ARTICLE | doi:10.20944/preprints202103.0696.v1
Subject: Life Sciences, Biochemistry Keywords: extracellular vesicles; enveloped viruses; lentiviral vectors; exosome; proteomic; lipidomic; transcriptomic
Online: 29 March 2021 (13:53:53 CEST)
Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purify with LVs during downstream processing, becoming “impurities” in the context of cell therapy. To characterize EVs from an inducible lentivirus producing cell line, two conditions were studied: non-induced and induced. EVs’ identity was confirmed by transmission electron microscopy and western blot. Seven proteins were identified by mass spectrometry as potential EV markers. Lipid composition of EVs and LVs showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in genes involved in viral processes and binding functions. Flow virometry, GTA and ddPCR results also confirmed the heterogenic nature of EVs and LVs populations. These findings provide insights on the product profile of lentiviral preparation and could help develop separation strategies of co-produced EVs.
ARTICLE | doi:10.20944/preprints201904.0264.v1
Subject: Life Sciences, Molecular Biology Keywords: human adipose stem cell; NANOG; cell cycle regulation; DNMT1; lentiviral transduction
Online: 24 April 2019 (10:45:01 CEST)
The core components of regenerative medicine are stem cells with high self-renewal and tissue regeneration potentials. Adult stem cells can be obtained from many organs and tissues. NANOG, SOX2 and OCT4 represent the core regulatory network that suppresses differentiation-associated genes, maintaining the pluripotency of mesenchymal stem cells. The roles of NANOG in maintaining self-renewal and undifferentiated status of adult stem cells are still not perfectly established. In this study we define the effects of downregulation of NANOG in maintaining self-renewal and undifferentiated state in mesenchymal stem cells (MSCs) derived from subcutaneous adipose tissue (hASCs). hASCs were expanded and transfected in vitro with short hairpin Lentivirus targeting NANOG. Gene suppressions were achieved at both transcript and proteome levels. The effect of NANOG knockdown on proliferation after 10 passages and on the cell cycle was evaluated by proliferation assay, colony forming unit (CFU), qRT-PCR and cell cycle analysis by flow-cytometry. Moreover, NANOG involvement in differentiation ability was evaluated. We report that downregulation of NANOG revealed a decrease in the proliferation and differentiation rate, inducing cell cycle arrest by increasing p27/CDKN1B (Cyclin-dependent kinase inhibitor 1B) and p21/CDKN1A(Cyclin-dependent kinase inhibitor 1A) through p53 and regulate DLK1/PREF1. Furthermore, NANOG induced downregulation of DNMT1, a major DNA methyltransferase responsible for maintaining methylation status during DNA replication probably involved in cell cycle regulation. Our study confirms that NANOG regulates the complex transcription network of plasticity of the cells, inducing cell cycle arrest and reducing differentiation potential.
REVIEW | doi:10.20944/preprints201804.0009.v1
Subject: Biology, Animal Sciences & Zoology Keywords: feline immunodeficiency virus; FIV; human immunodeficiency virus; HIV; animal models, opportunistic disease, lentiviral pathogenesis; molecular biology
Online: 2 April 2018 (07:54:28 CEST)
Feline immunodeficiency virus (FIV) is a naturally-occurring retrovirus that infects domestic and non-domestic feline species, producing progressive immune depletion that results in an acquired immunodeficiency syndrome (AIDS). Much has been learned about FIV since it was first described in 1987, particularly in regard to its application as a model to study the closely related lentivirus, human immunodeficiency virus (HIV). In particular, FIV and HIV share remarkable structure and sequence organization, utilize parallel modes of receptor-mediated entry, and result in a similar spectrum of immunodeficiency-related diseases due to analogous modes of immune dysfunction. This review summarizes current knowledge of FIV infection kinetics and mechanisms of immune dysfunction in relation to opportunistic disease, specifically in regard to studying HIV pathogenesis. Furthermore, we present data which highlight changes in the oral microbiota and oral immune system during FIV infection, and outline the potential for the feline model of oral AIDS manifestations to elucidate pathogenic mechanisms of HIV-induced oral disease. Finally, we discuss advances in molecular biology, vaccine development, neurologic dysfunction, and the ability to apply pharmacologic interventions and sophisticated imaging technologies to study experimental and naturally occurring FIV, which provide an excellent, but often overlooked resource for advancing therapies and management of HIV/AIDS.