A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.     

Complete genome sequence of Desulfocapsa sulfexigens,a marine deltaproteobacterium specialized in disproportionating inorganic sulfur compounds

Desulfocapsa sulfexigens SB164P1 (DSM 10523) belongs to the deltaproteobacterial family Desulfobulbaceae and is one of two validly described members of its genus. This strain was selected for genome sequencing, because it is the first marine bacterium reported to thrive on the disproportionation of elemental sulfur, a process with a unresolved enzymatic pathway in which elemental sulfur serves both as electron donor and electron acceptor. Furthermore, in contrast to its phylogenetically closest relatives, which are dissimilatory sulfate-reducers, D. sulfexigens is unable to grow by sulfate reduction and appears metabolically specialized in growing by disproportionating elemental sulfur, sulfite or thiosulfate with CO₂ as the sole carbon source. The genome of D. sulfexigens contains the set of genes that is required for nitrogen fixation. In an acetylene assay it could be shown that the strain reduces acetylene to ethylene, which is indicative for N-fixation. The circular chromosome of D. sulfexigens SB164P1 comprises 3,986,761 bp and harbors 3,551 protein-coding genes of which 78% have a predicted function based on auto-annotation. The chromosome furthermore encodes 46 tRNA genes and 3 rRNA operons.     

Seminal roots of wild and cultivated barley differentially respond to osmotic stress in gene expression,suberization, and hydraulic conductivity

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5-fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA-sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit.     

Connections Between Benthic Populations and Local Strandings of the Southern Bull Kelp Durvillaea Antarctica Along the Continental Coast of Chile1

Floating seaweeds are important dispersal vectors in marine ecosystems. However, the relationship between benthic populations and stranded seaweeds has received little attention. After detachment, a fraction of floating specimens returns to the shore, resulting in strandings that fluctuate in space and time. It has been hypothesized that the availability of stranded seaweeds is related to their benthic abundance on adjacent coasts. Using the large fucoid Durvillaea antarctica, we tested whether stranded biomasses are higher at sites with dense adjacent benthic populations. Benthic abundance of D. antarctica along the continental coast of Chile was estimated using three approximations: (i) availability of potentially suitable habitat (PSH), (ii) categorical visual abundance estimates in the field, and (iii) abundance measurements in the intertidal zone. Higher PSH for D. antarctica was observed between 31° S–32° S and 40° S–42° S than between 33° S and 39° S. Lowest benthic biomasses were estimated for the northern latitudes (31° S–32° S). Regression models showed that the association between stranded biomass and PSH was highest when only the extent of rocky shore 10 km to the south of each beach was included, suggesting relatively short-distance dispersal and asymmetrical transport of floating kelps, which is further supported by low proportions of rafts with Lepas spp. (indicator of rafting). The results indicate that stranded biomasses are mostly subsidized by nearby benthic populations, which can partly explain the low genetic connectivity among populations in the study region. Future studies should also incorporate other local factors (e.g., winds, currents, wave-exposure) that influence stranding dynamics.     

B lymphocyte inhibition of anti-tumor response depends on expansion of Treg but is independent of B-cell IL-10 secretion

The mechanisms by which B lymphocytes inhibit anti-tumor immunity remain poorly understood. Murine EMT-6 mammary tumors grow readily in immune competent mice (BALB/c), but poorly in B-cell-deficient μ^?/? BALB/c mice (BCDM). T regulatory cell (Treg) expansion and function were impaired in BCDM compared with BALB/c. In this study, we compared tumor growth, Treg cell proliferation, tumor lymphocyte infiltration and cytolytic T cell activity in BALB/c, BCDM and BCDM partially reconstituted with B cells by adoptive transfer (BCDM+B). Partial reconstitution of BCDM with adoptively transferred B cells restored EMT-6 tumor growth, which was independent of IL-10 secretion by B cells. Instead, high frequencies of intratumoral B cells were associated with increased recruitment and proliferation of Treg cells within the tumor microenvironment. The B-cell-dependent accumulation of Treg within the tumor microenvironment was associated with reduced tumor infiltration by CD49+ NK and CD8+ T cells and reduced cytotoxic T cell activity against EMT-6 targets. Our studies indicate that tumor-dependent immunosuppression of T-cell-mediated anti-tumor immunity is coordinated within the tumor microenvironment by B-cell-dependent cross talk with Treg cells, which does not require production of IL-10 by B cells.     

Regulation of Adhesion by Flexible Ectodomains of IgCAMs

To perform their diverse biological functions the adhesion activities of the cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) might be regulated by local clustering, proteolytical shedding of their ectodomains or rapid recycling to and from the plasma membrane. Another form of regulation of adhesion might be obtained through flexible ectodomains of IgCAMs which adopt distinct conformations and which in turn modulate their adhesion activity. Here, we discuss variations in the conformation of the extracellular domains of CEACAM1 and CAR that might influence their binding and signaling activities. Furthermore, we concentrate on alternative splicing of single domains and short segments in the extracellular regions of L1 subfamily members that might affect the organization of the N-terminal located Ig-like domains. In particular, we discuss variations of the linker sequence between Ig-like domains 2 and 3 (D2 and D3) that is required for the horseshoe conformation.     

144 Sculpting light with DNA origami

We used the DNA origami method (Rothemund, 2006) for the fabrication of self-assembled nanoscopic materials (Seeman, 2010). In DNA origami, a virus-based 8?kilobase-long DNA single-strand is folded into shape with the help of ～ 200 synthetic oligonucleotides. The resulting DNA nanostructures can be designed to adopt any three-dimensional shape and can be addressed through DNA hybridization or chemical modification with nanometer precision. We have realized that complex assemblies of nanoparticles, including magnetic, fluorescent, and plasmonic nanoparticles. Such nanoconstructs may exhibit striking optical properties such as strong optical activity in the visible range (Kuzyk et al., 2012). To this end, plasmonic particles were assembled in solution to form helices of controlled handedness. We achieved spatial control over particle placement better than 2?nm and attachment yields of 97% and above. As a collective optical response emerging from our dispersed nanostructures, we detected pronounced circular dichroism (CD) originating from the plasmon–plasmon interactions in the particle helices. In recent experiments, we were able to show that the optical response of chiral biomolecules can be transferred from the UV into the visible region in plasmonic hotspots. Thus, sensitive detection of chiral biomolecules may become feasible in the near future. We also found that the orientation of the helices in respect to the incoming light beam critically influences the resulting CD spectra. Our results can be explained with theoretical models based on plasmonic dipole interaction and demonstrate the potential of DNA origami for the assembly of metafluids with designed optical properties.     

Multiscale Metabolic Modeling: Dynamic Flux Balance Analysis on a Whole-Plant Scale

Plant metabolism is characterized by a unique complexity on the cellular, tissue, and organ levels. On a whole-plant scale, changing source and sink relations accompanying plant development add another level of complexity to metabolism. With the aim of achieving a spatiotemporal resolution of source-sink interactions in crop plant metabolism, a multiscale metabolic modeling (MMM) approach was applied that integrates static organ-specific models with a whole-plant dynamic model. Allowing for a dynamic flux balance analysis on a whole-plant scale, the MMM approach was used to decipher the metabolic behavior of source and sink organs during the generative phase of the barley (Hordeum vulgare) plant. It reveals a sink-to-source shift of the barley stem caused by the senescence-related decrease in leaf source capacity, which is not sufficient to meet the nutrient requirements of sink organs such as the growing seed. The MMM platform represents a novel approach for the in silico analysis of metabolism on a whole-plant level, allowing for a systemic, spatiotemporally resolved understanding of metabolic processes involved in carbon partitioning, thus providing a novel tool for studying yield stability and crop improvement.Plants are of vital significance as a source of food (Grusak and DellaPenna, 1999; Rogalski and Carrer, 2011), feed (Lu et al., 2011), energy (Tilman et al., 2006; Parmar et al., 2011), and feedstocks for the chemical industry (Metzger and Bornscheuer, 2006; Kinghorn et al., 2011). Given the close connection between plant metabolism and the usability of plant products, there is a growing interest in understanding and predicting the behavior and regulation of plant metabolic processes. In order to increase crop quality and yield, there is a need for methods guiding the rational redesign of the plant metabolic network (Schwender, 2009).Mathematical modeling of plant metabolism offers new approaches to understand, predict, and modify complex plant metabolic processes. In plant research, the issue of metabolic modeling is constantly gaining attention, and different modeling approaches applied to plant metabolism exist, ranging from highly detailed quantitative to less complex qualitative approaches (for review, see Giersch, 2000; Morgan and Rhodes, 2002; Poolman et al., 2004; Rios-Estepa and Lange, 2007).A widely used modeling approach is flux balance analysis (FBA), which allows the prediction of metabolic capabilities and steady-state fluxes under different environmental and genetic backgrounds using (non)linear optimization (Orth et al., 2010). Assuming steady-state conditions, FBA has the advantage of not requiring the knowledge of kinetic parameters and, therefore, can be applied to model detailed, large-scale systems. In recent years, the FBA approach has been applied to several different plant species, such as maize (Zea mays; Dal’Molin et al., 2010; Saha et al., 2011), barley (Hordeum vulgare; Grafahrend-Belau et al., 2009b; Melkus et al., 2011; Rolletschek et al., 2011), rice (Oryza sativa; Lakshmanan et al., 2013), Arabidopsis (Arabidopsis thaliana; Poolman et al., 2009; de Oliveira Dal’Molin et al., 2010; Radrich et al., 2010; Williams et al., 2010; Mintz-Oron et al., 2012; Cheung et al., 2013), and rapeseed (Brassica napus; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011), as well as algae (Boyle and Morgan, 2009; Cogne et al., 2011; Dal’Molin et al., 2011) and photoautotrophic bacteria (Knoop et al., 2010; Montagud et al., 2010; Boyle and Morgan, 2011). These models have been used to study different aspects of metabolism, including the prediction of optimal metabolic yields and energy efficiencies (Dal’Molin et al., 2010; Boyle and Morgan, 2011), changes in flux under different environmental and genetic backgrounds (Grafahrend-Belau et al., 2009b; Dal’Molin et al., 2010; Melkus et al., 2011), and nonintuitive metabolic pathways that merit subsequent experimental investigations (Poolman et al., 2009; Knoop et al., 2010; Rolletschek et al., 2011). Although FBA of plant metabolic models was shown to be capable of reproducing experimentally determined flux distributions (Williams et al., 2010; Hay and Schwender, 2011b) and generating new insights into metabolic behavior, capacities, and efficiencies (Sweetlove and Ratcliffe, 2011), challenges remain to advance the utility and predictive power of the models.Given that many plant metabolic functions are based on interactions between different subcellular compartments, cell types, tissues, and organs, the reconstruction of organ-specific models and the integration of these models into interacting multiorgan and/or whole-plant models is a prerequisite to get insight into complex plant metabolic processes organized on a whole-plant scale (e.g. source-sink interactions). Almost all FBA models of plant metabolism are restricted to one cell type (Boyle and Morgan, 2009; Knoop et al., 2010; Montagud et al., 2010; Cogne et al., 2011; Dal’Molin et al., 2011), one tissue or one organ (Grafahrend-Belau et al., 2009b; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011; Mintz-Oron et al., 2012), and only one model exists taking into account the interaction between two cell types by specifying the interaction between mesophyll and bundle sheath cells in C4 photosynthesis (Dal’Molin et al., 2010). So far, no model representing metabolism at the whole-plant scale exists.Considering whole-plant metabolism raises the problem of taking into account temporal and environmental changes in metabolism during plant development and growth. Although classical static FBA is unable to predict the dynamics of metabolic processes, as the network analysis is based on steady-state solutions, time-dependent processes can be taken into account by extending the classical static FBA to a dynamic flux balance analysis (dFBA), as proposed by Mahadevan et al. (2002). The static (SOA) and dynamic optimization approaches introduced in this work provide a framework for analyzing the transience of metabolism by integrating kinetic expressions to dynamically constrain exchange fluxes. Due to the requirement of knowing or estimating a large number of kinetic parameters, so far dFBA has only been applied to a plant metabolic model once, to study the photosynthetic metabolism in the chloroplasts of C3 plants by a simplified model of five biochemical reactions (Luo et al., 2009). Integrating a dynamic model into a static FBA model is an alternative approach to perform dFBA.In this study, a multiscale metabolic modeling (MMM) approach was applied with the aim of achieving a spatiotemporal resolution of cereal crop plant metabolism. To provide a framework for the in silico analysis of the metabolic dynamics of barley on a whole-plant scale, the MMM approach integrates a static multiorgan FBA model and a dynamic whole-plant multiscale functional plant model (FPM) to perform dFBA. The performance of the novel whole-plant MMM approach was tested by studying source-sink interactions during the seed developmental phase of barley plants.     

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.