High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation

Two transgenic callus lines of rice, stably expressing a β-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.

The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67 348 polypeptide encoded by genome segment 8

The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSV S8 is 1 914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1 810 which is capable of encoding a protein of M(r) 67 348. The N-terminal amino acid sequence of a ~43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto- catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a ~43K major capsid protein.

Genome segment 5 of rice ragged stunt virus encodes a virion protein

The complete nucleotide sequence of the genome segment 5 (S5) of a Thai isolate of rice ragged stunt virus (RRSV) was determined. The 2682 nucleotide sequence contains a single long open reading frame capable of encoding a polypeptide with a molecular mass of ~91 kDa. Polypeptides encoded by various truncated cDNAs of S5 were expressed using the pGEX fusion protein vector and the highest level of fusion protein was obtained from a construct encoding a hydrophilic region of S5 protein. Antibodies raised against this fusion protein recognized a minor polypeptide, with a molecular mass of ~ 91 kDa, that was present in purified preparations of RRSV particles, infected insect vectors and infected rice plants. This indicates that RRSV S5 encodes a minor structural protein. Comparing the RRSV S5 sequence with sequences of other reo-viruses did not reveal any significant sequence similarities.

Putative full-length clones of the genomic DNA segments of subterranean clover stunt virus and identification of the segment coding for the viral coat protein

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

Sequence and identification of the barley yellow dwarf virus coat protein gene

The nucleotide sequence of the coat protein gene of barley yellow dwarf virus (BYDV, PAV serotype) was determined, and the amino acid sequence was deduced. The open reading frame, encoding a protein of relative molecular mass (Mr) 22,047, was confirmed as the coat protein gene by comparison with amino acid sequences of tryptic peptides derived from dissociated virions. In addition, a fragment of this gene expressed in Escherichia coli produced a product which was recognized by antibodies prepared against purified BYDV virions. An overlapping reading frame encoding an Mr 17,147 protein is contained completely within the coat protein gene. © 1988.

Urban acupuncture: Hybrid social and technological practices for hyperlocal placemaking

This paper considers an emerging planning practice that uses networked connections to interact with urban places and re-create enlivened cities. The paper presents “urban acupuncture” as a new planning approach that broadens communication and strategically targets interventions across the city. Defined as an approach, which, through the use of digital social networks and interactions, involves citizens and planners in place activations in order to stimulate and reinvigorate place, thus creating meaningful relationships between citizens and their urban settings. This paper uses the UR[BNE] Brisbane Festival 2012 as a qualitative case study of urban acupuncture, best defined as a hyper-localized healing treatment through place activation to enliven and recreate cities. It examines the challenges faced and opportunities embraced by a network of urban professionals. Their aim was to activate the underused urban spaces of central Brisbane through the festival's activities and events. The findings identify the key elements required to design public spaces using socially and technologically networked interactions.

A hybrid constructive heuristic and simulated annealing for railway crew scheduling

Railway crew scheduling problem is the process of allocating train services to the crew duties based on the published train timetable while satisfying operational and contractual requirements. The problem is restricted by many constraints and it belongs to the class of NP-hard. In this paper, we develop a mathematical model for railway crew scheduling with the aim of minimising the number of crew duties by reducing idle transition times. Duties are generated by arranging scheduled trips over a set of duties and sequentially ordering the set of trips within each of duties. The optimisation model includes the time period of relief opportunities within which a train crew can be relieved at any relief point. Existing models and algorithms usually only consider relieving a crew at the beginning of the interval of relief opportunities which may be impractical. This model involves a large number of decision variables and constraints, and therefore a hybrid constructive heuristic with the simulated annealing search algorithm is applied to yield an optimal or near-optimal schedule. The performance of the proposed algorithms is evaluated by applying computational experiments on randomly generated test instances. The results show that the proposed approaches obtain near-optimal solutions in a reasonable computational time for large-sized problems.

Role of Human Topoisomerase I in DNA Repair and Apoptosis

Human topoisomerase I (htopoI) is an enzyme that up to now was believed to function mainly in the removal of torsional stress generated during transcription and replication. In 1998, it was found that htopoI might play another important role in the cellular response to DNA damage -- the so-called htopoI damage response. Since this initial discovery, many studies have suggested that the htopoI damage response is involved in DNA repair as well as in apoptosis. Here we discuss the earliest as well as the latest results in this field. Combining all of the published and as yet unpublished results, we suggest and discuss a model of how htopoI may function during DNA repair and apoptosis. Furthermore, numerous results show that the htopoI damage response is not a spontaneous event, but is strictly regulated by cellular signalling pathways. We discuss which pathways may be involved and how the htopoI damage response is activated. Although the htopoI damage response was discovered several years ago, research in this area is just beginning. The future will surely bring more clarity regarding the precise mechanism behind the involvement of htopoI in DNA repair and apoptosis, as well as its implications for a broader understanding of how the human organism ensures genomic stability.

Cellular stress triggers the human topoisomerase I damage response independently of DNA damage in a p53 controlled manner

The 'human topoisomerase I (htopoI) damage response' was reported to be triggered by various kinds of DNA lesions. Also, a high and persistent level of htopoI cleavage complexes correlated with apoptosis. In the present study, we demonstrate that DNA damage-independent induction of cell death using colcemid and tumor necrosis factor is also accompanied by a strong htopoI response that correlates with the onset of apoptotic hallmarks. Consequently, these results suggest that htopoI cleavage complex formation may be caused by signaling pathways independent of the kind of cellular stress. Thus, protein interactions or signaling cascades induced by DNA damage or cellular stress might lead to the formation of stabilized cleavage complexes rather than the DNA lesion itself. Finally, we show that p53 not only plays a key role in the regulation of the htopoI response to UV-C irradiation but also to treatment with colcemid.

Tensile properties of graphene nanotube hybrid structures : a molecular dynamics study

Graphene has been reported with record-breaking properties which have opened up huge potential applications. A considerable research has been devoted to manipulate or modify the properties of graphene to target a more smart nanoscale device. Graphene and carbon nanotube hybrid structure (GNHS) is one of the promising graphene derivates, while their mechanical properties have been rarely discussed in literature. Therefore, such a studied is conducted in this paper basing on the large-scale molecular dynamics simulation. The target GNHS is constructed by considering two separate graphene layers that being connected by single-wall carbon nanotubes (SWCNTs) according to the experimental observations. It is found that the GNHSs exhibit a much lower yield strength, Young’s modulus, and earlier yielding comparing with a bilayer graphene sheet. Fracture of studied GNHSs is found to fracture located at the connecting region between carbon nanotubes (CNTs) and graphene. After failure, monatomic chains are normally observed at the front of the failure region, and the two graphene layers at the failure region without connecting CNTs will adhere to each other, generating a bilayer graphene sheet scheme (with a layer distance about 3.4 Å). This study will enrich the current understanding of the mechanical performance of GNHS, which will guide the design of GNHS and shed lights on its various applications.

Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients

Recent studies suggest that genetic and environmental factors do not account for all the schizophrenia risk and epigenetics also plays a role in disease susceptibility. DNA methylation is a heritable epigenetic modification that can regulate gene expression. Genome-Wide DNA methylation analysis was performed on post-mortem human brain tissue from 24 patients with schizophrenia and 24 unaffected controls. DNA methylation was assessed at over 485 000 CpG sites using the Illumina Infinium Human Methylation450 Bead Chip. After adjusting for age and post-mortem interval (PMI), 4 641 probes corresponding to 2 929 unique genes were found to be differentially methylated. Of those genes, 1 291 were located in a CpG island and 817 were in a promoter region. These include NOS1, AKT1, DTNBP1, DNMT1, PPP3CC and SOX10 which have previously been associated with schizophrenia. More than 100 of these genes overlap with a previous DNA methylation study of peripheral blood from schizophrenia patients in which 27 000 CpG sites were analysed. Unsupervised clustering analysis of the top 3 000 most variable probes revealed two distinct groups with significantly more people with schizophrenia in cluster one compared to controls (p = 1.74x10-4). The first cluster was composed of 88% of patients with schizophrenia and only 12% controls while the second cluster was composed of 27% of patients with schizophrenia and 73% controls. These results strongly suggest that differential DNA methylation is important in schizophrenia etiology and add support for the use of DNA methylation profiles as a future prognostic indicator of schizophrenia.

Pathway to sustainability : the Lady Elliot Island hybrid solar power station

The Lady Elliot Island eco-resort, on the Great Barrier Reef, operates with a strong sustainability ethic, and has broken away from its reliance on diesel generators, an initiative which has ongoing and substantial economic benefit. The first step was an energy audit that led to a 35% reduction in energy usage, to an average of 575 kWh per day. The eco-resort then commissioned a hybrid solar power station, in 2008, with energy storage in battery banks. Solar power is currently (2013) providing about 160 kWh of energy per day, and the eco-resort’s diesel fuel usage has decreased from 550 to 100 litres per day, enabling the power station to pay for itself in 3 years. The eco-resort plans to complete its transition to renewable energy by 2015, by installing additional solar panels, and a 10-15 kW wind turbine. This paper starts by discussing why the eco-resort chose a hybrid solar power station to transition to renewable energy, and the barriers to change. It then describes the power station, upgrades through to 2013, the power control system, the problems that were solved to realise the potential of a facility operating in a harsh and remote environment, and its performance. The paper concludes by outlining other eco-resort sustainability practices, including education and knowledge-sharing initiatives, and monitoring the island’s environmental and ecological condition.

Hybrid model for motorway travel time estimation considering increased detector spacing

Travel time estimation and prediction on motorways has long been a topic of research. Prediction modeling generally assumes that the estimation is perfect. No matter how good is the prediction modeling- the errors in estimation can significantly deteriorate the accuracy and reliability of the prediction. Models have been proposed to estimate travel time from loop detector data. Generally, detectors are closely spaced (say 500m) and travel time can be estimated accurately. However, detectors are not always perfect, and even during normal running conditions few detectors malfunction, resulting in increase in the spacing between the functional detectors. Under such conditions, error in the travel time estimation is significantly large and generally unacceptable. This research evaluates the in-practice travel time estimation model during different traffic conditions. It is observed that the existing models fail to accurately estimate travel time during large detector spacing and congestion shoulder periods. Addressing this issue, an innovative Hybrid model that only considers loop data for travel time estimation is proposed. The model is tested using simulation and is validated with real Bluetooth data from Pacific Motorway Brisbane. Results indicate that during non free flow conditions and larger detector spacing Hybrid model provides significant improvement in the accuracy of travel time estimation.

A hybrid genetic algorithm for the energy-efficient virtual machine placement problem in data centers

Server consolidation using virtualization technology has become an important technology to improve the energy efficiency of data centers. Virtual machine placement is the key in the server consolidation technology. In the past few years, many approaches to the virtual machine placement have been proposed. However, existing virtual machine placement approaches consider the energy consumption by physical machines only, but do not consider the energy consumption in communication network, in a data center. However, the energy consumption in the communication network in a data center is not trivial, and therefore should be considered in the virtual machine placement. In our preliminary research, we have proposed a genetic algorithm for a new virtual machine placement problem that considers the energy consumption in both physical machines and the communication network in a data center. Aiming at improving the performance and efficiency of the genetic algorithm, this paper presents a hybrid genetic algorithm for the energy-efficient virtual machine placement problem. Experimental results show that the hybrid genetic algorithm significantly outperforms the original genetic algorithm, and that the hybrid genetic algorithm is scalable.

Development of an integrated GIS and land use planning course : impacts of hybrid instructional methods

This study reports an action research undertaken at Queensland University of Technology. It evaluates the effectiveness of the integration of GIS within the substantive domains of an existing land use planning course in 2011. Using student performance, learning experience survey, and questionnaire survey data, it also evaluates the impacts of incorporating hybrid instructional methods (e.g., in-class and online instructional videos) in 2012 and 2013. Results show that: students (re)iterated the importance of GIS in the course justifying the integration; the hybrid methods significantly increased student performance; and unlike replacement, the videos are more suitable as a complement to in-class activity.