Geosmin reduction by algaecide application to drinking water: field scale efficacy and mechanistic insights.

Ten years of field data from an Oklahoma drinking water utility were analyzed for the effects of an acid-stabilized, ionic copper algaecide/bactericide called EarthTec on geosmin concentrations in the water traveling by pipeline from the source lake to a water treatment plant. The data show that geosmin already present in the raw water is reduced more during periods of applying algaecide than when not. Median reduction in geosmin concentration from pipe intake to pipe outfall by natural degradation without addition of algaecide was 5.6 ng/L removed (56.7% reduction) and improved to 126 ng/L removed (83% reduction) during periods the algaecide was being dosed at 1 µL/L, equivalent to 0.06 mg/L as copper. A laboratory study to replicate the phenomenon at bench-scale showed that either the algaecide itself or its copper-free acidic carrier can be used to depress pH and drive a reaction converting geosmin to an odorless dehydration product, argosmin. Algaecides intuitively reduce the organisms that produce geosmin, but this study shows that geosmin already present in the water is also being reduced through chemical conversion to the odorless argosmin, representing a novel means of geosmin removal in drinking water.

An example of population-level risk assessments for small mammals using individual-based population models.

This article presents a case study demonstrating the application of 3 individual-based, spatially explicit population models (IBMs, also known as agent-based models) in ecological risk assessments to predict long-term effects of a pesticide to populations of small mammals. The 3 IBMs each used a hypothetical fungicide (FungicideX) in different scenarios: spraying in cereals (common vole, Microtus arvalis), spraying in orchards (field vole, Microtus agrestis), and cereal seed treatment (wood mouse, Apodemus sylvaticus). Each scenario used existing model landscapes, which differed greatly in size and structural complexity. The toxicological profile of FungicideX was defined so that the deterministic long-term first tier risk assessment would result in high risk to small mammals, thus providing the opportunity to use the IBMs for risk assessment refinement (i.e., higher tier risk assessment). Despite differing internal model design and scenarios, results indicated in all 3 cases low population sensitivity unless FungicideX was applied at very high (×10) rates. Recovery from local population impacts was generally fast. Only when patch extinctions occured in simulations of intentionally high acute toxic effects, recovery periods, then determined by recolonization, were of any concern. Conclusions include recommendations for the most important input considerations, including the selection of exposure levels, duration of simulations, statistically robust number of replicates, and endpoints to report. However, further investigation and agreement are needed to develop recommendations for landscape attributes such as size, structure, and crop rotation to define appropriate regulatory risk assessment scenarios. Overall, the application of IBMs provides multiple advantages to higher tier ecological risk assessments for small mammals, including consistent and transparent direct links to specific protection goals, and the consideration of more realistic scenarios.

Predicting the spread of aquatic invaders: insight from 200 years of invasion by zebra mussels.

Understanding factors controlling the introduction and spread of species is crucial to improving the management of both natural populations and introduced species. The zebra mussel, Dreissena polymorpha, is considered the most aggressive freshwater invader in the Northern Hemisphere, and is a convenient model system for invasion biology, offering one of the best aquatic examples for examining the invasion process. We used data on 553 of the 1040 glacial lakes in the Republic of Belarus that were examined for the presence of zebra mussels. We used these data to build, test, and construct modified models to predict the spread of this invader, including selection of important parameters that could limit the spread of this invader. In spite of 200 years of continuous invasion, by 1996, zebra mussels were found in only 16.8% of all lakes studied. Of those lakes without zebra mussels in 1996, 66% were predicted to be susceptible to invasion by zebra mussels in the future, and 33% were predicted to be immune to successful invasion due to their water chemistry. Eighty lakes free of zebra mussels in 1996 were reexamined from 1997 to 2008. Of these, zebra mussels successfully invaded an additional 31 lakes, all of which were classified initially as suitable for zebra mussels; none of the lakes previously classified as unsuitable were invaded. We used the Random Forests classification algorithm with 16 environmental variables to determine the most important factors that differed between invaded lakes and those lakes suitable for invasion that have not yet been invaded. Distance to the nearest infested lakes was found to be the most important variable, followed by the lake area, color, average depth, and concentration of chloride, magnesium, and bicarbonate. This study provides a useful approach for predicting the spread of an invader across a landscape with variable habitat suitability that can be applied to a variety of species and systems.

Aquastella gen. nov.: a new genus of saprolegniaceous oomycete rotifer parasites related to Aphanomyces, with unique sporangial outgrowths.

The oomycete genus Aquastella is described to accommodate two new species of parasites of rotifers observed in Brooktrout Lake, New York State, USA. Three rotifer species--Keratella taurocephala, Polyarthra vulgaris, and Ploesoma truncatum--were infected, and this is the first report of oomycete infection in these species. Aquastella attenuata was specific to K. taurocephala and Aquastella acicularis was specific to P. vulgaris and P. truncatum. The occurrence of infections correlated with peak host population densities and rotifers were infected in the upper layers of the water column. Sequencing of 18S rRNA and phylogenetic analysis of both species placed them within the order Saprolegniales, in a clade closely related to Aphanomyces. The Aquastella species were morphologically distinct from other rotifer parasites as the developing sporangia penetrated out through the host body following its death to produce unique tapered outgrowths. Aquastella attenuata produced long, narrow, tapering, finger-like outgrowths, whilst A. acicularis produced shorter, spike-like outgrowths. We hypothesize that the outgrowths serve to deter predation and slow descent in the water column. Spore cleavage was intrasporangial with spore release through exit tubes. Aquastella attenuata produced primary zoospores, whereas A. acicularis released spherical primary aplanospores, more typical of other genera in the Aphanomyces clade.

Cell competition is a tumour suppressor mechanism in the thymus.

Cell competition is an emerging principle underlying selection for cellular fitness during development and disease. Competition may be relevant for cancer, but an experimental link between defects in competition and tumorigenesis is elusive. In the thymus, T lymphocytes develop from precursors that are constantly replaced by bone-marrow-derived progenitors. Here we show that in mice this turnover is regulated by natural cell competition between 'young' bone-marrow-derived and 'old' thymus-resident progenitors that, although genetically identical, execute differential gene expression programs. Disruption of cell competition leads to progenitor self-renewal, upregulation of Hmga1, transformation, and T-cell acute lymphoblastic leukaemia (T-ALL) resembling the human disease in pathology, genomic lesions, leukaemia-associated transcripts, and activating mutations in Notch1. Hence, cell competition is a tumour suppressor mechanism in the thymus. Failure to select fit progenitors through cell competition may explain leukaemia in X-linked severe combined immune deficiency patients who showed thymus-autonomous T-cell development after therapy with gene-corrected autologous progenitors.

Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing.

Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS is associated with TKI resistance. Accordingly, EGFR mutation status is routinely examined in NSCLC specimens, but the employed methods may have a dramatic impact on the interpretation of results and, consequently, therapeutic decisions. Specimens with low tumour cell content are at particular risk for false-negative EGFR mutation reporting by sequencing with Sanger chemistry. To improve reliability of detecting clinically relevant mutant variants of EGFR and KRAS, we took full advantage of 454 deep sequencing and developed a two-step amplification protocol for the analysis of EGFR exons 18-21 and KRAS exons 2 and 3. We systematically addressed the sensitivity, reproducibility and specificity of the developed assay. Mutations could be reliably identified down to an allele frequency of 0.2-1.5 %, as opposed to 10-20 % detection limit of Sanger sequencing. High reproducibility (0-2.1 % variant frequency) and very low background level (0.4-0.8 % frequency) further complement the reliability of this assay. Notably, re-evaluation of 16 NSCLC samples with low tumour cell content ≤40 % and EGFR wild type status according to Sanger sequencing revealed clinically relevant EGFR mutations at allele frequencies of 0.9-10 % in seven cases. In summary, this novel two-step amplification protocol with 454 deep sequencing is superior to Sanger sequencing with significantly increased sensitivity, enabling reliable analysis of EGFR and KRAS in NSCLC samples independent of the tumour cell content.

Municipal wastewater treatment plants as pathogen removal systems and as a contamination source of noroviruses and Enterococcus faecalis.

Municipal wastewater treatment plants play a crucial role in reducing the microbial and pathogen load of human wastes before the end-products are discharged to surface waters (final effluent) or land spread (biosolids). This study investigated the occurrence frequency of noroviruses, Enterococcus faecalis and Enterococcus faecium in influent, final effluent and biosolids from four secondary wastewater treatment plants in northwestern Ireland (plants A-D) and observed the seasonal and spatial variation of the plant treatment efficiencies in the pathogen removals. It was noted that norovirus genogroup II was more resistant to the treatment processes than the norovirus genogroup I and other active viral particles, especially those in the discharge effluents. The percolating biofilm system at plant D resulted in better effluent quality than in the extended aerated activated sludge systems (plants A and B); primary biosolids produced at plant D may pose a higher health risk to the locals. The spread of norovirus genogroup II into the environment, irrespective of the wastewater treatment process, coincides with its national clinical predominance over norovirus genogroup I. This study provides important evidence that municipal wastewater treatment plants not only achieve pathogen removal but can also be the source of environmental pathogen contamination.

Concurrent epigenetic silencing of wnt/ß-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia.

BACKGROUND: The Wnt/ß-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/ß-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples. METHODS: Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and ß-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models. RESULTS: For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/ß-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of ß-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with ß-catenin and thus demonstrating its epigenetically regulated inhibition effect. CONCLUSIONS: The results suggest an epigenetic silencing mechanism of the Wnt/ß-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

Whole-genome analysis of gene expression associates the ubiquitin-proteasome system with the cardiomyopathy phenotype in disease-sensitized congenic mouse strains.

AIMS: Penetrance and phenotypic expressivity of cardiomyopathies are modulated by modifier genes both in model systems and patients. We aimed to dissect the disease-modifying mechanisms by examining genome-wide gene expression in a new set of mouse (Mus musculus) congenic strains. METHODS AND RESULTS: Mutant alleles of the genes calsarcin-1 (Myoz2), sarcoglycan-delta (Sgcd), and muscle LIM protein (Csrp2) were each transferred onto inbred strain backgrounds C57BL/6, C3H/He, 129S1/Sv, and FVB/N, respectively. At 9-10 weeks of age, left ventricular pump function (fractional shortening, FS) was determined by echocardiography in non-sedated congenic animals. Gene expression was then analysed in myocardial tissue using the Affymetrix Mouse 430.2 microarray platform. Variance stabilization, linear mixed-effects modelling, correlations, gene functional classification, and pathway analysis were conducted using the standard software. Strain background FVB/N appeared to protect against the consequences of gene inactivation. Sgcd-deficient congenics showed normal FS, which was consistent with their hypertrophic cardiomyopathy phenotype. Animals with other allele/background combinations developed an impaired ventricular pump function (FS <65%). Gender did not influence FS significantly, yet it determined the sets of genes that were differentially expressed in mice with low FS. In particular, genes encoding the elements of the ubiquitin-proteasome system (UPS) were strongly correlated with the cardiac impairment (absolute Spearman r ≥ 0.7) in both males and females. CONCLUSION: Gene expression profiling in a novel set of congenic strains revealed an association between the UPS and myocardial contractile function, indicating that the UPS may be an important modifier of phenotypic variability in cardiomyopathies.

Cross-effects of nickel contamination and parasitism on zebra mussel physiology.

Aquatic organisms are exposed to pollution which may make them more susceptible to infections and diseases. The present investigation evaluated effects of nickel contamination and parasitism (ciliates Ophryoglena spp. and intracellular bacteria Rickettsiales-like organisms), alone and in combination, on biological responses of the zebra mussel Dreissena polymorpha, and also the infestation abilities of parasites, under laboratory controlled conditions. Results showed that after 48 h, more organisms were infected in nickel-exposed groups, which could be related to weakening of their immune system. Acting separately, nickel contamination and infections were already stressful conditions; however, their combined action caused stronger biological responses in zebra mussels. Our data, therefore, confirm that the parasitism in D. polymorpha represents a potential confounding factor in ecotoxicological studies that involve this bivalve.

Municipal wastewater treatment plants as removal systems and environmental sources of human-virulent microsporidian spores.

Municipal wastewater treatment plants play a vital role in reducing the microbial load of sewage before the end-products are discharged to surface waters (final effluent) or local environments (biosolids). This study was to investigate the presence of human-virulent microsporidian spores (Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon hellem) and enterococci during treatment processes at four Irish municipal secondary wastewater treatment plants (plants A-D). Microsporidian abundance was significantly related to seasonal increase in water temperature. Plant A had the least efficient removal of E. intestinalis spores (32%) in wastewater, with almost 100% removal at other plants both in April and July. Some negative removal efficiencies were obtained for E. bieneusi (at plants C and D, -100%) and for E. hellem (at plants A and D, -90% and -50%). In addition, a positive correlation was found between the levels of enterococci and E. bieneusi in July (r (s) = 0.72, P < 0.05). In terms of the dewatered biosolids, a median concentration as high as 32,000 spores/Kg of E. hellem was observed at plant D in July. Plant C sewage sludge contained the lowest microsporidian loadings (E. bieneusi; 450 spores/L and 1,000 spores/L in April and July, respectively). This study highlights the seasonal variation in concentrations of microsporidian spores in the incoming sewage. Spores in final effluents and dewatered biosolids can be the source of human-virulent microsporidian contamination to the local environment. This emphasizes a considerably high public health risk when sewage-derived biosolids are spread during summer months. This study also suggested enterococci as a potential indicator of the presence of microsporidian spores in wastewater, especially for E. bieneusi.

Field evidence for a parasite spillback caused by exotic mollusc Dreissena polymorpha in an invaded lake.

This study provides field evidence in support of the "parasite spillback hypothesis" that predicts a positive numerical response of native parasites to the presence of an exotic host that is competing for these parasites with native hosts. We examined the role that the exotic mollusc Dreissena polymorpha (zebra mussel) plays as the second intermediate host of the echinostomatid trematode Echinoparyphium recurvatum in Lake Naroch, Belarus. We found that due likely to the disproportionately high abundance of its novel exotic host, the metacercarial stage of this parasite has become substantially more abundant than that of other echinostomatid trematodes recorded in native molluscs. This finding suggests an increased load (the "spillback") of E. recurvatum on native waterfowl that serve as its final hosts. The risk of aquatic birds becoming infected with this trematode via feeding on D. polymorpha was found to vary both spatially and seasonally. This risk was particularly high in the autumn at a depth of 2 m and at sites of bird gatherings.

Seasonal dynamics of endosymbiotic ciliates and nematodes in Dreissena polymorpha.

We report the results of a two-year study in the Svisloch River (Minsk, Belarus) on the dynamics of infection in Dreissena polymorpha by nematodes and three ciliate species Conchophthirus acuminatus, Ophryoglena sp., and Ancistrumina limnica. Although these endosymbionts were present in most of the samples, their prevalence and infection intensity differed significantly. C. acuminatus and A. limnica infection intensities in both years of the study had a maximum in summer and were positively correlated with water temperature. In contrast, Ophryoglena sp. and nematode infection intensities were considerably lower in summer versus winter and were negatively correlated with temperature. In the first long-term study to monitor the size and reproductive rate of C. acuminatus, we found that mean length was negatively correlated with temperature and that temperature was positively correlated with asexual reproduction, with a peak of cell division in April as water temperatures increased.