9 research outputs found

    Receptomics, design of a microfluidic receptor screening technology

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    This thesis describes the development of a G Protein-Coupled Receptor (GPCR) screening technology that combines a receptor cell array (~300 spots) with microfluidics. This technology was developed for the purpose of sensing the taste of, or active components in complex samples. GPCR activation was monitored using a genetically encoded calcium indicator (GECI) which was based on a change in Förster Resonance Energy Transfer (FRET) between two fluorescent proteins linked by a calcium binding domain which, upon binding of calcium, induces a conformational change between the fluorophores. The receptor cell arrays were created by reverse transfection of printed plasmid DNA. The arrays were assembled in a flowcell, connected to a microfluidic system, and mounted on a stereo fluorescence microscope. This setup allowed for controlled and importantly, repeated sample exposure while monitoring the changes in intracellular calcium in real-time. GPCRs play an important role in many physiological or disease-related processes. These membrane proteins have evolved to sense a wide range of molecules that can be of either exogenous or endogenous origin. Their sensing mechanisms are complex and potentially involve many cellular signalling events depending the cell type. The introductory chapter of this thesis presents a brief overview of the GPCR types and their signalling pathways with a focus on taste signalling. This chapter also places the microfluidic receptomics technology within the framework of existing receptor screening technologies. The second chapter explores the general principles, setup and characterization of the microfluidic biosensor to measure GPCR activation via imaging of [Ca2+] changes in recombinant human HEK293 cells. These cells expressed a combination of the Neurokinin 1-receptor and Cameleon YC3.6 protein as calcium indicator. Here, a stable cell line was employed for robust expression with little variation Next to GPCRs, the system was also used for the detection of transient receptor potential channel Vanilloid 1 (TRPV1) ion channel activation by means of the Cameleon YC3.6 calcium sensor as is reported in Chapter 3. This assay was performed with LCMS fractions and whole extracts of chilli pepper fruits which led to the identification of new ion channel agonists. This chapter also discusses the possibility of coupling the receptomics assay directly to an LCMS as an additional on-line bioactivity detector. The general discussion of this thesis (Chapter 7) elaborates on this topic with additional perspectives on the feasibility of coupling the two systems. Chapter 4 provides an extensive technical characterization of the preparation and measurement of reverse transfected cell arrays using fluorescent proteins. The response of the Neurokinin 1-receptor in relation to its gene dose in reverse transfection was studied, as well as response reproducibility during repeated activations. These results led to a study of bitter taste receptors in relation to sensitivity-determining parameters such as sensor type and calcium buffering (Chapter 5). This chapter aimed to enhance the sensitivity and robustness of the receptor assay and showed proof of concept with bitter receptor arrays that performed in the same range as existing state-of-the-art platforms. Such bitter taste receptor arrays may be employed for future screenings of new bitter taste agonists or modulators and the identification of bitter principles in foods. Development of software and statistical models -the linear mixed model, as presented in Chapter 6- to analyse this new type of data showed that a spot-based comparison of sequentially-tested samples yielded the most reliable data and largely eliminated inter-spot differences in signal strength. The method could also visualize receptor specific differences between samples in the presence of a simulated host cell response. A host cell response, induced by ATP, was used to show that specific bitter receptor responses from compound spikes were cumulative to the host cell response and can be retrieved from a host cell response signal by means of comparative analysis. The general discussion (Chapter 7) critically discusses the advantages and limitations of this new micro-fluidics approach and details which additional developments are needed to advance the technology further. The receptomics technology as described in this thesis is argued to be complementary to microplate screening technologies and represents a new analytical paradigm. The microfluidics aspect and overall assay size reduction are more cost efficient and allow both a high content dynamics analysis as well as the development of novel applications such as direct identification of bioactive compounds by coupling of LCMS to receptomics. All in all, this thesis presents an enabling receptor screening technology that is based on new design principles. This receptomics technology offers novel applications and has potential in the bioactivity screening of crude extracts.</p

    A Broad Set of Different Llama Antibodies Specific for a 16 kDa Heat Shock Protein of Mycobacterium tuberculosis

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    Background Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. Methodology/Principal Findings Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10-10 M. Conclusions/Significance A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria

    Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays.

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    Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed

    Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System

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    Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries

    Calcium imaging of GPCR activation using arrays of reverse transfected HEK293 cells in a microfluidic system

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    Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries

    <i>M. tuberculosis</i> binding VHH antibody fragments.

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    <p>Protein sequence of 62 selected VHH antibody fragments selected by phage display for binging to <i>M. tuberculosis</i>. Dots indicate sequence identity, and dashes indicate gaps. The three complementarity determining regions CDR1, CDR2 and CDR3 are shaded. Characteristic VH-VHH hallmark substitutions (Leu12Ser, Val42Phe or Val42Tyr, Gly49Glu, Leu50Arg or Leu50Cys and Trp52Gly or Trp52Leu (the last substitution is less well conserved) (the ImMunoGeneTics system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026754#pone.0026754-Lefranc1" target="_blank">[52]</a> was followed) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026754#pone.0026754-Muyldermans2" target="_blank">[15]</a> are underlined. The 12 clones selected for further investigations are underlined. VHH protein sequences labeled A-x (with x = 1–96) resulted from direct selection using semi-purified protein antigen, protein sequences labeled B-yx (with y = A–F and x = 1–12) were achieved by depletion method.</p

    Western blot to discover antigen of VHHs.

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    <p>A) Western blot using VHH A-23 as a probe. 9 µg <i>M. tb 1</i> lysate were run on a 15% SDS-PAGE gel in lanes A–D, and transferred to a nitrocellulose membrane. Lane A: incubated with VHH A-23 and detected using anti-VSV-HRP; Lane B: incubated with VHH A-23 and detected using anti-HIS-HRP; Lane C: incubated with detection antibody anti-VSV-HRP; Lane D: incubated with detection antibody anti-HIS-HRP. B) Western blot analysis to confirm the specificity of VHH A-23. Lane 1: 9 µg <i>M. tb 22</i> lysate detected by monoclonal mouse 16 kDa antibody, using anti-mouse-HRP secondary antibody; Lane 2: 9 µg <i>M. tb 22</i> lysate detected by VHH A-23, using anti-VSV-HRP secondary antibody. Lane 3: 3 µg of purified recombinant 16 kDa protein detected by VHH A-23, using anti-VSV-HRP secondary antibody. Due to the tags added for purification and detection purposes the calculated mass of the recombinant 16 kDa protein is 21 kDa. Indicated are the positions of relevant size markers in kDa. C) Western blot analysis of native PAGE analysis. Lane 1: 5 µg <i>M. tb 27</i> lysate; Lane 2: 1 µg of purified recombinant 16 kDa protein. Detection was by VHH A-23, using anti-VSV-HRP secondary antibody.</p

    Direct ELISA to confirm specific binging of VHH antibody fragments to tuberculosis lysate.

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    <p>VHH antibody fragments A-23 and B-F10 with A) <i>M. tuberculosis</i> whole cells, <i>M. tuberculosis</i> cell lysate and media in which <i>M. tuberculosis</i> bacteria were grown. B) different <i>M. tuberculosis</i> lysates as well as lysates of <i>M. bcg</i>, <i>M. avium</i>, <i>M. kansasii</i>, <i>M. smegmatis, S. pneumoniae and H. influenzae</i>. Measurements were performed in duplicate, expressed as means ± SD.</p
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