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.

Perceived obstacles to multi-storey timber-frame construction : an Australian study

The contemporary default materials for multi-storey buildings – namely concrete and steel – are all significant generators of carbon and the use of timber products provides a technically, economically and environmentally viable alternative. In particular, timber’s sustainability can drive increased use and subsequent evolution of the Blue economy as a new economic model. National research to date, however, indicates a resistance to the uptake of timber technologies in Australia. To investigate this further, a preliminary study involving a convenience sample of 15 experts was conducted to identify the main barriers involved in the use of timber frames in multi-storey buildings. A closed-ended questionnaire survey involving 74 experienced construction industry participants was then undertaken to rate the relative importance of the barriers. The survey confirmed the most significant barriers to be a perceived increase in maintenance costs and fire risk, together with a limited awareness of the emerging timber technologies available. It is expected that the results will benefit government and the timber industry, contributing to environmental improvement by developing strategies to increase the use of timber technologies in multi-storey buildings by countering perceived barriers in the Australian context.

Fault diagnosis of low speed bearing based on acoustic emission signal and multi-class relevance vector machine

This study presents an acoustic emission (AE) based fault diagnosis for low speed bearing using multi-class relevance vector machine (RVM). A low speed test rig was developed to simulate the various defects with shaft speeds as low as 10 rpm under several loading conditions. The data was acquired using anAEsensor with the test bearing operating at a constant loading (5 kN) andwith a speed range from20 to 80 rpm. This study is aimed at finding a reliable method/tool for low speed machines fault diagnosis based on AE signal. In the present study, component analysis was performed to extract the bearing feature and to reduce the dimensionality of original data feature. The result shows that multi-class RVM offers a promising approach for fault diagnosis of low speed machines.

Weak conformance of process models with respect to data objects

Process models specify behavioral aspects by describing ordering constraints between tasks which must be accomplished to achieve envisioned goals. Tasks usually exchange information by means of data objects, i.e., by writing information to and reading information from data objects. A data object can be characterized by its states and allowed state transitions. In this paper, we propose a notion which checks conformance of a process model with respect to data objects that its tasks access. This new notion can be used to tell whether in every execution of a process model each time a task needs to access a data object in a particular state, it is ensured that the data object is in the expected state or can reach the expected state and, hence, the process model can achieve its goals.

A multi-genome analysis approach enables tracking of the invasion of a single Russian wheat aphid (Diuraphis noxia) clone throughout the New World

This study investigated the population genetics, demographic history and pathway of invasion of the Russian wheat aphid (RWA) from its native range in Central Asia, the Middle East and Europe to South Africa and the Americas. We screened microsatellite markers, mitochondrial DNA and endosymbiont genes in 504 RWA clones from nineteen populations worldwide. Following pathway analyses of microsatellite and endosymbiont data, we postulate that Turkey and Syria were the most likely sources of invasion to Kenya and South Africa, respectively. Furthermore, we found that one clone transferred between South Africa and the Americas was most likely responsible for the New World invasion. Finally, endosymbiont DNA was found to be a high resolution population genetic marker, extremely useful for studies of invasion over a relatively short evolutionary history time frame. This study has provided valuable insights into the factors that may have facilitated the recent global invasion by this damaging pest.

A multi-hierarchical motorway ramp signalling strategy

Ramp signalling is an access control for motorways, in which a traffic signal is placed at on-ramps to regulate the rate of vehicles entering the motorway and thus to preserve the motorway capacity. In general, ramp signalling algorithms fall into two categories: local control and coordinated control by their effective scope. Coordinated ramp signalling strategies make use of measurements from the entire motorway network to operate individual ramp signals for the optimal performances at the network level. This study proposes a multi-hierarchical strategy for coordinated ramp signalling. The strategy is structured in two layers. At the higher layer with a longer update interval, coordination group is assembled and disassembled based on the location of high-risk breakdown flow. At the lower layer with a shorter update interval, individual ramps are hired to serve the coordination and are also released based on the prevailing congestion level on the ramp. This strategy is modelled and applied to the northbound Pacific Motorway micro-simulation platform (AIMSUN). The simulation results show an effective congestion mitigation of the proposed strategy.

Proton-transfer and non-transfer compounds of the multi-purpose drug dapsone [4-(4-Aminophenylsulfonyl)aniline] with 3,5-dinitrosalicylic acid and 5-nitroisophthalic acid

The structures of the compounds from the reaction of the drug dapsone [4-(4-aminophenylsulfonyl)aniline] with 3,5-dinitrosalicylic acid, the salt hydrate [4-(4-aminohenylsulfonyl)anilinium 2-carboxy-4,6-dinitrophenolate monohydrate] (1) and the 1:1 adduct with 5-nitroisophthalic acid [4-(4-aminophenylsulfonyl)aniline 5-nitrobenzene-1,3-dicarboxylic acid] (2) have been determined. Crystals of 1 are triclinic, space group P-1, with unit cell dimensions a = 8.2043(3), b = 11.4000(6), c = 11.8261(6)Å, α = 110.891(5), β = 91.927(3), γ = 98.590(4)deg. and Z = 4. Compound 2 is orthorhombic, space group Pbcn, with unit cell dimensions a = 20.2662(6), b = 12.7161(4), c = 15.9423(5)Å and Z = 8. In 1, intermolecular analinium N-H…O and water O-H…O and O-H…N hydrogen-bonding interactions with sulfone, carboxyl, phenolate and nitro O-atom and aniline N-atom acceptors give a two-dimensional layered structure. With 2, the intermolecular interactions involve both aniline N-H…O and carboxylic acid O-H…O and O-H…N hydrogen bonds to sulfone, carboxyl, nitro and aniline acceptors, giving a three-dimensional network structure. In both structures π--π aromatic ring associations are present.

The IKBALS project: multi-modal reasoning in legal knowledge based systems

In attempting to build intelligent litigation support tools, we have moved beyond first generation, production rule legal expert systems. Our work integrates rule based and case based reasoning with intelligent information retrieval. When using the case based reasoning methodology, or in our case the specialisation of case based retrieval, we need to be aware of how to retrieve relevant experience. Our research, in the legal domain, specifies an approach to the retrieval problem which relies heavily on an extended object oriented/rule based system architecture that is supplemented with causal background information. We use a distributed agent architecture to help support the reasoning process of lawyers. Our approach to integrating rule based reasoning, case based reasoning and case based retrieval is contrasted to the CABARET and PROLEXS architectures which rely on a centralised blackboard architecture. We discuss in detail how our various cooperating agents interact, and provide examples of the system at work. The IKBALS system uses a specialised induction algorithm to induce rules from cases. These rules are then used as indices during the case based retrieval process. Because we aim to build legal support tools which can be modified to suit various domains rather than single purpose legal expert systems, we focus on principles behind developing legal knowledge based systems. The original domain chosen was theAccident Compensation Act 1989 (Victoria, Australia), which relates to the provision of benefits for employees injured at work. For various reasons, which are indicated in the paper, we changed our domain to that ofCredit Act 1984 (Victoria, Australia). This Act regulates the provision of loans by financial institutions. The rule based part of our system which provides advice on the Credit Act has been commercially developed in conjunction with a legal firm. We indicate how this work has lead to the development of a methodology for constructing rule based legal knowledge based systems. We explain the process of integrating this existing commercial rule based system with the case base reasoning and retrieval architecture.

Engaging creativity through an action learning and action research process to develop an Indigenous art exhibition

In most art exhibitions, the creative part of the exhibition is assumed to be the artworks on display. But for the Capricornia Arts Mob’s first collective art exhibition in Rockhampton during NAIDOC Week in 2012, the process of developing the exhibition became the focus of creative action learning and action research. In working together to produce a multi-media exhibition, we learned about the collaborative processes and time required to develop a combined exhibition. We applied Indigenous ways of working – including yarning, cultural respect, cultural protocols, mentoring young people, providing a culturally safe working environment and sharing both time and food – to develop our first collective art exhibition. We developed a process that allowed us to ask deep questions, engage in a joint journey of learning, and develop our collective story. This paper explores the processes that the Capricornia Arts Mob used to develop the exhibition for NAIDOC 2012.

The value of respect in human research ethics: A conceptual analysis and a practical guide

In order to continue to maintain public trust and confidence in human research, participants must be treated with respect. Researchers and Human Research Ethics Committee members need to be aware that modern considerations of this value include: the need for a valid consenting process, the protection of participants who have their capacity for consent compromised; the promotion of dignity for participants; and the effects that human research may have on cultures and communities. This paper explains the prominence of respect as a value when considering the ethics of human research and provides practical advice for both researchers and Human Research Ethics Committee members in developing respectful research practices.

Interpretation of association rules with multi-tier granule mining

This study was a step forward to improve the performance for discovering useful knowledge – especially, association rules in this study – in databases. The thesis proposed an approach to use granules instead of patterns to represent knowledge implicitly contained in relational databases; and multi-tier structure to interpret association rules in terms of granules. Association mappings were proposed for the construction of multi-tier structure. With these tools, association rules can be quickly assessed and meaningless association rules can be justified according to the association mappings. The experimental results indicated that the proposed approach is promising.

The relevance of shared experiences : a multi-level study of collective efficacy as a moderator of job control in the stressor-strain relationship

Karasek's Job Demand-Control model proposes that control mitigates the positive effects of work stressors on employee strain. Evidence to date remains mixed and, although a number of individual-level moderators have been examined, the role of broader, contextual, group factors has been largely overlooked. In this study, the extent to which control buffered or exacerbated the effects of demands on strain at the individual level was hypothesized to be influenced by perceptions of collective efficacy at the group level. Data from 544 employees in Australian organizations, nested within 23 workgroups, revealed significant three-way cross-level interactions among demands, control and collective efficacy on anxiety and job satisfaction. When the group perceived high levels of collective efficacy, high control buffered the negative consequences of high demands on anxiety and satisfaction. Conversely, when the group perceived low levels of collective efficacy, high control exacerbated the negative consequences of high demands on anxiety, but not satisfaction. In addition, a stress-exacerbating effect for high demands on anxiety and satisfaction was found when there was a mismatch between collective efficacy and control (i.e. combined high collective efficacy and low control). These results provide support for the notion that the stressor-strain relationship is moderated by both individual- and group-level factors.

Matching image sets via adaptive multi convex hull

Traditional nearest points methods use all the samples in an image set to construct a single convex or affine hull model for classification. However, strong artificial features and noisy data may be generated from combinations of training samples when significant intra-class variations and/or noise occur in the image set. Existing multi-model approaches extract local models by clustering each image set individually only once, with fixed clusters used for matching with various image sets. This may not be optimal for discrimination, as undesirable environmental conditions (eg. illumination and pose variations) may result in the two closest clusters representing different characteristics of an object (eg. frontal face being compared to non-frontal face). To address the above problem, we propose a novel approach to enhance nearest points based methods by integrating affine/convex hull classification with an adapted multi-model approach. We first extract multiple local convex hulls from a query image set via maximum margin clustering to diminish the artificial variations and constrain the noise in local convex hulls. We then propose adaptive reference clustering (ARC) to constrain the clustering of each gallery image set by forcing the clusters to have resemblance to the clusters in the query image set. By applying ARC, noisy clusters in the query set can be discarded. Experiments on Honda, MoBo and ETH-80 datasets show that the proposed method outperforms single model approaches and other recent techniques, such as Sparse Approximated Nearest Points, Mutual Subspace Method and Manifold Discriminant Analysis.

Physical activity recognition from accelerometer data using a multi-scale ensemble method

Accurate and detailed measurement of an individual's physical activity is a key requirement for helping researchers understand the relationship between physical activity and health. Accelerometers have become the method of choice for measuring physical activity due to their small size, low cost, convenience and their ability to provide objective information about physical activity. However, interpreting accelerometer data once it has been collected can be challenging. In this work, we applied machine learning algorithms to the task of physical activity recognition from triaxial accelerometer data. We employed a simple but effective approach of dividing the accelerometer data into short non-overlapping windows, converting each window into a feature vector, and treating each feature vector as an i.i.d training instance for a supervised learning algorithm. In addition, we improved on this simple approach with a multi-scale ensemble method that did not need to commit to a single window size and was able to leverage the fact that physical activities produced time series with repetitive patterns and discriminative features for physical activity occurred at different temporal scales.