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The purpose of this study was to investigate the effect of new azoloazine derivatives on the level of DNA damage in Vero cells in vitro using alkaline gel electrophoresis (DNA comet assay). The objects of research were (1) 8-(piperidinocarbonyl)-3-cyclohexylimidazo[5,1-d][1,2,3,5]tetrazine-4(3N)-one, (2) 4‑aminoimidazo[5,1-c][1,2,4]triazine-3,8-dicarboxylic acid, and (3) 4-amino-8-ethoxycarbonylimidazo[5,1-c][1,2,4]triazine-3-N-(P-toluyl)carboxamide. Epirubicin was chosen as a comparison drug. Compounds were used in doses of 1/2, 1/10, and 1/50 IC50. The Vero cell line, cultured according to standard protocol was used as the cell model. To assess genotoxicity, an alkaline version of the comet assay, which is highly sensitive and allows the detection of DNA damage was used. Analysis of the obtained data indicates that tested compounds 1–3 increase the amount of DNA damage in nontumor cells. Compound 1 has the most pronounced genotoxic effect. Thus, using this compound at a dose of 1/2 IC50 led to a significant increase in the comet tail length by 1.5 times. It was noted that the amount of DNA damage caused by the action of compound 1 at the studied doses and epirubicin was on the same level. The results obtained show that compounds 1–3 may be potentially carcinogenic. However, this conclusion requires further in-depth experimental stud-ies. [ABSTRACT FROM AUTHOR]

Objective: Most research on the therapeutic effects of wild pistachio species has focused on bacteria and fungi. This study investigated the flavonoid and phenolic contents of different leaves and hulls of Pistacia atlantica (P. atlantica) subsp. Kurdica (P. atlantica subsp. Kurdica) extracts and their effect on herpes simplex virus 1 (HSV1). Methods: In this study, aqueous, ethanolic, and methanolic extracts of the leaf and hull of P. atlantica subsp. Kurdica were prepared by the percolation method. The total phenolic and flavonoid contents in different extracts were measured by UV/Vis spectrophotometry using the Folin-Ciocâlteu reagent and the colorimetric method of aluminum chloride, respectively. On African green monkey kidney cells (VERO), the ethanolic extract of P. atlantica subsp. Kurdica was tested for toxicity. Furthermore, 50% cytotoxic and inhibitory concentrations (CC50 and IC50) were identified. Results: Compared with aqueous and methanolic extracts, the ethanolic extract of the leaf and hull of P. atlantica subsp. Kurdica had the highest phenolic and flavonoid concentrations. In addition, our study showed that CC50 values were 661.81 and 795.21 μg/mL. Also, IC50 values were 97.51 and 110.82 μg/mL for leaf and hull extracts, respectively. The selectivity index for the ethanolic leaf and hull extracts were 6.79 and 7.18, respectively. Conclusion: Our results showed that P. atlantica subsp. Kurdica had an anti-HSV1 effect in a dose-dependent manner but at a higher dose than acyclovir. Ethanolic extract of P. atlantica subsp. Kurdica is probably a suitable herbal medicine with anti-herpetic effects. [ABSTRACT FROM AUTHOR]

Background. The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, originated in Wuhan, China, has claimed millions of lives around the world. In this regard, the search for effective drugs, including the repurposing of existing ones, has become an urgent task. A promising treatment strategy appears to be drug disruption of viral reproduction. RNA-dependent RNA polymerase (RdRp) is the central subunit of the RNA synthesis process for all positive-strand RNA viruses and is therefore an attractive target for antiviral inhibitors.The aim of this work is an experimental study of the antiviral activity of various drugs based on 6-fluoro-3-hydroxy-2-pyrazinecarboxamide (Favipiravir) in vitro and in vivo against the SARS-CoV-2 coronavirus (COVID-19).Material and methods. The experiments were carried out on a permanent culture of African green monkey kidney cells — Vero Cl008. The effectiveness of the drugs was assessed by suppressing the reproduction of the virus in vitro. Biological activity was assessed by titration of the virus-containing suspension in Vero Cl008 cell culture by the formation of negative colonies. Syrian golden hamsters orally infected with the SARS-CoV-2 virus, variant B, were used in the study. The effectiveness of the drug was assessed by the coefficient of therapeutic action.Results. The results of the study revealed that the compounds FP-1 and Avifavir in the concentration range of 100–400 µg/ml almost completely suppress the reproduction of the SARS-CoV-2 virus; the CTI index for the drug FP-1 was 4, for Avifavir it was 2. The ED₅₀ value for FP-1 was 26 µg/ml, for Avifavir it was 36 µg/ml. Preparations T-705 and Coronavir revealed antiviral activity only at extremely high concentrations. The CTI was 1. During the study on Syrian golden hamsters orally infected with the SARS-CoV-2 virus, variant B, at a dose of 5×105 PFU, it was shown that the use of Avifavir and FP-1 has a high protective efficacy, while Coronavir and T-705 cause a moderate suppression of virus reproduction in the target organ. According to the complex of clinical-virological, biochemical, and hematological indicators, the disease severity index (DSI) and the therapeutic index (TI) were calculated. For the drug Avifavir, the DSI was 0.269; the TI was 71.3% with a probability of 99.9%.Conclusion. Of the studied compounds, Avifavir and FP-1 showed the highest antiviral activity.

Lung cancer ranks as the 2nd most common cancer globally. It’s the most prevalent cancer in men and the 2nd most common in women. The prominent events in EGFR-mutated non-small-cell lung cancer (NSCLC) include the emergence of the L858R mutation within EGFR exon 21. Despite the promising efficacy of EGFR inhibitors in managing lung cancer, the development of acquired resistance poses a significant hurdle. In the current investigation, we focused on the screening of two phytochemicals, namely Dehydrocostus lactone and Mokkolactone, derived from the Saussurea lappa plant, as potential inhibitors targeting EGFR L858R mutant lung cancer. The chloroform and ethanol extract of the plant demonstrated anti-proliferative activity through the Resazurin chemosensitivity assay, exhibiting an IC50 value of 37.90 ± 0.29 µg/ml with selectivity index 2.4. Through a GC–MS study, we identified 11 phytochemicals for further insilico analysis. These compounds underwent ADMET assessment followed by drug likeliness analysis before being subjected to molecular docking against EGFR L858R, identified through protein–protein interaction network analysis. All phytochemicals exhibited binding energy scores ranging from −6.9 to −8.1 kcal/mol. Dehydrocostus lactone and Mokkolactone were specifically identified for their binding profile. Findings from 100 ns molecular dynamics simulations demonstrated their enhanced stability compared to the reference ligand DJK. This was evident in the root mean square deviation (RMSD) values, ranging from 0.23 ± 0.01 nm to 0.30 ± 0.05 nm, the radius of gyration values, from 1.71 ± 0.01 nm to 1.72 ± 0.01 nm, and the solvent accessible surface area values, from 155.39 ± 2.40 nm2 to 159.32 ± 2.14 nm2. Additionally, favourable characteristics were observed in terms of hydrogen bonding, principal component analysis, and free energy landscape analysis. Examination of their electronic structure via density functional theory revealed efficient properties, with the highest occupied molecular orbital-least unoccupied molecular orbital energy gap values ranging from −3.984 eV to −6.547 eV. Further, in vivo analysis is required to gain a more comprehensive understanding and efficacy of these identified phytochemicals against lung cancer.

The aim of the work is to study the effect of new azoloazine derivatives on the level of DNA damage to Vero cells (DNA comet test) in vitro by alkaline gel electrophoresis.Material and methods. The objects of the study are 8-(piperidinocarbonyl)3-cyclohexylimidazo[5,1-d][1,2,3,5]tetrazine-4(3H)-one (1), diethyl ether 4-aminoimidazo[5,1-c][1,2,4]triazine3,8-dicarboxylic acid (2), 4-amino-8-ethoxycarbonylimidazo[5,1-c][1,2,4]triazine-3-N-(p-toluyl)carboxamide (3). Epirubicin was chosen as a comparison drug. The compounds were used in doses of 1/2, 1/10 and 1/50 IC50. The Vero cell line cultured according to the standard protocol was selected as the cell model. To assess genotoxicity, an alkaline version of the DNA comet method was used, which has a high sensitivity and allows detecting DNA damage.Results. Analysis of the data obtained indicates that the tested compounds 1-3 enhance DNA damage in non-tumor cells. Compound 1 has the most pronounced genotoxic effect. Thus, the use of this substance in a dose of ½ IC50 led to a significant increase in the length of the comet’s tail by 1.5 times. It was noted that DNA damage under the action of compound 1 in the studied doses and of epirubicin was on the same level.Conclusions. The results obtained prove that compounds 1-3 may have a potentially carcinogenic effect. However, this assumption requires further in-depth experimental studies.

A new method for the synthesis of azido-propargyloxy derivatives of 1,3,5-triazine has been developed utilizing the nitrosation of hydrazyno-1,3,5-triazines. New hydrazines (2-hydrazino-4,6-bis(propargyloxy)-1,3,5-triazine and 2,4-dihydrazino-6-propargyloxy-1,3,5-triazine) were synthesized and characterized via FTIR, NMR spectroscopy and elemental analysis. The hyperbranched polymers with azide (diazide monomer) and propargyloxy terminal groups were obtained via the azide-alkyne polycycloaddition reaction of diazide and monoazide AB2-type monomers. The antibacterial activity against Escherichia coli bacteria of 2,4,6-trispropargyloxy-1,3,5-triazine, 2-azido-4,6-bispropargyloxy-1,3,5-triazine, and 2,4-diazido-6-propargyloxy-1,3,5-triazine and their hyperbranched polymers was studied. Only 2,4-diazido-6-propargyloxy-1,3,5-triazine has weak antibacterial activity in comparison with ampicillin. The cytotoxicity of these compounds against M-HeLa, FetMSC, and Vero cell lines was also studied. 2,4,6-trispropargyloxy-1,3,5-triazine does not show any cytotoxic effect (IC50 ≥ 280 µM). It was shown that the presence of an azide group in the compound directly affects the cytotoxic effect. Hyperbranched polymers have a less cytotoxic effect against M-HeLa (IC50 > 100) in comparison with monomers (IC50 = 90–99 µM). This makes it possible to use these polymers as the basis for biocompatible materials in biomedical applications. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)–vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×106 cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×108 plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 106 PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.

Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1-4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1-4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS). Key features • An improved focus-forming assay that has the potential to quantify the DENV-4 strain, which was previously hard to plaque. • Improvements have been made to reduce the possibility of false positives. • Improved workflow using automated digital imaging process and counting of foci. • Applicable to antiviral compounds screening and is amenable to high-throughput screening., Competing Interests: Competing interestsAuthors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Lung cancer ranks as the 2nd most common cancer globally. It's the most prevalent cancer in men and the 2nd most common in women. The prominent events in EGFR-mutated non-small-cell lung cancer (NSCLC) include the emergence of the L858R mutation within EGFR exon 21. Despite the promising efficacy of EGFR inhibitors in managing lung cancer, the development of acquired resistance poses a significant hurdle. In the current investigation, we focused on the screening of two phytochemicals, namely Dehydrocostus lactone and Mokkolactone, derived from the Saussurea lappa plant, as potential inhibitors targeting EGFR L858R mutant lung cancer. The chloroform and ethanol extract of the plant demonstrated anti-proliferative activity through the Resazurin chemosensitivity assay, exhibiting an IC50 value of 37.90 ± 0.29 µg/ml with selectivity index 2.4. Through a GC–MS study, we identified 11 phytochemicals for further insilico analysis. These compounds underwent ADMET assessment followed by drug likeliness analysis before being subjected to molecular docking against EGFR L858R, identified through protein–protein interaction network analysis. All phytochemicals exhibited binding energy scores ranging from −6.9 to −8.1 kcal/mol. Dehydrocostus lactone and Mokkolactone were specifically identified for their binding profile. Findings from 100 ns molecular dynamics simulations demonstrated their enhanced stability compared to the reference ligand DJK. This was evident in the root mean square deviation (RMSD) values, ranging from 0.23 ± 0.01 nm to 0.30 ± 0.05 nm, the radius of gyration values, from 1.71 ± 0.01 nm to 1.72 ± 0.01 nm, and the solvent accessible surface area values, from 155.39 ± 2.40 nm2 to 159.32 ± 2.14 nm2. Additionally, favourable characteristics were observed in terms of hydrogen bonding, principal component analysis, and free energy landscape analysis. Examination of their electronic structure via density functional theory revealed efficient properties, with the highest occupied molecular orbital-least unoccupied molecular orbital energy gap values ranging from −3.984 eV to −6.547 eV. Further, in vivo analysis is required to gain a more comprehensive understanding and efficacy of these identified phytochemicals against lung cancer. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×10 6 cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×10 8 plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 10 6 PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)