Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

Network-based high level data classification

Traditional supervised data classification considers only physical features (e. g., distance or similarity) of the input data. Here, this type of learning is called low level classification. On the other hand, the human (animal) brain performs both low and high orders of learning and it has facility in identifying patterns according to the semantic meaning of the input data. Data classification that considers not only physical attributes but also the pattern formation is, here, referred to as high level classification. In this paper, we propose a hybrid classification technique that combines both types of learning. The low level term can be implemented by any classification technique, while the high level term is realized by the extraction of features of the underlying network constructed from the input data. Thus, the former classifies the test instances by their physical features or class topologies, while the latter measures the compliance of the test instances to the pattern formation of the data. Our study shows that the proposed technique not only can realize classification according to the pattern formation, but also is able to improve the performance of traditional classification techniques. Furthermore, as the class configuration's complexity increases, such as the mixture among different classes, a larger portion of the high level term is required to get correct classification. This feature confirms that the high level classification has a special importance in complex situations of classification. Finally, we show how the proposed technique can be employed in a real-world application, where it is capable of identifying variations and distortions of handwritten digit images. As a result, it supplies an improvement in the overall pattern recognition rate.

High-level languages for small devices: A case study

In this paper we study, through a concrete case, the feasibility of using a high-level, general-purpose logic language in the design and implementation of applications targeting wearable computers. The case study is a "sound spatializer" which, given real-time signáis for monaural audio and heading, generates stereo sound which appears to come from a position in space. The use of advanced compile-time transformations and optimizations made it possible to execute code written in a clear style without efñciency or architectural concerns on the target device, while meeting strict existing time and memory constraints. The final executable compares favorably with a similar implementation written in C. We believe that this case is representative of a wider class of common pervasive computing applications, and that the techniques we show here can be put to good use in a range of scenarios. This points to the possibility of applying high-level languages, with their associated flexibility, conciseness, ability to be automatically parallelized, sophisticated compile-time tools for analysis and verification, etc., to the embedded systems field without paying an unnecessary performance penalty.

High-level concept annotation using ontology and probabilistic inference

Image annotation is a significant step towards semantic based image retrieval. Ontology is a popular approach for semantic representation and has been intensively studied for multimedia analysis. However, relations among concepts are seldom used to extract higher-level semantics. Moreover, the ontology inference is often crisp. This paper aims to enable sophisticated semantic querying of images, and thus contributes to 1) an ontology framework to contain both visual and contextual knowledge, and 2) a probabilistic inference approach to reason the high-level concepts based on different sources of information. The experiment on a natural scene database from LabelMe database shows encouraging results.

EMI issues in high power and high level diode-clamped converters

Planar busbar is a good candidate to reduce interconnection inductance in high power inverters compared with cables. However, power switching components with fast switching combined with hard switched-converters produce high di/dt during turn off time and busbar stray inductance then becomes an important issue which creates overvoltage. It is necessary to keep the busbar stray inductance as low as possible to decrease overvoltage and Electromagnetic Interference (EMI) noise. In this paper, the effect of different transient current loops on busbar physical structure of the high-voltage high-level diode-clamped converters will be highlighted. Design considerations of proper planar busbar will also be presented to optimise the overall design of diode-clamped converters.

Development of a high-level gene expression system for the production of bioplastics in sugarcane

Despite various approaches, the production of biodegradable plastics such as polyhydroxybutyrate (PHB) in transgenic plants has met with limited success due largely to low expression levels. Even in the few instances where high levels of protein expression have been reported, the transgenic plants have been stunted indicating PHB is phytotoxic (Poirier 2002). This PhD describes the application of a novel virus-based gene expression technology, termed InPAct („In Plant Activation.), for the production of PHB in tobacco and sugarcane. InPAct is based on the rolling circle replication mechanism by which circular ssDNA viruses replicate and provides a system for controlled, high-level gene expression. Based on these features, InPAct was thought to represent an ideal system to enable the controlled, high-level expression of the three phb genes (phbA, phbB and phbC) required for PHB production in sugarcane at a preferred stage of plant growth. A Tobacco yellow dwarf virus (TbYDV)-based InPAct-phbA vector, as well as linear vectors constitutively expressing phbB and phbC were constructed and different combinations were used to transform tobacco leaf discs. A total of four, eight, three and three phenotypically normal tobacco lines were generated from discs transformed with InPAct-phbA, InPAct-phbA + p1300-TaBV P-phbB/phbC- 35S T, p1300-35S P-phbA-NOS T + p1300-TaBV P-phbB/phbC-35S T and InPAct-GUS, respectively. To determine whether the InPAct cassette could be activated in the presence of the TbYDV Rep, leaf samples from the eight InPActphbA + p1300-TaBV P-phbB/phbC-35S T plants were agroinfiltrated with p1300- TbYDV-Rep/RepA. Three days later, successful activation was indicated by the detection of episomes using both PCR and Southern analysis. Leaf discs from the eight InPAct-phbA + p1300-TaBV P-phbB/phbC-35S T transgenic plant lines were agroinfiltrated with p1300-TbYDV-Rep/RepA and leaf tissue was collected ten days post-infiltration and examined for the presence of PHB granules. Confocal microscopy and TEM revealed the presence of typical PHB granules in five of the eight lines, thus demonstrating the functionality of InPActbased PHB production in tobacco. However, analysis of leaf extracts by HPLC failed to detect the presence of PHB suggesting only very low level expression levels. Subsequent molecular analysis of three lines revealed low levels of correctly processed mRNA from the catalase intron contained within the InPAct cassette and also the presence of cryptic splice sites within the intron. In an attempt to increase expression levels, new InPAct-phb cassettes were generated in which the castorbean catalase intron was replaced with a synthetic intron (syntron). Further, in an attempt to both increase and better control Rep/RepA-mediated activation of InPAct cassettes, Rep/RepA expression was placed under the control of a stably integrated alc switch. Leaf discs from a transgenic tobacco line (Alc ML) containing 35S P-AlcR-AlcA P-Rep/RepA were supertransformed with InPAct-phbAsyn or InPAct-GUSsyn using Agrobacterium and three plants (lines) were regenerated for each construct. Analysis of the RNA processing of the InPAct-phbAsyn cassette revealed highly efficient and correct splicing of the syntron, thus supporting its inclusion within the InPAct system. To determine the efficiency of the alc switch to activate InPAct, leaf material from the three Alc ML + InPAct-phbAsyn lines was either agroinfiltrated with 35S P-Rep/RepA or treated with ethanol. Unexpectedly, episomes were detected not only in the infiltrated and ethanol treated samples, but also in non-treated samples. Subsequent analysis of transgenic Alc ML + InPAct-GUS lines, confirmed that the alc switch was leaky in tissue culture. Although this was shown to be reversible once plants were removed from the tissue culture environment, it made the regeneration of Alc ML + InPAct-phbsyn plant lines extremely difficult, due to unintentional Rep expression and therefore high levels of phb expression and phytotoxic PHB production. Two Alc ML + InPAct-phbAsyn + p1300-TaBV P-phbB/phbC-35S T transgenic lines were able to be regenerated, and these were acclimatised, alcohol-treated and analysed. Although episome formation was detected as late as 21 days post activation, no PHB was detected in the leaves of any plants using either microscopy or HPLC, suggesting the presence of a corrupt InPAct-phbA cassette in both lines. The final component of this thesis involved the application of both the alc switch and the InPAct systems to sugarcane in an attempt to produce PHB. Initial experiments using transgenic Alc ML + InPAct-GUS lines indicated that the alc system was not functional in sugarcane under the conditions tested. The functionality of the InPAct system, independent of the alc gene switch, was subsequently examined by bombarding the 35S Rep/RepA cassette into leaf and immature leaf whorl cells derived from InPAct-GUS transgenic sugarcane plants. No GUS expression was observed in leaf tissue, whereas weak and irregular GUS expression was observed in immature leaf whorl tissue derived from two InPAct- GUS lines and two InPAct-GUS + 35S P-AlcR-AlcA P-GUS lines. The most plausible reason to explain the inconsistent and low levels of GUS expression in leaf whorls is a combination of low numbers of sugarcane cells in the DNA replication-conducive S-phase and the irregular and random nature of sugarcane cells bombarded with Rep/RepA. This study details the first report to develop a TbYDV-based InPAct system under control of the alc switch to produce PHB in tobacco and sugarcane. Despite the inability to detect quantifiable levels of PHB levels in either tobacco or sugarcane, the findings of this study should nevertheless assist in the further development of both the InPAct system and the alc system, particularly for sugarcane and ultimately lead to an ethanol-inducible InPAct gene expression system for the production of bioplastics and other proteins of commercial value in plants.

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals. © 2009 Blackwell Publishing Ltd.

Submission to the inquiry into the high level of involvement of Indigenous juveniles and young adults in the criminal justice system

Introduction • The Australian Institute of Criminology (AIC) is Australia's national research and knowledge centre on crime and justice. • The Institute seeks to promote justice and reduce crime by undertaking and communicating evidence-based research to inform policy and practice. • The AIC is governed by the Criminology Research Act and has been in operation since 1973. • The AIC is pleased to have the opportunity to contribute to the Committee's Inquiry into the high level of involvement of Indigenous juveniles and young adults in the criminal justice system. • There is a great deal of evidence to demonstrate that Indigenous young people are significantly over-represented at every stage of the criminal justice system in Australia.

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.

Evidence of effectiveness : high level synthesis of research to prevent obesity and increase physical activity in children

Background Prevention of childhood obesity is a public health priority for Malaysia and many other countries. Physical activity for children is also decreasing at an alarming rate. Both conditions are associated with non-communicable diseases and with significant morbidity and mortality in later life. Systematic reviews of public health interventions provide a useful summary to inform public health practice by combining the results of a range of research studies on a specific intervention into a single report. Systematic reviews are deemed most valuable for health program development and evidence based practice. Unfortunately, many policy makers and practitioners are simply unaware of the evidence: which strategies which are most likely to provide benefit; and which strategies are known to be harmful or useless. This presentation provides a “birds eye” overview based upon recent (since 2007 to present) high quality systematic reviews of public health interventions. Method HealthEvidece.org and the Cochrane Library were searched for systematic reviews which evaluated interventions targeting obesity prevention and increasing physical activity for children. The findings of the included reviews were themed and summarized. Results Seven reviews were identified addressing obesity in the early years, and fifteen reviews addressing obesity more broadly in childhood. Additional reviews were identified aimed at increasing physical activity. The synthesis shows several strategies to be effective, however many popular strategies clearly are not. Several of the reviews were inconclusive due to an absence of robust primary studies. Amongst the findings, interventions undertaken in the school setting appear very promising. Conclusions There is significant evidence from systematic reviews to guide public health practice and policy, and to inform future research.

High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

High level of MT-MMP expression is associated with invasiveness of cervical cancer cells

MMP-2 (gelatinase A) has been associated with the invasive potential of many cancer cells both in vitro and in vivo. It is now becoming clear that the activation of this enzyme might be a key step in tumor invasion. This activation process has been shown to be a membrane-associated pathway inducible by various agents such as collagen type I, concanavalin A or TGF-β, but its physiological regulation is still largely unresolved. MT-MMP was recently discovered and described as a potential gelatinase-A activator. In the present study, we investigated the expression of MT-MMP (membrane-type metalloproteinase) in cervical cancer cells both in vitro and in vivo. Comparing several in vitro-transformed cervical cell lines, previously shown to display different invasive potentials, our results showed that the ability of cells to overexpress MT-MMP mRNA following ConA induction correlated with their ability to activate gelatinase A and with a highly invasive behavior. Moreover, using immunohistochemistry and in situ hybridization, we found a higher level of MT-MMP expression in invasive cervical carcinoma and lymphnode metastases compared to its expression in non-invasive CIN III lesions. Our in vivo observations also clearly demonstrated a cooperation between stromal and tumor cells for the production of MT-MMP. Taken together, our results clearly correlated high level MT-MMP expression with invasiveness, and thus suggested that MT-MMP might play a crucial role in cervical tumor invasion.

No genetic bottleneck in Plasmodium falciparum wild-type Pfcrt alleles reemerging in Hainan Island, China, following high-level chloroquine resistance

Chloroquine-resistant Plasmodium falciparum was highly prevalent in Hainan, China, in the 1970s. Twenty-five years after cessation of chloroquine therapy, the prevalence of P. falciparum wild-type Pfcrt alleles has risen to 36% (95% confidence interval, 22.1 to 52.4%). The diverse origins of wild-type alleles indicate that there was no genetic bottleneck caused by high chloroquine resistance.