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2D cryo-EM image of a solution of membrane fractions carrying the OR1740 olfactory receptor at TPC 1⁄4 5000 m g mL À 1 after sonication (a). Results of DLS (b) and NTA (c) measurements of membrane fractions carrying the OR1740 olfactory receptor at room temperature. The size distributions in cyan refer to sonicated samples (TPC 1⁄4 60 m g mL À 1 ) while the ones in purple refer to samples, at the same concentration, which were first sonicated and then filtered (see the Experimental section). Data in (c) are fitted with the sum of 3 (sonicated sample) and 2 (sonicated + filtered sample) Gaussian functions in order to extract the contribution of each population to the size distribution. Calibration curve of nanosomes concentration ( C ), obtained from the total area of NTA size distributions at different TPC concentrations (c, inset).
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Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for sensing biodevices. This is particularly true for the case of G-protein coupled receptors (GPCRs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. Howe...
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Partial Fulfilment Dissertation’s Poster Presentation
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... For example, conjugating a luciferase-encoding EV (189) with a cancerspecific antibody may provide such an in vivo diagnostic. EVs may also serve as in vitro diagnostics and biosensors, such as has been reported for EVs with G protein-coupled receptors (GPCRs) on chips to detect GPCR agonists or antagonists (190). ...
The promise of drug repurposing is to accelerate the translation of knowledge to treatment of human disease, bypassing common challenges associated with drug development to be more time- and cost-efficient. Repurposing has an increased chance of success due to the previous validation of drug safety and allows for the incorporation of omics. Hypothesis-generating omics processes inform drug repurposing decision-making methods on drug efficacy and toxicity. This review summarizes drug repurposing strategies and methodologies in the context of the following omics fields: genomics, epigenomics, transcriptomics, proteomics, metabolomics, microbiomics, phenomics, pregomics, and personomics. While each omics field has specific strengths and limitations, incorporating omics into the drug repurposing landscape is integral to its success.
Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 60 is January 9, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Of these various systems, human em- bryonic kidney (HEK)− 293 cells [45] showed especially high expres- sion levels [46,47]. Also, these cells contain all of the essential proteins for signal transduction such as G proteins [48,49], adenylyl cyclases [50], and ion channels [51]; hence, they have been broadly used for the expression of olfactory receptors. In addition, modification of HEK293 cells with 3 transmembrane proteins (RTP1, RTP2 and REEP1) pro- motes functional cell surface expression [52]. ...
... By comparing the Biacore signal obtained upon binding of odorant onto olfactory receptors to that obtained upon NVs immobilization on sensor chips, and using the number of olfactory receptors in a NV, we were able to estimate for the first time the approximate number of odorant molecules interacting with an olfactory receptor to be between 100 and 1000. Furthermore, this is a first step towards developing an array 9 for high throughput recognition of multiple odorant [10][11][12] , which would require the use of a set of different specific OR-carrying NVs. ...
... Prior to use, the stock suspension of membrane fractions was diluted in the corresponding phosphate buffer according to the corresponding method. Further homogenization steps, as well as the characterization of the NVs (size, concentration, etc.), were performed following the protocol described in previous works 10,25 . ELISA assays: The stock suspension of the membrane fractions was diluted in PBT at a total protein concentration (TPC) of 60 μg/mL. ...
This study aims to improve our understanding of the interaction between olfactory receptors and odorants to develop highly selective biosensing devices. Natural nanovesicles (NVs) from Saccharomyces cerevisiae, ~100 nm in diameter, carrying either the human OR17-40 or the chimpanzee OR7D4 olfactory receptor (OR) tagged with the c-myc epitope at their N-terminus, are presented as model systems to quantify the interaction between odorant and olfactory receptors. The level of expression of olfactory receptors was determined at individual NVs using a novel competitive ELISA immunoassay comparing the values obtained against those from techniques involving the solubilization of cell membrane proteins and the identification of c-myc-carrying receptors. Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips indicate that cognate odorants bind to their Ors, thereby quantifying the approximate number of odorants that interact with a given olfactory receptor. The selectivity of OR17-40-carrying NVs towards helional and OR7D4-carrying NVs towards androstenone has been proven in cross-check experiments with non-specific odorant molecules (heptanal and pentadecalactone, respectively) and in control receptors.
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... ORs can then be purified in the presence of surfactants, possibly in the form of micelles or nanodiscs [22], or prepared as membrane fractions, natural nanoliposomes [30][31][32] or nanovesicles [33,34], and their functional response monitored by physical or biophysical measurements, at submillimetric to micrometric scales. ...
Two olfactory receptors (ORs), mouse M71 and chimpanzee OR7D4, were immobilized onto synthetic diamond transducers surfaces. 6His tagged M71 (6His-M71) was grafted using covalent attachment of nitriloacetic acid (NTA) as chelating agent, which could bind the 6His tagged receptor through nickel ions. OR7D4 was grafted through covalent bonding of hexanoic acid radical on diamond followed by EDC/NHS peptidic coupling to the receptor. Both grafting procedures were monitored by electrochemical impedance spectroscopy (EIS) on boron doped diamond (BDD) electrodes. Then the grafting protocols were applied to the surface of bulk diamond micro-cantilevers. The resulting sensors were assessed for odorant detection in the liquid phase using a Laser Doppler read-out system. The 6His-M71 based sensor was found to exhibit a good sensitivity to acetophenone, with a typical frequency shift near 100. Hz for 1. μM exposure, with a good selectivity against negative control 2-octanone. The OR7D4 based sensor showed a sensitivity of 200. Hz for exposures to 1 or 10. μM androstenone with a good selectivity against both non-ligands acetophenone and 2-octanone.
... Two TMV particles at different orientations are visible in the images presented in Fig. 2. Our sample substrate was not completely flat, due to the presence of protein aggregates, presumably single disks and/or vertically stacked disks of 17 coat proteins, having a diameter of 18 nm. This is a quite usual feature in the case of biological samples 31 . The experimental conditions (scan rate, amplitude set-point and feedback gains) were optimized to have a minimum error signal as seen from the A 1 image (see Supplementary Fig. S1). ...
High-resolution microscopy techniques have been extensively used to investigate the structure of soft, biological matter at the nanoscale, from very thin membranes to small objects, like viruses. Electron microscopy techniques allow for obtaining extraordinary resolution by averaging signals from multiple identical structures. In contrast, atomic force microscopy (AFM) collects data from single entities. Here, it is possible to finely modulate the interaction with the samples, in order to be sensitive to their top surface, avoiding mechanical deformations. However, most biological surfaces are highly curved, such as fibers or tubes, and ultimate details of their surface are in the vicinity of steep height variations. This limits lateral resolution, even when sharp probes are used. We overcome this problem by using multifrequency force microscopy on a textbook example, the Tobacco Mosaic Virus (TMV). We achieved unprecedented resolution in local maps of amplitude and phase shift of the second excited mode, recorded together with sample topography. Our data, which combine multifrequency imaging and Fourier analysis, confirm the structure deduced from averaging techniques (XRD, cryoEM) for surface features of single virus particles, down to the helical pitch of the coat protein subunits, 2.3 nm. Remarkably, multifrequency AFM images do not require any image postprocessing.
... In addition, for all experiments the membrane fraction was sonicated prior to use, for 10 min at 2 Â 160 W, 35 kHz (Sonoclean S2600 sonicator, Labomoderne, Paris, France) in ice-cold water. The average diameter of nanosomes is between 89 ± 23 nm, as evaluated by Dynamic Light Scattering (Calo et al., 2012), which would correspond to (4 ± 1) Â 10 9 nanosomes per cm 2 for a dense immobilized monolayer. Nanosomes were filtered prior to preparing the working solutions at 80 lg/ml, assuming that after sonication all nanosomes were recovered (Minic et al., 2005;Vidic et al., 2008). ...
... Her team found that if they burst open yeast cells genetically engineered to produce ORs, the cells' membranes reformed robust spherical 'nanovesicles' with ORs on the membrane. 5 The collaboration was also able to put these nanovesicles onto gold electrodes, making prototype e-noses that could successfully identify the presence and measure the concentration of specific odorants for use in monitoring food quality. ...
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... However, examples of transmembrane proteins used in biosensors (f.e. [2,3]) are still rare because they require a lipid or lipid-like environment, carefully optimized for both supporting the native protein conformation and long-term stability of the biosensor. Transport proteins, particularly ABC transporters, pose even more difficulties because a compartmentalized lipid bilayer environment of low permeability is necessary to facilitate transport. ...
