A genome-wide strategy for the identification of essential genes in Staphylococcus aureus

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.     

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.     

Long-term creatine supplementation does not significantly affect clinical markers of health in athletes

Creatine has been reported to be an effective ergogenic aid for athletes. However, concerns have been raised regarding the long-term safety of creatine supplementation. This study examined the effects of long-term creatine supplementation on a 69-item panel of serum, whole blood, and urinary markers of clinical health status in athletes. Over a 21-month period, 98 Division IA college football players were administered in an open label manner creatine or non-creatine containing supplements following training sessions. Subjects who ingested creatine were administered 15.75 g/day of creatine monohydrate for 5 days and an average of 5 g/day thereafter in 5–10 g/day doses. Fasting blood and 24-h urine samples were collected at 0, 1, 1.5, 4, 6, 10, 12, 17, and 21 months of training. A comprehensive quantitative clinical chemistry panel was determined on serum and whole blood samples (metabolic markers, muscle and liver enzymes, electrolytes, lipid profiles, hematological markers, and lymphocytes). In addition, urine samples were quantitatively and qualitative analyzed to assess clinical status and renal function. At the end of the study, subjects were categorized into groups that did not take creatine (n = 44) and subjects who took creatine for 0–6 months (mean 4.4 ± 1.8 months, n = 12), 7–12 months (mean 9.3 ± 2.0 months, n = 25), and 12–21 months (mean 19.3 ± 2.4 months, n = 17). Baseline and the subjects' final blood and urine samples were analyzed by MANOVA and 2 × 2 repeated measures ANOVA univariate tests. MANOVA revealed no significant differences (p = 0.51) among groups in the 54-item panel of quantitative blood and urine markers assessed. Univariate analysis revealed no clinically significant interactions among groups in markers of clinical status. In addition, no apparent differences were observed among groups in the 15-item panel of qualitative urine markers. Results indicate that long-term creatine supplementation (up to 21-months) does not appear to adversely effect markers of health status in athletes undergoing intense training in comparison to athletes who do not take creatine.     

Model of ionic transport for bovine ciliary epithelium: effects of acetazolamide and HCO

The possible existence of transepithelial bicarbonate transport across the isolated bovine ciliary body was investigated by employing a chamber that allows for the measurement of unidirectional, radiolabeled fluxes of CO2 + HCO. No net flux of HCO was detected. However, acetazolamide (0.1 mM) reduced the simultaneously measured short-circuit current (I(sc)). In other experiments in which (36)Cl- was used, a net Cl- flux of 1.12 microeq. h(-1). cm(-2) (30 microA/cm(2)) in the blood-to-aqueous direction was detected. Acetazolamide, as well as removal of HCO from the aqueous bathing solution, inhibited the net Cl- flux and I(sc). Because such removal should increase HCO diffusion toward the aqueous compartment and increase the I(sc), this paradoxical effect could result from cell acidification and partial closure of Cl- channels. The acetazolamide effect on Cl- fluxes can be explained by a reduction of cellular H+ and HCO (generated from metabolic CO2 production), which exchange with Na+ and Cl- via Na+/H+ and Cl-/HCO exchangers, contributing to the net Cl- transport. The fact that the net Cl- flux is about three times larger than the I(sc) is explained with a vectorial model in which there is a secretion of Na+ and K+ into the aqueous humor that partially subtracts from the net Cl- flux. These transport characteristics of the bovine ciliary epithelium suggest how acetazolamide reduces intraocular pressure in the absence of HCO transport as a driving force for fluid secretion.     

Substrates of Energy Metabolism Attenuate Methamphetamine-Induced Neurotoxicity in Striatum

Abstract: High doses of methamphetamine (METH) produce a long-term depletion in striatal tissue dopamine content. The mechanism mediating this toxicity has been associated with increased concentrations of dopamine and glutamate and altered energy metabolism. In vivo microdialysis was used to assess and alter the metabolic environment of the brain during high doses of METH. METH significantly increased extracellular concentrations of lactate in striatum and prefrontal cortex. This increase was significantly greater in striatum and coincided with the greater vulnerability of this brain region to the toxic effects of METH. To examine the effect of supplementing energy metabolism on METH-induced dopamine content depletions, the striatum was perfused directly with decylubiquinone or nicotinamide to enhance the energetic capacity of the tissue during or after a neurotoxic dosing regimen of METH. When decylubiquinone or nicotinamide was perfused into striatum during the administration of METH, there was no significant effect on METH-induced striatal dopamine efflux, glutamate efflux, or the long-term dopamine depletions measured 7 days later. However, a delayed perfusion with decylubiquinone or nicotinamide for 6 h beginning immediately after the last METH injection attenuated the METH-induced striatal dopamine depletions measured 1 week later. These results support the hypothesis that the compromised metabolic state produced by METH administration predisposes dopamine terminals to the neurotoxic effects of glutamate, dopamine, and/or free radicals.     

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.     

Disentangling host,pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research

The amphibian fungal disease chytridiomycosis, which affects species across all continents, recently emerged as one of the greatest threats to biodiversity. Yet, many aspects of the basic biology and epidemiology of the pathogen, Batrachochytrium dendrobatidis (Bd), are still unknown, such as when and from where did Bd emerge and what is its true ecological niche? Here, we review the ecology and evolution of Bd in the Americas and highlight controversies that make this disease so enigmatic. We explore factors associated with variance in severity of epizootics focusing on the disease triangle of host susceptibility, pathogen virulence, and environment. Reevaluating the causes of the panzootic is timely given the wealth of data on Bd prevalence across hosts and communities and the recent discoveries suggesting co‐evolutionary potential of hosts and Bd. We generate a new species distribution model for Bd in the Americas based on over 30,000 records and suggest a novel future research agenda. Instead of focusing on pathogen “hot spots,” we need to identify pathogen “cold spots” so that we can better understand what limits the pathogen''s distribution. Finally, we introduce the concept of “the Ghost of Epizootics Past” to discuss expected patterns in postepizootic host communities.     

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.