Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C

In Silico Screening Based on Predictive Algorithms as a Design Tool for Exon Skipping Oligonucleotides in Duchenne Muscular Dystrophy

The use of antisense 'splice-switching' oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2'O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R² 0.89) and 53 (R² 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each position of a target exon

An adaptive P1 finite element method for two-dimensional Maxwell\u27s equations

Recently a new numerical approach for two-dimensional Maxwell\u27s equations based on the Hodge decomposition for divergence-free vector fields was introduced by Brenner et al. In this paper we present an adaptive P finite element method for two-dimensional Maxwell\u27s equations that is based on this new approach. The reliability and efficiency of a posteriori error estimators based on the residual and the dual weighted-residual are verified numerically. The performance of the new approach is shown to be competitive with the lowest order edge element of Nédélec\u27s first family. © Springer Science+Business Media New York 2012.

An Adaptive P1 Finite Element Method for Two-Dimensional Transverse Magnetic Time Harmonic Maxwell’s Equations with General Material Properties and General Boundary Conditions

We present an adaptive P finite element method for two-dimensional transverse magnetic time harmonic Maxwell’s equations with general material properties and general boundary conditions. It is based on reducing the boundary value problems for Maxwell’s equations to standard second order scalar elliptic problems through the Hodge decomposition. We allow inhomogeneous and anisotropic electric permittivity, sign changing magnetic permeability, and both the perfectly conducting boundary condition and the impedance boundary condition. The optimal convergence of the adaptive finite element method is demonstrated by numerical experiments. We also present results for a semiconductor simulation, a cloaking simulation and a flat lens simulation that illustrate the robustness of the method.

Towards characterization of palmoplantar keratoderma caused by gain-of-function mutation in loricrin: analysis of a family and review of the literature

Loricrin keratoderma is an autosomal dominant palmoplantar keratoderma heterogeneous in clinical appearance. We report a family with diffuse ichthyosis and honeycomb palmoplantar keratoderma but no occurrence of pseudoainhums or autoamputations. All patients were born as collodion babies and displayed prominent knuckle pads. We identified the previously reported mutation 730insG in LOR, which elongates loricrin by 22 amino acids because of delayed termination. As pseudoainhums are missing in all patients of the family reported, we propose two compulsory features of loricrin keratoderma: (i) honeycomb palmoplantar keratoderma and (ii) diffuse ichthyosiform dermatosis. Therefore we suggest that the condition should be described clinically as ‘honeycomb palmoplantar keratoderma with ichthyosis’. Furthermore, we have assessed the amounts of transcript of LOR using pyrosequencing. This revealed an equal expression of mutant and wild-type alleles of LOR in an affected individual. These findings further underline the gain-of-function theory for mutant LOR in loricrin keratoderma

Application of an eremomycin chiral stationary phase for the separation of DL-methionine using simulated moving bed chromatography

Preparative chromatography has become an accepted tool in pharmaceutical industry for the production of enantiopure drugs and intermediates. An important reason is the still increasing availability of efficient chiral stationary phases (CSP). Recently, a new CSP of the class of macrocyclic glycopeptides was introduced, based on the immobilization of eremomycin to epoxy-activated silica1. This new phase has been shown to have a good selectivity with regard to amino acids in the analytical concentration range2. The application of this new CSP to preparative enantioseparation using simulated moving bed chromatography (SMB) will be presented. For preparative enantioseparations, besides selectivity, the long term stability of stationary phases is of importance. A critical test for the stability is the application in an SMB-unit, where the stationary phase is subject to high sample concentrations. The separation of the enantiomers of methionine is used as a model system. The mobile phase consists of 20/80v/v Methanol/Water (0.1M NaH2PO4). Competitive adsorption isotherms of D- and L-methionine were measured up to the solubility limit in the mobile phase by means of a perturbation method. Extensive perturbation experiments were performed, in order to test column stability and column to column reproducibility. The obtained adsorption isotherms were validated with breakthrough and overloaded elution experiments. In order to test the stability of the stationary phase small injections were performed before and after the perturbation experiments. 8 columns (0.8x6cm) were used for long term simulated moving bed experiments in a lab scale Prochrom SMB unit (Novasep, France). On the poster the results of the determination of the adsorption isotherms, the evaluation of the stability of the columns and the performed SMB-runs will be presented and discussed. 1 S.M. Staroverov, M.A. Kuznetsov, P.N. Nesterenko, G.G. Vasiarov, G.S. Katrukha, G.B. Fedorova, J. Chromatogr. A 1108 (2006) 263–267 2 K. Petrusevska, M.A. Kuznetsov, K. Gedicke, V. Meshko, S.M. Staroverov, A. Seidel-Morgenstern, J. Sep. Science, accepte

Application of an eremomycin-chiral stationary phase for the separation of DL-methionine using simulated moving bed technology

Recently a new chiral stationary phase (CSP) was introduced, based on the immobilization of the macrocyclic glycopeptide eremomycin to epoxy-activated silica. The application of this new CSP to preparative enantioseparation using simulated moving bed chromatography (SMB) will be presented. In such delicate continuous separation processes, besides selectivity, the long-term stability of the applied stationary phases is of importance. Column to column fluctuations of the bed homogeneity and long-term stability of the preparative stationary phase was satisfactory according to the results of perturbation experiments performed before and after long-term SMB runs. Copyright © 2007 Elsevier B.V. All rights reserved. [accessed 2013 November 26th

Chromatographic enantioseparation of amino acids using a new chiral stationary phase based on a macrocyclic glycopeptide antibiotic

The separation of the enantiomers of several α-amino acids was studied on a new chiral stationary phase (CSP) which is based on the macrocyclic glycopeptide antibiotic eremomycin attached to silica particles. Retention and separation factors were determined under analytical conditions at ambient temperature for different mobile phase compositions. In order to evaluate the potential with respect to preparative separations the adsorption isotherms of D- and L-methionine were determined for one mobile phase composition applying the elution by characteristic point method. The isotherms were validated by comparing experimentally determined elution profiles with predictions based on the equilibrium dispersive model. Finally, the performance of the eremomycin CSP was compared with a commercially available CSP based on the macrocyclic antibiotic teicoplanin. After determining the isotherms of D- and L-methionine also for the teicoplanin phase, the equilibrium dispersive model was used for both CSP to identify optimal operating conditions. For the separation and conditions considered the new eremomycin CSP revealed a better performance compared to the teicoplanin CSP. Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim [accessed February 8th 2013

Enantioseparation of racemic mixtures of amino acids using high performance liquid chromatography

According to the fact that the need of pure enantiomers in pharmaceutical industry, food industry, cosmetics and agrochemical industry is in tremendous increase, different separation techniques have been under investigation for production of pure enantiomers on a commercial scale. In the last years attention is focused on the development of new highly selective chiral stationary phases (CSP's) used as packing material in chromatographic columns. This work, which deals with the separation of racemic mixtures of amino acids using High Performance Liquid Chromatography (HPLC), can be divided in two parts. The separation of the following α-amino acids: DL-Methionine, DL-Asparagine, DL-Leucine, DL-Serine, DL-Glutamine and DL-Valine was investigated experimentally. Three chiral CSP's were applied: Chirobiotic T (macrocyclic antibiotic-Teicoplanin CSP, Astec, USA) Diaspher- Chirasel-E (macrocyclic antibiotic-scientific sorbent Eremomycin CSP, BioChemMack S&T, Russia) and Chirex (ligand exchange-Cu CSP, Phenomenex, USA). Temperature and flow rate were kept constant, only the composition of the mobile phase was changed. By taking into consideration the solubility of the amino acids, the cost of the solvent and the time needed for the separation the following systems were chosen for further work: Chirobiotic T column (DL-Methionine, mobile phase: EtOH:H2O=40:60v/v and DL-Asparagine, mobile phase: EtOH:H2O=60:40v/v); Diaspher-Chirasel-E column (DL-Methionine, mobile phase: MeOH:H2O (0,1M NaH2PO4)=20:80v/v). To estimate adsorption equilibria, the ECP method [1] was applied for determination of the singleenantiomer adsorption isotherms. Langmuir and bi-Langmuir isotherm model parameters were estimated. With the determined isotherms, simulation of the elution profiles was performed using the equilibrium-dispersive model[1]. The last goal of this work was the estimation of the production of D and L Methionine enantiomers (purity of 99%) and comparison between Chirobiotic T and Diaspher-Chirasel-E. Results obtained by this work form a good base for further intentions, like the application of these columns in a Simulated Moving Bed [1] plant. References: [1[ G. Guiochon, S.G. Shirazi, A.M. Katti, Fundamentals of Preparative and Nonlinear Chromatography, Academic Press, New York, 199