Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models.

To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states

Gli1 mediates lung cancer cell proliferation and sonic hedgehog-dependent mesenchymal cell activation.

Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options

QuateXelero : an accelerated exact network motif detection algorithm

Finding motifs in biological, social, technological, and other types of networks has become a widespread method to gain more knowledge about these networks’ structure and function. However, this task is very computationally demanding, because it is highly associated with the graph isomorphism which is an NP problem (not known to belong to P or NP-complete subsets yet). Accordingly, this research is endeavoring to decrease the need to call NAUTY isomorphism detection method, which is the most time-consuming step in many existing algorithms. The work provides an extremely fast motif detection algorithm called QuateXelero, which has a Quaternary Tree data structure in the heart. The proposed algorithm is based on the well-known ESU (FANMOD) motif detection algorithm. The results of experiments on some standard model networks approve the overal superiority of the proposed algorithm, namely QuateXelero, compared with two of the fastest existing algorithms, G-Tries and Kavosh. QuateXelero is especially fastest in constructing the central data structure of the algorithm from scratch based on the input network

Application of Petri net based analysis techniques to signal transduction pathways

BACKGROUND: Signal transduction pathways are usually modelled using classical quantitative methods, which are based on ordinary differential equations (ODEs). However, some difficulties are inherent in this approach. On the one hand, the kinetic parameters involved are often unknown and have to be estimated. With increasing size and complexity of signal transduction pathways, the estimation of missing kinetic data is not possible. On the other hand, ODEs based models do not support any explicit insights into possible (signal-) flows within the network. Moreover, a huge amount of qualitative data is available due to high-throughput techniques. In order to get information on the systems behaviour, qualitative analysis techniques have been developed. Applications of the known qualitative analysis methods concern mainly metabolic networks. Petri net theory provides a variety of established analysis techniques, which are also applicable to signal transduction models. In this context special properties have to be considered and new dedicated techniques have to be designed. METHODS: We apply Petri net theory to model and analyse signal transduction pathways first qualitatively before continuing with quantitative analyses. This paper demonstrates how to build systematically a discrete model, which reflects provably the qualitative biological behaviour without any knowledge of kinetic parameters. The mating pheromone response pathway in Saccharomyces cerevisiae serves as case study. RESULTS: We propose an approach for model validation of signal transduction pathways based on the network structure only. For this purpose, we introduce the new notion of feasible t-invariants, which represent minimal self-contained subnets being active under a given input situation. Each of these subnets stands for a signal flow in the system. We define maximal common transition sets (MCT-sets), which can be used for t-invariant examination and net decomposition into smallest biologically meaningful functional units. CONCLUSION: The paper demonstrates how Petri net analysis techniques can promote a deeper understanding of signal transduction pathways. The new concepts of feasible t-invariants and MCT-sets have been proven to be useful for model validation and the interpretation of the biological system behaviour. Whereas MCT-sets provide a decomposition of the net into disjunctive subnets, feasible t-invariants describe subnets, which generally overlap. This work contributes to qualitative modelling and to the analysis of large biological networks by their fully automatic decomposition into biologically meaningful modules

Bekämpfung der Blutlaus (Eriosoma lanigerum Hausm.) in ökologischen Obstanlagen durch Etablierung von Ohrwürmern (Forficula auricularia L.) und Bewertung unterstützender Bekämpfungsverfahren

Das Forschungsprojekt „Bekämpfung der Blutlaus (Eriosoma lanigerum Hausm.) in ökologischen Obstanlagen durch Etablierung von Ohrwürmern (Forficula auricularia L.) und Bewertung unterstützender Bekämpfungsverfahren“ lief in Zusammenarbeit verschiedener Forschungs- und Beratungseinrichtungen von April 2007 bis Dezember 2009. Ziel des Projektes war es, auf Basis von Versuchsergebnissen eine betriebliche Behandlungsstrategie zur Bekämpfung der Blutlaus durch den gezielten Ohrwurmeinsatz und Ölbehandlungen zu entwickeln. Der Einfluss der Ohrwürmer erwies sich als schwer nachweisbar. Dies kann mehrere Gründe haben. Zum einen verteilen sich die Ohrwürmer über die Anlage, so dass ein Effekt in den einzelnen Varianten dadurch ausgeglichen wird, dass die Ohrwürmer nun auch in der Kontrolle zu finden waren. Zum anderen sind zum Zeitpunkt der Ansiedlung schon zahlreiche andere Nützlinge wie Marienkäfer und deren Larven, Schwebfliegen- und Florfliegenlarven sowie Blutlauszehrwespen in den Anlagen anzutreffen, die ihren Teil zur Eindämmung der Blutlauspopulation beitragen. Gerade das Massenhafte Auftreten des Asiatischen Marienkäfers (Harmonis axyridids) lässt eine Überlagerung des Nützlingseinflusses vermuten, da die Asiatischen Marienkäfer eine enorme Fraßleistung zeigen. Der Vergleich der Applikation von Öl mit dem Pinsel bzw. per Gebläsespritze zeigt bei einem hohen Befall deutliche Vorteile für das Pinseln. Die Behandlung ist direkter und wesentlich effektiver bei hohem Befall, allerdings auch Zeit aufwendig und hat keine Zulassung in Deutschland. Bei niedrigem Befallsdruck zeigte eine Frühjahrsspritzung durchaus zufrieden stellende Ergebnisse. Als Maßnahme bei akutem Befall kann eine Ansiedlung von Ohrwürmern also nicht dienen, eine langfristige Förderung ist aber sinnvoll. Bei Applikation oder Pinseln von Öl als Bekämpfungsmaßnahme im Frühjahr muss zwischen Befallsstärke und Arbeitszeitaufwand abgewogen werden. Durch Ohrwurmkot verschmutzte Äpfel treten durchaus auf, verursachten aber in keiner der Versuchsanlagen ökonomische Schäden. Ein Versuch zum Einfluss der mechanischen Bodenbearbeitung auf die Überwinternden Ohrwürmer zeigte keinen negativen Einfluss der Bodenbearbeitung auf die Ohrwurmpopulation. Es wird davon ausgegangen, dass die Tiere zwischen die Wurzeln der Pflanzen in den Fahrgassen ausweichen oder ihre Gelege so tief im Boden anlegen, dass sie nicht mehr von der Bodenbearbeitung beeinflusst werden. Die Untersuchungen zum Gesundheitszustand der Ohrwürmer ergaben den Nematoden Mermis nigrescens sowie Vertreter der Raupenfliegen (Tachinidae) als Hauptparasiten. Insgesamt lag der Anteil parasitierter oder kranker Tiere zwischen drei und acht Prozent

Columba: an integrated database of proteins, structures, and annotations

BACKGROUND: Structural and functional research often requires the computation of sets of protein structures based on certain properties of the proteins, such as sequence features, fold classification, or functional annotation. Compiling such sets using current web resources is tedious because the necessary data are spread over many different databases. To facilitate this task, we have created COLUMBA, an integrated database of annotations of protein structures. DESCRIPTION: COLUMBA currently integrates twelve different databases, including PDB, KEGG, Swiss-Prot, CATH, SCOP, the Gene Ontology, and ENZYME. The database can be searched using either keyword search or data source-specific web forms. Users can thus quickly select and download PDB entries that, for instance, participate in a particular pathway, are classified as containing a certain CATH architecture, are annotated as having a certain molecular function in the Gene Ontology, and whose structures have a resolution under a defined threshold. The results of queries are provided in both machine-readable extensible markup language and human-readable format. The structures themselves can be viewed interactively on the web. CONCLUSION: The COLUMBA database facilitates the creation of protein structure data sets for many structure-based studies. It allows to combine queries on a number of structure-related databases not covered by other projects at present. Thus, information on both many and few protein structures can be used efficiently. The web interface for COLUMBA is available at

Evaluation of the importance of mixing during preparation of antibiotic infusions

BACKGROUND: The mixing step after medication addition to the infusion bag is frequently omitted during the preparation of drug infusions. However, the importance of mixing when preparing antibiotic infusions is still unknown. METHODS: The primary aim of this study was to assess the importance of the mixing step by comparing the concentrations of unmixed antibiotic infusions (cefuroxime, flucloxacillin, meropenem, and vancomycin) with the declared concentration at regular intervals during infusion. The secondary aim was to compare concentrations between preparation sites (hospital pharmacy versus clinical ward). Infusion bags were run through electronic infusion pumps. For cefuroxime, flucloxacillin, and meropenem, samples were collected 1, 15, and 20 min after starting the administration (infusion duration: 30 min). For vancomycin, samples were collected after 1, 60, and 110 min (infusion duration: 120 min). Vancomycin concentrations were measured using the Architect c4000 analyser and other concentrations using a validated UPC(2)-MS–MS multimethod. RESULTS: The median concentrations of the four antibiotics were comparable to the declared concentration at all three time points. No significant differences were found between preparation sites. CONCLUSIONS: Spontaneous mixing occurred in the examined antibiotic solutions during normal handling

JProGO: a novel tool for the functional interpretation of prokaryotic microarray data using Gene Ontology information

A novel program suite was implemented for the functional interpretation of high-throughput gene expression data based on the identification of Gene Ontology (GO) nodes. The focus of the analysis lies on the interpretation of microarray data from prokaryotes. The three well established statistical methods of the threshold value-based Fisher's exact test, as well as the threshold value-independent Kolmogorov–Smirnov and Student's t-test were employed in order to identify the groups of genes with a significantly altered expression profile. Furthermore, we provide the application of the rank-based unpaired Wilcoxon's test for a GO-based microarray data interpretation. Further features of the program include recognition of the alternative gene names and the correction for multiple testing. Obtained results are visualized interactively both as a table and as a GO subgraph including all significant nodes. Currently, JProGO enables the analysis of microarray data from more than 20 different prokaryotic species, including all important model organisms, and thus constitutes a useful web service for the microbial research community. JProGO is freely accessible via the web at the following address