Vectors and methods for hairpin RNA and artificial microRNA-mediated gene silencing in plants

In plant cells, DICER-LIKE4 processes perfectly double-stranded RNA (dsRNA) into short interfering (si) RNAs, and DICER-LIKE1 generates micro (mi) RNAs from primary miRNA transcripts (pri-miRNA) that form fold-back structures of imperfectly dsRNA. Both si and miRNAs direct the endogenous endonuclease, ARGONAUTE1 to cleave complementary target single-stranded RNAs and either small RNA (sRNA)-directed pathway can be harnessed to silence genes in plants. A routine way of inducing and directing RNA silencing by siRNAs is to express self-complementary single-stranded hairpin RNA (hpRNA), in which the duplexed region has the same sequence as part of the target gene's mRNA. Artificial miRNA (amiRNA)-mediated silencing uses an endogenous pri-miRNA, in which the original miRNA/miRNA* sequence has been replaced with a sequence complementary to the new target gene. In this chapter, we describe the plasmid vector systems routinely used by our research group for the generation of either hpRNA-derived siRNAs or amiRNAs.

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d/r) or inverted-repeat (i/r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i/r T-DNA insertions is a primary inducer of transgene silencing in plants. © CSIRO 2005.

Humanities and social scientific research methods in porn studies

Porn studies researchers in the humanities have tended to use different research methods from those in social sciences. There has been surprisingly little conversation between the groups about methodology. This article presents a basic introduction to textual analysis and statistical analysis, aiming to provide for all porn studies researchers a familiarity with these two quite distinct traditions of data analysis. Comparing these two approaches, the article suggests that social science approaches are often strongly reliable – but can sacrifice validity to this end. Textual analysis is much less reliable, but has the capacity to be strongly valid. Statistical methods tend to produce a picture of human beings as groups, in terms of what they have in common, whereas humanities approaches often seek out uniqueness. Social science approaches have asked a more limited range of questions than have the humanities. The article ends with a call to mix up the kinds of research methods that are applied to various objects of study.

Evaluation of two-view geometry methods with automatic ground-truth generation

A large number of methods have been published that aim to evaluate various components of multi-view geometry systems. Most of these have focused on the feature extraction, description and matching stages (the visual front end), since geometry computation can be evaluated through simulation. Many data sets are constrained to small scale scenes or planar scenes that are not challenging to new algorithms, or require special equipment. This paper presents a method for automatically generating geometry ground truth and challenging test cases from high spatio-temporal resolution video. The objective of the system is to enable data collection at any physical scale, in any location and in various parts of the electromagnetic spectrum. The data generation process consists of collecting high resolution video, computing accurate sparse 3D reconstruction, video frame culling and down sampling, and test case selection. The evaluation process consists of applying a test 2-view geometry method to every test case and comparing the results to the ground truth. This system facilitates the evaluation of the whole geometry computation process or any part thereof against data compatible with a realistic application. A collection of example data sets and evaluations is included to demonstrate the range of applications of the proposed system.

A comparison of methods to identify alcohol involvement in youth injury-related emergency department presentation data

Aims: To compare different methods for identifying alcohol involvement in injury-related emergency department presentation in Queensland youth, and to explore the alcohol terminology used in triage text. Methods: Emergency Department Information System data were provided for patients aged 12-24 years with an injury-related diagnosis code for a 5 year period 2006-2010 presenting to a Queensland emergency department (N=348895). Three approaches were used to estimate alcohol involvement: 1) analysis of coded data, 2) mining of triage text, and 3) estimation using an adaptation of alcohol attributable fractions (AAF). Cases were identified as ‘alcohol-involved’ by code and text, as well as AAF weighted. Results: Around 6.4% of these injury presentations overall had some documentation of alcohol involvement, with higher proportions of alcohol involvement documented for 18-24 year olds, females, indigenous youth, where presentations occurred on a Saturday or Sunday, and where presentations occurred between midnight and 5am. The most common alcohol terms identified for all subgroups were generic alcohol terms (eg. ETOH or alcohol) with almost half of the cases where alcohol involvement was documented having a generic alcohol term recorded in the triage text. Conclusions: Emergency department data is a useful source of information for identification of high risk sub-groups to target intervention opportunities, though it is not a reliable source of data for incidence or trend estimation in its current unstandardised form. Improving the accuracy and consistency of identification, documenting and coding of alcohol-involvement at the point of data capture in the emergency department is the most desirable long term approach to produce a more solid evidence base to support policy and practice in this field.

Comparing methods for modelling spreading cell fronts

Spreading cell fronts play an essential role in many physiological processes. Classically, models of this process are based on the Fisher-Kolmogorov equation; however, such continuum representations are not always suitable as they do not explicitly represent behaviour at the level of individual cells. Additionally, many models examine only the large time asymptotic behaviour, where a travelling wave front with a constant speed has been established. Many experiments, such as a scratch assay, never display this asymptotic behaviour, and in these cases the transient behaviour must be taken into account. We examine the transient and asymptotic behaviour of moving cell fronts using techniques that go beyond the continuum approximation via a volume-excluding birth-migration process on a regular one-dimensional lattice. We approximate the averaged discrete results using three methods: (i) mean-field, (ii) pair-wise, and (iii) one-hole approximations. We discuss the performace of these methods, in comparison to the averaged discrete results, for a range of parameter space, examining both the transient and asymptotic behaviours. The one-hole approximation, based on techniques from statistical physics, is not capable of predicting transient behaviour but provides excellent agreement with the asymptotic behaviour of the averaged discrete results, provided that cells are proliferating fast enough relative to their rate of migration. The mean-field and pair-wise approximations give indistinguishable asymptotic results, which agree with the averaged discrete results when cells are migrating much more rapidly than they are proliferating. The pair-wise approximation performs better in the transient region than does the mean-field, despite having the same asymptotic behaviour. Our results show that each approximation only works in specific situations, thus we must be careful to use a suitable approximation for a given system, otherwise inaccurate predictions could be made.

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods TXS and VEGF metabolite levels were measured in NSCLC serum samples (n=46) by EIA. TXB2 levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results Serum TXB2 levels were correlated with VEGF (p<0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p<0.0001) and microvessel density (CD-31; p<0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p<0.0001) and female patients (p<0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p<0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.

Improved rural health through a collaborative model of care – qualitative findings

INTRODUCTION Health disparity between urban and rural regions in Australia is well-documented. In the Wheatbelt catchments of Western Australia there is higher incidence and rate of avoidable hospitalisation for chronic diseases. Structured care approach to chronic illnesses is not new but the focus has been on single disease state. A recent ARC Discovery Project on general practice nurse-led chronic disease management of diabetes, hypertension and stable ischaemic heart disease reported improved communication and better medical administration.[1] In our study we investigated the sustainability of such a multi-morbidities general practice –led collaborative model of care in rural Australia. METHODS A QUAN(qual) design was utilised. Eight pairs of rural general practices were matched. Inclusion criteria used were >18 years and capable of giving informed consent, at least one identified risk factor or diagnosed with chronic conditions. Patients were excluded if deemed medically unsuitable. A comprehensive care plan was formulated by the respective general practice nurse in consultation with the treating General Practitioner (GP) and patient based on the individual’s readiness to change, and was informed by available local resource. A case management approach was utilised. Shediaz-Rizkallah and Lee’s conceptual framework on sustainability informed our evaluation.[2] Our primary outcome on measures of sustainability was reduction in avoidable hospitalisation. Secondary outcomes were patients and practitioners acceptance and satisfaction, and changes to pre-determined interim clinical and process outcomes. RESULTS The qualitative interviews highlighted the community preference for a ‘sustainable’ local hospital in addition to general practice. Costs, ease of access, low prioritisation of self chronic care, workforce turnover and perception of losing another local resource if underutilised influenced the respondents’ decision to present at local hospital for avoidable chronic diseases regardless. CONCLUSIONS Despite the pragmatic nature of rural general practice in Australia, the sustainability of chronic multi-morbidities management in general practice require efficient integration of primary-secondary health care and consideration of other social determinants of health. What this study adds: What is already known on this subject: Structured approach to chronic disease management is not new and has been shown to be effective for reducing hospitalisation. However, the focus has been on single disease state. What does this study add: Sustainability of collaborative model of multi-morbidities care require better primary-secondary integration and consideration of social determinants of health.

Stability of corneal topography and wavefront aberrations in young Singaporeans

Purpose To investigate the differences between and variations across time in corneal topography and ocular wavefront aberrations in young Singaporean myopes and emmetropes. Methods We used a videokeratoscope and wavefront sensor to measure the ocular surface topography and wavefront aberrations of the total eye optics in the morning, mid-day and late afternoon on two separate days. Topography data were used to derive the corneal surface wavefront aberrations. Both the corneal and total wavefronts were analysed up to the 4th radial order of the Zernike polynomial expansion, and were centred on the entrance pupil (5 mm). The participants included 12 young progressing myopes, 13 young stable myopes and 15 young age-matched emmetropes. Results For all subjects considered together there were significant changes in some of the aberrations terms across the day, such as spherical aberration ( ) and vertical coma ( ) (repeated measures ANOVA, p<0.05). The magnitude of positive spherical aberration ( ) was significantly lower in the progressing myope group than that of the stable myopes (p=0.04) and emmetrope group (p=0.02). There were also significant interactions between refractive group and time of day for with/against-the-rule astigmatism ( ). Significantly lower 4th order RMS of ocular wavefront aberrations were found in the progressing myope group compared with the stable myopes and emmetropes (p<0.01). Conclusions These differences and variations in the corneal and total aberrations may have significance for our understanding of refractive error development and for clinical applications requiring accurate wavefront measurements.

Development of an integrated GIS and land use planning course : impacts of hybrid instructional methods

This study reports an action research undertaken at Queensland University of Technology. It evaluates the effectiveness of the integration of GIS within the substantive domains of an existing land use planning course in 2011. Using student performance, learning experience survey, and questionnaire survey data, it also evaluates the impacts of incorporating hybrid instructional methods (e.g., in-class and online instructional videos) in 2012 and 2013. Results show that: students (re)iterated the importance of GIS in the course justifying the integration; the hybrid methods significantly increased student performance; and unlike replacement, the videos are more suitable as a complement to in-class activity.

Nutrition screening and assessment in Parkinson's disease : a comparison of methods

Background & Aims Nutrition screening and assessment enable early identification of malnourished people and those at risk of malnutrition. Appropriate assessment tools assist with informing and monitoring nutrition interventions. Tool choice needs to be appropriate to the population and setting. Methods Community-dwelling people with Parkinson’s disease (>18 years) were recruited. Body mass index (BMI) was calculated from weight and height. Participants were classified as underweight according to World Health Organisation (WHO) (≤18.5kg/m2) and age specific (<65 years,≤18.5kg/m2; ≥65 years,≤23.5kg/m2) cut-offs. The Mini-Nutritional Assessment (MNA) screening (MNA-SF) and total assessment scores were calculated. The Patient-Generated Subjective Global Assessment (PG-SGA), including the Subjective Global Assessment (SGA), was performed. Sensitivity, specificity, positive predictive value, negative predictive value and weighted kappa statistic of each of the above compared to SGA were determined. Results Median age of the 125 participants was 70.0(35-92) years. Age-specific BMI (Sn 68.4%, Sp 84.0%) performed better than WHO (Sn 15.8%, Sp 99.1%) categories. MNA-SF performed better (Sn 94.7%, Sp 78.3%) than both BMI categorisations for screening purposes. MNA had higher specificity but lower sensitivity than PG-SGA (MNA Sn 84.2%, Sp 87.7%; PG-SGA Sn 100.0%, Sp 69.8%). Conclusions BMI lacks sensitivity to identify malnourished people with Parkinson’s disease and should be used with caution. The MNA-SF may be a better screening tool in people with Parkinson’s disease. The PG-SGA performed well and may assist with informing and monitoring nutrition interventions. Further research should be conducted to validate screening and assessment tools in Parkinson’s disease.  

Different in vitro activation methods for latent transforming growth factors (TGF)–β : considerable exogenous factors to promote higher mesenchymal-origin cell proliferation in a bioprocessing platform

Regenerative medicine includes two efficient techniques, namely tissue-engineering and cell-based therapy in order to repair tissue damage efficiently. Most importantly, huge numbers of autologous cells are required to deal these practices. Nevertheless, primary cells, from autologous tissue, grow very slowly while culturing in vitro; moreover, they lose their natural characteristics over prolonged culturing period. Transforming growth factors-beta (TGF-β) is a ubiquitous protein found biologically in its latent form, which prevents it from eliciting a response until conversion to its active form. In active form, TGF-β acts as a proliferative agent in many cell lines of mesenchymal origin in vitro. This article reviews on some of the important activation methods-physiochemical, enzyme-mediated, non-specific protein interaction mediated, and drug-induced- of TGF-β, which may be established as exogenous factors to be used in culturing medium to obtain extensive proliferation of primary cells.

The effect of a supervised exercise training programme on sleep quality in recently discharged heart failure patients

Background Sleep disturbances, including insomnia and sleep-disordered breathing, are a common complaint in people with heart failure and impair well-being. Exercise training (ET) improves quality of life in stable heart failure patients. ET also improves sleep quality in healthy older patients, but there are no previous intervention studies in heart failure patients. Aim The aim of this study was to examine the impact of ET on sleep quality in patients recently discharged from hospital with heart failure. Methods This was a sub-study of a multisite randomised controlled trial. Participants with a heart failure hospitalisation were randomised within six weeks of discharge to a 12-week disease management programme including exercise advice (n=52) or to the same programme with twice weekly structured ET (n=54). ET consisted of two one-hour supervised aerobic and resistance training sessions, prescribed and advanced by an exercise specialist. The primary outcome was change in Pittsburgh Sleep Quality Index (PSQI) between randomisation and week 12. Results At randomisation, 45% of participants reported poor sleep (PSQI≥5). PSQI global score improved significantly more in the ET group than the control group (–1.5±3.7 vs 0.4±3.8, p=0.03). Improved sleep quality correlated with improved exercise capacity and reduced depressive symptoms, but not with changes in body mass index or resting heart rate. Conclusion Twelve weeks of twice-weekly supervised ET improved sleep quality in patients recently discharged from hospital with heart failure.

Sensitive methods for studying the environmental performance of protective coatings

The safe working lifetime of a structure in a corrosive or other harsh environment is frequently not limited by the material itself but rather by the integrity of the coating material. Advanced surface coatings are usually crosslinked organic polymers such as epoxies and polyurethanes which must not shrink, crack or degrade when exposed to environmental extremes. While standard test methods for environmental durability of coatings have been devised, the tests are structured more towards determining the end of life rather than in anticipation of degradation. We have been developing prognostic tools to anticipate coating failure by using a fundamental understanding of their degradation behaviour which, depending on the polymer structure, is mediated through hydrolytic or oxidation processes. Fourier transform infrared spectroscopy (FTIR) is a widely-used laboratory technique for the analysis of polymer degradation and with the development of portable FTIR spectrometers, new opportunities have arisen to measure polymer degradation non-destructively in the field. For IR reflectance sampling, both diffuse (scattered) and specular (direct) reflections can occur. The complexity in these spectra has provided interesting opportunities to study surface chemical and physical changes during paint curing, service abrasion and weathering, but has often required the use of advanced statistical analysis methods such as chemometrics to discern these changes. Results from our studies using this and related techniques and the technical challenges that have arisen will be presented.