A Mutational Analysis of the Structure and Function of the Herpes Simplex Virus Immediate Early Protein Vmw175

Herpes simplex virus type 1 (HSV-1) expresses three main classes of genes (immediate-early [IE], early [E] and late [L]) in a temporal cascade upon infection of tissue culture cells. These three groups of genes can also be defined by the sensitivity of their expression to metabolic inhibitors of protein or DNA synthesis. IE genes are transcribed in the absence of de novo protein synthesis and their transcription is stimulated by a component of the virus particle, Vmw65. IE gene products are required for the activation of later classes of viral genes, whose expression varies in sensitivity to inhibitors of viral DNA replication. Early genes are expressed at maximal levels in the absence of DNA synthesis, whilst true late genes are critically dependent on replication for expression. The predominant transcriptional regulatory protein specified by HSV-1 is the IE protein Vmw175, whose functions are essential for virus growth. Studies of viruses with temperature sensitive lesions in this protein have shown that Vmw175 is a complex multifunctional protein required for the transcriptional activation of many HSV-1 promoters and the repression of its own transcription. Cloned Vmw175 is a promiscuous transactivator of transcription of RNA polymerase type II promoters. In addition cloned Vmw175 represses transcription from its own promoter, probably by binding to a specific target sequence at the start site for transcription. The aim of the work described in this thesis was to investigate the relationship between the structure and function of this protein, and to define which regions of the protein are involved in each of its various activities. A panel of plasmid-borne in-frame insertion and deletion mutants of the gene encoding Vmw175 were constructed and assayed for their ability to regulate transcription in transient transfection assays. Fusions of HSV promoters to the chloramphenicol acetyl transferase gene were used to assay the ability of each mutant to transactivate an HSV early promoter (that of the gene encoding glycoprotein gD) and to repress the promoter of its own gene, IE3, in transiently transfected cells. By this approach it was possible to define the regions of the Vmw175 amino acid sequence that are required for transcriptional activation and repression. Large stretches of the protein are relatively unimportant for either function, while the regions most sensitive to disruption correlate to sequences conserved between Vmw175 and VZV 140K, the corresponding transactivating protein of another alphaherpesvirus, varicella-zoster virus. The region from amino acids 275 to 495 is particularly important for both repression and transactivation; whilst that from around 840 to 1100 seems to be more important for transactivation than repression. A monoclonal antibody directed against Vmw175 was used to visualize expression and localization of Vmw175 in transfected cells by indirect immunofluorescence. This provided confirmation that each of the mutant plasmids expressed Vmw175. Each of the 39 insertion mutant proteins exhibited a pattern of nuclear localization indistinguishable from that of wild-type, but when amino acids 682-774 were deleted a signal essential for nuclear localization was lost. A strong candidate for a nuclear localization signal centres around amino acid 728 and is strongly conserved in the VZV homologue. This sequence PREGRKRKSP contains four consecutive arginine (R) and lysine (K) residues and is related to a signal in the SV40 large T antigen required for nuclear localization. Vmw175 is known to bind directly to a number of HSV-1 sequences, some of which contain the consensus sequence ATCGTC. The binding of Vmw175 to this sequence at the transcriptional start site of IE gene 3 is thought to be involved in the mechanism of autoregulation. In order to correlate the transcriptional activity of each mutant with its ability to bind to DNA, the site specific DNA binding activity of each mutant was assayed. In these experiments nuclear extracts of transfected cells were incubated with a DNA probe spanning the IE3 cap site and protein complexes detected using the gel retardation technique. The results show that a critical region of Vmw175, amino acid residues 275-495, includes structures which are essential for specific DNA binding, transactivation and repression

Community-based responses to climate hazards: typology and global analysis

Published versionThe severity and frequency of climate change hazards are increasing around the world. Because the impacts are most acutely felt in local communities, it is critical to improve understanding of the response options that are available for and being chosen by communities. We conducted a mixed methods analysis of case studies reporting community-based responses to climate change hazards. Based on content analysis of published case studies, we generated an emergent evidence-based typology of such responses according to their nature and goals. Using this typology, we quantitatively analysed more than 1500 response examples and determined the patterns with which community-level climate change adaptation and disaster mitigation strategies vary across world regions and across economic and governance conditions. Specifically, diversity of responses is lower in developing countries, and implementation of local-level policy and planning responses is less frequent in countries characterized by low governance quality. Our results confirm that, although there is much that local communities can do to respond to the challenges of climate change, there is also a need for increased support of local activities. By synthesizing data from many local studies, our research provides a first global evidence base for local-level climate change adaptation policy

A global comparison of community-based responses to natural hazards

Published VersionCommunity-based disaster preparedness is an important component of disaster management. Knowledge of interventions that communities utilize in response to hazards is important to develop local-level capacity and increase community resilience. This paper systematically examines empirical information about local-level responses to hazards based on peer-reviewed, published case studies. We developed a data set based on 188 articles providing information from 318 communities from all regions of the world. We classified response examples to address four key questions: (i) what kinds of responses are used by communities all over the world? (ii) Do communities in different parts of the world use different kinds of responses? (iii) Are communities using hazard-specific responses? (iv) Are communities using a multi-hazard approach? We found that within an extensive literature on hazards, there is relatively little empirical information about community-based responses to hazards. Across the world, responses aiming at securing basic human needs are the most frequently reported kinds of responses. Although the notion of community-based disaster preparedness is gaining importance, very few examples of responses that draw on the social fabric of communities are reported. Specific regions of the world are lacking in their use of certain hazard responses classes. Although an all-hazard approach for disaster preparedness is increasingly recommended, there is a lack of multi-hazard response approaches on the local level

A standard data model representation for taxonomic information.

The names used by biologists to label the observations they make are imprecise. This is an issue as workers increasingly seek to exploit data gathered from multiple, unrelated sources on line. Even when the international codes of nomenclature are followed strictly the resultingnames (Taxon Names) do not uniquely identify the taxa (Taxon Concepts) that have been described by taxonomists but merely groups of type specimens. A standard data model for exchange of taxonomic information is described. It addresses this issue by facilitating explicit communication of information about Taxon Concepts and their associated names. A representation of this model as a XML Schema is introduced and the implications of the useof Globally Unique Identifiers discussed

Visualising errors in animal pedigree genotype data

Genetic analysis of a breeding animal population involves determining the inheritance pattern of genotypes for multiple genetic markers across the individuals in the population pedigree structure. However, experimental pedigree genotype data invariably contains errors in both the pedigree structure and in the associated individual genotypes, which introduce inconsistencies into the dataset, rendering them useless for further analysis. The resolution of these errors requires consideration of the genotype inheritance patterns in the context of the pedigree structure. Existing visualisations of pedigree structures are typically more suited to human pedigrees and are less suitable for large complex animal pedigrees which may exhibit cross generational inbreeding. Similarly, current table-based viewers of genotype marker information can highlight where errors become apparent but lack the functionality and interactive visual feedback to enable users to locate the underlying source of errors within the pedigree. In this paper, we detail a design study steered by biologists who work with pedigree data, and describe successive iterations through approaches and prototypes for viewing genotyping errors in the context of a displayed pedigree. We describe how each approach performs with real pedigree genotype data and why eventually we deemed them unsuitable. Finally, a novel prototype visualisation for pedigrees, which we term the ‘sandwich view’, is detailed and we demonstrate how the approach effectively communicates errors in the pedigree context, supporting the biologist in the error identification task

VIPER: a visualisation tool for exploring inheritance inconsistencies in genotyped pedigrees

Pedigree genotype datasets are used for analysing genetic inheritance and to map genetic markers and traits. Such datasets consist of hundreds of related animals genotyped for thousands of genetic markers and invariably contain multiple errors in both the pedigree structure and in the associated individual genotype data. These errors manifest as apparent inheritance inconsistencies in the pedigree, and invalidate analyses of marker inheritance patterns across the dataset. Cleaning raw datasets of bad data points (incorrect pedigree relationships, unreliable marker assays, suspect samples, bad genotype results etc.) requires expert exploration of the patterns of exposed inconsistencies in the context of the inheritance pedigree. In order to assist this process we are developing VIPER (Visual Pedigree Explorer), a software tool that integrates an inheritance-checking algorithm with a novel space-efficient pedigree visualisation, so that reported inheritance inconsistencies are overlaid on an interactive, navigable representation of the pedigree structure

An XML transfer schema for exchange of genomic and genetic mapping data: implementation as a web service in a Taverna workflow

<p>Abstract</p> <p>Background</p> <p>Genomic analysis, particularly for less well-characterized organisms, is greatly assisted by performing comparative analyses between different types of genome maps and across species boundaries. Various providers publish a plethora of on-line resources collating genome mapping data from a multitude of species. Datasources range in scale and scope from small bespoke resources for particular organisms, through larger web-resources containing data from multiple species, to large-scale bioinformatics resources providing access to data derived from genome projects for model and non-model organisms. The heterogeneity of information held in these resources reflects both the technologies used to generate the data and the target users of each resource. Currently there is no common information exchange standard or protocol to enable access and integration of these disparate resources. Consequently data integration and comparison must be performed in an <it>ad hoc </it>manner.</p> <p>Results</p> <p>We have developed a simple generic XML schema (GenomicMappingData.xsd – GMD) to allow export and exchange of mapping data in a common lightweight XML document format. This schema represents the various types of data objects commonly described across mapping datasources and provides a mechanism for recording relationships between data objects. The schema is sufficiently generic to allow representation of any map type (for example genetic linkage maps, radiation hybrid maps, sequence maps and physical maps). It also provides mechanisms for recording data provenance and for cross referencing external datasources (including for example ENSEMBL, PubMed and Genbank.). The schema is extensible via the inclusion of additional datatypes, which can be achieved by importing further schemas, e.g. a schema defining relationship types. We have built demonstration web services that export data from our ArkDB database according to the GMD schema, facilitating the integration of data retrieval into Taverna workflows.</p> <p>Conclusion</p> <p>The data exchange standard we present here provides a useful generic format for transfer and integration of genomic and genetic mapping data. The extensibility of our schema allows for inclusion of additional data and provides a mechanism for typing mapping objects via third party standards. Web services retrieving GMD-compliant mapping data demonstrate that use of this exchange standard provides a practical mechanism for achieving data integration, by facilitating syntactically and semantically-controlled access to the data.</p

Quantitative Differential Phase Contrast Imaging of the Magnetostructural Transition and Current-driven Motion of Domain Walls in FeRh Thin Films

No abstract available

Treponema spp. spirochetes and keratinopathogenic fungi isolated from keratomas in donkeys

Keratoma is an aberrant keratin mass thought to originate from epidermal horn-producing cells interposed between the stratum medium of the hoof wall and the underlying third phalanx. The cause is unknown, although the presence of keratomas is frequently associated with chronic irritation, focal infection, or trauma. A total of 167 donkeys with keratomas were presented in this study. The diagnosis of a keratoma was based on clinical signs, radiography, and histopathologic examination. Surgical excision was attempted on all donkeys with lameness unless euthanasia was advised. Histopathologic examination, including Giemsa, periodic acid Schiff, and Young's silver special histochemical stains, was performed and showed the presence of fungal hyphae and spirochete bacteria within the degenerate keratin. Polymerase chain reaction (PCR) for treponeme bacteria was performed on 10 keratoma lesions and 9 healthy pieces of hoof (controls). All healthy donkey tissues were negative for the 3 recognized digital dermatitis (DD) treponeme phylogroups, whereas 3 of 10 (30%) donkey keratoma samples were positive for one of the DD treponeme phylogroups. Routine fungal culture and PCR for fungi were performed on 8 keratoma lesions and 8 healthy pieces of hoof (controls). Keratinopathogenic fungi were detected in 1 of 8 (12.5%) keratomas, while only non-keratinopathogenic, environmental fungi were detected in 8 control healthy hoof samples. This is the first time the DD treponemes phylogroup and keratinopathogenic fungi have been detected in keratomas. Further studies are required to assess the significance of this finding