A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d/r) or inverted-repeat (i/r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i/r T-DNA insertions is a primary inducer of transgene silencing in plants. © CSIRO 2005.

A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance genes

A very simple leaf assay is described that rapidly and reliably identifies transgenic plants expressing the hygromycin resistance gene, hph or the phosphinothricin resistance gene, bar. Leaf tips were cut from plants propagated either in the glasshouse or in tissue culture and the cut surface embedded in solid medium containing the appropriate selective agent. Non-transgenic barley or rice leaf tips had noticeable symptoms of either bleaching or necrosis after three days on the medium and were completely bleached or necrotic after one week. Transgenic leaf tips remained green and healthy over this period. This gave unambiguous discrimination between transgenic and non-transgenic plants. The leaf assay was also effective for dicot plants tested (tobacco and peas).

How transit route passenger load and distance can together influence quality of service

This paper investigates: - correlation between transit route passenger loading and travel distance - its implications on quality of service (QoS) and resource productivity. It uses Automatic Fare Collection (AFC) data across a weekday on a premium bus line in Brisbane, Australia. A composite load-distance factor is proposed as a new measure for profiling transit route on-board passenger comfort QoS. Understanding these measures and their correlation is important for planning, design, and operational activities.

How transit route passenger load and distance can together influence quality of service

This paper investigates quality of service and resource productivity implications of transit route passenger loading and travel distance. Weekday Automatic Fare Collection data for a premium radial bus route in Brisbane, Australia, is used to investigate correlation between load factor and distance factor. Relationships between boardings and transit work indicate that distance factor generally increases with load factor. Time series analysis is then presented by examining each direction on an hour by hour basis. Inbound correlation is medium to strong across the entire span of service and strong for daytime services up to 19:30, while outbound correlation is strong across the entire span. Passengers tend to be making longer distance, peak direction commuter trips under the least comfortable conditions under stretched peak schedules than off-peak. Therefore productivity gains may be possible by adjusting fleet utilization during off-peak times. Weekday profiles by direction are established for a composite load-distance factor. A threshold corresponding to standing passengers on the Maximum Load Segment reveals that on-board loading and travel distance combined are more severe during the morning inbound peak than evening outbound peak, although the sharpness of the former suggests that encouraging shoulder peak travel during the morning would be more effective than evening peak. Further research suggested includes: consideration of travel duration factor, relating noise within hour to Peak Hour Factor, profiling load-distance factor across a range of case studies, and relating load-distance factor threshold to line length.

In vitro assessment of migratory behavior of two cell populations in a simple multichannel microdevice

Recent literature suggests that mesenchymal stem/stromal cells (MSC) could be used as Trojan Horses to deliver “death-signals” to cancer cells. Herein, we describe the development of a novel multichannel cell migration device, and use it to investigate the relative migration rates of bone marrow-derived MSC and breast cancer cells (MCF-7) towards each other. Confluent monolayers of MSC and MCF-7 were established in adjacent chambers separated by an array of 14 microchannels. Initially, culture chambers were isolated by air bubbles (air-valves) contained within each microchannel, and then bubbles were displaced to initiate the assay. The MCF-7 cells migrated preferentially towards MSC, whilst the MSC did not migrate preferentially towards the MCF-7 cells. Our results corroborate previous literature that suggests MSC migration towards cancer cells in vivo is in response to the associated inflammation rather than directly to signals secreted by the cancer cells themselves.

The “virtual” producer in the recording studio : media networks in long distance peripheral performances

The producer has for many years been a central agent in recording studio sessions; the validation of this role was, in many ways, related to the producer’s physical presence in the studio, to a greater or lesser extent. However, improvements in the speed of digital networks have allowed studio sessions to be produced long-distance, in real-time, through communication programs such as Skype or REDIS. How does this impact on the role of the producer, a “nexus between the creative inspiration of the artist, the technology of the recording studio, and the commercial aspirations of the record company” (Howlett 2012)? From observations of a studio recording session in Lisbon produced through Skype from New York, this article focuses on the role of the producer in these relatively new recording contexts involving long distance media networks. Methodology involved participant observation carried out in Estúdios Namouche in Lisbon (where the session took place), as part of doctoral research. This ethnographic approach also included a number of semi-directed ethnographic interviews of the different actors in this scenario—musicians, recording engineers, composers and producers. As a theoretical framework, the research of De Zutter and Sawyer on Distributed Creativity is used, as the recording studio sets an example of “a cognitive system where […] tasks are not accomplished by separate individuals, but rather through the interactions of those individuals” (DeZutter 2009:4). Therefore, creativity often emerges as a result of this interaction.

Using a video camera to measure the radius of the Earth

A simple but accurate method for measuring the Earth’s radius using a video camera is described. A video camera was used to capture a shadow rising up the wall of a tall building at sunset. A free program called ImageJ was used to measure the time it took the shadow to rise a known distance up the building. The time, distance and length of the sidereal day were used to calculate the radius of the Earth. The radius was measured as 6394.3 +/- 118 km, which is within 1.8% of the accepted average value of 6371 km and well within the experimental error. The experiment is suitable as a high school or university project and should produce a value for Earth’s radius within a few per cent at latitudes towards the equator, where at some times of the year the ecliptic is approximately normal to the horizon.

Characterisation of the ionic products arising from electron photodetachment of simple dicarboxylate dianions

Much of what we currently understand about the structure and energetics of multiply charged anions in the gas phase is derived from the measurement of photoelectron spectra of simple dicarboxylate dianions. Here we have employed a modified linear ion-trap mass spectrometer to undertake complementary investigations of the ionic products resulting from laser-initiated electron photodetachment of two model dianions. Electron photodetachment (ePD) of the \[M-2H](2-) dianions formed from glutaric and adipic acid were found to result in a significant loss of ion signal overall, which is consistent with photoelectron studies that report the emission of slow secondary electrons (Xing et al., 2010 \[201). The ePD mass spectra reveal no signals corresponding to the intact \[M-2H](center dot-) radical anions, but rather \[M-2H-CO2](center dot-) ions are identified as the only abundant ionic products indicating that spontaneous decarboxylation follows ejection of the first electron. Interestingly however, investigations of the structure and energetics of the \[M-2H-CO2](center dot-) photoproducts by ion-molecule reaction and electronic structure calculation indicate that (i) these ions are stable with respect to secondary electron detachment and (ii) most of the ion population retains a distonic radical anion structure where the radical remains localised at the position of the departed carboxylate moiety. These observations lead to the conclusion that the mechanism for loss of ion signal involves unimolecular rearrangement reactions of the nascent \[M-2H](center dot-) carbonyloxyl radical anions that compete favourably with direct decarboxylation. Several possible rearrangement pathways that facilitate electron detachment from the radical anion are identified and are computed to be energetically accessible. Such pathways provide an explanation for prior observations of slow secondary electron features in the photoelectron spectra of the same dicaboxylate dianions. (C) 2013 Elsevier B.V. All rights reserved.

A rule-based hybrid method for anomaly detection in online-social-network graphs

Detecting anomalies in the online social network is a significant task as it assists in revealing the useful and interesting information about the user behavior on the network. This paper proposes a rule-based hybrid method using graph theory, Fuzzy clustering and Fuzzy rules for modeling user relationships inherent in online-social-network and for identifying anomalies. Fuzzy C-Means clustering is used to cluster the data and Fuzzy inference engine is used to generate rules based on the cluster behavior. The proposed method is able to achieve improved accuracy for identifying anomalies in comparison to existing methods.

Cryptanalysis of the convex hull click human identification protocol

Recently, a convex hull-based human identification protocol was proposed by Sobrado and Birget, whose steps can be performed by humans without additional aid. The main part of the protocol involves the user mentally forming a convex hull of secret icons in a set of graphical icons and then clicking randomly within this convex hull. While some rudimentary security issues of this protocol have been discussed, a comprehensive security analysis has been lacking. In this paper, we analyze the security of this convex hull-based protocol. In particular, we show two probabilistic attacks that reveal the user’s secret after the observation of only a handful of authentication sessions. These attacks can be efficiently implemented as their time and space complexities are considerably less than brute force attack. We show that while the first attack can be mitigated through appropriately chosen values of system parameters, the second attack succeeds with a non-negligible probability even with large system parameter values that cross the threshold of usability.

Analysis of distance learner value assessment of distance education in engineering

Numerous research studies have evaluated whether distance learning is a viable alternative to traditional learning methods. These studies have generally made use of cross-sectional surveys for collecting data, comparing distance to traditional learners with intent to validate the former as a viable educational tool. Inherent fundamental differences between traditional and distance learning pedagogies, however, reduce the reliability of these comparative studies and constrain the validity of analyses resulting from this analytical approach. This article presents the results of a research project undertaken to analyze expectations and experiences of distance learners with their degree programs. Students were given surveys designed to examine factors expected to affect their overall value assessment of their distance learning program. Multivariate statistical analyses were used to analyze the correlations among variables of interest to support hypothesized relationships among them. Focusing on distance learners overcomes some of the limitations with assessments that compare off- and on-campus student experiences. Evaluation and modeling of distance learner responses on perceived value for money of the distance education they received indicate that the two most important influences are course communication requirements, which had a negative effect, and course logistical simplicity, which revealed a positive effect. Combined, these two factors accounted for approximately 47% of the variability in perceived value for money of the educational program of sampled students. A detailed focus on comparing expectations with outcomes of distance learners complements the existing literature dominated by comparative studies of distance and nondistance learners.

Reactions of simple and peptidic alpha-carboxylate radical anions with dioxygen in the gas phase

alpha-Carboxylate radical anions are potential reactive intermediates in the free radical oxidation of biological molecules (e. g., fatty acids, peptides and proteins). We have synthesised well-defined alpha-carboxylate radical anions in the gas phase by UV laser photolysis of halogenated precursors in an ion-trap mass spectrometer. Reactions of isolated acetate ((center dot)CH(2)CO(2)) and 1-carboxylatobutyl (CH(3)CH(2)CH(2)(center dot)CHCO(2)(-)) radical anions with dioxygen yield carbonate (CO(3)(center dot-)) radical anions and this chemistry is shown to be a hallmark of oxidation in simple and alkyl-substituted cross-conjugated species. Previous solution phase studies have shown that C(alpha)-radicals in peptides, formed from free radical damage, combine with dioxygen to form peroxyl radicals that subsequently decompose into imine and keto acid products. Here, we demonstrate that a novel alternative pathway exists for two alpha-carboxylate C(alpha)-radical anions: the acetylglycinate radical anion (CH(3)C(O)NH(center dot)CHCO(2)(-)) and the model peptide radical anion, YGGFG(center dot-). Reaction of these radical anions with dioxygen results in concerted loss of carbon dioxide and hydroxyl radical. The reaction of the acetylglycinate radical anion with dioxygen reveals a two-stage process involving a slow, followed by a fast kinetic regime. Computational modelling suggests the reversible formation of the C(alpha) peroxyl radical facilitates proton transfer from the amide to the carboxylate group, a process reminiscent of, but distinctive from, classical proton-transfer catalysis. Interestingly, inclusion of this isomerization step in the RRKM/ME modelling of a G3SX level potential energy surface enables recapitulation of the experimentally observed two-stage kinetics.

More than simple living : a cultural perspective on sustainable living

This paper provides a cultural perspective to the sustainability research within HCI. I draw on the results from an ethnographic field study of sustainable household practices of ten women belonging to the so-called middle class in India. Focusing on their reuse practices, I provide examples of domestic artefacts that are creatively and resourcefully reused from worn-out artefacts. My initial findings show that the rationale behind creating such artefacts is not limited to the practicality and usefulness, but how cultural and religious beliefs are incorporated into such practices.

A simple model for the establishment of tick-borne pathogens of Ixodes scapularis : a global sensitivity analysis of R0

The basic reproduction number of a pathogen, R 0, determines whether a pathogen will spread (R0>1R 0>1), when introduced into a fully susceptible population or fade out (R0<1R 0<1), because infected hosts do not, on average, replace themselves. In this paper we develop a simple mechanistic model for the basic reproduction number for a group of tick-borne pathogens that wholly, or almost wholly, depend on horizontal transmission to and from vertebrate hosts. This group includes the causative agent of Lyme disease, Borrelia burgdorferi, and the causative agent of human babesiosis, Babesia microti, for which transmission between co-feeding ticks and vertical transmission from adult female ticks are both negligible. The model has only 19 parameters, all of which have a clear biological interpretation and can be estimated from laboratory or field data. The model takes into account the transmission efficiency from the vertebrate host as a function of the days since infection, in part because of the potential for this dynamic to interact with tick phenology, which is also included in the model. This sets the model apart from previous, similar models for R0 for tick-borne pathogens. We then define parameter ranges for the 19 parameters using estimates from the literature, as well as laboratory and field data, and perform a global sensitivity analysis of the model. This enables us to rank the importance of the parameters in terms of their contribution to the observed variation in R0. We conclude that the transmission efficiency from the vertebrate host to Ixodes scapularis ticks, the survival rate of Ixodes scapularis from fed larva to feeding nymph, and the fraction of nymphs finding a competent host, are the most influential factors for R0. This contrasts with other vector borne pathogens where it is usually the abundance of the vector or host, or the vector-to-host ratio, that determine conditions for emergence. These results are a step towards a better understanding of the geographical expansion of currently emerging horizontally transmitted tick-borne pathogens such as Babesia microti, as well as providing a firmer scientific basis for targeted use of acaricide or the application of wildlife vaccines that are currently in development.