178,614 research outputs found
Nobel Lecture: A forty-year journey
No full text© 2022 Nobel Foundation, Published by the American Physical Society. The 2020 Nobel Prize for Physics was shared by Roger Penrose, Reinhard Genzel, and Andrea Ghez. This paper is the text of the address given in conjunction with the award
Limits and benefits of the interpretation of extracellular metabolite data in mammalian cell culture
No full textBefore considering intracellular metabolites clear ideas on the extracellular conditions of experiments are obligatory. Many interesting questions on understanding metabolism of cells are generated under process conditions and not under defined steady state conditions with defined media.
Here, we discuss limits and benefits of the interpretation of extracellular metabolite data for a process of influenza vaccine production in mammalian cells. Therefore, the production process is introduced together with relevant details on medium composition and cultivation conditions. Typical issues of metabolism during cell growth and viral infection are presented. Data on basic metabolites (glucose, lactate, glutamine, ammonia) are shown together with amino acid profiles measured from the cell culture medium [1]. Comparing different media (serum-containing, glutamine-free, serum-free [2-4]) as well as different cultivation methods (roller bottles, stirred tank bioreactor, wave bioreactor) it is demonstrated, what can be learned from extracellular metabolites and what questions remain.
[1] Genzel Y.; König S.; Reichl U.; "Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection (IPAD)." , Anal. Biochem. 2004, 335, 119-125.
[2] Genzel Y.; Behrendt I.; König S.; Sann H.; Reichl U.; "Metabolism of MDCK cells during cell growth and influence virus production in large-scale microcarrier culture" , Vaccine 2004, 22(17-18), 2202-2208.
[3] Genzel Y.; Ritter JB.; König S.; Alt R.; Reichl U.; "Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells." , Biotechnol. Progr. 2005, 21 (1), 58-69.
[4] Genzel Y.; Fischer M.; Reichl U.; "Serum-free influenza virus production avoiding washing steps and medium exchange in large-scale microcarrier culture.", Vaccine 2006, 24(16), 3261-3272
Limits and benefits of the interpretation of extracellular metabolite data in mammalian cell culture
No full textBefore considering intracellular metabolites clear ideas on the extracellular conditions of experiments are obligatory. Many interesting questions on understanding metabolism of cells are generated under process conditions and not under defined steady state conditions with defined media. Here, we discuss limits and benefits of the interpretation of extracellular metabolite data for a process of influenza vaccine production in mammalian cells. Therefore, the production process is introduced together with relevant details on medium composition and cultivation conditions. Typical issues of metabolism during cell growth and viral infection are presented. Data on basic metabolites (glucose, lactate, glutamine, ammonia) are shown together with amino acid profiles measured from the cell culture medium [1]. Comparing different media (serum-containing, glutamine-free, serum-free [2-4]) as well as different cultivation methods (roller bottles, stirred tank bioreactor, wave bioreactor) it is demonstrated, what can be learned from extracellular metabolites and what questions remain. [1] Genzel Y.; König S.; Reichl U.; "Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection (IPAD)." , Anal. Biochem. 2004, 335, 119-125. [2] Genzel Y.; Behrendt I.; König S.; Sann H.; Reichl U.; "Metabolism of MDCK cells during cell growth and influence virus production in large-scale microcarrier culture" , Vaccine 2004, 22(17-18), 2202-2208. [3] Genzel Y.; Ritter JB.; König S.; Alt R.; Reichl U.; "Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells." , Biotechnol. Progr. 2005, 21 (1), 58-69. [4] Genzel Y.; Fischer M.; Reichl U.; "Serum-free influenza virus production avoiding washing steps and medium exchange in large-scale microcarrier culture.", Vaccine 2006, 24(16), 3261-3272
Appropriate Similarity Measures for Author Cocitation Analysis
No full textWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Changes in specific enzyme activities of MDCK cells during growth under different cultivation conditions
No full textVarious producer cell lines used for large scale production of biopharmaceuticals like monoclonal antibodies, hormones or viral vaccines often show inefficient use of nutrients for growth. They have a very high demand in substrates, mainly glucose and glutamine, which results in release of large amounts of lactate and ammonium. This metabolic imbalance often not only decreases cell productivity and viability but also inhibits growth to high cell densities. Thus, new strategies including optimization of cultivation conditions, design of media and modification of specific properties of cells by systems biology approaches should be developed to increase cell densities and process yields.
For the growth of adherent MDCK cells distinct differences in glycolysis and tricarboxylic acid cycle were found for extra- and intracellular metabolite concentrations and metabolic fluxes when comparing the influence of glutamine or pyruvate addition to cell culture media [1-4].
The objective of this study was to further elucidate these differences by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [5]. This method, which uses four cycling assays, allowed the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Whereas, a comparison of Pyr and Gln-containing medium showed an up-regulation of glutamine synthetase activity by a factor of 4, the activity of glutaminase was down-regulated by a factor of 6 in GMEM medium. Decreased activities were also found for ATP-citrate lyase, phosphoenolpyruvate carboxykinase and the glutaminolytic enzymes aspartate- and alanine transaminase with Pyr as carbon source. Under all conditions, very low activities of pyruvate dehydrogenase and pyruvate carboxylase were measured and confirmed by comparing normal mouse kidney tissue with MDCK cells. Other enzymes like lactate dehydrogenase (LDH) and malate dehydrogenase showed less distinct changes in activity. In all media the activities of the glycolytic enzymes hexokinase and phosphofructokinase were relatively low, whereas maximum activities of LDH and pyruvate kinase were comparatively high. In contrast, most enzyme activities measured in serum-free Episerf medium were increased.
Based on the established assay metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization.
[1] Genzel, Y., Ritter, J.B., König, S., Alt, R. and Reichl, U. 2005. Biotechnol. Prog., 21, 58-69.
[2] Ritter, J.B., Genzel, Y. and Reichl, U. 2006. Journal of Chromatography B, 843, 216-226.
[3] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y. and Reichl, U. 2008. Biotechnol Prog, 24(2), 311-320.
[4] Wahl, A., Sidorenko, Y., Dauner, M., Genzel, Y. & Reichl, U. 2008. Biotechnology and Bioengineering, 101(1), 135-152.
[5] Janke, R., Genzel, Y., Wahl, A & Reichl, U. 2010. Metabolic Engineering, submitted
Effects of different growth conditions on the catalytic activities of central metabolic enzymes in MDCK cells
No full textIt is of common knowledge that Madin-Darby canine kidney (MDCK) cells are very suitable for the propagation of different influenza strains and, therefore, for the production of cell culture-based vaccines [1]. However, during growth in glutamine-containing media, the glycolytic and glutaminolytic fluxes of most cell lines are up-regulated and large amounts of toxic by-products, such as lactate and ammonia, are secreted into the medium. This metabolic imbalance often not only affects cell viability and productivity but also can prevent growth to high cell densities [2,3]. A promising approach to reduce waste-products is the substitution of one or several components in the culture medium [4,5]. In glutamine-free medium with pyruvate as carbon source, MDCK cells not only released no ammonia during cell growth but glucose consumption and lactate production was also reduced significantly [4]. In previous work with MDCK cells, several assays were developed, to determine the extra- and intracellular metabolite concentrations [6]. Furthermore, mathematical models were established to analyze the switch from glutamine-containing to glutamine-free (pyruvate) medium [7]. However, concerning the interpretation of experimental data and corresponding flux distributions, still some open questions remain. The objective of this study was to further elucidate the impact of media changes on metabolism by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [8]. The method established uses four sensitive enzymatic cycling assays, and allows the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Adherent MDCK cells were grown to stationary and exponential phases in 6-well plates in serum-containing GMEM supplemented with glutamine or pyruvate as well as in serum-free EPISERF medium, and key metabolic enzyme activities of cell extracts were analyzed. Significant differences were found in maximal enzyme activities from cells grown with pyruvate-containing medium compared to glutamine-containing medium. In particular, the overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium, which suggests that more glucose 6-phosphate was channeled into the oxidative branch and therefore more NADPH was required. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate, indicating an increased flux into the TCA cycle. An increase was also found for NAD+-dependent isocitrate dehydrogenase, glutamate dehydrogenase and glutamine synthetase, which is a strong indicator for an increased flux through the right part of the citrate cycle in MDCK cells grown with pyruvate. It can be assumed that extracellular pyruvate was directly shunted into the TCA cycle, and that the increase in enzyme activities was most likely required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of the glutaminolytic enzymes aspartate transaminase, alanine transaminase, malic enzyme and phosphoenolpyruvate carboxykinase were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism. Based on the established enzyme assays metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization. [1] Genzel, Y., Reichl, U., (2009). Expert Rev Vaccines 8, 1681-1692. [2] Glacken, M.W., (1988). Bio-Technol 6, 1041-1050. [3] Ozturk, S.S., Riley, M.R., Palsson, B.O., (1992). Biotechnol and Bioeng 39, 418-431. [4] Genzel, Y., Ritter, J.B., König, S., Alt, R., Reichl, U., (2005). Biotechnol Prog 21, 58-69. [5] Butler, M., Christie, A., (1994). Cytotechnology 15, 87-94. [6] Ritter, J.B., Genzel, Y., Reichl, U., (2008). Anal Biochem 373, 349-369. [7] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y., Reichl, U., (2008). Biotechnol Progr 24, 311-320. [8] Janke, R., Genzel, Y., Wahl, A., Reichl, U., (2010). Biotechnol and Bioeng 107, 566-581
Effects of different growth conditions on the catalytic activities of central metabolic enzymes in MDCK cells
No full textIt is of common knowledge that Madin-Darby canine kidney (MDCK) cells are very suitable for the propagation of different influenza strains and, therefore, for the production of cell culture-based vaccines [1]. However, during growth in glutamine-containing media, the glycolytic and glutaminolytic fluxes of most cell lines are up-regulated and large amounts of toxic by-products, such as lactate and ammonia, are secreted into the medium. This metabolic imbalance often not only affects cell viability and productivity but also can prevent growth to high cell densities [2,3]. A promising approach to reduce waste-products is the substitution of one or several components in the culture medium [4,5]. In glutamine-free medium with pyruvate as carbon source, MDCK cells not only released no ammonia during cell growth but glucose consumption and lactate production was also reduced significantly [4]. In previous work with MDCK cells, several assays were developed, to determine the extra- and intracellular metabolite concentrations [6]. Furthermore, mathematical models were established to analyze the switch from glutamine-containing to glutamine-free (pyruvate) medium [7]. However, concerning the interpretation of experimental data and corresponding flux distributions, still some open questions remain.
The objective of this study was to further elucidate the impact of media changes on metabolism by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [8]. The method established uses four sensitive enzymatic cycling assays, and allows the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Adherent MDCK cells were grown to stationary and exponential phases in 6-well plates in serum-containing GMEM supplemented with glutamine or pyruvate as well as in serum-free EPISERF medium, and key metabolic enzyme activities of cell extracts were analyzed.
Significant differences were found in maximal enzyme activities from cells grown with pyruvate-containing medium compared to glutamine-containing medium. In particular, the overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium, which suggests that more glucose 6-phosphate was channeled into the oxidative branch and therefore more NADPH was required. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate, indicating an increased flux into the TCA cycle. An increase was also found for NAD+-dependent isocitrate dehydrogenase, glutamate dehydrogenase and glutamine synthetase, which is a strong indicator for an increased flux through the right part of the citrate cycle in MDCK cells grown with pyruvate. It can be assumed that extracellular pyruvate was directly shunted into the TCA cycle, and that the increase in enzyme activities was most likely required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of the glutaminolytic enzymes aspartate transaminase, alanine transaminase, malic enzyme and phosphoenolpyruvate carboxykinase were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.
Based on the established enzyme assays metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization.
[1] Genzel, Y., Reichl, U., (2009). Expert Rev Vaccines 8, 1681-1692.
[2] Glacken, M.W., (1988). Bio-Technol 6, 1041-1050.
[3] Ozturk, S.S., Riley, M.R., Palsson, B.O., (1992). Biotechnol and Bioeng 39, 418-431.
[4] Genzel, Y., Ritter, J.B., König, S., Alt, R., Reichl, U., (2005). Biotechnol Prog 21, 58-69.
[5] Butler, M., Christie, A., (1994). Cytotechnology 15, 87-94.
[6] Ritter, J.B., Genzel, Y., Reichl, U., (2008). Anal Biochem 373, 349-369.
[7] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y., Reichl, U., (2008). Biotechnol Progr 24, 311-320.
[8] Janke, R., Genzel, Y., Wahl, A., Reichl, U., (2010). Biotechnol and Bioeng 107, 566-581
Changes in specific enzyme activities of MDCK cells during growth under different cultivation conditions
No full textVarious producer cell lines used for large scale production of biopharmaceuticals like monoclonal antibodies, hormones or viral vaccines often show inefficient use of nutrients for growth. They have a very high demand in substrates, mainly glucose and glutamine, which results in release of large amounts of lactate and ammonium. This metabolic imbalance often not only decreases cell productivity and viability but also inhibits growth to high cell densities. Thus, new strategies including optimization of cultivation conditions, design of media and modification of specific properties of cells by systems biology approaches should be developed to increase cell densities and process yields. For the growth of adherent MDCK cells distinct differences in glycolysis and tricarboxylic acid cycle were found for extra- and intracellular metabolite concentrations and metabolic fluxes when comparing the influence of glutamine or pyruvate addition to cell culture media [1-4]. The objective of this study was to further elucidate these differences by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [5]. This method, which uses four cycling assays, allowed the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Whereas, a comparison of Pyr and Gln-containing medium showed an up-regulation of glutamine synthetase activity by a factor of 4, the activity of glutaminase was down-regulated by a factor of 6 in GMEM medium. Decreased activities were also found for ATP-citrate lyase, phosphoenolpyruvate carboxykinase and the glutaminolytic enzymes aspartate- and alanine transaminase with Pyr as carbon source. Under all conditions, very low activities of pyruvate dehydrogenase and pyruvate carboxylase were measured and confirmed by comparing normal mouse kidney tissue with MDCK cells. Other enzymes like lactate dehydrogenase (LDH) and malate dehydrogenase showed less distinct changes in activity. In all media the activities of the glycolytic enzymes hexokinase and phosphofructokinase were relatively low, whereas maximum activities of LDH and pyruvate kinase were comparatively high. In contrast, most enzyme activities measured in serum-free Episerf medium were increased. Based on the established assay metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization. [1] Genzel, Y., Ritter, J.B., König, S., Alt, R. and Reichl, U. 2005. Biotechnol. Prog., 21, 58-69. [2] Ritter, J.B., Genzel, Y. and Reichl, U. 2006. Journal of Chromatography B, 843, 216-226. [3] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y. and Reichl, U. 2008. Biotechnol Prog, 24(2), 311-320. [4] Wahl, A., Sidorenko, Y., Dauner, M., Genzel, Y. & Reichl, U. 2008. Biotechnology and Bioengineering, 101(1), 135-152. [5] Janke, R., Genzel, Y., Wahl, A & Reichl, U. 2010. Metabolic Engineering, submitted
"Closing the R&D Gap, Evaluating the Sources of R&D Spending"
No full textBoth spending and tax policies have been implemented in the United States with the goal of stimulating private sector research and development (R&D). Karier questions whether current R&D policy, especially the research and experimentation tax credit, can contribute to closing the gap between nondefense expenditures on R&D in the United States and such expenditures in other countries, such as Japan and Germany. He also explores possible changes to our current R&D policy to make it more effective.
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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