Daniel Gygax

The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

This study identifies a GPCR, S1PR2, as a receptor for the Nogo-A-Δ20 domain of the membrane protein Nogo-A, which inhibits neuronal growth and synaptic plasticity.

Cytokine signaling in the human brain capillary endothelial cell line hCMEC/D3

Brain microvascular endothelial cells are part of the blood-brain barrier and participate actively in immunological processes including cytokine-mediated inflammatory reactions. Using the human brain capillary endothelial cell line hCMEC/D3, activation of JAK/STAT signaling pathways were studied in response to stimulation by cytokines. The phenotype of hCMEC/D3 cells was confirmed by flow cytometry analysis of cell adhesion factors (cluster of differentiation molecules CD31 and CD34) and the von Willebrand factor endothelial marker was detected by immunofluorescence. Strong STAT1, STAT6 and STAT3 activation was observed in response to interferon-gamma (IFN-gamma), interleukin 4 (IL-4) and interleukin 6 (IL-6), respectively. Nuclear translocation of phosphorylated STAT proteins was visualized by confocal microscopy. Treatment of hCMEC/D3 cells with IFN-gamma resulted in interferon-induced upregulation of major histocompatibility complex (MHC) class I within 48h. Interferon-alpha (IFN-alpha) did not activate STAT1 or STAT3 nor did it induce MHC class I upregulation. Therefore, hCMEC/D3 cells were judged to be non-responsive to IFN-alpha. We also observed that hCMEC/D3 cells exhibit functional expression of alternative cytokine signal transduction pathways (i.e. TNF-alpha mediated activation of NF-kappaB). Together these results indicate that human blood-brain barrier hCMEC/D3 cells are responsive towards stimulation with various cytokines. We conclude that this unique cell line can be used to explore in vitro human blood-brain barrier functionality under proinflammatory conditions.

DNA surface coating of calixarene-based nanoparticles: a sequence-dependent binding mechanism

An amphiphilic calix[4]arene derivative bearing four guanidino moieties at the upper rim and four dodecyl chains at the lower rim was shown to form stable solid lipid nanoparticles (SLNs) in water. The study of the interactions of these cationic SLNs with DNA revealed a sequence-dependent groove binding mechanism.

Immunoassays in monitoring biotechnological drugs.

For the evaluation and interpretation of pharmacokinetic data reliable quantitative determinations are a requirement that can only be met by well-characterized and fully validated analytical methods. To cope with these requirements a method is being established that is based on an integrated and automated fiber-optic biospecific interaction analysis system (FOBIA) for immunoassays. Performance characteristics of this system used in monitoring of recombinant hirudin (CGP 39 393) are presented. Recombinant hirudin is a highly potent and selective inhibitor of human thrombin. Owing to its size and charge, recombinant hirudin is mainly eliminated by glomerular filtration. But only a fraction of the hirudin dose seems to be reabsorbed at the proximal tubule by luminal endocytosis and hydrolyzed by lysosomal enzymes, leaving approximately 50% of the dose to be extracted in the urine. Thus, renal clearance of recombinant hirudin in the absence of renal insufficiency appears to depend primarily on the glomerular filtration rate. During a 3-month i.v. tolerability study in dogs, some of the dogs developed antibodies against recombinant hirudin. The hirudin-antibody complex accumulated in plasma and apparent hirudin plasma concentrations were therefore much higher than expected from single-dose kinetics. Hirudin captured by antibodies showed an extended half-life and the hirudin-antibody complex is still pharmacologically active, as demonstrated by the observed increase in thrombin time. In conclusion, only appropriate analytical methods allow adequate monitoring and pharmacokinetic characterization of biotechnology drugs in biological materials.

An enzymatic method to determine γ-hydroxybutyric acid in serum and urine.

Background: Gamma-hydroxybutyric acid (GHB) has become one of the most dangerous illicit drugs of abuse today. It is used as a recreational and date rape drug because of its depressant effect on the central nervous system, which may cause euphoria, amnesia, respiratory arrest, and coma. There is an urgent need for a simple, easy-to-use assay for GHB determination in urine and blood. In this article, a rapid enzymatic assay adapted to clinical chemistry analyzers for the detection of GHB is presented. Methods: The described GHB enzymatic assay is based on a recombinant GHB dehydrogenase. The full validation of the assay was performed on a Konelab 30 analyzer (Thermo Fisher Scientific). Results: The analytical sensitivity was <1.5 mg/L, whereas the functional sensitivity was 4.5 mg/L in serum and 2.8 mg/L in urine. The total imprecision coefficient of variation (CV) was <9.8% in serum and <7.9% in urine. The within-run imprecision showed a CV of <3.8% in serum and <4.6% in urine. The assay was linear within the range 5–250 mg/L. Mean recoveries were 109% in serum and 105% in urine. No cross-reactivity was observed for tested GHB analogues and precursors. Comparison of GHB-positive samples showed an excellent correlation with ion chromatography, gas chromatography–mass spectrometry, and liquid chromatography associated to tandem mass spectrometry. Except for ethanol, no substantial interference from serum constituents and some drugs was observed. Conclusions: This automated GHB assay is fully quantitative and allows the accurate measurement of GHB in serum and urine. It can be used as a rapid screening assay for the determination of GHB in intoxicated or overdosed patients.

Reverse protein arrays as novel approach for protein quantification in muscular dystrophies

The definite molecular diagnosis in patients with muscular dystrophies often requires the assessment of muscular expression of multiple proteins in small amounts of muscle tissue. The sample material obtained in muscle biopsies is limited and the measurement of multiple proteins is often restricted to conventional, non-quantitative assays, i.e. immunohistochemistry and immunoblotting. Here, we demonstrate that reverse protein arrays are a novel and excellent material-saving method for the measurement and quantification of changes in protein expression between healthy and diseased muscle tissue as well as cultured primary myotubes. We evaluated a set of antibodies and found reproducible differences between Duchenne muscular dystrophy/limb-girdle muscular dystrophy patients and control samples for dystrophin, the sarcoglycans and the dystroglycans. As little as 10 mg of tissue is sufficient for the analysis of all diagnostically relevant proteins. The average coefficient of variation calculated for the sample signals confirmed that the method is highly reproducible. Thus, our experiments provide strong evidence that quantitative protein detection from very small amounts of muscle tissue is possible using reverse protein arrays. This technology may not only be of interest for diagnostic purposes, but also for protein quantification of multiple, follow-up biopsies during clinical trials when protein expression in muscle is considered an important outcome measure or biomarker.

Establishment of a miniaturized enzyme-linked immunosorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate.

A miniaturized enzyme-linked immunosorbent assay (ELISA) with a reaction volume of 5 microl for human transferrin quantification has successfully been developed using an intelligent multifunctional analytical plate (IMAPlate 5RC96), the first miniature analytical platform capable of manually performing parallel liquid transfer, reaction, and analysis. This is the first article to validate the platform for the ELISA application. The data obtained from the standards in this miniaturized ELISA can well be fitted by a one-site binding reaction mode, the coefficient of variation (CV) of the whole plate for an artificial sample (spiking a known concentration of human transferrin into the assay diluent) is 7.0%, and the mean recovery is between 94 and 114% (n=96), comparable to the values from conventional ELISA in a 96-well format plate. The IMAPlate 5RC96-based miniaturized ELISA not only can reduce sample and reagent consumption to 5% of the conventional ELISA but also can shorten the reaction time. Combined with the advantages brought by miniaturization, the easy-to-handle, parallel, and simultaneous liquid transfer features of the IMAPlate 5RC96 provide a completely new lab tool for manually performing high-throughput ELISA. Our results demonstrate that the IMAPlate 5RC96 is a convenient, robust, high-throughput lab device feasible for miniaturized ELISA in an ordinary laboratory.

Activation of the JAK–STAT intracellular pathway in human retinal pigment epithelial cell line ARPE-19

Background: Retinal pigment epithelial cells constitute an important component of theblood–retinal barrier and play a pivotal role in the development of age-related maculardegeneration (AMD). Understanding the underlying molecular mechanisms is a prerequisitefor developing therapeutic strategies for the treatment of this disease. This study investigatedcytokine-induced changes of the JAK–STAT (Janus tyrosine kinase–signal transducers andactivators of transcription) signaling pathway in the human retinal pigment epithelial cell lineARPE-19 and potential implications for AMD.Methods: Electromobility shift assay, immunofluorescence staining, and flow cytometry wereused to evaluate the JAK–STAT pathway in the ARPE-19 cell line.Results: We examined cytokine-induced expression of STATs in the ARPE-19 cell line. StrongSTAT1 activation determined by electromobility shift assay and flow cytometry was demonstratedupon exposure to interferon-γ. Interferon-α upregulated STAT1, STAT2, and STAT3 inARPE-19 cells, while interleukin-6 (IL-6) and IL-4 activated STAT3 and STAT6, respectively.Confocal microscopy identified the nuclear translocation of the STAT proteins. Flow cytometryanalysis demonstrated the upregulation of major histocompatibility complex molecules onARPE-19 cells as responses to interferon-α and interferon-γ.Conclusion: Our data demonstrate the upregulation of members of the JAK–STAT signalingpathway in the ARPE-19 cells upon stimulation with interferon-α, interferon-γ, IL-4, and IL-6.We present a model for these signaling pathways potentially relevant for AMD, which mayprove useful for screening of AMD therapeutics.

Synthetic single domain antibodies for the conformational trapping of membrane proteins

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.

A simple approach to improve the sensitivity of enzyme-linked immunosorbent assay: Using the IMAPlate 5RC96 for result readout

In this article, we describe a new enzyme-linked immunosorbent assay (ELISA) setup to improve the sensitivity of commercial or homemade ELISAs. In the new ELISA setup, an IMAPlate 5RC96, a disposable multi-utility lab device developed by NCL New Concept Lab is used as a self-uptaking microcuvette array to read out the result of the ELISA that is performed in the normal 96-well plate with reduced substrate solution and stop solution. A commercial interleukin-6 (IL-6) ELISA reagent kit was used for the evaluation. Compared with the conventional ELISA setup, the new ELISA setup could easily increase the absorbance values by up to more than 10-fold. Therefore, the sensitivity (change in absorbance/change in concentration [DeltaAbs/DeltaConc]) is increased accordingly. In addition, methods to extend the upper detection limit of plate readers for the IMAPlate 5RC96 are described. This new ELISA setup may be more notable for the approach employed than for the specific analyte. It should generally be applicable to any conventional ELISA and should serve as an example of a simple solution that increases the detection sensitivity and/or detection range of other assays as well. We expect the approach to have a substantial practical impact on analytical methods and to accelerate discovery, research, and application of analytes at low concentration in life sciences and diagnostics.