Search

Hepatitis B virus (HBV) remains an important healthcare challenge, leading to liver diseases like cirrhosis and cancer. In response, we created a prophylactic and therapeutic HBV vaccine by integrating HBcAg and HBsAg from HBV genotype B into Norovirus (NoV) GII.2 P domain (PdomGII.2-HBV) for enhanced intranasal delivery. This vaccine also aimed to simultaneously prevent NoV infection, which causes gastroenteritis. Since the selected HBV epitopes have undergone extensive research and are tailored to the Indonesian population, this study focused on identifying NoV epitopes and assessing T cell epitopes coverage of the PdomGII.2-HBV for the Indonesian population. Following that, we expressed the PdomGII.2-HBV protein using Escherichia coli BL21(DE3) and employed a gentle solubilization technique for protein purification. Our in-silico analysis identified two B cell epitopes, along with 15 CD4+T cell epitopes and 35 CD8+T cell epitopes within the GII.2 P domain. These T cell epitopes cover 100% of the Javanese-Sundanese population's HLA allele variations, which constituted the largest demographic group in Indonesia. Subsequently, we successfully purified the presumed PdomGII.2-HBV protein, revealing a molecular weight of 39.5 kDa. Following the successful expression and purification of the presumed PdomGII.2-HBV protein, it is evident that this vaccine design has significant potential, warranting further study. [ABSTRACT FROM AUTHOR]

The investigation of mRNA development has gained substantial interest, particularly in the ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium and harvest time to increase plasmid DNA production as part of mRNA production. This study modified used a medium modification approach to achieve high density culture of Escherichia coli TOP10 pGEMT-N in batch cultivation method. Various media formulations were assessed, including LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer on culture density was measured through OD600 and wet cell weight analysis. The highest OD600 and wet cell weight was observed when LB with glycerol and phosphate buffer was used, with OD600 of 4.78±0.14 and wet cell weight of 36.00±0.63 mg/ml. Plasmid DNA was subsequently isolated from these cultures following 5- and 7.5-hour incubation periods. The utilization of LB medium with glycerol and phosphate buffer resulted in a substantial increase in the volumetric concentration of plasmid DNA of 1,516.97±385.00 ng/ml after 5 hours of incubation. In conclusion, a remarkable enhancement in plasmid DNA volumetric yield within 5 hours was achieved by addition of glycerol and phosphate buffer to LB medium, leading to incubation period. [ABSTRACT FROM AUTHOR]

Staphylococcal enterotoxin B (SEB) from Staphylococcus aureus is a potential therapeutic agent for cancer. SEB can activate the immune response, which could induce apoptosis of various cancer cells. This study aimed to design and produce a recombinant SEB protein using Escherichia coli BL21(DE3) expression system. We designed and optimized the codon of the inserted gene for E. coli and then transformed it into E. coli BL21(DE3). The transformants were verified by selective media containing kanamycin followed by colony PCR with T7 promoter and T7 terminator primers and DNA sequencing. The results showed that the SEBsyn encoding gene could be synthesized and cloned into pET-28a(+) expression plasmid. This recombinant plasmid that carried the SEB encoding gene (pET-28a_SEBsyn) was successfully transformed into E. coli BL21(DE3). The amplicons of colony PCR visualized in 2% agarose gel showed that transformants carrying the recombinant plasmid had an inserted gene length of about 1 kb. Gene inserts sequence verification by DNA sequencing resulted in 1,148 bp sequence consensus. The blastx analysis showed that it had the best hit with enterotoxin B in the NCBI database (accession id CAC6284025.1). The recombinant SEB protein was also successfully overexpressed in E. coli BL21(DE3) with 0.1 mM IPTG. Our recombinant SEB protein was dominantly insoluble in the inclusion body; however, some SEB protein was expressed as soluble. The molecular weight of the target protein is about 29 kDa. [ABSTRACT FROM AUTHOR]

The development of breast cancer involves many processes, including angiogenesis and metastasis. Some factors play a major role in angiogenesis, such as HIF-1α, and in metastasis, such as FAK and Wnt2. The aim of this study was to observe the effect of propolis and mangostin on the development of mammary gland cancer and on the expression of Wnt2 and FAK in Balb/C mice. Mammary gland tumors were induced in Balb/C mice by DMBA. The mice were divided into 5 treatment groups: negative control (K-); positive control treated with doxorubicin (14.04 mg/kg bw) (Dx); mice treated with propolis (0.32 mg/kg bw) (P) or mangostin (0.128 mg/kg bw) (M); and mice treated with a combination of propolis (0.32 mg/kg bw) and mangostin (0.128 mg/kg bw) (MP). Both mangostin and propolis did not affect the body weight of the mice. Treatment with propolis or treatment with propolis combined with mangostin was able to reduce tumor development activity in Balb/C mice. Moreover, the combination of mangostin and propolis was able to lower Wnt2, FAK and HIF- 1α expression. It can be concluded that the combination of propolis and mangostin has potential to inhibit cancer development through downregulation of Wnt2, FAK, and HIF1α expression. [ABSTRACT FROM AUTHOR]

Cryptocarya pulchrinervia is an Indonesian indigenous plant that grows in Sumatra, Kalimantan and Papua. One of the new compounds extracted from this plant was cryptobrachytone C, which was known to be cytotoxic against cancer cells of Murine leukemia P388 with IC 50 10.52 μM. In this study, the cytotoxicity and anticancer properties of cryptobrachytone C on proliferation, apoptosis, migration and clone formation of MCF-7 and T47D breast cancer cell lines were examined, which had not previously been done before. The cytotoxicity of the compound was measured using an MTT (3- (4,5-dimethylthiazol-2- yl) -2,5-di-phenyl-tetrazolium bromide) assay. The cell proliferation was measured using a BrdU assay, and the cell apoptosis was measured using annexin-V FITC, while the cell migration was measured using a transwell filter. The cytotoxic test result demonstrated that cryptobrachytone C was cytotoxic against MCF-7 cells with IC 50 12.94 ± 0.32 μM but not against T47D cells with IC 50 65.33 ± 2.33 μM nor against normal MRC-5 cells with IC 50 122.57 ± 19.84 μM. The cell proliferation assay showed that cryptobrachytone C at IC 50 concentration had antiproliferative properties against MCF-7 cancer cell lines ( p < 0.05) but did not significantly reduce T47D cell proliferation ( p < 0.07). Although the results of the cell apoptosis test showed that cryptobrachytone C could induce the apoptosis of the MCF-7 and T47D cells, it was insignificant ( p > 0.05). The cell migration test showed that cryptobrachytone at IC 50 concentrations could inhibit the migration of the MCF-7 and T47D cells. The clonogenic test showed that cryptobrachytone C at IC 50 concentration can induce the inhibition of the formation of MCF-7 and T47D cell colonies. The cryptobrachytone C anti-cancer character was more signi icant on the MCF-7 cell line compared to the T47D. This study showed that cryptobrachytone C was cytotoxic and had potential as an anti-cancer compound against MCF-7 and T47D breast cancer cell lines., Competing Interests: CONFLICT OF INTEREST: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, and there is also no conflict of interest between the authors. This paper is purely based on our research., (© Penerbit Universiti Sains Malaysia, 2023.)

Objective: This study aimed to determine the most effective method in establishing primary cell culture from epithelial serous ovarian cancer tissues with the highest yield of cells and percentage of epithelial cells., Methods: Primary and metastasis tissues from three serous ovarian cancer patients were processed using 18 different combinations of methods based on different factors: the source of tissue (primary site or metastasis site), the cell suspension method (explant method, enzymatic methods, or the addition of Percoll), and the alternatives of three different media. We compared the total count of cells, the percentage of epithelial cells, and the estimated number of epithelial cells per observation field. The calculation of cells from primary tissues were compared to metastasis tissues, and the difference was statistically analyzed using Mann Whitney-U test on SPSS software., Result: The groups that were processed using dispase and trypsin resulted higher number of cells and higher percentage of epithelial cells when compared to the explant method. Among all media, we found that DMEM:F12 and McCoy's 5A media as equally useful in isolating and culturing epithelial cells. Statistically, the metastasis tissue derived more epithelial cells when compared to the primary tissue (102.32±82.65 vs 22.6±23.81, p=0.001)., Conclusion: The use of metastasis tissue processed with trypsin or dispase and cultured in DMEM:F12 or McCoy's 5A media was found to be the most efficient way to produce the highest amount of cells with high percentage of epithelial cells.