Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities

BACKGROUND: Low concentrations of free magnesium in the intracellular environment can present critical limitations for hammerhead ribozymes, especially for those that are designed for intermolecular (trans) cleavage of a host or pathogen RNA. Tertiary stabilizing motifs (TSM's) from natural and artificial ribozymes with a "type I" topology have been exploited to stabilize trans-cleaving hammerheads. Ribozymes with "type II" or "type III" topologies might seem incompatible with conversion to trans-cleavage designs, because opening the loop at the end of stem 1 or stem 2 to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization. RESULTS: Stem 1, together with single-stranded segments capping or internal to this stem, contains both the substrate-binding and tertiary stabilization functions. This stem was made discontinuous within the sTRSV hammerhead ribozyme, thereby separating the two functions into discrete structural segments. The resulting ribozyme, designated "RzC," cleaved its 13 nucleotide target substrate at MgCl(2 )concentrations as low as 0.2 mM at 25°C and 0.5 mM at 37°C. Under multiple-turnover conditions, nearly thirty turnovers were observed at the highest substrate:RzC ribozyme ratios. Similar stabilization was observed for several derivatives of RzC. Catalytic activity was diminished or eliminated at sub-millimolar MgCl(2 )concentrations for ribozymes with weakened or deleted tertiary interactions. Eadie-Hofstee analysis revealed that the stabilized and non-stabilized ribozymes bind their substrates with equivalent affinities, suggesting that differences in observed activity are not the result of diminished binding. Some of the stabilized and non-stabilized ribozymes appear to fold into a heterogeneous collection of conformers, only a subset of which are catalytically active. CONCLUSION: Hammerhead ribozymes with the "type III" topology can be converted to a tertiary, trans-cleavage design. Separating the stabilization and substrate recognition functions of stem 1 increases cleavage activity at physiological concentrations of divalent magnesium while retaining recognition of exogenous targets. Trans-cleaving ribozymes that exploit the tertiary stabilizing motifs of all natural hammerhead topologies can therefore be used in intracellular applications

Biogeographic analysis of the Tortugas Ecological Reserve: Examining the refuge effect following reserve establishment

Almost 120 days at sea aboard three NOAA research vessels and one fishing vessel over the past three years have supported biogeographic characterization of Tortugas Ecological Reserve (TER). This work initiated measurement of post-implementation effects of TER as a refuge for exploited species. In Tortugas South, seafloor transect surveys were conducted using divers, towed operated vehicles (TOV), remotely operated vehicles (ROV), various sonar platforms, and the Deepworker manned submersible. ARGOS drifter releases, satellite imagery, ichthyoplankton surveys, sea surface temperature, and diver census were combined to elucidate potential dispersal of fish spawning in this environment. Surveys are being compiled into a GIS to allow resource managers to gauge benthic resource status and distribution. Drifter studies have determined that within the ~ 30 days of larval life stage for fishes spawning at Tortugas South, larvae could reach as far downstream as Tampa Bay on the west Florida coast and Cape Canaveral on the east coast. Together with actual fish surveys and water mass delineation, this work demonstrates that the refuge status of this area endows it with tremendous downstream spillover and larval export potential for Florida reef habitats and promotes the maintenance of their fish communities. In Tortugas North, 30 randomly selected, permanent stations were established. Five stations were assigned to each of the following six areas: within Dry Tortugas National Park, falling north of the prevailing currents (Park North); within Dry Tortugas National Park, falling south of the prevailing currents (Park South); within the Ecological Reserve falling north of the prevailing currents (Reserve North); within the Ecological Reserve falling south of the prevailing currents (Reserve South); within areas immediately adjacent to these two strata, falling north of the prevailing currents (Out North); and within areas immediately adjacent to these two strata, falling south of the prevailing currents (Out South). Intensive characterization of these sites was conducted using multiple sonar techniques, TOV, ROV, diver-based digital video collection, diver-based fish census, towed fish capture, sediment particle-size, benthic chlorophyll analyses, and stable isotope analyses of primary producers, fish, and, shellfish. In order to complement and extend information from studies focused on the coral reef, we have targeted the ecotone between the reef and adjacent, non-reef habitats as these areas are well-known in ecology for indicating changes in trophic relationships at the ecosystem scale. Such trophic changes are hypothesized to occur as top-down control of the system grows with protection of piscivorous fishes. Preliminary isotope data, in conjunction with our prior results from the west Florida shelf, suggest that the shallow water benthic habitats surrounding the coral reefs of TER will prove to be the source of a significant amount of the primary production ultimately fueling fish production throughout TER and downstream throughout the range of larval fish dispersal. Therefore, the status and influence of the previously neglected, non-reef habitat within the refuge (comprising ~70% of TER) appears to be intimately tied to the health of the coral reef community proper. These data, collected in a biogeographic context, employing an integrated Before-After Control Impact design at multiple spatial scales, leave us poised to document and quantify the postimplementation effects of TER. Combined with the work at Tortugas South, this project represents a multi-disciplinary effort of sometimes disparate disciplines (fishery oceanography, benthic ecology, food web analysis, remote sensing/geography/landscape ecology, and resource management) and approaches (physical, biological, ecological). We expect the continuation of this effort to yield critical information for the management of TER and the evaluation of protected areas as a refuge for exploited species. (PDF contains 32 pages.

Bushmeat Hunting, Deforestation, and Prediction of Zoonotic Disease

Integrating virology, ecology, and other disciplines enhances prediction of new emerging zoonoses

Local variations in spatial synchrony of influenza epidemics

Background: Understanding the mechanism of influenza spread across multiple geographic scales is not complete. While the mechanism of dissemination across regions and states of the United States has been described, understanding the determinants of dissemination between counties has not been elucidated. The paucity of high resolution spatial-temporal influenza incidence data to evaluate disease structure is often not available. Methodology and Findings: We report on the underlying relationship between the spread of influenza and human movement between counties of one state. Significant synchrony in the timing of epidemics exists across the entire state and decay with distance (regional correlation = 62%). Synchrony as a function of population size display evidence of hierarchical spread with more synchronized epidemics occurring among the most populated counties. A gravity model describing movement between two populations is a stronger predictor of influenza spread than adult movement to and from workplaces suggesting that non-routine and leisure travel drive local epidemics. Conclusions: These findings highlight the complex nature of influenza spread across multiple geographic scales. © 2012 Stark et al

Spatial and multidimensional visualization of Indonesia's village health statistics

Background: A community health assessment (CHA) is used to identify and address health issues in a given population. Effective CHA requires timely and comprehensive information from a wide variety of sources, such as: socio-economic data, disease surveillance, healthcare utilization, environmental data, and health resource allocation. Indonesia is a developing country with 235 million inhabitants over 13,000 islands. There are significant barriers to conducting CHA in developing countries like Indonesia, such as the high cost of computing resources and the lack of computing skills necessary to support such an assessment. At the University of Pittsburgh, we have developed the Spatial OLAP (On-Line Analytical Processing) Visualization and Analysis Tool (SOVAT) for performing CHA. SOVAT combines Geographic Information System (GIS) technology along with an advanced multidimensional data warehouse structure to facilitate analysis of large, disparate health, environmental, population, and spatial data. The objective of this paper is to demonstrate the potential of SOVAT for facilitating CHA among developing countries by using health, population, healthcare resources, and spatial data from Indonesia for use in two CHA cases studies. Results: Bureau of Statistics administered data sets from the Indonesian Census, and the Indonesian village statistics, were used in the case studies. The data consisted of: healthcare resources (number of healthcare professionals and facilities), population (census), morbidity and mortality, and spatial (GIS-formatted) information. The data was formatted, combined, and populated into SOVAT for CHA use. Case study 1 involves the distribution of healthcare professionals in Indonesia, while case study 2 involves malaria mortality. Screen shots are shown for both cases. The results for the CHA were retrieved in seconds and presented through the geospatial and numerical SOVAT interface. Conclusion: The case studies show the potential of spatial and multidimensional analysis using SOVAT for community health assessment in developing countries. Since SOVAT is based primarily on open-source components and can be deployed using small personal computers, it is cost-effective for developing countries. Also, combining the strength in analysis and the ease of use makes tools like SOVAT ideal for healthcare professionals without extensive computer skills. © 2008 Parmanto et al; licensee BioMed Central Ltd

Occupant Dynamics in Rollover Crashes: Influence of Roof Deformation and Seat Belt Performance on Probable Spinal Column Injury

Motor vehicle crashes are the leading cause of death in the United States for people ages 3–33, and rollover crashes have a higher fatality rate than any other crash mode. At the request and under the sponsorship of Ford Motor Company, Autoliv conducted a series of dynamic rollover tests on Ford Explorer sport utility vehicles (SUV) during 1998 and 1999. Data from those tests were made available to the public and were analyzed in this study to investigate the magnitude of and the temporal relationship between roof deformation, lap–shoulder seat belt loads, and restrained anthropometric test dummy (ATD) neck loads

Interventions in measles outbreaks: the potential reduction in cases associated with school suspension and vaccination interventions

Background: Measles is resurgent in the US, with more cases in 2019 than any year since 1992. Many of the cases were concentrated in three outbreaks in New York and Washington states, where local governments enacted intervention strategies in an attempt to limit the spread of measles. Regulations differed by location, suggesting guidance on the optimal interventions may be beneficial. Methods: We simulate the daily interactions of the populations of six metropolitan areas of Texas, US, using an agent-based model. The real-life vaccination rates of each school in these metropolitan areas are applied to simulated equivalents. A single case of measles is introduced to the population and the resulting number of cases counted. A range of public health interventions, focused on suspending unvaccinated students and mandatory vaccinations, were simulated during measles outbreaks and the reduction in the number of measles cases, relative to no intervention, recorded. Interventions were simulated only in schools with measles cases and in all schools in each metropolitan area. Results: Suspending unvaccinated students from school was associated with the greatest reduction in measles cases. In a plausible worst-case outbreak scenario, the number of cases is forecast to reduce by 68-96%. Interventions targeting all schools in a metropolitan area is not found to be associated with fewer measles cases than only targeting schools with measles cases, at 2018 vaccination rates. Targeting all schools also increases the cumulative number of school days missed by suspended students by a factor of 10-100, depending on the metropolitan area, compared to targeting only schools with measles cases. If vaccination rates drop 5% in the schools which are under-vaccinated in 2018, metropolitan area-wide interventions are forecast to be associated with fewer cases than school-specific interventions. Conclusions: Interventions that are quickly implemented and widely followed may reduce the size of measles outbreaks by up 96%. If vaccination rates continue to fall in Texas, metropolitan area-wide interventions should be considered in the event of an outbreak

Ongoing monitoring of Tortugas Ecological Reserve: Assessing the consequences of reserve designation

Over the past five years, a biogeographic characterization of Tortugas Ecological Reserve(TER) has been carried out to measure the post-implementation effects of TER as a refuge for exploited species. Our results demonstrate that there is substantial microalgal biomass at depths between 10 and 30 m in the soft sediments at the coral reef interface, and that this community may play an important role in the food web supporting reef organisms. In addition, preliminary stable isotope data, in conjunction with prior results from the west Florida shelf, suggest that the shallow water benthic habitats surrounding the coral reefs of TER will prove to be an important source of the primary production ultimately fueling fish production throughout TER. The majority of the fish analyzed so far have exhibited a C isotope signature consistent with a food web which relies heavily on benthic primary production. Fish counts indicate a marked increase in the abundance of large fish (>20 cm) within the Reserve relative to the Out and Park strata, across years. Faunal collections from open and protected soft bottom habitat near the northern boundary of Tortugas North strongly suggest that relaxation of trawling pressure has increased benthic biomass and diversity in this area of TER. These data, employing an integrated Before - After Control Impact (BACI) design at multiple spatial scales, will allow us to continue to document and quantify the post-implementation effects of TER. (PDF contains 58 pages