Promotional Language (Hype) in Abstracts of Publications of National Institutes of Health-Funded Research, 1985-2020.

Importance: Investigators applying for National Institutes of Health (NIH) funding increasingly use promotional language (or hype) that has the potential to undermine objective evaluation. Whether or not the same investigators use hype in subsequent research reports has yet to be investigated. Objective: To assess changes in the use of hype in journal abstracts reporting research funded by the NIH and to compare those trends with previously reported trends in the associated NIH funding applications. Design, Setting, and Participants: This cross-sectional study assessed trends (from 1985 to 2020) in the use of promotional adjectives in abstracts of journal articles reporting NIH-funded research, and then compared those trends with previously reported trends for the associated NIH funding applications. Articles included in analyses had abstracts available in PubMed. Main Outcomes and Measures: Absolute change for the 139 adjective forms that have previously been identified as representing hype in NIH funding applications was measured as the difference in frequency between 1985 and 2020. Relative change was measured as the percentage change in frequency in 2020 relative to 1985, or the first year of occurrence. Consistency of change was measured by the rank order correlation (Kendall τ). Concordance between longitudinal trends in the journal abstracts and NIH funding applications was measured by the rank-order cross-correlation. Results: In a total of 2 394 480 journal abstracts, all 139 adjective forms were identified in 2 793 592 total occurrences. Among these adjectives, 133 increased in absolute frequency by 5335 words per million (wpm), with a mean (SD) relative increase of 1404% (2371%). The largest absolute increases were for novel (524 wpm), important (414 wpm), and key (378 wpm). The largest relative increases were for scalable (22 wpm [19 964%]), unmet (23 wpm [12 126%]), and tailored (40 wpm [8169%]). The mean (SD) correlation for all adjectives was 0.70 (0.30) with 95 adjectives showing a strong positive correlation (τ > 0.7; P < .001), 24 a moderate positive correlation (0.5 < τ < 0.7; P < .001), and 3 a moderate negative correlation (-0.5 < τ < -0.7; P < .001). The mean (SD) cross-correlation was 0.64 (0.19) with 61 of the 139 adjectives showing a strong positive cross-correlations (τ > 0.7; P < .001), 53 a moderate positive cross-correlations (0.5 < τ < 0.7; P < .001), and 3 a moderate negative cross-correlation (-0.7 < τ < -0.5; P < .001). Conclusions and Relevance: In this analysis of journal abstracts reporting NIH-funded research from 1985 to 2020, levels of promotional language were found to be increasing and trends were closely associated with previously reported trends in the related NIH funding applications. This suggests that increasing levels of salesmanship may in part be a downstream effect of salesmanship infused during earlier stages of the research cascade.

Impact of hype on clinicians' evaluation of trials - a pilot study.

Objective: The purpose of this study was to determine the practicality of using a teleconferencing platform to assess the effect of hype on clinicians' evaluations of reports of clinical trials in spinal care. Methods: Twelve chiropractic clinicians were interviewed via a videoconferencing application. Interviews were recorded and timed. Participant behaviour was monitored for compliance with the protocol. Differences between participants numerical ratings of hyped and non-hyped abstracts based on four measures of quality were analysed using pairwise comparisons (Wilcoxon signed rank test for independent samples). In addition, a linear mixed effects model was fitted with condition (i.e. hype vs. no hype) as a fixed effect and participant and abstract as random effects. Results: The interviews and data analysis were conducted without significant technical difficulty. Participant compliance was high, and no harms were reported. There were no statistically significant differences in the quality rankings of hyped versus non-hyped abstracts. Conclusion: The use of a videoconferencing platform to measure the effects of hype on clinicians' evaluations of abstracts of clinical trials is practical and an adequately powered study is justified. Lack of statistically significant results may well be due to low participant numbers.

Objectif: L'objectif de cette étude était de déterminer s'il était possible d'utiliser une plateforme de téléconférence pour mesurer l'effet du battage médiatique sur les évaluations par les cliniciens des rapports d'essais cliniques dans le domaine des soins de la colonne vertébrale. Méthodes: Douze chiropracticiens ont été interrogés par le biais d'une application de vidéoconférence. Les entretiens ont été enregistrés et chronométrés. Le comportement des participants a été contrôlé pour s'assurer qu'ils respectaient le protocole. Les différences entre les évaluations numériques des participants pour les résumés avec et sans publicité, basées sur quatre mesures de qualité, ont été analysées en utilisant des comparaisons par paire (test de rang signé de Wilcoxon pour les échantillons indépendants). En outre, un modèle linéaire à effets mixtes a été ajusté avec la condition (c'est-à-dire avec ou sans battage publicitaire) comme effet fixe et le participant et le résumé comme effets aléatoires. Résultats: Les entretiens et l'analyse des données se sont déroulés sans difficulté technique majeure. Les participants se sont montrés très coopératifs et aucun problème n'a été signalé. Il n'y a pas eu de différences statistiquement significatives dans le classement de la qualité des résumés avec ou sans battage médiatique. Conclusion: L'utilisation d'une plate-forme de vidéoconférence pour mesurer les effets du battage médiatique sur les évaluations des résumés d'essais cliniques par les cliniciens est pratique et une étude suffisamment puissante est justifiée. L'absence de résultats statistiquement significatifs pourrait bien être due au faible nombre de participants.

Synthesis and Antagonist Activity of Methyllycaconitine Analogues on Human α7 Nicotinic Acetylcholine Receptors.

Methyllycaconitine (MLA), 1, is a naturally occurring norditerpenoid alkaloid that is a highly potent (IC50 = 2 nM) selective antagonist of α7 nicotinic acetylcholine receptors (nAChRs). Several structural factors affect its activity such as the neopentyl ester side-chain and the piperidine ring N-side-chain. The synthesis of simplified AE-bicyclic analogues 14-21 possessing different ester and nitrogen side-chains was achieved in three steps. The antagonist effects of synthetic analogues were examined on human α7 nAChRs and compared to that of MLA 1. The most efficacious analogue (16) reduced α7 nAChR agonist responses [1 nM acetylcholine (ACh)] to 53.2 ± 1.9% compared to 3.4 ± 0.2% for MLA 1. This demonstrates that simpler analogues of MLA 1 possess antagonist effects on human α7 nAChRs but also indicates that further optimization may be possible to achieve antagonist activity comparable to that of MLA 1.

Potentiation and allosteric agonist activation of α7 nicotinic acetylcholine receptors: binding sites and hypotheses.

Considerable progress has been made in recent years towards the identification and characterisation of novel subtype-selective modulators of nicotinic acetylcholine receptors (nAChRs). In particular, this has focussed on modulators of α7 nAChRs, a nAChR subtype that has been identified as a target for drug discovery in connection with a range of potential therapeutic applications. This review focusses upon α7-selective modulators that bind to receptor sites other than the extracellular 'orthosteric' agonist binding site for the endogenous agonist acetylcholine (ACh). Such compounds include those that are able to potentiate responses evoked by orthosteric agonists such as ACh (positive allosteric modulators; PAMs) and those that are able to activate α7 nAChRs by direct allosteric activation in the absence of an orthosteric agonist (allosteric agonists or 'ago-PAMs'). There has been considerable debate about the mechanism of action of α7-selective PAMs and allosteric agonists, much of which has centred around identifying the location of their binding sites on α7 nAChRs. Based on a variety of experimental evidence, including recent structural data, there is now clear evidence indicating that at least some α7-selective PAMs bind to an inter-subunit site located in the transmembrane domain. In contrast, there are differing hypotheses about the site or sites at which allosteric agonists bind to α7 nAChRs. It will be argued that the available evidence supports the conclusion that direct allosteric activation by allosteric agonists/ago-PAMs occurs via the same inter-subunit transmembrane site that has been identified for several α7-selective PAMs.

Imaging and Molecular Annotation of Xenographs and Tumours (IMAXT): High throughput data and analysis infrastructure.

With the aim of producing a 3D representation of tumors, imaging and molecular annotation of xenografts and tumors (IMAXT) uses a large variety of modalities in order to acquire tumor samples and produce a map of every cell in the tumor and its host environment. With the large volume and variety of data produced in the project, we developed automatic data workflows and analysis pipelines. We introduce a research methodology where scientists connect to a cloud environment to perform analysis close to where data are located, instead of bringing data to their local computers. Here, we present the data and analysis infrastructure, discuss the unique computational challenges and describe the analysis chains developed and deployed to generate molecularly annotated tumor models. Registration is achieved by use of a novel technique involving spherical fiducial marks that are visible in all imaging modalities used within IMAXT. The automatic pipelines are highly optimized and allow to obtain processed datasets several times quicker than current solutions narrowing the gap between data acquisition and scientific exploitation.

Trends in the Use of Promotional Language (Hype) in National Institutes of Health Funding Opportunity Announcements, 1992-2020.

This cross-sectional study examines changes from 1992 to 2020 in the use of promotional language in National Institutes of Health (NIH) funding opportunity announcements in comparison with trends reported in NIH grant applications.

Research priorities to reduce the impact of musculoskeletal disorders: a priority setting exercise with the child health and nutrition research initiative method.

Involving research users in setting priorities for research is essential to ensure the outcomes are patient-centred and maximise its value and impact. The Musculoskeletal Disorders Research Advisory Group Versus Arthritis led a research priority setting exercise across musculoskeletal disorders. The Child Health and Nutrition Research Initiative (CHNRI) method of setting research priorities with a range of stakeholders was used, involving four stages and two surveys, to: (1) gather research uncertainties, (2) consolidate these, (3) score uncertainties against importance and impact, and (4) analyse scoring for prioritisation. 213 people responded to the first survey and 285 people to the second, representing clinicians, researchers, and people with musculoskeletal disorders. Key priorities included developing and testing new treatments, better treatment targeting, early diagnosis, prevention, and better understanding and management of pain, with an emphasis on understanding underpinning mechanisms. We present a call to action to researchers and funders to target these priorities.

Trends in the Use of Promotional Language (Hype) in Abstracts of Successful National Institutes of Health Grant Applications, 1985-2020.

Importance: The integrity of the grant application process is important to the success of the entire research enterprise. However, little information is available concerning the prevalence and evolution of subjective or promotional language ("hype") that has the potential to undermine objectivity in the writing and evaluation of grant applications. Objective: To assess changes over time in the use of hype in abstracts of National Institutes of Health (NIH) grant applications. Design, Setting, and Participants: This cross-sectional study assessed the prevalence of promotional adjectives in abstracts in the NIH archive from 1985 to 2020. Main Outcomes and Measures: From all abstracts in the NIH RePORTER (Research Portfolio Online Reporting Tools: Expenditures and Results) archive, adjectives were automatically extracted, and their frequencies in the most recent year (2020) were assessed relative to the start year (1985). Adjectives that shifted significantly in frequency and that carried a promotional sense (ie, hype) were retained, and patterns of change were assessed by plotting yearly frequencies (1985-2020). By grouping the adjectives based on shared semantic properties, broad meanings commonly expressed by hype were identified. Absolute change was measured as the difference in normalized frequency between 1985 and 2020. Relative change was measured as the percentage change in normalized frequency in 2020 relative to 1985, or the first year of occurrence. Results: In total, 901 717 abstracts were analyzed and 139 adjective forms were identified as hype. Among these 139 adjective forms, 130 hype adjectives increased in frequency by 7690 words per million (wpm) (mean [SD] relative increase, 1378% [3132%]), while 9 hype adjectives decreased in frequency by 686 wpm (mean [SD] relative decrease, 44% [18%]). The largest absolute increases were for the terms novel (1054 wpm), critical (555 wpm), and key (461 wpm), while the largest relative increases were for the terms sustainable (25 157%), actionable (16 114%), and scalable (13 029%). Hype most often serves to promote the significance, novelty, scale, and rigor of a project; the utility of the expected outcomes; the qualities of the investigators and research environment; and the gravity of the problem; as well as conveying the personal attitudes of the applicants. Conclusions and Relevance: Levels of hype in successful NIH grant applications have increased over time from 1985 to 2020. The findings in this study should serve to sensitize applicants, reviewers, and funding agencies to the increasing prevalence of subjective, promotional language in funding applications.

Stoichiometry-Selective Antagonism of α4ß2 Nicotinic Acetylcholine Receptors by Fluoroquinolone Antibiotics.

Quinolone antibiotics disrupt bacterial DNA synthesis by interacting with DNA gyrase and topoisomerase IV. However, in addition, they have been shown to act as inhibitors of pentameric ligand-gated ion channels such as GABAA receptors and the α7 nicotinic acetylcholine receptor (nAChR). In the present study, we have examined the effects of quinolone antibiotics on the human α4ß2 nAChR, an important subtype that is widely expressed in the central nervous system. A key feature of α4ß2 nAChRs is their ability to coassemble into two distinct stoichiometries, (α4)2(ß2)3 and (α4)3(ß2)2, which results in differing affinities for acetylcholine. The effects of nine quinolone antibiotics were examined on both stoichiometries of the α4ß2 receptor by two-electrode voltage-clamp recording. All compounds exhibited significant inhibition of α4ß2 nAChRs. However, all of the fluoroquinolone antibiotics examined (ciprofloxacin, enoxacin, enrofloxacin, difloxacin, norfloxacin, pefloxacin, and sparfloxacin) were significantly more potent inhibitors of (α4)2(ß2)3 nAChRs than of (α4)3(ß2)2 nAChRs. This stoichiometry-selective effect was most pronounced with pefloxacin, which inhibited (α4)2(ß2)3 nAChRs with an IC50 of 26.4 ± 3.4 µM but displayed no significant inhibition of (α4)3(ß2)2 nAChRs. In contrast, two nonfluorinated quinolone antibiotics (cinoxacin and oxolinic acid) exhibited no selectivity in their inhibition of the two stoichiometries of α4ß2. Computational docking studies suggest that pefloxacin interacts selectively with an allosteric transmembrane site at the ß2(+)/ß2(-) subunit interface, which is consistent with its selective inhibition of (α4)2(ß2)3. These findings concerning the antagonist effects of fluoroquinolones provide further evidence that differences in the subunit stoichiometry of heteromeric nAChRs can result in substantial differences in pharmacological properties.

Agonist and antagonist properties of an insect GABA-gated chloride channel (RDL) are influenced by heterologous expression conditions.

Pentameric ligand-gated ion channels (pLGICs) activated by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) are expressed widely in both vertebrate and invertebrate species. One of the best characterised insect GABA-gated chloride channels is RDL, an abbreviation of 'resistance to dieldrin', that was originally identified by genetic screening in Drosophila melanogaster. Here we have cloned the analogous gene from the bumblebee Bombus terrestris audax (BtRDL) and examined its pharmacological properties by functional expression in Xenopus oocytes. Somewhat unexpectedly, the sensitivity of BtRDL to GABA, as measured by its apparent affinity (EC50), was influenced by heterologous expression conditions. This phenomenon was observed in response to alterations in the amount of cRNA injected; the length of time that oocytes were incubated before functional analysis; and by the presence or absence of a 3' untranslated region. In contrast, similar changes in expression conditions were not associated with changes in apparent affinity with RDL cloned from D. melanogaster (DmRDL). Changes in apparent affinity with BtRDL were also observed following co-expression of a chaperone protein (NACHO). Similar changes in apparent affinity were observed with an allosteric agonist (propofol) and a non-competitive antagonist (picrotoxinin), indicating that expression-depended changes are not restricted to the orthosteric agonist binding site. Interestingly, instances of expression-dependent changes in apparent affinity have been reported previously for vertebrate glycine receptors, which are also members of the pLGIC super-family. Our observations with BtRDL are consistent with previous data obtained with vertebrate glycine receptors and indicates that agonist and antagonist apparent affinity can be influenced by the level of functional expression in a variety of pLGICs.

The passive voice and comprehensibility of biomedical texts: An experimental study with 2 cohorts of chiropractic students.

OBJECTIVE:: Authors in the health sciences are encouraged to write in the active voice in the belief that this enhances comprehensibility. Hence, the purpose of this study was to compare objectively measured and subjectively perceived comprehensibility of texts in which one voice or the other was highly prevalent. METHODS:: Objectively rated comprehensibility was obtained by presenting 161 2nd-year chiropractic students with questions pertaining to 2 methods sections of biomedical articles, each presented in its original form with high prevalence of the passive voice, and in a manipulated form with all main verbs in the active voice. The difficulties and sensitivities of questions were compared for the 2 forms of each text. Comprehensibility was obtained by asking students to rate the comprehensibility of authentic sentences from biomedical manuscripts and matched manipulated form in which the voice of the main verb had been changed. Differences in comprehensibility between the 2 texts were assessed with a dependent t test. RESULTS:: There were no significant differences in the difficulties or sensitivities of questions pertaining to the 2 original texts written in the passive voice versus the active voice ( p > .35 for all comparisons). Students rated sentences written in the passive voice as marginally more comprehensible than sentences written in the active voice ( p = .003 per 2-tailed paired t test). CONCLUSION:: The texts written in the active voice were not more comprehensible than texts written in the passive voice. The results of this study do not support editorial guidelines that favor active voice over passive voice.

Identification by virtual screening and functional characterisation of novel positive and negative allosteric modulators of the α7 nicotinic acetylcholine receptor.

Several previous studies have demonstrated that the activity of neurotransmitters acting on ligand-gated ion channels such as the nicotinic acetylcholine receptor (nAChR) can be altered by compounds binding to allosteric modulatory sites. In the case of α7 nAChRs, both positive and negative allosteric modulators (PAMs and NAMs) have been identified and have attracted considerable interest. A recent study, employing revised structural models of the transmembrane domain of the α7 nAChR in closed and open conformations, has provided support for an inter-subunit transmembrane allosteric binding site (Newcombe et al 2017). In the present study, we have performed virtual screening of the DrugBank database using pharmacophore queries that were based on the predicted binding mode of PAMs to α7 nAChR structural models. A total of 81 compounds were identified in the DrugBank database, of which the 25 highest-ranked hits corresponded to one of four previously-identified therapeutic compound groups (carbonic anhydrase inhibitors, cyclin-dependent kinase inhibitors, diuretics targeting the Na+-K+-Cl- cotransporter, and fluoroquinolone antibiotics targeting DNA gyrase). The top-ranked compound from each of these four groups (DB04763, DB08122, furosemide and pefloxacin, respectively) was tested for its effects on human α7 nAChR expressed in Xenopus oocytes using two-electrode voltage-clamp electrophysiology. These studies, conducted with wild-type, mutant and chimeric receptors, resulted in all four compounds exerting allosteric modulatory effects. While DB04763, DB08122 and pefloxacin were antagonists, furosemide potentiated ACh responses. Our findings, supported by docking studies, are consistent with these compounds acting as PAMs and NAMs of the α7 nAChR via interaction with a transmembrane site.

Nicotinic acetylcholine receptors.

This themed section of the British Journal of Pharmacology is the product of a conference that focussed on nicotinic ACh receptors (nAChRs) that was held on the Greek island of Crete from 7 to 11 May 2017. 'Nicotinic acetylcholine receptors 2017' was the fourth in a series of triennial international meetings that have provided a regular forum for scientists working on all aspects of nAChRs to meet and to discuss new developments. In addition to many of the regular participants, each meeting has also attracted a new group of scientists working in a fast-moving area of research. This themed section comprises both review articles and original research papers on nAChRs. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc/.

Diversity of Nicotinic Acetylcholine Receptor Positive Allosteric Modulators Revealed by Mutagenesis and a Revised Structural Model.

By combining electrophysiological and computational approaches we have examined a series of positive allosteric modulators (PAMs) acting on the human α7 nicotinic acetylcholine receptor (nAChR). Electrophysiological studies have focused on three α7-selective PAMs (A-867744, TBS-516, and TQS) that display similar effects on wild-type α7 nAChRs. In addition to potentiating agonist-evoked responses, all three compounds reduce receptor desensitization and, consequently, are classed as type II PAMs. Despite having similar effects on wild-type receptors, A-867744 was found to have profoundly differing effects on mutated receptors compared with TBS-516 and TQS, a finding that is consistent with previous studies indicating that A-867744 may have a different mechanism of action compare with other α7-selective type II PAMs. Due to evidence that these PAMs bind within the α7 nAChR transmembrane region, we generated and validated new structural models of α7. Importantly, we have corrected a previously identified error in the transmembrane region of the original cryo-electron microscopy Torpedo model; the only pentameric ligand-gated ion channel imaged in a native lipid membrane. Real-space refinement was used to generate closed and open conformations on which the α7 models were based. Consensus docking with an extended series of PAMs with chemical similarity to A-867744, TBS-516, and TQS suggests that all bind to a broadly similar intersubunit transmembrane site. However, differences in the predicted binding of A-867744, compared with TBS-516 and TQS, may help to explain the distinct functional effects of A-867744. Thus, our revised structural models may provide a useful tool for interpreting functional effects of PAMs.

Mutations on M3 helix of Plutella xylostella glutamate-gated chloride channel confer unequal resistance to abamectin by two different mechanisms.

Abamectin is one of the most widely used avermectins for agricultural pests control, but the emergence of resistance around the world is proving a major threat to its sustained application. Abamectin acts by directly activating glutamate-gated chloride channels (GluCls) and modulating other Cys-loop ion channels. To date, three mutations occurring in the transmembrane domain of arthropod GluCls are associated with target-site resistance to abamectin: A309V in Plutella xylostella GluCl (PxGluCl), G323D in Tetranychus urticae GluCl1 (TuGluCl1) and G326E in TuGluCl3. To compare the effects of these mutations in a single system, A309V/I/G and G315E (corresponding to G323 in TuGluCl1 and G326 in TuGluCl3) substitutions were introduced individually into the PxGluCl channel. Functional analysis using Xenopus oocytes showed that the A309V and G315E mutations reduced the sensitivity to abamectin by 4.8- and 493-fold, respectively. In contrast, the substitutions A309I/G show no significant effects on the response to abamectin. Interestingly, the A309I substitution increased the channel sensitivity to glutamate by one order of magnitude (∼12-fold). Analysis of PxGluCl homology models indicates that the G315E mutation interferes with abamectin binding through a steric hindrance mechanism. In contrast, the structural consequences of the A309 mutations are not so clear and an allosteric modification of the binding site is the most likely mechanism. Overall the results show that both A309V and G315E mutations may contribute to target-site resistance to abamectin and may be important for the future prediction and monitoring of abamectin resistance in P. xylostella and other arthropod pests.

Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function.

Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and ß subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a ß subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL.

Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance.

Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol.

Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies.

A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella.

Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella.

Allosteric modulation of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are receptors for the neurotransmitter acetylcholine and are members of the 'Cys-loop' family of pentameric ligand-gated ion channels (LGICs). Acetylcholine binds in the receptor extracellular domain at the interface between two subunits and research has identified a large number of nAChR-selective ligands, including agonists and competitive antagonists, that bind at the same site as acetylcholine (commonly referred to as the orthosteric binding site). In addition, more recent research has identified ligands that are able to modulate nAChR function by binding to sites that are distinct from the binding site for acetylcholine, including sites located in the transmembrane domain. These include positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), silent allosteric modulators (SAMs) and compounds that are able to activate nAChRs via an allosteric binding site (allosteric agonists). Our aim in this article is to review important aspects of the pharmacological diversity of nAChR allosteric modulators and to describe recent evidence aimed at identifying binding sites for allosteric modulators on nAChRs.