Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

Amateur Media : Social, Cultural and Legal Perspectives

The rise of Web 2.0 has pushed the amateur to the forefront of public discourse, public policy and media scholarship. Typically non-salaried, non-specialist and untrained in media production, amateur producers are now seen as key drivers of the creative economy. But how do the activities of citizen journalists, fan fiction writers and bedroom musicians connect with longer traditions of extra-institutional media production? This edited collection provides a much-needed interdisciplinary contextualisation of amateur media before and after Web 2.0. Surveying the institutional, economic and legal construction of the amateur media producer via a series of case studies, it features contributions from experts in the fields of law, economics and media studies based in the UK, Europe and Singapore. Each section of the book contains a detailed case study on a selected topic, followed by two further pieces providing additional analysis and commentary. Using an extraordinary array of case studies and examples, from YouTube to online games, from subtitling communities to reality TV, the book is neither a celebration of amateur production nor a denunciation of the demise of professional media industries. Rather, this book presents a critical dialogue across law and the humanities, exploring the dynamic tensions and interdependencies between amateur and professional creative production. This book will appeal to both academics and students of intellectual property and media law, as well as to scholars and students of economics, media, cultural and internet studies.

Money ruins everything

In the economy of the 21st century, economic and technical innovation is increasingly based on developments that don't rely on economic incentive or public provision. Unlike 20th century innovation, the most important developments in innovation have been driven not by research funded by governments or developed by corporations but by the collaborative interactions of individuals. In most cases, this modality of innovation has not been motivated by economic concerns or the prospect of profit. This raises the possibility of a world in which some of the sectors of the economy particularly the ones dealing with innovation and creativity are driven by social interactions of various kinds, rather than by profit-oriented investment. This Article examines the development of this amateur modality of creative production, and explains how it came to exist. It then deals with why this modality is different from and potentially inconsistent with the typical modalities of production that are at the heart of modern views of innovation policy. It provides a number of policy prescriptions that should be used by governments to recognize the significance of amateur innovation, and to further the development of amateur productivity.

Amateur-to-amateur

Copyright, it is commonly said, matters in society because it encourages the production of socially beneficial, culturally significant expressive content. Our focus on copyright's recent history, however, blinds us to the social information practices that have always existed. In this Article, we examine these social information practices, and query copyright's role within them. We posit a functional model of what is necessary for creative content to move from creator to user. These are the functions dealing with the creation, selection, production, dissemination, promotion, sale, and use of expressive content. We demonstrate how centralized commercial control of information content has been the driving force behind copyright's expansion. All of the functions that copyright industries once controlled, however, are undergoing revolutionary decentralization and disintermediation. Different aspects of information technology, notably the digitization of information, widespread computer ownership, the rise of the Internet, and the development of social software, threaten the viability and desirability of centralized control over every one of the content functions. These functions are increasingly being performed by individuals and disaggregated groups. This raises an issue for copyright as the main regulatory force in information practices: copyright assumes a central control requirement that no longer applies for the development of expressive content. We examine the normative implications of this shift for our information policy in this new post-copyright era. Most notably, we conclude that copyright law needs to be adjusted in order to recognize the opportunity and desirability of decentralized content, and the expanded marketplace of ideas it promises.

Evaluation of a pilot-scale oil extraction from microalgae for biodiesel production

Biodiesel derived from microalgae is one of a suite of potential solutions to meet the increasing demand for a renewable, carbon-neutral energy source. However, there are numerous challenges that must be addressed before algae biodiesel can become commercially viable. These challenges include the economic feasibility of harvesting and dewatering the biomass and the extraction of lipids and their conversion into biodiesel. Therefore, it is essential to find a suitable extraction process given these processes presently contribute significantly to the total production costs which, at this stage, inhibit the ability of biodiesel to compete financially with petroleum diesel. This study focuses on pilot-scale (100 kg dried microalgae) solvent extraction of lipids from microalgae and subsequent transesterification to biodiesel. Three different solvents (hexane, isopropanol (IPA) and hexane + IPA (1:1)) were used with two different extraction methods (static and Soxhlet) at bench-scale to find the most suitable solvent extraction process for the pilot-scale. The Soxhlet method extracted only 4.2% more lipid compared to the static method. However, the fatty acid profiles of different extraction methods with different solvents are similar, suggesting that none of the solvents or extraction processes were biased for extraction of particular fatty acids. Considering the cost and availability of the solvents, hexane was chosen for pilot-scale extraction using static extraction. At pilot-scale the lipid yield was found to be 20.3% of total biomass which is 2.5% less than from bench scale. Extracted fatty acids were dominated by polyunsaturated fatty acids (PUFAs) (68.94±0.17%) including 47.7±0.43 and 17.86±0.42% being docosahexaenoic acid (DHA) (C22:6) and docosapentaenoic acid (DPA) (C22:5, ω-3), respectively. These high amounts of long chain poly unsaturated fatty acids are unique to some marine microalgae and protists and vary with environmental conditions, culture age and nutrient status, as well as with cultivation process. Calculated physical and chemical properties of density, viscosity of transesterified fatty acid methyl esters (FAMEs) were within the limits of the biodiesel standard specifications as per ASTM D6751-2012 and EN 14214. The calculated cetane number was, however, significantly lower (17.8~18.6) compared to ASTM D6751-2012 or EN 14214-specified minimal requirements. We conclude that the obtained microalgal biodiesel would likely only be suitable for blending with petroleum diesel to a maximum of 5 to 20%.

A review on the production of levulinic acid and furanics from sugars

Sugarcane products represent an abundant and relatively low cost carbon resource that can be utilised to produce chemical intermediates such as levulinic acid and furanics. These chemicals can be easily upgraded to commodity and specialty chemicals and biofuels by high yielding and well established technologies. However, there are challenges and technical hurdles that need to be overcome before these chemical intermediates can be cost-effectively produced in commercial quantities. The paper reviews production of levulinic acid and furanics from sugars by homogeneous mineral acid catalysts, and reports on preliminary studies on the production of these compounds with environmentally friendly biodegradable sulfonic acids. The yields (>50% of theoretical) of levulinic acid, formic acid and furfural obtained with these organic acids are comparable to that of sulphuric acid currently used for their production.

Health governance : law, regulation and policy

This special issue of Public Health is devoted to health governance, examining the role of law, regulation and policy in safeguarding the public's health. Each of us has devoted a career to thinking carefully about the role of law as a tool to prevent injury and disease and to promote the population's health and wellbeing. 1, 2, 3 and 4 In this Guest Editorial we first explain what we mean by the term ‘governance’, as well as the role of law in a well-regulated society. Next, we explore the increasingly important, and challenging, concept of what we call national and global federalism—the inter-relationships among the various levels of governance (local, national, supranational and transnational) and among various actors in national and global health. Third, we explain the origins of this journal symposium, which arises from three conferences on the topic in Hong Kong and Sydney. Finally, we offer a brief introduction to the articles that follow.

A new national women's health policy : legal, ethical and policy initiatives to support women's health

In 1989 the first National Women's Health Policy was launched in Australia. Now, 20 years later, the Federal Government has announced plans for the development of a new National Women's Health Policy to address the health needs of Australian women. The Policy will be based on five principles: gender equity; health equity between women; a focus on prevention; an evidence base for interventions; and a life course approach. This editorial examines the role for law in the development of a new National Women's Health Policy. It considers the relevance of regulatory frameworks for health research in supporting an evidence base for health interventions and analyses the requirement in the National Health and Medical Research Council's National Statement on Ethical Conduct in Human Research for "fair inclusion" of research participants. The editorial argues for a holistic approach to women's health that includes regulatory frameworks for research, identification of funding priorities for research, and the need for a dedicated government department or agency to promote women's health.

Theory based production and project management

This paper gives an overview of an ongoing project endeavouring to advance theory-based production and project management, and the rationale for this approach is briefly justified. The status of the theoretical foundation of production management, project management and allied disciplines is discussed, with emphasis on metaphysical grounding of theories, as well as the nature of the heuristic solution method commonly used in these disciplines. Then, on-going work related to different aspects of production and project management is reviewed from both theoretical and practical orientation. Next, information systems agile project management is explored with a view to its re-use in generic project management. In production management, the consequences and implementation of a new, wider theoretical basis are analyzed. The theoretical implications and negative symptoms of the peculiarities of the construction industry for supply chains and supply chain management in construction are observed. Theoretical paths for improvements of inter-organisational relationships in construction which are fundamental for improvement of construction supply chains are described. To conclude, the observations made in this paper vis-à-vis production, project and supply chain management are related again to the theoretical basis of this paper, and finally directions for theory development and future research are given and discussed.

Reproductive technology, public policy and single motherhood

In mid 2000, the Australian community engaged in a national debate over access to infertility treatment services. The debate was sparked by a Federal Court decision in late July. That decision, by Justice Sundberg in the case of McBain v State of Victoria 1 held that the provisions of the Infertility Treatment Act 1995 (Vic) which limited eligibility for infertility treatment to women who were married or in heterosexual de facto relationships, were inconsistent with section 22 of the Commonwealth Sex Discrimination Act 1984 (Cth) which prohibits discrimination on the basis of marital status. Justice Sundberg held that, by virtue of section 109 of the Constitution, 2 the provisions of the Victorian Act were inoperative to the extent of the inconsistency between the State and Commonwealth legislation.

Effects of child care policy and environment on physical activity

Child care centers differ systematically with respect to the quality and quantity of physical activity they provide, suggesting that center-level policies and practices, as well as the center's physical environment, are important influences on children's physical activity behavior. Purpose To summarize and critically evaluate the extant peer-reviewed literature on the influence of child care policy and environment on physical activity in preschool-aged children. Methods A computer database search identified seven relevant studies that were categorized into three broad areas: cross-sectional studies investigating the impact of selected center-level policies and practices on moderate-to-vigorous physical activity (MVPA), studies correlating specific attributes of the outdoor play environment with the level and intensity of MVPA, and studies in which a specific center-level policy or environmental attribute was experimentally manipulated and evaluated for changes in MVPA. Results Staff education and training, as well as staff behavior on the playground, seem to be salient influences on MVPA in preschoolers. Lower playground density (less children per square meter) and the presence of vegetation and open play areas also seem to be positive influences on MVPA. However, not all studies found these attributes to be significant. The availability and quality of portable play equipment, not the amount or type of fixed play equipment, significantly influenced MVPA levels. Conclusions Emerging evidence suggests that several policy and environmental factors contribute to the marked between-center variability in physical activity and sedentary behavior. Intervention studies targeting these factors are thus warranted.

Decoupling 2 : technologies, opportunities and policy options

This report was produced by the Decoupling Working Group of the International Resource Panel. It explores technological possibilities and opportunities for both developing and developed countries to accelerate decoupling and reap the environmental and economic benefits of increased resource productivity. It also examines several policy options that have proved to be successful in helping different countries to improve resource productivity in various sectors of their economy, avoiding negative impacts on the environment. It does not seem possible for a global economy based on the current unsustainable patterns of resource use to continue into the future. The economic consequences of these patterns are already apparent in three areas: increases in resource prices, increased price volatility and disruption of environmental systems. The environment impacts of resource use are also leading to potentially irreversible changes to the world’s ecosystems, often with direct effects on people and the economy – for example through damage to health, water shortages, loss of fish stocks or increased storm damage. But there are alternatives to these scary patterns. Many decoupling technologies and techniques that deliver resource productivity increases as high as 5 to 10-fold are already available, allowing countries to pursue their development strategies while significantly reducing their resource footprint and negative impacts on the environment. This report shows that much of the policy design “know-how” needed to achieve decoupling is present in terms of legislation, incentive systems, and institutional reform. Many countries have tried these out with tangible results, encouraging others to study and where appropriate replicate and scale up such practices and successes.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

Production of levulinic acid and other chemicals from sugarcane fibre

Lignocellulosics represent a renewable resource for producing fuels and chemicals as an alternative to fossil resources. This study utilised an organic acid catalyst and a co-solvent to develop an environmentally friendly processing technology for the production of levulinic acid and furfural from a waste material, sugarcane fibre.