Detection and quantification of 8‐hydroxy‐2′‐deoxyguanosine in Alzheimer's transgenic mouse urine using capillary electrophoresis

8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) is one of the major forms of oxidative DNA damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8‐OHdG by using CE‐LIF detection. The method involved the use of specific antibody to detect the DNA lesion (8‐OHdG) and consecutive fluorescence labeling. Next, urinary 8‐OHdG fluorescently labeled along with other constituents were resolved by capillary electrophoretic system and the lesion of interest was detected using a fluorescence detector. The limit of detection was 0.18 fmol, which proved sufficient sensitivity for detection and quantification of 8‐OHdG in untreated urine samples. The relative standard deviation was found to be 11.32% for migration time and 5.52% for peak area. To demonstrate the utility of this method, the urinary concentration of 8‐OHdG in an Alzheimer's transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages, such as high separation efficiency, good selectivity, low limit of detection, simplicity and low cost of analysis.     

An Experimentally Established Key Intermediate in Benzene Nitration with Mixed Acid

Experimental evidence is reported for the first intermediate in the classic S_EAr reaction of benzene nitration with mixed acid. The UV/Vis spectroscopic investigation of the reaction showed an intense absorption at 320 nm (appearing as a band shoulder) arising from a reaction intermediate. Our theoretical modeling shows that the interaction between the two principal reactants with solvent (H₂SO₄) molecules significantly affects the structure of the initial complex. In this complex, a larger distance between the aromatic ring and nitronium ion precludes the possibility for electronic charge transfer from the benzene π‐system to the electrophile. The computational modeling of the potential energy surface reveals that the reaction favors a stepwise mechanism with intermediate formation of π‐ and σ‐ (arenium ion) complexes.     

Solubility limits and phase diagrams for fatty acids in anionic (SLES) and zwitterionic (CAPB) micellar surfactant solutions

The limiting solubility of fatty acids in micellar solutions of the anionic surfactant sodium laurylethersulfate (SLES) and the zwitterionic surfactant cocamidopropyl betaine (CAPB) is experimentally determined. Saturated straight-chain fatty acids with n=10, 12, 14, 16, and 18 carbon atoms were investigated at working temperatures of 25, 30, 35, and 40°C. The rise of the fatty acid molar fraction in the micelles is accompanied by an increase in the equilibrium concentration of acid monomers in the aqueous phase. Theoretically, the solubility limit is explained with the precipitation of fatty acid crystallites when the monomer concentration reaches the solubility limit of the acid in pure water. In agreement with theory, the experiment shows that the solubility limit is proportional to the surfactant concentration. For ideal mixtures, the plot of the log of solubility limit vs. the chainlength, n, must be a straight line, which is fulfilled for n=14, 16, and 18. For the fatty acids of shorter chains, n=10 and 12, a deviation from linearity is observed, which is interpreted as non-ideal mixing due to a mismatch between the chainlengths of the surfactant and acid. The data analysis yields the solubilization energy and the interaction parameter for the fatty acid molecules in surfactant micelles. By using the determined parameter values, phase diagrams of the investigated mixed solutions are constructed. The four inter-domain boundary lines intersect in a quadruple point, whose coordinates have been determined. The results can be applied for the interpretation and prediction of the solubility, and phase behavior of medium- and long-chain fatty acids and other amphiphiles that are solubilizable in micellar surfactant solutions, as well as for determining the critical micellization concentration (CMC) of the respective mixed solution.     

A stepwise approach for defining the applicability domain of SAR and QSAR models

A stepwise approach for determining the model applicability domain is proposed. Four stages are applied to account for the diversity and complexity of the current SAR/QSAR models, reflecting their mechanistic rationality (including metabolic activation of chemicals) and transparency. General parametric requirements are imposed in the first stage, specifying in the domain only those chemicals that fall in the range of variation of the physicochemical properties of the chemicals in the training set. The second stage defines the structural similarity between chemicals that are correctly predicted by the model. The structural neighborhood of atom-centered fragments is used to determine this similarity. The third stage in defining the domain is based on a mechanistic understanding of the modeled phenomenon. Here, the model domain combines the reliability of specific reactive groups hypothesized to cause the effect and the domain of explanatory variables determining the parametric requirements in order for functional groups to elicit their reactivity. Finally, the reliability of simulated metabolism (metabolites, pathways, and maps) is taken into account in assessing the reliability of predictions, if metabolic activation of chemicals is a part of the (Q)SAR model. Some of the stages of the proposed approach for defining the model domain can be eliminated depending on the availability and quality of the experimental data used to derive the model, the specificity of (Q)SARs, and the goals of their ultimate application. The performance of the proposed definition of the model domain is tested using several examples of (Q)SARs that have been externally validated, including models for predicting acute toxicity, skin sensitization, and biodegradation. The results clearly showed that credibility in predictions of QSAR models for chemicals belonging to their domain is much higher than for chemicals outside this domain.     

Extension of the ladder model of self-assembly from cylindrical to disclike surfactant micelles

The ladder model of growth of cylindrical micelles gives expressions for the micellar size distribution and for the mean aggregation number, which are in good agreement with the experiment. Here, we consider this model and its extension to the case of disclike micelles. In analogy with the modeling of elongated micelles as sphero-cylinders, the disclike micelles can be modeled as toro-discs. Upon micelle growth, the hemispherical caps of a cylindrical aggregate remain unchanged, whereas the semitoroidal periphery of a disclike micelle expands. This effect can be taken into account in the expression for the size distribution of the disclike micelles, which predicts the dependence of the micelle mean aggregation number on the surfactant concentration. It turns out that disclike micelles could form in a limited range of surfactant concentrations, and that their mean aggregation number cannot exceed a certain maximal value. Large disclike micelles can exist only near the border with the domain of cylindrical micelles. Then, small variations in the experimental conditions could induce a transformation of the disclike micelles into cylindrical ones.     

Thermoelectric method for sequencing DNA

This study describes a novel, thermoelectric method for DNA sequencing in a microfluidic device. The method measures the heat released when DNA polymerase inserts a deoxyribonucleoside triphosphate into a primed DNA template. The study describes the principle of operation of a laminar flow microfluidic chip with a reaction zone that contains DNA template/primer complex immobilized to the inner surface of the device's lower channel wall. A thin-film thermopile attached to the external surface of the lower channel wall measures the dynamic change in temperature that results when Klenow polymerase inserts a deoxyribonucleoside triphosphate into the DNA template. The intrinsic rejection of common-mode thermal signals by the thermopile in combination with hydrodynamic focused flow allows for the measurement of temperature changes on the order of 10(-4) K without control of ambient temperature. To demonstrate the method, we report the sequencing of a model oligonucleotide containing 12 bases. Results demonstrate that it is feasible to sequence DNA by measuring the heat released during nucleotide incorporation. This thermoelectric method for sequencing DNA may offer a novel new method of DNA sequencing for personalized medicine applications.     

Electrophilic aromatic sulfonation with SO3: concerted or classic S(E)Ar mechanism?

The electrophilic sulfonation of several arenes with SO(3) was examined by electronic structure computations at the M06-2X/6-311+G(2d,2p) and SCS-MP2/6-311+G(2d,2p) levels of theory. In contrast to the usual interpretations, the results provide clear evidence that in nonpolar media and in the absence of catalysts the mechanism of aromatic sulfonation with a single SO(3) is concerted and does not involve the conventionally depicted 1:1 σ complex (Wheland) intermediate. Moreover, the computed activation energy for the 1:1 process is unrealistically high; barriers for alternative 2:1 mechanisms involving attack by two SO(3) molecules are 12-20 kcal/mol lower! A direct 2:1 sulfonation mechanism, involving a single essential transition state, but no Wheland type intermediate, is preferred generally at MP2 as well as at M06-2X in isolation (gas phase) or in noncomplexing solvents (such as CCl(4) or CFCl(3)). However, in polar, higher dielectric SO(3)-complexing media, M06-2X favors an S(E)Ar mechanism for the 2:1 reaction involving a Wheland-type arene-(SO(3))(2) dimer intermediate. The reaction is slower in complexing solvents, since the association energy, e.g., with nitromethane, must be overcome. But, in accord with the experimental kinetics (second-order in SO(3)), attack by two sulfur trioxide molecules is still favored energetically over reaction with a single SO(3) in CH(3)NO(2). The theoretical results also reproduce the experimental reactivity and regioselectivity trends for benzene, toluene, and naphthalene sulfonation accurately.     

A mathematical model and numerical method for thermoelectric DNA sequencing

Single nucleotide polymorphisms (SNPs) are single base pair variations within the genome that are important indicators of genetic predisposition towards specific diseases. This study explores the feasibility of SNP detection using a thermoelectric sequencing method that measures the heat released when DNA polymerase inserts a deoxyribonucleoside triphosphate into a DNA strand. We propose a three-dimensional mathematical model that governs the DNA sequencing device with a reaction zone that contains DNA template/primer complex immobilized to the surface of the lower channel wall. The model is then solved numerically. Concentrations of reactants and the temperature distribution are obtained. Results indicate that when the nucleoside is complementary to the next base in the DNA template, polymerization occurs lengthening the complementary polymer and releasing thermal energy with a measurable temperature change, implying that the thermoelectric conceptual device for sequencing DNA may be feasible for identifying specific genes in individuals.     

Surface shear rheology of adsorption layers from the protein HFBII hydrophobin: effect of added β-casein

The surface shear rheology of hydrophobin HFBII adsorption layers is studied in angle-ramp/relaxation regime by means of a rotational rheometer. The behavior of the system is investigated at different shear rates and concentrations of added β-casein. In angle-ramp regime, the experimental data comply with the Maxwell model of viscoelastic behavior. From the fits of the rheological curves with this model, the surface shear elasticity and viscosity, E(sh) and η(sh), are determined at various fixed shear rates. The dependence of η(sh) on the rate of strain obeys the Herschel-Bulkley law. The data indicate an increasing fluidization (softening) of the layers with the rise of the shear rate. The addition of β-casein leads to more rigid adsorption layers, which exhibit a tendency of faster fluidization at increasing shear rates. In relaxation regime, the system obeys a modified Andrade's (cubic root) law, with two characteristic relaxation times. The fact that the data comply with the Maxwell model in angle-ramp regime, but follow the modified Andrade's low in relaxation regime, can be explained by the different processes occurring in the viscoelastic protein adsorption layer in these two regimes: breakage and restoration of intermolecular bonds at angle-ramp vs solidification of the layer at relaxation.