Gi- and Gs-coupled GPCRs show different modes of G-protein binding.

More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants

Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations.

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A2A GPCR. While Na+ reinforces an inactive ensemble and a partial-agonist stabilized state, Ca2+ and Mg2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu

Efficient Iterative Algorithms for the Stochastic Finite Element Method with Application to Acoustic Scattering

In this study, we describe the algebraic computations required to implement the stochastic finite element method for solving problems in which uncertainty is restricted to right hand side data coming from forcing functions or boundary conditions. We show that the solution can be represented in a compact outer product form which leads to efficiencies in both work and storage, and we demonstrate that block iterative methods for algebraic systems with multiple right hand sides can be used to advantage to compute this solution. We also show how to generate a variety of statistical quantities from the computed solution. Finally, we examine the behavior of these statistical quantities in one setting derived from a model of acoustic scattering. UMIACS-TR-2002-10

Ernst Freund as Precursor of the Rational Study of Corporate Law

Gindis, David, Ernst Freund as Precursor of the Rational Study of Corporate Law (October 27, 2017). Journal of Institutional Economics, Forthcoming. Available at SSRN: https://ssrn.com/abstract=2905547, doi: https://dx.doi.org/10.2139/ssrn.2905547The rise of large business corporations in the late 19th century compelled many American observers to admit that the nature of the corporation had yet to be understood. Published in this context, Ernst Freund's little-known The Legal Nature of Corporations (1897) was an original attempt to come to terms with a new legal and economic reality. But it can also be described, to paraphrase Oliver Wendell Holmes, as the earliest example of the rational study of corporate law. The paper shows that Freund had the intuitions of an institutional economist, and engaged in what today would be called comparative institutional analysis. Remarkably, his argument that the corporate form secures property against insider defection and against outsiders anticipated recent work on entity shielding and capital lock-in, and can be read as an early contribution to what today would be called the theory of the firm.Peer reviewe

Genotypic and phenotypic analyses of a Pseudomonas aeruginosa chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains

Background Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized. Results This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to β-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4. Conclusions This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

The use of pure and impure placebo interventions in primary care - a qualitative approach

Background: Placebos play an important role in clinical trials and several surveys have shown that they are also common in daily practice. Previous research focused primarily on the frequency of placebo use in outpatient care. Our aim was to explore physicians' views on the use of placebos in daily practice, whereby distinction was made between pure placebos (substances with no pharmacological effect, e.g. sugar pills) and impure placebos (substances with pharmacological effect but not on the condition being treated, e.g. antibiotics in viral infections or vitamins). Methods: We performed semi-structured interviews with a sample of twelve primary care physicians (PCPs). The interview addressed individual definitions of a placebo, attitudes towards placebos and the participants' reasons for prescribing them. The interviews were transcribed and analysed using qualitative content analysis. Results: The definition of a placebo given by the majority of the PCPs in our study was one which actually only describes pure placebos. This definition, combined with the fact that most impure placebos were not regarded as placebos at all, means that most of the participating PCPs were not aware of the extent to which placebos are used in daily practice. The PCPs stated that they use placebos (both pure and impure) mainly in the case of non-severe diseases for which there was often no satisfactory somatic explanation. According to the PCPs, cases like this are often treated by complementary and alternative therapies and these, too, are associated with placebo effects. However, all PCPs felt that the ethical aspects of such treatment were unclear and they were unsure as to how to communicate the use of placebos to their patients. Most of them would appreciate ethical guidelines on how to deal with this issue. Conclusions: Many PCPs seem to be unaware that some of the drugs they prescribe are classified as impure placebos. Perceptions of effectiveness and doubts about the legal and ethical aspects of the use of placebos by PCPs may discourage their application. Dissemination of guidelines and consensus papers may be one approach, but it has to be acknowledged that the topic itself is in conflict with the PCPs' perception of themselves as professional and reliable physicians