Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications. This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics in the live cell setting.

Adaptive immunity induces mutualism between commensal eukaryotes.

Pathogenic fungi reside in the intestinal microbiota but rarely cause disease. Little is known about the interactions between fungi and the immune system that promote commensalism. Here we investigate the role of adaptive immunity in promoting mutual interactions between fungi and host. We find that potentially pathogenic Candida species induce and are targeted by intestinal immunoglobulin A (IgA) responses. Focused studies on Candida albicans reveal that the pathogenic hyphal morphotype, which is specialized for adhesion and invasion, is preferentially targeted and suppressed by intestinal IgA responses. IgA from mice and humans directly targets hyphal-enriched cell-surface adhesins. Although typically required for pathogenesis, C. albicans hyphae are less fit for gut colonization1,2 and we show that immune selection against hyphae improves the competitive fitness of C. albicans. C. albicans exacerbates intestinal colitis3 and we demonstrate that hyphae and an IgA-targeted adhesin exacerbate intestinal damage. Finally, using a clinically relevant vaccine to induce an adhesin-specific immune response protects mice from C. albicans-associated damage during colitis. Together, our findings show that adaptive immunity suppresses harmful fungal effectors, with benefits to both C. albicans and its host. Thus, IgA uniquely uncouples colonization from pathogenesis in commensal fungi to promote homeostasis.

Sub-Lethal Effects of Copper on Salmonids: An Avoidance Evaluation Using a Direct Test Method.

Avoidance of copper (Cu) by rainbow trout (Oncorhynchus mykiss) was evaluated using a Y-maze exposure system, with data collected over a 1-h exposure period using a digital camcorder. In exposures to five measured concentrations of dissolved copper (<0.3, 1.2, 9.8, 48.3, and 98.6 µg Cu/L), plus control, significant avoidance behavior (p < 0.05) relative to the control was observed at ≥9.8 µg Cu/L, but not at 1.2 µg Cu/L. The chronic value (i.e., geometric mean of these concentrations) was 3.43 µg Cu/L. Estimates of EC50 values for avoidance of Cu ranged from 4.81 to 9.15 µg Cu/L over four 15-min time intervals of exposure to the metal. Based on water quality characterization of the control/diluent water, the U.S. Environmental Protection Agency (USEPA) water hardness- and biotic ligand model (BLM)-based chronic criteria for dissolved Cu were 8.03 and 2.26 µg Cu/L, respectively. This study suggested that enforcement of the BLM-based criterion would provide a higher level of protection of trout for this sensitive response than the hardness-based criterion.

Survival, Reproduction and Growth of the Marine Amphipod, Leptocheirus plumulosus, Following Laboratory Exposure to Copper-Spiked Sediment.

Leptocheirus plumulosus was exposed for 28 days to Cu-spiked sediment at mean concentrations ranging from 44.4 to 605 mg Cu/kg dry sediment in a sediment/water test system designed to simulate natural conditions. The NOEC (no observed effect concentration)-LOEC (lowest observed effect concentration) range for the most sensitive endpoint of growth was 199-414 mg Cu/kg sediment. An IC50 for reproduction was estimated at 187 mg Cu/kg sediment. Mean Cu concentrations in pore water (PW) where significant effects were observed were 25.8 and 59.0 µg/L, while their respective concentrations in overlying water (OW) were 22.1 and 28.0 µg Cu/L. Copper concentrations were ≤19.1 and <16.6 µg/L in PW and OW, respectively, at lower exposures where effects were not evident. Concentrations of Cu in marine sediment lower than sediment quality guidelines based on geochemical factors of acid volatile sulfide, organic carbon content (f OC), and sediment grain size (i.e., silt + clay) would appear not to result in adverse effects toward L. plumulosus.

Survival and Growth of the Marine Polychaete, Neanthes arenaceodentata, Following Laboratory Exposure to Copper-Spiked Sediment.

Juvenile marine polychaetes, Neanthes arenaceodentata, were exposed for 28 days to copper (Cu)-spiked sediment at six concentrations ranging from 48.3 to 2380 mg Cu/kg dry sediment, plus control. Survival was reduced (p ≤ 0.05) at concentrations ≥1190 mg Cu/kg. Growth was inhibited at Cu concentrations ≥506 mg Cu/kg. Dose-response relationships yielded LC10 and LC50 estimates of 514 and 1230 mg Cu/kg, respectively. The growth effect EC50 estimate was 409 mg Cu/kg. Ranges for the no observable effect concentration and lowest observable effect concentration were 506-1190 mg Cu/kg for survival, and 230-506 mg Cu/kg for growth. Pore water concentrations of Cu were 38.7-65.8 µg Cu/L in exposures where toxic effects were observed, compared to a range of 15.1-22.4 µg Cu/L in exposures where significant effects were not evident. The results of the study were compared with empirical and mechanistic sediment quality guidelines for the protection of benthic organisms.

Energy technologies evaluated against climate targets using a cost and carbon trade-off curve.

Over the next few decades, severe cuts in emissions from energy will be required to meet global climate-change mitigation goals. These emission reductions imply a major shift toward low-carbon energy technologies, and the economic cost and technical feasibility of mitigation are therefore highly dependent upon the future performance of energy technologies. However, existing models do not readily translate into quantitative targets against which we can judge the dynamic performance of technologies. Here, we present a simple, new model for evaluating energy-supply technologies and their improvement trajectories against climate-change mitigation goals. We define a target for technology performance in terms of the carbon intensity of energy, consistent with emission reduction goals, and show how the target depends upon energy demand levels. Because the cost of energy determines the level of adoption, we then compare supply technologies to one another and to this target based on their position on a cost and carbon trade-off curve and how the position changes over time. Applying the model to U.S. electricity, we show that the target for carbon intensity will approach zero by midcentury for commonly cited emission reduction goals, even under a high demand-side efficiency scenario. For Chinese electricity, the carbon intensity target is relaxed and less certain because of lesser emission reductions and greater variability in energy demand projections. Examining a century-long database on changes in the cost-carbon space, we find that the magnitude of changes in cost and carbon intensity that are required to meet future performance targets is not unprecedented, providing some evidence that these targets are within engineering reach. The cost and carbon trade-off curve can be used to evaluate the dynamic performance of existing and new technologies against climate-change mitigation goals.

Toxicity of silver in water and sediment to the freshwater amphipod Hyalella azteca.

Hyalella azteca was exposed to Ag as AgNO3 over a 10-d period in water and two lake sediments that were selected on the basis of their differences in metal-binding properties. The median lethal concentrations (LC50s) for waterborne exposures were 5.4 and 4.9 microg/L for total and dissolved Ag, respectively. In the sediment containing a lesser quantity of total Ag-binding ligands (i.e., Bond Lake, Douglas County, WI, USA, sediment), an Ag-amended sediment toxicity test resulted in a 10-d LC50 of 0.084 g (i.e., 84,000 microg) Ag/kg dry sediment or 8.6 microg Ag/L of pore water (PW). The no-observed-effect concentration (NOEC) to lowest-observed-effect concentration (LOEC) range was 0.012 to 0.031 g Ag/kg dry sediment, or less than 5.0 to 6.0 microg Ag/L of PW. In the sediment with a greater quantity of total Ag-binding ligands (i.e., West Bearskin Lake, Cook County, MN, USA, sediment), the 10-d LC50 was 2.98 g Ag/kg dry sediment, and the NOEC to LOEC range was 2.15 to 4.31 g Ag/kg dry sediment. Because "dissolved" concentrations of Ag in PW were less than 5.0 microg/L at the critical exposures in the latter test, the bioavailable and toxic form of Ag may have been a weakly associated coprecipitate or colloidal complex with hydrous iron oxides that competitively partitioned to the surface of the gills.

Regional VA, Q, and VA/Q during PLV: effects of nitroprusside and inhaled nitric oxide.

Partial liquid ventilation (PLV) with high-specific-weight perfluorocarbon liquids has been shown to improve oxygenation in acute lung injury, possibly by redistributing perfusion from dependent, injured regions to nondependent, less injured regions of the lung. Our hypothesis was that during PLV in normal lungs, a shift in perfusion away from dependent lung zones might, in part, be due to vasoconstriction that could be reversed by infusing sodium nitroprusside (NTP). In addition, delivering inhaled NO during PLV should improve gas exchange by further redistributing blood flow to well-ventilated lung regions. To examine this, we used a single transverse-slice positron emission tomography camera to image regional ventilation and perfusion at the level of the heart apex in six supine mechanically ventilated sheep during five conditions: control, PLV, PLV + NTP, and PLV + NO at 10 and 80 ppm. We found that PLV shifted perfusion from dependent to middle regions, and the dependent region demonstrated marked hypoventilation. The vertical distribution of perfusion changed little when high-dose intravenous NTP was added during PLV, and inhaled NO tended to shift perfusion toward better ventilated middle regions. We conclude that PLV shifts perfusion to the middle regions of the lung because of the high specific weight of perflubron rather than vasoconstriction.