Detection of DNA sequence variations in homo- and heterozygous samples via molecular mass measurements by electrospray ionization time-of-flight mass spectrometry

The potential of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry for the characterization of polymerase chain reaction (PCR) amplified nucleic acids was evaluated. For that purpose, a "SNP toolbox" was constructed by cloning and PCR-mediated site-directed in vitro mutagenesis at nucleotide position (ntp) 16,519 of a sequence-verified fragment of the human mitochondrial genome (ntps 15,900-599). Confirmatory sequencing demonstrated that within the sequences of the clones one and the same base was mutated to all other bases. Using these clones or equimolar mixtures of these clones as PCR templates, 51-401-bp-long amplicons were generated, which were used to determine the upper size limits of PCR products for the unequivocal detection of sequence variations in homo- and heterozygous samples. Based on the high mass spectrometric performance of the applied time-of-flight mass spectrometer, the unequivocal genotyping of all kinds of single base exchanges in PCR amplicons from heterozygous samples with lengths up to 254 base pairs (bp) was demonstrated. Considering homozygous samples, the successful genotyping of single base substitutions in up to 401-bp-long PCR products was possible. Consequently, the described hyphenated technique represents one of the most powerful mass spectrometric genotyping assays available today.     

Extension Language Automation of Embedded System Debugging

Embedded systems contain several layers of target processing abstraction. These layers include electronic circuit, binary machine code, mnemonic assembly code, and high-level procedural and object-oriented abstractions. Physical and temporal constraints and artifacts within physically embedded systems make it impossible for software engineers to operate at a single layer of processor abstraction. The Luxdbg embedded system debugger exposes these layers to debugger users, and it adds an additional layer, the extension language layer, that allows users to extend both the debugger and its target processor capabilities. Tcl is Luxdbg's extension language. Luxdbg users can apply Tcl to automate interactive debugging steps, to redirect and to interconnect target processor input-output facilities, to schedule multiple processor execution, to log and to react to target processing exceptions, to automate target system testing, and to prototype new debugging features. Inclusion of an extension language like Tcl in a debugger promises additional advantages for distributed debugging, where debuggers can pass extension language expressions across computer networks.     

Profiling 627 mitochondrial nucleotides via the analysis of a 23-plex polymerase chain reaction by liquid chromatography-electrospray ionization time-of-flight mass spectrometry

We present a rapid and informative mitochondrial DNA profiling system, which has high forensic impact. The assay is based on the analysis of a 23-plex PCR by ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS). In a single 25-min run, an overall number of 627 nucleotide positions were screened. The vast majority of observed sequence variations were explainable by alterations of the allelic states of the 23 target SNPs, which were selected on their ability to increase forensic discrimination within West Eurasian populations. Within an Austrian population sample comprising 90 unrelated men, 14 different, nontarget SNP-related sequence variations--13 base substitutions and 1 deletion--were detected by ICEMS and confirmed by sequencing. All amplified sequences were located outside of the routinely sequenced hypervariable segments (HVS-I and HVS-II) of the noncoding control region. Accordingly, the genetic information obtained by the 23-plex PCR-ICEMS assay could be combined with HVS-I/HVS-II sequencing results to one highly discriminating mtDNA profile, which covered approximately 7.5% of the total mtDNA genome. With the 23-plex PCR-ICEMS assay, DNA mixtures were detected and the allelic ratios were accurately quantified. The observed robustness and sensitivity underlined the practical applicability of the assay in forensic science, which was proven by typing eight representative casework samples.     

Science-based MEMS reliability methodology

In cases where device numbers are limited, large statistical studies to verify reliability are impractical. Instead, an approach incorporating a solid base of modelling, simulation, and material science into a standard reliability methodology makes more sense and leads to a science-based reliability methodology. The basic reliability method is (a) design, model and fabricate, (b) test structures and devices, (c) identify failure modes and mechanisms, (d) develop predictive reliability models (accelerated aging), and (e) develop qualification methods. At various points in these steps technical data is required on MEMS material properties (residual stress, fracture strength, fatigue, etc.), MEMS surface characterization (stiction, friction, adhesion, role of coatings, etc.) or MEMS modelling and simulation (finite element, analysis, uncertainty analysis, etc.). This methodology is discussed as it relates to reliability testing of a micro-mirror array consisting of 144-piston mirrors. In this case, 140 mirrors were cycled full stroke (1.5 μm) 26 billion times with no failure. Using our technical science base, fatigue of the springs was eliminated as a mechanism of concern. Eliminating this wear-out mechanism allowed use of the exponential statistical model to predict lower bound confidence levels for failure rate in a “no-fail” condition.     

Biological variation of prostate specific antigen levels in serum: an evaluation of day-to-day physiological fluctuations in a well-defined cohort of 24 patients

PURPOSE: We evaluated the daily biological variation of serum prostate specific antigen (PSA) concentrations to determine the critical difference required between 2 consecutive PSA measurements that would indicate a significant elevation. MATERIALS AND METHODS: A total of 24 men, grouped according to clinical diagnosis and PSA, underwent phlebotomy for 10 consecutive weekdays. Duplicate serum samples were measured using 3 separate lots of Tandem-E and IMx PSA assays. The biological variation was calculated and the 2 PSA assay systems were compared. The critical difference was examined to determine the percent elevation necessary to indicate (with 95% confidence) that PSA had increased beyond what would be expected from biological and analytical variation. RESULTS: The biological variation, defined in terms of percent coefficient of variation, had a log-normal distribution with a geometric mean of 7.3% coefficient of variation and a 95th percentile value of 19.2% coefficient of variation using the Tandem-E PSA assay. Assuming an analytical variation of 5% coefficient of variation, the median critical difference was 20.5% and the 95th percentile critical difference was 45.8%. There was no significant difference between the 2 PSA assay systems in biological variation. However, PSA concentrations measured by the IMx assay were consistently lower compared to values measured by the Tandem-E assay. CONCLUSIONS: Characterizing the biological variation of serum PSA assists in evaluating the significance of changes in serial PSA measurements. The degree of biological variation differs among patients, such that an increase between 2 consecutive PSA levels that is less than 20 to 46% may be due to biological and analytical variation. These data influence interpretation of repeated measurements of serum PSA with time.     

Coagulation of NOM: linking character to treatment.

The paper presents an overview and discussion on the link between natural organic matter (NOM) character and its treatability by coagulation. Trials were conducted on a series of bench scale and pilot plant trials on three source waters: two from UK moorland catchments and one from a US snow melt source. Overall the work demonstrates the importance of the polarity balance and the charge density of the NOM contained within the source water. The hydrophobic content controls the coagulant demand such that variation in the demand between sources or sampling periods can be accounted for by changes in the hydrophobic content and its charge density. The raw water hydrophilic content, and specifically the nonacid fraction, provides a useful indicator of the achievable residual. Analysis of coagulation performance revealed a clear relationship between zeta potential and residual DOC. For each source an operational zeta potential range exists within which the residual concentration is optimal. Comparison of the ranges achieved during each trial demonstrated that a communal range between -10 and +3 mV exist for all waters thus providing a useful guide range for operational control.     

The Effect of Bridge Geometry on Microstructure and Texture Evolution During Porthole Die Extrusion of an Al–Mg–Si–Mn–Cr Alloy

Porthole die extrusion is used to produce complex hollow aluminum cross-sections for automotive applications. In a porthole die, the material is first divided into multiple streams which are separated by a bridge, before rejoining in the weld chamber and finally passing through the die orifice. The rejoining of the material in the weld chamber produces lines known as weld lines in the final extruded product. The microstructure along the weld line and its associated quality are strongly influenced by the thermal-mechanical history the material experiences as it passes through the portholes, the weld chamber, and the die orifice, which can be altered by die design and, in particular, the bridge geometry. To study the influence of bridge geometry on weld line microstructure and final quality, a series of porthole die extrusion experiments was conducted using an Al–Mg–Si–Mn–Cr alloy and two different types of bridge geometry (streamlined and flat). The experimental results showed that bridge geometry had a significant effect on the local microstructure and crystallographic texture at the weld line. Specifically, EBSD analysis indicated that the weld line texture associated with a streamlined bridge geometry consisted of a deformation texture (mainly the copper component), while the local texture produced by a flat bridge was a recrystallization texture consisting of Cube, Goss, and Cube_RD texture components. Simulation of the extrusion process, using DEFORM 3D, indicated that the weld line produced using a flat bridge experienced a slightly higher temperature, but much higher equivalent strains than the streamlined case. Material away from the weld line was very similar for both cases, indicating that the effect of the die bridge geometry is localized to the region close to the weld line.