Fast Structure-Based Assignment of 15N HSQC Spectra of Selectively 15N-Labeled Paramagnetic Proteins

A novel strategy for fast NMR resonance assignment of N-15 HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively N-15-labeled samples. Comparison of sensitive undecoupled N-15 HSQC spectra recorded of paramagnetic and diamagnetic samples yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole-dipole relaxation, and residual dipolar coupling. Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center. The method is demonstrated with the 30 kDa complex between the N-terminal domain of the epsilon subunit and the theta subunit of Escherichia Coll DNA polymerase III. The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy.

A PCR-based method of screening individuals of all ages, from neonates to the elderly, for familial hyperaldosteronism type I

Aim: Unless specifically treated (glucocorticoids in low doses), Familial Hyperaldosteronism Type I(FH-I) may result in early death from stroke. We report the successful application of a rapid, polymerase chain reaction (PCR)-based method of detecting the 'hybrid' 11 beta-hydroxylase (11 beta-OHase)/aldosterone synthase (AS) gene as a screening test for FH-I. Methods: 'Long-PCR' was used to amplify, concurrently, a 4 kb fragment of AS gene (both primers AS-specific) and a 4 kb fragment of the hybrid gene (5' primer 11 beta-OHase-specific, 3'primer AS-specific) from DNA extracted from blood either collected locally or transported from elsewhere. Sample collection and transport were straightforward. This 4 kb fragment contains all the currently recognised hybrid gene 'crossover' points. Results: Within a single family, long-PCR identified all 21 individuals known to have FH-I. Hypertension was corrected in all 11 treated with glucocorticoids. Nine with normal blood pressure are being closely followed for development of hypertension. Long-PCR cord blood analysis excluded FH-I in three neonates born to affected individuals. Long-PCR newly identified two other affected families: (1) a female (60 years) with a personal and family history of stroke and her normotensive daughter (40 years), and (2) a female (51 years) previously treated for primary aldosteronism with amiloride, her two hypertensive sons (14 and 16 years) and her hypertensive mother (78 years). No false negative or false positive results have yet been encountered. At least seven other centres have successfully performed this test. Conclusion: Long-PCR is a reliable method of screening individuals of all ages for FH-I.

Improved one-step solid-phase extraction method for morphine, morphine-3-glucuronide, and morphine-6-glucuronide from plasma and quantitation using high-performance liquid chromatography with electrochemical detection

This communication describes an improved one-step solid-phase extraction method for the recovery of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) from human plasma with reduced coextraction of endogenous plasma constituents, compared to that of the authors' previously reported method. The magnitude of the peak caused by endogenous plasma components in the chromatogram that eluted immediately before the retention time of M3G has been reduced (similar to 80%) significantly (p < 0.01) while achieving high extraction efficiencies for the compounds of interest, viz morphine, M6G, and M3G (93.8 +/- 2.5, 91.7 +/- 1.7, and 93.1 +/- 2.2%, respectively). Furthermore, when the improved solid-phase extraction method was used, the extraction cartridge-derived late-eluting peak (retention time 90 to 100 minutes) reported in our previous method, was no longer present in the plasma extracts. Therefore the combined effect of reducing the recovery of the endogenous components of plasma that chromatographed just before the retention time of M3G and the removal of the late-eluting, extraction cartridge-derived peak has resulted in a decrease in the chromatographic run-time to 20 minutes, thereby increasing the sample throughput by up to 100%.

A mathematical model for estimating the extent of solute- and water-flux heterogeneity in multiple sample percolation experiments

Multiple sampling is widely used in vadose zone percolation experiments to investigate the extent in which soil structure heterogeneities influence the spatial and temporal distributions of water and solutes. In this note, a simple, robust, mathematical model, based on the beta-statistical distribution, is proposed as a method of quantifying the magnitude of heterogeneity in such experiments. The model relies on fitting two parameters, alpha and zeta to the cumulative elution curves generated in multiple-sample percolation experiments. The model does not require knowledge of the soil structure. A homogeneous or uniform distribution of a solute and/or soil-water is indicated by alpha = zeta = 1, Using these parameters, a heterogeneity index (HI) is defined as root 3 times the ratio of the standard deviation and mean. Uniform or homogeneous flow of water or solutes is indicated by HI = 1 and heterogeneity is indicated by HI > 1. A large value for this index may indicate preferential flow. The heterogeneity index relies only on knowledge of the elution curves generated from multiple sample percolation experiments and is, therefore, easily calculated. The index may also be used to describe and compare the differences in solute and soil-water percolation from different experiments. The use of this index is discussed for several different leaching experiments. (C) 1999 Elsevier Science B.V. All rights reserved.

Implications of specimen preparation and of surface contamination for the measurement of the grain boundary carbon concentration of steels using x-ray microanalysis in an UHVFESTEM

The purpose of the present investigation was to gain an understanding of the nature of the carbon contamination on the surface of standard steel transmission electron spectroscopy (TEM) specimens, the effect of exposure of a clean specimen to normal laboratory air, and the efficacy of plasma-cleaning treatments. This knowledge is a necessary prerequisite to the development of appropriate specimen preparation and/or specimen cleaning methods. X-ray photoelectron spectroscopy in combination with argon ion beam profiling was used to characterize the specimen surfaces of X65 steel and 316 stainless steel. The only clean carbon-free surface obtained was that during argon etching of the sample in the surface analysis chamber. Any exposure of a previously cleaned sample to laboratory air resulted in a rapid carbon (hydrocarbon) contamination of the sample surface and the development of surface oxidation, Plasma cleaning with subsequent exposure of the specimen to the laboratory air also resulted in a carbon-contaminated surface. This suggests that procedures of preparation of TEM specimens of steels outside an ultrahigh vacuum chamber are unlikely to result in the lowering of contamination rates on specimens to levels where measurements for carbon in the grain boundaries are possible. What is needed is a cleaning system as an integral part of the specimen insertion system into the field-emission scanning transmission electron microscope. This cleaning could be carried out by argon ion etching. Copyright (C) 2000 John Wiley & Sons, Ltd.

Novel silica gel supported TiO2 photocatalyst synthesized by CVD method

TiO2 in anatase crystal phase is a very effective catalyst in the photocatalytic oxidation of organic compounds in water. To improve the recovery rate of TiO2 photocatalysts, which in most cases are in fine powder form, the chemical vapor deposition (CVD) method was used to load TiO2 onto a bigger particle support, silica gel. The amount of titania coating was found to depend strongly on the synthesis parameters of carrier gas flow rate and coating time. XPS and nitrogen ads/desorption results showed that most of the TiO2 particles generated from CVD were distributed on the external surface of the support and the coating was stable. The photocatalytic activities of TiO2/silica gel with different amounts of titania were evaluated for the oxidation of phenol aqueous solution and compared with that of Degussa P25. The optimum titania loading rate was found around 6 wt % of the TiO2 bulk concentration. Although the activity of the best TiO2/silica gel sample was still lower than that of P25, the synthesized TiO2/silica gel catalyst can be easily separated from the treated water and was found to maintain its TiO2 content and catalytic activity.

Determining complicated winding patterns for shim coils using stream functions and the target-field method

In a magnetic resonance imaging equipment, gradient and shim coils are needed to produce a spatially varying magnetic field throughout the sample being imaged. Such coils consist of turns of wire wound on the surface of a cylindrical tube. Shim coils in particular, must sometimes be designed to produce complicated magnetic fields to correct for impurities. Streamline patterns for shim coils are much more complicated than those for gradient coils, In this work we present a detailed analysis of streamline methods and their application to shim coil design, A method is presented for determining the winding patterns to generate these complicated fields. (C) 2002 John Wiley & Sons, Inc.

The preparation and readiness for voluntary movement: a high-field event-related fMRI study of the Bereitschafts-BOLD response

Activity within motor areas of the cortex begins to increase 1 to 2 s prior to voluntary self-initiated movement (termed the Bereitschaftspotential or readiness potential). There has been much speculation and debate over the precise source of this early premovement activity as it is important for understanding the roles of higher order motor areas in the preparation and readiness for voluntary movement. In this study, we use high-field (3-T) event-related fMRI with high temporal sampling (partial brain volumes every 250 ms) to specifically examine hemodynamic response time courses during the preparation, readiness, and execution of purely self-initiated voluntary movement. Five right-handed healthy volunteers performed a rapid sequential finger-to-thumb movement performed at self-determined times (12-15 trials). Functional images for each trial were temporally aligned and the averaged time series for each subject was iteratively correlated with a canonical hemodynamic response function progressively shifted in time. This analysis method identified areas of activation without constraining hemodynamic response timing. All subjects showed activation within frontal mesial areas, including supplementary motor area (SMA) and cingulate motor areas, as well as activation in left primary sensorimotor areas. The time courses of hemodynamic responses showed a great deal of variability in shape and timing between subjects; however, four subjects clearly showed earlier relative hemodynamic responses within SMA/cingulate motor areas compared with left primary motor areas. These results provide further evidence that the SMA and cingulate motor areas are major contributors to early stage premovement activity and play an important role in the preparation and readiness for voluntary movement. (C) 2003 Elsevier Inc. All rights reserved.

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

in the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 mu g of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 mu g of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach. (c) 2008 Elsevier Ltd. All rights reserved.

Operational regimes for a simplified one-step artificial chaperone refolding method

The artificial chaperone method for protein refolding developed by Rozema et al. (Rozema, D.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117 (8), 2373-2374) involves the sequential dilution of denatured protein into a buffer containing detergent (cetyltrimethylammonium bromide, CTAB) and then into a refolding buffer containing cyclodextrin WD). In this paper a simplified one-step artificial chaperone method is reported, whereby CTAB is added directly to the denatured solution, which is then diluted directly into a refolding buffer containing P-cyclodextrin (P-CD). This new method can be applied at high protein concentrations, resulting in smaller processing volumes and a more concentrated protein solution following refolding. The increase in achievable protein concentration results from the enhanced solubility of CTAB at elevated temperatures in concentrated denaturant. The refolding yields obtained for the new method were significantly higher than for control experiments lacking additives and were comparable to the yields obtained with the classical two-step approach. A study of the effect of beta-CD and CTAB concentrations on refolding yield suggested two operational regimes: slow stripping ( beta-CDXTABsimilar to1), most suited for higher protein concentrations, and fast stripping (beta-CD/CTABsimilar to2.7), best suited for lower protein concentrations. An increased chaotrope concentration resulted in higher refolding yields and an enlarged operational regime.

Fast and efficient synthesis of pyrano[3,2-c]quinolines catalyzed by niobium(V) chloride

A highly efficient two-step method for the synthesis of pyranoquinoline derivatives from imino-Diels-Alder reactions between aldimines and 3,4-dihydro-2H-pyran using niobium(V) chloride as catalyst under mild conditions is described.

Morphological and electrochemical investigation of RuO(2)-Ta(2)O(5) oxide films prepared by the Pechini-Adams method

Preparation methods can profoundly affect the structural and electrochemical properties of electrocatalytic coatings. In this investigation, RuO(2)-Ta(2)O(5) thin films containing between 10 and 90 at.% Ru were prepared by the Pechini-Adams method. These coatings were electrochemically and physically characterized by cyclic voltammetry, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). The composition and morphology of the oxide were investigated before and after accelerated life tests (ALT) by EDX and SEM. SEM results indicate typical mud-flat-cracking morphology for the majority of the films. High resolution SEMs reveal that pure oxide phases exhibit nanoporosity while binary compositions display a very compact structure. EDX analyses reveal considerable amounts of Ru in the coating even after total deactivation. XRD indicated a rutile-type structure for RuO(2) and orthorhombic structure for Ta(2)O(5). XPS data demonstrate that the binding energy of Ta is affected by Ru addition in the thin films, but the binding energy of Ru is not likewise influenced by Ta. The stability of the electrodes was evaluated by ALT performed at 750 mA cm(-2) in 80 degrees C 0.5 mol dm(-3) H(2)SO(4). The performance of electrodes prepared by the Pechini-Adams method is 100% better than that of electrodes prepared by standard thermal decomposition.

Rifampicin determination in plasma by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of rifampicin in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. This miniaturized method can result in faster analysis, higher sample throughput, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. Important factors in the optimization of SBSE efficiency such as pH, temperature, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) were optimized recoveries ranging from 75 to 80%. Separation was obtained using a reverse phase C(8) column with UV detection (254 nm). The mobile phase consisted of methanol:0.25 N sodium acetate buffer, pH 5.0 (58:42, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.125-50.0 mu g mL(-1). The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.25, 6.25 and 25.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 10% and all inter-CVs were less than 10%. Limits of quantification were 0.125 mu g mL(-1). Stability studies showed rifampicin was stable in plasma for 12 h after thawing; the samples were also stable for 24 h after preparation. Based on the figures of merit results, the SBSE/HPLC-UV proved to be adequate to the rifampicin analyses from therapeutic to toxic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (C) 2009 Elsevier B.V. All rights reserved.

Fast-convergent iterative scheme for filtering velocity signals and finding Kolmogorov scales

The present fundamental knowledge of fluid turbulence has been established primarily from hot- and cold-wire measurements. Unfortunately, however, these measurements necessarily suffer from contamination by noise since no certain method has previously been available to optimally filter noise from the measured signals. This limitation has impeded our progress of understanding turbulence profoundly. We address this limitation by presenting a simple, fast-convergent iterative scheme to digitally filter signals optimally and find Kolmogorov scales definitely. The great efficacy of the scheme is demonstrated by its application to the instantaneous velocity measured in a turbulent jet.

A computerized tomography scan method for calculating the hernia sac and abdominal cavity volume in complex large incisional hernia with loss of domain

Preoperative progressive pneumoperitoneum (PPP) is a safe and effective procedure in the treatment of large incisional hernia (size > 10 cm in width or length) with loss of domain (LIHLD). There is no consensus in the literature on the amount of gas that must be insufflated in a PPP program or even how long it should be maintained. We describe a technique for calculating the hernia sac volume (HSV) and abdominal cavity volume (ACV) based on abdominal computerized tomography (ACT) scanning that eliminates the need for subjective criteria for inclusion in a PPP program and shows the amount of gas that must be insufflated into the abdominal cavity in the PPP program. Our technique is indicated for all patients with large or recurrent incisional hernias evaluated by a senior surgeon with suspected LIHLD. We reviewed our experience from 2001 to 2008 of 23 consecutive hernia surgical procedures of LIHLD undergoing preoperative evaluation with CT scanning and PPP. An ACT was required in all patients with suspected LIHLD in order to determine HSV and ACV. The PPP was performed only if the volume ratio HSV/ACV (VR = HSV/ACV) was a parts per thousand yen25% (VR a parts per thousand yen 25%). We have performed this procedure on 23 patients, with a mean age of 55.6 years (range 31-83). There were 16 women and 7 men with an average age of 55.6 years (range 31-83), and a mean BMI of 38.5 kg/m(2) (range 23-55.2). Almost all patients (21 of 23 patients-91.30%) were overweight; 43.5% (10 patients) were severely obese (obese class III). The mean calculated volumes for ACV and HSV were 9,410 ml (range 6,060-19,230 ml) and 4,500 ml (range 1,850-6,600 ml), respectively. The PPP is performed by permanent catheter placed in a minor surgical procedure. The total amount of CO(2) insufflated ranged from 2,000 to 7,000 ml (mean 4,000 ml). Patients required a mean of 10 PPP sessions (range 4-18) to achieve the desired volume of gas (that is the same volume that was calculated for the hernia sac). Since PPP sessions were performed once a day, 4-18 days were needed for preoperative preparation with PPP. The mean VR was 36% (ranged from 26 to 73%). We conclude that ACT provides objective data for volume calculation of both hernia sac and abdominal cavity and also for estimation of the volume of gas that should be insufflated into the abdominal cavity in PPP.