Cell and Tissue Journal

Abstract Introduction: Concerns about fossil energy costs, environmental deterioration, and energy security has created strong motivation for the research and development of routes to provide sustainable and renewable fuels. In recent years, the use of biomass to produce highly valued chemicals has attracted widespread attention. Lignocellulosic biomass, as a promising renewable resource for biofuel production, has distinct advantages in terms of economic and environmental benefits. The conversion of renewable raw materials to hydrocarbon fuels is an attractive alternative to fossil fuels from economic and environmental perspectives. The production process of lignocellulosic biomass mainly consists of biomass accumulation, biomass decomposition, simple sugars, and conversion of sugars to biofuel. One of the crucial steps for the economic success of lignocellulosic biofuels depends on the inhibition of competitive metabolism in microorganisms to achieve high productivity. To date, there has been a growing focus on the use of S. cerevisiae and E. coli as cell lines. These two cellular factories have well known advantages. They are genetically transmissible and several tools are available for genetic manipulation. In order to produce xylonate, the engineered xylose is first converted by a dehydrogenase into the intermediate xylonolactone, which is then slowly converted to xylonate in a nonenzymatic reaction.
Aim: The organic compound D-1,2,4-Butanetriol (BT) is a valuable chemical with wide-ranging applications in various fields such as pharmaceuticals, paper, polymer materials, and military applications. However, the chemical synthesis routes for BT have many drawbacks. By genetically modifying microorganisms, the metabolic pathway for producing many substances, including BT, can be engineered. When D-xylose is supplied to the bacterium, it is first converted into an intermediate compound called xylonolactone. This compound slowly converts into xylonate through a non-enzymatic reaction. To produce xylonate, the engineered bacteria receive xylose, which is initially converted by a dehydrogenase reaction catalysed by the xylose dehydrogenase enzyme into an intermediate compound, xylonolactone. Xylonolactone is slowly converted to xylonate in a nonenzymatic reaction. Xylonate is a five-carbon organic acid. Over the past few years, xylonate has increasingly been considered as an important chemical due to its potential as an important chemical component. Xylonate has many applications in the food, chemical, and pharmaceutical industries. Specifically, xylonate can act as a precursor for the synthesis of D-1,2,4-Butanetriol and as a concrete water reducing agent. E. coli was chosen as the target strain for genetic and metabolic engineering due to its fast growth in inexpensive culture media, the presence of two enzymes for BT synthesis, and product formation in less than 24 hours of fermentation. This study aimed to clone and express xylose dehydrogenase from Caulobacter vibrioides in E.coli.
Materials and Methods: At first, to access the bacterial gene sequence, the genome of the target bacterium was extracted. Then, to create a strain expressing the enzymes xylose dehydrogenase and xylonolactonase, the genes for these proteins were amplified from Caulobacter vibrioides CB1 and transferred into E. coli. For this purpose, the target genes were amplified using specifically designed primers via the Polymerase Chain Reaction (PCR) method and initially cloned into a pTZ57cloning vector and then subcloned into pET 26b expression vector. At the final step, the expression of the enzyme was assessed by SDS-PAGE, and the other confirmation was the reduction of NAD+ to NADH, which was used as an activity indicator of the enzyme, as investigated by a change in NADH absorbance at 340 nm.
Results: Confirmatory tests were performed to ensure the presence of the gene in the vectors (using restriction enzymes and colony PCR for gene amplification). The expression and activity of the enzyme were analyzed. The recombinant protein's presence was confirmed by SDS-PAGE for the xylose dehydrogenase gene, with a molecular weight of 52.2 kDa. The estimated expression level of the recombinant protein was approximately 25%.
Conclusion: The objective of this research was solely to establish the metabolic pathway for xylonate production in E. coli by surface expression of enzymes in this pathway (xylose dehydrogenase). The results obtained in this study confirm that half of the pathway is active at the cell surface, but further experiments are required to determine the precise production levels and complete the pathway.

Abstract Introduction: Pancreatic cancer is the fourth leading cause of cancer-related deaths worldwide. 5-Fluorouracil is one of the commonly used chemotherapeutic drugs. The plant Artemisia absinthium has attracted attention as a potential herbal anticancer agent. This study investigated the effect of the methanolic extract of this plant on the expression of miR-34a and the apoptotic genes BAX, Caspase-3, and Caspase-9 in AsPC-1 pancreatic cancer cells.
Aims: This study aimed to evaluate the cytotoxic and pro-apoptotic effects of Artemisia absinthium methanol extract on pancreatic cancer cells. The research specifically investigated the molecular mechanism by analyzing expression changes in the tumor suppressor miR-34a and key apoptotic genes BAX, Caspase-3, and Caspase-9 to elucidate the extract's anti-cancer mode of action.
Materials and methods: To evaluate the cytotoxic effects of the plant extract on cancer cells, an MTT assay was performed to determine the viability and survival rate of the cells following treatment with various concentrations of the extract, the chemotherapeutic drug fluorouracil (5-FU), and the combined treatment of the extract and the drug. This assay measures cellular metabolic activity and allows quantification of live and dead cells after exposure to different treatments. Based on the obtained results, the IC₅₀ value for each treatment was calculated, representing the concentration at which 50% of the cells were inhibited or killed.
After determining the IC₅₀ value, cells were treated with concentrations equal to, lower, and higher than the IC₅₀ to further investigate the cytotoxic effects and the mode of cell death induced by the treatments. To distinguish between apoptotic and necrotic cell death, the Annexin V-FITC/PI assay was employed. This assay detects phosphatidylserine externalization on the cell membrane and enables differentiation between live, early apoptotic, late apoptotic, and necrotic cells.

In addition to the morphological and physiological assessments, molecular analyses were conducted to examine the expression levels of key apoptosis-related genes, including Caspase-3, Caspase-9, and BAX, as well as the regulatory microRNA miR-34a. Gene expression analysis was performed using Real-time PCR (qPCR).

Results: The results of the MTT assay demonstrated that the proliferation of AsPC-1 pancreatic cancer cells was inhibited by treatment with the extract of Artemisia absinthium and the chemotherapeutic drug fluorouracil (5-FU) in a concentration-dependent manner. As the concentration of each treatment increased, cell viability significantly decreased, indicating a marked cytotoxic effect of both the plant extract and the drug. Moreover, a possible synergistic effect between the extract and fluorouracil in suppressing. To determine the mode of cell death induced by these treatments, the Annexin V-FITC/PI assay was performed. The results revealed that a considerable proportion of treated cells underwent programmed cell death (apoptosis), while the percentage of necrotic cells remained relatively low. These findings suggest that the observed reduction in cell viability is mainly mediated through the activation of apoptotic pathways rather than necrosis. Furthermore, Real-time PCR analysis showed a significant upregulation in the expression of the regulatory microRNA miR-34a and the apoptosis-related genes Caspase-3, Caspase-9, and BAX in the treated groups compared to the control group.
Discussion: The obtained results suggest that Artemisia absinthium extract exerts its cytotoxic effect primarily through the induction of apoptosis rather than necrosis in AsPC-1 pancreatic cancer cells. The observed upregulation of miR-34a, Caspase-3, Caspase-9, and BAX implies activation of intrinsic apoptotic pathways. These findings are consistent with previous studies reporting pro-apoptotic properties of A. absinthium and other Artemisia species. Therefore, the extract may enhance the therapeutic response of pancreatic cancer cells when combined with conventional chemotherapeutic agents such as fluorouracil.
Conclusion: In conclusion, Artemisia absinthium extract demonstrated strong antiproliferative and apoptosis-inducing effects on AsPC-1 cancer cells in a dose-dependent manner. Its combination with fluorouracil produced a synergistic cytotoxic impact, significantly enhancing cell death through apoptotic signaling. The molecular findings support the potential of this extract as a complementary therapeutic agent. Further studies are recommended to explore its mechanisms and evaluate its efficacy in in vivo models of pancreatic cancer.

Abstract Introduction: Concern over fossil energy costs and environmental deterioration, along with energy security, has created a strong motivation for research and development of routes to provide sustainable, renewable fuels. In recent years, the use of biomass to produce highly valued chemicals has attracted widespread attention. lignocellulosic biomass, as a promising renewable resource for biofuel production, has distinct advantages in terms of economic and environmental aspects. The conversion of renewable raw materials to hydrocarbon fuels is an attractive alternative to fossil fuels from an economic and environmental point of view. The production process of lignocellulosic biomass mainly consists of biomass accumulation, biomass decomposition, simple sugars, and conversion of sugars to biofuel. One of the crucial steps in the economic success of lignocellulosic biofuels depends on the inhibition of competitive metabolism in microorganisms to achieve high productivity. To date, a growing focus is on the use of S. cerevisiae and E. coli as cell lines. These two cellular factories have the benefits that are well known.

Aim: The organic compound D-1,2,4-Butanetriol is a valuable chemical with wide-ranging applications in various fields. The chemical synthesis routes for BT have many drawbacks. By genetically modifying microorganisms, the metabolic pathway for producing many substances, including BT, can be engineered. The organic compound D-1,2,4-butanediol (BT) is an important intermediate chemical widely used in fields such as pharmaceuticals, paper, polymer materials, and military applications. When D-xylose sugar is provided to the bacterium, it is first converted to an intermediate compound called xylonolactone. This compound itself slowly converts into xylonate through a non-enzymatic reaction. To produce xylonate, the engineered bacteria have received xylose and initially, by a dehydrogenase reaction by the xylose dehydrogenase enzyme, that converts it into an intermediate substance: xylonolactone. The xylonolactone is converted slowly and in a nonenzymatic reaction to xylonate. Xylonate is a five-carbon organic acid. Over the past few years, xylonate has been increasingly being considered as an important chemical due to its potential as an important chemical component. Xylonate has many applications that can be used in the food, chemical, and pharmaceutical industries. Specifically, xylonate can act as a precursor for the synthesis of D-1,2,4-Butanetriol and a decrease in concrete water. E.coli due to fast growth in cheap culture medium, having two enzymes for the bto synthesis and product production in lower 24 hours of fermentation was chosen as the target strain of genetic engineering and metabolism. This study aimed to clone and express xylose dehydrogenase from Caulobacter vibrioides in E.coli. Materials and Methods: At first, to access the bacterial gene sequence, the genome of the target bacterium was extracted. Then, to create a strain expressing the enzymes xylose dehydrogenase and xylonolactonase, the genes for these proteins were amplified from Caulobacter vibrioides CB1 and transferred to E. coli. For this purpose, the target genes were amplified using specifically designed primers via the Polymerase Chain Reaction (PCR) method and initially cloned into a pTZ57cloning vector and then subcloned into pET 26b expression vector. At the final step, the expression of the enzyme was assessed by SDS-PAGE, and the other confirmation was the reduction of NAD+ to NADH, which was used as an activity indicator of the enzyme, as investigated by a change in NADH absorbance at 340 nm. Results: Confirmatory tests were performed to ensure the presence of the gene in the vectors (using restriction enzymes and colony PCR for gene amplification). The expression and activity of the enzyme were analysed. The recombinant protein's presence was confirmed by SDS-PAGE for the xylose dehydrogenase gene, with a molecular weight of 52.2 kDa. The estimated recombinant protein expression levels were approximately 25%. Conclusion: The objective of this research was solely to establish the metabolic pathway for xylonate production in E. coli by surface expression of enzymes in this pathway (xylose dehydrogenase). The results obtained in this study confirm that half of the pathway is active at the cell surface, but further experiments are required to determine the precise production levels and complete the pathway. This study aimed to create a metabolic pathway for producing xylonate in E.coli.