Advanced Engineering of Lipid Metabolism in Nicotiana benthamiana Using a Draft Genome and the V2 Viral Silencing-Suppressor Protein

The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18:1 to elongation reactions beginning with 18:1 as substrate. These V2-based leaf assays produced ~50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays. © 2012 Naim et al.

Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis

In plants, silencing of mRNA can be transmitted from cell to cell and also over longer distances from roots to shoots. To investigate the long-distance mechanism, WT and mutant shoots were grafted onto roots silenced for an mRNA. We show that three genes involved in a chromatin silencing pathway, NRPD1a encoding RNA polymerase IVa, RNA-dependent RNA polymerase 2 (RDR2), and DICER-like 3 (DCL3), are required for reception of long-distance mRNA silencing in the shoot. A mutant representing a fourth gene in the pathway, argonaute4 (ago4), was also partially compromised in the reception of silencing. This pathway produces 24-nt siRNAs and resulted in decapped RNA, a known substrate for amplification of dsRNA by RDR6. Activation of silencing in grafted shoots depended on RDR6, but no 24-nt siRNAs were detected in mutant rdr6 shoots, indicating that RDR6 also plays a role in initial signal perception. After amplification of decapped transcripts, DCL4 and DCL2 act hierarchically as they do in antiviral resistance to produce 21- and 22-nt siRNAs, respectively, and these guide mRNA degradation. Several dcl genotypes were also tested for their capacity to transmit the mobile silencing signal from the rootstock. dcl1-8 and a dcl2 dcl3 dcl4 triple mutant are compromised in micro-RNA and siRNA biogenesis, respectively, but were unaffected in signal transmission. © 2007 by The National Academy of Sciences of the USA.

Correlating flow induced shear stress and chondrocytes activity in micro-porous scaffold using computational fluid dynamics and rapid prototyping

Flow induced shear stress plays an important role in regulating cell growth and distribution in scaffolds. This study sought to correlate wall shear stress and chondrocytes activity for engineering design of micro-porous osteochondral grafts based on the hypothesis that it is possible to capture and discriminate between the transmitted force and cell response at the inner irregularities. Unlike common tissue engineering therapies with perfusion bioreactors in which flow-mediated stress is the controlling parameter, this work assigned the associated stress as a function of porosity to influence in vitro proliferation of chondrocytes. D-optimality criterion was used to accommodate three pore characteristics for appraisal in a mixed level fractional design of experiment (DOE); namely, pore size (4 levels), distribution pattern (2 levels) and density (3 levels). Micro-porous scaffolds (n=12) were fabricated according to the DOE using rapid prototyping of an acrylic-based bio-photopolymer. Computational fluid dynamics (CFD) models were created correspondingly and used on an idealized boundary condition with a Newtonian fluid domain to simulate the dynamic microenvironment inside the pores. In vitro condition was reproduced for the 3D printed constructs seeded by high pellet densities of human chondrocytes and cultured for 72 hours. The results showed that cell proliferation was significantly different in the constructs (p<0.05). Inlet fluid velocity of 3×10-2mms-1 and average shear stress of 5.65×10-2 Pa corresponded with increased cell proliferation for scaffolds with smaller pores in hexagonal pattern and lower densities. Although the analytical solution of a Poiseuille flow inside the pores was found insufficient for the description of the flow profile probably due to the outside flow induced turbulence, it showed that the shear stress would increase with cell growth and decrease with pore size. This correlation demonstrated the basis for determining the relation between the induced stress and chondrocyte activity to optimize microfabrication of engineered cartilaginous constructs.

A Literature Review of Wellness, Wellbeing and Quality of Life Issues as they impact upon the Australian Mining Sector

Australia’s mining boom Global demand for minerals and energy products has fuelled Australia’s recent resources boom and has led to the rapid expansion of mining projects not only in remote locations but increasingly in settled traditionally agricultural rural areas. A fundamental shift has also occurred in the provisioning of skilled and semi-skilled workers. The huge acceleration in industry demand for labour has been accompanied by the entrenchment of workforce arrangements largely dependent on fly-in, fly-out (FIFO) and drive–in, drive–out (DIDO) non-resident workers (NRWs). While NRWs are working away from their homes, they are usually accommodated in work camps or ‘villages’ for the duration of their work cycle which are normally comprised of many consecutive days of 12-hour day- and night-shifts. The health effects of this form of employment and the accompanying lifestyle is increasingly becoming contentious. Impacts on personal wellness, wellbeing and quality of life essentially remain under-researched and thus misunderstood. Sodexo in Australia Sodexo began operations in Australia in 1982, and has since become a leader in providing Quality of Life (QOL) services to businesses across the country. The 6,000 Australian employees are part of a global Sodexo team of 413,000 people. Sodexo in Australia designs, delivers and manages on-site their QOL services at 320 diverse site locations, including remote sites. Sodexo operates in a range of sectors, including the mining industry. Service plans are tailored to suit the individual needs of organisations. Sodexo Remote Sites has previously conducted unpublished research among mining workers in Australia. The results highlighted needs and expectations of Australian mining workers. Main insights about workers’ requirements were directed towards: • contacts with closest; • warm rest time around proper and varied meals; • additional services to help them better enjoy their life onsite and/or make the most of it; • organise their transportation; • promote community living; and • finding balance between professional and personal life. The brief for this current research is aimed at building upon this knowledge. Research brief Expectations for quality of life and wellness and wellbeing services are increasing dramatically. It's getting costlier and more difficult to retain valuable employees. This is particularly the case in the Australian mining sector. Given the level of interest in ensuring healthy workplaces in Australia, Sodexo has commissioned QUT to conduct a literature review. The objectives as specified by Sodexo are: Objective 1: To define the concepts of wellness and wellbeing and quality of life in Australia Objective 2: To examine how wellness and wellbeing are developed within organisations in Australia and how they impact on employee and organizational performance. More specifically, to review the literature that could be sourced about: • challenges of the mining environment; • the mining lifestyle – implications for health, wellness and daily life; • personal health and wellness of Australian mining workers; • factors affecting health in mines and perceived support for health and wellness; and • the impact of employer investment in health on perceptions and behaviour of employees. Objective 3: To determine what impact employee wellness and well-being has on the performance of mining workers. More specifically, to review the literature that could be sourced about: • impact of obesity, alcohol, tobacco use on companies; and • links between employee engagement and satisfaction and company productivity. Accordingly this review has attempted to ascertain what factors an organisation should focus on in order to reduce absenteeism and turnover and increase commitment, satisfaction, safety and productivity, with specific reference to the mining industry in Australia. The structure of the report aligns with the stated objectives in that each of the first three parts address an objective. Part IV summarises prominent issues that have arisen and offers some concluding observations and comments.

Immunity against a Chlamydia infection and disease may be determined by a balance of IL-17 signaling

Most vaccines developed against Chlamydia using animal models provide partial protection against a genital tract infection. However, protection against the oviduct pathology associated with infertility is highly variable and often has no defining immunological correlate. When comparing two adjuvants (CTA1-DD and a combination of Cholera toxin plus CpG- oligodeoxynucleotide–CT/CpG) combined with the chlamydial major outer membrane protein (MOMP) antigen and delivered via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, we identified two vaccine groups with contrasting outcomes following infection. SL immunization with MOMP/CTA1-DD induced a 70% reduction in the incidence of oviduct pathology, without significantly altering the course of infection. Conversely, IN immunization with MOMP/CT/CpG prevented an ascending infection, but not the oviduct pathology. This anomaly presented a unique opportunity to study the mechanisms by which vaccines can prevent oviduct pathology, other than by controlling the infection. The IL-17 signaling in the oviducts was found to associate with both the enhancement of immunity to infection and the development of oviduct pathology. This conflicting role of IL-17 may provide some explanation for the discordance in protection between infection and disease and suggests that controlling immunopathology, as opposed to the rapid eradication of the infection, may be essential for an effective human chlamydial vaccine that prevents infertility.

Comparison of high level FPGA hardware design for solving tri-diagonal linear systems

Reconfigurable computing devices can increase the performance of compute intensive algorithms by implementing application specific co-processor architectures. The power cost for this performance gain is often an order of magnitude less than that of modern CPUs and GPUs. Exploiting the potential of reconfigurable devices such as Field-Programmable Gate Arrays (FPGAs) is typically a complex and tedious hardware engineering task. Re- cently the major FPGA vendors (Altera, and Xilinx) have released their own high-level design tools, which have great potential for rapid development of FPGA based custom accelerators. In this paper, we will evaluate Altera’s OpenCL Software Development Kit, and Xilinx’s Vivado High Level Sythesis tool. These tools will be compared for their per- formance, logic utilisation, and ease of development for the test case of a Tri-diagonal linear system solver.

Unsupervised temporal ensemble alignment for rapid annotation

This paper presents a novel framework for the unsupervised alignment of an ensemble of temporal sequences. This approach draws inspiration from the axiom that an ensemble of temporal signals stemming from the same source/class should have lower rank when "aligned" rather than "misaligned". Our approach shares similarities with recent state of the art methods for unsupervised images ensemble alignment (e.g. RASL) which breaks the problem into a set of image alignment problems (which have well known solutions i.e. the Lucas-Kanade algorithm). Similarly, we propose a strategy for decomposing the problem of temporal ensemble alignment into a similar set of independent sequence problems which we claim can be solved reliably through Dynamic Time Warping (DTW). We demonstrate the utility of our method using the Cohn-Kanade+ dataset, to align expression onset across multiple sequences, which allows us to automate the rapid discovery of event annotations.

Simultaneous estimation of hunting pressure, harvest and hunter success rates using WinBUGS

The use of hierarchical Bayesian spatial models in the analysis of ecological data is increasingly prevalent. The implementation of these models has been heretofore limited to specifically written software that required extensive programming knowledge to create. The advent of WinBUGS provides access to Bayesian hierarchical models for those without the programming expertise to create their own models and allows for the more rapid implementation of new models and data analysis. This facility is demonstrated here using data collected by the Missouri Department of Conservation for the Missouri Turkey Hunting Survey of 1996. Three models are considered, the first uses the collected data to estimate the success rate for individual hunters at the county level and incorporates a conditional autoregressive (CAR) spatial effect. The second model builds upon the first by simultaneously estimating the success rate and harvest at the county level, while the third estimates the success rate and hunting pressure at the county level. These models are discussed in detail as well as their implementation in WinBUGS and the issues arising therein. Future areas of application for WinBUGS and the latest developments in WinBUGS are discussed as well.

An analysis for the causes of accidents of repair, maintenance, alteration and addition works in Hong Kong

This study unveils causes of accidents in repair, maintenance, alteration and addition (RMAA) work. RMAA work is playing an increasingly important role in developed societies, including Hong Kong. Safety problems associated with RMAA work in Hong Kong has reached an alarming level. In view of rapid expansion of the RMAA sector and rising proportion of accidents in the construction industry, there is a pressing need to investigate causes of RMAA accidents. Structured interviews were conducted with RMAA contractors to explore causes of accidents in the RMAA sector. A two-round Delphi method with 13 safety experts was subsequently employed to verify the interview findings and rank the relative degree of importance for various causes of accidents. Accidents happen in RMAA work due to intersection of reasons. One of the root causes of accidents in RMAA works is low safety awareness of RMAA workers; however, wider organizational and industrial factors are not negligible. This study sheds light on why accidents happen in the RMAA sector. Only when the factors leading to accidents are identified can effective measures be made.

Residual stresses in rail-ends from the in-service insulated rail joints using neutron diffraction

Insulated rail joints (IRJs) are an integral part of the rail track signaling system and pose significant maintenance and replacement costs due to their low and fluctuating service lives. Failure occurs mainly in rail head region, bolt- holes of fishplates and web-holes of the rails. Propagation of cracks is influenced by the evolution of internal residual stresses in rails during rail manufacturing (hot-rolling, roller-straightening, and head-hardening process), and during service, particularly in heavy rail haul freight systems where loads are high. In this investigation, rail head accumulated residual stresses were analysed using neutron diffraction at the Australian Nuclear Science and Technology Organisation (ANSTO). Two ex-service two head-hardened rail joints damaged under different loading were examined and results were compared with those obtained from an unused rail joint reference sample in order to differentiate the stresses developed during rail manufacturing and stresses accumulated during rail service. Neutron diffraction analyses were carried out on the samples in longitudinal, transverse and vertical directions, and on 5mm thick sliceed samples cut by Electric Discharge Machining (EDM). For the rail joints from the service line, irrespective of loading conditions and in-service times, results revealed similar depth profiles of stress distribution. Evolution of residual stress fields in rails due to service was also accompanied by evidence of larger material flow based on reflected light and scanning electron microscopy studies. Stress evolution in the vicinity of rail ends was characterised by a compressive layer, approximately 5 mm deep, and a tension zone located approximately 5- 15mm below the surfaces. A significant variation of d0 with depth near the top surface was detected and was attributed to decarburization in the top layer induced by cold work. Stress distributions observed in longitudinal slices of the two different deformed rail samples were found to be similar. For the undeformed rail, the stress distributions obtained could be attributed to variations associated with thermo-mechanical history of the rail.

Analysis of damage to buildings following the 2010–11 Eastern Australia floods

This research analyses the extent of damage to buildings in Brisbane, Ipswich and Grantham during the recent Eastern Australia flooding and explore the role planning and design/construction regulations played in these failures. It highlights weaknesses in the current systems and propose effective solutions to mitigate future damage and financial loss under current or future climates. 2010 and early 2011 saw major flooding throughout much of Eastern Australia. Queensland and Victoria were particularly hard hit, with insured losses in these states reaching $2.5 billion and many thousands of homes inundated. The Queensland cities of Brisbane and Ipswich were the worst affected; around two-thirds of all inundated property/buildings were in these two areas. Other local government areas to record high levels of inundation were Central Highlands and Rockhampton Regional Councils in Queensland, and Buloke, Campaspe, Central Gold Fields and Loddon in Victoria. Flash flooding was a problem in a number of Victorian councils, but the Lockyer Valley west of Ipswich suffered the most extensive damage with 19 lives lost and more than 100 homes completely destroyed. In all more than 28,000 properties were inundated in Queensland and around 2,500 buildings affected in Victoria. Of the residential properties affected in Brisbane, around 90% were in areas developed prior to the introduction of floodplain development controls, with many also suffering inundation during the 1974 floods. The project developed a predictive model for estimating flood loss and occupant displacement. This model can now be used for flood risk assessments or rapid assessment of impacts following a flood event.

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.

Characterization of ENU-induced mutations in red blood cell structural proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

An overview of the marine systems simulator (MSS) : a Simulink toolbox for marine control systems

The Marine Systems Simulator (MSS) is an environment which provides the necessary resources for rapid implementation of mathematical models of marine systems with focus on control system design. The simulator targets models¡Xand provides examples ready to simulate¡Xof different floating structures and its systems performing various operations. The platform adopted for the development of MSS is Matlab/Simulink. This allows a modular simulator structure, and the possibility of distributed development. Openness and modularity of software components have been the prioritized design principles, which enables a systematic reuse of knowledge and results in efficient tools for research and education. This paper provides an overview of the structure of the MSS, its features, current accessability, and plans for future development.

Evaluation of BMP-2 gene-activated muscle grafts for cranial defect repair

Large, osseous, segmental defects heal poorly. Muscle has a propensity to form bone when exposed to an osteogenic stimulus such as that provided by transfer and expression of cDNA encoding bone morphogenetic protein-2 (BMP-2). The present study evaluated the ability of genetically modified, autologous muscle to heal large cranial defects in rats. Autologous grafts (8 mm � 2 mm) were punched from the biceps femoris muscle and transduced intraoperatively with recombinant adenovirus vector containing human BMP-2 or green fluorescent protein cDNA. While the muscle biopsies were incubating with the vector, a central parietal 8 mm defect was surgically created in the calvarium of the same animal. The gene-activated muscle graft was then implanted into the cranial defect. After 8 weeks, crania were examined radiographically, histologically, and by micro-computed tomography and dual energy X-ray absorptiometry. Although none of the defects were completely healed in this time, muscle grafts expressing BMP-2 deposited more than twice as much new bone as controls. Histology confirmed the anatomical integrity of the newly formed bone, which was comparable in thickness and mineral density to the original cranial bone. This study confirms the in vivo osteogenic properties of genetically modified muscle and suggests novel strategies for healing bone. � 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1095–1102, 2012