Use of a new proximity assay (NanoBRET) to investigate the ligand binding characteristics of three fluorescent ligands to the human β1-adrenoceptor expressed in HEK-293 cells

Previous research has indicated that allosteric interactions across the dimer interface of β1-adrenoceptors may be responsible for a secondary low affinity binding conformation. Here we have investigated the potential for probe dependence, in the determination of antagonist pKi values at the human β1-adenoceptor, which may result from such allosterism interactions. Three fluorescent β1-adrenoceptor ligands were used to investigate this using bioluminescence energy transfer (BRET) between the receptor-bound fluorescent ligand and the N-terminal NanoLuc tag of a human β1-adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high affinity specific binding to the NanoLuc-β1-adrenoceptor with each of the three fluorescent ligands yielding KD values of 87.1 ± 10nM (n=8), 38.1 ± 12nM (n=7), 13.4 ± 2nM (n=14) for propranolol-Peg8-BY630, propranolol-3(Ala-Ala)-BY630 and CGP-12177- TMR respectively. Parallel radioligand-binding studies with 3H-CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc- 31-adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein-1). Following a 1h incubation with fluorescent ligands and β1-adrenoceptor competing antagonists, there were significant differences (p < 0.001) in the pKi values obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and 3H-CGP 12177. However, increasing the incubation time to 2h removed these significant differences. The data obtained show that the NanoBRET assay can be applied successfully to study ligand-receptor interactions at the human β1-adrenoceptor. However, the study also emphasizes the importance of ensuring that both the fluorescent and competing ligands are in true equilibrium before interpretations regarding probe dependence can be made

The Mississippi delta health collaborative medication therapy management model: Public health and pharmacy working together to improve population health in the Mississippi delta

© 2020. Introduction The Mississippi Delta has high rates of chronic disease and is known for its poor health outcomes and health disparities. University of Mississippi School of Pharmacy (UMSOP) and the Mississippi State Department of Health partnered in 2009 through the Mississippi Delta Health Collaborative to reduce health disparities and improve clinical outcomes by expanding the UMSOP\u27s evidence-based medication therapy management (MTM) initiative, focused in Mississippi\u27s 18-county Delta region, to federally qualified health centers (FQHCs) in 4 of those counties. Methods Between January 2009 and August 2018, the MTM initiative targeted FQHC patients aged 18 years or older with a diagnosis of diabetes, hypertension, and/or dyslipidemia. Pharmacists initially met face-to-face with patients to review all medications, provide education about chronic diseases, identify and resolve drug therapy problems, and take appropriate actions to help improve the effectiveness of medication therapies. Clinical parameters evaluated were systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, and hemoglobin A1c (HbA1c). Results The analysis included 335 patients with hypertension (n = 287), dyslipidemia (n = 131), and/or diabetes (n = 331). Significant mean reductions occurred in the following metrics: SBP (7.1 mm Hg), DBP (6.3 mm Hg), LDL cholesterol (24.9 mg/dL), triglycerides (45.5 mg/dL), total cholesterol (37.7 mg/dL), and HbA1c (1.6% [baseline ≥6%] and 1.9% [baseline ≥9%]). Conclusion Despite the cultural and environmental disadvantages present in the Mississippi Delta, the integrated MTM treatment program demonstrated significant health improvements across 3 chronic diseases: hypertension, dyslipidemia, and diabetes. This model demonstrates that a partnership between public health and pharmacy is a successful and innovative approach to care

Daily mood, partner support, sexual interest, and sexual activity among adolescent women

This is a post print version of the article. The official published version can be accessed from the link below.Objective: to examine day-to-day associations of coitus, sexual interest, partner emotional support, negative mood and positive mood among adolescent women. Methods: Women (ages 14 – 17 at enrollment; N=146) enrolled from one of three adolescent primary care clinics completed up to five 84-day diaries over a 27-month period. The diaries assessed partner interactions, sexual activity, substance use and mood. Partner-specific measures assessed on each day included partner emotional support (4 items; alpha = 0.94), argument with a partner (no/yes) and coitus (no/yes). Within-day measures assessed marijuana use (no/yes), Positive Mood (3-items; alpha = 0. 86); Negative Mood (3-items; alpha = 0.82) and Sexual Interest (1-item). Lagged measures of mood and sexual activity were included in multivariate models to control for recent mood and sexual behavior effects on current day mood and coitus. Two main analyses were conducted: coitus as a predictor of positive and negative mood; and the role of positive and negative mood as predictors of coitus. Analyses were conducted by multivariate mixed effect regression and mixed effect logistic regression models. Results: Data represent 28,376 days from 146 participants. The average number of diary days was 194 days per participant. Sexual activity was reported on 8.3% of days, with condoms used for 27.0% of these coital events. Marijuana was used on 11% of days. Significant predictors of positive mood on a given day included partner support, marijuana use, and coitus. Negative mood was associated with having an argument with a partner and with prior day coitus. Predictors of coitus on a given day included age (Odds ratio = 1.22), increased coital frequency in previous week (OR = 1.49), coitus on the previous day (1.21), increased same-day sexual interest (OR = 2.8) and decreased same-day negative mood (OR = 0.92). Conclusions: The data demonstrate complex associations of sexual interest, mood, partner interactions and sexual activity

GALA: an international multicentre randomised trial comparing general anaesthesia versus local anaesthesia for carotid surgery

Background: Patients who have severe narrowing at or near the origin of the internal carotid artery as a result of atherosclerosis have a high risk of ischaemic stroke ipsilateral to the arterial lesion. Previous trials have shown that carotid endarterectomy improves long-term outcomes, particularly when performed soon after a prior transient ischaemic attack or mild ischaemic stroke. However, complications may occur during or soon after surgery, the most serious of which is stroke, which can be fatal. It has been suggested that performing the operation under local anaesthesia, rather than general anaesthesia, may be safer. Therefore, a prospective, randomised trial of local versus general anaesthesia for carotid endarterectomy was proposed to determine whether type of anaesthesia influences peri-operative morbidity and mortality, quality of life and longer term outcome in terms of stroke-free survival. Methods/design: A two-arm, parallel group, multicentre randomised controlled trial with a recruitment target of 5000 patients. For entry into the study, in the opinion of the responsible clinician, the patient requiring an endarterectomy must be suitable for either local or general anaesthesia, and have no clear indication for either type. All patients with symptomatic or asymptomatic internal carotid stenosis for whom open surgery is advised are eligible. There is no upper age limit. Exclusion criteria are: no informed consent; definite preference for local or general anaesthetic by the clinician or patient; patient unlikely to be able to co-operate with awake testing during local anaesthesia; patient requiring simultaneous bilateral carotid endarterectomy; carotid endarterectomy combined with another operation such as coronary bypass surgery; and, the patient has been randomised into the trial previously. Patients are randomised to local or general anaesthesia by the central trial office. The primary outcome is the proportion of patients alive, stroke free ( including retinal infarction) and without myocardial infarction 30 days post-surgery. Secondary outcomes include the proportion of patients alive and stroke free at one year; health related quality of life at 30 days; surgical adverse events, re-operation and re-admission rates; the relative cost of the two methods of anaesthesia; length of stay and intensive and high dependency bed occupancy

Classification of hyperbolic Dynkin diagrams, root lengths and Weyl group orbits

We give a criterion for a Dynkin diagram, equivalently a generalized Cartan matrix, to be symmetrizable. This criterion is easily checked on the Dynkin diagram. We obtain a simple proof that the maximal rank of a Dynkin diagram of compact hyperbolic type is 5, while the maximal rank of a symmetrizable Dynkin diagram of compact hyperbolic type is 4. Building on earlier classification results of Kac, Kobayashi-Morita, Li and Sa\c{c}lio\~{g}lu, we present the 238 hyperbolic Dynkin diagrams in ranks 3-10, 142 of which are symmetrizable. For each symmetrizable hyperbolic generalized Cartan matrix, we give a symmetrization and hence the distinct lengths of real roots in the corresponding root system. For each such hyperbolic root system we determine the disjoint orbits of the action of the Weyl group on real roots. It follows that the maximal number of disjoint Weyl group orbits on real roots in a hyperbolic root system is 4.Comment: J. Phys. A: Math. Theor (to appear

Molecular phylodynamics of the heterosexual HIV epidemic in the United Kingdom

The heterosexual risk group has become the largest HIV infected group in the United Kingdom during the last 10 years, but little is known of the network structure and dynamics of viral transmission in this group. The overwhelming majority of UK heterosexual infections are of non-B HIV subtypes, indicating viruses originating among immigrants from sub-Saharan Africa. The high rate of HIV evolution, combined with the availability of a very high density sample of viral sequences from routine clinical care has allowed the phylodynamics of the epidemic to be investigated for the first time. Sequences of the viral protease and partial reverse transcriptase coding regions from 11,071 patients infected with HIV of non-B subtypes were studied. Of these, 2774 were closely linked to at least one other sequence by nucleotide distance. Including the closest sequences from the global HIV database identified 296 individuals that were in UK-based groups of 3 or more individuals. There were a total of 8 UK-based clusters of 10 or more, comprising 143/2774 (5%) individuals, much lower than the figure of 25% obtained earlier for men who have sex with men (MSM). Sample dates were incorporated into relaxed clock phylogenetic analyses to estimate the dates of internal nodes. From the resulting time-resolved phylogenies, the internode lengths, used as estimates of maximum transmission intervals, had a median of 27 months overall, over twice as long as obtained for MSM (14 months), with only 2% of transmissions occurring in the first 6 months after infection. This phylodynamic analysis of non-B subtype HIV sequences representing over 40% of the estimated UK HIV-infected heterosexual population has revealed heterosexual HIV transmission in the UK is clustered, but on average in smaller groups and is transmitted with slower dynamics than among MSM. More effective intervention to restrict the epidemic may therefore be feasible, given effective diagnosis programmes

Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection

Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals