Cell death analysis of recombinant mature epsilon toxin on the kidney cell line

Background and Objectives: Epsilon toxin is the third hazardous bacterial toxin causing ABS enterotoxaemia in domestic animal. In addition, epsilon toxin is known as a biological warfare agent. The aim of this study was to produce the recombi- nant mature epsilon toxin to evaluate cell death impact on the kidney cell line. Materials and Methods: For this purpose, the sequence of mature epsilon toxin (46-328 aa) in pET28a was cloned and expressed in Escherichia coli BL21 (DE3) and purified by nickel-nitrilotriacetic acid (Ni-NTA) column and confirmed by western blot analysis using HRP conjugated anti-His antibody. Then, to assess the anti-proliferative effects of different con- centrations of recombinant epsilon toxin, the MTT assay was done on the HEK293 cell line. The annexin V/PI staining was done to investigate the apoptotic and necrotic cell populations after exposure to epsilon toxin. Results: Induction by 1 mM IPTG for 4 h at 37°C was an optimized condition for expressing mature epsilon toxin in E. coli strain BL21 (DE3). Electrophoresis on SDS-PAGE 12% gel showed the desired band approximately at 38 KDa. Our results showed that recombinant epsilon toxin is mainly expressed as an inclusion body. Furthermore, 100, 150, and 200 µg/mL of mature epsilon toxin are significantly reduced the cell viability (P≤0.05). The considerable increase of necrotic cell percent- age was shown after exposing to 100, 150, and 200 µg/mL of mature epsilon toxin (P≤0.05). Conclusion: The recombinant mature epsilon toxin had cytotoxic effects and could induce necrosis.

Evaluation of the genotoxic potential of water impacted by acid mine drainage from a coal mine in Mpumalanga, South Africa, using the Ames test and Comet assay

Several potential genotoxins found in water samples arise from anthropogenic activities. Acid mine effluent resulting from coal mining poses serious environment concerns all over the world. The use of toxicity tests to evaluate the quality of streams add value by providing site-specific toxicological data. Treatment systems such as the use of natural wetlands (passive) or conventional physical and chemical pH-neutralised processes (active) are employed mainly to meet certain water quality guidelines. Nonetheless, potential genotoxins or residues remain which influence the quality of discharged effluents. The objective of this study was to evaluate the genotoxic potential of acid mine drainage (AMD) released into a natural stream following treatment by passive and active methods. This study aimed to identify the extent of AMD mutagenicity and genotoxicity to African Vero monkey kidney cell line and a fish gill cell line (RTgill-W1) using two assays, the Ames test, and the comet assay, as a rapid and effective screening tool. The Ames test performed without metabolic activation using Salmonella typhimurium TA98 and TA100 strains showed no indication of mutagenicity in the water samples tested. Differing results were however obtained for the comet assay using the African Vero monkey kidney cell line and a fish gill cell line (Rtgill-W1), which revealed DNA fragmentation and variations in morphologies indicative of genotoxicity in the water samples following the two treatment processes. A significant reduction in DNA damage was observed in water samples following active treatment of the AMD, evidenced by reduced damage frequency and a lowered comet score. This bioassay confirms the urgency of integrating high-throughput screening in aquatic toxicity assessment at genetic levels, giving further evidence that in-vitro bioassays can be incorporated for use in short-term genotoxicity assays. The result suggests that the comet assay proved sensitive at detecting genotoxicity, supporting the integration of this into environmental monitoring frameworks targeted at AMD-contaminated sites.

Rehabilitation of culture cell lines from mycoplasma infection

From the early 50's of last century and to nowadays the cell culture lines are a cornerstone in virological, cytological, biochemical and other biological studies. Cell cultures are essential in studying of cellular regulation mechanisms, virus interaction processes during their replication in sensitive cells, and in production of biologically active materials, including vaccines, enzymes, hormones, and monoclonal antibodies. However, today there is very common and often catastrophic problem that connected with cell line contamination by other microorganisms. Contamination with mycoplasma is of particular concern to scientists around the world. Due to difficulties in its detection, it stays unnoticed in cell cultures, having detrimental effect on cell function and morphological status. Cell cultures contamination with mycoplasmas can lead to degenerative changes in cells and their complete loss and represents a potential source of artifacts in cytological, virological and biochemical studies. Mycoplasma-contaminated cell lines are a major problem in research center laboratories and biotechnology facilities. Mycoplasmas are ultramicroscopic free-living prokaryotes whose sizes range from 200 to 400 nm. They lack a cell wall, which makes it impossible to detect them even with an ordinary microscope. In addition, mycoplasmas do not cause turbidity in cell culture media, which often accompanies bacterial or fungal cell culture contamination. The most important thing is that mycoplasma infection usually does not lead to cell death. Therefore, they can multiply and go unnoticed in a cell culture flask for a long period, becoming a major obstacle to reliable and standard in vitro experiments. Nowadays, the rehabilitation of the cell line from mycoplasmas is a very complex and urgent problem. According to many researchers, their attempts to decontaminate cell culture from mycoplasmas have been ineffective. Due to conclusions from their work, all cell lines infected with mycoplasmas are subject to immediate destruction. At the same time, the purchase of new cell lines requires considerable material costs. Therefore, there is a need to find real approaches in the rehabilitation of cell culture lines in research and production laboratories. The article represents the results of experimental studies focus on the decontamination of 12 cultures cell lines of animal origin from mycoplasmas. The research was conducted during 2019-2020 at the department of biotechnology and viral vaccine control (culture line sector) of State scientific control institute of biotechnology and strains of microorganisms. The cell culture lines with PCR-detected mycoplasma infection were used in the experiments, namely: FLK - sheep embryonic kidney cell line; PO - sheep kidney cell line; RK-13- rabbit kidney cell line; RK-15 - pig kidney cell line; MDBK- bovine kidney embryo cells; Vero- African green monkey kidney cells; Marc-145 - African green monkey kidney-derived MA-104 cells; BGM - African green monkey kidney cells; BHK-21 / clone 13 - Syrian hamster kidney cells; KST (KST) - cells of coronary vessels of cattle; SPEV (original name - SNEV) - pig kidney cells; A-72 - culture of subcutaneous tumor cells in dogs. On the early stages of the study, the optimal allowable concentration of the drug ciprofloxacin, which was used in our laboratory to rehabilitate cell cultures from mycoplasmas, was determined. It has been experimentally established that ciprofloxacin at concentration of 20 μg/cm3 completely inhibits the reproduction and life cycle of mycoplasmas and does not cause a significant effect on the cells themselves. Ciprofloxacin in concentration of 20 μg/cm3 was added into the growth and maintenance medium for five consecutive passages to the flasks with mycoplasma-contaminated cell cultures. After forming a cell monolayer in the flasks, it was always thoroughly washed five times with Hanks' solution, and only then the medium was replaced with a supportive one. Thus, the full cycle of cell culture remediation, from the moment of seeding the cells in the flasks until the next reseeding of cells took 3-4 days. 5 cycles were performed with each cell culture affected by mycoplasmas. At the completion of rehabilitation process, the samples of cell culture supernatant from each cell culture line were collected and examined by PCR for the presence of mycoplasmas. As a result, after remediation process of cell cultures mycoplasma infection was detected in only one sample. The efficiency is 88.9% and it proves that the drug ciprofloxacin can be successfully used for decontamination of cell cultures in scientific and industrial virological laboratories.

Genome Analysis and Replication Studies of the African Green Monkey Simian Foamy Virus Serotype 3 Strain FV2014

African green monkey (AGM) spumaretroviruses have been less well-studied than other simian foamy viruses (SFVs). We report the biological and genomic characterization of SFVcae_FV2014, which was the first foamy virus isolated from an African green monkey (AGM) and was found to be serotype 3. Infectivity studies in various cell lines from different species (mouse, dog, rhesus monkey, AGM, and human) indicated that like other SFVs, SFVcae_FV2014 had broad species and cell tropism, and in vitro cell culture infection resulted in cytopathic effect (CPE). In Mus dunni (a wild mouse fibroblast cell line), MDCK (Madin-Darby canine kidney cell line), FRhK-4 (a fetal rhesus kidney cell line), and MRC-5 (a human fetal lung cell line), SFVcae_FV2014 infection was productive resulting in CPE, and had delayed or similar replication kinetics compared with SFVmcy_FV21 and SFVmcy_FV34[RF], which are two Taiwanese macaque isolates, designated as serotypes 1 and 2, respectively. However, in Vero (AGM kidney cell line) and A549 (a human lung carcinoma cell line), the replication kinetics of SFVcae_FV2014 and the SFVmcy viruses were discordant: In Vero, SFVcae_FV2014 showed rapid replication kinetics and extensive CPE, and a persistent infection was seen in A549, with delayed, low CPE, which did not progress even upon extended culture (day 55). Nucleotide sequence analysis of the assembled SFVcae_FV2014 genome, obtained by high-throughput sequencing, indicated an overall 80–90% nucleotide sequence identity with SFVcae_LK3, the only available full-length genome sequence of an AGM SFV, and was distinct phylogenetically from other AGM spumaretroviruses, corroborating previous results based on analysis of partial env sequences. Our study confirmed that SFVcae_FV2014 and SFVcae_LK3 are genetically distinct AGM foamy virus (FV) isolates. Furthermore, comparative infectivity studies of SFVcae_FV2014 and SFVmcy isolates showed that although SFVs have a wide host range and cell tropism, regulation of virus replication is complex and depends on the virus strain and cell-specific factors.

AUJESZKY’S DISEASE IN A DOG - CASE REPORT

This article reports on the occurrence and diagnosis of Aujeszky’s disease in a dog. The procedure for isolation and identification of Aujeszky’sdisease virus was described. A dog of unknown breed aged about two years died of Aujeszky’s disease after consuming animal offal (internal organs: lungs, spleen, kidneys) fed by the owner after slaughtering piglets and preparing meat for cooking. As early as 24 hours after consuming the offal, the dog manifested characteristic symptoms of Aujeszky’s disease, which were immediately recognized by the veterinarian. The death occurred within less than 24 hours upon first clinical signs of disease. Aujeszky’s disease virus was isolated and identified from brain and internal organ (lung and spleen) samples of the dog at the Department of Virology of the Scientific Veterinary Institute „Novi Sad“. Isolation and identification of the virus was performed on PK-15 porcine kidney cell line and using nested PCR technique.

Synthesis of Novel Ibuprofen-Tranexamic Acid Codrug: Estimation of The Clinical Activity Against HCT116 Colorectal Carcinoma Cell Line and The Determination of Toxicity Profile Against MDCK Normal Kidney Cell Line

Objective: Both ibuprofen and tranexamic acid were tried to treat colorectal carcinoma, however, combined drugs were not. Accordingly, with the aid of SciFinder®, online absence of the mutual prodrug (codrug) was affirmed. This persuade the authors to conduct this research. Methods: The ibuprofen-tranexamic acid codrug was synthesized and characterized with 83% yield. The purified white powder codrug was tested against HCT116 colorectal cancerous cell line and MDCK normal non-cancerous cell line. Results: The newly synthesized and characterized ibuprofen-tranexamic acid codrug has significant parameters. One of which it absolutely obeyes the Lipinski rule of five. Moreover, like the Lipinski rule of five, the number of rotatable bonds (7 rotatable bonds) and the topological polar surface area tPSA (66.4 A2) shows a very favourable oral absorption drug candidate. The IC50 of the mutual prodrug against HCT116 colorectal cells was 5.33 mg/ml, while the IC50 for the MDCK normal kidney cell line was 6.4 g/ml. Conclusion: The authors conclude that the newly synthesized ibuprofen-tranexamic acid codrug has fair anticancer activity against HCT116 colorectal cancer cell line with tolerated toxicity profile acquired with the MDCK normal kidney cell line.