Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation, bioRxiv, 2019-03-08
AbstractKnowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating 40S recruitment. Our data suggest that G3BP1 functions by providing viral RNA a competitive advantage over capped cellular RNAs, uncovering a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifying the first host factor with pan-norovirus pro-viral activity.
biorxiv microbiology 0-100-users 2019Whisking asymmetry signals motor preparation and the behavioral state of mice, bioRxiv, 2019-03-08
AbstractA central function of the brain is to plan, predict and imagine the effect of movement in a dynamically changing environment. Here we show that in mice head fixed in a plus-maze, floating on air, and trained to pick lanes based on visual stimuli, the asymmetric movement and position of whiskers on the two sides of the face signals whether the animal is moving, turning, expecting reward or licking. We show that 1) we can decode and predict the behavioral state of the animal based on this asymmetry, 2) that tactile input from whiskers indicates little about the behavioral state, and 3) that movement of the nose correlates with asymmetry, indicating that facial expression of the mouse is itself correlated with behavioral state. Amazingly, the movement of whiskers – a behavior that is not instructed or necessary in the task--informs an observer about what a mouse is doing in the maze. Thus, these mobile tactile sensors reflect a behavioral and movement-preparation state of the mouse.
biorxiv neuroscience 0-100-users 2019A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis, bioRxiv, 2019-03-07
ABSTRACTNucleic acid stains are necessary for Agarose Gel Electrophoresis (AGE). The commonly used but mutagenic Ethidium Bromide is being usurped by a range of safer but more expensive alternatives. These safe stains vary in cost, sensitivity and the impedance of DNA as it migrates through the gel. Modified protocols developed to reduce cost increase this variability. In this study, five Gel stains (GelRed™, GelGreen™, SYBR™ safe, SafeView and EZ-Vision®In-Gel Solution) two premixed loading dyes (SafeWhite, EZ-Vision®One) and four methods (pre-loading at 100x, pre-loading at 10x, precasting and post-staining) are evaluated for sensitivity and effect on DNA migration. GelRed™ was found to be the most sensitive while the EZ-Vision® dyes and SafeWhite had no discernible effect on DNA migration. Homemade loading dyes were as effective as readymade ones at less than 4% of the price. This method used less than 1% of the dye needed for the manufacturer recommended protocols. Thus, with careful consideration of stain and method, Gel stain expenditure can be reduced by over 99%.
biorxiv molecular-biology 0-100-users 2019Absence of entourage Terpenoids commonly found in Cannabis sativa do not modulate the functional activity of Δ9-THC at human CB1and CB2 receptors, bioRxiv, 2019-03-06
AbstractIntroductionCompounds present in Cannabis sativa such as phytocannabinoids and terpenoids, may act in concert to elicit therapeutic effects. Cannabinoids such as Δ9-tetrahydrocannabinol (Δ9-THC) directly activate cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), however, it is not known if terpenoids present in Cannabis also affect cannabinoid receptor signalling. Therefore, we examined 6 common terpenoids alone, and in combination with cannabinoid receptor agonists, on CB1 and CB2 signalling in vitro.Materials and MethodsPotassium channel activity in AtT20 FlpIn cells transfected with human CB1 or CB2 receptors was measured in real-time using FLIPR® membrane potential dye in a FlexStation 3 plate reader. Terpenoids were tested individually and in combination for periods up to 30 minutes. Endogenous somatostatin receptors served as a control for direct effects of drugs on potassium channels.Resultsα-Pinene, β-pinene, β-caryophyllene, linalool, limonene and β-myrcene (up to 30-100 µM) did not change membrane potential in AtT20 cells expressing CB1 or CB2, or affect the response to a maximally effective concentration of the synthetic cannabinoid CP55,940. The presence of individual or a combination of terpenoids did not affect the hyperpolarization produced by Δ9-THC (10µM) (CB1 control, 59±7%; with terpenoids (10 µM each) 55±4%; CB2 Δ9-THC 16±5%, with terpenoids (10 µM each) 17±4%). To investigate possible effect on desensitization of CB1 responses, all six terpenoids were added together with Δ9-THC and signalling measured continuously over 30 min. Terpenoids did not affect desensitization, after 30 minutes the control hyperpolarization recovered by 63±6%, in the presence of the terpenoids recovery was 61±5%.DiscussionNone of the six of the most common terpenoids in Cannabis directly activated CB1 or CB2, or modulated the signalling of the phytocannabinoid agonist Δ9-THC. These results suggest that if a phytocannabinoid-terpenoid entourage effect exists, it is not at the CB1 or CB2 receptor level. It remains possible that terpenoids activate CB1 and CB2 signalling pathways that do not involve potassium channels, however, it seems more likely that they may act at different molecular target(s) in the neuronal circuits important for the behavioural effect of Cannabis.
biorxiv pharmacology-and-toxicology 0-100-users 2019Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome, bioRxiv, 2019-03-06
AbstractCRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because they are difficult to identify, the natural diversity and impact of Acrs on microbial ecosystems is underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection that isolates acr genes based on their ability to inhibit CRISPR-Cas function. Using this selection, we discovered ten DNA fragments from human oral and fecal metagenomes that antagonize Streptococcus pyogenes Cas9 (SpyCas9). The most potent acr discovered, acrIIA11, was recovered from a Lachnospiraceae phage and is among the strongest known SpyCas9 inhibitors. AcrIIA11 homologs are distributed across multiple bacterial phyla and many divergent homologs inhibit SpyCas9. We show that AcrIIA11 antagonizes SpyCas9 using a different mechanism than that of previously characterized inhibitors. Our study highlights the power of functional selections to uncover widespread Cas9 inhibitors within diverse microbiomes.
biorxiv microbiology 0-100-users 2019A robust and efficient method for Mendelian randomization with hundreds of genetic variants unravelling mechanisms linking HDL-cholesterol and coronary heart disease, bioRxiv, 2019-03-05
Mendelian randomization (MR) investigations with large numbers of genetic variants are becoming increasingly common. However, the reliability of findings from a MR investigation is dependent on the validity of the genetic variants as instrumental variables. We developed a method to identify groups of genetic variants with similar causal effect estimates, which may represent distinct mechanisms by which the risk factor influences the outcome. Our contamination mixture method is a robust and efficient method for valid MR in the presence of invalid IVs. Compared to other robust methods, our method had the lowest mean squared error across a range of realistic scenarios. The method is fast and efficient, and can perform analysis with hundreds of variants in a fraction of a second. In a MR analysis for high-density lipoprotein (HDL) cholesterol and coronary heart disease (CHD) risk, the method identified 11 variants associated with increased HDL-cholesterol, decreased triglyceride levels, and decreased CHD risk that had the same directions of associations with platelet distribution width and other blood cell traits, suggesting a shared mechanism linking lipids and CHD risk relating to platelet aggregation.
biorxiv genetics 0-100-users 2019