In this study we compare the cellular control of recombinant human IgG 4 monoclonal antibody (Mab) synthesis in different CHO cell lines. Based on comprehensive empirical analyses of mRNA and polypeptide synthetic intermediates we constructed cell line-specific mathematical models of recombinant Mab manufacture in seven GS-CHO cell lines varying in specific production rate (qMab) over 350-fold. This comparative analysis revealed that control of qMab involved both genetic construct and cell line-specific factors. With respect to the former, all cell lines exhibited excess production of light chain (LC) mRNA and polypeptide relative to heavy chain (HC) mediated by more rapid LC transcription and enhanced LC mRNA stability. Downstream of this, cell lines differed markedly in their relative rates of recombinant mRNA translation, Mab assembly and secretion although HC mRNA abundance and the rate of HC translation generally exerted most control over qMabthe latter being directly proportional to qMab. This study shows that (i) cell lines capable of high qMab exceed a threshold functional competency in all synthetic processes, (ii) the majority of cells in parental and transfected cell populations are functionally limited and (iii) cell engineering strategies to increase Mab production should be cell line specific. Biotechnol. Bioeng. 2010;106: 938-951.

Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of different engineering interventions.

Cholesterol plays a vital role in the human body as a precursor of steroid hormones and bile acids, in addition to providing structure to cell membranes. Whole body cholesterol metabolism is maintained by a highly coordinated balancing act between cholesterol ingestion, synthesis, absorption, and excretion. The aim of this review is to discuss how ageing interacts with these processes. Firstly, we will present an overview of cholesterol metabolism. Following this, we discuss how the biological mechanisms which underpin cholesterol metabolism are effected by ageing. Included in this discussion are lipoprotein dynamics, cholesterol absorption/synthesis and the enterohepatic circulation/synthesis of bile acids. Moreover, we discuss the role of oxidative stress in the pathological progression of atherosclerosis and also discuss how cholesterol biosynthesis is effected by both the mammalian target of rapamycin and sirtuin pathways. Next, we examine how diet and alterations to the gut microbiome can be used to mitigate the impact ageing has on cholesterol metabolism. We conclude by discussing how mathematical models of cholesterol metabolism can be used to identify therapeutic interventions.

The maternal tract plays a critical role in the success of early embryonic development providing an optimal environment for establishment and maintenance of pregnancy. Preparation of this environment requires an intimate dialogue between the embryo and her mother. However, many intriguing aspects remain unknown in this unique communication system. To advance our understanding of the process by which a blastocyst is accepted by the endometrium and better address the clinical challenges of infertility and pregnancy failure, it is imperative to decipher this complex molecular dialogue. The objective of the present work is to define the local response of the maternal tract towards the embryo during the earliest stages of pregnancy. We used a novel in vivo experimental model that eliminated genetic variability and individual differences, followed by Affymetrix microarray to identify the signals involved in this embryo-maternal dialogue. Using laparoscopic insemination one oviduct of a sow was inseminated with spermatozoa and the contralateral oviduct was injected with diluent. This model allowed us to obtain samples from the oviduct and the tip of the uterine horn containing either embryos or oocytes from the same sow. Microarray analysis showed that most of the transcripts differentially expressed were down-regulated in the uterine horn in response to blastocysts when compared to oocytes. Many of the transcripts altered in response to the embryo in the uterine horn were related to the immune system. We used an in silico mathematical model to demonstrate the role of the embryo as a modulator of the immune system. This model revealed that relatively modest changes induced by the presence of the embryo could modulate the maternal immune response. These findings suggested that the presence of the embryo might regulate the immune system in the maternal tract to allow the refractory uterus to tolerate the embryo and support its development.

Systems Biology is the science that aims to understand how biological function absent from macromolecules in isolation, arises when they are components of their system. Dedicated to the memory of Reinhart Heinrich, this paper discusses the origin and evolution of the new part of systems biology that relates to met- abolic and signal-transduction pathways and extends mathematical biology so as to address postgenomic experimental reality. Various approaches to modeling the dynamics generated by metabolic and signal-transduction pathways are compared. The silicon cell approach aims to describe the intracellular network of interest precisely, by numerically integrating the precise rate equations that characterize the ways macromolecules' interact with each other. The non-equilibrium thermodynamic or 'lin-log' approach approximates the enzyme rate equations in terms of linear functions of the logarithms of the concentrations. Biochemical Systems Analysis approximates in terms of power laws. Importantly all these approaches link system behavior to molecular interaction properties. The latter two do this less precisely but enable analytical solutions. By limiting the questions asked, to optimal flux patterns, or to control of fluxes and concentrations around the (patho)physiological state, Flux Balance Analysis and Metabolic/Hierarchical Control Analysis again enable analytical solutions. Both the silicon cell approach and Metabolic/Hierarchical Control Analysis are able to highlight where and how system function derives from molecular interactions. The latter approach has also discovered a set of fundamental principles underlying the control of biological systems. The new law that relates concentration control to control by time is illustrated for an important signal transduction pathway, i.e. nuclear hormone receptor signaling such as relevant to bone formation. It is envisaged that there is much more Mathematical Biology to be discovered in the area between molecules and Life.

Available online xxxKeywords: Microalgae Carbon dioxide Mixotrophic growth Synergistic Biodiesel Dissolved inorganic carbon a b s t r a c tIn recent years microalgae have attracted significant interest as a potential source of sustainable biofuel. Mixotrophic microalgae are able to simultaneously photosynthesise while assimilating and metabolising organic carbon. By combining autotrophic and heterotrophic metabolic pathways biomass productivity can be significantly increased. In this study, acetate-fed mixotrophic Micractinium inermum cultures were found to have a specific growth rate 1.74 times the sum of autotrophic and heterotrophic growth. It was hypothesised that gas exchange between the two metabolic pathways within mixotrophic cultures may have prevented growth limitation and enhanced growth. To determine the extent of synergistic gas exchange and its influence on metabolic activity, dissolved inorganic carbon (DIC), dissolved oxygen (DO) and photosynthesis and respiration rates were measured under different trophic conditions. A 32.7 fold and 2.4 fold increase in DIC and DO concentrations, relative to autotrophic and heterotrophic cultures respectively, were coupled with significant increases in rates of photosynthesis and respiration. These data strongly support the hypothesis of mixotrophic gas exchange within M. inermum cultures. In addition to enhanced growth, this phenomenon may provide reductions in aeration and oxygen stripping costs related to microalgae production.Please cite this article in press as: Smith RT, et al., Synergistic carbon metabolism in a fast growing mixotrophic freshwater microalgal species Micractinium inermum, Biomass and Bioenergy (2015), http://dx.

Background Large improvements in productivity are required to make massive scale biodiesel production from microalgae an economic reality. Although the maximum neutral lipid content of microalgae has received much attention as a target for optimization, there are other factors that are equally important. These are (1) the rates of accumulation of both biomass and lipids and (2) the maximum densities of algal cells that can be sustained in continuous cultivation. The combined effect of these factors for lipid production has not been thoroughly examined in Dunaliella species. Hence this study examines the rates of growth and lipid accumulation in Dunaliella salina using Fourier transform infrared spectroscopy (FTIR) under several combinations of temperatures and cell densities. Results The FTIR signal at 2926 cm−1 (rather than 1740 cm−1) is better for measuring lipids and the PCA of the full spectrum showed a clear separation between the nitrogen replete and nitrogen depleted cells. As expected, cells subjected to nitrogen starvation (N‐depleted) showed very little growth compared to the N‐replete cells. N‐depleted cells achieved a final lipid content that was 78% more than the N‐replete samples at 26 °C, while the differential for 16 °C was 28%. However, the slower growth rates caused by the stress of nitrogen starvation meant that the total lipid production over the starvation period was lower for many samples. Indeed, the only stress condition that gave significantly higher total lipid production was the highest cell density studied at 26 °C. Conclusion For optimization of lipid productivity for biodiesel, the trade‐off between lipid content, growth rate and cell density needs to be considered. © 2013 Society of Chemical Industry

Microbubbles increase the mixing efficiency in airlift bioreactors. Dispersal of gas phase throughout the ALR occurs with decreasing the bubble size. Phase slip velocity decreases with smaller bubble size as gas rise rate decreases. a b s t r a c tAirlift bioreactors can provide an attractive alternative to stirred tanks, particularly for bioprocesses with gaseous reactants or products. Frequently, however, they are susceptible to being limited by gas-liquid mass transfer and by poor mixing of the liquid phase, particularly when they are operating at high cell densities. In this work we use CFD modelling to show that microbubbles generated by fluidic oscillation can provide an effective, low energy means of achieving high interfacial area for mass transfer and improved liquid circulation for mixing.The results show that when the diameter of the microbubbles exceeded 200 mm, the "downcomer" region, which is equivalent to about 60% of overall volume of the reactor, is free from gas bubbles. The results also demonstrate that the use of microbubbles not only increases surface area to volume ratio, but also increases mixing efficiency through increasing the liquid velocity circulation around the draft tube. In addition, the depth of downward penetration of the microbubbles into the downcomer increases with decreasing bubbles size due to a greater downward drag force compared to the buoyancy force. The simulated results indicate that the volume of dead zone increases as the height of diffuser location is increased. We therefore hypothesise that poor gas bubble distribution due to the improper location of the diffuser may have a markedly deleterious effect on the performance of the bioreactor used in this work.