Modares Journal of Biotechnology

Abstract Background: Carotenoids are biological antioxidants and play important roles in protecting the body from diseases and aging. Canthaxanthin is one of the most widely used carotenoids in the industry and medicine. This study aimed to investigate the biological properties of canthaxanthin pigment as well as its production optimization in a low-cost medium using a radioresistant microbial strain named Dietzia maris.
Materials and methods: Bacterial carotenoids were extracted and its antibacterial, anti-tumor, and cytotoxicity properties were investigated. Then, the effect of Krebs intermediates and pH on the production of pigment and microbial biomass in the whey medium was investigated using the response surface methodology.
Results: Maximum pigment production was found to be 92/54 mg/l in whey culture medium at pH 8 and in the presence of 12.5 mM of each of citrate, glutamate, malate, and succinate by the response surface method. The pigment did not show any cytotoxic effect on Hela, HFB, and MCF-7 cell lines. Besides, the pigment did not have any antibacterial properties.
Conclusion: Radioresistant microbial strains are better candidates for microbial pigment production due to their stability and high antioxidant activity. In this study, a whey culture medium was used to reduce the production cost of canthaxanthin. The addition of Krebs intermediaries in the fermentation medium increased the pigment production by Dietzia maris significantly.

Abstract In the present work, we describe the synthesis of silver nanoparticles (Ag-NPs) using seed aqueous extract of Psidium guajava (PG) and its antibacterial activity. UV–visible spectroscopy, X-ray diffraction (XRD), Fourier transform infra red spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray energy dispersive spectrophotometer (EDAX) were performed to ascertain the formation of Ag-NPs. It was observed that the growths of Ag-NPs are stopped within 35 min of reaction time. The synthesized Ag-NPs were characterized by a peak at 446 nm in the UV–visible spectrum. XRD confirmed the crystalline nature of the nanoparticles of 10-20 nm size. The XRD peaks at 38◦, 44◦, 64◦ and 77◦ can be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) Bragg’s reflections of cubic structure of metallic silver, respectively. The FTIR result clearly showed that the extracts containing OH as a functional group act in capping the nanoparticles synthesis. Antibacterial activities of Ag-NPs were tested against the growth of Gram-positive (S. aureus) using SEM. The inhibition was observed in the Ag-NPs against S. aureus. The results suggest that the synthesized Ag-NPs act as an effective antibacterial agent. It is confirmed that Ag-NPs are capable of rendering high antibacterial efficacy and hence has a great potential in the preparation of used drugs against bacterial diseases. The results confirmed that the (PG) is a very good eco friendly and nontoxic source for the synthesis of Ag-NPs as compared to the conventional chemical/physical methods.

Abstract Graphene-based nanomaterials are being investigated for their biocompatibility and bioactivity, as well as their ability to improve osteogenic differentiation. In this research, the base material, reduced graphene oxide (rGO) sheets, were decorated with hydroxyapatite and strontium (rGO / HAp-Sr) to induce osteogenic differentiation in adipose-derived mesenchymal stem cells. Different techniques were used to determine the properties of the nanocomposite such as diffraction analysis techniques (XRD) and transmission electron microscopy (to evaluate the size and morphology of HAp-Sr on rGO plates), FT-IR (to analyze the nanocomposite functional group), Raman spectroscopy (to investigate possible disorders in nanocomposite structure and number of layers), induced dual plasma emission spectroscopy (to assess atomic concentration of Ca and Sr), zeta potential(electrical potential of the nanocomposite) and MTT (nanocomposite cytotoxicity assessment) were used. The ossification potential of the synthesized nanocomposite was investigated and confirmed using the calcium deposition test in dipose-derived mesenchymal stem cells. According to the obtained results, osteogenic differentiation induction is possible using synthesized nanocomposites without the need for chemical inducers.

Abstract Enzymes play an essential role in catalyzing the reactions for multiple industrial applications. One of these critical industries with a worldwide application is paper and pulp, which is cost-effective in increasing attention. Xylanases are potential enzymes that proved their abilities in a broad range of applications, specifically in the paper and pulp industry as a biobleaching agent and dye removal biocatalyst. In these decades, the production of novel enzymes from natural sources is conceivable, especially with applying the culture-independent method of metagenome. This practical approach provides the opportunity to identify the novel enzymes from uncultivable microbial diversities. Concerning the importance of the thermostable enzymes for industrial applications and their better action in harsh conditions, this study aimed to identify novel thermostable xylanase from metagenomic data of sheep rumen by applying the in-silico screening. The thermostable xylanase was extracted from the ruminal DNA and after cloning and expression named PersiXyn5. The enzymeschr('39') kinetic parameters, including Km, Vmax, and its specific activity, were examined. The enzyme was optimally active at 80 and pH 8 and could retain 58% of its maximum activity after 2h of incubation at 90 . The thermostable, alkali PersiXyn5 was an efficient enzyme in the paper industry and poultry feed and fuel applications.

Abstract Introduction: The present study reports a case of familial episodic coma in which three girls manifesting refractory seizures followed by coma. Targeted gene panel of epilepsy using next-generation sequencing (NGS) technique was requested to identified disease -causing variant(s) in the patients.
Materials and Method: After obtaining a written informed consent from our patient, genomic DNA was extracted from venous blood, for identifying mutations in epilepsy genes, at first, coding regions as well as all intron–exon boundaries of the 72 genes were captured by Sure Select Target Enrichment System V4 kit (Agilent, Santa Clara, CA), then captured libraries were sequenced on an Illumina HiSeq 4000(Illumina Inc., San Diego, CA, USA), sequenced reads were aligned with a reference human genome and Picard tools was used to remove duplicated reads; variant calling was performed using the Genome Analysis Tool Kit (GATK). ANNOVAR was used to annotated variants, then all variants were filtered out based on minor allele frequency (MAF) <1 % according to data bases of nucleotide including dpSNP, 1000 Genome project. In silico tools was performed to evaluated pathogenicity of variant(s).
Result: According to databases of pathogenicity prediction of gene, no specific mutation in epilepsy genes was found in our patients, but several polymorphisms were reported.
Conclusion: Given polymorphisms in genes related to epilepsy were found in our study, failed to provide us with an acceptable diagnosis of this condition, further research is needed to reveal the cause of the disease.
Key word: episodic coma, NGS, gene panel, epilepsy

Abstract
Chitinases are essential enzymes in crustaceans that play an important role in the molting cycle and digestion of chitin. Based on the present study, the chitinase encoding cDNA of Penaeus mergueinsis with a length of 1440 bp containing 467 amino acids was sequenced by PCR and then its phylogenetic and bioinformatics analysis was performed. The new sequence was registered in the gene bank with the accessition number MT250539 and the molecular weight of the protein resulting from this sequence was predicted to be 51.84 KDa and the theoretical isoelectric point of 4.79. Comparison of amino acid sequences among penaeid chitinases showed the highest identification (about 97 to 92%) with P. mondon chi-3, F. chinensis, P. vannamei and P. japonicus chi-3, respectively. Phylogenetic studies showed that chitinase in the present study belongs to group 3 chitinases. Revealed protein pattern analyzes showed that chitinase from P. mergueinsis contained the catalytic domain Glyco-18 at position 2-347, a chitin-binding site of pritrophin A at position 403-456, a disulfide bridge formed by two cysteines at position 436-421 is a chitin-binding domain type 2, active site (¹¹7FDGLDMDWE¹²⁵), a proline / threonine-rich region at positions 376-412, and a putative N-glycosylation site at position 427-424 (NTSG). The present study shows that the P. mergueinsis sequence contains active chitinase motifs similar to previously sequenced chitinases, and in the case of cloning, expression and purification probably has functional and structural features similar to the enzymes of the above species.

Abstract Aim: During the uncontrolled development of cells in the body, a subset of neoplasms or tumors is formed, the abnormal proliferation of these cells leads to the formation of a mass and eventually cancer. This mass can spread throughout the body. Thus, inhibiting the abnormal growth of cancer cells will have a significant effect on preventing the spread of cancerous tumors and improving the disease. Therefore, in the present study, a new sulfonamide derivative was designed and synthesized (HB20) and its anti-cancer effects on human breast cancer cell line (MCF-7) were investigated.
Materials and Methods: For the synthesis of a sulfonamide derivative (HB20), dcriptiazonium salt was first made using a sulfamethoxazole base compound and then combined with a pyrimidine coupling agent. Concentrations of a new synthetic compound (HB20) against Cells (MCF-7) were used. MTT assay was also performed to measure survival and cell proliferation.
Results: The synthesized compound structure was confirmed by spectral analysis, such as FT- IR, and NMR. Also, Survival in MCF-7 cells treated with a synthetic compound (HB20) was significantly reduced compared to the control group (untreated). HB20 inhibits the proliferation of MCF-7 cancer cells with an IC₅₀ value of 75/23 μg/ml.
Conclusion: The new sulfonamide derivative (HB20) has the potential to inhibit proliferation and anti-cancer properties in the cell line (MCF-7).

Abstract Activin A, a member of the transforming growth factor-β (TGF-β) superfamily, plays a central role in numerous physiological processes such as cell differentiation, tissue repair, angiogenesis, differentiation of stem cells, cell adhesion and apoptosis. Because of its various clinical usages, recombinant production of it is beneficial. Since E. coli is one of the most popular hosts for recombinant protein production, in this study, cytoplasmic expression in this strain was used to produce high levels of Activin A. So, the cDNA of the Activin A mature region was amplified and then cloned in pET28a(+) vector. The resulting vector was transformed to BL21(DE3), BL21(DE3)plysS, and BL21(DE3)Rosetta-gami strains. After induction the promoter by using IPTG, Activin A production was confirmed by SDS-PAGE and Western blotting assays. The results showed that the expression of Activin A in the cytoplasm of all three strains was an efficient approach to obtain high levels of recombinant protein, but BL21(DE3) strain produced more protein. At the next step in order to achieve soluble form of Activin A, co-expression of cytoplasmic chaperones TF, GroEL/ES, and DnaK with pET28a (+) vector was used. The SDS-PAGE and Western blotting results showed that co-expression of Activin A with cytoplasmic plasmid pGro7 containing GroEL and GroES chaperones, in BL21(DE3) strain is an efficient approach for producing of soluble Activin A.

Abstract In most cancers, the expression level of heat shock proteins is increased, which makes cancer cells resistant to drug therapy and has a poor prognosis. They have also been linked to cell proliferation, invasion and metastasis. Lycopene, as a carotenoid, is an effective antioxidant used to prevent the growth of cancerous glands. Prostate cancer is one of the most common cancers seen in men. To treat this disease, surgery and chemotherapy are mostly used, which have many complications after treatment and are costly. In this study, prostate cancer was treated with lycopene and raw microarray big data were received from GEO section of NCBI database. Then, the expression changes of heat shock proteins (hsp27) were determined using bioinformatics tools and methods, and comparing the expression levels of lycopene-treated genes with non-treated ones showed that the expression level of HSPB8 gene was drastically reduced and also, no significant changes were observed in the expression level of other gene families in this group. Results show that lycopene can cause stress in cancer cells and this stress predisposes the cell to apoptosis.

Abstract Klebsiella pneumoniae is a gram-negative bacillus of the Enterobacteriaceae family. Despite being part of the natural human microflora, this is an opportunistic pathogen and a major cause of nosocomial infections. The increased emergence of multidrug resistance in Klebsiella pneumoniae has limited the treatment options for this bacterium. Carbon nanotubes (CNT), by improving the stability and solubulity of drugs, could increase the effectiveness of drugs for treatment. The aim of this study is to investigate the antibacterial effect of nanofluid containing functionalized multi-walled carbon nanotubes (f-CNT-NF) on Klebsiella pneumoniae isolated from clinical specimens. For the strain confirmation, biochemical ,API20E kit, and additional differential tests were performed, and antibiotic susceptibility test was performed by the disk diffusion method. The studied strain showed a resistance to all antibiotics such as cefepime.The minimum inhibitory concentration (MIC) was determined using the antibiotic micro dilution method. The MIC was determined in five effect modes including antibiotic (Ab), nanofluid containing functionalized multi-walled carbon nanotubes (f-CNT-NF) , nanofluid containing multi-walled carbon nanotubes (CNT-NF) ,Ab in combination with f-CNT-NF and Ab with CNT-NF. Nevertheless the individual effects of 10 µg mL^-1 cefepime or 80 µg of nanofluid with f-CNT-NF did not inhibit the growth of the bacteria, but the co-administration of 10 µg mL^-1 cefepime with 80 µg of the f-CNT-NF could inhibit the bacteria`s growth. It was concluded that f-CNT-NF could be more effective in drug delivery at lower concentrations than the free state, which could be used as a tool for optimal drug delivery.