Continuous Culture Systems for Mammalian Cells

Reovirus and Rotaviruses

Hoorieh Soleimanjahi , Fatemeh Hosseini Heydarabadi , in Encyclopedia of Infection and Immunity, 2022

Genome organization and replication

Generally, the replication cycle of several animal rotaviruses is completed in 12–15   h at 37 °C, in monkey kidneys' continuous cell cultures. TheVP4 is the viral attachment protein, and the N-acetyl-sialic acid residues on the cell surface in some animal viruses serve as a receptor behind several integrins and heat shock protein 70 as coreceptors. Recently, the published data mentioned that rotaviruses may enter cells either by receptor-mediated endocytosis or by direct membrane penetration. In both cases, virus entry primes the loss of the outer VP4 and VP7 protein layer and DLP containing transcriptionally active dsRNA was released into the cytoplasm. Within DLP 5′-capped, nonpolyadenylated mRNAs were generated using the full-length minus strand of each genome segment as a template. The regulation of gene expression was done by differences in the level of transcription, which took place from each genome segment. The viral mRNAs support two steps; primarily, the mRNAs act as templates for translation and producing viral proteins, and furthermore, viral mRNAs act as templates for minus-strand synthesis in order to produce dsRNA genome segments during packaging of progeny. Nowadays, evidence showed that the pools of mRNAs that serve as templates for translation (cytosolic +   RNA) and replication (viroplasmic +   RNA) are distinct (McDonald and Patton, 2011). Virus assembly begins in cytoplasmic inclusions termed viroplasms, which is mediated by NSP2 and NSP5 (Carreño-Torres et al., 2010). During assembly of progeny, the 11 different viral mRNAs interact with one another and with VP1 and VP3, that is followed by association with VP2, for triggering -RNA synthesis and consequently cause the establishment of cores comprising a complete set of 11 dsRNA segments. The most critical Cis-acting replication elements have been identified for minus-strand synthesis. In the intermediate phase of assembly, VP6 is added to core particles, forming DLPs. The next steps in the morphogenesis of progeny virions are unique to rotaviruses and involve recruitment of DLPs to the ER by the NSP4 (Bugarc`ić and Taylor, 2006). The VP6 protein of Rotavirus A, which forms inner capsid proteins, is highly conserved, immunogenic, and contains virus group and subgroup-specific determinants. Although this protein does not induce neutralizing antibodies, it may play a role in the initiation of protective immunity.

The VP4 and VP7 outer capsid proteins elicit neutralizing antibodies. Rotavirus strains were classified based on VP4 and VP7 proteins into P serotypes (VP4 is protease-sensitive) and G serotypes (VP7 is a glycoprotein). Classification of rotaviruses into P (VP4) or G (VP7) serotypes is performed by cross-neutralization assays. So far, 23   G (VP7) genotypes and 32 P(VP4) genotypes have been identified (Matthijnssens et al., 2009). The P genotype is denoted by a number within square brackets, which immediately follows the P serotype.

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Engineering Perspectives in Biotechnology

Katsutoshi Hori , Hajime Unno , in Comprehensive Biotechnology (Third Edition), 2019

2.44.3.1 Continuous Operation

Continuous operation is almost equal to integrated production and separation because it implies continuous bleeding of culture broth, from which products can be separated continuously by an external separation unit. In other words, continuous cell culture lends itself to online integration with the downstream processing. This also allows monitoring of changes as a function of product quality and therefore adjustment of cell conditions as necessary to maintain product consistency. ³ Batch processes are difficult to monitor, since the effects of any changes that occur are integrated throughout the entire volume of the vessel. Long-term continuous bioreactions are more economical, and real-time process monitoring is possible, which becomes increasingly important for a number of reasons. For example, mammalian cell culture gives product concentrations of typically 1–100   mg   L⁻¹. This high background makes it difficult to monitor product quality in the cell culture medium. Monitoring the purified product from the integrated separation and purification process, instead of the broth, is extremely advantageous.

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CELL SURFACE IMMUNOGLOBULIN OF MOUSE T LYMPHOMA CELLS AND CULTURED FETAL THYMOCYTES

D. Haustein , ... T.E. Mandel , in Leukocyte Membrane Determinants Regulating Immune Reactivity, 1976

Publisher Summary

This chapter presents the results of studies using two types of mouse T-cell populations that are free from contamination by B cells, plasma cells, or mouse serum proteins. The first type of population comprises Thy-1-antigen-bearing T lymphoma cells that have been cloned and grown in continuous cell culture. The second population of cells comprises thymocytes obtained from fetal thymuses taken early in gestation, before the appearance of small lymphocytes, and grown in vitro as organ cultures for one or two weeks. Thymuses cultured for these periods contain newly formed Thy-1-positive lymphocytes but no detectable plasma cells or B cells. Two types of cell populations have been used as models to demonstrate that Ig is exhibited on the surface of Thy-l-positive cells and that this Ig is synthesized by these cells. The chapter also describes certain physico-chemical properties of the surface immunoglobulin (Ig) isolated from these cells.

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Mycoplasma Contamination of Cell Cultures: Incidence, Source, Prevention, and Problems of Elimination

Michael F. Barile , in Tissue Culture, 1973

INCIDENCE OF MYCOPLASMA CONTAMINATION

Since mycoplasmas are frequently present in oral and genital tissues of man and animals, and in neoplastic tissues, cell cultures prepared from these tissues would theoretically have a greater contamination risk. Nonetheless, primary cell cultures are rarely contaminated (1–3%), and the original tissues are apparently not a major source of contamination. On the other hand, continuous cell cultures are frequently contaminated (45–92%). Most contamination occurs during cell propagation, and comes from outside sources. ¹

Contamination occurs in cell cultures derived from all animal species tested, including human, nonhuman primates (rhesus, vervet and grivet monkey, chimpanzee), swine, dog, rabbit, hamster, mouse, rat, chicken, quail, duck, snake, fish, and insect. Cell cultures derived from plant tissues and from all types of animal cells and tissue cultures are subject to mycoplasma contamination, including primary, continuous, diploid and heteroploid cell cultures, tissue cultures, fibroblast and epithelial cell cultures, lymphocyte and macrophage cultures, cells grown in monolayer and suspension, and cells derived from normal, infected, or neoplastic tissues. The mycoplasma titers in contaminated cell cultures range from 10⁵ to 10⁸ colony-forming units/ml (CFU/ml) of medium fluids, regardless of cell type and origin. Occasionally a cell culture line is found which may be resistant to a given mycoplasma. ¹

The lowest incidence of contamination has been found in cells grown in small volumes and observed carefully for morphological changes consistent with contamination. The highest incidence of contamination has been found in cells used to propagate microorganisms or in cells supplied by the cell culture producer. ² These cells are produced on a large scale, grown in large volumes, and in large numbers of containers, conditions which provide a high contamination risk. Contamination occurs more frequently in cell cultures grown in media containing antibiotics and/or serum. Antibiotics provide a false sense of security, and may be used in lieu of rigid sterile procedures. Serum may provide a better medium for the growth of mycoplasmas, or serum may be a source of contamination.

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CLOSTRIDIUM | Clostridium acetobutylicum

H. Janssen , ... H.P. Blaschek , in Encyclopedia of Food Microbiology (Second Edition), 2014

Development of Fermentation and Product Recovery

Continuous fermentation in a chemostat mode has proven to be an effective means to increase productivity in the ABE fermentation. Phosphate is an appropriate limiting factor, but cultivation without nutrient limitation is also possible as the accumulating products limit growth and give rise to steady states. Usually lower product concentrations are obtained than in batch culture, but by application of two stages, an acid-forming growth stage at high dilution rate and a solvent-forming fermentation stage at low dilution rate, a solvent concentration was achieved approaching the usual batch concentration of 20 g l⁻¹ solvents.

To increase the relatively low productivity of chemostat cultures (0.5–2 kg solvents per (h⁻¹ m³)) two techniques, both designed to operate at elevated cell densities, were studied. With cell immobilization, spores are entrapped in gel beads or attached to solid particles using a low-growth medium, which is preferably nitrogen limited. Calcium-alginate beads and beechwood shavings have been tested successfully. Cell recycling involves permanent withdrawal of cell-free culture liquid into an external filtration unit and a returning of the more concentrated culture to the fermenter. With both methods, a productivity increase of about fourfold was achieved in comparison to the free-cell continuous culture. The rates obtained vary according to the amount of added complex substances, such as yeast extract and peptone, the maximum being at 3 kg solvents per (h m³).

The low final solvent concentration attained in the ABE fermentation and the high energy requirement for distillation of butanol, the boiling point of which is greater than that of water, has initiated a search for alternative solvent recovery processes. The main emphasis was put on product removal procedures that are integrated in the fermentation and thus increase productivity by reducing the concentration of toxic products in the culture.

As suggested in relation to the industrial production, liquid–liquid extraction by a water-immiscible liquid in direct contact with the culture has the advantage of being simple to realize. Good results have been obtained with oleyl alcohol, diluted with decane to reduce viscosity. Octanol has also proved to be a useful extractant, but this compound is slightly toxic to the clostridia, and it was necessary to separate the cells from the culture liquid by microfiltration. The solvents are extracted selectively and can be recovered by distillation at a relatively low energy input. Nevertheless, liquid–liquid extraction has the disadvantage of being comparatively expensive and forming emulsions. Therefore, a modification of the liquid–liquid extraction, known as perstraction, was developed. Here, the culture is separated from the extractant by a solvent-permeable membrane. This strategy avoids formation of emulsions between the phases, and the extractant need not be sterilized and does not affect the culture.

Inert gas is used to remove the solvents in variants with and without membranes. Gas-stripping (i.e., direct sparging of gas through the fermenter) is likewise attractive because of its simplicity and low chance of clogging or fouling. The microorganisms are not affected, and the products are recovered easily by condensation, with less energy consumption than with distillation of liquid extractants. It has been suggested that the self-produced fermentation gases, carbon dioxide, and hydrogen, are used instead of expensive nitrogen. The membrane modification of gas-stripping, known as pervaporation, requires an extended tubing system that is immersed in the fermentation vessel. The solvents evaporate through the membrane and are drawn off by vacuum or sweep gas. As the available membranes only allow passage of the solvents, the acids accumulate in the culture and may stop the fermentation. This problem was solved by low-acid mutants that were able to reutilize all of the acids.

Adsorption to solid materials such as silicalite or polyvinylpyridine also has been tested. Relatively low loading capacity, high estimated costs for the adsorbants, and the heat for desorption of the solvents presently diminish the chances for this method. For external application, reverse osmosis has been evaluated and found to be more favorable than distillation.

Recently, a novel process with simultaneous ABE fermentation and in situ product recovery with vacuum was reported. Results indicated that fermentation coupled with in situ vacuum recovery led to complete substrate utilization, greater solvent productivity, and improved cell growth.

Generally speaking the in situ recovery methods are interesting, but require high capital expenditure and permanent monitoring by the operator, and although their technical feasibility has been established, they require further development at the engineering level.

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Homogeneous Reactions

Pauline M. Doran , in Bioprocess Engineering Principles, 1995

11.13 Effect of Maintenance on Yields

True yields such as Y _XS, Y _PX and Y _PS are often difficult to evaluate. Although true yields are essentially stoichiometric coefficients, the stoichiometry of biomass production and product formation is only known for relatively simple fermentations. If the metabolic pathways are complex, stoichiometric calculations become too complicated. However, theoretical yields can be related to observed yields such as Y' _XS, Y' _PX and Y'_PS, which are more easily determined.

11.13.1 Observed Yields

Expressions for observed yield coefficients can be obtained by applying Eq. (11.48):

(11.77) $Y{\prime }_{\text{X}\text{S}}=\frac{-\text{d}X}{\text{d}\text{S}}=\frac{r_X}{r_{\text{S}}}$

(11.78) $Y{\prime }_{\text{P}X}=\frac{\text{d}\text{P}}{\text{d}X}=\frac{r_{\text{P}}}{r_X}$

and

(11.79) $Y{\prime }_{\text{P}\text{S}}=\frac{-\text{d}\text{P}}{\text{d}\text{S}}=\frac{r_{\text{P}}}{r_{\text{S}}}$

where X, S and P are masses of cells, substrate and product, respectively, and r _X, r _S and r _P are observed rates evaluated from experimental data. Therefore, yield coefficients can be determined by plotting X, S or P against each other and evaluating the slope as illustrated in Figure 11.14. Alternatively, observed yield coefficients at a particular instant in time can be calculated as the ratio of rates evaluated at that instant. Observed yields are not necessarily constant throughout batch culture; in some cases they exhibit significant dependence on environmental parameters such as substrate concentration and growth rate. Nevertheless, for many cultures, the observed biomass yield Y'_XS is approximately constant. Because of the errors in experimental data, considerable uncertainty is usually associated with measured yield coefficients.

Figure 11.14. Evaluation of observed yields in batch culture from cell, substrate and product concentrations.

11.13.2 Biomass Yield From Substrate

Equations for true biomass yield can be determined for systems without extracellular product formation or when product synthesis is directly coupled to energy metabolism. Substituting expressions for r _x and r _s from Eqs (11.52) and (11.73) into Eq. (11.77) gives:

(11.80) $Y{\prime }_{\text{X}\text{S}}=\frac{\mu }{\left(\frac{\mu }{Y_{\text{X}\text{S}}}+m_{\text{S}}\right).}$

Inverting Eq. (11.80) produces the expression:

(11.81) $\frac{1}{Y{\prime }_{\text{X}\text{S}}}=\frac{1}{Y_{\text{X}\text{S}}}+\frac{m_{\text{S}}}{\mu }.$

Therefore, if Y _XS and m _S are relatively constant, a plot of $\frac{1}{Y{\prime }_{\text{X}\text{S}}}$ versus $\frac{1}{\mu }$ gives a straight line with slope m _S and intercept $\frac{1}{Y{\prime }_{\text{X}\text{S}}}$ Eq. (11.81) is not generally applied to batch growth data; under typical batch conditions, μ does not vary from μ_max for much of the culture period so it is difficult to plot Y'_XS as a function of specific growth rate. We will revisit Eq. (11.81) when we consider continuous cell culture in Chapter 13. As a rule of thumb, true biomass yield from glucose under aerobic conditions is around 0.5 g g⁻¹.

In processes such as production of bakers' yeast and single-cell protein where the required product is biomass, it is desirable to maximise the actual or observed yield of cells from substrate. The true yield Y _XS is limited by stoichiometric considerations. However, from Eq. (11.80), Y'_XS can be improved by decreasing the maintenance coefficient or increasing the growth rate. m _S may be reduced by lowering the temperature of fermentation, using a medium of lower ionic strength, or by applying a different organism or strain with lower maintenance-energy requirements. Assuming these changes do not reduce the growth rate, they can be employed to improve the biomass yield.

When the culture produces compounds not directly coupled with energy metabolism, Eq. (11.80) and Eq. (11.81) do not apply because a different expression for r _s must be used in Eq. (11.77). Determination of true yields and maintenance coefficients is more difficult in this case because of the number of terms involved.

11.13.3 Product Yield From Biomass

Observed yield of product from biomass Y'_PX is defined in Eq. (11.78). When product synthesis is directly coupled to energy metabolism, r _p is given by Eq. (11.69). Substituting this and Eq. (11.52) into Eq. (11.78) gives:

(11.82) $Y{\prime }_{\text{P}X}=Y_{\text{P}X}+\frac{{\text{m}}_{\text{P}}}{\mu }.$

The extent of deviation of Y'_PX from Y _PX depends on the relative magnitudes of m _P and μ. To increase the observed yield of product for a particular process, m _P should be increased and μ decreased. Eq. (11.82) does not apply to products not directly coupled with energy metabolism; we do not have a general expression for r_P in terms of true yield coefficients for this class of product.

11.13.4 Product Yield From Substrate

Observed product yield from substrate Y' _PS is defined in Eq. (11.79). For products coupled to energy generation, expressions for r _P and r _s are available from Eqs (11.69) and (11.73). Therefore:

(11.83) $Y{\prime }_{\text{P}\text{S}}=\frac{Y_{\text{P}X}\mu +{\text{m}}_{\text{P}}}{\left(\frac{\mu }{Y_{\text{X}\text{S}}}+m_{\text{S}}\right)}$

In many anaerobic fermentations such as ethanol production, yield of product from substrate is a critical factor affecting process economics. At high Y' _PS, more ethanol is produced per mass of carbohydrate consumed so that the cost of production is reduced. Growth rate has a strong effect on Y' _PS for ethanol. Because Y' _PS is low when μ = μ_max, it is desirable to reduce the specific growth rate of the cells. Low growth rate can be obtained by depriving the cells of some essential nutrient, e.g. a nitrogen source, or by immobilising the cells to prevent growth. Increasing the rate of maintenance activity relative to growth will also enhance product yield. This can be done by using a medium of high ionic strength, raising the temperature, or selecting a mutant or different organism with high maintenance requirements. Continuous culture provides more opportunity for manipulating rates of growth than batch culture.

The effect of growth rate and maintenance on Y' _PS is difficult to determine for products not directly coupled with energy metabolism unless information is available about the effect of these parameters on q _P.

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Sustainability in the biopharmaceutical industry: Seeking a holistic perspective

Fergal Lalor , ... Edmond Byrne , in Biotechnology Advances, 2019

3.3 Continuous downstream processing

While continuous cell culture processing in the form of perfusion cell culture is well-established in the biopharmaceutical manufacturing sphere, continuous downstream purification processes are uncommon, with few documented in literature. While the current paradigm is for large, fed-batch cell cultures feeding similarly large batch purification trains, the previously mentioned economic risks to the industry from biosimilars and unsustainable treatment costs has led to a desire for increased volumetric productivity, reduced capital expenditures and an overall reduction in cost of goods ( Klutz et al., 2015). While single-use technology satisfies this in part, by reducing facility footprint and capital expenditures, it does not impact the issue of volumetric productivity (Klutz et al., 2015). As a result, the adoption of continuous manufacturing becomes attractive. This can be accomplished by employing perfusion cell culture in tandem with continuous purification unit operations such as tangential flow filtration and chromatography. Fully continuous processes have been documented in the literature (Klutz et al., 2015; Klutz et al., 2015; Walther et al., 2015) and continuous chromatography operation also has been documented (Steinebach et al., 2016). Table 5 describes the benefits and challenges of implementing continuous purification.

Table 5. Continuous downstream purification: benefits and challenges.

Benefits	Challenges
Increased volumetric productivity	Potential economic benefits may not be realised
Increased separation efficiencies	Regulatory challenges from product licence changes
Increased product quality	Development of reliable methods for all unit operations
Reduced capital expenditures	Increased complexity
Debottlenecking the process

Continuous purification processing is plausible utilising current technologies (Jungbauer, 2013). Significant challenges exist to its implementation at a commercial scale, ranging from technical issues including the development of a reliable method of incubation for steps such as viral inactivation and diafiltration (Przybycien and Titchener-Hooker, 2015) to uncertainties surrounding the predicted economic benefits being realised at commercial scale (Jungbauer, 2013) and, perhaps most significantly, the regulatory requirements to change product registrations (Przybycien and Titchener-Hooker, 2015). Until commercial scale applications of continuous purification processing are implemented and analysed, its development and uptake may remain inhibited by established attitudes within the industry.

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Cancer and Aging at the Crossroads

Sarantis Gagos , Irmgard Irminger-Finger , in The International Journal of Biochemistry & Cell Biology, 2005

In mammalian tumors and immortalized cell cultures, continuous growth requires telomere maintenance provided either by the action of telomerase or by alternative recombination mediated pathways (ALT pathways) that can sustain telomeric length in the absence of telomerase. ALT is characterized by extreme telomeric length heterogeneity (Bryan, Englezou, Gupta, Bacchetti, & Reddel, 1995), high degrees of structural chromosomal rearrangements, presence of sub-nuclear compartments resembling promyelocytic leukemia (PML) bodies and extremely high rates of telomeric sister chromatid exchanges (Bechter, Zou, Walker, Wright, & Shay, 2004; Biessmann & Mason, 1997; Bryan, Englezou, Dalla-Pozza, Dunham, & Reddel, 1997; Londono-Vallejo, Der-Sarkissian, Cazes, Bacchetti, & Reddel, 2004). Approximately 10% of human tumors utilize the ALT pathway (Bryan et al., 1995; Kim et al., 1994). The ALT-associated PML bodies (APBs) contain PML protein, telomeric DNA, telomere-binding proteins, the recombination proteins RAD51 and RAD52, the MRE11/RAD50/NBS1 complex, the replication protein RPA, and the BLM and WRN DNA helicases implicated in Bloom's syndrome and Werner's progeria, respectively (Henson, Neumann, Yeager, & Reddel, 2002; Yeager et al., 1999).

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The Boltzmann equation in molecular biology

Jean-Marc Dubois , ... Béatrice Rouzaire-Dubois , in Progress in Biophysics and Molecular Biology, 2009

During the cell cycle, most cells double their size so that they maintain a constant mass or volume as they proliferate. However during continuous cell cultures, the rate of cell proliferation and the cell size decrease with time even if the culture medium is changed every day ( Rouzaire-Dubois et al., 2004). These concomitant decreases in cell size and proliferation were interpreted by the cell production of inhibitory factors (Couldwell et al., 1992). Moreover, when the cell volume was increased beyond an optimal value by K⁺ or Cl⁻ channel blockers or by chronic hypertonicity, the rate of cell proliferation was reduced (Habela and Sontheimer, 2007; Rouzaire-Dubois et al., 2004). The combination of these two effects determines a bell-shaped relationship between cell proliferation and volume (Rouzaire-Dubois et al., 2004, 2005), suggesting that proliferation (P) is governed by two processes: a positive (activation [A]) and a negative (inhibition [I]) mechanism according to the equation:

(20) $\text{P}={\text{P}}_{\text{max}}\text{AI}$

with

(21) $\text{A}=\frac{1}{1+\exp \left[\left({\text{V}}_{\text{A0}\text{.5}}-\text{V}\right)/k_{\text{A}}\right]}$

and

(22) $\text{I}=\frac{1}{1+\exp \left[\left(\text{V}-{\text{V}}_{\text{I0}\text{.5}}\right)/k_{\text{I}}\right]}\text{.}$

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Prospects on bio-based 2,3-butanediol and acetoin production: Recent progress and advances

Sofia Maina , ... Vinod Kumar , in Biotechnology Advances, 2022

8.7 Fermentation mode

The bioreactor operation mode is an important factor for optimal process design. Various bioreactor operation modes, including batch, fed-batch, continuous cultures, cell recycle and immobilized cell systems, have been implemented for BDO production (Tables 2 and 3). Batch and fed-batch operation modes have been widely studied. Fed-batch fermentation mode has been used to overcome the effect of initial substrate inhibition and it is considered the most favourable operation mode for industrial scale. Using a balanced feeding strategy could lead to high BDO concentration. Ma et al. (2009) compared different fed-batch strategies for BDO production, including constant feed rate, pulse, constant residual glucose concentration and exponential feeding. Maximum BDO and acetoin production of 160 g L^-1 with productivity of 4.21 g L^-1 h^-1 was obtained by constant residual glucose concentration feeding method. A similar method was applied in BDO fermentation by S. marcescens (Zhang et al., 2010b). Generally, constant feeding of substrate provides suitable environment for cell metabolism and BDO production.

Continuous fermentation with cell recycle by maintaining high biomass density allows high and stable process efficiency with high values of productivity. Contamination is still the limiting factor of this technique. Improved economic viability can be achieved by continuous fermentation (Ji et al., 2011a, 2011b). Zeng et al. (1991b) evaluated continuous culture with cell recycle system resulting in high productivity (14.6 g L^-1 h^-1), though the final production of BDO and acetoin were lower than in fed-batch mode (54.2 and 110 g L^-1, respectively).

A promising system employing immobilized cells has been also evaluated to increase BDO efficiency. Maximum BDO concentration of 118.3 g L^-1 was achieved by B. licheniformis immobilized cells. Compared to free cells, the immobilized system showed lower BDO concentration. However, using proper amount of nutrients could enhance the efficiency of the bioprocess (Jurchescu et al., 2013).

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