Device Thrombogenicity Emulator (DTE) − Design optimization methodology for cardiovascular devices: A study in two bileaflet MHV designs

Patients who receive prosthetic heart valve (PHV) implants require mandatory anticoagulation medication after implantation due to the thrombogenic potential of the valve. Optimization of PHV designs may facilitate reduction of flow-induced thrombogenicity and reduce or eliminate the need for post-implant anticoagulants. We present a methodology entitled Device Thrombogenicty Emulator (DTE) for optimizing the thrombo-resistance performance of PHV by combining numerical and experimental approaches. Two bileaflet mechanical heart valves (MHV) designs, St. Jude Medical (SJM) and ATS, were investigated by studying the effect of distinct flow phases on platelet activation. Transient turbulent and direct numerical simulations (DNS) were conducted, and stress loading histories experienced by the platelets were calculated along flow trajectories. The numerical simulations indicated distinct design dependent differences between the two valves. The stress loading waveforms extracted from the numerical simulations were programmed into a hemodynamic shearing device (HSD), emulating the flow conditions past the valves in distinct ‘hot-spot’ flow regions that are implicated in MHV thrombogenicity. The resultant platelet activity was measured with a modified prothrombinase assay, and was found to be significantly higher in the SJM valve, mostly during the regurgitation phase. The experimental results were in excellent agreement with the calculated platelet activation potential. This establishes the utility of the DTE methodology for serving as a test bed for evaluating design modifications for achieving better thrombogenic performance for such devices.     

Automated analysis of FISH and immunohistochemistry images: a review.

Fluorescent in-situ hybridization (FISH) and immunohistochemistry (IHC) constitute a pair of complimentary techniques for detecting gene amplification and overexpression, respectively. The advantages of IHC include relatively cheap materials and high sample durability, while FISH is the more accurate and reproducible method. Evaluation of FISH and IHC images is still largely performed manually, with automated or semiautomated techniques increasing in popularity. Here, we provide a comprehensive review of a number of (semi-) automated FISH and IHC image processing systems, focusing on the algorithmic aspects of each technique. Our review verifies the increasingly important role of such methods in FISH and IHC; however, manual intervention is still necessary in order to resolve particularly challenging or ambiguous cases. In addition, large-scale validation is required in order for these systems to enter standard clinical practice.     

Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors

A c-Myc epitope-tagged N-methyl-D-aspartate receptor NR1-2a subunit was generated, NR1-2a(c-Myc), where the tag was inserted after amino acid 81. NR1-2a(c-Myc) /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2a(c-Myc) was, however, shown to assemble with NR2A subunits by immunoprecipitation and [(3)H]MK801 radioligand binding assays. Immunoblots of cells co-transfected with wild-type NR1-2a/NR2A subunits yielded two NR1-2a immunoreactive species with molecular masses of 115 and 226 kDa. Two-dimensional electrophoresis under non-reducing and reducing conditions revealed that the 226-kDa band contained disulfide-linked NR1-2a subunits. Only the 115-kDa NR1-2a species was detected for NR1-2a(c-Myc)/NR2A. The c-Myc epitope is inserted adjacent to cysteine 79 of the NR1-2a subunit; therefore, it is possible that the tag may prevent the formation of NR1 disulfide bridges. A series of cysteine --> alanine NR1-2a mutants was generated, and the NR1-2a mutants were co-expressed with NR2A or NR2B subunits in mammalian cells and characterized with respect to cell surface expression, cell cytotoxicity post-transfection, co-association by immunoprecipitation, and immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. When co-expressed with NR2A in mammalian cells, NR1-2a(C79A)/NR2A displayed similar properties to NR1-2a(c-Myc)/NR2A in that the 226-kDa NR1 immunoreactive species was not detectable, and trafficking to the cell surface was impaired compared with wild-type NR1/NR2 receptors. These results provide the first biochemical evidence for the formation of NR1-NR1 intersubunit disulfide-linked homodimers involving cysteine 79. They suggest that disulfide bridging and structural integrity within the NR1 N-terminal domain is requisite for cell surface N-methyl-D-aspartate receptor expression.     

Mapping the binding domains of the alpha(IIb) subunit. A study performed on the activated form of the platelet integrin alpha(IIb)beta(3).

alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.     

Anharmonic analysis of arterial blood pressure and flow pulses

Fourier analysis is usually employed for the computation of blood flow in arteries. Although the orthogonality of Fourier eigenfunctions guarantees the accurate mathematical modeling of the blood pressure and flow waveforms, the physics behind this objective function is frequently missing. We propose a new method to account for the blood pressure and flow, single-cycle (systole-diastole) waveforms. It is based on the one dimensional hydrodynamic mass and momentum conservation equations for viscous flow. The similarity of the linear problem, under discussion, with related transmission line theory in electromagnetic wave propagation, permits expansion in anharmonic, non-separable eigenfunctions. In some cases one term in the expansion is adequate to fit the main peak of the observed waveforms. Analytical formulas are derived for the dependence of the pressure and flow main peaks on whole blood viscosity and distance from the heart, which interpret observations related to hypertension.     

How Variability and Effort Determine Coordination at Large Forces

Motor control is a challenging task for the central nervous system, since it involves redundant degrees of freedom, nonlinear dynamics of actuators and limbs, as well as noise. When an action is carried out, which factors does your nervous system consider to determine the appropriate set of muscle forces between redundant degrees-of-freedom? Important factors determining motor output likely encompass effort and the resulting motor noise. However, the tasks used in many previous motor control studies could not identify these two factors uniquely, as signal-dependent noise monotonically increases as a function of the effort. To address this, a recent paper introduced a force control paradigm involving one finger in each hand that can disambiguate these two factors. It showed that the central nervous system considers both force noise and amplitude, with a larger weight on the absolute force and lower weights on both noise and normalized force. While these results are valid for the relatively low force range considered in that paper, the magnitude of the force shared between the fingers for large forces is not known. This paper investigates this question experimentally, and develops an appropriate Markov chain Monte Carlo method in order to estimate the weightings given to these factors. Our results demonstrate that the force sharing strongly depends on the force level required, so that for higher force levels the normalized force is considered as much as the absolute force, whereas the role of noise minimization becomes negligible.     

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.     

Epistatic Role of the MYH9/APOL1 Region on Familial Hematuria Genes

Familial hematuria (FH) is explained by at least four different genes (see below). About 50% of patients develop late proteinuria and chronic kidney disease (CKD). We hypothesized that MYH9/APOL1, two closely linked genes associated with CKD, may be associated with adverse progression in FH. Our study included 102 thin basement membrane nephropathy (TBMN) patients with three known COL4A3/COL4A4 mutations (cohort A), 83 CFHR5/C3 glomerulopathy patients (cohort B) with a single CFHR5 mutation and 15 Alport syndrome patients (cohort C) with two known COL4A5 mild mutations, who were categorized as “Mild” (controls) or “Severe” (cases), based on renal manifestations. E1 and S1 MYH9 haplotypes and variant rs11089788 were analyzed for association with disease phenotype. Evidence for association with “Severe” progression in CFHR5 nephropathy was found with MYH9 variant rs11089788 and was confirmed in an independent FH cohort, D (cumulative p value = 0.001, odds ratio = 3.06, recessive model). No association was found with APOL1 gene. Quantitative Real time PCR did not reveal any functional significance for the rs11089788 risk allele. Our results derive additional evidence supporting previous reports according to which MYH9 is an important gene per se, predisposing to CKD, suggesting its usefulness as a prognostic marker for young hematuric patients.     

Spatio-temporal dynamics and management implications of the nightly appearance of Boops boops (Acanthopterygii,Perciformes) juvenile shoals in the anthropogenically modified Mediterranean littoral zone

A remarkable phenomenon of dense Boops boops shoals appearing almost adjacent to the shoreline during nighttime is known to the locals of island communities of the Aegean Sea (eastern Mediterranean). In this work, we investigated this appearance testing the hypotheses that (a) it may occur only in anthropogenically modified locations (as suggested by previous observations), (b) the migration pattern to the littoral is not arbitrary but synchronized to the sunset/sunrise, (c) fish abundance is affected by location, season and/or natural (moon) light fluctuations. Quantitative sampling included visual observations from the coast at five stations in Syros (Cyclades, Greece) from July 2009 to September 2010. Both hypotheses concerning occurrence only in anthropogenically modified locations and timing with sunset/sunrise were confirmed. Fish abundance was modelled using generalized additive models, demonstrating a seasonal pattern and revealing significant differences among sampling stations, but no moon-phase effects. The phenomenon investigated here has implications for fisheries management, as the shoal proximity to the shore renders them prone to illegal harvesting (seasonally at high abundances), aggravating the problem of illegal, unreported and unregulated fishing. Further considerations on the integrated management of the coastal zone arise, especially concerning the effects of habitat structural modification and light pollution.