Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a Km of 46 μM and a kcat of 3.5 s−1 with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a Ki of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.

A differential kinetic spectrophotometric method for determination of three sulphanilamide artificial sweeteners with the aid of chemometrics

A simple and sensitive spectrophotometric method for the simultaneous determination of acesulfame-K, sodium cyclamate and saccharin sodium sweeteners in foodstuff samples has been researched and developed. This analytical method relies on the different kinetic rates of the analytes in their oxidative reaction with KMnO4 to produce the green manganate product in an alkaline solution. As the kinetic rates of acesulfame-K, sodium cyclamate and saccharin sodium were similar and their kinetic data seriously overlapped, chemometrics methods, such as partial least squares (PLS), principal component regression (PCR) and classical least squares (CLS), were applied to resolve the kinetic data. The results showed that the PLS prediction model performed somewhat better. The proposed method was then applied for the determination of the three sweeteners in foodstuff samples, and the results compared well with those obtained by the reference HPLC method.

Simultaneous kinetic-spectrophotometric determination of maltol and ethyl maltol in food samples by using chemometrics

A fast and accurate procedure has been researched and developed for the simultaneous determination of maltol and ethyl maltol, based on their reaction with iron(III) in the presence of o-phenanthroline in sulfuric acid medium. This reaction was the basis for an indirect kinetic spectrophotometric method, which followed the development of the pink ferroin product (λmax = 524 nm). The kinetic data were collected in the 370–900 nm range over 0–30 s. The optimized method indicates that individual analytes followed Beer’s law in the concentration range of 4.0–76.0 mg L−1 for both maltol and ethyl maltol. The LOD values of 1.6 mg L−1 for maltol and 1.4 mg L−1 for ethyl maltol agree well with those obtained by the alternative high performance liquid chromatography with ultraviolet detection (HPLC-UV). Three chemometrics methods, principal component regression (PCR), partial least squares (PLS) and principal component analysis–radial basis function–artificial neural networks (PC–RBF–ANN), were used to resolve the measured data with small kinetic differences between the two analytes as reflected by the development of the pink ferroin product. All three performed satisfactorily in the case of the synthetic verification samples, and in their application for the prediction of the analytes in several food products. The figures of merit for the analytes based on the multivariate models agreed well with those from the alternative HPLC-UV method involving the same samples.

Simultaneous kinetic spectrophotometric determination of norfloxacin and rifampicin in pharmaceutical formulation and human urine samples by use of chemometrics approaches

A kinetic spectrophotometric method with aid of chemometrics is proposed for the simultaneous determination of norfloxacin and rifampicin in mixtures. The proposed method was applied for the simultaneous determination of these two compounds in pharmaceutical formulation and human urine samples, and the results obtained are similar to those obtained by high performance liquid chromatography.

Multicomponent kinetic spectrophotometric determination of pefloxacin and norfloxacin in pharmaceutical preparations and human plasma samples with the aid of chemometrics

A spectrophotometric method for the simultaneous determination of the important pharmaceuticals, pefloxacin and its structurally similar metabolite, norfloxacin, is described for the first time. The analysis is based on the monitoring of a kinetic spectrophotometric reaction of the two analytes with potassium permanganate as the oxidant. The measurement of the reaction process followed the absorbance decrease of potassium permanganate at 526 nm, and the accompanying increase of the product, potassium manganate, at 608 nm. It was essential to use multivariate calibrations to overcome severe spectral overlaps and similarities in reaction kinetics. Calibration curves for the individual analytes showed linear relationships over the concentration ranges of 1.0–11.5 mg L−1 at 526 and 608 nm for pefloxacin, and 0.15–1.8 mg L−1 at 526 and 608 nm for norfloxacin. Various multivariate calibration models were applied, at the two analytical wavelengths, for the simultaneous prediction of the two analytes including classical least squares (CLS), principal component regression (PCR), partial least squares (PLS), radial basis function-artificial neural network (RBF-ANN) and principal component-radial basis function-artificial neural network (PC-RBF-ANN). PLS and PC-RBF-ANN calibrations with the data collected at 526 nm, were the preferred methods—%RPET not, vert, similar 5, and LODs for pefloxacin and norfloxacin of 0.36 and 0.06 mg L−1, respectively. Then, the proposed method was applied successfully for the simultaneous determination of pefloxacin and norfloxacin present in pharmaceutical and human plasma samples. The results compared well with those from the alternative analysis by HPLC.

Accurate series solutions for gravity-driven Stokes waves

In the past, high order series expansion techniques have been used to study the nonlinear equations that govern the form of periodic Stokes waves moving steadily on the surface of an inviscid fluid. In the present study, two such series solutions are recomputed using exact arithmetic, eliminating any loss of accuracy due to accumulation of round-off error, allowing a much greater number of terms to be found with confidence. It is shown that higher order behaviour of series generated by the solution casts doubt over arguments that rely on estimating the series’ radius of convergence. Further, the exact nature of the series is used to shed light on the unusual nature of convergence of higher order Pade approximants near the highest wave. Finally, it is concluded that, provided exact values are used in the series, these Pade approximants prove very effective in successfully predicting three turning points in both the dispersion relation and the total energy.

Marital loss, mental health and the role of perceived social support : findings from six waves of an Australian population based panel study

Objectives: To investigate the impact of transitions out of marriage (separation, widowhood) on the self reported mental health of men and women, and examine whether perceptions of social support play an intervening role. ---------- Methods: The analysis used six waves (2001–06) of an Australian population based panel study, with an analytical sample of 3017 men and 3225 women. Mental health was measured using the MHI-5 scale scored 0–100 (α=0.97), with a higher score indicating better mental health. Perceptions of social support were measured using a 10-item scale ranging from 10 to 70 (α=0.79), with a higher score indicating higher perceived social support. A linear mixed model for longitudinal data was used, with lags for marital status, mental health and social support. ---------- Results: After adjustment for social characteristics there was a decline in mental health for men who separated (−5.79 points) or widowed (−7.63 points), compared to men who remained married. Similar declines in mental health were found for women who separated (−6.65 points) or became widowed (−9.28 points). The inclusion of perceived social support in the models suggested a small mediation effect of social support for mental health with marital loss. Interactions between perceived social support and marital transitions showed a strong moderating effect for men who became widowed. No significant interactions were found for women. ---------- Conclusion: Marital loss significantly decreased mental health. Increasing, or maintaining, high levels of social support has the potential to improve widowed men's mental health immediately after the death of their spouse.

Linear stern waves in finite depth channels

This paper formulates an analytically tractable problem for the wake generated by a long flat bottom ship by considering the steady free surface flow of an inviscid, incompressible fluid emerging from beneath a semi-infinite rigid plate. The flow is considered to be irrotational and two-dimensional so that classical potential flow methods can be exploited. In addition, it is assumed that the draft of the plate is small compared to the depth of the channel. The linearised problem is solved exactly using a Fourier transform and the Wiener-Hopf technique, and it is shown that there is a family of subcritical solutions characterised by a train of sinusoidal waves on the downstream free surface. The amplitude of these waves decreases as the Froude number increases. Supercritical solutions are also obtained, but, in general, these have infinite vertical velocities at the trailing edge of the plate. Consideration of further terms in the expansions suggests a way of canceling the singularity for certain values of the Froude number.

Numerical solution to the Saffman-Taylor finger problem with kinetic undercooling regularisation

The Saffman-Taylor finger problem is to predict the shape and,in particular, width of a finger of fluid travelling in a Hele-Shaw cell filled with a different, more viscous fluid. In experiments the width is dependent on the speed of propagation of the finger, tending to half the total cell width as the speed increases. To predict this result mathematically, nonlinear effects on the fluid interface must be considered; usually surface tension is included for this purpose. This makes the mathematical problem suffciently diffcult that asymptotic or numerical methods must be used. In this paper we adapt numerical methods used to solve the Saffman-Taylor finger problem with surface tension to instead include the effect of kinetic undercooling, a regularisation effect important in Stefan melting-freezing problems, for which Hele-Shaw flow serves as a leading order approximation when the specific heat of a substance is much smaller than its latent heat. We find the existence of a solution branch where the finger width tends to zero as the propagation speed increases, disagreeing with some aspects of the asymptotic analysis of the same problem. We also find a second solution branch, supporting the idea of a countably infinite number of branches as with the surface tension problem.

Vibration of L-shaped plates under a deterministic force or moment excitation : a case of statistical energy analysis application

Analytical and closed form solutions are presented in this paper for the vibration response of an L-shaped plate under a point force or a moment excitation. Inter-relationships between wave components of the source and the receiving plates are clearly defined. Explicit expressions are given for the quadratic quantities such as input power, energy flow and kinetic energy distributions of the L-shaped plate. Applications of statistical energy analysis (SEA) formulation in the prediction of the vibration response of finite coupled plate structures under a single deterministic forcing are examined and quantified. It is found that the SEA method can be employed to predict the frequency averaged vibration response and energy flow of coupled plate structures under a deterministic force or moment excitation when the structural system satisfies the following conditions: (1) the coupling loss factors of the coupled subsystems are known; (2) the source location is more than a quarter of the plate bending wavelength away from the source plate edges in the point force excitation case, or is more than a quarter wavelength away from the pair of source plate edges perpendicular to the moment axis in the moment excitation case due to the directional characteristic of moment excitations. SEA overestimates the response of the L-shaped plate when the source location is less than a quarter bending wavelength away from the respective plate edges owing to wave coherence effect at the plate boundary