Modares Journal of Biotechnology

Abstract Background:
HIV has at least six regulatory genes among which the Vif protein can control HIV replication. This study, as the first report, investigated the important mutations in VIF protein in sequences from Iranian patients and using immunoinformatics, conserved regions of this protein and B-Cell, T-Cell and CTL epitopes to stimulate the immune system, were determined.
Methods:
VIF sequences were obtained from NCBI GenBank, and tertiary structures, B-Cell, T-Cell and CTL epitopes were predicted by bioinformatics tools; besides, their antigenic and allergenic properties were studied.
Results:
The most prevalent mutations in Vif protein were related to S 49 P (90%), S 140 N and N 186 S (80%). Two substitutions at positions 41 and 42 were introduced which have effect on Vif binding to host factor. In addition, three regions were identified as the best epitope sequences with high potential to induce immune system and the lowest allergic properties, among which 5-32 region was suggested as the best vaccine candidate regions.
Conclusion:
This study as the first study from Iran using immunoinformatics tools to introduced a region with the high potential to induce humoral and cellular immune systems and lowest allergenic properties which can be used for further studies on HIV vaccines.

Abstract In this study, the genetic diversity of 10 different accessions of Andrographis paniculata was investigated using protein and SRAP markers. In the vegetative stage, protein and DNA were extracted from the leaves. The results of protein profile indicated a total of 20 bands with 64.15% polymorphism. To evaluate genetic diversity at the DNA level, 6 SRAP primers were used and a total of 583 scalable bands were observed. A total of 549 bands had polymorphism with an average of 91.5 for the studied primers. The highest polymorphism (99.12%) and the lowest polymorphism (84.21%) were observed in E1/M1 E2/M2 primers, respectively. Cluster analysis produced four main clusters. Genetic diversity indices were calculated for all gene loci, including the average genetic diversity of Nei’s (0.27) and the mean of Shannon’s index with a value of 0.41. High level of population differentiation (Gst = 0.79) and good level of gene flow (Nm = 1.3) were estimated between the grouped populations. Molecular analysis of variance showed that intra-population variance (58%) was higher than inter-population variance (42%). Overall, the results of study showed a high genetic diversity in both protein electrophoresis pattern and in polymorphic bands separated using SRAP markers with emphasis on the greater efficiency of SRAP markers than protein markers, which can be selected in parents with genetic distance. It is widely used to produce dispersing and mapping populations in hybridization programs and to breed or improve desirable traits, as well as to protect and manage the germplasm of this plant.

Abstract Betulin is a pentacyclic lupane-type triterpene, mainly obtained from Betula species plants with a variety of biological actions such as anti-HIV and anticancer properties. This study aimed to enhance the production of betulin in cultures of Betula litwinowii calli under the influence of concentrations and duration time of elicitors and precursors. Collected leaves from Sangdeh habitat in summer, were cultured in WPM medium containing 2,4-D and BAP hormones. Four month calli were sub-cultured in medium containing elicitors such as salicylic acid, chlorocholine chloride and cobalt chloride and precursors such as sucrose, vitamin and glucose. . Calli were harvested from new culture media after two, three and four weeks and their wet and dry weights were calculated. Data analysis was performed based on two-factor factorial experiment (the first factor, elicitors and precursors each in four levels and the second factor, time with three levels) in a completely randomized design with three replications with SPSS software. Duncan's multiple range test were performed to compare the mean (p≤0.01). The results showed that two week elicitation with chlorocholine chloride (1.5 mg/l for one week) was the best with a more than 3-fold increasing in betulin induction compared to the control treatment (0.068 mg / g). The use of vitamin precursor (ten-fold the normal amount in WPM culture medium) for three weeks, causes the highest increase in betulin induction compared to the control (0.1 mg / g, respectively). In general, in the present study, this treatment is introduced as the best.

Abstract Occurrence of various types of incidents such as road accidents, damage and injuries during sports activities as well as some diseases can lead to the destruction and resorption of osteochondral tissue and cause many problems in health and quality of life of the patient, therefore control and repairing these defects is one of the major challenges in the field of regenerative medicine. Since osteochondral defects involve damage to both articular cartilage and underlying subchondral bone, the demands of bone, cartilage, and bone cartilage interface should be taken into account for repair. Current clinical therapies are more palliative and less therapeutic. Hence, due to the limitations of existing treatment methods over the past decade, the use of tissue engineering as an effective and low-risk treatment method for the treatment of many diseases, especially bone-cartilage lesions has been introduced. In this approach, some of the limitations of previous methods could be overcome by transplanting osteochondral composite tissues, which have been obtained by combining patient's own cells with three-dimensional porous biomaterials of predetermined shape and size. So far, various strategies for scaffold fabrication have been used to repair osteochondral defects, including single-phase, multilayer, and graded structures. In this study, some common strategies in tissue engineering as well as the challenges ahead are briefly discussed.

Abstract These days biosensors have worthy applications in different fields such as biomedicine, disease diagnosis, treatment monitoring, various aspects of the environment, food control, drug production, and assorted sides of medical science. Recently, different types of biosensors such as enzyme biosensors, immune, tissue, DNA, and thermal biosensors have been studied precisely by some research groups. These biosensors have many advantages such as simplicity in implementation, very high sensitivity, automatic performance, intrinsic and natural small size. Another valuable benefit of biosensors is that their high-affinity paring with biomolecules allows sensitive (high-sensitivity) and selective detection from a wide range of analytes. Artificial intelligence (AI) due to its high potency, if combined with biotechnology, like biosensors, can be effective in accurate prediction, diagnosis and treatment of some diseases, including cancer. Today, Machine learning (ML) as one of the branches of AI has become a beneficial tool in analyzing and categorizing obtained data from biosensors for bioanalysis. Using ML algorithms automates the complicated processes of extraction, processing, and assaying data achieved from biosensors. This article is a review for introducing and survey of various biosensors, their applications, and ways to apply them, focusing on cancer and Covid19 which are important diseases in the world obtained from previous studies, as a summary and providing information for researchers which working in this field.

Abstract Aims: Programmed cell death is a vital cellular process that is highly conserved in evolution. Apoptosis, as a common mode of programmed cell death, is disturbed in the most human malignancies and leads the resistance of cancers to current treatment strategies. Caspase 9 is a key protein in mitochondrial apoptosis. Activated Caspase 9 leads to activation of Caspase 3/7, initiating a caspase cascade and killing cell. In this study, Caspase 9 gene was cloned into pcDNA3.1(+) and its expression and function evaluated in cell.
Methods: PCR amplification of Caspase 9 was performed by specific primers and ligated into pcDNA3.1(+) after double-digestion with KpnI and BamHI. After sequencing, pcDNA/Caspase 9 was transfected into SH-SY5Y cells and treated with doxorubicin. Caspase 9 function was determined by its effect on cell death level by trypan blue and PI staining, and Caspase 3 activity, and its expression in cells measured by western blotting.
Finding: Caspase 9 gene cloning was done and its expression in cell defined by western blot. Overexpression of Caspase 9 led to autoprocessing following homodimerization and induction of cell death and also increased cell sensitivity to doxorubicin treatment and declined cell viability.
Conclusion: The cloned Caspase 9 was functional in cell and enhanced apoptosis in the treated cells by doxorubicin through self-activation and subsequently amplification of Caspase 3 activation.

Abstract Background: Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Oxypeucedanin methanolate, a member of furanocoumarin, is a naturally occurring compound, which is isolated from Ferulago trifida Boiss, an endemic species in North-West of Iran.
Purpose: We attempt to uncover the capacities of oxypeucedanin methanolate to induce apoptosis and autophagy in NSCLC cells, as well as the underlying mechanism involved in this process.
Methods: The effect of oxypeucedanin methanolate on cell viability was evaluated on A549 cells by MTT assay. Flow cytometry assay was used to detect cellular apoptosis. Expression levels of BAX, caspase-3, BCL2 and LC3 in A549 cells were measured by Real time quantitative reverse transcription-polymerase chain reaction (Real time RT-PCR). A549 cells migration were analyzed using a wound‐healing assay.
Results: Oxypeucedanin methanolate inhibited A549 cell proliferation in dose- and time- dependent manner, as evaluated by MTT assay. The total apoptosis rate was (5.46%) for A549 cells not treated with oxypeucedanin methanolate. In contrast, the apoptosis rate was (29.6%) for A549 cells treated with oxypeucedanin methanolate at the concentration of 0.4 mM. Real time RT-PCR revealed that the mRNA expression of BAX, caspase-3 and LC3 were upregulated, while mRNA expression of BCL2 was downregulated. Untreated cell migration increased significantly after 72 hours.
Conclusion: Oxypeucedanin methanolate inhibits proliferation and it could induce apoptosis and autophagy of human non-small cell lung cancer cell line A549. Oxypeucedanin methanolate may be a good candidate for reducing of A549 cells metastasis.

Abstract Background: Modern science in drug delivery systems has long been paid to effectively design drug delivery systems, to reduce side effects, increasing bioavailability, targeted drug delivery, and passing through blood-brain barriers. Nanoparticles are very important as carriers because they carry different types of drugs to different parts of the body at the right time. Method: In the present study, thiolated nano chitosan loaded with Biperiden was synthesized by ion gel method using two types of crosslinkers (tripolyphosphate and genipin) and their efficiency was investigated. Results: FTIR analysis confirmed the successful synthesis of various stages of thiolated nano chitosan loaded with Biperiden by both crosslinkers. SEM images showed that as-synthesized nano-carriers have a nanorod structure and nanocarriers crosslinked by genipin had a more regular structure with a size of about 150 nm than nanocarrier crosslinked by TPP with a size of approximately 200 nm. In vitro drug release and cytotoxicity studies showed nano-carriers crosslinked by genipin have had a higher release and less cytotoxicity than nano-carriers crosslinked by TPP. Conclusion: Considering the lower toxicity and delayed release of nanoparticles synthesized with genipine than nanocarriers synthesized with TPP, the use of this nanocarrier increases the bioavailability of the drug and can be used as a suitable drug delivery system.

Abstract The human amniotic membrane (HAM) is one of the rare allograft tissues that are in use in clinical trials. Biocompatibility, antibacterial effect, low immunogenicity, and scar prevention are properties that have made HAM attractive for tissue engineering (TE) applications, for example, as a cell carrier, injectable hydrogel, and cell culture substrate. In this research, the effect of digestion time on the structure, gelation kinetics, rheological and biological properties of amniotic membrane-derived hydrogels was studied. The results determined that digestion with pepsin should be performed at least for 24 h. Prolonging the digestion time to 72 h increased the shear modulus, fiber diameter, and gelation rate. Cytocompatibility assays with L929 fibroblast cells showed that the digestion time had no effect on the cell toxicity of the hydrogels. However, cell proliferation was improved due to preserved constitutive bioactive molecules. The results of this research can be used to develop amniotic membrane-derived hydrogels for TE applications.

Abstract Ras signaling is an important intracellular signaling pathway that is key regulator of several aspects of normal cell growth and malignant transformation. The RAS gene family consists of three small G proteins; H-Ras, N-Ras, and K-Ras that play a central role in cell signaling for growth, proliferation, and migration. Mutation of the Ras oncogenes creates the malignant properties that are needed for cancer to grow and spread. MicroRNAs (miRNAs) that are encoded within the Ras genes might also have roles in cancer development. Here, novel microRNAs located in the human N-Ras gene were bioinformatically predicted. SSC profiler program was utilized to predict the stem-loop structures within the genomic area of interest. UCSC genome browser database was useed to analyze the conservation status of the putative miRNA and its precursor sequence. Furthermore, the N-Ras-miRs prediction was also performed by using MatureBayes online tool. In addition, RNAFOLD online software which applies the minimum-free energy (MFE) RNA structure prediction algorithm, was used for approximate prediction of the stem-loop structure. Our results demonstrate that N-Ras with about 5Kb length has some predicted miRNA stem-loop-like structures that have relatively conserved sequences. Overall, accumulative pieces of evidence indicated the presence of novel miRNAs encoded within the N-Ras oncogene.

Abstract Cardiovascular diseases (CVDs) are globally the number 1 cause of death. Despite improvement in treatment strategies, heart disorders are strongly increasing. Therefore, identification of new regulatory factors involved in the cardiac differentiation is very important. TRKC receptor, part of the large family of receptor tyrosine kinases, is involved in development of the heart and central nervous system. There are many contradictory functions related to the TRKC gene which might be attributed to the non-coding RNAs located in it. Recently, a novel miRNA, hsa-miR-11181-5p located in TRKC gene, has been reported which is involved in nervous differentiation. MiRNAs are small non-coding RNAs regulating their target genes via mRNA degradation or protein inhibition. The goal of the present study was to investigate the expression pattern of hsa-miR-11181-5p during the course of cardiosphere-derived cells (CDCs) differentiation.