scholarly journals Generation of a Sleeping Beauty Transposon-Based Cellular System for Rapid and Sensitive Screening for Compounds and Cellular Factors Limiting SARS-CoV-2 Replication

2021 ◽  
Vol 12 ◽  
Author(s):  
Marek Widera ◽  
Alexander Wilhelm ◽  
Tuna Toptan ◽  
Johanna M. Raffel ◽  
Eric Kowarz ◽  
...  
Keyword(s):  
Cell Culture ◽  
Transfection Efficiency ◽  
Signal To Noise Ratio ◽  
A549 Cells ◽  
Constitutive Expression ◽  
Sleeping Beauty ◽  
Interferon Response ◽  
Infection Model ◽  
Proliferation Capacity ◽  
Extensive Evaluation

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The model is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression (A549-AT). Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV-2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected A549-AT cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel model cell line allows rapid and sensitive monitoring of SARS-CoV-2 infection and the screening for host factors essential for viral replication.

scholarly journals Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

2021 ◽  
Author(s):  
Marek Widera ◽  
Alexander Wilhelm ◽  
Tuna Toptan ◽  
Johanna M. Raffel ◽  
Eric Kowarz ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
High Throughput ◽  
A549 Cells ◽  
Sleeping Beauty ◽  
Cellular System ◽  
Reporter Cell Line ◽  
Restriction Factors ◽  
Reporter Cell ◽  
Sleeping Beauty Transposon

SummaryThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication.Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection- The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays- Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors

scholarly journals Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families

2015 ◽  
Vol 89 (18) ◽  
pp. 9465-9476 ◽  
Author(s):  
Amelia K. Pinto ◽  
Graham D. Williams ◽  
Kristy J. Szretter ◽  
James P. White ◽  
José Luiz Proença-Módena ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antiviral Activity ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Ectopic Expression ◽  
A549 Cells ◽  
Host Protein ◽  
Interferon Response ◽  
Restriction Factor ◽  
Negative Sense

ABSTRACTInterferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5′-triphosphates (5′-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5′-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infectedIfit1−/−and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack ofIfit1gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical betweenIfit1−/−and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5′ ends of IAV gene segments. The affinity for 5′-ppp RNA was approximately 10-fold lower than that for non-2′-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses.IMPORTANCENegative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis bothin vitroandin vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses.

scholarly journals Study Cellular Hypoxic Response under Cyclic Oxygen Gradients Generated in Microfluidic Devices Using Real-Time Fluorescence Imaging

2021 ◽  
Author(s):  
Dao-Ming Chang ◽  
Yi-Chung Tung
Keyword(s):  
Cell Culture ◽  
Intracellular Calcium ◽  
Real Time ◽  
Oxygen Tension ◽  
Calcium Concentration ◽  
A549 Cells ◽  
Cell Types ◽  
Hypoxic Response ◽  
Oxygen Gradient ◽  
Oxygen Gradients

Abstract In this paper, an integrated approach to study cellular hypoxic response under cyclic oxygen gradients is developed. In the experiments, a cell culture system based on a microfluidic device is constructed to generate cyclic oxygen gradients with fast response. The oxygen gradient is generated by alternatively introducing gases with specific compositions by a computer-controlled gas supply system into the microfluidic channels next to the cell culture one separated by thin channel walls. Observation of cellular hypoxic responses is performed using real-time fluorescence imaging of dyes sensitive to extra-and intra-cellular oxygen tensions as well as intracellular calcium concentrations. The capability of consistent cyclic oxygen gradient generation is confirmed by characterizing spatiotemporal oxygen tension profiles within the device. In the cell experiments, cellular hypoxic responses of human aortic smooth muscle cells (AoSMCs) and lung carcinoma epithelium (A549) cells including intracellular oxygen and calcium levels are measured. The relationships between the oxygen probe intensity and extracellular oxygen tension for both cell types are highly linear, and difference between the intensity measured from the two cell types is observed between 4 to 8% O2 culture environment. In addition, both cell types show detectable intracellular calcium concentration variation when the environmental O2 varies between normoxia and lower than 4%. The AoSMCs and A549 cells show decrease and increase of the intracellular calcium concentration during hypoxic response when facing change from normoxia to hypoxia environments, respectively. With the capability of real-time cellular responses monitoring under cyclic oxygen gradients, the developed approach provides a useful scheme to investigate cellular hypoxic response in vitro under microenvironments mimicking various in vivo physiological and pathological conditions.

scholarly journals Anti-Inflammatory Properties of Fructo-Oligosaccharides in a Calf Lung Infection Model and in Mannheimia haemolytica-Infected Airway Epithelial Cells

Nutrients ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 3514
Author(s):  
Yang Cai ◽  
Myrthe S. Gilbert ◽  
Walter J. J. Gerrits ◽  
Gert Folkerts ◽  
Saskia Braber
Keyword(s):  
Epithelial Cells ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Mannheimia Haemolytica ◽  
Epithelial Barrier ◽  
Infection Model ◽  
Airway Epithelial ◽  
Clinical Scores ◽  
Lung Infections ◽  
Anti Inflammatory

Emerging antimicrobial-resistant pathogens highlight the importance of developing novel interventions. Here, we investigated the anti-inflammatory properties of Fructo-oligosaccharides (FOS) in calf lung infections and in airway epithelial cells stimulated with pathogens, and/or bacterial components. During a natural exposure, 100 male calves were fed milk replacer with or without FOS for 8 weeks. Then, immune parameters and cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF) and blood were measured, and clinical scores were investigated. Calf primary bronchial epithelial cells (PBECs) and human airway epithelial cells (A549) were treated with Mannheimia haemolytica, lipopolysaccharides (LPS), and/or flagellin, with or without FOS pretreatment. Thereafter, the cytokine/chemokine levels and epithelial barrier function were examined. Relative to the control (naturally occurring lung infections), FOS-fed calves had greater macrophage numbers in BALF and lower interleukin (IL)-8, IL-6, and IL-1β concentrations in the BALF and blood. However, FOS did not affect the clinical scores. At slaughter, FOS-fed calves had a lower severity of lung lesions compared to the control. Ex vivo, FOS prevented M. haemolytica-induced epithelial barrier dysfunction. Moreover, FOS reduced M. haemolytica- and flagellin-induced (but not LPS-induced) IL-8, TNF-α, and IL-6 release in PBECs and A549 cells. Overall, FOS had anti-inflammatory properties during the natural incidence of lung infections but had no effects on clinical symptoms.

Evaluation of low molecular weight polyethylenimine-introduced chitosan for gene delivery to mesenchymal stem cells

2020 ◽  
Vol 10 (7) ◽  
pp. 1170-1176
Author(s):  
Minchen Liu ◽  
Yulan Hu ◽  
Yi Feng
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Transfection Efficiency ◽  
A549 Cells ◽  
Safety Evaluation ◽  
Physicochemical Characteristics ◽  
Hatching Rate

This study aimed to examine the transfection ability of polyethylenimine (PEI) (1800 Da)-grafted chitosan (10 kDa) (CP), a newly synthesized PEI derivative, in mesenchymal stem cells (MSCs). The safety evaluation of the complex/DNA was studied in vitro and in vivo. In addition, CP/pGL3 was applied to investigate the effects of transfection efficiency. In this study, CP/DNA can be formed with compatible physicochemical characteristics for gene delivery. CP cytotoxicity decreased in A549 cells. Moreover, a zebrafish embryo model was used for evaluating the safety in vivo. Compared to the PEI (25 kDa) group, the zebrafish hatching rate increased and the mortality rate decreased in the CP/DNA group, which provided an indication of the safety of CP. In comparison with chitosan (100 kDa)-PEI (1200 Da), CP's transfection efficiency was higher in both A549 cells and MSCs. This study aimed to lay the foundation for further applications of CP in gene delivery. Therefore, further gene therapy investigations of CP by using MSCs need to be performed.

scholarly journals Mutational Analysis of the Chlamydia muridarum Plasticity Zone

2015 ◽  
Vol 83 (7) ◽  
pp. 2870-2881 ◽  
Author(s):  
Krithika Rajaram ◽  
Amanda M. Giebel ◽  
Evelyn Toh ◽  
Shuai Hu ◽  
Jasmine H. Newman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Genital Tract ◽  
Mutational Analysis ◽  
Genomic Diversity ◽  
Infection Model ◽  
Plasticity Zone ◽  
Content Type ◽  
Chlamydia Muridarum ◽  
Ifn Γ ◽  
Tract Infections

Pathogenically diverseChlamydiaspp. can have surprisingly similar genomes.Chlamydia trachomatisisolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strainChlamydia muridarumshare 99% of their gene content. A region of high genomic diversity betweenChlamydiaspp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance ofC. muridarumcompared toC. trachomatisin the murine genital tract. To test this hypothesis, we isolated and characterized a series ofC. muridarumPZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins,guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest thatC. muridarumPZ genes are transcribed—and some may produce functional proteins—but are dispensable for infection of the murine genital tract.

scholarly journals Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists

2015 ◽  
Vol 89 (15) ◽  
pp. 8011-8025 ◽  
Author(s):  
Cindy Chiang ◽  
Vladimir Beljanski ◽  
Kevin Yin ◽  
David Olagnier ◽  
Fethia Ben Yebdri ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Inflammatory Responses ◽  
Rna Viruses ◽  
Rna Virus ◽  
A549 Cells ◽  
Antiviral Response ◽  
Interferon Response

ABSTRACTThe cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5′-triphosphate (5′ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5′ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5′pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5′pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replicationin vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatmentin vivoprolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5′pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses.IMPORTANCEThe development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral responsein vitroandin vivoand was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5′pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.

A dendritic catiomer with an MOF motif for the construction of safe and efficient gene delivery systems

2017 ◽  
Vol 5 (42) ◽  
pp. 8322-8329 ◽  
Author(s):  
Shuqi Dong ◽  
Qixian Chen ◽  
Wei Li ◽  
Zhu Jiang ◽  
Jianbiao Ma ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Gold Standard ◽  
Transfection Efficiency ◽  
A549 Cells ◽  
Delivery Systems ◽  
The Core ◽  
Efficient Gene

The dendritic catiomer using biocompatible Zr-MOFs as the core exhibited a markedly higher transfection efficiency and lower cytotoxicity than the commercial gold standard branched PEI25k in A549 cells.

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