Iranian Journal of Biotechnology
Volume:21 Issue: 3, Summer 2023

Mesenchymal stem cell (MSC) derived exosomes (MSC-DE) have been demonstrated to be potential candidates for the treatment of rat spinal cord injury (SCI). The effect of AD-MSC and AD-MSC-DE encapsulated into collagen and fibrin hydrogels on the treatment of SCI in a rat animal model was investigated for introducing a new effective SCI treatment method The AD-MSC-DE was isolated using ultra-centrifugation at 100,000×g for 120 min and characterized by different methods. Fibrin and collagen hydrogels were synthesized and then mixed with AD-MSC-DE suspension. the characterized AD-MSC-DE were encapsulated into collagen and fibrin hydrogels. eighteen adult male Wister rats were randomly classified into 3 equal groups (n=6): the control group (SCI rat without treatment), SCI rat treated with either AD-MSC-DE encapsulated in collagen hydrogel or encapsulated in fibrin hydrogel groups. the treatment approaches were evaluated using clinical, histological, and molecular assays. The AD-MSC-DE encapsulated into fibrin and collagen groups showed better clinical function than the control group. The AD-MSC-DE encapsulated into fibrin and collagen also improved SCI-induced polio and leuko-myelomalacia and leads to higher expression of NF protein than the control group. In the AD-MSC-DE encapsulated into collagen and fibrin leads to up-regulation the mean levels of NEFL (23.82 and 24.33, respectively), eNOS (24.31 and 24.53, respectively), and CK19 mRNAs (24.23 and 23.98, respectively) compared to the control group. The AD-MSC-DE encapsulated within ECM-based hydrogel scaffolds such as collagen and fibrin can regenerate the injured nerve in SCI rats and reduce spinal cord lesion-induced central neuropathic pain.

Cerebral ischemia has been a hotpot in the prevention and treatment of cerebral ischemia. Dexmedetomidine (Dex) is a new type of highly selective α2 adrenergic receptor agonist with pharmacological properties. Quantitative studies have shown that Dex has a protective effect on glutamate (Glu)-induced neuronaldamage. however, its mechanism has not been fully elucidated. The purpose of this study was to explore the underlyingmolecular mechanism by which Dex ameliorates Glu-induced neuronal injury by regulating miR-433/JAK2/STAT3axis. A model of neuronal injury was constructed by Glu treatment and intervened with Dex.miRNA expression profiling assay was conducted to screen potential miRNAs affected by Dex. Cell viability, lactatedehydrogenase (LDH) release and apoptosis were detected by MTT assay, LDH kit, and TUNEL staining, respectively.Oxidative stress indicators were assessed by ELISA whereas mitochondrial membrane potential (MMP) was assessedby C11-BODIPY581/591 staining. The targeting relationship between the miR-433 and JAK2 was verified by dualluciferase reporter assay and gene expression was analyzed by quantitative PCR and Western blot. Glu treatment decreased cell viability and MMP and promoted LDH release, apoptosis and oxidative damage.Glu-induced changes in neurons were reversed after Dex treatment through upregulating the miR-433 expression toblock the activation of JAK2/STAT3 pathway. Dex protects against Glu-induced neuronal injury by regulating miR-433/JAK2/STAT3 pathway, whichprovides new insights into the treatment of neuronal injury.

Celiac disease (CD) is a gluten-sensitive chronic autoimmune enteropathy. A strict life-long gluten-free dietis the only efficient and accepted treatment until now. However, maintaining a truly gluten-free status is both difficult and costly, often resulting in a social burden for the person. Moreover, 2 to 5 percent of patients fail to improve clinically and histologically upon elimination of dietary gluten. Therefore, novel therapeutic approaches, including gluten degrading enzymes, are an unmet need of celiac patients.

To evaluate the function of sunn pest prolyl endoprotease for gluten and gliadin hydrolysis in vitro.

The spPEP was expressed as a recombinant protein in E. coli BL21 (DE3), and its catalyticactivity was assessed by SDS-PAGE and RP-HPLC analyses.

Production of a 100-kDa spPEP protein was confirmed by SDS-PAGE and western blot analysis. Also, wedemonstrate that spPEP efficiently degrades gluten and α-gliadin (30-40 kDa) in vitro under conditions similar to the GIand is resistant to pepsin and trypsin.

The gathered data demonstrated that spPEP might be a novel candidate for Oral Enzymatic Therapy (OET)in CD and other gluten-related disorders.

Breast cancer is a prevalent tumor with high aggressiveness among female populations. MiRNA-145-5pplays an important role in multiple cancers. qRT-PCR detected miRNA-145-5p and histone protein family member X (H2AFX) mRNAexpression in breast cancer cells, and western blot determined the protein expression of H2AFX. After predicting the target genes via the bioinformatics methods, the targeting relationship between miRNA-145-5p and H2AFX was verified by dualluciferase, RIP, and RNA pull-down assays. The relationship between H2AFX and clinical indexes was also analyzed. Furthermore, the effects of miRNA-145-5p/H2AFX regulatory axis on breast cancer cell progression were determined by colony formation, wound healing, CCK-8, and Transwell assays. The results suggested that miRNA-145-5p was markedly lowly-expressed in breast cancer tissue and cells,while H2AFX was upregulated, which had a positive correlation with T stages of breast cancer. Besides, overexpressedmiRNA-145-5p was found to remarkably suppress progression of breast cancer cells. As bioinformatic analysis predictedthat H2AFX was the potential target of miRNA-145-5p, the dual-luciferase assay was conducted, which demonstratedthat miRNA-145-5p negatively regulated the expression of H2AFX by targeting its 3’-UTR. The rescue experimentdemonstrated that overexpression of miRNA-145-5p could offset the promotion effects of oe-H2AFX on malignantprogression. Our study is aimed at exploring how miRNA-145-5p functions in breast cancer cells. Our findings confirmed that miRNA-145-5p hindered malignant progression of breast cancer by negativelyregulating H2AFX. MiRNA-145-5p/H2AFX axis may be a novel therapeutic target for breast cancer.

Lung cancer is one of the most common types of cancer and a leading cause of cancer-related deathsworldwide. Therefore, it is useful to know the biomarkers involved in the malignancy of lung cancer. This study aimed to show that SOX2-OT as a long non-coding RNA (IncRNA) regulates gene expression viathe SOX2-OT/miR-194-5p/SOX5 axis molecular pathway in lung cancer. A549 cells transfected with siRNA-SOX2-OT and the expression of SOX2-OT and miR-194-5pgenes were analyzed by real-time PCR before and after transfection. In addition, the expression of the B-catenin, MMP9,phosphorylated and activated STAT3 (p-STAT3), SOX5, and VEGF proteins before and after transfection was investigatedby Western blotting. After using siRNA-SOX2-OT, an increase in the expression of miR-194-5p and a decrease in the expression ofB-catenin, SOX5, p-STAT3 activated STAT3, VEGF, and MMP9 proteins was observed. According to the results of the present study, an increase in SOX2-OT in lung cancer seems to stimulate theexpression of beta-catenin, SOX5, MMP9, and VEGF thus support the malignancy of lung cancer cells.

The mortality rate of esophageal cancer is on the continuous increase. Fortunately, with the development ofimmunotherapy, the prognosis and survival rate of patients with esophageal cancer have been improved gradually. Immune markers have a crucial part in immunotherapy. Therefore, it is of great meaning to delve further intoimmune-related biomarkers of esophageal cancer for better treatment. In this study, gene co-expression networks were established using weighted gene co-expressionnetwork analysis, thus forming gene modules with different clusters. The tumor immune microenvironment was assessedwith the ESTIMATE algorithm. Analysis of the module Eigen gene -immune score trait indicated that the black module was markedly associatedwith immune score, with the top 80 genes regarding correlation ranking as the candidate hub gene set. Enrichment analysis revealed that genes within the black module were primarily enriched in tumor immune-related functions. To mine the hub genes that were closely connected with immunity, protein-protein interaction networks were constructed by STRING for genes within the black module, and genes with the interaction score top10 were retained. They were intersected with hub genes to finally obtain four hub genes: CCR5, LCP2, PTPRC and TYROBP. The samples were divided into highand low-expression groups by the median expression of hub gene, and survival analysis was performed in combination with clinical information. The results revealed that the high-expression groups of genes LCP2 and PTPRC had a poor prognosis. TIMER immune cell infiltration analysis revealed that the expression levels of the 4 hub genes were positively correlated with immune cell infiltration and negatively correlated with tumor purity. In addition, these 4 hub genes were correlated with the expression of immune checkpoint genes CTLA-4 and PDCD1 positively. Gene set enrichment analysis enrichment analysis demonstrated that there were differences in tumor immunity and cancer-related pathways between high and low expression of 4 hub genes. Altogether, we identified four biomarkers that may have connection with tumor immunity, and speculatedthat these genes may influence patient prognosis by affecting pathways related to esophageal cancer immunity. This study will pave the way for the research of immune mechanisms of esophageal cancer and the analysis of patient’s prognosis.

Salinity is one of the major abiotic stresses that limit the production and yields of agricultural cropsworldwide. In order to identify key barley genes under salinity stress, the available metadata were examined by twomethods of Cytoscape and R software. Next, the hub expression of the selected gene was evaluated under different salinity stress treatments and finally, this gene was cloned into cloning and expression vector and recombinant plasmid was made. In this study, we extracted salinity stress tolerant genes from several kinds of literature and alsomicroarray data related to barley under salinity conditions from various datasets. The list of genes related to literatureanalyzed using string and Cytoscape. The genes from the datasets were first filtered and then the hub genes were identified by Cytoscape and R methods. Next, these hub genes were analyzed for the promoter. Ten hub genes were selected and their promoters were analyzed, the cis-element of which was often cisacting regulatory element involved in the methyl jasmonate -responsiveness, common cis-acting element in promoterand enhancer regions and MYBHv1 binding site. Finally, the sedoheptulose-1,7-bisphosp gene (SBPase), which had thehighest interaction in both gene lists and both types of gene networks, was selected as hub gene. Next, the expression ofSBPase gene was examined in two variety of Youssef variety (salt tolerant) and Fajr variety (salt sensitive) under salinitystress (NaCl 100mM) at 0 (control), 3, 6, 12 and 24 hours after stress. The results showed that the expression of thisgene increased with increasing the duration of stress in both varieties. Comparison of the two varieties showed that theexpression of SBPase gene in the tolerant genotype was twice as high as sensitive. Finally, SBPase gene as a key gene forsalinity stress was cloned in both cloning (pTG19) and expression (pBI121) vectors. According to our results, SBPase gene increased growth and photosynthesis in barley under various abioticstresses, therefore, over-expression of this gene in barley is recommended to produce plants resistant to abiotic stresses.

Most herbs play significant roles in the treatment of various diseases. Because dopamine functions in theanti-inflammatory process and the presence of this substance in Portulaca Oleracea L. native plant, investigating thisplant’s anti-inflammatory properties in treating neurological diseases is interesting. The objective of this study was to estimate the NO production and the expression level of inflammatory genesin lipopolysaccharide (LPS)-treated microglial cells affected by P. oleracea L. extraction. P. oleracea L. hairy root extract was isolated, and the primary microglial cell of the rat wasisolated from glial cells and confirmed by immunocytochemistry analysis. Microglial cells were pretreated with differentconcentrations of P. oleracea L. extract and then treated with 1 μg.mL-1 LPS. The control group did not receive anytreatment. The NO level in culture supernatants was measured by the Griess method. The mRNA expression levels ofiNOS (inducible Nitric oxide synthase) and TNF-α (tumor necrosis factor-alpha) in LPS-treated microglial cells wereevaluated using Real-Time PCR. The present study determined that 0.1 mg. mL-1 of the P. oleracea L. extract decreased the NO production in ratmicroglial cells. Different concentrations of the P. oleracea L. extract had no prominent effects on LPS-treated cell viability. The results of real-time PCR indicated that P. oleracea L extracts suppressed the mRNA expression levels of iNOS and TNF-α in LPS-treated cells. MTT assay determined that P. oleracea L. extract was not cytotoxic, and the anti-inflammatory P. oleracea L. extract effects observed were not because of cell death. P. oleracea L. extract might be helpful as an anti-inflammatory agent in treating inflammatory diseases.

Biological nitrogen fixation (BNF) is a unique mechanism in which microorganisms utilize the nitrogenaseenzyme to catalyze the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Fe protein, encoded by the nifHgene, is an essential component of the nitrogenase in Klebsiella variicola DX120E. However, the function of this gene inregulating nitrogen fixing activity is still unclear. The objective of this study was to reveal the function of nifH gene in associative nitrogen-fixing bacteriaKlebsiella variicola DX120E and micro-sugarcane system by immunoassay and gene editing. In the current investigation, the nifH gene was cloned in a pET-30a (+) vector and expressedin Escherichia coli. The NifH protein was purified and used to immunize rabbit, and then the serum was collected andpurified to obtain rabbit anti-NifH polyclonal antibodies. The CRISPR-Cas9 system was applied to produce nifH mutantstrains, and the nitrogen-fixing enzyme activity, gene, and protein expression were analyzed. Both in vitro and in vivo NifH proteins were detected by Western blotting, which were 43 and 32 kDa respectively. The expression of nifD and nifK genes was decreased, and nitrogenase activity was reduced in the nifH mutant strain. The nifH gene mutant weakened the nitrogenase activity by regulating the expression of Fe protein, whichsuggests a potential strategy to study the nitrogen fixation-related genes and the interactions between endophytic nitrogenfixing bacteria and sugarcane.

Today, numerous antimicrobial and anticancer properties have been reported for plant lectins due to theirability to bind to carbohydrates. The Urtica dioica agglutinin (UDA lectin) is a monomeric, small, and low molecularweight glycoprotein. It has attracted the attention of many researchers for identification, treatment, and other clinicalpurposes.

The aim of this study is the optimization of the chitin affinity chromatography based on Sepharose 4B (CNBractivated Sepharose 4B) for the rapid purification of UDA lectin from Urtica dioica rhizome.

The chitin ligands were dissolved in 40% Trichloroacetic acid and attached to Sepharose4B according to the Amersham-Biosciences instructions. The attachment of the ligand to the Sepharose 4B beads wasinvestigated by Fourier transform infrared (FTIR) spectroscopy. An acidic crude extract of nettle rhizome passes fromchromatographic columns in two sizes with dimensions: 24 x 0.51 cm and 8.44 x 0.86 cm. Quantity and quality of purifiedlectin were calculated by the Bradford microplate method SDS-PAGE gel electrophoresis and human erythrocyte cell(RBC) hemagglutination, respectively.

The analysis of FTIR spectrograms showed that major changes were observed in the fingerprint regions. Besides,due to the dissolution of Sepharose 4B and chitin in the aqueous phase, this difference was not significant in the Imineand Nitrile regions. On the other hand, the comparative results of purification chromatograms showed that increasingthe column length causes a smaller half-width and increases the length of the purified peak. Also, it leads to high-qualitypurified UDA lectin, with a molecular weight of almost 12.5 kDa in gel electrophoresis. Hemagglutination activityon trypsinized red blood cells was displayed, and agglutination of purified UDA lectin started at least at 300 μg.mL-1concentration.

According to our findings, we suggested that dissolving chitin in the polar solvent of Trichloroacetic acid,using Sepharose 4B as the beads of a matrix, and increasing the column length might lead to a decrease in the half-widthof the peak. These can increase the purity and concentration of purified UDA lectin, and speed up the purification process. These findings could be used by researchers to accelerate the purification of UDA lectin in other studies, dealing with drug delivery systems, ELISA techniques, and cell growth.