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A recently developed inhibitor of retrograde transport, namely Retro-2.1, proved to be a potent and broad-spectrum lead in vitro against intracellular pathogens, such as toxins, parasites, intracellular bacteria and viruses. To circumvent its low aqueous solubility, a formulation in poly(ethylene glycol)-block-poly(D,L)lactide micelle nanoparticles was developed. This formulation enabled the study of the pharmacokinetic parameters of Retro-2.1 in mice following intravenous and intraperitoneal injections, revealing a short blood circulation time, with an elimination half-life of 5 and 6.7 h, respectively. To explain the poor pharmacokinetic parameters, the metabolic stability of Retro-2.1 was studied in vitro and in vivo, revealing fast cytochrome-P-450-mediated metabolism into a less potent hydroxylated analogue. Subcutaneous injection of Retro-2.1 formulated in a biocompatible and bioresorbable polymer-based thermosensitive hydrogel allowed for sustained release of the drug, with an elimination half-life of 19 h, and better control of its metabolism. This study provides a guideline on how to administer this promising lead in vivo in order to study its efficacy.

Enterobactin (ENT) is a tris-catechol siderophore used to acquire iron by multiple bacterial species. These ENT-dependent iron uptake systems have often been considered as potential gates in the bacterial envelope through which one can shuttle antibiotics (Trojan horse strategy). In practice, siderophore analogues containing catechol moieties have shown promise as vectors to which antibiotics may be attached. Bis- and tris-catechol vectors (BCVs and TCVs, respectively) were shown using structural biology and molecular modeling to mimic ENT binding to the outer membrane transporter PfeA in

Context Radioiodine-refractory thyroid cancers have poor outcomes and limited therapeutic options (tyrosine kinase inhibitors) due to transient efficacy and toxicity of treatments. Therefore, combinatorial treatments with new therapeutic approaches are needed. Many studies link G protein–coupled receptors (GPCRs) to cancer cell biology. Objective To perform a specific atlas of GPCR expression in progressive and refractory thyroid cancer to identify potential targets among GPCRs aiming at drug repositioning. Methods We analyzed samples from tumor and normal thyroid tissues from 17 patients with refractory thyroid cancer (12 papillary thyroid cancers [PTCs] and 5 follicular thyroid cancers [FTCs]). We assessed GPCR mRNA expression using NanoString technology with a custom panel of 371 GPCRs. The data were compared with public repositories and pharmacological databases to identify eligible drugs. The analysis of prognostic value of genes was also performed with TCGA datasets. Results With our transcriptomic analysis, 4 receptors were found to be downregulated in FTC (VIPR1, ADGRL2/LPHN2, ADGRA3, and ADGRV1). In PTC, 24 receptors were deregulated, 7 of which were also identified by bioinformatics analyses of publicly available datasets on primary thyroid cancers (VIPR1, ADORA1, GPRC5B, P2RY8, GABBR2, CYSLTR2, and LPAR5). Among all the differentially expressed genes, 22 GPCRs are the target of approved drugs and some GPCRs are also associated with prognostic factors. Discussion For the first time, we performed GPCR mRNA expression profiling in progressive and refractory thyroid cancers. These findings provide an opportunity to identify potential therapeutic targets for drug repositioning and precision medicine in radioiodine-refractory thyroid cancer.

International audience; Dichlorodiphenyltrichloroethane (p,p ' DDT) is an endocrine-disrupting chemical (EDC). Several studies showed an association between p,p ' DDT exposure and reprotoxic effects. We showed that p,p ' DDT was a positive allosteric modulator of human follitropin receptor (FSHR). In contrast, we demonstrated that p,p ' DDT decreased the cyclic AMP (cAMP) production induced by human choriogonadotropin (hCG). This study evaluated further the effects of p,p ' DDT on Gs-, beta-arrestin 2- and steroidogenesis pathways induced by hCG or luteinizing hormone (LH). We used Chinese hamster ovary cells line stably expressing hCG/LHR. The effects of 10-100 mu M p,p ' DDT on cAMP production and on beta-arrestin 2 recruitment were measured using bioluminescence and time-resolved resonance energy transfer technology. The impact of 100 mu M of p,p ' DDT on steroid secretion was analysed in murine Leydig tumor cell line (mLTC-1). In cAMP assays, 100 mu M p,p ' DDT increased the EC50 by more than 300% and reduced the maximum response of the hCG/LHR to hCG and hLH by 30%. This inhibitory effect was also found in human granulosa cells line and in mLTC-1 cells. Likewise, 100 mu M p,p ' DDT decreased the hCG- and hLH-promoted beta-arrestin 2 recruitment down to 14.2 and 26.6%, respectively. Moreover, 100 mu M p,p ' DDT decreased by 30 and 47% the progesterone secretion induced by hCG or hLH, respectively, without affecting testosterone secretion. This negative effect of p,p'DDT was independent of cytotoxicity. p,p ' DDT acted as a negative allosteric modulator of the hCG/LHR signalling. This emphasizes the importance of analyzing all receptor-downstream pathways to fully understand the deleterious effects of EDC on human health.

Background/objectives Maternal obesity impacts vascular functions linked to metabolic disorders in offspring, leading to cardiovascular diseases during adulthood. Even if the relation between prenatal conditioning of cardiovascular diseases by maternal obesity and vascular function begins to be documented, little is known about resistance arteries. They are of particular interest because of their specific role in the regulation of local blood flow. Then our study aims to determine if maternal obesity can directly program fetal vascular dysfunction of resistance arteries, independently of metabolic disorders. Methods With a model of rats exposed in utero to mild maternal diet-induced obesity (OMO), we investigated third-order mesenteric arteries of 4-month old rats in absence of metabolic disorders. The methylation profile of these vessels was determined by reduced representation bisulfite sequencing (RRBS). Vascular structure and reactivity were investigated using histomorphometry analysis and wire-myography. The metabolic function was evaluated by insulin and glucose tolerance tests, plasma lipid profile, and adipose tissue analysis. Results At 4 months of age, small mesenteric arteries of OMO presented specific epigenetic modulations of matrix metalloproteinases (MMPs), collagens, and potassium channels genes in association with an outward remodeling and perturbations in the endothelium-dependent vasodilation pathways (greater contribution of EDHFs pathway in OMO males compared to control rats, and greater implication of PGI2 in OMO females compared to control rats). These vascular modifications were detected in absence of metabolic disorders. Conclusions Our study reports a specific methylation profile of resistance arteries associated with vascular remodeling and vasodilation balance perturbations in offspring exposed in utero to maternal obesity, in absence of metabolic dysfunctions.

Iron is an essential nutriment for almost all organisms, but this metal is poorly bioavailable. During infection, bacteria access iron from the host by importing either iron or heme.

Iron is an essential nutriment for almost all organisms, but this metal is poorly bioavailable. During infection, bacteria access iron from the host by importing either iron or heme. Pseudomonas aeruginosa, a gram-negative pathogen, secretes two siderophores, pyoverdine (PVD) and pyochelin (PCH), to access iron and is also able to use many siderophores produced by other microorganisms (called xenosiderophores). To access heme, P. aeruginosa uses three distinct uptake pathways, named Has, Phu, and Hxu. We previously showed that P. aeruginosa expresses the Has and Phu heme uptake systems and the PVD- and PCH-dependent iron uptake pathways in iron-restricted growth conditions, using proteomic and RT-qPCR approaches. Here, using the same approaches, we show that physiological concentrations of hemin in the bacterial growth medium result in the repression of the expression of the proteins of the PVD- and PCH-dependent iron uptake pathways, leading to less production of these two siderophores. This indicates that the pathogen adapts its phenotype to use hemin as an iron source rather than produce PVD and PCH to access iron. Moreover, the presence of both hemin and a xenosiderophore resulted in (i) the strong induction of the expression of the proteins of the added xenosiderophore uptake pathway, (ii) repression of the PVD- and PCH-dependent iron uptake pathways, and (iii) no effect on the expression levels of the Has, Phu, or Hxu systems, indicating that bacteria use both xenosiderophores and heme to access iron.

A recently developed inhibitor of the retrograde transport, namely Retro-2.1, proved to be a potent and broad-spectrum lead in vitro against intracellular pathogens such as toxins, parasites, intracellular bacteria, and viruses. In order to circumvent its low aqueous solubility, a formulation in poly(ethylene glycol)-bloc-poly(D,L)lactide micelles was developed. This formulation allowed studying Retro-2.1 pharmacokinetic parameters in mice following intravenous or intraperitoneal injection, revealing a low blood circulation time. To explain these poor pharmacokinetic parameters, Retro-2.1 metabolic stability was studied in vitro and in vivo revealing a fast cytochrome P-450-mediated metabolism into a less potent hydroxylated analog. Subcutaneous injection of Retro-2.1 formulated in a biocompatible and bioresorbable polymer-based thermosensitive hydrogel allowed for a sustained-release of the drug and a better control of its metabolism. This study gives a guideline on how to administer this promising lead in vivo in order to study its efficacy.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

Endocrine-disrupting chemicals have received significant concern, since they ubiquitously persist in the environment and are able to induce adverse effects on health, and more particularly on reproductive function. Most of the studies focused on nuclear hormone receptors as mediators of sex steroid hormones signaling. However, there are increasing evidences that peptides hormones of the Hypothalamo-Pituitary-Gonadal axis are targets of endocrine-disrupting chemicals (as Gonadotropin-Releasing Hormone, Follicle-Stimulating Hormone, Luteinizing Hormone…). The majority of these hormones act on G protein-coupled membrane receptors. This review summarizes the effects of endocrine-disrupting chemicals on homeostasis of peptides hormone of Hypothalamo-Pituitary-Gonadal axis and on their G protein-coupled membrane receptors signaling revealed by experimental, clinical, and epidemiological studies in human.

Introduction Le cancer thyroidien refractaire represente la majorite des deces par cancer de la thyroide. Les traitements actuels etant limite aux inhibiteurs de tyrosine kinase avec des effets secondaires limitant son utilisation, il est necessaire de developper de nouveaux modeles in vitro adaptes, afin d’elargir l’arsenal therapeutique et s’orienter vers des traitements personnalises. L’objectif etait d’etablir une lignee derivee d’une ascite metastatique d’un cancer thyroidien refractaire et de mettre en place un systeme de criblage pharmacologique. Methode A partir de l’ascite tumorale d’un cancer papillaire de la thyroide refractaire, metastatique et evolutif, des cellules ont ete isolees et maintenues en culture. Apres selection clonale par dilution limite, la lignee obtenue a ete soumise a une caracterisation phenotypique (RT-qPCR et immunohistochimie) et a un sequencage de l’ADN. Un criblage pharmacologique reposant sur la mesure de la viabilite cellulaire (CellTiter-Glo) a ete ensuite realise testant des molecules eligibles a du repositionnement de medicaments. Resultat La lignee conservait les marqueurs thyroidiens (TTF1, PAX8, TSHR, thyroglobuline et TPO) hormis l’expression de NIS. Le systeme de criblage pharmacologique a permis d’identifier un antagoniste de HTR7, un agoniste de S1PR2 et un agoniste de HRH1 ayant des effets anti-proliferatifs. Conclusion Ce travail decrit pour la premiere fois la possibilite d’obtenir une lignee thyroidienne a partir de liquide d’ascite metastatique plutot que de la tumeur primitive. Ce modele preclinique pourrait etre tres prometteur pour etudier la reponse clinique aux medicaments anticancereux de maniere personnalisee. L’utilisation d’epanchement pleural, plus frequent que l’ascite metastatique, serait egalement une piste interessante.

(1) Background: Chronic increases in blood flow, as in cardiovascular diseases, induce outward arterial remodeling. Thrombospondin-1 (TSP-1) is known to interact with matrix proteins and immune cell-surface receptors, but its contribution to flow-mediated remodeling in the microcirculation remains unknown. (2) Methods: Mesenteric arteries were ligated in vivo to generate high- (HF) and normal-flow (NF) arteries in wild-type (WT) and TSP-1-deleted mice (TSP-1−/−). After 7 days, arteries were isolated and studied ex vivo. (3) Results: Chronic increases in blood flow induced outward remodeling in WT mice (increasing diameter from 221 ± 10 to 280 ± 10 µm with 75 mmHg intraluminal pressure) without significant effect in TSP-1−/−(296 ± 18 to 303 ± 14 µm), neutropenic or adoptive bone marrow transfer mice. Four days after ligature, pro inflammatory gene expression levels (CD68, Cox2, Gp91phox, p47phox and p22phox) increased in WT HF arteries but not in TSP-1−/− mice. Perivascular neutrophil accumulation at day 4 was significantly lower in TSP-1−/− than in WT mice. (4) Conclusions: TSP-1 origin is important, indeed, circulating TSP-1 participates in vasodilation, whereas both circulating and tissue TSP-1 are involved in arterial wall thickness and diameter expansion.

Dichlorodiphenyltrichloroethane (p,p'DDT) is an endocrine-disrupting chemical (EDC). Several studies showed an association between p,p'DDT exposure and reprotoxic effects. We showed that p,p'DDT was a positive allosteric modulator of human follitropin receptor (FSHR). In contrast, we demonstrated that p,p'DDT decreased the cyclic AMP (cAMP) production induced by human choriogonadotropin (hCG). This study evaluated further the effects of p,p'DDT on Gs-, β-arrestin 2- and steroidogenesis pathways induced by hCG or luteinizing hormone (LH). We used Chinese hamster ovary cells line stably expressing hCG/LHR. The effects of 10-100 µM p,p'DDT on cAMP production and on β-arrestin 2 recruitment were measured using bioluminescence and time-resolved resonance energy transfer technology. The impact of 100 µM of p,p'DDT on steroid secretion was analysed in murine Leydig tumor cell line (mLTC-1). In cAMP assays, 100 µM p,p'DDT increased the EC

Objective Somatostatin receptor ligands (SRL) are useful to control central hyperthyroidism in patients with thyrotropin-secreting pituitary adenoma (TSH pituitary adenoma). The aim of this study was to describe the frequency of thyrotropin deficiency (TSH deficiency) in patients with TSH pituitary adenoma treated by SRL. Design Retrospective study. Methods Patients with central hyperthyroidism due to TSH pituitary adenoma treated by short or long-acting SRL were retrospectively included. TSH deficiency was defined by a low FT4 associated with non-elevated TSH concentrations during SRL therapy. We analysed the frequency of TSH deficiency and the characteristics of patients with or without TSH deficiency. Results Forty-six patients were included. SRL were used as the first-line therapy in 21 of 46 patients (46%). Central hyperthyroidism was controlled in 36 of 46 patients (78%). TSH deficiency appeared in 7 of 46 patients (15%) after a median time of 4 weeks (4–7) and for a median duration of 3 months (2.5–3). The TSH deficiency occurred after one to three injections of long-acting SRL used as first-line therapy in 6/7 cases. There were no differences in terms of clinical and hormonal features, size of adenomas or doses of SRL between patients with or without TSH deficiency. Conclusions SRL can induce TSH deficiency in patients with central hyperthyroidism due to TSH pituitary adenoma. Thyrotropic function should be assessed before the first three injections of SRL in order to track TSH deficiency and reduce the frequency of injections when control of thyrotoxicosis rather than tumour reduction is the aim of the treatment.

Background: Somatostatin analogs (SSA) are efficiently used to control central hyperthyroidism in patients with thyrotropin-secreting pituitary neuroendocrine tumor (TSH-PitNET). The aim of this study was to describe the frequency of thyrotropin (TSH) deficiency under SSA in patients with TSH-PitNET. Methods: We retrospectively recruited patients presenting a central hyperthyroidism due to TSH-PitNET. Inclusion criteria were patients treated in first, second or third line by short or long-acting SSA, with central hyperthyroidism before SSA. Patients treated by radiotherapy or dopamine agonist were excluded. TSH deficiency was defined by either a low FT4 or low FT4 and FT3, associated with non-elevated TSH concentrations during SSA therapy. We analyzed the frequency of TSH deficiency and the characteristics of patients with or without TSH deficiency. Results: 46 patients were included in the study. SSA were used as the first-line therapy in 21 of 46 patients (46%). Central hyperthyroidism was controlled in 36 of 46 patients (78%). TSH deficiency appeared in 7 of 46 patients (15%), after a median time of 4 weeks (4–7) after the starting of SSA, and for a median duration of 3 months (2.5–3). The TSH deficiency occurred after 1 to 3 injections of long-acting SSA. There were no differences in terms of clinical and hormonal features and size of adenomas between patients with or without TSH deficiency. Conclusions: In patients with central hyperthyroidism due to TSH-PitNET, SSA can induce TSH deficiency. Thyrotropic function should be assessed before each injection of SSA in order to adapt the frequency of injection when control of thyrotoxicosis rather than tumor reduction is purpose of the treatment.

International audience; The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

Enterobactin (ENT) is a siderophore (iron-chelating compound) produced by Escherichia coli to gain access to iron, an indispensable nutrient for bacterial growth. ENT is used as an exosiderophore by Pseudomonas aeruginosa with transport of ferri-ENT across the outer membrane by the PfeA transporter. Next to the pfeA gene on the chromosome is localized a gene encoding for an esterase, PfeE, whose transcription is regulated, as for pfeA, by the presence of ENT in bacterial environment. Purified PfeE hydrolyzed ferri-ENT into three molecules of 2,3-DHBS (2,3-dihydroxybenzoylserine) still complexed with ferric iron, and complete dissociation of iron from ENT chelating groups was only possible in the presence of both PfeE and an iron reducer, such as DTT. The crystal structure of PfeE and an inactive PfeE mutant complexed with ferri-ENT or a nonhydrolyzable ferri-catechol complex allowed identification of the enzyme binding site and the catalytic triad. Finally, cell fractionation and fluorescence microscopy showed periplasmic localization of PfeE in P. aeruginosa cells. Thus, the molecular mechanism of iron dissociation from ENT in P. aeruginosa differs from that previously described in E. coli. In P. aeruginosa, siderophore hydrolysis occurs in the periplasm, with ENT never reaching the bacterial cytoplasm. In E. coli, ferri-ENT crosses the inner membrane via the ABC transporter FepBCD and ferri-ENT is hydrolyzed by the esterase Fes only once it is in the cytoplasm.

International audience; Differentiated thyroid cancers are more frequent in women than in men. These different frequencies may depend on differences in patient’s behavior and in thyroid investigations. However, an impact on sexual hormones is likely, although this has been insufficiently elucidated. Estrogens may increase the production of mutagenic molecules in the thyroid cell and favor the proliferation and invasion of tumoral cells by regulating both the thyrocyte enzymatic machinery and the inflammatory process associated with tumor growth. On the other hand, the worse prognosis of thyroid cancer associated with the male gender is poorly explained.

Iron acquisition pathways have often been considered to be gateways for the uptake of antibiotics into bacteria. Bacteria excrete chelators, called siderophores, to access iron. Antibiotic molecules can be covalently attached to siderophores for their transport into pathogens during the iron-uptake process. P. aeruginosa produces two siderophores and is also able to use many siderophores produced by other bacteria. We investigated the phenotypic plasticity of iron-uptake pathway expression in an epithelial cell infection assay in the presence of two different siderophore&ndash, antibiotic conjugates, one with a hydroxamate siderophore and the second with a tris-catechol. Proteomic and RT-qPCR approaches showed that P. aeruginosa was able to sense the presence of both compounds in its environment and adapt the expression of its iron uptake pathways to access iron via them. Moreover, the catechol-type siderophore&ndash, antibiotic was clearly more efficient in inducing the expression of its corresponding transporter than the hydroxamate compound when both were simultaneously present. In parallel, the expression of the proteins of the two iron uptake pathways using siderophores produced by P. aeruginosa was significantly repressed in the presence of both conjugates. Altogether, the data indicate that catechol-type siderophores are more promising vectors for antibiotic vectorization using a Trojan-horse strategy.

Le bisphenol A (BPA) est defini comme un perturbateur endocrinien. Il existe une association epidemiologique entre son exposition et des effets nefastes sur la sante humaine, notamment l’obesite ou l’hypertension. Le recepteur 4 de la melanocortine (MC4R) est un regulateur central majeur de la prise alimentaire et de la pression sanguine. Ce recepteur est couple a la proteine Gs (voie de l’AMP cyclique (cAMP)) ainsi qu’a la proteine Gq (voie calcique). Nous avons etudie les effets du BPA sur la signalisation intracellulaire de MC4R. La lignee de cellules HEK293 T a ete transfectee de facon transitoire avec le MC4R humain. La production d’AMPc et la mobilisation intracellulaire du calcium ont ete mesurees. Le BPA (10 nM) augmentait la production d’AMPc stimulee par l’αMSH (agoniste) avec 20 % d’augmentation et diminuait de 50 % la reponse de MC4R a l’AgRP (agoniste inverse naturel). Dans les cellules HEK293 T natives, le BPA n’avait pas d’effet. Ainsi, l’effet potentialisateur du BPA etait dependant et specifique de MC4R. A l’oppose, le BPA reduisait (−16 %) la mobilisation du calcium induite par la stimulation de MC4R. Or, il est suggere que l’activation de la proteine Gq est liee a la regulation du poids corporel, tandis que celle de la proteine Gs est impliquee dans la regulation de la pression sanguine. En conclusion, les effets opposes du BPA sur les voies de signalisation de MC4R pourraient potentiellement expliquer le lien entre l’exposition au BPA et le developpement de l’obesite et/ou de l’hypertension.

Introduction Le cancer de la thyroide refractaire a l’iode radioactif pose un probleme en termes d’arsenal therapeutique. L’utilisation des inhibiteurs de tyrosine kinases (ITK) est aujourd’hui limitee par d’importants effets secondaires et/ou l’apparition d’une chimioresistance. Certains recepteurs couples aux proteines G (RCPG) font partie des nouvelles cibles potentielles en cancerologie. Cependant, l’atlas des RCPG exprimes au sein du cancer thyroidien refractaire n’a jamais ete realise. Notre objectif etait d’identifier des RCPG exprimes de maniere differentielle entre les tissus thyroidiens tumoraux et sains. Methode Nous avons realise une analyse transcriptomique ciblee sur 371 RCPG avec la technologie NanoString a partir d’echantillons de tissus tumoraux et sains, inclus en paraffine de 17 patients presentant un cancer thyroidien refractaire a l’iode radioactif (12 cancers papillaires et 5 cancers folliculaires), suivis au CHU d’Angers. Resultats Au sein des cancers folliculaires, 4 RCPG etaient sous-exprimes dans les tissus tumoraux (VIPR1, ADGRL2, ADGRA3 et ADGRV1). Concernant les cancers papillaires, 24 RCPG presentaient une difference d’expression significative entre les echantillons tumoraux et sains (5 sous-exprimes, 19 surexprimes). Les analyses de survie issues des donnees du TCGA (The Cancer Genome Atlas) ont montre une valeur pronostique pour 5 d’entre eux (F2RL1, GPR132, GPRC5B, HRH4 et LPAR5) et pres d’un tiers des recepteurs identifies etaient cibles par la pharmacopee actuelle. Conclusion Il s’agit de la premiere etude a decrire un atlas des RCPGs non sensoriels au sein des cancers thyroidiens refractaires. Ces donnees peuvent aider a identifier des marqueurs pronostiques ou de potentielles cibles therapeutiques.

Les perturbateurs endocriniens (PE) ont ete definis par l’Agence de Protection et de l’environnement des Etats Unis comme substance exogene qui alterent les fonctions du systeme endocrinien et induisent des effets nefastes sur la sante d’un organisme intact. Le Bisphenol A (BPA) est un PE qui interfere avec la synthese, metabolisme, transport, affinite des hormones naturelles. L’utilisation du BPA est tellement repandue dans l’environnement qu’il provoque de nombreux effets secondaires (cancers, anomalies du developpement, reprotoxicite, …). Le BPA est ubiquitaire, il est retrouve dans les sols, l’air et l’eau. Lors du traitement de l’eau du robinet, la derniere etape consiste a traiter avec du chlore afin d’eliminer des bacteries, cependant cette chloration mene egalement a la formation de derives chlores du BPA (ClxBPA). De plus la presence d’ions bromures dans l’eau, conduit a la formation de derives bromes du BPA (BrxBPA). Nous presenterons la synthese de ces composes ClxBPA, BrxBPA qui servent de temoins analytiques dans l’analyse d’echantillons biologiques humains ou afin d’evaluer leur toxicite. Une cohorte de femmes enceintes a permis de recueillir leur eau de boisson, des fluides biologiques (urine, colostrum) dont les analyses LC/MS-MS et GC/MS-MS seront detaillees sur leurs teneurs en derives halogenes du BPA. Enfin, les derives ClxBPA, BrxBPA ont ete evalues in vitro sur le recepteur Follitropine (FSHR). Ces composes ne sont pas cytotoxiques (test MTT), ils demontrent un effet allosterique negatif en presence de l’hormone FSH. Ces derives sont consideres comme des perturbateurs endocriniens et susceptibles d’avoir des effets reprotoxiques.

International audience; The last decade has witnessed tremendous progress in the understanding of the mineralocorticoid receptor (MR), its molecular mechanism of action, and its implications for physiology and pathophysiology. After the initial cloning of MR, and identification of its gene structure and promoters, it now appears as a major actor in protein-protein interaction networks. The role of transcriptional coregulators and the determinants of mineralocorticoid selectivity have been elucidated. Targeted oncogenesis and transgenic mouse models have identified unexpected sites of MR expression and novel roles for MR in non-epithelial tissues. These experimental approaches have contributed to the generation of new cell lines for the characterization of aldosterone signaling pathways, and have also facilitated a better understanding of MR physiology in the heart, vasculature, brain and adipose tissues. This review describes the structure, molecular mechanism of action and transcriptional regulation mediated by MR, emphasizing the most recent developments at the cellular and molecular level. Finally, through insights obtained from mouse models and human disease, its role in physiology and pathophysiology will be reviewed. Future investigations of MR biology should lead to new therapeutic strategies, modulating cell-specific actions in the management of cardiovascular disease, neuroprotection, mineralocorticoid resistance, and metabolic disorders.

International audience; In the absence of crystal available for the full-length thyrotropin receptor, knowledge of its structure and functioning has benefitted from the identification and characterization of mutations in patients with various thyroid dysfunctions. The characterization of activating mutations has contributed to the elaboration of a model involving the extracellular domain of the receptor as an inverse tethered agonist which, upon binding of the ligand, relieves the transmembrane domain from an inhibiting interaction and activates it. The models derived from comparisons with other receptors, enriched with the information provided by the study of mutations, have proven useful for the design of small-molecule agonists and antagonists that may be used in the future to treat thyroid dysfunctions. In this review, extrathyroidal expression of the thyrotropin receptor is described, the role of which is still poorly defined.

A one-step chromatographic method for the purification of R-phycoerythrin (R-PE) of Grateloupia turuturu Yamada is described. Native R-PE was obtained with a purity index of 2.89 and a recovery yield of 27% using DEAE-Sepharose Fast Flow chromatography with a three-step increase in ionic strength. The analysis by SDS electrophoresis showed a broad band between 18 and 21kDa in size corresponding to subunits α and β and a low intensity band of 29kDa corresponding to the γ subunit. Two forms of R-PE were identified by gel filtration chromatography: a native form with a molecular weight of 260±5kDa and a dissociated form with a molecular weight of 60±2kDa. The native form presented the characteristic absorption spectrum of R-PE with three absorbance maxima at 498, 540 and 565nm, whereas the dissociated form presented only the 498 and 540nm peaks. Moreover, the two forms displayed two different fluorescence maxima.

Hydroxamate analogs of fosfoxacin, the phosphate homolog of fosmidomycin, have been synthesized and their activity tested on Escherichia coli and Mycobacterium smegmatis DXRs. Except for compound 4b, the IC50 values of phosphate derivatives are approximately 10-fold higher than those of the corresponding phosphonates. Although their inhibitory activity on Escherichia coli DXR is less efficient than their phosphonate analogs, we report the ability of phosphate compounds to inhibit the growth of Escherichia coli. This work points out that the uptake of fosfoxacin and its analogs is taking place via the GlpT and UhpT transporters. As expected, these compounds are inefficient to inhibit the growth of M. smegmatis growth inhibition probably due to a lack of uptake.

National audience; Plusieurs données épidémiologiques associent l’exposition au p,p’DDT (perturbateur endocrinien [PE] persistant dans l’environnement) avec des anomalies de la fonction de reproduction. Nous avons montré, in vitro, que ce composé potentialise la réponse de l’hormone FSH sur son récepteur (FSHR) via une interaction avec le domaine transmembranaire du FSHR. Néanmoins, en plus de la FSH, la LH et l’hCG contrôlent aussi la fonction gonadique via le récepteur LH/hCGR. Ce dernier présente une forte homologie avec le FSHR. Ainsi, le but de ce travail est de déterminer les effets du p,p’DDT sur la fonction du LH/hCGR. Dans un premier temps, nous avons utilisé une lignée de cellules CHO exprimant de façon stable le LH/hCGR humain, dont l’activité est évaluée en mesurant la concentration d’AMP cyclique (AMPc). Le p,p’DDT réduit la production d’AMPc stimulée par l’hCG de façon dose-dépendante, en diminuant de 7 fois la puissance de l’hCG, suggérant un effet allostérique négatif. De plus, le p,p’DDT réduit de 50 % l’activité basale du LH/hCGR. Par une approche de Bioluminescence Resonance Energy Transfer (BRET), nous avons aussi montré que des doses croissantes de p,p’DDT antagonisent l’interaction de LH/hCGR avec la β-arrestine. Ces données, ainsi que celles sur le FSHR, démontrent une activité différentielle du p,p’DDT sur le FSHR (positive) et sur le LH/hCGR (négative), qui pourrait être liée aux effets reprotoxiques du p,p’DDT et qui reste à mieux caractériser in vivo.

Background: 1-chloro-4-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl]benzene (p,p′-DDT) is a persistent environmental endocrine disruptor (ED). Several studies have shown an association between p,p′-DDT exposure and reproductive abnormalities. Objectives: To investigate the putative effects of p,p′-DDT on the human follitropin receptor (FSHR) function. Methods and Results: We used Chinese hamster ovary (CHO) cells stably expressing human FSHR to investigate the impact of p,p′-DDT on FSHR activity and its interaction with the receptor. At a concentration of 5 μM p,p′-DDT increased the maximum response of the FSHR to follitropin by 32 ± 7.45%. However, 5 μM p,p′-DDT decreased the basal activity and did not influence the maximal response of the closely related LH/hCG receptor to human chorionic gonadotropin (hCG). The potentiating effect of p,p′-DDT was specific for the FSHR. Moreover, in cells that did not express FSHR, p,p′-DDT had no effect on cAMP response. Thus, the potentiating effect of p,p′-DDT was dependent on the FSHR. In addition, p,p′-DDT increased the sensitivity of FSHR to hCG and to a low molecular weight agonist of the FSHR, 3-((5methyl)-2-(4-benzyloxy-phenyl)-5-{[2-[3-ethoxy-4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-benzamide (16a). Basal activity in response to p,p′-DDT and potentiation of the FSHR response to FSH by p,p′-DDT varied among FSHR mutants with altered transmembrane domains (TMDs), consistent with an effect of p,p′-DDT via TMD binding. This finding was corroborated by the results of simultaneously docking p,p′-DDT and 16a into the FSHR transmembrane bundle. Conclusion: p,p′-DDT acted as a positive allosteric modulator of the FSHR in our experimental model. These findings suggest that G protein–coupled receptors are additional targets of endocrine disruptors. Citation: Munier M, Grouleff J, Gourdin L, Fauchard M, Chantreau V, Henrion D, Coutant R, Schiøtt B, Chabbert M, Rodien P. 2016. In vitro effects of the endocrine disruptor p,p′-DDT on human follitropin receptor. Environ Health Perspect 124:991–999; http://dx.doi.org/10.1289/ehp.1510006

Context: Gestational thyrotoxicosis, whether associated with hyperemesis gravidarum or not, is thought to be due to excessive human chorionic gonadotropin (hCG) secretion. Case Description: We report here the second case of gestational thyrotoxicosis associated with hyperemesis gravidarum due to a mutation of the TSH receptor, providing thyroid hypersensitivity to hCG. Conclusion: Severe and lasting gestational thyrotoxicosis with normal hCG concentration should lead to sequencing of the TSH receptor gene.

Seaweeds are traditionally used as sea vegetables in Asian countries but their consumption by western consumers is minimal. The nutritional value of algae and their potential use as functional ingredients in foods is discussed. The addition of seaweeds to the diet of marine animals produced by aquaculture is suggested. This would be a new opportunity to introduce seaweeds indirectly into the human food chain in western countries, especially in Europe. The treatment of wastewater produced by aquaculture facilities with macroalgae is an additional argument for the use of this marine resource in food production and the development of a new method for marine aquaculture.

Mineralocorticoid receptor (MR) signaling is pivotal for numerous physiological processes and implicated in various pathological conditions concerning among others, tight epithelia, central nervous and cardiovascular systems. For decades, the pleiotropic actions of MR have been investigated using animal and cellular models as well as by clinical studies. Here is reviewed and contextualized the utilization of a strategy that recently emerged to analyze the complexity of MR signaling: the derivation and differentiation of mouse embryonic stem (ES) cell models. ES cells were derived from wild-type or transgenic MR overexpressing animals. Undifferentiated ES cells were differentiated into cardiomyocytes, neurons and adipocytes, these cell types being important pathophysiological targets of MR. These approaches have already brought new insights concerning MR effect on cardiomyocyte contractility and ionic channel remodeling, in the regulation of neuronal MR expression and its positive role on neuron survival. Differentiated ES cell models thus constitute powerful and promising tools to further decipher the molecular mechanisms of cell-specific MR actions.

Introduction Des etudes epidemiologiques et experimentales in vivo mettent en evidence des effets reprotoxiques de perturbateurs endocriniens (phtalates (DBP, DEHP) et bisphenol A (BPA)). Ainsi, une association inverse entre la concentration de ces PE et celle en INSL3 a ete retrouvee. Le recepteur de cette hormone sexuelle est RXFP2. La voie INSL3/RXFP2 est impliquee dans la fonction gonadique. Nous avons donc souhaite caracteriser le role de RXFP2, dans le mode d’action de ces PE. Materiel et methode L’etude des effets des PE a ete realisee dans un systeme d’expression heterologue de surexpression de RXFP2 humain dans des cellules HEK293. L’activite du recepteur a ete analysee en mesurant la production d’AMPc intracellulaire. Resultats Nous avons tout d’abord verifie que les differents PE testes n’induisent pas de toxicite cellulaire (test MTT). Le DEHP et le DBP a des concentrations comprises entre 10−10 M et 10−4 M augmentaient de 40 % l’action de l’INSL3. De la meme facon, le BPA entre 10−9 et 10−5 M potentialisait de 10 a 25 % la reponse a l’hormone. Ces composes amplifiaient d’environ 20 % l’activite constitutive de RXFP2. Cet effet etait specifique de RXFP2 car ils n’agissaient pas sur l’activite de l’adenylate cyclase (synthese AMPc) ou des phosphodiesterases (PDE) (degradation AMPc). L’analyse des courbes dose-reponse a montre qu’ils pourraient agir comme des modulateurs allosteriques positifs, en augmentant la puissance et/ou l’efficacite de l’INSL3. Conclusion Nous montrons donc que RXFP2 intervient dans le mode d’action reprotoxique du DEHP, DBP et BPA.

Plusieurs donnees epidemiologiques associent l’exposition au bisphenol A (BPA) avec des anomalies de la fonction de reproduction. Notre equipe a recemment montre que le BPA inhibe l’action de la FSH sur son recepteur (FSHR). La decontamination de l’eau est realisee par chloration ce qui entraine la formation de derives chlores du BPA (mono, di, tri et tetra chlores) (BPA-Clx), qui se retrouvent dans les milieux naturels et dans les matrices biologiques. L’objectif de ce travail est de determiner les effets des BPA-Clx sur l’activite du FSHR. Nous avons utilise une lignee de cellules CHO exprimant de facon stable le FSHR humain, dont l’activite est evaluee en mesurant la production d’AMP cyclique (AMPc). Les derives chlores sont obtenus dans un melange CH2Cl2/THF en presence de chlorure de sulfuryle et purifies par LC/MS/MS. Tout d’abord nous avons verifie la non-cytotoxicite des composes (test MTT). Environs 10−7 M de BPA reduit la production d’AMPc stimulee par la FSH de facon dose-dependante d’environ 40 %. De la meme facon, les BPA-Clx diminuent de 30 % la reponse a la FSH, sans modifier l’activite basale du recepteur, suggerant un effet allosterique negatif. Le BPA-Cl4 (10−8 M) est le modulateur negatif le plus puissant de la reponse FSH, suivi par le BPA-Cl3 (10−7 M) et les BPA-Cl2 et BPA-Cl1 (10−5 M). Ces resultats montrent pour la premiere fois que, comme le BPA, les BPA-Clx peuvent egalement etre consideres comme perturbateurs endocriniens et avoir des effets reprotoxiques qu’il reste a mieux caracteriser.

Introduction Le recepteur de l’INSL3 (RXFP2) joue un role majeur dans la fonction de reproduction (migration testiculaire), mais egalement dans le metabolisme osseux ou dans le developpement de cancers (thyroide, prostate). RXFP2 appartient a la famille des recepteurs couples aux proteines G (RCPG). Recemment, il a ete mis en evidence que le RXFP2 de la drosophile possede une activite constitutive, c’est-a-dire, independante de son ligand. De plus, RXFP2 presente une forte homologie de structure avec RXFP1, un recepteur constitutivement actif. Dans ce contexte, nous avons donc cherche a caracteriser l’activite constitutive de RXFP2 humain. Materiel et methode Des cellules HEK293 et COS-7 ont ete transfectees de facon transitoire avec le RXFP2 humain ou avec le vecteur vide pcDNA3.1Zeo. L’activite du recepteur a ete analysee en mesurant la production d’AMPc intracellulaire dans differentes conditions. Resultats Nous avons ainsi montre qu’en absence d’INSL3 la production basale d’AMPc etait en moyenne 13,5 ± 1,2 fois plus importante dans les cellules transfectees avec hRXFP2 en comparaison aux cellules controles (p Conclusion Pour la premiere fois, nous mettons en evidence l’activite constitutive de RXFP2 chez l’homme. Le role physiologique de cette activite constitutive reste a definir, en particulier, son implication eventuelle dans la tumorigenese.

International audience; Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, beta-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.