Selective and wash‐resistant fluorescent dihydrocodeinone derivatives allow single‐molecule imaging of μ‐opioid receptor dimerization

μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Entry deterrence and mergers under price competition in pharmaceutical markets

After patent expirations in pharmaceutical markets, brand-name laboratories are threatened by generic firms' entry. To fill the gap in the theoretical literature on this topic, we study brand-name firms' incentives either to deter entry, or to merge with the entrant. These strategies are considered along with the possibility of the brand-name firm producing its own generic drug, called a pseudo-generic drug. Using a vertical differentiation model with Bertrand-Stackelberg competition, we show that each strategy, merging and deterring entry, may be Nash equilibrium, according to the generic firm's setup cost level and to the rate of discount.

Pleiotropy and cryptic genetic variation govern the phenotypic space in Arabidopsis response to water deficit

Background / Purpose:Pleiotropic genes have been identified to control simultaneously many processes involved in key functions like growth and reproduction. Mutations in these central genes affect numerous traits, but their impact on response to selection in natural populations, in which the genetic background and environments vary, remains poorly understood. We developed a modeling approach to uncover the variation of a critical trait related to fitness, the vegetative rosette surface development of Arabidopsis thaliana, at different levels of organization and for different processes such as flowering, cellular proliferation and expansion.Main conclusion:Plant growth, phenology and leaf anatomy were analyzed under well-watered and water-deficient conditions. In the joint population, ERECTA gene had common effects on cell patterning. However, differential trade-off effects of ERECTA on whole rosette development were found in each population induced by genotype-dependent epistatic interactions, revealing cryptic genetic variations at different levels. In response to water deficit, ERECTA affected the coordinated response of rosette development, through interaction with the quantitative trait loci (QTL), depending on the population in which the mutation segregate

Molecular insights into the biased signaling mechanism of the mu-opioid receptor

GPCR functional selectivity opens new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the beta-arrestin pathways through the mu-opioid receptor (mu OR). We combine functional assays in living cells, solution NMR spectroscopy, and enhanced-sampling molecular dynamic simulations to identify the specific mu OR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs, Most strikingly, the biased agonists trigger mu OR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair beta-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.1

Plant growth control by water deficit: which process(es) to lead the game?

National audienceThe nature of plant growth limitation by environmental stresses such as water deficit is a central question for physiologists and breeders because this knowledge could help to target key processes for breeding programs and help designing plants able to maintain growth under stressful conditions. In order to grow, plant cells must loosen their walls, absorb water, reduce and process enough C and minerals to match the plant demand. Therefore, plant cell growth can be limited by cell wall rheological properties, cell or tissue hydraulics or by metabolism. In addition, cell division can be an important process to consider as cell number, together with cell size, contributes to the whole organ size. Over the past few years, our group has questioned the importance of these limitations using combinations of ecophysiological tools, spatio-temporal growth analysis and modelling in ranges of genotypes (including natural variants and mutants). Among the outcomes of these studies, I will show that (i) hydraulic limitation plays a great role on organ growth on the short term, (ii) distinct members of the cell wall loosening expansins family are downstream, unspecific targets of a range of converging developmental, genetic, and environmental cues (iii) metabolism and growth are tightly connected, possibly through a remote control of leaf expansion by starch metabolism and (iv) leaf cell size is more a consequence of growth control at higher levels of organization than vice-vers

Structure of the µ-opioid receptor-Gi protein complex.

The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts. Keywords: FRET screening assay; ceramidase; intramembrane enzyme inhibition; lipid metabolism; structural dynamics + Graphical Abstrac