Super-resolution imaging as a method to study GPCR dimers and higher-order oligomers

The study of G protein-coupled receptor (GPCR) dimers and higher-order oligomers has unveiled mechanisms for receptors to diversify signaling and potentially uncover novel therapeutic targets. The functional and clinical significance of these receptor–receptor associations has been facilitated by the development of techniques and protocols, enabling researchers to unpick their function from the molecular interfaces, to demonstrating functional significance in vivo, in both health and disease. Here we describe our methodology to study GPCR oligomerization at the single-molecule level via super-resolution imaging. Specifically, we have employed photoactivated localization microscopy, with photoactivatable dyes (PD-PALM) to visualize the spatial organization of these complexes to <10 nm resolution, and the quantitation of GPCR monomer, dimer, and oligomer in both homomeric and heteromeric forms. We provide guidelines on optimal sample preparation, imaging parameters, and necessary controls for resolving and quantifying single-molecule data. Finally, we discuss advantages and limitations of this imaging technique and its potential future applications to the study of GPCR function

Evaluation and diagnostic potential of circulating extracellular vesicle-associated microRNAs in adrenocortical tumors

There is no available blood marker for the preoperative diagnosis of adrenocortical malignancy. The objective of this study was to investigate the expression of extracellular vesicle-associated microRNAs and their diagnostic potential in plasma samples of patients suffering from adrenocortical tumors. Extracellular vesicles were isolated either by using Total Exosome Isolation Kit or by differential centrifugation/ultracentrifugation. Preoperative plasma extracellular vesicle samples of 6 adrenocortical adenomas (ACA) and 6 histologically verified adrenocortical cancer (ACC) were first screened by Taqman Human Microarray A-cards. Based on the results of screening, two miRNAs were selected and validated by targeted quantitative real-time PCR. The validation cohort included 18 ACAs and 16 ACCs. Beside RNA analysis, extracellular vesicle preparations were also assessed by transmission electron microscopy, flow cytometry and dynamic light scattering. Significant overexpression of hsa-miR-101 and hsa-miR-483-5p in ACC relative to ACA samples has been validated. Receiver operator characteristics of data revealed dCT hsa-miR-483-5p normalized to cel-miR-39 to have the highest diagnostic accuracy (area under curve 0.965), the sensitivity and the specifity were 87.5 and 94.44, respectively. Extracellular vesicle-associated hsa-miR-483-5p thus appears to be a promising minimally invasive biomarker in the preoperative diagnosis of ACC but needs further validation in larger cohorts of patients

Preoperative medical treatment in Cushing&apos;s syndrome : frequency of use and its impact on postoperative assessment : data from ERCUSYN

Background: Surgery is the definitive treatment of Cushing's syndrome (CS) but medications may also be used as a first-line therapy. Whether preoperative medical treatment (PMT) affects postoperative outcome remains controversial. Objective: (1) Evaluate how frequently PMT is given to CS patients across Europe; (2) examine differences in preoperative characteristics of patients who receive PMT and those who undergo primary surgery and (3) determine if PMT influences postoperative outcome in pituitary-dependent CS (PIT-CS). Patients and methods: 1143 CS patients entered into the ERCUSYN database from 57 centers in 26 countries. Sixty-nine percent had PIT-CS, 25% adrenal-dependent CS (ADR-CS), 5% CS from an ectopic source (ECT-CS) and 1% were classified as having CS from other causes (OTH-CS). Results: Twenty per cent of patients took PMT. ECT-CS and PIT-CS were more likely to receive PMT compared to ADR-CS (P < 0.001). Most commonly used drugs were ketoconazole (62%), metyrapone (16%) and a combination of both (12%). Median (interquartile range) duration of PMT was 109 (98) days. PIT-CS patients treated with PMT had more severe clinical features at diagnosis and poorer quality of life compared to those undergoing primary surgery (SX) (P < 0.05). Within 7 days of surgery, PIT-CS patients treated with PMT were more likely to have normal cortisol (P < 0.01) and a lower remission rate (P < 0.01). Within 6 months of surgery, no differences in morbidity or remission rates were observed between SX and PMT groups. Conclusions: PMT may confound the interpretation of immediate postoperative outcome. Follow-up is recommended to definitely evaluate surgical results

REGLES DE SELECTION DANS LES REACTIONS DE DOUBLE TRANSFERT. APPLICATION AUX REACTIONS (p, t) ET (p, 3He) SUR LE 48Ca

The selection rules for two nucleons transfer reactions on pair - pair target nuclei are discussed. An application is made to the 48Ca (p, t) 46 and 48Ca (p, 3He) 46K reactions. Small violations of those selection rules have been observed, they may be explained by the intervention of more complex mechanisms. Furthermore disagreements are observed between assignments for the same final level depending on wether it is seen through (p, t) or (t, p) reaction. That can also be explained by two step mechanisms.L'utilisation des règles de sélection pour les réactions de double transfert sur les noyaux pair - pair est discutée. L'application est faite aux réactions 48Ca (p, t) 46Ca et 48Ca (p, 3He) 46K. De faibles violations à ces règles de sélection ont été observées et peuvent s'expliquer en faisant intervenir des mécanismes complexes. Par ailleurs un désaccord est noté entre les résultats des réactions (p, t) et (t, p) conduisant au même noyau final. Il peut aussi s'expliquer par des mécanismes à plusieurs étapes

REACTIONS DE PICK UP AVEC EXCITATION DU COEUR SUR LE 28Si ET LE 40Ca

Les réactions 28Si (p, d) 27Si et 40Ca (p, d) 39Ca ont été étudiées entre 30 et 45 MeV pour rechercher les transitions J interdites nécessitant une excitation du coeur à un état 2+ (dans le 28Si) ou 3- (dans le 40Ca). Un calcul de ce processus avec excitation dans la voie d'entrée rend compte de 50 % de la section efficace observée pour la transition au niveau 7/2+ à 2,17 MeV dans le 27Si.The 28Si (p, d) 27Si and 40Ca (p, d) 39Ca reactions have been studied between 30 et 45 MeV to look for J forbidden transitions involving a 2+ (in 28Si)or 3- (in 40Ca) core excitation. A calculation of this process in the entrance channel accounts for 50 % the cross section observed for the transition to the 7/2+ state at 2.17 MeV in 27Si