مجله سلول و بافت
سال چهاردهم شماره 3 (پاییز 1402)

 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.

 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.

 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.

 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.

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.

 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.

 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.

 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.

After pines, Ors (Juniperus) is the second most diverse genus of Coniferals, consisting of approximately 75 species and comprising the majority of evergreen shrubs and trees. Several studies have pointed out the importance of the genus in soil stabilization, and its ornamental, medicinal, and industrial applications. Ors or sarve kuhi (Juniperus seravschanica) has a wide distribution in Kerman province. However, in most areas, seedlings or young plants have very little distribution, which indicates the difficulty of seed germination due to various reasons. Also, rooting of its microshoots in micro culture techniques is considered to be difficult due to poor development and low frequency of rooting. Therefore, to overcome these problems, it needs to optimize an in vitro shoot multiplication and root induction and growth protocol. The purpose of this study was in vitro culture of apical shoots to induce their multiplication and rooting. Further, seeds were planted on micro culture media to study seed germination, callus induction and shoot and root regeneration from calli. The studied species was a population of J. seravschanica grown in Glochar (Raber, Kerman province).

Young shoots were collected from Glochar reserve of Rabor city (Kerman province) and kept at 4°C temperature for 3-7 days. The apical shoots with a size of 1.5 to 1.5 cm were separated and after sterilization, were cultured in woody plant medium (WPM) and Morashig and Skoog (MS) culture media without hormones to establish the samples. For shoot multiplication and growth, all samples were transferred to the medium containing plant growth regulators with optimized levels. Young shoots were transferred to rooting media (WPM, MS and ½MS) with different levels of IBA hormones. Seeds were separated from mature female cones, and full seeds (there are many empty seeds in cones) were exposed to cold treatment for 1-2 weeks. Then, they were sterilized and cultivated in MS (containing hormone) medium to study seed germination and callus formation from embryos. Callus regeneration and shoot formation and their rooting were investigated under appropriate hormone concentrations.

In the WPM medium, the highest percentage of shoot proliferation (40%) was obtained in the combined treatment of 3 mg/L BAP and 2 mg/L NAA. The shoot proliferation percentage was higher in MS media than WPM with 57% in concentrations of 0.2 and 3 mg/L of BAP and NAA, respectively. In both media, Proliferated shoots did not form any root in both media until four months after planting. The highest percentage of seed germination in MS medium was 33% at the concentrations of 5 mg/L BAP and 0.2 mg/L NAA. The highest rate of callus formation (40%) from embryos (seeds), as explants, was recorded in MS culture medium at the concentration of 0.2 and 3 mg/L of BAP and NAA, respectively. Calli produced shoots in in 3 mg/L BAP and 2 mg/L NAA in WPM medium. Root formation was not observed until six months, but about 10% of these regenerated shoots produced roots eight months after shoot proliferation.

It seems that various factors such as genotype, polyploidy or hybrid formation, slow plant growth (which is common in coniferal), or secondary compounds are the reasons for the low rate of regeneration, especially rooting. Also, the duration of cultivation can be effective in rooting.

 Nowadays, one of the great challenges of mankind is to find a suitable way for the treatment of cancer. Recent developments show that nanotechnology has a significant impact on the prevention, diagnosis and treatment of cancer. In nanotechnology, there are different physical and chemical methods to produce nanoparticles but mostly chemical methods are used. The nanoparticles produced using these methods are harmful to health due to the absorption of chemical species on their surface. In order to eliminate this big problem, biological methods such as the use of fungi, bacteria and plants have replaced other methods for the synthesis of nanoparticles which is called green synthesis. It is cheaper, simpler and non-toxic than other methods. The use of plants for the synthesis of nanoparticles has become more important than other biological methods due to the elimination of the difficult step of cell culture maintenance.

 In this study, gold nanoparticles were formed after treating the HAuCl4 solution with the aqueous extract of Anarijeh plant with the scientific name Pimpinella affinis. In this research, different amounts of aqueous extracts of leaves and stems of Pimpinella affinis were used for the synthesis of gold nanoparticles. UV-Visible spectroscopy and DLS methods were used to evaluate nanoparticles and determine their average size and stability. Also, SEM method was used to investigate their morphology. FTIR method was used to determine the functional groups of the aqueous extract and synthesized gold nanoparticles; in addition GC/MS device was used to determine the composition of the extract. Finally, MTT analysis was used to investigate the cytotoxic effects of nanoparticles on MCF-7 cell line along with treatments of 24, 48 and 72 hours.

 The results of the analysis were showed that the optimal concentration of the Pimpinella affinis extract for the synthesis of gold nanoparticles is 1 mg/ml. The synthesized gold nanoparticles using the mentioned concentration of the extract have an average size of 20 nanometers with a zeta potential of -61 mV which has the best stability. They had a spherical shape with a polydispersity equal to 0.7. The results of FTIR analysis show the presence of hydroxyl, simple carbonyl groups and hydrocarbon functional groups in the compounds of the extract. Experiments showed that phenolic and carboxylic compounds play the most important role in the reduction of gold salt. Also, proteins and lipids have made gold nanoparticles stable. The essential compounds identified in the extract by GC/MS method include Phenolic and flavonoid compounds and terpenes which are confirmed by the FTIR spectrum. The treatment time of 48 hours for MCF-7 cell line is the best time for the toxicity of nanoparticles with IC50 equal to 384.2 µg/ml, which was determined by MTT analysis.

 According to the above findings, it can be concluded that Pimpinella affinis is one of the suitable options for the synthesis of gold nanoparticles, and it can be done with more studies on nanoparticles synthesized using this plant, considered it as one of the efficient methods to achieve the goal of proper treatment for breast cancer.

 A multitude of devastating human diseases arise from genetic mutations that lead to cellular dysfunction, as well as from infectious diseases and cell transformation. These diseases have significantly impacted individuals and communities worldwide and led to challenges in medical and biological sciences to address their causes and improve treatment approaches. Since identifying genes as the main heredity unit, generating targeted alterations in specific gene loci for treatment and perception of disease function has been a crucial concern. The discovery of nuclease enzymes revolutionized this field and transformed the concept of genome editing from a dream to a tangible reality.
Currently, targeted gene editing and modification through techniques such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regulatory interspaced short palindromic repeat Cas-associated nuclease (CRISPR/Cas) offers robust methods to assess gene function, also precisely and efficiently manipulate cells behavior.
ZFN and the TALEN were the earliest gene editing approaches that developed, which rely on the FokI enzyme and engineered protein that binds to a specific sequence in the genome and induces double-strand breakage, stimulating the cell's repair mechanisms. Although these techniques led to many significant discoveries and developments in gene editing, they had limitations such as complexity and high prices.
The emergence of CRISPR/Cas technology propelled gene editing to a new stage of efficiency and accessibility. This technique utilizes RNA to guide the Cas enzyme to the target gene sequence and, like the ZFN and TALEN, creates a double-strand breakage, which triggers DNA repair mechanisms. CRISPR/Cas is a versatile, simple, and cost-effective revolutionary gene-manipulating tool that enabled researchers to explore the potential of gene editing in a wide application range.
Base and Prime editors are the latest systems in the genome manipulation area, which, based on the CRISPR/Cas technique, represent the impressive development and improvement in gene editing capabilities. Despite the others, these two technologies induce the single-strand breakage and modify at the nucleotide level in target sequences.
The ability to edit genes indicates new possibilities for treating genetic diseases and even preventing them before they appear. Nowadays, a growing number of clinical trials utilizing genome editing for therapeutic purposes are underway, thanks to the substantial advancements in these tools. These remarkable improvements hold promise in treating various diseases and improving patient’s outcomes.
However, despite the outstanding progress made in gene editing technology, there are still several challenges and obstacles, such as ethical considerations, off-target effects, and safety concerns, which still need more investigation and studies. Consequently, ongoing researches are focused on developing the accuracy and efficacy of these editing tools.

 The advancement of gene editing technology has opened up a new way in medical and biological sciences to modify and manipulate the genome, also exploring the various diseases caused.

 Hairy roots induced by inoculation with Agrobacterium rhizogenes are suitable for the production and accumulation of plant secondary metabolites. Genetically modified hairy roots produce valuable secondary metabolites to a greater amount and with more genetic stability than cell suspension cultures and normal roots. Catharanthus roseus is one of the most important medicinal plants that produces valuable secondary metabolites such as indole alkaloids. The purpose of this research was to investigate the effect of factors influencing the induction of hairy roots and to investigate the amount of production of secondary metabolites (vindoline and catharanthine) in the hairy roots produced by inoculation of C. roseus plant with A. rhizogenes.

 In order to produce sterile C. roseus seedlings and to prepare explants, seeds cultured on MS culture medium. The effect of the type of culture medium (MS, 1/2 MS and B5), the type of explant (leaf and stem), the type of bacterial strain (A4, 1132 and 2656) and the type of plant genotype (white and red) on the amount of hairy root induction in a completely randomized design was investigated. Extraction of samples was done using methanol solvent, then the amount of indole alkaloids was calculated using HPLC method and standard curve. The nature of transgenic hairy roots was confirmed after DNA extraction from different samples using PCR method with specific primers (rolB and virD).

 According to the results, the most efficient strain in transferring T-DNA to the explants and the appearance of hairy roots was related to A4 strain (53.8%). 1/2 MS culture medium with 77.69% of root induction was the best medium combination for the appearance of hairy roots. Also, white genotype (62% rooting) and leaf explant (77% rooting) were identified as the best genotype and explant for the production of hairy roots. After the specific amplification of the rolB gene and the lack of amplification of the virD gene in the PCR reaction in the samples, the molecular confirmation of the transgenic nature of the hairy root clones was done. Based on the results of HPLC experiment, the average amount of vindoline and catharanthine in hairy roots (0.24 and 0.615 mg/g dry weight) is higher than aerial parts (0.106 and 0.488 mg/g dry weight) and normal root (0.021 and 0.013 mg/g dry weight). The amount of these alkaloids in the aerial parts was higher than in the normal roots of the plant.

 The production of hairy roots in response to the inoculation of plant explants with A. rhizogenes is influenced by various factors such as the type of explant, the genotype of the plant, the strain of bacteria and the composition of culture medium. These factors must be optimized to establish effective hairy root cultur in a plant. It seems that with the introduction of rol genes from bacteria to the plant genome (hairy root) and as a result of the change in the amount of endogenous hormone production and the response of the plant cell to the entry of bacterial genes, the amount and profile of the production of the secondary metabolites of the plant will change. As a result, by selecting clones with increased production of medicinally important alkaloids and optimizing the conditions of large scale production of secondary metabolites in bioreactors, we can move towards the commercial production of these compounds through cell and plant tissue culture.