Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Public health and clinical dilemmas resulting from imprecise vitamin D tests

Objective: To determine whether there are clinical and public health dilemmas resulting from the reproducibility of routine vitamin D assays. Methods: Blinded agreement studies were conducted in eight clinical laboratories using two commonly used assays to measure serum 25-hydroxyvitamin D (25(OH)D) levels in Australasia and Canada (DiaSorin Radioimmunoassay (RIA) and DiaSorin LIAISON® one). Results: Only one laboratory measured 25(OH)D with excellent precision. Replicate 25(OH)D measurements varied by up to 97% and 15% of paired results differed by more than 50%. Thirteen percent of subjects received one result indicating insufficiency [25-50 nmol/l] and another suggesting adequacy [>50 nmol/l]). Agreement ranged from poor to excellent for laboratories using the manual RIA, while the precision of the semi-automated Liaison® system was consistently poor. Conclusions: Recent interest in the relevance of vitamin D to human health has increased demand for 25(OH)D testing and associated costs. Our results suggest clinicians and public health authorities are making decisions about treatment or changes to public health policy based on imprecise data. Clinicians, researchers and policy makers should be made aware of the imprecision of current 25(OH)D testing so that they exercise caution when using these assays for clinical practice, and when interpreting the findings of epidemiological studies based on vitamin D levels measured using these assays. Development of a rapid, reproducible, accurate and robust assay should be a priority due to interest in populationbased screening programs and research to inform public health policy about the amount of sun exposure required for human health. In the interim, 25(OH)D results should routinely include a statement of measurement uncertainty.

Rejoinder to James Hamilton

Hamilton (2001) makes a number of comments on our paper (Harding and Pagan, 2002b). The objectives of this rejoinder are, firstly, to note the areas in which we agree; secondly, to define with greater clarity the areas in which we disagree; and, thirdly, to point to other papers, including a longer version of this response, where we have dealt with some of the issues that he raises. The core of our debate with him is whether one should use an algorithm with a specified set of rules for determining the turning points in economic activity or whether one should use a parametric model that features latent states. Hamilton begins his criticism by stating that there is a philosophical distinction between the two methods for dating cycles and concludes that the method we use “leaves vague and intuitive exactly what this algorithm is intended to measure”. Nothing is further from the truth. When seeking ways to decide on whether a turning point has occurred it is always useful to ask the question, what is a recession? Common usage suggests that it is a decline in the level of economic activity that lasts for some time. For this reason it has become standard to describe a recession as a decline in GDP that lasts for more than two quarters. Finding periods in which quarterly GDP declined for two periods is exactly what our approach does. What is vague about this?

The use of visual analogue scales to assess motivation to eat in human subjects: a review of their reliability and validity with an evaluation of new hand-held computerized stsytems for temporal tracking of appetite ratings.

This present paper reviews the reliability and validity of visual analogue scales (VAS) in terms of (1) their ability to predict feeding behaviour, (2) their sensitivity to experimental manipulations, and (3) their reproducibility. VAS correlate with, but do not reliably predict, energy intake to the extent that they could be used as a proxy of energy intake. They do predict meal initiation in subjects eating their normal diets in their normal environment. Under laboratory conditions, subjectively rated motivation to eat using VAS is sensitive to experimental manipulations and has been found to be reproducible in relation to those experimental regimens. Other work has found them not to be reproducible in relation to repeated protocols. On balance, it would appear, in as much as it is possible to quantify, that VAS exhibit a good degree of within-subject reliability and validity in that they predict with reasonable certainty, meal initiation and amount eaten, and are sensitive to experimental manipulations. This reliability and validity appears more pronounced under the controlled (but more arti®cial) conditions of the laboratory where the signal : noise ratio in experiments appears to be elevated relative to real life. It appears that VAS are best used in within-subject, repeated-measures designs where the effect of different treatments can be compared under similar circumstances. They are best used in conjunction with other measures (e.g. feeding behaviour, changes in plasma metabolites) rather than as proxies for these variables. New hand-held electronic appetite rating systems (EARS) have been developed to increase reliability of data capture and decrease investigator workload. Recent studies have compared these with traditional pen and paper (P&P) VAS. The EARS have been found to be sensitive to experimental manipulations and reproducible relative to P&P. However, subjects appear to exhibit a signi®cantly more constrained use of the scale when using the EARS relative to the P&P. For this reason it is recommended that the two techniques are not used interchangeably

Characterising the homeostatic and hedonic markers of the susceptible phenotype

Over the years, approaches to obesity prevention and treatment have gone from focusing on genetic and other biological factors to exploring a diversity of diets and individual behavior modification interventions anchored primarily in the power of the mind, to the recent shift focusing on societal interventions to design ";temptation-proof"; physical, social, and economic environments. In spite of repeated calls to action, including those of the World Health Organization (WHO), the pandemic continues to progress. WHO recently projected that if the current lifestyle trend in young and adult populations around the world persist, by 2012 in countries like the USA, health care costs may amount to as much as 17.7% of the GDP. Most importantly, in large part due to the problems of obesity, those children may be the first generation ever to have a shorter life expectancy than that of their parents. This work presents the most current research and proposals for addressing the pandemic. Past studies have focused primarly on either genetic or behavioral causes for obesity, however today's research indicates that a strongly integrated program is the best prospect for success in overcoming obesity. Furthermore, focus on the role of society in establishing an affordable, accessible and sustainable program for implementing these lifestyle changes is vital, particularly for those in economically challenged situations, who are ultimately at the highest risk for obesity. Using studies from both neuroscience and behavioral science to present a comprehensive overview of the challenges and possible solutions, The brain-to-society approach to obesity prevention focuses on what is needed in order to sustain a healthy, pleasurable and affordable lifestyle.

Identifying the presence of psychopathy in the community

Aim: Researchers have suggested that approximately 1% of individuals with psychopathic tendencies can successfully function within the community, although there has been a lack of research to support this claim. The current study aimed to identify individuals with psychopathic tendencies within a community sample and furthermore the socio-demographic correlates of this community integrated psychopath (e.g. relationship stability, substance use, and employment status). Procedure: 300 participants completed the Self-Reported Psychopathy scale – version 3 which contains four core psychopathy subfactors: (a) Interpersonal Manipulation, (b) Callous Affect, (c) Erratic Lifestyle and (d) Criminal Tendencies as well as the Paulhus Deception Scales to explore the effect of impression management and self-deception on the identification of psychopathy. Findings: Results indicated that at least 1% of the current community displayed characteristics consistent with psychopathic tendencies. A series of bivariate and multivariate statistical analyses were conducted which indicated that gender, age and alcohol misuse were predictive of psychopathy scores for this sample. More specifically, younger males who tend to misuse alcohol were found to be most likely to have psychopathic tendencies. Interestingly, impression management and self-deception was not associated with such tendencies. Discussion: The results provide some support for the assertion that individuals with psychopathic tendencies can be identified within the community (regardless of impression management techniques) and that such tendencies are associated with specific socio-demographic characteristics.

Highly efficient, stoichiometric radical exchange reactions using isoindoline profluorescent nitroxides

Exchange reactions between the isoindoline profluorescent nitroxide 1,1,3,3-tetramethyldibenzo[e,g]isoindolin-2-yloxyl (TMDBIO) and a TEMPO capped polystyrene were carried out. High conversions to the desired products were achieved using only stoichiometric ratios of nitroxide relative to polymer. The scope of this study was expanded by exploiting a di-nitroxide 9,10-bis(5-[1,1,3,3-tetramethylisoindolin-2-yloxy])anthracene (BTMIOA) as a connector between two polymer chains forming PS–nitroxide–PS systems.

Improving road safety through deterrence-based initiatives : a review of research

The efficacy of road safety countermeasures to deter motorists from engaging in illegal behaviours is extremely important when considering the personal and economic impact of road accidents on the community. Within many countries, deterrence theory has remained a cornerstone to criminology and criminal justice policy, particularly within the field of road safety, as policy makers and enforcement agencies attempt to increase perceptions regarding the certainty, severity and swiftness of sanctions for those who engage in illegal motoring behaviours. Using the Australian experience (particularly the tremendous amount of research into drink driving), the current paper reviews the principles underpinning deterrence theory, the utilisation of the approach within some contemporary road safety initiatives (e.g., Random Breath Testing) as well as highlights some methods to enhance a deterrent effect. The paper also provides direction for future deterrence-based research, in particular, considering the powerful impact of non-legal sanctions, punishment avoidance as well as creating culturally embedded behavioural change.

James Street market, Brisbane : a subtropical retail case study

Climate change mitigation is driving demand for energy-efficient and environmentally conscious commercial buildings in Australia. In the Australian subtropics, high rainfall, warm weather and humidity present unique challenges and opportunities for the architects tasked with designing eco-sensitive projects. The case of the James Street Market in Brisbane’s Fortitude Valley shows that climate-responsive design is an effective approach for reducing the environmental impact of commercial developments. The James Street Market combines climate-responsiveness, environmentally sensitive design strategies and smart planning to create a more sustainable retail precinct. This paper details the design strategies featured in the James Street Market, the project that kicked off a renaissance in climate-responsive commercial building design in Brisbane.

Encyclopedia contributions: entries C-G in Oxford Companion to Australian Gardens

Caulfield, Harold William; p.131 Cowan, Alexander; p.164 Cowley, Ebenezer; p.164 East Talgai Station; p.193 Eaves, S.H.; p.193-194 Edgar, J.S.; p.196 Everist, Selwyn; p.206 Experimental Farms and Gardens; pp.207-208 Government Houses - Queensland; pp.267-268

Fusing curricula : science, technology and ICT

Curriculum demands continue to increase on school education systems with teachers at the forefront of implementing syllabus requirements. Education is reported frequently as a solution to most societal problems and, as a result of the world’s information explosion, teachers are expected to cover more and more within teaching programs. How can teachers combine subjects in order to capitalise on the competing educational agendas within school timeframes? Fusing curricula requires the bonding of standards from two or more syllabuses. Both technology and ICT complement the learning of science. This study analyses selected examples of preservice teachers’ overviews for fusing science, technology and ICT. These program overviews focused on primary students and the achievement of two standards (one from science and one from either technology or ICT). These primary preservice teachers’ fused-curricula overviews included scientific concepts and related technology and/or ICT skills and knowledge. Findings indicated a range of innovative curriculum plans for teaching primary science through technology and ICT, demonstrating that these subjects can form cohesive links towards achieving the respective learning standards. Teachers can work more astutely by fusing curricula; however further professional development may be required to advance thinking about these processes. Bonding subjects through their learning standards can extend beyond previous integration or thematic work where standards may not have been assessed. Education systems need to articulate through syllabus documents how effective fusing of curricula can be achieved. It appears that education is a key avenue for addressing societal needs, problems and issues. Education is promoted as a universal solution, which has resulted in curriculum overload (Dare, Durand, Moeller, & Washington, 1997; Vinson, 2001). Societal and curriculum demands have placed added pressure on teachers with many extenuating education issues increasing teachers’ workloads (Mobilise for Public Education, 2002). For example, as Australia has weather conducive for outdoor activities, social problems and issues arise that are reported through the media calling for action; consequently schools have been involved in swimming programs, road and bicycle safety programs, and a wide range of activities that had been considered a parental responsibility in the past. Teachers are expected to plan, implement and assess these extra-curricula activities within their already overcrowded timetables. At the same stage, key learning areas (KLAs) such as science and technology are mandatory requirements within all Australian education systems. These systems have syllabuses outlining levels of content and the anticipated learning outcomes (also known as standards, essential learnings, and frameworks). Time allocated for teaching science in obviously an issue. In 2001, it was estimated that on average the time spent in teaching science in Australian Primary Schools was almost an hour per week (Goodrum, Hackling, & Rennie, 2001). More recently, a study undertaken in the U.S. reported a similar finding. More than 80% of the teachers in K-5 classrooms spent less than an hour teaching science (Dorph, Goldstein, Lee, et al., 2007). More importantly, 16% did not spend teaching science in their classrooms. Teachers need to learn to work smarter by optimising the use of their in-class time. Integration is proposed as one of the ways to address the issue of curriculum overload (Venville & Dawson, 2005; Vogler, 2003). Even though there may be a lack of definition for integration (Hurley, 2001), curriculum integration aims at covering key concepts in two or more subject areas within the same lesson (Buxton & Whatley, 2002). This implies covering the curriculum in less time than if the subjects were taught separately; therefore teachers should have more time to cover other educational issues. Expectedly, the reality can be decidedly different (e.g., Brophy & Alleman, 1991; Venville & Dawson, 2005). Nevertheless, teachers report that students expand their knowledge and skills as a result of subject integration (James, Lamb, Householder, & Bailey, 2000). There seems to be considerable value for integrating science with other KLAs besides aiming to address teaching workloads. Over two decades ago, Cohen and Staley (1982) claimed that integration can bring a subject into the primary curriculum that may be otherwise left out. Integrating science education aims to develop a more holistic perspective. Indeed, life is not neat components of stand-alone subjects; life integrates subject content in numerous ways, and curriculum integration can assist students to make these real-life connections (Burnett & Wichman, 1997). Science integration can provide the scope for real-life learning and the possibility of targeting students’ learning styles more effectively by providing more than one perspective (Hudson & Hudson, 2001). To illustrate, technology is essential to science education (Blueford & Rosenbloom, 2003; Board of Studies, 1999; Penick, 2002), and constructing technology immediately evokes a social purpose for such construction (Marker, 1992). For example, building a model windmill requires science and technology (Zubrowski, 2002) but has a key focus on sustainability and the social sciences. Science has the potential to be integrated with all KLAs (e.g., Cohen & Staley, 1982; Dobbs, 1995; James et al., 2000). Yet, “integration” appears to be a confusing term. Integration has an educational meaning focused on special education students being assimilated into mainstream classrooms. The word integration was used in the late seventies and generally focused around thematic approaches for teaching. For instance, a science theme about flight only has to have a student drawing a picture of plane to show integration; it did not connect the anticipated outcomes from science and art. The term “fusing curricula” presents a seamless bonding between two subjects; hence standards (or outcomes) need to be linked from both subjects. This also goes beyond just embedding one subject within another. Embedding implies that one subject is dominant, while fusing curricula proposes an equal mix of learning within both subject areas. Primary education in Queensland has eight KLAs, each with its established content and each with a proposed structure for levels of learning. Primary teachers attempt to cover these syllabus requirements across the eight KLAs in less than five hours a day, and between many of the extra-curricula activities occurring throughout a school year (e.g., Easter activities, Education Week, concerts, excursions, performances). In Australia, education systems have developed standards for all KLAs (e.g., Education Queensland, NSW Department of Education and Training, Victorian Education) usually designated by a code. In the late 1990’s (in Queensland), “core learning outcomes” for strands across all KLA’s. For example, LL2.1 for the Queensland Education science syllabus means Life and Living at Level 2 standard number 1. Thus, a teacher’s planning requires the inclusion of standards as indicated by the presiding syllabus. More recently, the core learning outcomes were replaced by “essential learnings”. They specify “what students should be taught and what is important for students to have opportunities to know, understand and be able to do” (Queensland Studies Authority, 2009, para. 1). Fusing science education with other KLAs may facilitate more efficient use of time and resources; however this type of planning needs to combine standards from two syllabuses. To further assist in facilitating sound pedagogical practices, there are models proposed for learning science, technology and other KLAs such as Bloom’s Taxonomy (Bloom, 1956), Productive Pedagogies (Education Queensland, 2004), de Bono’s Six Hats (de Bono, 1985), and Gardner’s Multiple Intelligences (Gardner, 1999) that imply, warrant, or necessitate fused curricula. Bybee’s 5 Es, for example, has five levels of learning (engage, explore, explain, elaborate, and evaluate; Bybee, 1997) can have the potential for fusing science and ICT standards.

Professor Emeritus Leo G Carney Optometry's quiet achiever

Perhaps more than any other sub-discipline in optometry and vision science, the academic field of cornea and contact lenses is populated by an assortment of extroverted and flamboyant characters who constantly travel the world, entertaining clinicians with dazzling audiovisual presentations, informing them about the latest advances in the field and generally promoting their own scientific agendas. The antithesis of this is Leo Carney (Figure 1), a highly accomplished researcher, teacher, mentor and administrator, who has quietly and with great dignity carved out an impressive career in academic optometry. Indeed, Leo Carney is optometry's quintessential ‘quiet achiever’