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 Aim: The aim of this study was to investigate different concentrations of iron oxide nanoparticles in and benzylaminopurine (BAP) bell pepper anther culture on the traits of callus formation, embryogenesis, regeneration and rooting.
Material and methods: The experiment was conducted as a factorial experiment in a completely randomized design under in vitro culture conditions. Flower buds of appropriate size (equal sepal to petal ratio or slightly longer petal) were collected from the greenhouse and acetocarmine solution was used to determine the growth and development stage of microspores. The results showed that the most suitable stage for embryogenesis induction was the late mononuclear stage and the early binuclear stage. In order to sterilize the flower buds, 70% ethanol for 30 seconds and 5% sodium hypochlorite for 20 minutes were used and after each stage they were washed three times with sterile distilled water. Then, the anthers were separated from the flower bud and placed in C medium containing 2 mg/L naphthalene acetic acid (NAA), different concentrations of BAP (0, 0.1, 0.5, and 1 mg/L) and different concentrations of iron oxide nanoparticles (0, 1, 10, and 20 mg/L). After that, the explant cultured in C medium were kept at 35°C in the dark for 8 days in order to apply heat treatment. Then, they were transferred to 25°C in the light for 4 days. After this period, in order to induce embryogenesis, the explants were transferred from C medium to R medium and were subcultured every three weeks until embryos emerged. For further growth and root development, the embryos were transferred to V medium. Results: The results of the analysis of variance of the data showed that using different concentrations of iron oxide nanoparticles had a significant effect on the percentage of embryogenesis, regeneration and rooting of the plant, but did not have a significant effect on the percentage of callus formation. The results of comparing the average data showed that among the different concentrations of iron oxide nanoparticles, the 1 mg/L treatment had the highest percentage of embryogenesis (11.11). Also, the results of comparing the average effect of the nanoparticles on the percentage of regeneration showed that the 1 mg/L treatment had the highest percentage of regeneration (16.66). The results of the interaction effects showed that among the different concentrations of iron oxide nanoparticles and BAP, the highest percentage of embryogenesis was observed in the treatment of 20 mg/L iron oxide nanoparticles and 0 mg/L BAP. Also, the treatment of 20 mg/L iron oxide nanoparticles and 0 mg/L BAP had the highest percentage of regeneration (33.33%). After sufficient growth and root formation, the obtained plants were removed from the glass culture containers and transferred to pots containing sterilized culture medium and watered for adaptation. The tops of the pots were covered with plastic cups, and after three days, the cups were pierced and the plastic was gradually removed from the plant for further adaptation. Ploidy levels were determined by chromosome counting by staining the root tip cells. The results showed that out of the 23 obtained plants, 21 were diploid and had 2n=2x=24 chromosomes and 2 were haploid and had n=x=12 chromosomes. Conclusion: In bell pepper anther culture, different concentrations of iron oxide nanoparticles along with plant growth regulators at different concentrations showed a great effect on embryogenesis, regeneration, and rooting.

Abstract Introduction: Global climate change has significantly increased the frequency and intensity of abiotic stresses, thereby limiting plant growth, development, and overall yield by damaging physiological systems. In response to environmental stressors, plants have evolved various adaptive mechanisms, including the accumulation of compatible solutes such as glycine betaine (GB), which plays a pivotal role in protecting cellular functions under adverse conditions. Also known simply as betaine, this compound is a methylated glycine derivative recognized across plant species for its ability to mitigate the deleterious effects of stressful environments. Its zwitterionic structure; comprising a positively charged trimethylammonium group and a negatively charged carboxyl group, confers high solubility and chemical stability to the molecule.
Owing to its excellent biocompatibility, favorable carbon-to-nitrogen ratio, and high-concentration accumulation, glycine betaine can enhance plant tolerance against a wide spectrum of abiotic stresses. Specifically, GB contributes to photosynthetic recovery and the alleviation of oxidative stress by reducing the accumulation and facilitating the detoxification of reactive oxygen species (ROS). Furthermore, it plays a crucial role in stabilizing membranes and macromolecules, while protecting key components of the photosynthetic apparatus, such as the Rubisco enzyme, Photosystem II (PSII), quaternary enzymes, and complex protein structures. Notably, glycine betaine can accumulate at high concentrations within plant cells without interfering with normal metabolic processes, thereby significantly increasing resilience to various osmotic stresses, extreme temperatures (heat and cold), and oxidative damage. The biosynthesis of glycine betaine occurs through distinct metabolic pathways, including the choline oxidation pathway (prevalent in plants and mammals), the direct glycine methylation pathway (specific to certain bacteria and halophytes), the choline dehydrogenase pathway, and the serine metabolism pathway. Such diversity underscores the vital importance of this osmolyte in mediating responses to environmental stresses. Recently, biotechnological interventions, such as Agrobacterium-mediated transformation, have successfully enhanced stress tolerance in susceptible species by overexpressing key genes, most notably codA, BADH, GSMT, and SDMT. Given the functional diversity of genes involved in the glycine betaine biosynthetic pathway, extensive efforts have been made to develop transgenic plants capable of effective accumulation of this metabolite; however, serious challenges such as unstable and weak transgene expression remain as key obstacles in this path. Factors including promoter type, genomic integration site, and epigenetic factors can influence the final performance. Furthermore, the overexpression of enzymes in the glycine betaine biosynthetic pathway may potentially impair growth by disrupting metabolic stability. Therefore, future research should focus on decoding the molecular networks regulating the biosynthesis, signaling, and transport of glycine betaine, particularly its crosstalk with phytohormones, transcription factors, and the identification of stress-inducible promoters. Additionally, optimizing transformation protocols and synchronizing glycine betaine gene expression with the overall plant metabolism are essential.
Aims: This review examines the biosynthesis, physiological functions, and molecular regulation of glycine betaine (GB). It highlights genetic engineering techniques to boost GB production, offering sustainable strategies for enhancing crop tolerance to environmental stresses.
Conclusion: In-planta biosynthesis of glycine betaine (GB) offers a more sustainable alternative to exogenous application, aligning closely with the principles of green agriculture. By integrating GB synthesis pathways, genetically engineered crops can autonomously boost metabolite production and bolster stress resilience, thereby eliminating the logistical costs of external treatments. Furthermore, a more profound investigation into these biosynthetic pathways will facilitate the identification and cloning of novel target genes, ultimately maximizing GB accumulation and enhancing environmental tolerance.