Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses

Natural isolates and laboratory strains of West Nile virus (WNV) and Japanese encephalitis virus (JEV) were attenuated for neuroinvasiveness in mouse models for flavivirus encephalitis by serial passage in human adenocarcinoma (SW13) cells. The passage variants displayed a small-plaque phenotype, augmented affinity for heparin-Sepharose, and a marked increase in specific infectivity for SW13 cells relative to the respective parental viruses, while the specific infectivity for Vero cells was not altered. Therefore, host cell adaptation of passage variants was most likely a consequence of altered receptor usage for virus attachment-entry with the involvement of cell surface glycosaminoglycans (GAG) in this process. In vivo blood clearance kinetics of the passage variants was markedly faster and viremia was reduced relative to the parental viruses, suggesting that affinity for GAG (ubiquitously present on cell surfaces and extracellular matrices) is a key determinant for the neuroinvasiveness of encephalitic flaviviruses. A difference in pathogenesis between WNV and JEV, which was reflected in more efficient growth in the spleen and liver of the WNV parent and passage variants, accounted for a less pronounced loss of neuroinvasiveness of GAG binding variants of WNV than JEV. Single gain-of-net-positive-charge amino acid changes at E protein residue 49, 138, 306, or 389/390, putatively positioned in two clusters on the virion surface, define molecular determinants for GAG binding and concomitant virulence attenuation that are shared by the JEV serotype flaviviruses.

Regulated Cleavages at the West Nile Virus NS4A-2K-NS4B Junctions Play a Major Role in Rearranging Cytoplasmic Membranes and Golgi Trafficking of the NS4A Protein

A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS213-34A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.

Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3,site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

West (side) and south (front) elevations, public housing, Cotton Tree Housing Estate, Maroochydore

This pilot project at Cotton Tree, Maroochydore, on two adjacent, linear parcels of land has one of the properties privately owned while the other is owned by the public housing authority. Both owners commissioned Lindsay and Kerry Clare to design housing for their separate needs which enabled the two projects to be governed by a single planning and design strategy. This entailed the realignment of the dividing boundary to form two approximately square blocks which made possible the retention of an important stand of mature paperbark trees and gave each block a more useful street frontage. The scheme provides seven two-bedroom units and one single-bedroom unit as the private component, with six single-bedroom units, three two-bedroom units and two three-bedroom units forming the public housing. The dwellings are deployed as an interlaced mat of freestanding blocks, car courts, courtyard gardens, patios and decks. The key distinction between the public and private parts of the scheme is the pooling of the car parking spaces in the public housing to create a shared courtyard. The housing climbs to three storeys on its southern edge and falls to a single storey on the north-western corner. This enables all units and the principal private outdoor spaces to have a northern orientation. The interiors of both the public and private units are skilfully arranged to take full advantage of views, light and breeze.

Practice report: the role of environmental factors in industrial site selection activities: a case of limestone quarry expansion in Barbados, West Indies

Site selection is a key activity for quarry expansion to support cement production, and is governed by factors such as resource availability, logistics, costs, and socio-environmental factors. Adequate consideration of all factors facilitates both industrial productivity and sustainable economic growth. This study illustrates the site selection process that was undertaken for the expansion of limestone quarry operations to support cement production in Barbados. First, alternate sites with adequate resources to support a 25-year development horizon were identified. Second, socio-environmental conditions were described and potential impacts identified. Third, a comparative matrix was constructed to evaluate relative site characteristics with respect to physical, ecological, socio-cultural and economic factors. The study shows that environmental factors were essential to the final site recommendation.

Negotiating regional futures:the successes and failures of the West Midlands Regional Development Agency Network

The introduction of Regional Development Agencies (RDAs) in the English regions in 1999 presented a new set of collaborative challenges to existing local institutions. The key objectives of the new policy impetus emphasise increased joined-up thinking and holistic regional governance. Partners were enjoined to promote cross-sector collaboration and present a coherent regional voice. This study aims to evaluate the impact of an RDA on the partnership infrastructure of the West Midlands. The RDA network incorporates a wide spectrum of interest and organisations with diverse collaborative histories, competencies and capacities. The study has followed partners through the process over an eighteen-month period and has sought to explore the complexities and tensions of partnership working 'on the ground'. A strong qualitative methodology has been employed in generating 'thick descriptions' of the policy domain. The research has probed beyond the 'rhetoric' of partnerships and explores the sensitivities of the collaboration process. A number of theoretical frameworks have been employed, including policy network theory; partnership and collaboration theory; organisational learning; and trust and social capital. The structural components of the West Midlands RDA network are explored, including the structural configuration of the network and stocks of human and social capital assets. These combine to form the asset base of the network. Three sets of network behaviours are then explored, namely, strategy, the management of perceptions, and learning. The thesis explores how the combination of assets and behaviours affect, and in turn are affected by, each other. The findings contribute to the growing body of knowledge and understanding surrounding policy networks and collaborative governance.

Marketing in indigenous and Asian small firms in the West Midlands.

This study is concerned with examining the application of marketing during the start-up, development and growth of small firms in the West Midlands. As an exploratory study, it provides evidence to support the central hypothesis of the thesis that whilst many small firms have the potential to progress through to the successful growth stage of development, they fail to do so because of their owner-managers' orientation towards production and selling and because they do not apply formal marketing during the initial stages of business development. A comparative approach to studying marketing in indigenous and Asian firms is adopted in an attempt to fill a gap in the literature on the characteristics and differences in the formation and development processes of these two groups of enterprises. The study has three main objectives and is based on qualitative research techniques of in-depth interviews, case studies and longitudinal studies among sixty-six firms representing the key activities of the small firms sector of the local economy. Firstly, it investigates owner-managers' orientation in developing and managing new and established businesses and explores the sources of, and changes in their orientation during the various stages of development. Secondly, it assesses the owner-manager's awareness and understanding of what constitutes the marketing function and investigates what aspects of marketing are applied during the different stages of business development. Finally, the study monitors and evaluates the outcomes and implications of applying formal marketing techniques in a small sample of firms over a period of two years. The thesis concludes by using the findings of the study to contribute additions to existing models of growth and by proposing new models of evolution and application of marketing in small firms.

Access to start up finance for ethnic minority graduate entrepreneurs in the West Midlands: a gendered perspective

This aim of this paper, from a study funded by the National Council for Graduate Entrepreneurship (NCGE), is to explore access to finance for ethnic minority graduate entrepreneurs (EMGEs) with a particular focus on comparisons between different ethnic groups, and men and women. The authors interviewed selected individuals based upon a review of literature on finance for ethnic minority enterprise. A number of key results from the survey, in that EMGEs: • use external finance significantly (more so than non graduates) and encounter barriers in accessing finance at start-up, in particular those belonging to poor families. • rely excessively on personal savings and family finance, at the start-up and long after the start-up stage, that has implications for the optimal capital structure. • start up businesses that are, on average, larger than non-graduate enterprises and have the potential to reduce economic inactivity amongst the ethnic population. • have, in contrast to general graduate start-ups, a high level of unemployment, take a longer period of time to enter employment and there is a higher level of dissatisfaction with career progression. These findings raise the question whether the right financial advice is taken and whether this behaviour constrains EMGEs' expansion.

Physicians' perceptions, attitudes and expectations regarding the role of hospital-based pharmacists in the West Bank, Palestine

Objective - To evaluate the perceptions, expectations and experiences of physicians with regard to hospital-based pharmacists in the West Bank, Palestine. Methods - A self-administered questionnaire was distributed to 250 physicians practising in four general hospitals in the West Bank, Palestine. The main sections of the questionnaire comprised a series of statements pertaining to physicians' perceptions, expectations and experiences with pharmacists. Key findings - One hundred and fifty seven questionnaires were completed and returned (response rate, 62.8%). The majority of respondents were most comfortable with pharmacists detecting and preventing prescription errors (76.4%; 95% confidence interval (CI) 69.5–81.2%) and patient education (57.9%; CI 51.2–63.4%) but they were not comfortable with pharmacists suggesting the use of prescription medications to patients (56.7%; CI 49.8–62.4%). Most physicians (62.4%; CI 56.8–69.1%) expected the pharmacist to educate their patients about the safe and appropriate use of their medication. However, approximately one-third (31.7%; CI 26.0–39.6%) did not expect pharmacists to be available for consultation during rounds. Physicians' experiences with pharmacists were less favourable; whereas 77% (CI 70.2–81.5%) of the physicians agreed that pharmacists were always a reliable source of information, only 11.5% (CI 6.2–16.4%) agreed that pharmacists appeared to be willing to take responsibility for solving any drug-related problems. Conclusion -The present study showed that hospital physicians are more likely to accept traditional pharmacy services than newer clinical services for hospital-based pharmacists in the West Bank, Palestine. Pharmacists should therefore interact more positively and more frequently with physicians. This will close the gap between the physicians' commonly held perceptions of what they expect pharmacists to do and what pharmacists can actually do, and gain support for an extended role of hospital-based pharmacists in future patient therapy management.

The role of environmental factors in industrial site selection activities:a case of limestone quarry expansion in Barbados, West Indies

Site selection is a key activity for quarry expansion to support cement production, and is governed by factors such as resource availability, logistics, costs, and socio-environmental factors. Adequate consideration of all factors facilitates both industrial productivity and sustainable economic growth. This study illustrates the site selection process that was undertaken for the expansion of limestone quarry operations to support cement production in Barbados. First, alternate sites with adequate resources to support a 25-year development horizon were identified. Second, socio-environmental conditions were described and potential impacts identified. Third, a comparative matrix was constructed to evaluate relative site characteristics with respect to physical, ecological, socio-cultural and economic factors. The study shows that environmental factors were essential to the final site recommendation.

A key role for PTP1B in dendritic cell maturation, migration and T cell activation

Acknowledgements We thank the Iain Fraser Flow Cytometry Centre and the Medical Research Facility of the University of Aberdeen. We are grateful to Drs West, Zaru, and Davidson (University of Dundee) for the scientific discussion and technical assistance. Wethank Derek Mitchell (University of Dundee) for aiding with the quantification of focal contacts. Funding This work was supported by Saving Sight in Grampian and the Development Trust of the UoA (both to J.V.F.). Work on this project was partly funded by project grants from British Heart Foundation and European Foundation for the Study of Diabetes/Lilly diabetes programme grant (to M.D.).

A key role for PTP1B in dendritic cell maturation, migration and T cell activation

Acknowledgements We thank the Iain Fraser Flow Cytometry Centre and the Medical Research Facility of the University of Aberdeen. We are grateful to Drs West, Zaru, and Davidson (University of Dundee) for the scientific discussion and technical assistance. Wethank Derek Mitchell (University of Dundee) for aiding with the quantification of focal contacts. Funding This work was supported by Saving Sight in Grampian and the Development Trust of the UoA (both to J.V.F.). Work on this project was partly funded by project grants from British Heart Foundation and European Foundation for the Study of Diabetes/Lilly diabetes programme grant (to M.D.).

Hydrological and meteorological investigations in a lake near Kangerlussuaq, west Greenland

Few hydrological studies have been made in Greenland, other than on glacial hydrology associated with the ice sheet. Understanding permafrost hydrology and hydroclimatic change and variability, however, provides key information for understanding climate change effects and feedbacks in the Arctic landscape. This paper presents a new extensive and detailed hydrological and meteorological open access dataset, with high temporal resolution from a 1.56 km**2 permafrost catchment with a lake underlain by a through talik close to the ice sheet in the Kangerlussuaq region, western Greenland. The paper describes the hydrological site investigations and utilized equipment, as well as the data collection and processing. The investigations were performed between 2010 and 2013. The high spatial resolution, within the investigated area, of the dataset makes it highly suitable for various detailed hydrological and ecological studies on catchment scale.