Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.     

Ferritin, Lipid Peroxidation and Redox-Cycling Xenobiotics

A number of xenobiotics are toxic because they rcdox cycle and generate free radicals. Interaction with iron, either to produce reactive species such as the hydroxyl radical, or to promote lipid peroxidation, is an important factor in this toxicity. A potential biological source of iron is ferritin. The cytotoxic pyrimidines, dialuric acid, divicine and isouramil, readily release iron from ferritin and promote ferritin-dependent lipid peroxidation. Superoxide dismutase and GSH, which maintain the pyrimidines in their reduced form, enhance both iron release and lipid peroxidation. Microsomes plus NADPH can reduce a number of iron complexes, although not ferritin. Reduction of Adriamycin. paraquat or various quinones to their radicals by the microsomes enhances reduction of the iron complexes, and in some cases, enables iron release from ferritin. Adriamycin stimulates iron-dependent lipid peroxidation of the microsomes. Ferritin can provide the iron, and peroxidation is most pronounced at low PO₂. Compiexing agents that supress intraccllular iron reduction and lipid peroxidation may protect against the toxicity of Adriamycin.     

Methods for assessing the burden of parasitic zoonoses: echinococcosis and cysticercosis

Cysticercosis and echinococcosis cause illness and productivity losses in human and agricultural animal populations. Recent studies suggest that these diseases have large societal impacts on endemic areas. Estimates of burden provide essential, evidence-based data for conducting cost-benefit and cost-utility analyses that will secure political will, and financial and technical resources. To evaluate the burden, the monetary and non-monetary impacts of these zoonoses on human health, agriculture and society must be considered comprehensively. In this article, we review the framework used to assess the burden of cysticercosis and echinococcosis, and the data needed to estimate the extent of the problem for societies.     

Anthropogenic nitrogen sources and relationships to riverine nitrogen export in the northeastern U.S.A.

Human activities have greatly altered the nitrogen (N) cycle, accelerating the rate of N fixation in landscapes and delivery of N to water bodies. To examine relationships between anthropogenic N inputs and riverine N export, we constructed budgets describing N inputs and losses for 16 catchments, which encompass a range of climatic variability and are major drainages to the coast of the North Atlantic Ocean along a latitudinal profile from Maine to Virginia. Using data from the early 1990's, we quantified inputs of N to each catchment from atmospheric deposition, application of nitrogenous fertilizers, biological nitrogen fixation, and import of N in agricultural products (food and feed). We compared these inputs with N losses from the system in riverine export.The importance of the relative sources varies widely by catchment and is related to land use. Net atmospheric deposition was the largest N source (>60%) to the forested basins of northern New England (e.g. Penobscot and Kennebec); net import of N in food was the largest source of N to the more populated regions of southern New England (e.g. Charles & Blackstone); and agricultural inputs were the dominant N sources in the Mid-Atlantic region (e.g. Schuylkill & Potomac). Over the combined area of the catchments, net atmospheric deposition was the largest single source input (31%), followed by net imports of N in food and feed (25%), fixation in agricultural lands (24%), fertilizer use (15%), and fixation in forests (5%). The combined effect of fertilizer use, fixation in crop lands, and animal feed imports makes agriculture the largest overall source of N. Riverine export of N is well correlated with N inputs, but it accounts for only a fraction (25%) of the total N inputs. This work provides an understanding of the sources of N in landscapes, and highlights how human activities impact N cycling in the northeast region.     

Hydrogel design for supporting neurite outgrowth and promoting gene delivery to maximize neurite extension

Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration.     

On the streets of San Francisco: highlights from the ISSCR Annual Meeting 2010

The 2010 Annual Meeting of the International Society for Stem Cell Research (ISSCR) was held in San Francisco in June with an exciting program covering a wealth of stem cell research from basic science to clinical research.     

Postthaw survival of rhesus macaque sperm: variation in the response of individual males to different freezing protocols

This study was designed to assess the effects of various cryopreservation protocols on the postthaw survival of rhesus macaque sperm. Multiple ejaculates of five mature males were collected and frozen by each of four different methods. Cryopreservation significantly reduced motility in all samples, regardless of the method, and the response of different ejaculates of the same male to each method proved to be consistent. The freezing method was a significant factor in determining the immediate postthaw motility, although this effect disappeared after 4 hr. Moreover, there was a significant interaction between individual males and freezing method suggesting that developing a single freezing protocol that is universally suitable may be difficult.     

Silvopastoralism in the Alps: Native plant species selection under different grazing pressure

To evaluate the suitability of wood pastures as a managing tool in subalpine regions it is essential to know more about the influence of grazing on the ground vegetation. This study assessed native plant species selection by cattle at different stocking rates, feeding habits and site preferences of cattle. Based on the results, conclusions concerning the value of silvopastoral systems in the Alps were drawn. A field study on six different wood pasture areas, grazed by cattle at different stocking rates, was accompanied by an experiment on three adjoining areas of 0.51 ha each, stocked with either three, six, or nine heifers. Plant species were recorded in plots of 20 cm × 20 cm before and after grazing, and the intensity of grazing on each species was assessed. At low stocking rates, grasses and tall species were most intensely grazed, while at higher stocking rates the intake of forbs and small species increased. Since no relationship was found between nutritional value and species preference, other factors such as accessibility of a plant seem to be important for the feeding preferences of cattle. The preference for grasses at low and medium stocking rates suggests that an increased growth of forbs might lead to an increase in plant species diversity.