Slides of my thesis defence on 14th of July 2015.
As part of my PhD thesis, I analyzed β₂-adrenoceptor-mediated signal transduction. The β₂-adrenoceptor is pathophysiologically relevant e.g. in case of bronchial asthma.
My studies add to the receptor-pharmacological concept of functional selectivity. Functional selective (or biased ) ligands may be exploited to achieve highly selective drug effects and, thus, improved drug safety with less adverse effects.
Further, my studies included a collaboration with allergy geneticists. Here we linked genomic and pharmacological profiling of the β₂-adrenoceptor in 60 volunteers. Such pharmacogenomical data may help to stratify treatment groups and create a basis for a more effective, personalized asthma therapy.
(German title: "Molekulare, physiologische und pathophysiologische Analyse des β₂-Adrenorezeptors")
This document discusses the roles of O-linked β-N-acetylglucosamine (O-GlcNAc) in physiology and the analytical challenges of studying it. O-GlcNAc is a post-translational modification found on nuclear and cytosolic proteins that is involved in nutrient sensing and the regulation of many cellular processes. It has extensive crosstalk with phosphorylation and over 3000 protein sites have been mapped to date. Increased global O-GlcNAcylation, even over just 2.5 hours, affects the occupancy of nearly every phosphorylated site that is actively cycling. Many kinases are also regulated by O-GlcNAcylation, with over 40 synaptic kinases identified as being O-
This study examines the effect of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of lipid bilayer membranes composed of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE), and dimyristoylphosphatidylglycerol (DMPG) using differential scanning calorimetry. The results show that GS interacts more strongly with anionic DMPG bilayers than with zwitterionic DMPC or DMPE bilayers, reducing the temperature and cooperativity of DMPG's phase transition to a greater degree. In contrast, GS has little effect on DMP
NHERF1 regulates the membrane retention and recycling of the parathyroid hormone receptor PTH1R. Specifically:
1) NHERF1 inhibits the endocytosis of PTH1R in response to parathyroid hormone by binding to the receptor via its PDZ domains.
2) This prevents the internalization and delays the recycling of PTH1R after endocytosis.
3) Both the PDZ and MERM domains of NHERF1, as well as the PDZ-binding domain of PTH1R, are required for this effect of reduced endocytosis and delayed recycling.
This document discusses translational models of drug-induced liver injury. It begins by outlining the integrated mechanistic drug safety approach, which investigates chemicals, patients, and the interaction between the two. A key part of this approach is understanding the mechanisms of drug bioactivation and the consequences for cellular toxicity. Paracetamol is used as a case study, as it is a common cause of drug-induced liver injury. The document explores the mechanisms of paracetamol toxicity, including how its reactive metabolite NAPQI causes oxidative stress and depletion of glutathione, leading to liver cell damage through both necrosis and inflammation. It also discusses the role of the Nrf2 transcription factor in regulating the antioxidant response and adapting cells to
Differential regulation of transient receptor potential melastatin 6 and 7 ca...Aliny Vasconcelos
This document discusses a study examining the regulation of transient receptor potential melastatin 6 and 7 (TRPM6 and TRPM7) cation channels in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) compared to Wistar Kyoto (WKY) rats. The study found that while TRPM6 expression was similar between SHR and WKY VSMCs, TRPM7 expression and activity were lower in SHR cells. This was associated with reduced intracellular magnesium levels in SHR VSMCs. Angiotensin II increased TRPM6 expression similarly in both cell types but increased TRPM7 expression only in WKY cells, not SHR cells. The findings suggest down
The document discusses a study investigating the effects of food restriction on gene expression. It was previously found that 15 genes were upregulated in the brain during food restriction, suggesting they are part of an ancient stress response pathway. The current study aims to test if these genes are also induced in other tissue types under food restriction. Mice were either food restricted or not for 5 days, then gene expression was analyzed using qPCR in various tissues including the kidney. It was found that Angptl4, Mertk, Arrdc2 and Cdkn1a were significantly upregulated in the kidney of food restricted mice compared to controls, providing further evidence they are part of a general stress response pathway activated by food restriction across multiple
1. The document describes the design and synthesis of molecules that mimic the natural selectin ligand sialyl Lewis X (sLeX) to function as selectin antagonists.
2. Key modifications to the sLeX structure included replacing the GlcNAc saccharide unit with an acyclic tether, and installing benzoate groups at different positions on the galactose and fucose units.
3. The new molecules were evaluated for their ability to inhibit selectin interactions using assays with E-selectin and P-selectin. Compounds with benzoate groups at both C2 and C4 positions on galactose showed the highest potency as selectin antagonists.
This opinion article aims to highlight the use of the Word Association technique (WA) as a food safety tool, as evidenced in the article by J.M. Latorres and coauthors.
This document discusses the roles of O-linked β-N-acetylglucosamine (O-GlcNAc) in physiology and the analytical challenges of studying it. O-GlcNAc is a post-translational modification found on nuclear and cytosolic proteins that is involved in nutrient sensing and the regulation of many cellular processes. It has extensive crosstalk with phosphorylation and over 3000 protein sites have been mapped to date. Increased global O-GlcNAcylation, even over just 2.5 hours, affects the occupancy of nearly every phosphorylated site that is actively cycling. Many kinases are also regulated by O-GlcNAcylation, with over 40 synaptic kinases identified as being O-
This study examines the effect of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of lipid bilayer membranes composed of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE), and dimyristoylphosphatidylglycerol (DMPG) using differential scanning calorimetry. The results show that GS interacts more strongly with anionic DMPG bilayers than with zwitterionic DMPC or DMPE bilayers, reducing the temperature and cooperativity of DMPG's phase transition to a greater degree. In contrast, GS has little effect on DMP
NHERF1 regulates the membrane retention and recycling of the parathyroid hormone receptor PTH1R. Specifically:
1) NHERF1 inhibits the endocytosis of PTH1R in response to parathyroid hormone by binding to the receptor via its PDZ domains.
2) This prevents the internalization and delays the recycling of PTH1R after endocytosis.
3) Both the PDZ and MERM domains of NHERF1, as well as the PDZ-binding domain of PTH1R, are required for this effect of reduced endocytosis and delayed recycling.
This document discusses translational models of drug-induced liver injury. It begins by outlining the integrated mechanistic drug safety approach, which investigates chemicals, patients, and the interaction between the two. A key part of this approach is understanding the mechanisms of drug bioactivation and the consequences for cellular toxicity. Paracetamol is used as a case study, as it is a common cause of drug-induced liver injury. The document explores the mechanisms of paracetamol toxicity, including how its reactive metabolite NAPQI causes oxidative stress and depletion of glutathione, leading to liver cell damage through both necrosis and inflammation. It also discusses the role of the Nrf2 transcription factor in regulating the antioxidant response and adapting cells to
Differential regulation of transient receptor potential melastatin 6 and 7 ca...Aliny Vasconcelos
This document discusses a study examining the regulation of transient receptor potential melastatin 6 and 7 (TRPM6 and TRPM7) cation channels in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) compared to Wistar Kyoto (WKY) rats. The study found that while TRPM6 expression was similar between SHR and WKY VSMCs, TRPM7 expression and activity were lower in SHR cells. This was associated with reduced intracellular magnesium levels in SHR VSMCs. Angiotensin II increased TRPM6 expression similarly in both cell types but increased TRPM7 expression only in WKY cells, not SHR cells. The findings suggest down
The document discusses a study investigating the effects of food restriction on gene expression. It was previously found that 15 genes were upregulated in the brain during food restriction, suggesting they are part of an ancient stress response pathway. The current study aims to test if these genes are also induced in other tissue types under food restriction. Mice were either food restricted or not for 5 days, then gene expression was analyzed using qPCR in various tissues including the kidney. It was found that Angptl4, Mertk, Arrdc2 and Cdkn1a were significantly upregulated in the kidney of food restricted mice compared to controls, providing further evidence they are part of a general stress response pathway activated by food restriction across multiple
1. The document describes the design and synthesis of molecules that mimic the natural selectin ligand sialyl Lewis X (sLeX) to function as selectin antagonists.
2. Key modifications to the sLeX structure included replacing the GlcNAc saccharide unit with an acyclic tether, and installing benzoate groups at different positions on the galactose and fucose units.
3. The new molecules were evaluated for their ability to inhibit selectin interactions using assays with E-selectin and P-selectin. Compounds with benzoate groups at both C2 and C4 positions on galactose showed the highest potency as selectin antagonists.
This opinion article aims to highlight the use of the Word Association technique (WA) as a food safety tool, as evidenced in the article by J.M. Latorres and coauthors.
This study investigated whether S-methylcysteine (SMC), a metabolite of monohalomethanes, contributes to their neurotoxicity. The researchers found that:
1) High concentrations of SMC (10-2 M) reduced synaptic responses in hippocampal slices, an effect that was partially reversible.
2) In organotypic hippocampal cultures, 24 hour exposure to 5x10-5 M SMC compromised membrane integrity in the dentate gyrus, while lower concentrations increased population spike amplitudes and repetitive discharges without affecting membrane integrity.
3) In dissociated hippocampal neurons, SMC reduced GABA-induced currents, acting as a competitive GABAA receptor antagonist with
This document presents a study using differential scanning calorimetry (DSC) to examine the thermal stability of S-protein and its complexes with S-peptide at pH 7.0. DSC measurements showed that S-protein denatures through a reversible two-state transition with a denaturation temperature between 38.5-40.0°C and enthalpy of 165-180 kJ/mol, demonstrating its lower stability without S-peptide. A two-dimensional nonlinear regression analysis of excess heat capacity curves at varying temperatures and S-peptide concentrations was used to determine the binding thermodynamic parameters, yielding values of Kb = 1.10 × 106 M-1, ΔbH = -185 kJ
This study compared the contractile response patterns of ergot alkaloids isolated from tall fescue in bovine lateral saphenous veins. The ergoline alkaloids lysergic acid, lysergol, and ergonovine produced quicker contractile responses that began relaxing immediately, while the ergopeptine alkaloids ergovaline, ergotamine, ergocristine, ergocryptine, and ergocornine had slower, more persistent contractile responses with little to no relaxation over 120 minutes. The different classes of alkaloids produced significantly different contractile response patterns in the veins. Persistent vasoconstriction by certain ergot alkaloids may
This document describes the purification and characterization of a trypanothione-glutathione thioltransferase enzyme from Trypanosoma cruzi. Key findings include:
1) A 52 kDa protein was purified from T. cruzi using affinity chromatography columns containing either S-hexylglutathione or a trypanothione disulfide analogue as ligands.
2) Partial amino acid sequencing showed this protein is identical to one encoded by the previously described TcAc2 cDNA.
3) While it does not have significant glutathione or trypanothione transferase activity, it was found to catalyze thiol-disulfide exchange between dihydrotrypanothione and glutathione dis
- Replacing the chlorophenyl group of WR 194,965 with pyridines or pyrimidines improved antimalarial activity. Optimization of the pyridine series afforded JPC-3210 which displayed potent in vitro activity against chloroquine-sensitive and chloroquine-resistant parasites. JPC-3210 showed exceptional in vivo antimalarial activity in a mouse model and overcame the hERG liability of the lead compound with a favorable pharmacokinetic profile.
N-methyl-D-aspartate receptors (NMDARs) mediate a slow component of excitatory synaptic transmission that plays important roles in normal brain function and development. A large number of disease-associated variants in the GRIN gene family encoding NMDAR GluN subunits have been identified in patients with various neurological and neuropsychiatric disorders. Many of these variants reduce the function of NMDARs by a range of different mechanisms, including
reduced glutamate potency, reduced glycine potency, accelerated deactivation time course, reduced current response amplitude, decreased surface expression, and/or reduced open probability. This talk will focus on three positive allosteric modulators of NMDAR receptor function (24(S)-hydroxycholesterol, pregnenolone sulfate, tobramycin) and two co-agonists (D-serine and D-cycloserine) on their ability to mitigate the diminished function of NMDARs harboring GRIN variants. We examined the effects of these modulators on NMDARs that contained 24 different loss-of-function missense variants in GRIN1, GRIN2A, or GRIN2B. The clinical phenotypes of the patients hosting these variants include epilepsy/seizures and/or developmental delay/intellectual disability. Some patients have autistic behavior or show symptoms of ADHD. For all variants, some aspect of the
reduced function was partially restored. Moreover, some variants showed enhanced sensitivity to positive allosteric modulators compared to wild type receptors. These results raise the possibility that enhancement of NMDAR function by positive allosteric modulators may be a useful therapeutic strategy.
1) The document discusses various determinants of pharmacokinetic parameters like absorption, distribution, metabolism and elimination of drugs in the body.
2) It describes factors influencing absorption like drug properties, dosage forms, patient factors and routes of administration. It also discusses volume of distribution and factors affecting it like plasma protein binding, tissue affinity and physiological barriers.
3) The document covers different pathways of drug metabolism like phase I and II reactions and factors influencing drug metabolism like age, genetics, disease states, nutrition and drug-drug interactions.
El capítulo 2 trata sobre los principales eventos que ocurren. Se describe cómo los personajes principales interactúan entre sí y los desafíos a los que se enfrentan. Además, se establecen las bases para los conflictos y giros de la trama que se desarrollarán en los capítulos posteriores.
Usability session @ SEI Universidade do MinhoRuben Goncalves
Usability: n. The degree to which an object, device, software application, etc. is easy to use with no specific training.
In this session we'll start by understanding what is usability and what are the risks (and costs) of creating non-usable apps. Then we'll focus into understand a bit of the science behind usability and how we can use it efficiently.
This session was created for the SEI 2016 of Minho University.
Plugin smilk : données liées et traitement de la langue pour améliorer la nav...SemWebPro
Pour nourrir leur stratégie marketing et leur veille concurrentielle, les entreprises doivent surveiller le Web et donner un sens à cette grande quantité d'informations. Cette information est éparpillée et nécessite beaucoup de temps pour analyser les différentes sources et compiler les connaissances recueillies de manière intelligente. SMILK est un laboratoire commun entre l'Institut de recherche Inria et la société VISEO pour étudier le couplage fort d'algorithmes et de modèles linguistiques au niveau sémantique, l'extraction et le liage de connaissances issues des ressources du Web et la combinaison de différentes techniques de raisonnement (inférences logiques, des approximations et similitude, etc.). Dans ce contexte, nous allons présenter un prototype permettant d'enrichir les connaissances des utilisateurs naviguant sur le Web à l'aide de résultats issus du Traitement Automatique du Langage Naturel, du Web de Données et des réseaux sociaux. Notre présentation se concentrera sur la démonstration d'un plugin de navigateur facile à installer et à utiliser, qui permet d’enrichir l’expérience utilisateur avec quatre fonctions : - la démo commence par montrer comment il est possible d'identifier dans une page les entités pertinentes selon les intérêts de l'utilisateur et comment structurer les données connexes à l'aide d'une analyse linguistique dédiée ; - la deuxième partie de la démo montre comment nous lions les entités reconnues dans le texte ; - la troisième étape de la démo traite du liage d’entités pour connecter les données figurant dans le texte avec les données obtenues à partir de bases de connaissances du Web ; - enfin, dans une dernière étape la démo montre l’intégration de connaissances issues des médias sociaux pour fournir aux utilisateurs des opinions et les idées clés liées au sujet exploré. Le prototype que nous présenterons intègre entièrement les quatre fonctions précédemment définies et, dans le cadre de cette démonstration, est appliqué au domaine des cosmétiques.
Auteurs : Elena Cabrio, Jordan Calvi, Fabien Gandon, Cédric Lopez, Farhad Nooralahzadeh, Thibault Parmentier, Frédérique Segond Laboratoire Commun SMILK, Inria, VISEO
Remerciements : ces travaux de R&D et transfert sont soutenus par l’ANR au travers du laboratoire commun SMILK ANR-13-LAB2-0001-01
Experience either makes or breaks your app. In this session, we'll show how OutSystems 10 empowers you to create amazing applications. We're going without a safety net, so you will get to see us doing it live, in real time.
Talk performed in OutSystems NextStep Benelux 2016
This document discusses the role of echocardiography in assessing hearts for transplantation and monitoring hearts after transplantation. It covers how echo is used to evaluate donor hearts for abnormalities and the condition of the coronary arteries. Echo is also vital for monitoring transplant recipients for signs of rejection by assessing left ventricular mass, valve performance, and ventricular function. The document introduces a new echo rejection score using tissue Doppler imaging metrics that shows promise as a non-invasive alternative to biopsy for detecting rejection.
This document summarizes several post-harvest research institutions in India and around the world. It discusses the Central Institute for Research on Cotton Technology in Mumbai, which conducts research on cotton post-harvest processing and technologies. It also mentions the Central Institute of Fisheries Technology in Kochi, which focuses on fisheries from harvest to post-harvest. Additionally, it outlines the Division of Post Harvest Technology at IARI in New Delhi and the AICRP on Post Harvest Technology project coordinated in Ludhiana, which aims to reduce food losses. Finally, it briefly describes the Canadian Wheat Board Centre for Grain Storage Research in Manitoba, which takes a multi-disciplinary approach to preserving stored grain.
This document analyzes and summarizes the music video for Jessie J's song "Thunder" through the lens of film theory. It breaks the video down into individual shots, analyzing elements like camera movement, lighting, editing techniques, and how they relate to the song's lyrics and overall themes. The video utilizes a dark, sinister aesthetic and slow movements that match the somber tone of the album it promotes. Imagery of Jessie floating and being "lifted to the heavens" represents a core theme of being uplifted that runs throughout the song and video.
Effective post harvest management of highly perishable horticultural crops rests on following appropriate primary processing protocols. But in the absence of appropriate and crop specific equipment it is not possible. This presentation explains various appropriate equipment developed for this purpose.......
At Social Shared we take teamwork and project management to another level.Social Shared
Increase the profitability of your company. Improve communication with your employees, clients and suppliers. Manage your projects and collaborate from anywhere and any device.
Eli des identifiants pour le croisement des sources ouvertes du droit SemWebPro
Le projet ELI, consiste à créer un identificateur européen et des métadonnées standardisés pour la législation des Etats membres par le biais du web sémantique et de rendre disponible ces données dans le Linked Open Data via l’annotation sémantique des pages des Journaux Officiels. ELI est entré dans la phase de réalisation technique pour différents Journaux Officiels, notamment la France (DILA), l’Union Européenne, le Royaume Uni, le Luxembourg. Cette infrastructure de connaissance sur les ressources législatives des différents états de l’Union Européenne ouvre de nombreuses perspectives de réutilisation et de création de services innovants.
Par Jean Delahousse
This study investigated whether S-methylcysteine (SMC), a metabolite of monohalomethanes, contributes to their neurotoxicity. The researchers found that:
1) High concentrations of SMC (10-2 M) reduced synaptic responses in hippocampal slices, an effect that was partially reversible.
2) In organotypic hippocampal cultures, 24 hour exposure to 5x10-5 M SMC compromised membrane integrity in the dentate gyrus, while lower concentrations increased population spike amplitudes and repetitive discharges without affecting membrane integrity.
3) In dissociated hippocampal neurons, SMC reduced GABA-induced currents, acting as a competitive GABAA receptor antagonist with
This document presents a study using differential scanning calorimetry (DSC) to examine the thermal stability of S-protein and its complexes with S-peptide at pH 7.0. DSC measurements showed that S-protein denatures through a reversible two-state transition with a denaturation temperature between 38.5-40.0°C and enthalpy of 165-180 kJ/mol, demonstrating its lower stability without S-peptide. A two-dimensional nonlinear regression analysis of excess heat capacity curves at varying temperatures and S-peptide concentrations was used to determine the binding thermodynamic parameters, yielding values of Kb = 1.10 × 106 M-1, ΔbH = -185 kJ
This study compared the contractile response patterns of ergot alkaloids isolated from tall fescue in bovine lateral saphenous veins. The ergoline alkaloids lysergic acid, lysergol, and ergonovine produced quicker contractile responses that began relaxing immediately, while the ergopeptine alkaloids ergovaline, ergotamine, ergocristine, ergocryptine, and ergocornine had slower, more persistent contractile responses with little to no relaxation over 120 minutes. The different classes of alkaloids produced significantly different contractile response patterns in the veins. Persistent vasoconstriction by certain ergot alkaloids may
This document describes the purification and characterization of a trypanothione-glutathione thioltransferase enzyme from Trypanosoma cruzi. Key findings include:
1) A 52 kDa protein was purified from T. cruzi using affinity chromatography columns containing either S-hexylglutathione or a trypanothione disulfide analogue as ligands.
2) Partial amino acid sequencing showed this protein is identical to one encoded by the previously described TcAc2 cDNA.
3) While it does not have significant glutathione or trypanothione transferase activity, it was found to catalyze thiol-disulfide exchange between dihydrotrypanothione and glutathione dis
- Replacing the chlorophenyl group of WR 194,965 with pyridines or pyrimidines improved antimalarial activity. Optimization of the pyridine series afforded JPC-3210 which displayed potent in vitro activity against chloroquine-sensitive and chloroquine-resistant parasites. JPC-3210 showed exceptional in vivo antimalarial activity in a mouse model and overcame the hERG liability of the lead compound with a favorable pharmacokinetic profile.
N-methyl-D-aspartate receptors (NMDARs) mediate a slow component of excitatory synaptic transmission that plays important roles in normal brain function and development. A large number of disease-associated variants in the GRIN gene family encoding NMDAR GluN subunits have been identified in patients with various neurological and neuropsychiatric disorders. Many of these variants reduce the function of NMDARs by a range of different mechanisms, including
reduced glutamate potency, reduced glycine potency, accelerated deactivation time course, reduced current response amplitude, decreased surface expression, and/or reduced open probability. This talk will focus on three positive allosteric modulators of NMDAR receptor function (24(S)-hydroxycholesterol, pregnenolone sulfate, tobramycin) and two co-agonists (D-serine and D-cycloserine) on their ability to mitigate the diminished function of NMDARs harboring GRIN variants. We examined the effects of these modulators on NMDARs that contained 24 different loss-of-function missense variants in GRIN1, GRIN2A, or GRIN2B. The clinical phenotypes of the patients hosting these variants include epilepsy/seizures and/or developmental delay/intellectual disability. Some patients have autistic behavior or show symptoms of ADHD. For all variants, some aspect of the
reduced function was partially restored. Moreover, some variants showed enhanced sensitivity to positive allosteric modulators compared to wild type receptors. These results raise the possibility that enhancement of NMDAR function by positive allosteric modulators may be a useful therapeutic strategy.
1) The document discusses various determinants of pharmacokinetic parameters like absorption, distribution, metabolism and elimination of drugs in the body.
2) It describes factors influencing absorption like drug properties, dosage forms, patient factors and routes of administration. It also discusses volume of distribution and factors affecting it like plasma protein binding, tissue affinity and physiological barriers.
3) The document covers different pathways of drug metabolism like phase I and II reactions and factors influencing drug metabolism like age, genetics, disease states, nutrition and drug-drug interactions.
El capítulo 2 trata sobre los principales eventos que ocurren. Se describe cómo los personajes principales interactúan entre sí y los desafíos a los que se enfrentan. Además, se establecen las bases para los conflictos y giros de la trama que se desarrollarán en los capítulos posteriores.
Usability session @ SEI Universidade do MinhoRuben Goncalves
Usability: n. The degree to which an object, device, software application, etc. is easy to use with no specific training.
In this session we'll start by understanding what is usability and what are the risks (and costs) of creating non-usable apps. Then we'll focus into understand a bit of the science behind usability and how we can use it efficiently.
This session was created for the SEI 2016 of Minho University.
Plugin smilk : données liées et traitement de la langue pour améliorer la nav...SemWebPro
Pour nourrir leur stratégie marketing et leur veille concurrentielle, les entreprises doivent surveiller le Web et donner un sens à cette grande quantité d'informations. Cette information est éparpillée et nécessite beaucoup de temps pour analyser les différentes sources et compiler les connaissances recueillies de manière intelligente. SMILK est un laboratoire commun entre l'Institut de recherche Inria et la société VISEO pour étudier le couplage fort d'algorithmes et de modèles linguistiques au niveau sémantique, l'extraction et le liage de connaissances issues des ressources du Web et la combinaison de différentes techniques de raisonnement (inférences logiques, des approximations et similitude, etc.). Dans ce contexte, nous allons présenter un prototype permettant d'enrichir les connaissances des utilisateurs naviguant sur le Web à l'aide de résultats issus du Traitement Automatique du Langage Naturel, du Web de Données et des réseaux sociaux. Notre présentation se concentrera sur la démonstration d'un plugin de navigateur facile à installer et à utiliser, qui permet d’enrichir l’expérience utilisateur avec quatre fonctions : - la démo commence par montrer comment il est possible d'identifier dans une page les entités pertinentes selon les intérêts de l'utilisateur et comment structurer les données connexes à l'aide d'une analyse linguistique dédiée ; - la deuxième partie de la démo montre comment nous lions les entités reconnues dans le texte ; - la troisième étape de la démo traite du liage d’entités pour connecter les données figurant dans le texte avec les données obtenues à partir de bases de connaissances du Web ; - enfin, dans une dernière étape la démo montre l’intégration de connaissances issues des médias sociaux pour fournir aux utilisateurs des opinions et les idées clés liées au sujet exploré. Le prototype que nous présenterons intègre entièrement les quatre fonctions précédemment définies et, dans le cadre de cette démonstration, est appliqué au domaine des cosmétiques.
Auteurs : Elena Cabrio, Jordan Calvi, Fabien Gandon, Cédric Lopez, Farhad Nooralahzadeh, Thibault Parmentier, Frédérique Segond Laboratoire Commun SMILK, Inria, VISEO
Remerciements : ces travaux de R&D et transfert sont soutenus par l’ANR au travers du laboratoire commun SMILK ANR-13-LAB2-0001-01
Experience either makes or breaks your app. In this session, we'll show how OutSystems 10 empowers you to create amazing applications. We're going without a safety net, so you will get to see us doing it live, in real time.
Talk performed in OutSystems NextStep Benelux 2016
This document discusses the role of echocardiography in assessing hearts for transplantation and monitoring hearts after transplantation. It covers how echo is used to evaluate donor hearts for abnormalities and the condition of the coronary arteries. Echo is also vital for monitoring transplant recipients for signs of rejection by assessing left ventricular mass, valve performance, and ventricular function. The document introduces a new echo rejection score using tissue Doppler imaging metrics that shows promise as a non-invasive alternative to biopsy for detecting rejection.
This document summarizes several post-harvest research institutions in India and around the world. It discusses the Central Institute for Research on Cotton Technology in Mumbai, which conducts research on cotton post-harvest processing and technologies. It also mentions the Central Institute of Fisheries Technology in Kochi, which focuses on fisheries from harvest to post-harvest. Additionally, it outlines the Division of Post Harvest Technology at IARI in New Delhi and the AICRP on Post Harvest Technology project coordinated in Ludhiana, which aims to reduce food losses. Finally, it briefly describes the Canadian Wheat Board Centre for Grain Storage Research in Manitoba, which takes a multi-disciplinary approach to preserving stored grain.
This document analyzes and summarizes the music video for Jessie J's song "Thunder" through the lens of film theory. It breaks the video down into individual shots, analyzing elements like camera movement, lighting, editing techniques, and how they relate to the song's lyrics and overall themes. The video utilizes a dark, sinister aesthetic and slow movements that match the somber tone of the album it promotes. Imagery of Jessie floating and being "lifted to the heavens" represents a core theme of being uplifted that runs throughout the song and video.
Effective post harvest management of highly perishable horticultural crops rests on following appropriate primary processing protocols. But in the absence of appropriate and crop specific equipment it is not possible. This presentation explains various appropriate equipment developed for this purpose.......
At Social Shared we take teamwork and project management to another level.Social Shared
Increase the profitability of your company. Improve communication with your employees, clients and suppliers. Manage your projects and collaborate from anywhere and any device.
Eli des identifiants pour le croisement des sources ouvertes du droit SemWebPro
Le projet ELI, consiste à créer un identificateur européen et des métadonnées standardisés pour la législation des Etats membres par le biais du web sémantique et de rendre disponible ces données dans le Linked Open Data via l’annotation sémantique des pages des Journaux Officiels. ELI est entré dans la phase de réalisation technique pour différents Journaux Officiels, notamment la France (DILA), l’Union Européenne, le Royaume Uni, le Luxembourg. Cette infrastructure de connaissance sur les ressources législatives des différents états de l’Union Européenne ouvre de nombreuses perspectives de réutilisation et de création de services innovants.
Par Jean Delahousse
‘Whizzing words’, a platform which is so indeed simple for anyone to put their thoughts, stories forward. Because we believe every story speaks for it.
http://www.whizzingwords.com
Mobile device integration is hard. But not anymore. In this session you'll be able to see how you can easily use device capabilities and integrate with Beacons and Geofence. This presentation consisted in a live demo, which will be shared later on, and on a small video, to be shared as well later.
Presentation made at OutSystems NextStep Lisbon 2016.
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive functioning. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms.
This summary provides an overview of post-harvest institutions around the world from the given document:
The document discusses several prominent post-harvest research and education institutions from India, the United States, and Israel. These include the ADM Institute for the Prevention of Postharvest Loss in Illinois, USA which focuses on reducing losses in staple crops. It also mentions the Central Food Technological Research Institute in Mysore, India and the Central Institute of Post Harvest Engineering and Technology in Ludhiana, India as leading Indian institutions. Finally, it briefly discusses the Postharvest Technology Center at UC Davis and the Volcani Institute Post-Harvest Innovations in Israel.
The document discusses the functional interaction between mGlu1a and GABAB receptors. It first provides background on G protein-coupled receptors and their importance as drug targets. It then reviews evidence that mGlu1a and GABAB receptors physically interact in cortical neurons and Purkinje cells based on co-localization and co-immunoprecipitation studies. The study aims to further investigate the physical interaction between the receptors and the mechanism underlying their functional cross-talk using biophysical techniques like BRET, TR-FRET, and cell-surface co-immunoprecipitation. Preliminary data suggests mGlu1a and GABAB do not form complexes at the cell surface but may oligomerize intracellularly.
The DIONESUS algorithm provides a scalable and accurate method to reconstruct dynamic phosphoproteomic networks and reveal new drug targets. The document describes an experiment where the DIONESUS algorithm was applied to phosphorylation kinetics data from panels of growth factors, small molecule inhibitors, and antioxidants tested on A431 cells. Inhibition of SRC family kinases or PLCγ pathways attenuated EGF-stimulated cell death, while inhibition of PI3K, autocrine signaling pathways, or antioxidants decreased cell viability. The phosphorylation profiles informed dynamic network modeling to infer signaling pathways and potential drug targets.
Managerial evolution and Adchievements at ServierDidier Cussac
Managerial evolution of a research scientist from 1997-2003:
- Junior Research Scientist from 1997-1999 developing assays for D3 receptor binding and MAP kinase activation, and characterizing the D3 receptor antagonist S33084.
- Research Scientist from 1999-2001 managing a team of 2-4 technicians and developing new assays for G protein activation and PLC activity.
- Group Leader from 2001-2003 managing a team of 5 people and a drug screening program on 20 GPCRs, as well as studies on receptor regulation and signaling in cell lines and brain tissue.
The document provides an overview of computational chemistry methods for structure-activity relationship analysis, pharmacophore modeling, and protein-ligand docking. It discusses topics like SAR, QSAR, molecular alignment, conformational analysis, homology modeling of protein targets, and docking programs. Examples are given of applying these methods to study benzodiazepine ligands and GABA receptor subtypes.
Glutamatergic neurotransmission involves glutamate, the major excitatory neurotransmitter in the brain. There are two pathways for glutamate synthesis from precursors and multiple receptor types including NMDA, AMPA, KA, and metabotropic receptors. The different receptor subunits provide diversity in function. Glutamate signaling is involved in many brain pathways and clinical implications include roles in schizophrenia, Parkinson's disease, and drug mechanisms of action.
The document describes research into developing new ligands that bind to the α2δ1 subunit of voltage-gated calcium channels for the treatment of neuropathic pain. Several chemical series were discovered that showed binding affinity for α2δ1 including pyrrolopyridazines, triazolophthalazines, and N-acridinylbutanediamines. The researchers aimed to improve potency, metabolic stability, and physicochemical properties. High-throughput methods identified new analogs and established that binding was specific to the α2δ1 target.
Introduction to the phenomenon of Biased agonism with few examples of receptors exhibiting this phenomenon and an example of drug developed on the basis of biased agonism.
Deepak Pandey, PG Pharmacology, VMMC
This document discusses G protein-coupled receptors (GPCRs), which are the largest family of membrane receptors in the human genome. GPCRs have seven transmembrane domains and signal by interacting with G proteins. They regulate many important physiological processes and are involved in many diseases. The document outlines the structure and function of GPCRs and G proteins, including how GPCRs activate G proteins, the different classes of GPCRs, mechanisms of GPCR regulation like phosphorylation and desensitization, and the roles of GTPases in general.
This document summarizes research on molecular mechanisms of pain conducted by Dr. Nana Voitenko and colleagues. It discusses 4 parts: 1) classification and structure of glutamate receptors including AMPA receptors, 2) involvement of spinal AMPA receptors in inflammatory pain transmission, 3) role of extrasynaptic AMPA receptors in maintaining persistent pain, and 4) targeting of protein kinase C alpha using antisense oligonucleotides to treat inflammatory pain. The research finds that peripheral inflammation induces internalization of GluR2-containing AMPA receptors from synapses and insertion of GluR1-containing receptors at extrasynaptic sites, contributing to persistent pain states.
GAPDH, a well-known glycolytic enzyme, mediatesPei-Ju Chin
This document proposes experiments to investigate how the glycolytic enzyme GAPDH (TDH3 in yeast) mediates apoptosis through epigenetic mechanisms. The first aim is to test if the protein interaction between SET and GAPDH regulates caspase-independent apoptosis by regulating granzyme A activity. The second aim is to determine if GAPDH-mediated expression of histone H2B, influenced by redox status, exerts apoptotic potential in cadmium-stressed yeast cells. Experiments include in vitro and in vivo assays of granzyme A activity and use of yeast mutants and complementation to assess the roles of TDH3, SET and H2B in apoptosis.
Nicholas Young, Sphingosine 1-phosphate Receptor Subtype Influence over Gliob...Nicholas Young
My talk presenting my thesis work per invitation of Research and Development Division of Genzyme Corporation to the Lipid Storage Disorders Department. October 5, 2007. Boston, MA: "Sphingosine 1-phosphate Receptor Subtype Influence over Glioblastoma Multiforme Pathology". My PhD was earned through the Integrated Biomedical Sciences, September 2007, The Ohio State University, College of Medicine Columbus, OH. Area of focus for my PhD: Biochemical and Molecular Disease Mechanisms. Dissertation title: Sphingosine 1-phosphate Receptor Subtype Influence Over Glioblastoma Multiforme Malignant Behavior"
Elevation of Brain Magnesium Prevents and Reverses Cognitive Deficits and Syn...Balogun Wasiu Gbolahan
This study investigated the effects of elevated brain magnesium on cognitive deficits and synaptic loss in an Alzheimer's disease mouse model. The study found that elevating brain magnesium levels through supplementation prevented and reversed cognitive impairment and synaptic loss in transgenic Alzheimer's mouse models. Specifically, magnesium treatment prevented learning and memory deficits, preserved synapse density, and reversed impairments in NMDA receptor signaling pathways involved in learning and memory. The treatment also reduced amyloid plaque levels and decreased expression of the amyloid precursor protein cleaving enzyme BACE1. The results suggest elevated brain magnesium as a potential therapeutic approach for Alzheimer's disease.
Receptors are binding sites located on cells that bind specific molecules and initiate responses. There are several types of receptors including ionotropic receptors that act as ligand-gated ion channels, G protein-coupled receptors that activate intracellular signaling pathways via G proteins, and intracellular or nuclear receptors that directly influence gene expression. Receptor binding involves various interaction forces and leads to responses by altering cellular functions. Understanding receptor pharmacology is crucial for explaining how drugs produce their effects.
The document discusses various strategies for targeting protein-protein interactions (PPIs) for drug development, including small molecule inhibitors, peptides, and allosteric modulation. PPIs are important for many cellular pathways but have been challenging to target with drugs. Examples are given of peptides being developed into peptidomimetics to inhibit PPIs like integrins. Fragment-based approaches and structure-based design have also produced PPI inhibitors for targets like interleukin-2 receptor. Covalent modifiers and stabilizing allosteric sites are additional strategies discussed.
Plant epigenetic memory in plant growth behavior and stress response. Sally M...CIAT
Speaker: Sally Mackenzie, Lloyd and Dottie Huck Chair for Functional Genomics, Department of Biology, Pennsylvania State University. Fellow in the American Society of Plant Biologists and the American Association for the Advancement of Science (AAAS).
Event: Robert D. Havener Seminar on “Innovations for Crop Productivity”.
http://ciat.cgiar.org/event/robert-d-havener-seminar-on-innovations-for-crop-productivity/
The document summarizes a study that investigated the role of glycosyltransferases in regulating leukocyte interactions with selectins. Key findings include:
1) A PSGL-1 variant called 19Fc containing a single O-glycan site was used to determine glycan distribution at the N-terminus of PSGL-1 via mass spectrometry. This found core-2 sLeX and disialylated T-antigen glycans.
2) Overexpression of ST6GalNAc-2 in HL-60 cells reduced sLeX expression and binding to P-, L-, and E-selectins, increasing rolling velocities.
3) ST6GalNAc-2 compet
Functional Analysis Of Heterologous Gpcr Signaling Pathways In Yeastbeneshjoseph
The document discusses using yeast as a model system to study heterologous G protein-coupled receptors (GPCRs). Yeast have signaling pathways that can be exploited to study GPCRs. Chimeric Gα proteins were developed to efficiently couple yeast and mammalian GPCRs. Methods like modifying receptor sequences or co-expressing receptor activity modifying proteins allow functional expression of GPCRs in yeast. This enables large-scale screens that can define receptor-G protein specificity and identify novel ligands, improving drug discovery. Studies in yeast have characterized receptors, G proteins, and their regulators like receptor kinases and RGS proteins. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast are discussed
G-protein coupled receptors (GPCRs) are the largest family of membrane receptors that are linked to intracellular effector proteins via G-protein activation. There are several classes of GPCRs classified based on sequence homology. All GPCRs have seven transmembrane domains and signal through heterotrimeric G proteins. When a ligand binds a GPCR, it activates an associated G protein which then regulates downstream effectors like adenylyl cyclase or phospholipase C. These pathways mediate many physiological processes such as vision, smell, immune response, and neuronal signaling.
Similar to Molecular, physiological and pathophysiological analysis of the β₂-adrenoreceptor (20)
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
Unlocking the mysteries of reproduction: Exploring fecundity and gonadosomati...AbdullaAlAsif1
The pygmy halfbeak Dermogenys colletei, is known for its viviparous nature, this presents an intriguing case of relatively low fecundity, raising questions about potential compensatory reproductive strategies employed by this species. Our study delves into the examination of fecundity and the Gonadosomatic Index (GSI) in the Pygmy Halfbeak, D. colletei (Meisner, 2001), an intriguing viviparous fish indigenous to Sarawak, Borneo. We hypothesize that the Pygmy halfbeak, D. colletei, may exhibit unique reproductive adaptations to offset its low fecundity, thus enhancing its survival and fitness. To address this, we conducted a comprehensive study utilizing 28 mature female specimens of D. colletei, carefully measuring fecundity and GSI to shed light on the reproductive adaptations of this species. Our findings reveal that D. colletei indeed exhibits low fecundity, with a mean of 16.76 ± 2.01, and a mean GSI of 12.83 ± 1.27, providing crucial insights into the reproductive mechanisms at play in this species. These results underscore the existence of unique reproductive strategies in D. colletei, enabling its adaptation and persistence in Borneo's diverse aquatic ecosystems, and call for further ecological research to elucidate these mechanisms. This study lends to a better understanding of viviparous fish in Borneo and contributes to the broader field of aquatic ecology, enhancing our knowledge of species adaptations to unique ecological challenges.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...Leonel Morgado
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
Molecular, physiological and pathophysiological analysis of the β₂-adrenoreceptor
1. 1
Molekulare, physiologische und
pathophysiologische Analyse
des β2-Adrenorezeptors
Rezeptorpharmakologie / Pharmakogenetik
In vitro / Ex vivo
Funktionelle Selektivität / Individuelle Arzneimittelreaktion
Disputation
Michael T. Reinartz
10. 11
Fight-or-Flight reaction via β2-adrenergic receptors
Selective activation of β2AR-specific effects
cardiac output
broncho dilation
glycogenolysis
immune-suppression
digestion
... utilized for
...
http://www.openclipart.org
tocolysis
11. 12
Broncho-dilative effect of β2AR-selective agonists
Pathology of bronchial asthma
relaxed
smooth
muscles
air trapped
in alveoli
tightened
smooth
muscles
normal airway asthmatic airway
during attack
asthmatic airway
wall inflamed
and
thickened
http://www.ocallergy.com
12. 13
Ligand-specific pharmacology
of 14 β2AR-ligands
Reinartz MT et al., Naunyn Schmiedebergs Arch Pharmacol. 388:1 (2015)
β2AR-
specific
information
Part 1
13. 14
β2AR ligands can be functionally selective
Ligand classification
Simmons MA Mol Interv 5:3 (2005)
14. 15
β2AR ligands can be functionally selective
Ligand classification
Gstimulatory Ginhibitory β-arrestin
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
Signal transduction pathways via β2AR
15. 16
β2AR ligands can be functionally selective
Ligand classification
Gstimulatory Ginhibitory β-arrestin
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
Signal transduction pathways via β2AR
16. 17
β2AR ligands can be functionally selective
Ligand classification
Functional selectivity or “ligand bias“ is the ligand-dependent selectivity for
certain signal transduction pathways in one and the same receptor.
This can be present when a receptor has several possible signal transduction pathways.
Gstimulatory Ginhibitory β-arrestin
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
Signal transduction pathways via β2AR
Simmons MA Mol Interv 5:3 (2005)
17. 18
Relevance of functional selectivity via β2AR
Gstimulatory
AC
cAMP
Ginhibitory
AC MAPK
(fast)
cAMP
β-arrestin
MAPK
(delayed)
other
signals
?
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
airway smooth muscle
relaxation contractile sensitization desensitization
18. 19
Relevance of functional selectivity via β2AR
Gstimulatory
AC
cAMP
Ginhibitory
AC MAPK
(fast)
cAMP
β-arrestin
MAPK
(delayed)
other
signals
?
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
immune cells
immune-suppression pro-inflammatory? immune-modulation?
desensitization
airway smooth muscles
relaxation contractile sensitization desensitization
24. 25
Extensive pharmacological profiling
In vitro, in cell and ex vivo
Gi-GTPase
Gs-GTPase
AC
Binding
β2AR-Gxα- fusion proteins
β-arrestin-2 recruitment
HEK293-cells expressing β2AR
Takakura H et al., ACS Chem Biol 7:5 (2012)
25. 26
Extensive pharmacological profiling
In vitro, in cell and ex vivo
cAMP accumulation
inhibition of ROS production
(radical oxygen species)
Neutrophils expressing β2AR
Gi-GTPase
Gs-GTPase
AC
Binding
β2AR-Gxα- fusion proteins
β-arrestin-2 recruitment
HEK293-cells expressing β2AR
Takakura H et al., ACS Chem Biol 7:5 (2012)
AC
NOX
IP3
DAG
ROS
PKC
FPR
b2AR
Gi b
b
cAMP
PIP2
Gs
29. 30
Quantification of receptor activation & ligand bias
log(τ/KA)Gsα → each agonist
log(τ/KA)Giα → each agonist
Δlog(τ/KA) → agonist – (R)-EPI
Condensing efficacy and potency to log(τ/KA)
Normalization cancels system bias
ΔΔlog(τ/KA) = Gs - Gi
Comparison of two pathways
31. 32
Bias analysis: Gs- vs. Gi-coupling at β2AR
Gs-bias of
(S,S')-methoxy-
fenoterol
(R,S')-
methoxy-
naphthyl-
fenoterol
OH OO
32. 33
Bias analysis: Gs- vs. Gi-coupling at β2AR
Gs-bias of
(S,S')-methoxy-
fenoterol
(R,S')-
methoxy-
naphthyl-
fenoterol
extreme Gs-bias
not quantifiable
no detectable
Gi-activation by
(S,S')-3
OH OO
33. 34
Summary as functional “fingerprints”
Pairwise comparison of six assays per ligand
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
Six β2AR assays
15 pair-wise comparisons
Single fingerprint per ligand
34. 35
Heatmap of 14 functional “fingerprints”
Sums up 84 concentration-response curves (with n ≥ 3)
Heatmap to compare
14 ligand fingerprints
35. 36
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
36. 37
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
37. 38
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
modification at
aminoalkyl-tail
increases Gs-bias
> >
> >
> >
> >
> >
> >
> >
38. 39
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
modification at
aminoalkyl-tail
increases Gs-bias
disfavored / silenced
Gi-coupling
β-arr-2 recruitment
39. 40
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
modification at
aminoalkyl-tail
increases Gs-bias
disfavored / silenced
Gi-coupling
β-arr-2 recruitment
(R,S')- and (S,S')-
methoxy-naphthyl-
fenoterol (3) are
strong / extreme Gs-
biased
40. 41
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
modification at
aminoalkyl-tail
increases Gs-bias
disfavored / silenced
Gi-coupling
β-arr-2 recruitment
(R,S')- and (S,S')-
methoxy-naphthyl-
fenoterol (3) are
strong / extreme Gs-
bias
Naphthyl-moiety AND (X,S‘)-chirality
Structure-bias relationship
CH3
O
NHOH
OH
OH
*
S
41. 42
TM 4
TM 5
TM 6
TM 1
TM 2
TM 3
OH
N+
OH
OH
OH
TM 7
site 1 site 2
adopted from Jozwiak, K et al., Chirality, 23 (2011)
Specific interactions crucial for Gi and β-arr-2 signaling
Orthosteric ligand binding site of β2AR
42. 43
TM 4
TM 5
TM 6
TM 1
TM 2
TM 3
OH
N+
OH
OH
OH
TM 7
site 1 site 2
interactions with transmembrane domain 7 (TM 7)
stabilisation of an inactive conformation
selective β-arrestin-2 activation
Structure Bias Relationship
Specific interactions crucial for Gi and β-arr-2 signaling
OMe
adopted from Jozwiak, K et al., Chirality, 23 (2011)
Orthosteric ligand binding site of β2AR
43. 44
confirmation of β2AR functional selectivity
aminoalkyl-derivatization and stereochemistry of
fenoterol modify Gi and β-arr-2 signalling
insights into Gs-biased β2AR agonism
Value for Gs-biased drug development
Conclusions – β2AR-specific information
β2AR-
specific
information
Part 1
CH3
O
NHOH
OH
OH
*
S
51. 52
“Caucasian“ background
comparable to HAPMAP-CEPH
healthy volunteers recruited at MHH
22 – 56 yrs old
Cohort characteristics (n=60)
60 healthy volunteers – background check
0 0,2 0,4 0,6
Thr283Ser
Thr164Ile
Gln27Glu
Gly16Arg
this study
HAPMAP-
CEPH
/ MAF 0% 50% 100%
asthma
atopic
smoking
male
yes
no
HAPMAP-CEPH: haplotype map of the human genome, CEPH (809
“Caucasian” individuals); MAF: minor allele frequency
52. 53
No Influence of relevant assay parameter
no relevant confounding effect on
formylpeptide (fMLP)-induced ROS (radical oxygen species) production
β2AR-mediated inhibition of fMLP-induced ROS-production
One-way ANOVA; p-value > 0.05 = n.s.
Statistical testing for difference between sub-populations
sex asthma atopy smoking age
53. 54
multivariate regression analysis:
description of
dependent variable (responsiveness)
using
influencing factors (ligand, SNPs)
stepwise improvement of model
Influence of Glycine-16-Arginine Polymorphism
small decrease if carrying Arg-allele (p < 0.002)
0.146 on the logarithmic concentration scale
SNP as possible marker or cause for decreased responsiveness
in silico analysis suggests deleterious effect of non-synonymous amino acid exchange
on biological function
Influence on pooled responsiveness
Multivariate regression detects decrease in responsiveness
estimated influence: -0.40 ± 0.13
54. 55
Conclusions – Subject-specific information
ROS-inhibition assay in
neutrophils is
a sensitive and robust ex vivo
test model for individual
β2AR responsiveness
not influence by sex, age,
smoking, atopy, or asthma
Gly16Arg SNP
as a genetic marker for
decreased
β2AR responsiveness
Inter-individual variability of β2AR responsiveness
Subject-
specific
Information
Part 2
AC
NOX
IP3
DAG
ROS
PKC
FPR
b2AR
Gi b
b
cAMP
PIP2
Gs
56. 57
β2AR-specific information
Gs-biased ligands based on fenoterol scaffold
more precise therapy (e. g. bronchial asthma)
improved tools to research 7TMR functional selectivity
Perspectives
more structural information on 7TMR-ligand-signalling-protein
complexes needed
insights from new techniques looking at receptor
conformations
rational medicinal-chemical design of biased ligands
increased complexity of drug development
Challenges
Outlook
Improved
therapy
57. 58
Patient-specific information
ex vivo ROS assay results as parameter in clinical studies on
β2AR-pharmacogenetics
e.g. with neutrophils isolated from patients
stratification of patient groups
Perspectives
further verification, that SNPs influence responsiveness
more and more individual data ((epi-)genomic, microbiomic,
life-style (wearables), ...)
employment of “BigData”-analyses for personalized medicine
Challenges
Outlook
Improved
therapy
58. 59
Thank You
Ideas, advice, assistance, collaboration, material & any support
... and to all study-participants!!
Prof. M. Gaestel (Physiological Chemistry)
Prof. E. Ponimaskin (Neurophysiology)
Prof. R. Seifert (Pharmacology)
Dr. C. Happle
Prof. M. Kabesch
Dr. M. Wetzke
(Clinic for Paediatric Pneumology and Neonatology)
Pharmacogenetic Studies
A. Garbe (ZFA Metabolomics)
S. Kälble (Pharmacology)
Prof. V. Kaever (ZFA Metabolomics)
Technical Assistance / Analyses
Review & Supervision
Dr. I. R. Wainer (National Institute on Aging)
Dr. A. Schnapp (Boehringer Ingelheim)
Enantiopure Ligands
T. Littmann (Pharmacology)
Prof. T. Ozawa (University of Tokyo)
β-arrestin data & cell line
Prof. S. Dove (University of Regensburg)
Prof. A. Koch (Biometry)
R. Scherer (Biometry)
Statistical Advice
59. 60
Questions and Answers
β2-AR
functional selectivity
in vitro, in cell, ex vivo
bias quantification
Receptor Pharmacology Pharmacogenetics
Inter-individual variability
ex vivo
study with 60 volunteers
Naunyn Schmiedebergs Arch
Pharmacol. 2015 May;388(5):517-24
Naunyn Schmiedebergs Arch
Pharmacol. 2015 Jan;388(1):51-65
PLoS One. 2013 May 31;8(5):e64556
Review on β2AR Functional Selectivity:
BIOspektrum, 2014 March; 20(2):130-
135
Submitted to Allergy
Rasmussen et al., Nature 2011 Jan.; 469:175-180
61. 62
Model of Multiple Receptor Conformations
A complex composed melody and NOT only a single tune
62. 63
Interaction of fenoterol stereoisomers with β2-
adrenoceptor-Gsα fusion proteins:
antagonist and agonist competition binding
Reinartz, MT et al., Naunyn Schmiedebergs Arch Pharmacol. 388:5 (2015)
β2AR-
specific
information
Part 1B
63. 64
β2AR-Gs fusion protein – binding assays
Competition with radioactive labeled agonist or antagonist
analysis of the binding affinity of ligands using β2AR-Gs fusion protein
displacement of fenoterol ligands by radio-actively labeled ligands
[3H]-DHA AND [3H]-(R,R‘)-Methoxynaphthyl-Fenoterol (2)
Analysis of ternary complex (ligand + receptor + G-protein)
with AND without GTP
Concentration-dependent inhibition of radio-ligand binding
64. 65
12 Fenoterol ligands –
pick-locking tool to probe the binding site
Seifert, R & Dove, S, Mol Pharmacol, 2009, 75
detektei-schutzdienst-shop.de, wikihow.com/Pick-a-Lock
D113
S203
S204
S207
F290
C191
Y316
H93
W109
F193
Molecular Modeling mit
(R,R') und (S,R')-Fenoterol
65. 66
Association of
ligand binding to the receptor ([3H]DHA or [3H](R,R‘)-2)
activation of proximal downstream signalling (GTPase)
Potency vs. Affinity
Gs-GTPase vs. competition binding
A closer look at the coupling of β2AR with Gs
4 5 6 7 8 9
4
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521 pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pEC50,GTPase
4 5 6 7 8 9
4
5
6
7
8
9
10
pEC50,GTPase = 2.1 + 0.719 pKi,low,(R,R')-2
R² = 0.77
pKi,low,(R,R')-2
pEC50,GTPase
66. 67
Association of
ligand binding to the receptor ([3H]DHA or [3H](R,R‘)-2)
activation of proximal downstream signalling (GTPase)
Potency vs. Affinity
Gs-GTPase vs. competition binding
[3H]-(R,R‘)-Methoxy-Fenoterol probes active conformation
4 5 6 7 8 9
4
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521 pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pEC50,GTPase
4 5 6 7 8 9
4
5
6
7
8
9
10
pEC50,GTPase = 2.1 + 0.719 pKi,low,(R,R')-2
R² = 0.77
pKi,low,(R,R')-2
pEC50,GTPase
agonist as radio-ligand
reflects active
receptor
conformation
fenoterol derivative as
radio-ligand
structural-similar to
the other „fenoterols“
Higher Association for
pEC50 vs. pKi,low,(R,R‘)-2
67. 68
Association of
ligand binding to the receptor ([3H]DHA or [3H](R,R‘)-2)
activation of proximal downstream signalling (GTPase)
Potency vs. Affinity
Gs-GTPase vs. competition binding
Information on structure-(bias)/activity-relationship
4 5 6 7 8 9
4
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521 pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pEC50,GTPase
4 5 6 7 8 9
4
5
6
7
8
9
10
pEC50,GTPase = 2.1 + 0.719 pKi,low,(R,R')-2
R² = 0.77
pKi,low,(R,R')-2
pEC50,GTPase
agonist as radio-ligand
reflects active
receptor
conformation
fenoterol derivative as
radio-ligand
structural-similar to
the other „fenoterols“
„clustering“ by
stereoconfiguration
antagonist as radio
ligand
(R,S‘)- and (S,S‘)-3
step-out
Higher Association for
pEC50 vs. pKi,low,(R,R‘)-2
68. 69
Binding assays (antagonist and agonist)
Functional assays (GTPase and AC)
agonist competition at β2AR-Gs
fusion proteins fits in silico data best
(not shown)
supports medicinal-chemical
rationale (Wainer et al.)
Insights into stereoselective
interaction & functional selectivity
Various Levels in the G-protein cycle:
binding, transmission, effector
Taken together – Binding Assays
Adds to functional data and published analyses
CH3
OH
NHOH
OH
OH
* *
69. 70
Binding assays (antagonist and agonist)
Functional assays (GTPase and AC)
agonist competition at β2AR-Gs
fusion proteins fits in silico data best
(not shown)
supports medicinal-chemical
rationale (Wainer et al.)
Insights into stereoselective
interaction & functional selectivity
Various Levels in the G-protein cycle:
binding, transmission, effector
Taken together – Binding Assays
Adds to functional data and published analyses
CH3
OH
NHOH
OH
OH
* *
O
70. 71
Binding assays (antagonist and agonist)
Functional assays (GTPase and AC)
agonist competition at β2AR-Gs
fusion proteins fits in silico data best
(not shown)
supports medicinal-chemical
rationale (Wainer et al.)
Insights into stereoselective
interaction & functional selectivity
Various Levels in the G-protein cycle:
binding, transmission, effector
Taken together – Binding Assays
Adds to functional data and published analyses
CH3
OH
NHOH
OH
OH
* *
O
71. 72
Binding assays (antagonist and agonist)
Functional assays (GTPase and AC)
agonist competition at β2AR-Gs
fusion proteins fits in silico data best
(not shown)
supports medicinal-chemical
rationale (Wainer et al.)
(R,S‘)- and (S,S‘)-3 depict unique
dissociation of binding and GTPase
activity
supports (own) functional
analyses
Insights into stereoselective
interaction & functional selectivity
Various Levels in the G-protein cycle:
binding, transmission, effector
Taken together – Binding Assays
Adds to functional data and published analyses
CH3
OH
NHOH
OH
OH
* *
O
72. 73
EX VIVO ASSAY IN NEUTROPHILS OF 60
VOLUNTEERS
Supplemental Results: Study
73. 74
Study with neutrophils of 60 volunteers
Connecting β2AR SNPs and ex vivo responsiveness
96-well format
1 x 105 cells/well, just 4-8 ml of blood needed
4 hrs from blood collection to assay completion
ROS assay and pharmacological analysis
60 volunteers
180 96-well-plate assays
240 concentration-response
curves
Numbers
74. 75
healthy volunteers recruited at MHH
„caucasian“ background
Cohort characteristics (n=60)
Sixty Volunteers – Background check
Receptor polymorphisms (SNP) vs. β2AR pharmacology
0% 50% 100%
asthma
atopic
smoking
male
yes
no
0 0,2 0,4 0,6
Thr283Ser
Thr164Ile
Gln27Glu
Gly16Arg
this study
HAPMAP-
CEPH
/ MAF
Cell isolation & ex vivo „performance“
neutrophils eosinophils basophils
0
1000
2000
3000
4000
granulocytes count / mL blood
75. 76
Influence of factors despite SNPs
No significant difference in responsiveness
One-way ANOVA; p-value > 0.05 = n.s.
possible confounders are excluded from the model or have no significant effect
comparing β2AR-responsiveness from fitting concentration-response data
standardized
pooled for all four ligands β2AR-agonists
Statistical modeling for difference between sub-populations
76. 77
Ligand-specific responsiveness (potency)
60 volunteers ~ 180 Assays
concentration-response data yielded pIC50 values on the β2AR-mediated ROS-inhibition
Formoterol (FORM) most potent, Salbutamol (SAL) least potent
looking at individual -> ligand-specific responsiveness (color-coded)
77. 78
Standardization of response values of 4 ligands
Substraction of the ligand-group mean
Apple and Oranges ?
(difficult to pool & compare)
1110987
pIC50,ROS
(R)-ISO (R,R)-FEN (R)-SAL (R,R)-FORM
78. 79
Standardization of response values of 4 ligands
Substraction of the ligand-group mean
Standardized responsiveness
(values directly comparable)
Apple and Oranges ?
(difficult to pool & compare)
1110987
pIC50,ROS
(R)-ISO (R,R)-FEN (R)-SAL (R,R)-FORM
-2-1012
(R)-ISO
79. 80
Low-concentration or high-concentration responders
Correlation of ligand-specific responsiveness
significant correlation between the seperate pIC50 values
ISO vs. FEN, ISO vs. SAL, ISO vs. FORM
allowed pooling of values
researching the association with the genetic background (ADRB2 gene)
Ligand-independency of inter-individual variability
80. 81
ADRB2 exon
Analysis of known and unknown SNPs
-468 -406 -367
-376 -262
-47
-26
+66 +659
+46
+79 +252
+491
+523
+1053
+1098
+1239
+1268
+1269
+1275
+1277
+1278
+1629
+1678
ADRB2
Chr. 5
85. 86
Extraction of recombinant β2AR from insect cells
Sf9 cells – a well-established baculoviral expression system
Sf9 cells are suitable to culture and
prepare human recombinant receptors
3 x 106 cells/mL are inoculated with high-
titer virus encoding β2AR-Gxα fusion
protein
harvesting of cells after 48 h
(very-late phase of infection)
washing, lysis, homogenization and
sedimentation
membrane pellets are resuspended in
binding buffer and stored at -80°C
Infection and Membrane Preparation
86. 87
β2AR-Gs/-Gi fusion protein - GTPase Assay
Turn-over at the mediator of the G-Protein cycle
Sf9 membranes expressing β2AR-Gsα or β2AR-Giα
activation of the coupled Gxα
GTPase activity produces GDP and anorganic phosphate
radiometric analysis of [γ-32P]GTP turnover (20 min at 25°C)
concentration-response data
GTPase
+H2O
87. 88
β2AR-Gs/-Gi fusion protein - GTPase Assay
Radiometric analysis using [γ-32P]GTP
GTPase
+H2O
+ GDP
in 50 mM Tris/HCl buffer
100 µM adenosine-5‘-[b,g-imido]triphosphate
100 nM GTP
100 µM ATP
1 mM MgCl2
100 µM EDTA
0.2% BSA
5 mM creatine phosphate and 0.4 µg creatine kinase
Buffered reaction mixture with a regenerative component
88. 89
β2AR-Gs fusion protein – Adenylyl Cyclase Assay
Production of the 2nd messenger cAMP
Sf9 membranes expressing β2AR-Gsα
activation of endogenous AC
cAMP production
radiometric analysis of [α-32P]ATP turn-over (20 min at 37 °C)
concentration-response data
89. 90
β2AR-Gs fusion protein – AC Assay
Radiometric analysis of cAMP generation
AC
high-speed centrifugation
single-column, gravity-driven seperation
Al2O3 packing restrains [α-32P]ATP
[32P]cAMP eluted to scintillation vials (0.1 M NH4-AcO)
followed by liquid scintillation counting (Cerenkov)
Chromatographic separation of [α-32P]ATP and [32P]cAMP
+ PPi
90. 91
β2AR-Gs fusion protein – AC Assay
Turn-over of [α-32P]ATP (20 min @ 37°C)
AC
+ PPi
in binding buffer
10 µM GTP
40 µM [α-32P]ATP
0.1 mM cAMP
2.7 mM mono(cyclohexyl)ammonium phosphoenolpyruvate
0.125 IU pyruvate kinase and 1 IU myokinase
Buffered reaction mixture with a regenerative component
91. 92
β-arrestin-2 recruitment assays
Analysis of G-protein-independent signalling
stimulation of seeded cells @ 37°C
stopped after 10 min by addition of detection reagent
reading luminescence counts (2s/well)
HEK293-cells expressing β2AR
Takakura et al.
complementation of luciferase fragments
CHO-cells expressing β2AR
DiscoverX PathHunter®
complementation of β-gal fragments
92. 93
β-arrestin-2 recruitment assays
Commercial buy-in vs. academic collaboration
own data (DiscoverX, N=1) comparable to data by Timo Littmann (Takakura Assay, N=3)
decision against costly assay ready kit (~ 400 € per 96 well plate)
future publication (Littmann et al) on β-arr-1 vs. β-arr-2 recruitment in preparation
includes comparison of assays (no difference)
Comparison of DiscoverX PathHunter® and Takakura Assay
93. 94
Neutrophils from human whole blood (EDTA)
Gentle cell isolation without pre-activation
Ficoll seperating solution
density 1.077 g/mL
30 min @ 400 x g seperates into layers of
plasma
lymphocytes (white)
erythrocytes / granulocytes (red)
followed by
selective lysis of erythrocytes (ddH2O)
washing (PBS)
resuspension in cold PBS
> 98% viable neutrophils
Density Gradient Centrifugation
Q: Nature Protocols
94. 95
cAMP production in human neutrophils
The second messenger in a cellular context
stimulation of 5 x 105 cells/tube
in 100 µL PBS
with 1 mM CaCl2, 100 µM IBMX
10 min @ 37°C
stopped @ 95°C
addition of 100 µL eluent A
3/97 MeOH/H20, 50 mM NH4OAc, 0.1%
HOAc
100 ng tenofovir/mL (internal standard)
hand-over to the Core Facility
Metabolomics
quantification by reversed-phase HPLC
mass spectrometry
cAMP extraction
AC
b2AR b
Gib
cAMP
Gs
b2AR
95. 96
O2
.- + Ferricytochrome C (Fe(III)) → O2 + Ferrocytochrome C (Fe(II))
reduction alters absorbance at 550 nm
isosbestic point at 557 nm (no change)
A550(t=30 min) - A550(t=0) = ΔA550
concentration-response data
Inhibition of ROS production in human neutrophils
A robust assay of pathophysiologically-relevant signalling
stimulation of 105 cells/well
in a coated 96-well plate
PBS with
1 CaCl2, 100 µM ferricytochrome c,
0.3 µg/mL cytochalasin b
30 min @ 37°C
absorbance at 550 nm (A550)
Colormetric cytochrome c assay
AC
NOX
IP3
DAG
ROS
PKC
FPR
b2AR
Gi b
b
cAMP
PIP2
Gs
97. 98
Gs vs. Gi coupling at β2AR – quantitatively
Condensing efficacy and potency to the transducer coefficient
log(τ/KA)Gsα → each agonist
log(τ/KA)Giα → each agonist
Δlog(τ/KA) → agonist – (R)-EPI
1st Step: Fitting the
transducer coefficient
2nd Step:
Normalization to reference
ΔΔlog(τ/KA) = Gs - Gi
3rd Step:
Comparison
agonist β2AR KA,Gsα agonist-β2AR effectorGsα signalGsα
τGsαlog(τGsα/KA,Gsα)
modulator conduit guest
transducer coefficient
98. 100
Gs vs. Gi coupling at β2AR – bias plot (still qualitatively)
Epinephrine and ISO as reference
0 50 100
0
50
100
(R)-EPI
(R)-ISO
GTPase activity of b2AR-Gs
(equimolar response)
GTPaseactivityofb2AR-Gi2
(equimolarresponse)
endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
99. 101
Gs vs. Gi coupling at β2AR – bias plot (still qualitatively)
(R,R')-stereoisomers more Gs-biased as reference
0 50 100
0
50
100
(R)-EPI
(R)-ISO
(R,R')-1
(R,R')-2
(R,R')-3
GTPase activity of b2AR-Gs
(equimolar response)
GTPaseactivityofb2AR-Gi2
(equimolarresponse)
endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
relatively more bending towards x-
axis depicts Gs bias
100. 103
Gs vs. Gi coupling at β2AR
Tendency to Gs clearly dominates – no Gi-bias
0 50 100
0
50
100
(R)-EPI
(R)-ISO
(R,R')-1
(R,S')-1
(S,R')-1
(S,S')-1
(R,R')-2
(R,S')-2
(S,R')-2
(S,S')-2
(R,R')-3
(R,S')-3
(S,R')-3
(S,S')-3
GTPase activity of b2AR-Gs
(equimolar response)
GTPaseactivityofb2AR-Gi2
(equimolarresponse)
endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
relatively more bending towards x-
axis depicts Gs bias
no curve „near“ y-axis = no Gi-
biased ligand
101. 104
Gs vs. Gi coupling at β2AR – bias plot
Bending curves towards x-axis depict Gs bias
0 50 100
0
50
100
(R)-EPI
(R)-ISO
(S,S')-2
(R,S')-3
(S,S')-3
GTPase activity of b2AR-Gs
(equimolar response)
GTPaseactivityofb2AR-Gi2
(equimolarresponse)
endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
relatively more bending towards x-
axis depicts Gs bias
no curve „near“ y-axis = no Gi-
biased ligand
(S,S')-Methoxy and (R,S')-
Methoxy-naphthyl-fenoterol partial
agonists at Gi (< 50%)
strong Gs-bias
(S,S')-Methoxy-naphthyl-fenoterol
extremely Gs-biased
no Gi-GTPase activity at all
103. 111
Structure-biological techniques
analysis of single structure elementslemente
energy landscape
mechanical flexibility
conformative variability
single-molecule force spectroscopy
Zocher et al Chem Soc 42:19 (2013)
104. 112
Structure-biological techniques
analysis of different receptor conformations
agonist / antagonist bound ..
detects chemical shifts -> distance between 1H- / 13C- / 15N-labeled residues
Solid state nuclear magnetic resonance
Bokoch et al Nature 463 (2010)
105. 113
Structure-biological techniques
analysis of different receptor conformations
agonist / antagonist bound ..
detects chemical shifts -> distance between 1H- / 13C- / 15N-labeled residues
Solid state nuclear magnetic resonance
Bokoch et al Nature 463 (2010)
106. 114
Functional Selectivity via β2-Adrenoreceptor
Relevance of signalling during chronic heart failure
Gs
AC
cAMP ↑
Gi
AC MAPK
(fast)
cAMP ↓
β-arrestin
MAPK
(delayed)
other
signals?
PTX
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
immune cell
immunosuppression pro-inflammatory?
immune-modulation?
desensitization
cardiomyocyte
contractile support
cardio-protective
compromised contractile support
cardio-protective
cardiac remodeling
airway smooth muscle
relaxation contractile sensitization desensitization
openclipart.org & http://hsc.uwe.ac.uk/rcp/rs-rt-bronchioles.aspx
109. 117
- +
not studied
Institute of Pharmacology
Roland Seifert
Effects of fenoterol stereoiomers on ERK activation
Differential phosphoprofiles
(R,R’)-Fenoterol (S,R’)-Fenoterol
110. 118
control 1 µM (R,R)-Fenoterol 10 µM (S,R)-Fenoterol
Effects of fenoterol stereoiomers on ERK activation
Phosphoprofiler in HL-60 promyelocytes