Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy3-(p- nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30-42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.

Human identification from video using advanced gait recognition techniques

The solutions proposed in this thesis contribute to improve gait recognition performance in practical scenarios that further enable the adoption of gait recognition into real world security and forensic applications that require identifying humans at a distance. Pioneering work has been conducted on frontal gait recognition using depth images to allow gait to be integrated with biometric walkthrough portals. The effects of gait challenging conditions including clothing, carrying goods, and viewpoint have been explored. Enhanced approaches are proposed on segmentation, feature extraction, feature optimisation and classification elements, and state-of-the-art recognition performance has been achieved. A frontal depth gait database has been developed and made available to the research community for further investigation. Solutions are explored in 2D and 3D domains using multiple images sources, and both domain-specific and independent modality gait features are proposed.

An enhanced procedure for the rapid digestion of high silicate archeological specimens followed by ICP-MS determination of traces of rare earth elements (REE's)

A straightforward procedure for the acid digestion of geological samples with SiO2 concentrations ranging between about 40 to 80%, is described. A powdered sample (200 mesh) of 500 mg was used and fused with 1000 mg spectroflux at about 1000 degreesC in a platinum crucible. The molten was subsequently digested in an aqueous solution of HNO3 at 100 degreesC. Several systematic digestion procedures were followed using various concentrations of HNO3. It was found that a relationship could be established between the dissolution-time and acid concentration. For an acid concentration of 15% an optimum dissolution-time of under 4 min was recorded. To verify that the dissolutions were complete, they were subjected to rigorous quality control tests. The turbidity and viscosity were examined at different intervals and the results were compared with that of deionised water. No significant change in either parameter was observed. The shelf-life of each solution lasted for several months, after which time polymeric silicic acid formed in some solutions, resulting in the presence of a gelatinous solid. The method is cost effective and is clearly well suited for routine applications on a small scale, especially in laboratories in developing countries. ICP-MS was applied to the determination of 13 Rare Earth Elements and Hf in a set of 107 archaeological samples subjected to the above digestion procedure. The distribution of these elements was examined and the possibility of using the REE's for provenance studies is discussed.

Rapid and highly efficient growth of graphene on copper by chemical vapor deposition of ethanol

The growth of graphene by chemical vapor deposition on metal foils is a promising technique to deliver large-area films with high electron mobility. Nowadays, the chemical vapor deposition of hydrocarbons on copper is the most investigated synthesis method, although many other carbon precursors and metal substrates are used too. Among these, ethanol is a safe and inexpensive precursor that seems to offer favorable synthesis kinetics. We explored the growth of graphene on copper from ethanol, focusing on processes of short duration (up to one min). We investigated the produced films by electron microscopy, Raman and X-ray photoemission spectroscopy. A graphene film with high crystalline quality was found to cover the entire copper catalyst substrate in just 20 s, making ethanol appear as a more efficient carbon feedstock than methane and other commonly used precursors.

Identification of eusynstyelamide B as a potent cell cycle inhibitor following the generation and screening of an ascidian-derived extract library using a real time cell analyzer

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 μM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Identification of novel antigens in blood vessels in rectovaginal endometriosis

To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. (C) 2012 Elsevier B.V. All rights reserved.

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

A little learning is a dangerous thing: Factors influencing the increased identification of special educational needs from the perspective of education policymakers and school practitioners

This article considers the increased identification of special educational needs in Australia’s largest education system from the perspectives of senior public servants, regional directors, principals, school counsellors, classroom teachers, support class teachers, learning support teachers and teaching assistants (n = 30). While their perceptions of an increase generally align with the story told by official statistics, participants’ narratives reveal that school-based identification of special educational needs is neither art nor science. This research finds that rather than an objective indication of the number and nature of children with SEN, official statistics may be more appropriately viewed as a product of funding eligibility and the assumptions of the adults who teach, refer and assess children who experience difficulties in school and with learning.

MIMO and SISO identification of radiation force terms for models of marine structures in waves

This paper presents a discussion on the use of MIMO and SISO techniques for identification of the radiation force terms in models for surface vessels. We compare and discuss two techniques recently proposed in literature for this application: time domain identification and frequency domain identification. We compare the methods in terms of estimates model order, accuracy of the fit, use of the available information, and ease of use and implementation.

Identification of pre-spike network in patients with mesial temporal lobe epilepsy

Background: Seizures and interictal spikes in mesial temporal lobe epilepsy (MTLE) affect a network of brain regions rather than a single epileptic focus. Simultaneous electroencephalography and functional magnetic resonance imaging (EEG-fMRI) studies have demonstrated a functional network in which hemodynamic changes are time-locked to spikes. However, whether this reflects the propagation of neuronal activity from a focus, or conversely the activation of a network linked to spike generation remains unknown. The functional connectivity (FC) changes prior to spikes may provide information about the connectivity changes that lead to the generation of spikes. We used EEG-fMRI to investigate FC changes immediately prior to the appearance of interictal spikes on EEG in patients with MTLE. Methods/principal findings: Fifteen patients with MTLE underwent continuous EEG-fMRI during rest. Spikes were identified on EEG and three 10 s epochs were defined relative to spike onset: spike (0–10 s), pre-spike (−10 to 0 s), and rest (−20 to −10 s, with no previous spikes in the preceding 45s). Significant spike-related activation in the hippocampus ipsilateral to the seizure focus was found compared to the pre-spike and rest epochs. The peak voxel within the hippocampus ipsilateral to the seizure focus was used as a seed region for FC analysis in the three conditions. A significant change in FC patterns was observed before the appearance of electrographic spikes. Specifically, there was significant loss of coherence between both hippocampi during the pre-spike period compared to spike and rest states. Conclusion/significance: In keeping with previous findings of abnormal inter-hemispheric hippocampal connectivity in MTLE, our findings specifically link reduced connectivity to the period immediately before spikes. This brief decoupling is consistent with a deficit in mutual (inter-hemispheric) hippocampal inhibition that may predispose to spike generation.

Near infrared spectroscopy for rapid determination of Mankin score components : a potential tool for quantitative characterization of articular cartilage at surgery

PURPOSE The purpose of this study was to demonstrate the potential of near infrared (NIR) spectroscopy for characterizing the health and degenerative state of articular cartilage based on the components of the Mankin score. METHODS Three models of osteoarthritic degeneration induced in laboratory rats by anterior cruciate ligament (ACL) transection, meniscectomy (MSX), and intra-articular injection of monoiodoacetate (1 mg) (MIA) were used in this study. Degeneration was induced in the right knee joint; each model group consisted of 12 rats (N = 36). After 8 weeks, the animals were euthanized and knee joints were collected. A custom-made diffuse reflectance NIR probe of 5-mm diameter was placed on the tibial and femoral surfaces, and spectral data were acquired from each specimen in the wave number range of 4,000 to 12,500 cm(-1). After spectral data acquisition, the specimens were fixed and safranin O staining (SOS) was performed to assess disease severity based on the Mankin scoring system. Using multivariate statistical analysis, with spectral preprocessing and wavelength selection technique, the spectral data were then correlated to the structural integrity (SI), cellularity (CEL), and matrix staining (SOS) components of the Mankin score for all the samples tested. RESULTS ACL models showed mild cartilage degeneration, MSX models had moderate degeneration, and MIA models showed severe cartilage degenerative changes both morphologically and histologically. Our results reveal significant linear correlations between the NIR absorption spectra and SI (R(2) = 94.78%), CEL (R(2) = 88.03%), and SOS (R(2) = 96.39%) parameters of all samples in the models. In addition, clustering of the samples according to their level of degeneration, with respect to the Mankin components, was also observed. CONCLUSIONS NIR spectroscopic probing of articular cartilage can potentially provide critical information about the health of articular cartilage matrix in early and advanced stages of osteoarthritis (OA). CLINICAL RELEVANCE This rapid nondestructive method can facilitate clinical appraisal of articular cartilage integrity during arthroscopic surgery.

A collaborative rapid persona-building workshop : creating design personas with health researchers

In the HealthMap project for People With HIV, (PWHIV) designers employed a collaborative rapid ‘persona-building' workshop with health researchers to develop patient personas that embodied patient-centred design goals and contextual awareness from a variety of qualitative and quantitative data. On reflection, this collaborative rapid workshop was a process for drawing together the divergent user research insights and expertise of stakeholders into focus for a chronic disease self-management design. This paper discusses, (i) an analysis of the transcript of the workshop and, (ii) interviews with five practising senior designers, in order to reflect on how the persona-building process was enacted and its role in the HealthMap design evolution. The collaborative rapid persona-building methodology supported: embedding user research insights, eliciting domain expertise, introducing design thinking, facilitating stakeholder collaboration and defining early design requirements. The contribution of this paper is to model the process of collaborative rapid persona-building and to introduce the collaborative rapid persona-building framework as a method to generate design priorities from domain expertise and user research data.

Short-data recursive HMM parameter estimation for rapid vision-based aircraft heading estimation

Rapid recursive estimation of hidden Markov Model (HMM) parameters is important in applications that place an emphasis on the early availability of reasonable estimates (e.g. for change detection) rather than the provision of longer-term asymptotic properties (such as convergence, convergence rate, and consistency). In the context of vision- based aircraft (image-plane) heading estimation, this paper suggests and evaluates the short-data estimation properties of 3 recursive HMM parameter estimation techniques (a recursive maximum likelihood estimator, an online EM HMM estimator, and a relative entropy based estimator). On both simulated and real data, our studies illustrate the feasibility of rapid recursive heading estimation, but also demonstrate the need for careful step-size design of HMM recursive estimation techniques when these techniques are intended for use in applications where short-data behaviour is paramount.

Superstars as drivers of organizational identification : empirical findings from professional soccer

This paper examines the effect of superstars on external stakeholders’ organizational identification through the lens of sport. Drawing on social identity theory and the concept of organizational identification, as well as on role model theories and superstar economics, several hypotheses are developed regarding the influence of soccer stars on their fans’ degree of team identification. Using a proprietary data set that combines archival data on professional German soccer players and clubs with survey data on more than 1,400 soccer fans, this study finds evidence for a positive effect of superstar characteristics and role model perception. Moreover, it is found that players who qualify for the definition of a superstar are more important to fans of established teams than to fans of unsuccessful teams. The player's club tenure, however, seems to have no influence on fans’ team identification. It is further argued that the effect of soccer stars on their fans is comparable to that of executives on external stakeholders, and hence, the results are applied to the business domain. The results of this study contribute to existing research by extending the list of personnel-related determinants of organizational identification.