An improved method for surface immobilisation of RNA: application to small Non-Coding RNA - mRNA pairing

Characterisation of RNA and its intermolecular interactions is increasing in importance as the inventory of known RNA functions continues to expand. RNA-RNA interactions are central to post-transcriptional gene regulation mechanisms in bacteria, and the interactions of bacterial small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. The technology of surface plasmon resonance (SPR) is an attractive approach to studying these interactions since it is highly sensitive, and allows interaction measurements to be recorded in real-time. Whilst a number of approaches exist to label RNAs for surface-immobilisation, the method documented here is simple, quick, efficient, and utilises the high-affinity streptavidin-biotin interaction. Specifically, we ligate a biotinylated nucleotide to the 3' end of RNA using T4 RNA ligase. Although this is a previously recognised approach, we have optimised the method by our discovery that the incorporation of four or more adenine nucleotides at the 3' end of the RNA (a poly-A-tail) is required in order to achieve high ligation efficiencies. We use this method within the context of investigating small non-coding RNA (sRNA)-mRNA interactions through the application of surface technologies, including quantitative SPR assays. We first focus on validating the method using the recently characterised Escherichia coli sRNA-mRNA pair, MicA-ompA, specifically demonstrating that the addition of the poly-A-tail to either RNA does not affect its subsequent binding interactions with partner molecules. We then apply this method to investigate the novel interactions of a Vibrio cholerae Qrr sRNA with partner mRNAs, hapR and vca0939; RNA-RNA pairings that are important in mediating pathogenic virulence. The calculated binding parameters allow insights to be drawn regarding sRNA-mRNA interaction mechanisms

Evaluation of frameworks of analysis employed in studies of exclusion zones

The purpose of this Work Package is to examine the various approaches to analysing fishery exclusion zones (FEZs) and to identify the circumstances in which one approach might be preferred to another. Our concern here is not so much with answering questions about exclusion zones - these being dealt with in later Work Packages - as with articulating the questions themselves and in understanding how in principle they could be addressed. An important theme is the precision with which questions need to be answered, since this will determine the type of information collected and how such information is analysed. The question ' is an exclusion zone likely to improve the condition of this fishery ?' is less precise and less demanding of data than the question ' by how much will an exclusion zone improve this fishery ?' since it could in principle be answered by expert judgement rather than quantitative analysis. In practice fisheries managers may be confronted with situations where decisions have to be made quickly, and qualitative answers may be the only thing possible in circumstances where data cannot be obtained in the available time. The Work Package will look at FEZs from a number of different perspectives, but its dominant concern is with the information – principally in the form of socio-economic and biological indicators - needed by fisheries managers in order to evaluate the effectiveness of FEZs. To contextualise the discussion we start by outlining a paradigm for understanding the linkages between human activities and the environment, showing how it can be applied to fisheries and marine resources. The Work Package then considers the substantive information requirements of fisheries managers, commencing with socio-economic assessment and moving on to a review of biological assessment and the progress which has been made in the development of mathematical models of FEZs. Bio-economic modelling, which is essentially a specialised type of socio-economic assessment in which explicit account is taken of the interaction between the biological and economic components of the fishing system, is dealt with in the final section

Fisheries Exclusion Zones: Value and its sensitivity to data uncertainty

This paper focuses on the findings of the project that pertain particularly to the determination of the value of fisheries exclusion zones and the sensitivity of value estimation to data uncertainty. It necessarily draws on both the lite rature review and the case studies and in terms of its content extracts, presents and su mmarises the pertinent material therein covered. By collating this material a more directed consid eration of the topic of value is facilitated and the findings of the study as a whole more clear ly highlighted, and through consideration of uncertainty, also qualified

Real world evaluation of three models of NHS smoking cessation service in England

<p>Abstract</p> <p>Background</p> <p>NHS Stop Smoking Services provide various options for support and counselling. Most services have evolved to suit local needs without any retrospective evaluation of their efficiency.</p> <p>Three local service evaluations were carried out at Bournemouth & Poole Teaching Primary Care Trust (PCT) (PCT1), NHS South East Essex (PCT2) and NHS Warwickshire (PCT3) to describe the structure and outcomes associated with different services.</p> <p>Result</p> <p>Standardised interviews with key personnel in addition to analysis of data from 400 clients accessing the service after 1^stApril 2008 in each PCT. The PCTs varied in geography, population size and quit rate (47%-63%). Services were delivered by PCT-led specialist teams (PCT1), community-based healthcare providers (PCT3) and a combination of the two (PCT2) with varying resources and interventions in each.</p> <p>Group support resulted in the highest quit rates (64.3% for closed groups v 42.6% for one-to-one support (PCT1)). Quit rates were higher for PCT (75.0%) v GP (62.0%) and pharmacist-delivered care (41.0%) where all existed in the same model (PCT2). The most-prescribed therapy was NRT (55.8%-65.0%), followed by varenicline (24.5%-34.3%), counselling alone (6.0%-7.8%) and bupropion (2.0%-4.0%).</p> <p>Conclusion</p> <p>The results suggest that service structure, method of support, healthcare professional involved and pharmacotherapy all play a role in a successful quit. Services must be tailored to support individual needs with patient choice and access to varied services being key factors.</p

Selecting Indicator Portfolios for Marine Species and Food Webs: A Puget Sound Case Study

Ecosystem-based management (EBM) has emerged as a promising approach for maintaining the benefits humans want and need from the ocean, yet concrete approaches for implementing EBM remain scarce. A key challenge lies in the development of indicators that can provide useful information on ecosystem status and trends, and assess progress towards management goals. In this paper, we describe a generalized framework for the methodical and transparent selection of ecosystem indicators. We apply the framework to the second largest estuary in the United States – Puget Sound, Washington – where one of the most advanced EBM processes is currently underway. Rather than introduce a new method, this paper integrates a variety of familiar approaches into one step-by-step approach that will lead to more consistent and reliable reporting on ecosystem condition. Importantly, we demonstrate how a framework linking indicators to policy goals, as well as a clearly defined indicator evaluation and scoring process, can result in a portfolio of useful and complementary indicators based on the needs of different users (e.g., policy makers and scientists). Although the set of indicators described in this paper is specific to marine species and food webs, we provide a general approach that could be applied to any set of management objectives or ecological system

PNA HyBeacons for analysis of human mutations related to statin-induced myopathy.

Aminoalkyl and alkyne-tagged PNA HyBeacons have been synthesized, labeled with fluorescein via conventional amide bond or triazole formation (click chemistry) and used to detect single nucleotide polymorphisms (SNPs) implicated in statin-induced myopathy. The PNA HyBeacons gave much better mismatch/mutant discrimination than conventional DNA HyBeacons but smaller fluorescence changes on melting