Defining the role of phytoene synthase in carotenoid accumulation of high provitamin A bananas

Vitamin A deficiency (VAD) is a serious problem in developing countries, affecting approximately 127 million children of preschool age and 7.2 million pregnant women each year. However, this deficiency is readily treated and prevented through adequate nutrition. This can potentially be achieved through genetically engineered biofortification of staple food crops to enhance provitamin A (pVA) carotenoid content. Bananas are the fourth most important food crop with an annual production of 100 million tonnes and are widely consumed in areas affected by VAD. However, the fruit pVA content of most widely consumed banana cultivars is low (~ 0.2 to 0.5 ìg/g dry weight). This includes cultivars such as the East African highland banana (EAHB), the staple crop in countries such as Uganda, where annual banana consumption is approximately 250 kg per person. This fact, in addition to the agronomic properties of staple banana cultivars such as vegetative reproduction and continuous cropping, make bananas an ideal target for pVA enhancement through genetic engineering. Interestingly, there are banana varieties known with high fruit pVA content (up to 27.8 ìg/g dry weight), although they are not widely consumed due to factors such as cultural preference and availability. The genes involved in carotenoid accumulation during banana fruit ripening have not been well studied and an understanding of the molecular basis for the differential capacity of bananas to accumulate carotenoids may impact on the effective production of genetically engineered high pVA bananas. The production of phytoene by the enzyme phytoene synthase (PSY) has been shown to be an important rate limiting determinant of pVA accumulation in crop systems such as maize and rice. Manipulation of this gene in rice has been used successfully to produce Golden Rice, which exhibits higher seed endosperm pVA levels than wild type plants. Therefore, it was hypothesised that differences between high and low pVA accumulating bananas could be due either to differences in PSY enzyme activity or factors regulating the expression of the psy gene. Therefore, the aim of this thesis was to investigate the role of PSY in accumulation of pVA in banana fruit of representative high (Asupina) and low (Cavendish) pVA banana cultivars by comparing the nucleic acid and encoded amino acid sequences of the banana psy genes, in vivo enzyme activity of PSY in rice callus and expression of PSY through analysis of promoter activity and mRNA levels. Initially, partial sequences of the psy coding region from five banana cultivars were obtained using reverse transcriptase (RT)-PCR with degenerate primers designed to conserved amino acids in the coding region of available psy sequences from other plants. Based on phylogenetic analysis and comparison to maize psy sequences, it was found that in banana, psy occurs as a gene family of at least three members (psy1, psy2a and psy2b). Subsequent analysis of the complete coding regions of these genes from Asupina and Cavendish suggested that they were all capable of producing functional proteins due to high conservation in the catalytic domain. However, inability to obtain the complete mRNA sequences of Cavendish psy2a, and isolation of two non-functional Cavendish psy2a coding region variants, suggested that psy2a expression may be impaired in Cavendish. Sequence analysis indicated that these Cavendish psy2a coding region variants may have resulted from alternate splicing. Evidence of alternate splicing was also observed in one Asupina psy1 coding region variant, which was predicted to produce a functional PSY1 isoform. The complete mRNA sequence of the psy2b coding regions could not be isolated from either cultivar. Interestingly, psy1 was cloned predominantly from leaf while psy2 was obtained preferentially from fruit, suggesting some level of tissue-specific expression. The Asupina and Cavendish psy1 and psy2a coding regions were subsequently expressed in rice callus and the activity of the enzymes compared in vivo through visual observation and quantitative measurement of carotenoid accumulation. The maize B73 psy1 coding region was included as a positive control. After several weeks on selection, regenerating calli showed a range of colours from white to dark orange representing various levels of carotenoid accumulation. These results confirmed that the banana psy coding regions were all capable of producing functional enzymes. No statistically significant differences in levels of activity were observed between banana PSYs, suggesting that differences in PSY activity were not responsible for differences in the fruit pVA content of Asupina and Cavendish. The psy1 and psy2a promoter sequences were isolated from Asupina and Cavendish gDNA using a PCR-based genome walking strategy. Interestingly, three Cavendish psy2a promoter clones of different sizes, representing possible allelic variants, were identified while only single promoter sequences were obtained for the other Asupina and Cavendish psy genes. Bioinformatic analysis of these sequences identified motifs that were previously characterised in the Arabidopsis psy promoter. Notably, an ATCTA motif associated with basal expression in Arabidopsis was identified in all promoters with the exception of two of the Cavendish psy2a promoter clones (Cpsy2apr2 and Cpsy2apr3). G1 and G2 motifs, linked to light-regulated responses in Arabidopsis, appeared to be differentially distributed between psy1 and psy2a promoters. In the untranscribed regulatory regions, the G1 motifs were found only in psy1 promoters, while the G2 motifs were found only in psy2a. Interestingly, both ATCTA and G2 motifs were identified in the 5’ UTRs of Asupina and Cavendish psy1. Consistent with other monocot promoters, introns were present in the Asupina and Cavendish psy1 5’ UTRs, while none were observed in the psy2a 5’ UTRs. Promoters were cloned into expression constructs, driving the â-glucuronidase (GUS) reporter gene. Transient expression of the Asupina and Cavendish psy1 and psy2a promoters in both Cavendish embryogenic cells and Cavendish fruit demonstrated that all promoters were active, except Cpsy2apr2 and Cpsy2apr3. The functional Cavendish psy2a promoter (Cpsy2apr1) appeared to have activity similar to the Asupina psy2a promoter. The activities of the Asupina and Cavendish psy1 promoters were similar to each other, and comparable to those of the functional psy2a promoters. Semi-quantitative PCR analysis of Asupina and Cavendish psy1 and psy2a transcripts showed that psy2a levels were high in green fruit and decreased during ripening, reinforcing the hypothesis that fruit pVA levels were largely dependent on levels of psy2a expression. Additionally, semi-quantitative PCR using intron-spanning primers indicated that high levels of unprocessed psy2a and psy2b mRNA were present in the ripe fruit of Cavendish but not in Asupina. This raised the possibility that differences in intron processing may influence pVA accumulation in Asupina and Cavendish. In this study the role of PSY in banana pVA accumulation was analysed at a number of different levels. Both mRNA accumulation and promoter activity of psy genes studied were very similar between Asupina and Cavendish. However, in several experiments there was evidence of cryptic or alternate splicing that differed in Cavendish compared to Asupina, although these differences were not conclusively linked to the differences in fruit pVA accumulation between Asupina and Cavendish. Therefore, other carotenoid biosynthetic genes or regulatory mechanisms may be involved in determining pVA levels in these cultivars. This study has contributed to an increased understanding of the role of PSY in the production of pVA carotenoids in banana fruit, corroborating the importance of this enzyme in regulating carotenoid production. Ultimately, this work may serve to inform future research into pVA accumulation in important crop varieties such as the EAHB and the discovery of avenues to improve such crops through genetic modification.

Breaching the standardised assessment boundary : assessment for learning as a field of exchange

The introduction of the Australian curriculum, the use of standardised testing (e.g. NAPLAN) and the My School website are couched in a context of accountability. This circumstance has stimulated and in some cases renewed a range of boundaries in Australian Education. The consequences that arise from standardised testing have accentuated the boundaries produced by social reproduction in education which has led to an increase in the numbers of students disengaging from mainstream education and applying for enrolment at the Edmund Rice Education Australia Flexible Learning Centre Network (EREAFLCN). Boundaries are created for many young people who are denied access to credentials and certification as a result of being excluded from or in some way disengaging from standardised education and testing. Young people who participate at the EREAFLCN arrive with a variety of forms of cultural capital that are not valued in current education and employment fields. This is not to say that these young people’s different forms of cultural capital have no value, but rather that such funds of knowledge, repertoires and cultural capital are not valued by the majority of powerful agents in educational and employment fields. How then can the qualitative value of traditionally unorthodox - yet often intricate, ingenious, and astute - versions of cultural capital evident in the habitus of many young people be made to count, be recognised, be valuated? Can a process of educational assessment be a field of capital exchange and a space which breaches boundaries through a valuating process? This paper reports on the development of an innovative approach to assessment in an alternative education institution designed for the re-engagement of ‘at risk’ youth who have left formal schooling. A case study approach has been used to document the engagement of six young people, with an educational approach described as assessment for learning as a field of exchange across two sites in the EREAFLCN. In order to capture the broad range of students’ cultural and social capital, an electronic portfolio system (EPS) is under trial. The model draws on categories from sociological models of capital and reconceptualises the eportfolio as a sociocultural zone of learning and development. Results from the trial show a general tendency towards engagement with the EPS and potential for the attainment of socially valued cultural capital in the form of school credentials. In this way restrictive boundaries can be breached and a more equitable outcome achieved for many young Australians.

Towards general capabilities through technology : assessment for learning as a field of exchange

The introduction of the Australian curriculum, the use of standardised testing (e.g. NAPLAN) and the My School website have stimulated and in some cases renewed a range of boundaries for young people in Australian Education. Standardised testing has accentuated social reproduction in education with an increase in the numbers of students disengaging from mainstream education and applying for enrolment at the Edmund Rice Education Australia Flexible Learning Centre Network (EREAFLCN). Many young people are denied access to credentials and certification as they become excluded from standardised education and testing. The creativity and skills of marginalised youth are often evidence of general capabilities and yet do not appear to be recognised in mainstream educational institutions when standardised approaches are adopted. Young people who participate at the EREAFLCN arrive with a variety of forms of cultural capital, frequently utilising general capabilities, which are not able to be valued in current education and employment fields. This is not to say that these young people‟s different forms of cultural capital have no value, but rather that such funds of knowledge, repertoires and cultural capital are not valued by the majority of powerful agents in educational and employment fields. How then can the inherent value of traditionally unorthodox - yet often intricate, ingenious, and astute-versions of cultural capital evident in the habitus of many young people be made to count, be recognised, be valuated?Can a process of educational assessment be a field of capital exchange and a space which crosses boundaries through a valuating process? This paper reports on the development of an innovative approach to assessment in an alternative education institution designed for the re engagement of „at risk‟ youth who have left formal schooling. A case study approach has been used to document the engagement of six young people, with an educational approach described as assessment for learning as a field of exchange across two sites in the EREAFLCN. In order to capture the broad range of students‟ cultural and social capital, an electronic portfolio system (EPS) is under trial. The model draws on categories from sociological models of capital and reconceptualises the eportfolio as a sociocultural zone of learning and development. Results from the trial show a general tendency towards engagement with the EPS and potential for the attainment of socially valued cultural capital in the form of school credentials. In this way restrictive boundaries can be breached and a more equitable outcome achieved for many young Australians.

Engaging bodily with video in design

Video is commonly used as a method for recording embodied interaction for purposes of analysis and design and has been proposed as a useful ‘material’ for interaction designers to engage with. But video is not a straight forward reproduction of embodied activity – in themselves video recordings ‘flatten’ the space of embodied interaction, they impose a perspective on unfolding action, and remove the embodied spatial and social context within which embodied interaction unfolds. This does not mean that video is not a useful medium with which to engage as part of a process of investigating and designing for embodied interaction – but crucially, it requires that as people attempting to engage with video, designers own bodies and bodily understandings must be engaged with and brought into play. This paper describes and reflects upon our experiences of engaging with video in two different activities as part of a larger research project investigating the design of gestural interfaces for a dental surgery context.

Defining standards for modelling the biomechanics of the foot and ankle : a systematic review

The complex interaction of the bones of the foot has been explored in detail in recent years, which has led to the acknowledgement in the biomechanics community that the foot can no longer be considered as a single rigid segment. With the advance of motion analysis technology it has become possible to quantify the biomechanics of simplified units or segments that make up the foot. Advances in technology coupled with reducing hardware prices has resulted in the uptake of more advanced tools available for clinical gait analysis. The increased use of these techniques in clinical practice requires defined standards for modelling and reporting of foot and ankle kinematics. This systematic review aims to provide a critical appraisal of commonly used foot and ankle marker sets designed to assess kinematics and thus provide a theoretical background for the development of modelling standards.

The case for ICT work-integrated learning from graduates in the workplace

An online survey of recent ICT graduates in the workplace was carried out as part of a recent project funded by the Australian Learning and Teaching Council. The survey was concerned with the ICT curriculum in relation to workplace job requirements and university preparation for these requirements. The survey contained quantitative and qualitative components and findings from the former have been published (Koppi et al., 2009). This paper reports on a quantitative comparison of responses from graduates who had workplace experience and those who did not, and a qualitative analysis of text responses from all ICT graduates to open-ended questions concerning the curriculum and their perceived university preparation for the workplace. The overwhelming response from ICT graduates in the workplace was for more industry related learning. These industry relationships included industry involvement, workplace learning and business experience, up-to-date teaching and technologies, practical applications, and real-world activities. A closer relationship of academia and industry was strongly advocated by ICT graduates in the workplace.

'Online' geriatric assessment procedure for older adults referred for geriatric assessment during an acute care episode for consideration of reliability of triage decisions

Background Comprehensive geriatric assessment has been shown to improve patient outcomes, but the geriatricians who deliver it are in short-supply. A web-based method of comprehensive geriatric assessment has been developed with the potential to improve access to specialist geriatric expertise. The current study aims to test the reliability and safety of comprehensive geriatric assessment performed “online” in making geriatric triage decisions. It will also explore the accuracy of the procedure in identifying common geriatric syndromes, and its cost relative to conventional “live” consultations. Methods/Design The study population will consist of 270 acutely hospitalized patients referred for geriatric consultation at three sites. Paired assessments (live and online) will be conducted by independent, blinded geriatricians and the level of agreement examined. This will be compared with the level of agreement between two independent, blinded geriatricians each consulting with the patient in person (i.e. “live”). Agreement between the triage decision from live-live assessments and between the triage decision from live-online assessments will be calculated using kappa statistics. Agreement between the online and live detection of common geriatric syndromes will also be assessed using kappa statistics. Resource use data will be collected for online and live-live assessments to allow comparison between the two procedures. Discussion If the online approach is found to be less precise than live assessment, further analysis will seek to identify patient subgroups where disagreement is more likely. This may enable a protocol to be developed that avoids unsafe clinical decisions at a distance. Trial registration Trial registration number: ACTRN12611000936921

‘Show Me Your Wiki and I’ll Show you Mine’: Using Online Interactive Media to Improve Academic Writing and Research in a Public Health Under-Graduate Cohort

The number of Internet users in Australia has been steadily increasing, with over 10.9 million people currently subscribed to an internet provider (ABS, 2011). Over the past year, the most avid users of the Internet were 15 – 24 year olds, with approximately 95% accessing the internet on a regular basis (ABS, Social Trends, 2011). While the internet has been described as fundamental to higher education students, social and leisure internet tools are also increasingly being used by these students to generate and maintain their social and professional networks and interactions (Duffy & Bruns 2006). Rapid technological advancements have enabled greater and faster access to information for learning and education (Hemmi et al, 2009; Glassman and Kang, 2011). As such, we sought to integrate interactive, online social media into the assessment profile of a Public Health undergraduate cohort at the Queensland University of Technology (QUT). The aim of this exercise was to engage students to both develop and showcase their research on a range of complex, contemporary health issues within the online forum of Wikispaces (http://www.wikispaces.com/) for review and critique by their peers. We applied Bandura’s Social Learning Theory (SLT) to analyse the interactive processes from which students developed deeper and more sustained learning, and via which their overall academic writing standards were raised. This paper outlines the assessment task, and the students’ feedback on their learning outcomes in relation to the Attentional, Retentional, Motor Reproduction, and Motivational Processes outlined by Bandura in SLT. We conceptualise the findings in a theoretical model, and discuss the implications for this approach within the broader tertiary environment.

Assessment of function and clinical utility of alcohol and other drug web sites : an observational, qualitative study

Background The increasing popularity and use of the internet makes it an attractive option for providing health information and treatment, including alcohol/other drug use. There is limited research examining how people identify and access information about alcohol or other drug (AOD) use online, or how they assess the usefulness of the information presented. This study examined the strategies that individuals used to identify and navigate a range of AOD websites, along with the attitudes concerning presentation and content. Methods Members of the general community in Brisbane and Roma (Queensland, Australia) were invited to participate in a 30-minute search of the internet for sites related to AOD use, followed by a focus group discussion. Fifty one subjects participated in the study across nine focus groups. Results Participants spent a maximum of 6.5 minutes on any one website, and less if the user was under 25 years of age. Time spent was as little as 2 minutes if the website was not the first accessed. Participants recommended that AOD-related websites should have an engaging home or index page, which quickly and accurately portrayed the site’s objectives, and provided clear site navigation options. Website content should clearly match the title and description of the site that is used by internet search engines. Participants supported the development of a portal for AOD websites, suggesting that it would greatly facilitate access and navigation. Treatment programs delivered online were initially viewed with caution. This appeared to be due to limited understanding of what constituted online treatment, including its potential efficacy. Conclusions A range of recommendations arise from this study regarding the design and development of websites, particularly those related to AOD use. These include prudent use of text and information on any one webpage, the use of graphics and colours, and clear, uncluttered navigation options. Implications for future website development are discussed.

How Does Information Processing Speed Relate to the Attentional Blink?

Background When observers are asked to identify two targets in rapid sequence, they often suffer profound performance deficits for the second target, even when the spatial location of the targets is known. This attentional blink (AB) is usually attributed to the time required to process a previous target, implying that a link should exist between individual differences in information processing speed and the AB. Methodology/Principal Findings The present work investigated this question by examining the relationship between a rapid automatized naming task typically used to assess information-processing speed and the magnitude of the AB. The results indicated that faster processing actually resulted in a greater AB, but only when targets were presented amongst high similarity distractors. When target-distractor similarity was minimal, processing speed was unrelated to the AB. Conclusions/Significance Our findings indicate that information-processing speed is unrelated to target processing efficiency per se, but rather to individual differences in observers' ability to suppress distractors. This is consistent with evidence that individuals who are able to avoid distraction are more efficient at deploying temporal attention, but argues against a direct link between general processing speed and efficient information selection.

Change Point Estimation in Monitoring Survival Time

Precise identification of the time when a change in a hospital outcome has occurred enables clinical experts to search for a potential special cause more effectively. In this paper, we develop change point estimation methods for survival time of a clinical procedure in the presence of patient mix in a Bayesian framework. We apply Bayesian hierarchical models to formulate the change point where there exists a step change in the mean survival time of patients who underwent cardiac surgery. The data are right censored since the monitoring is conducted over a limited follow-up period. We capture the effect of risk factors prior to the surgery using a Weibull accelerated failure time regression model. Markov Chain Monte Carlo is used to obtain posterior distributions of the change point parameters including location and magnitude of changes and also corresponding probabilistic intervals and inferences. The performance of the Bayesian estimator is investigated through simulations and the result shows that precise estimates can be obtained when they are used in conjunction with the risk-adjusted survival time CUSUM control charts for different magnitude scenarios. The proposed estimator shows a better performance where a longer follow-up period, censoring time, is applied. In comparison with the alternative built-in CUSUM estimator, more accurate and precise estimates are obtained by the Bayesian estimator. These superiorities are enhanced when probability quantification, flexibility and generalizability of the Bayesian change point detection model are also considered.

Sri Lanka in global medical research: a scientific analysis of the Sri Lankan research output during 2000-2009

Background Scientific research is an essential component in guiding improvements in health systems. There are no studies examining the Sri Lankan medical research output at international level. The present study evaluated the Sri Lankan research performance in medicine as reflected by the research publications output between years 2000-2009. Methods This study was based on Sri Lankan medical research publication data, retrieved from the SciVerse Scopus® from January 2000 to December 2009. The process of article selection was as follows: Affiliation - 'Sri Lanka' or 'Ceylon', Publication year - 'January 2000 to December 2009' and Subject area - 'Life and Health Sciences'. The articles identified were classified according to disease, medical speciality, institutions, major international collaborators, authors and journals. Results Sri Lanka's cumulative medical publications output between years 2000-2009 was 1,740 articles published in 160 different journals. The average annual publication growth rate was 9.1%. Majority of the articles were published in 'International' (n = 950, 54.6%) journals. Most articles were descriptive studies (n = 611, 35.1%), letters (n-345, 19.8%) and case reports (n = 311, 17.9%). The articles were authored by 148 different Sri Lankan authors from 146 different institutions. The three most prolific local institutions were Universities of; Colombo (n = 547), Kelaniya (n = 246) and Peradeniya (n = 222). Eighty four countries were found to have published collaborative papers with Sri Lankan authors during the last decade. UK was the largest collaborating partner (n = 263, 15.1%). Malaria (n = 75), Diabetes Mellitus (n = 55), Dengue (n = 53), Accidental injuries (n = 42) and Lymphatic filariasis (n = 40) were the major diseases studied. The 1,740 publications were cited 9,708 times, with an average citation of 5.6 per paper. The most cited paper had 203 citations, while there were 597 publications with no citations. The Sri Lankan authors' contribution to the global medical research output during the last decade was only 0.086%. Conclusion The Sri Lankan medical research output during the last decade is only a small fraction of the global research output. There it is a necessity to setup an enabling environment for research, with a proper vision, support, funds and training. In addition, collaborations across the region need to be strengthened to face common regional health challenges. Keywords: Sri Lanka, Medical research, Publication, Analysis