High density culturing of porcine hepatocytes immobilized on nonwoven polyurethane-based biomatrices

Objective: Hepatocytes are increasingly used as functional units in bioartificial liver devices. The objective of the present study was to investigate the feasibility of culturing porcine hepatocytes in high density on a novel polyurethane-based nonwoven three-dimensional matrix. We investigated (1) the optimal cell density within this culture configuration, (2) the maintenance of liver-specific morphology and cell functions over long-term periods and (3) the necessity to apply an additional extracellular matrix component (collagen gel). Methods: Nonwoven polyurethane matrices were manufactured by a specially developed fiber extrusion technology. Pig hepatocytes were cultured at various cell densities of 0.1, 0.25, 0.5, 0.75, 1 and 2 x 10(6) cells/cm(2) on three-dimensional networks of nonwoven polyurethane matrices and cell adhesion as well as functional parameters (DNA of nonattached/attached cells, lactate dehydrogenase release and cytochrome P450 activity) were determined. To assess the performance of cells within this configuration albumin and urea excretion was measured over 8 days. The potentially beneficial effect of an additional extracellular matrix configuration was evaluated by comparing the average albumin synthesis in groups of identical cell numbers. Results: The optimal cell density in this three-dimensional culture configuration was 1 x 10(6) cells/cm(2). The functional capacity of hepatocytes was stable for 8 days at an average level of 53.7 +/- 5.6 ng/h/mug DNA and of 1.8 +/- 0.14 mug/h/mug DNA for albumin and urea excretion, respectively. The supplementation of an extracellular matrix configuration did not improve functional activity of cells. Average albumin synthesis was 35.6 ng/h/mug DNA (28.7, 42.8) and 32.7 ng/h/mug DNA (23.4, 49.2) for collagen-immobilized and control cultures, respectively, Conclusion: The results of the study indicate that nonwoven polyurethane sheets supply a biocompatible support structure for functionally active high density cultures. Thus, nonwoven polyurethane matrices should be further investigated on with respect to their role in the development, optimization and design of bioartificial liver systems. Copyright (C) 2001 S.Karger AG, Basel

Sleep and wake affect glycogen content and turnover at perisynaptic astrocytic processes

Astrocytic glycogen represents the only form of glucose storage in the brain, and one of the outcomes of its breakdown is the production of lactate that can be used by neurons as an alternative energetic substrate. Since brain metabolism is higher in wake than in sleep, it was hypothesized that glycogen stores are depleted during wake and replenished during sleep. Furthermore, it was proposed that glycogen depletion leads to the progressive increase in adenosine levels during wake, providing a homeostatic signal that reflects the buildup of sleep pressure. However, previous studies that measured glycogen dynamics across the sleep/wake cycle obtained inconsistent results, and only measured glycogen in whole tissue. Since most energy in the brain is used to sustain synaptic activity, here we employed tridimensional electron microscopy to quantify glycogen content in the astrocytic processes surrounding the synapse. We studied axon-spine synapses in the frontal cortex of young mice after ~7 h of sleep, 7–8 h of spontaneous or forced wake, or 4.5 days of sleep restriction. Relative to sleep, all wake conditions increased the number of glycogen granules around the synapses to a similar extent. However, progressively longer periods of wake were associated with progressively smaller glycogen granules, suggesting increased turnover. Despite the increased number of granules, in all wake conditions the estimated amount of glucose within the granules was lower than in sleep, indicating that sleep may favor glucose storage. Finally, chronic sleep restriction moved glycogen granules closer to the synaptic cleft. Thus, both short and long wake lead to increased glycogen turnover around cortical synapses, whereas sleep promotes glycogen accumulation

Pixel and Voxel Representations of Graphs

We study contact representations for graphs, which we call pixel representations in 2D and voxel representations in 3D. Our representations are based on the unit square grid whose cells we call pixels in 2D and voxels in 3D. Two pixels are adjacent if they share an edge, two voxels if they share a face. We call a connected set of pixels or voxels a blob. Given a graph, we represent its vertices by disjoint blobs such that two blobs contain adjacent pixels or voxels if and only if the corresponding vertices are adjacent. We are interested in the size of a representation, which is the number of pixels or voxels it consists of. We first show that finding minimum-size representations is NP-complete. Then, we bound representation sizes needed for certain graph classes. In 2D, we show that, for $k$-outerplanar graphs with $n$ vertices, $\Theta(kn)$ pixels are always sufficient and sometimes necessary. In particular, outerplanar graphs can be represented with a linear number of pixels, whereas general planar graphs sometimes need a quadratic number. In 3D, $\Theta(n^2)$ voxels are always sufficient and sometimes necessary for any $n$-vertex graph. We improve this bound to $\Theta(n\cdot \tau)$ for graphs of treewidth $\tau$ and to $O((g+1)^2n\log^2n)$ for graphs of genus $g$. In particular, planar graphs admit representations with $O(n\log^2n)$ voxels

Desingularization of vortices for the Euler equation

We study the existence of stationary classical solutions of the incompressible Euler equation in the plane that approximate singular stationnary solutions of this equation. The construction is performed by studying the asymptotics of equation -\eps^2 \Delta u^\eps=(u^\eps-q-\frac{\kappa}{2\pi} \log \frac{1}{\eps})_+^p with Dirichlet boundary conditions and $q$ a given function. We also study the desingularization of pairs of vortices by minimal energy nodal solutions and the desingularization of rotating vortices.Comment: 40 page

Geometry of Polynomials and Root-Finding via Path-Lifting

Using the interplay between topological, combinatorial, and geometric properties of polynomials and analytic results (primarily the covering structure and distortion estimates), we analyze a path-lifting method for finding approximate zeros, similar to those studied by Smale, Shub, Kim, and others. Given any polynomial, this simple algorithm always converges to a root, except on a finite set of initial points lying on a circle of a given radius. Specifically, the algorithm we analyze consists of iterating $$z - \frac{f(z)-t_kf(z_0)}{f'(z)}$$ where the $t_k$ form a decreasing sequence of real numbers and $z_0$ is chosen on a circle containing all the roots. We show that the number of iterates required to locate an approximate zero of a polynomial $f$ depends only on $\log|f(z_0)/\rho_\zeta|$ (where $\rho_\zeta$ is the radius of convergence of the branch of $f^{-1}$ taking $0$ to a root $\zeta$) and the logarithm of the angle between $f(z_0)$ and certain critical values. Previous complexity results for related algorithms depend linearly on the reciprocals of these angles. Note that the complexity of the algorithm does not depend directly on the degree of $f$, but only on the geometry of the critical values. Furthermore, for any polynomial $f$ with distinct roots, the average number of steps required over all starting points taken on a circle containing all the roots is bounded by a constant times the average of $\log(1/\rho_\zeta)$. The average of $\log(1/\rho_\zeta)$ over all polynomials $f$ with $d$ roots in the unit disk is ${\mathcal{O}}({d})$. This algorithm readily generalizes to finding all roots of a polynomial (without deflation); doing so increases the complexity by a factor of at most $d$.Comment: 44 pages, 12 figure

Hepatic levels of bile acids in end-stage chronic cholestatic liver disease

In chronic cholestatic liver disease hydrophobic and potentially cytotoxic bile acids are assumed to accumulate in the liver. To test this hypothesis we investigated bile acid levels and pattern in livers and serum of patients with, (A) end-stage chronic cholestatic liver disease, and with (B) end-stage cirrhosis of alcoholic/chronic hepatitic origin who underwent liver transplantation. Bile acids were also analyzed in (C) normal liver tissue. Levels of bile acids were 215 +/- 39.1 nmol/g liver (wet weight) in chronic cholestasis and 120 +/- 32.7 and 56.1 +/- 24.2 nmol/g liver in group B and group C (P <0.01 and P <0.005), respectively. Cholic acid was the prevailing bile acid in chronic cholestasis (51%) and was elevated eight-fold as compared to group C (P <0.005). Chenodeoxycholic acid contributed 41% to total bile acids and was elevated four-fold (P <0.005). Deoxycholic acid contributed only 1.5% to bile acids in chronic cholestasis as compared to 27% in group C (P <0.01) and was absent in group B. Levels of lithocholic acid tended to be increased in chronic cholestasis as compared to group C and its sulfation was impaired (P <0.05). The pattern of serum bile acids in chronic cholestasis agreed well with the bile acid pattern in the explanted livers. We conclude that hepatic accumulation of hydrophobic chenodeoxycholic acid and impaired sulfation of lithocholic acid might contribute to tissue degeneration in chronic cholestatic liver disease due to the detergent effects of these bile acid