Genome sequencing: a grin without a cat

In some types of unicellular algae, the chloroplasts have their own nucleus — a legacy of the time when the chloroplast was a free-living cell. The sequence of the genome in one such nucleus is now revealed.

The type II secretion system and its ubiquitous lipoprotein substrate, SsIE are required for biofilm formation and virulence of enteropathogenic escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.

A parallel DNA fragment assembly algorithm based on eulerian superpath approach

Fragments assembly is among the core problems in the research of Genome. Although many assembly tools based on the "overlap-layout-consensus" paradigm are widely used such as in the Human Genome Project currently, they still can not resolve the "repeats problem" in the DNA sequencing. For the purpose of resolving such problem, Pevzner et al. put forward a new Euler Superpath assembly algorithm. But it needs a big and complex de Bruijin graph which consumes large amounts of memories i.e. becomes the bottleneck of the performance. We present a parallel DNA fragment assembly algorithm based on the Eularian Superpath theory and solve the bottleneck in the current assembly program. The experimental results demonstrate that our approach has a good scalability, and can be used in DNA assembly of middle and large size of eukaryote genome.

Identification of novel therapeutics for complex diseases from genome-wide association data

Human genome sequencing has enabled the association of phenotypes with genetic loci, but our ability to effectively translate this data to the clinic has not kept pace. Over the past 60 years, pharmaceutical companies have successfully demonstrated the safety and efficacy of over 1,200 novel therapeutic drugs via costly clinical studies. While this process must continue, better use can be made of the existing valuable data. In silico tools such as candidate gene prediction systems allow rapid identification of disease genes by identifying the most probable candidate genes linked to genetic markers of the disease or phenotype under investigation. Integration of drug-target data with candidate gene prediction systems can identify novel phenotypes which may benefit from current therapeutics. Such a drug repositioning tool can save valuable time and money spent on preclinical studies and phase I clinical trials.

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

Background
The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.
Results
To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.
Conclusion
We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.

CIDB: Chlamydia Interactive Database for cross-querying genomics, transcriptomics and proteomics data

Chlamydiae are important pathogens of humans, birds and a wide range of animals. They are a unique group of bacteria, characterized by their developmental cycle. Chlamydia has been difficult to study because of their obligate intracellular growth habit and lack of a genetic transformation system. However, the past 5 years has seen the full genome sequencing of seven strains of Chlamydia and a rapid expansion of genomic, transcriptomic (RT-PCR, microarray) and proteomic analysis of these pathogens. The Chlamydia Interactive Database (CIDB) described here is the first database of its type that holds genomic, RT-PCR, microarray and proteomics data sets that can be cross-queried by researchers for patterns in the data. Combining the data of many research groups into a single database and cross-querying from different perspectives should enhance our understanding of the complex cell biology of these pathogens. The database is available at: http://www3.it.deakin.edu.au:8080/CIDB/.

Backward modelling and look-ahead fuzzy energy management controller for a parallel hybrid vehicle

This paper focuses on a parallel hybrid electric vehicle. It first develops a model for the vehicle using the backward-looking approach where the flow of energy starts from wheels and spreads towards engine and electric motor. Next, a fuzzy logic-based strategy is developed to control the operation of the vehicle. The objectives of the controller include managing the energy flow from engine and electric motor, controlling transmission ratio, adjusting speed, and sustaining battery's state of charge. The controller examines current vehicle speed, demand torque, slope difference, state of charge of battery, and engine and electric motor rotation speeds. Then, it determines the best values for continuous variable transmission ratio, speed, and torque. A slope window scheme is also developed to take into account the look-ahead slope information and determine the best vehicle speed for better fuel economy. The developed model and control strategy are simulated. The simulation results are presented and discussed. It is shown that the use of the proposed fuzzy controller reduces fuel consumption.

Adaptive intelligent energy management system of plug-in hybrid electric vehicle

Efficient energy management in hybrid vehicles is the key for reducing fuel consumption and emissions. To capitalize on the benefits of using PHEVs (Plug-in Hybrid Electric Vehicles), an intelligent energy management system is developed and evaluated in this paper. Models of vehicle engine, air conditioning, powertrain, and hybrid electric drive system are first developed. The effect of road parameters such as bend direction and road slope angle as well as environmental factors such as wind (direction and speed) and thermal conditions are also modeled. Due to the nonlinear and complex nature of the interactions between PHEV-Environment-Driver components, a soft computing based intelligent management system is developed using three fuzzy logic controllers. The crucial fuzzy engine controller within the intelligent energy management system is made adaptive by using a hybrid multi-layer adaptive neuro-fuzzy inference system with genetic algorithm optimization. For adaptive learning, a number of datasets were created for different road conditions and a hybrid learning algorithm based on the least squared error estimate using the gradient descent method was proposed. The proposed adaptive intelligent energy management system can learn while it is running and makes proper adjustments during its operation. It is shown that the proposed intelligent energy management system is improving the performance of other existing systems. © 2014 Elsevier Ltd.

Yield optimization of a heterologously expressed novel mouse macrophage protein

The large scale whole-genome sequencing projects have resulted in large numbers of un-characterized and un-annotated protein sequences. This presents an opportunity and a challenge to characterise these novel protein sequences. Structural biology has become a widely accepted methodology to help assign functions to such proteins, complementing other cellular and biochemical studies. However, most of these studies require the target protein to be produced in large quantities and in a highly pure and soluble state. The present study is an attempt to maximise production of a recombinant mouse macrophage protein (rMMP) over-expressed heterologously in Escherichia coli. Highest production of biomass and total protein (6.6 mg mL-1) was observed at 37 °C. Maximum cell disruption (89%) was observed during freeze-thawing and subsequent ultrasonication.

Mass balance for a novel RO/FO hybrid system in seawater desalination

Seawater Reverse Osmosis (SWRO) desalination is being used by several countries to aid the current demand for fresh water, hence numerous large scale and small scale desalination plants have been built during last decade. Despite major advancements in SWRO technology, the desalination industry is still facing significant practical issues. Two of the major issues are (1) generation of higher volumes of pre-treatment sludge, and (2) overall water recovery. This paper proposes a novel hybrid reverse osmosis (RO) - forward osmosis (FO) system to overcome the above two drawbacks. Mass balance calculations based on laboratory experiments have been used to predict increased water recovery and reduced pre-treatment sludge volume arising from large scale (340,000 m3/day of intake) and small scale (15,000 m3/day of intake) hybrid SWRO desalination plants. The percentage reduction of pre-treatment sludge volume, increase in overall RO water recovery, FO membrane area required and dilution in RO reject have been estimated.

Characterization of haemolytic phospholipase A2 activity in clinical isolates of Campylobacter concisus

A membrane-bound, haemolytic phospholipase A2 (PLA2) activity was detected in clinical strains of Campylobacter concisus isolated from children with gastroenteritis. The clinical strains were assigned into two molecular groups (genomospecies) based on PCR amplification of their 23S rDNA. This calcium-dependent, heat-stable, haemolytic PLA2 activity was detected in strains from both genomospecies. A crude haemolysin extract (CHE) was initially prepared from cellular outer-membrane proteins of these isolates and was further fractionated by ultrafiltration. The haemolytic activity of the extracted fraction (R30) was retained by ultrafiltration using a 30 kDa molecular mass cut-off filter, and was designated haemolysin extract (HE). Both CHE and HE had PLA2 activity and caused stable vacuolating and cytolytic effects on Chinese hamster ovary cells in tissue culture. Primers for the conserved region of pldA gene (phospholipase A gene) from Campylobacter coli amplified a gene region of 460 bp in all tested isolates, confirming the presence of a homologous PLA gene sequence in C. concisus. The detection of haemolytic PLA₂ activity in C. concisus indicates the presence of a potential virulence factor in this species and supports the hypothesis that C. concisus is a possible opportunistic pathogen.

Ontogeny of putative GABAergic neurons and their receptors in the nervous system of the crayfish Cherax destructor

Inhibitory neurons exert control the expression of many aspects of behaviour by regulating the effectiveness of excitatory neural function. By comparison with excitatory neural systems, relatively little is known about the development of inhibitory neurons and the influence which these neurons exert on the development of other neural systems. Two issues which relate to the development of inhibitory neurons are of particular interest. First, a paradox arises when inhibitory neurons are considered in terms of modern models of synaptic development which involve activity-dependent mechanisms of synaptic plasticity. Second, there is some evidence that inhibitory neurotransmitters may act in a special trophic manner during the early development of nervous systems. Investigations of these issues would be greatly facilitated in a neural system in which it was possible to experimentally control aspects of the development of individual pre- and postsynaptic cells. The aim of the results presented in this thesis was to characterise the normal development of one such system: the GABAergic inhibitory system of the Australian freshwater crayfish, Cherax destructor. The ontogeny of the inhibitory neurotransmitter GABA across the embryonic period of 30% to 100% development was investigated using immunohistochemical techniques. GABA-like immunoreactive cells and fibres were first detected in the embryonic brain region. The expression of GABA-like immunoreactivity progressed along a rostro-caudal gradient, with GABA-like immunoreactive cells being detected in the most anterior thoracic ganglia at 45% development and in all ganglia by 65% development. GABA-like immunoreactive fibres were evident in peripheral nerves as early as 55% development and ramified extensively throughout the neuropil of the nervous system by 65% development. By contrast, immunoreactivity to the primary excitatory neurotransmitter, glutamate, was not detected until 60-65% development. Glutamate-like immunoreactivity at 60-65% development was evident only in the form of punctate staining in the midline of the ventral nerve cord. Cell body staining was observed only at 90% development and was restricted to only a few cells on the periphery of the ventral nerve cord. Radio-labelled ligand binding methods and autoradiography were used to study the expression of putative GABA receptors in the Cherax embryos from 30% to 100% development. Specific binding was evident in the earliest embryos studies at 30% development. There was an initial increase in binding from 30% to 40% development, followed by a dramatic drop to almost zero binding at 50-55% development. This was followed by a gradual increase in binding levels with age, reaching a plateau at 85% development. Preliminary pharmacological evaluation of binding indicated that at least three GABA receptor types were expressed during embryonic development. Methods for culturing, dissociated neural tissues explanted form Cherax embryos at 85% development were established. The success of cultures was demonstrated by neurite extension, and neuronal networks in which neurons appeared to form connections with other neurons and with explanted muscle cells after two days in culture. Immunohistochemical studies demonstrated that some explanted neurons expressed GABA-like immunoreactivity within two days of explanting. These studies have provided a comprehensive description of the development of GABAergic neurons and their receptors in Cherax destructor embryos. The very early expression of GABA-like immunoreactivity, coupled with the early onset of specific GABA binding, strongly indicates that the GABAergic neurons are functional and able to exert an effect on other cells during much of the period of nervous system development in crayfish embryos. These results support the hypothesis that inhibitory neurons may play an important role as regulators of the overall process of assembly and maturation of the nervous system and provide a substantial basis for future experimental studies in which the specific action of inhibitory neurons on the development of discrete components of the crayfish nervous system may be investigated.

Plasmodium falciparum possesses two GRASP proteins that are differentially targeted to the Golgi complex via a higher- and lower-eukaryote-like mechanism

Plasmodium falciparum, the causative agent of malaria, relies on a complex protein-secretion system for protein targeting into numerous subcellular destinations. Recently, a homologue of the Golgi re-assembly stacking protein (GRASP) was identified and used to characterise the Golgi organisation in this parasite. Here, we report on the presence of a splice variant that leads to the expression of a GRASP isoform. Although the first GRASP protein (GRASP1) relies on a well-conserved myristoylation motif, the variant (GRASP2) displays a different N-terminus, similar to GRASPs found in fungi. Phylogenetic analyses between GRASP proteins of numerous taxa point to an independent evolution of the unusual N-terminus that could reflect unique requirements for Golgi-dependent protein sorting and organelle biogenesis in P. falciparum. Golgi association of GRASP2 depends on the hydrophobic N-terminus that resembles a signal anchor, leading to a unique mode of Golgi targeting and membrane attachment.

Haptics and virtual reality in modern manufacturing

Product assembly is one of the most studied processes in modern manufacturing. In recent years a number of computer-based virtual reality systems have been proposed, developed and adopted by the manufacturing industries. Such systems have major advantages over conventional training practices for product assembly. Significant cost savings can be realized due to shorter training-scenario development times and reuse of existing engineering math models. In addition, the time span from the product design to full production can be shortened due to non-reliance on actual components and subsystems for training. Such training systems are effective if the knowledge required to be transferred is just process sequence such as assembly sequence. However, knowledge transfer for procedural and cognitive learning as well as skills development is very limited, due to the lack of user interactivity and immersion.

This talk will focus on a research technology platform where haptics and virtual reality are integrated to create an effective environment for production assembly operators’ training. In this system virtual reality provides the grounds for realistic visualization, as well as immersion, whereas haptics enforces physical constraints within the virtual world generating the feelings of realistic interaction, making it accessible for formal learning and better understanding during task performance.

The developed research technology platform imitates real physical training scenarios by providing comprehensive user interaction, constrained within the physical limitations of the real world. Through the utilization of a haptics device, providing realistic force feedback, users are able to engage in product assembly training with a stronger sense of ‘reality’.