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Exploring the molecular content of CHO exosomes during bioprocessing.

ABSTRACT: In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species. Graphical Abstract KEY POINTS: • First-time comprehensive protein and miRNA characterization of CHO exosomes. • Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles. • CHO-derived exosomes also contain numerous piRNA species of yet unknown function.

SUBMITTER: Keysberg C

PROVIDER: S-EPMC8102462 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDespite advocating for male involvement in antenatal education, there is unmet need for antenatal education information for expectant couples. The objective of this study was to gain a deeper understanding of the education content for couples during antenatal education sessions in Malawi. This is needed for the development of a tailor-made curriculum for couple antenatal education in the country, later to be tested for acceptability, feasibility and effectiveness.MethodsAn exploratory cross sectional descriptive study using a qualitative approach was conducted in semi-urban areas of Blantyre District in Malawi from February to August 2016. We conducted four focus group discussions (FGDs) among men and women independently. We also conducted one focus group discussion with nurses/ midwives, 13 key informant interviews whose participants were drawn from both health-related and non-health related institutions; 10 in-depth interviews with couples and 10 separate in-depth interviews with men who had attended antenatal clinics before with their spouses. All the interviews were audiotaped, transcribed verbatim and translated from Chichewa, the local language, into English. We managed data with NVivo 10.0 and used the thematic content approach as a guide for analysis.ResultsWe identified one overarching theme: couple antenatal education information needs. The theme had three subthemes which were identified based on the three domains of the maternity cycle which are pregnancy, labour and delivery and postpartum period. Preferred topics were; description of pregnancy, care of pregnant women, role of men during perinatal period, family life birth preparedness and complication readiness plan, coitus during pregnancy and after delivery, childbirth and baby care.ConclusionAntenatal education is a potential platform to disseminate information and discuss with male partners the childbearing period and early parenting. Hence, if both men and women were to participate in antenatal education, their information needs should be prioritized. Men and women had similar choices of topics to be taught during couple antenatal education, with some minor variations.Electronic supplementary materialThe online version of this article (10.1186/s12884-018-2137-y) contains supplementary material, which is available to authorized users.

Project description:Protozoan parasites of Leishmania genus are able to successfully infect their host macrophage due to multiple virulence strategies that result in its deactivation. Recent studies suggest Leishmania GP63 to be a critical virulence factor in modulation of many macrophage molecules, including protein tyrosine phosphatases (PTPs) and transcription factors (TFs). Additionally, we and others recently reported that Leishmania-released exosomes can participate in pathogenesis. Exosomes are 40-100 nm vesicles that are freed by many eukaryotic cells. To better understand the GP63-dependent immune modulation of the macrophage by Leishmania parasites and their exosomes, we compared the immunomodulatory properties of Leishmania major (WT) and L. major gp63-/- (KO) as well as their exosomes in vitro and in vivo. Importantly, we observed that Leishmania exosomes can modulate macrophage PTPs and TFs in a GP63-dependent manner. In addition, our qRT-PCR analyses showed that WT parasites were able to downregulate multiple genes involved in the immune response, especially cytokines and pattern recognition receptors. KO parasites showed a strongly reduced modulatory capacity compared to WT parasites. Furthermore, comparison of WT versus KO exosomes also showed divergences in alteration of gene expression, especially of chemokine receptors. In parallel, studying the in vivo inflammatory recruitment using a murine air pouch model, we found that exosomes have stronger proinflammatory properties than parasites and preferentially induce the recruitment of neutrophils. Finally, comparative proteomics of WT and KO exosomes surprisingly revealed major differences in their protein content, suggesting a role for GP63 in Leishmania exosomal protein sorting. Collectively our data clearly establish the crucial role of GP63 in dampening the innate inflammatory response during early Leishmania infection, and also provides new insights in regard to the role and biology of exosomes in Leishmania host-parasite interactions.

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Exploring the molecular content of CHO exosomes during bioprocessing.

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