An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

Reflections of ecological differences? Stress responses of sympatric Alpine chamois and red deer to weather,forage quality,and human disturbance

Depending on the habitats they live in, temperate ungulates have adapted to different degrees to seasonally changing forage and weather conditions, and to specific escape strategies from predators. Alpine chamois, a mountain ungulate, and red deer, originally adapted to open plains, would therefore be expected to differ in their physiological responses to potential stressors. Based on 742 chamois and 1557 red deer fecal samples collected year‐round every 2 weeks for 4 years at the same locations within a strictly protected area in the Swiss Alps, we analyzed glucocorticoid metabolite (FGM) concentrations for both species. Results from linear mixed effects models revealed no physiological stress response to changing visitor numbers, but instead to drought conditions for both species during summer. In winter, FGM concentrations increased with increasing snow height in both species, but this response was modulated by temperature in red deer. Chamois showed a stronger stress response to increasing snow height during November and December than between January and March, while FGM concentrations increased with decreasing temperature throughout winter. An increase in FGM concentrations with decreasing forage digestibility during winter was found only for red deer. The results are thus partly in contradiction to expectations based on feeding type and adaptations to different habitats between the two species. The lack of a response to forage digestibility in chamois may reflect either better adaptation to difficult feeding conditions in subalpine forests, or, by contrast, strong constraints imposed by forage quality. The similar responses of both species to weather conditions in winter suggest that climatic factors at the elevations examined here are sufficiently harsh to be limiting to temperate ungulates regardless of their specific adaptations to this environment.     

Negative regulation of RPE cell attachment by carbohydrate-dependent cell surface binding of galectin-3 and inhibition of the ERK-MAPK pathway

Adhesion and spreading of retinal pigment epithelial (RPE) cells on fibronectin-rich extracellular matrices is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). In the present study we explored the capacity of galectin-3, a β-galactoside-binding endogenous lectin, to inhibit early PVR-associated cellular events from a therapeutic perspective. We assessed the relative expression levels of galectin-3 in native RPE and dedifferentiated, cultured RPE. Galectin-3 was constitutively expressed under in vivo and in vitro conditions and was abundant in cultured cells. Treatment of human RPE cells with soluble galectin-3 disclosed no toxicity within control limits up to 250 μg/ml. When added to the medium, galectin-3 dose-dependently inhibited attachment and spreading of the cells on fibronectin by more than 75%. When coated on the plastic surface, galectin-3 alone impaired attachment and spreading of RPE cells, and reduced attachment but not spreading on fibronectin. Galectin-3 bound to the cell surface, and, as determined by the use of the competing sugar β-lactose, galectin-3-mediated effects were dependent on carbohydrate binding. To ascertain the role of the ability of galectin-3 to form pentamers, we proteolytically removed the N-terminal, cross-linking section. The remaining C-terminal carbohydrate-binding domain alone failed to bind to cells and was functionally inactive. These results emphasize the relevance of both properties, i.e., glycan-binding and cross-linking of glycan moieties, for the inhibitory activity of galectin-3. Incubation of mobilized RPE cells with galectin-3 significantly disturbed microfilament assembly and, in correlation with decreased attachment, inhibited ERK phosphorylation. Therefore, galectin-3, acting as a cross-linking lectin on the cell surface, negatively regulates attachment and spreading of RPE cells in vitro. This effect, at least in part, is attributed to an inhibition of the ERK-MAPK pathway, which prevents cytoskeletal rearrangements needed for RPE cell attachment and spreading. Further investigation at this pathway may disclose a promising nouveau perspective for treatment and prophylaxis of early PVR.     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

Testing the threat-sensitive predator avoidance hypothesis: physiological responses and predator pressure in wild rabbits

Predation is a strong selective force with both direct and indirect effects on an animal’s fitness. In order to increase the chances of survival, animals have developed different antipredator strategies. However, these strategies have associated costs, so animals should assess their actual risk of predation and shape their antipredator effort accordingly. Under a stressful situation, such as the presence of predators, animals display a physiological stress response that might be proportional to the risk perceived. We tested this hypothesis in wild European rabbits (Oryctolagus cuniculus), subjected to different predator pressures, in Doñana National Park (Spain). We measured the concentrations of fecal corticosterone metabolites (FCM) in 20 rabbit populations. By means of track censuses we obtained indexes of mammalian predator presence for each rabbit population. Other factors that could modify the physiological stress response, such as breeding status, food availability and rabbit density, were also considered. Model selection based on information theory showed that predator pressure was the main factor triggering the glucocorticoid release and that the physiological stress response was positively correlated with the indexes of the presence of mammalian carnivore predators. Other factors, such as food availability and density of rabbits, were considerably less important. We conclude that rabbits are able to assess their actual risk of predation and show a threat-sensitive physiological response.     

Molecular dynamics simulations of the chemokine CCL2 in complex with pull down-derived heparan sulfate hexasaccharides

Background

Binding of chemokines to glycosaminoglycans (GAGs) is a crucial step in leukocyte recruitment to inflamed tissues.

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.     

Structure of the Large Subunit rDNA from a Diatom, and Comparison Between Small and Large Subunit Ribosomal RNA for Studying Stramenopile Evolution

The aim of this study was to compare the usefulness of complete small and large subunit rRNA, and a combination of both molecules, for reconstructing stramenopile evolution. To this end, phylogenies from species of which both sequences are known Acre constructed with the neighbor-joining, maximum parsimony, and maximum likelihood methods. Also the use of structural features of the rRNAs was evaluated. The large subunit rRNA from the diatom Skeletonema pseudocostatum was sequenced in order to have a more complete taxon sampling, and a group I intron was identified. Our results indicated that heterokont algae are monophyletic, with diatoms diverging first. However, as the analysis was restricted to a particular data set containing merely six taxa, the outcome has limited value for elucidating stramenopile relationships. On the other hand, this approach permits comparison of the performance of both rRNA molecules without interference from other factors, such as a different species selection for each molecule. For the taxa used, the large subunit rRNA clearly contained more phylogenetic information than the small subunit rRNA. Although this result can definitely not be generalized and depends on the phvlogeny to be studied, in some cases determining complete large subunit rRNA sequences certainly seems worthwhile.