Use of multi-staining flow cytometry to characterise the physiological state of Escherichia coli W3110 in high cell density fed-batch cultures.

High cell density fed-batch fermentations of Escherichia coli W3110 have been carried out at specific growth rates of less than 0.3 h-1, to investigate the effect of glucose limitation on the physiological state of individual cells. After an initial exponential batch phase, the feed rate was held constant and a final dry cell weight of approximately 50 g per litre was achieved. The fermentations were monitored by mass spectrometry whilst measurements of pH, DOC, CFU/mL, TCN, OD500nm and residual glucose concentrations were made. Satisfactory and reproducible results were obtained. Flow cytometric analysis of cells in broth samples, based on either of two multi-staining protocols, revealed a progressive change in cell physiological state throughout the course of the fermentations. From these measurements it was concluded that the loss in reproductive viability towards the end of the fed-batch process is due to cell death and not due to the formation of a "viable but nonculturable state" as had previously been reported. Since the presence of a high proportion of dead or dying cells at any time during a fermentation has a detrimental effect on the synthesis of any desired product it is proposed that an on-line flow cytometric analysis and control strategy could be used as a means of increasing overall process efficiency.     

Further studies related to the scale-up of high cell density Escherichia coli fed-batch fermentations: the additional effect of a changing microenvironment when using aqueous ammonia to control pH

In this work, we report on the further development of the scale-down, two-compartment (STR + PFR) experimental simulation model. For the first time, the effect on high cell density Escherichia coli fed-batch fermentations of a changing microenvironment with respect to all three of the major spatial heterogeneities that may be associated with large-scale processing (pH, glucose, and dissolved oxygen concentration) were studied simultaneously. To achieve this, we used traditional microbiological analyses as well as multiparameter flow cytometry to monitor cell physiological response at the individual cell level. It was demonstrated that for E. coli W3110 under such conditions in a 20 m(3) industrial fed-batch fermentation, the biomass yield is lower and final cell viability is higher than those found in the equivalent well-mixed, 5L laboratory scale case. However, by using a combination of the well-mixed 5L stirred tank reactor (STR) with a suitable plug flow reactor (PFR) to mimic the changing microenvironment at the large scale, very similar results to those in the 20 m(3) reactor may be obtained. The similarity is greatest when the PFR is operated with a mean residence time of 50 sec with a low level of dO(2) and a high glucose concentration with either a pH of 7 throughout the two reactors or with pH controlled at 7 in the STR by addition into the PFR where the pH is > 7.     

Application of multi-parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene bioconversion

The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.     

A comparison of high cell density fed-batch fermentations involving both induced and non-induced recombinant Escherichia coli under well-mixed small-scale and simulated poorly mixed large-scale conditions

In this work, multi-parameter flow cytometric techniques, coupled with dual colour fluorescent staining, have been used to study the metabolic consequences of inclusion body formation in high cell density fed-batch cultures of the recombinant E. coli strain MSD3735, producing the IPTG inducible model mammalian protein, AP50. Further, we report on the development of the scale-down, two compartment (STR + PFR) experimental simulation model to study, for the first time, the effect of a changing microenvironment with respect to three of the major spatial heterogeneities that may be associated with large-scale bioprocessing (pH, glucose and dissolved oxygen concentration) on a recombinant bacterial system. Using various time points for induction and various scale-down configurations, it has been shown that inclusion body formation is followed immediately by a detrimental progressive change in individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability, resulting in a lower final biomass yield. However, the extent of this change was found to be dependent on whether the AP50 protein was induced or not, on the time of induction and on which combination of heterogeneities was being simulated. From this and previous work, it is clear that the scale-down two-compartment model can be used to study the impact of genetically modifying an organism to produce inclusion bodies and any range and combination of potential heterogeneities known to exist at the large scale.     

Isolation and characterization of 12 tetranucleotide repeat microsatellite loci from the green‐backed tit (Parus monticolus)

From a partial genomic library enriched for GATA short tandem repeats, we developed 12 polymorphic microsatellite loci from the green‐backed tit (Parus monticolus). We characterized these loci by genotyping 30 adult individuals with unknown relationship. The number of alleles ranged from four to 17 per locus (mean = 9.3 alleles) and the observed heterozygosity for each locus ranged from 0.633 to 0.933 (mean = 0.789). All loci conformed to Hardy–Weinberg expectations. Four of 66 possible pairwise comparisons between loci showed significant gametic disequilibrium.     

Bioprocess development for scalable production of cultivated meat

Traditional farm-based products based on livestock are one of the main contributors to greenhouse gas emissions. Cultivated meat is an alternative that mimics animal meat, being produced in a bioreactor under controlled conditions rather than through the slaughtering of animals. The first step in the production of cultivated meat is the generation of sufficient reserves of starting cells. In this study, bovine adipose-derived stem cells (bASCs) were used as starting cells due to their ability to differentiate towards both fat and muscle, two cell types found in meat. A bioprocess for the expansion of these cells on microcarriers in spinner flasks was developed. Different cell seeding densities (1,500, 3,000, and 6,000 cells/cm²) and feeding strategies (80%, 65%, 50%, and combined 80%/50% medium exchanges) were investigated. Cell characterization was assessed pre- and postbioprocessing to ensure that bioprocessing did not negatively affect bASC quality. The best growth was obtained with the lowest cell seeding density (1,500 cells/cm²) with an 80% medium exchange performed (p < .0001) which yielded a 28-fold expansion. The ability to differentiate towards adipogenic, osteogenic, and chondrogenic lineages was retained postbioprocessing and no significant difference (p > .5) was found in clonogenicity pre- or postbioprocessing in any of the feeding regimes tested.     

Prevention of recurrence and prolonged survival in primary central nervous system lymphoma (PCNSL) patients treated with adjuvant high-dose methylprednisolone

Five patients at risk for primary central nervous system lymphoma (PCNSL) recurrence were treated with high-dose methylprednisolone, (HDMP) to prevent ‘trafficking’ of malignant lymphocytes into the central nervous system (CNS). HDMP was chosen because of its ability to stabilize the ‘blood brain barrier (BBB)’. Three men with newly diagnosed PCNSL, ages 62, 76 and 78 y, whose survival was projected to be 6.6 months, began treatment after achieving complete response (CR) to initial radiation therapy alone and survived 27, 37 and 59 months after treatment. In none was death from recurrent disease in CNS but one patient did die of systemic non-Hodgkin’s lymphoma (NHL) five years after PCNSL diagnosis. A 20 y old man was treated with HDMP after successful combined modality therapy and is alive 75+months after initial diagnosis without evidence of disease recurrence. A 34 y old man relapsed after combined modality initial treatment and failed to respond to HDMP when treatment was begun after unsuccessful salvage therapy; he died of disease 12 months after initial diagnosis. There were no treatment complications. The promising results in this pilot study from the basis for a North Central Cancer Treatment Group (NCCTG) 96-73-51, a Phase 2 clinical trial of brain radiotherapy and HDMP for PCNSL patients 70 y of age and older, a group of patients at high risk for toxicity from intensive combined modality therapy.     

Functional and Structural Characterization of a Eurytolerant Calsequestrin from the Intertidal Teleost Fundulus heteroclitus

Calsequestrins (CSQ) are high capacity, medium affinity, calcium-binding proteins present in the sarcoplasmic reticulum (SR) of cardiac and skeletal muscles. CSQ sequesters Ca²⁺ during muscle relaxation and increases the Ca²⁺-storage capacity of the SR. Mammalian CSQ has been well studied as a model of human disease, but little is known about the environmental adaptation of CSQ isoforms from poikilothermic organisms. The mummichog, Fundulus heteroclitus, is an intertidal fish that experiences significant daily and seasonal environmental fluctuations and is an interesting study system for investigations of adaptation at the protein level. We determined the full-length coding sequence of a CSQ isoform from skeletal muscle of F. heteroclitus (FCSQ) and characterized the function and structure of this CSQ. The dissociation constant (K_d) of FCSQ is relatively insensitive to changes in temperature and pH, thus indicating that FCSQ is a eurytolerant protein. We identified and characterized a highly conserved salt bridge network in FCSQ that stabilizes the formation of front-to-front dimers, a process critical to CSQ function. The functional profile of FCSQ correlates with the natural history of F. heteroclitus suggesting that the eurytolerant function of FCSQ may be adaptive.