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.

New parameters for daytime wandering

This study aimed to describe wandering using new parameters and to evaluate parameters as a function of cognitive impairment and mobility. Forty-four wanderers in long-term care settings were videotaped 12 times. Rate and duration of wandering episodes were plotted and used to derive parameters from values above and below case medians, proportion of hours wandering, and time of day. Participants wandered during 47% of observations; on average, the hourly rate was 4.3 episodes, the peak hourly rate was 18 episodes, and the peak hourly duration was 19.9 minutes. Mini-Mental State Examination (MMSE) scores was negatively correlated with overall duration and number of observations during which duration exceeded 15 minutes per hour, was positively correlated with number of observations without wandering, and was not significantly correlated with rate-related parameters. Mobility correlated positively with rate and duration parameters. Interaction of MMSE score and mobility was the strongest predictor of wandering duration. Parameters derived from repeated measures provide a new view of daytime wandering and insight into relationships between MMSE score and mobility status with specific parameters of wandering.

Effect of inaccuracies in anthropometric data and linked-segment inverse dynamic modeling on kinetics of gait in persons with partial foot amputation

The accuracy of data derived from linked-segment models depends on how well the system has been represented. Previous investigations describing the gait of persons with partial foot amputation did not account for the unique anthropometry of the residuum or the inclusion of a prosthesis and footwear in the model and, as such, are likely to have underestimated the magnitude of the peak joint moments and powers. This investigation determined the effect of inaccuracies in the anthropometric input data on the kinetics of gait. Toward this end, a geometric model was developed and validated to estimate body segment parameters of various intact and partial feet. These data were then incorporated into customized linked-segment models, and the kinetic data were compared with that obtained from conventional models. Results indicate that accurate modeling increased the magnitude of the peak hip and knee joint moments and powers during terminal swing. Conventional inverse dynamic models are sufficiently accurate for research questions relating to stance phase. More accurate models that account for the anthropometry of the residuum, prosthesis, and footwear better reflect the work of the hip extensors and knee flexors to decelerate the limb during terminal swing phase.

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.

Same-sex intimate partner violence : exploring the parameters

Intimate partner violence (IPV) is not only a problem for heterosexual couples. Although research in the area is beset by methodological and definitional problems, studies generally demonstrate that IPV also affects those who identify as non-heterosexual; that is, those sexualities that are typically categorized as lesbian, gay, bisexual, transgender, or intersex (LGBTI). IPV appears to be at least as prevalent in LGBTI relationships as it is in heterosexual couples, and follows similar patterns (e.g. Australian Research Centre on Sex, Health and Society 2006; Donovan et al. 2006; Chan 2005; Craft and Serovich 2005; Burke et al. 2002; Jeffries and Ball 2008; Kelly and Warshafsky 1987; Letellier 1994; Turrell 2000; Ristock 2003; Vickers 1996). There is, however, little in the way of specific community or social services support available to either victims or perpetrators of violence in same-sex relationships (see Vickers 1996). In addition, there are important differences in the experience of IPV between LGBTI and non-LGBTI victims, and even among LGBTI individuals; for example, among transgender populations (Chan 2005), and those who are HIV sero-positive (Craft and Serovich 2005). These different experiences of IPV include the use of HIV and the threat of “outing” a partner as tools of control, as just two examples (Jeffries and Ball 2008; Salyer 1999; WA Government 2008b). Such differences impact on how LGBTI victims respond to the violence, including whether or not and how they seek help, what services they are able to avail themselves of, and how likely they are to remain with, or return to, their violent partners (Burke et al. 2002). This chapter explores the prevalent heteronormative discourses that surround IPV, both within the academic literature, and in general social and government discourses. It seeks to understand how same-sex IPV remains largely invisible, and suggests that these dominant discourses play a major role in maintaining this invisibility. In many respects, it builds on work by a number of scholars who have begun to interrogate the criminal justice and social discourses surrounding violent crime, primarily sexual violence, and who problematize these discourses (see for example Carmody 2003; Carmody and Carrington 2000; Marcus 1992). It will begin by outlining these dominant discourses, and then problematize these by identifying some of the important differences between LGBTI IPV and IPV in heterosexual relationships. In doing so, this chapter will suggest some possible reasons for the silence regarding IPV in LGBTI relationships, and the effects that this can have on victims. Although an equally important area of research, and another point at which the limitations of dominant social discourses surrounding IPV can be brought to light, this chapter will not examine violence experienced by heterosexual men at the hands of their intimate female partners. Instead, it will restrict itself to IPV perpetrated within same-sex relationships.