Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Damping structure selection in nonlinear ship manoeuvring models

This paper presents the application of a statistical method for model structure selection of lift-drag and viscous damping components in ship manoeuvring models. The damping model is posed as a family of linear stochastic models, which is postulated based on previous work in the literature. Then a nested test of hypothesis problem is considered. The testing reduces to a recursive comparison of two competing models, for which optimal tests in the Neyman sense exist. The method yields a preferred model structure and its initial parameter estimates. Alternatively, the method can give a reduced set of likely models. Using simulated data we study how the selection method performs when there is both uncorrelated and correlated noise in the measurements. The first case is related to instrumentation noise, whereas the second case is related to spurious wave-induced motion often present during sea trials. We then consider the model structure selection of a modern high-speed trimaran ferry from full scale trial data.

An exploratory study into director selection : who do directors want on their boards and how do they select them?

This exploratory study into director selection involved in-depth interviews with Australian non-executive directors to identify what directors consider as important criteria when selecting new members and the approach taken to identify and select candidates. The findings indicate boards select new members based not only on their ability to contribute complementary skills and experience but also on a perceived compatibility with incumbent board members. While these two selection criteria are considered equal in importance, not all selection approaches are able to adequately assess both criteria. As a result many selections fail to realise their selection criteria.

Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

Who selects the 'right' directors? An examination of the association between board selection, gender diversity and outcomes

Using a sample of companies from the top 500 listed firms in Australia, we investigate whether the presence of a designated nomination committee and female representation on the nomination committee affect board gender diversity. We also examine whether gender diversity on the board affects firm risk and financial performance. We find that board gender diversity is significantly and positively associated with the presence of a designated nomination committee and that female representation on the nomination committee is a significant explanatory factor of increasing board gender diversity following the release of the 2010 Australian Securities Exchange Corporate Governance Council (ASXCGC) recommendations. Further, our results support the business case for board gender diversity as we find greater gender diversity moderates excessive firm risk which in turn improves firms’ financial performance. Our results are robust after correcting for selection bias and controlling for other board, firm and industry characteristics.

Phosphoprotein pathway mapping: Akt/Mammalian target of rapamycin activation is negatively associated with childhood rhabdomyosarcoma survival

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains ∼60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Children's Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser473 (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr37/46 (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser1108 (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr389 (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy.

Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers

This study investigated the clinicopathologic roles of mammalian target of rapamycin (mTOR) expression and its relationship to carcinogenesis and tumor progression in a colorectal adenoma-adenocarcinoma model. Two colon cancer cell lines with different pathologic stages (SW480 and SW48) and 1 normal colonic epithelial cell line (FHC) were used, in addition to 119 colorectal adenocarcinomas and 32 adenomas. mTOR expression profiles at messenger RNA (mRNA) and protein levels were investigated in the cells and tissues using real-time quantification polymerase chain reaction and immunohistochemistry. The findings were correlated with the clinicopathologic features of the tumors. The colon cell line from stage III cancer (SW48) showed higher expression of mTOR mRNA than that from stage II cancer (SW480). At the tissue level, mTOR showed higher mRNA and protein expression in colorectal carcinoma than in adenoma. The mRNA and protein expression was correlated with each other in approximately one-third of the carcinomas and adenomas. High levels of mTOR mRNA expression were noted more in carcinoma or adenoma arising from the distal portion of the large intestine (P = .025 and .019, respectively). Within the colorectal cancer population, a high level of expression of mTOR mRNA was related to the presence of lymph node metastases (P = .031), advanced pathologic stage (P = .05), and presence of persistent disease or tumor recurrence (P = .035). To conclude, the study has indicated that mTOR is likely to be involved in the development and progression of colorectal cancer and is linked to cancer initiation, invasiveness, and progression.

Mechanisms of mono- and poly-ubiquitination : ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/ E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.

A review selection method using product feature taxonomy

As of today, online reviews have become more and more important in decision making process. In recent years, the problem of identifying useful reviews for users has attracted significant attentions. For instance, in order to select reviews that focus on a particular feature, researchers proposed a method which extracts all associated words of this feature as the relevant information to evaluate and find appropriate reviews. However, the extraction of associated words is not that accurate due to the noise in free review text, and this affects the overall performance negatively. In this paper, we propose a method to select reviews according to a given feature by using a review model generated based upon a domain ontology called product feature taxonomy. The proposed review model provides relevant information about the hierarchical relationships of the features in the review which captures the review characteristics accurately. Our experiment results based on real world review dataset show that our approach is able to improve the review selection performance according to the given criteria effectively.

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

Biolistic transformation of elite indica rice (Oryza sativa L.) cultivars through semi-solid and liquid medium selection systems

Following microprojectile mediated delivery of a plasmid construct (pAHC-25) encoding bar (bialophos resistance) gene into five-day-old scutellar calli derived from mature embryos, the effectiveness of selection procedure for bar-gene expressing tissue was compared for two indica rice cultivars (IR-64 and Karnal Local). While IR-64 transformants could be selected through the generally used semi-solid selection medium, the same procedure was not effective in the basmati cultivar Karnal Local. In the latter case, while lower concentrations (2–4 mg 1−1) of the selective agent phosphinothricin (PPT) yielded only escapes, higher concentrations (6–8 mg l−1) inhibited proliferation of transformed as well as untransformed sectors. For Karnal Local, a liquid medium based selection system was successfully utilized for recovering transformed sectors and, eventually, regenerants. The study demonstrates the generation of transformants of two elite indica cultivars using the environment-independent system of mature embryos from seeds.

Reflections on issues of power in packaged software selection

The adoption of packaged software is becoming increasingly common in a variety of organizations and much of the packaged software literature presents this as a straightforward, linear process based on rationalistic evaluation. This paper applies the framework of power relations developed by Markus and Bjørn-­‐Anderson (1987) to a longitudinal study concerning the adoption of a customer relationship management package in a small organization. This is used to highlight both overt and covert power issues within the selection and procurement of the product and illustrate the interplay of power between senior management, IT managers, IT vendors and consultants, and end-­‐users. The paper contributes to the growing body of literature on packaged software and also to our understanding of how power is deeply embedded within the surrounding processes.

IT consultants, salesmanship and the challenges of packaged software selection in SMEs

Purpose – This paper seeks to analyse the process of packaged software selection in a small organization, focussing particularly on the role of IT consultants as intermediaries in the process. Design/methodology/approach – This is based upon a longitudinal, qualitative field study concerning the adoption of a customer relationship management package in an SME management consultancy. Findings – The authors illustrate how the process of “salesmanship”, an activity directed by the vendor/consultant and focussed on the interests of senior management, marginalises user needs and ultimately secures the procurement of the software package. Research limitations/implications – Despite the best intentions the authors lose something of the rich detail of the lived experience of technology in presenting the case study as a linear narrative. Specifically, the authors have been unable to do justice to the complexity of the multifarious ways in which individual perceptions of the project were influenced and shaped by the opinions of others. Practical implications – Practitioners, particularly those from within SMEs, should be made aware of the ways in which external parties may have a vested interest in steering projects in a particular direction, which may not necessarily align with their own interests. Originality/value – This study highlights in detail the role of consultants and vendors in software selection processes, an area which has received minimal attention to date. Prior work in this area emphasises the necessary conditions for, and positive outcomes of, appointing external parties in an SME context, with only limited attention being paid to the potential problems such engagements may bring.

The social shaping of packaged software selection

As organisations increasingly engage in the selection, purchase, and adoption of packaged software products, how these activities are carried out in practice becomes increasingly relevant for researchers and practitioners. Our focus in this paper is to propose a framework for understanding the packaged software selection process. The functionalist literature on this area of study suggests a number of generic recommendations, which are based on rational assumptions about the process and view the decision making that takes place as producing the “best technology solution.’” To explore this, we conducted a longitudinal, in-depth study of packaged software selection in a small organisation. For interpretation of the case, we draw upon the Social Construction of Technology, a theoretical framework arguing that technology is socially constituted and regarding the process of development as contradictory and uncertain. We offer a number of contributions. First, we further our understanding of packaged software selection with the critique that we offer of the functionalist literature, drawing insights from the emerging critical/constructivist literature and expanding our domain of interest to encompass the wider environment. Second, we weave this together with our experiences in the field, drawing on social constructivism for theoretical support, to develop a framework of packaged software selection that shows how various actors shape the process.

The automation of radiotherapy treatment planning assessment

Aim The assessment of treatment plans is an important component in the education of radiation therapists. The establishment of a grade for a plan is currently based on subjective assessment of a range of criteria. The automation of assessment could provide a number of advantages including faster feedback, reduced chance of human error, and simpler aggregation of past results. Method A collection of treatments planned by a cohort of 27 second year radiation therapy students were selected for quantitative evaluation. Treatment sites included the bladder, cervix, larynx, parotid and prostate, although only the larynx plans had been assessed in detail. The plans were designed with the Pinnacle system and exported using the DICOM framework. Assessment criteria included beam arrangement optimisation, volume contouring, target dose coverage and homogeneity, and organ-at-risk sparing. The in-house Treatment and Dose Assessor (TADA) software1 was evaluated for suitability in assisting with the quantitative assessment of these plans. Dose volume data were exported in per-student and per-structure data tables, along with beam complexity metrics, dose volume histograms, and reports on naming conventions. Results The treatment plans were exported and processed using TADA, with the processing of all 27 plans for each treatment site taking less than two minutes. Naming conventions were successfully checked against a reference protocol. Significant variations between student plans were found. Correlation with assessment feedback was established for the larynx plans. Conclusion The data generated could be used to inform the selection of future assessment criteria, monitor student development, and provide useful feedback to the students. The provision of objective, quantitative evaluations of plan quality would be a valuable addition to not only radiotherapy education programmes but also for staff development and potentially credentialing methods. New functionality within TADA developed for this work could be applied clinically to, for example, evaluate protocol compliance.