scholarly journals High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 462
Author(s):  
Jeffrey W. Ecker ◽  
Greg A. Kirchenbaum ◽  
Spencer R. Pierce ◽  
Amanda L. Skarlupka ◽  
Rodrigo B. Abreu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Influenza Viruses ◽  
Scale Production ◽  
Vaccine Platform ◽  
Mammalian Cell Lines

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

scholarly journals In VitroandIn VivoAntimalarial Evaluations of Myrtle Extract, a Plant Traditionally Used for Treatment of Parasitic Disorders

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Farzaneh Naghibi ◽  
Somayeh Esmaeili ◽  
Noor Rain Abdullah ◽  
Mehdi Nateghpour ◽  
Mahdieh Taghvai ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Methanolic Extract ◽  
Cytotoxic Activities ◽  
Traditional Use ◽  
Aerial Parts ◽  
Mammalian Cell Lines ◽  
Phytochemical Investigation

Based on the collected ethnobotanical data from the Traditional Medicine and Materia Medica Research Center (TMRC), Iran,Myrtus communisL. (myrtle) was selected for the assessment ofin vitroandin vivoantimalarial and cytotoxic activities. Methanolic extract of myrtle was prepared from the aerial parts and assessed for antiplasmodial activity, using the parasite lactate dehydrogenase (pLDH) assay against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains ofPlasmodium falciparum. The 4-day suppressive test was employed to determine the parasitemia suppression of the myrtle extract againstP. berghei  in vivo. The IC50values of myrtle extract were 35.44 µg/ml against K1 and 0.87 µg/ml against 3D7. Myrtle extract showed a significant suppression of parasitaemia (84.8 ± 1.1% at 10 mg/kg/day) in mice infected withP. bergheiafter 4 days of treatment. Cytotoxic activity was carried out against mammalian cell lines using methyl thiazol tetrazolium (MTT) assay. No cytotoxic effect on mammalian cell lines up to 100 µg/mL was shown. The results support the traditional use of myrtle in malaria. Phytochemical investigation and understanding the mechanism of action would be in our upcoming project.

scholarly journals In Vitro and In Vivo Toxicity Evaluation of Natural Products with Potential Applications as Biopesticides

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 805
Author(s):  
Felicia Sangermano ◽  
Marco Masi ◽  
Amrish Kumar ◽  
Ravindra Peravali ◽  
Angela Tuzi ◽  
...  
Keyword(s):  
Natural Products ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Toxic Effects ◽  
Mammalian Cell Lines ◽  
Dittrichia Viscosa ◽  
Potential Applications

The use of natural products in agriculture as pesticides has been strongly advocated. However, it is necessary to assess their toxicity to ensure their safe use. In the present study, mammalian cell lines and fish models of the zebrafish (Danio rerio) and medaka (Oryzias latipes) have been used to investigate the toxic effects of ten natural products which have potential applications as biopesticides. The fungal metabolites cavoxin, epi-epoformin, papyracillic acid, seiridin and sphaeropsidone, together with the plant compounds inuloxins A and C and ungeremine, showed no toxic effects in mammalian cells and zebrafish embryos. Conversely, cyclopaldic and α-costic acids, produced by Seiridium cupressi and Dittrichia viscosa, respectively, caused significant mortality in zebrafish and medaka embryos as a result of yolk coagulation. However, both compounds showed little effect in zebrafish or mammalian cell lines in culture, thus highlighting the importance of the fish embryotoxicity test in the assessment of environmental impact. Given the embryotoxicity of α-costic acid and cyclopaldic acid, their use as biopesticides is not recommended. Further ecotoxicological studies are needed to evaluate the potential applications of the other compounds.

scholarly journals Development of pyridyl thiosemicarbazones as highly potent agents for the treatment of malaria after oral administration

2019 ◽  
Vol 74 (10) ◽  
pp. 2965-2973 ◽  
Author(s):  
Christopher J Parkinson ◽  
Geoffrey W Birrell ◽  
Marina Chavchich ◽  
Donna Mackenzie ◽  
Richard K Haynes ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Low Cost ◽  
In Vitro Cytotoxicity ◽  
Pharmacokinetic Analysis ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Mammalian Cell Lines

AbstractObjectivesDrug resistance exists to all current and investigational antimalarial drug classes. Consequently, we have set out to develop chemically and mechanistically discrete antimalarials. Here we report on the development of thiosemicarbazone (TSC) antimalarials, with TSC3 as the most advanced lead.MethodsThiosemicarbazones were generated through simple condensation reactions of thiosemicarbazides and ketones. TSC3 was selected and tested for in vitro antimalarial activities against MDR Plasmodium falciparum lines using the [3H]hypoxanthine growth assay, in vitro cytotoxicity against mammalian cell lines using the alamarBlue fluorescence cell viability assay, in vivo potency in the mouse–Plasmodium berghei model and blood exposure in mice measured by LC-MS for pharmacokinetic analysis.ResultsTSC3 showed potent in vitro activity against atovaquone-, dihydroartemisinin-, chloroquine- and mefloquine-resistant P. falciparum lines (EC50 <15 nM). The selectivity index (EC50 cells/EC50Pf W2 line) of TSC3 was >500 in two of three mammalian cell lines. In P. berghei-infected mice, TSC3 showed potent activity in the Peters 4 day suppression test (ED50 1.2 mg/kg/day) and was as potent as artesunate and chloroquine in the curative modified Thompson test. A single oral dose of TSC3 at 16 mg/kg in healthy mice achieved a mean maximum blood concentration of 1883 ng/mL at 1 h after dosing and an elimination half-life of 48.7 h in groups of five mice.ConclusionsTSC3 shows promise as a persistent, potent and orally effective antimalarial. This, coupled with the extremely low cost of synthesis, suggests that the further development of antimalarial thiosemicarbazones is clearly warranted.

scholarly journals Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

scholarly journals N-linked glycosylation plays an important role in budding of NA protein and virulence of influenza viruses

2020 ◽  
Author(s):  
Danqi Bao ◽  
Ruixue Xue ◽  
Min Zhang ◽  
Chenyang Lu ◽  
Tianxin Ma ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Immune Escape ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
H1n1 Influenza Virus ◽  
Protein Loss ◽  
Knock Out

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of Hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knock-out significantly inhibited the budding of NA. Further studies showed that the NLG knock-out caused attenuation of virus in vitro and in vivo. Notably the NLG at 219 position plays an important role in budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, unfolded protein response (UPR) was determined in NLG knock-out NA overexpressed cells, which showed that the mutant NA was mainly located in ER, and the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knock-out NA was stacked in ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding. IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. Especially the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

scholarly journals The Molecular Pharmacology of Pateamine A

2021 ◽  
Author(s):  
◽  
James Henry Matthews
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mode Of Action ◽  
Protein Synthesis Inhibitors ◽  
Primary Target ◽  
Dead Box ◽  
Mammalian Cell Lines ◽  
Pateamine A

<p>Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found.</p>

scholarly journals LACK OF IDENTITY IN NEUTRALIZING AND HEMAGGLUTINATION-INHIBITING ANTIBODIES AGAINST INFLUENZA VIRUSES

1950 ◽  
Vol 91 (1) ◽  
pp. 65-86 ◽  
Author(s):  
Duard L. Walker ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Hemagglutination Inhibition ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Immune Serum ◽  
Influenza Viruses ◽  
In Ovo ◽  
Neutralization Line

There is an exponential linear relationship between the quantity of influenza virus neutralized and the quantity of immune serum employed in in ovo neutralization. The slope of the neutralization line is extremely steep. The concentration of neutralizing antibody can be measured with considerable precision in ovo if the constant virus-varying serum technique is utilized. The amounts of hemagglutination-inhibiting and neutralizing antibodies which are absorbed by a given quantity of influenza virus (PR8) were found to be predictable and the degree of reactivity of these two antibodies was shown to be directly related to the extent of immunization. It was demonstrated that there are marked discrepancies in correlation between antibody titers obtained by in vitro hemagglutination-inhibition and in vivo neutralization techniques and that neutralizing antibody is preferentially absorbed by a given quantity of virus. Inasmuch as the results were found not to be attributable to peculiarities of the techniques employed, it appears that the antibodies measured by hemagglutination-inhibition in vitro and by neutralization in vivo are not identical.

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