Mitochondrial multiplex real-time PCR as a source tracking method in fecal-contaminated effluents

Multiplex real-time PCR amplifying fecal mitochondrial DNA (mtDNA) combined with rapid, crude DNA preparations are promising additions to surface water source tracking methods. Amplification of eukaryotic mitochondrial DNA identifies the fecal source directly and can be used in conjunction with other intestinal microbial methods to characterize effluents. Species-specific primers and dual-labeled probes for human, swine, and bovine NADH dehydrogenase subunit 5 (ND5) genes were created for multiplex real-time PCR in feces and effluent slurries. The linear range of the multiplex assay was 10(2)-10(7) mtDNA copies for human, bovine, and swine effluent in combination (equal volumes). PCR amplification efficiencies for bovine, human, and swine mtDNA when assayed in combination were 93, 107, and 92% respectively. Linear regression correlation coefficients (r2) were 0.99 for all standard curves except for human mtDNA in combination (r2 = 0.95). Multiplex amplification of bovine, human, and swine mtDNA (ND5) exhibited no cross-reactions between the effluents from three species of interest. Also, no cross-reactions were observed with effluents of other vertebrates: sheep, goat, horse, dog, cat, Canada goose, broiler, layer, turkey, and tilapia. Performed as a blind test, the PCR operator was able to correctly identify all but two effluent challenge samples (10/12 or 83% correct) with no false positives (22/22 or 100% correct). The multiplex assay had a tendency to detect the species of highest mtDNA concentration only. Better detection of all three species in a combination of human, bovine, and swine effluents was accomplished by running each real-time PCR primer/ probe set singly. Real-time PCR detection limit was calculated as 2.0 x 10(6) mitochondrial copies or 0.2 g of human feces per 100 mL effluent. Some carry-over mtDNA PCR signal from consumed beef, but not pork, was found in feces of human volunteers.     

The Oxidative Precipitation of Thallium at Alkaline pH for Treatment of Mining Influenced Water

Thallium (Tl) may exceed regulatory limits in mining-influenced water (MIW) associated with processing cadmium, copper, gold, lead, and zinc ores. It is a toxic metal that is soluble over a wide pH range, resulting in both persistence in the environment and poor removal by conventional lime precipitation. This study evaluated the effect of potassium permanganate (KMnO₄) at alkaline pH on Tl removal from MIW in batch experiments. The oxidation of Tl⁺ to Tl³⁺ by KMnO₄ and subsequent Tl removal was explored at Tl concentrations of ≤1 mg/L in synthetic and actual MIW. In addition to Tl, the synthetic MIW contained ≈5 mg/L of Mn, while the actual MIW contained >10 mg/L of Al, Cu, Fe, Mn, and Zn and had a pH ≈ 2.5. Dissolved Tl <2 μg/L in synthetic MIW was achieved at a pH ≈ 9 (CaO addition) and ≥5 mg/L of KMnO₄. In the actual MIW, dissolved Tl <2 μg/L was achieved at pH ≈ 9 and ≥12 mg/L of KMnO₄. The Tl removal mechanism is complicated due to the presence of reduced Mn in the synthetic MIW and multiple metals in the actual MIW. However, effective Tl removal was achieved by adding KMnO₄ to synthetic and actual MIW at alkaline pH.     

Reduction of error in spectrophotometry of scattering media using polarization techniques

Scattering can result in erroneous determination of the concentrations of constituent absorbers in spectrophotometry. This is due to the relationship between attenuation and absorption coefficient becoming nonlinear; hence, the use of the Lambert-Beer law becomes invalid. It has previously been shown that application of polarization techniques can reduce these effects, resulting in a more linear relationship. Here we quantify the impact of this improvement on measurement of the ratio of concentrations for two general absorbing species and show that measurement using polarization-maintaining light is more accurate. This is performed using a generalized version of theory previously dependent on selection of isosbestic wavelengths. For the absorbing species and geometries considered here, the mean error on the estimation of absorber concentration ratio is 18.2% for the case of detection without polarization discrimination. When polarization-maintaining light is extracted, mean errors of 1.2% and 5.1% are achieved for linear and circular polarizations, respectively. The improvement provided by the polarization techniques is observed regardless of the illuminating wavelengths but is achieved at the expense of a reduced signal-to-noise ratio. Taking this into account, for the detection scheme considered with a detector well capacity of 4 x 10(5) electrons the improvement provided by linear polarization-maintaining light is reduced to a factor of 3.6 and for circular polarizations a factor of 2.2.     

Analytical electric field and sensitivity analysis for two microfluidic impedance cytometer designs

Microfabricated impedance cytometers have been developed to measure the electrical impedance of single biological particles at high speed. A general approach to analytically solve the electric field distributions for two different designs of cytometers: parallel facing electrodes and coplanar electrodes, using the Schwarz-Christoffel Mapping method is presented. Compared to previous analytical solutions, our derivations are more systematic and solutions are more straightforward. The solutions have been validated by comparison with numerical simulations performed using the finite element method. The influences on the electric field distribution due to the variations in the geometry of the devices have been discussed. A simple method is used to determine the impedance sensitivity of the system and to compare the two electrode designs. For identical geometrical parameters, we conclude that the parallel electrodes design is more sensitive than the coplanar electrodes.     

Fragmentation characteristics of collision-induced dissociation in MALDI TOF/TOF mass spectrometry

The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/TOF mass spectrometry, collecting statistics using a curated set of 2459 MS/MS spectra and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest mass peaks. The amino acids P, W, D, and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K, and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation.     

Rapid rotation of micron and submicron dielectric particles measured using optical tweezers

Abstract

We demonstrate the use of a laser trap (‘optical tweezers’) and back-focal-plane position detector to measure rapid rotation in aqueous solution of single particles with sizes in the vicinity of 1 μm. Two types of rotation were measured: electrorotation of polystyrene microspheres and rotation of the flagellar motor of the bacterium Vibrio alginolyticus. In both cases, speeds in excess of 1000 Hz (rev s^?1) were measured. Polystyrene beads of diameter about 1 μm labelled with smaller beads were held at the centre of a microelectrode array by the optical tweezers. Electrorotation of the labelled beads was induced by applying a rotating electric field to the solution using microelectrodes. Electrorotation spectra were obtained by varying the frequency of the applied field and analysed to obtain the surface conductance of the beads. Single cells of V. alginolyticus were trapped and rotation of the polar sodium-driven flagellar motor was measured. Cells rotated more rapidly in media containing higher concentrations of Na⁺, and photodamage caused by the trap was considerably less when the suspending medium did not contain oxygen. The technique allows single-speed measurements to be made in less than a second and separate particles can be measured at a rate of several per minute.     

Epitaxial La0.5Sr0.5CoO3 electrode films for ferroelectric device applications

Abstract

We have grown high quality La_0.5Sr_0.5CoO₃ thin films by Pulsed Laser Deposition. This material is a electrically conducting perovskite which can be used as structurally compatible electrodes for devices using ferroelectric materials such as Pb-La-Zr-Ti-O (PLZT). Oxygen stability was investigated under various annealing conditions. LSCO/PLZT/LSCO heterostructures were grown epitaxially on SrTiO₃ (STO) and MgO substrates. P-E hysteresis loop was observed with P_r = 20 μC/cm² and E_c = 24 kV/cm. Direct observation of the piezoelectric effect in PLZT was made by measuring the PLZT c-axis lattice constant under various electric fields. LSCO/STO superlattice was also grown by PLD. The superlattice showed excellent crystallinity with a Rutherford Backscattering (RBS) channeling minimum yield of only 3.6%.     

Lean Product Development as a System: A Case Study of Body and Stamping Development at Ford

Abstract:

A systems view of lean product development is presented integrating people, process, and tools. This systems approach was derived from intensive study of Toyota's product development system. Principles and methods based on this systems model of lean product development were applied with great success at Ford Motor Company helping to bring to market a record number of products that helped fuel a rebuilding of the company to financial success and award winning world-class product quality.     

Effectiveness of aeration and mixing in the remediation of a saline stratified river

This study examines the use of an aeration scheme to remediate low oxygen conditions in a saline stratified system. The Tawe estuary was impounded in 1992 and quickly developed saline stratification during the summer months which led to an anoxic hypolimnon. In 1998 trials began in which a suite of aerators was applied to remediate the water quality; the trial was later extended to a full aeration scheme. This study examines pre-aeration conditions in order to delineate conditions under which poor water quality would develop, and would therefore be the conditions when aeration would be necessary. Furthermore, the study compared identical periods within the impoundment during which the following conditions existed: no aeration; and aeration with first 44, then 88, aerators. The study shows that (i) destratification occurred naturally under flows of >10 m3/s, and no low dissolved oxygen conditions were observed at higher flows; (ii) the presence of all levels of aeration had a statistically significant effect upon dissolved oxygen (DO) levels; the effect of increasing the number of aerators was approximately linear; (iii) the average effect of aeration was an increase of up to 3 mg/L DO in the deepest water; (iv) the frequency of low DO conditions decreased from 19% to 3% with the operation of aerators; and (v) aeration is most effective during periods of no tidal incursion and further from the saline water source. This study is the first to demonstrate the effectiveness of aeration in a saline stratified system.