Development and in vitro Profiling of Dual FXR/LTA4H Modulators

Designed polypharmacology presents as an attractive strategy to increase therapeutic efficacy in multi-factorial diseases by a directed modulation of multiple involved targets with a single molecule. Such an approach appears particularly suitable in non-alcoholic steatohepatitis (NASH) which involves hepatic steatosis, inflammation and fibrosis as pathological hallmarks. Among various potential pharmacodynamic mechanisms, activation of the farnesoid X receptor (FXRa) and inhibition of leukotriene A4 hydrolase (LTA4Hi) hold promise to counteract NASH according to preclinical and clinical observations. We have developed dual FXR/LTA4H modulators as pharmacological tools, enabling evaluation of this polypharmacology concept to treat NASH and related pathologies. The optimized FXRa/LTA4Hi exhibits well-balanced dual activity on the intended targets with sub-micromolar potency and is highly selective over related nuclear receptors and enzymes rendering it suitable as tool to probe synergies of dual FXR/LTA4H targeting.     

Clarifications of the Carbonyl and Water Absorptions in Fourier Transform Near Infrared Spectra from Extra Virgin Olive Oil

The Fourier transform near infrared (FT-NIR) spectrum of extra virgin olive oils (EVOO) shows two minor carbonyl absorptions at 5280 and 5180 cm^–1 that has been used to assess their authenticity. To establish components absorbing at 5280 cm^–1, volatile aldehydes and ketones, triacylglycerol (TAG), diacylglycerols (DAG), free fatty acids (FFA), phenolics, and water are investigated and sometimes added to refined olive oil (ROO). Except TAG, the remaining carbonyls contribute to 5280 cm^–1 by broadening peak. Water absorption is demonstrated by its removal using Na₂SO₄ or deuterium oxide addition; FT-NIR spectral changes are reconstituted by water addition. Water absorption depends on being free or complexed with polar compounds in oil. The size of absorption is not related to abundance, but on unique absorption specificity of components; water shows the strongest absorption. Heat removes water and volatiles, leaving behind DAG, FFA, and phenolics, and makes it possible to differentiate absorption of water, volatile and non-volatile carbonyls. Cloudy olive oils are analyzed using FT-NIR methodology after warming for 3 min at 50 °C. FT-NIR index values are replaced by a new calibration model based on correlating gravimetric mass loss of water plus volatiles with spectral changes. The FT-NIR methodology is expanded to include EVOOs with 15.5% to 21% linoleic acid. Practical Applications: Testing for authenticity of EVOOs remains a challenge because adulterations continue to be a problem due to economic gains. Spectroscopy methods, specifically FT-NIR, are much preferred to targeted chemical methods because they measure all constituents in products and are non-destructive and fast. The current universal FT-NIR methodology assesses 13 different parameters: five major FAs, and the DAG and FFA contents. The FT-NIR index value measuring the content of moisture plus volatiles is now replaced by a gravimetric determination. The methodology identifies four major types of adulterants, high in oleic acid, linoleic acid, palm olein or ROO. The composition of olive oils makes it necessary to develop five oil-specific groups, but cloudy samples still need to be clarified by slight warming before measuring. The value of this universal FT-NIR methodology will increase after being adopted by commercial and in regulatory settings.     

Systematic investigation of pipe flows and installation effects using laser Doppler anemometry—Part I. Profile measurements downstream of several pipe configurations and flow conditioners

The first part of this paper reports on an automated facility designed to investigate the influence of disturbed flows in pipes on the shift of the error curves of gas flowmeters in situ. This facility can be equipped with several pipe configurations (single and double bends, convergent and divergent sections, straight pipes up to 40 diameters in length etc.) as well as with various types of flow conditioners. It works with atmospheric air at flowrates of up to 5500 m³/h. A two-component semiconductor Laser Doppler Anemometer (LDA) is used to measure the spatial velocity and turbulence fields of the flow along the entire cross section in front of the flowmeter to be investigated. More than 150 velocity distributions have been determined for different pipe configurations at several flowrates and data have been collected to describe the corresponding flowmeter's behaviour. Some typical velocity profiles for the most usual pipe elements and flow conditioners are shown.

The second part of the publication, which will be presented in the following, compares the changes in the meter behaviour with the specific development of the flow characteristics downstream of the pipe configurations investigated. The model found to explain these metering effects will be described, evaluated and verified.     

Arsenate and arsenite adsorption and desorption behavior on coprecipitated aluminum:iron hydroxides

Although arsenic adsorption/desorption behavior on aluminum and iron (oxyhydr)oxides has been extensively studied, little is known about arsenic adsorption/desorption behavior by bimetal Al:Fe hydroxides. In this study, influence of the Al:Fe molar ratio, pH, and counterion (Ca2+ versus Na+) on arsenic adsorption/desorption by preformed coprecipitated Al:Fe hydroxides was investigated. Adsorbents were formed by initial hydrolysis of mixed Al3+/ Fe3+ salts to form coprecipitated Al:Fe hydroxide products. At Al:Fe molar ratios < or = 1:4, Al3+ was largely incorporated into the iron hydroxide structure to form a poorly crystalline bimetal hydroxide; however, at higher Al:Fe molar ratios, crystalline aluminum hydroxides (bayerite and gibbsite) were formed. Although approximately equal As(V) adsorption maxima were observed for 0:1 and 1:4 Al:Fe hydroxides, the As(III) adsorption maximum was greater with the 0:1 Al: Fe hydroxide. As(V) and As(III) adsorption decreased with further increases in Al:Fe molar ratio. As(V) exhibited strong affinity to 0:1 and 1:4 Al:Fe hydroxides at pH 3-6. Adsorption decreased at pH > 6.5; however, the presence of Ca2+ compared to Na+ as the counterion enhanced As( retention by both hydroxides. There was more As(V) and especially As(III) desorption by phosphate with an increase in Al:Fe molar ratio.     

Identification of black carbon derived structures in a volcanic ash soil humic acid by Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), coupled with cross-polarization magic angle spinning 13C nuclear magnetic resonance (NMR) spectroscopy and Kendrick mass defect analysis, was used to study the molecular composition of an aromatic carbon-rich humic acid extracted from a dark black soil from Iwata, Japan. Black carbon, produced by the incomplete combustion of fossil fuels and organic matter, has been suggested as a major component of humic acids having intense peaks in the aromatic and carboxyl regions of the 13C NMR spectrum. Taking advantage of the high resolving power of FT-ICR MS to make precise formula assignments, three different types of highly carboxylated polycyclic aromatic compounds were identified in the sample: linearly fused aromatic structures, aromatic structures linked by carbon-carbon single bonds, and highly condensed aromatic structures. These carboxylated aromatic structures have a low mass defect in their mass spectra due to their abundance of oxygen and deficiency of hydrogen. This mass defect is observed in the vast majority of peaks present in the entire mass spectrum, differentiating them from structures that are hydrogen-rich (e.g., fatty acids, proteins, carbohydrates). Thus, we conclude that the bulk of the sample analyzed is comprised of these heavily carboxylated, hydrogen-deficient, condensed aromatic structures, features believed to be characteristic of black carbon-like material.     

Influence of triglycerides and free fatty acids in milk replacers on calf performance, blood plasma, and adipose lipids

In a 4-wk study of 48 3-day-old calves we compared effects of feeding various fats or their free fatty acids in skim milk-powder based milk replacer, on calf performance, feed utilization, and blood plasma and adipose lipids. When fat was fed, calf performance and feed utilization were equivalent for tallow and coconut oil diets but markedly poorer for corn oil. Complete replacement (tallow) or one-half replacement (coconut and corn oils) of the fats with their free fatty acids reduced calf gains and feed efficiency. Tallow free fatty acids gave lower digestibilities of palmitic and stearic acid and reduced calcium absorption. Free fatty acids from both coconut and corn oils reduced diet palatability and intake; those from tallow and coconut oil markedly interfered (in vitro) with rennet clotting of milk replacers. The main lipid classes in blood plasma for all treatments were cholesteryl esters and phosphatidylcholine. High concentrations (56 to 87%) of linoleic acid occurred in cholesteryl esters for all diets despite low concentrations of linoleic acid in the tallow and coconut oil diets.     

A complete separation of lipids by three-directional thin layer chromatography

A novel three-directional thin layer chromatography (TLC) method is reported by which all the polar and neutral lipids are isolated on a single TLC plate. Following resolution of the phospholipids by two-directional TLC, lipids are visualized by ultraviolet light after spraying with 2′,7′-dichloro-fluorescein. A line is drawn across the plate, parallel to the second direction of development, separating the resolved phospholipids and the neutral lipids concentrated along the solvent front. The TLC plate is then chromatographed in the reverse direction of the second development to resolve the neutral lipids. By exposing the lipids to HCl fumes after the first development, the plasmalogen content of the lipids may also be determined. This new technique is rapid and lends itself to qualitative and quantitative analyses of total lipids. Contribution no. 1163 from the Animal Research Centre.     

Evolutionary affinity and selectivity optimization of a pesticide-selective antibody utlizing a hapten-selective immunoglobulin repertoire

Selectivity and sensitivity are considered as pivotal criteria for the quality of immunochemical assay designs in environmental analysis. They are essentially determined by the variable domains of the implemented antibody. The variable domains of a triazine-selective single-chain Fv (scFv) were genetically engineered by stringent molecular evolution in order to optimize analytical characteristics of the corresponding atrazine immunoassay. Gene variation of the template antibody by sequential shuffling against the variable heavy and light chain repertoire of a triazine-selective immunoglobulin library was enhanced by introducing additional point mutatons. Improved scFv variants were selected by phage display employing an atrazine derivative. By this means the paramounting affinity of the initial scFv to sebuthylazine was shifted for the mutant antibodies toward a preferential recognition of the envisaged target analyte atrazine. In addition, the detection limit of the atrazine assaywas significantly improved by factor 25 from 5.1 microg/L for the initial template antibody to 0.2 microg/L for the mutant antibodies. The contribution of the engineered antibody variants to the assay improvement is also reflected by a shift of the equilibrium dissociation constant KD from 1.27 x 10(-8) M of the template antibody to 7.46 x 10(-10) M of the optimized variant. Sequence analysis revealed a bias of amino acid substitutions in the first two complementarity-determining regions (CDR) and the flanking framework regions of both variable chains for the shuffled clones as well as a deletion in the CDR3 of the light chain. Particularly the mutations of the VL domain turned out to have a decisive impact on the alterations in the analytical performance of the engineered scFv mutants. The application of the mutant antibodies for the atrazine determination of soil samples revealed consistency with HPLC data within the experimental error.     

A family of compact genetic algorithms for intrinsic evolvable hardware

For many evolvable hardware applications, small size and power efficiency are critical design considerations. One manner in which significant memory, and thus, power and space savings can be realized in a hardware-based evolutionary algorithm is to represent populations of candidate solutions as probability vectors rather than as sets of bit strings. The compact genetic algorithm (CGA) is a probability vector-based evolutionary algorithm that can be efficiently and elegantly implemented in digital hardware. Unfortunately, the CGA is a very weak, first order, evolutionary algorithm that is unlikely to possess sufficient search power to enable intrinsic evolvable hardware applications. In this paper, we further develop a number of modifications to the basic CGA that significantly improve its search efficacy without substantially increasing the size and complexity of its hardware implementation. The paper provides both benchmark results demonstrating increased efficacy and a conceptual data path/microcontroller design suitable for implementation in digital hardware. Following, it demonstrates efficient implementation by making a head-to-head comparison of field programmable gate array implementations of both the classic CGA and a member of our family of modifications. The paper concludes with a discussion of future research, including several additional extensions that we expect will further increase search efficacy without increasing implementation cost.