Human cytomegalovirus plasmid-based amplicon vector system for gene therapy

We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage

Distribution and Movements of the Teshekpuk Caribou Herd 1990–2005: Prior to Oil and Gas Development

Four caribou (Rangifer tarandus grantii) herds calve on the North Slope of Alaska, three of which have been exposed to little or no resource development. We present 15 years of baseline data on the distribution and movements of 72 satellite-collared and 10 GPS-collared caribou from the Teshekpuk caribou herd (TCH) that have had little to no exposure to oil and gas activities. Fixed-kernel home range analyses of collared caribou revealed that calving grounds were concentrated (i.e., 50% kernel utilization distribution) along the northeastern, eastern, and southeastern shores of Teshekpuk Lake. During the postcalving period, 51% and 35% of caribou moved through two constricted zones to the east and west of Teshekpuk Lake, respectively, and accessed insect-relief habitat along the Beaufort Sea coast. During late summer and early fall, TCH caribou were concentrated to the southeast and southwest of Teshekpuk Lake. Although 65% of the Teshekpuk caribou wintered in two areas on the central coastal plain around the village of Atqasuk and south of Teshekpuk Lake, other TCH animals wintered in a great variety of places, including the Seward Peninsula, the eastern and southern Brooks Range, and the Arctic National Wildlife Refuge. We detected an apparent emigration rate of 6.9%. One male and five female TCH caribou joined the breeding populations of the Western Arctic and Central Arctic herds. TCH caribou traveled an average distance of 2348 ± 190 km annually. Movement rates were at a maximum in midsummer, lowest in winter, and intermediate during spring and fall migrations. Restrictions on oil and gas leasing and surface occupancy have been in place to protect calving, migratory corridors, and insect-relief habitat for the TCH, but these protections are likely to be removed. These data will provide a good baseline that can be used to compare predevelopment distribution and movement patterns of TCH caribou to distribution and movement patterns during and after petroleum development.Quatre hardes de caribous (Rangifer tarandus grantii) vêlent sur la côte nord de l’Alaska, dont trois de ces hardes ont été exposées à peu ou pas d’aménagement des ressources. Nous présentons des données de base échelonnées sur 15 ans relativement à la répartition et aux déplacements de 72 caribous dotés d’un collier émetteur par satellite et de 10 caribous munis d’un collier émetteur GPS de la harde de caribous de Teshekpuk (HCT), caribous qui ont été peu ou pas du tout frottés aux activités pétrolières et gazières. L’analyse du noyau fixe des domaines vitaux des caribous à collier a révélé que les lieux de vêlage étaient concentrés (c’est-à-dire 50 % de la répartition de l’utilisation du noyau) le long des côtes nord-est, est et sud-est du lac Teshekpuk. Après la période de vêlage, 51 pour cent et 35 pour cent des caribous se déplaçaient au sein de deux zones de constriction à l’est et à l’ouest du lac Teshekpuk, respectivement, et accédaient un habitat où se trouvait moins d’insectes sur la côte de la mer de Beaufort. Vers la fin de l’été et le début de l’automne, les caribous de la HCT étaient concentrés au sud-est et au sud-ouest du lac Teshekpuk. Bien que 65 pour cent des caribous de Teshekpuk passaient l’hiver dans deux régions de la plaine côtière centrale autour du village d’Atqasuk et au sud du lac Teshekpuk, les autres bêtes de la HCT passaient l’hiver dans divers endroits, dont la péninsule de Seward, les versants est et sud des montagnes de Brooks et la Réserve faunique nationale de l’Arctique. Nous avons détecté un taux d’émigration apparent de 6,9 pour cent. Un caribou mâle et cinq caribous femelles de la HCT ont rejoint les populations de reproduction des hardes de l’ouest et du centre de l’Arctique. En moyenne, le caribou de la HCT parcourait une distance de 2348 ± 190 km annuellement. Les taux de déplacement étaient à leur point le plus élevé au milieu de l’été, tandis qu’ils étaient à leur niveau le plus bas l’hiver et à un niveau intermédiaire pendant les migrations du printemps et de l’automne. Il existe des restrictions en matière de location et d’occupation en surface pour le pétrole et le gaz afin de protéger le vêlage, les corridors de migration et les habitats à faible taux d’insectes pour la HCT, mais il est vraisemblable que ces restrictions soient éliminées. Ces données fourniront une bonne base pour comparer la répartition et les déplacements du caribou de la HCT avant la mise en valeur des ressources à la répartition et aux déplacements du caribou de la HCT pendant et après la mise en valeur pétrolière

Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA

BACKGROUND: Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. RESULTS: A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. CONCLUSION: These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis

Use of satellite telemetry to evaluate movements of caribou within subsistence hunting areas in northern Alaska

Caribou from the Teshekpuk Herd (TH) are an important subsistence resource for residents of Inupiaq villages in northern Alaska. In recent years the use of satellite telemetry has increased the understanding of the herd's annual movements and interactions with other herds. Most caribou of the TH are within the National Petroleum Reserve—Alaska (NPRA) throughout the year. The northeastern portion of NPRA has undergone two lease sales for oil and gas exploration, and lease sales are tentatively scheduled for the central/northwest portion of the NPRA in 2004. During 1990—1999, the movements of 27 caribou from the TH were tracked using satellite collars. We evaluated the proportion of time caribou were available to Inupiaq hunters by incorporating maps depicting subsistence-use areas for each of seven Inupiaq villages, and then examining seasonal and annual movements of caribou relative to those areas. By combining caribou locations with subsistence hunting areas, we were able to explore spatial and temporal patterns in caribou availability to subsistence hunters. This information is useful for managers to set appropriate hunting regulations and for devising sensible alternatives and mitigation of likely petroleum development in NPRA

Fantasies of subjugation: a discourse theoretical account of British policy on the European Union

The decision by the UK government to hold a referendum on Britain’s membership of the European Union (EU) marks an important development in policy towards the EU. Policy changes of this kind must be understood in the historical and political context in which they occur. This includes the framing of the policy issues within public discourse. In the UK, policies are formed in a discursive environment which is overwhelmingly hostile towards the EU. Debates are structured by a predominantly Euroskeptic discourse which emphasizes the UK’s separation and heterogeneity from the rest of the EU. Drawing on the logics of critical explanation, this article examines the structure and affective power of Euroskeptic discourses which dictate the terms of the EU debate. It presents a case study of the recent EU treaty revision process, culminating in the Treaty of Lisbon. In so doing, it enables a deeper understanding of recent policy developments

Use of satellite telemetry data, GIS, and HTML to create an interactive display of caribou movements

The use of animation clearly reveals the large annual variation in wintering areas and large differences in daily movement rates for this herd. This interactive display can be adapted for school groups, subsistence hunters, the general public, or scientists

Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

Background The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system. Results The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci. Conclusion This archived set of Gateway® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome

Implementation of Antibody Rapid Diagnostic Testing versus Real-Time Reverse Transcription-PCR Sample Pooling in the Screening of COVID-19: a Case of Different Testing Strategies in Africa

COVID-19 has wreaked havoc across the globe, although cases in Africa remain lower than 50 other regions but they are gradually on an upward trajectory. To date, COVID-19 cases have 51 been reported in 54 countries. However, due to limited SARS-COV-2 rRT-PCR testing 52 capacity and scarcity of testing reagents, it is probable that the total number of cases could 53 far exceed published statistics. In this viewpoint, using Ghana, Malawi, South Africa and 54 Zimbabwe as examples of countries that have implemented different testing strategies, we 55 argue that the implementation of sample pooling for rRT-PCR over antibody rapid diagnostic 56 testing could have a greater impact in assessing disease burden. Sample pooling offers huge 57 advantages compared to single test rRT-PCR, as it lowers experimental costs, personnel 58 time, reduces burnout and analytical run-times. Africa is already strained in terms of testing 59 resources for COVID-19, hence cheaper alternative ways need to be implemented to 60 conserve resources, maximise on mass testing and reduce transmission in the wider 61 population