In vivo bioluminescence imaging for integrated studies of infection

Understanding biological processes in the context of intact organ systems with fine temporal resolution has required the development of imaging strategies that reveal cellular and molecular changes in the living body. Reporter genes that confer optical signatures on a given biological process have been used widely in cell biology and have been used more recently to interrogate biological processes in living animal models of human biology and disease. The use of internal biological sources of light, luciferases, to tag cells, pathogens, and genes has proved to be a versatile tool to provide in vivo indicators that can be detected externally. The application of this technology to the study of animal models of infectious disease has not only provided insights into disease processes, but has also revealed new mechanisms by which pathogens may avoid host defences during infection.     

Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite

AMP-activated kinase (AMPK) is a fuel-sensing enzyme present in most mammalian tissue. In response to a decrease in the energy state of a cell AMPK is phosphorylated and activated by still poorly characterized upstream events. Exposure of bovine aortic endothelial cells (BAEC) to chemically synthesized ONOO- acutely and significantly increased phosphorylation of c-Src, PDK1, AMPK, and its downstream target, acetyl-CoA carboxylase (ACC), without affecting cellular AMP. This novel pathway for AMPK activation was confirmed by the use of pharmacological inhibitors and dominant-negative mutants. Exposure of BAEC to hypoxia-reoxygenation (H/R) caused a biphasic increase in AMPK and ACC phosphorylation, which was prevented by adenoviral overexpression of superoxide dismutase (SOD) or inhibition of nitric-oxide synthase (NOS) implicating a role of ONOO- formed during H/R. Furthermore, dominant-negative mutants of c-Src or kinase-defective PDK1 also blocked H/R-induced AMPK activation indicating that, as with addition of exogenous ONOO-, both c-Src and PI 3-kinase are upstream of AMPK. Moreover, H/R, like ONOO-, significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO- favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO- activates AMPK in a c-Src-mediated, PI 3-kinase-dependent manner, and suggest that ONOO--induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC.     

For uptake of yolk precursors,epithelial cell-oocyte gap junctional communication is required by insects representing six different orders

For uptake of vitellogenin protein into nascent yolk spheres, communication through open gap junction channels between the follicle epithelium and oocyte is required by six different insects representing six different orders. It was recently shown in the hemipteran, Oncopeltus fasciatus, that endocytic uptake of yolk protein resulting in the formation of nascent yolk spheres depended upon an intact epithelium communicating with the oocyte through patent gap junctions. Following treatment with octanol, which down-regulated gap junctions below the level of dye coupling, vitellogenin uptake was terminated. Yet, for another hemipteran, Dysdercus intermedius, it has been shown that yolk spheres can form even when all epithelial cells have been stripped from the oocyte. To determine if the mechanism seen in Oncopeltus is present in other insects, we utilized the same techniques to study nascent yolk sphere production in a dipteran, Drosophila melanogaster, a lepidopteran, Actias luna, a hymenopteran, Xylocopa virginica, a coleopteran, Tenebrio molitor and an orthopteran, Acheta domesticus. In each of these, when gap junctions were down-regulated yolk uptake quickly stopped. That six different insects from six different orders all required a gap junctionally transmitted chemical signal of epithelial cell origin suggests that this mechanism is widespread throughout the insects.     

Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains

Several human pathogens (e.g., Bacillus anthracis, Yersinia pestis, Bordetella pertussis, Plasmodium falciparum, and Mycobacterium tuberculosis) have very restricted unselected allelic variation in structural genes, which hinders study of the genetic relationships among strains and strain-trait correlations. To address this problem in a representative pathogen, 432 M. tuberculosis complex strains from global sources were genotyped on the basis of 230 synonymous (silent) single nucleotide polymorphisms (sSNPs) identified by comparison of four genome sequences. Eight major clusters of related genotypes were identified in M. tuberculosis sensu stricto, including a single cluster representing organisms responsible for several large outbreaks in the United States and Asia. All M. tuberculosis sensu stricto isolates of previously unknown phylogenetic position could be rapidly and unambiguously assigned to one of the eight major clusters, thus providing a facile strategy for identifying organisms that are clonally related by descent. Common clones of M. tuberculosis sensu stricto and M. bovis are distinct, deeply branching genotypic complexes whose extant members did not emerge directly from one another in the recent past. sSNP genotyping rapidly delineates relationships among closely related strains of pathogenic microbes and allows construction of genetic frameworks for examining the distribution of biomedically relevant traits such as virulence, transmissibility, and host range.     

The kinetics of translocation of Smac/DIABLO from the mitochondria to the cytosol in HeLa cells

Smac (second mitochondrial activator of caspases) is released from the mitochondria during apoptosis to relieve inhibition of caspases by the inhibitor of apoptosis proteins (IAPs). The release of Smac antagonizes several IAPs and assists the initiator caspase-9 and effector caspases (caspase-3, caspase-6, and caspase-7) in becoming active, ultimately leading to death of the cell. Translocation of Smac along with cytochrome c and other mitochondrial pro-apoptotic proteins represent important regulatory checkpoints for mitochondria-mediated apoptosis. Whether Smac and cytochrome c translocate by the same mechanism is not known. Here, we show that the time required for Smac efflux from the mitochondria of cells subjected to staurosporine-induced apoptosis is approximately four times longer than the time required for cytochrome c efflux. These results suggest that Smac and cytochrome c may exit the mitochondria by different pathways.     

Predicting fine‐scale distributions of peripheral aquatic species in headwater streams

Headwater species and peripheral populations that occupy habitat at the edge of a species range may hold an increased conservation value to managers due to their potential to maximize intraspecies diversity and species' adaptive capabilities in the context of rapid environmental change. The southern Appalachian Mountains are the southern extent of the geographic range of native Salvelinus fontinalis and naturalized Oncorhynchus mykiss and Salmo trutta in eastern North America. We predicted distributions of these peripheral, headwater wild trout populations at a fine scale to serve as a planning and management tool for resource managers to maximize resistance and resilience of these populations in the face of anthropogenic stressors. We developed correlative logistic regression models to predict occurrence of brook trout, rainbow trout, and brown trout for every interconfluence stream reach in the study area. A stream network was generated to capture a more consistent representation of headwater streams. Each of the final models had four significant metrics in common: stream order, fragmentation, precipitation, and land cover. Strahler stream order was found to be the most influential variable in two of the three final models and the second most influential variable in the other model. Greater than 70% presence accuracy was achieved for all three models. The underrepresentation of headwater streams in commonly used hydrography datasets is an important consideration that warrants close examination when forecasting headwater species distributions and range estimates. Additionally, it appears that a relative watershed position metric (e.g., stream order) is an important surrogate variable (even when elevation is included) for biotic interactions across the landscape in areas where headwater species distributions are influenced by topographical gradients.     

Managing Small-Scale Commercial Fisheries for Adaptive Capacity: Insights from Dynamic Social-Ecological Drivers of Change in Monterey Bay

Globally, small-scale fisheries are influenced by dynamic climate, governance, and market drivers, which present social and ecological challenges and opportunities. It is difficult to manage fisheries adaptively for fluctuating drivers, except to allow participants to shift effort among multiple fisheries. Adapting to changing conditions allows small-scale fishery participants to survive economic and environmental disturbances and benefit from optimal conditions. This study explores the relative influence of large-scale drivers on shifts in effort and outcomes among three closely linked fisheries in Monterey Bay since the Magnuson-Stevens Fisheries Conservation and Management Act of 1976. In this region, Pacific sardine (Sardinops sagax), northern anchovy (Engraulis mordax), and market squid (Loligo opalescens) fisheries comprise a tightly linked system where shifting focus among fisheries is a key element to adaptive capacity and reduced social and ecological vulnerability. Using a cluster analysis of landings, we identify four modes from 1974 to 2012 that are dominated (i.e., a given species accounting for the plurality of landings) by squid, sardine, anchovy, or lack any dominance, and seven points of transition among these periods. This approach enables us to determine which drivers are associated with each mode and each transition. Overall, we show that market and climate drivers are predominantly attributed to dominance transitions. Model selection of external drivers indicates that governance phases, reflected as perceived abundance, dictate long-term outcomes. Our findings suggest that globally, small-scale fishery managers should consider enabling shifts in effort among fisheries and retaining existing flexibility, as adaptive capacity is a critical determinant for social and ecological resilience.