Rapid parallel expression in E-coli and insect cells: Analysis of five lef gene products of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)

A number of strategies are emerging for the high throughput (HTP) expression of recombinant proteins to enable structural and functional study. Here we describe a workable HTP strategy based on parallel protein expression in E. coli and insect cells. Using this system we provide comparative expression data for five proteins derived from the Autographa californica polyhedrosis virus genome that vary in amino acid composition and in molecular weight. Although the proteins are part of a set of factors known to be required for viral late gene expression, the precise function of three of the five, late expression factors (lefs) 6, 7 and 10, is unknown. Rapid expression and characterisation has allowed the determination of their ability to bind DNA and shown a cellular location consistent with their properties. Our data point to the utility of a parallel expression strategy to rapidly obtain workable protein expression levels from many open reading frames (ORFs).

Identification of lipophilic flavones and flavonols by comparative HPLC, TLC and UV spectral analysis

The identification of lipophilic flavones and flavonols using a combination of high performance liquid chromatography, thin layer chromatography and UV spectral analysis is discussed. Data are provided for the flavones, apigenin, luteolin and tricetin and twelve of their methyl ethers, 8-hydroxyluteolin, 6-hydroxyluteolin and scutellarein and fourteen of their methyl ethers, and some 6,8-dihydroxyapigenin and 6,8-dihydroxyluteolin derivatives. Data for some forty two flavonols with extra 6- and/or 8-hydroxylation, mostly 6-hydroxykaempferol and quercetagetin derivatives, are also presented. The remaining compounds analysed include fourteen 5-deoxyflavones, four 5-methoxyflavones and five 5-deoxyflavonols plus further 5-hydroxylated flavones and flavonols without B-ring oxidation or with 2-, 5- or 6-hydroxylation. Copyright © 2003 John Wiley & Sons, Ltd.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INF alpha and INF gamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Potential energy surface and MULTIMODE vibrational analysis of C2H3+

A full dimensional, ab initio-based semiglobal potential energy surface for C2H3+ is reported. The ab initio electronic energies for this molecule are calculated using the spin-restricted, coupled cluster method restricted to single and double excitations with triples corrections [RCCSD(T)]. The RCCSD(T) method is used with the correlation-consistent polarized valence triple-zeta basis augmented with diffuse functions (aug-cc-pVTZ). The ab initio potential energy surface is represented by a many-body (cluster) expansion, each term of which uses functions that are fully invariant under permutations of like nuclei. The fitted potential energy surface is validated by comparing normal mode frequencies at the global minimum and secondary minimum with previous and new direct ab initio frequencies. The potential surface is used in vibrational analysis using the "single-reference" and "reaction-path" versions of the code MULTIMODE. (c) 2006 American Institute of Physics.

Rapid quality assessment of wheat cultivars registered in Poland using the 2-g mixograph and multivariate statistical analysis

Baking and 2-g mixograph analyses were performed for 55 cultivars (19 spring and 36 winter wheat) from various quality classes from the 2002 harvest in Poland. An instrumented 2-g direct-drive mixograph was used to study the mixing characteristics of the wheat cultivars. A number of parameters were extracted automatically from each mixograph trace and correlated with baking volume and flour quality parameters (protein content and high molecular weight glutenin subunit [HMW-GS] composition by SDS-PAGE) using multiple linear regression statistical analysis. Principal component analysis of the mixograph data discriminated between four flour quality classes, and predictions of baking volume were obtained using several selected mixograph parameters, chosen using a best subsets regression routine, giving R-2 values of 0.862-0.866. In particular, three new spring wheat strains (CHD 502a-c) recently registered in Poland were highly discriminated and predicted to give high baking volume on the basis of two mixograph parameters: peak bandwidth and 10-min bandwidth.

Computer generation and quantitative morphometric analysis of virtual neurons

An important goal in computational neuroanatomy is the complete and accurate simulation of neuronal morphology. We are developing computational tools to model three-dimensional dendritic structures based on sets of stochastic rules. This paper reports an extensive, quantitative anatomical characterization of simulated motoneurons and Purkinje cells. We used several local and global algorithms implemented in the L-Neuron and ArborVitae programs to generate sets of virtual neurons. Parameters statistics for all algorithms were measured from experimental data, thus providing a compact and consistent description of these morphological classes. We compared the emergent anatomical features of each group of virtual neurons with those of the experimental database in order to gain insights on the plausibility of the model assumptions, potential improvements to the algorithms, and non-trivial relations among morphological parameters. Algorithms mainly based on local constraints (e.g., branch diameter) were successful in reproducing many morphological properties of both motoneurons and Purkinje cells (e.g. total length, asymmetry, number of bifurcations). The addition of global constraints (e.g., trophic factors) improved the angle-dependent emergent characteristics (average Euclidean distance from the soma to the dendritic terminations, dendritic spread). Virtual neurons systematically displayed greater anatomical variability than real cells, suggesting the need for additional constraints in the models. For several emergent anatomical properties, a specific algorithm reproduced the experimental statistics better than the others did. However, relative performances were often reversed for different anatomical properties and/or morphological classes. Thus, combining the strengths of alternative generative models could lead to comprehensive algorithms for the complete and accurate simulation of dendritic morphology.

Self-assembled molecular complexes and coordination polymers of CdII, hexamine, and monocarboxylates: structural analysis and theoretical studies of supramolecular interactions

Four new cadmium(II) complexes [Cd-2(bz)(4)(H2O)(4)(mu 2-hmt)]center dot Hbz center dot H2O (1), [Cd-3(bz)(6)(H2O)(6)(mu 2-hmt)(2)]center dot 6H(2)O (2), [Cd(pa)(2)(H2O)(mu(2)-hmt)](n) (3), and {[Cd-3(ac)(6)(H2O)(3)(mu(3)-hmt)(2)]center dot 6H(2)O}(n) (4) with hexamine (hmt) and monocarboxylate ions, benzoate (bz), phenylacetate (pa), or acetate (ac) have been synthesized and characterized structurally. Structure determinations reveal that 1 is dinuclear, 2 is trinuclear, 3 is a one-dimensional (1D) infinite chain, and 4 is a two-dimensional (2D) polymer with fused hexagonal rings consisting of Cd-II and hmt. All the Cd-II atoms in the four complexes (except one CdII in 2) possess seven-coordinate pentagonal bipyramidal geometry with the various chelating bidentate carboxylate groups in equatorial sites. One of the CdII ions in 2, a complex that contains two monodentate carboxylates is in a distorted octahedral environment. The bridging mode of hmt is mu 2- in complexes 1-3 but is mu 3- in complex 4. In all complexes, there are significant numbers of H-bonds, C-H/pi, and pi-pi interactions which play crucial roles in forming the supramolecular networks. The importance of the noncovalent interactions in terms of energies and geometries has been analyzed using high level ab initio calculations. The effect of the cadmium coordinated to hmt on the energetic features of the C-H/pi interaction is analyzed. Finally, the interplay between C-H/pi and pi-pi interactions observed in the crystal structure of 3 is also studied.

Principal-agent issues in asset acquisition: UK institutions and their investment agents

This paper explores principal‐agent issues in the stock selection processes of institutional property investors. Drawing upon an interview survey of fund managers and acquisition professionals, it focuses on the relationships between principals and external agents as they engage in property transactions. The research investigated the extent to which the presence of outcome‐based remuneration structures could lead to biased advice, overbidding and/or poor asset selection. It is concluded that institutional property buyers are aware of incentives for opportunistic behaviour by external agents, often have sufficient expertise to robustly evaluate agents’ advice and that these incentives are counter‐balanced by a number of important controls on potential opportunistic behaviour. There are strong counter‐incentives in the need for the agents to establish personal relationships and trust between themselves and institutional buyers, to generate repeat and related business and to preserve or generate a good reputation in the market.

Judgments, forecasts and decisions: an analysis of fund managers over time

Decision theory is the study of models of judgement involved in, and leading to, deliberate and (usually) rational choice. In real estate investment there are normative models for the allocation of assets. These asset allocation models suggest an optimum allocation between the respective asset classes based on the investors’ judgements of performance and risk. Real estate is selected, as other assets, on the basis of some criteria, e.g. commonly its marginal contribution to the production of a mean variance efficient multi asset portfolio, subject to the investor’s objectives and capital rationing constraints. However, decisions are made relative to current expectations and current business constraints. Whilst a decision maker may believe in the required optimum exposure levels as dictated by an asset allocation model, the final decision may/will be influenced by factors outside the parameters of the mathematical model. This paper discusses investors' perceptions and attitudes toward real estate and highlights the important difference between theoretical exposure levels and pragmatic business considerations. It develops a model to identify “soft” parameters in decision making which will influence the optimal allocation for that asset class. This “soft” information may relate to behavioural issues such as the tendency to mirror competitors; a desire to meet weight of money objectives; a desire to retain the status quo and many other non-financial considerations. The paper aims to establish the place of property in multi asset portfolios in the UK and examine the asset allocation process in practice, with a view to understanding the decision making process and to look at investors’ perceptions based on an historic analysis of market expectation; a comparison with historic data and an analysis of actual performance.

Evaluation of satellite-based and model re-analysis rainfall estimates for Uganda

The dependence of much of Africa on rain fed agriculture leads to a high vulnerability to fluctuations in rainfall amount. Hence, accurate monitoring of near-real time rainfall is particularly useful, for example in forewarning possible crop shortfalls in drought-prone areas. Unfortunately, ground based observations are often inadequate. Rainfall estimates from satellite-based algorithms and numerical model outputs can fill this data gap, however rigorous assessment of such estimates is required. In this case, three satellite based products (NOAA-RFE 2.0, GPCP-1DD and TAMSAT) and two numerical model outputs (ERA-40 and ERA-Interim) have been evaluated for Uganda in East Africa using a network of 27 rain gauges. The study focuses on the years 2001 to 2005 and considers the main rainy season (February to June). All data sets were converted to the same temporal and spatial scales. Kriging was used for the spatial interpolation of the gauge data. All three satellite products showed similar characteristics and had a high level of skill that exceeded both model outputs. ERA-Interim had a tendency to overestimate whilst ERA-40 consistently underestimated the Ugandan rainfall.

Coverage-dependent molecular tilt of carbon monoxide chemisorbed on Pt{110}: A combined LEED and DFT structural analysis

The adsorption of carbon monoxide on the Pt{110} surface at coverages of 0.5 ML and 1.0 ML was investigated using quantitative low-energy electron diffraction (LEED IV) and density-functional theory (DFT). At 0.5 ML CO lifts the reconstruction of the clean surface but does not form an ordered overlayer. At the saturation coverage, 1.0 ML, a well-ordered p(2×1) superstructure with glide line symmetry is formed. It was confirmed that the CO molecules adsorb on top of the Pt atoms in the top-most substrate layer with the molecular axes tilted by ±22° with respect to the surface normal in alternating directions away from the close packed rows of Pt atoms. This is accompanied by significant lateral shifts of 0.55 Å away from the atop sites in the same direction as the tilt. The top-most substrate layer relaxes inwards by −4% with respect to the bulk-terminated atom positions, while the consecutive layers only show minor relaxations. Despite the lack of long-range order in the 0.5 ML CO layer it was possible to determine key structural parameters by LEED IV using only the intensities of the integer-order spots. At this coverage CO also adsorbs on atop sites with the molecular axis closer to the surface normal (b10°). The average substrate relaxations in each layer are similar for both coverages and consistent with DFT calculations performed for a variety of ordered structures with coverages of 1.0 ML and 0.5 ML.

A pharmacist-led information technology intervention for medication errors (PINCER): a multicentre, cluster randomised, controlled trial and cost-effectiveness analysis

Background: Medication errors are common in primary care and are associated with considerable risk of patient harm. We tested whether a pharmacist-led, information technology-based intervention was more effective than simple feedback in reducing the number of patients at risk of measures related to hazardous prescribing and inadequate blood-test monitoring of medicines 6 months after the intervention. Methods: In this pragmatic, cluster randomised trial general practices in the UK were stratified by research site and list size, and randomly assigned by a web-based randomisation service in block sizes of two or four to one of two groups. The practices were allocated to either computer-generated simple feedback for at-risk patients (control) or a pharmacist-led information technology intervention (PINCER), composed of feedback, educational outreach, and dedicated support. The allocation was masked to general practices, patients, pharmacists, researchers, and statisticians. Primary outcomes were the proportions of patients at 6 months after the intervention who had had any of three clinically important errors: non-selective non-steroidal anti-inflammatory drugs (NSAIDs) prescribed to those with a history of peptic ulcer without co-prescription of a proton-pump inhibitor; β blockers prescribed to those with a history of asthma; long-term prescription of angiotensin converting enzyme (ACE) inhibitor or loop diuretics to those 75 years or older without assessment of urea and electrolytes in the preceding 15 months. The cost per error avoided was estimated by incremental cost-eff ectiveness analysis. This study is registered with Controlled-Trials.com, number ISRCTN21785299. Findings: 72 general practices with a combined list size of 480 942 patients were randomised. At 6 months’ follow-up, patients in the PINCER group were significantly less likely to have been prescribed a non-selective NSAID if they had a history of peptic ulcer without gastroprotection (OR 0∙58, 95% CI 0∙38–0∙89); a β blocker if they had asthma (0∙73, 0∙58–0∙91); or an ACE inhibitor or loop diuretic without appropriate monitoring (0∙51, 0∙34–0∙78). PINCER has a 95% probability of being cost eff ective if the decision-maker’s ceiling willingness to pay reaches £75 per error avoided at 6 months. Interpretation: The PINCER intervention is an effective method for reducing a range of medication errors in general practices with computerised clinical records. Funding: Patient Safety Research Portfolio, Department of Health, England.