Determination of Transgene Copy Number by Real-Time Quantitative PCR

Efficient methods to characterize transgenic plants are important to quickly understand the state of the transformant. Determining transgene copy number is an important step in transformant characterization and can differentiate between complex and simple transformation events. This knowledge can be extremely useful when determining what future experiments and uses the transgenic lines can be utilized for. The method described here uses real-time quantitative PCR to determine the transgene copy number present in the genome of the transformant. Specifically, this method measures the relative transgene copy number by comparing it with an endogenous gene with a known copy number. This method is a quick alternative to the Southern blot, a method that is commonly used to determine gene copy number, and is effective when screening large numbers of transformants

Providing healthcare for people with chronic illness: the views of Australian GPs

The document attached has been archived with permission from the editor of the Medical Journal of Australia (26 April 2007). An external link to the publisher’s copy is included.OBJECTIVES: To explore general practitioners' views on chronic-disease care: the difficulties and rewards, the needs of patients, the impact of government incentive payments, and the changes needed to improve chronic-disease management. DESIGN: Qualitative study, involving semi-structured questions administered to 10 focus groups of GPs, conducted from April to October 2002. PARTICIPANTS AND SETTING: 54 GPs from both urban and rural practices in New South Wales and South Australia. RESULTS: Consistent themes emerged about the complex nature of chronic-disease management, the tension between patients' and GPs' goals for care, the time-consuming aspects of care (exacerbated by federal government requirements), and the conflicting pressures that prevent GPs engaging in structured multidisciplinary care (ie, team-based care involving systems for patient monitoring, recall, and care planning). CONCLUSIONS: Structured multidisciplinary care for people with chronic conditions can be difficult to provide. Barriers include the lack of fit between systems oriented towards acute care and the requirements of chronic-disease care, and between bureaucratic, inflexible structures and the complex, dynamic nature of GP–patient relationships. These problems are exacerbated by administrative pressures associated with federal government initiatives to improve chronic-illness management. Changes are needed in both policies and attitudes to enable GPs to move from episodic care to providing structured long-term care as part of a multidisciplinary team.John Oldroyd, Judith Proudfoot, Fernando A Infante, Gawaine Powell Davies, Tanya Bubner, Chris Holton, Justin J Beilby and Mark F Harri

Quality improvement activities associated with organisational capacity in general practice

Copyright © 2007 Australian College of General Practitioners Copyright to Australian Family Physician. Reproduced with permission. Permission to reproduce must be sought from the publisher, The Royal Australian College of General Practitioners.BACKGROUND Clinical audit is recognised worldwide as a useful tool for quality improvement. METHODS A feedback report profiling capacity for chronic disease care was sent to 97 general practices. These practices were invited to complete a clinical audit activity based on that feedback. Data were analysed quantitatively and case studies were developed based on the free text responses. RESULTS Eighty-two (33%) of 247 general practitioners participated in the clinical audit process, representing 57 (59%) of 97 general practices. From the data in their feedback report, 37 (65%) of the 57 practices recognised the area most in need of improvement. This was most likely where the need related to clinical practice or teamwork, and least likely where the need related to linkages with other services, and business and finance. Only 25 practices (46%) developed an action plan related to their recognised area for improvement, and 22 (39%) practices implemented their chosen activity. Participating GPs judged that change activity focused on teamwork was most successful. DISCUSSION The clinical audit process offered participating GPs and practices an opportunity to reflect on their performance across a number of key areas and to implement change to enhance the practice’s capacity for quality chronic disease care. The relationship between need and action was weak, suggesting a need for greater support to overcome barriers.Cheryl Amoroso, Judy Proudfoot, Tanya Bubner, Edward Swan, Paola Espinel, Christopher Barton, Justin Beilby and Mark Harri

Species identification of European forest pathogens of the genus Milesina (Pucciniales) using urediniospore morphology and molecular barcoding including M. woodwardiana sp. nov

Species of rust fungi of the genus Milesina (Pucciniastraceae, Pucciniales) are distributed mainly in northern temperate regions. They host-alternate between needles of fir (Abies spp.) and fronds of ferns (species of Polypodiales). Milesina species are distinguished based on host taxonomy and urediniospore morphology. In this study, 12 species of Milesina from Europe were revised. Specimens were examined by light and scanning electron microscopy for urediniospore morphology with a focus on visualising germ pores (number, size and position) and echinulation. In addition, barcode loci (ITS, nad6, 28S) were used for species delimitation and for molecular phylogenetic analyses. Barcodes of 72 Milesina specimens were provided, including 11 of the 12 species. Whereas urediniospore morphology features were sufficient to distinguish all 12 Milesina species except for 2 (M. blechni and M. kriegeriana), ITS sequences separated only 4 of 11 species. Sequencing with 28S and nad6 did not improve species resolution. Phylogenetic analysis, however, revealed four phylogenetic groups within Milesina that also correlate with specific urediniospore characters (germ pore number and position and echinulation). These groups are proposed as new sections within Milesina (sections Milesina, Vogesiacae M. Scholler & Bubner, sect. nov., Scolopendriorum M. Scholler & Bubner, sect. nov. and Carpaticae M. Scholler & Bubner, sect. nov.). In addition, Milesina woodwardiana Buchheit & M. Scholler, sp. nov. on Woodwardia radicans, a member of the type section Milesina, is newly described. An identification key for European Milesina species, based on urediniospore features, is provided

Statistical tools for transgene copy number estimation based on real-time PCR

Background As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. Results Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination. Conclusion These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification

Organisational capacity and chronic disease care - An Australian general practice perspective

Copyright © 2007 Australian Family Physician Copyright to Australian Family Physician. Reproduced with permission. Permission to reproduce must be sought from the publisher, The Royal Australian College of General Practitioners.Although we are rapidly improving our understanding of how to manage patients with chronic illness in Australian general practice, many patients are still receiving suboptimal care. General practices have limited organisational capacity to provide the structured care that is required for managing chronic conditions: regular monitoring, decision support, patient recall, supporting patient self management, team work, and information management. This requires a shift away from episodic, acute models. Overseas research has shown that areas such as team work, clinical information systems, decision support, linkages and leadership are also important in managing chronic illness, but we do not know which of these are most important in Australia.Judith Proudfoot, Fernando Infante, Christine Holton, Gawaine Powell-Davies, Tanya Bubner, Justin Beilby, Mark Harri

ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations

Repeat prenatal corticosteroid prior to preterm birth: a systematic review and individual participant data meta-analysis for the PRECISE study group (prenatal repeat corticosteroid international IPD study group: assessing the effects using the best level of evidence) - study protocol

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.BACKGROUND The aim of this individual participant data (IPD) meta-analysis is to assess whether the effects of repeat prenatal corticosteroid treatment given to women at risk of preterm birth to benefit their babies are modified in a clinically meaningful way by factors related to the women or the trial protocol. METHODS/DESIGN The Prenatal Repeat Corticosteroid International IPD Study Group: assessing the effects using the best level of Evidence (PRECISE) Group will conduct an IPD meta-analysis. The PRECISE International Collaborative Group was formed in 2010 and data collection commenced in 2011. Eleven trials with up to 5,000 women and 6,000 infants are eligible for the PRECISE IPD meta-analysis. The primary study outcomes for the infants will be serious neonatal outcome (defined by the PRECISE International IPD Study Group as one of death (foetal, neonatal or infant); severe respiratory disease; severe intraventricular haemorrhage (grade 3 and 4); chronic lung disease; necrotising enterocolitis; serious retinopathy of prematurity; and cystic periventricular leukomalacia); use of respiratory support (defined as mechanical ventilation or continuous positive airways pressure or other respiratory support); and birth weight (Z-scores). For the children, the primary study outcomes will be death or any neurological disability (however defined by trialists at childhood follow up and may include developmental delay or intellectual impairment (developmental quotient or intelligence quotient more than one standard deviation below the mean), cerebral palsy (abnormality of tone with motor dysfunction), blindness (for example, corrected visual acuity worse than 6/60 in the better eye) or deafness (for example, hearing loss requiring amplification or worse)). For the women, the primary outcome will be maternal sepsis (defined as chorioamnionitis; pyrexia after trial entry requiring the use of antibiotics; puerperal sepsis; intrapartum fever requiring the use of antibiotics; or postnatal pyrexia). DISCUSSION Data analyses are expected to commence in 2011 with results publicly available in 2012.Caroline A Crowther ... Tanya K Bubner ... Philippa F Middleton ... Lisa Yelland ... Sasha Zhang ... et al

A new perspective in bio-refining : Levoglucosenone and cleaner lignin from waste biorefinery hydrolysis lignin by selective conversion of residual saccharides

An unexpected opportunity is reported to improve the sustainability of biorefineries whereby 8 wt% levoglucosenone (LGE) can be derived from unconverted saccharides in a lignin-rich biorefinery waste stream in a highly selective fashion (>90%). Additionally, in the process a purer lignin is obtained which can be used for further processing or materials applications. LGE is a valuable and versatile product with a plethora of applications

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs