Error propagation from prime variables into specific rates and metabolic fluxes for mammalian cells in perfusion culture

Error propagation from prime variables into specific rates and metabolic fluxes was quantified for high‐concentration CHO cell perfusion cultivation. Prime variable errors were first determined from repeated measurements and ranged from 4.8 to 12.2%. Errors in nutrient uptake and metabolite/product formation rates for 5–15% error in prime variables ranged from 8–22%. The specific growth rate, however, was characterized by higher uncertainty as 15% errors in the bioreactor and harvest cell concentration resulted in 37.8% error. Metabolic fluxes were estimated for 12 experimental conditions, each of 10 day duration, during 120‐day perfusion cultivation and were used to determine error propagation from specific rates into metabolic fluxes. Errors of the greater metabolic fluxes (those related to glycolysis, lactate production, TCA cycle and oxidative phosphorylation) were similar in magnitude to those of the related greater specific rates (glucose, lactate, oxygen and CO₂ rates) and were insensitive to errors of the lesser specific rates (amino acid catabolism and biosynthesis rates). Errors of the lesser metabolic fluxes (those related to amino acid metabolism), however, were extremely sensitive to errors of the greater specific rates to the extent that they were no longer representative of cellular metabolism and were much less affected by errors in the lesser specific rates. We show that the relationship between specific rate and metabolic flux error could be accurately described by normalized sensitivity coefficients, which were readily calculated once metabolic fluxes were estimated. Their ease of calculation, along with their ability to accurately describe the specific rate‐metabolic flux error relationship, makes them a necessary component of metabolic flux analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Investigating sensitivity coefficients characterizing the response of a model of tau protein transport in an axon to model parameters

Evaluating the sensitivity of biological models to various model parameters is a critical step towards advancing our understanding of biological systems. In this paper, we investigated sensitivity coefficients for a model simulating transport of tau protein along the axon. This is an important problem due to the relevance of tau transport and agglomeration to Alzheimer’s disease and other tauopathies, such as some forms of parkinsonism. The sensitivity coefficients that we obtained characterize how strongly three observables (the tau concentration, average tau velocity, and the percentage of tau bound to microtubules) depend on model parameters. The fact that the observables strongly depend on a parameter characterizing tau transition from the retrograde to the anterograde kinetic states suggests the importance of motor-driven transport of tau. The observables are sensitive to kinetic constants characterizing tau concentration in the free (cytosolic) state only at small distances from the soma. Cytosolic tau can only be transported by diffusion, suggesting that diffusion-driven transport of tau only plays a role in the proximal axon. Our analysis also shows the location in the axon in which an observable has the greatest sensitivity to a certain parameter. For most parameters, this location is in the proximal axon. This could be useful for designing an experiment aimed at determining the value of this parameter. We also analyzed sensitivity of the average tau velocity, the total tau concentration, and the percentage of microtubule-bound tau to cytosolic diffusivity of tau and diffusivity of bound tau along the MT lattice. The model predicts that at small distances from the soma the effect of these two diffusion processes is comparable.     

Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway

Production of chemicals directly from carbon dioxide using light energy is an attractive option for a sustainable future. The 1,3-propanediol (1,3-PDO) production directly from carbon dioxide was achieved by engineered Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. Glycerol dehydratase catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde in a coenzyme B₁₂-dependent manner worked in S. elongatus PCC 7942 without addition of vitamin B₁₂, suggesting that the intrinsic pseudovitamin B₁₂ served as a substitute of coenzyme B₁₂. The highest titers of 1,3-PDO (3.79±0.23 mM; 288±17.7 mg/L) and glycerol (12.62±1.55 mM; 1.16±0.14 g/L), precursor of 1,3-PDO, were reached after 14 days of culture under optimized conditions in this study.     

Optimization-based metabolic control analysis

In this work, a novel optimization-based metabolic control analysis (OMCA) method is introduced for reducing data requirement for metabolic control analysis (MCA). It is postulated that using the optimal control approach, the fluxes in a metabolic network are correlated to metabolite concentrations and enzyme activities as a state-feedback control system that is optimal with respect to a homeostasis objective. It is then shown that the optimal feedback gains are directly related to the elasticity coefficients (ECs) of MCA. This approach requires determination of the relative "importance" of metabolites and fluxes for the system, which is possible with significantly reduced experimental data, as compared with typical MCA requirements. The OMCA approach is applied to a top-down control model of glycolysis in hepatocytes. It is statistically demonstrated that the OMCA model is capable of predicting the ECs observed experimentally with few exceptions. Further, an OMCA-based model reconciliation study shows that the modification of four assumed stoichiometric coefficients in the model can explain most of the discrepancies, with the exception of elasticities with respect to the NADH/NAD ratio.     

基于小波高频系数基因芯片数据的特征提取

结合小波分析理论与支持向量机理论,构造分类器模型,将前列腺癌基因芯片数据分成癌症和正常两种。本文着重研究小波高频系数基因芯片数据的特征提取,并通过实验对比小波高频系数和低频系数特征提取对分类器性能的影响。其中haar小波3层分解提取高频系数,送入分类器分类后,得到的正确分类率为93.31%。db1小波4层分解提取低频系数,送入分类器分类后,得到的正确分类率为93.53%。小波低频系数特征提取分类效果总体上好于高频系数,分类器性能稳定。     

Interpretation of metabolic flux maps by limitation potentials and constrained limitation sensitivities

Two new concepts, "Limitation Potential" and "Constraint Limitation Sensitivity" are introduced that use definitions derived from metabolic flux analysis (MFA) and metabolic network analysis (MNA). They are applied to interpret a measured flux distribution in the context of all possible flux distributions and thus combine MFA with MNA. The proposed measures are used to quantify and compare the influence of intracellular fluxes on the production yield. The methods are purely based on the stoichiometry of the network and constraints that are given from irreversible fluxes. In contrast to metabolic control analysis (MCA), within this approach no information about the kinetic mechanisms are needed. A limitation potential (LP) is defined as the reduction of the reachable (theoretical) maximum by a measured flux. Measured fluxes that strongly narrow the reachable maximum are assumed to be limiting as the network has no ability to counterbalance the restriction due to the observed flux. In a second step, the sensitivity of the reduced maximum is regarded. This measure provides information about the necessitated changes to reach higher yields. The methods are applied to interpret the capabilities of a network based on measured fluxes for a L-phenylalanine producer. The strain was examined by a series of experiments and three flux maps of the production phase are analyzed. It can be shown that the reachable yield is drastically reduced by the measured efflux into the TCA cycle, while the oxidative pentose-phosphate pathway only plays a secondary role on the reachable maximum.     

Drag coefficients of stream bryophytes: experimental determinations and ecological significance

1. Drag coefficients ( C D) of bryophyte-covered rocks were measured to see whether these differed between species. Replicate rocks, each supporting one of six bryophyte taxa, were attached to a base plate mounted on Teflon bearings in a flow tank. The drag force exerted on rocks with and without bryophyte material was measured by a strain gauge at different water velocities.
2. The difference in C D between rocks with and without bryophyte material was calculated for each plant, and expressed as a percentage change ( δC D). This varied significantly from 0 in three of the six taxa. The cushion-shaped moss Bryum blandum increased the C D by around 10%. The moss Blindia lewinskyae and liverwort Cryptochila grandiflora decreased the C D by around 40 and 30%, respectively, presumably reflecting their streamlined growth.
3. Drag characteristics of aquatic bryophytes may help explain differences in their resistance to fast flows. Furthermore, we suggest that some aquatic bryophytes can increase substrate stability by streamlining rocks, rendering them less prone to movement.     

Insulin sensitivity in experimental cirrhosis

Summary To investigate insulin action in muscle and adipose tissue in hepatic cirrhosis, a recently described animal model was used. Dimethylnitrosamine administration induced histologically proven cirrhosis. Contrary to expectation, muscle strips from cirrhotic rats displayed increased insulin sensitivity both with respect to glycogen synthesis (ED₅₀ 0.11 ± 0.01 vs 0.23 ± 0.04 nmol/1; p < 0.03) and glucose oxidation (ED₅₀ 0.36 ±0.07 vs 0.97 ± 12 nmol/1; p < 0.02). As the cirrhotic rats had failed to gain weight normally, it is postulated that a state of relative starvation accounted for the enhanced insulin sensitivity. These data demonstrate that the severe insulin resistance characteristically associated with cirrhosis is reversible. Control of nutritional state in future studies upon DMNA induced cirrhosis should permit detailed examination of the cellular mechanisms controlling insulin sensitivity in hepatic cirrhosis.     

Sensitivity analysis of metabolic cascades catalyzed by bifunctional enzymes

Covalent modification/demodification cycles are common in metabolism. When the modification and demodification steps are carried out by two independent enzymes, the degree of modification can be ultrasensitive to the total concentration of either catalyst. We recently showed that the degree of modification of a target molecule cannot exhibit ultrasensitivity to the free concentrations of effectors that decide whether a bifunctional enzyme acts as modifier or demodifier. However, here we can now demonstrate that the degree of modification of a target molecule can display ultrasensitivity to the total, rather than free, concentrations of such effectors. Our results clarify some general aspects of ultrasensitive responses to effectors, including competitive inhibitors, in mono-cyclic cascades.     

Application of the transition time of metabolic system as a criterion for optimization of metabolic processes

Transition time of metabolic systems in introduced as a suitable optimization criterion for biotechnological processes in which it is desirable to reduce the lag time and minimize the mass contained within the system. Lag time is the time needed for the system to attain the steady state. Results obtained from the sensitivity analysis of this steady state response are presented within the metabolic control analysis and applied to 3 case studies. In all of them the information provided by the transition time control profile allows the implementation of a strategy for biotechnological manipulations aimed at the improvement of the process. (c) 1994 John Wiley & Sons, Inc.     

Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via regulation of pyruvate dehydrogenase phosphorylation

A high-salt diet significantly impacts various diseases, ilncluding cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, we determined that the pyruvate dehydrogenase phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmosensing mechanism. Additionally, we demonstrate that Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Collectively, our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of pyruvate dehydrogenase phosphorylation by direct osmosensing within mitochondria.     

Estimating stochastic elasticities directly from longitudinal data

The elasticities of long-run population growth rate with respect to vital rates are useful in studying selection on vital rates, and in evaluating management policy that aims to control vital rates. In temporally varying environments, elasticity is often calculated from simulations that assume a probability distribution for the environmental states. Here we develop a method to estimate elasticities directly from demographic data. Using a time-series of demographic matrices and age-structure we construct a consistent statistical estimator of elasticity that converges to the correct limiting value as the sample length increases. We also construct confidence intervals for elasticities from temporal data and suggest tools for testing hypotheses about the strength of selection. We use data on a natural population to show that our method can indeed accurately estimate elasticities using relatively short time series.     

Beta diversity as the variance of community data: dissimilarity coefficients and partitioning

Beta diversity can be measured in different ways. Among these, the total variance of the community data table Y can be used as an estimate of beta diversity. We show how the total variance of Y can be calculated either directly or through a dissimilarity matrix obtained using any dissimilarity index deemed appropriate for pairwise comparisons of community composition data. We addressed the question of which index to use by coding 16 indices using 14 properties that are necessary for beta assessment, comparability among data sets, sampling issues and ordination. Our comparison analysis classified the coefficients under study into five types, three of which are appropriate for beta diversity assessment. Our approach links the concept of beta diversity with the analysis of community data by commonly used methods like ordination and anova . Total beta can be partitioned into Species Contributions (SCBD: degree of variation of individual species across the study area) and Local Contributions (LCBD: comparative indicators of the ecological uniqueness of the sites) to Beta Diversity. Moreover, total beta can be broken up into within‐ and among‐group components by manova , into orthogonal axes by ordination, into spatial scales by eigenfunction analysis or among explanatory data sets by variation partitioning.     

In situ metabolic flux analysis to quantify the liver metabolic response to experimental burn injury

Trauma such as burns induces a hypermetabolic response associated with altered central carbon and nitrogen metabolism. The liver plays a key role in these metabolic changes; however, studies to date have evaluated the metabolic state of liver using ex vivo perfusions or isotope labeling techniques targeted to specific pathways. Herein, we developed a unique mass balance approach to characterize the metabolic state of the liver in situ, and used it to quantify the metabolic changes to experimental burn injury in rats. Rats received a sham (control uninjured), 20% or 40% total body surface area (TBSA) scald burn, and were allowed to develop a hypermetabolic response. One day prior to evaluation, all animals were fasted to deplete glycogen stores. Four days post-burn, blood flow rates in major vessels of the liver were measured, and blood samples harvested. We combined measurements of metabolite concentrations and flow rates in the major vessels entering and leaving the liver with a steady-state mass balance model to generate a quantitative picture of the metabolic state of liver. The main findings were: (1) Sham-burned animals exhibited a gluconeogenic pattern, consistent with the fasted state; (2) the 20% TBSA burn inhibited gluconeogenesis and exhibited glycolytic-like features with very few other significant changes; (3) the 40% TBSA burn, by contrast, further enhanced gluconeogenesis and also increased amino acid extraction, urea cycle reactions, and several reactions involved in oxidative phosphorylation. These results suggest that increasing the severity of injury does not lead to a simple dose-dependent metabolic response, but rather leads to qualitatively different responses.     

Vesicle membrane-water partition coefficients determined from Fourier transform pulsed-gradient spin-echo NMR based self-diffusion data. Application to anesthetic binding in tetracaine-phosphatidylcholine-water systems

Multicomponent self-diffusion data on dioleoyl(DOL)- and dipalmitoyllecithin (DPL) vesicle-water systems have been determined using a Fourier transform NMR technique. The self-diffusion of vesicles is characterized by diffusion coefficients two magnitudes lower than that of small molecules in solution. Consequently, the degree of binding of small molecules is strongly reflected in their time-averaged self-diffusion coefficient in vesicle-water systems. This provides a new basis for the determination of vesicle-water partition equilibria. The feasibility of the technique has been investigated in one anesthetic-lipid system and is found to be very good. The binding of the hydrochloride form of tetracaine to DOL vesicles at pH 3 and 7 (K_p = 30–50) is found to be very much lower than that of the neutral molecule at pH 9 (K_p = 800–900). No significant difference in the tetracaine binding characteristics was found between DOL, DOL-cholesterol and DPL systems.     

The individual and mean activity coefficients of an electrolyte from the inverse GCMC simulation

The grand-canonical Monte Carlo (GCMC) technique, which is primarily used to calculate the concentration of a solute for a given activity coefficient, can be inverted and applied to calculate the activity coefficients corresponding to a given concentration of a solute. The changes needed to be introduced in the GCMC algorithm are discussed in the paper. The new method called the inverse GCMC (IGCMC) technique is applied to calculate the mean activity coefficient of 1:1, 2:2, 2:1, 3:1 salts with equal and unequal ion sizes and over a wide range of the electrolyte concentrations. The results for equal ion sizes are compared with the corresponding data obtained from the GCMC of Valleau and Cohen. It is shown that the IGCMC technique, after some extensions, can be used to compute the individual ionic activity coefficients. A comparison is made with the Sloth and Sørensen individual activity results and with the theoretical predictions for the 1:1 electrolyte with unequal ion sizes.     

Challenges in experimental data integration within genome-scale metabolic models

A report of the meeting "Challenges in experimental data integration within genome-scale metabolic models", Institut Henri Poincaré, Paris, October 10-11 2009, organized by the CNRS-MPG joint program in Systems Biology.