simian virus 40
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Biomimetics ◽  
2021 ◽  
Vol 7 (1) ◽  
pp. 5
Author(s):  
Hieu M. Pham ◽  
Yuli Zhang ◽  
Jose G. Munguia-Lopez ◽  
Simon D. Tran

Saliva production by salivary glands play a crucial role in oral health. The loss of salivary gland function could lead to xerostomia, a condition also known as dry mouth. Significant reduction in saliva production could lead to further complications such as difficulty in speech, mastication, and increased susceptibility to dental caries and oral infections and diseases. While some palliative treatments are available for xerostomia, there are no curative treatments to date. This study explores the use of Egg White Alginate (EWA), as an alternative scaffold to Matrigel® for culturing 3D salivary gland cells. A protocol for an optimized EWA was established by comparing cell viability using 1%, 2%, and 3% alginate solution. The normal salivary simian virus 40-immortalized acinar cell (NS-SV-AC) and the submandibular gland-human-1 (SMG-hu-1) cell lines were also used to compare the spheroid formation and cell viability properties of both scaffold biomaterials; cell viability was observed over 10 days using a Live–Dead Cell Assay. Cell viability and spheroid size in 2% EWA was significantly greater than 1% and 3%. It is evident that EWA can support salivary cell survivability as well as form larger spheroids when compared to cells grown in Matrigel®. However, further investigations are necessary as it is unclear if cultured cells were proliferating or aggregating.


Author(s):  
Manipa Saha ◽  
Neha Deshpande ◽  
Abhinav Dubey ◽  
Debnath Pal ◽  
Hanudatta S. Atreya ◽  
...  

Attachment to the matrix is critical for the survival of adherent cells, whereas detachment triggers death by apoptosis. Therefore, solid tumors must acquire the ability to survive the stress of matrix-detachment to transit through circulation and seed metastases. Although a central role for energy metabolism in cancer progression is well established, what distinguishes its role in the cellular state of the matrix-deprived form compared to the matrix-attached form is not fully understood yet. Using an in vitro transformation model dependent on simian virus 40 (SV40) small t (ST) antigen for cellular survival and proliferation in matrix-deprived conditions, we demonstrate that 5′-adenosine monophosphate-activated protein kinase (AMPK) activity is elevated and sustained under matrix-deprived conditions in ST-expressing fibroblasts. Additionally, these cells display elevated energy (ATP) levels under matrix-deprived conditions in contrast to cells lacking ST expression. The elevated ATP levels are coupled to increased levels of proline in ST-expressing cells, as revealed by metabolomics studies. The AMPK-dependent upregulation of proline oxidase, an enzyme of proline degradation, is a key link for elevated ATP levels. This functional link is further established by proline supplementation concomitant with AMPK activation in matrix-deprived cells lacking ST antigen, yielding ATP and enhancing survival. Thus, our data establishes a key role for AMPK-dependent regulation of proline metabolism in mediating energy homeostasis and promoting survival of matrix-deprived cells. These findings identify key markers that distinguish the metabolic states of matrix-detached and matrix-attached transformed cells and have implications in developing novel therapeutic strategies for specifically targeting matrix-detached metastasizing cancer cells.


2021 ◽  
Author(s):  
Jason Nomburg ◽  
Wei Zou ◽  
Thomas C Frost ◽  
Chandreyee Datta ◽  
Shobha Vasudevan ◽  
...  

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.


2021 ◽  
Vol 22 (19) ◽  
pp. 10667
Author(s):  
María Piñeiro-Ramil ◽  
Clara Sanjurjo-Rodríguez ◽  
Silvia Rodríguez-Fernández ◽  
Rocío Castro-Viñuelas ◽  
Tamara Hermida-Gómez ◽  
...  

Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.


Author(s):  
Marta Bally ◽  
Stephan Block ◽  
Fredrik Höök ◽  
Göran Larson ◽  
Nagma Parveen ◽  
...  

AbstractThe objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.


Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2064
Author(s):  
Yang Ge ◽  
Anke M. Smits ◽  
Jia Liu ◽  
Juan Zhang ◽  
Thomas J. van Brakel ◽  
...  

Rationale: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. Aim: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. Methods and results: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor β3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. Conclusion: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.


2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Kai Furuya ◽  
Tao Wu ◽  
Ai Orimoto ◽  
Eriko Sugano ◽  
Hiroshi Tomita ◽  
...  

AbstractCellular immortalization enables indefinite expansion of cultured cells. However, the process of cell immortalization sometimes changes the original nature of primary cells. In this study, we performed expression profiling of poly A-tailed RNA from primary and immortalized corneal epithelial cells expressing Simian virus 40 large T antigen (SV40) or the combination of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). Furthermore, we studied the expression profile of SV40 cells cultured in medium with or without serum. The profiling of whole expression pattern revealed that immortalized corneal epithelial cells with SV40 showed a distinct expression pattern from wild-type cells regardless of the presence or absence of serum, while corneal epithelial cells with combinatorial expression showed an expression pattern relatively closer to that of wild-type cells.


EP Europace ◽  
2021 ◽  
Vol 23 (Supplement_3) ◽  
Author(s):  
N Harlaar ◽  
SO Dekker ◽  
J Zhang ◽  
MJ Schalij ◽  
RJM Klautz ◽  
...  

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): LUMC Background Current in vitro models of atrial fibrillation have limited translational potential due to a lack of relevant human physiology or the inability to reach the high activation frequencies present in human atrial fibrillation. Absence of relevant models is the result of a general deficit of readily available and standardized sources of well-differentiated human atrial cardiomyocytes. Therefore, we aimed to immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells. Methods Human fetal atrial cardiomyocytes were transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen. Addition of doxycycline to the culture medium pushed cardiomyocytes towards a highly proliferative phenotype (proliferation up to 10^12 cells). These cells were labelled hiAMs (human immortalised Atrial Myocytes). After differentiation upon doxycycline removal, hiAM cells were characterized using various molecular, biological and electrophysiological assays. Results Following cardiomyogenic differentiation, hiAMs no longer expressed the proliferation marker Ki67, revealed striated α-actinin and troponin T staining patterns and displayed synchronous contractions. Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22.5 ± 3.1 cm/s with a mean APD80 of 139 ± 22 ms. Addition of flecainide (10 µM) to hiAM monolayers decreased the conduction velocity by 35% and increased the APD80 by 107%. Dofetilide (10 nM) addition had no effect on the conduction velocity, but did increase the APD80 by 81%. Due to their scalability, monolayers of hiAMs as big as 10 cm2 showing homogenous action potential propagation could easily be created. Following high-frequency electrical pacing, rotors could be induced with an average activation frequency of 7.5 ± 0.9 Hz. Infusion of flecainide during arrhythmic activity resulted in termination of the rotor in 18 of 24 attempts (75%), whereas addition of 0.1% DMSO (vehicle control) did not result in termination in any of the attempts. Dofetilide infusion did not result in termination. However, it did lower the average activation frequency to 2.1 ± 0.7 Hz. Conclusion We have generated first-of-a-kind lines of human atrial cardiomyocytes, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. These characteristics allow, for the first time, the modelling, at a large-scale, of human atrial arrhythmias with frequencies similar to human atrial fibrillation. With the generation of hiAMs, a user-friendly, clinically-relevant and much-anticipated human atrial research model has been produced. Abstract Figure. hiAM AF Model


PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250605
Author(s):  
Xiaoxia Shi ◽  
Giulia Bortolussi ◽  
Lysbeth ten Bloemendaal ◽  
Suzanne Duijst ◽  
Andrés F. Muro ◽  
...  

In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy.


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