The use of proteomic techniques to explore the holistic effects of nutrients in vivo

The availability of ‘omics’ technologies is transforming scientific approaches to physiological problems from a reductionist viewpoint to that of a holistic viewpoint. This is of profound importance in nutrition, since the integration of multiple systems at the level of gene expression on the synthetic side through to metabolic enzyme activity on the degradative side combine to govern nutrient availability to tissues. Protein activity is central to the process of nutrition from the initial absorption of nutrients via uptake carriers in the gut, through to distribution and transport in the blood, metabolism by degradative enzymes in tissues and excretion through renal tubule exchange proteins. Therefore, the global profiling of the proteome, defined as the entire protein complement of the genome expressed in a particular cell or organ, or in plasma or serum at a particular time, offers the potential for identification of important biomarkers of nutritional state that respond to alterations in diet. The present review considers the published evidence of nutritional modulation of the proteome in vivo which has expanded exponentially over the last 3 years. It highlights some of the challenges faced by researchers using proteomic approaches to understand the interactions of diet with genomic and metabolic–phenotypic variables in normal populations.

Solvent extraction of phosphoric acid

This work investigated the purification of phosphoric acid using a suitable organic solvent, followed by re-extraction of the acid from the solvent using water. The work consisted of practical batch and continuous studies and the economics and design of a full scale plant, based on the experimental data. A comprehensive literature survey on the purification of wet process phosphoric acid by organic solvents is presented and the literature describing the design and operation of mixer-settlers has also been reviewed. In batch studies, the equilibrium and distribution curves for the systems water-phosphoric acid-solvent for Benzaldehyde, Cyclohexanol and Methylisobutylketone (MIBK) were determined together with hydrodynamic characteristics for both pure and impure systems. The settling time increased with acid concentration, but power input had no effect. Drop size was found to reduce with acid concentration and power input. For the continuous studies a novel horizontal mixer~settler cascade was designed, constructed and operated using pure and impure acid with MIBK as the solvent. The cascade incorporates three air turbine agitated, cylindrical 900 ml mixers, and three cylindrical 200 ml settlers with air-lift solvent interstage transfer. Mean drop size in the fully baffled mixer was correlated. Drop size distributions were log-normal and size decreased with acid concentration and power input and increased with dispersed phase hold-up. Phase inversion studies showed that the width of the ambivalent region depended upon rotor speed, hold-up and acid concentration. Settler characteristics were investigated by measuring wedge length. Distribution coefficients of impurities and acid were also investigated. The following optimum extraction conditions were found: initial acid concentration 63%, phase ratio of solvent to acid 1:1 (v/v), impeller speed recommended 900 r.p.m. In the washing step the maximum phase ratio of solvent to water was 8:1 (v/v). Work on phosphoric acid concentration involved constructing distillation equipment consisting of a 10& spherical still. A 100 T/d scale detailed process design including capital cost, operating cost and profitability was also completed. A profit model for phosphoric acid extraction was developed and maximised. Recommendations are made for both the application of the results to a practical design and for extensions of the study.

A detailed design study of leaching and ancillary processes in the hydrometallurgical extraction of iron from iron bearing feedstocks

A systematic survey of the possible methods of chemical extraction of iron by chloride formation has been presented and supported by a comparable study of :feedstocks, products and markets. The generation and evaluation of alternative processes was carried out by the technique of morphological analysis vihich was exploited by way of a computer program. The final choice was related to technical feasibility and economic viability, particularly capital cost requirements and developments were made in an estimating procedure for hydrometallurgjcal processes which have general applications. The systematic exploration included the compilation of relevant data, and this indicated a need.to investigate precipitative hydrolysis or aqueous ferric chloride. Arising from this study, two novel hydrometallurgical processes for manufacturing iron powder are proposed and experimental work was undertaken in the following .areas to demonstrate feasibility and obtain basic data for design purposes: (1) Precipitative hydrolysis of aqueous ferric chloride. (2) Gaseous chloridation of metallic iron, and oxidation of resultant ferrous chloride. (3) Reduction of gaseous ferric chloride with hydrogen. (4) Aqueous acid leaching of low grade iron ore. (5) Aqueous acid leaching of metallic iron. The experimentation was supported by theoretical analyses dealing with: (1) Thermodynamics of hydrolysis. (2) Kinetics of ore leaching. (3) Kinetics of metallic iron leaching. (4) Crystallisation of ferrous chloride. (5) Oxidation of anhydrous ferrous chloride. (6) Reduction of ferric chloride. Conceptual designs are suggested fbr both the processes mentioned. These draw attention to areas where further work is necessary, which are listed. Economic analyses have been performed which isolate significant cost areas, und indicate total production costs. Comparisons are mode with previous and analogous proposals for the production of iron powder.

Steps and bumps:precision extraction of discrete states of molecular machines

We report statistical time-series analysis tools providing improvements in the rapid, precision extraction of discrete state dynamics from time traces of experimental observations of molecular machines. By building physical knowledge and statistical innovations into analysis tools, we provide techniques for estimating discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of steplike rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, periodicities and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a step in the direction toward automated analysis of high-sample-rate, molecular-machine dynamics. Modular, open-source software that implements these techniques is provided.

Independent component analysis and feature extraction of financial time series

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Dynamical embedding and feature extraction of electroencephalographic data

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An adaptive hierarchical approach to the extraction of high resolution medial surfaces

We introduce a novel algorithm for medial surfaces extraction that is based on the density-corrected Hamiltonian analysis. The approach extracts the skeleton directly from a triangulated mesh and adopts an adaptive octree-based approach in which only skeletal voxels are refined to a lower level of the hierarchy, resulting in robust and accurate skeletons at extremely high resolution. The quality of the extracted medial surfaces is confirmed by an extensive set of experiments. © 2012 IEEE.

The design of a high pressure solvent extraction process using liquid ammonia as solvent

The literature on the potential use of liquid ammonia as a solvent for the extraction of aromatic hydrocarbons from mixtures with paraffins, and the application of reflux, has been reviewed. Reference is made to extractors suited to this application. A pilot scale extraction plant was designed comprising a Scm. diameter by 12Scm. high, 50 stage Rotating Disc Contactor with 2 external settlers. Provision was made for operation with, or without, reflux at a pressure of 10 bar and ambient temperature. The solvent recovery unit consisted of an evaporator, compressor and condenser in a refrigeration cycle. Two systems were selected for study, Cumene-n-Heptane-Ammonia and Toluene-Methylcyclohexane-Ammonia. Equlibrium data for the first system was determined experimentally in a specially-designed, equilibrium bomb. A technique was developed to withdraw samples under pressure for analysis by chromatography and titration. The extraction plant was commissioned with a kerosine-water system; detailed operating procedures were developed based on a Hazard and Operability Study. Experimental runs were carried out with both ternary ammonia systems. With the system Toluene-Methylcyclohexane-Ammonia the extraction plant and the solvent recovery facility, operated satisfactorily, and safely,in accordance with the operating procedures. Experimental data gave reasonable agreement with theory. Recommendations are made for further work with plant.

A novel process for the production of castor oil by direct solvent extraction

This research was undertaken to: develop a process for the direct solvent extraction of castor oil seeds. A literature survey confirmed the desirability of establishing such a process with emphasis on the decortication, size, reduction, detoxification-deallergenization, and solvent·extraction operations. A novel process was developed for the dehulling of castor seeds which consists of pressurizing the beans and then suddenly releasing the pressure to vaccum. The degree of dehulling varied according to the pressure applied and the size of the beans. Some of the batches were difficult-to-hull, and this phenomenon was investigated using the scanning electron microscope and by thickness and compressive strength measurements. The other variables studied to lesser degrees included residence time, moisture, content, and temperature.The method was successfully extended to cocoa beans, and (with modifications) to peanuts. The possibility of continuous operation was looked into, and a mechanism was suggested to explain the method works. The work on toxins and allergens included an extensive literature survey on the properties of these substances and the methods developed for their deactivation Part of the work involved setting up an assay method for measuring their concentration in the beans and cake, but technical difficulties prevented the completion of this aspect of the project. An appraisal of the existing deactivation methods was made in the course of searching for new ones. A new method of reducing the size of oilseeds was introduced in this research; it involved freezing the beans in cardice and milling them in a coffee grinder, the method was found to be a quick, efficient, and reliable. An application of the freezing technique was successful in dehulling soybeans and de-skinning peanut kernels. The literature on the solvent extraction, of oilseeds, especially castor, was reviewed: The survey covered processes, equipment, solvents, and mechanism of leaching. three solvents were experimentally investigated: cyclohexane, ethanol, and acetone. Extraction with liquid ammonia and liquid butane was not effective under the conditions studied. Based on the results of the research a process has been suggested for the direct solvent extraction of castor seeds, the various sections of the process have analysed, and the factors affecting the economics of the process were discussed.

Acanthamoebal surface properties and the modulation of phagocytosis

The surface nature of Acanthamoeba trophozoites and cysts was investigated with respect to cell surface charge, hydrophobicity and surface carbohydrate composition. Particulate microelectrophoresis revealed a marked negative charge for both morphological forms, though less for cyst surfaces. Hydrophobicity was determined by adhesion to n-hexadecane and indicated a relatively low hydrophobic nature of both forms, though less so for cysts. Surface carbohydrate composition was studied by the use of fluorescent lectins and flow cytometry, using a ligand-receptor approach for further in depth analysis of binding of particular lectins. These studies showed trophozoite and cyst surfaces to be rich in N-acetylglucosamine, N-acteylneuraminic acid, mannose and glucose, with the addition of N-acetylgalactosamine on cysts. The importance of such surface properties was investigated with respect to phagocytosis of polystyrene latex microspheres, of different surface types and size. Investigations into the optimum conditions of uptake of beads indicated a preference for a medium devoid of nutrients, such as saline, though temperature was not a factor. An amoebal predilection for beads of lower charge and greater hydrophobicity was demonstrated. Furthermore, a preference for the largest bead size used (2.0 m) was observed. The influence of either Con A or mannose or glucose on bead association was apparently limited. The fate of foreign DNA ingested by Acanthamoeba appeared to indicate that such DNA was destroyed, as it could not be detected following extraction procedures and PCR amplification.

Enterotoxigenic Escherichia coli infection of pigs: expression, extraction, purification and the adhesive properties of the K88 fimbrial adhesin

The ability of Escherichia coli to express the K88 fimbrial adhesin was satisfactorily indicated by the combined techniques of ELISA, haemagglutination and latex agglutination. Detection of expression by electron microscopy and the ability to metabolize raffinose were unsuitable. Quantitative expression of the K88 adhesin was determined by ELISA. Expression was found to vary according to the E.coli strain examined, media type and form. In general it was found that the total amount was greater, while the amount/cfu was less on agar than in broth cultures. Expression of the K88 adhesin during unshaken batch culture was related to the growth rate and was maximal during late logarithmic to early stationary phase. A combination of heat extraction, ammonium sulphate and isoelectric precipitation was found suitable for both large and small scale preparation of purified K88ab adhesin. Extraction of the K88 adhesin was sensitive to pH and it was postulated that this may affect the site of colonisation of by ETEC in vivo. Results of haemagglutination experiments were consistent with the hypothesis that the K88 receptor present on erythrocytes is composed of two elements, one responsible for the binding of K88ab and K88ac and a second responsible for the binding of the K88ad adhesin. Comparison of the haemagglutinating properties of cell-free and cell-bound K88 adhesin revealed some differences probably indicating a minor conformational change in the K88 adhesin on its isolation. The K88ab adhesin was found to bind to erythrocytes over a wide pH range (PH 4-9) and was inhibited by αK88ab and αK88b antisera. Inhibition of haemagglutination was noted with crude heparin, mannan and porcine gastric mucin, chondrosine and several hexosamines, glucosamine in particular. The most potent inhibitor of haemagglutination was n-dodecyl-β-D-glucopyranoside, one of a series of glucosides found to have inhibitory properties. Correlation between hydrophobicity of glucosides tested and degree of inhibition observed suggested hydrophobic forces were important in the interaction of the K88 adhesin with its receptor. The results of Scatchard and Hill plots indicated that binding of the K88ab adhesin to porcine enterocytes in the majority of cases is a two-step, three component system. The first K88 receptor (or site) had a K2. of 1.59x1014M-1 and a minimum of 4.3x104 sites/enterocyte. The second receptor (or site) had a K2 of 4.2x1012M-1 with a calculated 1.75x105 sites/enterocyte. Attempts to inhibit binding of cell-free K88 adhesin to porcine enterocytes by lectins were unsuccessful. However, several carbohydrates including trehalose, lactulose, galactose 1→4 mannopyranoside, chondrosine, galactosamine, stachyose and mannan were inhibitory. The most potent inhibitor was found to be porcine gastric mucin. Inhibition observed with n-octyl-α-D-glucopyranose was difficult to interpret in isolation because of interference with the assay, however, it agreed with the results of haemagglutination inhibition experiments.

A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

Lipidome profile of fluids and tissues is a growing field as the role of lipids as signaling molecules is increasingly understood, relying on an effective and representative extraction of the lipids present. A number of solvent systems suitable for lipid extraction are commonly in use, though no comprehensive investigation of their effectiveness across multiple lipid classes has been carried out. To address this, human LDL from normolipidemic volunteers was used to evaluate five different solvent extraction protocols [Folch, Bligh and Dyer, acidified Bligh and Dyer, methanol (MeOH)-tert-butyl methyl ether (TBME), and hexane-isopropanol] and the extracted lipids were analyzed by LC-MS in a high-resolution instrument equipped with polarity switching. Overall, more than 350 different lipid species from 19 lipid subclasses were identified. Solvent composition had a small effect on the extraction of predominant lipid classes (triacylglycerides, cholesterol esters, and phosphatidylcholines). In contrast, extraction of less abundant lipids (phosphatidylinositols, lyso-lipids, ceramides, and cholesterol sulfates) was greatly influenced by the solvent system used. Overall, the Folch method was most effective for the extraction of a broad range of lipid classes in LDL, although the hexane-isopropanol method was best for apolar lipids and the MeOH-TBME method was suitable for lactosyl ceramides. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.