De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila

The bacterial pathogen Legionella pneumophila exploits host cell vesicle transport by transiently manipulating the activity of the small guanosine triphosphatase (GTPase) Rab1. The effector protein SidM recruits Rab1 to the Legionella-containing vacuole (LCV), where it activates Rab1 and then AMPylates it by covalently adding adenosine monophosphate (AMP). L. pneumophila GTPase-activating protein LepB inactivates Rab1 before its removal from LCVs. Because LepB cannot bind AMPylated Rab1, the molecular events leading to Rab1 inactivation are unknown. We found that the effector protein SidD from L. pneumophila catalyzed AMP release from Rab1, generating de-AMPylated Rab1 accessible for inactivation by LepB. L. pneumophila mutants lacking SidD were defective for Rab1 removal from LCVs, identifying SidD as the missing link connecting the processes of early Rab1 accumulation and subsequent Rab1 removal during infection.     

Adaptation and evolutionary rescue in metapopulations experiencing environmental deterioration

It is not known whether evolution will usually be rapid enough to allow a species to adapt and persist in a deteriorating environment. We tracked the eco-evolutionary dynamics of metapopulations with a laboratory model system of yeast exposed to salt stress. Metapopulations experienced environmental deterioration at three different rates and their component populations were either unconnected or connected by local dispersal or by global dispersal. We found that adaptation was favored by gradual deterioration and local dispersal. After further abrupt deterioration, the frequency of evolutionary rescue depended on both the prior rate of deterioration and the rate of dispersal. Adaptation was surprisingly frequent and rapid in small peripheral populations. Thus, evolutionary dynamics affect both the persistence and the range of a species after environmental deterioration.     

Negative effects of energy supplementation at peak lactation of sheep can be offset by the addition of Lactobacillus-fermented plant extracts

Energy supplementation may reduce oxidative stress by correcting a negative energy balance, but in some contexts, it has been shown to increase oxidative stress, especially at peak lactation. The current experiment examined if a pelleted energy supplement with or without the addition of Lactobacillus-fermented seaweed or seaweed plus terrestrial plants extracts affected oxidative stress of ewes from late gestation through to weaning and ewe and lamb production from lambing to weaning. Treatments were either no supplement (CON−), a pelleted supplement only (CON+, 100 g/ewe per d), CON+ with seaweed extract only (SWO, 10 mL/ewe per day), or CON+ with seaweed plus an arrangement of terrestrial plant extract (SWP, 10 mL/ewe per d). Ewes (n = 160; mean initial BW = 72.3 ± 9.5 kg [mean ± SD]) were randomized to pastures (n = 4 pastures per treatment with 10 ewes each). After lambing, ewes with twins were reallocated to pastures (n = 3 pastures per treatment with 10 ewes each) according to lambing date. At 4 wk in milk, supplementation tended to reduce total antioxidant status (TAS; P = 0.10) and increased glutathione peroxidase (GPx) activity compared with nonsupplemented ewes (P = 0.04). The addition of seaweed and terrestrial plants extracts to the concentrate, that is, SWO and SWP, increased TAS and reduced GPx activity compared with CON+ (P < 0.01). Supplementation increased milk yield at weeks 4, 6, and 8 of lactation, and protein, lactose, and total milk solids yield at peak lactation (week 4; P < 0.05). The CON− ewes had greater somatic cell count than the supplemented ewes at weeks 4, 8, and 10 of lactation (P = 0.03). Our results suggest that energy supplementation, alone, increases oxidative stress of lactating ewes, which may relate to increased oxidative phosphorylation. Most importantly, these results indicate that in situations where energy supplementation is needed to increase animal performance, negative effects of energy supplementation around peak lactation can be offset by the addition of Lactobacillus-fermented plant extracts (SWO and SWP) to improve antioxidant status.     

Membrane Dialysis Extraction (MDE): A Novel Approach for Extracting Toxicologically Relevant Hydrophobic Organic Compounds from Soils and Sediments for Assessment in Biotests

Goal, Scope and Background   Organic solvents are routinely used to extract toxicants from polluted soils and sediments prior to chemical analysis or bioassay. Conventional extraction methods often require the use of heated organic solvents, in some cases under high pressure. These conditions can result in loss of volatile compounds from the sample and the degradation of thermally labile target analytes. Moreover, extracts of soils and sediments also frequently contain substantial quantities of organic macromolecules which can act as sorbing phases for target analytes and in doing so interfere with both chemical analysis and bioassays. Membrane dialysis extraction (MDE) is described as a simple, passive extraction method for selectively extracting toxicologically relevant hydrophobic organic compounds (HOCs) from polluted soils and sediments and anaylzed for its applicability in ecotoxicological investigations. Methods   Toxicologically relevant hydrophobic organic compounds were extracted from wet and dry sediments by sealing replicate samples in individual lengths of pre-cleaned low-density polyethylene (LD-PE) tubing and then dialysing in n-hexane. The efficacy of the MDE method for use in ecotoxicological investigations was assessed by testing the concentrated extracts in the neutral red assay for acute cytotoxicity, in the EROD assay for the presence of dioxin-like compounds and in the Danio rerio fish egg assay for embryotoxic and teratogenic effects. Conditions of the sediment sample (with or without water content), dialysis membrane length and duration of dialysis were analyzed with respect to their impact on three endpoints. Results of the MDE investigations were compared to data obtained in samples prepared using conventional Soxhlet extraction. Results and Discussion   The membrane dialysis extraction was found to be at least as efficient as Soxhlet methodology to extract toxicologically relevant HOCs from sediment samples. In most cases, MDE-derived extracts showed a higher toxicological potential than the Soxhlet extracts. Lack of any significant effects in any MDE controls indicated these differences were not caused by contamination of the LD-PE membrane used. The elevated toxicological potential of MDE extracts is most likely the result of enhanced bioavailability of toxic compounds in consequence of lower amounts of organic macromolecules (i.e. sorbing phases) in the MDE extracts. This effect is probably the result of a size-selective restriction by the LD-PE membrane. Conclusion   Membrane dialysis extraction was found to be a simple, efficient and cost-effective method for the extraction of sediment samples. MDE can be used to extract toxicologically relevant hydrophobic organic compounds from both wet and dry sediments without the risk of loosing volatile and thermally labile target analytes. The size-selectivity of the LD-PE membrane also appears to have the capacity to increase the bioavailablity of potential target analytes in the resulting extracts by retaining much of the organic macromolecules present in the sample. Thus, results suggest that MDE may be particularly useful for the extraction of toxicologically relevant hydrophobic organic compounds from soils and sediments for bioassays and other ecotoxicological investigations. Recommendation and Perspective   Further validation of MDE has been initiated and the applicability of the methodology to other sample types will be investigated. Of particular interest is the potential application of MDE to recover hydrophobic target analytes from biological samples such as muscle, other soft tissues and blood.     

Fate of Amoxicillin In Two Water Reclamation Systems

The overall objective of this research was to determine the fate of amoxicillin in two wastewater reclamation systems containing biological and physical/chemical treatment processes. The results of this study indicate that amoxicillin is easily removed in a biological wastewater treatment system (<0.15 and 0.10 mg/L). Further evaluation of the physical/chemical treatment process, which included reverse osmosis, ion exchange, and UV oxidation and disinfection, indicated that amoxicillin will be removed (<0.10 mg/L) should it breakthrough the biological system. The concern of amoxicillin is not its presence in high concentrations in the effluent of a water reclamation system, but low antibiotic concentrations, which are known to encourage antibiotic resistance development.     

Attempts to derive EC50 values for heavy metals from land-applied Cu-, Ni-, and Zn-spiked sewage sludge

We established a field trial to assess the impacts on soil biological properties of application of heavy metal-spiked sewage sludge, with the aim of determining toxicity threshold concentrations of heavy metals in soil. Plots were treated with sludges containing increasing concentrations of Cu, Ni and Zn in order to raise the metal concentrations in the soil by 0-200 mg Cu kg⁻¹, 0-60 mg Ni kg⁻¹ and 0-400 mg Zn kg⁻¹, and were then cultivated and sown in ryegrass-clover pasture and monitored annually for 6 years. All biological properties measured (soil basal respiration, microbial biomass C, and sulphatase enzyme activities), except phosphatase activity, increased in all plots over the duration of the experiment. Consequently, it was only possible to assess effects of heavy metals across time if, each year, all data for each metal were normalised by expressing them as percentages of the activities measured in an un-sludged control plot. When this was done, no significant effects of increasing heavy-metal concentrations on basal respiration, microbial biomass C or respiratory quotient (qCO₂) were observed, although total Cu and soil solution Cu were significantly negatively related to microbial biomass C when it was expressed as a proportion of soil total C. None of the properties measured were affected by increasing Ni concentrations. Phosphatase and sulphatase activities were significantly negatively related to increasing Zn concentrations, but not usually to increasing Cu unless they were expressed as a proportion of total C. A sigmoidal dose-response model was used to calculate EC₂₀ and EC₅₀ values using the normalised data, but generally, the model parameters had very large 95% confidence intervals and/or the fits to the model had small R² values. The factors primarily responsible for confounding these results were site and sample variations not accounted for by the normalisation process and the absence of any data points at metal concentrations beyond the calculated EC₅₀ values. In the few instances where reasonable EC₂₀ values could be calculated, they were relatively consistent across properties, e.g., EC₂₀ for total Zn and phosphatase (330 mg kg⁻¹), total Zn and sulphatase (310 mg kg⁻¹), and EC₂₀ for total Cu and sulphatase (140 mg kg⁻¹) and total Cu and microbial biomass C (140 mg kg⁻¹), when both sulphatase and microbial biomass C were expressed as a proportion of total C. Our results suggest that Cu and Zn at the upper concentrations used in this experiment were possibly having adverse effects on some soil biological properties. However, much higher metal concentrations will be needed to accurately calculate EC₂₀ and EC₅₀ and this may not be easily achievable without many applications of sewage sludge, even if the sludge is spiked with heavy metals.     

Elemental concentrations in fresh snowfall across a regional transect in the northeastern U.S.: Apparent sources and contribution to acidity

Fresh snowfall was collected on the surface of 8 lakes across a 350 km west-east transect from northeastern New York state to the coast of Maine after a single storm. In addition, every snowfall event during the winter of 1993 was collected on a single lake near the center of the transect. Across the transect, midwestern sources appear to dominate Pb and Cd concentrations, while Sb appears to be derived from midwestern sources as well as local and/or industrial East Coast sources. In all samples, the highest Na, Cl and Mg concentrations reflect a marine influence, but at some transect sites roadspray aerosol appears to contribute to Na and Cl concentrations. The regional pattern of Ca, K, Mn and Sr concentrations and Mn/Sr ratios indicate that woodsmoke may be an important winter source of these elements at some sites. In all samples, H⁺ is strongly correlated with NO₃ ^? (R² = 0.97) and mean NO₃ ^?/SO₄ ^2? molar ratios of 6.4 for transect samples, and 4.7 for temporal samples, are higher than mean NO₃ ^?/SO₄ ^2? reported for other precipitation studies in the same region. The contribution of NO₃ ^? to the snowpack greatly exceeds that of SO₄ ^2?, and may be a major source of acidity in aquatic ecosystems during snowmelt.