Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

BACKGROUND: We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector. RESULTS: Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized. CONCLUSION: Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed

Users’ encounter with normative discourses on Facebook:A three-pronged analysis of user agency as power structure, nexus and reception

This study asks whether users’ encounter with normative discourses of lifestyle, consumption, and health on social media such as Facebook gives rise to agency. The theoretical framework draws on reception analysis, for its implied, but central interest in agency that lies at the intersection of texts and audiences. Based on a critique of the “participatory paradigm,” a paradigm that situates the locus of agency in the structural opposition between senders and users, in the norms of rational deliberation or in the figure of the activist, gaps are identified which can be filled by adopting an explicit focus on the socio-cultural practices of ordinary audiences in their encounters with media discourses. The study investigates user agency on seven Facebook groups and pages with the help of a three-pronged perspective based on the notion of the media–audience relationship as (1) power structure, (2) nexus, and (3) reception. The analysis reveals that the structure at play on these Facebook groups and pages does not encourage user agency. However, user agency manifests itself through user interactions and expressive sense-making processes associated with reception. The benefits of such audience agency are a public, collective, and communicative sense-making process and an expansion of the professionally controlled text

Large‐scale calculations of excitation energies in coupled cluster theory : The singlet excited states of benzene

Algorithms for calculating singlet excitation energies in the coupled cluster singles and doubles (CCSD) model are discussed and an implementation of an atomic‐integral direct algorithm is presented. Each excitation energy is calculated at a cost comparable to that of the CCSD ground‐state energy. Singlet excitation energies are calculated for benzene using up to 432 basis functions. Basis‐set effects of the order of 0.2 eV are observed when the basis is increased from augmented polarized valence double‐zeta (aug‐cc‐pVDZ) to augmented polarized valence triple‐zeta (aug‐cc‐pVTZ) quality. The correlation problem is examined by performing calculations in the hierarchy of coupled cluster models CCS, CC2, CCSD, and CC3, as well as by using the CCSDR(3) perturbative triples corrections. The effect of triple excitations are less than 0.2 eV for all excitations except for the 2 1E2g state. The calculated excitation energies are compared with experiment and other theoretical [email protected]

Destination development in Western Siberia:Tourism governance and evolutionary economic geography

Tourism development has often been identified as a tool for balancing negative effects of economic restructuring, especially in peripheral regions. Tourism-based activities often utilize the availability of abundant nature, but although most English language studies of destination development are presented from western contexts, examples from post-Soviet Russia are rare. Western Siberia is a periphery with access to natural resources and heavy industrialization but remotely located from domestic (Russian) and international markets, where tourism is often considered a saviour, especially for the regional economies. Stakeholders in this Russian resource periphery face challenges in managing governance and cooperation in destinations development due to frequent institutional, economic and social changes. Using evolutionary economic geography and based on primary sources and interview data, tourism development and stakeholder relations are assessed in three Western Siberia regions: Tomsk, Kemerovo and Altai Krai. Findings show that for tourism to make a significant contribution, it must be more central to the economic development agenda in all three regions. However, it is currently only achieving a permanent high-profile in one of them, being crowded out by other (mostly primary) industries in the other two. Although the specific tourism governance set-up varies between the three regions, it is clear that public tourism governance still sits somewhat uneasily between state control and the market economy. Tourism receives substantial public subsidies, especially in large-scale investment projects, which depend on federal support within a governance system where decentralization seems to be somewhat limited and unstable. As a result, the tourism path development in the Siberian periphery is highly dependent on state intervention and success in other sectors.</p

Metabolic analysis of the interaction between plants and herbivores

Insect herbivores by necessity have to deal with a large arsenal of plant defence metabolites. The levels of defence compounds may be increased by insect damage. These induced plant responses may also affect the metabolism and performance of successive insect herbivores. As the chemical nature of induced responses is largely unknown, global metabolomic analyses are a valuable tool to gain more insight into the metabolites possibly involved in such interactions. This study analyzed the interaction between feral cabbage (Brassica oleracea) and small cabbage white caterpillars (Pieris rapae) and how previous attacks to the plant affect the caterpillar metabolism. Because plants may be induced by shoot and root herbivory, we compared shoot and root induction by treating the plants on either plant part with jasmonic acid. Extracts of the plants and the caterpillars were chemically analysed using Ultra Performance Liquid Chromatography/Time of Flight Mass Spectrometry (UPLCT/MS). The study revealed that the levels of three structurally related coumaroylquinic acids were elevated in plants treated on the shoot. The levels of these compounds in plants and caterpillars were highly correlated: these compounds were defined as the ‘metabolic interface’. The role of these metabolites could only be discovered using simultaneous analysis of the plant and caterpillar metabolomes. We conclude that a metabolomics approach is useful in discovering unexpected bioactive compounds involved in ecological interactions between plants and their herbivores and higher trophic levels.

Electron affinities of the first- and second- row atoms: benchmark ab initio and density functional calculations

A benchmark ab initio and density functional (DFT) study has been carried out on the electron affinities of the first- and second-row atoms. The ab initio study involves basis sets of $spdfgh$ and $spdfghi$ quality, extrapolations to the 1-particle basis set limit, and a combination of the CCSD(T), CCSDT, and full CI electron correlation methods. Scalar relativistic and spin-orbit coupling effects were taken into account. On average, the best ab initio results agree to better than 0.001 eV with the most recent experimental results. Correcting for imperfections in the CCSD(T) method improves the mean absolute error by an order of magnitude, while for accurate results on the second-row atoms inclusion of relativistic corrections is essential. The latter are significantly overestimated at the SCF level; for accurate spin-orbit splitting constants of second-row atoms inclusion of (2s,2p) correlation is essential. In the DFT calculations it is found that results for the 1st-row atoms are very sensitive to the exchange functional, while those for second-row atoms are rather more sensitive to the correlation functional. While the LYP correlation functional works best for first-row atoms, its PW91 counterpart appears to be preferable for second-row atoms. Among ``pure DFT'' (nonhybrid) functionals, G96PW91 (Gill 1996 exchange combined with Perdew-Wang 1991 correlation) puts in the best overall performance. The best results overall are obtained with the 1-parameter hybrid modified Perdew-Wang (mPW1) exchange functionals of Adamo and Barone [J. Chem. Phys. {\bf 108}, 664 (1998)], with mPW1LYP yielding the best results for first-row, and mPW1PW91 for second-row atoms. Indications exist that a hybrid of the type $a$ mPW1LYP + $(1-a)$ mPW1PW91 yields better results than either of the constituent functionals.Comment: Phys. Rev. A, in press (revised version, review of issues concerning DFT and electron affinities added

Knowledge dynamics in the tourism-social entrepreneurship nexus

Tourism is often employed as a vehicle for facilitating social-economic development, however its usefulness has been somewhat limited in relation to addressing social issues, and in particular, those issues relating to poverty. This is partly due to the lack of cross-sectoral interactions and knowledge exchange between private, public and third sectors that are needed to create effective and appropriate initiatives to leverage tourism for social benefits. Such traditional sectoral boundaries can be broken down through social entrepreneurship approaches which concomitantly, facilitate the creation and synergizing of social innovation that addresses persistent social issues. Yet to date, the utility of cross-sectoral knowledge dynamics still remains largely under-researched in both the social entrepreneurship and tourism literature. This chapter introduces readers to the concept of knowledge dynamics and discusses knowledge dynamics in the tourism and social entrepreneurship nexus via a case study of community-based tourism in Mai Hich, Vietnam. We argue that by gaining an enhanced understanding of cross-sectoral knowledge dynamics, we can strengthen the overall praxis of tourism and social entrepreneurship, and in particular, assist policymakers in fostering conditions that generate increased innovation.Griffith Business School, Department of Tourism, Sport and Hotel ManagementNo Full Tex

‘Change Today, Choose Fairtrade’ Fairtrade Fortnight and the citizen-consumer

The Fairtrade consumer is widely represented as an individual who intentionally and reflexively consumes Fairtrade goods in order to register their support for the plight of producers in the developing world. This figure is imagined to ‘vote’ with her/his pocket every time they visit the supermarket thus demonstrating their commitment to the Fairtrade trading model. However, this image of the Fairtrade citizen-consumer does not emerge automatically as a response to the increasing availability of Fairtrade goods in the market-place but has to be made by various intermediary actors and organizations. This paper examines how the Fairtrade consumer was constructed and called to action by the Fairtrade Fortnight promotional campaign that occurred within the UK in 2008 and was co-ordinated by the Fairtrade Foundation. This annual event offers a unique window into the processes and actors involved in the mobilization of the Fairtrade citizen-consumer. Through a close focus on the promotional material distributed to different audiences and the events that occurred during this Fortnight, this paper reveals the contingent and shifting nature of the citizen-consumer identity. In so doing, it highlights how varying degrees of reflexivity and action are demanded of different audiences and how this shapes the way that Fairtrade goods are qualified and distributed in the market