Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery

Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs.     

CYTOCENTERING: a novel technique enabling automated cell-by-cell patch clamping with the CYTOPATCH chip

Automats for patch clamping suspended cells in whole-cell configuration must (1) bring isolated cells in contact with patch contacts, (2) form gigaseals, and (3) establish stable intracellular access that allows for high quality recording of ionic currents. Single openings in planar substrates seem to be intriguing simple solutions for these problems, but due to the low rate of formation of whole-cell configurations we discarded this approach. Single openings are not suited for both attracting cells to the opening by suction and forming gigaseals with subsequent membrane rupture. To settle the three tasks with a mechanical microstructure we developed the socalled CYTOCENTERING technique to apply to suspended cells the same operation sequence as in conventional patch clamping. With this method we immobilized selected cells from a flowing suspension on the tip of a patch pipette by suction with a success rate of 97% and formed gigaseals with a success rate of 68%. Subsequent whole-cell recordings and intracellular staining with Lucifer yellow proved the stable access to the cytoplasm. Currently, a chip with an embedded suction opening in glass surrounding the microstructured contact pipette is under development. The processing of this CYTOPATCH chip is compatible to large-volume production. The CYTOPATCH automat will allow for fully automated, parallel, and asynchronous whole-cell recordings.     

Datenschutz im IT-Grundschutz

Zusammenfassung  Zur Pr?zisierung der Sicherheitsanforderungen der Anlage zum § 9 BDSG wird der Ansatz verfolgt, das IT-Grundschutzhandbuch des BSI um einen Baustein „Datenschutz“ zu erg?nzen. Claus Simon berichtet über Hintergrund, Zielsetzung und Stand dieser Erg?nzung. Claus Simon Referatsleiter Technik beim Landesbeauftragten für Datenschutz und Informationsfreiheit Saarland     

Parallel processing in a multifeature whole-report paradigm.

Observers given brief exposures of pairs of colored bars and asked to report both the color and the orientation of each bar showed evidence of stochastic independence between reports of the 4 features (2 colors and 2 orientations). The authors also found virtually perfect stochastic independence between reports of colors and directions of motion of pairwise presented circular disks at each of 3 levels of exposure duration that varied unpredictably from trial to trial. Stimulus triples, rather than pairs, yielded more complex results. However, the findings provide strong evidence that the relevant features of the 2-3 stimuli were identified and localized in parallel across the display. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Low temperature transient liquid phase bonding of Au/Sn and Cu/Sn electroplated material systems for MEMS wafer-level packaging

This paper presents the development of a low temperature transient liquid phase bonding process for 8″ wafer-level packaging of micro-electro-mechanical systems. Cu/Sn and Au/Sn material systems have been investigated under varying bonding temperatures from 240 to 280 °C and different dwell times from 8 to 30 min. The used bond frame had a width of 80 μm and lateral dimensions of 1.5 mm × 1.55 mm. The sealing frame of the cap wafer consisted of Au and Cu, respectively, and Sn. The MEMS wafer only holds the parent metal of Au or Cu. High quality bonds were confirmed by shear tests, cleavage analysis, polished cross-section analysis using optical and electron microscope, energy dispersive X-ray spectroscopy and pressure cocker test. The samples showed high shear strength (>80 MPa), nearly perfect bond regions and no main failure mode in the cleavage analyses. Non-corroded Cu test structures confirmed the hermeticity.     

Is LRP2 Involved in Leptin Transport over the Blood-Brain Barrier and Development of Obesity?

The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.     

Nonproteolytic Clostridium botulinum toxigenesis in cooked turkey stored under modified atmospheres

The ability of nonproteolytic Clostridium botulinum type B spores to grow and produce toxin in cooked, uncured turkey packaged under modified atmospheres was investigated at refrigeration and mild to moderate abuse temperatures. Cook-in-bag turkey breast was carved into small chunks, surface-inoculated with a mixture of nonproteolytic C. botulinum type B spores, packaged in O2-impermeable bags under two modified atmospheres (100% N2 and 30% CO2:70% N2), and stored at 4, 10, and 15 degrees C. Samples were analyzed for botulinal toxin and indigenous microorganisms, as well as subjected to sensory evaluation, on days 0, 7, 14, 28, 42, and 60. Given sufficient incubation time, nonproteolytic C. botulinum type B grew and produced toxin in all temperature and modified atmosphere treatment combinations. At moderate temperature abuse (15 degrees C), toxin was detected by day 7, independent of packaging atmosphere. At mild temperature abuse (10 degrees C), toxin was detected by day 14, also independent of packaging atmosphere. At refrigeration temperature (4 degrees C), toxin was detected by day 14 in product packaged under 100% N2 and by day 28 in product packaged under 30% CO2:70% N2. Reduced storage temperature significantly delayed toxin production and extended the period of sensory acceptability of cooked turkey, but even strict refrigeration did not prevent growth and toxigenesis by nonproteolytic C. botulinum. At all three storage temperatures, toxin detection preceded or coincided with development of sensory characteristics of spoilage, demonstrating the potential for consumption of toxic product when spoilage-signaling sensory cues are absent.     

Assessment of drug release from oil depot formulations using an in vitro model -- potential applicability in accelerated release testing

In vitro drug release and transport rates from oil depot formulations under nonsink conditions have been investigated in the rotating dialysis cell model. Eight model drug compounds and eight oil vehicle compositions were used for the releaseexperiments. The experimentally obtained apparent first-order rate constants related to the drug appearance in the acceptor phase after initial instillation of a drug-containing oil solution were found to be in excellent agreement with the rate constants obtained from a theoretically derived expression. It was observed that the drug oil-water distribution coefficient was the key parameter influencing the release characteristics. As compared with ketoprofen, flurbiprofen exhibited a higher affinity for the oil, resulting in a significantly lower and more slowly decreasing drug concentrations in the aqueous donor compartment. Release profiles for prilocaine and the more lipophilic agent bupivacaine after incorporation of both drugs in fractionated coconut oil were characterized by a fast release of prilocaine, whereas bupivacaine was liberated much slower to the acceptor phase. The high oil-buffer interfacial area generated in vitro by rotation of the donor cell tends to overestimate release rates in comparison to those expected in vivo, for example, after intra-articular administration of oil solutions. The present in vitro method may constitute a valuable tool in accelerated in vitro release testing of parenteral oil depot formulations in areas comprising formulation design and product quality control.     

Quantitative microfluidic separation of DNA in self-assembled magnetic matrixes

We present an experimental study of the microfluidic electrophoresis of long DNA in self-assembling matrixes of magnetic bead columns. Results are presented for the rapid separation of lambda-phage, 2lambda-DNA, and bacteriophage T4 DNA, where separation resolutions greater than 2 between lambda and T4 are achieved in times as short as 150 s. The use of a computer-piloted flow control system and injection results in high reproducibility between separations. We compare the experimentally measured mobility and dispersion with an exactly solvable lattice Monte Carlo model. The theory predicts that the mean velocity scales linearly with the field, the band broadening scales with the inverse of the field, and the resolution is independent of the field for intermediate fields-all of which are in accord with the experimental results. Moreover, reasonable quantitative agreement is achieved for band broadening for longer DNA (2lambda and T4) when the average postengagement time is measured experimentally. This work demonstrates the possibility of achieving fast microfluidic separation of large DNA on a routine basis.