Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.

The common G-allele of interleukin-18 single-nucleotide polymorphism is a genetic risk factor for atopic asthma. The SAPALDIA Cohort Study

BACKGROUND: IL-18 is a pleiotrophic cytokine involved in both, T-helper type 1 (Th1) and Th2 differentiation. Recently genetic variants in the IL-18 gene have been associated with increased risk of atopy and asthma. OBJECTIVE: To examine the relationship of a genetic, haplotype-tagging promotor variant -137G/C in the IL-18 gene with atopic asthma in a large, well-characterized and population-based study of adults. METHODS: Prospective cohort study design was used to collect interview and biological measurement data at two examination time-points 11 years apart. Multivariate logistic regression analysis was used to assess the association of genotype with asthma and atopy. RESULTS: The G-allele of the IL-18 promotor variant (-137G/C) was associated with a markedly increased risk for the prevalence of physician-diagnosed asthma with concomitant skin reactivity to common allergens. Stratification of the asthma cases by skin reactivity to common allergens revealed an exclusive association of IL-18 -137 G-allele with an increased prevalence of atopic asthma (adjusted odds ratio (OR): 3.63; 95% confidence interval: (1.64-8.02) for GC or GG carriers vs. CC carriers), and no according association with asthma and concomitant negative skin reactivity (adjusted OR: 1.13; 0.66-1.94). The interaction between IL-18 -137G/C genotype and positive skin prick test was statistically significant (P=0.029). None of 74 incident asthma cases with atopy at baseline exhibited the CC genotype. CONCLUSION: Our results strongly suggest that this variant of the IL-18 gene is an important genetic determinant involved in the development of atopic asthma.

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts

VE-cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE-PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor-type phosphatase, co-precipitates with VE-cadherin, but not with beta-catenin, from cell lysates of transfected COS-7 cells and of endothelial cells. Co-precipitation of VE-cadherin and VE-PTP required the most membrane-proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 cells and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells transfected with VE-cadherin and VEGFR-2 increased the VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VE-cadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VE-cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE-cadherin and cell layer permeability independently of its enzymatic activity.

High variability and non-neutral evolution of the mammalian avpr1a gene

BACKGROUND: The arginine-vasopressin 1a receptor has been identified as a key determinant for social behaviour in Microtus voles, humans and other mammals. Nevertheless, the genetic bases of complex phenotypic traits like differences in social and mating behaviour among species and individuals remain largely unknown. Contrary to previous studies focusing on differences in the promotor region of the gene, we investigate here the level of functional variation in the coding region (exon 1) of this locus. RESULTS: We detected high sequence diversity between higher mammalian taxa as well as between species of the genus Microtus. This includes length variation and radical amino acid changes, as well as the presence of distinct protein variants within individuals. Additionally, negative selection prevails on most parts of the first exon of the arginine-vasopressin receptor 1a (avpr1a) gene but it contains regions with higher rates of change that harbour positively selected sites. Synonymous and non-synonymous substitution rates in the avpr1a gene are not exceptional compared to other genes, but they exceed those found in related hormone receptors with similar functions. DISCUSSION: These results stress the importance of considering variation in the coding sequence of avpr1a in regards to associations with life history traits (e.g. social behaviour, mating system, habitat requirements) of voles, other mammals and humans in particular.

Entscheidungsprozesse in Sportvereinen in Zusammenhang mit externer Beratung

Introduction: Verbunden mit den steigenden Mitgliederzahlen, fällt es Sportvereinen zunehmend schwer, die vielfältige Nachfrage zu bedienen und die hohen Erwartungen zu erfüllen. Viele Schweizer Sportvereine bekunden Probleme, insbesondere im Bereich der Gewinnung und Bindung von ehrenamtlichen Mitarbeitern (Lamprecht, Fischer & Stamm, 2012). Da ca. 90 % der Schweizer Sportvereine ehrenamtlich organisiert sind und die Erfüllung des Vereinszwecks direkt mit dem ehrenamtlichen Engagement der Vereinsmitglieder zusammenspielt, führt der Mangel an Ehrenamtlichen zu erheblichen Problemen. Sportvereine können diese skizzierten Herausforderungen aufgrund deren Komplexität und den eigenen begrenzten Ressourcen nicht mehr alleine bewältigen und sind deshalb auf Wissen von aussen angewiesen (Bette, 2009). Damit stellt sich zugleich die Frage, inwieweit von aussen an Sportvereine herangetragene Steuerungspraktiken und Beratungsprogramme (z.B. durch Sportverbände) in Bezug auf den Umgang mit personalen Problemlagen wirksam sind oder nicht. So lässt sich vielfach beobachten, dass standardisierte Beratungsinputs im Verein zu unterschiedlichen Konsequenzen führen. Demnach ist zu vermuten, dass externe Impulse vereinsintern in unterschiedlicher Art und Weise interpretiert und in Vereinsrealität übersetzt (programmiert) werden. Solche Prozesse sind in hohem Masse an die vereinsspezifischen Reproduktionsbedingungen, also Entscheidungsprozesse gebunden. Deshalb stellt sich die Frage: Welche organisationalen Entscheidungsprozesse im Allgemeinen und speziell in Zusammenhang mit externer Beratung sind in Sportvereinen zu beobachten? Methods: Die Daten zur Analyse der Entscheidungsprozesse wurden im Rahmen des Projekts „Mehr Freiwillige im Fussballverein“ (MFiF) in Kooperation mit dem Schweizerischen Fussballverband (SFV) in elf Fussballvereinen erhoben. Die Interventionsstudie umfasst vier Aspekte: (1) systematischer Ansatz, (2) Einbezug aller Vereinsmitglieder, (3) konsequente Implementation in den Fussballvereinen und (4) eine längerfristige Bindungsstrategie für ehrenamtliche Mitarbeiter. Die Daten wurden einerseits über Fragebogen zur Struktur des Vereins und den Ergebnissen des Projekts erhoben, andererseits wurden leitfadengestützte Interviews mit den führenden Vereinsvertretern in den Projektgruppen durchgeführt und anschliessend anhand qualitativer Inhaltsanalyse ausgewertet (Mayring, 2010). Results: Die Auswertung der Interviews zeigt auf, dass verschiedene Faktoren für eine erfolgreiche Umsetzung einer Gewinnungs- und Bindungsstrategie entscheidend sind. Einerseits wird die Rolle der zuständigen Personen unterschiedlich interpretiert und deshalb fällt das Engagement im Entscheidungsprozess unterschiedlich aus. Die Bandbreite reicht vom Informator über den Moderator bis hin zum aktiven Promotor. Mit Blick auf die externe Beratung lässt sich erkennen, dass die Fussballvereine die Unterstützung unterschiedlich einordnen. Dem traditionellen Beratungskonzept folgend anerkennen Vereine die externe Beratung als bewährte Musterlösung und verfolgen unreflektiert die vorgeschlagene Lösungsstrategie. Gleichzeitig lässt sich eine zweite Gruppe von Vereinen entsprechend dem systemischen Beratungskonzept durch die externe Beratung irritieren und hinterfragt die eigene Struktur und sucht nach eine passenden Lösungsstrategie. Discussion/Conclusion: Die Untersuchung zeigt auf, dass externe Vereinsberatung sowohl mimetisch, wie auch als systemische Beratung interpretiert zu Verbesserungen im Bereich des Ehrenamtmanagements in Sportvereinen führen kann. References: Bette, K.-H. (2009). Beratung von Sportorganisationen: Konzepte und Voraussetzungen. In C. Breuer & A. Thiel (Hrsg.), Handbuch Sportmanagement (2. Aufl., S. 139-155). Schorndorf: Hofmann. Lamprecht, M., Fischer, A., & Stamm, H.-P. (2012). Sportvereine in der Schweiz. Strukturen, Leistungen, Herausforderungen. Zürich: Seismo. Mayring, P. (2010). Qualitative Inhaltsanalyse: Grundlagen und Techniken (11. Aufl.). Hemsbach: Beltz.

Salicylic Acid, a Plant Defense Hormone, Is Specifically Secreted by a Molluscan Herbivore

Slugs and snails are important herbivores in many ecosystems. They differ from other herbivores by their characteristic mucus trail. As the mucus is secreted at the interface between the plants and the herbivores, its chemical composition may play an essential role in plant responses to slug and snail attack. Based on our current knowledge about host-manipulation strategies employed by pathogens and insects, we hypothesized that mollusks may excrete phytohormone-like substances into their mucus. We therefore screened locomotion mucus from thirteen molluscan herbivores for the presence of the plant defense hormones jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). We found that the locomotion mucus of one slug, Deroceras reticulatum, contained significant amounts of SA, a plant hormone that is known to induce resistance to pathogens and to suppress plant immunity against herbivores. None of the other slugs and snails contained SA or any other hormone in their locomotion mucus. When the mucus of D. reticulatum was applied to wounded leaves of A. thaliana, the promotor of the SA-responsive gene pathogenesis related 1 (PR1) was activated, demonstrating the potential of the mucus to regulate plant defenses. We discuss the potential ecological, agricultural and medical implications of this finding.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490