Evaluation of different operating modes of an autosampler for portable capillary electrophoresis.

A compact, inexpensive sampler instrument for portable capillary electrophoresis (CE) was developed and tested to monitor common inorganic ions in drinking water samples. The sampler uses peristaltic and vacuum pumps and pinch and check valves to control liquid flows. The paper also addresses various aspects of CE associated with portability, open access instrumentation and prospects of CE for citizen science. The extensive use of items provided by the electronic and computer industry contributes to this trend.

Autosampler for portable capillary electrophoresis.

A compact, inexpensive sampler instrument for portable capillary electrophoresis was developed for monitoring illegal drugs in human body fluids and evaluated for γ-hydroxybutyric acid (GHB) in simulated saliva samples. The sampler uses peristaltic pumps and pinch and check valves to activate liquid flows. This short communication addresses aspects of CE associated with portability, open access instrumentation, and prospects of CE for citizen science. The extensive use of items provided by the electronic and computer industry contributes to this trend.

Portable fully automated oral fluid extraction device for illegal drugs.

Currently used methods for in-field determination of illegal drugs involve various test kits based mainly on the immunoassay technique, where the presence of a compound of interest is assessed by antibody-antigen reaction and manifested by observable color change. Despite being accepted and widely used by police forces to test the presence of illegal drugs in a suspect person, these tests often suffer from unreliable results (high level of false-positive and/or false-negative) due to the cross-reactivity and difficulties with quantification. Therefore, we have developed a portable capillary electrophoresis instrument to determine illegal drugs in oral fluid collected from a suspected person. However, this drug analyzer has still required manual sample preparation. Therefore, this research aimed to develop, test, and validate a fully automated sample pretreatment (purification, extraction, pre-concentration) prototype compatible with the capillary electrophoresis drug of abuse analyzer and suitable for confirmatory analysis by mass spectrometry. The cotton swab from Salivette® oral fluid collector was examined and integrated into the fully automated extractor prototype. The recoveries for the automated extractor were between 18 and 20%, with repeatabilities within 5-11% for 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), cocaine (COC), and cocaethylene (COET). The developed extraction device was easy to use even for unskilled persons, required minimal liquid handling, and was applicable to use in field conditions.

Sensitive Versatile Fluorogenic Transmembrane Peptide Substrates for Rhomboid Intramembrane Proteases.

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

Interactions of helquats with chiral acidic aromatic analytes investigated by partial-filling affinity capillary electrophoresis.

Noncovalent molecular interactions between helquats, a new class of dicationic helical extended diquats, and several chiral acidic aromatic drugs and catalysts have been investigated using partial-filling affinity capillary electrophoresis (PF-ACE). Helquats dissolved at 1mM concentration in the aqueous background electrolyte (40mM Tris, 20mM acetic acid, pH 8.1) were introduced as ligand zones of variable length (0-130mm) into the hydroxypropylcellulose coated fused silica capillary whereas 0.1mM solutions of negatively charged chiral drugs or catalysts (warfarin, ibuprofen, mandelic acid, etodolac, binaphthyl phosphate and 11 other acidic aromatic compounds) were applied as a short analyte zone at the injection capillary end. After application of electric field, analyte and ligand migrated against each other and in case of their interactions, migration time of the analyte was increasing with increasing length of the ligand zone. From the tested compounds, only isomers of those exhibiting helical chirality and/or possessing conjugated aromatic systems were enantioselectively separated through their differential interactions with helquats. Some compounds with conjugated aromatic groups interacted with helquats moderately strongly but non-enantiospecifically. Small compounds with single benzene ring exhibited no or very weak non-enantiospecific interactions. PF-ACE method allowed to determine binding constants of the analyte-helquat complexes from the changes of migration times of the analytes. Binding constants of the weakest complexes of the analytes with helquats were less than 50L/mol, whereas binding constants of the strongest complexes were in the range 1 000-1 400L/mol.

Study of deoxyribonucleic acid-ligand interactions by partial filling affinity capillary electrophoresis.

In this work, a new partial filling affinity capillary electrophoresis (PF-ACE) method has been developed and applied to investigation of non-covalent molecular interactions between double stranded DNA oligonucleotide (Dickerson dodecamer) and classical DNA intercalator ligand-ethidiumbromide (EtBr) or oligophenylene derivatives-based potential new type of DNA ligands. Binding constants of DNA-ligand complexes were determined from the dependence of migration time changes of DNA oligomer (applied as analyte) on the length of ligand zones introduced beforehand as plugs of various lengths (0-75mm with 12.5mm step) in hydroxypropylcellulose coated fused silica capillary of 50/375µm I.D./O.D. and 400/300mm total/effective length. PF-ACE experiments were performed in two background electrolytes, Tris-borate, pH 8.0, ionic strength 14.3mM (BGE1), and sodium phosphate, pH 7.5, ionic strength 133mM (BGE2). Binding constants of DNA-EtBr complex (ca 15300L/mol in the BGE1 and 4200L/mol in the BGE2) were found to be significantly higher than those of DNA complexes with oligophenylene derivatives (ca 2200-3600L/mol in the BGE1 and 1600-2300L/mol in the BGE2).

Toward Evaluating the Usefulness of GlobalIllumination for Novices in Lighting Design Tasks.

Thanks to its ability to improve the realism of computer-generated imagery, the use of global illumination has recently become widespread among digital lighting artists. It remains unclear, though, what impact it has on the lighting design workflows, especially for novice users. In this paper we present a user study which investigates the use of global illumination, large area lights, and non-physical fill lights in lighting design tasks, where 26 novice subjects design lighting with these tools. The collected data suggest that global illumination is not significantly harder to control for novice users that direct illumination, and when given the possibility, most users opt to use it in their designs. The use of global illumination together with large area lights leads to simpler lighting setups with fewer non-physical fill lights. Interestingly, global illumination does not supersede fill lights: users still include them into their globally illuminated lighting setups. We believe that our results will find use in the development of lighting design tools for non-expert users.

The apparent and potential effects of climate change on the inferred concentration of dissolved organic matter in a temperate stream (the Malse River, South Bohemia).

Long-term and seasonal changes in concentration of dissolved organic matter (DOM) and their possible drivers were evaluated for an upland stream in central Europe during 1969-2000. Two periods have been detected within this data set-years with decreased DOM until the middle of 1980s and then years with increased DOM until 2000. Temperature, hydrological regime of runoff from the catchment (namely the amount of interflow), and changes in atmospheric deposition of acidity coincided with the variations in DOM concentrations. The analysis of single runoff events confirmed the relation between the export of increased DOM concentrations from the catchment and interflow. A multiple linear regression model based on monthly averages of temperature and interflow explained 67% of DOM variability. This model suggested a 7% increase in DOM concentration under the scenarios of possible future climate change related to doubled CO(2) concentration in the atmosphere. The scenarios were based on results of several global circulation models.