Large-volume methacrylate monolith for plasmid purification : process engineering approach to synthesis and application

The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 °C. Maximum radial temperature recorded at the centre of the monolith was 62.3 °C, which was only 2.3 °C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5α-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.

Mesoporous silica spheres from colloids

A novel method has been developed to synthesize mesoporous silica spheres using commercial silica colloids (SNOWTEX) as precursors and electrolytes (ammonium nitrate and sodium chloride) as destabilizers. Crosslinked polyacrylamide hydrogel was used as a temporary barrier to obtain dispersible spherical mesoporous silica particles. The influences of synthesis conditions including solution composition and calcination temperature on the formation of the mesoporous silica particles were systematically investigated. The structure and morphology of the mesoporous silica particles were characterized via scanning electron microscopy (SEM) and N2 sorption technique. Mesoporous silica particles with particle diameters ranging from 0.5 to 1.6 μm were produced whilst the BET surface area was in the range of 31-123 m2 g-1. Their pore size could be adjusted from 14.1 to 28.8 nm by increasing the starting particle diameter from 20-30 nm up to 70-100 nm. A simple and cost effective method is reported that should open up new opportunities for the synthesis of scalable host materials with controllable structures.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase

A monolithic stationary phase was prepared via free radical co-polymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalized. with 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) for anion-exchange purification of plasmid DNA (pDNA) from clarified lysate obtained from E. coli DH5α-pUC19 culture in a ribonuclease/ protease-free environment. Characterization of the monolithic resin showed a porous material, with 68% of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single-stage 5 min anion-exchange purification was a pure and homogeneous supercoiled (SC) pDNA with no gDNA, RNA and protein contamination as confirmed by ethidium bromide agarose gel electrophoresis (EtBr-AGE), enzyme restriction analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This non-toxic technique is cGMP compatible and highly scalable for production of pDNA on a commercial level.

Internet-wide scanning taxonomy and framework

Industrial control systems (ICS) have been moving from dedicated communications to switched and routed corporate networks, making it probable that these devices are being exposed to the Internet. Many ICS have been designed with poor or little security features, making them vulnerable to potential attack. Recently, several tools have been developed that can scan the internet, including ZMap, Masscan and Shodan. However, little in-depth analysis has been done to compare these Internet-wide scanning techniques, and few Internet-wide scans have been conducted targeting ICS and protocols. In this paper we present a Taxonomy of Internet-wide scanning with a comparison of three popular network scanning tools, and a framework for conducting Internet-wide scans.

Application of anomaly technique in wind turbine bearing fault detection

Bearing faults are the most common cause of wind turbine failures. Unavailability and maintenance cost of wind turbines are becoming critically important, with their fast growing in electric networks. Early fault detection can reduce outage time and costs. This paper proposes Anomaly Detection (AD) machine learning algorithms for fault diagnosis of wind turbine bearings. The application of this method on a real data set was conducted and is presented in this paper. For validation and comparison purposes, a set of baseline results are produced using the popular one-class SVM methods to examine the ability of the proposed technique in detecting incipient faults.

Instructional objectives to core-gameplay : a serious game design technique

This paper explores a gap within the serious game design research. That gap is the ambiguity surrounding the process of aligning the instructional objectives of serious games with their core-gameplay i.e. the moment-to-moment activity that is the core of player interaction. A core-gameplay focused design framework is proposed that can work alongside existing, more broadly focused serious games design frameworks. The framework utilises an inquiry-based approach that allows the serious game designer to use key questions as a means to clearly outline instructional objectives with the core-gameplay. The use of this design framework is considered in the context of a small section of gameplay from an educational game currently in development. This demonstration of the framework brings shows how instructional objectives can be embedded into a serious games core-gameplay.

A modified Density-Based Scanning Algorithm with Noise for spatial travel pattern analysis from Smart Card AFC data

Smart Card Automated Fare Collection (AFC) data has been extensively exploited to understand passenger behavior, passenger segment, trip purpose and improve transit planning through spatial travel pattern analysis. The literature has been evolving from simple to more sophisticated methods such as from aggregated to individual travel pattern analysis, and from stop-to-stop to flexible stop aggregation. However, the issue of high computing complexity has limited these methods in practical applications. This paper proposes a new algorithm named Weighted Stop Density Based Scanning Algorithm with Noise (WS-DBSCAN) based on the classical Density Based Scanning Algorithm with Noise (DBSCAN) algorithm to detect and update the daily changes in travel pattern. WS-DBSCAN converts the classical quadratic computation complexity DBSCAN to a problem of sub-quadratic complexity. The numerical experiment using the real AFC data in South East Queensland, Australia shows that the algorithm costs only 0.45% in computation time compared to the classical DBSCAN, but provides the same clustering results.

The role of non-thermal plasma technique in {NOx} treatment: A review

Non-thermal plasma (NTP) has been introduced over the past several years as a promising method for nitrogen oxide (NOx) removal. The intent, when using NTP, is to selectively transfer input electrical energy to the electrons, and to not expend this in heating the entire gas stream, which generates free radicals through collisions, and promotes the desired chemical changes in the exhaust gases. The generated active species react with the pollutant molecules and decompose them. This paper reviews and summarizes relevant literature regarding various aspects of the application of {NTP} technology on {NOx} removal from exhaust gases. A comprehensive description of available scientific literature on {NOx} removal using {NTP} technology is presented, including various types of NTP, e.g. dielectric barrier discharge, corona discharge and electron beam. Furthermore, the combination of {NTP} with catalyst and adsorbent for better {NOx} removal efficiency is presented in detail. The removal of {NOx} from both simulated gases and real diesel engines is also considered in this review paper. As {NTP} is a new technique and is not yet commercialized, there is a need for more studies to be performed in this field.

Template-based circuit understanding

When verifying or reverse-engineering digital circuits, one often wants to identify and understand small components in a larger system. A possible approach is to show that the sub-circuit under investigation is functionally equivalent to a reference implementation. In many cases, this task is difficult as one may not have full information about the mapping between input and output of the two circuits, or because the equivalence depends on settings of control inputs. We propose a template-based approach that automates this process. It extracts a functional description for a low-level combinational circuit by showing it to be equivalent to a reference implementation, while synthesizing an appropriate mapping of input and output signals and setting of control signals. The method relies on solving an exists/forall problem using an SMT solver, and on a pruning technique based on signature computation.

Blast response of segmented bored tunnel using coupled SPH–FE method

Underground transport tunnels are vulnerable to blast events. This paper develops and applies a fully coupled technique involving the Smooth Particle Hydrodynamics and Finite Element techniques to investigate the blast response of segmented bored tunnels. Findings indicate that several bolts failed in the longitudinal direction due to redistribution of blast loading to adjacent tunnel rings. The tunnel segments respond as arch mechanisms in the transverse direction and suffered damage mainly due to high bending stresses. The novel information from the present study will enable safer designs of buried tunnels and provide a benchmark reference for future developments in this area.

Engagement [Reference entry]

Engagement is an important multi-dimensional concept with relevance for corporate reputation and brand management, particularly in focal areas such as corporate social responsibility, stakeholder relations, and issues or crisis management. This entry examines the levels of analysis where engagement occurs, three types of engagement, the different contextual and disciplinary perspectives of engagement, and the implications of engagement for corporate reputation.

A technique for analysing GOOSE packets when testing relays in an IEC 61850-8-1 environment

While the implementation of the IEC 61850 standard has significantly enhanced the performance of communications in electrical substations, it has also increased the complexity of the system. Subsequently, these added elaborations have introduced new challenges in relation to the skills and tools required for the design, test and maintenance of 61850-compatible substations. This paper describes a practical experience of testing a protection relay using a non-conventional test equipment; in addition, it proposes a third party software technique to reveal the contents of the packets transferred on the substation network. Using this approach, the standard objects can be linked and interpreted to what the end-users normally see in the IED and test equipment proprietary software programs.

Wide-area damping control for inter-area oscillations using inverse filtering technique

In this study, a non-linear excitation controller using inverse filtering is proposed to damp inter-area oscillations. The proposed controller is based on determining generator flux value for the next sampling time which is obtained by maximising reduction rate of kinetic energy of the system after the fault. The desired flux for the next time interval is obtained using wide-area measurements and the equivalent area rotor angles and velocities are predicted using a non-linear Kalman filter. A supplementary control input for the excitation system, using inverse filtering approach, to track the desired flux is implemented. The inverse filtering approach ensures that the non-linearity introduced because of saturation is well compensated. The efficacy of the proposed controller with and without communication time delay is evaluated on different IEEE benchmark systems including Kundur's two area, Western System Coordinating Council three-area and 16-machine, 68-bus test systems.

Empirical reference points for Bernstein’s model of pedagogic rights: Recontextualising the reconciliation agenda to Australian schooling

In this chapter we use Bernstein’s (2000) model of pedagogic rights to examine the learning experiences for non-Indigenous teachers in two reconciliation projects. In the context within which we write, reconciliation is the process of establishing a culture of mutual respect between Aboriginal and Torres Strait Islander peoples and non-Indigenous Australians. In 1991, the Royal Commission into Aboriginal Deaths in Custody linked the continuation of racism in Australian society to the weak coverage of Aboriginal and Torres Strait Islander content in the school curriculum (Reconciliation Australia 2010). Nearly two decades later, the Melbourne Declaration on Educational Goals for Young Australians issued by the council of Federal, State and Territory Ministers of Education proclaimed that curriculum should enable all students to ‘understand and acknowledge the value of Indigenous cultures and possess the knowledge, skills and understanding to contribute to, and benefit from reconciliation between Indigenous and non-Indigenous Australians’ (MCEETYA 2008, 9). Education holds out promise not only of better life chances for Indigenous young people, but also of replacing myths with understanding and tackling prejudice and racism within the non-Indigenous population. Bernstein’s (2000) model of pedagogic rights promises some purchase on this pedagogic work by providing concepts for looking systematically at the participation of non-Indigenous teachers in education. As observed by Frandji and Vitale (Chapter 2, this volume), the model is not sufficient to achieve a democratic reality, ‘but simply provides a basis for problematizing reality and considering possibilities’.