Journal of Cell and Molecular Research
Volume:14 Issue: 1, Summer and Autumn 2022

Cellulose which is extremely produced by plants, can be used for biofuel production but this function needs chemical or enzymatic digestion. Cellulose hydrolysis of plant wastes for ethanol production requires a mixture of three enzyme groups, including endoglucanases, exoglucanases, and beta-glucosidases. The cellobiohydrolase enzyme (Cel6B) from Thermobifidia fusca has been used for cellulase activity extensively. This research aimed to express recombinant Cel6B enzyme in Pichia pastoris. For this purpose, cel6B gene in control of AOX1 promoter (methanol inducible) was introduced into Pichia pastoris. Amplification of cel6B gene was performed by PCR technique and then introduced into the Phil-S1 yeast vector. The recombinant construct contained the cel6B gene sequence and PHO1 signal sequence as secretion signal was transferred into Pichia pastoris GS115 strain. The transformed yeast cells expressed the recombinant Cel6B to yield 2.104 U (µmol/min)/ml of culture medium. Purified recombinant enzyme showed the best activity at 60 °C and pH 4.5 and this was agreed with optimum conditions for recombinant Cel6B enzymes which were produced in other systems. The purity of the enzyme was examined by SDS–PAGE technique, and a single band with a molecular weight about 59.6 kDa was observed. As cel6B gene sequence was not optimized for expression in the Pichia pastoris yeast, this could be one of the reasons for low level activity of recombinant Cel6B enzyme. This thermostable enzyme can be used for cellulolytic digestion of biomaterials in biofuel production research and other uses.

The present study was conducted to determine the volatile and non-volatile fractions and the antioxidant and anti-cancer activities of ethanolic extracts of Dracocephalum polychaetum and D. kotschyi. The volatile and non-volatile fractions were investigated by gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The cytotoxicity effect of two ethanol extracts and the major phenolic components has been evaluated on breast and colon cancer cells by the MMT assay. GC-MS of the essential oils identified about 50 compounds, and perillylaldehyde and D-limonene were the main constituents in the essential oils of the two species. Moreover, high-performance liquid chromatography- Diode array detector analysis demonstrated that the ethanolic extract of  D. polychaetum and D. kotschyi were the source of phenolic compounds such as rosmarinic acid, protocatechuic acid, naringin, apigenin, syringic acid, epicatechin, chlorogenic acid, thymol, carvacrol, rutin, p-coumaric acid, gallic acid, benzoic acid, cinnamic acid, resorcinol, quercetin, salicylic acid, 4-hydroxybenzoic acid, and ferulic acid. Rosmarinic acid and thymol were the main predominant phenolic constituents in D. kotschyi and D. polychaetum ethanolic extracts. The cytotoxicity effect of D. kotschyi and D. polychaetum ethanol extracts and the major phenolic components including rosmarinic acid, thymol, apigenin, quercetin, and nariginin has been evaluated on breast and colon cancer cells by MMT assay and results indicated IC50 values in the range of 90 to 140 (µg.ml-1) after 48 hours of treatment with ethanol extracts. Among phenolic components, thymol caused the lowest cell viability and Narengin showed the lowest anti-proliferative activity. Both extracts also showed antioxidant activity using DPPH assay. The findings of this research suggest that the Dracocephalum have precious bioactive and natural compounds with significant antioxidant and in vitro anti-cancer activities.

Myostatin (MSTN) is primarily expressed in skeletal muscle tissue and acts as a negative regulator of skeletal muscle growth by inhibiting differentiation and proliferation of myoblasts. Inhibition of MSTN expression could result in muscular hypertrophy. An effective therapeutic approach based on specific silencing of a target gene is provided by RNA interference. The distribution of biologically active small interfering RNAs (siRNAs) inside the target cells/ tissue, is a significant problem due to the limited stability and delivery of siRNAs. Strategies depending on vector delivery have also a limited clinical utility due to safety concerns. Thus direct application of active siRNAs in vivo is the preferred strategy. We described the efficiency of intramuscular and intraperitoneal injections of MSTN-siRNA conjugated with cholesterol into the skeletal muscle of mice. The designed siRNA molecule was complementary to the exon II of the mouse MSTN gene. Mice were injected with a weekly dose of 10 μg/kg conjucated siRNA-cholesterol intraperitoneally or intramuscularly. Our findings suggested that within a few weeks of application, siRNA-treated mice showed a significant increase in muscle mass and suppressed MSTN gene expression. Even though both types of injections increased muscle weight, intramuscular siRNA injections suppressed the MSTN gene more effectively, whereas intraperitoneal RNA injections had a more significant impact on total body weight. The cholesterol-conjugated siRNA platform discussed here may hold promise for treating several skeletal muscle-related diseases, such as atrophic muscle disease, muscular dystrophy, and type II diabetes.

Somatic embryogenesis encompasses the same set of various developmental processes similar to zygotic embryogenesis. The conversion of somatic cells to embryos also requires stages of differentiation and reprogramming of cells. Since somatic embryogenesis is a complex process, a comprehensive investigation is required to identify the effective gene networks and their interactions with environmental factors. As part of this study, bioinformatics tools and molecular studies were used to gain a better understanding of Arabidopsis thaliana somatic embryogenesis. The enriched pathways of somatic embryogenesis and their core-enriched genes were identified using gene set enrichment analysis. The results indicated that significant interaction between hormones helps to induce and develop somatic embryos. The gene ontology (including biological process, molecular function and cellular compartment) of core-enriched genes revealed that lipid storage and metabolism as well as stress response are the active biological pathways during somatic embryogenesis. In the protein-protein interaction network, TIR1/AFBs as auxin receptors exhibited the greatest number of interactions and proteins involved in lipid storage and metabolism acted as mediators between auxin receptors and ethylene perception. Also, Kyoto encyclopedia of genes and genomes analysis indicated that the metabolism of membrane lipids during somatic embryogenesis of Arabidopsis is primarily related to the biosynthesis of jasmonates and their derivatives. This process is initiated by Lypooxygenase proteins in the chloroplast, while Acyl-CoA oxidase 1 (ACX1) and Oxophytodienoate reductase 3 (OPR3) proceed this process in the peroxisome. The qRT-PCR analysis also confirmed the role of these genes during somatic embryogenesis, as the activity of these genes decreased at the beginning of 2,4-D treatment, but it increased during somatic embryogenesis. According to these results, jasmonates play an important role during somatic embryogenesis by mediating auxin signaling and stress response.

Colorectal cancer (CRC) and gastric cancer (GC) are multifactorial diseases likely influenced by genetic susceptibility. Gastric cancer is also the fourth most common cancer in the world and the second leading cause of cancer-related mortality. CD86 (B7-2) is a costimulatory molecule found on antigen-presenting cells (APCs) that is important in autoimmune, transplantation, and tumor immunity. This protein is expressed in the immune system cells and is involved in the pathogenesis of various inflammatory disorders and inflammation. Rs17281995 polymorphism is located in section 3’ UTR, and given the regulatory role of 3' UTR gene sequences, SNPs located in these regions can affect the expression and function of the corresponding protein. In the present study, the relationship between rs17281995 polymorphism located in the 3' UTR regulatory region of the CD86 gene sequence and the risk of colorectal and gastric cancer in Iranian patients was analyzed. Polymorphism was identified in 26 patients with colorectal cancer, 30 patients with gastric cancer, and 36 healthy controls using the high-resolution DNA melting curve analysis (HRM) technique. The Data was then analyzed using SPSS software. There was no significant relationship between rs17281995 polymorphism and colorectal (P = 0.75) and gastric cancers (P = 0.97) in the Iranian population. In addition, genotypic distribution analysis showed no significant difference between the patient and control groups (P˃0.05). Among people with colorectal cancer, 0.577 had the G allele and 0.423 had the C allele. In the control group, 0.639 had the G allele and 0.361 had the C allele. In conclusion, our data indicate that the CD86 rs17281995 gene polymorphism does not seem to be a risk factor for colorectal and gastric cancers in the Iranian population.

Genetic engineering is a powerful technology of the present century that has revolutionized the agricultural, health, pharmaceutical and food industries worldwide. It is important to identify changes caused by transgenes that may be attributed to unintended traits in the risk assessment of genetically modified (GM) crops. Rhizomania, which is caused by beet necrotic yellow vein virus (BNYVV) infection, is considered to be a significant constraint in order to produce sugar beet worldwide. The resistance of transgenic sugar beet plants to the BNYVV was previously developed through RNA silencing by expression of hairpin RNA (hpRNA) structures. In the present study, the RNA sequencing (RNA-seq) analysis was performed in order to evaluate the transcriptional changes of an event of transgenic sugar beet plants, named 219-T3:S3-13.2 (S3), with the non-transgenic parental plants grown in virus-infected soil. The results of the present study indicate that there are only 0.9% differentially expressed genes (DEGs) at significant levels. The functional analysis shows alterations of transcription in lipids, amino acids, and carbohydrates metabolisms, cellular processes (autophagy), hormone signal transduction, and biosynthesis of secondary metabolites in the transgenic event, which are related to stress-adaption for which most of the genes were up-regulated. All in all, we conclude that the presence of the transgenes does not have substantial effects on the plant gene expression patterns. This work also indicates that RNA-seq analysis can be useful to evaluate the unintended effects and risk assessment of GM sugar beet plants.

Glutamine (Gln) is an essential amino acid with a wide range of cellular functions and is necessary for cell proliferation. It is usually added to the culture media in the form of L-glutamine, which is highly unstable and degrades in a temperature-dependent manner during the culture period. Although, Gln is beneficial for the cells, its degradation produces ammonia which is toxic and negatively affect cell culture. Pheochromocytoma cells (PC-12), originating from cancerous cells of the rat adrenal gland, are considered as a suitable model to study the differentiating effects of different factors. Previous studies showed the importance of Gln in the normal growth and differentiation of the cells. Alginate, is one of the biomaterials currently used as a natural scaffold for the induction of neuronal differentiation. In the present experimental research, the effect of stable and elevated levels of Gln on the growth and neuronal differentiation of PC-12 cells was compared under 2D- and 3D- (sodium alginate hydrogel beads) culture conditions. The cells’ viabilities were determined and compared between experimental groups using live/dead cell staining by Acridine orange/Propidium iodide (AO/PI), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Furthermore, cells were stained using cresyl violet to detect neuronal Nissl bodies. The induction of differentiation was confirmed using immunocytochemical analysis of Nestin and β-tubulin III proteins and Cells’ nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Results showed that high concentration of Gln can induce neuronal differentiation in PC-12 cells under both 2D- and 3D- culture conditions and increases the expression of progenitor and mature neuronal markers Nestin and β-tubulin III, respectively.

Long non-coding RNAs (lncRNAs) have recently emerged as effective regulatory agents in biological processes as well as in the formation of tumors. LncRNAs are important regulators of cell transformation and cancer progression. LncRNA NEAT1 is one of the most important lncRNAs, and its deregulation has been reported in a variety of human cancers. Ovarian cancer has an inverse relationship with the number of reported pregnancies and deliveries, while it has a direct relationship with infertility. This study aimed to investigate NEAT1 expression in ovarian cancer. A total of 140 tissue samples, including 70 ovarian tumors and 70 marginal samples, were included in the study. Total RNA was extracted using the RNXplus solution. The quality and quantity of the extracted RNAs were determined using gel electrophoresis and a NanoDrop device. The complementary DNA was synthesized by the reverse transcriptase enzyme, and quantitative reverse transcriptase PCR was used to quantify the expression of NEAT1. A comparison between the mean expression of NEAT1 in ovarian tumors and marginal samples showed an increase in NEAT1 expression in tumor tissue that was not statistically significant (P-value = 0.2). ROC curve analysis also showed that NEAT1 expression level might not be an informative biomarker for ovarian cancer.