Iron Is Critical for Mucosal-Associated Invariant T Cell Metabolism and Effector Functions.

Mucosal-Associated Invariant T (MAIT) cells are a population of innate T cells that play a critical role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells can rapidly respond via both TCR-dependent and -independent mechanisms, resulting in robust cytokine production. The metabolic and nutritional requirements for optimal MAIT cell effector responses are still emerging. Iron is an important micronutrient and is essential for cellular fitness, in particular cellular metabolism. Iron is also critical for many pathogenic microbes, including those that activate MAIT cells. However, iron has not been investigated with respect to MAIT cell metabolic or functional responses. In this study, we show that human MAIT cells require exogenous iron, transported via CD71 for optimal metabolic activity in MAIT cells, including their production of ATP. We demonstrate that restricting iron availability by either chelating environmental iron or blocking CD71 on MAIT cells results in impaired cytokine production and proliferation. These data collectively highlight the importance of a CD71-iron axis for human MAIT cell metabolism and functionality, an axis that may have implications in conditions where iron availability is limited.

GPCR kinase subtype requirements for arrestin-2 and -3 translocation to the cannabinoid CB1 receptor and the consequences on G protein signalling.

Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of 'phosphorylation barcode,' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB1 receptor (CB1) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB1, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB1 must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.

Determinants of Mortality and Mid-Term Outcomes After Transcarotid Artery Revascularization and Transfemoral Carotid Artery Stenting.

OBJECTIVES: The potential benefit of transcarotid artery revascularization (TCAR) over transfemoral carotid artery stenting (tfCAS) has been studied in the perioperative period with lower rates of stroke and death; however, data on mid-term outcomes are limited. We aimed to evaluate 3-year outcomes after TCAR and tfCAS and determine the primary predictors of 30-day and 1-year mortality following TCAR. METHODS: Data from the Vascular Quality Initiative for patients undergoing TCAR or tfCAS from January 2016 to December 2022 were analyzed. 1:1 propensity score matching using the nearest-neighbor method was used to adjust baseline demographics and clinical characteristics. Kaplan-Meier survival analysis and Cox Proportional Hazard Regression were used to evaluate long-term outcomes. Iterative stepwise multiple logistic regression analysis and Cox Proportional Hazard Regression were used to identify predictors of 30-day and 1-year mortality, respectively, based upon preoperative, intraoperative, and postoperative factors. RESULTS: A total of 70 237 patients were included in analysis (TCAR=58.7%, tfCAS=41.3%). Transcarotid artery revascularization patients were older and had higher rates of comorbid conditions and high-risk medical and anatomic features than tfCAS patients. Propensity score matching yielded 22 322 pairs with no major differences between groups except that TCAR patients were older (71.6 years vs 70.8 years). At 3 years, TCAR was associated with a 24% reduction in hazard of death compared with tfCAS (hazard ratio [HR]=0.76, 95% confidence interval [CI]=0.71-0.82, p<0.001), for both symptomatic and asymptomatic patients. This survival advantage was established in the first 6 months (HR=0.59, 95% CI=0.53-0.62, p<0.001), with no difference in mortality risk from 6 months to 36 months (HR=0.95, 95% CI=0.86-1.05, p=0.31). Transcarotid artery revascularization was also associated with decreased hazard for 3-year stroke (HR=0.81, 95% CI=0.66-0.99, p=0.04) and stroke or death (HR=0.81, 95% CI=0.76-0.87, p<0.001) compared with tfCAS. The top predictors for 30-day and 1-year mortality were postoperative complications. The primary independent predictor was the occurrence of postoperative stroke. CONCLUSIONS: Transcarotid artery revascularization had a sustained mid-term survival advantage associated over tfCAS, with the benefit being established primarily within the first 6 months. Notably, our findings highlight the importance of postoperative stroke as the primary independent predictor for 30-day and 1-year mortal. CLINICAL IMPACT: The ongoing debate over the superiority of TCAR compared to tfCAS and CEA has been limited by a lack of comparative studies examining the impact of pre-operative symptoms on outcomes. Furthermore, data are scarce on mid-term outcomes for TCAR beyond the perioperative period. As a result, it remains uncertain whether the initial benefits of stroke and death reduction observed with TCAR over tfCAS persist beyond one year. Our study addresses these gaps in the literature, offering evidence to enable clinicians to assess the efficacy of TCAR for up to three years. Additionally, our study seeks to identify risk factors for postoperative mortality following TCAR, facilitating optimal patient stratification.

Metformin and feeding increase levels of the appetite-suppressing metabolite Lac-Phe in humans.

Metformin, a widely used first-line treatment for type 2 diabetes (T2D), is known to reduce blood glucose levels and suppress appetite. Here we report a significant elevation of the appetite-suppressing metabolite N-lactoyl phenylalanine (Lac-Phe) in the blood of individuals treated with metformin across seven observational and interventional studies. Furthermore, Lac-Phe levels were found to rise in response to acute metformin administration and post-prandially in patients with T2D or in metabolically healthy volunteers.

Investigating selectivity and bias for G protein subtypes and ß-arrestins by synthetic cannabinoid receptor agonists at the cannabinoid CB1 receptor.

The cannabinoid CB1 receptor (CB1) is a G protein-coupled receptor (GPCR) with widespread expression in the central nervous system. This canonically G⍺i/o-coupled receptor mediates the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoid receptor agonists (SCRAs). Recreational use of SCRAs is associated with serious adverse health effects, making pharmacological research into these compounds a priority. Several studies have hypothesised that signalling bias may explain the different toxicological profiles between SCRAs and THC. Previous studies have focused on bias between G protein activation measured by cyclic adenosine monophosphate (cAMP) inhibition and ß-arrestin translocation. In contrast, the current study characterises bias between G⍺ subtypes of the G⍺i/o family and ß-arrestins; this method facilitates a more accurate assessment of ligand bias by assessing signals that have not undergone major amplification. We have characterised G protein dissociation and translocation of ß-arrestin 1 and 2 using real-time BRET reporters. The responses produced by each SCRA across the G protein subtypes tested were consistent with the responses produced by the reference ligand AMB-FUBINACA. Ligand bias was probed by applying the operational analysis to determine biases within the G⍺i/o family, and between G protein subtypes and ß-arrestins. Overall, these results confirm SCRAs to be balanced, high-efficacy ligands compared to the low efficacy ligand THC, with only one SCRA, 4CN-MPP-BUT7IACA, demonstrating statistically significant bias in one pathway comparison (towards ß-arrestin 1 when compared with G⍺oA/oB). This suggests that the adverse effects caused by SCRAs are due to high potency and efficacy at CB1, rather than biased agonism.

Determination of the Cannabinoid CB1 Receptor's Positive Allosteric Modulator Binding Site through Mutagenesis Studies.

Positive allosteric modulators (PAMs) of the cannabinoid CB1 receptor (CB1) offer potential therapeutic advantages in the treatment of neuropathic pain and addiction by avoiding the adverse effects associated with orthosteric CB1 activation. Here, molecular modeling and mutagenesis were used to identify residues central to PAM activity at CB1. Six putative allosteric binding sites were identified in silico, including novel sites previously associated with cholesterol binding, and key residues within each site were mutated to alanine. The recently determined ZCZ011 binding site was found to be essential for allosteric agonism, as GAT228, GAT229 and ZCZ011 all increased wild-type G protein dissociation in the absence of an orthosteric ligand; activity that was abolished in mutants F191A3.27 and I169A2.56. PAM activity was demonstrated for ZCZ011 in the presence of the orthosteric ligand CP55940, which was only abolished in I169A2.56. In contrast, the PAM activity of GAT229 was reduced for mutants R220A3.56, L404A8.50, F191A3.27 and I169A2.56. This indicates that allosteric modulation may represent the net effect of binding at multiple sites, and that allosteric agonism is likely to be mediated via the ZCZ011 site. This study underlines the need for detailed understanding of ligand receptor interactions in the search for pure CB1 allosteric modulators.

Insight into the mechanism of action of ORG27569 at the cannabinoid type one receptor utilising a unified mathematical model.

Allosteric modulation of CB1 is therapeutically advantageous compared to orthosteric activation as it potentially offers reduced on-target adverse effects. ORG27569 is an allosteric modulator that increases orthosteric agonist binding to CB1 but decreases functional signalling. ORG27569 is characterised by a delay in disinhibition of agonist-induced cAMP inhibition (lag); however, the mechanism behind this kinetic lag is yet to be identified. We aimed to utilise a mathematical model to predict data and design in vitro experiments to elucidate mechanisms behind the unique signalling profile of ORG27569. The established kinetic ternary complex model includes the existence of a transitional state of CB1 bound to ORG27569 and CP55940 and was used to simulate kinetic cAMP data using NONMEM 7.4 and Matlab R2020b. These data were compared with empirical cAMP BRET data in HEK293 cells stably expressing hCB1. The pharmacometric model suggested that the kinetic lag in cAMP disinhibition by ORG27569 is caused by signal amplification in the cAMP assay and can be reduced by decreasing receptor number. This was confirmed experimentally, as reducing receptor number through agonist-induced internalisation resulted in a decreased kinetic lag by ORG27569. ORG27569 was found to have a similar interaction with CP55940 and the high efficacy agonist WIN55,212-2, and was suggested to have lower affinity for CB1 bound by the partial agonist THC compared to CP55940. Allosteric modulators have unique signalling profiles that are often difficult to interrogate exclusively in vitro. We have used a combined mathematical and in vitro approach to prove that ORG27569 causes a delay in disinhibition of agonist-induced cAMP inhibition due to large receptor reserve in this pathway. We also used the pharmacometric model to investigate the common phenomenon of probe dependence, to propose that ORG27569 binds with higher affinity to CB1 bound by high efficacy orthosteric agonists.

A quantitative pharmacology model for cannabinoid CB1 receptor mediated by Gi/Gs protein competition.

BACKGROUND AND PURPOSE: Orthosteric agonism of the CB1 receptor normally associates with Gi signalling resulting in a net inhibition of cAMP production. Empirical evidence shows CB1 causes a net cAMP stimulation through Gs coupling under two conditions: co-stimulation with the D2 receptor and high-level CB1 expression. Two hypotheses have been proposed to account for these paradoxical effects, (1) Gi is consumed by coupling to D2 or extra CB1 and excess CB1 binds to Gs and (2), the formation of dimers CB1 -CB1 or CB1 -D2 switches Gi/Gs preference. This study explored the mechanisms of Gi/Gs preference based on a mathematical model of the CB1 receptor. EXPERIMENTAL APPROACH: The model was based on Hypothesis 1 and known mechanisms. The model was calibrated to align with multiple types of data (cAMP, Gi dissociation and internalisation). The key step of Hypothesis 1 was examined by simulation from the model. An experiment was proposed to distinguish Hypothesis 1 and 2. KEY RESULTS: The model successfully descripted multiple types of data under Hypothesis 1. Simulations from the model indicated that precoupling of G protein with receptors is necessary for this hypothesis. The model designed experiments to distinguish Hypothesis 1 and 2 by increasing Gi & Gs in parallel with CB1 overexpression. The two hypotheses result in distinct cAMP responses. CONCLUSION AND IMPLICATIONS: A mathematical model of CB1 -regulated Gi/Gs pathways was developed. It indicated Hypothesis 1 is feasible and G protein precoupling is a key step causing cAMP signalling switch. The model-designed experiments provided guides for future experimentation.

Botulism in Two Juvenile Wild Red Foxes (Vulpes vulpes) in Somerset, England, UK.

Two juvenile red foxes (Vulpes vulpes) were euthanased because of severe nervous signs and paralysis. Detailed postmortem examinations were carried out with bacteriology, histology, and Clostridium botulinum toxin screening, which confirmed botulism as the cause of the clinical signs.

Fatty acids are crucial to fuel NK cells upon acute retrovirus infection.

Natural killer (NK) cells are cytotoxic innate immune cells, able to recognize and eliminate virus-infected as well as cancer cells. Metabolic reprogramming is crucial for their activity as they have enhanced energy and nutritional demands for their functions during an infection. Fatty acids (FAs) represent an important source of cellular energy and are essential for proliferation of immune cells. However, the precise role of FAs for NK cells activity in retrovirus infection was unknown. Here we show that activated NK cells increase the expression of the FA uptake receptor CD36 and subsequently the uptake of FAs upon acute virus infection. We found an enhanced flexibility of NK cells to utilize FAs as source of energy compare to naïve NK cells. NK cells that were able to generate energy from FAs showed an augmented target cell killing and increased expression of cytotoxic parameters. However, NK cells that were unable to generate energy from FAs exhibited a severely decreased migratory capacity. Our results demonstrate that NK cells require FAs in order to fight acute virus infection. Susceptibility to severe virus infections as it is shown for people with malnutrition may be augmented by defects in the FA processing machinery, which might be a target to therapeutically boost NK cell functions in the future.

Evaluating signaling bias for synthetic cannabinoid receptor agonists at the cannabinoid CB2 receptor.

The rapid structural evolution and emergence of novel synthetic cannabinoid receptor agonists (SCRAs) in the recreational market remains a key public health concern. Despite representing one of the largest classes of new psychoactive substances, pharmacological data on new SCRAs is limited, particularly at the cannabinoid CB2 receptor (CB2 ). Hence, the current study aimed to characterize the molecular pharmacology of a structurally diverse panel of SCRAs at CB2 , including 4-cyano MPP-BUT7AICA, 4F-MDMB-BUTINACA, AMB-FUBINACA, JWH-018, MDMB-4en-PINACA, and XLR-11. The activity of SCRAs was assessed in a battery of in vitro assays in CB2 -expressing HEK 293 cells: G protein activation (Gαi3 and GαoB ), phosphorylation of ERK1/2, and ß-arrestin 1/2 translocation. The activity profiles of the ligands were further evaluated using the operational analysis to identify ligand bias. All SCRAs activated the CB2 signaling pathways in a concentration-dependent manner, although with varying potencies and efficacies. Despite the detection of numerous instances of statistically significant bias, compound activities generally appeared only subtly distinct in comparison with the reference ligand, CP55940. In contrast, the phytocannabinoid THC exhibited an activity profile distinct from the SCRAs; most notably in the translocation of ß-arrestins. These findings demonstrate that CB2 is able to accommodate a structurally diverse array of SCRAs to generate canonical agonist activity. Further research is required to elucidate whether the activation of CB2 contributes to the toxicity of these compounds.

The impact of high intensity interval training in a diverse group of cancer survivors: CAPABLE, a pilot study.

Purpose: Given the well-documented benefits of regular exercise to cancer survivors, current American Cancer Society guidelines recommend that patients engage in a minimum of 150 min per week of moderate-to-vigorous physical activity with a minimum of two days of strength training. However, few survivors meet this goal, particularly among minorities. Methods: The CAPABLE study is a single-arm, pilot exercise intervention that introduced 48 cancer survivors to a high intensity interval and strength training program three days a week for 12 weeks. We evaluated the impact of this unique training method on bodyweight, % body fat, serum markers correlated with an adverse cardiometabolic profile and health-related quality of life (HRQoL). Measures were summarized at baseline and program exit. Paired t-tests were used to assess change in each of these measures over time. Results: We observed losses in weight, body mass index, and % body fat, and glycosylated hemoglobin (HbA1c) levels over 12-weeks. There were also clinically meaningful improvements in reported overall HRQoL (FACTG total change +9.5 (95% CI, 4.6, 14.4)) and in each one of the individual domains (physical, social, emotional, and functional well-being). Conclusions: We observed meaningful improvements in body composition, HbA1c and quality of life over 12 weeks among cancer survivors participating in a high-intensity interval training program. Future work will include a control arm for comparison and address barriers to participation and adherence which will be important in using this intervention and others like it to improve outcomes and reduce cancer health disparities.

QUAS-R: An SLC1A5-mediated glutamine uptake assay with single-cell resolution reveals metabolic heterogeneity with immune populations.

System-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to understand how and when immune cells access these nutrients. Here, we describe a new approach for single-cell analysis of nutrient uptake where we use in-cell biorthogonal labeling of a functionalized amino acid after transport into the cell. In this manner, the bona fide active uptake of glutamine via SLC1A5/ASCT2 could be quantified. We used this assay to interrogate the transport capacity of complex immune subpopulations, both in vitro and in vivo. Taken together, our findings provide an easy sensitive single-cell assay to assess which cells support their function via SLC1A5-mediated uptake. This is a significant addition to the single-cell metabolic toolbox required to decode the metabolic landscape of complex immune microenvironments.

A kinetic model for positive allosteric modulator (PAM)-antagonists for the type 1 cannabinoid (CB1 ) receptor.

BACKGROUND AND PURPOSE: The cannabinoid (CB1 ) receptor is among the most abundant G protein-coupled receptors in brain. Allosteric ligands bind to a different site on receptors than the orthosteric ligand can have effects that are unique to the allosteric ligand and modulate orthosteric ligand activity. We propose a unified mathematical model describing the interaction effects of the allosteric ligand Org27569 and the orthosteric agonist CP55940 on CB1 receptor. EXPERIMENTAL APPROACH: A ternary complex model was constructed, which incorporated kinetic properties to describe the time course of effects of Org27569 and CP55940 reported in the literature: (i) enhanced receptor binding of CP55940, (ii) reduced internalisation and (iii), time-dependent modulation of cAMP. Underlying mechanisms of time-dependent modulation by Org27569 were evaluated by simulation. KEY RESULTS: A hypothetical transitional state of CP55940-CB1 -Org27569, which can internalise but cannot inhibit cAMP, was shown to be necessary and was sufficient to describe the allosteric modulation by Org27569, prior to receptors adopting an inactive conformation. The model indicated that the formation of this transitional CP55940-CB1 -Org27569 state and final inactive CP55940-CB1 -Org27569 state contributes to the enhanced CP55940 binding. The inactive CP55940-CB1 -Org27569 cannot internalise or inhibit cAMP, leading to reduced internalisation and cessation of cAMP inhibition. CONCLUSIONS AND IMPLICATIONS: In conclusion, a kinetic mathematical model for CB1 receptor allosteric modulation was developed. However, a standard ternary complex model was not sufficient to capture the data and a hypothetical transitional state was required to describe the allosteric modulation properties of Org27569.

Signaling-specific inhibition of the CB1 receptor for cannabis use disorder: phase 1 and phase 2a randomized trials.

Cannabis use disorder (CUD) is widespread, and there is no pharmacotherapy to facilitate its treatment. AEF0117, the first of a new pharmacological class, is a signaling-specific inhibitor of the cannabinoid receptor 1 (CB1-SSi). AEF0117 selectively inhibits a subset of intracellular effects resulting from Δ9-tetrahydrocannabinol (THC) binding without modifying behavior per se. In mice and non-human primates, AEF0117 decreased cannabinoid self-administration and THC-related behavioral impairment without producing significant adverse effects. In single-ascending-dose (0.2 mg, 0.6 mg, 2 mg and 6 mg; n = 40) and multiple-ascending-dose (0.6 mg, 2 mg and 6 mg; n = 24) phase 1 trials, healthy volunteers were randomized to ascending-dose cohorts (n = 8 per cohort; 6:2 AEF0117 to placebo randomization). In both studies, AEF0117 was safe and well tolerated (primary outcome measurements). In a double-blind, placebo-controlled, crossover phase 2a trial, volunteers with CUD were randomized to two ascending-dose cohorts (0.06 mg, n = 14; 1 mg, n = 15). AEF0117 significantly reduced cannabis' positive subjective effects (primary outcome measurement, assessed by visual analog scales) by 19% (0.06 mg) and 38% (1 mg) compared to placebo (P < 0.04). AEF0117 (1 mg) also reduced cannabis self-administration (P < 0.05). In volunteers with CUD, AEF0117 was well tolerated and did not precipitate cannabis withdrawal. These data suggest that AEF0117 is a safe and potentially efficacious treatment for CUD.ClinicalTrials.gov identifiers: NCT03325595 , NCT03443895 and NCT03717272 .

Canagliflozin impairs T cell effector function via metabolic suppression in autoimmunity.

Augmented T cell function leading to host damage in autoimmunity is supported by metabolic dysregulation, making targeting immunometabolism an attractive therapeutic avenue. Canagliflozin, a type 2 diabetes drug, is a sodium glucose co-transporter 2 (SGLT2) inhibitor with known off-target effects on glutamate dehydrogenase and complex I. However, the effects of SGLT2 inhibitors on human T cell function have not been extensively explored. Here, we show that canagliflozin-treated T cells are compromised in their ability to activate, proliferate, and initiate effector functions. Canagliflozin inhibits T cell receptor signaling, impacting on ERK and mTORC1 activity, concomitantly associated with reduced c-Myc. Compromised c-Myc levels were encapsulated by a failure to engage translational machinery resulting in impaired metabolic protein and solute carrier production among others. Importantly, canagliflozin-treated T cells derived from patients with autoimmune disorders impaired their effector function. Taken together, our work highlights a potential therapeutic avenue for repurposing canagliflozin as an intervention for T cell-mediated autoimmunity.

Effects of front-of-package nutrition labelling systems on understanding and purchase intention in Jamaica: results from a multiarm randomised controlled trial.

OBJECTIVE: To assess the effects of three different front-of-package labelling (FOPL) schemes on objective understanding and intention to purchase of products, in Jamaica. SETTING: Supermarkets in Jamaica. PARTICIPANTS: Adult supermarket shoppers in Jamaica (n=1206) aged 18 years old or older were included in the study, except for those visually impaired, or unable to give informed consent. DESIGN: Multiarm parallel-group randomised trial. INTERVENTIONS: Participants were randomly allocated to one of the three intervention groups or the control group. They were exposed to two-dimensional images of 12 mock-up products presented in random and balanced order. Participants assigned to the intervention groups were exposed to one FOPL scheme: black octagonal warning labels (OWL), magnifying glass high-in single icon (MGG) or traffic-light labelling (TFL). The control group was exposed to the nutrition facts up front. OUTCOME MEASURES: OR for correctly understanding nutritional information (correctly selecting the least harmful option, correctly identifying sugars, sodium and/or saturated fats found to be in excess) and choosing to purchase the least harmful option (purchase intention), more often. RESULTS: Compared with the control group, the odds for correctly selecting the least harmful option more often were 107% higher in the OWL group (OR 2.07, 95% CI 1.54 to 2.78; p<0.001), whereas the MGG (1.18, 95% CI 0.89 to 1.57; p=0.24) and the TFL (1.13, 95% CI 0.85 to 1.51; p=0.39) were inefficacious in improving such odds. OWL also resulted in the highest odds for correctly identifying a product with excessive amounts of sugars, sodium and/or saturated fats and for deciding to purchase the least harmful option or none of the options. CONCLUSIONS: Octagonal warning labels performed best at improving the ability of adult shoppers in Jamaica to understand the nutrition information and at encouraging them to purchase the least harmful option more often.

Small Caliber Distal Cephalic Veins Undergo Significant Dilation under Anesthesia and Can Successfully Be Used for Arteriovenous Fistula Creation.

BACKGROUND: Successful arteriovenous fistula (AVF) maturation and use for dialysis is highly dependent on preoperative diameter. Small veins (<2 mm) exhibit high failure rates and are typically avoided. This study investigates the effects of anesthesia on the distal cephalic vein diameter as compared to preoperative outpatient vein mapping for the purpose of hemodialysis access creation. METHODS: One hundred eight consecutive procedures for dialysis access placement met inclusion criteria and were reviewed. All patients received preoperative venous mapping and postanesthesia ultrasound mapping (PAUS). All patients received either regional and/or general anesthesia. A multiple regression was conducted to determine predictors of venous dilatation. The independent variables included both demographical and operative-specific variables such as the type of anesthesia. Outcomes of fistula maturation (successful cannulation and dialysis) were analyzed. RESULTS: In this cohort, the mean preoperative vein diameter was 1.85 mm and the mean PAUS diameter was 3.45 mm, a 2.21× increase, with only 2 patient veins failing to increase in diameter. Smaller veins (<2 mm) exhibited significantly more dilation than larger veins after anesthesia (2.73 vs. 1.47×, P < 0.001). In the multiple regression analysis, smaller vein diameter was correlated with a significantly greater degree of dilation (P < 0.001). The degree of venous dilation was not affected by patient demographic-specific factors or by the type of anesthesia (regional block versus general) in the multiple regression analysis. 6 month follow-up data for fistula maturation was available for 75 of 108 patients. Small veins (<2 mm) on preoperative ultrasound matured at a similar rate as larger veins (90% vs. 91.4%, P = 0.833). CONCLUSIONS: Small caliber distal cephalic veins experience a significant degree of dilation under regional and general anesthesia and can successfully be used for AVF creation. Consideration should be made to perform a postanesthesia vein mapping for all patients undergoing access placement despite preoperative venous mapping results.

RAMP and MRAP accessory proteins have selective effects on expression and signalling of the CB1 , CB2 , GPR18 and GPR55 cannabinoid receptors.

BACKGROUND AND PURPOSE: Receptor activity-modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB1 and CB2 , and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies. We investigated whether interactions with RAMPs or MRAPs could explain these phenomena. EXPERIMENTAL APPROACH: Receptors and accessory proteins were co-expressed in HEK-293 cells. Selected receptors were studied at basal expression levels and also with enhanced expression produced by incorporation of a preprolactin signal sequence/peptide (pplss). Cell surface and total expression of receptors and accessory proteins were quantified using immunocytochemistry. Signalling was measured using cAMP (CAMYEL) and G protein dissociation (TRUPATH Gα13 ) biosensors. KEY RESULTS: MRAP2 enhanced surface and total expression of GPR18. Pplss-GPR18 increased detection of cell surface MRAP2. MRAP1α and MRAP2 reduced GPR55 surface and total expression, correlating with reduced constitutive, but not agonist-induced, signalling. GPR55, pplss-CB1 and CB2 reduced detection of MRAP1α at the cell surface. Pplss-CB1 agonist potency was reduced by MRAP2 in Gα13 but not cAMP assays, consistent with MRAP2 reducing pplss-CB1 expression. Some cannabinoid receptors increased RAMP2 or RAMP3 total expression without influencing surface expression. CONCLUSIONS AND IMPLICATIONS: Mutual influences on expression and/or function for specific accessory protein-receptor pairings raises the strong potential for physiological and disease-relevant consequences. Sequestration and/or hetero-oligomerisation of cannabinoid receptors with accessory proteins is a possible novel mechanism for receptor crosstalk.

The piperazine analogue para-fluorophenylpiperazine alters timing of the physiological effects of the synthetic cannabinoid receptor agonist AMB-FUBINACA, without changing its discriminative stimulus, signalling effects, or metabolism.

AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA), which has been associated with substantial abuse and health harm since 2016 in many countries including New Zealand. A characteristic of AMB-FUBINACA use in New Zealand has included the observation that forensic samples (from autopsies) and drugs seized by police have often been found to contain para-fluorophenylpiperazine (pFPP), a relatively little-characterised piperazine analogue that has been suggested to act through 5HT1a serotonin receptors. In the current study, we aimed to characterise the interactions of these two agents in rat physiological endpoints using plethysmography and telemetry, and to examine whether pFPP altered the subjective effects of AMB-FUBINACA in mice trained to differentiate a cannabinoid (THC) from vehicle. Though pFPP did not alter the ability of AMB-FUBINACA to substitute for THC, it did appear to abate some of the physiological effects of AMB-FUBINACA in rats by delaying the onset of AMB-FUBINACA-mediated hypothermia and shortening duration of bradycardia. In HEK cells stably expressing the CB1 cannabinoid receptor, 5HT1a, or both CB1 and 5HT1a, cAMP signalling was recorded using a BRET biosensor (CAMYEL) to assess possible direct receptor interactions. Although low potency pFPP agonism at 5HT1a was confirmed, little evidence for signalling interactions was detected in these assays: additive or synergistic effects on potency or efficacy were not detected between pFPP and AMB-FUBINACA-mediated cAMP inhibition. Experiments utilising higher potency, classical 5HT1a ligands (agonist 8OH-DPAT and antagonist WAY100635) also failed to reveal evidence for mutual CB1/5HT1a interactions or cross-antagonism. Finally, the ability of pFPP to alter the metabolism of AMB-FUBINACA in rat and human liver microsomes into its primary carboxylic acid metabolite via carboxylesterase-1 was assessed by HPLC; no inhibition was detected. Overall, the effects we have observed do not suggest that increased harm/toxicity would result from the combination of pFPP and AMB-FUBINACA.