Design, synthesis, and evaluation of peptide-imidazo[1,2-a]pyrazine bioconjugates as potential bivalent inhibitors of the VirB11 ATPase HP0525

Helicobacter pylori (H. pylori) infections have been implicated in the development of gastric ulcers and various cancers: however, the success of current therapies is compromised by rising antibiotic resistance. The virulence and pathogenicity of H. pylori is mediated by the type IV secretion system (T4SS), a multiprotein macromolecular nanomachine that transfers toxic bacterial factors and plasmid DNA between bacterial cells, thus contributing to the spread of antibiotic resistance. A key component of the T4SS is the VirB11 ATPase HP0525, which is a hexameric protein assembly. We have previously reported the design and synthesis of a series of novel 8-amino imidazo[1,2-a]pyrazine derivatives as inhibitors of HP0525. In order to improve their selectivity, and potentially develop these compounds as tools for probing the assembly of the HP0525 hexamer, we have explored the design and synthesis of potential bivalent inhibitors. We used the structural details of the subunit-subunit interactions within the HP0525 hexamer to design peptide recognition moieties of the subunit interface. Different methods (cross metathesis, click chemistry, and cysteine-malemide) for bioconjugation to selected 8-amino imidazo[1,2-a]pyrazines were explored, as well as peptides spanning larger or smaller regions of the interface. The IC50 values of the resulting linker-8-amino imidazo[1,2-a]pyrazine derivatives, and the bivalent inhibitors, were related to docking studies with the HP0525 crystal structure and to molecular dynamics simulations of the peptide recognition moieties

Ambiguity, multiple streams, and EU policy

The multiple streams framework draws insight from interactions between agency and institutions to explore the impact of context, time, and meaning on policy change and to assess the institutional and issue complexities permeating the European Union (EU) policy process. The authors specify the assumptions and structure of the framework and review studies that have adapted it to reflect more fully EU decision-making processes. The nature of policy entrepreneurship and policy windows are assessed to identify areas of improvement. Finally, the authors sketch out a research agenda that refines the logic of political manipulation which permeates the lens and the institutional complexity which frames the EU policy process

Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry

P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed “donor-strand exchange (DSE)” in which an N-terminal extension (Nte) of one subunit donates a β-strand to an adjacent subunit, completing its Ig fold. Despite structural determination of the different subunits, the mechanism determining specificity of subunit ordering in pilus assembly remained unclear. Here, we have used noncovalent mass spectrometry to monitor DSE between all 30 possible pairs of P pilus subunits and their Ntes. We demonstrate a striking correlation between the natural order of subunits in pili and their ability to undergo DSE in vitro. The results reveal insights into the molecular mechanism by which subunit ordering during the assembly of this complex is achieved

Mind the (yield) gap(s)

This paper explores the origin of the notion of “yield gap” and its use as a framing device for agricultural policy in sub-Saharan Africa. The argument is that while the yield gap of policy discourse provides a simple and powerful framing device, it is most often used without the discipline or caveats associated with the best examples of its use in crop production ecology and microeconomics. This argument is developed by examining how yield gap is used in a selection of recent and influential agricultural policy documents. The message for policy makers and others is clear: “mind the (yield) gap(s)”, for they are seldom what they appear

How does the tobacco industry attempt to influence marketing regulations? A systematic review

BACKGROUND: The Framework Convention on Tobacco Control makes a number of recommendations aimed at restricting the marketing of tobacco products. Tobacco industry political activity has been identified as an obstacle to Parties' development and implementation of these provisions. This study systematically reviews the existing literature on tobacco industry efforts to influence marketing regulations and develops taxonomies of 1) industry strategies and tactics and 2) industry frames and arguments. METHODS: Searches were conducted between April-July 2011, and updated in March 2013. Articles were included if they made reference to tobacco industry efforts to influence marketing regulations; supported claims with verifiable evidence; were written in English; and concerned the period 1990-2013. 48 articles met the review criteria. Narrative synthesis was used to combine the evidence. RESULTS: 56% of articles focused on activity in North America, Europe or Australasia, the rest focusing on Asia (17%), South America, Africa or transnational activity. Six main political strategies and four main frames were identified. The tobacco industry frequently claims that the proposed policy will have negative unintended consequences, that there are legal barriers to regulation, and that the regulation is unnecessary because, for example, industry does not market to youth or adheres to a voluntary code. The industry primarily conveys these arguments through direct and indirect lobbying, the promotion of voluntary codes and alternative policies, and the formation of alliances with other industrial sectors. The majority of tactics and arguments were used in multiple jurisdictions. CONCLUSIONS: Tobacco industry political activity is far more diverse than suggested by existing taxonomies of corporate political activity. Tactics and arguments are repeated across jurisdictions, suggesting that the taxonomies of industry tactics and arguments developed in this paper are generalisable to multiple jurisdictions and can be used to predict industry activity

Systematizing Policy Learning: From Monolith to Dimensions

notes: The authors wish to express their gratitude to the Norwegian Political Science Association Annual Conference, 6 January 2010, University of Agder, Kristiansand, participants of the ‘Establishing Causality in Policy Learning’ panel at the American Political Science Association (APSA) annual meeting,2–5 September 2010,Washington DC, and the European Consortium of Political Research (ECPR) Joint Sessions, St Gallen, 12–17 April 2011, workshop 2. Dunlop and Radaelli gratefully acknowledge the support of the European Research Council, grant on Analysis of Learning in Regulatory Governance, ALREG, http://centres.exeter.ac.uk/ceg/research/ALREG/index.php.publication-status: AcceptedThe definitive version is available at www.blackwell-synergy.com and also from DOI: 10.1111/j.1467-9248.2012.00982.xThe field of policy learning is characterised by concept stretching and lack of systematic findings. To systematize them, we combine the classic Sartorian approach to classification with the more recent insights on explanatory typologies. At the outset, we classify per genus et differentiam – distinguishing between the genus and the different species within it. By drawing on the technique of explanatory typologies to introduce a basic model of policy learning, we identify four major genera in the literature. We then generate variation within each cell by using rigorous concepts drawn from adult education research. Specifically, we conceptualize learning as control over the contents and goals of knowledge. By looking at learning through the lenses of knowledge utilization, we show that the basic model can be expanded to reveal sixteen different species. These types are all conceptually possible, but are not all empirically established in the literature. Up until now the scope conditions and connections among types have not been clarified. Our reconstruction of the field sheds light on mechanisms and relations associated with alternatives operationalizations of learning and the role of actors in the process of knowledge construction and utilization. By providing a comprehensive typology, we mitigate concept stretching problems and aim to lay the foundations for the systematic comparison across and within cases of policy learning.European Research Council, grant no 230267 on Analysis of Learning in Regulatory Governance, ALREG

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies