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The signalling of the D 2 receptor (D 2 R), a G protein-coupled receptor (GPCR), is a complex process consisting of various components. For the screening of D 2 R ligands, methods quantifying distinct second messengers such as cAMP or the interaction of the receptor with β-arrestin, are commonly employed. In contrast, a label-free biosensor technology like dynamic mass redistribution (DMR), where it is mostly unknown how the individual signalling pathways contribute to the DMR signal, provides a holistic readout of the complex cellular response. In this study, we report the successful application of the DMR technology to CHO-K1 cells stably expressing the human dopamine D 2long receptor. In real-time kinetic experiments, studies of D 2 R reference compounds yielded results for agonists and antagonists that were consistent with those obtained by conventional methods and also allowed a discrimination between partial and full agonists. Furthermore, investigations on the signalling pathway in CHO-K1 hD 2long R cells identified the Gα i/o protein as the main proximal trigger of the observed DMR response. The present study has shown that the DMR technology is a valuable method for the characterisation of putative new ligands and, due to its label-free nature, suggests its use for deorphanisation studies of GPCRs.

The Asian Citrus Psyllid (ACP), Diaphorina citri, can transmit the bacterium Candidatus Liberibacter while feeding on citrus flush shoots. This bacterium causes Huanglongbing (HLB), a major disease of citrus cultivation worldwide necessitating the development of new tools for ACP surveillance and control. The olfactory system of ACP is sensitive to variety of odorants released by citrus plants and offers an opportunity to develop new attractants and repellents. In this study, we performed single-unit electrophysiology to identify odorants that are strong activators, inhibitors, and prolonged activators of ACP odorant receptor neurons (ORNs). We identified a suite of odorants that activated the ORNs with high specificity and sensitivity, which may be useful in eliciting behavior such as attraction. In separate experiments, we also identified odorants that evoked prolonged ORN responses and antagonistic odorants able to suppress neuronal responses to activators, both of which can be useful in lowering attraction to hosts. In field trials, we tested the electrophysiologically identified activating odorants and identified a 3-odor blend that enhances trap catches by ∼230%. These findings provide a set of odorants that can be used to develop affordable and safe odor-based surveillance and masking strategies for this dangerous pest insect.

Health degree programs provide opportunities to reduce disparities in care for LGBTQ patients by exposing students to LGBTQ communities and current health issues. However, LGBTQ content is mostly absent from medical school curricula. This mixed method assessment study, conducted during the 2018 to 2019 academic year, examined the feasibility of implementing a medical student journal club focused specifically on LGBTQ health issues as a complementary training tool to support efforts to create an inclusive educational environment. Compared to the pre-test, mean response scores increased for most of the parameters including familiarity with LGBTQ healthcare issues, confidence in the ability to identify harmful medical provider practices, and reading and assessing scientific literature. Qualitative data showed increased confidence, comfort and knowledge about LGBTQ health barriers. This study offers a framework for using a journal club to provide an effective platform for enhancing students’ LGBTQ cultural humility and research literacy.

Background The Mind, Exercise, Nutrition … Do it! (MEND) childhood obesity intervention was implemented in British Columbia (B.C.), Canada from April 2013 to June 2017. The study objective was: a) to describe and explore program reach, attendance, satisfaction, acceptability, fidelity, and facilitators and challenges during scale-up and implementation of MEND in B.C. while b) monitoring program effectiveness in improving children’s body mass index (BMI) z-score, waist circumference, dietary and physical activity behaviours, and psychological well-being. Methods This prospective, pragmatic implementation evaluation (Hybrid Type 3 design) recruited families with children and adolescents aged 7–13 with a BMI ≥ 85th percentile for age and sex. The 10-week MEND B.C. program was delivered in 27 sites, throughout all five B.C. health regions (Northern, Interior, Island, Fraser, and Vancouver Coastal) over 4 years. Families attended two weekly in-person group sessions aimed to increase physical activity and promote healthy eating. BMI z-score and waist circumference were measured at baseline and follow-up. Dietary and physical activity behaviours and psychological well-being were measured using validated questionnaires. A mixed-method approach was used to collect and analyze the data. Results One hundred thirty-six MEND B.C. programs were delivered over 4 years. The program reached 987 eligible participants. 755 (76.5%) children and adolescents completed the program. The average program attendance was 81.5%. Parents reported the program content was easy to understand, culturally suitable, respectful of family’s financial situation, and provided adequate information to build a healthy lifestyle. Children achieved significant positive changes across all four evaluation years in BMI z-score ( d  = − 0.13), nutrition behaviours ( d  = 0.64), physical activity levels ( d  = 0.30), hours of screen time per week ( d  = − 0.38) and emotional distress ( d  = − 0.21). Challenges to continued program implementation included: recruitment, resource requirement for implementation, and the need to tailor the program locally to be more flexible and culturally relevant. Conclusions The program reached a broad demographic of children and adolescents in B.C. Families were highly satisfied with the program delivery. MEND. B.C. at scale was effective across all four evaluation years in improving BMI z-score, lifestyle behaviours and psychological well-being among children. Future interventions need to explore strategies to enhance program delivery flexibility.

The signalling of the D receptor (D R), a G protein-coupled receptor (GPCR), is a complex process consisting of various components. For the screening of D R ligands, methods quantifying distinct second messengers such as cAMP or the interaction of the receptor with β-arrestin, are commonly employed. In contrast, a label-free biosensor technology like dynamic mass redistribution (DMR), where it is mostly unknown how the individual signalling pathways contribute to the DMR signal, provides a holistic readout of the complex cellular response. In this study, we report the successful application of the DMR technology to CHO-K1 cells stably expressing the human dopamine D receptor. In real-time kinetic experiments, studies of D R reference compounds yielded results for agonists and antagonists that were consistent with those obtained by conventional methods and also allowed a discrimination between partial and full agonists. Furthermore, investigations on the signalling pathway in CHO-K1 hD R cells identified the Gα protein as the main proximal trigger of the observed DMR response. The present study has shown that the DMR technology is a valuable method for the characterisation of putative new ligands and, due to its label-free nature, suggests its use for deorphanisation studies of GPCRs.

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of β-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify β-arrestin2 recruitment to D2long and D3 receptors and measure time-resolved β-arrestin2 recruitment to the D2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no β-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the β-arrestin recruitment to the D2longR and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/β-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.

Ergometrine (6a R ,9 R )- N -(( S )-1-hydroxypropan-2-yl)-7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo-[4,3- fg ]chinolin-9-carboxamide or lysergide acid β-ethanolamide or ergonovine) activates several types of serotonin and histamine receptors in the animal heart. We thus examined whether ergometrine can activate human serotonin 5-HT 4 receptors (h5-HT 4 R) and/or human histamine H 2 receptors (hH 2 R) in the heart of transgenic mice and/or in the human isolated atrium. Force of contraction or beating rates were studied in electrically stimulated left atrial or spontaneously beating right atrial preparations or spontaneously beating isolated retrogradely perfused hearts (Langendorff setup) of mice with cardiac specific overexpression of the h5-HT 4 R (5-HT 4 -TG) or of mice with cardiac specific overexpression of the hH 2 R (H 2 -TG) or in electrically stimulated human right atrial preparations obtained during cardiac surgery. Western blots to assess phospholamban (PLB) phosphorylation on serine 16 were performed. Ergometrine exerted concentration- and time-dependent positive inotropic effects and positive chronotropic effects in atrial preparations starting at 0.3 µM and reaching a plateau at 10 µM in H 2 -TGs ( n  = 7). This was accompanied by an increase in PLB phosphorylation at serine 16. Ergometrine up 10 µM failed to increase force of contraction in left atrial preparations from 5-HT 4 -TGs ( n  = 5). Ten micrometer ergometrine increased the force of contraction in isolated retrogradely perfused spontaneously beating heart preparations (Langendorff setup) from H 2 -TG but not 5-HT 4 -TG. In the presence of the phosphodiesterase inhibitor cilostamide (1 µM), ergometrine at 10 µM exerted positive inotropic effects in isolated electrically stimulated human right atrial preparations, obtained during cardiac surgery, and these effects were eliminated by 10 µM of the H 2 R antagonist cimetidine but not by 10 µM of the 5-HT 4 R antagonist tropisetron. Furthermore, ergometrine showed binding to human histamine H 2 receptors (at 100 µM and 1 mM) using HEK cells in a recombinant expression system (p K i  < 4.5, n  = 3). In conclusion, we suggest that ergometrine is an agonist at cardiac human H 2 Rs.

Clonidine has various clinical effects mediated by agonism of α 1 - or α 2 -adrenoceptors and the blocking of hyperpolarization-activated-nucleotide-gated pacemaker channels (HCN). It is unknown whether clonidine can also stimulate human cardiac histamine H 2 receptors (hH 2 Rs). We used isolated electrically stimulated left and spontaneously beating right atrial preparations from mice overexpressing the hH 2 R specifically in the heart (H 2 -TG), and spontaneously beating right atrial preparations of guinea pigs for comparison. Moreover, we studied isolated electrically stimulated muscle strips from the human right atrium. Clonidine (1, 3, and 10 µM) increased force of contraction in isolated left atrial preparations from H 2 -TG mice. In contrast, clonidine reduced the spontaneous beating rate in right atrial preparations from H 2 -TG. Clonidine raised the beating rate in guinea pig right atrial preparations. Clonidine failed to increase the force of contraction but reduced beating rate in wild-type litter mate mice (WT). In WT, histamine failed to increase the force of contraction in left atrial preparations and beating rate in right atrial preparations. Clonidine (10 µM) increased the force of contraction in isolated human right atrial preparations. The positive inotropic effect in the human atrium was attenuated by cimetidine (10 µM). Clonidine increased the beating rate of the isolated spontaneously beating guinea pig right atrium and acted as a H 2 R partial agonist. Furthermore, clonidine showed binding to the guinea pig H 2 R (100 µM) using HEK cells in a recombinant expression system (p K i  < 4.5) but hardly to the human H 2 R. These data suggest that clonidine can functionally activate cardiac human H 2 R.

Clonidine has various clinical effects mediated by agonism of α - or α -adrenoceptors and the blocking of hyperpolarization-activated-nucleotide-gated pacemaker channels (HCN). It is unknown whether clonidine can also stimulate human cardiac histamine H receptors (hH Rs). We used isolated electrically stimulated left and spontaneously beating right atrial preparations from mice overexpressing the hH R specifically in the heart (H -TG), and spontaneously beating right atrial preparations of guinea pigs for comparison. Moreover, we studied isolated electrically stimulated muscle strips from the human right atrium. Clonidine (1, 3, and 10 µM) increased force of contraction in isolated left atrial preparations from H -TG mice. In contrast, clonidine reduced the spontaneous beating rate in right atrial preparations from H -TG. Clonidine raised the beating rate in guinea pig right atrial preparations. Clonidine failed to increase the force of contraction but reduced beating rate in wild-type litter mate mice (WT). In WT, histamine failed to increase the force of contraction in left atrial preparations and beating rate in right atrial preparations. Clonidine (10 µM) increased the force of contraction in isolated human right atrial preparations. The positive inotropic effect in the human atrium was attenuated by cimetidine (10 µM). Clonidine increased the beating rate of the isolated spontaneously beating guinea pig right atrium and acted as a H R partial agonist. Furthermore, clonidine showed binding to the guinea pig H R (100 µM) using HEK cells in a recombinant expression system (pK  < 4.5) but hardly to the human H R. These data suggest that clonidine can functionally activate cardiac human H R.

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of β-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify β-arrestin2 recruitment to D and D receptors and measure time-resolved β-arrestin2 recruitment to the D receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D R and D R subtypes, whereas for the D R, no β-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the β-arrestin recruitment to the D R and D R, as well as at the D R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/β-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.