Lactate increases coenzyme Q10 production by Agrobacterium tumefaciens

By the optimization of nitrogen source for coenzyme Q₁₀ (ubiquinone, CoQ₁₀) production in Agrobacterium tumefaciens KCCM 10413 culture, the highest CoQ₁₀ production was achieved in medium containing corn steep powder (CSP). Components for a stimulatory effect on the production of CoQ₁₀ in CSP were screened, and lactate was found to increase dry cell weight (DCW) and the specific CoQ₁₀ content. In a fed-batch culture of A. tumefaciens, supplementation with 1.5 g of lactate l⁻¹ further improved DCW, the specific CoQ₁₀ content, and CoQ₁₀ production by 16.0, 5.8, and 22.8%, respectively. It has been reported that lactate stimulates cell growth and acts as an accelerator driving the tricarboxylic acid (TCA) cycle (Roberto et al. 2002, Biotechnol Let 24:427–431; Matsuoka et al. 1996, Biosci Biotechnol Biochem 60:575–579). In this study, lactate supplementation increased DCW and the specific CoQ₁₀ content in A. tumefaciens culture, probably by accelerating TCA cycle and energy production as reported previously, leading to the increase of CoQ₁₀ production.     

Aeration strategy: a need for very high ethanol performance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> fed-batch process

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions—oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)—on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l^–1), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l^–1 in 45 h) and average productivity (3.3 vs 2.6 g l^–1 h^–1) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l^–1. We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l^–1 in 45 h) in order to achieve a highly competitive dynamic process.     

Cytosolic NADPH balancing in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary ¹³C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD⁺ dependent. Metabolic modeling shows that changing the NAD⁺-aldehyde dehydrogenase to NADP⁺-aldehyde dehydrogenase can increase the penicillin yield on substrate.     

Modeling lipid accumulation in oleaginous fungi in chemostat cultures. II: Validation of the chemostat model using yeast culture data from literature

A model that predicts cell growth, lipid accumulation and substrate consumption of oleaginous fungi in chemostat cultures (Meeuwse et al. in Bioproc Biosyst Eng. doi:, 2011) was validated using 12 published data sets for chemostat cultures of oleaginous yeasts and one published data set for a poly-hydroxyalkanoate accumulating bacterial species. The model could describe all data sets well with only minor modifications that do not affect the key assumptions, i.e. (1) oleaginous yeasts and fungi give the highest priority to C-source utilization for maintenance, second priority to growth and third priority to lipid accumulation, and (2) oleaginous yeasts and fungi have a growth rate independent maximum specific lipid production rate. The analysis of all data showed that the maximum specific lipid production rate is in most cases very close to the specific production rate of membrane and other functional lipids for cells growing at their maximum specific growth rate. The limiting factor suggested by Ykema et al. (in Biotechnol Bioeng 34:1268–1276, 1989), i.e. the maximum glucose uptake rate, did not give good predictions of the maximum lipid production rate.     

Chain elongation of pectic β-(1→4)-galactan by a partially purified galactosyltransferase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> Merr.) hypocotyls

Pectin is one of the major cell wall polysaccharides found in dicotyledonous plants. We have solubilized and partially purified a β-(1→4)-galactosyltransferase (GalT) involved in the synthesis of the β-(1→4)-galactan side chains of pectin. The enzyme protein was almost completely solubilized by mixing a crude microsomal preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls with a detergent, Triton X-100 (0.75%, w/v), in buffer. The solubilized enzyme was partially purified by ion-exchange chromatography. The crude membrane-bound GalT transferred Gal from UDP-Gal onto 2-aminobenzamide (AB)-derivatized β-(1→4)-galactoheptaose (Gal₇-AB), leading to the formation of Gal_8–11-AB by attachment of a series of one to four galactosyl residues; this is similar to what has previously been observed for 2-aminopyridine-derivatized β-(1→4)-galactooligomer acceptors (Konishi et al. in Planta 218:833–842, 2004). The partially purified GalT, by contrast, was able to transfer more than 25 galactosyl residues and elongated the chains to about Gal₃₅-AB, thus almost reaching the length (43–47 Gal units) of native β-(1→4)-galactan side chains found in pectic polysaccharides from soybean cotyledons (Nakamura et al. in Biosci Biotechnol Biochem 66:1301–1313, 2002). Enzyme activity increased with increasing chain length of β-(1→4)-galactooligomers and reached maximal activity at heptaose, whereas galactooligomers higher than heptaose showed lower acceptor efficiency. Sugars described in this paper belong to the d-series unless otherwise noted.     

Analysis of kinetic parameters of gas phase bioreactors

The dependence of toluene elimination capacity on its load was obtained in five small-scale reactors filled with glass beads carrying biocatalyst cells. With increase in operation time the calculated maximal elimination capacity was shown to increase along with biomass density in the biocatalyst bed. Fivefold increase in trickling intensity did not affect the reactor performance. A simplified mathematical model for evaluation of minimal required biocatalyst bed volume at certain loading was developed based on experimental dependence of elimination capacity vs. loading.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Dynamic formation of oriented patches in chondrocyte cell cultures

Growth factors have a significant impact not only on the growth dynamics but also on the phenotype of chondrocytes (Barbero et al. in J. Cell. Phys. 204:830–838, 2005). In particular, as chondrocytes approach confluence, the cells tend to align and form coherent patches. Starting from a mathematical model for fibroblast populations at equilibrium (Mogilner et al. in Physica D 89:346–367, 1996), a dynamic continuum model with logistic growth is developed. Both linear stability analysis and numerical solutions of the time-dependent nonlinear integro-partial differential equation are used to identify the key parameters that lead to pattern formation in the model. The numerical results are compared quantitatively to experimental data by extracting statistical information on orientation, density and patch size through Gabor filters.     

A general linear framework for the comparison and evaluation of models of sensorimotor synchronization

Sensorimotor synchronization (SMS), the temporal coordination of a rhythmic movement with an external rhythm, has been studied most often in tasks that require tapping along with a metronome. Models of SMS use information about the timing of preceding stimuli and responses to predict when the next response will be made. This article compares the theoretical structure and empirical predictions of four two-parameter models proposed in the literature: Michon (Timing in temporal tracking, Van Gorcum, Assen, 1967), Hary and Moore (Br J Math Stat Psychol 40:109–124, 1987b), Mates (Biol Cybern 70:463–473, 1994a; Biol Cybern 70:475–484, 1994b), and Schulze et al. (Mus Percept 22:461–467, 2005). By embedding these models within a general linear framework, the mathematical equivalence of the Michon, Hary and Moore, and Schulze et al. models is demonstrated. The Mates model, which differs from the other three, is then tested empirically with new data from a tapping experiment in which the metronome alternated between two tempi. The Mates model predictions are found to be invalid for about one-third of the trials, suggesting that at least one of the model’s underlying assumptions is incorrect. The other models cannot be refuted as easily, but they do not predict some features of the data very accurately. Comparison of the models’ predictions in a training/test procedure did not yield any significant differences. The general linear framework introduced here may help in the formulation of new models that make better predictions.     

Use of the valine biosynthetic pathway to convert glucose into isobutanol

Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259–2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also—as has been demonstrated previously (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009)—used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used.     

Evaluation of the Kinetic Parameters of Trickle Bed Vapor Phase Bioreactors

The dependence of toluene elimination capacity on its load was determined in five small-scale reactors filled with glass beads carrying biocatalyst cells. With an increase in the operation time, the calculated maximum elimination capacity was shown to increase in parallel with the biomass density in the biocatalyst bed. A fivefold increase in the trickling rate did not affect the reactor performance. A simplified mathematical model for evaluating the minimal required biocatalyst bed volume at a certain loading was developed based on the experimental curve of elimination capacity versus loading.     

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

A Computational Analysis of Localized Ca<Superscript>2+</Superscript>-Dynamics Generated by Heterogeneous Release Sites

We investigate the role of heterogeneous expression of IP₃R and RyR in generating diverse elementary Ca²⁺ signals. It has been shown empirically (Wojcikiewicz and Luo in Mol. Pharmacol. 53(4):656–662, 1998; Newton et al. in J. Biol. Chem. 269(46):28613–28619, 1994; Smedt et al. in Biochem. J. 322(Pt. 2):575–583, 1997) that tissues express various proportions of IP₃ and RyR isoforms and this expression is dynamically regulated (Parrington et al. in Dev. Biol. 203(2):451–461, 1998; Fissore et al. in Biol. Reprod. 60(1):49–57, 1999; Tovey et al. in J. Cell Sci. 114(Pt. 22):3979–3989, 2001). Although many previous theoretical studies have investigated the dynamics of localized calcium release sites (Swillens et al. in Proc. Natl. Acad. Sci. U.S.A. 96(24):13750–13755, 1999; Shuai and Jung in Proc. Natl. Acad. Sci. U.S.A. 100(2):506–510, 2003a; Shuai and Jung in Phys. Rev. E, Stat. Nonlinear Soft Matter Phys. 67(3 Pt. 1):031905, 2003b; Thul and Falcke in Biophys. J. 86(5):2660–2673, 2004; DeRemigio and Smith in Cell Calcium 38(2):73–86, 2005; Nguyen et al. in Bull. Math. Biol. 67(3):393–432, 2005), so far all such studies focused on release sites consisting of identical channel types. We have extended an existing mathematical model (Nguyen et al. in Bull. Math. Biol. 67(3):393–432, 2005) to release sites with two (or more) receptor types, each with its distinct channel kinetics. Mathematically, the release site is represented by a transition probability matrix for a collection of nonidentical stochastically gating channels coupled through a shared Ca²⁺ domain. We demonstrate that under certain conditions a previously defined mean-field approximation of the coupling strength does not accurately reproduce the release site dynamics. We develop a novel approximation and establish that its performance in these instances is superior. We use this mathematical framework to study the effect of heterogeneity in the Ca²⁺-regulation of two colocalized channel types on the release site dynamics. We consider release sites consisting of channels with both Ca²⁺-activation and inactivation (“four-state channels”) and channels with Ca²⁺-activation only (“two-state channels”) and show that for the appropriate parameter values, synchronous channel openings within a release site with any proportion of two-state to four-state channels are possible, however, the larger the proportion of two-state channels, the more sensitive the dynamics are to the exact spatial positioning of the channels and the distance between channels. Specifically, the clustering of even a small number of two-state channels interferes with puff/spark termination and increases puff durations or leads to a tonic response.     

Asymptotics of Conduction Velocity Restitution in Models of Electrical Excitation in the Heart

We extend a non-Tikhonov asymptotic embedding, proposed earlier, for calculation of conduction velocity restitution curves in ionic models of cardiac excitability. Conduction velocity restitution is the simplest non-trivial spatially extended problem in excitable media, and in the case of cardiac tissue it is an important tool for prediction of cardiac arrhythmias and fibrillation. An idealized conduction velocity restitution curve requires solving a non-linear eigenvalue problem with periodic boundary conditions, which in the cardiac case is very stiff and calls for the use of asymptotic methods. We compare asymptotics of restitution curves in four examples, two generic excitable media models, and two ionic cardiac models. The generic models include the classical FitzHugh–Nagumo model and its variation by Barkley. They are treated with standard singular perturbation techniques. The ionic models include a simplified “caricature” of Noble (J. Physiol. Lond. 160:317–352, 1962) model and Beeler and Reuter (J. Physiol. Lond. 268:177–210, 1977) model, which lead to non-Tikhonov problems where known asymptotic results do not apply. The Caricature Noble model is considered with particular care to demonstrate the well-posedness of the corresponding boundary-value problem. The developed method for calculation of conduction velocity restitution is then applied to the Beeler–Reuter model. We discuss new mathematical features appearing in cardiac ionic models and possible applications of the developed method.     

A Mathematical Model of Schistosoma mansoni in Biomphalaria glabrata with Control Strategies

We describe and analyze a mathematical model for schistosomiasis in which infected snails are distinguished from susceptible through increased mortality and no reproduction. We based the model on the same derivation as Anderson and May (J. Anim. Ecol. 47:219–247, 1978), Feng and Milner (A New Mathematical Model of Schistosomiasis, Mathematical Models in Medical and Health Science, Nashville, TN, 1997. Innov. Appl. Math., Vanderbilt Univ. Press, Nashville, pp. 117–128, 1998), and May and Anderson (J. Anim. Ecol. 47:249–267, 1978), but used logistic growth both in human and snail hosts. We introduce a parameter r, the effective coverage of medical treatment/prevention to control the infection. We determine a reproductive number for the disease directly related to its persistence and extinction. Finally, we obtain a critical value for r that indicates the minimum treatment effort needed in order to clear out the disease from the population.     

Estimation of Cell Proliferation Dynamics Using CFSE Data

Advances in fluorescent labeling of cells as measured by flow cytometry have allowed for quantitative studies of proliferating populations of cells. The investigations (Luzyanina et al. in J. Math. Biol. 54:57–89, 2007; J. Math. Biol., 2009; Theor. Biol. Med. Model. 4:1–26, 2007) contain a mathematical model with fluorescence intensity as a structure variable to describe the evolution in time of proliferating cells labeled by carboxyfluorescein succinimidyl ester (CFSE). Here, this model and several extensions/modifications are discussed. Suggestions for improvements are presented and analyzed with respect to statistical significance for better agreement between model solutions and experimental data. These investigations suggest that the new decay/label loss and time dependent effective proliferation and death rates do indeed provide improved fits of the model to data. Statistical models for the observed variability/noise in the data are discussed with implications for uncertainty quantification. The resulting new cell dynamics model should prove useful in proliferation assay tracking and modeling, with numerous applications in the biomedical sciences.     

1H, 13C and 15N NMR assignments of the C1A and C1B subdomains of PKC-delta

The Protein Kinase C family of enzymes is a group of serine/threonine kinases that play central roles in cell-cycle regulation, development and cancer. A key step in the activation of PKC is translocation to membranes and binding of membrane-associated activators including diacylglycerol (DAG). Interaction of novel and conventional isotypes of PKC with DAG and phorbol esters occurs through the two C1 regulatory domains (C1A and C1B), which exhibit distinct ligand binding selectivity that likely controls enzyme activation by different co-activators. PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα (Slater et al. J Biol Chem 272(10):6167–6173, 1997; Slater et al. Biochemistry 43(23):7601–7609, 2004), PKCε (Das et al. Biochem J 421(3):405–413, 2009) and PKCδ (Das et al. J Biol Chem 279(36):37964–37972, 2004; Das et al. Protein Sci 15(9):2107–2119, 2006) contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC. Here we present the ¹H, ¹⁵N and ¹³C NMR chemical shift assignments for the Rattus norvegicus PKCδ C1A and C1B proteins.     

Strategic Treatment Interruptions During Imatinib Treatment of Chronic Myelogenous Leukemia

Although imatinib is an effective treatment for chronic myelogenous leukemia (CML), and nearly all patients treated with imatinib attain some form of remission, imatinib does not completely eliminate leukemia. Moreover, if the imatinib treatment is stopped, most patients eventually relapse (Cortes et al. in Clin. Cancer Res. 11:3425–3432, 2005). In Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008), the authors presented a mathematical model for the dynamics of CML under imatinib treatment that incorporates the anti-leukemia immune response. We use the mathematical model in Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008) to study and numerically simulate strategic treatment interruptions as a potential therapeutic strategy for CML patients. We present the results of numerous simulated treatment programs in which imatinib treatment is temporarily stopped to stimulate and leverage the anti-leukemia immune response to combat CML. The simulations presented in this paper imply that treatment programs that involve strategic treatment interruptions may prevent leukemia from relapsing and may prevent remission for significantly longer than continuous imatinib treatment. Moreover, in many cases, strategic treatment interruptions may completely eliminate leukemic cells from the body. Thus, strategic treatment interruptions may be a feasible clinical approach to enhancing the effects of imatinib treatment for CML. We study the effects of both the timing and the duration of the treatment interruption on the results of the treatment. We also present a sensitivity analysis of the results to the parameters in the mathematical model.     

Photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity

The photocatalytic properties of titanium dioxide are well known and have many applications including the removal of organic contaminants and production of self-cleaning glass. There is an increasing interest in the application of the photocatalytic properties of TiO₂ for disinfection of surfaces, air and water. Reviews of the applications of photocatalysis in disinfection (Gamage and Zhang 2010; Chong et al., Wat Res 44(10):2997–3027, 2010) and of modelling of TiO₂ action have recently been published (Dalrymple et al. , Appl Catal B 98(1–2):27–38, 2010). In this review, we give an overview of the effects of photoactivated TiO₂ on microorganisms. The activity has been shown to be capable of killing a wide range of Gram-negative and Gram-positive bacteria, filamentous and unicellular fungi, algae, protozoa, mammalian viruses and bacteriophage. Resting stages, particularly bacterial endospores, fungal spores and protozoan cysts, are generally more resistant than the vegetative forms, possibly due to the increased cell wall thickness. The killing mechanism involves degradation of the cell wall and cytoplasmic membrane due to the production of reactive oxygen species such as hydroxyl radicals and hydrogen peroxide. This initially leads to leakage of cellular contents then cell lysis and may be followed by complete mineralisation of the organism. Killing is most efficient when there is close contact between the organisms and the TiO₂ catalyst. The killing activity is enhanced by the presence of other antimicrobial agents such as Cu and Ag.