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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]

Coronaviridae is a family of single-stranded RNA (ssRNA) viruses that can cause diseases with high mortality rates. SARS-CoV-1 and MERS-CoV appeared in 2002‒2003 and 2012, respectively. A novel coronavirus, SARS-CoV-2, emerged in 2019 in Wuhan (China) and has caused more than 5 million deaths in worldwide. The entry of SARS-CoV-1 into the cell is due to the interaction of the viral spike (S) protein and the cell protein, angiotensin-converting enzyme 2 (ACE2). After infection, virus assembly occurs in Golgi apparatus-derived vesicles during exocytosis. One of the possible participants in this process is LAMP1 protein. We established transgenic Vero cell lines with increased expression of human LAMP1 gene and evaluated SARS-CoV-1 and SARS-CoV-2 production. An increase in the production of both viruses in LAMP1-expressing cells when compared with Vero cells was observed, especially in the presence of trypsin during infection. From these results it can be assumed that LAMP1 promotes SARS-CoV-1 and SARS-CoV-2 production due to enhanced exocytosis. [ABSTRACT FROM AUTHOR]

The synthesis and evaluation of some bioisosteres of thalidomide metabolites for antiangiogenic and anticancer activity on multiple myeloma are described in this article. Four compounds, diethyl 2-(thiophene-2-sulfonamido) pentanedioate, N1,N5-diethyl-2-(thiophene-2-sulfonamido) pentanediamide, N1,N5-dihexyl-2-(thiophene-2-sulfonamido) pentanediamide, and N-(1,5-dioxo-1,5-bis(2-phenylhydrazinyl)pentan-2-yl)thiophene-2-sulfonamide exhibit cytotoxic effect on human multiple myeloma cell line (RPMI8226) with IC50 (in μM) values 3.33, 0.75, 1.23, and 3.28, respectively. The in vitro studies on human umbilical vein endothelial cells (HUVEC) and normal African green monkey kidney epithelial cells (Vero cell line) concluded that these compounds bear antiangiogenic properties and are selectively cytotoxic to cancer cells. Except for N-(1,5-dioxo-1,5-bis(2-phenylhydrazinyl)pentan-2-yl)Thiophene-2-sulfonamide, it was found to be toxic to normal epithelial cells. The "trypan blue" dye exclusion method was used to conduct an antiproliferative assay of the active compounds on HUVECs. The phosphorylation inhibition assay on Vascular Endothelial Growth Factor Receptor-2 Tyr-1175 (VEGFR-2 Tyr-1175) revealed that N1,N5-diethyl-2-(thiophene-2-sulfonamido) pentanediamide and N1,N5-dihexyl-2-(thiophene-2-sulfonamido) pentanediamide are active, indicating that the molecules have antiangiogenic activity by targeting VEGFR-2 Tyr-1175, a key autophosphorylation site that regulates pro-angiogenic responses. On CDOCKER, a molecular docking study of the most active compound N1,N5-diethyl-2-(thiophene-2-sulfonamido) pentanediamide with VEGFR-2 revealed that they might interact with the essential amino acid residues, with a binding energy of –105.304 kcal/mol. [ABSTRACT FROM AUTHOR]

Various biological models are used to isolate West Nile virus, but their role as a selection factor that facilitates selection of isolates with certain properties is usually not evaluated. We compared pathogenic properties of three strains of the West Nile virus obtained from one sample of virus-containing material using different models: WNV Volgograd 900m/18 (on the model of suckling mice), WNV Volgograd 900a/18 (on C6/36 cells) and WNV Volgograd 900v/18 (on Vero cells). WNV Volgograd 900m/18 strain demonstrated virulent (LD50 5×103±0.005×104 PFU, p≤0.05) and neuroinvasive properties, induced viremia and pathomorphological changes in the liver, lymph nodes, and brain of nonlinear white mice. WNV Volgograd 900v/18 strain had similar characteristics except for neuroinvasiveness. WNV Volgograd 900a/18 variant demonstrated minimum virulence (LD50 5×104±0.005×104 PFU, p≤0.05), did not cause neurological symptoms, and was not isolated from the blood of infected animals. [ABSTRACT FROM AUTHOR]

Main conclusion: The similarity of IREH1 (Incomplete Root Hair Elongation 1) and animal MAST kinases was confirmed; IREH1cDNA was cloned while expressing in cultured animal cells co-localized with the centrosome. In mammals and fruit flies, microtubule-associated serine/threonine-protein kinases (MAST) are strongly involved in the regulation of the microtubule system. Higher plants also possess protein kinases homologous to MASTs, but their function and interaction with the cytoskeleton remain unclear. Here, we confirmed the sequence and structural similarity of MAST-related putative protein kinase IREH1 (At3g17850) and known animal MAST kinases. We report the first cloning of full-length cDNA of the IREH1 from Arabidopsis thaliana. Recombinant GFP-IREH1 protein was expressed in different cultured animal cells. It revealed co-localization with the centrosome without influencing cell morphology and microtubule arrangement. Structural N-terminal region of the IREH1 molecule co-localized with centrosome as well. [ABSTRACT FROM AUTHOR]

Ticks transmit many different pathogens to animals, humans and their pets. Rickettsia slovaca, as a member of the spotted-fever-group rickettsiae is an agent of the human disease Tick-borne lymphadenopathy (TIBOLA), also called Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL), which occurs from the Mediterranean to central Europe, transmitted by Dermacentor reticulatus and Dermacentor marginatus (Acari: Ixodidae). In this study, quantitative real time PCR was used to characterize the growth of R. slovaca, strain B in static (mammalian L929 and Vero cells without replacement of growth medium) and dynamic (D. marginatus and Ixodes ricinus ticks) cultivation systems. Curves of bacterial growth in static cultivations were modeled with exponential, stationary and death phases, whereas in dynamic systems the stationary phase was absent. The highest point of multiplication of R. slovaca was recorded on the 4th day post infection in both cell lines and the rickettsial DNA copy number in L929 and Vero cells at this point was 21 and 27 times greater than rickettsial DNA copy number of inoculum, respectively. In the dynamic system, the highest point of multiplication was on the 21th and 12th day after feeding of ticks and rickettsial DNA copy numbers were 7,482 and 865 times greater than the inoculum in D. marginatus and I. ricinus, respectively. Life cycle of R. slovaca in mammalian cell lines was shorter; supposedly, bacteria destroyed these cells and ticks, especially D. marginatus, were considered a more appropriate environment. [ABSTRACT FROM AUTHOR]

Disabled Infectious Single Cycle (DISC) HSV-2 has been cultured in the complimentary cell line CR2 to provide high titre bulk material suitable for the purification of the virus as a live viral vaccine. CR2 cells are cultured on the microcarrier Cytodex-1 at 5 g l-1 in small scale (1 l) and larger scale (15 l) reactors. The cells are infected at an MOI of 0.01 pfu cell-1 and the culture harvested 60–72 h later. The infected cells are removed from the microcarriers by the addition of a hypotonic saline and the virus released by low-pressure disruption techniques. Virus titres achieved are compared to the standard roller bottle process. The resulting material is the starting point for the purification of the DISC-HSV virus. [ABSTRACT FROM AUTHOR]

The development of media free of serum and animal or human proteins is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. In order to reduce the risk of contamination, we have modified the serum free medium MDSS2, a very efficient serum free medium for the production of various biologicals including experimental vaccines using different cell lines (Merten et al., 1994), by replacing the animal derived products by plant extracts. The new serum and animal protein free medium (MDSS2N) can be efficiently used for biomass production of various cell lines. These cells grow equally well or better in this new serum-free medium than in the old formulation (MDSS2): • BHK-21/BRS cells, adapted to MDSS2N, showed an overall specific growth rate of 0.0197 h-1 (μ_max = 0.0510±0.0058 h-1), whereas those cultivated in MDSS2 grew with an average specific growth rate of 0.0179 h-1 (μ_max = 0.0305±0.0177 h-1). • Vero cells grew with an average specific growth rate of 0.0159 h-1 and 0.0153 h-1 in MDSS2 and MDSS2N, respectively. Very similar growth rates were obtained in microcarrier cultures in stirred tank reactors: the specific growth rates were 0.0161 h-1 and 0.0166 h-1 for MDSS2 and MDSS2N cultures, respectively. • For MDCK cells, when cultured on microcarriers in bioreactors, a higher average specific growth rate was observed in MDSS2N than in MDSS2; values of 0.0248 h-1 and 0.0168 h-1, respectively, were obtained. The capacity of MDSS2N to support the production of different viruses was equally evaluated and it could be established that for certain viruses there are no or insignificant differences between MDSS2N and MDSS2 (influenza and polio virus), whereas, the production of rabies virus is somewhat reduced in MDSS2N when compared to MDSS2. The use of MDSS2N for cell culture and the production of various viruses is discussed. [ABSTRACT FROM AUTHOR]

Photoelectron spectroscopic examination of treated plastic surfaces showed that surface oxidation, primarily as carboxyl groups, was responsible for formation of good growth surfaces. Gas-plasma studies indicated that only very short exposures were required and that the effect was confined to a thin surface layer that produced adhesive surfaces. Highly adhesive surfaces were produced using oxidizing chemicals. Studies with a polymeric ester demonstrated the importance of unesterified carboxyl groups for high adhesiveness. [ABSTRACT FROM AUTHOR]

A centrifugal method has been evaluated for measuring the strength of Vero Green Monkey kidney cell adhesion to growth surfaces. The centrifugal force necessary to remove cells gave a quantitative measure of cell adhesion and hence the quality of the growth surface. After being subjected to high gravity forces, both the remaining attached cells and the detached cells were viable, indicating the detachment process did not simply rupture the cell. Electron microscope examination of growth surfaces after cell detachment suggested that remnants related to filopodia remained. [ABSTRACT FROM AUTHOR]