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Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.

We present a protocol for building, validating, and simulating models of signal transduction networks. These networks are challenging modeling targets due to the combinatorial complexity and sparse data, which have made it a major challenge even to formalize the current knowledge. To address this, the community has developed methods to model biomolecular reaction networks based on site dynamics. The strength of this approach is that reactions and states can be defined at variable resolution, which makes it possible to adapt the model resolution to the empirical data. This improves both scalability and accuracy, making it possible to formalize large models of signal transduction networks. Here, we present a method to build and validate large models of signal transduction networks. The workflow is based on rxncon, the reaction-contingency language. In a five-step process, we create a mechanistic network model, convert it into an executable Boolean model, use the Boolean model to evaluate and improve the network, and finally export the rxncon model into a rule-based format. We provide an introduction to the rxncon language and an annotated, step-by-step protocol for the workflow. Finally, we create a small model of the insulin signaling pathway to illustrate the protocol, together with some of the challenges-and some of their solutions-in modeling signal transduction.

In the context of ongoing criticisms of the lack of pluralism in economics, the present article aims to discuss the development of ‘heterodox’ economics since the 1970s. Following Lakatos’s concept of scientific research programs (srp), and concentrating on the situation in Germany, the article will discuss classifications of economics, and will specify the understanding of diversity in the light of ‘axiomatic variations’ of the economic mainstream. This will form the basis for the subsequent description of the development of heterodoxy in Germany, with special reference to the founding of new universities and the reform movements in the 1970s. It can be shown that the heterodox scene flourished in this period, but that this pluralization remained fragmented and short-lived; by the 1980s at the latest heterodoxy was again on its way to marginalization. The history of heterodoxy in Germany thus presents itself as an unequal ‘battle of the paradigms,’ and can only be told as the story of a failure.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5′ homology arm (HA) of 8 kb and 3′HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3′HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.

The metabolic modelling community has established the gold standard for bottom-up systems biology with reconstruction, validation and simulation of mechanistic genome-scale models. Similar methods have not been established for signal transduction networks, where the representation of complexes and internal states leads to scalability issues in both model formulation and execution. While rule- and agent-based methods allow efficient model definition and execution, respectively, model parametrisation introduces an additional layer of uncertainty due to the sparsity of reliably measured parameters. Here, we present a scalable method for parameter-free simulation of mechanistic signal transduction networks. It is based on rxncon and uses a bipartite Boolean logic with separate update rules for reactions and states. Using two generic update rules, we enable translation of any rxncon model into a unique Boolean model, which can be used for network validation and simulation—allowing the prediction of system-level function directly from molecular mechanistic data. Through scalable model definition and simulation, and the independence of quantitative parameters, it opens up for simulation and validation of mechanistic genome-scale models of signal transduction networks., Modelling: signal transduction at the genome scale Mechanistic models are like road maps—they help us understand how signals travel through cells and guide cellular decisions. We developed a method to turn these models executable at the genome scale, and show that this method can explain and predict cellular physiology directly from molecular mechanisms. The bottom line is that the regulatory structure of the network is encoded in the molecular mechanisms, and we capture this knowledge in two generic update rules. These can be used to assemble molecular mechanisms, LEGO-brick style, into a functional model—without parametrisation or model fitting. This makes it possible to use the method for network validation and simulation of systems that are far too large to be parametrised, opening the door to genome-scale mechanistic models of cellular signal transduction.

The new coaxial fine wire deposition optic presents reduced weight (~10 kg without fiber and wire feed) and has extended functionality. The optical components are designed for wavelengths of 450 – 550 nm and 890 – 1100 nm. The head is prepared for the processing of even thin wires down to 100 μm in diameter. The new system is equipped with an integrated mini camera, sensors and interfaces to control units and networks. As a result the new head exhibits potential to produce very precise metallic structures as low as 200 - 300 μm. Suitable materials are steel, titanium, nickel and cobalt base alloys as well as materials on the basis of copper.

The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

Acute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral loss-of-function RNA interference (RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was toxic to this cell fraction in feeder coculture and xenotransplant experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient-derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling.

Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1β, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4(+) T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells.

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

Motivation: Fusion genes result from genomic rearrangements, such as deletions, amplifications and translocations. Such rearrangements can also frequently be observed in cancer and have been postulated as driving event in cancer development. to detect them, one needs to analyze the transition region of two segments with different copy number, the location where fusions are known to occur. Finding fusion genes is essential to understanding cancer development and may lead to new therapeutic approaches. Results: Here we present a novel method, the Genomic Fusion Detection algorithm, to predict fusion genes on a genomic level based on SNP-array data. This algorithm detects genes at the transition region of segments with copy number variation. With the application of defined constraints, certain properties of the detected genes are evaluated to predict whether they may be fused. We evaluated our prediction by calculating the observed frequency of known fusions in both primary cancers and cell lines. We tested a set of cell lines positive for the BCR-ABL1 fusion and prostate cancers positive for the TMPRSS2-ERG fusion. We could detect the fusions in all positive cell lines, but not in the negative controls. Availability: The algorithm is available from the supplement. Contact: philip.groth@bayer.com Supplementary information: Supplementary data are available at Bioinformatics online.

Part 3: Additive Manufacturing; International audience; In the field of Laser Additive Manufacturing, modern wire-based laser deposition techniques offer advantageous solutions for combining the high quality level of layer-by-layer fabrication of high value parts with the industry’s economical requirements regarding productivity and energy efficiency. A newly developed coaxial wire head allows for omni-directional welding operation and, thus, the use of wire even for complex surface claddings as well as the generation of three-dimensional structures. Currently, several metallic alloys as steel, titanium, aluminium, and nickel are available for the generation of defect-free structures. Even cored wires containing carbide hardmetals can be used for the production of extra wear-resistant parts. Simultaneous heating of the wire using efficient electric energy increases significantly the deposition rate and energy efficiency. Examples of application are light-weight automotive parts, turbine blades of Nickel super alloys, and complex inserts of injection moulds.

In der Schweiz betreibt eine Initiative aktuell ganz konkret einen Volksentscheid mit dem Ziel, ein bedingungsloses Grundeinkommen einzuführen. In Deutschland wird das Konzept lebhaft diskutiert. Allerdings wird seine Wirkung auf Beschäftigung, Besteuerung, Einkommensverteilung und die Bereitstellung von staatlichen Leistungen ganz unterschiedlich beurteilt. The introduction of an unconditional basic income has been discussed in Switzerland for many years. Germany, however, takes a sceptical view of this idea. The authors of this 'Zeitgespräch' express disparate opinions on how broadly the concept should be defined, but they agree on the motive behind the concept: respect for human dignity. Moreover, it may make many social policy rules obsolete. There are conflicting opinions concerning the reaction of labour supply to the unconditional basic income. Some authors fear that it will shrink substantially. But its financing will be the main problem. There are proposals to increase the value added tax rate. The economic impacts of funding it by income tax revenues are analysed using a macroeconomic model. Ultimately, the implementation of the concept will have distributional effects which are very difficult to predict.

Two brothers in their fifties presented with a medical history of suspected fungal allergy, allergic bronchopulmonary aspergillosis, alveolitis, and invasive aspergillosis and pulmonary fistula, respectively. Eventually, after a delay of 50 years, chronic granulomatous disease (CGD) was diagnosed in the index patient. We found a new splice mutation in the NCF2 (p67-phox) gene, c.1000 + 2T → G, that led to several splice products one of which lacked exons 11 and 12. This deletion was in frame and allowed for remarkable residual NADPH oxidase activity as determined by transduction experiments using a retroviral vector. We conclude that p67-phox which lacks the 34 amino acids encoded by the two exons can still exert considerable functional activity. This activity can partially explain the long-term survival of the patients without adequate diagnosis and treatment, but could not prevent progressing lung damage.

The marine mussel Mytilus edulis and its closely related sister species are distributed world-wide and play an important role in coastal ecology and economy. The diversification in different species and their hybrids, broad ecological distribution, as well as the filter feeding mode of life has made this genus an attractive model to investigate physiological and molecular adaptations and responses to various biotic and abiotic environmental factors. In the present study we investigated the immune system of Mytilus, which may contribute to the ecological plasticity of this species. We generated a large Mytilus transcriptome database from different tissues of immune challenged and stress treated individuals from the Baltic Sea using 454 pyrosequencing. Phylogenetic comparison of orthologous groups of 23 species demonstrated the basal position of lophotrochozoans within protostomes. The investigation of immune related transcripts revealed a complex repertoire of innate recognition receptors and downstream pathway members including transcripts for 27 toll-like receptors and 524 C1q domain containing transcripts. NOD-like receptors on the other hand were absent. We also found evidence for sophisticated TNF, autophagy and apoptosis systems as well as for cytokines. Gill tissue and hemocytes showed highest expression of putative immune related contigs and are promising tissues for further functional studies. Our results partly contrast with findings of a less complex immune repertoire in ecdysozoan and other lophotrochozoan protostomes. We show that bivalves are interesting candidates to investigate the evolution of the immune system from basal metazoans to deuterostomes and protostomes and provide a basis for future molecular work directed to immune system functioning in Mytilus.

Phylogenies of multi-domain proteins have to incorporate macro-evolutionary events, which dramatically increases the complexity of their construction. We present an application to infer ancestral multi-domain proteins given a species tree and domain phylogenies. As the individual domain phylogenies are often incongruent, we provide diagnostics for the identification and reconciliation of implausible topologies. We implement and extend a suggested algorithmic approach by Behzadi and Vingron (2006).

Objective Internal tandem duplication (ITD) mutations of the FLT3 receptor are associated with a high incidence of relapse in acute myeloid leukemia (AML). Expression of the CXCR4 receptor in FLT3-ITD−positive AML is correlated with poor outcome, and inhibition of CXCR4 was shown to sensitize AML blasts toward chemotherapy. The aim of this study was to evaluate the impact of FLT3-ITD on cell proliferation and CXCR4-dependent migration in human hematopoietic progenitor cells and to investigate their response to CXCR4 inhibition. Materials and Methods We used primary blasts from patients with FLT3-ITD or FLT3 wild-type AML. In addition, human CD34 + hematopoietic progenitor cells were transduced to >70% with retroviral vectors containing human FLT3-ITD. Results We found that FLT3-ITD transgene overexpressing human hematopoietic progenitor cells show strongly reduced migration toward stromal-derived factor−1 in vitro and display significantly reduced bone marrow homing in nonobese diabetic severe combined immunodeficient mice. Cocultivation of FLT3-ITD−positive AML blasts or hematopoietic progenitor cells on bone marrow stromal cells resulted in a strong proliferation advantage and increased early cobblestone area−forming cells compared to FLT3−wild-type AML blasts. Addition of the CXCR4 inhibitor AMD3100 to the coculture significantly reduced both cobblestone area−forming cells and proliferation of FLT3-ITD−positive cells, but did not affect FLT3−wild-type cells—highlighting the critical interaction between CXCR4 and FLT3-ITD. Conclusion CXCR4 inhibition to decrease cell proliferation and to control the leukemic burden may provide a novel therapeutic strategy in patients with advanced FLT3-ITD−positive AML.

Objective The Notch signaling pathway has been shown to play a role in bone marrow−derived stromal cell differentiation, however, the precise outcome of Notch activation remains controversial. The aim of this study was to evaluate the effect of Notch signaling in primary human bone marrow−derived stromal cells (hBMSCs). Materials and Methods hBMSCs were transduced to >90% with lentiviral vectors containing either human notch1 intracellular domain ( NICD ), jagged1 , or dominant negative mastermind1 . Cells were exposed to adipogenic and osteogenic differentiation stimuli and differentiation was quantified by oil red or alizarin red staining, alkaline phosphatase liver/bone/kidney (ALPL) activity and expression of adipogenic or osteogenic marker genes. Results NICD and jagged1 transgene−expressing hBMSCs demonstrated enhanced mineralization, nodule formation, and ALPL activity in osteogenic differentiation media. These findings correlated with increased gene expression of bone morphogenetic protein 2 and ALPL . In contrast, NICD or jagged1 transgene expression strongly inhibited adipocyte formation and reduced peroxisome proliferator-activated receptor-γ , fatty acid binding protein 4 , and adiponectin precursor gene expression. Co-overexpression of dominant negative mastermind1 and NICD or jagged1 led to a partial rescue of the differentiation phenotypes. In addition, high endogenous jagged1 expression levels were observed in hBMSCs samples with strong ALPL activity compared to a group of samples with low ALPL activity. Conclusion In summary, our data suggest that induction of Notch signaling enhances the osteogenic differentiation of hBMSCs while inhibiting the adipogenic fate.

In the field of laser additive manufacturing, modern hybrid technologies offer advantageous solutions for combining the high quality level of laser surface claddings with the industry's economical requirements regarding productivity and energy efficiency. The technical approach is to supply energy sources in two fundamentally different ways. First, welding material in the form of wire is directly heated, i.e., by electrical resistance, almost to its melting point and is fed to the process region simultaneously with the laser beam. A newly developed coaxial wire head allows for omni-directional welding operation and, thus, the use of wire even for complex surface claddings as well as layer-by-layer fabrication of metallic parts. Second, an additional energy source is used to heat the substrate in order to compensate for the heat conduction losses. This technical variant is suitable for use with both wire and powder as deposition materials. Additionally, heating and cooling gradients can be precisely adjusted, thus improving the build-up of structures of hard and brittle metallic super-alloys. Currently, deposition rates up to 18 kg/h of metal alloys and metal matrix composites have been demonstrated using these laser hybrid techniques. Industrial applications are, among others, large hydraulic cylinders and tools of the oil and mining industries.

In seinem Beitrag zur Sozialen Marktwirtschaft versuchte Florian Josef Hoffmann u. a. eine Antwort auf die Frage „Soziale Marktwirtschaft, was ist das?“ zu geben und dabei das Konzept der Sozialen Marktwirtschaft kritisch zu wurdigen. Daran anknupfend will der vorliegende Beitrag zeigen, dass sich die Unscharfe, die der Sozialen Marktwirtschaft haufig kritisch unterstellt wird, als eigentliche Starke entpuppt. Mit Bezug zu Alfred Muller-Armack soll gezeigt werden, dass es sich bei der Sozialen Marktwirtschaft um eine ethische und moderne Wirtschaftskonzeption handelt.

Notch, expressed on hematopoietic progenitors plays a crucial role in hematopoiesis. Mesenchymal stem cells (MSC) express both, Notch and its ligand Jagged and are known to support self renewal of hematopoietic progenitors via cell-cell contact and cytokine secretion. The Jagged/Notch signaling pathway has been implicated in the differentiation process of MSC, however it is not completely understood and current observations are contradictory. In order to analyze the effect of Notch signaling on human MSC differentiation we constructed lentiviral vectors that contained either the GFP-marker gene, hJagged1 IRES GFP, hNotch1 intracellular domain (NICD) IRES GFP or a gene fusion between dominant negative Mastermind1 (MAML1dn - inhibitor of Notch signaling) and the Cherry reporter gene. Primary hMSC that were obtained from bone marrow of 3 different donors were transduced with respective lentivirus vectors to greater than 98%. After exposure to adipogenic and osteogenic differentiation stimuli hMSC differentiation was quantified by Alizarin red or oil red staining, alkaline phosphatase (AP) activity and expression levels of adipogenic or osteogenic markers by Real-time PCR. Jagged1 transduced hMSC demonstrated enhanced calcium phosphate deposits and enhanced AP activity and expression levels in osteogenic differentiation medium, while adipogenic differentiation was strongly inhibited as quantified by oil red staining and low mRNA expression of genes upregulated during adipogenic differentiation (pprY, Fabp4). Similarly, overexpression of NICD induced strong and rapid osteogenic differentiation while inhibiting adipogenic differentiation and reducing cell viability. Moreover, NICD overexpression upregulates the expression of endogenous Jagged1 up to 5-fold. Inhibition of Notch signaling via overexpression of MAML1dn partially blocked the effect of hJagged1 and NICD in co-transduction experiments. In another approach MSC samples obtained from 20 donors with various osteogenic differentiation potential as measured by AP activity were analyzed for Notch1 and Jagged1 expression. While there was no correlation between AP activity and Notch1 levels we observed a significant positive correlation for AP activity and Jagged1 expression. In summary, our data strongly suggest that increased Jagged/Notch signaling enhances the osteogenic differentiation of hMSC while inhibiting their adipogenic fate.

mRNA transfection is a safe alternative to DNA transfection for transient transgene overexpression in mammalian cells as genomic integration of the transferred nucleic acid is impossible. We developed a new mRNA electroporation protocol for transfection of primary mesenchymal stem cells (MSCs) using a hypoosmolar buffer system. Polyadenylated and capped mRNA was in vitro transcribed from DNA templates by T7 RNA polymerase. The DNA template generation has been simplified by our group using PCR amplification of the transgene sequence with primers providing the T7 RNA polymerase promoter and optimized 5′ and 3′ UTR sequences for better translation and RNA stability. This manoever allows to generate the DNA template from any plasmid vector and bypasses the need for additonal cloning steps. Canine primary MSCs were isolated by selection of adherent cells from bone marrow aspirates in a low glucose medium. Transfection of GFP or CXCR4 mRNA into primary MSCs resulted in transgene overexpression in more than 90% of target cells with excellent viabilities (>95%). Expression of CXCR4 was well detectable for up to 96 hours after transfection. SDF-1 dependent Ca-flux was only present in CXCR4-transgene overexpressing MSCs suggesting functional expression of the chemokine receptor. Transwell migration experiments demonstrated a 2 fold increased migration of CXCR4 mRNA transfected MSCs towards a gradient of SDF-1 compared to background migration without SDF-1. GFP mRNA transfection did not result in any SDF-1 stimulated migration. Our data demonstrate efficient and safe transient genetic engineering of primary MSCs based on mRNA transfection. mRNA transfection induced chemotaxis will facilitate receptor mediated targeting of transplanted MSCs at specific body compartments.