A novel nanobody-based bio-assay using functional complementation of a split nanoluciferase to monitor Mu- opioid receptor activation

The Mu opioid receptor (MOR) has been the subject of intense research over the past decades, especially in the field of analgesic therapeutics. It is the primary target for both clinical and recreational opioids. Recently, camelid-derived nanobodies have received significant attention due to their applicability in stabilizing the crystal structure of activated MOR, via specific recognition of and binding to the active receptor conformation. In the present study, we developed and applied a novel bio-assay to monitor MOR activation, utilizing intracellular expression of one such nanobody, Nb39. The principle of functional complementation of a split nanoluciferase was used to assess recruitment of Nb39 to MOR, following activation by a set of five synthetic opioids. The obtained pharmacological parameters—negative logarithm of EC50 (pEC50, as a measure of potency) and maximal response provoked by a ligand (Emax, as a measure of efficacy; relative to hydromorphone)—were compared with those obtained using a G protein recruitment assay, in which a mini-Gi protein (engineered GTPase domain of Gαi subunit) is recruited to activated MOR. Similar EC50 but distinct Emax values were obtained with both bio-assays, with lower Emax values for the Nbbased bio-assay. Both bio-assays may assist to gain better insight into activation of the MOR

A comparative study of verbal IQ, performance IQ and verbal IQ-performance IQ disparity among Turner syndrome patients and patients with primary amenorrhoea due to other aetiologies

Background: Assessing disparity in IQ is important in selecting an occupation and thus helping people lead a productive life. In Turner syndrome patients, this shall be more important as assessment of verbal IQ- and performance IQ disparity could be used in helping them select an occupation so that productivity and quality of life is not grossly compromised.Methods: Based on karyotyping, 30 patients with turner syndrome and 30 patients with primary amenorrhoea due to other aetiologies were selected for the study. Cytogenetic analysis was done for every case using G-banding technique. Assessment of intellectual functions was done using Wechsler Adult Intelligence Scale (WAIS).Results: Mean value of PIQ of X0 (turner) was 74.67 and that of XX (other amenorrhoea patients) was 90.30. Mean value of VIQ of X0 (turner) was 93.67 and that of XX (other amenorrhoea patients) was 93.60. Mean value of VIQ and PIQ disparity of X0 (turner) was 18.67 and that of XX (other amenorrhoea patients) was 3.Conclusions: Turner syndrome patients have high VIQ-PIQ disparity when compared to other primary amenorrhoea patients

Luminescence- and fluorescence-based complementation assays to screen for GPCR oligomerization : current state of the art

G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR-GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein-protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR-GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR-GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays

Simultaneous removal of Co, Cu, and Cr from water by electrocoagulation

This study provides an electrocoagulation process for the removal of metals such as cobalt, copper, and chromium from water using magnesium as anode and galvanized iron as cathode. The various parameters like pH, current density, temperature, and inter electrode distance on the removal efficiency of metals were studied. The results showed that maximum removal efficiency was achieved for cobalt, copper, and chromium with magnesium as anode and galvanized iron as cathode at a current density of 0.025 A dm À2 at pH 7.0. First-and second-order rate equations were applied to study adsorption kinetics. The adsorption process follows second-order kinetics model with good correlation. The Langmuir and Freundlich adsorption isotherm models were studied using the experimental data. The Langmuir adsorption isotherm favors monolayer coverage of adsorbed molecules for the adsorption of cobalt, copper, and chromium. Temperature studies showed that adsorption was endothermic and spontaneous in nature

Design, synthesis, and biological evaluation of bivalent ligands targeting dopamine D2-like receptors and the μ-opioid receptor

Currently, there is mounting evidence that intermolecular receptor-receptor interactions may result in altered receptor recognition, pharmacology and signaling. Heterobivalent ligands have been proven useful as molecular probes for confirming and targeting heteromeric receptors. This report describes the design and synthesis of novel heterobivalent ligands for dopamine D-2-like receptors (D-2-likeR) and the -opioid receptor (OR) and their evaluation using ligand binding and functional assays. Interestingly, we identified a potent bivalent ligand that contains a short 18-atom linker and combines good potency with high efficacy both in -arrestin2 recruitment for OR and MAPK-P for D4R. Furthermore, this compound was characterized by a biphasic competition binding curve for the D4R-OR heterodimer, indicative of a bivalent binding mode. As this compound possibly bridges the D4R-OR heterodimer, it could be used as a pharmacological tool to further investigate the interactions of D4R and OR

On the balance of D2R-MOR and D4R-MOR in the dorsal and ventral striatum. Putative link to morphine dependence and addiction

Este resumen forma parte de la conferencia invitada (título del resumen) del Symposium S19 - Understanding the role of GPCR heteroreceptor complexes and their adaptor proteins in the neuronal networks of the brain in health and mental disordersThe widespread distribution of heteroreceptor complexes with allosteric receptor-receptor interactions in the CNS represents a novel integrative molecular mechanism in the plasma membrane of neurons and glial cells. It was proposed that they form the molecular basis for learning and short-and long-term memories. This is also true for drug memories formed during the development of substance use disorders like morphine and cocaine use disorders. Herein, we discuss and propose how an increase in opioid heteroreceptor complexes, containing MOR-DOR, MOR-D4R and MOR-D2R, and their balance with each other and A2AR-D2R complexes in the striatal-pallidal enkephalin positive GABA antireward neurons, may represent markers for development of morphine use disorders. We suggest that increased formation of MOR-DOR complexes takes place in the striatal-pallidal enkephalin positive GABA antireward neurons after chronic morphine treatment in part through recruitment of MOR from the MOR-D2R and/or MOR-D4R complexes due to the possibility that MOR upon morphine treatment can develop a higher affinity for DOR. As a result, increased numbers of D2R monomers/homomers in these neurons become free to interact with the A2ARs found in high densities within such neurons. Increased numbers of A2AR-D2R heteroreceptor complexes are formed and contribute to enhanced firing of these antireward neurons due to loss of inhibitory D2R protomer signaling which finally leads to the development of morphine use disorder. Altogether, we propose that these altered complexes could be pharmacological target to modulate the reward and the development of substance use disorders.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Defluoridation of drinking water using a new flow column-electrocoagulation reactor (FCER) - Experimental, statistical, and economic approach.

A new batch, flow column electrocoagulation reactor (FCER) that utilises a perforated plate flow column as a mixer has been used to remove fluoride from drinking water. A comprehensive study has been carried out to assess its performance. The efficiency of fluoride removal (R%) as a function of key operational parameters such as initial pH, detention time (t), current density (CD), inter-electrode distance (ID) and initial concentration (C0) has been examined and an empirical model has been developed. A scanning electron microscopy (SEM) investigation of the influence of the EC process on morphology of the surface of the aluminium electrodes, showed the erosion caused by aluminium loss. A preliminary estimation of the reactor's operating cost is suggested, allowing for the energy from recycling of hydrogen gas hydrogen gas produced amount. The results obtained showed that 98% of fluoride was removed within 25 min of electrolysis at pH of 6, ID of 5 mm, and CD of 2 mA/cm(2). The general relationship between fluoride removal and operating parameters could be described by a linear model with R(2) of 0.823. The contribution of the operating parameters to the suggested model followed the order: t > CD > C0 > ID > pH. The SEM images obtained showed that, after the EC process, the surface of the anodes, became non-uniform with a large number of irregularities due to the generation of aluminium hydroxides. It is suggested that these do not materially affect the performance. A provisional estimate of the operating cost was 0.379 US $/m(3). Additionally, it has been found that 0.6 kW/m(3) is potentially recoverable from the H2 gas

Iron removal, energy consumption and operating cost of electrocoagulation of drinking water using a new flow column reactor

The goal of this project was to remove iron from drinking water using a new electrocoagulation (EC) cell. In this research, a flow column has been employed in the designing of a new electrocoagulation reactor (FCER) to achieve the planned target. Where, the water being treated flows through the perforated disc electrodes, thereby effectively mixing and aerating the water being treated. As a result, the stirring and aerating devices that until now have been widely used in the electrocoagulation reactors are unnecessary. The obtained results indicated that FCER reduced the iron concentration from 20 to 0.3 mg/L within 20 min of electrolysis at initial pH of 6, inter-electrode distance (ID) of 5 mm, current density (CD) of 1.5 mA/cm2, and minimum operating cost of 0.22 US $/m3. Additionally, it was found that FCER produces H2 gas enough to generate energy of 10.14 kW/m3. Statistically, it was found that the relationship between iron removal and operating parameters could be modelled with R2 of 0.86, and the influence of operating parameters on iron removal followed the order: C0>t>CD>pH. Finally, the SEM (scanning electron microscopy) images showed a large number of irregularities on the surface of anode due to the generation of aluminium hydroxides

Electrocoagulation as a green technology for phosphate removal from River water

The current study investigates the removal of phosphate from water using a new baffle plates aluminium-based electrochemical cell (PBPR) taking consideration the influence of key operating parameters. This new cell utilises perforated baffle plates as a water mixer rather than magnetic stirrers that require extra power to work. As this unit is new, a comprehensive study has been carried to assess it performance. This study also includes preliminary estimates of the reactor’s operating costs, the amount of H2 gas produced and the yieldable energy from it. SEM (scanning electron microscope) was used to investigate the influence of the electrocoagulation process on the morphology of the surface of aluminium electrodes, and an empirical model developed to reproduce the phosphate removal process. The results showed that 99% of phosphate was removed within 60 minutes of electrolysis at an initial pH (ipH) of 6, inter-electrode distance (ID) of 0.5 cm, current density (J) of 6 mA/cm2, initial concentration of phosphate (IC) of 100 mg/L, and minimum operating cost of 0.503 US $/m3. The electrochemical cell produced enough H2 gas to generate 4.34 kWh/m3 of power. Statistically, it was proved that the influence of the operating parameters on phosphate removal could be modelled with an R2 of 0.882, the influence of these operating parameters on phosphate removal following the order: t>J>IC>ipH >ID. Finally, SEM images showed that after several electrolysing runs, the Al anode became rough and nonuniform which could be related to the production of aluminium hydroxides