Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

The use of headings and marginal notes in interpreting Queensland legislation : appearances can be deceptive

This article considers the rules relating to the use of marginal notes and headings in interpreting Queensland legislation, both in its original form and in reprinted legislation. It also examines the application of the principles of construction as to the use of section headings in the interpretation of provisions of the Criminal Code 1899 (Qld). Finally, it suggests that amendments should be made to the Acts Interpretation Act 1954 (Qld) in order to clarify the position as to the status of marginal notes and headings in statutory interpretation.

Bone regeneration based on tissue engineering conceptions – a 21st century perspective

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteo- conductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineer- ing and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.

Encapsulation of nanoparticles into single-crystal ZnO nanorods and microrods

One-dimensional single crystal incorporating functional nanoparticles of other materials could be an interesting platform for various applications. We studied the encapsulation of nanoparticles into single-crystal ZnO nanorods by exploiting the crystal growth of ZnO in aqueous solution. Two types of nanodiamonds with mean diameters of 10 nm and 40 nm, respectively, and polymer nanobeads with size of 200 nm have been used to study the encapsulation process. It was found that by regrowing these ZnO nanorods with nanoparticles attached to their surfaces, a full encapsulation of nanoparticles into nanorods can be achieved. We demonstrate that our low-temperature aqueous solution growth of ZnO nanorods do not affect or cause degradation of the nanoparticles of either inorganic or organic materials. This new growth method opens the way to a plethora of applications combining the properties of single crystal host and encapsulated nanoparticles. We perform micro-photoluminescence measurement on a single ZnO nanorod containing luminescent nanodiamonds and the spectrum has a different shape from that of naked nanodiamonds, revealing the cavity effect of ZnO nanorod.

Use of genetically modified muscle and fat grafts to repair defects in bone and cartilage

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified (“gene activated”) tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

Retooling Chinese primary school teachers to use technology creatively to promote innovation and problem solving skills in science classrooms

This paper reports on the initial phase of a Professional Learning Program (PLP) undertaken by 100 primary school teachers in China that aimed to facilitate the development of adaptive expertise in using technology to facilitate innovative science teaching and learning such as that envisaged by the Chinese Ministry of Education’s (2010-2020) education reforms. Key principles derived from literature about professional learning and scaffolding of learning informed the design of the PLP. The analysis of data revealed that the participants had made substantial progress towards the development of adaptive expertise. This was manifested not only by advances in the participants’ repertoires of Subject Matter Knowledge and Pedagogical Content Knowledge but also in changes to their levels of confidence and identities as teachers. By the end of the initial phase of the PLP, the participants had coalesced into a professional learning community that readily engaged in the sharing, peer review, reuse and adaption, and collaborative design of innovative science learning and assessment activities. The findings from the study indicate that those engaged in the development of PLPs for teachers in China need to take cognizance of certain cultural factors and traditions idiosyncratic to the Chinese educational system. A set of revised principles is then presented to inform the future design and implementation of PLPs for teachers in China.

Evaluating the use of a very large-scale presentation and collaboration framework

In this paper we describe the use and evaluation of CubIT, a multi-user, very large-scale presentation and collaboration framework. CubIT is installed at the Queensland University of Technology’s (QUT) Cube facility. The “Cube” is an interactive visualisation facility made up of five very large-scale interactive multi-panel wall displays, each consisting of up to twelve 55-inch multi-touch screens (48 screens in total) and massive projected display screens situated above the display panels. The paper outlines the unique design challenges, features, use and evaluation of CubIT. The system was built to make the Cube facility accessible to QUT’s academic and student population. CubIT enables users to easily upload and share their own media content, and allows multiple users to simultaneously interact with the Cube’s wall displays. The features of CubIT are implemented via three user interfaces, a multi-touch interface working on the wall displays, a mobile phone and tablet application and a web-based content management system. The evaluation reveals issues around the public use and functional scope of the system.

The chef as designer : classifying the techniques that chefs use in creating innovative dishes

This qualitative study explores the methods that chefs use to create innovative marketable product and compares these findings to other design tools. This study is based on a series of interviews with locally recognised chefs in Minnesota and observations of them in their kitchens in order to understand the details of how they conceive and develop dishes from preliminary concept to final plating and user consumption. This paper focuses on idea generation and discusses two key findings: first, the variety of idea generation techniques presented by the chefs can be classified into the creativity tool SCAMPER (substitute, combine, adapt, modify/magnify, put to other use, eliminate, reverse/rearrange); second, chefs evoke the theory of MAYA or Most Advanced Yet Acceptable when innovating new dishes, which implies making novel changes while remaining relatable to the consumer. Other reoccurring topics in the interview discussion of food innovation include play, surprise, and humour.

Beyond rule based reasoning - the meaning and use of cases

Commercial legal expert systems are invariably rule based. Such systems are poor at dealing with open texture and the argumentation inherent in law. To overcome these problems we suggest supplementing rule based legal expert systems with case based reasoning or neural networks. Both case based reasoners and neural networks use cases-but in very different ways. We discuss these differences at length. In particular we examine the role of explanation in existing expert systems methodologies. Because neural networks provide poor explanation facilities, we consider the use of Toulmin argument structures to support explanation (S. Toulmin, 1958). We illustrate our ideas with regard to a number of systems built by the authors

Determining epithelial contribution to in vivo mesenchymal tumour expression signature using species-specific microarray profiling analysis of xenografts

Gene expression profiling using microarrays and xenograft transplants of human cancer cell lines are both popular tools to investigate human cancer. However, the undefined degree of cross hybridization between the mouse and human genomes hinders the use of microarrays to characterize gene expression of both the host and the cancer cell within the xenograft. Since an increasingly recognized aspect of cancer is the host response (or cancer-stroma interaction), we describe here a bioinformatic manipulation of the Affymetrix profiling that allows interrogation of the gene expression of both the mouse host and the human tumour. Evidence of microenvironmental regulation of epithelial mesenchymal transition of the tumour component in vivo is resolved against a background of mesenchymal gene expression. This tool could allow deeper insight to the mechanism of action of anti-cancer drugs, as typically novel drug efficacy is being tested in xenograft systems.

Knowledge ecosystems of early career academics : a grounded theory of experiencing information use for learning in developmental networks

This study explored early career academics' experiences in using information to learn while building their networks for professional development. A 'knowledge ecosystem' model was developed consisting of informal learning interactions such as relating to information to create knowledge and engaging in mutually supportive relationships. Findings from this study present an alternative interpretation of information use for learning that is focused on processes manifesting as human interactions with informing entities revolving around the contexts of reciprocal human relationships.

Falling behind : business use of social media

Australians are turning onto social media in their droves - nearly 90 per cent of our citizenry are online and more than 50 per cent of the population has a Facebook account.

Clinical use of objective measures of physical activity

With measurement of physical activity becoming more common in clinical practice, it is imperative that healthcare professionals become more knowledgeable about the different methods available to objectively measure physical activity behaviour. Objective measures do not rely on information provided by the patient, but instead measure and record the biomechanical or physiological consequences of performing physical activity, often in real time. As such, objective measures are not subject to the reporting bias or recall problems associated with self-report methods. The purpose of this article was to provide an overview of the different methods used to objectively measure physical activity in clinical practice. The review was delimited to heart rate monitoring, accelerometers and pedometers since their small size, low participant burden and relatively low cost make these objective measures appropriate for use in clinical practice settings. For each measure, strengths and weakness were discussed; and whenever possible, literature-based examples of implementation were provided.

Polysubstance use in cannabis users referred for treatment: drug use profiles, psychiatric comorbidity and cannabis-related beliefs

Background: Population-based surveys demonstrate cannabis users are more likely to use both illicit and licit substances, compared with non-cannabis users. Few studies have examined the substance use profiles of cannabis users referred for treatment. Co-existing mental health symptoms and underlying cannabis-related beliefs associated with these profiles remains unexplored. Methods: Comprehensive drug use and dependence severity (Severity of Dependence Scale-Cannabis) data were collected on a sample of 826 cannabis users referred for treatment. Patients completed the General Health Questionnaire, Cannabis Expectancy Questionnaire, Cannabis Refusal Self-Efficacy Questionnaire, and Positive Symptoms and Manic-Excitement subscales of the Brief Psychiatric Rating Scale. Latent class analysis was performed on last month use of drugs to identify patterns of multiple drug use. Mental health comorbidity and cannabis beliefs were examined by identified drug use pattern. Results: A three-class solution provided the best fit to the data: (1) cannabis and tobacco users (n = 176), (2) cannabis, tobacco, and alcohol users (n = 498), and (3) wide-ranging sub- stance users (n = 132). Wide-ranging substance users (3) reported higher levels of cannabis dependence severity, negative cannabis expectancies, lower opportunistic, and emotional relief self-efficacy, higher levels of depression and anxiety and higher manic-excitement and positive psychotic symptoms. Conclusion: In a sample of cannabis users referred for treatment, wide-ranging substance use was associated with elevated risk on measures of cannabis dependence, co-morbid psychopathology, and dysfunctional cannabis cognitions. These findings have implications for cognitive-behavioral assessment and treatment.