Dynamic metabolic models of CHO cell cultures through minimal sets of elementary flux modes.

The concept of Elementary Flux Modes (EFMs) has been of central importance in a number of studies involving the analysis of metabolism. In Provost and Bastin (2007) this concept is used to translate the metabolic networks of the different phases of CHO cell cultures into macroscopic bioreactions linking extracellular substrates to products. However, a critical issue concerns the calculation of these elementary flux vectors, as their number combinatorially increases with the size of the metabolic network. In this study, a detailed metabolic network of CHO cells is considered, where the above-mentioned combinatorial explosion makes the computation of the elementary flux modes impossible. To alleviate this problem, a methodology proposed in Jungers et al. (2011) is used to compute a decomposition of admissible flux vectors in a minimal number of elementary flux modes without explicitly enumerating all of them. As a result, a set of macroscopic bioreactions linking the extracellular measured species is obtained at a very low computational expense. The procedure is repeated for the several cell culture phases and a global model is built using a multi-model approach, which is able to successfully predict the evolution of experimental data.

A detailed metabolic flux analysis of an underdetermined network of CHO cells.

In this article the metabolic flux analysis of growing CHO-320 cells is performed for a detailed metabolic network which involves 100 reactions and embraces all the significant pathways describing the metabolism of CHO cells. The purpose is to investigate the efficiency of the flux analysis when it is based on a relatively small set of extracellular measurements that can be easily achieved in most laboratories. In this case the flux analysis problem leads to a generally underdetermined mass balance system, as data are not sufficient to uniquely define the metabolic fluxes. Our main contribution is to show that, provided the system of mass balance equations is well-posed, although it is underdetermined, very narrow intervals may be found for most fluxes. The importance of checking the well-posedness of the problem is emphasized and the influence of the number of available measurements on the accuracy of the metabolic flux intervals is systematically investigated. In all cases the computed flux intervals are bounded and a single well defined value is obtained for the formation rates of the cellular macromolecules (proteins, DNA, RNA, lipids) that are not measured. The potential gain of a simple theoretical assumption regarding the metabolism of Threonine is also discussed and compared with an optimal solution calculated by maximizing the biomass formation rate. Alternative network structures obtained by inverting the direction of reversible reactions are also considered. Finally, the results of the metabolic flux analysis are exploited to estimate the total energy production resulting from the metabolism of growing CHO-320 cells.

Wavelength- and energy-dispersive Electron Probe Microanalysis (EPMA) measurements with non-perpendicular incidence of the electron beam.

A database of 416 wavelength- and energy-dispersive EPMA measurements on tilted specimens of NiAl, TiO(2) and Ti(3)Al is presented. The analyses were performed between 10 and 30 kV and the tilt angles were varied between 0 degrees and 60 degrees in seven steps. The necessary hardware modifications for the specimen holder are discussed, as well as the various focusing techniques used in the measurements. A comparison between the experimental data, the calculations of our proza96t program and the results of Monte Carlo simulations shows that up to 50 degrees tilt the predictions of our software are more than satisfactory. At larger tilt angles some deviations become noticeable. The Monte Carlo simulations appear to produce deviations at a somewhat earlier stage already, for reasons as yet unknown.

Metabolic design of macroscopic bioreaction models: application to Chinese hamster ovary cells.

The aim of this paper is to present a systematic methodology to design macroscopic bioreaction models for cell cultures based upon metabolic networks. The cell culture is seen as a succession of phases. During each phase, a metabolic network represents the set of reactions occurring in the cell. Then, through the use of the elementary flux modes, these metabolic networks are used to derive macroscopic bioreactions linking the extracellular substrates and products. On this basis, as many separate models are obtained as there are phases. Then, a complete model is obtained by smoothly switching from model to model. This is illustrated with batch cultures of Chinese hamster ovary cells.

Design of an efficient medium for insect cell growth and recombinant protein production.

We report the development of a new serum-free medium based on the use of factorial experiments. At first, a variety of hydrolysates were screened using a fractional factorial approach with High-Five cells. From this experiment yeastolate ultrafiltrate was found to have, by far, the most important effect on cell growth. Furthermore, Primatone RL was found to remarkably prolong the stationary phase of Sf-9 and High-Five cell cultures. The optimal concentrations for yeastolate and Primatone were determined to be 0.6 and 0.5%, respectively, on the basis of a complete factorial experiment. This new medium, called YPR, supported good growth of both Sf-9 and High-Five cells in batch cultures, with maximal densities of 5.4 and 6.1 x 10(6) cells/ml, respectively. In addition, both cell lines achieved good growth in bioreactor batch culture and had a prolonged stationary phase of 3-4 d in YPR medium compared to Insect-XPRESS medium. The ability of the new medium to support recombinant protein expression was also tested by infecting Sf-9 or High-Five cells at high density (2 x 10(6) cells/ml) with a baculovirus expressing secreted placental alkaline phosphatase (SEAP). The maximum total SEAP concentration after 7 d was about 43 lU/ml (58 mg/L) and 28 lU/ml (39 mg/L) for High-Five and Sf-9 cells, respectively.

Design of a fungal bioprocess for vanillin production from vanillic acid at scalable level by Pycnoporus cinnabarinus.

The biotechnological process of vanillin production from vanillic acid by Pycnoporus cinnabarinus was scaled-up at the laboratory level. Vanillin production was studied in two types of bioreactors, a mechanically agitated and an air-lift bioreactor. In the mechanically agitated bioreactor where vanillin was produced in greater quantities, oxygen availability was studied during the growth and production phases. A maximal aeration rate (90l/h equivalent to 0.83 volume of air/volume of medium/min or vvm) during the growth phase and a minimal aeration rate (30 l/h equivalent to 0.28 vvm) during the production phase were necessary to increase vanillin production to 1260 mg/l. Vanillic acid bioconversion to vanillin occurred under the conditions of reduced dissolved oxygen concentration, gentle agitation, high carbon dioxide production and low specific growth rate. However, under these conditions, vanillin production was accompanied by a significant amount of methoxyhydroquinone. Vanillin over a concentration of 1000 mg/l was shown to be highly toxic to the growth of P. cinnabarinus on agar medium. The application of selective XAD-2 resin led to a reduction of vanillin concentration in the medium, thus limiting its toxicity towards the fungal biomass as well as the formation of unwanted by-products such as methoxyhydroquinone and allowed the concentration of vanillin produced to reach 1575 mg/l.

Mass balance modeling of vanillin production from vanillic acid by cultures of the fungus Pycnoporus cinnabarinus in bioreactors.

A systematic two-step procedure for the structural identification of bioprocesses is followed in order to establish a mechanistic model for vanillin production by Pycnoporus cinnabarinus. The first step is devoted to the identification of the underlying reaction structure and the development of a validated mass balance model for the growth of P. cinnabarinus and the biotransformation of vanillic acid into vanillin. The second step is devoted to the kinetic modeling, namely, the estimation of the reaction rates and the calibration of the kinetic parameters. The whole procedure leads to the final set up of a simulation model of the process. The results are supported by the data from five cultures of P. cinnabarinus in bioreactors.

Autologous stem cell infusion for acute myeloblastic leukemia in an HIV-1 carrier.

We present the case of an asymptomatic HIV carrier, who presented with acute myeloblastic leukemia in third relapse and successfully underwent autologous stem cell transplantation as a rescue treatment. This observation supports the conclusion that tolerance of autologous bone marrow or stem cell transplant in patients with HIV may correlate with a low viral burden and relatively good immune function.

Importance of ionized magnesium measurement for monitoring of citrate-anticoagulated plateletpheresis.

BACKGROUND: The infusion of citrate during apheresis may affect the levels of ionized magnesium in the blood. Hypomagnesemia and concomitant hypocalcemia could influence the parathormone response and could be responsible for some of the symptoms observed during apheresis. STUDY DESIGN AND METHODS: The study reports measurement of ionized magnesium by the new ion-selective electrode technique in response to citrate infusion in 15 donors undergoing continuous flow high-yield plateletpheresis. The monitoring included measurement of ionized calcium and parathormone every 30 minutes during the 120-minute apheresis (plus the next 30 minutes to assess recovery). RESULTS: Ionized magnesium fell by 30 +/- 4 percent (mean +/- SD, p<0.01), which contrasts with minor changes in total concentrations. Comparison of variations in the levels of ionized and total magnesium found major formation of complexes during citrate infusion. Ionized calcium fell by 15 +/- 3 percent (p<0.01), while parathormone peaked at 356 +/- 114 percent (p<0.01) of initial value after 30 minutes. Ionized cations and parathormone recovered by more than 50 percent within 30 minutes of the end of apheresis. CONCLUSION: An acute and steep drop in ionized magnesium occurs during citrate administration. The measurement of ionized magnesium should be included in future prospective studies of donor safety and parathormone regulation during apheresis.

Simplification of the blood stem cell transplantation (BSCT) procedure: large volume apheresis and uncontrolled rate cryopreservation at -80 degrees C.

Very high-dose chemotherapy with autologous blood stem cell (BSC) rescue becomes more and more widely performed. In order to simplify the technique, a large volume apheresis programme combined with an uncontrolled rate cryopreservation at -80 degrees C was developed. Twenty-six patients suffering from multiple myeloma (n = 8), non-Hodgkin's lymphoma (n = 7), dysgerminoma (n = 4), breast cancer (n = 3), Hodgkin's disease (n = 2), acute lymphoblastic leukaemia (n = 1) and acute myelocytic leukaemia (n = 1) were autografted after a classical high-dose chemotherapy regimen. A single large volume apheresis was sufficient to obtain the threshold value of CD34+ BSC in 24/26 transplantations. The haematological recovery was favourably comparable with the previously published data obtained with controlled rate frozen BSC: median time to granulocytes > 1000/microL and to a self-supporting platelet count > 20,000/microL, respectively, 10.5 and 12 d. The treatment-related mortality was confined to 1/26 BSCT. These results indicate that this easy and cost-saving policy of BSCT is efficacious and safe: sustained long-term haematopoiesis, reduced morbidity and mortality were observed.

Structural identifiability of the yield coefficients in bioprocess models when the reaction rates are unknown.

The identification problem of bioprocesses in a stirred tank reactor essentially concerns the determination of the yield coefficients and the reaction rates involved in a general state-space model. This paper deals with the analysis of structural identifiability of the yield coefficients issuing from a two-step identification procedure. In fact, the identification of the yield coefficients can be completely decoupled from that of the reaction rates by means of an appropriate transformation of the dynamical model. It is shown that the identifiability properties of these coefficients can be drawn from the structure of the underlying reaction network. Necessary and sufficient identifiability conditions are given in the form of some simple algebraic tests.

[Novel autograft method using positive selection of CD34 stem cells]. / Méthode nouvelle d'autogreffe par sélection positive de cellules souches sanguines CD34.

High dose chemotherapy with autologous blood stem cell rescue becomes widely used for patients with hematologic malignancies and solid tumors. Recently, it has been demonstrated that stem cells characterized by the CD34 antigenic marker could be positively selected using an anti CD34 monoclonal antibody and an avidin biotin immunoabsorption device. We report our experience of twelve selections and ten grafts. A CD34+ cells enrichment of 1.9 log (purity: 72%) and a CFU-GM cells concentration of 1.6 log have been obtained. In ten transplanted patients, the hematological recovery was similar to that obtained with non selected blood stem cells. The CD34+ cells purification allows mini graft infusion and purge of residual tumor cells implicated in relapse after autologous stem cells transplantation.

Optimal blood stem cell mobilization using 10 micrograms/kg granulocyte colony-stimulating factor (G-CSF) alone for high-dose melphalan intensification in multiple myeloma: an intrapatient controlled study.

A recent randomized multicentric French study has shown that intensification with stem cell rescue improves the response rate and progression-free survival in multiple myeloma. Transplantation with primed peripheral blood stem cells (PBSC) displays a faster hematological recovery, especially for platelets, as compared with a bone marrow stem cell graft. In multiple myeloma, the optimal mobilization method for PBSC is unknown. The present study compares mobilization with cyclophosphamide 4 g/m2 + G-CSF 5 micrograms/kg versus G-CSF 5 micrograms/kg alone versus G-CSF 10 micrograms/kg alone in two cases of multiple myeloma, using an intrapatient controlled evaluation of the amount of CD34-positive cells obtained during each leukapheresis. In both cases, the highest CD34-positive cells yield was obtained with G-CSF at 10 micrograms/kg. Despite the low number of cases, this method, devoid of life-threatening toxicity, might be of greatest interest in multiple myeloma.

Nonlinear control of biotechnological processes with growth-production decoupling.

Nonlinear control design techniques for a class of continuous biological processes with growth and production decoupling are investigated. We establish, under realistic assumptions on the kinetics, that though neither the inlet substrate concentration nor the dilution rate can achieve linearization of the global dynamics, we can maximize the dimension of the linear system obtained after feedback and get stable zero dynamics by choosing output functions having a physical meaning. More precisely, if the manipulated variable is the inlet substrate concentration, then the output can be chosen as the biomass concentration. But if the chosen input is the dilution rate, then a suitable output corresponds to yields.

Optimal control of biomass growth in a mixed culture.

It is shown that, in a mixed culture, under realistic assumptions, the optimal temperature profile maximizing the final biomass production under the constraint of the final relative proportions of the two populations is constant. This result is illustrated with a simulation experiment on a lactic fermentation model.

Central venous access for haemodialysis using the Hickman catheter.

One hundred and seven Hickman catheters for haemodialysis were inserted in 90 end-stage chronic renal failure patients, and were used for 1-448 days (median 45 days). Sixty-nine per cent of the patients were treated without any problem for 1-165 days (median 34 days). Clinically evident complications occurred in 44 catheters inserted in 28 patients, and included outflow obstruction (16.8% of the catheters) and thrombosis (13.1% of the catheters). However, many episodes of clotting or insufficient flow could be corrected by simple manoeuvres. Other less frequent complications were recorded: sepsis, mainly in patients with increased risk factors (4.1% of the catheters), laceration of the catheter (3.7%) and occasional cases of jugular-vein phlebitis, transient palsy of a vocal cord, haematoma of the wound, and bleeding of the cutaneous orifice. No clinical sign of subclavian or innominate-vein thrombosis was observed. Nevertheless, a prospective study conducted in 50 asymptomatic patients demonstrated a 12% rate of anomalies of the venous system, although two-thirds of these alterations were mild and had no consequence. When the present series is compared to the results obtained with currently available percutaneous haemodialysis catheters, it is concluded that the Hickman catheter is a safe, comfortable and efficient vascular access device.

Hand-held computer program for field-capture and analysis of herbage yield and composition data using a modified dry-weight-rank and yield estimate method.

A BASIC program is described which is used to collect, check and analyse rank estimates of plant yield in the field. The program operates in a portable, battery-powered Sharp PC1500A hand-held computer than can be used in a field environment. Data are collected using a modified dry-weight-rank method and comparative yield estimates. Much of the software is designed to trap incorrect data entry. Raw data or summary data may be printed, displayed, and stored on cassette tape or transferred to another computer through a communications interface. The program can be easily modified to run on other models of the Sharp PC series or other portable computers that use a similar BASIC interpreter.

Adaptive control of anaerobic digestion processes-a pilot-scale application.

A simple adaptive control algorithm, for which theoretical stability and convergence properties had been previously demonstrated, has been successfully implemented on a biomethanation pilot reactor. The methane digester, operated in the CSTR mode was submitted to a shock load, and successfully computer controlled during the subsequent transitory state.