Evaluation and ranking of enzyme designs

In 2008, a successful computational design procedure was reported that yielded active enzyme catalysts for the Kemp elimination. Here, we studied these proteins together with a set of previously unpublished inactive designs to determine the sources of activity or lack thereof, and to predict which of the designed structures are most likely to be catalytic. Methods that range from quantum mechanics (QM) on truncated model systems to the treatment of the full protein with ONIOM QM/MM and AMBER molecular dynamics (MD) were explored. The most effective procedure involved molecular dynamics, and a general MD protocol was established. Substantial deviations from the ideal catalytic geometries were observed for a number of designs. Penetration of water into the catalytic site and insufficient residue‐packing around the active site are the main factors that can cause enzyme designs to be inactive. Where in the past, computational evaluations of designed enzymes were too time‐extensive for practical considerations, it has now become feasible to rank and refine candidates computationally prior to and in conjunction with experimentation, thus markedly increasing the efficiency of the enzyme design process.     

Water quality and herbivory interactively drive coral-reef recovery patterns in American Samoa

Background

Compared with a wealth of information regarding coral-reef recovery patterns following major disturbances, less insight exists to explain the cause(s) of spatial variation in the recovery process.

Methodology/Principal Findings

This study quantifies the influence of herbivory and water quality upon coral reef assemblages through space and time in Tutuila, American Samoa, a Pacific high island. Widespread declines in dominant corals (Acropora and Montipora) resulted from cyclone Heta at the end of 2003, shortly after the study began. Four sites that initially had similar coral reef assemblages but differential temporal dynamics four years following the disturbance event were classified by standardized measures of ‘recovery status’, defined by rates of change in ecological measures that are known to be sensitive to localized stressors. Status was best predicted, interactively, by water quality and herbivory. Expanding upon temporal trends, this study examined if similar dependencies existed through space; building multiple regression models to identify linkages between similar status measures and local stressors for 17 localities around Tutuila. The results highlighted consistent, interactive interdependencies for coral reef assemblages residing upon two unique geological reef types. Finally, the predictive regression models produced at the island scale were graphically interpreted with respect to hypothesized site-specific recovery thresholds.

Conclusions/Significance

Cumulatively, our study purports that moving away from describing relatively well-known patterns behind recovery, and focusing upon understanding causes, improves our foundation to predict future ecological dynamics, and thus improves coral reef management.     

Serological response of cats to experimental Besnoitia darlingi and Besnoitia neotomofelis infections and prevalence of antibodies to these parasites in cats from Virginia and Pennsylvania

Besnoitia darlingi and Besnoitia neotomofelis are cyst-forming tissue apicomplexan parasites that use domestic cats (Felis domesticus) as definitive hosts and opossums (Didelphis virginiana ) and Southern Plains woodrats (Neotoma micropus) as intermediate hosts, respectively. Nothing is known about the prevalence of B. darlingi or B. neotomofelis in cats from the United States. Besnoitia darlingi infections have been reported in naturally infected opossums from many states in the United States, and B. neotomofelis infections have been reported from Southern Plains woodrats from Texas, but naturally infected cats have not been identified. The present study examined the IgG antibody response of cats to experimental infection (B. darlingi n = 1 cat; B. neotomofelis n = 3 cats). Samples from these cats were used to develop an indirect immunofluorescent antibody test (IFAT), which was then used to examine seroprevalence of IgG antibodies to tachyzoites of B. darlingi and B. neotomofelis in a population of domestic cats from Virginia (N = 232 cats) and Pennsylvania (N = 209). The serum from cats inoculated with B. darlingi or B. neotomofelis cross-reacted with each other's tachyzoites. The titers to heterologous tachyzoites were 1 to 3 dilutions lower than to homologous tachyzoites. Sera from B. darlingi- or B. neotomofelis-infected cats did not react with tachyzoites of Toxoplasma gondii or Neospora caninum or merozoites of Sarcocystis neurona using the IFAT. Antibodies to B. darlingi were found in 14% and 2% of cats from Virginia and Pennsylvania, respectively. Antibodies to B. neotomofelis were found in 5% and 4% of cats from Virginia and Pennsylvania, respectively. Nine cats from Virginia and 1 cat from Pennsylvania were positive for both.     

Applying a ridge-to-reef framework to support watershed,water quality,and community-based fisheries management in American Samoa

Water quality and fisheries exploitation are localized, chronic stressors that impact coral reef condition and resilience. Yet, quantifying the relative contribution of individual stressors and evaluating the degree of human impact to any particular reef are difficult due to the inherent variation in biological assemblages that exists across and within island scales. We developed a framework to first account for island-scale variation in biological assemblages, and then evaluate the condition of 26 reefs adjacent to watersheds in Tutuila, American Samoa. Water quality data collected over 1 year were first linked with watershed characteristics such as land use and human population. Dissolved inorganic nitrogen (DIN) concentrations were best predicted by total human population and disturbed land for watersheds with over 200 humans km⁻², providing a predictive threshold for DIN enrichment attributed to human populations. Coral reef assemblages were next partitioned into three distinct reeftypes to account for inherent variation in biological assemblages and isolate upon local stressors. Regression models suggested that watershed characteristics linked with DIN and fishing access best predicted ecological condition scores, but their influences differed. Relationships were weakest between coral assemblages and watershed-based proxies of DIN, and strongest between fish assemblages and distances to boat harbors and wave energy (i.e., accessibility). While we did not explicitly address the potential recursivity between fish and coral assemblages, there was a weak overall correlation between these ecological condition scores. Instead, the more complex, recursive nature between reef fish and habitats was discussed with respect to bottom-up and top-down processes, and several ongoing studies that can better help address this topic into the future were identified. The framework used here showed the spatial variation of stressor influence, and the specific assemblage attributes influenced by natural and anthropogenic drivers which aims to guide a local ridge-to-reef management strategy.

     

The Micronesia Challenge: Assessing the Relative Contribution of Stressors on Coral Reefs to Facilitate Science-to-Management Feedback

Fishing and pollution are chronic stressors that can prolong recovery of coral reefs and contribute to ecosystem decline. While this premise is generally accepted, management interventions are complicated because the contributions from individual stressors are difficult to distinguish. The present study examined the extent to which fishing pressure and pollution predicted progress towards the Micronesia Challenge, an international conservation strategy initiated by the political leaders of 6 nations to conserve at least 30% of marine resources by 2020. The analyses were rooted in a defined measure of coral-reef-ecosystem condition, comprised of biological metrics that described functional processes on coral reefs. We report that only 42% of the major reef habitats exceeded the ecosystem-condition threshold established by the Micronesia Challenge. Fishing pressure acting alone on outer reefs, or in combination with pollution in some lagoons, best predicted both the decline and variance in ecosystem condition. High variances among ecosystem-condition scores reflected the large gaps between the best and worst reefs, and suggested that the current scores were unlikely to remain stable through time because of low redundancy. Accounting for the presence of marine protected area (MPA) networks in statistical models did little to improve the models’ predictive capabilities, suggesting limited efficacy of MPAs when grouped together across the region. Yet, localized benefits of MPAs existed and are expected to increase over time. Sensitivity analyses suggested that (i) grazing by large herbivores, (ii) high functional diversity of herbivores, and (iii) high predator biomass were most sensitive to fishing pressure, and were required for high ecosystem-condition scores. Linking comprehensive fisheries management policies with these sensitive metrics, and targeting the management of pollution, will strengthen the Micronesia Challenge and preserve ecosystem services that coral reefs provide to societies in the face of climate change.     

Isolation and characterization of Brush border fragments from mosquito mesenterons

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca²⁺ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.