Local and global inverse modelling strategies to estimate parameters for pesticide leaching from lysimeter studies.

BACKGROUND: As an option for higher-tier leaching assessment of pesticides in Europe according to FOCUS, pesticide properties can be estimated from lysimeter studies by inversely fitting parameter values (substance half-life DT50 and sorption coefficient to organic matter kom ). The aim of the study was to identify adequate methods for inverse modelling. RESULTS: Model parameters for the PEARL (Pesticide Emission Assessment at Regional and Local scales) model were estimated with different inverse optimisation algorithms - the Levenberg-Marquardt (LM), PD_MS2 (PEST Driver Multiple Starting Points 2) and SCEM (Shuffled Complex Evolution Metropolis) algorithms. Optimisation of crop factors and hydraulic properties was found to be an ill-posed problem, and all algorithms failed to identify reliable global minima for the hydrological parameters. All algorithms performed equally well in estimating pesticide sorption and degradation parameters. SCEM was in most cases the only algorithm that reliably calculated uncertainties. CONCLUSION: The most reliable approach for finding the best parameter set in the stepwise approach of optimising evapotranspiration, soil hydrology and pesticide parameters was to run only SCEM or a combined approach with more than one algorithm using the best fit of each step for further processing. PD_MS2 was well suited to a quick parameter search. The linear parameter uncertainty intervals estimated by LM and PD_MS2 were usually larger than by the non-linear method used by SCEM. With the suggested methods, parameter optimisation, together with reliable estimation of uncertainties, is possible also for relatively complex systems.

Assessment of rare dietary risks based on analysis of food combinations.

Probabilistic methods, in particular Monte Carlo methods, have become widely used in assessment of dietary risks from plant protection products. However, if the critical exposure occurs rarely, estimating its probability with commonly used Monte Carlo approaches can require an unrealistically big number of iterations. A simple method proposed in this paper, referred to as food combination analysis (FCA), finds out subsets of input values necessary for occurrence of a critical exposure event. In particular, for a critical event to occur consumption of a certain combination of contaminated foods could be required. Sometimes by finding the probability that such a food combination is consumed one could directly get an acceptable estimate of the risk, without Monte Carlo simulations. The method performs especially well if available data sets of consumed amounts of foods and residue concentrations of a chemical contain a large fraction of zeros. Based on a literature example, it is shown that the probability of the critical exposure estimated with the FCA could be more than 10 times lower than the estimate of a Monte Carlo approach with 50,000 iterations. The present approach also provides a platform for adaptation and development of more sophisticated methods to estimate low dietary risks.

Metabolic flux model for an anchorage-dependent MDCK cell line: characteristic growth phases and minimum substrate consumption flux distribution.

Up to now cell-culture based vaccine production processes only reach low productivities. The reasons are: (i) slow cell growth and (ii) low cell concentrations. To address these shortcomings, a quantitative analysis of the process conditions, especially the cell growth and the metabolic capabilities of the host cell line is required. For this purpose a MDCK cell based influenza vaccine production process was investigated. With a segregated growth model four distinct cell growth phases are distinguished in the batch process. In the first phase the cells attach to the surface of the microcarriers and show low metabolic activity. The second phase is characterized by exponential cell growth. In the third phase, preceded by a change in oxygen consumption, contact inhibition leads to a decrease in cell growth. Finally, the last phase before infection shows no further increase in cell numbers. To gain insight into the metabolic activity during these phases, a detailed metabolic model of MDCK cell was developed based on genome information and experimental analysis. The MDCK model was also used to calculate a theoretical flux distribution representing an optimized cell that only consumes a minimum of carbon sources. Comparing this minimum substrate consumption flux distribution to the fluxes estimated from experiments unveiled high overflow metabolism under the applied process conditions.

Comparison of metabolic flux distributions for MDCK cell growth in glutamine- and pyruvate-containing media.

In mammalian cell cultures, ammonia that is released into the medium as a result of glutamine metabolism and lactate that is excreted due to incomplete glucose oxidation are both known to essentially inhibit the growth of cells. For some cell lines, for example, hybridoma cells, excreted ammonia also has an effect on product formation. Although glutamine has been generally considered as the major energy source for mammalian cells, it was recently found that various adherent cell lines (MDCK, CHO-K1, and BHK21) can grow as well in glutamine-free medium, provided glutamine is substituted with pyruvate. In such a medium the level of both ammonia and lactate released was significantly reduced. In this study, metabolic flux analysis (MFA) was applied to Madin Darby Canine Kidney (MDCK) cells cultivated in glutamine-containing and glutamine-free medium. The results of the MFA allowed further investigation of the influence of glutamine substitution with pyruvate on the metabolism of MDCK cells during different growth stages of adherent cells, e.g., early exponential and late contact-inhibited phase. Pyruvate seemed to directly enter the TCA cycle, whereas most of the glucose consumed was excreted as lactate. Although the exact mechanisms are not clear so far, this resulted in a reduction of the glucose uptake necessary for cellular metabolism in glutamine-free medium. Furthermore, consumption of ATP by futile cycles seemed to be significantly reduced when substituting glutamine with pyruvate. These findings imply that glutamine-free medium favors a more efficient use of nutrients by cells. However, a number of metabolic fluxes were similar in the two cultivations considered, e.g., most of the amino acid uptake and degradation rates or fluxes through the branch of the TCA cycle converting alpha-ketoglutarate to malate, which is responsible for the mitochondrial ATP synthesis. Besides, the specific rate of cell growth was approximately the same in both cultivations. Thus, the switch from glutamine-containing to glutamine-free medium with pyruvate provided a series of benefits without dramatic changes of cellular metabolism.