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.

Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research

Cell proliferation is a critical and frequently studied feature of molecular biology in cancer research. Therefore, various assays are available using different strategies to measure cell proliferation. Metabolic assays such as AlamarBlue, WST-1, and MTT, which were originally developed to determine cell toxicity, are being used to assess cell numbers. Additionally, proliferative activity can be determined by quantification of DNA content using fluorophores, such as CyQuant and PicoGreen. Referring to data published in high ranking cancer journals, 945 publications applied these assays over the past 14 years to examine the proliferative behaviour of diverse cell types. Within this study, mainly metabolic assays were used to quantify changes in cell growth yet these assays may not accurately reflect cellular proliferation rates due to a miscorrelation of metabolic activity and cell number. Testing this hypothesis, we compared metabolic activity of different cell types, human cancer cells and primary cells, over a time period of 4 days using AlamarBlue and fluorometric assays CyQuant and PicoGreen to determine their DNA content. Our results show certain discrepancies in terms of over-estimation of cell proliferation with respect to the metabolic assay in comparison to DNA binding fluorophores.

What works elsewhere? A review of physical activity intervention studies

Obesity and type 2 diabetes mellitus (T2D) have reached epidemic proportions in many parts of the world with numbers projected to rise dramatically in coming decades (Wang and Lobstein, 2006; Zaninotto et al., 2006). In Australia, and consistent with much of the developed world, the problem has been described as a ‘juggernaut’ that is out of control (Zimmet and James, 2007). Unfortunately the burgeoning problem of non-communicable diseases, including obesity and T2D, is also impacting developing nations as populations are undergoing a nutrition transition (Caballero, 2005). The increased prevalence of overweight and obesity in children, adolescents and adults in both the developed and developing world is consistent with reductions in all forms of physical activity (Brownson et al., 2005). This brief paper provides an overview of the importance of physical activity and an outline of physical activity intervention studies with particular reference to the growing years. As many interventions studies involving physical activity have been undertaken in the context of childhood obesity prevention (Lobstein et al., 2004), and an increasing proportion of the childhood population is overweight or obese, this is a major focus of discussion.

Parental physical activity : exploring the role of social support

Objectives: To explore the influence of social support on parental physical activity (PA). Methods: Forty parents (21 mothers, 19 fathers) participated in semistructured individual or group interviews. Data were analyzed using thematic content analysis.---------- Results: Instrumental (eg, providing child care, taking over chores), emotional (eg, encouragement, companionship), and informational support (eg, ideas and advice) as well as reciprocal support (eg, giving as well as receiving support) and autonomy support (eg, respecting one’s choices) are important for parents’ PA behavior. However, having support for being active is not straightforward in that many parents discussed issues that inhibited the facilitative nature of social support for PA performance (eg, guilt in getting help). Conclusions: Results highlight the complex nature of social support in facilitating parental PA.

Website physical activity interventions : preferences of potential users

Information and communication technologies (particularly websites and e-mail) have the potential to deliver health behavior change programs to large numbers of adults at low cost. Controlled trials using these new media to promote physical activity have produced mixed results. User-centered development methods can assist in understanding the preferences of potential participants for website functions and content, and may lead to more effective programs. Eight focus group discussions were conducted with 40 adults after they had accessed a previously trialed physical activity website. The discussions were audio taped, transcribed and interpreted using a themed analysis method. Four key themes emerged: structure, interactivity, environmental context and content. Preferences were expressed for websites that include simple interactive features, together with information on local community activity opportunities. Particular suggestions included online community notice boards, personalized progress charts, e-mail access to expert advice and access to information on specific local physical activity facilities and services. Website physical activity interventions could usefully include personally relevant interactive and environmentally focused features and services identified through a user-centered development process.

Neighborhood disadvantage and physical activity : baseline results from the HABITAT multilevel longitudinal study

PURPOSE: To examine the association between neighborhood disadvantage and physical activity (PA). ---------- METHODS: We use data from the HABITAT multilevel longitudinal study of PA among mid-aged (40-65 years) men and women (n=11, 037, 68.5% response rate) living in 200 neighborhoods in Brisbane, Australia. PA was measured using three questions from the Active Australia Survey (general walking, moderate, and vigorous activity), one indicator of total activity, and two questions about walking and cycling for transport. The PA measures were operationalized using multiple categories based on time and estimated energy expenditure that were interpretable with reference to the latest PA recommendations. The association between neighborhood disadvantage and PA was examined using multilevel multinomial logistic regression and Markov Chain Monte Carlo simulation. The contribution of neighborhood disadvantage to between-neighborhood variation in PA was assessed using the 80% interval odds ratio. ---------- RESULTS: After adjustment for sex, age, living arrangement, education, occupation, and household income, reported participation in all measures and levels of PA varied significantly across Brisbane’s neighborhoods, and neighborhood disadvantage accounted for some of this variation. Residents of advantaged neighborhoods reported significantly higher levels of total activity, general walking, moderate, and vigorous activity; however, they were less likely to walk for transport. There was no statistically significant association between neighborhood disadvantage and cycling for transport. In terms of total PA, residents of advantaged neighborhoods were more likely to exceed PA recommendations. ---------- CONCLUSIONS: Neighborhoods may exert a contextual effect on residents’ likelihood of participating in PA. The greater propensity of residents in advantaged neighborhoods to do high levels of total PA may contribute to lower rates of cardiovascular disease and obesity in these areas

Electromyographic activity in lower limb muscles is temporally associated with the slow phase of oxygen uptake during cycling

Although the "slow" phase of pulmonary oxygen uptake (Vo2) appears to represent energetic processes in contracting muscle, electromyographic evidence tends not to support this. The present study assessed normalized integrated electromyographic (NIEMG) activity in eight muscles that act about the hip, knee and ankle during 8 min of moderate (ventilatory threshold) cycling in six male cyclists. (Vo2) was measured breath by breath during four repeated trials at each of the two intensities. Moderate and very heavy exercise followed a 4-min period of light exercise (50 W). During moderate exercise the slow (Vo2) phase was absent and NIEMG in all muscles did not increase after the first minute of exercise. During very heavy exercise, the slow phase emerged (time delay=58 ± 16 s) and increased progressively (time constant=120 ± 35 s) to an amplitude (0.83 ± 0.16 L/min) that was approximately 21% of the total (Vo2) response. This slow (Vo2) phase coincided with a significant increase in NIEMG in most muscles, and differences in NIEMG activities between the two intensities revealed "slow" muscle activation profiles that differed between muscles in terms of the onset, amplitude and shape of these profiles. This supports the hypothesis that the slow (Vo2) phase is a function of these different slow muscle activation profiles.

Paediatric obesity, physical activity and the musculoskeletal system

The current epidemic of paediatric obesity is consistent with a myriad of health-related comorbid conditions. Despite the higher prevalence of orthopaedic conditions in overweight children, a paucity of published research has considered the influence of these conditions on the ability to undertake physical activity. As physical activity participation is directly related to improvements in physical fitness, skeletal health and metabolic conditions, higher levels of physical activity are encouraged, and exercise is commonly prescribed in the treatment and management of childhood obesity. However, research has not correlated orthopaedic conditions, including the increased joint pain and discomfort that is commonly reported by overweight children, with decreases in physical activity. Research has confirmed that overweight children typically display a slower, more tentative walking pattern with increased forces to the hip, knee and ankle during 'normal' gait. This research, combined with anthropometric data indicating a higher prevalence of musculoskeletal malalignment in overweight children, suggests that such individuals are poorly equipped to undertake certain forms of physical activity. Concomitant increases in obesity and decreases in physical activity level strongly support the need to better understand the musculoskeletal factors associated with the performance of motor tasks by overweight and obese children.

Incidental walking activity is sufficient to induce time-dependent conditioning of the Achilles tendon

The Achilles tendon has been seen to exhibit time-dependent conditioning when isometric muscle actions were of a prolonged duration, compared to those involved in dynamic activities, such as walking. Since, the effect of short duration muscle activation associated with dynamic activities is yet to be established, the present study aimed to investigate the effect of incidental walking activity on Achilles tendon diametral strain. Eleven healthy male participants refrained from physical activity in excess of the walking required to carry out necessary daily tasks and wore an activity monitor during the 24 h study period. Achilles tendon diametral strain, 2 cm proximal to the calcaneal insertion, was determined from sagittal sonograms. Baseline sonographic examinations were conducted at ∼08:00 h followed by replicate examinations at 12 and 24 h. Walking activity was measured as either present (1) or absent (0) and a linear weighting function was applied to account for the proximity of walking activity to tendon examination time. Over the course of the day the median (min, max) Achilles tendon diametral strain was −11.4 (4.5, −25.4)%. A statistically significant relationship was evident between walking activity and diametral strain (P < 0.01) and this relationship improved when walking activity was temporally weighted (AIC 131 to 126). The results demonstrate that the short yet repetitive loads generated during activities of daily living, such as walking, are sufficient to induce appreciable time-dependant conditioning of the Achilles tendon. Implications arise for the in vivo measurement of Achilles tendon properties and the rehabilitation of tendinopathy.

Elective foot and ankle surgery : activity and perioperative complications in Queensland public hospitals, Australia

Objective: To conduct an audit of elective foot and ankle surgery in Queensland public hospitals and to compare the frequency of these procedures performed to other states and territories of Australia. ---------- Methods: ICD-10-AM data was used to extract elective foot and ankle procedures from the Data Services Unit of Queensland Health, and the Australian Institute of Health and Welfare between the years of 2000 and 2004. ---------- Results During the 4-year audit period 3846 primary procedures were performed during the 4-year period with a complication rate of 2.2% during the hospital admission period. Mean length of stay was 1.7 days. Post-operative infection rates were 0.26%. With the exception of Tasmania and the Northern Territory, Queensland performs the least number of elective foot and ankle procedures per capita per year in Australia. ---------- Conclusions This is the first reported audit of elective foot and ankle surgery for Queensland public hospitals. Complication rates cannot be directly compared to the literature as this data could only capture complications within hospital admission period. Fewer elective foot and ankle procedures were performed in Queensland public hospitals compared to all other mainland states of Australia during the data collection period.

Understanding parental physical activity : meanings, habits, and social role influence

Objectives: The research aimed to explore parents’ understandings of physical activity (PA), patterns of PA-related behaviour, and how constructions of social role expectations might influence their PA behaviour. Design and Method: Using a qualitative descriptive design and adopting a social constructionism approach to broaden interpretations of parents’ understandings, 40 adults (21 mothers, 19 fathers; aged 23 to 49 years) living in South East Queensland, Australia participated in semi-structured individual and group interviews. The interviews were analysed using thematic analysis. Results: Parents had clear understandings of what constitutes PA and engaged in various activities which were integrated with or independent of the children. Being active with children, however, was not always constructed favourably in which many parents described the difficulties of being active with their children. All individuals experienced changes in their PA behaviours after having children. For most, a decline in PA level, intensity, and structure was experienced; however, some did experience parenthood as a time to be active. A level of acceptance for the lack of activity performed was also expressed as were feelings of resentment and envy for those who maintained previous activity habits. Parenting and partner roles were considered most influential on PA-related behaviour and were constructed in ways that had both positive and negative influences on activity performance. Parents, however, were empowered to construct strategies to resolve conflicts between social role performance and being active. Conclusion: Results show that parents experience unique difficulties that intervention work should consider when designing programs aimed at increasing parental PA.