Iranian Journal of Biotechnology
Volume:20 Issue: 3, Summer 2022

Although epidermal growth factor (EGF) controls many crucial processes in the human body, it can increase the risk of developing cancer when overexpresses. This study focused on detecting cancer-associated genes that are dysregulated by EGF overexpression. To identify differentially expressed genes (DEGs), two independent meta-analyses with normal and cancer RNA-Seq samples treated by EGF were conducted. The new DEGs detected only via two meta-analyses were used in all downstream analyses. To reach count data, the tools of FastQC, Trimmomatic, HISAT2, SAMtools, and HTSeq-count were employed. DEGs in each individual RNA-Seq study and the meta-analysis of RNA-Seq studies were identified using DESeq2 and metaSeq R package, respectively. MCODE detected densely interconnected top clusters in the protein-protein interaction (PPI) network of DEGs obtained from normal and cancer datasets. The DEGs were then introduced to Enrichr and ClueGO/CluePedia, and terms, pathways, and hub genes enriched in Gene Ontology (GO) and KEGG and Reactome were detected. The meta-analysis of normal and cancer datasets revealed 990 and 541 new DEGs, all upregulated. A number of DEGs were enriched in protein K48-linked deubiquitination, ncRNA processing, ribosomal large subunit binding, and protein processing in endoplasmic reticulum. Hub genes overexpression (DHX33, INTS8, NMD3, OTUD4, P4HB, RPS3A, SEC13, SKP1, USP34, USP9X, and YOD1) in tumor samples were validated by TCGA and GTEx databases. Overall survival and disease-free survival analysis also confirmed worse survival in patients with hub genes overexpression. The detected hub genes could be used as cancer biomarkers when EGF overexpresses.

As the most prevalent form of liver cancer, hepatocellular carcinoma (HCC) ranks the fifth highest cause of cancer-related death worldwide. Despite recent advancements in diagnostic and therapeutic techniques, the prognosis for HCC is still unknown. This study aimed to identify potential genes contributing to HCC pathogenicity. To this end, we examined the GSE39791 microarray dataset, which included 72 HCC samples and 72 normal samples. An investigation of co-expression networks using WGCNA found a highly conserved blue module with 665 genes that were strongly linked to HCC. APOF, NAT2, LCAT, TTC36, IGFALS, ASPDH, and VIPR1 were the blue module’s top 7 hub genes. According to the results of hub gene enrichment, the most related issues in the biological process and KEGG were peroxisome organization and metabolic pathways, respectively. In addition, using the drug-target network, we discovered 19 FDA-approved medication candidates for different reasons that might potentially be employed to treat HCC patients through the modulation of 3 hub genes of the co-expression network (LCAT, NAT2, and VIPR1). Our findings also demonstrated that the 3 scientifically validated miRNAs regulated the co-expression network by the VIPR1 hub gene. We found co-expressed gene modules and hub genes linked with HCC advancement, offering insights into the mechanisms underlying HCC progression as well as some potential HCC treatments.

The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic “master switch” in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

Studies have shown that MS results from synergism between genetic and environmental factors. As a genetic factor, the rs9267649 variant through the regulatory effect on the HLA-DRB1 expression is involved in the MS development. In addition, vitamin D deficiency through involvement of rs2248359 variant of CYP24A1 has shown to play important role in the risk of MS.

The aim of this study was to investigate both the HLA rs9267649 and CYP24A1 rs2248359 variants with risk of multiple sclerosis (MS) in Iranian population.

The rs9267649 and rs2248359 variants were genotyped in 82 Iranian Relapsing-Remitting Multiple Sclerosis (RRMS) patients and 100 matched healthy controls, using the PCR-RFLP method. The genotype and allele frequencies were calculated and statistically analyzed.

A significant difference was found in the allele distribution for the both rs9267649 and rs2248359 variants, such that the A allele of rs9267649 and the C allele of rs2248359 were found to be more frequent in MS patients than in the healthy controls (p-value: 0.009, OR: 2.264, 95% CI: 1.211-4.231 and p-value: 0.028 OR: 1.594, 95% CI: 1.052-2.415), respectively.

The present research results provide further evidence on the association of the two variants: rs9267649 of the HLA and rs2248359 of the CYP24A1 gene with MS etiology and an increased risk of MS in Iranian RRMS patients. However, further large-scale investigations in various ethnicities and in the functional genomics level are demanded to confirm our findings.

Microalgae have the potential to generate high-value products. The design of photobioreactors (PBRs), in which microalgae are cultured, is crucial because alterations in their configuration and operational conditions can affect the biomass production and productivity. The objective of this study was to optimize the diameter of the internal tube of an airlift PBR and to characterize the growth of Spirulina maxima in an optimized design. S. maxima was cultured in a mineral medium without an organic carbon source. The PBR consisted of an acrylic cylinder with an operational volume of 7 L. Daily determinations of biomass (by filtration), chlorophyll, N-NO3 and P-PO4 (spectrophotometrically) were carried out. The use of a concentric tube with a diameter of 3 inches led to an increased biomass concentration of 1.14 ± 0.136 g.L-1, allowing a global biomass productivity of 153 mg.L-1.d-1. The culture reached a volumetric consumption velocity of27.34 ± 1.596 and 2.29 ± 0.353 mg.L-1.d-1 for N and P, respectively. It was concluded that operational conditions must be specifically selected for each cultivated strain and that this configuration of airlift PBR can produce Spirulina biomass under laboratory conditions with a high biomass productivity.

Autolysate products from yeast origin are very interesting for food, feed, cosmetic, pharmaceutical, and fermentation industries. The lysis process greatly influences the quality and efficiency of the final autolysates. Here, we have compared four lysis methods based on autolysis, plasmolysis (with ethanol 1.5% (v/v) and coconut fatty acids 1% (w/w)) and hydrolysis (with alkaline protease 0.4 % (v/w)) on degrading the baker’s yeast Saccharomyces cerevisiae. The efficiency of processes was evaluated according to the recovered solid and protein contents, release of intracellular materials, cell viability, microscopy imaging, degree of hydrolysis and electrophoresis studies. Results showed that the increased recovered solids and proteins, as well as a higher degree of hydrolysis (DH) were obtained for the enzymatic hydrolyzed cells using alkaline protease. SDS-PAGE analysis also confirmed the results. Further, functionality of the final products by agglutination test showed that the hydrolyzed cells could effectively bind pathogenic bacteria compared to the other cell lysates. In conclusion, this work provides adequate evidence for efficiency of alkaline protease for producing the nutritional cell lysates from baker’s S. cerevisiae used in food, feed, cosmetic, and pharmaceutical applications. Moreover, this was the first report on using coconut fatty acids and alkaline protease in lysis of baker’s yeast.

Cyanobacteria can produce compounds absorbing ultraviolet irradiation. Mycosporine like amino acids (MAAs) are some of these important metabolites, which can be potentially considered as a sunscreen agent in the pharmaceutical and cosmetic industry. Different factors have been reported that can affect the biosynthesis of MAA. In this study, the influence of different concentrations of phosphate and nitrate under different environmental conditions on MAA production by Chlorella vulgaris was investigated using an experimental design method, in order to enhance MAAs production in this specious. A 23 full factorial design (FFD) using Design-Expert v7.0.0 software was used to optimize simultaneously all the three factors of nitrate and phosphate concentration and condition of incubation environment on the MAA production by this species of C. vulgaris. Two milliliter of organism stock were grown in 200 mL BG11 medium and after 21 days, the biomasses of all samples were separated. Then, the MAA was extracted from dried biomass using methanol extraction. The extracts were analyzed by reverse-phase high performance liquid chromatography (RP-HPLC). After complete analysis, four samples were then cultured at the optimized conditions and analyzed by liquid chromatohraphy coupled to mass spectrometry (LC/MS). The results showed that this microalga could produce compounds with λmax of 330 nm and a retention time of about 2 min. According to the central composite analysis, phosphate at 0.51 g.L-1 and nitrate at 2.5 g.L-1 can be considered as the optimum concentrations, resulting to the preferable conditions concerning the culture in germinator. Based on LC/MSS analysis, the major compound had a m/z of 332 at the optimum condition. Thus, this species is expected to have the capability of MAA production (maybe Shinorine) or one of its glycosylated derivatives.

Methyl-Tert-Butyl Ether (MTBE) as a gasoline modifier is frequently added to fuels and used in plenty of worldwide applications. MTBE biodegradation in groundwater occurs slowly and produces water miscibility; therefore, it causes diverse environmental and human health concerns. The interaction of MTBE with bovine serum albumin (BSA) as a model protein at physiological conditions is investigated to illustrate the possible interactions of MTBE with the body’s proteins. Uv–visible, fluorescence, circular dichroism (CD) spectroscopy methods, and molecular modeling were used to analyze the MTBE’s effect on BSA structure and dynamics. The constant protein concentration and various MTBE contents were used for possible interactions. The protein structural analysis shows that MTBE binds to BSA via positive enthalpy and entropy via hydrophobic interactions. Molecular docking shows the participation of several amino acids in the MTBE-BSA interaction. The CD spectroscopy results show that the BSA structure was not changed in the MTBE concentrations utilized in the study. Molecular dynamics (MD) simulation results suggest that MTBE can slightly change protein structure in the last 50ns. Comparing experimental and MD simulation results demonstrated that the BSA secondary structure was maintained in the low concentration of the MTBE. The entropy and enthalpy parameters asserted the hydrophobic interaction was the major force in the interaction between the BSA and MTBE.

Chitosan nanoparticles (CSNP) are becoming a popular alternative for delivering nucleic acids to tissues for gene transfer (gene therapy). The size and morphology of these biodegradable nano-carriers are adjustable, and their positive charge allows them to interact strongly with negatively charged nucleic acids. This study aimed to fabricate and characterize pcDNA3.1 (+) plasmid (pDNA) and CSNP complexes and determine the plasmid location in these vehicles. The characteristics of the pDNA/CSNP complex after production were investigated by SEM, XRD, DLS, TGA, and FTIR. The capacity of CSNP to form complexes with pDNA was investigated by labeling free plasmids with the fluorescent intercalating dye OliGreen. The stability of pDNA/CSNP in the presence of chitosanase was evaluated. Surface-Enhanced Raman Spectroscopy (SERS) for pDNA localization was performed, and absorption rate in BALB/c mice was assessed by real-time PCR. The optimum pDNA/CSNP ratio for plasmid complex formation was established to be 1:2 (w.w) by measuring spectroscopy. At these optimum complex formation ratios, spectroscopy, and gel digest experiments, SERS indicated that a part of the pDNA was present on the complex outer surface. The findings of plasmid absorption in mouse thigh tissue by real-time PCR revealed that the rate of gene uptake was significantly greater at a dose of 1:2 (w.w) of pDNA/CSNP than in other groups (P< 0.001). The findings of this study reveal exactly pDNA fits into polymer nanostructured delivery systems, allowing the formulation to be adjusted for selective distribution. This understanding will aid future research into the system’s functioning in vitro and in vivo.

In Escherichia coli (E. coli) culture, acetate accumulates as an undesirable by-product of aerobic fermentation on glucose and inhibits cell growth and recombinant protein production. We examined whether the heterologous expression of a eukaryotic heat shock protein (Hsp) can confer tolerance to acetate in E. coli. Transgenic cell lines (TCLs) heterologously expressing a small heat shock protein (sHsp) from carrot (Daucus carota L.), DcHsp17.7, were exposed to heat, sodium acetate, and alkaline conditions. The cell growth and cell viability were examined by measuring O.D.600 and colony-forming units (CFU), respectively. The His-tagged recombinant alcohol dehydrogenase (ADH) gene cloned in a pET11a expression vector was introduced into TCL1 and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. After purifying using Ni-NTA affinity chromatography, its accumulation levels were examined using SDS-PAGE in the presence of acetate. TCLs constitutively expressing DcHsp17.7 showed improved growth, cell density, and cell viability under the stress conditions of heat, acetate, and alkaline compared to an empty vector control line. In acetate stress conditions, TCL1 accumulated more cellular proteins (approximately 130%) than the control. The recombinant ADH accumulated to a higher level in TCL1 (2.2-fold at 16 °C) than the control. The addition of acetate reduced the recombinant ADH level by 70% in the control when compared with the absence of acetate. In contrast, recombinant ADH accumulation was not affected by acetate in TCL1. In the presence of acetate, TCL1 accumulated 6.4-fold more recombinant ADH than did the control. Furthermore, recombinant ADH produced in TCL1 showed 1.5-fold higher enzyme activity than that produced in the control in the presence or absence of acetate. Our study showed that heterologously expressed DcHsp17.7 from carrot can alleviate the negative effects of acetate on E. coli.