In this study we compare the cellular control of recombinant human IgG 4 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 qMabthe 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.

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.

Interdisciplinary work has the potential to advance public health science but the numerous challenges should not be underestimated. Use of a checklist, such as BASICS, when planning and managing projects may help future collaborations to avoid some of the common pitfalls of interdisciplinary research.

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.

gamma-Secretase cleaves amyloid precursor protein (APP) to generate amyloid-beta (Abeta) peptides, which aggregate in the brain in Alzheimer's disease (AD). gamma-Secretase also cleaves molecules that regulate osteoblast activity, such as Notch and ephrinB2. However, the role of APP in bone is unknown. In this study, the expression, cleavage, and function of APP were investigated during osteogenesis in vitro and in vivo. Expression of all gamma-secretase subunits was confirmed in human primary osteoprogenitors cells, and a significant increase in enzyme activity was observed during osteogenic differentiation using a specific fluorimetric assay. Application of selective inhibitors confirmed gamma-secretase-dependent cleavage of APP within osteogenic cells, and secretion of Abeta by mature osteoblasts was demonstrated with the use of a chemiluminescent immunoassay. Osteoprogenitors showed a selective and significant increase in adhesion to extracellular matrices containing aged Abeta plaques compared with nonaged Abeta peptide controls. Abeta on the endosteal and periosteal surfaces of adult rat ulnae were identified by immunohistochemistry. MicroCT analysis of vertebrae from an AD mouse model, Tg2576, identified a decrease in bone volume, surface area, and thickness compared with wild-type controls. These findings indicate that APP functions as a novel regulator of osteoblast activity and suggest that the mechanisms underlying the pathogenesis of AD may also influence bone.

Delayed heart repolarisation is marked by prolonged QT interval on electrocardiography and may be complicated by ventricular arrhythmias, syncope, and sudden death.' Such events have recently been associated with severe malnutrition, as occurs in anorexia nervosa2 and after ileojejunal bypass.3 These reports have prompted us to measure QT interval in adult patients with coeliac disease, a condition which often presents with nutritional impairment.

Background Studies on the determinants of the development of substance use disorders are done across the natural and social sciences. However, research is usually conducted by individual or closely related disciplines, so evidence on how determinants from multiple disciplines interact has been slow to emerge. Within the Addiction and Lifestyles In Contemporary Europe Reframing Addictions Project (ALICE RAP) we have brought together experts from 11 disciplines in the humanities, social sciences, and medical sciences to identify the determinants of risky and harmful substance use and gambling. The aim of the present research was to explore ALICE RAP members' experiences of working with experts from diverse disciplines and to make recommendations for best practice in such projects.Methods All members of the interdisciplinary team (n=18) were invited to participate in an in-depth interview in early 2014. The aims were to explore the experience of working in an interdisciplinary manner; to identify perceived strengths and weaknesses in our approach; and to provide recommendations for future interdisciplinary collaborations. 14 experts representing ten disciplines participated. Data were analysed thematically, and recommendations were derived from suggestions made by participants and discussion among the authors on the implications of the fi ndings. Ethics approval for this study was granted by the ScHARR Research Ethics Review Committee at the University of Sheffi eld in December, 2013.Findings Participants described fi ve strengths of our approach: inclusiveness, generosity and openness of experts, fl exibility, diverse forms of interaction, and the value of a dedicated facilitator or facilitators. However, the widespread view was that we had underestimated the challenge of interdisciplinary working. Specifi c concerns were diverse approaches to scientifi c evidence, diff ering expectations of discipline experts, ambiguity of some disciplines' roles, confl icting commitments, and a vague project endpoint. We have identifi ed four recommendations for interdisciplinary working: to have a blueprint for the integration of disciplines, to be clear about expectations, to ensure resilient project staffi ng, and to talk frequently to maintain relationships and progress work.Interpretation Interdisciplinary work is challenging; however, our recommendations for interdisciplinary collaborations, identifi ed through expert interviews, could be used to facilitate the successful planning and management of interdisciplinary work in the specialty of substance use disorders and beyond.

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