Iranian Journal of Biotechnology
Volume:20 Issue: 4, Autumn 2022

Electrospinning has been widely used to prepare nanofibrous scaffolds for bone tissue engineering. However, owing to the small pore size of electrospun scaffolds, cellular infiltration and tissue ingrowth are impossible. The use of sacrificial fibers (like poly ethylene oxide (PEO)) in combination with ultrasonication has been an appropriate way to increase the pore size of the electrospun scaffolds in our previous work. However, it is uneconomical due to the high cost of PEO. In this study, gelatin was chosen as a novel sacrificial agent in co-electrospun with polycaprolacton-nanohydroxyapatite (PCL-nHA). After electrospinnig, gelatin was washed with water, and the prepared scaffold was ultrasonicated. Morphological and structural properties of the prepared scaffolds were studied by SEM. Fourier transform infrared (FTIR) spectroscopy and water contact angle analysis were used to evaluate the removal of gelatin. According to the SEM results, the pore size of the modified scaffolds was increased 3-folds compared to the control sample. For PCL-nHA: gelatin (80:20) after the treatment, the average cell infiltration was 42.7 μm, while there was no infiltration for the control group. The modified electrospun scaffold significantly enhanced the osteogenic differentiation of hBMSCs as verified by increased ALP activity and upregulation of runt-related transcription factor 2 (RUNX2), collagen type 1 (COL1) and osteocalcin (OCN) genes. Co-electrospun PCL-nHA with gelatin as a sacrificial agent in combination with ultrasonication may be an effective, economic and controllable method to increase the pore size in electrospun scaffolds for bone tissue engineering applications.

Owing to the fact that the heart tissue is not able to repair itself. Biomaterial-based scaffolds are important cues in tissue engineering (TE) applications. Recent advances in TE have led to the development of suitable scaffold architecture for various tissue defects. Given the importance of cellular therapy, it was the aim of the present study to differentiate cardio myocyte cells from human adipose-derived mesenchymal stem cells (Ad-MSCs) using suitable induction reagents (namely, 5-azacytidine and transforming growth factor beta (TGF-β)) on poly-caprolactone (PCL)/Poly aniline (PANI) Nano fibrous scaffolds prepared by electrospinning. For this purpose, the adipose-derived mesenchymal stem cells (Ad-MSCs) were initially isolated and characterized before cultivation on the PCL/PANI Nano fibrous scaffold to be treated for 21 days with 5-azacytidine either singly or in combination with TGF-β in medium. The scaffold’s morphological and cell attachment properties were investigated using electron microscopy (SEM). Finally, the cardio myocyte differentiation of Ad-MSCs on the scaffold was studied using both quantitative Real-time PCR (qPCR) and flow-cytometry while the expression rates of the cardio myocytes’ specific genes (Gata4, NKX2.5, MYH-7, and Troponin I) were also determined. The results of Ad-MSCs culture, MTT assay, and SEM indicated that the cells had well proliferated on the PCL/PANI scaffolds, showing the biocompatibility of the nanofibers for cellular growth and adhesion. After 21 days of induced cardio myocyte differentiation by both agents, Real-time PCR revealed increases in the expressions of Gata4, Troponin I, MYH-7, and NKX2.5 genes in the cells cultured on the PCL/PANI scaffolds while the flow-cytometry test approved the expression of troponin I. The data obtained showed that the PCL/PANI Nano fibrous scaffolds were able to promote and support mesenchymal stem cell transformation to cardio myocyte cells. Generally speaking, the results of the study might be exploited in future in vitro and in vivo experimental model studies of cardio myocyte differentiation using co-polymer scaffolds.

Background:

 One neurodegenerative disorder that is caused by a mutation in the hSOD1 gene is Amyotrophic lateral sclerosis (ALS).

The current study was developed in order to evaluate the effect exerted by two ALS-associated point mutations, L67P and D76Y are located in the metal-binding loop, on structural characterization of hSOD1 protein using molecular dynamics (MD) simulations and computational predictions.

In this study, GROMACS was utilized to perform molecular dynamics simulations along with 9 different algorithms such as Predict SNP, PhD-SNP, MAPP, PolyPhen-1, Polyphen-2, SNP, SIFT, SNP&GO, and PMUT for predicting and also evaluating the mutational effect on the structural and conformational characterization of hSOD1.

Our study was done by several programs predicting the destabilizing and harmful effect exerted by mutant hSOD1. The deleterious effect of L67P mutation was predicted by MAPP and PhD-SNP algorithms, and D76Y mutation was predicted by 9 algorithms. Comparative studies that were conducted on mutants and wild-type indicated the altar in flexibility and protein conformational stability influenced the metal-binding loop’s conformation. The outcomes of the MD exhibited an increase and decrease of flexibility for D76Y and L67P mutants compared to the wild type, respectively. On the other hand, analysis of the gyration radius indicated lower and higher compactness for D76Y and L67P, respectively, suggesting that replacing amino acid at the metal-binding loop can alter the protein compactness compared with the protein the wild type.

Overall, these findings provided insight into the effect of mutations on the hSOD1, which leads to neurodegeneration disorders in humans. The results show that the mutations of L67P and D76Y influence the stability of protein conformational and flexibility associated with ALS disease. Thus, results of such mutations are can be a prerequisite to achieve a thorough understanding of ALS pathogenicity.

A disintegrin and metalloproteinase (ADAM) cell surface proteins are expressed in different cells and are involved in biological processes such as cell-cell interactions, cell differentiation, sperm attachment and fertilization. A significant number of ADAM isoforms are expressed in the reproductive tracts of male mice and other mammals, which shows the importance of this gene family in reproduction. The role of ADAM27 protein in reproduction was investigated. ADAM27 knock-out mutant mice were generated using the blastocyst microinjection technique. The knock-out mice were analyzed genetically and phenotypically to discover any abnormalities. The results of this study revealed that the homozygote mutant male mice were fertile and showed no significant differences compared to wild-type male mice. A histological exam, sperm analysis and in-vitro fertilization experiments showed no statistical differences. We can conclude that the role of deficient ADAM27 protein is probably compensated mainly by other ADAM isoforms which are expressed in the reproductive system.

The second most common cause of mortality is cancer. Increased NOX4 expression is linked to cancer development and metastasis. However, the significance of NOX4 in cell growth and assault, remains unclear. This study aimed to evaluate the effect of NOX4 knockouts in MCF7, UM-RC-6, HCA-7 cell lines. The NOX4 gene was knocked out in MCF7, UM-RC-6, and HCA-7 cell lines through using CRISPR Cas-9 genetic engineering techniques. After transfection, the CRISPR Cas-9 cassette, the T7 endonuclease I, qPCR, and western blotting assay detected the NOX4 knockouts. MTT and Annexin assessed the percentage of cell proliferation and apoptosis. Real-time PCR was used to measure the expression of pro- and anti-apoptotic genes. Occurrence of NOX4 gene knockout in the examined cell lines, was confirmed by q-PCR and Western blot (P<0.001). The NOX4-deleted cell lines with increased sub-G1 caused lowered cell proliferation and population at S / G2/ M phases. In Vitro, NOX4 silencing caused lowered expressions of anti-apoptosis genes BCL-2 and SURVIVIN (P<0.0001), leading to increased tendency of apoptosis in the cell lines (P<0.0001) of the apoptotic genes BAX, P53, FAS. Additionally, the MTT and Annexin results of the target gene NOX4 knockout inhibited proliferation, increased mortality rates (P<0.01), and increased apoptosis. The findings of this study indicate that using NOX4 as a target can have therapeutic value for creating potential treatments against breast, colorectal, and kidney cancers which shows a need for a deeper understanding of the biology of these cancers with direct clinical outcomes for developing novel treatment strategies.

Cholangiocarcinoma is a primary malignant tumor, and its progression involves oncogene activation, the absence of tumor suppressor gene, abnormal signaling pathways and miRNA expression. MiRNAs are abnormally expressed in many types of tumors.This study aims to observe the effects of miR-582 on cholangiocarcinoma cell proliferation, S-phase arrest, migration and invasion and to analyze the regulation of miR-582 on LIS1 to clarify the real role of miR-582 in cholangiocarcinoma development. TCGA database of cholangiocarcinoma samples was analyzed. Dual fluorescence reporter and TargetScan were conducted to confirm whether LIS1 was the target gene of miR-582. Effects of miR-582 and LIS1 on HCC-9810 cell proliferation, S-phase cell ratio, migration and invasion were determined by CCK-8, Flow cytometry and Transwell, respectively, whereas the function of miR-582 on MMP-2 and P-Akt expression was identified by Western blotting. Nude mice xenograft model of cholangiocarcinoma was established to detect what miR-582 did for tumor growth.TCGA showed that miR-582 was lowly expressed and LIS1 was highly expressed in tumor tissues compared with adjacent tissues. MiR-582 targeted LIS1 to inhibit MMP-2 and p-AKT expression. Transfection of miR-582 mimics could suppress HCC-9810 cell proliferation, S-stage arrest, migration and invasion, while LIS1 worked oppositely. MiR-582 inhibitors promoted cell biological behavior, whereas LIS1 siRNA was opposite. In nude mice xenograft model, miR-582 overexpression inhibited tumor growth.It implies that miR-582 could negatively regulate LIS1 to inhibit MMP-2 and P-Akt expression, thus suppressing cell invasion and proliferation in cholangiocarcinoma.

Nanoparticles can be chemically, physically, or biologically synthesized. Biosynthesis of silver nanoparticles (AgNPs) utilizing microbes is a promising process due to the low toxicity and high stability of AgNPs. Here, AgNPs were fabricated by Gram-negative Raoultella planticola. This study aimed to assess the ability of Raoultella planticola to produce nanoparticles (NPs) and evaluate their antibacterial potential against multidrug-resistant pathogens (MDR). Additionally, the study aimed to compare the antibacterial activity of biosynthesized nanoparticles to well-known conventional antibiotics Azithromycin and Tetracycline. AgNPs were characterized using visual observation, UV–visible spectroscopy (UV-vis), X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier-transform infrared spectroscopy (FTIR). The TEM and SEM were used to determine the size and shape of the nanoparticles. The XRD data were recorded in the 2θ ranging from 20-80° to analyze the crystalline structure of nanoparticles. The antibacterial activity was detected using a 96-well microtiter plate. The UV–vis absorption recorded from the 300 – 900 nm spectrum was well defined at 420 nm, and the XRD pattern was compatible with Braggs’s reflection of the silver nanocrystals. FTIR showed absorbance bands corresponding to different functional groups. TEM and SEM images showed non-uniform spherical and AgNPs of 10-80 nm. XRD data confirmed that the resultant particles are AgNPs. The AgNPs showed effective activity against multi-drug resistant (MDR) Pseudomonas aeruginosa, Salmonella sp., Shigella sp., E. coli, Enterobacter sp., Staphylococcus aureus, and Bacillus cereus. The AgNPs demonstrated effectiveness in lower concentrations compared to broad-spectrum antibiotics. These data reveal that AgNP generated by R. planticola was more efficient against MDR microorganisms than commercial antibiotics. However, the cytotoxicity of these nanoparticles must be further studied.

Fungal extracts have received increased attention due to their great medicinal applications including antitumor, immune-modulating, antioxidant, radical scavenging, antiviral, antibacterial, antifungal and detoxificating properties.

This study is the first report on a novel bioactive compound, namely Childinan SF-2 which was isolated from soil ascomycete fungus. The significant antibacterial, antioxidant and cytotoxic properties of the extract may lead to development of novel, safe and useful substances.

The isolate was identified on the basis of molecular approach. Spore suspension was inoculated in the culture medium and the bioactive compound was isolated from the viscous fermented broth via ethanol precipitation of the extracellular compound. The basic chemical composition of the extract including protein, carbohydrate, sulfate radical and uronic acid content were measured. FTIR (Fourier-transform infrared spectroscopy) and GC–MS (Gas chromatography–mass spectrometry) analysis were used for further structural characterization. The extract was utilized for treatment of AGS and MDA cell lines to assess the cell cycle and apoptosis. The antioxidant activity was examined using DPPH, hydroxyl radicals scavenging, β-carotene bleaching inhibition and ferric reducing power assay methods. The extract was tested for evaluation of antibacterial activity using different Gram-positive and Gram-negative bacterial strains

The fungal isolate was identified as the new strain Daldinia childiae SF-2. Initial biochemical characterization of the extract showed that the fungal biopolymer was composed of total sugars, protein, uronic acids and sulfated groups with values of 91.6%, 2.15%, 2.25% and 1.05% (w/w), respectively. FTIR and GC–MS analysis revealed that Childinan SF-2 might be mainly constructed from D-glucose, D-mannitol and D-galactofuranose. The in vitro experiments revealed that Childinan SF-2 enhanced the percentage of necrosis and apoptosis of cancer cells and blocked the cell cycle progression as shown by flowcytometry. Childinan SF-2 represented a considerable antioxidant and antibacterial activity.

These results indicated that Childinan SF-2 can serve as a potential source in medicinal applications.

The Forumad chromite area from Sabzevar ophiolite belt, Northeastern Iran, is an environment with high concentration of heavy metals, particularly chromite and magnesite minerals, containing chromium and magnesium.

In this study for the first time, we analyzed and report the diversity of microbial (bacterial and archaeal) community inhabiting in Forumad chromite mine environment using metagenomics approach.

Samples were obtained from different areas of the mine, and total DNA was extracted from water and soil samples. 16S rDNA was amplified using universal primers and the PCR products were cloned in pTz57R/T plasmid. Then, 43% of the positive clones were randomly sequenced. BLAST program in NCBI and EzTaxon databases were used to identify similar 16S rDNA sequences. Phylogenetic analysis was performed using the MEGA5 software and multiple alignments of sequences.

In the phylogenetic analyses, proteobacteria, which contains many heavy metals tolerant bacteria especially chromium, were the dominant population in bacterial libraries with Rheinheimera and Cedecaeas the most abundant genuses. Other phyla were Bacteroidetes, Firmicutes, Verrucomicrobia, Chloroflexi, Actinobacteria, Acidobacteria, Cyanobacteria, Gemmatimonadetes, and Planctomycetes. In the archaeal clone library, all the sequences were related to the phylum Thaumarchaeota. Further, 68.6% of the sequences had less than 98.7℅ similarity with the recorded strains which could represent new taxons.

The results showed that there was a high microbial diversity in the Forumad chromite area. These results can be used for detoxification and bioremediation of regions contaminated with heavy metals, although more studies are needed.

Soybean is an important oilseed crop that its development and production are affected by environmental stresses (such as saline-alkaline and water deficit). This experiment was performed with the aim of identifying candidate genes in saline-alkaline stress and water-deficit stress conditions using transcriptome analysis and to investigate the expression of these genes under water deficit stress conditions using RTqPCR. In this experiment, soybean transcriptome data under saline-alkaline and water-deficit stress were downloaded from the NCBI website, and then the co-expression modules were determined for them and the gene network was plotted for each module, and finally, the hub genes were identified. To compare the expression of genes in saline-alkaline and water deficit conditions, soybean plants were subjected to water deficit stress and their gene expression was determined using RTqPCR. The filtered (Log FC above +2 and below -2) genes of soybean were grouped under saline-alkaline stress in 15 modules and under water-deficit stress in 2 different modules. Within each module, the interaction of genes was identified using the gene network, then three genes of ann11, cyp450 and zfp selected as hub genes. These hub genes are highly co-expression with other network genes, which not only display differential expression but also differential co-expression. The results of RT-PCR indicated that cyp450 gene expression was not significantly different from the control, while ann11 gene expression significantly increased under water deficit stress, but zfp gene expression decreased significantly under water deficit stress. We identified three genes, ann11, cyp450 and zfp, as hub genes. According to our results, ann11 gene had a significant increase in expression under water deficit stress, which can indicate the importance of this gene under drought conditions. Therefore, according to the results of this experiment as well as other researchers, we introduce this gene as a key gene in water deficit tolerance and recommend its use in genetic engineering to increase the tolerance of other plants.