Stoichiometric analysis of animal cell growth and its application in medium design

Animal cell cultivation in vitro has been studied for more than 40 years. However, the culture medium composition has not been designed on the basis of the stoichiometric nutritional demands for animal cell growth. In this article, a model was developed to study the stoichiometric demands for nutrients (including glucose, 20 amino acids, and 10 vitamins)for the synthesis of cell mass and product. The coefficients for these nutrients in the stoichiometric equation governing animal cell growth were determined based on cell composition. In addition, a detailed analysis of the nutrients' roles in the synthesis of cell mass and product was also performed. Applications of the stoichiometric analysis in animal cell cultivation, such as culture medium design, supplemental medium formulation, and feeding strategy will also be discussed. The stoichiometric analysis can be potentially employed to analyze results from animal cell cultures, to improve the performance of culture processes, and to design new process rationally. It can also help to provide a better understanding of animal cell metabolism. Simplifications on the cellular energy metabolism were made in order to simplify the model and to provide the preliminary bases to test the process performance. However, this could introduce inaccuracies for the model and results in errors in the calculations of glucose and glutamine concentrations when employed in medium design. (c) 1994 John Wiley & Sons, Inc.     

Control of fatty acid and glycerol release in adipose tissue lipolysis

Adipose tissue lipolysis is the catabolic process leading to the breakdown of triglycerides stored in fat cells and the release of fatty acids and glycerol. Recent work has revealed that lipolysis is not a simple metabolic pathway stimulated by catecholamines and inhibited by insulin. New discoveries on the regulation of lipolysis by endocrine and paracrine factors and on the proteins involved in triglyceride hydrolysis have led to a reappraisal of the complexity of the various signal transduction pathways. The steps involved in the dysregulation of lipolysis observed in obesity have partly been identified.     

Metabolic targeted therapy of cancer: current tracer technologies and future drug design strategies in the old metabolic network

Targeted drugs tailored against genes and signaling proteins have formed the new era termed Targeted Therapies. Although the field is relatively young, since only about 5 years ago clinical trials started showing promise, there have are already been significant setbacks due to drug resistance caused by point mutations, alterations in gene expression or complete loss of target proteins with disease progression. Although new drugs are continuously designed and tried, it seems inevitable that genetic and signal protein targets pose too broad flexibility and variability, often changing target characteristics and thus escape treatments turning “magic bullets” into rather “wondering bullets”. This is especially true in cancer, where old and new targeted therapies continue to fail and the most recent ones do not offer much improvement on clinical outcome parameters. Metabolic targeted therapies are aimed at control points of the metabolic network by targeting particular enzymes of major macromolecule synthesis pathways in cancer. This review summarizes the potential benefits of targeted therapies in the metabolic network as applied with genetic and proteomic approaches. The metabolic target approach is most efficient if and when pathway flux information is available for drug target development using the stable isotope based dynamic metabolic profile (SIDMAP) of tumor cells, in vitro or in vivo.This revised version was published online in June 2005. The previous version did not contain colour images.     

Evolution of hemocyte concentration in the melanogaster subgroup species

We explore two possible hypotheses about the ecological factors driving the evolution of hemocyte load in insects. The first is parasite infection risk, its variability and the genetic diversity of parasites. The other supposes that all the other factors of environmental stress drive the evolution through a pleiotropic selection on metabolic rate. The two hypotheses are tested with data on hemocyte load and ecology of six Drosophila species of the melanogaster subgroup. Hemocyte load correlates significantly with the parasite-driven selection index, but not with the stress selection index. The result being based on too little data to conclude definitely, this work must be considered more as a methodological research based on published results on insect physiology immunity and ecology rather than an empirical evidence of such a relationship.     

Metabolic flux analysis in plants: coping with complexity

Theory and experience in metabolic engineering both show that metabolism operates at the network level. In plants, this complexity is compounded by a high degree of compartmentation and the synthesis of a very wide array of secondary metabolic products. A further challenge to understanding and predicting plant metabolic function is posed by our ignorance about the structure of metabolic networks even in well-studied systems. Metabolic flux analysis (MFA) provides tools to measure and model the functioning of metabolism, and is making significant contributions to coping with their complexity.
This review gives an overview of different MFA approaches, the measurements required to implement them and the information they yield. The application of MFA methods to plant systems is then illustrated by several examples from the recent literature. Next, the challenges that plant metabolism poses for MFA are discussed together with ways that these can be addressed. Lastly, new developments in MFA are described that can be expected to improve the range and reliability of plant MFA in the coming years.     

Exploiting binding-site arginines in drug design: Recent examples

Active or allosteric site arginines can form diverse interactions with ligands including different types of cation-π interactions, H-bond interactions and non-bond, non-canonical interactions. This provides many opportunities for creative structure-based drug design to improve potency, introduce novelty, and modulate MoA (mode of action), and even to achieve selectivity. This digest will use some recent drug targets of interest as examples to illustrate different types of interactions and how these interactions impact on potency, MoA, and selectivity.     

GPCR structures in drug design,emerging opportunities with new structures

In recent years, GPCR targets from diverse regions of phylogenetic space have been determined. This effort has culminated this year in the determination of representatives of all major classes of GPCRs (A, B, C, and F). Although much of the now well established knowledge on GPCR structures has been known for some years, the new high-resolution structures allow structural insight into the causes of ligand efficacy, biased signaling, and allosteric modulation. In this digest the structural basis for GPCR signaling in the light of the new structures is reviewed and the use of the new non-class A GPCRs for drug design is discussed.     

Docking, virtual high throughput screening and in silico fragment-based drug design

The drug discovery process has been profoundly changed recently by the adoption of computational methods helping the design of new drug candidates more rapidly and at lower costs. In silico drug design consists of a collection of tools helping to make rational decisions at the different steps of the drug discovery process, such as the identification of a biomolecular target of therapeutical interest, the selection or the design of new lead compounds and their modification to obtain better affinities, as well as pharmacokinetic and pharmacodynamic properties. Among the different tools available, a particular emphasis is placed in this review on molecular docking, virtual high-throughput screening and fragment-based ligand design.     

Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis

Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h(-1)) and high (0.44 h(-1)) dilution rates. Using a mixture of 10% [U-(13)C] and 90% glucose labeled at natural abundance, (13)C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [(13)C, (1)H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a chi(2) test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.     

Trehalose synthesis inhibitor: A molecular in silico drug design

Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.     

Dynamic simulation of pollutant effects on the threonine pathway in Escherichia coli

The enzymatic activities of threonine pathway in Escherichia coli are sensitive to pollutants such as cadmium, copper and mercury, which, even at low concentration, can substantially decrease or even block the pathway at several steps. Our aim was to investigate the complex effects on a metabolic pathway of such general enzyme inhibitors with several sites of action, using a previously developed computer simulation of the pathway. For this purpose, the inhibition parameters were experimentally determined and incorporated in the model. The calculation of the flux control coefficient distribution between the five steps of the threonine pathway showed that control remains shared between the three first steps under most inhibition conditions. Response coefficient analysis shows that the inhibition of aspartate semialdehyde dehydrogenase is quantitatively dominant in most circumstances.     

Metabolic analysis of antibody producing CHO cells in fed‐batch production

Chinese hamster ovary (CHO) cells are commonly used for industrial production of recombinant proteins in fed batch or alternative production systems. Cells progress through multiple metabolic stages during fed‐batch antibody (mAb) production, including an exponential growth phase accompanied by lactate production, a low growth, or stationary phase when specific mAb production increases, and a decline when cell viability declines. Although media composition and cell lineage have been shown to impact growth and productivity, little is known about the metabolic changes at a molecular level. Better understanding of cellular metabolism will aid in identifying targets for genetic and metabolic engineering to optimize bioprocess and cell engineering. We studied a high expressing recombinant CHO cell line, designated high performer (HP), in fed‐batch productions using stable isotope tracers and biochemical methods to determine changes in central metabolism that accompany growth and mAb production. We also compared and contrasted results from HP to a high lactate producing cell line that exhibits poor growth and productivity, designated low performer (LP), to determine intrinsic metabolic profiles linked to their respective phenotypes. Our results reveal alternative metabolic and regulatory pathways for lactate and TCA metabolite production to those reported in the literature. The distribution of key media components into glycolysis, TCA cycle, lactate production, and biosynthetic pathways was shown to shift dramatically between exponential growth and stationary (production) phases. We determined that glutamine is both utilized more efficiently than glucose for anaplerotic replenishment and contributes more significantly to lactate production during the exponential phase. Cells shifted to glucose utilization in the TCA cycle as growth rate decreased. The magnitude of this metabolic switch is important for attaining high viable cell mass and antibody titers. We also found that phosphoenolpyruvate carboxykinase (PEPCK1) and pyruvate kinase (PK) are subject to differential regulation during exponential and stationary phases. The concomitant shifts in enzyme expression and metabolite utilization profiles shed light on the regulatory links between cell metabolism, media metabolites, and cell growth. Biotechnol. Bioeng. 2013; 110: 1735–1747. © 2013 Wiley Periodicals, Inc.     

Intérêt de la TEP/TDM au 18F-FDG dans la neurofibromatose de type 1, expérience du centre national de référence Henri-Mondor sur 10 ans

ObjectiveMalignant peripherals nerve sheath tumours (MPNST) are one of the leading causes of death in neurofibromatosis type 1 (NF1). Given the temporal and spatial multiplicity of suspicious lesions, the histological diagnosis of certainty might require iterative and decaying surgical procedure. Our objective was to determine the threshold values of metabolic metrics determined on ¹⁸F-FDG PET/CT (SUV_max, total lesion glycolysis [TLG], total metabolic tumour volume [TMTV], tumour-to-liver [T/L] ratio and heterogeneity index [HI]) in order to distinguish at best MPNST from benign lesion (simple neurofibromas [NF] or dysplastic NF).Patients and methodsHundred and seven patients from the national reference centre of NF1 Henri-Mondor, clinically suspects of TMGN, underwent 160 ¹⁸F-FDG PET/CT over a period of 10 years, between 2005 and 2015. The hypermetabolics lesions identified on ¹⁸F-FDG PET/CT were confronted with pathological analysis or clinico-radiological and metabolic follow-up of more than 6 months.ResultsFour hundred and eight hypermetabolics lesions were identified, of which 112 were histologically confronted with 38 simple NF, 29 dysplastic NF, 39 TMGN and 6 incidentalomas. The remaining 296 hypermetabolics lesions experienced a median follow-up of 40.4 months [13.5–137 months]. The optimal cut-off values for malignant lesions, determined by ROC curves, were in order of decreasing performance: T/L ratio > 2.03 (sensitivity 96%, specificity 88%); SUVmax > 4.74 (sensitivity 93%, specificity 86%); TLG > 172 (sensitivity 84%, specificity 76%); TMTV > 53.5 (sensitivity 83%, specificity 68%); HI > 1.63 (sensitivity 84%, specificity 54%).Conclusions¹⁸F-FDG PET/CT is a powerful diagnostic and prognostic tool in the management of TMGN. Metabolic metrics allow with good sensitivity and specificity to identify lesions transformed into TMGN. The advent of PET/MRI will undoubtedly allow in the near future to reinforce the diagnostic and prognostic performances for the detection of transformations of neurofibromas in TMGN.     

Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data

The novel concept of isotopic dynamic 13C metabolic flux analysis (ID-13C MFA) enables integrated analysis of isotopomer data from isotopic transient and/or isotopic stationary phase of a 13C labeling experiment, short-time experiments, and an extended range of applications of 13C MFA. In the presented work, an experimental and computational framework consisting of short-time 13C labeling, an integrated rapid sampling procedure, a LC-MS analytical method, numerical integration of the system of isotopomer differential equations, and estimation of metabolic fluxes was developed and applied to determine intracellular fluxes in glycolysis, pentose phosphate pathway (PPP), and citric acid cycle (TCA) in Escherichia coli grown in aerobic, glucose-limited chemostat culture at a dilution rate of D = 0.10 h(-1). Intracellular steady state concentrations were quantified for 12 metabolic intermediates. A total of 90 LC-MS mass isotopomers were quantified at sampling times t = 0, 91, 226, 346, 589 s and at isotopic stationary conditions. Isotopic stationarity was reached within 10 min in glycolytic and PPP metabolites. Consistent flux solutions were obtained by ID-13C MFA using isotopic dynamic and isotopic stationary 13C labeling data and by isotopic stationary 13C MFA (IS-13C MFA) using solely isotopic stationary data. It is demonstrated that integration of dynamic 13C labeling data increases the sensitivity of flux estimation, particularly at the glucose-6-phosphate branch point. The identified split ratio between glycolysis and PPP was 55%:44%. These results were confirmed by IS-13C MFA additionally using labeling data in proteinogenic amino acids (GC-MS) obtained after 5 h from sampled biomass.     

A technique to study molecular recognition in drug design: Preliminary application of free energy derivatives to inhibition of a malarial cysteine protease

We present molecular dynamics studies on model complexes of inhibitors of a malarial cysteine protease. The initial model for such complexes came from the model building of the protein using its homology with other cysteine proteases and calculations using DOCK to generate new lead compounds. Some of the initial model-built structures were quite stable for 100 psec of dynamics; others moved significantly from their model-built orientation. We also calculated the free energy derivatives at each atom in the inhibitor, both in water and in the binding site. The results of these calculations suggest directions for the design of new, more potent enzyme inhibitors and agree qualitatively with some of the experimental findings. Nonetheless, we stress that we have only used this methodology in an interpretive rather than a predictive manner.     

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium

Haemophilus influenzae type b (Hib), a Gram‐negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl‐ribitol‐phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake‐flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (～500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α‐ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508–1519, 2017     

Stopped‐flow chemiluminescence determination of the anticancer drug capecitabine: Application in pharmaceutical analysis and drug‐delivery systems

Capecitabine is a chemotherapeutic agent used for the treatment of patients with metastatic cancers. This study aimed at determining the drug capecitabine in a simple chemiluminescence (CL) system of acidic potassium permanganate using the stopped‐flow injection technique. Statistical methods were used to detect optimum conditions. The method showed two linear calibration ranges from 6.7 × 10^?6 to 6.7 × 10^?5 mol L^?1 and from 6.7 × 10^?5 to 2.7 × 10^?3 mol L^?1 with a detection limit of 1.5 × 10^?6 mol L^?1. Chitosan‐modified magnetic nanoparticles were studied in the drug‐delivery experiments. According to the pH sensitivity of chitosan and low pH values in tumour cells, the chitosan‐coated magnetic nanoparticles could provide a good targeting drug‐delivery system to tumour sites. To evaluate the applicability of the method, the capecitabine‐loaded magnetic chitosan nanoparticles were synthesized with two different cross‐linkers; loading and releasing rates of the drug were investigated using the proposed CL method and an ultraviolet–visible light spectrophotometric method (absorption at 305 nm). The results showed a good correlation between the two methods, and it was found that the synthesized chitosan‐modified magnetic nanoparticles could be used for pH‐dependent release of capecitabine in cancer cells. Moreover, determination of capecitabine in tablets and synthetic samples was performed.     

Data Processing System (DPS) software with experimental design,statistical analysis and data mining developed for use in entomological research

Abstract A comprehensive but simple‐to‐use software package called DPS (Data Processing System) has been developed to execute a range of standard numerical analyses and operations used in experimental design, statistics and data mining. This program runs on standard Windows computers. Many of the functions are specific to entomological and other biological research and are not found in standard statistical software. This paper presents applications of DPS to experimental design, statistical analysis and data mining in entomology.