Validation of membrane protein topology models by oxidative labeling and mass spectrometry

Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

Do smokers with chronic obstructive pulmonary disease report their smoking status reliably?:A comparison of self-report and bio-chemical validation

Chronic obstructive pulmonary disease (COPD) is predominantly caused by cigarette smoking and is considered a worldwide preventable chronic illness. Smoking cessation is considered the primary intervention for disease management and nurses should play a major role in assisting patients to stop smoking. Currently there is a lack of professional consensus on how cessation interventions should be evaluated. The vast array of biochemical markers reported in the literature can be confusing and can make the comparisons of results difficult.

The technical merits of turbogenerating shown through the design, validation and implementation of a 1 dimensional engine model

Turbocompounding is the process of recovering a proportion of an engine’s fuel energy that would otherwise be lost in the exhaust process and adding it to the output power. This was first seen in the 1930s and is carried out by coupling an exhaust gas turbine to the crankshaft of a reciprocating engine. It has since been recognised that coupling the power turbine to an electrical generator instead of the crankshaft has the potential to reduce the fuel consumption further with the added flexibility of being able to decide how this recovered energy is used. The electricity generated can be used in automotive applications to assist the crankshaft using a flywheel motor generator or to power ancillaries that would otherwise have run off the crankshaft. In the case of stationary power plants, it can assist the electrical power output. Decoupling the power turbine from the crankshaft and coupling it to a generator allows the power electronics to control the turbine speed independently in order to optimise the specific fuel consumption for different engine operating conditions. This method of energy recapture is termed ‘turbogenerating’.

This paper gives a brief history of turbocompounding and its thermodynamic merits. It then moves on to give an account of the validation of a turbogenerated engine model. The model is then used to investigate what needs to be done to an engine when a turbogenerator is installed. The engine being modelled is used for stationary power generation and is fuelled by an induced biogas with a small portion of palm oil being injected into the cylinder to initiate combustion by compression ignition. From these investigations, optimum settings were found that result in a 10.90% improvement in overall efficiency. These savings relate to the same engine without a turbogenerator installed operating with fixed fuelling.

Comparison of consumer perception and acceptability for steaks cooked to different endpoints: Validation of photographic approach

Photographs have been used to enhance consumer reporting of preference of meat doneness, however, the use of photographs has not been validated for this purpose. This study used standard cooking methods to produce steaks of five different degrees of doneness (rare medium, medium well, well done and very well done) to study the consumer’s perception of doneness, from both the external and internal surface of the cooked steak and also from corresponding photographs of each sample. Consumers evaluated each surface of the cooked steaks in relation to doneness for acceptability, ‘just about right’ and perception of doneness. Data were analysed using a split plot ANOVA and least significant test. Perception scores (for both external and internal surfaces) between different presentation methods (steak samples and corresponding photos), were not significantly different (p > 0.05). The result indicates that photographs can be used as a valid approach for assessing preference for meat doneness.

Development and validation of an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic shellfish toxins in marine algae

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCß were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCß and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L. © 2012 Elsevier B.V. All rights reserved.

Anthelmintic drug residues in beef: UPLC-MS/MS method validation, European retail beef survey, and associated exposure and risk assessments

Anthelmintic drugs are widely used to control parasitic infections in cattle. The ProSafeBeef project addressed the need for data on the exposure of European consumers of beef to potentially harmful drug residues. A novel analytical method based on matrix solid-phase dispersive extraction and ultra-performance liquid chromatography-tandem mass spectrometry was validated for 37 anthelmintic drugs and metabolites in muscle (assay decision limits, CCa, = 0.15-10.2 µg kg -1). Seven European countries (France, Spain, Slovenia, Ireland, Italy, Belgium and Portugal) participated in a survey of retail beef purchased in local shops. Of 1061 beef samples analysed, 26 (2.45%) contained detectable residues of anthelmintic drugs (0.2-171 µg kg -1), none above its European Union maximum residue limit (MRL) or action level. Residues detected included closantel, levamisole, doramectin, eprinomectin, moxidectin, ivermectin, albendazole and rafoxanide. In a risk assessment applied to mean residue concentrations across all samples, observed residues accounted for less than 0.1% of the MRL for each compound. An exposure assessment based on the consumption of meat at the 99th percentile of consumption of adults in 14 European countries demonstrated that beef accounted for less than 0.02% of the acceptable daily intake for each compound in each country. This study is the first of its kind to apply such a risk-based approach to an extensive multi-residue survey of veterinary drug residues in food. It has demonstrated that the risk of exposure of the European consumer to anthelmintic drug residues in beef is negligible, indicating that regulation and monitoring is having the desired effect of limiting residues to non-hazardous concentrations. © 2012 Copyright Taylor and Francis Group, LLC.

Design and experimental validation of liquid crystal-based reconfigurable reflectarray elements with improved bandwith in F-band

A reconfigurable reflectarray which exploits the dielectric anisotropy of liquid crystals (LC) has been designed to operate in the frequency range from 96 to 104 GHz. The unit cells are composed of three unequal length parallel dipoles placed above an LC substrate. The reflectarray has been designed using an accurate model which includes the effects of anisotropy and inhomogeneity. An effective permittivity that accounts for the real effects of the LC has also been used to simplify the analysis and design of the unit cells. The geometrical parameters of the cells have been adjusted to simultaneously improve the bandwidth, maximize the tunable phase-range and reduce the sensitivity to the angle of incidence. The performance of the LC based unit cells has been experimentally evaluated by measuring the reflection amplitude and phase of a reflectarray consisting of 52x54 identical cells. The good agreement between measurements and simulations validate the analysis and design techniques and demonstrate the capabilities of the proposed reflectarray to provide beam scanning in F band.

Maximum Residue Level validation of triclabendazole marker residues in bovine liver, muscle and milk by UHPLC tandem mass spectrometry.

Triclabendazole is the only anthelmintic drug, which is active against immature, mature and adult stages of fluke. The objective of this work was to develop an analytical method to quantify and confirm the presence of triclabendazole residues around the MRL. In this work, a new analytical method was developed, which extended dynamic range to 1–100 and 5–1000 g kg-1 for milk and tissue, respectively. This was achieved using a mobile phase containing trifluoroacetic acid (pKa of 0.3), which resulted in the formation of the protonated pseudomolecular ions, [M+H]+, of triclabendazole metabolites. Insufficient
ionisation of common mobile phase additives due to low pKa values (<2) was identified as the cause of poor linearity. The new mobile phase conditions allowed the analysis of triclabendazole residues in liver, muscle and milk encompassing their EU maximum residue levels (MRL) (250, 225 and 10 g kg-1 respectively). Triclabendazole residues were extracted using a modified QuEChERS method and analysed by positive electrospray ionisation mass spectrometry with all analytes eluted by 2.23 min. The method was validated at the MRL according to Commission Decision (CD) 2002/657/EC criteria. The decision limit (CC) of the method was in the range of 250.8–287.2, 2554.9–290.8 and 10.9–12.1 g kg-1 for liver, muscle and milk, respectively. The performance of the method was successfully verified for triclabendazole in muscle by participating in a proficiency study, the method was also applied to incurred liver, muscle and milk samples.

Development and Validation of a Forklift Truck Powertrain Simulation

Fuel economy has become an important consideration in forklift truck design, particularly in Europe. A simulation of the fuel consumption and performance of a forklift truck has been developed, validated and subsequently used to determine the energy consumed by individual powertrain components during drive cycles.
The truck used in this study has a rated lifting capacity of 2500kg, and is powered by a 2.6 litre naturally aspirated diesel engine with a fuel pump containing a mechanical variable-speed governor. The drivetrain consisted of a torque convertor, hydraulic clutch and single speed transmission.
AVL Cruise was used to simulate the vehicle powertrain, with coupled Mathworks Simulink models used to simulate the hydraulic and control systems and governor. The vehicle has been simulated on several performance and fuel consumption drive cycles with the main focus being the VDI 2198 fuel consumption drive cycle.
To validate the model, a truck was instrumented and measurements taken to compare the performance and instantaneous fuel consumption to simulated values. The fuel injector pump was modified and calibrated to enable instantaneous fuel flow to be measured.
The model has been validated to within acceptable limits and has been used to investigate the effect four different torque converters have on the fuel consumption and performance of the forklift truck. The study demonstrates how the model can be used to compare the fuel consumption and performance trade-offs when selecting drivetrain components.

Initial validation of the Behavioral Indicators of Infant Pain (BIIP)

Accurate pain assessment in preterm infants in the neonatal intensive care unit (NICU) is complex. Infants who are born at early gestational ages (GA), and who have had greater early pain exposure, have dampened facial responses which may lead to under-treatment. Since behavioral and physiological responses to pain in infants are often dissociated, using multidimensional scales which combine these indicators into a single score may limit our ability to determine the effects of interventions on each system. Our aim was to design a unidimensional scale which would combine the relatively most specific, individual, behavioral indicators for assessing acute pain in this population. The Behavioral Indicators of Infant Pain (BIIP) combines sleep/wake states, 5 facial actions and 2 hand actions. Ninety-two infants born between 23 and 32 weeks GA were assessed during 3, 1 min Phases of blood collection. Outcome measures included changes in BIIP and in Neonatal Infant Pain Scale (NIPS) scores coded in real time from continuous bedside video recordings; changes in heart rate (HR) were obtained using custom physiological processing software. Scores on the BIIP changed significantly across Phases of blood collection (p

Organizational assessment in paediatric primary care:Development and initial validation of the primary care organizational questionnaire

Primary care in the United States is undergoing many changes. Reliable and valid instruments are needed to assess the effects of these changes. The Primary Care Organizational Questionnaire (PCOQ), a 56-item 5-point Likert scale survey that evaluates interactions among members of the clinic/practice and job-related attributes, was administered to clinicians and staff in 36 primary care practices serving paediatric populations in Connecticut. A priori scales were reliable (Cronbach alpha =0.7). Analysis of variance (ANOVA) showed greater heterogeneity across clinics than within clinics for 13 of 15 a priori scales, which were then included in a principal component factor analysis with varimax rotation. Eigenvalue analysis showed nine significant factors, largely similar to the a priori scales, indicating concurrent construct validity. Further research will ascertain the utility of the PCOQ in predicting the effectiveness of primary care practices in implementing disease management programmes. © 2006 Royal Society of Medicine Press.

Pre-validation methods for developing a patient reported outcome instrument

BACKGROUND: Measures that reflect patients' assessment of their health are of increasing importance as outcome measures in randomised controlled trials. The methodological approach used in the pre-validation development of new instruments (item generation, item reduction and question formatting) should be robust and transparent. The totality of the content of existing PRO instruments for a specific condition provides a valuable resource (pool of items) that can be utilised to develop new instruments. Such 'top down' approaches are common, but the explicit pre-validation methods are often poorly reported. This paper presents a systematic and generalisable 5-step pre-validation PRO instrument methodology.

METHODS: The method is illustrated using the example of the Aberdeen Glaucoma Questionnaire (AGQ). The five steps are: 1) Generation of a pool of items; 2) Item de-duplication (three phases); 3) Item reduction (two phases); 4) Assessment of the remaining items' content coverage against a pre-existing theoretical framework appropriate to the objectives of the instrument and the target population (e.g. ICF); and 5) qualitative exploration of the target populations' views of the new instrument and the items it contains.

RESULTS: The AGQ 'item pool' contained 725 items. Three de-duplication phases resulted in reduction of 91, 225 and 48 items respectively. The item reduction phases discarded 70 items and 208 items respectively. The draft AGQ contained 83 items with good content coverage. The qualitative exploration ('think aloud' study) resulted in removal of a further 15 items and refinement to the wording of others. The resultant draft AGQ contained 68 items.

CONCLUSIONS: This study presents a novel methodology for developing a PRO instrument, based on three sources: literature reporting what is important to patient; theoretically coherent framework; and patients' experience of completing the instrument. By systematically accounting for all items dropped after the item generation phase, our method ensures that the AGQ is developed in a transparent, replicable manner and is fit for validation. We recommend this method to enhance the likelihood that new PRO instruments will be appropriate to the research context in which they are used, acceptable to research participants and likely to generate valid data.