Fluorescence-based directed termination PCR: direct mutation characterization without sequencing

We describe a fluorescence-based directed termination PCR (fluorescent DT–PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT–PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT–PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT–PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT–PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.

New methods of structure refinement for macromolecular structure determination by NMR

Recent advances in multidimensional NMR methodology have permitted solution structures of proteins in excess of 250 residues to be solved. In this paper, we discuss several methods of structure refinement that promise to increase the accuracy of macromolecular structures determined by NMR. These methods include the use of a conformational database potential and direct refinement against three-bond coupling constants, secondary 13C shifts, 1H shifts, T1/T2 ratios, and residual dipolar couplings. The latter two measurements provide long range restraints that are not accessible by other solution NMR parameters.

Cell fate determination in the vertebrate retina.

In the vertebrate central nervous system, the retina has been a useful model for studies of cell fate determination. Recent results from studies conducted in vitro and in vivo suggest a model of retinal development in which both the progenitor cells and the environment change over time. The model is based upon the notion that the mitotic cells within the retina change in their response properties, or "competence", during development. These changes presage the ordered appearance of distinct cell types during development and appear to be necessary for the production of the distinct cell types. As the response properties of the cells change, so too do the environmental signals that the cells encounter. Together, intrinsic properties and extrinsic cues direct the choice of cell fate.

(Table 1) Age determination of sediment cores MD925-2002 and MD03-2690

The compilation of results obtained on three giant piston cores from the Whittard, Shamrock and Guilcher turbidite levees reveals a high-resolution stratigraphic record for the Bay of Biscay. Due to the abundance of reworked sediments in these sedimentary environments, a specific methodological approach, based on an X-ray-assisted subsampling phase associated with sedimentological, geochemical and micropalaeontological analyses, was implemented. With an accurate chronological framework, this multi-proxy investigation provides observations on the 'Fleuve Manche' palaeoriver and the British-Irish Ice Sheet (BIS) histories over the last 20,000 years. The results obtained highlight the direct influence of the decay of the BIS on the Bay of Biscay deep-sea clastic sedimentation during the last European deglacial phase. During this period, the annual BIS cycle of meltwater seems enough to generate seasonal turbidity currents associated with exceptional sedimentation rates in all the Celtic and Armorican turbidite systems. With very high sedimentation rates, the turbidite levees represent the main deep-sea clastic depositional area. Long coring combined with a very careful subsampling method can provide continuous high-resolution palaeoenvironmental signals.

Age determination of Antarctic marine sediments

We present the first circum-East Antarctic chronology for the Holocene, based on 17 radiocarbon dates generated by the accelerator method. Marine sediments from around East Antarctica contain a consistent, high-resolution record of terrigenous (ice-proximal) and biogenic (open-marine) sedimentation during Holocene time. This record demonstrates that biogenic sedimentation beneath the open-marine environment on the continental shelf has been restricted to approximately the past 4 ka, whereas a period of terrigenous sedimentation related to grounding line advance of ice tongues and ice shelves took place between 7 and 4 ka. An earlier period of open-marine (biogenic sedimentation) conditions following the late Pleistocene glacial maximum is recognized from the Prydz Bay (Ocean Drilling Program) record between 10.7 and 7.3 ka. Clearly, the response of outlet systems along the periphery of the East Antarctic ice sheet during the mid-Holocene was expansion. This may have been a direct consequence of climate warming during an Antarctic 'Hypsithermal'. Temperature-accumulation relations for the Antarctic indicate that warming will cause a significant increase in accumulation rather than in ablation. Models that predict a positive mass balance (growth) of the Antarctic ice sheet under global warming are supported by the mid-Holocene data presented herein.

(Table 1) Age determination of sediment core RC11-83

Correlation of Southern Ocean deep sea sediment core records with ice core records of polar climate delineates with unprecedented detail the relationship between high latitude climate and the ocean's thermohaline circulation over the last 80,000 years. Our observations suggest that, while North Atlantic Deep Water variability manifests itself clearly in Southern Ocean nutrient proxy records over periods as short as 500 yr, this deep water variability did not promote a direct link between climate variability in the high latitudes of the two hemispheres on millennial timescales. In particular, the proxy records indicate that, on average, northern hemisphere climate fluctuations lagged those of the southern hemisphere by 1500 yr.

Rapid determination of aluminum in plutonium peroxide cake /

A carrier distillation spectrochemical method has been successfully adapted to direct reading spectrometry. Direct reading techniques have been developed for the rapid analyses of aluminum in plutonium peroxide to demonstrate the capability of direct reading for process impurity control.

Electrochemical deposition of cupric ions for the determination of mass transfer rates to a stepped rotating cylinder electrode

The stepped rotating cylinder electrode (SRCE) geometry has been developed as a simple aid to the practical study of the flow-enhanced corrosion and applied electrochemistry problems commonly observed under conditions of disturbed, turbulent flow. The electrodeposition of cupric ions from an acid sulphate plating bath has been used to characterise differential rates of mass transfer to the SRCE. The variation in thickness of electrodeposited copperfilms has allowed the mapping of local rates of mass transfer over the active surface of this geometry. Both optical and scanning electron microscopy were used for the examination of metallographic sections to provide a high resolution evaluation of the distribution of mass transfer coefficient. Results are also discussed using the convective-diffusion model in combination with the existing direct numerical flow simulation (DNS) data for this geometry.

Direct quantification of rare earth element concentrations in natural waters by ICP-MS

A direct quadrupole ICP-MS technique has been developed for the analysis of the rare earth elements and yttrium in natural waters. The method has been validated by comparison of the results obtained for the river water reference material SLRS-4 with literature values. The detection limit of the technique was investigated by analysis of serial dilutions of SLRS-4 and revealed that single elements can be quantified at single-digit fg/g concentrations. A coherent normalised rare earth pattern was retained at concentrations two orders of magnitude below natural concentrations for SLRS-4, demonstrating the excellent inter-element accuracy and precision of the method. The technique was applied to the analysis of a diluted mid-salinity estuarine sample, which also displayed a coherent normalised rare earth element pattern, yielding the expected distinctive marine characteristics. (c) 2006 Published by Elsevier Ltd.

On the numerical solution of a Cauchy problem for the Laplace equation via a direct integral equation approach

We investigate the problem of determining the stationary temperature field on an inclusion from given Cauchy data on an accessible exterior boundary. On this accessible part the temperature (or the heat flux) is known, and, additionally, on a portion of this exterior boundary the heat flux (or temperature) is also given. We propose a direct boundary integral approach in combination with Tikhonov regularization for the stable determination of the temperature and flux on the inclusion. To determine these quantities on the inclusion, boundary integral equations are derived using Green’s functions, and properties of these equations are shown in an L2-setting. An effective way of discretizing these boundary integral equations based on the Nystr¨om method and trigonometric approximations, is outlined. Numerical examples are included, both with exact and noisy data, showing that accurate approximations can be obtained with small computational effort, and the accuracy is increasing with the length of the portion of the boundary where the additionally data is given.

Determination of a spacewise dependent heat source

This paper investigates the inverse problem of determining a spacewise dependent heat source in the parabolic heat equation using the usual conditions of the direct problem and information from a supplementary temperature measurement at a given single instant of time. The spacewise dependent temperature measurement ensures that the inverse problem has a unique solution, but this solution is unstable, hence the problem is ill-posed. For this inverse problem, we propose an iterative algorithm based on a sequence of well-posed direct problems which are solved at each iteration step using the boundary element method (BEM). The instability is overcome by stopping the iterations at the first iteration for which the discrepancy principle is satisfied. Numerical results are presented for various typical benchmark test examples which have the input measured data perturbed by increasing amounts of random noise.

On a Direct Approach to the Solution of Inverse Optical Problems

The evaluation from experimental data, of physical quantities, which enter into the electromagnetic Maxwell equations, is described as inverse optical problem. The functional relations between the dependent and independent variables are of transcendental character and numeric procedures for evaluation of the unknowns are largely used. Herein, we discuss a direct approach to the solution, illustrated by a specific example of determination of thin films optical constants from spectrophotometric data. New algorithm is proposed for the parameters evaluation, which does not need an initial guess of the unknowns and does not use iterative procedures. Thus we overcome the intrinsic deficiency of minimization techniques, such as gradient search methods, Simplex methods, etc. The price of it is a need of more computing power, but our algorithm is easily implemented in structures such as grid clusters. We show the advantages of this approach and its potential for generalization to other inverse optical problems.

The development of direct ultra-fast PCR for forensic genotyping using short channel microfluidic systems with enhanced sieving matrices

There are situations in which it is very important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. The goal of this project was to develop an ultra fast, direct PCR method for forensic genotyping of oral swabs. The procedure developed eliminates the need for cellular digestion and extraction of the sample by performing those steps in the PCR tube itself. Then, special high-speed polymerases are added which are capable of amplifying a newly developed 7 loci multiplex in under 16 minutes. Following the amplification, a postage stamp sized microfluidic device equipped with specially designed entangled polymer separation matrix, yields a complete genotype in 80 seconds. The entire process is rapid and reliable, reducing the time from sample to genotype from 1-2 days to under 20 minutes. Operation requires minimal equipment and can be easily performed with a small high-speed thermal-cycler, reagents, and a microfluidic device with a laptop. The system was optimized and validated using a number of test parameters and a small test population. The overall precision was better than 0.17 bp and provided a power of discrimination greater than 1 in 106. The small footprint, and ease of use will permit this system to be an effective tool to quickly screen and identify individuals detained at ports of entry, police stations and remote locations. The system is robust, portable and demonstrates to the forensic community a simple solution to the problem of rapid determination of genetic identity.

Online solid phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of sucralose in reclaimed and drinking waters and its photo degradation in natural waters from South Florida

Background Sucralose has gained popularity as a low calorie artificial sweetener worldwide. Due to its high stability and persistence, sucralose has shown widespread occurrence in environmental waters, at concentrations that could reach up to several μg/L. Previous studies have used time consuming sample preparation methods (offline solid phase extraction/derivatization) or methods with rather high detection limits (direct injection) for sucralose analysis. This study described a faster and sensitive analytical method for the determination of sucralose in environmental samples. Results An online SPE-LC–MS/MS method was developed, being capable to quantify sucralose in 12 minutes using only 10 mL of sample, with method detection limits (MDLs) of 4.5 ng/L, 8.5 ng/L and 45 ng/L for deionized water, drinking and reclaimed waters (1:10 diluted with deionized water), respectively. Sucralose was detected in 82% of the reclaimed water samples at concentrations reaching up to 18 μg/L. The monthly average for a period of one year was 9.1 ± 2.9 μg/L. The calculated mass loads per capita of sucralose discharged through WWTP effluents based on the concentrations detected in wastewaters in the U. S. is 5.0 mg/day/person. As expected, the concentrations observed in drinking water were much lower but still relevant reaching as high as 465 ng/L. In order to evaluate the stability of sucralose, photodegradation experiments were performed in natural waters. Significant photodegradation of sucralose was observed only in freshwater at 254 nm. Minimal degradation (<20%) was observed for all matrices under more natural conditions (350 nm or solar simulator). The only photolysis product of sucralose identified by high resolution mass spectrometry was a de-chlorinated molecule at m/z 362.0535, with molecular formula C12H20Cl2O8. Conclusions Online SPE LC-APCI/MS/MS developed in the study was applied to more than 100 environmental samples. Sucralose was frequently detected (>80%) indicating that the conventional treatment process employed in the sewage treatment plants is not efficient for its removal. Detection of sucralose in drinking waters suggests potential contamination of surface and ground waters sources with anthropogenic wastewater streams. Its high resistance to photodegradation, minimal sorption and high solubility indicate that sucralose could be a good tracer of anthropogenic wastewater intrusion into the environment.

(Table 1) Age determination of North Atlantic sediment cores

High-resolution sediment cores from the Vøring Plateau, the North Iceland shelf, and the East Greenland shelf have been studied to investigate the stability of major surface currents in the Nordic Seas during the Holocene. Results from diatom assemblages and reconstructed sea-surface temperatures (SSTs) indicate a division of the Holocene into three periods: the Holocene Climate Optimum (9500-6500 calendar (cal) years BP), the Holocene Transition Period (6500-3000 cal years BP) and the Cool Late Holocene Period (3000-0 cal years BP). The overall climate development is in step with the decreasing insolation on the Northern Hemisphere, but regional differences occur regarding both timing and magnitude of SST changes. Sites under the direct influence of the Norwegian Atlantic Current and the Irminger Current indicate SST cooling of 4-5°C from early Holocene to present, compared to 2°C recorded under the East Greenland Current. Superimposed on the general Holocene cooling trend, there is a high-frequency SST variability, which is in the order of 1-1.5°C for the Vøring Plateau and the East Greenland shelf and 2.5-3°C on the North Iceland shelf.