Towards the biofortification of banana fruit for enhanced micronutrient content

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major public health problems with between 15-32% of children under 5 years of age showing VAD and 73% being anaemic. This is largely due to the fact that the staple food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading to dietary deficiencies. Although worldwide progress has been made to control VAD and IDA through supplementation, food fortification and diet diversification, their long term sustainability and impact in developing countries such as Uganda is limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer acceptable banana varieties with significantly increased levels of pro-vitamin A and iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high â-carotene banana (F’ei cv Asupina). In addition, sequences of banana ferritin, an iron storage protein, have been isolated from Cavendish banana. The aim of the research described in this thesis was to evaluate the function of these genes to assess their suitability for the biofortification of banana fruit. In addition, a range of banana-derived promoters were characterised to determine their suitability for controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice was used as the model crop to investigate the functionality of the banana-derived APsy2a and ferritin genes. Using Agrobacterium-mediated transformation, rice callus was transformed with APsy2a +/- the bacterial-derived carotene desaturase gene (CrtI) each under the control of the constitutive maize poly-ubiquitin promoter (ZmUbi) or seed-specific rice glutelin1 (Gt1) promoter. The maize phytoene synthase (ZmPsy1) gene was included as a control. On selective media, with the exception of ZmUbi-CrtI-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of â-carotene was demonstrated using Raman spectroscopy. Although the regeneration of plants from yellow-orange callus was difficult, 16 transgenic plants were obtained and characterised from callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds developed a yellow-orange coloured callus which was found to contain levels of â-carotene ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice callus. Using the seed-specific Gt1 promoter, 38 transgenic rice plants were generated from APsy2a-CrtI-transformed callus while 32 plants were regenerated from ZmPsy1-CrtI-transformed callus. However, when analysed for presence of transgene by PCR, all transgenic plants contained the APsy2a, ZmPsy1 or CrtI transgene, with none of the plants found to be co-transformed. Using Raman spectroscopy, no â-carotene was detected in-situ in representative T1 seeds. To investigate the potential of the banana-derived ferritin gene (BanFer1) to enhance iron content, rice callus was transformed with constitutively expressed BanFer1 using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11 callus lines independently transformed with BanFer1 and SoyFer, respectively, were multiplied and transgene expression was verified by RT-PCR. Pearl’s Prussian blue staining for in-situ detection of ferric iron showed a stronger blue colour in rice callus transformed with BanFer1 compared to SoyFer. Using flame atomic absorption spectrometry, the highest mean amount of iron quantified in callus transformed with BanFer1 was 30-fold while that obtained using the SoyFer was 14-fold higher than the controls. In addition, ~78% of BanFer1-transgenic callus lines and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than the non-transformed controls. Since the genes used for enhancing micronutrient content need to be expressed in banana fruit, the activity of a range of banana-derived, potentially fruit-active promoters in banana was investigated. Using uidA (GUS) as a reporter gene, the function of the Expansin1 (MaExp1), Expansin1 containing the rice actin intron (MaExp1a), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO), Metallothionein (MaMT2a) and phytoene synthase (APsy2a) promoters were transiently analysed in intact banana fruit using two transformation methods, particle bombardment and Agrobacterium-mediated infiltration (agro-infiltration). Although a considerable amount of variation in promoter activity was observed both within and between experiments, similar trends were obtained using both transformation methods. The MaExp1 and MaExp1a directed high levels of GUS expression in banana fruit which were comparable to those observed from the ZmUbi and Banana bunchy top virus-derived BT4 promoters that were included as positive controls. Lower levels of promoter activity were obtained in both methods using the MaACO and MaExt promoters while the MaExp4, MaACS, and APsy2a promoters directed the lowest GUS activity in banana fruit. An attempt was subsequently made to use agro-infiltration to assess the expression of pVA biosynthesis genes in banana fruit by infiltrating fruit with constructs in which the ZmUbi promoter controlled the expression of APsy2a +/- CrtI, and with the maize phytoene synthase gene (ZmPsy1) included as a control. Unfortunately, the large amount of variation and inconsistency observed within and between experiments precluded any meaningful conclusions to be drawn. The final component of this research was to assess the level of promoter activity and specificity in non-target tissue. These analyses were done on leaves obtained from glasshouse-grown banana plants stably transformed with MaExp1, MaACO, APsy2a, BT4 and ZmUbi promoters driving the expression of the GUS gene in addition to leaves from a selection of the same transgenic plants which were growing in a field trial in North Queensland. The results from both histochemical and fluorometric GUS assays showed that the MaExp1 and MaACO promoters directed very low GUS activities in leaves of stably transformed banana plants compared to the constitutive ZmUbi and BT4 promoters. In summary, the results from this research provide evidence that the banana phytoene synthase gene (APsy2a) and the banana ferritin gene (BanFer1) are functional, since the constitutive over-expression of each of these transgenes led to increased levels of pVA carotenoids (for APsy2a) and iron content (for BanFer1) in transgenic rice callus. Further work is now required to determine the functionality of these genes in stably-transformed banana fruit. This research also demonstrated that the MaExp1 and MaACO promoters are fruit-active but have low activity in non-target tissue (leaves), characteristics that make them potentially useful for the biofortification of banana fruit. Ultimately, however, analysis of fruit from field-grown transgenic plants will be required to fully evaluate the suitability of pVA biosynthesis genes and the fruit-active promoters for fruit biofortification.

Prevalence of malnutrition in Parkinson's disease : a systematic review

Parkinson's disease (PD) patients may be at higher risk of malnutrition because of the symptoms associated with the disease and the side effects of the medication used to manage it. A decline in nutritional status is associated with many adverse outcomes related to health and quality of life. It is not clear, however, to what extent this population is currently affected by malnutrition. The objective of this review was to systematically assess the methodology and outcomes of studies reporting the prevalence of malnutrition in PD patients. Studies that attempted to classify participants with PD into nutritional risk and/or malnutrition categories using body mass index, weight change, anthropometric measures, and nutritional screening and assessment scores were included. The prevalence of malnutrition ranged from 0% to 24% in PD patients, while 3–60% of PD patients were reported to be at risk of malnutrition. There was a large degree of variation among studies in the methods chosen, the definition of malnutrition using those methods, and the detail in which the methodological protocols were reported. The true extent of malnutrition in the PD population has yet to be accurately quantified. It is important, however, to screen for malnutrition at the time of PD diagnosis.

Contribution of take-out food consumption to socioeconomic differences in fruit and vegetable intake : a mediation analysis

Lower fruit and vegetable intake among socioeconomically disadvantaged groups has been well documented, and may be a consequence of a higher consumption of take-out foods. This study examined whether, and to what extent, take-out food consumption mediated (explained) the association between socioeconomic position and fruit and vegetable intake. A cross-sectional postal survey was conducted among 1500 randomly selected adults aged 25–64 years in Brisbane, Australia in 2009 (response rate = 63.7%, N = 903). A food frequency questionnaire assessed usual daily servings of fruits and vegetables (0 to 6), overall take-out consumption (times/week) and the consumption of 22 specific take-out items (never to ≥once/day). These specific take-out items were grouped into “less healthy” and “healthy” choices and indices were created for each type of choice (0 to 100). Socioeconomic position was ascertained by education. The analyses were performed using linear regression, and a bootstrap re-sampling approach estimated the statistical significance of the mediated effects. Mean daily serves of fruits and vegetables was 1.89 (SD 1.05) and 2.47 (SD 1.12) respectively. The least educated group were more likely to consume fewer serves of fruit (B= –0.39, p<0.001) and vegetables (B= –0.43, p<0.001) compared with the highest educated. The consumption of “less healthy” take-out food partly explained (mediated) education differences in fruit and vegetable intake; however, no mediating effects were observed for overall and “healthy” take-out consumption. Regular consumption of “less healthy” take-out items may contribute to socioeconomic differences in fruit and vegetable intake, possibly by displacing these foods.

The Metapopulation Dynamics of an Infectious Disease: Tuberculosis in Possums

An SEI metapopulation model is developed for the spread of an infectious agent by migration. The model portrays two age classes on a number of patches connected by migration routes which are used as host animals mature. A feature of this model is that the basic reproduction ratio may be computed directly, using a scheme that separates topography, demography, and epidemiology. We also provide formulas for individual patch basic reproduction numbers and discuss their connection with the basic reproduction ratio for the system. The model is applied to the problem of spatial spread of bovine tuberculosis in a possum population. The temporal dynamics of infection are investigated for some generic networks of migration links, and the basic reproduction ratio is computed—its value is not greatly different from that for a homogeneous model. Three scenarios are considered for the control of bovine tuberculosis in possums where the spatial aspect is shown to be crucial for the design of disease management operations

Falls in Parkinson’s disease : evidence for altered stepping strategies on compliant surfaces

Background: Real-world environments comprise surfaces of different textures, densities and gradients, which can threaten postural stability and increase falls risk. However, there has been limited research that has examined how walking on compliant surfaces influences gait and postural stability in older people and PD patients. Methods: PD patients (n = 49) and age-matched controls (n = 32) were assessed using three dimensional motion analysis during self-paced walking on both firm and foam walkways. Falls were recorded prospectively over 12 months using daily falls calendars. Results: Walking on a foam surface influenced the temporospatial characteristics for all groups, but PD fallers adopted very different joint kinematics compared with controls. PD fallers also demonstrated reduced toe clearance and had increased mediolateral head motion(relative to walking velocity) compared with control participants. Conclusions: Postural control deficits in PD fallers may impair their capacity to attenuate surface-related perturbations and control head motion. The risk of falling for PD patients may be increased on less stable surfaces.