The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M₁ muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M₁ and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M₁-mEGFP was expressed at levels equal to the M₁ receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M₁-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M₁-mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M₁-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Measuring and understanding the extent and potential significance of quaternary organization of members of the class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs) have both fascinated and frustrated researchers for many years (1, 2). Over time, a wide range of methods have been applied to address this question, and many different GPCRs have been examined. Outcomes have ranged from assertions that such receptors are monomeric and that results consistent with other conclusions reflect either artifacts of the method of measurement or that studies have been performed at nonphysiological levels of expression of the receptor being studied, to those that have suggested rather stable dimeric or tetrameric complexes (1). Only in the case of rhodopsin, the photon receptor expressed at very high levels (in the range of 24,000–30,000 molecules/µm²) in rod outer segments of the eye, have detailed studies been conducted in situ on a class A GPCR. In this example, various studies have shown that rhodopsin is organized as rows of dimers (3, 4). However, to our knowledge, no other GPCR is expressed natively at levels akin to rhodopsin. As such, although a substantial number of studies, generally performed in transfected cell lines or in artificial bilayer systems, have provided evidence that other GPCRs can and do form dimeric and/or higher-order quaternary complexes in a concentration-dependent manner (1, 2), how levels of expression required to observe such complexes relate to expression levels in native cells and tissues has been poorly defined, as is the stability of such complexes and whether they are regulated by ligand binding.Developments in fluorescence fluctuation analysis (FFA) have facilitated efforts to define the oligomeric status of transmembrane receptor proteins (5, 6). Unlike methods based on resonance energy transfer, only a single fluorophore-linked protein is required to be expressed to use FFA. It is, therefore, more practical to use such methods in native cells and tissues if linked to genome-editing approaches and/or the generation of transgenic “knock-in” animal models in which a receptor of interest is replaced with a fluorophore-tagged, modified form of the receptor. Moreover, the recent introduction of fluorescence intensity fluctuation (FIF) spectrometry (7–10) has overcome issues with other methods based on FFA that result in information being compressed due to averaging of oligomer-size data from interrogated regions of interest (RoIs) in which complex mixtures of oligomers of different sizes may be present (7, 8).To define whether the class A M₁ muscarinic acetylcholine receptor is present in hippocampal and cortical neurons as strict monomers or as a range of monomeric, dimeric, and, potentially, oligomeric complexes, we applied FIF spectrometry to images of such neurons isolated from a line of transgenic mice in which we replaced the M₁ receptor with a form of the receptor that includes C-terminally linked monomeric enhanced green fluorescent protein (mEGFP). We first show that both expression levels and function of the introduced M₁-mEGFP construct appear equivalent to the native M₁ receptor in wild-type (WT) mice, using a range of methods and measures ranging from [³H]ligand binding and cell signaling assays to locomotion. We then demonstrate in hippocampal and cortical neurons that in the basal state, the M₁-mEGFP construct is present as a mixture of monomers and dimeric or oligomeric complexes. We also show that the presence of either an agonist or an antagonist ligand promotes monomerization of the receptor. In these studies, we combined analysis of images of a fluorophore-modified receptor in situ with calculation of receptor oligomer complexity. The studies provide a clear and unambiguous answer to a long-standing question that has been the subject of considerable debate (11–13) but that has previously been restricted to studies performed on transfected cell lines. Moreover, these studies are a model for subsequent studies for researchers who plan to explore the topic of dimerization of rhodopsin-family GPCRs.     

Efficacy of a spatial repellent for control of Aedes-borne virus transmission: A cluster-randomized trial in Iquitos,Peru

Over half the world’s population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito–human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = −9.11) and 12.4% (95% CI 4.2%, ∞); z = −2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Aedes-borne viral diseases (ABVDs) [e.g., dengue (DENV), chikungunya, Zika (ZIKV), and yellow fever] are devastating, expanding global public health threats that disproportionally affect low- and middle-income countries. DENV, one of the most rapidly increasing vector-borne infectious diseases, results in ∼400 million infections each year (1, 2), with 4 billion people at risk for infection annually (3). Currently, the primary means for ABVD prevention is controlling the primary mosquito vector, Aedes aegypti. Existing vector control interventions, however, have failed to prevent ABV transmission and epidemics (4–6).There is an urgent need to develop evidence-based guidance for the use of new and existing ABV vector control tools. The evidence base for vector control against ABVs is weak, despite considerable government investments in World Health Organization (WHO)-recommended control of larval habitats (larviciding, container removal) and ultra-low-volume insecticide spraying (4, 5, 7–9). These strategies continue to be implemented despite the lack of rigorously generated data from controlled clinical trials demonstrating they reduce ABV infection or disease (6). The only ABV intervention with a proven epidemiological impact in a cluster-randomized control trial (cRCT) assessed community mobilization to reduce mosquito larval habitats (10). A recent test-negative trial with Wolbachia-infected mosquitoes reported a significant reduction of DENV illness in Indonesia (11).Spatial repellents (SRs) are devices that contain volatile active ingredients that disperse in air. The active ingredients can repel mosquitoes from entering a treated space, inhibit attraction to human host cues, or disrupt mosquito biting and blood-feeding behavior and, thus, interfere with mosquito–human contact (12–14). Any of these outcomes reduce the probability of pathogen transmission. Pyrethroid-based SRs have shown efficacy in reducing malaria infections in China (15) and Indonesia (16). There have, however, been no clinical trials evaluating the protective efficacy (PE) of SRs against ABV infection or disease.To generate evidence for public health consideration, we conducted a double-blinded, parallel cRCT to demonstrate and quantify the PE of a transfluthrin-based SR to reduce ABV infection incidence over 2 y in a human cohort in Iquitos, Peru.     

The Unrealized Potential of Cervical Smears

We have a simple, efficient method for early detection of cervical cancer, yet there has been little change in mortality of this disease in 10 years. We need some new approach if we are to reach more women with this screening method, including prenatal patients under 25.     

Getting To Outcomes: a community-based participatory approach to preventive interventions

Bouget, D. (1998). The Juppé Plan and the future of the French social welfare system. Journal of European Social Policy, 8(2) 154-172.     

Evaluation of acute fixation strength of absorbable and nonabsorbable barrier coated mesh secured with fibrin sealant

Introduction  

The purpose of this study was to evaluate the acute fixation strength of fibrin sealant as an alternative fixation method for laparoscopic ventral hernia repair (LVHR) when utilized with absorbable and nonabsorbable barrier meshes.