Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.

Measuring employee innovation:a review of existing scales and the development of the innovative behavior and innovation support inventories across cultures

Purpose - The paper develops a model of employee innovative behavior conceptualizing it as distinct from innovation outputs and as a multi-faceted behavior rather than a simple count of ‘innovative acts’ by employees. It understands individual employee innovative behaviors as a micro-foundation of firm intrapreneurship that is embedded in and influenced by contextual factors such as managerial, organizational and cultural support for innovation. Building from a review of existing employee innovative behavior scales and theoretical considerations we develop and validate the Innovative Behavior Inventory (IBI) and the Innovation Support Inventory (ISI). Design/methodology/approach – Two pilot studies, a third validation study in the Czech Republic and a fourth cross-cultural validation study using population representative samples from Switzerland, Germany, Italy and the Czech Republic (N=2812 employees and 450 entrepreneurs) were conducted. Findings - Both inventories were reliable and showed factorial, criterion, convergent and discriminant validity as well as cross-cultural equivalence. Employee innovative behavior was supported as comprising of idea generation, idea search, idea communication, implementation starting activities, involving others and overcoming obstacles. Managerial support was the most proximal contextual influence on innovative behavior and mediated the effect of organizational support and national culture. Originality/value - The paper advances our understanding of employee innovative behavior as a multi-faceted phenomenon and the contextual factors influencing it. Where past research typically focuses on convenience samples within a particular country, we offer first robust evidence that our model of employee innovative behavior generalizes across cultures and types of samples. Our model and the IBI and ISI inventories enable researchers to build a deeper understanding of the important micro-foundation underpinning intrapreneurial behavior in organizations and allow practitioners to identify their organizations’ strengths and weaknesses related to intrapreneurship.

A climate profile indicator based comparative analysis of climate change scenarios with regard to maize cultures

Using ecological data compiled from scientific literature on pest, pathogen and weed species characteristic in maize cultures in Hungary, we defined monthly climate profile indicators and applied them to complete a comparative analysis of the historical and modelled climate change scenario meteorological data of the city of Debrecen. Our results call attention to a drastic decline of the competitive ability of maize as compared to several C4 and especially C3 plants. According to the stricter scenarios, the frequency of potential pest and pathogen damage emergency situations will grow significantly by the end of the century.

Gender across cultures: The immigration experience/Bulgarian immigrants in South Florida

During the last decade, the United States has witnessed a high increase in its immigration flow. Despite recent surge of interest in numerically small ethnic groups, data on Bulgarian immigrants in the United States is almost non-existent. This is the first comprehensive study on the Bulgarian ethnic group in the United States. ^ This study examined the impact of immigration upon the negotiation of cultural prescriptions by Bulgarian immigrants in the United States. Several fundamental Bulgarian prescriptions for daily behavior, such as emphasis on stability rather than mobility, preference for collectivism instead of individualism, and wide use of social critique rather than social approval were tested on U.S. soil. Furthermore, the impact of gender upon cultural appropriation and retention was considered. The study made use of a comprehensive historical analysis of Bulgarian immigration to the United States, statistical descriptions of contemporary Bulgarian immigrants in the United States, and over a three-year long qualitative study of Bulgarian immigrants in South Florida. ^ The results of the study indicated that the transferring of cultural prescriptions due to immigration did not occur unaffected by gender. Bulgarian and U.S. cultural models clashed, intermingled, and resisted each other in various ways depending on the immigrants' gender. At the same time, gender did not solely determine the validity of prescriptions for daily behavior, but it rather acted in concert with other principles of everyday life. In addition, gender impacted significantly the method, type, and structure of the immigration journey. Gender was a very significant variable in the determination of these features of the immigration process, but it did not account independently for historical variations. Other factors, such as larger contexts of reception and rejection also played an important role. ^

A study of dual cultures in dyadic relations

Elderly caregiving, a fact of life for millions of Americans, has gained significance with the increase in the elderly population. Over 25 million family caregivers in the US, most of whom are women rearing their own children, care for severely ill or disabled family members. The increased flow of women into the labor force has caused this traditional role to be entrusted to hired caregivers, mainly home health aides. ^ This case study describes and explains the dyadic experiences of elderly Jewish clients and their Jamaican home health aides in a mixed-culture environment. The inquiry was conducted with a purposive sample of four dyads, their case manager, and the placement officer, all of whom were selected through a home care agency in Fort Lauderdale, Florida. Strategies of data collection including non-participant observation, interviews, document analysis, and researcher's journals. Data from verbatim transcription of interviews and field notes were coded, sorted into emic categories, and reduced as linkages were identified via cross-case comparison. Three major themes—mixed-culture experiences, relationship building and maintenance, and agency role perceptions—emerged from the interpretative analysis of the data. ^ Assertions from these findings include that dyads have established a range of relationships to meet their personal needs, expectations, and desires in these goal-driven relationships. Relationally, they have reached interactional synchrony with some achieving the ideal family-type bond. Cultural difference is but one of the many contextual variables in the home care environment, which has its own cultural norms and expectations. Conflicts transcend cultural difference and seemed more a factor of individual relational disposition. ^

Identification and localization of polyketide synthase genes from cultures of diarrheic shellfish poisoning toxin producing dinoflagellates of the genus Prorocentrum

Aquatic toxins are responsible for a number of acute and chronic diseases in humans. Okadaic acid (OA) and other dinoflagellate derived polyketide toxins pose serious health risks on a global scale. Ingestion of OA contaminated shellfish causes diarrheic shellfish poisoning (DSP). Some evidence also suggests tumor promotion in the liver by OA. Microcystin-LR (MC-LR) is produced by cyanobacteria and is believed to be the most common freshwater toxin in the US. Humans may be exposed to this acute hepatotoxin through drinking or recreational use of contaminated waters. ^ OA producing dinoflagellates have not been cultured axenically. The presence of associated bacteria raises questions about the ultimate source of OA. Identification of the toxin-producing organism(s) is the first step in identifying the biosynthetic pathways involved in toxin production. Polyketide synthase (PKS) genes of toxic and non-toxic species were surveyed by construction of clonal libraries from PCR amplicons of various toxic and non-toxic species of Prorocentrum in an effort to identify genes, which may be part of the biosynthetic pathway of OA. Analysis of the PKS sequences revealed that toxic species shared identical PKS genes not present in non-toxic species. Interestingly, the same PKS genes were identified in a library constructed from associated bacteria. ^ Subsequent bacterial small subunit RNA (16S) clonal libraries identified several common bacterial species. The most frequent 16S sequences found were identified as species of the genus Roseobacter which has previously been implicated in the production of OA. Attempts to culture commonly occurring bacteria resulted in the isolation of Oceanicaulis alexandrii , a novel marine bacterium previously isolated from the dinoflagellate Alexandrium tamarense, from both P. lima, and P. hoffmanianum. ^ Metabolic studies of microcystin-LR, were conducted to probe the activity of the major human liver cytochromes (CYP) towards the toxin. CYPs may provide alternate routes of detoxification of toxins when the usual routes have been inhibited. For example, some research indicates that cyanobacterial xenobiotics, in particular, lipopolysaccharides may inhibit glutathione S-transferases allowing the toxin to persist long enough to be acted upon by other enzymes. These studies found that at least one human liver CYP was capable of metabolizing the toxin. ^

Hyphenated cultures: Ethnicity and nation in Trinidad

This dissertation attempts to unravel why and how postcolonial Trinidad has displayed relative stability in spite of the presence of the factors that have produced conflict and instability in other postcolonial societies.^ Trinidad's distinctive social formation began in the colonial period with a unique politics of culture among the landowning European groups, Anglican English and French Creole. Contrary to the materialist assumption of landowners' class solidarity, the development of Trinidad's plantation economy into two crops, each controlled by a separate European ethno-religious faction, impeded the integration and subsequent ideological domination of European-Christians. Throughout the nineteenth century neither group dominated the other, nor did they fuse into a single ruling class. The dynamics between them both generated recurring conflict while simultaneously creating mechanisms that limited conflict. ^ Based on original in-depth fieldwork and historical analysis, the dissertation proceeds to demonstrate that Trinidad's unique intra-class conflict within the dominant European population has produced hyphenated, as opposed to hybridized cultural elements. Supplementing the historical analysis with empirical examinations of contemporary inter-religious rituals and post-colonial politics this dissertation argues that social integration is inseparable from the question of inter-cultural mixture or articulation. In Trinidad, however, the resulting combination of distinct cultural elements is neither a "plural society" (M.G. Smith 1965; Despres 1967) nor an integrated totality in the structural-functionalistic sense (R.T. Smith 1962; Braithwaite 1967). Moreover, Trinidad does not conform to the post-structural framework's depiction of the social linkage between power and culture. The concept of cultural hybridization is equally misleading in the case of Trinidad. The underlying assumption of a monolithic European population's cultural hegemony and post-structural analysis's almost exclusive focus on the inter -class politics of culture seriously misrepresent and misunderstand Trinidadian cultural and its associated social and political relations. The dissertation examines this reflexive influence of culture not as an instrument of the powerful few but as an autonomous force that reproduces social divisions, yet restrains conflict.^

Collecting and Curating Cuban: Memory, Museum, and Material Cultures, Lecture by Raúl Rubio and Christian Larsen

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Woven cultures : New insights into Pictish and Viking culture contact using the implements of textile production

This paper is based on the undergraduate dissertation of Lindsey Stirling, which was supervised by Karen Milek, and which won the Society of Medieval Archaeology's John Hurst Memorial Dissertation Prize Acknowledgements This research would not have been possible without the assistance of Dr Martin Goldberg at the National Museum of Scotland, Lynda Aiano at Tankerness House Museum, Orkney, and Beverley Ballin Smith. The authors also wish to thank the two anonymous reviewers who provided valuable comments on earlier versions of this paper.

Woven cultures : New insights into Pictish and Viking culture contact using the implements of textile production

This paper is based on the undergraduate dissertation of Lindsey Stirling, which was supervised by Karen Milek, and which won the Society of Medieval Archaeology's John Hurst Memorial Dissertation Prize Acknowledgements This research would not have been possible without the assistance of Dr Martin Goldberg at the National Museum of Scotland, Lynda Aiano at Tankerness House Museum, Orkney, and Beverley Ballin Smith. The authors also wish to thank the two anonymous reviewers who provided valuable comments on earlier versions of this paper.

Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

Peer reviewed

Il faut défendre les cultures : une ethnographie de l'UNESCO

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Protection of primary cultures of mouse hepatocytes against fas-induced apoptosis : role of EGF receptor intrinsic activity and intracellular redox state

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Faire dialoguer les cultures : rencontre entre la bioéthique et l’art contemporain

Commentaire critique / Critical Commentary

Il faut défendre les cultures : une ethnographie de l'UNESCO

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.