Analysis of Ligand Bias in Functional Studies Involving the Allosteric Modulation of G Protein- Coupled Receptors

Introduction The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants. Methods Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands – including inverse agonists – and the efficacy of the unoccupied receptor (i.e., constitutive activity). Results and discussion We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor-signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe-dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration–response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor

Analysis of Functional Responses at G Protein-Coupled Receptors: Estimation of Relative Affinity Constants for the Inactive Receptor Sstate

We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (Kobs) and efficacy (fraction of ligand-receptor complex in the active state, ε) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, εi). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus. The latter function includes Kobs and the parameter τ, which is proportional to ε. We introduce the parameter τi, which is proportional to εi. We have previously shown that the product, Kobsτ, of one agonist, expressed relative to that of another (intrinsic relative activity, RAi), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, Kobsτi, of one agonist, expressed relative to that of another (intrinsic relative inactivity, RIi), is a relative measure of the affinity constant for the inactive state of the receptor. We use computer simulation techniques to verify our analysis and apply our method to the analysis of published data on agonist activity at the M3 muscarinic receptor. Our method should have widespread application in the analysis of agonist bias in drug discovery programs and in the estimation of a more fundamental relative measure of efficacy (RAi/RIi)

A Simple Method for Estimation of Agonist Activity at Receptor Subtypes: Comparison of Native and Cloned M3 Muscarinic Receptors in Guinea Pig Ileum and Transfected Cells

We describe a simple method for calculating the pharmacological activity of an agonist (A) relative to a standard agonist (S) using only the concentration-response curves of the two agonists. In most situations, we show that the product of the ratios of maximal responses (E max − A/E max − S) and potencies (EC50 − S/EC50 − A) is equivalent to the product of the affinity and intrinsic efficacy of A expressed relative to that of S. We refer to this term as the IRA value of A. In a cooperative system where the concentration-response curve of the standard agonist is steep and that of the test agonist is flatter with a lower maximal response, the simple calculation of IRA described above underestimates agonist activity; however, we also describe a means of correcting the IRA in this situation. We have validated our analysis with modeling techniques and have shown experimentally that the IRA values of muscarinic agonists for stimulating contractions in the guinea pig ileum (M3 response) are in excellent agreement with those measured in the phosphoinositide assay on Chinese hamster ovary cells expressing the M3 muscarinic receptor

Muscarinic Agonist-Mediated Heterologous Desensitization in Isolated Ileum Requires Activation of Both Muscarinic M2 and M3 Receptors

We investigated the subtypes of the muscarinic receptor mediating short-term heterologous desensitization in the isolated ileum. Treatment of the ileum from C57BL/6 mice with acetylcholine (30 μM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F2α (PGF2α) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7- and 3-fold increases in the EC50 values of the agonists, respectively, with no significant effect on the maximal response. The subsensitivity to PGF2α was prevented in both M2 and M3 muscarinic receptor knockout mice. Similarly, the subsensitivity to oxotremorine-M was prevented in M2 knockout mice. Acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum was inhibited by both M2- and M3-selective muscarinic antagonists with high potency, although careful analysis of the data suggested behavior more consistent with an M2 antagonistic profile. Modeling studies showed that the competitive antagonism of response contingent upon activation of two receptor subtypes should exhibit a pharmacological profile similar to that of the least sensitive signaling pathway. Our results demonstrate that muscarinic agonist-mediated short-term heterologous desensitization of intestinal smooth muscle is contingent upon activation of both M2 and M3 muscarinic receptors and that activation of either receptor by itself is insufficient to cause desensitization

Impact of submarine groundwater discharge on biogeochemistry and microbial communities in pockmarks

The impact of submarine groundwater discharge (SGD) on coastal sea biogeochemistry has been demonstrated in many recent studies. However, only a few studies have integrated biogeochemical and microbiological analyses, especially at sites with pockmarks of different degrees of groundwater influence. This study investigated biogeochemical processes and microbial community structure in sediment cores from three pockmarks in Hanko, Finland, in the northern Baltic Sea. Pockmark data were supplemented by groundwater and seawater measurements. Two active pockmarks showed SGD rates of 0.02 cm d-1 and 0.31 cm d-1, respectively, based on porewater Cl- profiles, while a third pockmark had no SGD influence. Reactive transport modelling (RTM) established that the porewater systems of these active pockmarks are dominated by advection, resulting in the focusing of biogeochemical reactions and the microbial community into a thin zone at the sediment surface. The advection further reduces the accumulation of organic matter in the surface sediments, resulting in the absence of a sulfate-methane transition zone (SMTZ) at these pockmarks. Furthermore, the RTM estimated low rates of consumption of SO42-, and low rates of production of CH4, NH4+, DIC at the active pockmarks. Archaeal communities in the active pockmarks were dominated by ammonia-oxidizing archaea of predominantly groundwater origin. In contrast, at the inactive pockmark, the lack of SGD has permitted rapid deposition of organic-rich mud. The porewater system in the inactive pockmark is dominated by diffusion, leading to orders of magnitude higher metabolite concentrations at depth compared to the active pockmarks. The biogeochemical environment in the inactive pockmark resembles typical organic-rich mud seafloor in the area, with sulphate reduction and methanogenesis dominating organic matter remineralization. Accordingly, methanogens dominate the archaeal community, whereas sulfate reducers dominate the bacterial community. RTM results suggest that sulfate-mediated anaerobic oxidation of methane (S-AOM) also occurs at this site. Although depth-integrated fluxes of SO42-, CH4, NH4, DIC at the inactive pockmark are orders of magnitude higher compared to the active pockmarks, processes at the inactive pockmark represent internal recycling in the coastal sea. Fluxes observed at the active pockmarks, although comparatively small in magnitude, are partly influenced by external inputs to the sea through SGD. Hence, effluxes across the sediment-water interface at these sites partly represent direct external fluxes to the marine environment, in addition to diagenetic recycling at the benthic interface. The study highlights that SGD can result in significant spatial heterogeneity of biogeochemical processes and microbial community structure in the coastal zone, and that the overall effects of SGD and associated solute fluxes at an SGD site are a function of the number of pockmarks, the rate of SGD, and the ratio of active to inactive pockmarks.Peer reviewe

A review of the stable isotope bio-geochemistry of the global silicon cycle and its associated trace elements

Silicon (Si) is the second most abundant element in the Earth's crust and is an important nutrient in the ocean. The global Si cycle plays a critical role in regulating primary productivity and carbon cycling on the continents and in the oceans. Development of the analytical tools used to study the sources, sinks, and fluxes of the global Si cycle (e.g., elemental and stable isotope ratio data for Ge, Si, Zn, etc.) have recently led to major advances in our understanding of the mechanisms and processes that constrain the cycling of Si in the modern environment and in the past. Here, we provide background on the geochemical tools that are available for studying the Si cycle and highlight our current understanding of the marine, freshwater and terrestrial systems. We place emphasis on the geochemistry (e.g., Al/Si, Ge/Si, Zn/Si, δ13C, δ15N, δ18O, δ30Si) of dissolved and biogenic Si, present case studies, such as the Silicic Acid Leakage Hypothesis, and discuss challenges associated with the development of these environmental proxies for the global Si cycle. We also discuss how each system within the global Si cycle might change over time (i.e., sources, sinks, and processes) and the potential technical and conceptual limitations that need to be considered for future studies.The work by JS was supported by the “Laboratoire d’Excellence” LabexMER (ANR-10-LABX-19) and co-funded by a grant from the French government under the program “Investissements d’Avenir,” and by a grant from the Regional Council of Brittany (SAD programme). DJC was partially supported by the Knut and Alice Wallenberg Foundation (KAW Wallenberg Scholar) and the Swedish Research Council. This review article has benefited from funding by the European Union Seventh Framework Programme under grant agreement n◦294146 (project MuSiCC, Marie Curie CIG to DC). GdS is supported by a Marie Skłodowska-Curie Research Fellowship under EU Horizon2020 (GA #708407). JuD was supported by the American Chemical Society Petroleum Research Fund (Grant # 53798-DNI2). CE acknowledges financial support by the Institute for Chemistry and Biology of the Marine Environment (Oldenburg, Germany) and the Max Planck Institute for Marine Microbiology (Bremen, Germany). KH is funded by The Royal Society (UF120084) and the European Research Council (ERC-2015-StG - 678371_ICY-LAB). PG acknowledges funding by the Collaborative Research Centre 754 “ClimateBiogeochemistry interactions in the Tropical Ocean” (www. sfb754.de), supported by the Deutsche Forschungsgemeinschaft (DFG)

Worldwide variations in artificial skyglow

Despite constituting a widespread and significant environmental change, understanding of artificial nighttime skyglow is extremely limited. Until now, published monitoring studies have been local or regional in scope, and typically of short duration. In this first major international compilation of monitoring data we answer several key questions about skyglow properties. Skyglow is observed to vary over four orders of magnitude, a range hundreds of times larger than was the case before artificial light. Nearly all of the study sites were polluted by artificial light. A non-linear relationship is observed between the sky brightness on clear and overcast nights, with a change in behavior near the rural to urban landuse transition. Overcast skies ranged from a third darker to almost 18 times brighter than clear. Clear sky radiances estimated by the World Atlas of Artificial Night Sky Brightness were found to be overestimated by ~25%; our dataset will play an important role in the calibration and ground truthing of future skyglow models. Most of the brightly lit sites darkened as the night progressed, typically by ~5% per hour. The great variation in skyglow radiance observed from site-to-site and with changing meteorological conditions underlines the need for a long-term international monitoring program

Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs

Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type

Galaxy And Mass Assembly (GAMA): The effect of galaxy group environment on active galactic nuclei

In galaxy clusters, efficiently accreting active galactic nuclei (AGN) are preferentially located in the infall regions of the cluster projected phase-space, and are rarely found in the cluster core. This has been attributed to both an increase in triggering opportunities for infalling galaxies, and a reduction of those mechanisms in the hot, virialised, cluster core. Exploiting the depth and completeness (98 per cent at r9.9 in 695 groups with 11.53≤log 10 (M 200 /M ⊙ )≤14.56 at z13.5 , AGN are preferentially found in the infalling galaxy population with 3.6σ confidence. At lower halo masses we observe no difference in AGN fraction between core and infalling galaxies. These observations support a model where a reduced number of low-speed interactions, ram pressure stripping and intra-group/cluster medium temperature, the dominance of which increase with halo mass, work to inhibit AGN in the cores of groups and clusters with log 10 (M 200 /M ⊙ )>13.5 , but do not significantly affect nuclear activity in cores of less massive structures