicated that previously derived cutoff points could be used with excellent sensitivity to exclude hypoadrenocorticism in this population of dogs when serum cortisol concentration was measured with the evaluated benchtop analyzer. An ACTH stimulation test may need to only be performed to diagnose hypoadrenocorticism if resting serum cortisol concentration is ≤ 0.58 μg/dL when measured with the evaluated benchtop analyzer.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order, an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.