Cell and Tissue Journal

Abstract Aim: Cell therapy using mesenchymal stem cells (MSCs) can be a promising tool in regenerative medicine. One of the richest sources of mesenchymal stem cells is fetal bone marrow. Silymarin has strong antioxidant and anti-inflammatory activities with a positive effect on the proliferation of some cells as well as anti-osteoporosis properties. This study aimed to show the effect of silymarin on the differentiation of mesenchymal stem cells derived from the bone marrow of sheep embryos into the osteogenic line.
Materials and Methods: Mesenchymal stem cells were isolated from the bone marrow of sheep embryos. MTT test was performed to investigate the cytotoxicity of silymarin on cells at different concentrations for 24 and 72 hours. Then, cells in one of 8 groups 1: negative control; 2: treated with 10 μmol/liter silymarin in the usual environment, 3: treated with 20 μmol/liter silymarin in the usual environment, 4: treated with 100 μmol/liter estradiol in the usual environment, 5: positive control, 6: treatment treated with 10 μmol/liter silymarin in the differentiation medium, 7: treated with 20 μmol/liter silymarin in the differentiation medium, 8: treated with 100 μmol/liter in the differentiation medium, were cultured for 21 days. To determine the osteogenic differentiation of cells, the deposition of hydroxyapatite ions was examined using alizarin staining, and also, the amount of ALP enzyme secretion was also measured in the studied groups. Results: Comparing the average optical absorption of cells at different concentrations between 24 and 72 hours after treatment showed that the average optical absorption of cells at zero concentration of silymarin after 72 hours of treatment decreased in comparison with those treated for 24 hours (P<0.05), but no significant difference was observed in other concentrations (P>0.05). Examining the level of ALP enzyme secretion, 21 days after treatment with silymarin in the studied groups showed that the highest level of enzyme secretion was in group 8 (P≤0.05). The lowest amount of enzyme secretion was observed in group 1 (negative control) and then in group 2 and group 3 respectively (P<0.05). No significant difference was observed between groups 4, 5, and 6 (P>0.05). Based on the alizarin red staining results, calcium ions deposition was observed in all the groups related to the differentiation medium, which increased in groups 8, 7, 6, and 5, respectively. In the groups cultured in the usual environment, there was no calcification in group 1 and the amount of calcification increased in groups 2, 3, and 4, respectively. In total, the amount of calcification in the differentiation environment groups was higher in comparison with the usual environment.
Conclusion: During this study, Silymarin had no toxic effect on the mesenchymal stem cells derived from the bone marrow of sheep embryos in the studied concentrations after 24 and 72 hours of treatment. It increased the differentiation of the cells into the osteogenic lineage in a concentration-dependent manner. Therefore, it seems that with further studies and identification of the molecular pathways of silymarin's effect, it can be used in cell therapy in order to repair bone lesions.

Abstract The electrical functions of the organs of the body, such as the nervous system and the bone marrow, has led to the use of one of the most widely used therapies called electrotherapy, especially in relieving pain. On the other hand, advancement of medical science in the field of stem cells and regenerative medicine has made many perspective in treatment. Electric field-based therapies have recently been widely used in the treatment of cancer. The main issues in regenerative medicine are the proliferation of stem cells to the required extent and their guidance towards differentiation into the target tissue. Electric field stimulation (EF) can also play an important role in generating appropriate stem cell responses and guiding stem cell differentiation through osteogenesis/neurogenesis/cardiomyogenesis. Nanosecond pulsed electric field as well as the tumor treating field have attracted a lot of attention today for the treatment of cancer. Major signaling pathways and cellular responses elicited by electrical stimulation are included reactive oxygen species and heat shock proteins, intracellular calcium ion fluctuation, so ATP production, clustering or re-accumulation of cell surface receptors, Skeletal regeneration and so on that they can affect the stem cell fate. Also, none invasive, ease of usage, and reasonable price have caused that the treatment of cancer with an electric field to be increasingly used. This study seeks to provide a brief overview of the effects of electrical signals on the behavior of stem cells, as well as examples of their therapeutic effects in the treatment of tissue lesions and cancer.
 

Abstract Aim: In this review article, we intend to investigate and review the factors that lead to embryonic developmental arrest and explore different strategies to address this phenomenon. We aim to pave the way for further research in this field and enhance the efficiency of the IVF method for infertility treatment.
 
Introduction: To ensure proper embryonic development, embryos need to progress through their developmental stages unimpeded. Any disruptions in these stages including oocyte factors, sperm factors, and embryonic factors can result in infertility due to pre-implantation developmental arrest. Therefore, it can be very helpful to understand the effective factors behind this phenomenon.
 
Topic: Among couples attempting to conceive, infertility persists despite their best efforts. Couples are typically considered infertile if they have tried to conceive for twelve months without success and without using protection; but if the woman is over 37 years old, this twelve months will be reduced to six months. Assisted reproductive techniques (ART) encompass various methods that are aimed at aiding these couples in overcoming infertility. This definition includes any manipulation of embryos and eggs for infertility treatment. Assisted reproductive techniques include in vitro maturation (IVM), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Among these, IVF/ICSI is particularly effective in treating couples infertility. However, one common challenge in this process is embryonic developmental arrest during the pre-implantation stages, a primary cause of infertility in treatment cycles. Approximately 40 to 50 percent of IVF cycles do not progress to the blastocyst stage and arrest. This arrest characterized by a lack of cell division for at least 24 hours. Most embryonic arrests occur on the second and third day after fertilization, during the two to eight-cell stages. These arrested embryos retain their developmental potential, and their gene expression program related to their arrested stage remains unaffected. Arrested embryos can be classified into three categories: those with impaired activation of the embryonic genome (EGA), those with low levels of glycolysis, and those with variable levels of oxidative phosphorylation. Solutions to overcome embryonic developmental arrest include maternal spindle transfer (MST)/nuclear transfer (NT), optimizing culture media, employing antioxidants, and synthesizing complementary RNA (cRNA)/small interfering RNA (siRNA).
 
Conclusion: Pre-implantation arrest can result from various factors, either of embryonic, maternal, and paternal origin. Among embryonic factors, disturbances in fields like gene expression, mitochondrial activity, methylation patterns, chromosomal abnormalities, small non-coding RNAs, and embryonic metabolic status have the most significant impact on embryonic arrest induction. Additionally, parental factors, like genetic factors, and infertility etiology can lead to embryonic developmental arrest. External factors, such as laboratory conditions, ART methods, and the role of the physician, also play a role in embryonic developmental arrest. While treatment studies for overcoming embryonic arrest are very limited, they are generally based on animal models, which is why it is necessary to conduct more studies and enter the human phase. A comprehensive understanding of the causes of embryonic arrest and solutions to address them can enhance infertility treatment technologies and improve the effectiveness of the IVF method for infertility treatment.

Abstract

Abstract Aim: Red blood cells and white blood cells are the main  cells of blood.These two types of cells have significant differences in number, nucleus, and other intracellular organelles such as mitochondria, ribosomes, and metabolic pathways. Enzymes are important proteins found in these cells . MAPKinase (Mitogen activated protein kinase) superfamily are protein kinases playing key role in phosphorylation of threonine , thyrosine and serine in the enzymes of the family and target proteins during kinase cascades  in metabolic pathways  of cells. They are found in nucleated cells from unicellular to multicellular. These enzymes have important roles in regulating various processes of eukaryotic cells, including cell proliferation, differentiation, survival , apoptosis  and  in various signaling pathways and gene expression as well. These enzymes are ubiquitously expressed and evolutionarily conserved in eukaryotes. In mammalian cells, there are three well-known MAPK   including  extracellular signal-regulated protein kinase (ERK) 1/2 , c-Jun N-terminal Kinase 1, 2, 3 (JNK1/2/3) and p38 MAPK α,β,δ,γ
ERK, JNK and p38 isoforms are grouped according to their motif, structure and function.  ERK 1/2 is related to  the response to growth factors, hormones and inflammatory stimuli, while JNK1/2/3 and p38 MAPK α, β, δ, and γ are activated through environmental or cellular stress and inflammatory stimuli.
JNK   enzyme is activated under stress. JNK pathway has role in apoptosis and cell survival. The presence of JNK is essential for stress-induced mitochondrial cytochrome c release. Cytochrome c together with Apaf activates the initiator caspase 9. If the defect in the release of cytochrome c from the mitochondrial membrane is likely to cause a defect in the release of pro-apoptotic molecules such Smac/DIABLO, AIF. The aim of the study is to investigate the JNK enzyme level in blood anucleated cell such as RBC compared to nucleated cell like WBC. 
Materials and Methods: RBCs were isolated from a fresh blood. WBCs (Mononuclear) were separated from blood using Ficoll solution.  Their suspensions were prepared in isotonic condition using 0.9 % NaCl. In next step, the number of cells counted by means of cell counter followed by lysis  RBCs by ultrasonic  homogenizer and  lysis of WBCs using  ultrasonic bath. Considering that, the presence of hemoglobin following the lysis of RBCs affects the assay of JNK level by ELISA immunoassay technique, hence 6 mM zinc sulfate used to remove the hemoglobin. Two kinds of lysates were centrifuged to separate the  lysed cells membranes before assay the level of JNK.  Then, the level of JNK in the RBC and WBC lysates were measured using ELISA technique.
Results: Regarding that the number of RBCs in sample was 1000 times more than WBCs one per sample volume, but the JNK enzyme level showing  1.72 x10 ^-2  ng/ml per cell and 6.2 x 10 ^-6  ng/ml per cell in WBCs and RBCs  respectively. As a result, JNK enzyme level in each WBC   is 2770 fold   more than each RBC.
Conclusion: In comparison with WBCs having nuclei and high level of   JNK enzyme,  RBCs due to losing their nuclei during differentiation from stem cells in bone marrow show low level of JNK enzyme denoting blocking of pathways related to MAPK enzymes. This is an evidence that the absence of nucleus does not support of MAPK family enzyme and related pathways.

Abstract Aim: In the past decades, many efforts have been made with the aim of searching for new tools to treat cancer. In this regard, the discovery, investigation and application of techniques related to small interfering RNAs (siRNA) has been one of the most significant advances in the field of cancer detection and treatment. Small interfering RNA, sometimes known as short interfering RNA or silencing RNA, is usually 21 bp long and interferes with the expression of specific genes with complementary nucleotide sequences and prevents translation by degrading mRNA after transcription. Many studies have shown that siRNAs affect the regulation of the expression of some genes that play a role in cancers. siRNAs are effective on Snail transcription factors, which play an important role in the invasion and metastasis of cancer cells, and miR-143, which plays an important role in the pathogenesis of cancers. miRNAs together with transcription factors can disrupt the biological pathways involved in carcinogenesis. However, the exact effect of siRNA on the expression of snail1 and miRNA-143 genes in breast cancer cells is not completely clear. Based on this, the present study investigated the effects of siRNA on snail1 and miRNA-143 on breast cancer cells.Material and Methods were purchased from Pasteur Institute of Iran. The cells were cultured in RPMI-1640 medium containing 10% FBS. Snail1 gene kit (Santacruz biotechnology, California, USA) was used to treat cancer cells with specific siRNA. The cells were divided into two groups: control (no treatment) and treated cells (transfected with siRNA). In order to determine the effective time, the cells were exposed to a dose of 60 picomoles of siRNA for 24, 48 and 72 hours. Beta actin gene was used as internal control gene. Morphology of MDA-MB-468 metastatic cells were examined using light microscopy before and after specific gene transfection. Cell proliferation was checked by trypan blue staining. Snail1 and miR-143 gene expression levels were evaluated by qRT-PCR. Data were analyzed using t-test. Results: In this study, in MDA-MB-468 breast cancer cells, the relative level of Snail1 gene expression was significantly decreased in the effective time of 48 hours and when exposed to the effective dose of 60 pmol (P < 0.0001). However, the knockdown of Snail1 gene by specific siRNA in MDA-MB-468 cancer cells when exposed to an effective dose of 60 pmol and an effective time of 48 hours caused an increase in the relative expression level of miR-143 gene compared to the control group (P < 0.0001). Also, the growth rate of MDA-MB-468 cancer cells decreased with Snail1 gene knockdown. Conclusion: The results of this research showed that the transfection of MDA-MB-468 breast cancer cells by specific siRNA can successfully reduce the expression level of Snail1 gene and miR-143 gene. Proliferation and invasion of breast cancer cells.

Abstract Aim: Spermatogonial stem cells (SSCs) are the basis of male spermatogenesis and fertility. SSCs are distinguished by their ability to self-renew and differentiate into spermatozoa throughout the male reproductive life and pass genetic information to the next generation. Immunohistochemical analysis showed PLZF-positive cells in the basement membrane of the seminiferous tubule. It seems that the PLZF germ cell marker is specifically expressed in the spermatogonial cells of the testis. In mice with genetic deletion of the VASA gene, males show reproductive deficiency with reduced sperm production. In this study, we looked at the expression of PLZF and VASA in spermatogenic tubules and germ cells in vivo and in vitro. Material and method: Isolation of spermatogonial stem cells, Cultivation of testis on STO feeder layer, Immunohistochemistry (IHC), Immunocytochemistry (ICC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR), Network analysis of protein-protein interactions (PPI), Pathway enrichment analysis and gene analysis (GO), were used to analyze the expression of PLZF and VASA in mice testis tissue. Results: In this experimental study, whereas undifferentiated spermatogonial cells sharply express PLZF, other types of germ cells located in the seminefrous tubul were negative for this marker. In other hand, the germ cells near the basal membrane of seminefrous tubul showed expression of VASA wheras the undifferentiated germ cells located on the basal membrane were negative. The ICC analysis indicated higher expression of PLZF in the isolated undifferetiated cells in compare to the differentiated germ cells. Fluidigm real-time RT-PCR result demonstrated a significant expression (P<0.05) of VASA in the spermatogonial stem cells compared to differentiated cells and also showed expression of PLZF in undifferentiated spermatogonia. In the present experiment, after the production of SSCs under the stimulation of growth factors FGF, EGF, and GDNF, immunocytochemical staining showed a clear expression of PLZF and VASA in SSCs compared to in-vivo conditions, and VASA is less expressed in these cells. The data obtained from IHC analysis showed that VASA is expressed in the center of testicular cords. The data set related to protein-protein interaction members was used in this research due to the lack of information about PLZF expression in different stages of spermatogenesis. The study of gene expression showed that several biological and functional pathways are involved in the expression of PLZF in different stages of spermatogenesis. Conclusion: These results clearly proved the role of PLZF as a specific marker for spermatogonial stem cells, and can be beneficial for advance reserach about in-vitro differentiation of SSCs to functional sperms.

Abstract Aim: In current study was aimed to investigate effects of different concentrations Genestein on osteogenic differentiation and proliferation of adipose driven mesenchymal stem cells (ADMSCs) from Turkman horses.
Material and Methods: In this experimental study, ADMSCs were treated with 10^-8,10^-7,10^-6,10^-5 μM concentrations of Genestein with osteogenic medium. The proliferation and cell viability of Genestein was evaluated by MTT test for 7 days after cell culture, and the role of It on osteogenic differentiation was investigated by alizarin red staining, alkaline phosphatase assay and calcium content for 21 days. The data were analyzed with One-way ANOVA.
Results: The results of MTT test showed after treatment of cells by Genestein, it had no toxic effect (p < 0.05). The activity of alkaline phosphatase in 10-5 concentration treated   cells was higher than the other which attributed to the end of differentiation process (21th day) (p < 0.05). The results of alizarin red staining and calcium content showed that the maximum level of Genestein has significantly increase of precipitation of mineral ions on extracellular matrix on 14th and 21st days compare other concertation.
Conclusion: According to the results of this study, Genestein promoted differentiation of these cells in to osteoblast cells without any toxicity effect.
 

Abstract

Abstract In daily life, stress in one of the important and potent modulators of behaviour. Inhibitory or faciliatory effects of acute and chronic stress exposure on memory performance (acquisition, consolidation and retrieval) have shown in previous researches. Under such circumstances, the levels of (nor) epinephrine (NE) rapidly increases in the memory related area including hippocampus and amygdala. Along with NE, the hypothalamic-pituitary-adrenocortical axis activates. Glocorticoids (GCs) hormones are the main end-products of the HPA axis activation. In different animal models have been shown that NE and glocorticoids mediate the modulatory effect of stress on memory. Microglia that originally form in the yolk sac are immune cells in the central nervous system and act as the brain's first line of cellular defense against various pathogens. These cells release inflammatory mediators and neurotrophic factors and also phagocytes cellular debris. In addition, are also shown to play a role in the development of brain. During embryonic development, microglia remove apoptotic cells and regulate synaptic pruning. These cells play an essential role in regulating of synapse regeneration, neurogenesis, synaptic function, angiogenesis and myelination. They are dynamic cells in the adult brain and have the ability to rapidly change their morphology to properly respond to the functional needs of the brain. Microglia is activated in M1 and M2 phenotype. M1 microglia activation is induced by gamma interferon and LPS and promotes inflammation via release of inflammatory mediators such as tumor necrosis factor alpha (αTNF) and interleukins. M2 activation mainly is related to secretion of glucocorticoids, extracellular matrix proteins and anti-inflammatory cytokines. It has been reported that microglia as a key regulator of neuronal function have NE and GCs receptors, suggesting a critical role of these brain cells in modulating stress effects. Several lines of studies indicates that microglia regulate learning and memory via the formation and stability of synapses. Microglia actively contribute in synaptic pruning via classical complement cascade mechanism. Apoptotic, immature or poorly growing synapses are labeled with complement components, C1q and C3. Microglia recognize these complement components through the complement receptor CR3 and eliminate C1q and C3-labeled synapses. Microglia also detect and remove inactive synapses by the triggering receptor expressed on myeloid cells 2 (TREM2) consequently regulate brain connectivity and activity. Moreover, microglia regulatory negative feedback mechanism prevents neuron hyperactivity. Microglia play an important role in the stability of long-term potentiation. In addition, microglial fractalkine signaling is potentially involved in LTD. The number and morphology of hippocampal microglia is altered in response to chronic stress exposure thus consequently becomes reactive phenotype. This effect is mediated via stress hormones. Evidence show that stress also affect expression of microglial genes (cytokines, TNF-α and interleukins) that have regulatory role in learning and memory. Microglial–neuronal crosstalk which is crucial for memory processing is another site for stress-induced memory changes. Moreover, stress exposure alters glutamate transmission through negative effect on kynurenine pathway. These effects support the involvement of microglia in destructive effect of stress on memory. In this review article, focusing on newly published articles, we examine the role of microglia in synaptic plasticity, learning and memory, and especially the role of activated microglia in the effects of stress on learning and memory. By examining these processes, our aim is to provide an overview of the role of microglia in synaptic plasticity and learning and memory, and the possibility of using microglia targeting as a therapeutic method to improve cognitive deficits associated with stressful conditions

Abstract Introduction: Heavy metal refers to any metal with relatively high density, with toxic effects even at ppb levels. Heavy metals are one of the main threats to human health. Arsenic (As) is a heavy metal found in our living environment due to its widespread use in industrial activities. Exposure to As and its absorption by different tissues may lead to damage to cardiovascular function, skin lesions, high blood pressure, and diabetes. As harmful effects on chromosome integrity have been proven a long time ago. It has also been classified as a potent carcinogen by the Environmental Protection Agency (EPA) and the International Agency for Research on Cancer (IARC).  One of the damages caused by As is chromosome breakages. These damages are the result of this heavy metal's ability in producing free radicals and their interaction with the DNA molecule. The role of aneuploidy in cancer induction has been suggested in several investigations. Aneuploidy can lead to drastic changes in the genetic balance of normal cells, potentially forcing them towards cancer formation.
Aims: Due to the close relation between chromosome instability and cancer induction and the ability of As to induce cancer, the question arises whether As can also play a role in chromosome instability? The design of this study is based on answering this question.
Materials and methods: In this study, to analyze potential cell toxicity and chromosome abnormalities induced in different treatments, MTT and Micronucleus assay on Binucleated cells were used, respectively. Cell toxicity of different doses of As was investigated on HDF cells. According to the results of MTT, three non-toxic doses of 100, 200, and 300 ng/ml were selected. To be able to understand the effect of As treatment on chromosome instability induced by aneugenic agent, vinblastine was used. Vinblastine is classified as an aneugen in several studies. Co-treatment of three doses of As and three doses of 0.75, 1.00, and 2.00 ng/ml of vinblastine (Vin) on the induction of chromosome abnormalities was investigated on the HDF cell line using the Micronucleus assay on Binucleated cells.
Results: According to the results of the MTT assay, cell toxicity induced by As at 24, 48, and 72 hours was at doses of 604.3, 397.3, and 228.6, respectively.   As also led to chromosomal abnormalities. Mean frequency of micronucleated binucleates (BiMn) was 0.296, 0497, and 7.270 for 100, 200, and 300 ng/ml, respectively, compared to the frequency of BiMn in the control (0.078). Vinblastine treatment also significantly increased the frequency of BiMn in all doses used compared to the control. Finally, Co-treatment with three doses of vinblastine and three doses of As significantly increased chromosomal abnormalities to almost three to seven times compared to solo vinblastine treatment.
Discussion: Results indicate that As can induce chromosomal abnormalities, which is represented by an increase in BiMn frequency. In addition, co-treatment with vinblastine and As leads to higher chromosomal abnormalities compared to solo treatment with vinblastine. Significant increase in frequency of BiMn in the later experiment suggests that As provides a suitable ground for chromosomal abnormalities induced by other aneugenic agents, here, vinblastine. This effect might be the result of direct interference of As with mechanisms of cell division control or inducing mutation in genes involved in such mechanisms through oxidative species, which leads to an increase in their errors. In both cases, the results of this study reveal an alternative mechanism for As-induced tumor formation.
Conclusion: As not only involves cancer formation by direct damage to chromosomes but also provides a suitable ground for chromosomal mis-segregation in cell divisions, which is believed to be a main step towards cancer formation.  

Abstract

Abstract Aim: b-Catenin is a known proto-oncoprotein which its upregulation is involved in tumorigenesis of several human cancers including colorectal cancer, melanoma, pancreatic, ovarian and many other human malignancies. It is believed that this protein is a very potential target for cancer prevention and therapy. Although b-Catenin was originally identified as a component of the canonical Wnt signaling, there are many reports that this protein can also be regulated by several other signaling pathways including those through receptor tyrosine kinases, PI3-kinses and heterotrimeric G-proteins. It is quite likely that regulation of b-Catenin by multiple signaling pathways is a result of the cross-talk among these pathways. We and others have already provided evidence that activation of almost all members of the Ga subunits of heteritrimeric G-proteins can regulate b-Catenin via different mechanisms. For example, by using several cell systems, we have shown that activation of the Gaq class of Ga proteins leads to inhibition of GSK-3b and cellular accumulation of b-Catenin, suggesting that Gaq signaling may somehow positively regulate the canonical Wnt signaling. We have also confirmed the positive role of Gaq on b-Catenin-mediated signaling by using a couple of specific Gaq blocking peptides. Receptors for G-proteins (G protein-coupled receptors, GPCRs) are the most diverse family of proteins in mammals which are involved in many critical cellular processes. Around one thousand different GPCRs are encoded by human genome and more than 30% of the approved therapeutic drugs, target these receptors. Calcitonin receptor (CTR) is a member of the GPCR family (B1 subfamily) which is shown to couple to Gaq or Gas containing trimeric G proteins involved in primarily activation of phospholipase Cb1 and adenylate cyclase respectively. The known ligand for Calcitonin receptor is a 32 amino acid peptide called Calcitonin which is produced by several tissues in the body including thyroid, prostate, and central nervous system. CTR-mediated signaling has very important biological roles in many tissues including bones in which this signaling pathway is involved in maturation of osteoclasts and bone homeostasis. Calcitonin receptor is a GPCR family member which functions through activation of Gs and Gq trimeric G proteins and therefore, in this study we have examined whether activation of this receptor has any effect on b-Catenin transcriptional activity by measuring the expression of several b-Catenin-target genes including a reporter luciferase gene harboring several b-Catenin/T-cell factor recognition elements.
Material and Methods: HEK293T cell culture and transfection with appropriate genetic constructs plus gene expression assays at the level of transcription including quantitative RT-PCR and real-time PCR have been used in this study.
Results: The results showed that cellular activation of Calcitonin receptor increases the expression of several b-Catenin-responsive genes. Expression of the reporter luciferase, CCND1 (the Cyclin D1 encoding gene), c-MYC, and FGF-20 (Fibroblast growth factor-20) was increased upon the expression of Calcitonin receptor in HEK293T cells or treatment of these cells with Calcitonin.
Conclusion: The Calcitonin receptor-mediated signaling may activate b-Catenin and (or) the Wnt/b-Catenin pathway. Further investigations are required to find out the exact mechanism of regulation of the Wnt/b-Catenin pathway by Calcitonin receptor-mediated signaling. Since b-Catenin is a potential oncogene in human cancers, Calcitonin receptor and its signaling partners can be considered for clinical studies.
 

Abstract Aim: The effect of  low- frequency electromagnetic waves on motor activity and brain tissue motor area in rats was investigated.
Material and Methods: In this experimental study, 20 rats were randomly divided into four groups. The first group was designated as the control group, and experimental groups received 900, 1800, and 2450 MHz waves, with an intensity of 2, 1, and 20 watts per kilogram, 4 hours per day for one month respectively. Behavioral evaluation of open field test and histomorphometric evaluation of cortical thickness and count of internal pyramidal cells were determined.
Results: The results showed that low- frequency electromagnetic waves significantly reduced the distance traveled and the time spent in the central open field test area of the experimental groups compared to the control group (p ˂ 0.05). The number of neurons in the inner pyramidal layer and the thickness of the cortical thickness of the frontal lobe showed a significant decrease in the experimental groups compared to the control group (p ˂ 0.05).
Conclusion: Low- frequency electromagnetic waves increased anxiety and decreased motor activity in the behavioral tests with a histomorphometric change of frontal cortex in the experimental groups.
 

Abstract Intruduction: Colorectal adenocarcinoma, a frequent malignancy of the large intestine, is often treated with chemotherapy. However, the significant toxicity of these drugs to healthy tissues poses a major challenge in achieving successful cancer treatment. Cell culture has enabled the development of in vitro models for drug and anti-cancer compound testing. However, traditional 2D monolayer cultures have limitations in studying cancer biology. To more accurately recapitulate in vivo cellular behavior, cells are now routinely cultured in three-dimensional environments, which closely resemble the native tissue in terms of morphology and gene expression profiles. Electrospun nanofibers, with their high surface area and porosity, closely mimic the extracellular matrix and promote cell-cell interactions. These scaffolds have revolutionized 3D cell culture, providing a powerful tool for evaluating anticancer drugs in a more physiologically relevant environment. In recent years, much research has focused on herbal medicines as potent drug candidates for inducing apoptosis. Terminalia chebula is a medicinal plant with a wide array of therapeutic applications, including anticancer effects. Although numerous studies have reported the growth of healthy tissues on nanofiber scaffolds, there is limited information regarding the growth of tumor cells on these scaffolds.
Aim: This study investigated the anti-proliferative and apoptotic effects of Terminalia chebula extract on HT-29 colorectal cancer cells cultured in a PCL/Gelatin nanofiber scaffold model.
Materials and Methods: Colorectal cancer cells (HT-29 cell line) were cultured in both conventional (2D) and nanofiber scaffold (3D) culture conditions. After 24 hours, the cells were treated with 10, 100, 200, and 500 μg/mL concentrations of the extract for 24 hours. Subsequently, the MTT assay was performed to determine cell viability and IC50. In addition, cells treated with the IC50 concentration were stained with acridine orange/ethidium bromide. The morphology of the scaffold and cell adhesion on it were also examined using scanning electron microscopy. The obtained data were statistically analyzed using SPSS software, one-way ANOVA, and Tukey's post-hoc test.
Results: Our findings indicated that Scanning electron microscopy images revealed that the 3D nanofibrous scaffolds, both cell-free and seeded with HT-29 cells, exhibited desirable mechanical properties and adequate porosity. The cells cultured on these scaffolds demonstrated robust growth, proliferation, and a 3D morphology closely resembling in vivo conditions, indicating successful cell-scaffold interactions.  The MTT assay revealed that cell viability decreased in a concentration-dependent manner following 24 hours of treatment with the extract.  The IC50 of the extract was 500 μg/mL in 2D culture and 200 μg/mL in 3D culture after 24 hours of treatment. Based on the morphological assessment using acridine orange/ethidium bromide staining, it was determined that treatment with the extract induced apoptosis in HT-29 cancer cell line.
Conclusion: Collectively, the findings of this study indicated that the hydroalcoholic extract of Terminalia chebula exhibits concentration-dependent cytotoxicity against cancer cells, with enhanced efficacy in a 3D culture model. This highlights the advantages of using nanofiber scaffolds to mimic the in vivo tumor microenvironment and to better understand the mechanisms of cancer progression. Future studies should further explore the potential of this model for drug discovery and mechanistic investigations.

Abstract

Abstract Aim: Plastics, as diverse polymeric materials, have become an integral part of modern life due to their unique properties, including high resistance to various chemicals, a high strength-to-weight ratio, ease of preparation, and low cost. Millions of tons of these materials are produced annually; however, only a small portion is recycled or reused. By leaving single-use plastics in the environment, these plastics are worn out and fragmented into smaller sizes due to various factors such as UV irradiation and oxidation and finally enter the aquatic and terrestrial ecosystems and based on their size are called Micro and Nanoplastics and may be uptaken by aquatic organisms such as fishes and also shrimps and accumulate in their tissues. They can also be absorbed by the roots of plants and enter the food chain of us humans. Although various in vitro and in vivo studies have been conducted to investigate the different effects of various nanoplastics, and various mechanisms such as oxidative stress and inflammation have been reported for their effects, but subchronic studies have been conducted to investigate the effects of polyethylene terephthalate nanoparticles. It is very important on the tissue of the heart in the body. As a result, it seems important to investigate their toxic effects in vital tissues such as heart tissue.
Material and methods: In this experimental study, polyethylene terephthalate Nanoplastics were prepared using aqueous solution and then characterized using Dynamic Light Scattering and Scanning Electronic Microscopy. Four weeks old 12 male wistar rats (Average weight=250 gr) were randomly divided into two groups. Animals in the treatment group received 200 ppm of polyethylene Terephthalate Nanoplastics by gavage daily for 90 days and Control group received only Phosphate Buffer Saline (pH=7.4). After this period, the rats were anesthetized and after collecting the serum, Lactate Dehydrogenase and Creatine Kinase activity levels were measured using commercial colorimetric kit. The level of Lipid Peroxidation was analyzed using Thiobarbituric acid reactive substance formation colorimetric assay, Total Antioxidant Capacity was assessed using Ferric reducing antioxidant power and Catalase Enzyme Activity were also measured in the heart tissue homogenate using specific kit, the results data were analyzed by one-way analysis of variance. Histopathological studies were also performed by hematoxylin and eosin staining using light microscopy.
 Results: The results demonstrated that long term exposure to polyethylene Terephthalate Nanoplastics caused a significant increase in the levels of Lactate Dehydrogenase and Creatine Kinase. Also, an increase in Lipid Peroxidation, and a decrement in Total Antioxidant Capacity, Catalase Activity were observed in the heart tissue of treatment group animals compared to the control group (P value < 0.05). In histological studies, intermuscular edema and degeneration of myofibrils were evident in the group that received Nanoplastics.
 Conclusion: Long-term exposure to polyethylene terephthalate nanoplastics causes a change in the redox state of the heart tissue of male Wistar rats, these nanoparticles cause the cardiac antioxidant enzymatic and non-enzymatic defense mechanisms to be overwhelmed, such as reducing the total antioxidant capacity and catalase.  which has finally led to irreversible damage to the cardiac tissue, which was confirmed by increasing serum levels of creatine kinase and lactate dehydrogenase enzymes and intramuscular edema and degeneration of myofibrils.
 

Abstract Aim: This study aimed to investigate the relationship between the expression of the antioxidant gene of the nuclear factor Erythroid 2 related to factor 2 (Nrf2) and sperm quality in asthenoteratozoospermia men.
Material and Methods: The study was conducted at the University Jihad Infertility Treatment Center in Qom, Iran. In this study, 50 infertile with asthenoteratozoospermia and 50 fertile individuals were enrolled as the control group. Sperm parameters were evaluated according to WHO (2010). Sperm DNA fragmentation, Nrf2 gene expression, and antioxidant enzyme levels were assessed with TUNEL, RT-PCR, and ELISA kits, respectively. The significance level was considered to be p < 0.05.
Results: The results showed that the expression level of the NRF2 gene in patients with asthenoteratozoospermia was lower than the control group (P <0.05). The quality of sperm parameters and the level of antioxidant enzymes were lower than the control group (P <0.05). A Significant association was observed between gene expression of Nrf2 gene, sperm parameters and levels of antioxidant enzymes (p < 0.05).
Conclusion: Our findings show that mRNA NRF2 expression is significantly associated with the quality of sperm parameters in asthenoteratozoospermia men. This suggests that NRF2 is important for spermatogenesis and may serve as a useful indicator in the diagnosis of male infertility.
 

Abstract Aim: The chlorophyll biosynthesis pathway is a main target for genetic modification to change plant photosynthesis and growth rate to support a greater demand for food in the growing world population. In this study, the effect of overexpression of GSA gene one of gene involved in biosynthesis pathway of chlorophyll on physiological condition of tobacco plant was investigated. 5-Aminolevulinate (ALA) is product of GSA gene. ALA is a precursor for all tetrapyrrole, these components have impotent roles in living cell, as pigments, light receptor (Phytochrome), prosthetic group of many different proteins (like cytochromes, hemoglobin, myoglobin, and leghemoglobin) and enzymes (for example Catalase, Ascorbate, Peroxidase and etc.). Nowadays, ALA has received wide attention for its widespread usage in agriculture, forestry and medication. ALA at low concentrations increases photosynthesis, growth, development, yield and productivity, also promoted fruit color appearance and quality and taste of products in treated plants under both normal and stressful conditions. ALA also improves antioxidant features, absorption of nutrient, water use efficiency and osmotic balance in plants.
Materials and methods: In this research, according to bioinformatics studies, MsGSA gene cDNA of Alfalfa (Medicago sativa L. cv. Isfahani) was selected to transfer to Xanthi tobacco (Nicotiana tabacum) plant, the binary expression vector pBI121 which has Kanamycin antibiotic resistance gene for selection in bacteria and plants, cutting sites for SacI and BamHI enzyme, CaMV35S promoter (cauliflower mosaic virus promoter), nos transcription termination sequences and ß-glucuronidase (GUS) reporter gene was used. After constructing the gene construct pBI121-GSA and confirming the transfer of the construct using PCR cloning methods, enzymatic digestion and sequencing were performed, then the corresponding construct was transferred to Agrobacterium tumefaciens strain LB4404 using Agrobacterium with the gene construct. The corresponding gene was transferred to the tobacco plant genome and the transgenic plants were selected on the medium containing kanamycin and the presence of the gene was confirmed by performing PCR in the regenerated plants. The rooted transgenic sprouts were transferred to the soil. The level of GSA gene expression in the resulting transgenic plants was evaluated by real-time PCR, and their growth rate and biochemical content were also evaluated.
Results: It was observed that the growth of transgenic plants increased significantly depending on the level of GSA gene expression, and the content of ALA (aminolevulinic acid) and chlorophyll a, b and total chlorophyll, which are the products of the corresponding gene expression. The results also showed the content of anthocyanin, flavonoids and phenol of plants have significantly increased in proportion to the increase in GSA gene expression compared to wild type tobacco plants.
Conclusion: The growth rate as well as the content of chlorophyll a, b, total chlorophyll, ALA, anthocyanin, flavonoids and phenol of transgenic GSA plants is proportional to the increase in the expression of the GSA gene. The results from this study indicate that an increase in transgenic growth rate as well as an increase in the secondary metabolites content in transgenic plants were influenced by GSA transferred gene and an increase in the content of ALA. These results imply that transgenic tobacco plants expressing MsGSA gene had higher resistance potential to stresses than the wild type tobacco plants.

Abstract Introduction: Colorectal cancer (CRC) is the second most commonly diagnosed malignant tumor in the world, with about 1,926,425 new cases estimated for 2022. The most therapeutic approaches for CRC include surgery, chemotherapy, targeted medicine, and radiation therapy. Each of these treatments exerts side effects such as fatigue, constipation, loss of appetite, and low blood cell counts. Natural products have provided valuable anticancer effects for years. An increasing number of studies have elucidated that plant-based natural compounds can act as an alternative chemotherapy option against CRC. It has been demonstrated that herbal bioactive compounds such as flavonoids, alkaloids, peptides, terpenoids, and steroids have the potential to effectively treat CRC. Sesquiterpenoid lactones, a subclass of terpenoid compounds, have been shown to induce anti-tumor effects. Aguerin B, a sesquiterpene lactone, exhibits cytotoxic effects against some types of tumors.
Aims: The current study aims to investigate the effect of Aguerin B on HT29 colorectal cancer, and assess the molecular mechanism of Aguerin B in colorectal cancer by evaluating the type of cell death it induces and its effect on the expression of the tumor suppressor gene p53 in HT29 colorectal cancer cells.
Materia and Methods: Colorectal cancer cells were purchased from the Iranian Biological Resource Center. HT29 cells were cultured in DMEM medium enriched with 10% fetal bovine serum and 1% antibiotics in 96-well plates. After 24 hours, the cells were treated with Aguerin B (variable concentrations from 1 to 8 μg/ml) for 24 and 48 hours. Cell viability was assessed using the MTT assay. Acridine orange-propidium iodide staining, caspase-3 and caspase-9 activity assays, using DCF-DA kits, were used to determine the type of cell death induced by Aguerin B. Additionally, qRT-PCR was used to evaluate p53 expression at the transcriptional level.
Results: The findings demonstrated that Aguerin B exerts cytotoxic effects on colorectal cancer cells in a dose- and time-dependent manner, with an IC50 (Inhibitory Concentration) value of 4.6 μg/ml. Acridine orange-propidium iodide staining confirmed apoptosis induction at the IC50 concentration of Aguerin B. The cultivated activity of caspase-3 and -9, along with elevated ROS levels, indicated apoptosis induction via the mitochondrial pathway. Furthermore, the upregulation of p53 suggests the tumor-suppressive effect of Aguerin B in colorectal cancer cells.
Discussion: Among the cell death mechanisms, the induction of apoptosis is a more profound mechanism of cell death mediated by anticancer bioactive compounds extracted from natural sources. Recent investigations have been focused on cancer therapeutic techniques that increase the apoptosis rate in tumor cells by disrupting mitochondrial biogenesis, leading to mitochondrial dysfunction. Caspases, as proteolytic enzymes, play a significant role as effector molecules in the apoptosis-induced cell death pathway. HT29 cells are sensitive to the chemotherapeutic drugs 5-fluorouracil and oxaliplatin, which are standard treatment options for colorectal cancer, and therefore were selected as chemotherapy-responsive colorectal cancer cells in this study. The results of this study exhibited that Aguerin B can induce cytotoxicity and anticancer effects in HT29 colorectal cancer cells in a dose- and time-dependent manner. This compound is also able to induce its properties through the recruitment of the intrinsic pathway, the caspase-dependent pathway in HT29 cells. Nevertheless, the role of p53 mutations in the pathogenesis of CRC has been recognized. The obtained data showed that Aguerin B can exert its anti-cancer potential. Previously, it was found that some natural products, including sesquiterpenoids, can also exert their suppressive effects against cancers by targeting the p53-MDM2 pathway.
Conclusion: Given its potent cytotoxicity and apoptosis-inducing effects, Aguerin B, which can suppress HT29 cells, appears to be an effective compound for obstructing human colorectal cancer cells, warranting further investigation in the pre-clinical phase and clinical studies.

Abstract Aim: In this study, the effect of TiO2 nanoparticles, as nanoparticles that are the most widely used in industry, on the morphological, and anatomical and biochemical properties of the baby sun rose Aptenia cordifolia was studied by method of spraying in four different concentrations.
Material and Methods: Titanium dioxide nanoparticles were sprayed on the baby sun rose plant twice a week. Growth parameters and chlorophyll content, phenol and flavonoids were measured. To study the anatomical structure of the plant; manual cutting, composite staining and Stomatal count were performed. Data analysis was performed using SPSS 16 software and the Duncan test.
Results: Treatment of titanium dioxide nanoparticles significantly increased photosynthetic pigments of chlorophyll a and b in the treated plants. The highest amount of phenol and flavonoids in the fourth leaf was related to the plant treated with 0.05% titanium dioxide nanoparticles. Also, the treatment of titanium dioxide nanoparticles increased the vascular diameter in the root and decreased the vascular diameter in the stem of some treatments.
Conclusion: The different morphological, biochemical, and anatomical responses of the baby sun rose to indicate the plant's genetic potential for growth in an environment contaminated with titanium dioxide nanoparticles.
 

Abstract

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 Introduction: The construction of synthetic pathways within the framework of metabolic engineering is considered a modern approach in biotechnology, enabling the production of valuable compounds from natural biological resources. This strategy focuses on utilizing abundant biomaterials—particularly carbohydrates—for the industrial production of chemical compounds by modifying metabolic pathways in microorganisms. These processes can convert biomass derived from biological sources into fuels, chemicals, and polymers, thereby opening new opportunities for the sustainable production of chemical substances from renewable resources.
Aim: This study specifically focuses on the enzymatic production of benzoylformate decarboxylase (BFD) with the overarching goal of completing the enzymatic pathway for the biosynthesis of BT. This intricate pathway initiates with xylose as the primary carbon source and proceeds through a cascade of four distinct enzymatic reactions. Notably, Escherichia coli (E. coli), possessing two endogenous enzymes integral to this pathway, holds the potential for complete BT biosynthesis upon the introduction of the remaining two requisite genes. This research thus seeks to engineer E. coli as a robust biocatalyst for sustainable BT production. The strategic implementation of a fully functional enzymatic pathway within a well-characterized microbial host, such as  E. coli, promises a more environmentally benign and potentially more efficient route to BT synthesis compared to traditional chemical methods. Furthermore, the ability to manipulate and optimize the expression of these key enzymatic components within E. coli offers opportunities to enhance the overall yield and productivity of the bioproduction process. The successful establishment of such a system could pave the way for large-scale, cost-effective, and sustainable production of this valuable chemical intermediate.
Materials and Methods: To construct an E. coli strain capable of expressing the benzoylformate decarboxylase enzyme, the mdlC gene originating from Pseudomonas putida was amplified and subsequently cloned into both pBAD and pET28 expression vectors. Following the confirmation of successful cloning through rigorous confirmatory assays, protein expression was evaluated using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and the enzymatic activity was assessed.
Results: Benzoylformate decarboxylase (BFD) is a pivotal enzyme within the engineered metabolic pathway for producing 1,2,4-butanetriol (BT) in   E.coli. In this study, the mdlC gene, encoding BFD from Pseudomonas putida, was successfully amplified and cloned into the versatile pBAD and the robust pET28 expression vectors. The pET28  system was preferred due to its ease of use and established track record in protein production, while the pBAD vector was strategically employed for its inducible expression capabilities, allowing for controlled protein synthesis. The expression of the  56 kDa target protein was confirmed through SDS-PAGE analysis, and the enzymatic function in the production of BT was subsequently verified using the sensitive and accurate HPLC method. This work lays a crucial foundation for the further optimization and development of a fully functional and efficient microbial cell factory for the sustainable production of this valuable chemical
Conclusion: The successful transfer of the expression construct into an appropriate E. coli host strain was confirmed by the presence of a distinct protein band at approximately 56 kDa on the SDS-PAGE gel, unequivocally verifying the expression of the mdlC gene. To evaluate the functional capacity of the expressed enzyme, the recombinant vector pBAD.mdlC was transformed into the E. coli TOP10 strain. The subsequent production of BT in the culture medium was meticulously analyzed using High-Performance Liquid Chromatography (HPLC).
               

Abstract Aim: In the past decades, nanotechnology has received much attention in order to develop new drug delivery systems to overcome the limitations of routine drugs in the treatment of diseases. Nanotechnology offers very useful applications in the diagnosis and treatment of cancer, as nanomaterials can penetrate into body tissues at the cellular and molecular levels.
Materials and methods: In the present study, samarium nanoparticles were synthesized by the extract of ginger using green chemistry synthesis method. The size of the synthesized nanoparticles was investigated by dynamic light scattering (DLS) technique and formation of new functional groups was investigated by FT_IR technique. The morphology of the nanoparticles was determined using FE-SEM scanning electron microscopy. Finally, the cytotoxicity and anticancer activity of samarium nanoparticles were studied against human colorectal cancer cell line of HCT116 after 24 and 48 hours incubation times using tetrazolium colorimetric assay (MTT assay).
Results: Dynamic light scattering data in agreement with Fe-SEM data revealed the formation of globular nanoparticles of 60 nm. Cell survival assay showed Ic50 values (the concentration of the compound that induces 50% death in cancer cells) of 90 (equal to 23.1 mg/ml) and 81 μM (equal to 20.7 mg/ml) of samarium nanoparticles on HCT116 cell line after 24 and 48 hours incubation times, respectively.
Conclusion: It is concluded that the newly green synthesized samarium nanoparticles with anticancer activity might be a good candidate for colon cancer therapy.