فهرست مطالب سامان حسینخانی

Gene delivery using the force of a magnetic field is called magnetofection. The purpose of this study is the synthesis and characterization of magnetic iron oxide nanoparticles (Fe3O4) as the core of the transfer agent and to investigate the effect of alternating magnetic field on transfection efficiency. For this purpose, the first magnetic nanoparticles (MNP) were synthesized by coprecipitation method. The magnetic properties of the synthesized MNP were investigated by vibrating sample magnetometer (VSM), appearance characteristics, and zeta potential of the synthesized particles were evaluated  using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Then, using magnetic nanoparticles (MNP), polyethylene imine (PEI) and plasmid DNA containing luciferase reporter gene (pDNA), PEI-pDNA binary complex and MNP-PEI-pDNA ternary complex were synthesized. The complexes were evaluated using DLS and gel retardation techniques. The results of DLS and gel retardation technique showed that the complexes have a suitable surface charge and polyethyleneimine is well joined to pDNA and neutralized its negative charge. Finaly, human breast cancer cell lines (MCF-7) and Hek293T cells were transfected by ternary complex in the presence of 50 Hz alternating magnetic field. Cell viability was measured using the MTT test. The obtained results showed that the transfection efficiency in the cells that were transfected with the ternary complex in the presence of alternating magnetic field increased significantly compared to the control group, without any additional toxicity (P ≤ 0.05).

SH-SY5Y is a neuroblastoma cell line which used as a cancer and neurodegenerative disorders model and its neuro-experimental studies. The different diseases cause by a defect in apoptosis pathway. Disruption of apoptotic proteins has an effect on the treatment process and response to drugs. In nerve cells, due to the high expression of apoptosis inhibitory proteins, the efficacy of drugs is low. Combination therapy is one of the developing treatment methods. The aim of this research is to evaluate the effectiveness of doxorubicin drug on apoptosis in SH-SY5Y cells under the conditions of high expression of caspase9. Caspase9 is a key enzyme in intrinsic apoptosis. First, cell viability was obtained through MTT assay under the different drug concentrations. Then, caspase9 gene was transfected in cells and affected by the concentration lower than IC50 of drug, and cell energy level and cell death were checked by different methods. ATP assay showed that the expression of caspase9 with drug lead to ATP decreases. Caspase3/7 activity indicated an increase in cell death by drug and caspase. Propidium staining to hoechst showed that the expression of caspase9 in combination with doxorubicin induce more death. To ensure the expression levels of protein that induces cell death, the amount of caspase3 protein was checked by western blotting, which showed a significant increase in combination of caspase9 and drug. Our findings showed that the induction of caspase9 expression intensifies the effect of drug and the combined treatment may be effective on the responsiveness of neuronal diseases.

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.

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.

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.

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.

The reduction of pathogenicity due to the biological structure of the virus envelope was targeted in this study. Therefore, considering the effect of the prepared solution (Nardin 19) on enveloped viruses, the aim of this study was to investigate the preventive effects of this solution on SARS co-2 virus and H1N1 flu on Vero cell line cells, respectively. And red blood cells The results of this study showed that nardin 19 solution significantly (P <0.001) reduced 2 logs of SARS Co-2 virus in approximately 2 minutes. Also, the results of hemagglutination test for H1N1 influenza virus showed that this solution was able to reduce the virus titer from 516 units to 2 units of hemagglutination in approximately 2 minutes (P <0.001). The results of cytotoxicity test for Nardin 19 solution showed that this solution had no toxicity or lethal effect on Vero cells and red blood cells in one hour. Considering that one of the main ways of penetration of SARS-2 virus and H1N1 flu is through the mouth and nose and also the significant effectiveness of nardin 19 solution in reducing the concentration of these viruses in approximately 2 minutes, according to this use Nardin 19 solution can be suggested as a suitable candidate for the prevention of Quid 19 and influenza as a mouthwash and nasal drops solution.

 Colorectal cancer (CRC) is one of the most common cancers in the world. The role of IL-17A in cancer begins in the early stages of tumorigenesis and appears to play an important role in tumorigenesis by causing inflammation. The present study was conducted to investigate the expression of KI67 and P53 genes in HT-29 colon epithelial cells with IL-17A.

IL-17A was purchased and 50 ng/ml was added to HT-29 culture medium and after 24 hours the cells were isolated from the culture medium and cell necrosis was examined by MTT. Then RNA was extracted and the expression levels of P53 and KI67 were analyzed using newly designed primers by Reverse transcription (RT) qPCR method and GeniX6 software.

 The MTT test showed that a concentration of 150 ng/ml for 24 hours had maximal necrosis rate in HT-29. After 24 hours of IL-17A incubation, the expression of KI67 (P = 0.003) and P53 (P = 0.001) genes in HT-29 cells in the IL-17A exposed group decreased and increased, respectively. Also, compared to the control group the number of examined HT-29 cells in the IL-17A treated group showed a significant decrease (P

Nowadays, the use of laboratory methods based on enzymatic reactions to monitor the health of individuals, especially in the field of military forces, has become widespread. One of the enzymatic methods is the use of creatinase enzyme for clinical measurement of creatine and creatinine in body fluids, through which the health of kidneys, muscles, and thyroid gland can be understood. This study aimed to optimize the expression of the creatinase enzyme and to investigate the effect of gold nanoparticles on the stability of the creatinase enzyme.

This research was conducted in the spring and summer of 2021 in the electrochemical institute located at the University of Tehran, Iran. In this study, E. coli BL21 containing pET28a amplification vector was cultured in TB medium and then induced at 18, 22, 28, 32, and 37°C. After sonication, purification of enzymes was performed by affinity chromatography. After confirmation of expression by SDS-PAGE, the concentrations of enzymes expressed at different induction temperatures were measured and compared by the Bradford method. Then the activity of enzymes was evaluated and compared by the colorimetric method. Finally, the effect of gold nanoparticles on the structure of the creatinase enzyme was investigated by fluorescence spectroscopy.

Examination of creatinase expression at different temperatures in the TB medium showed that at 28°C the highest amount of enzyme with the highest activity was obtained. On the other hand, fluorescence spectroscopy of creatinase enzyme with the presence and absence of gold nanoparticles at excitation wavelength of 295 nm showed a decrease in fluorescence intensity in the presence of gold nanoparticles.

The induction temperature of 28°C in the TB medium provides a more active and more concentrated enzyme. Also, the presence of gold nanoparticles reduced the fluorescence intensity of the creatinase enzyme, which could indicate a change in the fluorescent amino acid microenvironment of the creatinase enzyme structure.

Developing a technique for efficient and safe gene delivery to plant cells is a fundamental aim of plant biotechnology. Agrobacterium mediated transformation as the most common and practical method in plant gene delivery has considerable difficulties such as limitation in applicable for plant species. In recent years several new methods have been suggested, although none of them could be a good replacement. The use of nanotechnology has been provided new solutions to overcome the limitations of biotechnology. Designing biocompatible nanostructures for passing cell barriers and targeted delivery of cargo has improved the biological achievements. In this research the capability of arginine functionalized single-walled carbon nanotube (Arg-SWNT) as a new carrier to transfer plasmid DNA, which codes green fluorescent protein (GFP) to tobacco suspension cells, has been investigation. It is suggested that single-walled carbon nanotubes can pass through cell wall pores and plasma membrane while it carries plasmid DNA along with. The fluorescence microscopy images illustrate the success of gene delivery by Arg-SWNT

Piscidin has a wide range in killing microorganisms including bacteria, fungi, viruses, parasites and has strong anti-tumor activity and plays a role in increasing innate immunity and also does not provide resistance against bacteria; Therefore, it is of great importance in aquaculture. In this study, piscidin gene of Sparus aurata in vector pTZ57R / T was cloned. In this research, ligation product was transferred to component cell of E. coli DH5α strain. Plasmid extraction was performed from single colonies observed in ampicillin plate. Confirmation of the accuracy of single colonies grown in this research was performed by direct PCR and sequencing. The amplified cDNA fragment of the gilthead seabream piscidin gene consists of 310 nucleotides and 57 amino acids. The results of this research show that piscidin gene has been successfully cloned in pTZ57R / T vector. Comparison of nucleotide sequence of piscidin gene in this study showed high similarity with piscidin 5  of Morone chrysops. The comparison of the amino acid sequence of signal peptide piscidin is quite similar to Dicentracin-like of that species registered in the genebank, and mature peptide piscidin sequence is similar in only three amino acids to Pleorocidin-like of Poesila farmosa and Dicentracin-like of Sphaeramia orbicularis. This study could be a step towards further studies of piscidin peptide.

Cell penetrating peptides (CPPs) can enter a cell through the cell membrane, and used in the fields of drug delivery, gene therapy, and cancer therapy by their property transporting various molecules into cytoplasm. Gold nanospheres (GNSs) are a useful tool for molecular imaging, because they are not cytotoxic and have high solubility, excellent light scattering property and ease of synthesis. We constructed a drug delivery system by developing gold nanospheres conjugated to MCaUF1-9-C, a CPP derived from maurocalcine (MCa) scorpion toxin. We examined the applicability of this cell-selective anti-cancer drug delivery system by evaluating its cell-penetrating and cell death activities.

Cell viability of HeLa and MDA-MB-231 cells, against 500 nanomolar concentration of doxorubicin (DOX)-conjugated gold nanoparticles (DOX-GNPs, and DOX-MCaUF1–9-C-GNP) were evaluated using MTT assay. Cell penetrability of MCaUF1-9-C peptide conjugated to gold nanoparticles investigated using of dark field imaging and atomic absorption spectroscopy (AAS).

HeLa cell viability was about 87% when treated with DOX-GNP and DOX-MCaUF1-9-C-GNP, whereas DOX-MCaUF1–9-C-GNP induced high cytotoxicity in MDA-MB-231 cells (30%). Dark field imaging demonstrated higher affinity of MCaUF1–9-C-GNP for the MDA-MB-231 cell compared with HeLa cells. Moreover, atomic absorption spectrometry data analysis showed that 33% of the total amounts of the applied MCaUF1–9-C-GNP were internalized in MDA-MB-231 cells. 

These results demonstrated that peptide conjugated GNP would be a useful tool for the development of a cell-selective drug delivery system.

Inhibitor of apoptosis (IAP) are a family of proteins that block cell death through caspase activity. Survivin is smallest IAPs family member that overexpresses in different cancer types but not in normal tissue except embryonic tissue. Survivin may be used as a new marker to stratify cancer patients for more optimal treatment modalities. The aim of the current study was to investigate survivin DNA cloning into pET-28a and its expression in E.coli.The sequence of survivin gene was amplified by PCR using specific primers and pcDNA-survivin temple. PCR product and pET-28a plasmid were digested by HindIII/NheI restriction enzymes and survivin was ligated into the digested vector. Then, the ligation product was transformed into the E.coli DH5a competent cells and screened by antibiotic selection marker (kanamycin). Positive colonies were selected by colony PCR and screened by double digestion of isolated plasmid. One positive colony was sequenced and confirmed. The recombinant plasmid was transformed into the expression strain of E.coli (BL21) by chemical method. The expression of survivin was induced in the different conditions and expression level investigated by SDS-PAGE.The size of PCR product in agarose gel showed the correctness of amplification. The digested pET-28a plasmid also indicated the correctness of enzymatic reaction. The sequence of the cloned fragment revealed a 100% similarity to the human survivin. In expressing, adding IPTG increased the expression of survivin protein in all conditions, especially 37 ᵒC from 2 h after induction. At all conditions, most of survivin accumulated in the bacteria as inclusion body.

The bioluminescence process is a widespread phenomenon in Nature. These enzymes are identified in some domains of life, but the luciferases from the lampyridea genus are considered for biological applications. The molecular cloning of a new type of Iranian firefly luciferase from Lampyroidea maculata was reported, previously. In this study, we analyzed the rare codons of the Iranian insect luciferase gene using the computational databases as ATGme, RACC, LaTcOm, and Sherlocc. Also, the structural modeling process of this enzyme was performed. Next, the status of these rare codons in this structural model was studied using SPDBV and PyMOL software. In the following, the substrate binding site was studied using the AutoDock Vina. By molecular modeling, some rare codons were identified that may have a critical role in the structure and function of this luciferase. AutoDock Vina was used in the molecular docking that recognizes Asp531 that yield closely related to luciferin and AMP binding site.. This bioinformatics analyzes play an important role in the design of new drugs.

Hepcidins are a group of cysteine-rich antimicrobial peptides that are active in fish against gram-positive and gram-negative bacteria as well as viruses. The objective of the present study is to use the synthetic peptide of Caspian trout Hepcidin (CtHep) for controlling in vivo Streptococcus iniae infection and also to study the immunomodulatory effects of CtHep in Caspian trout. Juvenile Caspian trout with average weight of 20 g were injected with synthetic CtHep at a dose of 1 µg/g fish, and then exposed to S. iniae bacteria. After 24 hours, the expression of cytokine genes and total bacterial load were evaluated in kidney and spleen tissues of different groups. Fish survival rate was noticed until 10th day of infection. The expression of IL-1β, IL-6 and TNF-α mRNA in CtHep-receiving group significantly increased compared to the control group. In addition, total bacterial load in the kidney and spleen tissues of infected fish receiving CtHep, was significantly lower than infected fish which did not receive CtHep. Survival rate of infected fish receiving CtHep was considerably higher than that of the infected fish which did not receive CtHep. Our study shows that CtHep has the potential to be used as an antimicrobial agent as well as an immunostimulant in aquaculture industry.

The SPTBN4 gene, a part of the spectrin protein family, plays important roles in various cellular processes, including cell cycle, nerve cell development, and so on. Recently, a new miRNA has been found in this SPTBN4 gene, which was registered at the NCBI database. The aim of the present study was to investigate the expression of this miRNA, called SPTBN4-miR1, in the process of differentiation of human embryonal carcinoma cell line NT2 and also the overexpression effect of this miRNA on the differentiation of these cells. RT-qPCR results indicate that SPTBN4-miR1-5p and SPTBN4-miR1-3p show a significant increase in expression in the process of neural differentiation from day three until the 8th and 14th day of differentiation. Then, after overexpressing the SPTBN4-miR1 precursor in NT2 cells and retinoic acid treatment, the expression of pluripotent and differentiation revealed the role of SPTBN4-miR1-5p and SPTBN4-miR1-3p in promoting differentiation and exclusion from the pluripotent state. It seems that by making further studies and finding out the possible targets of these miRNAs, a distinctive marker can be achieved and used to improve the differentiation process.

Luciferase from firefly Photinus pyralis (P .py) is a peroxisomal enzyme that converts a heterocyclic substrate luciferin to an excited state oxyluciferin in the presence of Mg+2-ATP and O2. Excited oxyluciferin with the emission of visible light is changed to its ground state. The combination of rapidity, sensitivity, and convenience has led to the development of a broad range of luminescence applications. In spite of wide ranges applications, firefly luciferase is unstable against changes in chemical and physical conditions, thereby reduce its precision and sensitivity. The most undesirable instability of the luciferase is low thermostability and high susceptibility to proteolytic degradation. According to previous studies, limited proteolysis by trypsin of P .py luciferase indicated six cleavage sites on two accessible regions: 206-220 (Including K206, R213, and R218) and 329-341 (Including K329, R330, and R337) on N-terminal domain. In this study, we used site-directed mutagenesis to introduce one point mutation on the 329-341 accessible regions of P. py luciferase, in order to investigate the role of R330 on the enzyme structure and function which R330 changed to Q. Based on limited proteolysis data, R330Q mutant didn’t significantly change compared to wild type, but this mutation caused several alterations in enzymatic properties including shifting the pH optimum from 7.5 to 8 and increasing the thermal inactivation. Based on the results, it can be concluded that whilst Arg330 is a conserved residue but not effects on trypsinolysis stability.

​One of the main challenges in the treatment of genetic disorders, such as cancer, is of drug delivery systems and their inability to monitor and track delivered drug to the targeted site. Therefore, the design of novel with dual capabilities of nuclear drug delivery and tracking into a research priority for this field’s The aim of this study is to design based on both non-cytotoxic quantum dots and chimeric peptides, with dual tracking and delivering small genetic agents into the nucleus. The GQDs with green emission color were synthesized by Hummer’s and methods and characterized by UV-Vis, photoluminescence (PL), Raman spectroscopies, and scanning electron microscopy (SEM). conjugated with MPG-2H1 chimeric peptides through noncovalent interactions. Following conjugation step, the ζ-potential of the complex increased (From -38.6 to -11.1 in complex1, -9.6 in complex2 and -5.74 in complex3). The conjugation was confirmed by native acrylamide gel retardation assay. The of the GQDs was investigated by MTT assay and finally, was carried out. The results showed that MPG-2H1/ GQD complexes can enter cells; however, free-GQDs didn’t enter the cells significantly.

In this study, the effect of single and dual-frequency sonication on cell death of B16-F10 melanoma cells is investigated at constant temperature. Here, 20 groups were studied. The test groups consisted of: control and sham, 40 kHz (intensity: 0.24 W/cm2), 1 MHZ (intensity: 0.5 W/cm2) and the dual frequency groups which each frequency group included seven subgroups of 30, 120, 60, 150, 300, 600 and 1200 s. Cell viability was measured by MTT assay. The result demonstrated that the cell viability for 40 kHz with 30 s sonication time was 96%, which decreased to 6% by increasing the sonication time up to 1200 s. In dual frequency, cell viability decreased in all subgroups, and its amount ranges from 95% at a sonication time of 30 s to 3% at 1200 s sonication time. The same process happens for 1 MHz frequency with a lower relative slope (97% to 15% when sonication time increased from 30 s 1200 s). Ultrasound waves caused the B16-F10 melanoma cell death in constant temperature. Dual frequency sonication caused more cell death especially at higher sonication time, possibly due to cavitation.

The probability of establishing electrostatic interactions due to the abundance of charged hydrophilic residues and especially arginine is considered the most important thermal stabilizing factor of thermophilic enzymes. The current study was conducted with the aim of comparing thermodynamic stability and kinetic refolding of Lampyris turkestanicus and some of its mutants. In the present experimental thermal stability and the way of refolding Lampyris turkestanicus and 3 mutations, including ERR, ERR/I232R, ERR/Q35R/I182R/I232R were investigated by various spectroscopic techniques. In order to high expression of proteins, a single clone of each sample was selected and inoculated into 10ml of LB culture medium, containing Kanamycin at a concentration of 50μg/mg and incubated at 37°C with an ideal aeration for 12-15 hours. The culture medium was centrifuged for 5 minutes at 5000g at 4°C to provide the cellular contents of the bacteria. The results were obtained through spectroscopic methods of remote and near circular dichroism, intrinsic fluorescence, differential scanning calorimetry, and kinetics experiments, using fluorescence-stopped flow technique. Along with the increase in the number of arginine residues at the protein level, the stability and structural compression of the mutated enzymes in comparison with the wild enzyme were increased and the thermograms obtained from differential scanning calorimetry showed a slight increase in Tm and calorimetric enthalpy of mutated proteins in comparison with wild protein. The rate constant of refolding mutated enzymes has increased compared with the wild type. The improvement of thermodynamic and kinetic parameters results from the improvement of electrostatic interactions, which results in a higher degree of compression and structural density.

Aims: Among different nanosystems, polymeric nanoparticles are highly regarded because of their potential to be used as drug carrier. poly(ethylene glycol)-block-lactide-glycolide (PEG-PLGA) is an amphiphilic copolymer that can be used to carry water-soluble drugs and drugs and molecules insoluble in water. PEG-PLGA polymeric nanoparticles can reduce renal filtration and drug toxicity; they are also biodegradable and biocompatible. The aim of this study was to optimize preparation of PEG-PLGA nanoparticles by solvent evaporation method. In the present experimental study, PEG-PLGA nanoparticles with a diameter of 150nm and a zeta potential of -10 were prepared by solvent evaporation method. Then, the physicochemical properties of nanoparticles were carefully examined. Findings: By increasing the polymer concentration and the percentage of polyvinyl alcohol, particle size increased. The production of nanoparticles with a concentration of 5mg/ml copolymer, a 2% w/v polyvinyl alcohol concentration, and in a 12:1 volume ratio showed the best size and superficial load. Morphologically, the nanoparticles were structurally similar and spherical. According to the FTIR spectrum, the peak in 2900-13000cm region was in accordance with the tensile bond C-H in CH3. A strong peak in 1760cm-1 was related to the tensile-CO that showed the copolymer formation. The production of PEG-PLGA nanoparticles in a concentration of 5mg/ml copolymer, 2% w/v of polyvinyl alcohol concentration, and in a 12:1 volume ratio shows the best size and superficial load; also, the nanoparticles are structurally similar and spherical.

Deep eutectic solvents (DES) are significantly important in enzymatic reactions. These solvents increase the efficient contact between substrate and enzyme and therefore, may increase enzyme stability and activity. Bioluminescence is the production and emission of light by a living organism. The principal chemical reaction in bioluminescence involves the light-emitting pigment luciferin and the enzyme luciferase. The enzyme catalyzes the oxidation of luciferin, which requires the energy-carrying molecule adenosine triphosphate (ATP). Because of its easily detectable product, luciferase has found a wide range of application in biotechnology and molecular biology. However, its low thermostability, low turnover number and high Km for ATP restrict further use of luciferase based systems in commercial application. Newly developed method which can be used to increase the stability of proteins and biocatalysts is to take advantage of Deep eutectic solvents (DESs). One group of Deep eutectic solvents are generally composed of organic salts with hydrogen bond donors, as a result of which hydrogen bonds are formed. In this study, we investigated the effects of these solvents on kinetic properties of wild type and E354R/Arg356 mutant Lampyris turkestanicus luciferases in the presence of choline chloride: glycerol with molar ratio of 2:1 as DES. For this, both wild type and mutant, expressed in Escherichia coli BL21, the protein of interest purified through affinity chromatography and used for kinetic studies. Based on the obtained results, DES has positive effect on the thermostability of wild type and mutant luciferases.

Reduced toxicity and ease of modification make gold nanoparticles (GNPs) suitable for targeted delivery and bioimaging via conjugating cell-penetrating peptides (CPPs). This study aimed to assess the enhanced penetration of gold nanoparticles into cells via using TAT peptide as a cell-penetrating peptide.

Gold nanoparticles and TAT-conjugated gold nanoparticles (TAT-C-GNP) were characterized using ultraviolet-visible spectrophotometry, dynamic light scattering, zeta potential method, and transmission electron microscopy. The cytotoxicity effects of nanoparticles on HeLa, and A431 cell lines were studied. Uptake of TAT-conjugated gold nanoparticles was tested in cells, using dark field microscopy.

Gold nanoparticles and TAT-conjugated gold nanoparticles had little to no effect on cell viability. Upon exposure to gold nanoparticles, TAT-conjugated gold nanoparticles displayed higher uptake than gold nanoparticles in A431 cell line; whereas none of the nanoparticles showed penetration in HeLa cell.

Thus, TAT-conjugated gold nanoparticles have enhanced cellular internalization and are suitable for various biomedical applications as nanoconjugates.

Firefly luciferase is a light generating enzyme, which is used in different fields of biotechnology and molecular biology. Luciferase has found widespread applications in many areas of genetic analysis such as detecting gene expression, reporter gene assay and proteomics studies such as protein-protein interactions. Despite many advantages, there are some limitations in luciferase-based systems, the most important of which is its low stability. One of the newly developed methods to solve this problem is to take advantage of Deep Eutectic Solvents (DES). One group of DESs is those that composed of organic salts with hydrogen donor, due to which, intermolecular hydrogen bonds cause lower melting point in comparison with each of the component. In this study, we investigated the effects of DES on kinetic properties of wild type and I232R - E354R/Arg356 mutant Lampyris turkestanicus luciferases. For this, both enzymes, wild type and mutant, expressed in BL21, the protein of interest purified through affinity chromatography and used for kinetic studies. Here, we used choline chloride: glycerol as DES. According to the results, the wild type luciferase is much more thermostable in DES than I232R - E354R/Arg356 mutant. Furthermore, the remaining activity of both wild type and mutant luciferases are greater in the presence of DES than those in the absence of DES.

Lead is one of the four metals that has most complication on human health. Although there has been no known vital effect for it، lead can be collected in living systems to be toxic. Lead enters the environment by different approaches. One of the methods for heavy metals impurity detection in environment is cellular biosensor. Cellular biosensor is cell containing one reporting gene under the control of promoter that is sensitive to an element (such as heavy metals). One of the important operons in Ralstonia bacterium resistance to heavy metals is pbr. This research was done to design biosensor containing pbr promoter with Luciferase reporting gene in E. coli (DH5α) as host. The pbr promoter sequence with pbrR as regulator gene in 634 nucleotide size was synthesized and Luciferase reporting gene in pGL3 plasmid was located under this promoter. Then the recombinant plasmid was transferred to E. coli (DH5α). These bacteria were able to detect the lead in the environment and were used to detect different concentrations of lead in medium. The least concentration of lead that can significantly express Luciferase reporting gene was 1µM and the most concentration of it was 100µM. It seemed that the concentration above 100µM caused decrease in reporting gene expression through toxic effects on biosensor survival. The results indicated that this biosensor can detect 1-100 µMol concentration of the heavy metal «Lead» in aqueous solutions.

Cell-therapy provides a promising alternative for the treatment of type 1 diabetes. One of the most proper candidates is multipotent skin-derived precursors cells (SKPs)، which can be differentiated into insulin producing cells (IPCs). In this study، human skin-derived precursors (hSKPs) were isolated، expanded and differentiated into IPCs in vitro، through exposure to rat pancreatic extract (2 days after a 60% pancreatectomy). In order to differentiation، SKPs were Cultured in DMEM، containing FBS، pancreatic extract and glucose for 14 days. In order to verify insulin production in the cells، dithizone-staining، which is a method for insulin identification، was employed. In addition، insulin expression was examined immunocytochemically، and insulin secretion was examined using enzyme-linked immunosorbent assay. Cellular clusters appeared after approximately 14 days. The clusters were found to be immunoreactive to insulin. Cellular clusters were also able to secrete detectable amounts of insulin in glucose concentration dependent manner. The results of the current study showed that pancreatic extract treatment can differentiate human SKPs into functional IPCs. This may offer a safer cell source for future stem cell-based therapies.