Updated Perceptions on Polymer-Based Enhanced Oil Recovery toward High-Temperature High-Salinity Tolerance for Successful Field Applications in Carbonate Reservoirs.

The aging of the existing reservoirs makes the hydrocarbon extraction shift toward newer reserves, and harsh conditioned carbonates, which possess high temperature and high salinity (HTHS). Conventional polymer-flooding fails in these HTHS carbonates, due to precipitation, viscosity loss, and polymer adsorption. Therefore, to counteract these challenges, novel polymer-based cEOR alternatives employ optimized polymers, polymer-surfactant, and alkali-surfactant-polymer solutions along with hybrid methods, which have shown a potential to target the residual or remaining oils in carbonates. Consequently, we investigate novel polymers, viz., ATBS, Scleroglucan, NVP-based polymers, and hydrophobic associative polymers, along with bio-polymers. These selected polymers have shown low shear sensitivity, low adsorption, and robust thermal/salinity tolerance. Additionally, adding an alkali-surfactant to polymer solution produces a synergy effect of improved mobility control, wettability alteration, and interfacial-tension reduction. Thus, enhancing the displacement and sweep efficiencies. Moreover, low-salinity water can precondition high-salinity reservoirs before polymer flooding (hybrid method), to decrease polymer adsorption and viscosity loss. Thus, this paper is a reference for novel polymers, and their hybrid techniques, to improve polymer-based cEOR field applications under HTHS conditions in carbonates. Additionally, the recommendations can assist in project designs with reasonable costs and minimal environmental impact. The implication of this work will aid in supplementing the oil and gas energy sector growth, making a positive contribution to the Middle Eastern economy.

Vascular Inflammation in Cardiovascular Disease: Is Immune System Protective or Bystander?

Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Chronic atherosclerosis induced vascular inflammation and perturbation of lipid metabolism is believed to be a major cause of CVD. Interplay of innate and adaptive Immune system has been interwined with various risk factors associated with the initiation and progression of atherosclerosis in CVD. A large body of evidence indicates a correlation between immunity and atherosclerosis. Retention of plasma lipoproteins in arterial subendothelial wall triggers the T helper type 1 (Th1) cells and monocyte-derived macrophages to form atherosclerotic plaques. In the present review, we will discuss the pathogenesis of CVD in relation to atherosclerosis with a particular focus on pro-atherogenic role of immune cells. Recent findings have also suggested anti-atherogenic roles of different B cell subsets. Therapeutic approaches to target atherosclerosis risk factors have reduced the mortality, but a need exists for the novel therapies to treat arterial vascular inflammation. These insights into the immune pathogenesis of atherosclerosis can lead to new targeted therapeutics to abate cardiovascular mortality and morbidity.

BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes.

The type 1 angiotensin II (AngII) receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT1R was able to transactivate HER2, but not HER3. Mechanistically, AT1R-mediated ERK1/2 activation was dependent on Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/11. EGFR transactivation was also apparently independent of ß-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted AT1R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT1R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT1R and EGFR.

Integration of GPCR Signaling and Sorting from Very Early Endosomes via Opposing APPL1 Mechanisms.

Endocytic trafficking is a critical mechanism for cells to decode complex signaling pathways, including those activated by G-protein-coupled receptors (GPCRs). Heterogeneity in the endosomal network enables GPCR activity to be spatially restricted between early endosomes (EEs) and the recently discovered endosomal compartment, the very early endosome (VEE). However, the molecular machinery driving GPCR activity from the VEE is unknown. Using luteinizing hormone receptor (LHR) as a prototype GPCR for this compartment, along with additional VEE-localized GPCRs, we identify a role for the adaptor protein APPL1 in rapid recycling and endosomal cAMP signaling without impacting the EE-localized ß2-adrenergic receptor. LHR recycling is driven by receptor-mediated Gαs/cAMP signaling from the VEE and PKA-dependent phosphorylation of APPL1 at serine 410. Receptor/Gαs endosomal signaling is localized to microdomains of heterogeneous VEE populations and regulated by APPL1 phosphorylation. Our study uncovers a highly integrated inter-endosomal communication system enabling cells to tightly regulate spatially encoded signaling.

Recent advances in bioluminescence resonance energy transfer technologies to study GPCR heteromerization.

The field of G protein-coupled receptor (GPCR) research has undergone a transformation in recent years due to the notion of heteromerization. In order to progress our understanding of the functional implications of this phenomenon, as well as its applicability across the diversity of GPCR subtypes, we need to continually look to improve the technologies we use to evaluate protein-protein interactions in as near a physiological setting as possible. The bioluminescence resonance energy transfer (BRET) technology has been intimately associated with the study of GPCR-GPCR interactions for the past ten years, and over this period, both the tools and the methods of analysis have continually evolved. In this review, we highlight recent advances in the BRET technology and focus particularly on the drive to establish the specificity of GPCR heteromers.

The constitutively active V2 receptor mutants conferring NSIAD are weakly sensitive to agonist and antagonist regulation.

Patients having the nephrogenic syndrome of inappropriate antidiuresis present either the R137C or R137L V2 mutated receptor. While the clinical features have been characterized, the molecular mechanisms of functioning of these two mutants remain elusive. In the present study, we compare the pharmacological properties of R137C and R137L mutants with the wild-type and the V2 D136A receptor, the latter being reported as a highly constitutively active receptor. We have performed binding studies, second messenger measurements and BRET experiments in order to evaluate the affinities of the ligands, their agonist and antagonist properties and the ability of the receptors to recruit beta-arrestins, respectively. The R137C and R137L receptors exhibit small constitutive activities regarding the G(s) protein activation. In addition, these two mutants induce a constitutive beta-arrestin recruitment. Of interest, they also exhibit weak sensitivities to agonist and to inverse agonist in term of G(s) protein coupling and beta-arrestin recruitment. The small constitutive activities of the mutants and the weak regulation of their functioning by agonist suggest a poor ability of the antidiuretic function to be adapted to the external stimuli, giving to the environmental factors an importance which can explain some of the phenotypic variability in patients having NSIAD.

Two transmembrane Cys residues are involved in 5-HT4 receptor dimerization.

The 5-HT(4) receptor (5-HT(4)R) belongs to the G-protein-coupled receptor (GPCR) family and is of considerable interest for the development of new drugs to treat gastrointestinal diseases and memory disorders. The 5-HT(4)R exists as a constitutive dimer but its molecular determinants are still unknown. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer (BRET) techniques, we show here that 5-HT(4)R homodimerization but not 5-HT(4)R-beta(2) adrenergic receptor (beta(2)AR) heterodimerization is largely decreased under reducing conditions suggesting the participation of disulfide bonds in 5-HT(4)R dimerization. Molecular modeling and protein docking experiments identified four cysteine (Cys) residues potentially involved in the dimer interface through intramolecular or intermolecular disulfide bonds. We show that disulfide bridges between Cys112 and Cys145 located within TM3 and TM4, respectively, are of critical importance for 5-HT(4)R dimer formation. Our data suggest that two disulfide bridges between two transmembrane Cys residues are involved in the dimerization interface of a GPCR.

Real-time analysis of agonist-induced activation of protease-activated receptor 1/Galphai1 protein complex measured by bioluminescence resonance energy transfer in living cells.

G protein-coupled receptors transmit extracellular signals into the cells by activating heterotrimeric G proteins, a process that is often followed by receptor desensitization. Monitoring such a process in real time and in living cells will help better understand how G protein activation occurs. Energy transfer-based approaches [fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET)] were recently shown to be powerful methods to monitor the G protein-coupled receptors (GPCRs)-G protein association in living cells. Here, we used a BRET technique to monitor the coupling between the protease-activated receptor 1 (PAR1) and Galpha(i1) protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the Galpha(i1) protein expressed at a physiological level. This signal is insensitive to pertussis toxin (PTX) and probably reflects the preassembly of these two proteins. The BRET signal rapidly increases upon receptor activation in a PTX-sensitive manner. The BRET signal then returns to the basal level after few minutes. The desensitization of the BRET signal is concomitant with beta-arrestin-1 recruitment to the receptor, consistent with the known rapid desensitization of PARs. The agonist-induced BRET increase was dependent on the insertion site of fluorophores in proteins. Taken together, our results show that BRET between GPCRs and Galpha proteins can be used to monitor the receptor activation in real time and in living cells. Our data also revealed that PAR1 can be part of a preassembled complex with Galpha(i1) protein, resulting either from a direct interaction between these partners or from their colocalization in specific microdomains, and that receptor activation probably results in rearrangements within such complexes.

Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers.

G-protein-coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand-independent functions for these receptors. This emerging concept is connected with the observation that orphan GPCRs can heterodimerize with GPCRs that have identified ligands, and by so doing regulate the function of the latter. Pairing orphan GPCRs with their potential heterodimerization partners will have a major impact on our understanding of the extraordinary diversity offered by GPCR heterodimerization and, in addition, will constitute a novel strategy to elucidate the function of orphan receptors that needs to be added to the repertoire of 'deorphanization' strategies.

The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization.

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.

Are G protein-coupled receptor heterodimers of physiological relevance?--Focus on melatonin receptors.

In mammals, the circadian hormone melatonin targets two seven-transmembrane-spanning receptors, MT1 and MT2, of the G protein-coupled receptor (GPCR) super-family. Evidence accumulated over the last 15 yrs convincingly demonstrates that GPCRs, classically considered to function as monomers, are actually organized as homodimers and heterodimerize with other GPCR family members. These dimers are formed early in the biosynthetic pathway and remain stable throughout the entire life cycle. A growing number of observations demonstrate that GPCR oligomerization may occur in native tissues and may have important consequences on receptor function. The formation of MT1 and MT2 homodimers and MT1/MT2 heterodimers has been shown in heterologous expression systems at physiological expression levels. Formation of MT1/MT2 heterodimers remains to be shown in native tissues but is suggested by the documented co-expression of MT1 and MT2 in many melatonin-sensitive tissues, such as the hypothalamic suprachiasmatic nuclei, retina, arteries, and adipose tissue. Considering that multiple GPCRs are expressed simultaneously in most cells, the possible engagement into heterodimeric complexes has to be considered and taken into account for the interpretation of experimental data obtained from native tissues and knockout animals.

Reduced hippocampal MT2 melatonin receptor expression in Alzheimer's disease.

The aim of the present study was to identify the distribution of the second melatonin receptor (MT2) in the human hippocampus of elderly controls and Alzheimer's disease (AD) patients. This is the first report of immunohistochemical MT2 localization in the human hippocampus both in control and AD cases. The specificity of the MT2 antibody was ascertained by fluorescence microscopy using the anti-MT2 antibody in HEK 293 cells expressing recombinant MT2, in immunoblot experiments on membranes from MT2 expressing cells, and, finally, by immunoprecipitation experiments of the native MT2. MT2 immunoreactivity was studied in the hippocampus of 16 elderly control and 16 AD cases. In controls, MT2 was localized in pyramidal neurons of the hippocampal subfields CA1-4 and in some granular neurons of the stratum granulosum. The overall intensity of the MT2 staining was distinctly decreased in AD cases. The results indicate that MT2 may be involved in mediating the effects of melatonin in the human hippocampus, and this mechanism may be heavily impaired in AD.

Preferential formation of MT1/MT2 melatonin receptor heterodimers with distinct ligand interaction properties compared with MT2 homodimers.

Heterodimerization has been documented for several members of the G protein-coupled receptor (GPCR) superfamily, including the closely related MT(1) and MT(2) melatonin receptors. However, the relative abundance of hetero-versus homodimers and the specific properties, which can be attributed to each form, are difficult to determine. Using a bioluminescence resonance energy transfer (BRET) donor saturation assay, we show that half-maximal MT(1)/MT(2) heterodimer formation is reached for expression levels as low as approximately 4000 receptors per cell. The relative propensity of MT(1) homodimer and MT(1)/MT(2) heterodimer formation are similar, whereas that for the MT(2) homodimer formation is 3- to 4-fold lower. These data indicate that both the relative expression level of each receptor isoform and the affinities between monomers may determine the actual proportion of homo- and heterodimers. The specific interaction of ligands with the MT(1)/MT(2) heterodimer was studied using a BRET-based assay as a readout for the conformational changes of the heterodimer. An MT(1)/MT(2) heterodimer-specific profile and ligands selective for the MT(1)/MT(2) heterodimer compared with the MT(2) homodimer could be identified. Classic radioligand binding and BRET studies suggest that heterodimers contain two functional ligand binding sites that maintain their respective selectivity for MT(1) and MT(2) ligands. Occupation of either binding site is sufficient to induce a conformational change within the heterodimer. Taken together, these results show that the probability of GPCR heterodimer formation may be equal to or even higher than that of the corresponding homodimers and that specific properties of heterodimers can be revealed using a BRET-based ligand/receptor interaction assay.

Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis.

G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from several studies suggesting that GPCRs can form both homo- and heterodimers. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) approaches, we established that the vasopressin V1a, V2, and the oxytocin receptors exist as homo- and hetero-dimers in transfected human embryonic kidney 293T cells. Each receptor protomer had a similar propensity to form homo- and heterodimers, indicating that their relative expression levels may determine the homo-/heterodimer ratio. The finding that immature forms of the receptor can be immunoprecipitated as homo- and heterodimers and the detection by BRET of such oligomer in endoplasmic reticulum-enriched fractions suggest that the oligomerization processes take place early during biosynthesis. Treatment with agonists or antagonists did not modify the BRET among any of the vasopressin and oxytocin receptor pairs studied, indicating that the dimerization state of the receptors is not regulated by ligand binding once they have reached the cell surface. Taken together, these results strongly support the notion that GPCR dimerization is a constitutive process.

Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer.

Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo- and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view.