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.

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

Enforceability of leasehold covenants and the landlord and tenant (Covenants) Act 1995

Describes the impact of the English Landlord and Tenant (Covenants) Act 1995, reforming liability in the context of new leases, extending the 'touching and concerning' requirement so all covenants 'run with the land' (with some exceptions), and abolishing the enduring liability of the original tenants and landlords. Explains that landlords will have more freedom to prescribe in advance the circumstances in which they consent to an assignment, referring also to changes in default notices requiring an 'early warning' to defaulters, and overriding leases, with a remedy for former tenants. Expects future leases to be shorter as landlords realize they cannot hold original tenants liable any more.

Finding natural groups in data:an application to strategic group research

The best way of finding “natural groups” in management research remains subject to debate and within the literature there is no accepted consensus. The principle motivation behind this study is to explore the effect of choices of method upon strategic group research, an area that has suffered enduring criticism, as we believe that these method choices are still not fully exploited. Our study is novel in the use of a variety of more robust clustering and validation techniques, rarely used in management research, some borrowed from the natural sciences, which may provide a useful and more robust base for this type of research. Our results confirm that methods do exist to address the concerns over strategic group research and adoption of our chosen methods will improve the quality of management research.

The role of analogy in spelling in English primary school children - a partial intervention study

The English writing system is notoriously irregular in its orthography at the phonemic level. It was therefore proposed that focusing beginner-spellers’ attention on sound-letter relations at the sub-syllabic level might improve spelling performance. This hypothesis was tested in Experiments 1 and 2 using a ‘clue word’ paradigm to investigate the effect of analogy teaching intervention / non-intervention on the spelling performance of an experimental group and controls. The results overall showed the intervention to be effective in improving spelling, and this effect to be enduring. Experiment 3 demonstrated a greater application of analogy in spelling, when clue words, which participants used in analogy to spell test words, remained in view during testing. A series of regression analyses, with spelling entered as the criterion variable and age, analogy and phonological plausibility (PP) as predictors, showed both analogy and PP to be highly predictive of spelling. Experiment 4 showed that children could use analogy to improve their spelling, even without intervention, by comparing their performance in spelling words presented in analogous categories or in random lists. Consideration of children’s patterns of analogy use at different points of development showed three age groups to use similar patterns of analogy, but contrasting analogy patterns for spelling different words. This challenges stage theories of analogy use in literacy. Overall the most salient units used in analogy were the rime and, to a slightly lesser degree, the onset-vowel and vowel. Finally, Experiment 5 showed analogy and phonology to be fairly equally influential in spelling, but analogy to be more influential than phonology in reading. Five separate experiments therefore found analogy to be highly influential in spelling. Experiment 5 also considered the role of memory and attention in literacy attainment. The important implications of this research are that analogy, rather than purely phonics-based strategy, is instrumental in correct spelling in English.

The behaviour of granular material in pure shear, direct shear and simple shear

In biaxial compression tests, the stress calculations based on boundary information underestimate the principal stresses leading to a significant overestimation of the shear strength. In direct shear tests, the shear strain becomes highly concentrated in the mid-plane of the sample during the test. Although the stress distribution within the specimen is heterogeneous, the evolution of the stress ratio inside the shear band is similar to that inferred from the boundary force calculations. It is also demonstrated that the dilatancy in the shear band significantly exceeds that implied from the boundary displacements. In simple shear tests, the stresses acting on the wall boundaries do not reflect the internal state of stress but merely provide information about the average mobilised wall friction. It is demonstrated that the results are sensitive to the initial stress state defined by K0 = sh/sv. For all cases, non-coaxiality of the principal stress and strain-rate directions is examined and the corresponding flow rule is identified. Periodic cell simulations have been used to examine biaxial compression for a wide range of initial packing densities. Both constant volume and constant mean stress tests have been simulated. The characteristic behaviour at both the macroscopic and microscopic scales is determined by whether or not the system percolates (enduring connectivity is established in all directions). The transition from non-percolating to percolating systems is characterised by transitional behaviour of internal variables and corresponds to an elastic percolation threshold, which correlates well with the establishment of a mechanical coordination number of ca. 3.0. Strong correlations are found between macroscopic and internal variables at the critical state.

Stanley Hoffmann: a political life:a political life

Stanley Hoffmann is one of the most eminent political scholars of our age—a renowned authority in the study of French, European, and world politics over half a century, an influential theorist of international relations, a critical analyst of US foreign policy, and a voice of moral conscience in many public debates of his time. Hoffmann has always asked big questions—and to those questions he brings an encyclopedic mind that crosses boundaries between politics, history, sociology, law, philosophy, ethics, and literature. This brief article highlights some aspects of his life and work, and introduces a symposium in his honor bringing together five leading scholars on France, Europe, international relations, and international law—each with an enduring debt to the teaching, writings and example of Stanley Hoffmann.

The relative strength of affective commitment in securing loyalty in service relationships

While commitment is an important antecedent to customer retention, a broad consensus has yet to emerge on the impacts of constituent dimensions of commitment on loyalty in service relationships. This study explores the impacts of affective and continuance commitment on attitudinal and behavioral loyalty in a service context. Since affective commitment is more positive and governed by free choice, whereas continuance commitment is more the result of perceived economic and psychological benefits of being in a relationship, the results of this study suggest that emotional bonds with customers provide a more enduring source of loyalty as compared to economic incentives and switching costs.

Institutional work at field-configuring events:shaping industry change within sustainable transitions

Adopting an institutional approach from organization studies, this paper explores the role of key actors on “purposeful governance for sustainability” (Smith, Voss et al. 2010: 444) through the case of smart metering in the UK. Institutions are enduring patterns in social life, reflected in identities, routines, rules, shared meanings and social relations, which enable, and constrain, the beliefs and behaviours of individual and collective actors within a field (Thornton and Ocasio 2008). Large-scale external initiatives designed to drive regime-level change prompt ‘institutional entrepreneurs’ to perform ‘institutional work’ – “purposive action aimed at creating, maintaining and disrupting institutions” (Lawrence and Suddaby, 2006). Organization scholars are giving increasing attention to ‘field-configuring events’ (FCEs) which provide social spaces for diverse organizational actors to come together to collectively shape socio-technical pathways (Lampel and Meyer 2008). Our starting point for this exploratory study is that FCEs can offer important insights to the dynamics, politics and governance of sustainability transitions. Methodologically, FCEs allow us to observe and “link field evolution at the macro-level with individual action at the micro-level” (Lampel and Meyer, 2008: 1025). We examine the work of actors during a series of smart metering industry forums over a three-year period (industry presentations [n= 77] and panel discussions [n= 16]). The findings reveal new insights about how institutional change unfolds, alongside technological transitions, in ways that are partial and aligned with the interests of powerful incumbents whose voices are frequently heard at FCEs. The paper offers three contributions. First, the study responds to calls for more research examining FCEs and the role they play in transforming institutional fields. Second, the emergent findings extend research on institutional work by advancing our understanding of a specific site of institutional work, namely a face-to-face inter-organizational arena. Finally, in line with the research agenda for innovation studies and sustainability transitions elaborated by Smith et al (2010), the paper illustrates how actors in a social system respond to, translate, and enact interventions designed to promote industrial transformation, ultimately shaping the sustainability transition pathway.

Leadership propensity and sales performance among sales personnel and managers in a speciality retail store setting

We propose that specialty store managers, as well as outside sales personnel attached to the store, have selling responsibilities. In addition, we propose that sales personnel, as well as store managers, should have a propensity for leadership, which reflects an individual's enduring disposition to exhibit leadership within the context of his or her organizational roles. In two studies, we develop a new individual difference measure of propensity to lead and investigate its nomological validity within a specialty retail store environment. As predicted, leadership propensity was predictive of self-rated sales performance and a proclivity to identify prospects through cold calls to close sales, to reveal customer orientation, and to exhibit organizational citizenship behavior. We found that propensity to lead did not differ between salespeople and retail store managers, but we found that the respondent's role moderated the relationship between propensity to lead and supervisor performance ratings. Study limitations and managerial implications of this heretofore unidentified trait of salespeople are discussed.

Theoretical perspectives in operations management:an analysis of the literature

Purpose - It is important to advance operations management (OM) knowledge while being mindful of the theoretical developments of the discipline. The purpose of this paper is to explore which theoretical perspectives have dominated the OM field. This analysis allows the authors to identify theory trends and gaps in the literature and to identify fruitful areas for future research. A reflection on theory is also practical, given that it guides research toward important questions and enlightens OM practitioners. Design/methodology/approach - The authors provide an analysis of OM theory developments in the last 30 years. The study encompasses three decades of OM publications across three OM journals and contains an analysis of over 3,000 articles so as to identify which theories, over time, have been adopted by authors in order to understand OM topics. Findings - The authors find that the majority of studies are atheoretical, empirical, and focussed upon theory testing rather than on theory development. Some theories, such as the resource-based view and contingency theory, have an enduring relevance within OM. The authors also identify theories from psychology, economics, sociology, and organizational behavior that may, in the future, have salience to explain burgeoning OM research areas such as servitization and sustainability. Research limitations/implications - The study makes a novel contribution by exploring which main theories have been adopted or developed in OM, doing so by systematically analyzing articles from the three main journals in the field (the Journal of Operations Management, Production and Operations Management, and the International Journal of Operations and Production Management), which encompass three decades of OM publications. In order to focus the study, the authors may have missed important OM articles in other journals. Practical implications - A reflection on theories is important because theories inform how a researcher or practicing manager interprets and solves OM problems. This study allows the authors to reflect on the collective OM journey to date, to spot trends and gaps in the literature, and to identify fruitful areas for future research. Originality/value - As far as the authors are aware, there has not been an assessment of the main theoretical perspectives in OM. The research also identifies which topics are published in OM journals, and which theories are adopted to investigate them. The authors also reflect on whether the most cited papers and those winning best paper awards are theoretical. This gives the authors a richer understanding of the current state of OM research.

Corporate identity at the stakeholder group level:a conceptual model of front-line employee corporate identity

There is a paucity of literature regarding the construction and operation of corporate identity at the stakeholder group level. This article examines corporate identity from the perspective of an individual stakeholder group, namely, front-line employees. A stakeholder group that is central to the development of an organization’s corporate identity as it spans an organization’s boundaries, frequently interacts with both internal and external stakeholders, and influences a firm’s financial performance by building customer loyalty and satisfaction. The article reviews the corporate identity, branding, services and social identity literatures to address how corporate identity manifests within the front-line employee stakeholder group, identifying what components comprise front-line employee corporate identity and assessing what contribution front-line employees make to constructing a strong and enduring corporate identity for an organization. In reviewing the literature the article develops propositions that, in conjunction with a conceptual model, constitute the generation of theory that is recommended for empirical testing.