Development of process control strategies exploiting knowledge from systems biology: application to MDCK suspension cells

Madine Darby Canine Kidney (MDCK) cell lines have been extensively evaluated for their potential as host cells for influenza vaccine production. Recent studies allowed the cultivation of these cells in a fully defined medium and in suspension. However, reaching high cell densities in animal cell cultures still remains a challenge. To address this shortcoming, a combined methodology allied with knowledge from systems biology was reported to study the impact of the cell environment on the flux distribution. An optimization of the medium composition was proposed for both a batch and a continuous system in order to reach higher cell densities. To obtain insight into the metabolic activity of these cells, a detailed metabolic model previously developed by Wahl A. et. al was used. The experimental data of four cultivations of MDCK suspension cells, grown under different conditions and used in this work came from the Max Planck Institute, Magdeburg, Germany. Classical metabolic flux analysis (MFA) was used to estimate the intracellular flux distribution of each cultivation and then combined with partial least squares (PLS) method to establish a link between the estimated metabolic state and the cell environment. The validation of the MFA model was made and its consistency checked. The resulted PLS model explained almost 70% of the variance present in the flux distribution. The medium optimization for the continuous system and for the batch system resulted in higher biomass growth rates than the ones obtained experimentally, 0.034 h-1 and 0.030 h-1, respectively, thus reducing in almost 10 hours the duplication time. Additionally, the optimal medium obtained for the continuous system almost did not consider pyruvate. Overall the proposed methodology seems to be effective and both proposed medium optimizations seem to be promising to reach high cell densities.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Improvement of in silico strain engineering methods in Saccharomyces cerevisiae

PhD thesis in Bioengineering

On line pre-concentration for simultaneous determination of low molecular weight organic acids and inorganic anions in Amazonian river water samples employing ion chromatography with conductivity detection

An ion chromatography procedure, employing an IonPac AC15 concentrator column was used to investigate on line preconcentration for the simultaneous determination of inorganic anions and organic acids in river water. Twelve organic acids and nine inorganic anions were separated without any interference from other compounds and carry-over problems between samples. The injection loop was replaced by a Dionex AC15 concentrator column. The proposed procedure employed an auto-sampler that injected 1.5 ml of sample into a KOH mobile phase, generated by an Eluent Generator, at 1.5 mL min-1, which carried the sample to the chromatographic columns (one guard column, model AG-15, and one analytical column, model AS15, with 250 x 4mm i.d.). The gradient elution concentrations consisted of a 10.0 mmol l-1 KOH solution from 0 to 6.5 min, gradually increased to 45.0 mmol l-1 KOH at 21 min., and immediatelly returned and maintained at the initial concentrations until 24 min. of total run. The compounds were eluted and transported to an electro-conductivity detection cell that was attached to an electrochemical detector. The advantage of using concentrator column was the capability of performing routine simultaneous determinations for ions from 0.01 to 1.0 mg l-1 organic acids (acetate, propionic acid, formic acid, butyric acid, glycolic acid, pyruvate, tartaric acid, phthalic acid, methanesulfonic acid, valeric acid, maleic acid, oxalic acid, chlorate and citric acid) and 0.01 to 5.0 mg l-1 inorganic anions (fluoride, chloride, nitrite, nitrate, bromide, sulfate and phosphate), without extensive sample pretreatment and with an analysis time of only 24 minutes.

Nonhemocyte sources of selected lysosomal enzymes in Biomphalaria glabrata, B. tenagophila and B. straminea (Mollusca: Pulmonata)

The specific activities of acid phosphatase, alkaline phosphatase, β-glucuronidase, lysozymes, glutamate-oxalacetate transaminase and glutamate-pyruvate transaminate were determined in the head-foot and digestive gland of Brazilian Biomphalaria glabrata (Touros), B. tenagophila (Caçapava) and B. straminea (Monsenhor Gil). All six enzymes were detected inthe 3000g supernatant. Both cytoplasmic enzymes, glutamate-oxalacetate and glutamate-pyruvate transaminase exhibited the highest specific activities. In the case of the four hydrolytic enzymes assayed, β-glucuronidase exhibited the highest specific activity while lysozyme showed the lowest activity. All six enzymes are thought to be produced by cells within the head-foot and digestive gland of B. glabrata, B. tenagophila and B. straminea.

Sistema de secreción de tipo III en Aeromonas mesòfilas

Aeromonas hydrophila és un bacil gram-negatiu, patogen oportunista d’animal i humans. La patogènesi d’A. Hydrophila és multifactorial. A fi d'identificar gens implicats en la virulència de la soca PPD134/91 d’A. hydrophila, vam realitzar experiments de substracció gènica, que van dur a la detecció de 22 fragments d’ADN que codificaven 19 potencials factors de virulencia, incloent un gen que codificava una proteïna de sistema de secreció de tipus III (T3SS). La importància creixent del T3SS en la patogènesi de diversos bacteris, ens va dur a identificar i analitzar l'agrupació gènica del T3SS de les soques AH-1 i AH-3 d’A. hydrophila. La inactivació dels gens de T3SS aopB i aopD d’A. hydrophila AH-1, i ascV d’A. hydrophila AH-3, comporta una disminució de la citotoxicitat, un increment de la fagocitosi, i una reducció de la virulència en diferents models animals. Aquests resultats demostren que el T3SS és necessari per a la patogenicitat. També vam clonar i seqüenciar una ADP-ribosiltransferasa (AexT) a la soca AH-3 d’A. hydrophila, i vam demostrar que aquesta toxina és translocada via el T3SS, sistema que al seu torn sembla ser induïble in vitro en condicions de depleció de calci. El mutant en el gen aexT de la soca AH-3 d’A. hydrophila va mostrar una lleugera reducció de la virulència, assajada amb diferents mètodes. Mitjançant l'ús de diferents sondes d’ADN, vam determinar la presència del T3SS en soques tant clíniques com ambientals de diferents espècies del gènere Aeromonas: A. hydrophila, A. veronii, i A. caviae, i la codistribució d'aquesta agrupació gènica i el gen aexT. Finalment, amb la finalitat d'estudiar la regulació transcripcional de l'agrupació gènica de T3SS i de l’efector AexT A. hydrophila AH-3, vam aïllar els promotors predits per l’operó aopN-aopD i el gen aexT, i els vam fusionar amb el gen reporter gfp (Green Fluorescence Protein). A més, vam demostrar que l'expressió d'ambdós promotors depèn de diferents components bacterians, com per exemple el sistema de dos components PhoP/PhoQ, el sistema de quorum sensing AhyI/AhyR, o el complex piruvat deshidrogenasa.

Aggregating Brain Cell Cultures as a Model to Study Ischaemia-induced Neurodegeneration.

Rotation-mediated aggregating brain cell cultures at two different maturational stages (DIV 11 and DIV 20) were subjected for 1 or 2 hours to ischaemic conditions by transient immobilization (arrest of media circulation). During recovery, cell damage was evaluated by measuring changes in cell type-specific enzyme activities and total protein content. It was found that in immature cultures (DIV 11), immobilization for 1 or 2 hours did not affect the parameters measured. By contrast, at DIV 20, ischaemic conditions for 1 hour caused a pronounced decrease in the activities of glutamic acid decarboxylase and choline acetyltransferase. A significant decrease in these neuron-specific enzyme activities was found at post-ischaemic days 1-14, indicating immediate and irreversible neuronal damage. The activity of the astrocyte-specific enzyme, glutamine synthetase, was significantly increased at 4 days post-treatment; equal to control values at 6 days; and significantly decreased at 14 days after the ischaemic insult. Immobilization of DIV 20 cultures for 2 hours caused a drastic reduction in all the parameters measured at post-ischaemic day 6. Generally, the ischaemic conditions appeared to be more detrimental to neurons than to astrocytes, and GABAergic neurons were more affected than cholinergic neurons.

Compartmentalized Cerebral Metabolism of [1,6-(13)C]Glucose Determined by in vivo (13)C NMR Spectroscopy at 14.1 T.

Cerebral metabolism is compartmentalized between neurons and glia. Although glial glycolysis is thought to largely sustain the energetic requirements of neurotransmission while oxidative metabolism takes place mainly in neurons, this hypothesis is matter of debate. The compartmentalization of cerebral metabolic fluxes can be determined by (13)C nuclear magnetic resonance (NMR) spectroscopy upon infusion of (13)C-enriched compounds, especially glucose. Rats under light α-chloralose anesthesia were infused with [1,6-(13)C]glucose and (13)C enrichment in the brain metabolites was measured by (13)C NMR spectroscopy with high sensitivity and spectral resolution at 14.1 T. This allowed determining (13)C enrichment curves of amino acid carbons with high reproducibility and to reliably estimate cerebral metabolic fluxes (mean error of 8%). We further found that TCA cycle intermediates are not required for flux determination in mathematical models of brain metabolism. Neuronal tricarboxylic acid cycle rate (V(TCA)) and neurotransmission rate (V(NT)) were 0.45 ± 0.01 and 0.11 ± 0.01 μmol/g/min, respectively. Glial V(TCA) was found to be 38 ± 3% of total cerebral oxidative metabolism, accounting for more than half of neuronal oxidative metabolism. Furthermore, glial anaplerotic pyruvate carboxylation rate (V(PC)) was 0.069 ± 0.004 μmol/g/min, i.e., 25 ± 1% of the glial TCA cycle rate. These results support a role of glial cells as active partners of neurons during synaptic transmission beyond glycolytic metabolism.

Accuracy of Brain Multimodal Monitoring to Detect Cerebral Hypoperfusion After Traumatic Brain Injury.

OBJECTIVE:: To examine the accuracy of brain multimodal monitoring-consisting of intracranial pressure, brain tissue PO2, and cerebral microdialysis-in detecting cerebral hypoperfusion in patients with severe traumatic brain injury. DESIGN:: Prospective single-center study. PATIENTS:: Patients with severe traumatic brain injury. SETTING:: Medico-surgical ICU, university hospital. INTERVENTION:: Intracranial pressure, brain tissue PO2, and cerebral microdialysis monitoring (right frontal lobe, apparently normal tissue) combined with cerebral blood flow measurements using perfusion CT. MEASUREMENTS AND MAIN RESULTS:: Cerebral blood flow was measured using perfusion CT in tissue area around intracranial monitoring (regional cerebral blood flow) and in bilateral supra-ventricular brain areas (global cerebral blood flow) and was matched to cerebral physiologic variables. The accuracy of intracranial monitoring to predict cerebral hypoperfusion (defined as an oligemic regional cerebral blood flow < 35 mL/100 g/min) was examined using area under the receiver-operating characteristic curves. Thirty perfusion CT scans (median, 27 hr [interquartile range, 20-45] after traumatic brain injury) were performed on 27 patients (age, 39 yr [24-54 yr]; Glasgow Coma Scale, 7 [6-8]; 24/27 [89%] with diffuse injury). Regional cerebral blood flow correlated significantly with global cerebral blood flow (Pearson r = 0.70, p < 0.01). Compared with normal regional cerebral blood flow (n = 16), low regional cerebral blood flow (n = 14) measurements had a higher proportion of samples with intracranial pressure more than 20 mm Hg (13% vs 30%), brain tissue PO2 less than 20 mm Hg (9% vs 20%), cerebral microdialysis glucose less than 1 mmol/L (22% vs 57%), and lactate/pyruvate ratio more than 40 (4% vs 14%; all p < 0.05). Compared with intracranial pressure monitoring alone (area under the receiver-operating characteristic curve, 0.74 [95% CI, 0.61-0.87]), monitoring intracranial pressure + brain tissue PO2 (area under the receiver-operating characteristic curve, 0.84 [0.74-0.93]) or intracranial pressure + brain tissue PO2+ cerebral microdialysis (area under the receiver-operating characteristic curve, 0.88 [0.79-0.96]) was significantly more accurate in predicting low regional cerebral blood flow (both p < 0.05). CONCLUSION:: Brain multimodal monitoring-including intracranial pressure, brain tissue PO2, and cerebral microdialysis-is more accurate than intracranial pressure monitoring alone in detecting cerebral hypoperfusion at the bedside in patients with severe traumatic brain injury and predominantly diffuse injury.

Differentiation of rat striatal embryonic stem cells in vitro: monolayer culture vs. three-dimensional coculture with differentiated brain cells.

Several groups have demonstrated the existence of self-renewing stem cells in embryonic and adult mouse brain. In vitro, these cells proliferate in response to epidermal growth factor, forming clusters of nestin-positive cells that may be dissociated and subcultured repetitively. Here we show that, in stem cell clusters derived from rat embryonic striatum, cell proliferation decreased with increasing number of passages and in response to elevated concentrations of potassium (30 mM KCl). In monolayer culture, the appearance of microtubule-associated protein type-5-immunoreactive (MAP-5(+)) cells (presumptive neurons) in response to basic fibroblast growth factor (bFGF) was reduced at low cell density and with increasing number of passages. In the presence of bFGF, elevated potassium caused a more differentiated neuronal phenotype, characterized by an increased proportion of MAP-5(+) cells, extensive neuritic branching, and higher specific activity of glutamic acid decarboxylase. Dissociated stem cells were able to invade cultured brain cell aggregates containing different proportions of neurons and glial cells, whereas they required the presence of a considerable proportion of glial cells in the host cultures to become neurofilament H-positive. The latter observation supports the view that astrocyte-derived factors influence early differentiation of the neuronal cell lineage.

Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition

We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways. Indeed, if alternative (compensative) pathway(s) can operate after blockage of the de novo PC biosynthesis pathway this would indicate a potential mechanism for resistance acquisition. Up until now, there is no evidence of such a compensative process occurring in Plasmodium-infected erythrocytes under physiological conditions. Besides, the discovery of a highly parasite-specific pathway (serine decarboxylation and the presence of PS synthase) constitutes a very attractive and promising target, which could be attacked if resistances are built up against choline analogs. Indeed, potential inhibitions of the serine decarboxylase pathway could be very useful in acting instead of, or in surgery with, choline analogs.

Monoamine oxidase A down-regulation contributes to high metanephrine concentration in pheochromocytoma.

CONTEXT: The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes. OBJECTIVE: The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT. DESIGN: A multicenter comparative study was conducted. STUDY PARTICIPANTS: The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control. MAIN OUTCOME MEASURES: CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations. RESULTS: At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor. CONCLUSION: High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.

Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway.

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.

A Comparative Study of the Organic Acid Content of the Hemolymph of Schistosoma mansoni-Resistant and Susceptible Strains of Biomphalaria glabrata

The freshwater snail Biomphalaria glabrata is an intermediate host of the trematode Schistosoma mansoni. However, some strains of B. glabrata are resistant to successful infection by S. mansoni larvae. The present work examines the profile of organic acids present in S. mansoni-resistant and -susceptible strains of B. glabrata, in order to determine whether the type of organic acid present is related to susceptibility. The organic acids were extracted from the hemolymph of two susceptible B. glabrata strains (PR, Puerto Rico and Ba, Jacobina-Bahia from Brazil), and from the resistant strains 13-16-R1 and 10R2, using solid phase extraction procedures followed by high performance liquid chromatography. The organic acids obtained were analyzed and identified by comparison with known standards. Pyruvate, lactate, succinate, malate, fumarate, acetate, propionate, ß-hydroxybutyrate and acetoacetate were detected in all hemolymph samples. Under standard conditions, the concentration of each of these substances varied among the strains tested and appeared to be specific for each strain. An interesting variation was the low concentration of pyruvate in the hemolymph of PR-snails. Only the concentration of fumarate was consistently different (p&pound; 0.05) between resistant and susceptible strains