Anti-metabolic effects of Galanthus nivalis agglutinin and wheat germ agglutinin on nymphal stages of the common brown leafhopper using a novel artificial diet system

The common brown leafhopper, Orosius orientalis (Matsumura) (Homoptera: Cicadellidae), previously described as Orosius argentatus (Evans), is an important vector of several viruses and phytoplasmas worldwide. In Australia, phytoplasmas vectored by O. orientalis cause a range of economically important diseases, including legume little leaf (Hutton & Grylls, 1956), tomato big bud (Osmelak, 1986), lucerne witches broom (Helson, 1951), potato purple top wilt (Harding & Teakle, 1985), and Australian lucerne yellows (Pilkington et al., 2004). Orosius orientalis also transmits Tobacco yellow dwarf virus (TYDV; genus Mastrevirus, family Geminiviridae) to beans, causing bean summer death disease (Ballantyne, 1968), and to tobacco, causing tobacco yellow dwarf disease (Hill, 1937, 1941). TYDV has only been recorded in Australia to date. Both diseases result in significant production and quality losses (Ballantyne, 1968; Thomas, 1979; Moran & Rodoni, 1999). Although direct damage caused by leafhopper feeding has been observed, it is relatively minor compared to the losses resulting from disease (P Tr E bicki, unpubl.).

Response efficacy : the key to minimizing rejection and maximizing acceptance of emotion-based anti-speeding messages

This study sought to improve understanding of the persuasive process of emotion-based appeals not only in relation to negative, fear-based appeals but also for appeals based upon positive emotions. In particular, the study investigated whether response efficacy, as a cognitive construct, mediated outcome measures of message effectiveness in terms of both acceptance and rejection of negative and positive emotion-based messages. Licensed drivers (N = 406) participated via the completion of an on-line survey. Within the survey, participants received either a negative (fear-based) appeal or one of the two possible positive appeals (pride or humor-based). Overall, the study's findings confirmed the importance of emotional and cognitive components of persuasive health messages and identified response efficacy as a key cognitive construct influencing the effectiveness of not only fear-based messages but also positive emotion-based messages. Interestingly, however, the results suggested that response efficacy's influence on message effectiveness may differ for positive and negative emotion-based appeals such that significant indirect (and mediational) effects were found with both acceptance and rejection of the positive appeals yet only with rejection of the fear-based appeal. As such, the study's findings provide an important extension to extant literature and may inform future advertising message design.

Enhancing road safety for young drivers : how Graduate Driver Licensing initiatives can complement "anti-hooning" legislation

Street racing and associated (hooning) behaviours have attracted increasing concern in recent years. While New Zealand and all Australian jurisdictions have introduced “antihooning” legislation and allocated significant police resources to managing the problem, there is limited evidence of the road safety implications of hooning. However, international and Australian data suggests that drivers charged with a hooning offence tend to be young males who are accompanied by one or more peers, and hooning-related crashes tend to occur at night. In this regard, there is considerable evidence that drivers under the age of 25 are over-represented in crash statistics, and are particularly vulnerable soon after obtaining a Provisional licence, when driving at night, and when carrying peer-aged passengers. The similarity between the nature of hooning offenders, offences and crashes, and road safety risks for young drivers in general, suggests that hooning is an issue that may be viewed as part of the broader young driver problem. Many jurisdictions have recently implemented a range of evidence-based strategies to address young driver road safety, and this paper will present Queensland crash and offence data to highlight the potential benefit of Graduated Driver Licensing initiatives, such as night driving restrictions and peer-aged passenger restrictions, to related road safety issues, including hooning. An understanding of potential flow-on effects is important for evaluations of anti-hooning legislation and Graduated Driver Licensing programs, and may have implications for future law enforcement resource allocation and policy development.

Lyprinol : is it a useful anti-inflammatory agent?

The New Zealand green lipped mussel preparation Lyprinol is available without a prescription from a supermarket, pharmacy or Web. The Food and Drug Administration have recently warned Lyprinol USA about their extravagant anti-inflammatory claims for Lyprinol appearing on the web. These claims are put to thorough review. Lyprinol does have anti-inflammatory mechanisms, and has anti-inflammatory effects in some animal models of inflammation. Lyprinol may have benefits in dogs with arthritis. There are design problems with the clinical trials of Lyprinol in humans as an anti-inflammatory agent in osteoarthritis and rheumatoid arthritis, making it difficult to give a definite answer to how effective Lyprinol is in these conditions, but any benefit is small. Lyprinol also has a small benefit in atopic allergy. As anti-inflammatory agents, there is little to choose between Lyprinol and fish oil. No adverse effects have been reported with Lyprinol. Thus, although it is difficult to conclude whether Lyprinol does much good, it can be concluded that Lyprinol probably does no major harm.

Anti-cancer activity of zoledronic acid in breast cancer

Background: Zoledronic acid is used to prevent the bone loss associated with antioestrogen treatments in subjects with breast cancer. Preclinical studies suggest that zoledronic acid may have anticancer activity in its own right. This anticancer possibility with zoledronic acid has not been investigated extensively in clinical trials. Objectives/methods: This evaluation is of a large clinical trial that investigated the effect of zoledronic acid on cancer outcomes in premenopausal women with breast cancer. Results: The trial showed that after 4 years, 94.0% of subjects who were treated with zoledronic acid were disease-free compared with 90.8% of those not treated with zoledronic acid. Recurrence survival was a secondary end point; this occurred in 94.0% with, and 90.9% without, zoledronic acid treatment. Conclusions: Zoledronic acid does have anticancer activity in premenopausal women with cancer.

Microbial risk from rainwater tanks in South East Queensland

Quantitative Microbial Risk Assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to pathogens from potable and non-potable uses of roof-harvested rainwater in South East Queensland (SEQ). A total of 84 rainwater samples were analysed for the presence of faecal indicators (using culture based methods) and zoonotic bacterial and protozoan pathogens using binary and quantitative PCR (qPCR). The concentrations of Salmonella invA, and Giardia lamblia β-giradin genes ranged from 65-380 genomic units/1000 mL and 9-57 genomic units/1000 mL of water, respectively. After converting gene copies to cell/cyst number, the risk of infection from G. lamblia and Salmonella spp. associated with the use of rainwater for bi-weekly garden hosing was calculated to be below the threshold value of 1 extra infection per 10,000 persons per year. However, the estimated risk of infection from drinking the rainwater daily was 44-250 (for G. lamblia) and 85-520 (for Salmonella spp.) infections per 10,000 persons per year. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically discussed. Nevertheless, it would seem prudent to disinfect rainwater for potable use.

New insights into appetite, inflammation and the use of fish oil in hemodialysis patients

Protein-energy wasting (PEW) is commonly seen in patients with chronic kidney disease (CKD). The condition is characterised by chronic, systemic low-grade inflammation which affects nutritional status by a variety of mechanisms including reducing appetite and food intake and increasing muscle catabolism. PEW is linked with co-morbidities such as cardiovascular disease, and is associated with lower quality of life, increased hospitalisations and a 6-fold increase in risk of death1. Significant gender differences have been found in the severity and effects of several markers of PEW. There have been limited studies testing the ability of anti-inflammatory agents or nutritional interventions to reduce the effects of PEW in dialysis patients. This thesis makes a significant contribution to the understanding of PEW in dialysis patients. It advances understanding of measurement techniques for two of the key components, appetite and inflammation, and explores the effect of fish oil, an anti-inflammatory agent, on markers of PEW in dialysis patients. The first part of the thesis consists of two methodological studies conducted using baseline data. The first study aims to validate retrospective ratings of hunger, desire to eat and fullness on visual analog scales (VAS) (paper and pen and electronic) as a new method of measuring appetite in dialysis patients. The second methodological study aims to assess the ability of a variety of methods available in routine practice to detect the presence of inflammation. The second part of the thesis aims to explore the effect of 12 weeks supplementation with 2g per day of Eicosapentaenoic Acid (EPA), a longchain fatty acid found in fish oil, on markers of PEW. A combination of biomarkers and psychomarkers of appetite and inflammation are the main outcomes being explored, with nutritional status, dietary intake and quality of life included as secondary outcomes. A lead in phase of 3 months prior to baseline was used so that each person acts as their own historical control. The study also examines whether there are gender differences in response to the treatment. Being an exploratory study, an important part of the work is to test the feasibility of the intervention, thus the level of adherence and factors associated with adherence are also presented. The studies were conducted at the hemodialysis unit of the Wesley Hospital. Participants met the following criteria: adult, stage 5 CKD on hemodialysis for at least 3 months, not expected to receive a transplant or switch to another dialysis modality during the study, absence of intellectual impairment or mental illness impairing ability to follow instructions or complete the intervention. A range of intermediate, clinical and patient-centred outcome measures were collected at baseline and 12 weeks. Inflammation was measured using five biomarkers: c-reactive protein (CRP), interleukin-6 (IL6), intercellular adhesion molecule (sICAM-1), vascular cell adhesion molecule (sVCAM-1) and white cell count (WCC). Subjective appetite was measured using the first question from the Appetite and Dietary Assessment (ADAT) tool and VAS for measurements of hunger, desire to eat and fullness. A novel feature of the study was the assessment of the appetite peptides leptin, ghrelin and peptide YY as biomarkers of appetite. Nutritional status/inflammation was assessed using the Malnutrition-Inflammation Score (MIS) and the Patient-Generated Subjective Global Assessment (PG-SGA). Dietary intake was measured using 3-day records. Quality of life was measured using the Kidney Disease Quality of Life Short Form version 1.3 (KDQOL-SF™ v1.3 © RAND University), which combines the Short-Form 36 (SF36) with a kidney-disease specific module2. A smaller range of these variables was available for analysis during the control phase (CRP, ADAT, dietary intake and nutritional status). Statistical analysis was carried out using SPSS version 14 (SPSS Inc, Chicago IL, USA). Analysis of the first part of the thesis involved descriptive and bivariate statistics, as well as Bland-Altman plots to assess agreement between methods, and sensitivity analysis/ROC curves to test the ability of methods to predict the presence of inflammation. The unadjusted (paired ttests) and adjusted (linear mixed model) change over time is presented for the main outcome variables of inflammation and appetite. Results are shown for the whole group followed by analyses according to gender and adherence to treatment. Due to the exploratory nature of the study, trends and clinical significance were considered as important as statistical significance. Twenty-eight patients (mean age 61±17y, 50% male, dialysis vintage 19.5 (4- 101) months) underwent baseline assessment. Seven out of 28 patients (25%) reported sub-optimal appetite (self-reported as fair, poor or very poor) despite all being well nourished (100% SGA A). Using the VAS, ratings of hunger, but not desire to eat or fullness, were significantly (p<0.05) associated with a range of relevant clinical variables including age (r=-0.376), comorbidities (r=-0.380) nutritional status (PG-SGA score, r=-0.451), inflammatory markers (CRP r=-0.383; sICAM-1 r=-0.387) and seven domains of quality of life. Patients expressed a preference for the paper and pen method of administering VAS. None of the tools (appetite, MIS, PG-SGA, albumin or iron) showed an acceptable ability to detect patients who are inflamed. It is recommended that CRP should be tested more frequently as a matter of course rather than seeking alternative methods of measuring inflammation. 27 patients completed the 12 week intervention. 20 patients were considered adherent based on changes in % plasma EPA, which rose from 1.3 (0.94)% to 5.2 (1.1)%, p<0.001, in this group. The major barriers to adherence were forgetting to take the tablets as well as their size. At 12 weeks, inflammatory markers remained steady apart from the white cell count which decreased (7.6(2.5) vs 7.0(2.2) x109/L, p=0.058) and sVCAM-1 which increased (1685(654) vs 2249(925) ng/mL, p=0.001). Subjective appetite using VAS increased (51mm to 57mm, +12%) and there was a trend towards reduction in peptide YY (660(31) vs 600(30) pg/mL, p=0.078). There were some gender differences apparent, with the following adjusted change between baseline and week 12: CRP (males -3% vs females +17%, p=0.19), IL6 (males +17% vs females +48%, p=0.77), sICAM-1 (males -5% vs females +11%, p=0.07), sVCAM-1 (males +54% vs females +19%, p=0.08) and hunger ratings (males 20% vs females -5%, p=0.18). On balance, males experienced a maintainence or reduction in three inflammatory markers and an improvement in hunger ratings, and therefore appeared to have responded better to the intervention. Compared to those who didn’t adhere, adherent patients maintained weight (mean(SE) change: +0.5(1.6) vs - 0.8(1.2) kg, p=0.052) and fat-free mass (-0.1 (1.6) vs -1.8 (1.8) kg, p=0.045). There was no difference in change between the intervention and control phase for CRP, appetite, nutritional status or dietary intake. The thesis makes a significant contribution to the evidence base for understanding of PEW in dialysis patients. It has advanced knowledge of methods of assessing inflammation and appetite. Retrospective ratings of hunger on a VAS appear to be a valid method of assessing appetite although samples which include patients with very poor appetite are required to confirm this. Supplementation with fish oil appeared to improve subjective appetite and dampen the inflammatory response. The effectiveness of the intervention is influenced by gender and adherence. Males appear to be more responsive to the primary outcome variables than females, and the quality of response is improved with better adherence. These results provide evidence to support future interventions aimed at reducing the effects of PEW in dialysis patients.

CTA1-DD is an effective adjuvant for targeting anti-chlamydial immunity to the murine genital mucosa

Chlamydia trachomatis is a significant human pathogen with potentially severe disease sequelae in the genital tract, including infertility. A successful vaccine will need to effectively target immunity to the genital mucosa. Intranasal immunisation with cholera toxin (CT) can target immunity to the genital tract, but has the potential to cause neurological side effects. CTA1-DD is a non-toxic potent mucosal adjuvant which combines the enzymatic properties of CT, with a B cell targeting moiety. Here, we demonstrate that intranasal immunisation with CTA1-DD and chlamydial Major Outer Membrane Protein (MOMP) results in the induction of neutralising systemic and mucosal antibodies, and reduces the level of chlamydial shedding following intravaginal challenge with Chlamydia muridarum. Thus, CTA1-DD is an effective adjuvant for vaccine development against Chlamydia trachomatis, and possibly also a range of other genital pathogens.

Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.