69 research outputs found

    Characterization of a site on PAI-1 that binds to vitronectin outside of the somatomedin B domain

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    Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are proteins that interact in the circulatory system and pericellular region to regulate fibrinolysis, cell adhesion, and migration. The interactions between the two proteins have been attributed primarily to binding of the somatomedin B (SMB) domain, which comprises the N-terminal 44 residues of vitronectin, to the flexible joint region of PAI-1, including residues Arg-103, Met-112, and Gln-125 of PAI-1. A strategy for deletion mutagenesis that removes the SMB domain demonstrates that this mutant form of vitronectin retains PAI-1 binding (Schar, C. R., Blouse, G. E., Minor, K. M., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309). In the current study, the complementary binding site on PAI-1 was mapped by testing for the ability of a battery of PAI-1 mutants to bind to the engineered vitronectin lacking the SMB domain. This approach identified a second, separate site for interaction between vitronectin and PAI-1. The binding of PAI-1 to this site was defined by a set of mutations in PAI-1 distinct from the mutations that disrupt binding to the SMB domain. Using the mutations in PAI-1 to map the second site suggested interactions between α-helices D and E in PAI-1 and a site in vitronectin outside of the SMB domain. The affinity of this second interaction exhibited a KD value ∼100-fold higher than that of the PAI-1-somatomedin B interaction. In contrast to the PAI-1-somatomedin B binding, the second interaction had almost the same affinity for active and latent PAI-1. We hypothesize that, together, the two sites form an extended binding area that may promote assembly of higher order vitronectin-PAI-1 complexes. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc

    Platelet Function During Extracorporeal Membrane Oxygenation in Adult Patients: A Systematic Review

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    Background: Hemorrhagic and thromboembolic complications are common during treatment with extracorporeal membrane oxygenation (ECMO), resulting in considerable morbidity and mortality. This emphasizes the clinical relevance of understanding hemostatic changes occurring during ECMO treatment. As platelets are key players in hemostasis, detailed knowledge on how ECMO treatment affects platelet function is of great importance. We therefore aimed to systematically summarize and discuss existing knowledge on platelet function during ECMO treatment in adult patients.Methods: Systematic review complying with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Objectives and methods were specified in a PROSPERO protocol (ID no CRD42018084059). The MEDLINE/PubMed, EMBASE, and Web of Science databases were systematically searched on September 10, 2018. A standardized quality assessment tool was used to assess the risk of bias in included studies. Primary outcome was platelet function during ECMO treatment, measured as platelet adhesion, activation or aggregation. Secondary outcomes were thrombosis, bleeding, and mortality during ECMO treatment.Results: A total of 591 studies were identified, of which seven were eligible for inclusion in the qualitative synthesis. Of these, one study investigated expression of platelet adhesion receptors and found them to be reduced during ECMO treatment; two studies reported a decrease in platelet activation markers during ECMO treatment; and five studies demonstrated reduced platelet aggregation during ECMO treatment. Three studies reported on thrombosis, mortality and/or bleeding during ECMO treatment; no thromboembolic events were reported; all three studies reported frequent bleeding episodes defined on basis of transfusion requirements. An in-hospital mortality of 35–40% and a 30-day mortality of roughly 30% were reported in three different studies.Conclusions: The present systematic review reveals a substantial knowledge gap regarding platelet function during ECMO treatment in adult patients and underscores the demand for more and well-designed studies on this topic. There is suggested evidence of reduced platelet adhesion, decreased platelet activation, and reduced platelet aggregation in adult patients during ECMO treatment. Importantly, platelet aggregation results need to be interpreted in the light of low platelet counts. The associations of platelet function and bleeding and/or thromboembolic complications during ECMO treatment remain to be fully elucidated

    miRNA profiling of circulating EpCAM(+) extracellular vesicles:promising biomarkers of colorectal cancer

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    Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. These contain biomolecules, including proteins and microRNAs (miRNAs). Both circulating EVs and miRNAs have received much attention as biomarker candidates for non-invasive diagnostics. Here we describe a sensitive analytical method for isolation and subsequent miRNA profiling of epithelial-derived EVs from blood samples of patients with colorectal cancer (CRC). The epithelial-derived EVs were isolated by immunoaffinity-capture using the epithelial cell adhesion molecule (EpCAM) as marker. This approach mitigates some of the specificity issues observed in earlier studies of circulating miRNAs, in particular the negative influence of miRNAs released by erythrocytes, platelets and non-epithelial cells. By applying this method to 2 small-scale patient cohorts, we showed that blood plasma isolated from CRC patients prior to surgery contained elevated levels of 13 EpCAM+-EV miRNAs compared with healthy individuals. Upon surgical tumour removal, the plasma levels of 8 of these were reduced (miR-16-5p, miR-23a-3p, miR-23b-3p, miR-27a-3p, miR-27b-3p, miR-30b-5p, miR-30c-5p and miR-222-3p). These findings indicate that the miRNAs are of tumour origin and may have potential as non-invasive biomarkers for detection of CRC. This work describes a non-invasive blood-based method for sensitive detection of cancer with potential for clinical use in relation to diagnosis and screening. We used the method to study CRC; however, it is not restricted to this disease. It may in principle be used to study any cancer that release epithelial-derived EVs into circulation

    A cyclic peptidic serine protease inhibitor:increasing affinity by increasing peptide flexibility

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    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden

    Hemmelighedernes kammer

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    Analysis of Vortex Induced Motions for Floating Wind Turbines

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    Et økende fokus på globale oppvarming har vært den drivende kraften bak forsking på fornybar energi i flere år. Vindkraft er en viktig bidragsyter blant de fornybare energiprodusentene, og markedet for vindkraft er i stadig vekst. Den første offshore vindturbinparken med bunnfaste vindturbiner ble installert i 1991. I 2017 begynte den første flytende offshore vindparken, Hywind Scotland, å operere. Flytende strukturer kan oppleve bevegelse på grunn av virvelavløsning og dette kan føre til store krefter og påvirke levetiden til forankringssystemet. Dette prosjektet omhandler en spar plattform og en delvis nedsenkbare plattformen som er designet for å være flytende understell for to vindturbiner som skal operere Vest for Barra, utenfor Skottland. Strømningen i området er den viktigste miljøbetraktningen når bevegelse på grunn av virvelavløsning skal analyseres. DNV offshore standarder har blitt brukt til å regne ut en 50 års strømningsprofil og gjennomsnittsverdien er bruk som en øvre grense for strømningshastigheten. Dette gir en gjennomsnittlig maksimal strømningshastighet på 1.4 m/s for sparen og 2.07 m/s for den delvis nedsenkbare plattformen. Den naturlige egenperioden for sparen og den delvis nedsenkbare plattformen er 128.87 s og 104.17 s. 2D tverrsnittsareal for de to plattformene er studert ved bruk av computational fluid dynamic (CFD) and OpenFOAM er programvaren. Turbulent strømning i området 7.65e5 ≤ Re ≤ 1.58e7, med turbulent intensitet på 1% og turbulent lengde på 3% av diameteren er analysert. En gridkonvergensstudie for en fast sylinder var først gjennomført for et strukturerte grid som ble generert med blockMesh. Numeriske utfordringer førte til at det ble bestemt at et ustrukturert grid ville egne seg bedre spesielt når flere sylindere skal studeres. Det ustrukturerte gridet ble generert med snappyHexMesh for en sylinder før et grid for den delvis nedsenkbare plattformen ble generert. Sparen og den delvis nedsenkbare plattformen ble først studert som fastholdt og hydrodynamiske verdier som Cd, Cl and St ble analysert sammen med strømningskarakteristikken. Effekten av strømningsinteraksjon har også blitt studert for den delvis nedsenkbare plattformen og kanseleringseffekt har blitt observert for løft kreftene for de to sylindrene ved siden av hverandre. Den bakerste sylinderen opplever størst løft og drag krefter. En økning i drag er observert for økende Re for begge strukturer og dette kan være et resultat av implementasjonen av vegg funksjonene. To dynamiske grid har blitt analysert for spar fundamentet. En fri bevegelses test og en tvungen bevegelses test har blitt utført for å kartlegge egenperioden og tilleggsmassen til systemet. To overlappende grids og et deformerende grid ble testet. Det deformerende gridet ga en egenperiode på 125 s og det overlappende gridet med ustrukturert grid ga egenperiode på 129.87 s. Disse to gridene er testet med innkommende strømningshastighet U=0.4 m/s som korresponderer til Ur ≈ 3.6 for begge gridene. Resonans ble observert i begge tilfeller da sylinderen svinger med en periode nær den naturlige perioden, men svingningsamplituden er rundt 0.7D og høyere enn forventet. Bevegelsene er for store for det deformerende gridet og de fleste simulieringene med inkommende srøming ble utført med det overlappende gridet. Den nedre lock-in regionen er blitt identifisert og forventede svingningsamplituder ble observert. Det er behov for mer forskning på den øvre lock-in regionen. To høye hastigheter ble testet, men urealistiske høye svingningsamplituder ble observert, på grunn av en stor løftekraft i starten av simuleringen. En rampefunksjon og demping ble testet og en reduksjon i amplituden ble observert. Gridet trenger imidlertid ytterligere tilpasninger, og et grid konvergensstudie bør gjennomføres før resultatene kan valideres ytterligere
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