Glycosylation: impact,control and improvement during therapeutic protein production

Abstract

The emergence of the biopharmaceutical industry represented a major revolution for modern medicine, through the development of recombinant therapeutic proteins that brought new hope for many patients with previously untreatable diseases. There is a ever-growing demand for these therapeutics that forces a constant technological evolution to increase product yields while simultaneously reducing costs. However, the process changes made for this purpose may also affect the quality of the product, a factor that was initially overlooked but which is now a major focus of concern. Of the many properties determining product quality, glycosylation is regarded as one of the most important, influencing, for example, the biological activity, serum half-life and immunogenicity of the protein. Consequently, monitoring and control of glycosylation is now critical in biopharmaceutical manufacturing and a requirement of regulatory agencies. A rapid evolution is being observed in this context, concerning the influence of glycosylation in the efficacy of different therapeutic proteins, the impact on glycosylation of a diversity of parameters/processes involved in therapeutic protein production, the analytical methodologies employed for glycosylation monitoring and control, as well as strategies that are being explored to use this property to improve therapeutic protein efficacy (glycoengineering). This work reviews the main findings on these subjects, providing an up-to-date source of information to support further studies.     

Organotypic Suprachiasmatic Nuclei Cultures of Adult Voles Reflect Locomotor Behavior: Differences in Number of Vasopressin Cells

This study is the first to demonstrate organotypic culturing of adult suprachiasmatic nuclei (SCN). This approach was used to obtain organotypic SCN cultures from adult vole brain with a previously determined state of behavioral circadian rhythmicity. We examined vasopressin (AVP) immunoreactivity in these organotypic slice cultures. AVP is one of the major neuropeptides produced by the SCN, the main mammalian circadian pacemaker. AVP immunoreactivity in the SCN of adult common voles in vivo has been shown to correlate with the variability in expression of circadian wheel-running behavior. Here, cultures prepared from circadian rhythmic and nonrhythmic voles were processed immunocytochemically for AVP. Whereas in all cultures AVP could be observed, AVP immunoreactivity differed considerably between vole SCN cultures. SCN cultures from rhythmic voles contained significantly lower numbers of AVP immunoreactive (AVPir) cells per surface area than cultures from nonrhythmic voles. The correlation between timing of behavior and AVP immunoreactivity in vitro is similar to the correlation found earlier in vivo. Apparently, such correlation depends on intrinsic AVP regulation mechanisms of SCN tissue, and not on neural or hormonal input from the environment, as present in intact brain.     

Scanning electron microscopy of glochidia and juveniles of the freshwater mussel,Hyriopsis myersiana

Summary

The ultrastructure of early stages of the mussel, Hyriopsis (Limnoscapha) myersiana (Lea, 1856), was observed by scanning electron microscopy from the glochidial period until the onset of the juvenile stage 10 days later. Further observations were performed for an additional 13 days to assess juvenile development. Glochidia extracted from the brood chambers have a hookless, semi-oval and equivalve calcareous shell with numerous pores in the internal surface, pits in the external surface and cuticular spines in the ventral region. Keratin fibers with a random arrangement in the cuticle of the glochidial shell were also detected. The appearance of the foot within 10 days of in vitro glochidial culture was considered the main feature of metamorphosis to the juvenile stage. Another change during the following 13 days was the formation of a new periostracum exhibiting growth lines under the old glochidial shell. This development occurs mainly in the anterior region and is followed by hardening of the periostracum matrix by calcium deposition. Periostracum growth gradually became apparent in the lateral and posterior regions at the end of this period. The retraction of spines and the alteration of the external surface of the old shell are also described. It is speculated that transcuticular filaments identified in the juvenile stage may have sensory or metabolic exchange functions. The prominent foot, gradually covered by long dense cilia, shows rhythmical movements which suggest a role in feeding. Similarly, cilia present in the mantle may also be involved in the capture of food, while microvilli may facilitate absorption of dissolved materials. Longer cilia, sparsely distributed in the mantle, may function as chemo- or tactile sensors.     

Wild plants eaten in childhood: a retrospective of Estonia in the 1970s–1990s

In this ethnobotanical study, the authors provide the first quantitative analysis of the use of wild edible plants in Estonia, describing the domains and assessing the food importance of different species. The information was collected using free‐listing written questionnaires and concerned plants used by the respondents in their childhood. As part of a major study, this article covers the responses of professionals with some botanical education at vocational or university level, to ensure the greatest possible reliability without using voucher specimens. Fifty‐eight respondents provided information on the use of 137 plant taxa, corresponding to approximately 6% of the native and naturalized vascular plants of Estonia. According to use frequency, the most typical wild food plant of Estonia is a fruit, eaten raw as a snack. The results clearly signal that the majority of famine and food shortage plants had already been forgotten by the end of the 20^th century, but new plants have been introduced as green vegetables for making salads. Despite changes in the nomenclature of the plants, the use of wild food plants in Estonia was still thriving at the turn of the 20^th century, covering many domains already forgotten in urbanized modern Europe. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 239–253.     

Ethnic identity on display: West Indian youth and the creation of ethnic boundaries in high school

The black immigrant population in New York City has grown exponentially since 1990, such that West Indians now compose the majority of the black population in several neighbourhoods. This article examines how this ethnic density manifests among youth in high school, and how it has influenced ethnic identity formation among second-generation West Indians. My findings are based on twenty-four interviews and eight months of participant observation in two Brooklyn high schools from 2003 to 2004. The results show that in both schools, Caribbean island identities have become a ‘cool’ commodity within peer groups. Further, although it was important to express pride in one's island identity, these young people often blurred their national origin boundaries by drawing on Jamaican popular culture as way of projecting a unified ‘West Indian identity’. The research also uncovers evidence of a de-stigmatization of Haitianness as a way to incorporate them as cultural insiders into the larger Caribbean collective.     

Assimilation without groups?

In “Origins and Destinations”, Luthra, Soehl and Waldinger aim at “extending the canon” in research on immigrant integration. They do so by studying group and individual level characteristics across a large number of origin groups, thereby replacing group labels with theoretically relevant variables at the group level. In this review, three challenges in this endeavour are discussed: First of all, the analyses do not allow to disentangle the impact of group and individual level characteristics for all variables alike. Secondly, a primary data collection more tailored to the specifics of their conceptual framework is needed. This would many conclusions about how contexts of emigration and immigration truly matter above and beyond individual characteristic on a more solid ground. And third, the authors’ aim to abandon the use of origin group categories in empirical research on immigrant integration may come at a price.     

Colour-blindness and diversity: race frames and their consequences for white undergraduates at elite US universities

In this paper we bring together the literatures on frame analysis, the meaning of race and campus racial climate to analyse the race frames – lenses through which individuals understand the role of race in society – held by white students attending elite US universities. For most, the elite university experience coincides with a strengthening or emergence of the diversity frame, which emphasizes the positive benefits of cultural diversity. Still, many also hold a colour-blind frame, which sees race groups as equivalent and racial identities as insignificant. We highlight the ambivalence that these divergent frames create for student perspectives on affirmative action and interracial contact on campus. Our findings demonstrate the mutability of race frames. We also highlight the impact that institutions may have on individuals' race frames. The paper is based on in-depth interviews with forty-seven US-born white undergraduates attending Brown University and Harvard University.     

Continuous hydrogen peroxide production by organic buffers in phytoplankton culture media

We investigated the production of hydrogen peroxide (HOOH) in illuminated seawater media containing a variety of zwitterionic buffers. Production rates varied extensively among buffers, with 4‐(2‐hydroxyethyl)1‐piperazineethanesulfonic acid (HEPES) highest and N‐Tris(hydroxymethyl)methyl‐3‐aminopropanesulfonic acid (TAPS) among the lowest. The rate of HOOH accumulation was remarkably consistent over many days, and increased linearly with buffer concentration, natural seawater concentration, and light level. Concentrations of HEPES commonly used in culture media (1–10 mM) generated enough HOOH to kill the axenic Prochlorococcus strain VOL1 during growth in enriched seawater media at lower, environmentally realistic cell concentrations and/or under high light exposure. We also demonstrated that HEPES can be used experimentally to study the biological effects of chronic exposure to sublethal levels of HOOH such as may be experienced by light‐exposed microorganisms.     

Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium.     

Refractions through culture: the new genomics in Iceland

This paper explores the processes whereby complex scientific developments are rendered into locally intelligible idioms as part of strategies to incorporate a ‘public’ in decisions which may have profound implications in the longer term. We focus in particular on the recent developments in genetic technologies and take as our example the deCode biogenetic project in Iceland. We trace how the images and metaphors drawn upon by all sides of the debate created powerful resonances with Icelandic history and culture. We analyse these representations and the broader themes of ethnicity and nationalism which they invoke.