Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

Crystallization and preliminary X-ray diffraction studies of the soluble 14 kDa beta-galactoside-binding lectin from bovine heart

The soluble 14 kDa beta-galactoside-binding lectin from bovine heart, a member of the S-type lectin family, has been crystallized in a form suitable for X-ray diffraction analysis. The crystals, in the absence of a saccharide ligand, diffract beyond 2.5 A resolution. They are obtained from polyethylene glycol 6000 at pH 6.0. Crystals grow as monoclinic plates, space group P2(1), with cell dimensions: a = 35.47 A, b = 64.33 A, c = 58.78 A and beta = 91.7 degrees. The asymmetric unit contains two molecules related by a 2-fold non-crystallographic axis. Two lectin monomers in the asymmetric unit give a Vm of 2.4 A3/Da, i.e. a solvent content of approximately 50%. The complex of lectin with the saccharide ligand, N-acetyllactosamine, crystallizes in the space group P2(1)2(1)2 with cell dimensions: a = 63.55 A, b = 82.13 A and c = 62.39 A. Crystals of this complex diffract beyond 2.0 A resolution. Two complexes in the asymmetric unit lead to a Vm value of 2.8 A3/Da (57% solvent).     

Uncertainty analysis based on sensitivity applied to angle-ply composite structures

This article describes a finite element-based formulation for the statistical analysis of the response of stochastic structural composite systems whose material properties are described by random fields. A first-order technique is used to obtain the second-order statistics for the structural response considering means and variances of the displacement and stress fields of plate or shell composite structures. Propagation of uncertainties depends on sensitivities taken as measurement of variation effects. The adjoint variable method is used to obtain the sensitivity matrix. This method is appropriated for composite structures due to the large number of random input parameters. Dominant effects on the stochastic characteristics are studied analyzing the influence of different random parameters. In particular, a study of the anisotropy influence on uncertainties propagation of angle-ply composites is carried out based on the proposed approach.     

The cholinergic system in Alzheimer's disease

The past decade has witnessed an enormous increase in our knowledge of the variety and complexity of neuropathological and neurochemical changes in Alzheimer's disease. Although the disease is characterized by multiple deficits of neurotransmitters in the brain, this overview emphasizes the structural and neurochemical localization of the elements of the acetylcholine system (choline acetyltransferase, acetylcholinesterase, and muscarinic and nicotinic acetylcholine receptors) in the non-demented brain and in Alzheimer's disease brain samples. The results demonstrate a great variation in the distribution of acetylcholinesterase, choline acetyltransferase, and the nicotinic and muscarinic acetylcholine receptors in the different brain areas, nuclei and subnuclei. When stratification is present in certain brain regions (olfactory bulb, cortex, hippocampus, etc.), differences can be detected as regards the laminar distribution of the elements of the acetylcholine system. Alzheimer's disease involves a substantial loss of the elements of the cholinergic system. There is evidence that the most affected areas include the cortex, the entorhinal area, the hippocampus, the ventral striatum and the basal part of the forebrain. Other brain areas are less affected. The fact that the acetylcholine system, which plays a significant role in the memory function, is seriously impaired in Alzheimer's disease has accelerated work on the development of new drugs for treatment of the disease of the 20th century.     

Effects of different cooking methods on the formation of food mutagens in meat

Hamburgers and chicken fillets were cooked in convection ovens, deep‐fried or contact fried and analysed for mutagenic activity using the Ames test. For the three different convection ovens, the cooking parameters studied included the presence of steam, air velocity, air temperature and holding time. For deep‐frying and contact frying, the cooking parameters included cooking temperature and cooking time. In cooked hamburgers, mutagenic activity was only detected in those that had been deep‐fried. In chicken fillets, mutagenic activity was detected in samples prepared with all cooking methods, being highest in the deep‐fried samples. Factorial analysis indicated that heat transfer was the most important factor affecting mutagenic activity. High temperature and high air velocity in the convection ovens enhanced mutagenic activity. The presence of steam reduced the mutagenic activity, except when high temperature was used in combination with high air velocity. In chicken fillets, high mutagenic activity correlated to high weight loss during cooking. Pan‐fried chicken fillets were analysed using high performance liquid chromatography (HPLC) and the mutagenic/carcinogenic heterocyclic amines 2‐amino‐3,8‐dimethylimidazo[4,5‐f ]‐quinoxaline (MeIQx), 2‐amino‐1‐methyl‐6‐phenylimidazo‐[4,5‐b]‐pyridine (PhIP) and the co‐mutagenic heterocyclic amine Norharman (9H‐pyrido[3,4‐b]‐indole) were identified. The HPLC fractions were tested for mutagenic activity and, apart from the mutagenic fractions corresponding to MeIQx and PhIP, several mutagenic fractions were detected that did not correspond to known heterocyclic amines.     

Secondary Metabolic Profile as a Tool for Distinction and Characterization of Cultivars of Black Pepper (Piper nigrum L.) Cultivated in Pará State,Brazil

This study evaluated the chemical compositions of the leaves and fruits of eight black pepper cultivars cultivated in Pará State (Amazon, Brazil). Hydrodistillation and gas chromatography–mass spectrometry were employed to extract and analyze the volatile compounds, respectively. Sesquiterpene hydrocarbons were predominant (58.5–90.9%) in the cultivars “Cingapura”, “Equador”, “Guajarina”, “Iaçará”, and “Kottanadan”, and “Bragantina”, “Clonada”, and “Uthirankota” displayed oxygenated sesquiterpenoids (50.6–75.0%). The multivariate statistical analysis applied using volatile composition grouped the samples into four groups: γ-Elemene, curzerene, and δ-elemene (“Equador”/“Guajarina”, I); δ-elemene (“Iaçará”/“Kottanadan”/“Cingapura”, II); elemol (“Clonada”/“Uthirankota”, III) and α-muurolol, bicyclogermacrene, and cubebol (“Bragantina”, IV). The major compounds in all fruit samples were monoterpene hydrocarbons such as α-pinene, β-pinene, and limonene. Among the cultivar leaves, phenolics content (44.75–140.53 mg GAE·g⁻¹ FW), the enzymatic activity of phenylalanine-ammonia lyase (20.19–57.22 µU·mL⁻¹), and carotenoids (0.21–2.31 µg·mL⁻¹) displayed significant variations. Due to black pepper’s susceptibility to Fusarium infection, a molecular docking analysis was carried out on Fusarium protein targets using each cultivar’s volatile components. F. oxysporum endoglucanase was identified as the preferential protein target of the compounds. These results can be used to identify chemical markers related to the susceptibility degree of black pepper cultivars to plant diseases prevalent in Pará State.     

Photocurrents and degradation rates on particulate TiO2 layers: Effect of layer thickness, concentration of oxidizable substance and illumination direction

Different routes for immobilization of TiO₂ on conducting substrates were compared to find active and stable photocatalysts, which could either be operated in open circuit (photocatalysis) or with applied electric potential (photoelectrocatalysis). The advantage of applying an electric potential was investigated. Polarization curves and photoelectrochemical degradation reactions served to characterize catalysts and to find a way of predicting the optimum synthesis conditions. The difference between front side (EE) and back side (SE) illumination is discussed as a function of layer thickness, as well as the influence of oxidizable substance concentration on photocurrents. Stability of catalysts was investigated in repetitive degradation experiments.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Paradoxical effect of n−3-containing vegetable oils on long-chain n−3 fatty acids in rat heart

Flaxseed, echium, and canola oils contain α-linolenic acid (18∶3n−3, ALA) in a range of concentrations. To examine their effect on elevating cardiac levels of long-chain n−3 FA, diets based on these n−3-containing vegetable oils were fed to rats for 4 wk. Sunflower oil, which contains little ALA, was a comparator. Despite canola oil having the lowest ALA content of the three n−3-containing vegetable oils, it was the most potent for elevating DHA (22∶6n−3) levels in rat hearts and plasma. However, the relative potencies of the dietary oils for elevation of EPA (20∶5n−3) in heart and plasma followed the same rank order as their ALA content, i.e., flaxseed>echium>canola>sunflower oil. This paradox may be explained by lower ALA intake leading to decreased competition for Δ6 desaturase activity between ALA and the 24∶5n−3 FA precursor to DHA formation.     

An empirical study of control logic specifications for programmable logic controllers

This paper presents an empirical study of control logic specifications used to document industrial control logic code in manufacturing applications. More than one hundred input/output related property specifications from ten different reusable function blocks were investigated. The main purpose of the study was to provide understanding of how the specifications are expressed by industrial practitioners, in order to develop new tools and methods for specifying control logic software, as well as for evaluating existing ones. In this paper, the studied specifications are used to evaluate linear temporal logic in general and the specification language ST-LTL, tailored for functions blocks, in particular. The study shows that most specifications are expressed as implications, that should always be fulfilled, between input and output conditions. Many of these implications are complex since the input and output conditions may be mixed and involve sequences, timer issues and non-boolean variables. Using ST-LTL it was possible to represent all implications of this study. The few non-implication specifications could be specified in ST-LTL as well after being altered to suit the specification language. The paper demonstrates some advantages of ST-LTL compared to standard linear temporal logic and discusses possible improvements such as support for automatic rewrite of complex specifications.