Self-assembled artificial cilia

Due to their small dimensions, microfluidic devices operate in the low Reynolds number regime. In this case, the hydrodynamics is governed by the viscosity rather than inertia and special elements have to be introduced into the system for mixing and pumping of fluids. Here we report on the realization of an effective pumping device that mimics a ciliated surface and imitates its motion to generate fluid flow. The artificial biomimetic cilia are constructed as long chains of spherical superparamagnetic particles, which self-assemble in an external magnetic field. Magnetic field is also used to actuate the cilia in a simple nonreciprocal manner, resulting in a fluid flow. We prove the concept by measuring the velocity of a cilia-pumped fluid as a function of height above the ciliated surface and investigate the influence of the beating asymmetry on the pumping performance. A numerical simulation was carried out that successfully reproduced the experimentally obtained data.     

Antithrombotic potential of new direct thrombin inhibitors built on the azaphenylalanine scaffold in two rat venous thrombosis models

The antithrombotic potential of new direct thrombin inhibitors built on the azaphenylalanine scaffold (LK-732, LK-639 and LK-731) and their amidoxime prodrugs (LK-658, LK-633 and LK-730) was studied in comparison to argatroban and nadroparin in two rat models of venous thrombosis, induced either by complete stasis combined with hypercoagulability (model 1) or by partial stasis combined with vessel injury (model 2). In initial experiments LK-732 was established as the most promising antithrombotic of the LK inhibitors and as such was further tested. In model 1, intravenous bolus administration of LK-732 produced a dose-dependent inhibition of thrombus formation with an ID50 value of 1.3 mg/kg. This ID50 value was approximately four times higher than the ID50 value of argatroban (0.3 mg/kg; p=0.011). However, in model 2, LK-732 and argatroban decreased thrombus weight by 50% at similar ID50 values (3.8 mg/kg vs 3.0 mg/kg, respectively; p=0.726). The ex vivo anticoagulant effect of LK-732 was substantially weaker compared to argatroban at doses that produced comparable antithrombotic effects. After subcutaneous administration, in vivo thrombus weight reduction of LK inhibitors (10 mg/kg) ranged between 22 to 48%. However, their oral antithrombotic effect at a dose of 30 mg/kg was rather low. LK amidoxime prodrugs failed to produce a substantial antithrombotic effect after subcutaneous (10 mg/kg) as well as after oral administration (30 mg/kg). In conclusion, thrombin inhibitors built on the azaphenylalanine scaffold represent a new group of intravenously effective antithrombotics. However, optimisation of the oral antithrombotic effect of amidoxime prodrug LK-658 of the lead inhibitor LK-732 is required for justifying further development of these inhibitors.     

Novel non-covalent thrombin inhibitors incorporating P(1) 4,5,6,7-tetrahydrobenzothiazole arginine side chain mimetics

The design, synthesis and biological activity of a series of novel non-covalent D-Phe-Pro-Arg motif-based thrombin inhibitors incorporating 4,5,6,7-tetrahydrobenzothiazol-2-amine as a novel arginine surrogate are described. Compound 9, the most potent in the series of thrombin inhibitors, exhibited an in vitro K(i) of 128 nM and 342-fold selectivity against trypsin. The binding mode of this novel class of thrombin inhibitors in the enzyme active site, based on the X-ray crystal structure of compound 9 co-crystallized with human alpha-thrombin, is discussed.     

The - 429 T/C and - 374 T/A gene polymorphisms of the receptor of advanced glycation end products gene are not risk factors for diabetic retinopathy in Caucasians with type 2 diabetes

OBJECTIVE: The aim of this study was to look for a relationship between the - 429 T/C and the - 374 T/A gene polymorphisms of the receptor of advanced glycation end products (RAGE) gene and the development of diabetic retinopathy in Caucasians with type 2 diabetes. MATERIALS AND METHODS: One hundred and sixteen subjects with type 2 diabetes and diabetic retinopathy were compared to 70 diabetic subjects without diabetic retinopathy. Additionally, 76 subjects with proliferative diabetic retinopathy (a subgroup of diabetic retinopathy) were compared to 70 diabetic subjects without diabetic retinopathy. RESULTS: The - 429 T/C and the - 374 T/A RAGE gene distributions in patients with diabetic retinopathy (- 429 T/C: CC 0.9%, TC 25.9%, TT 73.2%; - 374 T/A: AA 9.5%, TA 47.4%, TT 43.1%) were not significantly different from those of diabetic subjects without retinopathy (- 429 T/C: CC 0%, TC 25.7%, TT 74.3%; - 374 T/A: AA 15.7%, TA 42.9%, TT 41.4%). Moreover, the - 429 T/C and the - 374 T/A RAGE gene distributions in patients with proliferative diabetic retinopathy were not statistically significantly different from those in diabetic subjects without retinopathy. CONCLUSION: Our study failed to demonstrate an association between either - 429 T/C or - 374 T/A gene polymorphism of the RAGE gene and diabetic retinopathy in Caucasians with type 2 diabetes. Additionally, we failed to demonstrate an association between either - 429 T/C or - 374 T/A gene polymorphism of the RAGE gene and proliferative diabetic retinopathy in Caucasians with type 2 diabetes.     

High D-dimer levels predict cardiovascular events in patients with chronic atrial fibrillation during oral anticoagulant therapy

Atrial fibrillation (AF) is associated with hemostatic abnormalities and increased risk of thrombotic cardiovascular events even during oral anticoagulant therapy (OAT). The aim of our study was to evaluate the predictive value of hemostatic markers for the risk of major cardiovascular events during OAT. The study group comprised 113 patients with chronic AF (70.2 +/- 5.4 years old, 60% men), referred for OAT. Established clinical risk factors and levels of prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complexes (TAT), D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) antigen and activity, before and during OAT (after 3.9 +/- 0.7 months; INR 2.57 +/- 0.57) were determined. In all patients OAT significantly suppressed levels of F1+2 by 67%,TAT by 30% and D-dimer by 48% (all p <0.001). During an average follow-up of 44 months 22/111 (20%) patients suffered a combined cardiovascular event (stroke, myocardial infarction, peripheral vascular occlusion or vascular death). Patients with cardiovascular events were significantly older, had more frequent heart failure/systolic dysfunction and had significantly increased levels of D-dimer at entry (63 vs 39 ng/mL, p = 0.005) and during OAT (33 vs 18 ng/mL, p = 0.002), and of t-PA antigen at entry (14.3 vs 10.9 ng/mL, p = 0.02) and during OAT (15.0 vs 11.2 ng/mL, p = 0.05) (all values are medians). In multivariate Cox proportional hazard models, heart failure/systolic dysfunction (hazard ratio 2.91; 95% CI 1.17-7.26; p = 0.02), high levels of D-dimer on OAT (top vs. lower two quartiles) (hazard ratio 4.78, 95% CI 1.39-16.41; p = 0.01) and t-PA antigen levels (continuous variable) (hazard ratio 1.09; 95% CI 1.01-1.17; p = 0.02) were significantly associated with combined cardiovascular events. In conclusion, high levels of D-dimer and t-PA antigen during OAT are significant predictors of combined cardiovascular events in AF patients and, on this basis, could be useful additional markers of cardiovascular risk in such patients.     

Validation of an automated immunoturbidimetric assay for measurement of plasma D-dimer.

The performance characteristics and diagnostic accuracy of a new rapid automated quantitative immunoturbidimetric D-dimer assay, Auto-Dimer (Biopool, Ume?, Sweden) were evaluated in a population of 135 outpatients with suspected deep-vein thrombosis of a lower limb. Enzyme-linked immunosorbent assay was used as the reference method. The Auto-Dimer assay showed good reproducibility. The correlation between Auto-Dimer and enzyme-linked immunosorbent assay was high (r = 0.91, p < 0.05) and agreement in classifying patients above or below the cut-off was good (kappa coefficient 0.74, 95% CI 0.62-0.86). At a cut-off of 340 microg/l the sensitivity and specificity of Auto-Dimer were high (100% and 61%, respectively). These data show that Auto-Dimer is a reliable screening test for exclusion of deep-vein thrombosis. The assay could be included in prospective patient management studies in order to obtain further information on its clinical usefulness.     

Normothermic and hypothermic models for studying the deleterious effects of hypoxia-reoxygenation on EDHF-mediated relaxation in isolated porcine coronary arteries

IntroductionThe vasomotor response of the coronary artery is altered by hypoxia–reoxygenation (H–R) induced damage. The aim of our study was to compare and evaluate normothermic and hypothermic models which are suitable for future drug studies of vasoprotective action against H–R injury.MethodsPorcine coronary arterial rings were isolated and placed in Krebs–Henseleit (K–H) solution. Rings were exposed to normoxic conditions (control group) and two different H–R conditions: the first induced by a 95% N₂–5% CO₂ gas mixture (40- and 60-min hypoxia) in a normothermic protocol, and the second induced by hypothermic (4 °C) hypoxia–reoxygenation in an air-tight beaker filled with K–H solution (24- and 48-hours hypoxia). Reoxygenation was applied by introducing K–H solution aerated with a 95% O₂–5% CO₂ mixture under normothermic (37 °C) conditions. To test the EDHF-mediated relaxation by substance P, rings were first incubated in L-NNA, nitric oxide synthase inhibitor, and indomethacin, cyclooxygenase inhibitor, and then pre-contracted with thromboxane analogue U-46619. Analysis of the maximum relaxation of the arterial rings was performed by one-way ANOVA, followed by Bonferroni's post-test.ResultsDistal segments of the coronary artery responded faster to contraction induced by U-46619 and were relaxed by substance P to a greater extent than proximal segments. Maximal relaxations of arterial rings induced by a 10 nM solution of substance P were significantly reduced (p < 0.001) from the values for normoxic rings (81.0 ± 1.0%, n = 30) after 40-min H–R (50.5 ± 5.3%, n = 30), 60-min H–R (32.1 ± 3.5%, n = 30), 24-hours hypothermic H–R (56.0 ± 2.3%, n = 30) and after 48-hours hypothermic H–R (38.5 ± 5.1%, n = 30).ConclusionsThe model employing 40-min normothermic H–R is as effective as 24-hours hypothermic H–R, and 60-min normothermic H–R as 48-hours hypothermic H–R for studying the deleterious effects of H–R on EDHF-mediated relaxation.