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.

Ability of recombinant human bone morphogenetic protein 2 to enhance bone healing in the presence of tobramycin : evaluation in a rat segmental defect model

Objective To determine whether locally applied tobramycin influences the ability of recombinant human bone morphogenetic protein 2 (rhBMP-2) to heal a segmental defect in the rat femur. Methods The influence of tobramycin on the osteogenic differentiation of mesenchymal stem cells was first evaluated in vitro. For the subsequent, in vivo experiments, a 5-mm segmental defect was created in the right femur of each of 25 Sprague-Dawley rats and stabilized with an external fixator and four Kirschner wires. Rats were divided in four groups: empty control, tobramycin (11 mg)/absorbable collagen sponge, rhBMP-2 (11 μg)/absorbable collagen sponge, and rhBMP-2/absorbable collagen sponge with tobramycin. Bone healing was monitored by radiography at 3 and 8 weeks. Animals were euthanized at 8 weeks and the properties of the defect were compared with the intact contralateral femur. Bone formation in the defect region was assessed by dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing. Results Tobramycin exerted a dose-dependent inhibition of alkaline phosphatase induction and calcium deposition by mesenchymal stem cells cultured under osteogenic conditions. The inhibition was reversed in the presence of 500 ng/mL of rhBMP-2. Segmental defects in the rat femora failed to heal in the absence of rhBMP-2. Tobramycin exerted no inhibitory effects on the ability of rhBMP-2 to heal these defects and increased the bone area of the defects treated with rhBMP-2. Data obtained from all other parameters of healing, including dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing, were unaffected by tobramycin. Conclusions Although our in vitro results suggested that tobramycin inhibits the osteogenic differentiation of mesenchymal stem cells, this could be overcome by rhBMP-2. Tobramycin did not impair the ability of rhBMP-2 to heal critical-sized femoral defects in rats. Indeed, bone area was increased by nearly 20% in the rhBMP-2 group treated with tobramycin. This study shows that locally applied tobramycin can be used in conjunction with rhBMP-2 to enhance bone formation at fracture sites.

Recombinant cellulase accumulation in the leaves of mature, vegetatively propagated transgenic sugarcane

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.

Inheritance of qualitative and quantitative trypsin inhibitor variants in Pisum

A trypsin inhibitor locus (Tri) has been mapped close to Vc-2 on Pisum (pea) linkage group 5 using recombinant inbred lines derived from crosses of genotypes showing qualitative variation in seed trypsin inhibitors. F2 seed populations derived from crosses between lines showing qualitative variation in trypsin inhibitors as well as quantitative variation in inhibitor activity showed an association between the segregation of the structural variation and relative activity levels. Clones complementary to Pisum trypsin inhibitor mRNA were used in hybridization analyses which showed that the segregation of protein polymorphisms reflected directly the segregation of polymorphisms associated with the structural genes.

Biological performance of a polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

PURPOSE. We develop a sheep thoracic spine interbody fusion model to study the suitability of polycaprolactone-based scaffold and recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within the thoracic spine. The surgical approach is a mini- open thoracotomy with relevance to minimally invasive deformity correction surgery for adolescent idiopathic scoliosis. To date there are no studies examining the use of this biodegradable implant in combination with biologics in a sheep thoracic spine model. METHODS. In the present study, six sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone-based scaffold plus 0.54μg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion was assessed at six months post-surgery. RESULTS. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL- based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluations of the respective groups. Biomechanical testing revealed significantly higher stiffness for the rhBMP-2 plus PCL- based scaffold group in all loading directions in comparison to the other two groups. CONCLUSION. The results of this study demonstrate that rhBMP-2 plus PCL- based scaffold is a viable bone graft substitute, providing an optimal environment for thoracic interbody spinal fusion in a large animal model.

6 versus 12 month performance of polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

Introduction Well-designed biodegradable scaffolds in combination with bone growth factors offer a valuable alternative to the current gold standard autograft in spinal fusion surgery Yong et al. (2013). Here we report on 6- vs 12- month data set evaluating the longitudinal performance of a CaP coated polycaprolactone (PCL) scaffold loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within a large preclinical animal model. Methods Twelve sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone based scaffold plus 0.54µg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion assessments were performed via high resolution clinical computed tomography and histological evaluation were undertaken at six (n=6) and twelve (n=6) months post-surgery using the Sucato grading system (Sucato et al. 2004). Results The computed tomography fusion grades of the 6- and 12- months in the rhBMP-2 plus PCL- based scaffold group were 1.9 and 2.1 respectively, in the autograft group 1.9 and 1.3 respectively, and in the scaffold alone group 0.9 and 1.17 respectively. There were no statistically significant differences in the fusion scores between 6- and 12- month for the rhBMP plus PCL- based scaffold or PCL – based scaffold alone group however there was a significant reduction in scores in the autograft group. These scores were seen to correlate with histological evaluations of the respective groups. Conclusions The results of this study demonstrate the efficacy of scaffold-based delivery of rhBMP-2 in promoting higher fusion grades at 6- and 12- months in comparison to the scaffold alone or autograft group within the same time frame. Fusion grades achieved at six months using PCL+rhBMP-2 are not significantly increased at twelve months post-surgery.

Effect of hydrophilicity of carbon nanotube arrays on the release rate and activity of recombinant human bone morphogenetic protein-2

Novel nanostructures such as vertically aligned carbon nanotube (CNT) arrays have received increasing interest as drug delivery carriers. In the present study, two CNT arrays with extreme surface wettabilities are fabricated and their effects on the release of recombinant human bone morphogenetic protein-2 (rhBMP-2) are investigated. It is found that the superhydrophilic arrays retained a larger amount of rhBMP-2 than the superhydrophobic ones. Further use of a poloxamer diffusion layer delayed the initial burst and resulted in a greater total amount of rhBMP-2 released from both surfaces. In addition, rhBMP-2 bound to the superhydrophilic CNT arrays remained bioactive while they denatured on the superhydrophobic surfaces. These results are related to the combined effects of rhBMP-2 molecules interacting with poloxamer and the surface, which could be essential in the development of advanced carriers with tailored surface functionalities.

The game of clones and the Australia tax : divergent views about copyright business models and the willingness of Australian consumers to infringe

It is often said that Australia is a world leader in rates of copyright infringement for entertainment goods. In 2012, the hit television show, Game of Thrones, was the most downloaded television show over bitorrent, and estimates suggest that Australians accounted for a plurality of nearly 10% of the 3-4 million downloads each week. The season finale of 2013 was downloaded over a million times within 24 hours of its release, and again Australians were the largest block of illicit downloaders over BitTorrent, despite our relatively small population. This trend has led the former US Ambassador to Australia to implore Australians to stop 'stealing' digital content, and rightsholders to push for increasing sanctions on copyright infringers. The Australian Government is looking to respond by requiring Internet Service Providers to issue warnings and potentially punish consumers who are alleged by industry groups to have infringed copyright. This is the logical next step in deterring infringement, given that the operators of infringing networks (like The Pirate Bay, for example) are out of regulatory reach. This steady ratcheting up of the strength of copyright, however, comes at a significant cost to user privacy and autonomy, and while the decentralisation of enforcement reduces costs, it also reduces the due process safeguards provided by the judicial process. This article presents qualitative evidence that substantiates a common intuition: one of the major reasons that Australians seek out illicit downloads of content like Game of Thrones in such numbers is that it is more difficult to access legitimately in Australia. The geographically segmented way in which copyright is exploited at an international level has given rise to a ‘tyranny of digital distance’, where Australians have less access to copyright goods than consumers in other countries. Compared to consumers in the US and the EU, Australians pay more for digital goods, have less choice in distribution channels, are exposed to substantial delays in access, and are sometimes denied access completely. In this article we focus our analysis on premium film and television offerings, like Game of Thrones, and through semi-structured interviews, explore how choices in distribution impact on the willingness of Australian consumers to seek out infringing copies of copyright material. Game of Thrones provides an excellent case study through which to frame this analysis: it is both one of the least legally accessible television offerings and one of the most downloaded through filesharing networks of recent times. Our analysis shows that at the same time as rightsholder groups, particularly in the film and television industries, are lobbying for stronger laws to counter illicit distribution, the business practices of their member organisations are counter-productively increasing incentives for consumers to infringe. The lack of accessibility and high prices of copyright goods in Australia leads to substantial economic waste. The unmet consumer demand means that Australian consumers are harmed by lower access to information and entertainment goods than consumers in other jurisdictions. The higher rates of infringement that fulfils some of this unmet demand increases enforcement costs for copyright owners and imposes burdens either on our judicial system or on private entities – like ISPs – who may be tasked with enforcing the rights of third parties. Most worryingly, the lack of convenient and cheap legitimate digital distribution channels risks undermining public support for copyright law. Our research shows that consumers blame rightsholders for failing to meet market demand, and this encourages a social norm that infringing copyright, while illegal, is not morally wrongful. The implications are as simple as they are profound: Australia should not take steps to increase the strength of copyright law at this time. The interests of the public and those of rightsholders align better when there is effective competition in distribution channels and consumers can legitimately get access to content. While foreign rightsholders are seeking enhanced protection for their interests, increasing enforcement is likely to increase their ability to engage in lucrative geographical price-discrimination, particularly for premium content. This is only likely to increase the degree to which Australian consumers feel that their interests are not being met and, consequently, to further undermine the legitimacy of copyright law. If consumers are to respect copyright law, increasing sanctions for infringement without enhancing access and competition in legitimate distribution channels could be dangerously counter-productive. We suggest that rightsholders’ best strategy for addressing infringement in Australia at this time is to ensure that Australians can access copyright goods in a timely, affordable, convenient, and fair lawful manner.

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

Detecting approximate clones in business process model repositories

Empirical evidence shows that repositories of business process models used in industrial practice contain significant amounts of duplication. This duplication arises for example when the repository covers multiple variants of the same processes or due to copy-pasting. Previous work has addressed the problem of efficiently retrieving exact clones that can be refactored into shared subprocess models. This article studies the broader problem of approximate clone detection in process models. The article proposes techniques for detecting clusters of approximate clones based on two well-known clustering algorithms: DBSCAN and Hi- erarchical Agglomerative Clustering (HAC). The article also defines a measure of standardizability of an approximate clone cluster, meaning the potential benefit of replacing the approximate clones with a single standardized subprocess. Experiments show that both techniques, in conjunction with the proposed standardizability measure, accurately retrieve clusters of approximate clones that originate from copy-pasting followed by independent modifications to the copied fragments. Additional experiments show that both techniques produce clusters that match those produced by human subjects and that are perceived to be standardizable.

Longitudinal performance of polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in large preclinical animal model : 6- versus 12 months

There is strong current interest in the use of biodegradable scaffolds in combination with bone growth factors as a valuable alternative to the current gold standard autograft in spinal fusion surgery Yong et al. (2013). Here we report on 6- vs 12- month data set evaluating the longitudinal performance of a CaP coated polycaprolactone (PCL) scaffold loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within a preclinical ovine thoracic spine. The results of this study demonstrate the efficacy of scaffold-based delivery of rhBMP-2 in promoting higher fusion grades at 6- and 12- months in comparison to the scaffold alone or autograft group within the same time frame. Fusion grades achieved at six months using PCL+rhBMP-2 are not significantly increased at twelve months post surgery.

The attack of the clones: Patent law and stem cell research

This article considers the integral role played by patent law in respect of stem cell research. It highlights concerns about commercialization, access to essential medicines and bioethics. The article maintains that there is a fundamental ambiguity in the Patents Act 1990 (Cth) as to whether stem cell research is patentable subject matter. There is a need to revise the legislation in light of the establishment of the National Stem Cell Centre and the passing of the Research Involving Embryos Act 2002 (Cth). The article raises concerns about the strong patent protection secured by the Wisconsin Alumni Research Foundation and Geron Corporation in respect of stem cell research in the United States. It contends that a number of legal reforms could safeguard access to stem cell lines, and resulting drugs and therapies. Finally, this article explores how ethical concerns are addressed within the framework of the European Biotechnology Directive. It examines the decision of the European Patent Office in relation to the so-called Edinburgh patent, and the inquiry of the European Group on Ethics in Science and New Technologies into The Ethical Aspects of Patenting Involving Human Stem Cells.

Incorporation of human recombinant tropoelastin into silk fibroin membranes with the view to repairing Bruch’s membrane

Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

Induction of a Th1 immune response and suppression of IgE via immunotherapy with a recombinant hybrid molecule encapsulated in liposome-protamine-DNA nanoparticles in a model of experimental allergy

Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.