Modares Journal of Biotechnology

Abstract Vitamins D and E are two common medicines for diabetes treatment. Among the main issues in this field is the release of insulin into the circulatory system. Increasing the stability of insulin hexamer is an evolving strategy in improving insulin secretion efficiency. Insulin protein is commonly found in three forms: monomer, dimer, and hexamer. In this study, for the first time, computational approaches were used to investigate the effect of vitamins D3 and E on the stability of insulin hexamer. The molecular docking results indicate six specific binding sites for these vitamins. These bind to the hydrophobic sites of insulin subunits due to their structural rings and hydrophobic properties. The G-mmpbsa analysis indicates the stabilizing role of both vitamins. The binding of these vitamins to the hexamer has significantly increased the binding energy between insulin subunits. Also, the number of hydrogen bonds between monomeric subunits of each insulin homodimer increased in the presence of the vitamins. It also significantly increases the number of internal hydrogen bonds of hexamer protein. Accordingly, vitamins D3 and E bind to and stabilize the insulin hexamer, resulting in a slower and more balanced insulin release as well as a longer half-life for the dimer in the bloodstream. These findings will pave the way to design a new strategy to regulate insulin release and increase its half-life in the blood for type II diabetes treatment. Besides, hexamer stabilization can be an effective treatment strategy for type I diabetes through slow release from an implanted biosensor system.

Abstract The development and function of mammalian cells, like other multicellular animals, requires cell to cell interactions, which are carried out directly via cellular junctions or indirectly by secretion of secretory molecules such as hormones. During the last two decades, exosomes have been introduced as the third mechanism for cellular interactions. Exosomes are small vesicles with membranes and 30 to 100 nm in size that exist in blood, urine, saliva, semen, and serum. Exosomes play an important role in a variety of biological processes such as immune response and inflammation, pregnancy, tissue generalization, blood coagulation, and angiogenesis. Exosomes are also involved in pathologic process such as neurological disorders, cancer, infectious diseases, and cardiovascular diseases. Because of their small size, exosomes are able to cross the cell membrane and protect the proteins from degradation. They also have the potential of transferring different compounds into the cell. Due to their receiver specificity, lack of inducing immune system, and more importantly having the capacity to be engineered as drug carriers, exosomes have been introduced as new agents for the transfer of genetic material and disease treatment.

Abstract Aspergillus has many species that are important in medicine, agriculture, and various industries. The genus has 446 identified species, which are difficult to distinguish from each other with the use of morphological characteristics. Xylan 1,4-beta-xylosidase is an enzyme that catalyzes the hydrolysis process of xylose in xylooligosaccharides and is produced by different species of Aspergillus. This research was conducted with the aim of a bioinformatics study of the gene region related to this enzyme and to evaluate its similarities and differences in some Aspergillus species. The results showed that this gene region, due to the presence of conserved motifs, was able to distinguish some species studied in this research.

Abstract In this study, the wheat germ was fermented with industrial bakery yeast powder to produce FWGE with high 2,6-DMBQ content in a Bench-scale bioreactor by scale-up approach. The 2,6-DMBQ content of FWGE was increased by optimizing the three initial variables of pH, fermentation temperature, and agitation rate at two levels using the Taguchi method. The 2,6-DMBQ content of the samples was determined at 14, 16, and 18 hours of the fermentation process. Then, the results were analyzed by Qualitek software. The effect of centrifugation speed on turbidity and the yeast's number in the final supernatant was then investigated. Finally, the supernatant was dried by spray dryer with an inlet temperature of 120 °C and outlet temperature of 70°C, and the amount of active 2,6-DMBQ, pH, moisture, and ash was determined. Under optimal conditions: initial pH of 6, fermentation temperature of 32 °C, and agitation rate of 80 rpm, maximum 1.527 mg of 2,6-DMBQ per gram of FWGE obtained. The separation results showed that the centrifugation rate doesn't have a significant effect on the final turbidity and the number of yeasts left, and thus 3000 g was selected as the optimal speed. However, because of the high content of yeast in the supernatant, filtration was required after centrifugation. Due to the high speed of sample drying, the low moisture of the final product, and high efficiency on an industrial scale, the samples were dried using a spray dryer. Finally, the moisture, protein, ash, and pH of the final product were measured.

Abstract Fibrinogen is a major component of the coagulation cascade following tissue damage and rapidly forms an insoluble fibrin scaffold. Fibrin is a filamentous biopolymer that naturally forms from fibrinogen polymerization during blood clotting. After tissue damage and coagulation cascade initiation, soluble fibrinogen polymerization by thrombin enzymebegins and forms an insoluble fibrin network and blood clots with platelets. This fibrin network is crucial for the development of homeostasis after tissue damage. This biopolymer also plays a key role in the wound healing as a temporary scaffoldand due to its unique structural properties and physiological function; it is used in reconstructive medicine. Fibrin is able to absorb extracellular matrix proteins (ECM) such as fibronectin and growth factors. The main types of fibrin scaffolds like platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) are being used as autologous biomaterials in reconstructive medicine, wound healing, orthopedics and skin reconstruction and cosmetic sciences. Fibrin derivatives and degradation products also play an important role in the process of wound healing by stimulating cell infiltration and tissue regeneration and they are being widely used in developing new products as a biological material for over a century.

Abstract Datura (Datura stramonium) is known as a rich source of tropane alkaloids, including scopolamine and hyoscyamine as parasympatholytics that competitively antagonize acetylcholine. Production of secondary metabolites often occurs in plants against the various elicitors or signal molecules. In this study, the effect of yeast extract as a biotic elicitor on tropane alkaloid production was evaluated. Surface-sterilized Datura seeds were cultured on 1/2 MS medium supplemented with different concentrations of yeast extract (0, 1.5, and 3g/L). One month after germination, alkaloid yield of Datura plantlets was measured by the use of High-Performance Liquid Chromatography (HPLC). Also, total protein content and antioxidative enzymes activity were determined by spectrophotometry method. According to the results, the fresh weight of root and shoot parts of Datura plantlets at 1.5g/L yeast extract was increased about 2 and 4 times, respectively. Yeast extract (1.5g/L) caused to 1.7 times increase of hyoscyamine amount of root and shoot parts and 2.5 folds scopolamine of Datura plantlets. Moreover, on yeast extract (1.5g/L)-treated plantlets, total protein content, and activity of catalase and guaiacol peroxidase were almost the same as the control group. Altogether, yeast extract (1.5g/L) can be used as a good candidate for enhancement production of tropane alkaloids especially hyoscyamine and scopolamine with high medicinal value.

Abstract Introduction: Macrophages are considered a particularly challenging cell type to transfect. Given their importance as therapeutic targets, developing a successful transfection method for these cells is highly desirable.
Materials and Methods: The efficiency of lentiviral transfection was compared to three commercially transfection reagents (Xfect™ Transfection Reagent, FuGENE® HD, and Lipofectamine ^TM 3000) in RAW264.7 macrophage cells. Following optimization and production of lentiviral particles in 293T cells, RAW264.7 cells were infected with varying MOI. Transduction efficiency, cell viability, and metabolic activity were measured and compared to the transfection efficiency of the chemical methods.
Results: None of the three chemical transfection reagents successfully transfected RAW264.7 cells. In contrast, the lentiviral method achieved transduction even at the lowest concentration of viral stock, with a green fluorescent signal observable under a fluorescence microscope. Increasing the viral stock concentration and using higher MOIs (up to 30) significantly (p≤0.0001) increased transduction efficiency.
Discussion: Despite requiring more time and effort than chemical methods, lentiviral transduction exhibited superior efficiency in transfecting hard-to-transfect cells and further improvements were achieved through some modifications such as virus concentration, the use of polybrene, no viral freezing and O/N incubation with concentrated viral particles. Since other parameters, especially the use of retronectin and spinoculation, are effective on the efficiency of the virus infection process, it is suggested that they be considered in future studies and given the encouraging data of this research, this completed method can also be applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.