17,004 research outputs found
Glycan-analysis in influenza vaccine production based on cgelif: influence of host cell and virus on the glycosylationpattern of viral hemagglutinin
No full textMammalian cell culture processes, are commonly used for production of recombinant glycoproteins, antibodies and viral vaccines. Since several years there is an increasing interest in cell culture-based influenza vaccine production to overcome limitations of egg-based production systems, to improve vaccine supply and to increase flexibility in vaccine manufacturing. With the switch of the production system several key questions concerning the possible impact of host cell lines on antigen quality, passage-dependent selection of certain viral phenotypes or changes in hemagglutinin (HA) conformation have to be addressed to guarantee safety and efficiency of vaccines. Within this study, a capillary DNA-sequencer (based on CGE-LIF technology: capillary-gelelectrophoresis coupled online to laser-induced fluorescence detection) was utilized [1] for Nglycan analysis of different influenza virus strains, replicated in different mammalian cell lines. Detailed results concerning the influence of the host cell line on complexity and composition of the HA N-glycosylation pattern, are presented. Besides a strong host cell dependence of HA N-glycosylation that could be shown, a significant change in N-glycan type attached to HA was observed, comparing different virus types and subtypes [2]. [1] Schwarzer, J.; Rapp, E.; Reichl, U. Electrophoresis, 2008, 29, 4203-4214. [2] Schwarzer, J.; Rapp, E.; Hennig, R.; Genzel, Y.; Reichl U. Vaccine, 2008, submitted
Towards High Throughput Characterization of Glycosylation-Patterns Using a Capillary-DNA-Sequencer
No full textWithin this work, the utilization of a capillary DNA-sequencer for N-glycan analysis [1] is presented. With this highly sensitive nano-separator, based on capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF), the N-glycans can be analyzed in two stages: first - comparing “glycan-pool fingerprints” and second - structural elucidation via database matching. The developed procedure allows monitoring and elucidation of N-glycosylation patterns of relevant glycoproteins. Besides workflow and proof of principle, suitability and potential of the method are depicted. The methods performance is exemplarily shown via elucidation of glycosylation patterns of pharmaceutical relevant glycoproteins (e.g. interferons, immunoglobulins, …). In more detail, feasibility and performance of this method is demonstrated exemplarily for hemagglutinin (HA), an important glycoprotein of the influenza virus membrane. The HA glycosylation patterns of different influenza virus strains, replicated in different mammalian cell lines for vaccine production were elucidated. Results concerning the influence of the host cell line on complexity and composition of the HA N-glycosylation pattern, are presented. Besides a strong host cell dependence of HA N-glycosylation that could be shown, a significant change in N-glycan type attached to HA was observed, comparing different virus types and subtypes [2]. [1] Schwarzer, J.; Rapp*, E.; Reichl, U. Electrophoresis, 2008, 29, 4203-4214. [2] Schwarzer, J.; Rapp*, E.; Hennig, R.; Genzel, Y.; Jordan, I.; Sandig, V.; Reichl U. Vaccine, 2009, in press
In vitro proliferation of human large granular lymphocytes with v-raf/v-myc recombinant retrovirus.
No full textThe effect of infection with a retrovirus carrying v-raf/v-myc oncogenes (J2 virus) on the in vitro proliferation of human large granular lymphocytes (LGL) was investigated. LGL infected with J2 virus (J2LGL), unlike uninfected cells, grew with a proliferation peak eight days after infection. Such cells retained the morphology and functional properties typical of LGL. Furthermore, 5% of J2LGL produced virus the day after infection, whereas non-virus production was detectable five days later. These data indicate that J2 virus provides a transient mitogenic signal for LGL
Real-space Manifestations of Bottlenecks in Turbulence Spectra
No full textAn energy-spectrum bottleneck, a bump in the turbulence spectrum between the inertial and dissipation ranges, is shown to occur in the non-turbulent, one-dimensional, hyperviscous Burgers equation and found to be the Fourier-space signature of oscillations in the real-space velocity, which are explained by boundary-layer-expansion techniques. Pseudospectral simulations are used to show that such oscillations occur in velocity correlation functions in one- and three-dimensional hyperviscous hydrodynamical equations that display genuine turbulence
Reynolds number dependence of the dimensionless dissipation rate in stationary magnetohydrodynamic turbulence
No full textResults on the Reynolds number dependence of the dimensionless total dissipation rate C_ε are presented, obtained from medium to high resolution direct numerical simulations (DNSs) of mechanically forced stationary homogeneous magnetohydrodynamic (MHD) turbulence in the absence of a mean magnetic field, showing that C_ε -> const with increasing Reynolds number. Furthermore, a model equation for the Reynolds number dependence of the dimensionless dissipation rate is derived from the real-space energy balance equation by asymptotic expansion in terms of Reynolds number of the second- and third-order correlation functions of the Elsässer fields z± = u ± b. At large Reynolds numbers we find that a model of the form C_ε = C_ε,∞ + C/R describes the data well, while at lower Reynolds numbers the model needs to be extended to second order in 1/R in order to obtain a good fit to the data, where R is a generalised Reynolds number with respect to the Elsässer field z-
Universal Statistical Properties of Inertial-particle Trajectories in Three-dimensional, Homogeneous, Isotropic, Fluid Turbulence
No full textWe obtain new universal statistical properties of heavy-particle trajectories in three-dimensional, statistically steady, homogeneous, and isotropic turbulent flows by direct numerical simulations. We show that the probability distribution functions (PDFs) P(Φ), of the angle Φ between the Eulerian velocity u and the particle velocity v, at a point and time, scales as P(Φ) ∼Φ−, with a new universal exponent ≃ 4
Turbulent stratified shear flow experiments: Length scale comparison
No full textStratified shear flows are ubiquitous in geophysical systems such as oceanic overflows, wind-driven thermoclines, and atmo- spheric inversion layers. The stability of such flows is governed by the Richardson Number Ri which represents a balance between the stabilizing influence of stratification and the destabilizing influence of shear. For a shear flow with velocity difference U, density difference ∆ρ and characteristic length H, one has Ri = g(∆ρ/ρ)H/U^2 which is often used when detailed information about the flow is not available. A more precise definition is the gradient Richardson Number Rig = N^2/S^2 where the buoyancy frequency N = ((g/ρ)∂ρ/∂z)^{1/2}, the mean strain S = ∂U/∂z in which z is parallel to gravity and suitable ensemble or time averages define the gradients. We explore the stability and mixing properties of a wall-bounded shear flow over a range 0.1< Rig <1 using simultaneous planar measurements of density and velocity fields using Planar Laser-Induced Fluorescence (PLIF) and Particle Image Velocimetry (PIV), respectively. The flow, confined from the top by glass horizontal boundary, is a lighter alcohol-water mixture injected from a nozzle into quiescent heavier salt-water fluid with velocity between 5 and 10 cm/s and with a relative fractional density difference of 0.0026 or 0.0052. The injected flow is turbulent with Taylor Reynolds number between 50 and 100. We compare a set of length scales that characterize the mixing properties of our turbulent stratified shear flow including the Thorpe Length L_T, the Ozmidov Length L_o, the Ellison Length L_E, and turbulent mixing lengths L_m and L_ρ
A software tool for automated high-throughput processing of CGE-LIF based glycoanalysis data, generated by a multiplexing capillary DNA sequencer
No full textGlycomics is a rapidly emerging field that can be viewed as a continuation of the genomic and proteomic era. Hence, there is a dramatic dynamic increase in the demand for sophisticated databases and analytical tools in glycobiology respectively glycobiotechnology. In order to enhance and improve the comparatively small existing glycoanalytical toolbox, a fully automated, high-throughput (HTP) and high-resolution (HR) analysis method, with a fully automated data evaluation, is required. Besides several mass spectrometry and liquid chromatography based analysis techniques, electromigrative separation techniques for the analysis of oligosaccharides became apparent during the recent past. Especially capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF) as one feasible electromigrative separation technique - using standard DNA sequencer equipment - has been developed for HTP glycoprofiling of APTS-labeled glycans [1,2]. The application of this technique with up to 96 capillaries in parallel, results in massive reduction of the effective separation time per sample combined with an impressive sensitivity achieved due to LIF detection [3]. Due to the lack of appropriated software, the Matlab® based software tool “glyXtool V3.0” was developed for the data analysis of CGE-LIF generated electropherogram data. Thereby, glyXtool V3.0 provides automated raw data smoothing, background adjustment and normalization to in internal standard in HTP. Furthermore, glyXtool V3.0 includes an automated peak picking algorithm, a peak comparison function for biomarker screening and a peak annotation with access to a continuous growing oligosaccharide database. A graphic user interface makes this tailor made analysis tool also easy to handle. This novel modular glycoanalysis software-tool for data-processing and automated structural elucidation by interfacing a corresponding oligosaccharide-database (initiated and to be further built-up) will allow fully automated, highly sensitive instrument-, lab- and operator-independent high-throughput HTP-glycoanalysis, even when operated by non-experts. This is groundbreaking and in contrast to the currently prevailing methods, where multiplexing with respect to high-throughput is highly cost and lab-space intensive and ties up a lot of manpower and experts hands-on-time. [1]Laroy, W.; Contreras, R.; Callewaert, N.; Glycome mapping on DNA sequencing equipment. Nature Protocols (2006) 1, 397 - 405. [2]Schwarzer, J.; Rapp, E.; Reichl, U.; N-glycan analysis by CGE–LIF: Profiling influenza A virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis (2008) 29, 4203 – 4214. [3]Ruhaak, L.R.; Hennig, R.; Huhn, C.; Borowiak, M.; Dolhain, R. J. E. M.; Deelder, A. M.; Rapp, E.; Wuhrer, M.; Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis o
Tailored sample preparation methods for xCGE-LIF based carbohydrate analysis
No full textCarbohydrates are some of the most common biomolecules in nature. Being nearly ubiquitous present, in their simplest form they are acting as a universal energy source. Furthermore, these carbohydrates forming various sets of complex linear or branched homo- and hetero-oligo/polymers. E.g. they are building a big part of the extracellular matrix or alter protein properties as posttranslational modifications. For that reason carbohydrate analysis became of increasing importance in the recent past. To enhance the knowledge in glycobiology, respectively glycobiotechnology – “high-performance” glycoanalytical methods are required that allow in-depth glycoanalysis on large sample sets in an acceptable time. Using mass spectrometry and liquid chromatography in various setups, as well as electrokinetic separation techniques, detailed information on glycosylation can be obtained. Especially, multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) was recently established for “real” HT-profiling of APTS labeled N-glycans from glycoproteins in complex samples [1,2,3]. By using standard DNA sequencing equipment with up to 96 capillaries in parallel, a massive reduction of the effective separation time per sample can be achieved. To ensure its HT-character, xCGE-LIF requires a fast and flexible sample preparation, optimized for ease of use. For that reason we developed a modular sample preparation toolbox with respect to the different needs, caused by the unique characteristics of each type of sample. Starting with optional protein enrichment and/or protein separation, enzymatic glycan-release has to be performed, followed by glycan labeling with a fluorescent dye. Before xCGE-LIF measurement, optionally, post-labeling sample clean-up can be applied, based on size exclusion chromatography or hydrophilic interaction solid phase extraction. By using this sample preparation toolbox glycoanalysis of complex samples like citrate plasma, human milk oligosaccharides (HMOS), as well as glycoanalysis of more modest samples like Immunoglobuline G (IgG), Erythropoetin (EPO) and the influenza virus antigen Hemagglutinin is feasible with a minimal hands-on time. The presented xCGE-LIF based glycoanalysis method, in combination with an automated data-processing via “glyXtool”, enables in-depth and “real” HT-analysis of complex carbohydrates like glycans. [1]Laroy, W.; Contreras, R.; Callewaert, N.; Glycome mapping on DNA sequencing equipment. Nature Protocols (2006) 1, 397 - 405. [2]Schwarzer, J.; E. Rapp; U. Reichl; N-glycan analysis by CGE-LIF: Profiling influenza A virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis (2008) 29, 4203-4214. [3]Ruhaak, L.R.; Hennig, R.; Huhn, C.; Borowiak, M.; Dolhain, R. J. E. M.; Deelder, A. M.; Rapp, E.; Wuhrer, M.; Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. Journal of Proteome Research (2010) 9, 6655 – 6664
Selective immortalization of murine macrophages from fresh bone marrow by a raf/myc recombinant murine retrovirus
No full textMyeloid precursors can be grown in vitro in the presence of specific growth factors; however, their expansion is limited by a competing process of terminal differentiation. Proto-oncogenes seem to be involved in cellular proliferation and/or differentiation and may also play a role in the myelopoietic process. Murine myeloid precursors which are grown in vitro with growth factors respond with augmented self-renewal upon infection with recombinant retroviruses carrying the v-myc or v-src oncogenes, suggesting a synergism or complementation between some viral oncogenes (v-onc) and certain growth factors. We now show that the combination of two v-onc genes (raf and myc) induces the selective proliferation of monocytic cells from fresh murine bone marrow (BM) in the absence of a specific growth factor supplement. Depending on the culture conditions these cells can either differentiate and cease to proliferate or grow continuously, thus mimicking the alternative pathways that can be followed by committed BM stem cells in vivo
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