Iranian Journal of Biotechnology
Volume:21 Issue: 2, Spring 2023

STK11 mutation in LUAD affects immune cell infiltration in tumor tissue, and is associated with tumor prognosis. This study aimed to construct a STK11 mutation and immune-related LUAD prognostic model. The mutation frequency of STK11 in LUAD was queried via cBioPortal in TCGA and PanCancer Atlas databases. The degree of immune infiltration was analyzed by CIBERSORT analysis. DEGs in STK11mut and STK11wt samples were analyzed. Metascape, GO and KEGG methods were adopted for functional and signaling pathway enrichment analysis of DEGs. Genes related to immune were overlapped with DEGs to acquire immune-related DEGs, whose Cox regression and LASSO analyses were employed to construct prognostic model. Univariate and multivariate Cox regression analyses verified the independence of riskscore and clinical features. A nomogram was established to predict the OS of patients. Additionally, TIMER was introduced to analyze relationship between infiltration abundance of 6 immune cells and expression of feature genes in LUAD. The mutation frequency of STK11 in LUAD was 16%, and the degrees of immune cell infiltration were different between the wild-type and mutant STK11. DEGs of STK11 mutated and unmutated LUAD samples were mainly enriched in immune-related biological functions and signaling pathways. Finally, 6 feature genes were obtained, and a prognostic model was established. Riskscore was an independent immuno-related prognostic factor for LUAD. The nomogram diagram was reliable. Collectively, genes related to STK11 mutation and immunity were mined from the public database, and a 6-gene prognostic prediction signature was generated.

In animals and plants, antimicrobial peptides (AMPs) are crucial components of defense mechanisms, as they play a crucial role in innate immunity, which protects hosts from pathogenic bacteria. The CM15 has attracted considerable interest as a novel antibiotic against gram-negative and positive pathogens. The aim of this study was to investigate the permeation potential of the CM15 with membrane bilayers of Staphylococcus aureus and Escherichia coli. The bilayer membranes of Escherichia coli and Staphylococcus aureus were modelled with the resemblance in lipid composition to its biological sample. This study followed Protein-Membrane Interaction (PMI) through successive applications of molecular dynamics simulation by GROMACS and CHARMM36 force field for two sets of 120-ns simulations. Significant results were obtained from analyzing the trajectory of the unsuccessful insertion of CM15 during simulation. Our data suggested that Lysine residues in CM15 and Cardiolipins in membrane leaflets play a crucial role in stability and interaction terms. The obtained results strengthen the insertion possibility through the toroidal model, which should consider for further studies on AMPs interaction.

Bioleaching is a practical method to recover metals from low-grade mineral sulfides. The most frequent bacteria involved in the bioleaching of metals from ores are Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Experimental design is a method through which the optimum activity condition will be obtained, avoiding numerous trials and errors. This study aimed to optimize the bioleaching condition of two indigenous iron- and sulfur-oxidizing bacteria from the Meydouk mine, Iran, and evaluate their function in a semi-pilot operation in pure and mixed cultures. After treatment with sulfuric acid, the bacterial DNA was extracted, and further 16S rRNA was sequenced to characterize the bacterial species. The cultivation condition of these bacteria was optimized using Design-expert (6.1.1 version) software. The copper recovery rate and the differentiation in the ORP rate in the percolation columns were also investigated. These strains were isolated from the Meydouk mine for the first time. 16S rRNA analysis revealed that both bacteria belong to the Acidithiobacillus genus. The factors with the most significant impact on Acidithiobacillus ferrooxidans with their optimum level were temperature=35 °C, pH=2.5, and initial FeSO4 concentration=25 g.L-1. Also, initial sulfur concentration had the most significant impact on Acidithiobacillus thiooxidans with the optimum level of 35 g.L-1. Moreover, the mixed culture determined higher bioleaching efficiency compared with the case of employing the pure cultures. Utilizing a mixture of both bacteria, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans elevated the Cu recovery rate due to the synergetic function of the strains. Also, introducing an initial dosage of sulfur and pre-acidification could elevate metal recovery efficiency.

Q-PCR is the method of choice for PCR- based transcriptomics and validating microarray-based and RNA-seq results. Proper application of this technology requires proper normalization to correct as much as possible errors propagating during RNA extraction and cDNA synthesis

The investigation was performed to find stable reference genes in sunflower under shifting in ambient temperature.

Sequences of five well-known reference genes of Arabidopsis (Actin, Ubiquitin, Elongation factor-1, GAPDH, and SAND) and one well-known reference gene in اhuman, Importin, were subjected to BLASTX against sunflower databases and the relevant genes were subjected to primer designing for q-PCR. Two sunflower inbred lines were cultivated at two dates so that anthesis occurred at nearly 30 °C and 40 °C (heat stress). The experiment was repeated for two years. Q-PCR was run on samples taken for two planting date separately at the beginning of anthesis for each genotype from leaf, taproots, receptacle base, immature and mature disc flowers and on pooled samples comprising of the tissues for each genotype, planting dates and also all tissues for both genotypes and both planting dates. Basic statistical properties of each candidate gene across all the samples were calculated. Furthermore, gene expression stability analysis was done for six candidate reference genes on Cq mean of two years using three independent algorithms, geNorm, Bestkeeper, and Refinder.

Designed primers for Actin2, SAND, GAPDH, Ubiquitin, EF-1a, and Importin yielded a single peak in melting curve analysis indicating specificity of the PCR reaction. Basic statistical analysis showed that Actin2 and EF-1a had the highest and lowest expression levels across all the samples, respectively. Actin2 appeared to be the most stable reference gene across all the samples based on the three used algorithms. Pairwise variation analysis revealed that for samples taken under ambient temperature of 30 °C, Actin2, EF-1a, SAND and for those taken under ambient temperature of 40 °C, Actin2, EF-1a, Importin and SAND have to be used for normalization in q-PCR studies. Moreover, it is suggested that normalization to be based on Actin2, SAND and EF-1a for vegetative tissues and Actin2, EF-1a, SAND and Importin for reproductive tissues.

In the present research, proper reference genes for normalization of gene expression studies under heat stress conditions were introduced. Moreover, the presence of genotype-by- planting date interaction effects and tissue specific gene expression pattern on the behavior of the most three stable reference genes was indicated

In the CNS, glial cells are involved in neuroinflammation and neuropathic pain. The glial cells are activated by a variety of pathological conditions and release pro-inflammatory mediators, including nitric oxide (NO). Overexpression of iNOS (inducible nitric oxide synthase) and extra NO is detrimental to neurophysiology and neuronal viability. This study aimed to examine the effect of Gnidilatimonein isolated from D. mucronata and its leaves extract (as natural phytochemicals) on NO production in the LPS-induced primary glial cells. A preparative HPLC method was used to isolate gnidilatimonoein from leaves ethanolic extract. Various doses of Gnidilatimonoein, the ethanolic extract were applied to primary glial cells inflamed by lipopolysaccharide. A Colorimetric test, an MTT assay, and a RT-PCR analysis were then performed to analyze and compare NO production, cell viability, and iNOS expression. Gnidilatimonoein treatment of pretreated primary glial cells significantly inhibited iNOS expression and decreased NO synthesis. Plant extracts also reduced NO production in inflamed microglial and glial at 0.1-3 mg.mL-1. At these concentrations, none of these compounds exerted a cytotoxic effect, suggesting that their anti-inflammatory effects were not due to the death of cells. This study indicates that D. mucronata and its active compound, Gnidilatimonoein, could have restrained effects on the expression of iNOS on the induced glial cells; however, further investigation is warranted.

SRC is a member of the membrane-associated non-receptor protein tyrosine kinase superfamily. It has been reported to mediate inflammation and cancer. However, the exact molecular mechanism involved is still not clear. The current study was designed to explore the prognostic landscape of SRC and further investigate the relationship between SRC and immune infiltration in pan-cancer. Kaplan-Meier Plotter was used to detect the prognostic value of SRC in pan-cancer. Then using TIMER2.0 and CIBERSORT, the relationship between SRC and immune infiltration in pan-cancer was evaluated. Furthermore, the LinkedOmics database was used to screen SRC co-expressed genes, followed by functional enrichment of SRC co-expressed genes by Metascape online tool. STRING database and Cytoscape software were applied to construct and visualise the protein-protein interaction network of SRC co-expressed genes. MCODE plug-in was used to screen hub modules in the PPI network. The SRC co-expressed genes in hub modules were extracted, and the correlation analysis between interested SRC co-expressed genes and immune infiltration was conducted via TIMER2.0 and CIBERSORT. Our study demonstrated that SRC expression was significantly associated with overall survival and relapse-free survival in multiple cancer types. In addition, SRC expression was significantly correlated with the immune infiltration of B cells, dendritic cells, CD4+ T cells, macrophages, and neutrophils in pan-cancer. The expression of SRC had shown to have close correlations with M1 macrophage polarisation in LIHC, TGCT, THCA, and THYM. Moreover, the genes that co-expressed with SRC in LIHC, TGCT, THCA, and THYM were mainly enriched in lipid metabolism. Besides, correlation analysis showed that SRC co-expressed genes associated with lipid metabolism were also significantly correlated with the infiltration and polarisation of macrophages. These results indicate that SRC can serve as a prognostic biomarker in pan-cancer and is related to macrophages infiltration and interacts with genes involved in lipid metabolism.

Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis.

Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster′s ovary cells producing erythropoietin.

The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein.

BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 μM.ml-1 Vs. 2505 μM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 μM oleuropein compared to the control cell line (p-value=0.0002).

CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.

In this study, chitosan with various deacetylation degrees was extracted from crayfish (Astacus leptodactylus) shells with the purpose of examining the effect of deacetylation on the characterization of chitosan. Recycling of wastes has become an important issue with the advancement of shellfish processing technology. Therefore, this study examined the most important and conventional characterization parameters of chitosan extracted from crayfish shells and investigated whether crayfish chitosan can be an alternative to commercial products. In order to determine the characterization of the chitosan; degree of deacetylation, yield, molecular weight, apparent viscosity, water binding capacity, fat binding capacity, moisture content, ash content, color properties, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and X-ray diffraction analyses (XRD) were applied. The low (LDD) and high (HDD) deacetylated crayfish chitosan characterization results in terms of yield, molecular weight, apparent viscosity, water binding capacity, fat binding capacity, moisture content, ash content were 17.50%, 424.03-334.66 kDa, 16.82-9.63 cP, 481.29-428.04%, 419.30-355.75%, 3.32-1.03%, 0.98-1.01%, respectively. As detected by two different methods, potentiometric titration and elemental analysis, the deacetylation degrees of low and high crayfish chitosan were found close to each other, which were 76.98-94.98% and 73.79-92.06%, respectively. As the deacetylation period extended, acetyl groups were removed, and the degree of the deacetylation of crayfish chitosan increased while the apparent viscosity, molecular weight, water and fat binding capacity decreased. The findings of the present study are important to obtain the chitosan having various physicochemical characteristics from unevaluated crayfish wastes and to use it in many different sectors, especially biotechnology, medicine, pharmaceutical, food, and agriculture.

The occurrence of aflatoxins in food products is a silent threat to human health worldwide. A range of strategies has been introduced to address the bioavailability of aflatoxins, which are considered microbial tools to provide a low-cost and promising approach. The present study focused on the separation of yeast strains from the homemade cheese rind layer to investigate the ability of native yeasts to eliminate AB1 and AM1 from simulated gastrointestinal fluids. Homemade cheese samples were prepared from different locations in Tehran provinces and yeast strains were isolated and identified through the biochemical methods and molecular analysis of internal transcribed spacer and D1/D2 domain of 26S rDNA regions. Isolated strains were screened using simulated gastrointestinal fluids, and the ability of yeast strains to absorb aflatoxin was evaluated. Out of 13 strains, 7 yeast strains were not affected by 5 ppm AFM1 while 11 strains did not show any significant response to 5 mg.L-1 (ppm) of AFB1. On the other hand, 5 strains were able to successfully tolerate 20 ppm AFB1. Candidate yeasts showed different abilities to remove aflatoxins B1 and M1. In addition, C. lusitaniae, G. geotrichum, G. candidum, and C. sanyaensis exhibited a significant ability to detoxify aflatoxins from the gastrointestinal fluid, respectively. Our data suggest that yeast communities with essential effects on the quality of homemade cheese appear to be precise candidates for the potential elimination of aflatoxins from the gastrointestinal fluid.

Over expression of Reteplase enzyme has already been studies in the periplasmic space of Escherichia coli (E. coli). However, the role different factors in its expresssin rate remained to be elucidated. Optical cell density (OD), IPTG concentration, and expression time are highly effective in the protein expression rates. Therefore, we aimed to determine the optimum levels of these factors for reteplase expression using response surface methodology (RSM). The pET21b plasmid was used to sub-clone the designed reteplase gene. Then, the gene was transformed into E. coli BL21 strain. Induction of expression was done by IPTG and analyzed by the SDS page. experiments were designed using the RMS, while the effects of different conditions were evaluated using the Real time-PCR. Sequence optimization removed all undesirable sequences of the designed gene. Transformation into E. coli BL21 was confirmed with an 1152 bp band on the agarose gel. A 39 kDa expression band on the SDS gel confirmed the gene expression. Performing 20 RSM-designed experiments, the optimum levels for IPTG concentration and OD were determined as 0.34mM and 5.6, respectively. Moreover, the optimum level of expression time was demonstrated to be 11.91 hours.The accuracy of the regression model for reteplase overexpression was confirmed by an F-value equal to 25.31 and a meager probability value [(Prob > F) < 0.0001]. The real-time-PCR results indicated that the performed calculations were highly accurate. The obtained results indicate that IPTG concentration, OD, and expression time are significantly involved in the augmentation of recombinant reteplase expression. To the best of our knowledge, this is the first study to assess the combined effect of these factors on reteplase expression. Further RSM-based experiments would bring about new insights regarding the best conditions for reteplase expression.