SITE-SPECIFIC RECOMBINATION OF THE MYCOBACTERIUM TUBERCULOSISPROPHAGE-LIKE ELEMENT PHIRV1

The site-specific recombination systems of bacteriophages and other mobile elements fall into two categories that are named for the integrase protein that catalyzes integration and excision of the phage genome. These integrases utilize either a tyrosine or a serine residue to carry out the nucleophilic attack of the DNA. In many cases the directionality of the reaction, that is whether the enzyme catalyzes integration or excision, is determined by an additional phage encoded protein referred to as RDF, or recombination directionality factor.Two prophage-like elements, phiRv1 and phiRv2, were found through sequencing Mycobacterium tuberculosis strain H37Rv. These are absent from the vaccine bacillus M. bovis BCG, and are often found in virulent strains of M. bovis and M. tuberculosis. Through the work presented here, one of these elements, phiRv1, was found to encode an active recombination system, with a serine integrase and RDF. The phiRv1 element is found within a degenerate repeated element, REP13E12, which is present in seven non-identical copies in M. tuberculosis and M. bovis BCG. In vivo studies have revealed that four of the seven 13E12 elements can serve as integration sites (attBs) for a plasmid carrying a reconstructed attP and integrase, and that multiple integrations can occur. The fast growing saprophyte, M. smegmatis, also supports integration, although inefficiently. In M. smegmatis, the phiRv1 plasmid integrates into at least two 13E12 repeats that are quite different from those found in M. bovis BCG and M. tuberculosis. Inefficiency is overcome by providing M. smegmatis with an attB site from BCG. These integrated plasmids are stable, and excision occurs in the presence of the phiRv1 RDF encoded by Rv1584c. In vitro assays were developed for both integration and excision. All the substrate site requirements are relatively small. Integration occurs slowly but efficiently in the presence of excess attB or on an intramolecular attP-attB plasmid. In the presence of RDF integration is inhibited and excision is stimulated. The RDF binds to a specific sequence in both attB and attL, although the way in which it functions may be through protein-protein interactions with integrase

Funding Indigenous organisations: improving governance performance through innovations in public finance management in remote Australia

This review explains the context and past experience of public finance reform and its effects on governance in remote Indigenous communities. Preamble The poor development standards experienced by Indigenous Australians, especially in places remote from urban areas, are regularly characterised in public and academic discourse as a crisis, with calls for ‘new approaches, new thinking and new commitment’. This paper focuses on the modalities used to manage the conversion of public financing of Indigenous organisations into activities designed to impact on these standards. By modalities we mean the policies and instruments that structure and govern how funding is delivered and aligned with government priorities, including administrative, financing and accountability mechanisms. In this review, block funding was identified for its potential to reform the public finance system to create enabling conditions for enhanced Indigenous governance. Building a devolved accountability framework around the organisation, rather than the centralised grant program, is a sensible alternative to multiple grants and ineffective cycles of grant risk management and attendant accountability measures. As block funding has never been explicitly trialled in Australia, there is a lack of evaluations and other evidence for its efficacy in remote Indigenous contexts. In comparison, the international development literature documents a wealth of experience of the success and shortcoming of generically similar financing modalities. The paper therefore considers the circumstances under which block funding could be usefully adapted to the unique context of remote Indigenous communities in Australia. This review examines the literature and evidence from two principal sources. First and foremost, lessons are distilled and the context defined from a wide array of experience over the past two decades across remote Australia. This is then compared with the evidence from similar contexts abroad; that is, countries and regions that are remote from centres of economic wealth and political power, where populations are generally relatively isolated, scattered and highly diverse. These are often poorly served by administrative and service delivery arrangements due to the impost of great distances and high costs. In these settings, whether abroad or in Australia, local authorities are often referred to as being ‘fragile’ and ‘weak’. Two quite different approaches to handling public finances can be found in these contexts: One is to centralise responsibilities to govern public finances and to institute a host of compliance and reporting obligations on local authorities to manage perceived risks to fiduciary standards. This approach can be an effective way to respond to crises in the short term, but over time, this response tends to corrode local capability and introduce perverse incentives to ‘break the rules’ and ‘game the system’ to respond to local needs and demands. A second, contending approach has developed, particularly in the past decade. This approach shifts responsibility in the direction of local authorities and organisations for a specific range of services and functions. It also negotiates mutually acceptable agreements about the conditions under which public monies can be used and how performance will be jointly assessed. This paper synthesises Australian and international experiences, then suggests avenues for future engagement, including both new experimentation and upscaling of already promising precedents

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacI-galR family of regulatory genes

malR of Strepomyces coelicolor A3(2) encodes a homologue of the Lacl/Galr family of repressor proteins, and is divergently transcribed from the malEFG gene cluster, which encodes components of an ATP-dependent transport system that is required for maltose utilization. Transcription of malE was induced by maltose and repressed by glucose. Disruption or deletion of malR resulted in constitutive, glucose-insensitive malE transcription at a level markedly above that observed in the parental malR+ strain, and overproduction of MalR prevented growth on maltose as carbon source. Consequently, MalR plays a crucial role in both substrate induction and glucose repression of maltose utilization. MalR is expressed from a single promoter with transcription initiating at the first G of the predicted GTG translataion start codon

Towards Additive Manufacture of Next Generation Prosthetics, Assessing Emerging CAD Strategies for Improving the Existing CAD Process

The research project that this work comes from aims to address the issues and inefficiencies of current CAD systems in regards to working on multiple scales, with a particular focus on improving prosthetic design. Working from the micron scale to the macro scale and following on from work that defined a criteria of necessary material properties, this paper is a continuation of previous work that attempts to answer the research question ‘How can new CAD strategies be applied to improve the efficiency of producing parts with these necessary material properties?’ A selection of emerging CAD strategies from the last five years have been selected with a view of improving the hybrid process order created in the previous study. Each of these processes is introduced, and their pros and cons compared before identifying the areas of the CAD criteria that they can improve efficiency. Testing was performed using the software if it is available to see areas of improvement first-hand

Development of the Orion Crew Module Static Aerodynamic Database

The Orion aerodynamic database provides force and moment coefficients given the velocity, attitude, configuration, etc. of the Crew Exploration Vehicle (CEV). The database is developed and maintained by the NASA CEV Aerosciences Project team from computational and experimental aerodynamic simulations. The database is used primarily by the Guidance, Navigation, and Control (GNC) team to design vehicle trajectories and assess flight performance. The initial hypersonic re-entry portion of the Crew Module (CM) database was developed in 2006. Updates incorporating additional data and improvements to the database formulation and uncertainty methodologies have been made since then. This paper details the process used to develop the CM database, including nominal values and uncertainties, for Mach numbers greater than 8 and angles of attack between 140deg and 180deg. The primary available data are more than 1000 viscous, reacting gas chemistry computational simulations using both the Laura and Dplr codes, over a range of Mach numbers from 2 to 37 and a range of angles of attack from 147deg to 172deg. Uncertainties were based on grid convergence, laminar-turbulent solution variations, combined altitude and code-to-code variations, and expected heatshield asymmetry. A radial basis function response surface tool, NEAR-RS, was used to fit the coefficient data smoothly in a velocity-angle-of-attack space. The resulting database is presented and includes some data comparisons and a discussion of the predicted variation of trim angle of attack and lift-to-drag ratio. The database provides a variation in trim angle of attack on the order of +/-2deg, and a range in lift-to-drag ratio of +/-0.035 for typical vehicle flight conditions

Sketch-To-Solution: An Exploration of Viscous CFD with Automatic Grids

Numerical simulation of the Reynolds-averaged NavierStokes (RANS) equations has become a critical tool for the design of aerospace vehicles. However, the issues that affect the grid convergence of three dimensional RANS solutions are not completely understood, as documented in the AIAA Drag Prediction Workshop series. Grid adaption methods have the potential for increasing the automation and discretization error control of RANS solutions to impact the aerospace design and certification process. The realization of the CFD Vision 2030 Study includes automated management of errors and uncertainties of physics-based, predictive modeling that can set the stage for ensuring a vehicle is in compliance with a regulation or specification by using analysis without demonstration in flight test (i.e., certification or qualification by analysis). For example, the Cart3D inviscid analysis package has automated Cartesian cut-cell gridding with output-based error control. Fueled by recent advances in the fields of anisotropic grid adaptation, error estimation, and geometry modeling, a similar work flow is explored for viscous CFD simulations; where a CFD application engineer provides geometry, boundary conditions, and flow parameters, and the sketch-to-solution process yields a CFD simulation through automatic, error-based, grid adaptation

Integrated Flush Air Data Sensing System Modeling for Planetary Entry Guidance with Direct Force Control

Flush air data sensing (FADS) systems have been previously used at both Earth and Mars to provide onboard estimates of angle of attack, sideslip angle, and dynamic pressure. However, these FADS data were often not used in an in-the-loop sense to inform the onboard guidance and control systems. A method to integrate FADS-derived density and wind estimates with a numerical predictor-corrector guidance algorithm is presented. The method is demonstrated in a high-fidelity simulation of a human-scale Mars entry vehicle that utilizes a hypersonic inflatable aerodynamic decelerator (HIAD) with direct force control. Effects on guidance commands and state uncertainties both with and without FADS system modeling are presented and discussed

Watasemycin biosynthesis in Streptomyces venezuelae : thiazoline C-methylation by a type B radical-SAM methylase homologue

2-Hydroxyphenylthiazolines are a family of iron-chelating nonribosomal peptide natural products that function as virulence-conferring siderophores in various Gram-negative bacteria. They have also been reported as metabolites of Gram-positive Streptomyces species. Transcriptional analyses of Streptomyces venezuelae ATCC 10712 revealed that its genome contains a putative 2-hydroxyphenylthiazoline biosynthetic gene cluster. Heterologous expression of the gene cluster in Streptomyces coelicolor M1152 showed that the mono- and dimethylated derivatives, thiazostatin and watasemycin, respectively, of the 2-hydroxyphenylthiazoline enantiopyochelin are two of its metabolic products. In addition, isopyochelin, a novel isomer of pyochelin containing a C-methylated thiazolidine, was identified as a third metabolic product of the cluster. Metabolites with molecular formulae corresponding to aerugine and pulicatins A/B were also detected. The structure and stereochemistry of isopyochelin were confirmed by comparison with synthetic standards. The role of two genes in the cluster encoding homologues of PchK, which is proposed to catalyse thiazoline reduction in the biosynthesis of enantiopyochelin in Pseudomonas protegens, was investigated. One was required for the production of all the metabolic products of the cluster, whereas the other appears not to be involved in the biosynthesis of any of them. Deletion of a gene in the cluster encoding a type B radical-SAM methylase homologue abolished the production of watasemycin, but not thiazostatin or isopyochelin. Feeding of thiazostatin to the mutant lacking the functional PchK homologue resulted in complete conversion to watasemycin, demonstrating that thiazoline C-methylation by the type B radical-SAM methylase homologue is the final step in watasemycin biosynthesis

Designing end use components for additive manufacturing: navigating an emerging field

Despite much excitement, research and development, Additive Manufacturing (AM) as a series production process for end-use components and products is not yet widespread or considered mainstream. However, there is a clear potential for AM to form a viable alternative to many conventional manufacturing processes, especially in low to medium production volumes. A key enabler for this transformation is the capacity to design components and products that are both able to exploit AM capabilities and avoid its limitations. In recent years, many studies have explored the topic of Design for Additive Manufacturing (DfAM). This report presents an overview of the state of the art of this research area. A systematic review has been carried out to identify the most significant academic studies on the topic. The review resulted in 66 key resources being identified and critically reviewed. These resources have been reviewed and categorised using a generic model of the design process. This categorisation provides and easy and immediate way to map and navigate this emerging field. Consequently, five major research areas are presented: 1. Process planning 2. Detail design 3. Embodiment design 4. Conceptual design 5. Design processes In the discussion, these research areas are examined with the aim of highlighting shortcomings and providing future research directions