Developing a scalable model of recombinant protein yield from Pichia pastoris:the influence of culture conditions, biomass and induction regime

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

A mathematical model of coal-fired fluidised bed boilers

A mathematical model of a large coal-fired fluidized bed boiler for power generation is synthesised. The effect of variations in the main parameters of the model on variables such as the background carbon concentrations in the bed, and the transient response of heat evolution are studied. The mechanisms of solids mixing within the bed, combustion and the flow of heat to the boiler tubes are shown to result in a characteristic dynamic response, knowledge of which is essential for the proper control and regulation of a practical system.

Meta-analytic model comparisons of surface- and deep-level relational diversity effects on social integration and individual effectiveness

While diversity might give an organization a competitive advantage, individuals have a tendency to prefer homogenous group settings. Prior research suggests that group members who are dissimilar (vs. similar) to their peers in terms of a given diversity attribute (e.g. demographics, attitudes, values or traits) feel less attached to their work group, experience less satisfying and more conflicted relationships with their colleagues, and consequently are less effective. However, prior empirical findings tend to be weak and inconsistent, and it remains unclear when, how and to what extent such differences affect group members’ social integration (i.e. attachment with their work group, satisfaction and conflicted relationships with their peers) and effectiveness. To address these issues the current study conducted a meta-analysis and integrated the empirical results of 129 studies. For demographic diversity attributes (such as gender, ethnicity, race, nationality, age, functional background, and tenure) the findings support the idea that demographic dissimilarity undermines individual member performance via lower levels of social integration. These negative effects were more pronounced in pseudo teams – i.e. work groups in which group members pursue individual goals, work on individual tasks, and are rewarded for their individual performance. These negative effects were however non-existent in real teams - i.e. work groups in which groups members pursue group goals, work on interdependent tasks, and are rewarded (at least partially) based on their work group’s performance. In contrast, for underlying psychological diversity attributes (such as attitudes, personality, and values), the relationship between dissimilarity and social integration was more negative in real teams than in pseudo teams, which in return translated into even lower individual performance. At the same time however, differences in underlying psychological attributes had an even stronger positive effect on dissimilar group member’s individual performance, when the negative effects of social integration were controlled for. This implies that managers should implement real work groups to overcome the negative effects of group member’s demographic dissimilarity. To harness the positive effects of group members’ dissimilarity on underlying psychological attributes, they need to make sure that dissimilar group members become socially integrated.

The planning and management of health for development:a model for Zimbabwe

In this thesis, I view the historical background of Zimbabwe to show the patterns of traditional life that existed prior to settlerism. The form, nature, pace and impact of settlerism and colonialism up to the time of independence are also discussed to show how they affected the health of the population and the pace of development of the country. The political, social and economic underdevelopment of the African people that occurred in Zimbabwe prior to independence was a result of deliberate, politically motivated and controlled policy initiatives. These led to inequatable, inadequate, inappropriate and inaccessible health care provision. It is submitted that since it was the politics that determined the pace of underdevelopment, it must be the politics that must be at the forefront of the development strategy adopted. In the face of the amed conflict that existed in Zimbabwe, existing frameworks of analyses are shown to be inadequate for planning purposes because of their inability to provide indications about the stability of future outcomes. The Metagame technique of analysis of options is proposed as a methology that can be applied in such situations. It rejects deterministic predicative models as misleading and advocates an interactive model based on objective and subjective valuation of human behaviour. In conclusion, the search for stable outcomes rather than optimal and best solutions strategies is advocated in decision making in organisations of all sizes.

Development of a multicellular co-culture model of normal and cystic fibrosis human airways in vitro

Cystic fibrosis (CF) is the most common lethal inherited disease among Caucasians and arises due to mutations in a chloride channel, called cystic fibrosis transmembrane conductance regulator. A hallmark of this disease is the chronic bacterial infection of the airways, which is usually, associated with pathogens such as Pseudomonas aeruginosa, S. aureus and recently becoming more prominent, B. cepacia. The excessive inflammatory response, which leads to irreversible lung damage, will in the long term lead to mortality of the patient at around the age of 40 years. Understanding the pathogenesis of CF currently relies on animal models, such as those employing genetically-modified mice, and on single cell culture models, which are grown either as polarised or non-polarised epithelium in vitro. Whilst these approaches partially enable the study of disease progression in CF, both types of models have inherent limitations. The overall aim of this thesis was to establish a multicellular co-culture model of normal and CF human airways in vitro, which helps to partially overcome these limitations and permits analysis of cell-to-cell communication in the airways. These models could then be used to examine the co-ordinated response of the airways to infection with relevant pathogens in order to validate this approach over animals/single cell models. Therefore epithelial cell lines of non-CF and CF background were employed in a co-culture model together with human pulmonary fibroblasts. Co-cultures were grown on collagen-coated permeable supports at air-liquid interface to promote epithelial cell differentiation. The models were characterised and essential features for investigating CF infections and inflammatory responses were investigated and analysed. A pseudostratified like epithelial cell layer was established at air liquid interface (ALI) of mono-and co-cultures and cell layer integrity was verified by tight junction (TJ) staining and transepithelial resistance measurements (TER). Mono- and co-cultures were also found to secrete the airway mucin MUC5AC. Influence of bacterial infections was found to be most challenging when intact S. aureus, B. cepacia and P. aeruginosa were used. CF mono- and co-cultures were found to mimic the hyperinflammatory state found in CF, which was confirmed by analysing IL-8 secretions of these models. These co-culture models will help to elucidate the role fibroblasts play in the inflammatory response to bacteria and will provide a useful testing platform to further investigate the dysregulated airway responses seen in CF.

An X-ray micro-fluorescence study to investigate the distribution of Al, Si, P and Ca ions in the surrounding soft tissue after implantation of a calcium phosphate-mullite ceramic composite in a rabbit animal model

Synthetic calcium phosphates, despite their bioactivity, are brittle. Calcium phosphate-mullite composites have been suggested as potential dental and bone replacement materials which exhibit increased toughness. Aluminium, present in mullite, has however been linked to bone demineralisation and neurotoxicity: it is therefore important to characterise the materials fully in order to understand their in vivo behaviour. The present work reports the compositional mapping of the interfacial region of a calcium phosphate-20 wt% mullite biocomposite/soft tissue interface, obtained from the samples implanted into the long bones of healthy rabbits according to standard protocols (ISO-10993) for up to 12 weeks. X-ray micro-fluorescence was used to map simultaneously the distribution of Al, P, Si and Ca across the ceramic-soft tissue interface. A well defined and sharp interface region was present between the ceramic and the surrounding soft tissue for each time period examined. The concentration of Al in the surrounding tissue was found to fall by two orders of magnitude, to the background level, within similar to 35 mu m of the implanted ceramic.

Ontology-based protein-protein interactions extraction from literature using the hidden vector state model

This paper proposes a novel framework of incorporating protein-protein interactions (PPI) ontology knowledge into PPI extraction from biomedical literature in order to address the emerging challenges of deep natural language understanding. It is built upon the existing work on relation extraction using the Hidden Vector State (HVS) model. The HVS model belongs to the category of statistical learning methods. It can be trained directly from un-annotated data in a constrained way whilst at the same time being able to capture the underlying named entity relationships. However, it is difficult to incorporate background knowledge or non-local information into the HVS model. This paper proposes to represent the HVS model as a conditionally trained undirected graphical model in which non-local features derived from PPI ontology through inference would be easily incorporated. The seamless fusion of ontology inference with statistical learning produces a new paradigm to information extraction.

Model thrombi formed under flow reveal the role of factor XIII-mediated cross-linking in resistance to fibrinolysis

Background: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin-fibrin and fibrin-inhibitor cross-links, resulting in more mechanically stable clots. The impact of cross-linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively. Methods: We used a whole blood model thrombus system to characterize the role of cross-linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute. Results: A site-specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet-rich head and fibrin-rich tail. Model thrombi from an FXIII-deficient patient lysed more quickly than normal thrombi; replacement therapy with FXIII concentrate normalized lysis. In vitro addition of purified FXIII to the patient's preprophylaxis blood, but not to normal control blood, resulted in more stable thrombi, indicating no further efficacy of supraphysiologic FXIII. However, addition of tissue transglutaminase, which is synthesized by endothelial cells, generated thrombi that were more resistant to fibrinolysis; this may stabilize mural thrombi in vivo. Conclusions: Model thrombi formed under flow, even those prepared as plasma 'thrombi', reveal the effect of FXIII on fibrinolysis. Although very low levels of FXIII are known to produce mechanical clot stability, and to achieve ?-dimerization, they appear to be suboptimal in conferring full resistance to fibrinolysis.

Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis

Background - Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size. Methods - The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology. Results - Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028). Conclusion - Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring.

A model to assess the cost effectiveness of statins in achieving the UK National Service Framework target cholesterol levels

Background: Coronary heart disease (CHD) is a public health priority in the UK. The National Service Framework (NSF) has set standards for the prevention, diagnosis and treatment of CHD, which include the use of cholesterol-lowering agents aimed at achieving targets of blood total cholesterol (TC) < 5.0 mmol/L and low density lipoprotein-cholesterol (LDL-C) < 3.0 mmol/L. In order to achieve these targets cost effectively, prescribers need to make an informed choice from the range of statins available. Aim: To estimate the average and relative cost effectiveness of atorvastatin, fluvastatin, pravastatin and simvastatin in achieving the NSF LDL-C and TC targets. Design: Model-based economic evaluation. Methods: An economic model was constructed to estimate the number of patients achieving the NSF targets for LDL-C and TC at each dose of statin, and to calculate the average drug cost and incremental drug cost per patient achieving the target levels. The population baseline LDL-C and TC, and drug efficacy and drug costs were taken from previously published data. Estimates of the distribution of patients receiving each dose of statin were derived from the UK national DIN-LINK database. Results: The estimated annual drug cost per 1000 patients treated with atorvastatin was £289 000, with simvastatin £315 000, with pravastatin £333 000 and with fluvastatin £167 000. The percentages of patients achieving target are 74.4%, 46.4%, 28.4% and 13.2% for atorvastatin, simvastatin, pravastatin and fluvastatin, respectively. Incremental drug cost per extra patient treated to LDL-C and TC targets compared with fluvastafin were £198 and £226 for atorvastatin, £443 and £567 for simvastatin and £1089 and £2298 for pravastatin, using 2002 drug costs. Conclusions: As a result of its superior efficacy, atorvastatin generates a favourable cost-effectiveness profile as measured by drug cost per patient treated to LDL-C and TC targets. For a given drug budget, more patients would achieve NSF LDL-C and TC targets with atorvastatin than with any of the other statins examined.

A framework for automated construction of resource space based on background knowledge

Resource Space Model is a kind of data model which can effectively and flexibly manage the digital resources in cyber-physical system from multidimensional and hierarchical perspectives. This paper focuses on constructing resource space automatically. We propose a framework that organizes a set of digital resources according to different semantic dimensions combining human background knowledge in WordNet and Wikipedia. The construction process includes four steps: extracting candidate keywords, building semantic graphs, detecting semantic communities and generating resource space. An unsupervised statistical language topic model (i.e., Latent Dirichlet Allocation) is applied to extract candidate keywords of the facets. To better interpret meanings of the facets found by LDA, we map the keywords to Wikipedia concepts, calculate word relatedness using WordNet's noun synsets and construct corresponding semantic graphs. Moreover, semantic communities are identified by GN algorithm. After extracting candidate axes based on Wikipedia concept hierarchy, the final axes of resource space are sorted and picked out through three different ranking strategies. The experimental results demonstrate that the proposed framework can organize resources automatically and effectively.©2013 Published by Elsevier Ltd. All rights reserved.

Smartphone-based digital government model:the case of the beyaz masa (white table) app in Turkey

M-Government services are now at the forefront of both user expectations and technology capabilities. Within the current setting, there is growing evidence that interoperability is becoming a key issue towards service sustainability. Thus, the objective of this chapter is to highlight the case of "Beyas Masa" - a Turkish application for infrastructure repair services. This application requires different stakeholders from different cultural background and geographically dispersed regions to work together. The major aim of this chapter to showcase experiences in as far as implementation and adoption of m-Government is concerned in the case of Turkey. The study utilizes the co-creation literature to investigate the factors influencing successful implementation of the Beyas Masa. This study reveals that initiatives are fragmented due to differences in the characteristics of the targeted audience, the marketing strategy, technology supply, distribution, and media utilized to promote its awareness. The chapter posits that in order to have affluent m-Government implementation in Turkey, it is important that many of the standalone applications are integrated to encourage interoperability and that socio-cultural behaviours should be re-shaped to encourage active engagement and interactive government service provisions that unlock the power of ICT.

Channel model and lower capacity bound for the transmission based on nonlinear Fourier transform

The integrability of the nonlinear Schräodinger equation (NLSE) by the inverse scattering transform shown in a seminal work [1] gave an interesting opportunity to treat the corresponding nonlinear channel similar to a linear one by using the nonlinear Fourier transform. Integrability of the NLSE is in the background of the old idea of eigenvalue communications [2] that was resurrected in recent works [3{7]. In [6, 7] the new method for the coherent optical transmission employing the continuous nonlinear spectral data | nonlinear inverse synthesis was introduced. It assumes the modulation and detection of data using directly the continuous part of nonlinear spectrum associated with an integrable transmission channel (the NLSE in the case considered). Although such a transmission method is inherently free from nonlinear impairments, the noisy signal corruptions, arising due to the ampli¯er spontaneous emission, inevitably degrade the optical system performance. We study properties of the noise-corrupted channel model in the nonlinear spectral domain attributed to NLSE. We derive the general stochastic equations governing the signal evolution inside the nonlinear spectral domain and elucidate the properties of the emerging nonlinear spectral noise using well-established methods of perturbation theory based on inverse scattering transform [8]. It is shown that in the presence of small noise the communication channel in the nonlinear domain is the additive Gaussian channel with memory and signal-dependent correlation matrix. We demonstrate that the effective spectral noise acquires colouring", its autocorrelation function becomes slow decaying and non-diagonal as a function of \frequencies", and the noise loses its circular symmetry, becoming elliptically polarized. Then we derive a low bound for the spectral effiency for such a channel. Our main result is that by using the nonlinear spectral techniques one can significantly increase the achievable spectral effiency compared to the currently available methods [9]. REFERENCES 1. Zakharov, V. E. and A. B. Shabat, Sov. Phys. JETP, Vol. 34, 62{69, 1972. 2. Hasegawa, A. and T. Nyu, J. Lightwave Technol., Vol. 11, 395{399, 1993. 3. Yousefi, M. I. and F. R. Kschischang, IEEE Trans. Inf. Theory, Vol. 60, 4312{4328, 2014. 4. Yousefi, M. I. and F. R. Kschischang, IEEE Trans. Inf. Theory, Vol. 60, 4329{4345 2014. 5. Yousefi, M. I. and F. R. Kschischang, IEEE Trans. Inf. Theory, Vol. 60, 4346{4369, 2014. 6. Prilepsky, J. E., S. A. Derevyanko, K. J. Blow, I. Gabitov, and S. K. Turitsyn, Phys. Rev. Lett., Vol. 113, 013901, 2014. 7. Le, S. T., J. E. Prilepsky, and S. K. Turitsyn, Opt. Express, Vol. 22, 26720{26741, 2014. 8. Kaup, D. J. and A. C. Newell, Proc. R. Soc. Lond. A, Vol. 361, 413{446, 1978. 9. Essiambre, R.-J., G. Kramer, P. J. Winzer, G. J. Foschini, and B. Goebel, J. Lightwave Technol., Vol. 28, 662{701, 2010.

The Policy Dystopia Model:an interpretive analysis of tobacco industry political activity

BACKGROUND: Tobacco industry interference has been identified as the greatest obstacle to the implementation of evidence-based measures to reduce tobacco use. Understanding and addressing industry interference in public health policy-making is therefore crucial. Existing conceptualisations of corporate political activity (CPA) are embedded in a business perspective and do not attend to CPA's social and public health costs; most have not drawn on the unique resource represented by internal tobacco industry documents. Building on this literature, including systematic reviews, we develop a critically informed conceptual model of tobacco industry political activity. METHODS AND FINDINGS: We thematically analysed published papers included in two systematic reviews examining tobacco industry influence on taxation and marketing of tobacco; we included 45 of 46 papers in the former category and 20 of 48 papers in the latter (n = 65). We used a grounded theory approach to build taxonomies of "discursive" (argument-based) and "instrumental" (action-based) industry strategies and from these devised the Policy Dystopia Model, which shows that the industry, working through different constituencies, constructs a metanarrative to argue that proposed policies will lead to a dysfunctional future of policy failure and widely dispersed adverse social and economic consequences. Simultaneously, it uses diverse, interlocking insider and outsider instrumental strategies to disseminate this narrative and enhance its persuasiveness in order to secure its preferred policy outcomes. Limitations are that many papers were historical (some dating back to the 1970s) and focused on high-income regions. CONCLUSIONS: The model provides an evidence-based, accessible way of understanding diverse corporate political strategies. It should enable public health actors and officials to preempt these strategies and develop realistic assessments of the industry's claims.