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Background: A promising strategy in cancer therapy involves the production of fusion proteins, which entail the fusion of two distinct proteins. This study aimed to produce and assess the cytotoxic effects of the Nisin-arginine deiminase (ADI) fusion protein on the SW480 cell lines, a common model for studying colorectal cancer (CRC).
Materials & Methods: The designed Nisin-ADI gene fragment sequence was sent to Biomatik Company for synthesis in pET-28a vectors between SacI and HindIII restriction enzyme sites. Escherichia coli (E. coli) DH5α and BL21 were utilized for cloning and protein expression, respectively. The recombinant fusion protein expression was induced by Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and purified using Ni2+-nitrilotriacetic acid (1) resin affinity chromatography. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were conducted to analyze the purified protein. The cytotoxic effect of the purified recombinant fusion protein on SW480 and NIH3T3 cells, as a control, was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay.
Findings: The results of this study showed that the fusion protein had a significant impact on the SW480 cell lines. The Half-maximal inhibitory concentration (IC50) of the fusion protein was 30 µg/mL, indicating that it effectively inhibited the growth of cancer cells. However, the fusion protein did not significantly affect the control group.
Conclusion: This study provides helpful insights into the potential application of recombinant Nisin-ADI fusion proteins as a potential treatment option for colorectal cancer. The potential for selective targeting of cancer cells is promising as normal cells are unaffected by this fusion protein.

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Objective: Different cryoprotectants are used for cryopreservation of ovarian tissue in patients at risk of infertility. Ethylene glycol (EG), dimethyl sulfoxide (DMSO) and propanediol (PROH) have been chosen as the basic permeable cryoprotectants due to their decreased glass-formation characteristics compared to other cryoprotectants. In the present study, the effects of two different vitrification methods on whole mouse ovarian tissue by the use of a novel staining method (trypan blue) has been evaluated. Methods: Ovaries of 8 day-old NMRI mice were isolated and divided among the control, vitrification 1 (Vit1) and vitrification 2 (Vit2) groups. The Vit1 solution was composed of α-MEM+ 20% FBS + 15% EG + 15% DMSO. The Vit 2 solution was composed of α-MEM+ 15% FBS +20% EG + 20% PROH. Vit1 and Vit2 procedures were performed at 4˚C and room temperature, respectively. Warming was performed in α-MEM+ 20% FBS supplemented with 1M sucrose in the Vit1 group and α-MEM+ 15% FBS with descending concentrations of sucrose (1, 0.5, 0.25 M) in the Vit2 group. Control and vitrified warmed ovaries were put in α-MEM supplemented by 0.4% trypan blue for 20 min, and then stained ovaries were fixed in Bouin’s fixative, serially sectioned in paraffin wax and finally quantitatively evaluated under a light microscope. Results: The highest percentage of primordial follicles was observed in the control group. There was a significant difference between the control and Vit1 groups, and between the Vit1 and Vit2 groups (p<0.05). No significant difference was observed in primary and preantral follicles between the control and vitrification groups. Conclusion: Vitrification with EG and PROH are more suitable for preservation of follicle reserves in ovaries. Trypan blue staining is a faster and easier method for evaluation of ovarian tissue.