On two-echelon inventory systems with Poisson demand and lost sales

We derive approximations for the service levels of two-echelon inventory systems with lost sales and Poisson demand. Our method is simple and accurate for a very broad range of problem instances, including cases with both high and low service levels. In contrast, existing methods only perform well for limited problem settings, or under restrictive assumptions.\u

M3 muscarinic acetylcholine receptor facilitates the endocytosis of mu opioid receptor mediated by morphine independently of the formation of heteromeric complexes

Morphine inefficiency to induce the internalization of mu opioid (MOP) receptors observed in numerous experimental models constitutes a paradigm of G-protein coupled receptor (GPCR) functional selectivity. We recently described that activation of Gαq/11 proteins through 5-HT2A serotonin receptors co-expressed in the same cells facilitates MOP receptor endocytosis promoted by morphine. In order to explore whether a different Gαq/11 coupled GPCR would emulate this effect, a double stable Flp-In T-REx HEK293 cell line permanently expressing MOP-YFP receptors along with FLAG-M3-Cerulean receptors expressed in an inducible manner was generated. Fluorescence microscopy examination of these cells revealed a co-distribution of both receptors mainly compartmentalized in plasma membrane. Concurrent stimulation with carbachol and morphine promoted MOP receptor internalization, desensitization and down-regulation and this facilitation was not dependent on PKC activation. Co-immunoprecipitation experiments demonstrated that FLAG-M3-Cerulean/MOP-YFP receptors interact forming heteromeric complexes in a time depending manner, i.e. the strongest interaction was detected after 96h of FLAG-M3-Cerulean induced expression. Under these experimental conditions, treatment of cells with carbachol plus morphine resulted in the internalization of both receptors within separated endocytic vesicles as visualized by confocal microscopy. This trafficking segregation observed for FLAG-M3-Cerulean and MOP-YFP receptors upon agonist stimulation suggests that this protein-protein interaction presents temporal and dynamic properties. Moreover, MOP-YFP receptor internalization facilitated by FLAG-M3-Cerulean receptors is independent of the constitution of heteromeric complexes. [Abstract copyright: Copyright © 2017. Published by Elsevier Inc.

Distinct agonist regulation of muscarinic acetylcholine M2-M3 heteromers and their corresponding homomers

Each subtype of the muscarinic receptor family of G protein-coupled receptors is activated by similar concentrations of the neurotransmitter acetylcholine or closely related synthetic analogs such as carbachol. However, pharmacological selectivity can be generated by the introduction of a pair of mutations to produce Receptor Activated Solely by Synthetic Ligand (RASSL) forms of muscarinic receptors. These display loss of potency for acetylcholine/carbachol alongside a concurrent gain in potency for the ligand clozapine N-oxide. Co-expression of a form of wild type human M2 and a RASSL variant of the human M3 receptor resulted in concurrent detection of each of M2-M2 and M3-M3 homomers alongside M2-M3 heteromers at the surface of stably transfected Flp-InTM T-RExTM 293 cells. In this setting occupancy of the receptors with a muscarinic antagonist was without detectable effect on any of the muscarinic oligomers. However, selective agonist occupancy of the M2 receptor resulted in enhanced M2-M2 homomer interactions but decreased M2-M3 heteromer interactions. By contrast, selective activation of the M3 RASSL receptor did not significantly alter either M3-M3 homomer or M2-M3 heteromer interactions. Selectively targeting closely related receptor oligomers may provide novel therapeutic opportunities

Muscarinic receptor oligomerization

G protein-coupled receptors (GPCRs) have been classically described as monomeric entities that function by binding in a 1:1 stoichiometric ratio to both ligand and downstream signalling proteins. However, in recent years, a growing number of studies has supported the hypothesis that these receptors can interact to form dimers and higher order oligomers although the molecular basis for these interactions, the overall quaternary arrangements and the functional importance of GPCR oligomerization remain topics of intense speculation. Muscarinic acetylcholine receptors belong to class A of the GPCR family. Each muscarinic receptor subtype has its own particular distribution throughout the central and peripheral nervous systems. In the central nervous system, muscarinic receptors regulate several sensory, cognitive, and motor functions while, in the peripheral nervous system, they are involved in the regulation of heart rate, stimulation of glandular secretion and smooth muscle contraction. Muscarinic acetylcholine receptors have long been used as a model for the study of GPCR structure and function and to address aspects of GPCR dimerization using a broad range of approaches. In this review, the prevailing knowledge regarding the quaternary arrangement for the various muscarinic acetylcholine receptors has been summarized by discussing work ranging from initial results obtained using more traditional biochemical approaches to those generated with more modern biophysical techniques

Ligand regulation of the quaternary organization of cell surface M3 muscarinic acetylcholine receptors analyzed by fluorescence resonance energy transfer (FRET) imaging and homogenous time-resolved FRET

Flp-In T-REx 293 cells expressing a wild type human M muscarinic acetylcholine receptor construct constitutively and able to express a Receptor Activated Solely by Synthetic Ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine-N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET-donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogenous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M receptor expressed stably in Flp-In TREx 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used

Defining the organizational structure of dopamine and muscarninic acetylcholine receptors

No abstract available

Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signalling

G protein-coupled receptors (GPCRs) can initiate intracellular signalling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signalling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells, together with the elimination of receptor phosphorylation sites, to define the relative contribution of G proteins, arrestins and receptor phosphorylation to the signalling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular [Ca2+] in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signalling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signalling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms whilst a substantially enhanced ERK1/2-response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signalling research and employ these cells to reveal a previously unappreciated interplay of signalling pathways where receptor phosphorylation can impact on ERK1/2 signalling through a mechanism that is likely independent of arrestins

Diseño de una metodología para estimar los consumos de la red de distribución de agua potable de Barquisimeto, Venezuela

En el presente trabajo se desarrolló una metodología para la estimación de consumos de una red de distribución de agua potable, cuando no se cuenta con equipos de medición instalados en la red para la cuantificación de los mismos. Para tal fin se analizó la red de distribución de agua potable de la ciudad de Barquisimeto, que abastece a una población de alrededor de un millón de habitantes. Dicha metodología se plantea en dos etapas, la primera de ellas destinada a la estimación de los consumos a través de variables que son también utilizadas en la estimación de caudales de aguas residuales como lo son: coeficientes de densidad poblacional y de superficie servida, y la otra, para la validación de los valores de presión obtenidos a través de la simulación al compararlos con los valores de presión medidos en algunos puntos de la red. Los resultados obtenidos indican que la calibración de la red, en cuanto al promedio de presiones, es satisfactorio, sin embargo el análisis puntual demuestra que se debe calibrar cada sector por separado para de esta forma lograr que la curva de comportamiento de presiones pueda ajustarse correctamente

Distinct phosphorylation clusters determines the signalling outcome of the free fatty acid receptor FFA4/GPR120

It is established that long-chain free fatty acids including ω-3 fatty acids mediate an array of biological responses through members of the free fatty acid receptor family, which includes FFA4. However, the signalling mechanisms and modes of regulation of this receptor class remain unclear. Here we employ mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C terminal tail, designated cluster 1 (Thr347, Thr349 and Ser350) and cluster 2 (Ser357 and Ser361). Mutation of these phospho-acceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to ERK1/2 activation. Rather an inhibitor of Gq/11 proteins completely prevented receptor signalling to ERK1/2. In contrast, the recruitment of arrestin 3, receptor internalization and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signalling was extended further by selective mutations of the phospho-acceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phospho-acceptor sites within cluster 1 had no effect on receptor internalization and a less extensive effect on arrestin 3 recruitment, but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signalling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signalling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C-terminus of the receptor