Systematic generation of in vivo G protein-coupled receptor mutants in the rat

G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies

Prevalence of mental disorders from adolescence through early adulthood in American Indian and First Nations communities

Indigenous communities lack representation in psychiatric epidemiology despite disproportionate exposure to risk factors. We document the cumulative and 12-month prevalence of psychiatric disorders across the early life course among a sample of Indigenous young adults and compare prospective and retrospective reporting of lifetime mental disorders. This community-based participatory research includes data from 735 Indigenous people from 8 reservations/reserves. Personal interviews were conducted between 2002–2010 and 2017–2018 totaling 9 waves; diagnostic assessments of DSM-IV-TR alcohol abuse/dependence, marijuana use/dependence, other substance abuse/dependence, generalized anxiety disorder, major depressive disorder, dysthymic disorder, and attention deficit/hyperactivity disorder occurred at waves 1 (mean age = 11.1 years), 4 (mean age = 14.3 years), 6 (mean age = 16.2 years), 8 (mean age = 18.3 years), and 9 (mean age = 26.3 years). Cumulative lifetime psychiatric disorders reached 77.3% and lifetime comorbidity 56.4% by wave 9. Past-year prevalence and comorbidity at wave 9 were 28.7% and 6.7%, respectively. Substance use disorders (SUDs) were most common with peak past-year prevalence observed when participants were on average 16.3 years old then declining thereafter. Trends in early life course psychiatric disorders in this study with Indigenous participants highlight cultural variations in psychiatric epidemiology including surprisingly low rates of internalizing disorders in the face of risk factors, disproportionately high rates of early-onset and lifetime SUD, and lower rates of past-year SUD in early adulthood compared with prior research.Peer reviewedSociolog

Down-Regulation of Honey Bee IRS Gene Biases Behavior toward Food Rich in Protein

Food choice and eating behavior affect health and longevity. Large-scale research efforts aim to understand the molecular and social/behavioral mechanisms of energy homeostasis, body weight, and food intake. Honey bees (Apis mellifera) could provide a model for these studies since individuals vary in food-related behavior and social factors can be controlled. Here, we examine a potential role of peripheral insulin receptor substrate (IRS) expression in honey bee foraging behavior. IRS is central to cellular nutrient sensing through transduction of insulin/insulin-like signals (IIS). By reducing peripheral IRS gene expression and IRS protein amount with the use of RNA interference (RNAi), we demonstrate that IRS influences foraging choice in two standard strains selected for different food-hoarding behavior. Compared with controls, IRS knockdowns bias their foraging effort toward protein (pollen) rather than toward carbohydrate (nectar) sources. Through control experiments, we establish that IRS does not influence the bees' sucrose sensory response, a modality that is generally associated with food-related behavior and specifically correlated with the foraging preference of honey bees. These results reveal a new affector pathway of honey bee social foraging, and suggest that IRS expressed in peripheral tissue can modulate an insect's foraging choice between protein and carbohydrate sources

Thermal Stability of the Human Immunodeficiency Virus Type 1 (HIV-1) Receptors, CD4 and CXCR4, Reconstituted in Proteoliposomes

BACKGROUND: The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4. METHODOLOGY/PRINCIPAL FINDINGS: We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E(a)) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T(i)) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E(a) of 278 kJ/mol (66.5 kcal/mol), and a T(i) of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules. CONCLUSIONS/SIGNIFICANCE: Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors

Computing Highly Correlated Positions Using Mutual Information and Graph Theory for G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions) were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families

Toxicity of, and development of predictive models for, substituted phenols to Ceridaphnia cf. dubia and Vibrio fischeri

Twelve substituted phenols that have a polar narcotic mode of action had their acute toxicity to the freshwater cladoceran Ceriodaphnia cf. dubia and the marine bacterium Vibrio fischeri determined. The 48-h EC50 (immobilisation) values of the chemicals to C. cf. dubia ranged from 6.13 to 183 Êmol/L while the 30-min EC50 (luminescence) values of the chemicals to V. fischeri ranged from 1.39 to 1031 Êmol/L. Seventy-five percent of the chemicals were classed as having moderate toxicity of to C. cf. dubia while 25 percent had below toxicity f. For V. fischeri the percentage of chemicals classified as having high moderate and below toxicity was approximately 17%, 33% and 50% respectively. Quantitative activity-activity relationships (QAARs) that could predict the toxicity of chemicals with a polar narcotic mode of action to C. cf. dubia were developed by regressing the toxicity data for polar narcotic chemicals to C. cf. dubia with that of eight non-Australasian species. Those QAARs based on the toxicity data for Tetrahymena pyriformis, Chlorella vulgaris, Poecilia reticulata and Daphnia magna were of high quality (r2 . 0.9). Only the QAARs for T. pyriformis, C. vulgaris and D. magna had sufficient data to test their validity. This revealed that the absolute percentage difference between experimentally derived EC50 values and those predicted by these QAARs were between 13 and 120%. These QAARs provide an easy, cost-effective means of estimating toxicity values for polar narcotic chemicals to C. cf. dubia

Toxicity of Herbicide Mixtures to Tropical Freshwater Microalgae Using a Multispecies Test.

Agriculture within the Great Barrier Reef catchment area has contributed to pesticide contamination of adjacent freshwater ecosystems that flow into the Great Barrier Reef World Heritage Area. A novel multispecies toxicity test was used to assess the toxicity of diuron and hexazinone, 2 herbicides commonly detected within the Great Barrier Reef catchment area, to a community of 3 tropical freshwater microalgae: Monoraphidium arcuatum, Nannochloropsis-like sp., and Pediastrum duplex. Diuron was the most toxic herbicide, with 10% inhibition concentration (IC10) values of 4.3, 7.1, and 29 µg/L for P. duplex, M. arcuatum, and Nannochloropsis-like sp., respectively, followed by hexazinone, with IC10 values of 15, 18, and 450 µg/L, respectively Toxicity testing on 2 commercial formulations (Barrage, 13.2% hexazinone and 48.6% diuron; Diurex, 90% diuron) showed that additives in the commercial formulations did not significantly increase the toxicity of diuron. Direct toxicity assessments were carried out on water samples from the herbicide-contaminated Sandy Creek, which discharges to the Great Barrier Reef lagoon, and a clean reference site, Tully Gorge in the Tully River. Toxicity was observed in several Sandy Creek samples. Artificial herbicide mixtures were assessed in synthetic soft water and natural freshwaters, with toxic responses being observed at environmentally relevant concentrations. The present study successfully applied a novel multispecies tropical microalgal toxicity test, indicating that it is an effective tool for the assessment of herbicide toxicity in both natural and synthetic freshwaters. Environ Toxicol Chem 2021;40:473-486. © 2020 SETAC