CVD accounted for 27 % of all deaths in the UK in 2014, and was responsible for 1·7 million hospital admissions in 2013/2014. This condition becomes increasingly prevalent with age, affecting 34·1 and 29·8 % of males and females over 75 years of age respectively in 2011. The dysregulation of cholesterol metabolism with age, often observed as a rise in LDL-cholesterol, has been associated with the pathogenesis of CVD. To compound this problem, it is estimated by 2050, 22 % of the world's population will be over 60 years of age, in culmination with a growing resistance and intolerance to pre-existing cholesterol regulating drugs such as statins. Therefore, it is apparent research into additional therapies for hypercholesterolaemia and CVD prevention is a growing necessity. However, it is also imperative to recognise this complex biological system cannot be studied using a reductionist approach; rather its biological uniqueness necessitates a more integrated methodology, such as that offered by systems biology. In this review, we firstly discuss cholesterol metabolism and how it is affected by diet and the ageing process. Next, we describe therapeutic strategies for hypercholesterolaemia, and finally how the systems biology paradigm can be utilised to investigate how ageing interacts with complex systems such as cholesterol metabolism. We conclude by emphasising the need for nutritionists to work in parallel with the systems biology community, to develop novel approaches to studying cholesterol metabolism and its interaction with ageing.

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.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the UK. This condition becomes increasingly prevalent during ageing; 34.1% and 29.8% of males and females respectively, over 75 years of age have an underlying cardiovascular problem. The dysregulation of cholesterol metabolism is inextricably correlated with cardiovascular health and for this reason low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) are routinely used as biomarkers of CVD risk. The aim of this work was to use mathematical modelling to explore how cholesterol metabolism is affected by the ageing process. To do this we updated a previously published whole-body mathematical model of cholesterol metabolism to include an additional 96 mechanisms that are fundamental to this biological system. Additional mechanisms were added to cholesterol absorption, cholesterol synthesis, reverse cholesterol transport (RCT), bile acid synthesis, and their enterohepatic circulation. The sensitivity of the model was explored by the use of both local and global parameter scans. In addition, acute cholesterol feeding was used to explore the effectiveness of the regulatory mechanisms which are responsible for maintaining whole-body cholesterol balance. It was found that our model behaves as a hypo-responder to cholesterol feeding, while both the hepatic and intestinal pools of cholesterol increased significantly. The model was also used to explore the effects of ageing in tandem with three different cholesterol ester transfer protein (CETP) genotypes. Ageing in the presence of an atheroprotective CETP genotype, conferring low CETP activity, resulted in a 0.6% increase in LDL-C. In comparison, ageing with a genotype reflective of high CETP activity, resulted in a 1.6% increase in LDL-C. Thus, the model has illustrated the importance of CETP genotypes such as I405V, and their potential role in healthy ageing.

This paper addresses the use of anaerobic bacteria to convert carbon dioxide to biomethane as part of the biodegradation process of organic waste. The current study utilises gaslift bioreactors with microbubbles generated by fluidic oscillation to strip the methane produced in the gaslift bioreactor. Removal of methane makes its formation thermodynamically more favourable. In addition, intermittent sparging of microbubbles can prevent thermal stratification, maintain uniformity of the pH and increase the intimate contact between the feed and microbial culture with lower energy requirements than traditional mixing. A gaslift bioreactor with microbubble sparging has been implemented experimentally, using a range of carrier gas, culminating in pure carbon dioxide, in the anaerobic digestion process. The results obtained from the experiments show that the methane production rate is approximately doubled with pure carbon dioxide as the carrier gas for intermittent microbubble sparging.

Short-chain fatty acids have distinct effects on cytoskeletal proteins at the level of expression and organisation. We report a new oscillatory, deterministic model which accounts for different actions and predicts response according to fatty acid chain length.

The underlying cellular mechanisms that characterize aging are complex and multifaceted. However, it is emerging that aging could be regulated by two distinct metabolic hubs. These hubs are the pathway defined by the mammalian target of rapamycin (mTOR) and that defined by the NAD+-dependent deacetylase enzyme, SIRT1. Recent experimental evidence suggests that there is crosstalk between these two important pathways; however, the mechanisms underpinning their interaction(s) remains poorly understood. In this review, we propose using computational modelling in tandem with experimentation to delineate the mechanism(s). We briefly discuss the main modelling frameworks that could be used to disentangle this relationship and present a reduced reaction pathway that could be modelled. We conclude by outlining the limitations of computational modelling and by discussing opportunities for future progress in this area.

Airlift 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 µm, 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.

Health and environmental factors as well as operational difficulties are major challenges facing the development of an anaerobic digestion process. Some of these problems relate to the use of sludge collected from primary and secondary clarifier units in wastewater treatment plants for laboratory purposes. The present study addresses the preparation of sludge for laboratory purposes by using a mixture that consists of the digested sludge, which is less pathogenic, compared to the collected sludge from the primary or secondary clarifier, and food wastes. The sludge has been tested experimentally for 19 and 32 days under mesophilic conditions. The results show a steady methane production rate from the anaerobic digester which used sludge with a rate of 1.5 l/day and concentration around 60%, with comparatively low H2S gas content (10 ppm). The methane produced from the digester that used only digested sludge decreases during the experimental period.

In 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.

Particle concentration and filtration is a key stage in a wide range of processing industries and also one that can be present challenges for high throughput, continuous operation. Here we demonstrate some features which increase the efficiency of ultrasound enhanced sedimentation and could enable the technology the potential to be scaled up. In this work, 20 mm piezoelectric plates were used to drive 100 mm high chambers formed from single structural elements. The coherent structural resonances were able to drive particles (yeast cells) in the water to nodes throughout the chamber. Ultrasound enhanced sedimentation was used to demonstrate the efficiency of the system (>99% particle clearance). Sub-wavelength pin protrusions were used for the contacts between the resonant chamber and other elements. The pins provided support and transferred power, replacing glue which is inefficient for power transfer. Filtration energies of ∼4 J/ml of suspension were measured. A calculation of thermal convection indicates that the circulation could disrupt cell alignment in ducts >35 mm high when a 1K temperature gradient is present; we predict higher efficiencies when this maximum height is observed. For the acoustic design, although modelling was minimal before construction, the very simple construction allowed us to form 3D models of the nodal patterns in the fluid and the duct structure. The models were compared with visual observations of particle movement, Chladni figures and scanning laser vibrometer mapping. This demonstrates that nodal planes in the fluid can be controlled by the position of clamping points and that the contacts could be positioned to increase the efficiency and reliability of particle manipulations in standing waves.

ABSTRACT 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 IgG 1 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. Biotechnol. Bioeng. 2014;111: 372–385. © 2013 Wiley Periodicals, Inc.

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

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.

This paper addresses further anaerobic digestion of already digested sludge by processing in an airlift bioreactor. Anaerobic digestion is commonly used for nutrient and energy recovery from biomass. It is used to breakdown organic matter into methane(CH4), carbon dioxide (CO2), hydrogen sulphate (H2S), and digested sludge, which is used for fertilizer, through four biodegradation stages. The rate of gas generation through mesophilic anaerobic digestion is generally higher, yet, the remaining dissolved gases in a digested sludge have a pejorative effect on the environment when they are eventually released, as well as causing operational difficulties. Moreover, the generation of biogas continuously in an already digested sludge increases cavitation phenomena and the accompanying pump load. Removal of these gases is investigated by a bubbling system. An airlift bioreactor (ALR) is used as anaerobic digester in the present research to remove produced methane, carbon dioxide and hydrogen sulphate (H2S) from digested sludge, consequently, reducing pathogens and odour as well as improvement digested sludge for fertilizer. ALRs have many valuable benefits in comparison with stirred tanks for instance: there are no moving parts inside the reactor, low cost of installation and maintenance, and low energy required. In addition, using an airlift reactor enhances the mixing efficiency. The process preferable to agitation as in conventional stirred tanks on power consumption grounds. The experimental data shows that the cumulative methane production of airlift anaerobic digester about 29% more than the observed in the conventional anaerobic digester. Through 170 hours of processing, there is a significantly greater removal of carbon dioxide and hydrogen sulphate in airlift digester over a conventional digester.

In this study, we systematically compare two vector design strategies for recombinant monoclonal antibody (Mab) synthesis by Chinese hamster ovary (CHO) cells; a dual open reading frame (ORF) expression vector utilizing separate cytomegalovirus (CMV) promoters to drive heavy chain (HC) and light chain (LC) expression independently, and a single ORF vector design employing a single CMV promoter to drive HC and LC polypeptide expression joined by a foot and mouth disease virus F2A polypeptide self‐cleaving linker sequence. Initial analysis of stable transfectants showed that transfectants utilizing the single ORF vector designs exhibited significantly reduced Mab production. We employed an empirical modeling strategy to quantitatively describe the cellular constraints on recombinant Mab synthesis in all stable transfectants. In all transfectants, an intracellular molar excess of LC polypeptide over HC polypeptide was observed. For CHO cells transfected with the single ORF vectors, model‐predicted, and empirical intracellular intermediate levels could only be reconciled by inclusion of nascent HC polypeptide degradation. Whilst a local sensitivity analysis showed that qMab of all transfectants was primarily constrained by recombinant mRNA translation rate, our data indicated that all single ORF transfectants exhibited a reduced level of recombinant gene transcription and that Mab folding and assembly reactions generically exerted greater control over qMab. We infer that the productivity of single ORF transfectants is limited by ER processing/degradation “capacity” which sets a limit on transcriptional input. We conclude that gene vector design for oligomeric recombinant proteins should be based on an understanding of protein‐specific synthetic kinetics rather than polypeptide stoichiometry. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

Systems biology combines experimental data with computational modelling to describe complex biological mechanisms and pathways. Short-chain fatty acids (SCFAs-chemopreventive compounds produced in the colon lumen) impair microtubule (MT) function in colon cancer cells by altering the relative expression of β-tubulin isotypes. The β-tubulin isotype composition along MT fibres is believed to contribute to a "tubulin code" defining which microtubule-associated proteins (MAPs) and kinesins are recruited and the arrangement of tubulin post-transcriptional modifications (PTMs) along the fibre, which in turn dictate many critical cellular functions. SCFAs drive acetylation of many proteins by virtue of being histone deacetylase inhibitors (HDACi's). Known acetyl-proteins include transcription factors and cytoplasmic cytoskeletal keratins as well as histones. Disruption of the MT cytoskeleton is a prime target of many cancer therapies including anti-microtubule drugs (AMD). This review focuses on SCFAs as HDACi's and how they might affect tubulin dynamics, modifications and isotypes. It discusses the evolution of mechanistic models that have helped improve understanding of tubulin-MT structure and dynamics and how to develop these models, combined with those describing transcription and the cell cycle, could provide hypotheses for how SCFAs disrupt cytoskeletal function. The review demonstrates how systems biology could offer potentially novel ideas for therapies in the prevention and treatment of cancers through the continued development and elaboration of such models.

In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG 4 monoclonal antibody production by GS‐CHO cells throughout a state‐of‐the‐art industrial fed‐batch culture process. Based on the calculation of a production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy‐intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of production. Instead, cell‐specific Mab production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole‐process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase production of a recombinant protein by a mammalian host cell—where control of productivity is inherently protein product and cell line specific. Biotechnol. Bioeng. 2011;108:2193–2204. © 2011 Wiley Periodicals, Inc.

An important aspect of systems biology research is the so-called “reverse engineering” of cellular metabolic dynamics from measured input-output data. This allows researchers to estimate and validate both the pathway’s structure as well as the kinetic constants. In this paper, the recently published ‘Proximate Parameter Tuning’ (PPT) method for the identification of biochemical networks is analysed. In particular, it is shown that the described PPT algorithm is essentially equivalent to a sequential linear programming implementation of a constrained optimization problem. The corresponding objective function consists of two parts, the first emphasises the data fitting where a residual 1-norm is used, and the second emphasises the proximity of the calculated parameters to the specified nominal values, using an ∞-norm. The optimality properties of PPT algorithm solution as well as its geometric interpretation are analyzed. The concept of optimal parameter locus is applied for the exploration of the entire family of optimal solutions. An efficient implementation of the parameter locus is also developed. Parallels are drawn with 1-norm parameter deviation regularization which attempt to fit the data with a minimal number of parameters. Finally, a small example is used to illustrate all of these properties.

In this study we compare the cellular control of recombinant human IgG4 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 qMab—the 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. © 2010 Wiley Periodicals, Inc.