Development & evaluation of chlorophyll a fluorescence as a bioanalytical tool for pollutant identification

University of Technology, Sydney. Faculty of Science.There is potential to improve water quality monitoring programs by generating pollution data that better represents the aquatic ecosystem being monitored. By incorporating rapid and cost-effective bioanalytical methods into water quality monitoring programs, risk associated with unrepresentative data can be reduced by increasing the number of samples collected without incurring additional costs. The rapid and cost-effective toxin-identification method presented here is based on quantifying patterns of change in chlorophyll a fluorescence (fluorescence fingerprints) associated with a toxicants mode of action (MoA). Chlorophyll a fluorescence yield is influenced by environmental factors and can be used to identify stress caused by light, nutrient status and the presence of pollutants. When the functional state of the photosynthetic apparatus changes, the yield of fluorescence emission also changes, generating a chlorophyll a fluorescence response that has previously been thought to be unique based on a toxicants mode of action. The toxin-identification method was developed as a bioanalytical system based on the chlorophyll a fluorescence responses of a microalgae (Dunaliella tertiolecta) to herbicide and nutrient impacts, measured using the Imaging-PAM fluorometer. The analysis of the fluorescence response was the novel method; a holistic approach was employed. Unlike previous approaches which measured one fluorescence parameter for toxicant identification, the method presented here assessed the temporal unity of change in energy dissipation, which was found to be unique depending on a chemical ' s mode of action (i.e. its physico-chemical properties and toxicokinetic relationship with the organism). The method was tested for two different uses: (1) as a non-specific biosensor able to identify herbicides (and their potency) in a water sample of unknown constituents, and (2) a method specific to the identification and potency of nutrients in a water sample. Seven herbicides were examined totaling three different MoAs; PSII inhibitors (DCMU, Irgarol, Bromacil and Simazine), uncoupling of phosphorylation (Dinoseb and PCP) and creation of reactive oxygen species (paraquat). By first generating a database of reference response patterns, the response patterns of laboratory derived test samples were then measured and quantitatively compared to the reference patterns. The unknown or test sample was compared to reference toxicants using a mean-square fit (MSF) software program. The MSF program tells the user how well the fingerprint of the test sample fits to each of the fingerprints of the reference chemicals. The method showed 93% accuracy in correctly identifying six herbicides, with false negative identifications occurring for only two toxicants, simazine (8% of samples) and Dinoseb (27% of samples). Phosphate induced fluorescence transients were also assessed to demonstrate that the toxin-identification method was versatile in its ability to also be used as a selective biomarker. By culturing P-limited D. tertiolecta cells, a unique fluorescence response was recorded upon additions of PO₄³⁻. The NIFT (nutrient induced fluorescent transient) response was specific to PO₄³⁻ additions compared to NH₄³⁺ and NO²⁻ additions. Quantification of the NIFT response showed high levels of precision and specificity for multiple fluorescence parameters. The toxin-identification method presented here is still in its preliminary stages and higher levels of validation are still necessary including testing environmental samples, and comparing results from the toxin-identification method to results from chemical analysis. However, this thesis presents the foundational work of a unique and powerful bioanalytical tool with the potential to greatly improve water quality management practices

Experimental autoimmune encephalomyelitis from a tissue energy perspective

Increasing evidence suggests a key role for tissue energy failure in the pathophysiology of multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE), a commonly used model of MS, have been instrumental in illuminating the mechanisms that may be involved in compromising energy production. In this article, we review recent advances in EAE research focussing on factors that conspire to impair tissue energy metabolism, such as tissue hypoxia, mitochondrial dysfunction, production of reactive oxygen/nitrogen species, and sodium dysregulation, which are directly affected by energy insufficiency, and promote cellular damage. A greater understanding of how inflammation affects tissue energy balance may lead to novel and effective therapeutic strategies that ultimately will benefit not only people affected by MS but also people affected by the wide range of other neurological disorders in which neuroinflammation plays an important role

Müller glia activation in response to inherited retinal degeneration is highly varied and disease-specific

Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1rd8/rd8, Prph2+/Δ307, Rho-/-, Pde6brd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1rd8/rd8 and Prph2+/Δ307, but decreased in the Prph2+/Δ307 and Pde6brd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6brd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent

Strategies for reducing nonresponse in a longitudinal panel survey

This article provides an evaluation of some of the fieldwork procedures and survey systems used on the British Household Panel Study (BHPS). The BHPS procedures for dealing with nonresponse through panel maintenance systems, tracking procedures, and refusal conversion during fieldwork are described. The analysis uses data from the first four waves of BHPS from 1991 to 1994, to examine longitudinal patterns of response and reasons for refusal. The reasons for refusal or for becoming a non-contact over the life of the panel are discussed. The process of refusal conversion is described together with conversion outcomes. Finally the effect of interviewer continuity on maintaining the co-operation of sample members is examined. The article argues that in the context of a longitudinal panel survey, having a relatively complex set of procedures in place is critically important to minimise nonresponse and maintain high response rates over time

Comparing badger (Meles meles) management strategies for reducing tuberculosis incidence in cattle

This is the final version of the article. Available from Public Library of Science via the DOI in this record.Bovine tuberculosis (bTB), caused by Mycobacterium bovis, continues to be a serious economic problem for the British cattle industry. The Eurasian badger (Meles meles) is partly responsible for maintenance of the disease and its transmission to cattle. Previous attempts to manage the disease by culling badgers have been hampered by social perturbation, which in some situations is associated with increases in the cattle herd incidence of bTB. Following the licensing of an injectable vaccine, we consider the relative merits of management strategies to reduce bTB in badgers, and thereby reduce cattle herd incidence. We used an established simulation model of the badger-cattle-TB system and investigated four proposed strategies: business as usual with no badger management, large-scale proactive badger culling, badger vaccination, and culling with a ring of vaccination around it. For ease of comparison with empirical data, model treatments were applied over 150 km(2) and were evaluated over the whole of a 300 km(2) area, comprising the core treatment area and a ring of approximately 2 km. The effects of treatment were evaluated over a 10-year period comprising treatment for five years and the subsequent five year period without treatment. Against a background of existing disease control measures, where 144 cattle herd incidents might be expected over 10 years, badger culling prevented 26 cattle herd incidents while vaccination prevented 16. Culling in the core 150 km(2) plus vaccination in a ring around it prevented about 40 cattle herd breakdowns by partly mitigating the negative effects of culling, although this approach clearly required greater effort. While model outcomes were robust to uncertainty in parameter estimates, the outcomes of culling were sensitive to low rates of land access for culling, low culling efficacy, and the early cessation of a culling strategy, all of which were likely to lead to an overall increase in cattle disease.Funding: The UK Department for Environment, Food and Rural Affairs (Defra, http://ww2.defra.gov.uk/) funded this work. Defra designed the three strategies to investigate, but had no role in data collection and analysis, decision to publish, or preparation of the manuscript

To remove or to replace traditional electronic games? A crossover randomised controlled trial on the impact of removing or replacing home access to electronic games on physical activity and sedentary behaviour in children aged 10-12 years.

This is the final version of the article. Available from BMJ Publishing Group via the DOI in this record.OBJECTIVE: To evaluate the impact of (1) the removal of home access to traditional electronic games or (2) their replacement with active input electronic games, on daily physical activity and sedentary behaviour in children aged 10-12 years. DESIGN: Crossover randomised controlled trial, over 6 months. SETTING: Family homes in metropolitan Perth, Australia from 2007 to 2010. PARTICIPANTS: 10-year-old to 12-year-old children were recruited through school and community media. From 210 children who were eligible, 74 met inclusion criteria, 8 withdrew and 10 had insufficient primary outcome measures, leaving 56 children (29 female) for analysis. INTERVENTION: A counterbalanced randomised order of three conditions sustained for 8 weeks each: no home access to electronic games, home access to traditional electronic games and home access to active input electronic games. MAIN OUTCOME MEASURES: Primary outcome was accelerometer assessed moderate/vigorous physical activity (MVPA). Secondary outcomes included sedentary time and diary assessed physical activity and sedentary behaviours. RESULTS: Daily MVPA across the whole week was not significantly different between conditions. However, compared with home access to traditional electronic games, removal of all electronic games resulted in a significant increase in MVPA (mean 3.8 min/day, 95% CI 1.5 to 6.1) and a decrease in sedentary time (4.7 min/day, 0.0 to 9.5) in the after-school period. Similarly, replacing traditional games with active input games resulted in a significant increase in MVPA (3.2 min/day, 0.9 to 5.5) and a decrease in sedentary time (6.2 min/day, 1.4 to 11.4) in the after-school period. Diary reports supported an increase in physical activity and a decrease in screen-based sedentary behaviours with both interventions. CONCLUSIONS: Removal of sedentary electronic games from the child's home and replacing these with active electronic games both resulted in small, objectively measured improvements in after-school activity and sedentary time. Parents can be advised that replacing sedentary electronic games with active electronic games is likely to have the same effect as removing all electronic games. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Registry (ACTRN 12609000279224).The study was funded by a National Health and Medical Research Council of Australia project grant (533526). The funding body had no influence on the analysis and reporting of the study. LMS had financial support from an NHMRC senior research fellowship (1019980) for the submitted work

Women's views on screening for Type 2 diabetes after gestational diabetes: a systematic review, qualitative synthesis and recommendations for increasing uptake.

AIM: Many women do not attend recommended glucose testing following a pregnancy affected by gestational diabetes (GDM). We aimed to synthesize the literature regarding the views and experiences of women with a history of GDM on postpartum glucose testing, focusing on barriers and facilitators to attendance. METHODS: We systematically identified qualitative studies that examine women's experiences following GDM relating to glucose testing (diabetes screening) or experience of interventions to promote uptake of testing. We conducted a thematic synthesis to develop descriptive and then analytical themes, then developed recommendations to increase uptake based on the findings. We evaluated the quality of each study and the confidence that we had in the recommendations using published checklists. RESULTS: We included 16 articles after screening 23 160 citations and 129 full texts. We identified four themes of influences relating to the healthcare system and personal factors that affected both ability and motivation to attend: relationship with health care, logistics of appointments and tests, family-related practicalities and concern about diabetes. We developed 10 recommendations addressing diabetes risk information and education, and changes to healthcare systems to promote increased attendance at screening in this population, most with high or moderate confidence. CONCLUSIONS: We have identified a need to improve women's understanding about Type 2 diabetes and GDM, and to adjust healthcare provision during and after pregnancy to decrease barriers and increase motivation for testing. Encouraging higher uptake by incorporating these recommendations into practice will enable earlier management of diabetes and improve long-term outcomes.R.D. is funded by a PhD studentship from the National Institute for Health Research (NIHR) School for Primary Care Research (SPCR; SPCR-S-S102). This paper presents independent research funded by the NIHR SPCR. The views expressed are those of the author(s) and not necessarily those of the NIHR, the NHS or the Department of Health. R.W. is funded by an NIHR Academic Clinical Fellowship. S.G. is supported by the Medical Research Council (MC_UU_12015/4). S.G. is an NIHR Senior Investigator. The University of Cambridge has received salary support in respect of S.G. from the NHS in the East of England through the Clinical Academic Reserve. J.U-S. is funded by a Cancer Research UK Cancer Prevention Fellowship (C55650/A21464)

ciliaFA : a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software

Background: Analysis of ciliary function for assessment of patients suspected of primary ciliary dyskinesia (PCD) and for research studies of respiratory and ependymal cilia requires assessment of both ciliary beat pattern and beat frequency. While direct measurement of beat frequency from high-speed video recordings is the most accurate and reproducible technique it is extremely time consuming. The aim of this study was to develop a freely available automated method of ciliary beat frequency analysis from digital video (AVI) files that runs on open-source software (ImageJ) coupled to Microsoft Excel, and to validate this by comparison to the direct measuring high-speed video recordings of respiratory and ependymal cilia. These models allowed comparison to cilia beating between 3 and 52 Hz. Methods: Digital video files of motile ciliated ependymal (frequency range 34 to 52 Hz) and respiratory epithelial cells (frequency 3 to 18 Hz) were captured using a high-speed digital video recorder. To cover the range above between 18 and 37 Hz the frequency of ependymal cilia were slowed by the addition of the pneumococcal toxin pneumolysin. Measurements made directly by timing a given number of individual ciliary beat cycles were compared with those obtained using the automated ciliaFA system. Results: The overall mean difference (± SD) between the ciliaFA and direct measurement high-speed digital imaging methods was −0.05 ± 1.25 Hz, the correlation coefficient was shown to be 0.991 and the Bland-Altman limits of agreement were from −1.99 to 1.49 Hz for respiratory and from −2.55 to 3.25 Hz for ependymal cilia. Conclusions: A plugin for ImageJ was developed that extracts pixel intensities and performs fast Fourier transformation (FFT) using Microsoft Excel. The ciliaFA software allowed automated, high throughput measurement of respiratory and ependymal ciliary beat frequency (range 3 to 52 Hz) and avoids operator error due to selection bias. We have included free access to the ciliaFA plugin and installation instructions in Additional file 1 accompanying this manuscript that other researchers may use

On modelling fluid/body interactions, impacts and lift-offs

A description is given of recent progress in the understanding of mechanisms in fluid-body interactions where the motion of a body and the motion of the surrounding fluid affect each other substantially. The mathematical modelling of such unsteady interactions is for internal channel and external near-wall flows in two spatial dimensions and time. The emphasis throughout is on analytical developments with accompanying reduced computation. The successive aspects studied here are interactions and impacts in inviscid flows, skimming and sinking, the lift-off, fly-away or bouncing of a body, and viscous effects including especially the interplay between viscous and inviscid contributions. The main findings are concerned with physical and mechanical insights into impact times, lift-off criteria, the borders between impact and fly-away, the principal parameters and their ranges and the influences from body shape and mass

Arginase from kiwifruit: properties and seasonal variation

The in vitro activity of arginase (EC 3.5.3.1) was investigated in youngest-mature leaves and roots (1-3 mm diameter) of kiwifruit vines (Actinidia deliciosa var. deliciosa) during an annual growth cycle, and enzyme from root material partially purified. No seasonal trend in the specific activity of arginase was observed in roots. Measurements in leaves, however, rose gradually during early growth and plateaued c. 17 weeks after budbreak. Changes in arginase activity were not correlated with changes in the concentration of arginine (substrate) or glutamine (likely end-product of arginine catabolism) in either tissue during the growth cycle. Purification was by (NH4)2SO4 precipitation and DEAE-cellulose chromatography. The kinetic properties of the enzyme, purified 60-fold over that in crude extracts, indicated a pH optimum of 8.8, and a Km (L-arginine) of 7.85 mM. Partially-purified enzyme was deactivated by dialysis against EDTA, and reactivated in the presence of Mn²⁺, Co²⁺, and Ni²⁺