Improvements in single-use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance

Single-use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO-K1, and CHO-DP12, cultured in media conditioned in an older single-use bioreactor (SUB) film (F-1) and a newer generation film (F-2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F-1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F-2 film. Proteomic profiling of CHO cells cultured in the F-1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F-2 films when compared to F-1 films including bDtBPP, potentially explaining improved CHO cell growth in F-2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess.     

Intraspecific genetic variations, fitness cost and benefit of RPW8, a disease resistance locus in Arabidopsis thaliana

The RPW8 locus of Arabidopsis thaliana confers broad-spectrum resistance to powdery mildew pathogens. In many A. thaliana accessions, this locus contains two homologous genes, RPW8.1 and RPW8.2. In some susceptible accessions, however, these two genes are replaced by HR4, a homolog of RPW8.1. Here, we show that RPW8.2 from A. lyrata conferred powdery mildew resistance in A. thaliana, suggesting that RPW8.2 might have gained the resistance function before the speciation of A. thaliana and A. lyrata. To investigate how RPW8 has been maintained in A. thaliana, we examined the nucleotide sequence polymorphisms in RPW8 from 51 A. thaliana accessions, related disease reaction phenotypes to the evolutionary history of RPW8.1 and RPW8.2, and identified mutations that confer phenotypic variations. The average nucleotide diversities were high at RPW8.1 and RPW8.2, showing no sign of selective sweep. Moreover, we found that expression of RPW8 incurs fitness benefits and costs on A. thaliana in the presence and absence of the pathogens, respectively. Our results suggest that polymorphisms at the RPW8 locus in A. thaliana may have been maintained by complex selective forces, including those from the fitness benefits and costs both associated with RPW8.     

Diagnosing idiopathic learning disability: a cost-effectiveness analysis of microarray technology in the National Health Service of the United Kingdom

Array based comparative genomic hybridisation (aCGH) is a powerful technique for detecting clinically relevant genome imbalance and can offer 40 to > 1000 times the resolution of karyotyping. Indeed, idiopathic learning disability (ILD) studies suggest that a genome-wide aCGH approach makes 10–15% more diagnoses involving genome imbalance than karyotyping. Despite this, aCGH has yet to be implemented as a routine NHS service. One significant obstacle is the perception that the technology is prohibitively expensive for most standard NHS clinical cytogenetics laboratories. To address this, we investigated the cost-effectiveness of aCGH versus standard cytogenetic analysis for diagnosing idiopathic learning disability (ILD) in the NHS. Cost data from four participating genetics centres were collected and analysed. In a single test comparison, the average cost of aCGH was ￡442 and the average cost of karyotyping was ￡117 with array costs contributing most to the cost difference. This difference was not a key barrier when the context of follow up diagnostic tests was considered. Indeed, in a hypothetical cohort of 100 ILD children, aCGH was found to cost less per diagnosis (￡3,118) than a karyotyping and multi-telomere FISH approach (￡4,957). We conclude that testing for genomic imbalances in ILD using microarray technology is likely to be cost-effective because long-term savings can be made regardless of a positive (diagnosis) or negative result. Earlier diagnoses save costs of additional diagnostic tests. Negative results are cost-effective in minimising follow-up test choice. The use of aCGH in routine clinical practice warrants serious consideration by healthcare providers. Copyright statement The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article (if accepted) to be published in BMJ editions and any other BMJPGL products and to exploit all subsidiary rights, as set out in our licence (bmj.com/advice/copyright.shtml). Authorship The authors included on this paper fulfil the criteria of authorship and no one who fulfils the criteria has been excluded from authorship. The authors made a substantial contribution to the conception, design, analysis and interpretation of data. They were involved in drafting the article or revising it critically for important intellectual content and approving the version to be published. Contributorship Sarah Wordsworth (Guarantor): Planning, conducting and reporting work, interpretation of data, drafting and revising article. James Buchanan: Conducting and reporting work, interpretation of data, revising article. Regina Regan: Completing costing questionnaire, providing protocol details, other costing information, interpretation of data, information about learning disability and genome imbalance and revising article. Val Davison: Completing costing questionnaire, providing protocol details, sharing overall laboratory experience and drafting article. Kim Smith: Completing costing questionnaire, providing protocol details, drafting article. Sara Dyer: Completing costing questionnaire and providing protocol details. Carolyn Campbell: Completing costing questionnaire and providing protocol details. Edward Blair: Critical appraisal of article for clinical content and revising article. Eddy Maher: Completing costing questionnaire, providing protocol details, sharing overall laboratory experience and drafting article. Jenny Taylor: Planning and facilitating work between centres. Drafting and revising article. Samantha JL Knight: Completing costing questionnaire, providing protocol details, other costing information, interpretation of data, providing information about learning disability and genome imbalance, drafting and revising article. Jenny Taylor and Samantha JL Knight contributed equally to the work presented.     

Mechanosensitive channels in bacteria: signs of closure?

Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.     

Pediatric measles vaccine expressing a dengue antigen induces durable serotype-specific neutralizing antibodies to dengue virus

Dengue disease is an increasing global health problem that threatens one-third of the world's population. Despite decades of efforts, no licensed vaccine against dengue is available. With the aim to develop an affordable vaccine that could be used in young populations living in tropical areas, we evaluated a new strategy based on the expression of a minimal dengue antigen by a vector derived from pediatric live-attenuated Schwarz measles vaccine (MV). As a proof-of-concept, we inserted into the MV vector a sequence encoding a minimal combined dengue antigen composed of the envelope domain III (EDIII) fused to the ectodomain of the membrane protein (ectoM) from DV serotype-1. Immunization of mice susceptible to MV resulted in a long-term production of DV1 serotype-specific neutralizing antibodies. The presence of ectoM was critical to the immunogenicity of inserted EDIII. The adjuvant capacity of ectoM correlated with its ability to promote the maturation of dendritic cells and the secretion of proinflammatory and antiviral cytokines and chemokines involved in adaptive immunity. The protective efficacy of this vaccine should be studied in non-human primates. A combined measles-dengue vaccine might provide a one-shot approach to immunize children against both diseases where they co-exist.     

Isolation and characterisation of polymorphic microsatellite loci in the vulnerable spectacled flying fox, Pteropus conspicillatus

The spectacled flying fox, Pteropus conspicillatus, is listed as vulnerable in Australia and is under threat from numerous impacts. Primers to amplify eight co-dominant microsatellite loci were designed for Pteropus conspicillatus, based on an enriched genomic library. Four loci were monomorphic in this species while the remaining four loci were highly polymorphic with 16–23 alleles. Two of the four monomorphic loci were found to be polymorphic in Pteropus alecto, a closely related congener. All but one of the six polymorphic loci were in Hardy Weinberg equilibrium. Additionally, six microsatellite loci isolated for Pteropus rodricensis were tested against individuals of P. conspicillatus with all loci amplifying reliably. These loci will be used to investigate population genetic structure in the vulnerable spectacled flying fox.     

Total Dental Occlusal Area as a Feeding Constraint Feature in Extant Walruses (Odobenus rosmarus), and Implications for the Evolution of Molluscivory in Odobenidae

Journal of Mammalian Evolution - Mammalian dental formulae often are highly conserved, at least at a generic level. In walruses (Odobenus rosmarus), the constraints of dentition in light of...