Expression of the chloride channel CLC-K in human airway epithelial cells

Airway submucosal gland function is severely disrupted in cystic fibrosis (CF), as a result of genetic mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane Cl– channel. To identify other Cl– channel types that could potentially substitute for lost CFTR function in these cells, we investigated the functional and molecular expression of Cl– channels in Calu-3 cells, a human cell line model of the submucosal gland serous cell. Whole cell patch clamp recording from these cells identified outwardly rectified, pH- and calcium-sensitive Cl– currents that resemble those previously ascribed to ClC-K type chloride channels. Using reverse transcription – polymerase chain reaction, we identified expression of mRNA for ClC-2, ClC-3, ClC-4, ClC-5, ClC-6, ClC-7, ClC-Ka, and ClC-Kb, as well as the common ClC-K channel b subunit barttin. Western blotting confirmed that Calu-3 cells express both ClC-K and barttin protein. Thus, Calu-3 cells express multiple members of the ClC family of Cl– channels that, if also expressed in native submucosal gland serous cells within the CF lung, could perhaps act to partially substitute lost CFTR function. Furthermore, this work represents the first evidence for functional ClC-K chloride channel expression within the lung

Molecular detection and quantification of slug parasitic nematodes from the soil and their hosts

Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques based on qPCR methods for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (>32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Alloionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras larvae compared to the resistant Arion vulgaris. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes.This work was funded by the Swiss National Science Foundation: Grants NRP68 and SNSF Div. I-III, reference 166632. The Government of Spain supports RCH with a Ramón y Cajal contract award: RYC-2016-19939.Peer reviewe

The evaluation of entomopathogenic nematode soil food web assemblages across Switzerland reveals major differences among agricultural, grassland and forest ecosystems

Entomopathogenic nematodes (EPNs) occur in natural and agricultural soils and contribute to the control of soil dwelling insect pests. Ecosystems differ in their soil parameters (pH, moisture, texture, etc.) and microhabitat conditions (presence of roots, plant cover, belowground fauna, etc.), which can affect the EPN soil food web assemblage in an unpredictable manner. By comparing soils that represent different ecological scenarios, we can link the natural distribution of the EPN to abiotic and biotic factors that shape the assemblages of soil food web to unravel the factors that determine EPN abundance and activity. We hypothesized that the EPN soil food web assemblages are affected by agricultural management practices and that this should be reflected when compared to natural ecosystems, such as forests and grasslands. By combining traditional and state-of-the-art molecular tools, we targeted 25 soil organisms to explore EPN food web in 40 Swiss georeferenced sites that can be categorized as “agricultural area”, “forest”, and “grassland”. EPNs abundance, richness and diversity did not vary among the different ecosystems. However, EPN activity (capability to kill insects) was higher in grasslands. Similarly, the free-living nematodes (FLNs) that compete with EPN for the cadavers were much more abundant in grassland, while reduced in the forest soils, suggesting contrasting conditions between these two natural areas. Nematophagous fungi (NF) were more diverse and present in agricultural areas, suggesting that the abiotic conditions in the agricultural areas may favor NF presence. Six soil properties and environmental factors shaped the EPN soil food web in Swiss soils (pH, elevation, clay content, soil water content, temperature, and rainfall), which explained 54% of the community variation in multivariate analysis. These observations were consistent with the key abiotic variables described for subtropical and Mediterranean regions. Identifying the links between the abiotic and biotic factors in very distinct areas can be the basis for predicting EPN soil food web assemblages. Such information can be of value for developing strategies to favour EPN resilience in a changing environment and enhance their capacity as biocontrol agents.This work was supported by an economic stimulus grant (51NF40-144621) and by the National Research Program 68 (NRP68) grant (406840_143065) from the Swiss National Science Foundation. GJ was supported by an assistantship from the University of Neuchâtel and XC was supported by a PhD fellowship from the Commission Fédérale des Bourses pour Etudiants Etrangères CFBE (Confédération Suisse). RCH was supported by The Government of Portugal (Investigator Program award, IF/00552/2014).Peer reviewe

Biocompatibility of amine-functionalized silica nanoparticles: The role of surface coverage.

Here, amorphous silica nanoparticles (NPs), one of the most abundant nanomaterials, are used as an example to illustrate the utmost importance of surface coverage by functional groups which critically determines biocompatibility. Silica NPs are functionalized with increasing amounts of amino groups, and the number of surface exposed groups is quantified and characterized by detailed NMR and fluorescamine binding studies. Subsequent biocompatibility studies in the absence of serum demonstrate that, irrespective of surface modification, both plain and amine-modified silica NPs trigger cell death in RAW 264.7 macrophages. The in vitro results can be confirmed in vivo and are predictive for the inflammatory potential in murine lungs. In the presence of serum proteins, on the other hand, a replacement of only 10% of surface-active silanol groups by amines is sufficient to suppress cytotoxicity, emphasizing the relevance of exposure conditions. Mechanistic investigations identify a key role of lysosomal injury for cytotoxicity only in the presence, but not in the absence, of serum proteins. In conclusion, this work shows the critical need to rigorously characterize the surface coverage of NPs by their constituent functional groups, as well as the impact of serum, to reliably establish quantitative nanostructure activity relationships and develop safe nanomaterials

A method of establishing a transect for biodiversity and ecosystem function monitoring across Europe

The establishment of the range of soil biodiversity found within European soils is needed to guide EU policy development regarding the protection of soil. Such a base-line should be collated from a wide-ranging sampling campaign to ensure that soil biodiversity from the majority of soil types, land-use or management systems, and European climatic (bio-geographical zones) were included. This paper reports the design and testing of a method to achieve the large scale sampling associated with the establishment of such a baseline, carried out within the remit of the EcoFINDERS project, and outlines points to consider when such a task is undertaken. Applying a GIS spatial selection process, a sampling campaign was undertaken by 13 EcoFINDERS partners across 11 countries providing data on the range of indicators of biodiversity and ecosystem functions including; micro and meso fauna biodiversity, extracellular enzyme activity, PLFA and community level physiological profiling (MicroResp™ and Biolog™). Physical, chemical and bio-geographical parameters of the 81 sites sampled were used to determine whether the model predicted a wide enough range of sites to allow assessment of the biodiversity indicators tested. Discrimination between the major bio-geographical zones of Atlantic and Continental was possible for all land-use types. Boreal and Alpine zones only allowed discrimination in the most common land-use type for that area e.g. forestry and grassland sites, respectively, while the Mediterranean zone did not have enough sites sampled to draw conclusions across all land-use types. The method used allowed the inclusion of a range of land-uses in both the model prediction stage and the final sites sampled. The establishment of the range of soil biodiversity across Europe is possible, though a larger targeted campaign is recommended. The techniques applied within the EcoFINDERS sampling would be applicable to a larger campaign