A PCR method for the identification of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs

Aim: The aim of this study was to assess the discriminatory power and potential turn around time ( TAT) of a PCR-based method for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs. Methods: Screening swabs were examined using the current laboratory protocol of direct culture on mannitol salt agar supplemented with oxacillin (MSAO-direct). The PCR method involved pre-incubation in broth for 4 hours followed by a multiplex PCR with primers directed to mecA and nuc genes of MRSA. The reference standard was determined by pre-incubation in broth for 4 hours followed by culture on MSAO (MSAO-broth). Results: A total of 256 swabs was analysed. The rates of detection of MRSA using MSAO-direct, MSAO-broth and PCR were 10.2, 13.3 and 10.2%, respectively. For PCR, the sensitivity, specificity, positive predictive value and negative predictive values were 66.7% (95% CI 51.9 - 83.3%), 98.6% ( 95% CI 97.1 - 100%), 84.6% ( 95% CI 76.2 - 100%) and 95.2% ( 95% CI 92.4 - 98.0%), respectively, and these results were almost identical to those obtained from MSAO-direct. The agreement between MSAO-direct and PCR was 61.5% ( 95% CI 42.8 - 80.2%) for positive results, 95.6% ( 95% CI 93.0 - 98.2%) for negative results and overall was 92.2% ( 95% CI 88.9 - 95.5%). Conclusions: ( 1) The discriminatory power of PCR and MSAO-direct is similar but the level of agreement, especially for true positive results, is low. ( 2) The potential TAT for the PCR method provides a marked advantage over conventional methods. ( 3) Further modifications to the PCR method such as increased broth incubation time, use of selective broth and adaptation to real-time PCR may lead to improvement in sensitivity and TAT.

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.

Laser frequency locking by direct measurement of detuning

We present a new method of laser frequency locking in which the feedback signal is directly proportional to the detuning from an atomic transition, even at detunings many times the natural linewidth of the transition. Our method is a form of sub-Doppler polarization spectroscopy, based on measuring two Stokes parameters (I-2 and I-3) of light transmitted through a vapor cell. It extends the linear capture range of the lock loop by as much as an order of magnitude and provides frequency discrimination equivalent to or better than those of other commonly used locking techniques. (C) 2004 Optical Society of America

Is the method of signal analysis and test selection important for measuring standing balance in subjects with persistent whiplash?

Dizziness and or unsteadiness, associated with episodes of loss of balance, are frequent complaints in those suffering from persistent problems following a whiplash injury. Research has been inconclusive with respect to possible aetiology, discriminative tests and analyses used. The aim of this pilot research was to identify the test conditions and the most appropriate method for the analysis of sway that may differentiate subjects with persistent whiplash associated disorders (WAD) from healthy controls. The six conditions of the Clinical Test for Sensory Interaction in Balance was performed in both comfortable and tandem stance in 20 subjects with persistent WAD compared to 20 control subjects. The analyses were carried out using a traditional method of measurement, total sway distance, to results obtained from the use of wavelet analysis. Subjects with WAD were significantly less able to complete the tandem stance tests on a firm surface than controls. In comfortable stance, using wavelet analysis, significant differences between subjects with WAD and the control group were evident in total energy of the trace for all test conditions apart from eyes open on the firm surface. In contrast, the results of the analysis using total sway distance revealed no significant differences between groups across all six conditions. Wavelet analysis may be more appropriate for detecting disturbances in balance in whiplash subjects because the technique allows separation of the noise from the underlying systematic effect of sway. These findings will be used to direct future studies on the aeitiology of balance disturbances in WAD. (c) 2004 Elsevier B.V. All rights reserved.

Piggery pond sludge as a nitrogen source for crops - 2. Assay of wet and stockpiled piggery pond sludge by successive cereal crops or direct measurement of soil available N

The appropriate use of wastes is a significant issue for the pig industry due to increasing pressure from regulatory authorities to protect the environment from pollution. Nitrogen contained in piggery pond sludge ( PPS) is a potential source of supplementary nutrient for crop production. Nitrogen contribution following the application of PPS to soil was obtained from 2 field experiments on the Darling Downs in southern Queensland on contrasting soil types, a cracking clay ( Vertosol) and a hardsetting sandy loam (Sodosol), and related to potentially mineralisable N from laboratory incubations conducted under controlled conditions and NO3- accumulation in the field. Piggery pond sludge was applied as-collected ( wet PPS) and following stockpiling to dry ( stockpiled PPS). Soil NO3- levels increased with increased application rates of wet and stockpiled PPS. Supplementary N supply from PPS estimated by fertiliser equivalence was generally unsatisfactory due to poor precision with this method, and also due to a high level of NO3- in the clay soil before the first assay crop. Also low recoveries of N by subsequent sorghum ( Sorghum bicolor) and wheat ( Triticum aestivum) assay crops at the 2 sites due to low in-crop rainfall in 1999 resulted in low apparent N availability. Over all, 29% ( range 12 - 47%) of total N from the wet PPS and 19% ( range 0 - 50%) from the stockpiled PPS were estimated to be plant-available N during the assay period. The high concentration of NO3- for the wet PPS application on sandy soil after the first assay crop ( 1998 barley, Hordeum vulgare) suggests that leaching of NO3- could be of concern when high rates of wet PPS are applied before infrequent periods of high precipitation, due primarily to the mineral N contained in wet PPS. Low yields, grain protein concentrations, and crop N uptake of the sorghum crop following the barley crop grown on the clay soil demonstrated a low residual value of N applied in PPS. NO3- in the sandy soil before sowing accounted for 79% of the variation in plant N uptake and was a better index than anaerobically mineralisable N ( 19% of variation explained). In clay soil, better prediction of crop N uptake was obtained when both anaerobically mineralisable N (39% of variation explained) and soil pro. le NO3- were used in combination (R-2 = 0.49).

Adsorption of pure fluids in activated carbon with a modified lattice gas model

In this article we study the effects of adsorbed phase compression, lattice structure, and pore size distribution on the analysis of adsorption in microporous activated carbon. The lattice gas approach of Ono-Kondo is modified to account for the above effects. Data of nitrogen adsorption at 77 K onto a number of activated carbon samples are analyzed to investigate the pore filling pressure versus pore width, the packing effect, and the compression of the adsorbed phase. It is found that the PSDs obtained from this analysis are comparable to those obtained by the DFT method. The discrete nature of the PSDs derived from the modified lattice gas theory is due to the inherent assumption of discrete layers of molecules. Nevertheless, it does provide interesting information on the evolution of micropores during the activation process.

Lifetime prevalence estimates of major depression: An indirect estimation method and a quantification of recall bias

The measurement of lifetime prevalence of depression in cross-sectional surveys is biased by recall problems. We estimated it indirectly for two countries using modelling, and quantified the underestimation in the empirical estimate for one. A microsimulation model was used to generate population-based epidemiological measures of depression. We fitted the model to 1-and 12-month prevalence data from the Netherlands Mental Health Survey and Incidence Study (NEMESIS) and the Australian Adult Mental Health and Wellbeing Survey. The lowest proportion of cases ever having an episode in their life is 30% of men and 40% of women, for both countries. This corresponds to a lifetime prevalence of 20 and 30%, respectively, in a cross-sectional setting (aged 15-65). The NEMESIS data were 38% lower than these estimates. We conclude that modelling enabled us to estimate lifetime prevalence of depression indirectly. This method is useful in the absence of direct measurement, but also showed that direct estimates are underestimated by recall bias and by the cross-sectional setting.

The performance of the maximum ratio combining method in correlated Rician-fading channels for antenna-diversity signal combining

The performance of the maximum ratio combining method for the combining of antenna-diversity signals in correlated Rician-fading channels is rigorously studied. The distribution function of the normalized signal-to-noise ratio (SNR) is expanded in terms of a power series and calculated numerically. This power series can easily take into account the signal correlations and antenna gains and can be applied to any number of receiving antennas. An application of the method to dual-antenna diversity systems produces useful distribution curves for the normalized SNR which can be used to find the diversity gain. It is revealed that signal correlation in Rician-fading channels helps to increase the diversity gain rather than to decrease it as in the Rayleigh fading channels. It is also shown that with a relative strong direct signal component, the diversity gain can be much higher than that without a direct signal component.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Beyond the sphere of the English facial approximation literature: Ramifications of German papers on western method concepts

In the English literature, facial approximation methods have been commonly classified into three types: Russian, American, or Combination. These categorizations are based on the protocols used, for example, whether methods use average soft-tissue depths (American methods) or require face muscle construction (Russian methods). However, literature searches outside the usual realm of English publications reveal key papers that demonstrate that the Russian category above has been founded on distorted views. In reality, Russian methods are based on limited face muscle construction, with heavy reliance on modified average soft-tissue depths. A closer inspection of the American method also reveals inconsistencies with the recognized classification scheme. This investigation thus demonstrates that all major methods of facial approximation depend on both face anatomy and average soft-tissue depths, rendering common method classification schemes redundant. The best way forward appears to be for practitioners to describe the methods they use (including the weight each one gives to average soft-tissue depths and deep face tissue construction) without placing them in any categorical classificatory group or giving them an ambiguous name. The state of this situation may need to be reviewed in the future in light of new research results and paradigms.

New method to determine PSD using supercritical adsorption: Applied to methane adsorption in activated carbon

Adsorption of supercritical fluids is increasingly carried out to determine the micropore size distribution. This is largely motivated by the advances in the use of supercritical adsorption in high energy applications, such as hydrogen and methane storage in porous media. Experimental data are reported as mass excess versus pressure, and when these data are matched against the theoretical mass excess, significant errors could occur if the void volume used in the calculation of the experimental mass excess is incorrectly determined [Malbrunot, P.; Vidal, D.; Vermesse, J.; Chahine, R.; Bose, T. K. Langmuir 1997, 13, 539]. 1 The incorrect value for the void volume leads to a wrong description of the maximum in the plot of mass excess versus pressure as well as the part of the isotherm over the pressure region where the isotherm is decreasing. Because of this uncertainty in the maximum and the decreasing part of the isotherm, we propose a new method in which the problems associated with this are completely avoided. Our method involves only the relationship between the amount that is introduced into the adsorption cell and the equilibrium pressure. This information of direct experimental data has two distinct advantages. The first is that the data is the raw data without any manipulation (i.e., involving further calculations), and the second one is that this relationship always monotonically increases with pressure. We will illustrate this new method with the adsorption data of methane in a commercial sample of activated carbon.

Analysis of transient eddy currents in MRI using a cylindrical FDTD method

Most magnetic resonance imaging (MRI) spatial encoding techniques employ low-frequency pulsed magnetic field gradients that undesirably induce multiexponentially decaying eddy currents in nearby conducting structures of the MRI system. The eddy currents degrade the switching performance of the gradient system, distort the MRI image, and introduce thermal loads in the cryostat vessel and superconducting MRI components. Heating of superconducting magnets due to induced eddy currents is particularly problematic as it offsets the superconducting operating point, which can cause a system quench. A numerical characterization of transient eddy current effects is vital for their compensation/control and further advancement of the MRI technology as a whole. However, transient eddy current calculations are particularly computationally intensive. In large-scale problems, such as gradient switching in MRI, conventional finite-element method (FEM)-based routines impose very large computational loads during generation/solving of the system equations. Therefore, other computational alternatives need to be explored. This paper outlines a three-dimensional finite-difference time-domain (FDTD) method in cylindrical coordinates for the modeling of low-frequency transient eddy currents in MRI, as an extension to the recently proposed time-harmonic scheme. The weakly coupled Maxwell's equations are adapted to the low-frequency regime by downscaling the speed of light constant, which permits the use of larger FDTD time steps while maintaining the validity of the Courant-Friedrich-Levy stability condition. The principal hypothesis of this work is that the modified FDTD routine can be employed to analyze pulsed-gradient-induced, transient eddy currents in superconducting MRI system models. The hypothesis is supported through a verification of the numerical scheme on a canonical problem and by analyzing undesired temporal eddy current effects such as the B-0-shift caused by actively shielded symmetric/asymmetric transverse x-gradient head and unshielded z-gradient whole-body coils operating in proximity to a superconducting MRI magnet.

Direct seeding to restore rainforest species: Microsite effects on the early establishment and growth of rainforest tree seedlings on degraded land in the wet tropics of Australia

Reforestation in tropical areas is usually attempted by planting seedlings but, direct seeding (the artificial addition or sowing of seed) may be an alternative way of accelerating forest recovery and successional processes. This study investigated the effects of various sowing treatments (designed to create different microsite conditions for seed germination) and seed sizes on the early establishment and growth of directly sown rainforest tree species in a variety of experimental plots at three sites in the wet tropical region of north-cast Queensland, Australia. The different sowing treatments were found to have significant effects on seedling establishment. Broadcast sowing treatments were ineffective and resulted in very poor seedling establishment and high seed wastage. Higher establishment rates occurred when seeds were buried. Seed size was found to be an important factor affecting establishment in relation to micro-site condition. In general, larger seeded species had higher establishment rates at all three sites than species of small and intermediate seed size, but only in sowing treatments where seeds were buried. Overall these results suggest that direct sowing of seed can be used as a too] to accelerate recolonisation of certain rainforest tree species on degraded tropical lands, but initial success will be dependent on the choice of sowing method and its suitability for the seed types selected. The results also indicate that the recruitment of naturally dispersed tree species at degraded sites is likely to be severely limited by the availability of suitable microsites for seed germination. Consequently the natural recovery of degraded sites via seed rain can be expected to be slow and unpredictable, particularly in areas where soil compaction has occurred. (c) 2006 Elsevier B.V. All rights reserved.

Prediction of vapor-moisture equilibriums for a wood-moisture system using a modified UNIQUAC model

A modified UNIQUAC model has been extended to describe and predict the equilibrium relative humidity and moisture content for wood. The method is validated over a range of moisture content from oven-dried state to fiber saturation point, and over a temperature range of 20-70 degrees C. Adjustable parameters and binary interaction parameters of the UNIQUAC model were estimated from experimental data for Caribbean pine and Hoop pine as well as data available in the literature. The two group-interaction parameters for the wood-moisture system were consistent with using function group contributions for H2O, -OH and -CHO. The result reconfirms that the main contributors to water adsorption in cell walls are the hydroxyl groups of the carbohydrates in cellulose and hemicelluloses. This provides some physical insight into the intermolecular force and energy between bound water and the wood material. (c) 2006 Elsevier Ltd. All rights reserved.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.