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: Male factors account for approximately half of infertility cases. Sperm DNA damage, including DNA fragmentation, abnormal chromatin packaging, and epigenetic alterations, is associated with impaired sperm function, recurrent miscarriage, and poor outcomes of assisted reproductive technologies such as IVF and ICSI. Increasing evidence indicates that sperm DNA integrity is a key determinant of fertilization and embryo development. Aims: Therefore, the purpose of conducting research to identify sperm DNA damage and study its effect on the success rate of assisted reproductive methods, including microinjection, in infertile couples is essential, with the aim of improving sperm quality before starting the treatment cycle and imposing excessive costs on couples. Materials and methods: In this experimental study, 60 infertile couples were enrolled, including 30 couples as the positive-control group and 30 couples as the test group, consisting of men with oligoasthenospermia who had referred to the Royesh Infertility Treatment Center in Karaj for ICSI treatment. All female partners had an anti-Müllerian hormone (AMH) level >2. Semen samples were collected, and sperm DNA fragmentation was assessed by DNA staining. On the day of ICSI, oocytes were retrieved, and morphologically normal spermatozoa were selected for intracytoplasmic injection. On day 3 after fertilization, embryo number and quality were evaluated, and the relationship between embryo quality and sperm DNA fragmentation was analyzed. Results: Embryo quality was significantly higher in the normal sperm group than in the sperm DNA-damaged group (93.35% vs. 75.74%). Sperm motility was markedly reduced in the DNA-damaged group, with significantly lower sperm velocity (5.53% vs. 36.2%) and a higher proportion of non-motile sperm (82.17% vs. 41.93%). Progressive motility was also significantly decreased in the DNA-damaged group. Furthermore, sperm DNA fragmentation was significantly associated with reduced embryo quality, impaired sperm motility, lower sperm count, and abnormal sperm morphology (P < 0.05). Conclusion: Sperm DNA fragmentation may adversely affect the outcomes of assisted reproductive techniques. Therefore, assessment of sperm DNA fragmentation before selecting the appropriate treatment strategy is recommended. SDFA (sperm DNA fragmentation rate) testing is a reliable tool for evaluating sperm DNA integrity and may improve the clinical management and treatment outcomes of male infertility.

Abstract Introduction: Global climate change has damaged plant systems, their growth, development and overall performance by increasing the frequency and intensity of abiotic stresses. In response to environmental stresses, plants have evolved diverse adaptive mechanisms, including the accumulation of adaptive solutes, such as glycine betaine (GB), which plays a pivotal role in protecting cellular function under adverse conditions. Also known as betaine, this substance is a methylated derivative of glycine that reduces the harmful effects of stressful environments in plant species. GB is a low molecular weight and highly soluble osmoprotectant. GB is mainly produced in roots and vascular tissues and accumulates mainly in plastids and chloroplasts of young leaf cells, where it plays a vital role in protecting thylakoid membranes and thus maintaining the efficiency of photosynthesis. The level of GB accumulation is often directly related to the level of plant resistance to abiotic stresses. Furthermore, the capacity for GB accumulation varies among different plant genotypes. Among the protective roles of glycine betaine are reducing the accumulation and detoxification of free radicals, stabilizing membranes and macromolecules, and protecting components of the photosynthetic system such as the Rubisco enzyme, enzymes, and tetramers, and complex protein structures. Also, GB systematically modulates growth through interaction with hormones and regulators such as auxin, gibberellin, cytokinin, abscisic acid, salicylic acid, and jasmonic acid; as a result, this strategy increases the plant's ability to cope with stress conditions by optimally allocating energy. GB production, which is mainly synthesized from choline or glycine, follows 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. Although the glycine betaine biosynthetic pathway is not active in all plants, the choline oxidation pathway is the main mechanism for the production of this metabolite in most higher plants. This pathway is carried out through two consecutive enzymatic steps. The first step involves the hydroxylation of choline by choline monooxygenase (CMO) to betaine aldehyde, and the second step involves the irreversible oxidation of betaine aldehyde to glycine betaine, catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH). Identification, transfer, and alteration of the expression pattern of genes involved in the biosynthetic pathways of osmoprotectants, including glycine betaine, lead to improved stress tolerance in plants. Currently, research on the use of engineered plants or bacterial strains to enhance the synthesis of this osmolyte has mainly focused on the strategy of transferring exogenous single genes encoding key enzymes. An effective strategy to enhance abiotic stress tolerance in plants is through the stimulation of GB biosynthesis. Recently, biotechnological interventions, such as Agrobacterium-mediated transfection, have successfully enhanced stress tolerance in susceptible species by overexpression of key genes, particularly codA, BADH, GSMT and SDMT.
Aims: This review evaluates the various biosynthetic pathways, physiological functions and molecular regulatory mechanisms of GB under environmental stress conditions. Furthermore, it highlights advanced genetic engineering strategies aimed at enhancing GB biosynthesis, thereby providing sustainable approaches to enhance crop resistance to diverse environmental stresses.
Conclusion: Enhancing GB biosynthesis in plants has been recognized as an effective strategy to improve plant tolerance to environmental stresses. In this regard, two main approaches are commonly used: exogenous application and genetic engineering of GB biosynthetic pathways. Both methods increase resistance to a range of abiotic stresses by increasing GB accumulation and activating stress response pathways. External application of GB is a simple and effective method to improve plant performance under stress. However, its effectiveness depends on its absorption and translocation efficiency, which varies depending on the plant species, tissue type, and application method. Given the diversity of GB synthesis pathways and the identification of choline and glycine as GB precursors in the choline oxidation and glycine methylation pathways and the high ATP consumption in the glycine methylation pathway, by identifying and studying the role of each of the related genes in these pathways, especially the genes involved in enzyme synthesis, they can be used in breeding programs to increase GB biosynthesis under abiotic stress conditions. Genetically engineered products can automatically increase metabolite production and thus increase overall stress resistance. Genetic engineering strategies focused on increasing glycine betaine production and its accumulation in plant tissues under stress have shown significant improvements in plant growth and development. Stimulating glycine betaine production through the production of transgenic plants is an emerging technology due to its ability to overcome the problems associated with conventional modification of glycine betaine synthesis. These methods are mainly focused on identifying target genes in the glycine betaine synthesis pathway in stress-tolerant plants and microorganisms and cloning and transferring them to susceptible species using various biotechnological methods such as Agrobacterium-mediated transfection. The genes that have been most widely used in this regard include codA, BADH, GSMT, SDMT. Furthermore, a deeper investigation of these biosynthetic pathways will facilitate the identification and cloning of new target genes, ultimately maximizing GB accumulation and increasing environmental resistance. However, the use of this method as a sustainable approach compatible with green agriculture requires more detailed and extensive research on the impact of genetic manipulation on human health and nutrition.

Abstract Introduction: Prostate cancer is one of the most prevalent malignancies among men worldwide and represents a major cause of cancer-related morbidity and mortality. Despite advances in screening and therapeutic approaches, resistance to standard treatments, including androgen‑deprivation therapy, remains a major clinical challenge in prostate cancer. As a result, identifying novel compounds that can suppress proliferation or modulate key molecular regulators of the cell cycle is of considerable interest. Papaverine is an isoquinoline alkaloid derived from the opium poppy, widely known for its long‑standing clinical use as a smooth‑muscle relaxant and vasodilator. In recent years, it has gained attention for its potential anticancer effects, including its influence on mitochondrial function, cellular energy metabolism, and the modulation of signaling pathways involved in cell cycle regulation. However, the specific effects of papaverine on prostate cancer cells have not yet been clearly elucidated. Tamoxifen, a selective estrogen receptor modulator widely used in breast cancer therapy, has also demonstrated off-target antiproliferative effects in various tumor models.
Aims: The present study aimed to evaluate the effects of papaverine and tamoxifen on the viability and molecular regulatory profile of the DU145 prostate cancer cell line. Specifically, the study investigated the cytotoxic potential of these compounds at different concentrations and incubation times, and their ability to alter the expression of the CDK4 gene, a key mediator of G1-to-S phase progression, along with two microRNAs, miR-146a and miR-22, known to influence cell cycle control and tumorigenic pathways. By integrating cellular and molecular findings, the study sought to clarify whether these compounds could serve as potential modulators of prostate cancer cell growth.
Materials and Methods: The DU145 human prostate cancer cell line was cultured under standard conditions and treated with different concentrations of papaverine and tamoxifen. Cell viability was assessed for 24, 48, and 72 hours using the MTT (3- (4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Dose–response curves were generated to calculate IC50 values for each compound at the specified time points. To investigate molecular changes, total RNA was extracted from treated and control cells, and the expression levels of the CDK4 gene, miR-146a, and miR-22 were quantified by Real-Time PCR with appropriate internal controls. Relative expression changes were determined using the 2^-ΔΔCt method. The data were analyzed using SPSS software. Results were presented as Mean ± SD, and a significance level of p ≤ 0.05 was considered statistically significant.
Results: Treatment of the DU145 prostate cancer cell line with various concentrations of papaverine and tamoxifen resulted in a significant reduction in cell viability in a concentration- and time-dependent manner, as measured by the MTT assay for 24, 48, and 72 hours. IC50 values were successfully determined for both compounds across the different time points.
Molecular analysis using Real-Time PCR showed that treatment with either papaverine or tamoxifen led to a marked downregulation of CDK4 expression compared with untreated controls. In contrast, both compounds induced a significant up‑regulation of miR‑146a and miR‑22 levels.
Discussion: The observed decrease in viability suggests that papaverine and tamoxifen exert notable antiproliferative effects on DU145 prostate cancer cells. The downregulation of the CDK4 gene provides a mechanistic link to cell cycle arrest, as CDK4 is a central regulator of G1‑to‑S phase progression. Such suppression is in line with reduced cellular proliferation and supports the therapeutic potential of these compounds.
The increased expression of miR‑146a and miR‑22 further highlights the involvement of tumor suppressive microRNAs in mediating these effects. Both miRNAs have been implicated in regulating pathways associated with proliferation, apoptosis, and oncogenic signaling. Their upregulation may contribute to the inhibition of cell growth through negative modulation of critical oncogenic targets.
These results align with prior evidence demonstrating tamoxifen’s antiproliferative effects, but also importantly emphasize that the molecular and cellular impacts of papaverine in prostate cancer remain poorly understood. The current findings provide initial insight into papaverine’s interaction with cell cycle regulatory genes and microRNAs, suggesting a potential role in prostate cancer growth inhibition. Considering the adverse effect profile associated with tamoxifen, papaverine may therefore represent a more favorable candidate for further investigation, particularly as a potential alternative with a potentially safer therapeutic window.
Conclusion: In conclusion, both papaverine and tamoxifen effectively reduced DU145 cell viability and modulated the expression of the CDK4 gene, miR‑146a, and miR‑22, indicating their capability to interfere with cell cycle regulatory mechanisms. While their antiproliferative effects were broadly comparable, the established side effects of tamoxifen suggest limitations in its clinical applicability. Given that papaverine produced similar molecular outcomes without the same known adverse profile, it may be considered a potentially more suitable candidate for further preclinical evaluation in prostate cancer research.

Abstract Introduction: Cancer is recognized as one of the leading causes of mortality worldwide and remains a major global health challenge. Among the various types of cancer, gastric cancer is considered one of the most aggressive and prevalent malignancies due to its high incidence rate, late diagnosis, and poor prognosis. The AGS cell line is widely used as a reliable in vitro model for studying human gastric adenocarcinoma because of its adhesive growth pattern, relatively rapid proliferation, and preservation of the invasive characteristics of primary tumor cells. Conventional therapeutic approaches for gastric cancer, including surgery, radiotherapy, and chemotherapy, have shown partial clinical success; however, these methods are frequently associated with serious limitations such as systemic toxicity, damage to healthy tissues, severe side effects, and the development of drug resistance. Consequently, the development of advanced drug delivery systems has attracted considerable attention as a promising strategy to improve therapeutic efficacy while minimizing adverse effects. In this regard, nanotechnology has emerged as a powerful platform for the design of smart nanocarriers capable of enhancing drug stability, improving bioavailability, and enabling controlled and targeted drug release. Cisplatin is one of the most important chemotherapeutic agents used in the treatment of gastric cancer and exerts its anticancer activity primarily through binding to DNA, disrupting replication and transcription processes, and ultimately inducing apoptosis. Nevertheless, its clinical application is limited by severe toxicity and acquired drug resistance. Cellulose nanocrystals have gained significant attention in drug delivery systems owing to their biodegradability, high mechanical strength, structural stability, and ability to provide sustained and controlled drug release. In addition, bovine serum albumin is considered an ideal biological carrier because of its excellent biocompatibility, low toxicity, biodegradability, and natural tendency to accumulate in tumor tissues. Polyvinyl alcohol also plays an important role as a biocompatible polymeric matrix in the fabrication of stable nanocarriers. Furthermore, hyaluronic acid serves as a targeting ligand through its specific interaction with CD44 receptors, which are overexpressed on the surface of many cancer cells, including gastric cancer cells. This targeted interaction facilitates the selective uptake of nanoparticles by tumor cells and significantly enhances the efficiency of targeted drug delivery systems.
Aims: The aim of this study was to design and evaluate a smart nanocarrier system based on cellulose nanocrystals, polyvinyl alcohol, bovine serum albumin, and hyaluronic acid for the targeted delivery of cisplatin to gastric cancer cells. This study sought to improve drug stability, controlled release behavior, selective cellular uptake, and reduced toxicity toward normal cells by utilizing the biocompatible and targeting properties of these components. In addition, the cytotoxicity and anticancer selectivity of the synthesized nanoparticles were investigated in the AGS gastric cancer cell line to evaluate the potential efficiency of this nanocarrier system in enhancing the therapeutic performance of gastric cancer treatment.
Materials and methods: In this study, cellulose nanocrystals were extracted from raw cellulose through controlled sulfuric acid hydrolysis using 64% sulfuric acid, followed by ultrasonication, centrifugation, and dialysis to obtain both stable suspensions and freeze-dried powders. Cisplatin was subsequently loaded onto cellulose nanocrystals at different concentrations, and the drug-loading efficiency was determined spectrophotometrically at 370 nm. Multifunctional BSA (Bovine Serum Albumin)-PVA (Polyvinyl Alcohol)-CNC (Cellulose Nanocrystals)-CDP (Cisplatin) nanoparticles coated with PEG-hyaluronic acid were then synthesized by initially coating the drug-loaded cellulose nanocrystals with bovine serum albumin, followed by the formation of a stable hydrogel network using polyvinyl alcohol. To improve targeting ability and biological stability, the nanoparticle surface was further functionalized with NH2-PEG-HA in the presence of EDC/NHS as a coupling agent, and the final suspension was purified by dialysis before further analyses. The physicochemical properties of the synthesized nanoparticles, including crystalline structure, thermal stability, particle size, and zeta potential, were characterized using X-ray Diffraction (XRD), Thermogravimetric Analysis (TGA), and Dynamic Light Scattering (DLS) analyses, respectively. In addition, the release kinetics of cisplatin from the nanocapsules were investigated under different pH conditions simulating normal and tumor microenvironments, and the obtained data were analyzed using the Korsmeyer–Peppas kinetic model. To evaluate biocompatibility and selective cytotoxicity, AGS gastric cancer cells and normal GES-1 gastric epithelial cells were treated with the synthesized nanocapsules and free cisplatin, followed by MTT assay for IC₅₀ determination. Furthermore, the selectivity index (SI) was calculated to assess preferential toxicity toward cancer cells. Finally, apoptosis induction was evaluated using Annexin V/PI staining and flow cytometry. All experiments were performed in triplicate, and the data were statistically analyzed using one-way ANOVA followed by Duncan’s multiple range test in SPSS22 software.
Results: The physicochemical characterization results demonstrated that all synthesized nanoparticles and nanocapsules possessed a predominantly amorphous structure. The XRD patterns exhibited broad diffraction peaks within the 15–30° range, while the absence of sharp crystalline peaks confirmed the successful incorporation of cellulose nanocrystal–cisplatin complexes into the BSA-PVA matrix and the formation of a homogeneous noncrystalline system. TGA analysis further revealed that increasing the concentration of cellulose nanocrystals from 50 to 200 mg significantly enhanced the thermal stability of CNC-CDP nanoparticles, as higher concentrations remained thermally stable up to nearly 300°C. Moreover, the BPC_{(100 mg)}CP-HA nanocapsules showed lower thermal degradation and greater structural stability compared with drug-free nanocapsules, indicating the reinforcing effect of CNC-CDP within the nanocapsule network. DLS analysis demonstrated that increasing the cellulose nanocrystal concentration increased particle size from 322 to 363 nm, while the zeta potential decreased from +13.05 to +2.14 mV. Surface coating with BSA, PVA, PEG, and hyaluronic acid further increased the particle size to 476 nm and elevated the PDI value, confirming the successful formation of multilayered nanocapsules. Drug release studies indicated that cisplatin release was significantly higher under acidic conditions (pH=5.8) than under physiological conditions (pH=7.4), and the release rate increased over time. In contrast, increasing the cellulose nanocrystal concentration reduced the drug release rate, suggesting a more compact and stable nanostructure. MTT assay results showed that drug-free nanocapsules exhibited negligible cytotoxicity against both AGS and GES-1 cells, whereas cisplatin-loaded nanocapsules significantly reduced the viability of cancer cells. Among all formulations, BPC_{(50 mg)}CP-HA exhibited the strongest anticancer activity, with an IC₅₀ value of 224.75 µg.mL^-1 against AGS cells and the highest selectivity index (SI=1.75), indicating superior selective toxicity toward cancer cells. Furthermore, flow cytometry analysis demonstrated that this formulation induced the highest percentages of early and late apoptotic cells, while free cisplatin caused the greatest level of necrotic cell death.
Discussion: In recent years, smart nanocarrier systems have emerged as promising strategies for targeted cancer therapy due to their ability to improve drug stability, provide controlled release, and reduce systemic side effects. In the present study, multifunctional nanocapsules composed of cellulose nanocrystals, BSA, PVA, PEG, and hyaluronic acid were designed for the targeted delivery of cisplatin to AGS gastric cancer cells. The obtained results demonstrated that drug-free nanocapsules exhibited negligible cytotoxicity against both AGS and GES-1 cells, indicating the favorable biocompatibility of the synthesized nanocarrier system. This biocompatibility may be attributed to the intrinsic properties of the nanocarrier components, which are unlikely to induce oxidative stress, membrane damage, or mitochondrial dysfunction in healthy cells. Furthermore, PEGylation and hyaluronic acid-mediated targeting reduced nonspecific nanoparticle accumulation in normal cells while improving selective uptake by cancer cells through CD44 receptor interactions. Physicochemical characterization revealed that increasing the concentration of cellulose nanocrystals resulted in larger particle sizes and lower zeta potential values, likely due to the formation of denser polymeric networks and stronger hydrogen-bonding interactions among the nanocarrier components. Nevertheless, the BPC_{(100 mg)}CP-HA formulation exhibited an appropriate particle size distribution and acceptable uniformity for drug delivery applications. Drug release studies further demonstrated that cisplatin release was significantly higher under acidic conditions than at physiological pH, while increasing cellulose nanocrystal concentration reduced the release rate. This behavior was associated with the formation of a more compact polymeric network that restricted drug diffusion and enabled sustained and controlled release. In addition, flow cytometry analysis revealed that the BPC_{(50 mg)}CP-HA nanocapsules induced the highest levels of apoptosis in AGS cells, whereas free cisplatin predominantly caused necrotic cell death. These findings suggest that encapsulation of cisplatin within the designed nanocarrier system not only enhances anticancer selectivity but also promotes apoptosis-mediated cell death, thereby potentially improving the therapeutic efficacy of cisplatin in gastric cancer treatment.
Conclusion: This study demonstrates that smart nanocapsules BPCCP-HA based on cellulose nanocrystals and a BSA-PVA hydrogel provide an effective strategy to overcome the therapeutic limitations of cisplatin. This system enhances physicochemical properties, structural stability, and pH-responsive drug release, thereby improving targeted delivery of the drug to AGS cancer cells. The designed formulation shows good biocompatibility and low toxicity toward healthy cells.