Biomarker discovery in asthma‐related inflammation and remodeling

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma‐related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) on lung samples from mouse models of allergen‐induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELMα (FIZZ‐1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.     

Unintended molecular interactions in transgenic plants expressing clinically useful proteins: The case of bovine aprotinin traveling the potato leaf cell secretory pathway

We assessed the impact of subcellular targeting on the heterologous expression of a clinically useful protease inhibitor, bovine aprotinin, in leaves of potato, Solanum tuberosum. Transgenic potato lines targeting aprotinin to the cytosol, the ER or the apoplast were first generated, and then assessed for their ability to accumulate the recombinant protein. On‐chip detection and quantitation of aprotinin variants by SELDI TOF MS showed the inhibitor to be absent in the cytosol, but present under different forms in the ER and the apoplast. No visible phenotypic effects of aprotinin were observed for the transgenic lines, but aprotinin retention in the ER was associated with a significant decrease of leaf soluble protein content. A 2‐D gel assessment of control and transgenic lines revealed a possible link between this altered protein content and the down‐regulation of proteins implicated in protein synthesis and maturation. These observations, supported by complementary 2‐DE analyses with potato lines targeting aprotinin to the apoplast, suggest an aprotinin‐mediated feedback in planta negatively altering protein anabolism. From a practical viewpoint, these data illustrate the importance of taking into account not only the characteristics of recombinant proteins expressed in heterologous environments, but also their possible effects on protein accumulation in the host plant factory.     

Mapping field-scale spatial patterns of size and activity of the denitrifier community

There is ample evidence that microbial processes can exhibit large variations in activity on a field scale. However, very little is known about the spatial distribution of the microbial communities mediating these processes. Here we used geostatistical modelling to explore spatial patterns of size and activity of the denitrifying community, a functional guild involved in N-cycling, in a grassland field subjected to different cattle grazing regimes. We observed a non-random distribution pattern of the size of the denitrifier community estimated by quantification of the denitrification genes copy numbers with a macro-scale spatial dependence (6–16 m) and mapped the distribution of this functional guild in the field. The spatial patterns of soil properties, which were strongly affected by presence of cattle, imposed significant control on potential denitrification activity, potential N₂O production and relative abundance of some denitrification genes but not on the size of the denitrifier community. Absolute abundance of most denitrification genes was not correlated with the distribution patterns of potential denitrification activity or potential N₂O production. However, the relative abundance of bacteria possessing the nosZ gene encoding the N₂O reductase in the total bacterial community was a strong predictor of the N₂O/(N₂ + N₂O) ratio, which provides evidence for a relationship between bacterial community composition based on the relative abundance of denitrifiers in the total bacterial community and ecosystem processes. More generally, the presented geostatistical approach allows integrated mapping of microbial communities, and hence can facilitate our understanding of relationships between the ecology of microbial communities and microbial processes along environmental gradients.     

Targeting and post‐translational processing of human α1‐antichymotrypsin in BY‐2 tobacco cultured cells†

The post‐translational processing of human α₁‐antichymotrypsin (AACT) in Bright Yellow‐2 (BY‐2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse‐chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non‐glycosylated form, in contrast with secreted variants undergoing multiple post‐translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N‐glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY‐2 tobacco cells led to the production of two protein products: (i) a stable, non‐glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post‐translational N‐glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.     

Niche-driven evolution of metabolic and life-history strategies in natural and domesticated populations of Saccharomyces cerevisiae

Background  

Variation of resource supply is one of the key factors that drive the evolution of life-history strategies, and hence the interactions between individuals. In the yeast Saccharomyces cerevisiae, two life-history strategies related to different resource utilization have been previously described in strains from different industrial origins. In this work, we analyzed metabolic traits and life-history strategies in a broader collection of yeast strains sampled in various ecological niches (forest, human body, fruits, laboratory and industrial environments).     

Multi-sensor plant imaging: Towards the development of a stress-catalogue

Agricultural production is limited by a wide range of abiotic (e.g. drought, waterlogging) and biotic (pests, diseases and weeds) stresses. The impact of these stresses can be minimized by appropriate management actions such as irrigation or chemical pesticide application. However, further optimization requires the ability to diagnose and quantify the different stresses at an early stage. Particularly valuable information of plant stress responses is provided by plant imaging, i.e. non-contact sensing with spatial resolving power: (i) thermal imaging, detecting changes in transpiration rate and (ii) fluorescence imaging monitoring alterations in photosynthesis and other physiological processes. These can be supplemented by conventional video imagery for study of growth. An efficient early warning system would need to discriminate between different stressors. Given the wide range of sensors, and the association of specific plant physiological responses with changes at particular wavelengths, this goal seems within reach. This is based on the organization of the individual sensor results in a matrix that identifies specific signatures for multiple stress types. In this report, we first review the diagnostic effectiveness of different individual imaging techniques and then extend this to the multi-sensor stress-identification approach.     

Direct In Vivo Cell Lineage Analysis in the Retrorsine and 2AAF Models of Liver Injury after Genetic Labeling in Adult and Newborn Rats

Backgrounds and Aims

When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.

Methodology/Principal Findings

We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats.

Conclusions

Our results srongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.