Profiling microRNAs in lung tissue from pigs infected with Actinobacillus pleuropneumoniae

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are a class of non-protein-coding genes that play a crucial regulatory role in mammalian development and disease. Whereas a large number of miRNAs have been annotated at the structural level during the latest years, functional annotation is sparse. Actinobacillus pleuropneumoniae (APP) causes serious lung infections in pigs. Severe damage to the lungs, in many cases deadly, is caused by toxins released by the bacterium and to some degree by host mediated tissue damage. However, understanding of the role of microRNAs in the course of this infectious disease in porcine is still very limited. RESULTS: In this study, the RNA extracted from visually unaffected and necrotic tissue from pigs infected with Actinobacillus pleuropneumoniae was subjected to small RNA deep sequencing. We identified 169 conserved and 11 candidate novel microRNAs in the pig. Of these, 17 were significantly up-regulated in the necrotic sample and 12 were down-regulated. The expression analysis of a number of candidates revealed microRNAs of potential importance in the innate immune response. MiR-155, a known key player in inflammation, was found expressed in both samples. Moreover, miR-664-5p, miR-451 and miR-15a appear as very promising candidates for microRNAs involved in response to pathogen infection. CONCLUSIONS: This is the first study revealing significant differences in composition and expression profiles of miRNAs in lungs infected with a bacterial pathogen. Our results extend annotation of microRNA in pig and provide insight into the role of a number of microRNAs in regulation of bacteria induced immune and inflammatory response in porcine lung.     

Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy

The generation and use of avian antibodies is of increasing interest in a wide variety of applications within the life sciences. Due to their phylogenetic distance, mechanisms of immune diversification and the way in which they deposit IgY immunoglobulin in the egg yolk, chickens provide a number of advantages compared to mammals as hosts for immunization. These advantages include: the one-step purification of antibodies from egg yolk in large amounts facilitates having a virtually continuous supply; the epitope spectrum of avian antibodies potentially grants access to novel specificities; the broad absence of cross-reactivity with mammalian epitopes avoids assay interference and improves the performance of immunological techniques. The polyclonal nature of IgY antibodies has limited their use since avian hybridoma techniques are not well established. Recombinant IgY, however, can be generated from mammalian monoclonal antibodies which makes it possible to further exploit the advantageous properties of the IgY scaffold. Moreover, cloning and selecting the immune repertoire from avian organisms is highly efficient, yielding antigen-specific antibody fragments. The recombinant approach is well suited to circumvent any limitations of polyclonal antibodies. This review presents comprehensive information on the generation, purification, modification and applications of polyclonal and monoclonal IgY antibodies.     

How do extreme drought and plant community composition affect host plant metabolites and herbivore performance?

Water availability and plant community composition alter plant nutrient availability and the accumulation of plant defence compounds therefore having an impact on herbivore performance. Combined effects of drought stress and plant community composition on leaf chemicals and herbivore performance are largely unexplored. The objective of our study was, therefore, to find out the impact of extreme drought and of plant community composition on plant–herbivore interactions. Larvae of the generalist butterfly Spodoptera littoralis were reared on leaves of the grass Holcus lanatus which was grown in experimental communities, differing in species- and functional group richness. These communities were either subjected to extreme drought or remained under ambient climatic conditions. Drought decreased relative water content, soluble protein content, nitrogen and total phenol content and increased the content of carbohydrates in the grass. As a consequence, the larvae feeding on drought-exposed plants revealed a longer larval stage, increased pupal weight and higher adult eclosion rates. Plant community composition mainly caused changes to the defensive compounds of the grass, but also marginally affected protein and carbohydrate content. Larvae feeding on species-richest communities without legumes showed the highest mortality. Our findings imply that climate change that is projected to increase the frequency of severe droughts, as well as alter plant community compositions, is likely to affect arthropod–plant interactions through an alteration of leaf chemicals.     

Selective non-steroidal glucocorticoid receptor agonists attenuate inflammation but do not impair intestinal epithelial cell restitution in vitro

Introduction

Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile.

Methods

The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis.

Results

Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348.

Conclusion

Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing. The fact that SEGRAs did not have a similar effect on cell restitution might be due to a different modulation of EGF/ERK1/2 MAPK signalling.     

Cruising specialists and accelerators--are different types of fish locomotion driven by differently structured myosepta?

Locomotor specialists, such as accelerators and cruisers, have clearly differing body designs. For physical reasons these designs are mutually exclusive, i.e. cruisers necessarily have poor accelerating capabilities and vice versa. For the first time, we examine whether differences in the anatomy of the musculo-tendinous system of the trunk are present in addition to the differences in external body design. We investigated the myoseptal series of two closely related locomotor specialists, the cruiser Scomber scombrus and the accelerator Channa obscura, by microdissections combined with polarized light microscopy and histology. Our comparison includes 3D-morphology of myosepta, spatial arrangement and length of myoseptal tendons, their relation to red and white muscles, rostrocaudal changes in all these aspects and the musculo-tendinous system of the caudal fin. Regarding all these features, Channa has retained the plesiomorphic condition of its actinopterygian ancestor. In contrast, the derived morphology of Scomber is characterized by (i) lateral (LT) and myorhabdoid tendons (MT) that are lengthened to up to 20% of body length (compared to a maximum of 8.2% in Channa), (ii) posterior myoseptal cones that are subsequently linked by horizontal projections of merged LTs and MTs, (iii) an increased area of red muscle fibers that insert to LTs of myosepta, (iv) the reduction of epineural (ENTs) and epipleural tendons (EPTs) that connect backbone and skin, (v) specific caudal tendons that are identified to be serial homologues of LTs and MTs of more anterior myosepta, (vi) and a partial reduction of intrinsic caudal muscles. These results suggest the following functional adaptations in the cruiser Scomber. Red muscle forces may be transmitted through LTs and posterior cones to the prominent tendons of the caudal fin. The length of LTs and the intersegmental connections along the posterior cones may facilitate posterior force transmission and may be correlated with the long propulsive wavelength generally observed in cruising carangiform swimmers. Epineural and epipleural tendons are interpreted to minimize lateral backbone displacement during high body curvatures. This is consistent with the lack of these tendons in Scomber, because high body curvatures are not displayed in stiffer-bodied carangiform swimmers. It remains to be tested whether the specializations revealed in this initial study for Scomber represent general specializations of carangiform swimmers. Taking into account the geometry of myoseptal tendons and the horizontal septum we evaluate how local bending according to beam-theory can be generated by white or red muscle activity in Channa and Scomber. In both species, the musculo-tendinous anatomy of the caudal fin explains the functional asymmetry of the caudal fin that was experimentally revealed in previous studies.     

Planktonic-Cell Yield of a Pseudomonad Biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10⁹ cells ml⁻¹, suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

Scenarios of freshwater fish extinctions from climate change and water withdrawal

Reductions in river discharge (water availability) like those from climate change or increased water withdrawal, reduce freshwater biodiversity. We combined two scenarios from the Intergovernmental Panel for Climate Change with a global hydrological model to build global scenarios of future losses in river discharge from climate change and increased water withdrawal. Applying these results to known relationships between fish species and discharge, we build scenarios of losses (at equilibrium) of riverine fish richness. In rivers with reduced discharge, up to 75% (quartile range 4–22%) of local fish biodiversity would be headed toward extinction by 2070 because of combined changes in climate and water consumption. Fish loss in the scenarios fell disproportionately on poor countries. Reductions in water consumption could prevent many of the extinctions in these scenarios.     

S-layer nanoglycobiology of bacteria

Cell surface layers (S-layers) are common structures of the bacterial cell envelope with a lattice-like appearance that are formed by a self-assembly process. Frequently, the constituting S-layer proteins are modified with covalently linked glycan chains facing the extracellular environment. S-layer glycoproteins from organisms of the Bacillaceae family possess long, O-glycosidically linked glycans that are composed of a great variety of sugar constituents. The observed variations already exceed the display found in eukaryotic glycoproteins. Recent investigations of the S-layer protein glycosylation process at the molecular level, which has lagged behind the structural studies due to the lack of suitable molecular tools, indicated that the S-layer glycoprotein glycan biosynthesis pathway utilizes different modules of the well-known biosynthesis routes of lipopolysaccharide O-antigens. The genetic information for S-layer glycan biosynthesis is usually present in S-layer glycosylation (slg) gene clusters acting in concert with housekeeping genes. To account for the nanometer-scale cell surface display feature of bacterial S-layer glycosylation, we have coined the neologism 'nanoglycobiology'. It includes structural and biochemical aspects of S-layer glycans as well as molecular data on the machinery underlying the glycosylation event. A key aspect for the full potency of S-layer nanoglycobiology is the unique self-assembly feature of the S-layer protein matrix. Being aware that in many cases the glycan structures associated with a protein are the key to protein function, S-layer protein glycosylation will add a new and valuable component to an 'S-layer based molecular construction kit'. In our long-term research strategy, S-layer nanoglycobiology shall converge with other functional glycosylation systems to produce 'functional' S-layer neoglycoproteins for diverse applications in the fields of nanobiotechnology and vaccine technology. Recent advances in the field of S-layer nanoglycobiology have made our overall strategy a tangible aim of the near future.