Profiling novel pharmacology of receptor complexes using Receptor-HIT.

Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the individual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. This review will discuss various applications of Receptor-HIT, including its use with different classes of receptors (e.g. G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and others), its use to monitor receptor interactions both intracellularly and extracellularly, and also its use with genome-edited endogenous proteins.

Investigation of Receptor Heteromers Using NanoBRET Ligand Binding.

Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the ß2 adrenergic receptor (AT1-ß2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).

AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts: Impact on the Therapeutic Targeting of Renal and Cardiac Fibrosis.

BACKGROUND: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT1R), which is expressed on myofibroblasts along with RXFP1 and AT2R, is unknown. METHODS: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo. RESULTS: The AT1R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT1Rs but that constitutive AT1R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT2R agonist (compound 21). CONCLUSIONS: These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.

Insights into the Interaction of LVV-Hemorphin-7 with Angiotensin II Type 1 Receptor.

Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII's kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.

Application of BRET to monitor ligand binding to GPCRs.

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.

A nationwide survey on producer and veterinarian perceptions of the painfulness of procedures and disease states in dairy and beef cattle.

Failure to adequately manage pain in cattle causes suffering and is thus a welfare concern for the livestock industry. The objectives of this study were to summarize caregiver perceptions of the painfulness of various procedures and disease conditions in cattle. This survey also assessed factors that impact the perception of painfulness and determined relationships between pain perception and mitigation in producers and veterinarians in the United States beef and dairy cattle industries. An online survey was distributed via organization listservs and social media groups representing beef and dairy veterinarians and producers. The survey included questions about respondent demographics and pain perception and frequency of pain mitigation use for a variety of common husbandry procedures and disease conditions in cattle less than 2 months, 2-12 months, and greater than 12 months of age. Descriptive statistics were generated, and ordinal logistic regressions were used to assess the relationship between perceived pain level, frequency of pain mitigation use, and respondent demographic factors (e.g., gender, age, and role). There was a relatively low percentage of respondents that identified there was "no pain" associated with the listed procedures and conditions. Across the majority of procedures and conditions and cattle age categories, men perceived procedures to be less painful than women (P < 0.05). Veterinarians and producer-veterinarians perceived procedures to be more painful than producers (P < 0.05) for the majority of procedures and conditions. There were some differences identified between respondent age groups in pain perception but the trends were not consistent across procedures and conditions. There was a significant positive linear trend, with greater perceived pain associated with greater likelihood of providing local and systemic analgesia for all procedures and conditions across all cattle age categories (P ≤ 0.02). Perception of pain is complex and multifactorial, and it influences the likelihood to treat pain in cattle. This research highlighted the importance of understanding how these factors may play a role in increasing the use of pain mitigation within the beef and dairy industries.

GPCR heteromers: An overview of their classification, function and physiological relevance.

G protein-coupled receptors (GPCRs) are capable of interacting to form higher order structures such as homomers and heteromers. Heteromerisation in particular has implications for receptor function, with research showing receptors can attain unique expression, ligand binding, signalling and intracellular trafficking upon heteromerisation. As such, GPCR heteromers represent novel drug targets with extensive therapeutic potential. Changes to ligand affinity, efficacy and G protein coupling have all been described, with alterations to these pharmacological aspects now well accepted as common traits for heteromeric complexes. Changes in internalisation and trafficking kinetics, as well as ß-arrestin interactions are also becoming more apparent, however, few studies to date have explicitly looked at the implications these factors have upon the signalling profile of a heteromer. Development of ligands to target GPCR heteromers both experimentally and therapeutically has been mostly concentrated on bivalent ligands due to difficulties in identifying and developing heteromer-specific ligands. Improving our understanding of the pharmacology and physiology of GPCR heteromers will enable further development of heteromer-specific ligands with potential to provide therapeutics with increased efficacy and decreased side effects.

Orexin Signaling: A Complex, Multifaceted Process.

The orexin system comprises two G protein-coupled receptors, OX1 and OX2 receptors (OX1R and OX2R, respectively), along with two endogenous agonists cleaved from a common precursor (prepro-orexin), orexin-A (OX-A) and orexin-B (OX-B). For the receptors, a complex array of signaling behaviors has been reported. In particular, it becomes obvious that orexin receptor coupling is very diverse and can be tissue-, cell- and context-dependent. Here, the early signal transduction interactions of the orexin receptors will be discussed in depth, with particular emphasis on the direct G protein interactions of each receptor. In doing so, it is evident that ligands, additional receptor-protein interactions and cellular environment all play important roles in the G protein coupling profiles of the orexin receptors. This has potential implications for our understanding of the orexin system's function in vivo in both central and peripheral environments, as well as the development of novel agonists, antagonists and possibly allosteric modulators targeting the orexin system.

Frequency of disagreements between producers and veterinarians about pain management in cattle.

Pain management is a key element of ensuring animal welfare. Although the opinions of both producers and veterinarians affect decisions about the use of pain mitigation on cattle operations, little is known about how they communicate about this topic. Given the importance of a veterinary-client-patient relationship for developing pain mitigation protocols, understanding the communication between veterinarians and producers is key to the implementation of robust, industry-wide pain management protocols. The objectives of this survey were to understand how producers and veterinarians may respond to disagreements about pain mitigation and to determine where respondents obtain their knowledge about pain recognition and treatment. Results presented herein are part of a larger study previously described. An online survey was distributed to 6 cattle industry groups. Surveys that were >80% complete were included for analysis (n = 1,066). Approximately half of the respondents identified as producers (497, 46.6%) and half as veterinarians (569, 53.4%). The majority of producers believed that disagreements about the use of pain management in cattle never affected their relationship with their veterinarians (349, 70.2%). The veterinarian respondents indicated more disagreements, although the frequency was relatively low, with 43.9% (250) indicated having a disagreement less than once a year. Most producers and veterinarians indicated they were either "extremely unlikely" or "somewhat unlikely" to dissolve the relationship completely if disagreements about pain management arose (veterinarians: 398/569, 70%; producers: 294/497, 59.1%). Veterinarians and producers reported gaining their knowledge about pain recognition from a variety of sources including personal experience and continuing education opportunities. Disagreements about pain mitigation occurred infrequently; however, this could be due to few discussions about pain management in general. These results indicated that there is opportunity for veterinarians to engage with their producers in more discussions about pain management.

Novel Pharmacology Following Heteromerization of the Angiotensin II Type 2 Receptor and the Bradykinin Type 2 Receptor.

The angiotensin type 2 (AT2) receptor and the bradykinin type 2 (B2) receptor are G protein-coupled receptors (GPCRs) that have major roles in the cardiovascular system. The two receptors are known to functionally interact at various levels, and there is some evidence that the observed crosstalk may occur as a result of heteromerization. We investigated evidence for heteromerization of the AT2 receptor and the B2 receptor in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, including the Receptor Heteromer Investigation Technology (Receptor-HIT) and the NanoBRET ligand-binding assay. The Receptor-HIT assay showed that Gαq, GRK2 and ß-arrestin2 recruitment proximal to AT2 receptors only occurred upon B2 receptor coexpression and activation, all of which is indicative of AT2-B2 receptor heteromerization. Additionally, we also observed specific coupling of the B2 receptor with the Gαz protein, and this was found only in cells coexpressing both receptors and stimulated with bradykinin. The recruitment of Gαz, Gαq, GRK2 and ß-arrestin2 was inhibited by B2 receptor but not AT2 receptor antagonism, indicating the importance of B2 receptor activation within AT2-B2 heteromers. The close proximity between the AT2 receptor and B2 receptor at the cell surface was also demonstrated with the NanoBRET ligand-binding assay. Together, our data demonstrate functional interaction between the AT2 receptor and B2 receptor in HEK293FT cells, resulting in novel pharmacology for both receptors with regard to Gαq/GRK2/ß-arrestin2 recruitment (AT2 receptor) and Gαz protein coupling (B2 receptor). Our study has revealed a new mechanism for the enigmatic and poorly characterized AT2 receptor to be functionally active within cells, further illustrating the role of heteromerization in the diversity of GPCR pharmacology and signaling.

Pharmacists' views and desires regarding pharmacist administration of vaccines in New Zealand.

OBJECTIVES: To explore pharmacists' views and experiences of pharmacist-administered vaccinations, motivators and barriers to pharmacists administering vaccinations and their preferences for expansions to such services. METHODS: All practising pharmacist members (n = 3400) of the Pharmaceutical Society of New Zealand were invited to participate in an online survey in 2017. KEY FINDINGS: A total of 468 pharmacists completed the survey (14%). Most (86%) strongly agreed/agreed that pharmacists should provide vaccinations, primarily citing patient benefit, for example, convenience, potential for increased vaccination uptake, easing general practice burden and better utilisation of the pharmacist. Half had completed vaccinator training, mainly for professional satisfaction, to help public or community health and/or to provide a new service for their community. Trained pharmacists had administered influenza (95%), pertussis (47%), zoster (45%) and/or meningococcal vaccines (13%), with patient cost limiting some vaccination uptake. Cost or workplace constraints were leading reasons for the 17% not planning to undertake vaccinator training. Key barriers for pharmacy owners not offering vaccinations were set-up or other costs, insufficient funding (62%) or staffing/time concerns (27%). Some trained vaccinators (39%) wanted the recipient age lowered below 13 years, and 44% wanted intern pharmacists to be able to administer vaccinations. CONCLUSION: This study found strong support for this service, including benefits for patients, and for customer relationships. Identified barriers including service setup and patient costs could be reduced by expanding the categories (e.g. pharmacy students and technicians) of staff able to vaccinate and having more government funded vaccines available through pharmacies, therefore, improving access for patients.

Current attitudes of veterinarians and producers regarding the use of local and systemic analgesia in beef and dairy cattle in the United States.

OBJECTIVE: To survey cattle producers and veterinarians about the use of analgesia on US cattle operations. SAMPLE: 1,187 members of the following database, electronic mailing lists, and social media groups: FarmProgress master file, American Association of Bovine Practitioners, Academy of Veterinary Consultants, National Milk Producers Federation Farm Evaluators, Dairy Moms Facebook group, and Dairy Girl Network Facebook group. PROCEDURES: An online survey was developed to gather information about the frequency of local and systemic analgesia use for common painful procedures and diseases in cattle < 2, 2 to 12, and > 12 months old. Respondents also rated their extent of agreement with each of 10 statements related to pain management in cattle. The survey was available from June 11 to August 10, 2018. Descriptive data were generated. Logistic regression was used for comparisons among cattle age groups and respondents on the basis of their industry role. RESULTS: In general, frequency of analgesia use increased as cattle age increased, regardless of the procedure or disease. The odds of analgesia use were lower for men, compared with women, and greater for veterinarians, compared with producers. Many respondents indicated they were cognizant of the benefits of analgesia use in cattle but perceived federal regulations and drug costs as impediments to the implementation of pain mitigation protocols on cattle operations. CONCLUSIONS AND CLINICAL RELEVANCE: Results provided insight into current perceptions and use of analgesia in cattle, which can be used to guide implementation of pain mitigation protocols on US beef and dairy cattle operations.

Producer and Veterinarian Perspectives towards Pain Management Practices in the US Cattle Industry.

Producers and veterinarians are considered responsible for improving animal welfare, as they are responsible for implementing practices that directly impact the animal's well-being. Most husbandry procedures performed in cattle do not include pain mitigation, and understanding challenges faced by these stakeholders to use analgesics is key in improving on-farm pain management strategies. Therefore, the objectives of this study were to explore producer and veterinarian perspectives on pain management practices by (1) exploring inquires received by Food Animal Residue Avoidance Databank (FARAD) regarding analgesic use in cattle and (2) using a survey instrument to identify factors that impact pain management implementation in the US cattle industry. Albeit analgesia use increased in the past ten years for some producers and the majority of veterinarians, administering analgesics for pain management on US cattle farms remains a challenge. From a producer perspective, drug cost, availability and logistics for administration. From a veterinarian perspective, lack of Food and Drug Administration (FDA) products hinders the support of on-farm protocols requiring extra-label drug use. Future steps to improve analgesic use on-farm include identifying and approving drugs that demonstrate efficacy for managing pain in cattle and disseminating educational resources to support stakeholders in both the implementation and drug withdrawal process.

Complex interactions between the angiotensin II type 1 receptor, the epidermal growth factor receptor and TRIO-dependent signaling partners.

Transactivation of the epidermal growth factor receptor (EGFR) by the angiotensin II (AngII) type 1 (AT1) receptor is involved in AT1 receptor-dependent growth effects and cardiovascular pathologies, however the mechanisms underpinning this transactivation are yet to be fully elucidated. Recently, a potential intermediate of this process was identified following the discovery that a kinase called TRIO was involved in AngII/AT1 receptor-mediated transactivation of EGFR. To investigate the mechanisms by which TRIO acts as an intermediate in AngII/AT1 receptor-mediated EGFR transactivation we used bioluminescence resonance energy transfer (BRET) assays to investigate proximity between the AT1 receptor, EGFR, TRIO and other proteins of interest. We found that AngII/AT1 receptor activation caused a Gαq-dependent increase in proximity of TRIO with Gγ2 and the AT1-EGFR heteromer, as well as trafficking of TRIO towards the Kras plasma membrane marker and into early, late and recycling endosomes. In contrast, we found that AngII/AT1 receptor activation caused a Gαq-independent increase in proximity of TRIO with Grb2, GRK2 and PKCζ, as well as trafficking of TRIO up to the plasma membrane from the Golgi. Furthermore, we confirmed the proximity between the AT1 receptor and the EGFR using the Receptor-Heteromer Investigation Technology, which showed AngII-induced recruitment of Grb2, GRK2, PKCζ, Gγ2 and TRIO to the EGFR upon AT1 coexpression. In summary, our results provide further evidence for the existence of the AT1-EGFR heteromer and reveal potential mechanisms by which TRIO contributes to the transactivation process.

The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer.

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.

Global Forum on Quality Assurance in CE/CPD: Assuring Quality across Boundaries.

As a result of the globalization of access and provision of continuing education and continuing professional development (CE/CPD), the national CE/CPD accreditation organizations of Australia, Canada, Ireland, New Zealand, South Africa, United Kingdom and United States formed the Global Forum on Quality Assurance of Continuing Education and Continuing Professional Development (GFQACE) to investigate and develop means of recognizing CE/CPD across boundaries. Two priorities were identified at their first meeting in 2016: (1) the development of an accreditation framework and (2) the identification of models and approaches to mutual recognition. The GFQACE approved an accreditation framework and facilitated review approach to mutual recognition in 2018 and is currently working on implementation guides. As background to the work of the GFQACE, this article provides a brief history of continuing education (CE) and continuing professional development (CPD) and discusses the value and benefits of CE/CPD to professional development of pharmacy professionals, innovation of pharmacy practice and the provision of quality patient care. Due to the essential role of CE/CPD accreditation in enabling recognition across boundaries, the nature and role of accreditation in defining, assuring and driving quality CE/CPD is described. Four conclusions regarding the broad sharing of perceptions of quality CE/CPD, the potential for expansion of the GFQACE and the benefits to pharmacy professionals, providers and pharmacy practice are discussed.

Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer.

Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers.

NanoBRET: The Bright Future of Proximity-Based Assays.

Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.

NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing.

Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A2B receptors under endogenous promotion tagged on their N-terminus with Nluc. As expected, we found relatively low levels of endogenous (gene-edited) Nluc/A2B receptor expression compared to cells transiently transfected with expression vectors coding for Nluc/A2B. However, in cells expressing gene-edited Nluc/A2B receptors we observed clear saturable ligand binding of a non-specific fluorescent adenosine receptor antagonist XAC-X-BY630 (Kd = 21.4 nM). Additionally, at gene-edited Nluc/A2B receptors we derived pharmacological parameters of ligand binding; Kd as well as Kon and Koff for binding of XAC-X-BY630 by NanoBRET association kinetic binding assays. Lastly, cells expressing gene-edited Nluc/A2B were used to determine the pKi of unlabelled adenosine receptor ligands in competition ligand binding assays. Utilising CRISPR/Cas9 genome engineering here we show that NanoBRET ligand binding assays can be performed at gene-edited receptors under endogenous promotion in live cells, therefore overcoming a fundamental limitation of NanoBRET ligand assays.

Knowledge and Opinions of Third Year Veterinary Students Relevant to Animal Welfare Before and After Implementation of a Core Welfare Course.

Although leading veterinary organizations emphasize the importance of animal welfare knowledge, there exists a gap in current veterinary student animal welfare education and training. A survey instrument was created to assess third-year Doctor of Veterinary Medicine (DVM) student knowledge of key animal welfare topics, opinions regarding the inclusion of welfare education in the veterinary curriculum, and views on veterinarian responsibilities as advocates. In Spring 2018, Colorado State University added a required animal welfare course to the DVM curriculum. Pre- and post-course paper surveys were distributed to the third-year students enrolled in the animal welfare course. One hundred thirty one completed pre-course surveys were collected and 125 completed post-course surveys were collected. Of the pre and post-course surveys collected, 61 were paired with identification codes and utilized for statistical comparison. Results indicated that the course led students to view the inclusion of an animal welfare course in the veterinary curriculum more favorably (p = 0.009) and improved their confidence in conducting research on animal welfare topics (p < 0.001). The course did not change students' sense of responsibility toward welfare advocacy. Associations were not found between attitudes toward these issues and demographic variables of home community, respondent gender, and track selection (p > 0.06). Veterinarians were consistently ranked by students as the most influential member of a community in matters of animal welfare. Future research on the lack of veterinary student knowledge of animal welfare should be done on a national scale to facilitate strategic development of mandatory animal welfare courses in veterinary curricula. Future research should be designed to gain knowledge regarding DVM students' opinions and attitudes regarding effective methods of incorporating animal welfare education into their professional training.