Die Rolle von TGF-beta bei der Regulation des Biglykan-Gens und bei der Tumorprogression des Pankreaskarzinoms

TGF-β1 ist ein Zytokin, das an der Regulation verschiedenster biologischer Prozesse beteiligt ist und u.a. die Bildung der extrazellulären Matrix durch die Induktion des kleinen leucin-reichen Proteoglykans Biglykan (BGN) fördert. Die intrazelluläre Signaltransduktion, die zur Aktivierung des BGN Gens führt, ist sehr komplex und abhängig von der Aktivierung des Smad- (Chen et al., 2002) und des p38-MAPK-Signalweges (Ungefroren et al., 2003), wobei beide über das „stress response“ Gen GADD45β miteinander verknüpft sind (Ungefroren et al., 2005). Unter Einsatz verschiedener kinase-defizienter und kinase-aktiver Mutanten sowie spezifischer pharmakologischer Inhibitoren in der TGF-β responsiven Pankreaskarzinomzelllinie Panc-1 konnte gezeigt werden, dass die Kinaseaktivität und die Smad-aktivierende Funktion des TGF-β Typ I Rezeptors (ALK5) für die Induktion der BGN-Expression erforderlich sind. Durch Experimente, in denen Panc-1-Zellen in Suspension kultiviert oder mit dem Zytoskelett zerstörenden Toxin Cytochalasin D behandelt wurden, konnte gezeigt werden, dass der TGF-β-Effekt auf BGN abhängig von der Zelladhäsion ist. In einer weiteren Versuchsreihe, in denen Panc-1-Zellen mit einem Rac1-Inhibitor behandelt wurden, konnte deutlich gemacht werden, dass der TGF-β-Effekt auf BGN durch die kleine GTPase Rac1, die auch an der Weiterleitung von Adhäsions-Signalen beteiligt ist, vermittelt wird. Außerdem konnte in dieser Arbeit mit Hilfe von Panc-1-Zellen, die unterschiedliche Rac1-Mutanten (dnRac1, WT-Rac1, caRac1) ektop exprimierten, unter verschiedenen Kultivierungsbedingungen der Zellen (adhärent oder in Suspension) gezeigt werden, dass Rac1 auf der Ebene von p38 wirkt und keinen Einfluss auf die Smad3-Aktivierung und die GADD45β-Induktion hat. Schließlich konnte durch die pharmakologische Inhibition des NAD(P)H-Oxidase-Komplexes mittels Diphenyleniodoniumchlorid (DPI) gezeigt werden, dass Rac1 seine Funktion bei der p38-Aktivierung als Untereinheit der reaktive Sauerstoffspezies (ROS: „reactive oxygen species“)-produzierenden NAD(P)H-Oxidase ausübt. Dass auch ROS selbst eine wichtige Rolle bei der TGF-β-Regulation von BGN spielen, konnte durch Experimente mit Radikalfängern und Antioxidantien demonstriert werden. Die Vermutung, dass die NAD(P)H-Oxidase als Quelle der ROS-Produktion an diesem Prozess beteiligt ist, wurde weiterhin dadurch unterstützt, dass in den Panc-1-Zellen verschiedene Nox und phox Untereinheiten dieses Enzymkomplexes exprimiert werden. Zusätzlich wurden in dieser Arbeit die Effekte der TGF-β-Signalwirkung auf das Wachstumsverhalten von Pankreaskarzinomzellen in vitro und auf ihre Tumorigenität in vivo analysiert. Dazu wurden die biochemischen und zellulären Effekte von zwei ALK5-Mutanten nach Überexpression in Panc-1-Zellen untersucht. Diese Mutanten besitzen beide eine intakte Kinase-Domäne, unterscheiden sich aber in ihrer Fähigkeit, Smads zu aktivieren, wobei konstitutiv-aktives RImL45 (caRImL45) aufgrund einer Mutation in der L45-Schleife, im Gegensatz zu konstitutiv-aktivem ALK5 (caALK) keine Smads binden kann. Es konnte ferner gezeigt werden, dass die stabile Expression von caALK5 (im Gegensatz zu caRImL45) den antiproliferativen Effekt von TGF-β nachahmen konnte, was auf der Fähigkeit dieser Mutante beruht, den Smad-Signalweg zu aktivieren. Im Gegensatz zu den caRImL45-transduzierten Panc-1-Zellen zeigten caALK5-transduzierte Zellen eine epitheliale-mesenchymale Transdifferenzierung (EMT) und eine erhöhte Expression von metastasierungs-assoziierten Genen. Zusätzlich kam es in diesen Zellen zu einem erhöhten Expressionsverhältnis von anti-angiogenen (TSP-1, PAI-1) zu pro-angiogenen Faktoren (VEGF-A). Nach einer orthotopen Transplantation dieser Zellen in immundefiziente Mäuse kam es durch caALK5 im Gegensatz zu caRImL45 zu einer Reduktion des Primärtumorwachstums kam. Allerdings förderte diese Mutante im Gegensatz zu caRImL45 die Bildung von Lebermetastasen, woraus geschlossen werden kann, dass der Smad-Signalweg in Bezug auf den Primärtumor zwar anti-onkogen, in Bezug auf die Metastasenbildung aber pro-onkogen wirkt

Zero-knowledge Argument for Polynomial Evaluation with Application to Blacklists

Verification of a polynomial’s evaluation in a secret committed value plays a role in cryptographic applications such as non-membership or membership proofs. We construct a novel special honest verifier zero-knowledge argument for correct polynomial evaluation. The argument has logarithmic communication cost in the degree of the polynomial, which is a significant improvement over the state of the art with cubic root complexity at best. The argument is relatively efficient to generate and very fast to verify compared to previous work. The argument has a simple public-coin 3-move structure and only relies on the discrete logarithm assumption. The polynomial evaluation argument can be used as a building block to construct zero-knowledge membership and non-membership arguments with communication that is logarithmic in the size of the blacklist. Non-membership proofs can be used to design anonymous blacklisting schemes allowing online services to block misbehaving users without learning the identity of the user. They also allow the blocking of single users of anonymization networks without blocking the whole network

Differential roles of Smad2 and Smad3 in the regulation of TGF-β1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1

<p>Abstract</p> <p>Background</p> <p>Progression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-β)/Smad signalling pathway, eventually resulting in loss of TGF-β-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-β are mediated solely or partially through the canonical Smad signalling pathway which commences with activation of receptor-regulated Smads (R-Smads) Smad2 and Smad3 by the TGF-β type I receptor. However, little is known on the relative contribution of each R-Smad, the possible existence of functional antagonism, or the crosstalk with other signalling pathways in the control of TGF-β1-induced growth inhibition and cell migration. Using genetic and pharmacologic approaches we have inhibited in PDAC cells endogenous Smad2 and Smad3, as well as a potential regulator, the small GTPase Rac1, and have analysed the consequences for TGF-β1-mediated growth inhibition and cell migration (chemokinesis).</p> <p>Results</p> <p>SiRNA-mediated silencing of Smad3 in the TGF-β responsive PDAC cell line PANC-1 reduced TGF-β1-induced growth inhibition but increased the migratory response, while silencing of Smad2 enhanced growth inhibition but decreased chemokinesis. Interestingly, siRNA-mediated silencing of the small GTPase Rac1, or ectopic expression of a dominant-negative Rac1 mutant largely mimicked the effect of Smad2 silencing on both TGF-β1-induced growth inhibition, via upregulation of the cdk inhibitor p21^WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-β1-induction of a Smad2-specific transcriptional reporter and Smad2 C-terminal phosphorylation in PDAC cells while Smad3-specific transcriptional activity and Smad3 C-terminal phosphorylation appeared increased. Disruption of autocrine TGF-β signalling in PANC-1 cells rendered cells less susceptible to the growth-suppressive effect of Rac1 inhibition, suggesting that the decrease in "basal" proliferation upon Rac1 inhibition was caused by potentiation of autocrine TGF-β growth inhibition.</p> <p>Conclusions</p> <p>In malignant cells with a functional TGF-β signalling pathway Rac1 antagonizes the TGF-β1 growth inhibitory response and enhances cell migration by antagonistically regulating Smad2 <it>and </it>Smad3 activation. This study reveals that Rac1 is prooncogenic in that it can alter TGF-β signalling at the R-Smad level from a tumour-suppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression.</p

Sulfation degree of glycosaminoglycans triggers distinct cytoskeleton organisation in mesenchymal stem cells

Glycosaminoglycans (GAGs) comprise the closest cellular environment: they are building elements of the ECM and can be also found on cells surface. Their biological activity depends on several parameters among which the negative charge is of prime importance[1]. This charge is generally associated with the presence of sulfate groups (-OSO3H). Sulfation is a dynamic modification: it can occur at various positions within the glycan and different sulfation patterns have been identified for the same organs and cells during their development. However, the mechanisms of coding and transferring information by these functionalities are not yet complete understood, mainly because of (i)the complex physiological microenvironment in which GAGs interactions occur and (ii)the inability to access homogeneous GAGs[2]. In this work, we propose model surfaces bearing GAGs with different sulfation degree as platform to investigate the pathways by which mesenchymal stem cells (MSCs) sense and respond to this peculiar functionality: the -OSO3H. We have selected two natural GAGs for this study: hyaluronic acid (HA) because it is the only non-sulfated glycan and heparin (HEP) as it is the GAG with the highest degree of sulfation. To obtain a larger range of sulfation degrees, we have also prepared a synthetic analogue of HA with a sulfation degree of 1.4 (sHA). All these GAGs were covalently bonded to aminothiols deposited on gold surfaces. MSCs, both from bone marrow and adipose tissue, adhered well to all surfaces. Formation of focal adhesions was observed after only 1h of culture for bone marrow derived MSCs regardless the used substrate. The presence of –OSO3H groups induced different morphology and cytoskeleton organisation: formation of longer filopodia and well pronounced actin fibers were visible for the MSCs from both sources. Moreover, cells were more spread after 24h in contact with – OSO3H containing surfaces. Cells behaved similarly on both sulfated surfaces (sHA and HEP) and differences in cell morphology were less obvious: higher sulfation degree induced less lamellipodia formation while filopodia number and length increased. In summary, the present study provides evidence that sulfation degree of GAGs triggers distinct cytoskeleton organisation in mesenchymal stem cells that may be related with the differentiation of those cells. However, further studies at the molecular level about the exact mechanism of these processes need to be carried out

Shaping sustainable nutrition through participation – instruments to support a transformation process

Die „regional-saisonale Bio-Abokiste“ als exemplarisches Prinzip didaktischer Überlegungen kann langfristig „Bottom-up“ einen schulischen Transformationsprozess initiieren, in dem sich Lehrende und Lernende mit der Komplexität nachhaltiger Ernährung auseinandersetzen. Veränderte Präkonzepte, erfahrene Selbstwirksamkeit sowie erfolgreiche Partizipation verändern das Setting Schule und seine Ernährungsumgebungen nachhaltig. Unterstützungsinstrumente sind Teil einer Gelingensbedingung, diese (mit) zu gestalten. (DIPF/Orig.)The “regional-seasonal organic subscription box” is an exemplary principle of didactic considerations that can initiate a long-term “bottom-up” school transformation process in which teachers and learners deal with the complexity of sustainable nutrition. Modified preconcepts, experienced self-efficacy, and successful participation change the school setting and its nutritional environment sustainably. Support tools are part of a condition for success in (co)shaping this. (DIPF/Orig.

Towards FAIR principles for research software

The FAIR Guiding Principles, published in 2016, aim to improve the findability, accessibility, interoperability and reusability of digital research objects for both humans and machines. Until now the FAIR principles have been mostly applied to research data. The ideas behind these principles are, however, also directly relevant to research software. Hence there is a distinct need to explore how the FAIR principles can be applied to software. In this work, we aim to summarize the current status of the debate around FAIR and software, as basis for the development of community-agreed principles for FAIR research software in the future. We discuss what makes software different from data with regard to the application of the FAIR principles, and which desired characteristics of research software go beyond FAIR. Then we present an analysis of where the existing principles can directly be applied to software, where they need to be adapted or reinterpreted, and where the definition of additional principles is required. Here interoperability has proven to be the most challenging principle, calling for particular attention in future discussions. Finally, we outline next steps on the way towards definite FAIR principles for research software

Visual Genome: Connecting language and vision using crowdsourced dense image annotations

Despite progress in perceptual tasks such as image classification, computers still perform poorly on cognitive tasks such as image description and question answering. Cognition is core to tasks that involve not just recognizing, but reasoning about our visual world. However, models used to tackle the rich content in images for cognitive tasks are still being trained using the same datasets designed for perceptual tasks. To achieve success at cognitive tasks, models need to understand the interactions and relationships between objects in an image. When asked “What vehicle is the person riding?”, computers will need to identify the objects in an image as well as the relationships riding(man, carriage) and pulling(horse, carriage) to answer correctly that “the person is riding a horse-drawn carriage.” In this paper, we present the Visual Genome dataset to enable the modeling of such relationships. We collect dense annotations of objects, attributes, and relationships within each image to learn these models. Specifically, our dataset contains over 108K images where each image has an average of (Formula presented.) objects, (Formula presented.) attributes, and (Formula presented.) pairwise relationships between objects. We canonicalize the objects, attributes, relationships, and noun phrases in region descriptions and questions answer pairs to WordNet synsets. Together, these annotations represent the densest and largest dataset of image descriptions, objects, attributes, relationships, and question answer pairs

COMT val158met is not associated with Aβ-amyloid and APOE ε4 related cognitive decline in cognitively normal older adults

The non-synonymous single nucleotide polymorphism (SNP), Val158Met within the Catechol-O-methyltransferase (COMT) gene has been associated with altered levels of cognition and memory performance in cognitively normal adults. This study aimed to investigate the independent and interactional effects of COMT Val158Met on cognitive performance. In particular, it was hypothesised that COMT Val158Met would modify the effect of neocortical Aβ-amyloid (Aβ) accumulation and carriage of the apolipoprotein E (APOE) ε4 allele on cognition in preclinical Alzheimer’s disease (AD). In 598 cognitively normal older adults with known neocortical Aβ levels, linear mixed modelling revealed no significant independent or interactional associations between COMT Val158Met and cognitive decline. These findings do not support previous associations between COMT Val158Met and cognitive performance and suggest this variant does not influence Aβ-amyloid or APOE ε4 driven cognitive decline in a well characterised cohort of cognitively normal older adults

SPON1 is associated with amyloid-β and APOE ϵ4-related cognitive decline in cognitively normal adults

Background: Genetic variation in Spondin-1, specifically rs11023139, has been associated with reduced rates of cognitive decline in individuals with Alzheimer\u27s disease. Objective: The aim of this study was to assess whether the association was present in cognitively normal older adults. Methods: Longitudinal cognitive decline was investigated using linear mixed modelling in a cohort of 590 cognitively normal older adults enrolled in the Australian Imaging, Biomarkers and Lifestyle Study. Results: No independent effect of Spondin-1 rs11023139 on cognitive decline was observed. However, significant associations were observed for the interaction between Apolipoprotein E (APOE) ϵ4 and rs11023139 in individuals with high amyloid-β burden. APOE ϵ4/rs11023139-A carriers declined significantly faster than APOE ϵ4/rs11023139-G-G carriers in measures of global cognition (p=0.011) and verbal episodic memory (p=0.020). Conclusion: These results suggest that carriage of the Spondin-1 rs11023139-A allele significantly contributes to a worsening of cognitive performance in APOE ϵ4 cognitively normal older adults with a high neocortical amyloid-β burden