A three-level problem-centric strategy for selecting NMR precursor labeling and analytes

We have developed a sequential set of computational screens that may prove useful for evaluating analyte sets for their ability to accurately report on metabolic fluxes. The methodology is problem-centric in that the screens are used in the context of a particular metabolic engineering problem. That is, flux bounds and alternative flux routings are first identified for a particular problem, and then the information is used to inform the design of nuclear magnetic resonance (NMR) experiments. After obtaining the flux bounds via MILP, analytes are first screened for whether the predicted NMR spectra associated with various analytes can differentiate between different extreme point (or linear combinations of extreme point) flux solutions. The second screen entails determining whether the analytes provide unique flux values or multiple flux solutions. Finally, the economics associated with using different analytes is considered in order to further refine the analyte selection process in terms of an overall utility index, where the index summarizes the cost-benefit attributes by quantifying benefit (contrast power) per cost (e.g., NMR instrument time required). We also demonstrate the use of an alternative strategy, the Analytical Hierarchy Process, for ranking analytes based on the individual experimentalist's-generated weights assigned for the relative value of flux scenario contrast, unique inversion of NMR data to fluxes, etc.     

Cholesterol: lecithin association at molecular ratios of up to 2 : 1.

X-ray diffraction studies of cholestol: egg lecithin mixtures have demonstrated that single phase systems with molecular ratios of up to 2 : 1 can be prepared from solutions in chloroform but that mixtures prepared from ethanol solutions form a single phase only up to a maximum molecular ratio of 1 : 1. The low angle X-ray patterns of the two mixtures (2 : 1 and 1 : 1) are quite distinctive but there is only a small difference in the wide angle spacings. Independent cholesterol reflections begin to appear in the X-ray diffraction pattern of the 2 : 1 mixture after a few days even when the dry sample is contained in a sealed glass capillary tube. Addition of water greatly accelerates this process. In contrast, a 2 : 1 mixture prepared from chloroform solutions can be maintained in sonicated dispersions in water for long periods.     

Comparison of two fluorescence immunoassay methods for the detection of endocrine disrupting chemicals in water

We describe two fluorescence immunoassays capable of detecting endocrine disrupting compounds in waste water. The first fluorescence method is a heterogeneous assay using total internal reflection fluorescence (TIRF) detection. The second method is a homogeneous assay that utilizes energy transfer (ETIA). Both fluorescence immunoassays are compared with respect to detection principle and ability to quantify the model analytes estrone, estradiol, and ethinylestradiol in a complex matrix regarding recovery rates and limits of detection. Calibrations were performed for the three analytes using both fluorescence methods. Limits of detection between 0.01 and 0.85 microg/l are achieved. In addition, measurements in synthetic waste water spiked with the analytes were performed. Both immunoassays allow the detection in waste water with recovery rates in the range of 70-112%.     

A one-dimensional model of vertical stratification of Lake Shira focussed on winter conditions and ice cover

In meromictic lakes such as Lake Shira, horizontal inhomogeneity is small in comparison with vertical gradients. To determine the vertical distribution of temperature, salinity, and density of water in a deep zone of a Lake Shira, or other saline lakes, a one-dimensional (in vertical direction) mathematical model is presented. A special feature of this model is that it takes into account the process of ice formation. The model of ice formation is based on the one-phase Stefan problem with the linear temperature distribution in the solid phase. A convective mixed layer is formed under an ice cover due to salt extraction in the ice formation process. To obtain analytical solutions for the vertical distribution of temperature, salinity, and density of water, we use a scheme of vertical structure in the form of several layers. In spring, the ice melts as top and bottom. These processes are taken into account in the model. The calculated profiles of salinity and temperature of Shira Lake are in good agreement with field measurement data for each season. Additionally, we focussed on the redox zone, which is the zone in which the aerobic layers of a water column meet the anaerobic ones. Hyperactivity of plankton communities is observed in this zone in lakes with hydrogen sulphide monimolimnion, and Lake Shira is among them. The location of the redox zone in the lake, which is estimated from field measurements, coincides with a sharp increase in density (the pycnocline) during autumn and winter. During spring and summer, the redox zone is deeper than the pycnocline. The location of pycnocline calculated with the hydro physical model is in good agreement with field measurement data.     

FLAVOR THRESHOLD AS AFFECTED BY INTERACTION AMONG THREE DAIRY‐RELATED FLAVOR COMPOUNDS*

The hypothesis of the present study was that the presence of one or more flavor compounds in a matrix would alter the perception (either enhance or suppress) of individual compounds as compared to a single flavor compound in a matrix. Thresholds were established for the flavor compounds (diacetyl, hexanal and δ‐decalactone) within each matrix; for each compound in the presence of a second compound; and for each compound in the presence of the remaining two compounds. An ascending order 2‐alternative forced choice test with 12 panelists was used for the evaluations. Results showed that diacetyl in water was suppressed by the presence of hexanal, but the presence of δ‐decalactone with hexanal reversed the trend. In the binary skim milk solutions diacetyl perception was enhanced, while being suppressed in the ternary solution. The perception of hexanal was enhanced in water (binary and ternary solutions). Hexanal and δ‐decalactone were suppressed in skim milk (binary and ternary solutions). δ‐Decalactone showed similar trends as hexanal in water, except that it was suppressed in the presence of diacetyl.     

Affinity capillary electrophoresis: important application areas and some recent developments

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting (`plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen–antibody, hapten–antibody, lectin–sugar, drug–protein, and enzyme–substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.     

Preparation and hydrolytic degradation of semi-interpenetrating networks of poly(3-hydroxyundecenoate) and poly(lactide-co-glycolide)

Semi-interpenetrating networks (semi-IPNs), where poly(lactide-co-glycolide) (PLGA) molecules were entrapped in the crosslinked matrices of poly(3-hydroxyundecenoate) (PHU), were prepared by irradiating homogeneous solutions of PHU and PLGA in chloroform with UV light. Attenuated total reflectance infrared spectroscopy showed that the PLGA chains were entrapped in PHU networks. The semi-IPNs showed enhanced mechanical strength as the PLGA content increased. The semi-IPNs were incubated at 37 °C in a 0.01N NaOH solution, and the extent of hydrolytic degradation was investigated by monitoring changes in various parameters such as water uptake, pH, mass, and morphology. Hydrolysis of semi-IPNs were significantly affected by the presence of PLGA. A semi-IPN prepared from a 9:1 (by weight) mixture of PHU and PLGA lost 25% of its original weight in 12 weeks while a PHU sample containing no PLGA lost only 5% of its weight during the same period under identical conditions. The hydrolysis was most likely accelerated when the pH of the medium was lowered by the hydrolyzed products of PLGA, 2-hydroxyalkanoic acids. These results showed that hydrolysis of PHA could be enhanced by incorporating a second component that lowered the pH of the hydrolysis system.     

Charged cyclodextrin-mediated sample stacking in micellar capillary electrophoresis: A simple method for enhancing the detection sensitivity of hydrophobic compounds

The development of on-line sample stacking techniques for enhancing limits of detection of neutral analytes in micellar capillary electrophoresis (MCE) has recently gained much attention. Utilizing high-conductivity sample matrices to invoke sample stacking is promising, but requires the limited use of sample solubilizing agents such as alcohols in the sample matrix. In this study, we show how simple replacement of the sample solvent (methanol) with a solution of sulfated β-cyclodextrin (sβ-CD) allows a significant increase in the sensitivity of detection of model hydrophobic analytes. This increase in sensitivity is accompanied by significant peak sharpening. Sulfated CDs in the sample matrix allow for effective solubilization of hydrophobic analytes without the use of organic solvents such as methanol. The testing of various sample matrix sβ-CD concentrations for their effect on peak sharpening identified 3 to 5% as optimal for the estrogen standards. The use of a sβ-CD sample matrix allowed for hydrostatic injections (3.5 kPa) of 297 s, compared with 4 s when the analytes were dissolved in methanol. A mechanism explaining the sβ-CD-induced effect involves an analyte transfer mechanism where the sβ-CDs, despite providing anodic mobility to analytes in the sample zone, are able to transfer analytes to trailing separation buffer micelles for “recycling” back into the sample zone without compromising the stacking process. The overall improvement in sensitivity allows detection of estrogens in the parts-per-billion range and stands to improve the utility of MCE as a bioanalytical technique.     

High aeration rate enhances flow stratification in full-scale oxidation ditch

Aerated channel reactors with a uniform field of aeration may display flow stratification and short-circuit phenomena in wastewater treatment systems. In this study, we present data suggesting that flow stratification is closely related to the aeration rate and the arrangement of aerators. A full-scale oxidation ditch, with a total volume of 6,500 m³ and a membrane-diffused aerated zone of 60 × 7 × 5 m (length–width–depth), was selected for water velocity measurements. Two profiles of the oxidation ditch were studied in detail: the first one was at the end of the aerated zone and the second one at the end of the anoxic zone. The results of this work demonstrate that the horizontal water velocity at the end of the aerated zone displayed significant stratification, with maximum velocity near the water surface (0.5–0.7 m/s) and almost zero velocity at a depth of 2.5 m. At the end of the anoxic zone, water velocity was uniform and equal to 0.27–0.31 m/s. Increasing the aeration rate from 1,800 to 4,300 m³/h, almost 90% of the water flow was found to discharge through the upper-half of the channel reactor profile. Different options to mitigate flow stratification of the oxidation ditch are discussed in this paper.     

Rearrangement of the Hydrogen-Bonded Network of the Clathrate Hydrates Encaging Polar Guest

Abstract

Rearrangement process of the hydrogen-bonded network of clathrate hydrate of the polar guest ethylamine is examined by the molecular dynamics simulation. The hydrogen-bonded network rearrangements with reorientation of water or migration of water are observed in the 10 ns trajectories and analyzed in term of a representative connectivity pattern of a time zone longer than a time scale of vibrational motion of molecules. The most frequent rearrangement is the reorientation of single water molecule rotating 180° around its twofold axis in the network unlike Bjerrum's picture of molecular rotation in ice. Migration of water in the host lattice rarely occurs and very long time (several hundred pico second) is required to complete the rearrangement process since cooperative reorientation of many neighboring water is necessarily accompanied. The correlation of reorientational motion of water appears to decay not with the Debye type but rather with a power-law behavior.     

Water molecule binding and lifetimes on the DNA duplex d(CGCGAATTCGCG)2

Summary Measurements of the water proton spin-lattice relaxation rate for aqueous solutions of the palindromic dodecamer, d(CGCGAATTCGCG)₂, are reported as a function of the magnetic field strength. The magnitude of the relaxation rates at low magnetic field strengths and the shape of the relaxation dispersion curve permit assessment of the number of water molecules which may be considered bound to the DNA for a time equal to or longer than the rotational correlation time of the duplex. The data are examined using limiting models that arbitrarily use the measured rotational correlation time of the polynucleotide complex as a reference point for the water molecule lifetime. If it is assumed that water molecules are bound at DNA sites for times as long as or longer than the rotational correlation time of the duplex, then the magnitude of the relaxation rates at low field require that there may be only two or three such water sites. However, if the lifetime constraints is relaxed, and we assume that the number of water molecules bound to the DNA is more nearly the number identified in the X-ray structures, then the average water molecule lifetime is on the order of 1 ns. Measurements of ¹H NOESY spectra demonstrate that some water molecules must have lifetimes sufficiently long that negative Overhauser effects are observed. Taken together, these results suggest a distribution of water molecule lifetimes in which most of the DNA-bound water molecule lifetimes are shorter than the rotational correlation time of the duplex, but where some have lifetimes of at least 1 ns under these concentrated conditions.Abbreviations DNA deoxyribonucleic acid - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy     

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e. samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.     

气候过渡带锐齿栎林土壤呼吸对降雨改变的响应

作为大气与陆地生态系统之间的第二大碳通量,土壤呼吸是评价陆地生态系统碳循环及碳汇能力的不确定性来源之一。降雨格局改变及其导致的土壤水分变化是调节土壤呼吸的重要驱动。气候过渡带的水热状况受全球降雨格局改变的影响更为明显,揭示该区域森林土壤呼吸对降雨改变的响应规律有助于改善碳循环模型的预测精度。然而,气候过渡区的土壤碳排放过程如何响应降雨格局改变尚不清楚。通过在亚热带-暖温带的过渡区(宝天曼)开展降雨改变实验,以阐明锐齿栎林土壤呼吸及其温度敏感性对降雨增加(50%)和减少(50%)的响应规律。结果表明,降雨增加显著提高土壤湿度(+8.92%)而不影响土壤温度。与对照相比,降雨增加导致土壤呼吸显著提高80.5%,其土壤呼吸的温度敏感性(4.07)显著高于对照样地(2.66)。增雨处理下的土壤呼吸与土壤湿度呈负相关。降雨减少则显著降低土壤湿度(-10.25%),并对土壤呼吸有促进趋势,然而,对土壤呼吸的温度敏感性(2.64)无显著影响。减雨处理下的土壤呼吸强度与土壤湿度呈正相关。这意味着在我国亚热带—暖温带过渡区,降雨增加或减少均对土壤呼吸有不同程度的刺激作用,进而很可能减弱该区域森林生态系统土壤的固碳潜力。     

Gradient and zonal analysis of understorey suppression by Eucalyptus wandoo

Gradients, zones and boundaries can be recognized on the basis of biotic, abiotic, field and experimentally-determined attributes along transects through isolated patches of Eucalyptus wandoo trees. The patch zone (corresponding to the tree canopy) could be defined on numerous environmental grounds. The suppression zone (with few shrubs) which embraced the patch zone, could only be distinguished from the surrounding scrub zone (with a dense shrub cover) on vegetation grounds. Site relationships, including identification of boundaries, were analyzed with the aid of a range-standardized dissimilarity measure and metric multidimensional scaling. Differences in availability of nutrients and phytotoxic substances from wandoo leaves, roots and litter had some effects on growth in pot trials but it is concluded that their role in explaining understorey suppression in the field is negligible. Competition for water at depth between the extensive lateral root system of wandoo trees and the roots of adult shrubs is shown to be the most likely explanation for genesis of the suppression zone and location of its boundary. Factors affecting seedling establishment, mainly surface water availability, are more concerned with maintenance of the suppression zone once it is formed.     

Tube gel isotachophoresis: A method for quantitative isolation of nucleic acids from diluted solutions

The technique of isotachophoresis is intended for separation of molecules having different electrophoretic mobilities in a nonhomogeneous electric field. Since the mobility of nucleic acids in water solutions is uniform and does not depend on their size (because of a uniform distribution of negatively charged phosphate groups along the molecule), isotachophoresis will concentrate rather than separate them in the mobile borderline zone between the rapid (Cl⁻) and the slow (β-alanine⁻) anions. This idea served as the basis for elaboration of a novel method for isolation of nucleic acids from diluted solutions. Advantages of the method include quantitative yield (regardless of molecule size), high degree of concentration, and the ability to visually monitor the process. The method may find applications in nucleic acid isolation from highly degraded forensic and clinical samples, from bodily fluids in particular, and thereby promote development of this important direction of diagnostics.     

Centrifugal precipitation chromatography

Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. This countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.     

Effects of sucrose and urea on soy hull pectic polysaccharide gel induced by d-glucono-1,5-lactone

Gelation properties of pectic polysaccharide extracted with ammonium oxalate from soybean hulls assisted by microwave were seldom studied. Water mobility in soy hull pectic polysaccharide (SHPP) was firstly studied by low field NMR. d-Glucono-1,5-lactone (GDL) and sucrose both could decrease spin–spin relaxation times (T₂) of SHPP solutions which indicated the SHPP network formed. Rheological analysis conformed that SHPP gel was formed induced by GDL and enhanced by sucrose. Urea can increase T₂ and collapse the network of SHPP. TGA was used to draw the profiles of water desorption from SHPP solutions or gels, during heating at a controlled rate. It was found that sucrose increased the bound water content and urea acted a conversely role. Hydrogen bond is the main force to maintain SHPP gel network.     

Magnetophoretic position detection for multiplexed immunoassay using colored microspheres in a microchannel

This paper demonstrates a new magnetophoretic position detection method for multiplexed immunoassay using colored microspheres as an encoding tool in a microchannel. Colored microspheres conjugated with respective capture molecules are incubated with a mixture of target analytes, followed by reaction with the probe molecules which had been conjugated with superparamagnetic nanoparticles (SMNPs). Under the magnetic field gradient, the resulting microspheres are deflected from their focused streamlines in a microchannel, and respective colored microspheres are detected using color charge-coupled device (CCD) in a specific detection region of the microchannel. The color and position of respective colored microspheres are automatically decoded and analyzed by MATLAB program, and the position was correlated with the concentration of corresponding target analytes. As a proof-of-concept, we attempted to assay simultaneously three types of biotinylated immunoglobuline Gs (IgGs), such as goat, rabbit and mouse IgGs, using colored microspheres (red, yellow and blue, respectively). As the capture molecules, corresponding anti-IgGs were employed and target analytes were probed using streptavidin-modified superparamagnetic nanoparticles. As a result, three analytes were simultaneously assayed using colored microspheres with high accuracy, and detection limits of goat IgG, rabbit IgG and mouse IgG were estimated to be 10.9, 30.6 and 12.1fM, respectively. In addition, with adjustment of the flow rate and detection zone, the dynamic range could be controlled by more than one order of magnitude.     

Sugar and fat conditioned flavor preferences in C57BL/6J and 129 mice: oral and postoral interactions

C57BL/6J (B6) mice consume more sugar and fat solutions than do 129 mice in 24-h preference tests. Previous studies have attributed this observation to strain differences in taste responsiveness to these nutrients. We tested the hypothesis that differences in postingestive responsiveness contribute to the strain differences. In experiment 1, B6 and 129 mice were trained to associate consumption of a flavored solution (CS+) with intragastric (IG) infusions of 16% sucrose and a different flavored solution (CS-) with IG water infusions (22 h/day). They were then retrained with new flavors paired with IG infusions of 5.6% soybean oil and water. Although both strains developed preferences for the nutrient-paired CS+ solutions, the B6 mice displayed significantly stronger preferences. The B6 mice consumed more CS+ during training, which may have contributed to their enhanced preference. In a second experiment, training intakes were equated by giving B6 and 129 mice "isosweet" CS solutions prepared with different amounts of sucrose and saccharin. The B6 and 129 mice consumed more of the sugar- or fat-paired CS+ than the water-paired CS- during training. The two strains also displayed equally strong preferences for the CS+ over CS- in choice tests, indicating that they had similar postingestive responsiveness to the sucrose and soybean oil. We propose that B6 mice consume more sugar and fat than 129 mice because their stronger orosensory response stimulates greater intake, which leads to greater stimulation of postingestive nutrient detectors and further enhancement of consumption.     

The role of metals in the enzymatic and nonenzymatic oxidation of epinephrine

The effects of transition metals on nonenzymatic and ceruloplasmin catalyzed epinephrine oxidation were investigated by studying rates of epinephrine oxidation in purified buffers and in the presence of metal chelating agents. We found that epinephrine does not “autoxidize” in sodium chloride solutions prepared with deionized water that was further purified by chromatography over Chelex 100 resin prior to use. Epinephrine was oxidized rapidly in sodium chloride prepared with tap water (1.20±0.12 nmoles/min) or in deionized water (0.40±0.80 nmoles/min), but this oxidation was prevented by the addition of Desferal, a potent metal chelating agent. Epinephrine oxidation was enhanced upon the addition of ceruloplasmin, and this oxidation rate could be slowed, but not eliminated, by the addition of Desferal. If epinephrine solutions were preincubated for 72 hours with Desferal prior to ceruloplasmin addition, however, no oxidation was observed. Epinephrine was shown to form colored complexes with both iron and copper at pH 7.0. The Fe(III)-epinephrine complex was much more stable than was the Cu(II)-epinephrine complex. Oxygen consumption studies of ceruloplasmin catalyzed epinephrine oxidation showed that copper was a better promoter of epinephrine oxidation than was iron, suggesting that ceruloplasmin-catalyzed epinephrine oxidation results from adventitious copper bound to the purified enzyme. In light of these results, the physiological relevance of ceruloplasmin catalyzed oxidation of biogenic amines may be minor.