Fast functional mapping of ligand-gated ion channels.

Ligand-gated ion channels are formed by three to five subunits that control the opening of the pore in a cooperative fashion. We developed a microfluidic chip-based technique for studying ion currents and fluorescence signals in either excised membrane patches or whole cells to measure activation and deactivation kinetics of the channels as well as ligand binding and unbinding when using confocal patch-clamp fluorometry. We show how this approach produces in a few seconds either unidirectional concentration-activation relationships at or near equilibrium and, moreover, respective time courses of activation and deactivation for a large number of freely designed steps of the ligand concentration. The short measuring period strongly minimizes the contribution of disturbing superimposing effects such as run-down phenomena and desensitization effects. To validate gating mechanisms, complex kinetic schemes are quantified without the requirement to have data at equilibrium. The new method has potential for functionally analyzing any ligand-gated ion channel and, beyond, also for other receptors.

Humans, but not animals, perceive the thermal grill illusion as painful.

Simultaneous presentation of alternating innocuous warm and cold stimuli induces in most humans a painful sensation called thermal grill illusion (TGI). Here, pain is elicited although nociceptors are not activated. Upon back-translation of behavioural correlates from humans to animals, we found that neither cats nor rodents show adverse reactions when exposed to TGI stimulation. These results question that a TGI observed as a pain-related change in behaviour can be elicited in animals. While distinct neuronal patterns as previously reported may be measurable in animals upon TGI stimulation, their translational meaning towards the sensation elicited in humans is unclear.

Simplified pulse reactor for real-time long-term in vitro testing of biological heart valves.

Long-term function of biological heart valve prostheses (BHV) is limited by structural deterioration leading to failure with associated arterial hypertension. The objective of this work was development of an easy to handle real-time pulse reactor for evaluation of biological and tissue engineered heart valves under different pressures and long-term conditions. The pulse reactor was made of medical grade materials for placement in a 37 degrees C incubator. Heart valves were mounted in a housing disc moving horizontally in culture medium within a cylindrical culture reservoir. The microprocessor-controlled system was driven by pressure resulting in a cardiac-like cycle enabling competent opening and closing of the leaflets with adjustable pulse rates and pressures between 0.25 to 2 Hz and up to 180/80 mmHg, respectively. A custom-made imaging system with an integrated high-speed camera and image processing software allow calculation of effective orifice areas during cardiac cycle. This simple pulse reactor design allows reproducible generation of patient-like pressure conditions and data collection during long-term experiments.

Searching biomarker candidates in serum using multidimensional native chromatography. II Method evaluation with Alport syndrome and severe inflammation.

Biomarker search using multidimensional native liquid fractionation of serum in microplates was evaluated. From different donors, homologous sample fractions with UV absorbance depending on state of illness were selected, and their constituents were identified and quantitated by MS. Analysis of sera of patients with Alport syndrome and severe inflammation proved the reliability of the method by confirming characteristic alterations. Moreover, 23 new marker candidates were detected for Alport syndrome, some of them being involved in matrix degradation and repair, and 33 new candidates for severe inflammation, among them alpha1B-glycoprotein cysteine-rich secretory protein and an apparently low molecular-weight albumin variant.

Searching biomarker candidates in serum using multidimensional native chromatography. I. Enhanced separation method.

The microplate-based method developed by our group for non-denaturing multidimensional proteome separation was improved on using improved column arrays and a newly developed robot. Currently size exclusion, anion exchange and lectin affinity chromatography are combined orthogonally. Different samples run simultaneously to enhance reliability of intercomparison. LC-ESI (electro-spray ionization) MS/MS analysis of selected fractions identified 32,288 peptides matching 2,669 serum proteins. The present contribution (I) shows the characteristics of the method, whereas "prove of principle" by applying it to search for biomarker candidates with model diseases is reported in an accompanying paper (II).

Multidimensional proteomics of human serum using parallel chromatography of native constituents and microplate technology.

A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags.