Cell and Tissue Journal

Abstract Aims: Nanoparticles due to their wide applications in medicine,industry,and biotechnology, have attracted many scientists’ attentions. Recently, nanoparticles especially selenium nanoparticles are widely used to diagnosis and cancer treatment. The aim of this study was to evaluate the cytotoxic and anticancer effects of selenium nanoparticles on colon cancer cell line and analysis of CAD (Caspase Activated DNase) gene expression.
Material and methods: In this study, colon cancer HT29 and normal HEK293 cell lines were purchased from the Pasteur Institute Cell Bank of Tehran and treated with selenium nanoparticles overnight. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Scotland) medium with 10% FBS serum and 1% streptomycin antibiotic (Gibco, Scotland). The cells were then stored at 37 ° C. In this study, cytotoxic effect of Selenium NPs was evaluated on HT29 and HEK293 cells using MTT (3-(4, 5-Dimethyltetrazollium Bromide) assay. Subsequently, they were treated with selenium nanoparticles in different concentrations (0, 7.81, 15.62, 31.25, 62.5, 125, 250 and 500 mg/mL) for 24 hours. To solubilize the viable cells formazan crystals production, we added 100 μl/well of dimethyl sulfoxide (DMSO) to them. After treatment of HT29 cells with IC₅₀ concentration, the total RNA was extracted and cDNA synthesized. Moreover, CAD gene expression was evaluated using Real Time PCR method. The data was evaluated by ABI StepOne utilizing the Applied Biosystems qRT-PCR (ABI 7300 system, Applied Biosystems). The quantification of the mode of Selenium NPs -induced cell death in the HT29 cells were ascertained using flow cytometry followed by staining with fluorescein isothiocyanate (FITC)‐Annexin V and propidium iodide (PI) staining. Finally, the study of apoptosis and necrosis of Selenium NPs was evaluated using flow cytometry method. Data analysis was statistically determined by using One-way analysis of variance (ANOVA) with SPSS/22 software followed by a Tukey test.
Results: The result showed that the treatment of Selenium NPs at 31.25 to 500 µg/mL concentration had maximum cytotoxic effect, revealed statistically significant (P˂0.001). The IC₅₀ value for Selenium NPs were measured at 75 µg/mL after 24 hours. In order to determine the effect of Selenium NPs on cancerous cells, alterations in the mRNA expression levels of CAD gene in HT29 cells were done by qRT-PCR technique followed by the exposure to nanoparticle. The CAD gene expression comparing to reference gene was up-regulated 4.04±0.125 fold. To determine the mechanism of cell death in the cancer cells, annexin V/PI flow cytometry was carried out. In the treatment of HT29 cells by IC₅₀ of selenium NPs, 10.43%, and, 24.28% of early and late stages’ apoptosis were observed, respectively
Conclusion: Our results suggest that selenium NPs can display some promising cytotoxic properties through inducing apoptosis pathway. Based on the results, up-regulated gene expression involved in apoptosis (CAD) and activating apoptosis, it can be concluded that the selenium NPs can be used as drug candidate in colon cancer treatment, but more studies are needed regarding the medicinal importance of nanoparticles.

Abstract Aim: Lignocellulosic biomass such as agricultural wastes (corn stover, sugar beet pulp and citrus peel) is a widely abundant and attractive source for the production of biofuels and chemicals.
biofuels are sources of clean and renewable energy that are considered as a potential substitute for non-renewable oil fuels. various methods and processes have been tested by scientists and researchers in this field and the most favorable conditions for producing biofuels from biomass. however, this biomass has not been fully exploited in many parts of the world for biofuel production, especially in developing countries, and there is little relation with crop residues and forest and waste in this area. so much work is still needed to replace fossil fuels with biofuels from biomass. lignocellulosic waste biomass such as cassava peels, sugar beet pulp, and Ulva lactuca are suitable materials for bioethanol synthesis.
D-xylose, is the second most abundant sugar in lignocellulosic hydrolysates. Nowadays, considerable efforts have been made to expand microbial cell factories to use D-xylose for the production of value-added chemicals. D-1,2,4-butanetriol (BT) is an extremely important intermediate chemical, is widely used in many fields, such as pharmaceuticals, paper, polymer materials, and military applications.A molecule 1,2,4-butanteriol (BT) is a polyol with unique chemical properties, which has a stereocenter and can be divided into D-BT (the S-enantiomer) and L-BT(the R-enantiomer). BT is widely used in the military industry, medicine, tobacco, polymer.  A synthetic pathway involving four enzymes—D-xylose dehydrogenase (XDH), D-xylonate dehydratase (XD), 2-keto acid decarboxylase (KDC), and aldehyde reductase (ALR)—has been proposed and implemented to produce BT from D-xylose, highlighting its significant role in bioproduction. And because in most studies, the xylonate dehydratase was used in the case of Caulobacter.crescentus, and given the very high genetic similarity between Caulobacter.crescentus and Caulobacter.vibrioides, the aim of this study is to clone and express xylonate dehydratase gene of C.vibrioides in the E.coli. For this purpose, the research was carried out with the aforementioned methods.
Material and methods:  The xylonate dehydratase gene was retrieved from the NCBI database and amplified using PCR with specific primers after extracting the C. vibrioides genome. The piece of the gene was cloned in the pET28 expression vector and then transferred to the E.coli prepared cells using chemical methods. After the induction of the cells, recombinant protein expression was examined using SDS-PAGE. Results: By using restriction enzymes, Colony PCR and sequencing, the cloning process and the entry of the gene into the pET28 expression vector was confirmed. The presence of the recombinant protein was tested by SDS-PAGE gel with a molecular weight of approximately 68 KDa and the expression rate of the recombinant protein, estimated by Image J software, was 54 percent.
Conclusion: The bioproduction of butanetriol requires the construction of a metabolic pathway consisting of several enzymes. The presence of the bacterium E.coli as the target strain and the use of cheap substrate such as xylose-containing biomass and the existence of the enzyme xylonate dehydratase are essential for the production of high-speed and high-volume D-1,2,4 butanetriol.   

Abstract Aim: Organotypic cultures of spinal cord slices from mammalian neonatal and fetal animals are powerful tools for studies of spinal cord injury, neuronal degeneration, and cell death but also motor neuron regeneration. Models in which adult slices are used would be very useful. However, adult spinal cord slices are notoriously difficult to maintain in culture and rapidly deteriorate in vitro. Degeneration of motor neurons in the spinal cord is a critical phenomenon in spinal cord injuries and certain neurodegenerative diseases such as amyotrophic lateral sclerosis, a neurodegenerative disorder in which motor neurons in the spinal cord and motor cortex are lost. A variety of mechanisms have been proposed as having a role in neuronal apoptosis during spinal cord injury. Oxidative stress has been reported as one of the mechanisms involved in the apoptosis of motor neurons in spinal cord injuries and neurodegenerative diseases. This study was conducted to determine whether quercetin, as a potent antioxidant, can delay apoptosis in motor neurons of cultured spinal cords by reducing oxidative stress.
 Material and methods: The thoracic regions of the spinal cord from adult NMRI mice were sliced using a tissue chopper and divided into three groups: 1) 0-hour, 2) control group, and 3) group treated with quercetin (100 µM). Spinal cord slices in the 2 and 3 groups were incubated for 6 hours at 37°C in a Co₂ incubator. The viability of the spinal cord slices was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The morphological features of apoptosis and the number of motor neurons were examined using Hoechst and propidium iodide staining. The malondialdehyde was measured to determine lipid peroxidation while the FRAP (ferric reducing antioxidant power) was assessed to evaluate total antioxidant capacity in fresh and cultured spinal cord slices. Results were expressed as mean±SD. One-way analysis of variance (ANOVA) followed by Tucky’s test was used to assess the statistical significances of the data. In all cases, a statistical probability of p<0.05 was considered significant.
Results: After 6 hours (control group) in culture, the viability of the spinal cord slices, the neuron diameter, and the number of healthy motor neurons significantly decreased compared to the 0-hour group. Also, motor neurons showed the morphological features of apoptosis including cell shrinkage, nuclear and chromatin condensation in the control group. A significant increase in the amount of malondialdehyde and a significant decrease in the total antioxidant capacity was also observed compared with the 0-hour group. After 6 hours, quercetin not only increased the viability and the number of healthy motor neurons in the cultured slices but also reduced the morphological features of apoptosis in the motor neurons compared with the control group. In addition, quercetin significantly reduced the amount of malondialdehyde and increased the total antioxidant power in slices cultured for 6 hours.
Conclusion: Oxidative stress might be considered as one of the mechanisms involved in the apoptosis of motor neurons in cultured spinal cord slices and quercetin, as a potent antioxidant, was able to increase the viability of the cultured spinal cord slices and delay the morphological features of apoptosis in the motor neurons through reducing lipid peroxidation and increasing the total antioxidant capacity.

Abstract Aim: Zinc nanoparticles have gained significant attention due to their wide-ranging applications in various fields, including industries, medicine, and nutrition. These nanoparticles are characterized by their small size, which allows for easy absorption into biological systems, enabling them to interact with different tissues effectively. One of the critical areas of concern regarding the use of zinc nanoparticles is their potential impact on kidney health. The kidneys are vital organs responsible for filtering blood, excreting waste, and regulating essential bodily functions. Due to their high blood flow and the capacity to excrete various compounds, kidneys may be particularly vulnerable to the effects of toxins, including nanoparticles. Therefore, this study aims to investigate the effects of nano zinc oxide on antioxidant activity and histological changes in the kidneys of rats. Understanding these effects is crucial for assessing the safety and potential therapeutic applications of zinc nanoparticles in medical and nutritional contexts.
Material and Methods: In this research, male Wistar rats were utilized as the experimental model. The rats were randomly divided into six groups, with seven rats in each group to ensure statistical validity. Group 1 served as the normal control group, while the remaining groups received varying doses of nano zinc oxide. Specifically, group 2 received 5 mg/kg of nano zinc, group 3 received 10 mg/kg, group 4 received 25 mg/kg, group 5 received 50 mg/kg, and group 6 received 100 mg/kg of nano zinc. The administration of these doses continued for one month to observe both short-term and potential cumulative effects. After this period, the rats were sacrificed, and their kidneys were isolated for further analysis. Various biochemical assays were conducted to assess total antioxidant capacity (TAC), total oxidant capacity (TOS), glutathione levels, and malondialdehyde (MDA) concentration. Additionally, liver enzymes were measured, and kidney histopathology was examined to evaluate any structural changes resulting from nano zinc exposure.
Results: The findings revealed significant alterations in antioxidant activity among the groups that received nano zinc. Specifically, total antioxidant capacity (TAC) and glutathione levels decreased markedly in the rats that received higher doses of nano zinc, particularly at 10, 25, 50, and 100 mg/kg. In contrast, there was a sharp increase in malondialdehyde (MDA) concentrations and total oxidant capacity (TOS) in these groups, indicating heightened oxidative stress. Interestingly, the group that received the lowest dose of 5 mg/kg of nano zinc exhibited no harmful effects on tissue changes or antioxidant capacity, suggesting a threshold below which nano zinc may not pose significant risks. Conversely, in the groups receiving higher doses, observable tissue damage was noted, and these changes appeared to be dose-dependent, emphasizing the importance of dosage in determining the safety and efficacy of nano zinc.
Conclusion:The results of this study underscore the potential harmful effects of nano zinc, particularly at elevated doses, on kidney function and histopathology. The observed increase in oxidative stress markers and accompanying tissue changes highlight the need for caution in the use of zinc nanoparticles, especially in medical and nutritional applications. While nano zinc in low concentrations may offer beneficial effects, such as potential therapeutic properties, the risks associated with higher doses cannot be overlooked. Further research is warranted to explore the mechanisms underlying these effects and to establish safe dosage guidelines for the use of zinc nanoparticles in various applications. Overall, this study contributes valuable insights into the safety profile of nano zinc, particularly concerning kidney health, and emphasizes the need for careful consideration of dosage in therapeutic contexts.

Abstract Aim: Infertility is a complex issue that affects many couples worldwide. In vitro fertilization (IVF) has been a groundbreaking technique in addressing this challenge, but it comes with its own set of problems in the development of embryos during the pre-implantation stages. Understanding the processes involved in embryo development is crucial in overcoming these hurdles. During the pre-implantation stage, one of the challenges of treating infertility through in vitro fertilization is the developmental arrest of the embryo. In embryonic arrest, cell division stops for at least 24 hours, which, if it occurs in infertility treatments, leads to failure in ART cycles.
Cell signaling pathways play a vital role in the development and progression of embryos. The phosphatidylinositol 3-kinase (PI3K) pathway, known for its efficiency in regulating the cell cycle and various cellular processes, was the aim of this study. By targeting this pathway, we aimed to explore the effects of specific factors - ITS (Insulin, Transferrin, Selenium), Essential E8 medium (E8), and the CHIR99021 small molecule - on the resumption of development in type I arrested human embryos under in vitro conditions.
Material and Methods: In this study, firstly, after receiving ethics approval and patient consent, day 3 embryos (2-3 cell stage) from the Embryology Department of Royan Research Institute were utilized. The experimental groups consisted of control, CHIR99021, ITS, and E8. The optimum concentrations were chosen 1 for CHIR99021, 0.5% for ITS, and 0.1% for E8 + 10% serum. The culture medium for these groups was prepared and covered with liquid paraffin before being incubated at 37°C and 5% CO2 for 4 hours. Subsequently, the embryos were transferred randomly to either experimental or control groups and cultured in an incubator for 48 to 72 hours. Morphological evaluations of the embryos were conducted using an inverted microscope. Data analysis was performed using SPSS software and chi-square test, with a significance threshold set at P<0.05.
Results: The findings of the study revealed that the rate of arrest in the CHIR99021 and ITS groups showed a significant reduction compared to the control group. Moreover, all three experimental groups (ITS, CHIR99021, and E8) exhibited a notable increase in development rate up to the pre-morula stage when compared to the control group. Interestingly, while none of the embryos in the control group progressed to the blastocyst stage, two embryos in each of the CHIR99021 and ITS groups reached this advanced developmental stage.
Conclusion: In conclusion, the study's outcomes indicate the notable effect of ITS and CHIR99021 in modulating the phosphatidylinositol 3-kinase pathway to stimulate cell cycle progression in type I arrested embryos. ITS factor is probably able to regulate this pathway by activating insulin receptors and small molecule CHIR99021 by inhibiting Glycogen synthase kinase 3 (GSK-3). These findings hold promise for further research and potential applications in improving the success rates of IVF treatments and addressing infertility challenges. Understanding the mechanisms of embryo development is crucial for advancing reproductive medicine. It is important to note that the statistical population used in this study was limited and it is believed that more research is needed. 

Abstract Aim: Addiction is an important social and health problem in many Middle Eastern countries. Studies show that opium is the most commonly used substance after tobacco in many countries, especially in Iran. Opium is obtained from the seeds of the Papaver somniferum plant and contains more than 40 different alkaloids, the most important of which are morphine, codeine, papaverine, noscapine, and thebaine. Various studies show that morphine, which is one of the most important substances in opium, increases the production of free radicals in the body and reduces the antioxidant capacity. Opium consumption has various adverse health effects on the body. Therefore, long-term use of this combination can be related to some pathological consequences, including neurological disorders, liver toxicity, kidney dysfunction, oxidative stress, and apoptosis. Most drugs are metabolized by liver hepatocytes and excreted by kidney cells. Therefore, opium consumption can directly cause damage to liver cells. Free radicals play an important role in liver diseases. The increase of free radicals and on the other hand the decrease of antioxidants in the body caused liver toxicity. Oxidative stress is caused by an imbalance between the production of free radicals and their neutralization inside the body by antioxidant defense mechanisms. Oxidative stress is produced in various diseases, consumption of toxic substances, and old age, since it is not possible to investigate the effect of opium on human liver cells, and no study has been conducted in this field. The liver is the most important organ that is directly responsible for the metabolism and excretion of opium. Therefore, in this study, the effects of opium on oxidative stress markers in HepG2 cell line were determined.
Material and methods: Human liver cancer cell lines (HepG2) were used in this experimental study. At first, the cell line was placed in a 25 ml flask in the culture medium. DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum), and 1% penicillin and streptomycin antibiotics were incubated in a 37°C incubator with 5% CO_{2 .} The cells were treated with different concentrations of opium (0-100ug/ml) for 24 hours. Then IC₅₀ was determined and then a lower concentration, IC₅₀, and a higher concentration (three concentrations in total) were used for further studies.
The lipid peroxidation was measured by thiobarbituric acid method. The total oxidant was measured using xylenol orange. The amount of total antioxidants was determined by the FRAP (Ferric Reducing / Antioxidant Power) method. The catalase, glutathione peroxidase, and superoxide dismutase enzyme activities were measured by colorimetry methods. the data was entered into SPSS software (Version 20)  and analyzed using ANOVA and Tukey statistical tests. P less than 0.05 was considered a significant level. Results: Statistical analysis results indicated a significant difference in the amount of oxidative stress factors compared with control (p<0.001). In the group receiving opium with a dose of 60 and 70 mg/ml compared to the control group, the amount of TOS and MDA increased significantly (p<0.001), while the amount of TAC decreased (p<0.001). Also, the activity of catalase, glutathione peroxidase, and superoxide dismutase enzymes decreased in opium-treated groups (p<0.001). Conclusion: The results of this research showed that opium had a lethal effect on HepG2 cells. Also, opium caused an increase in oxidative stress in this cell line, which indicates the vulnerability of the liver against this compound. The results of this study show that although opium has analgesic effects, it can seriously damage the liver tissue.