Journal of Applied Biotechnology Reports
Volume:5 Issue: 1, Winter 2018

The production and purification cost of a recombinant protein in plants is lower than other conventional systems, such as bacteria. Comparing different parts of the plant, the hairy root is known to be a suitable bioreactor to produce recombinant proteins including vaccine candidate immunogens. Among the most important causes of diarrhea, E. coli and Vibrio cholerae cause the disease by producing a toxin. The B subunit of this toxin is an appropriate candidate for vaccine development because of its non-toxicity and exposure to the immune system. To investigate the efficiency of transgenic hairy roots in the production of recombinant protein, two methods were considered: direct (induction of hairy root with recombinant Agrobacterium rhizogenes) and indirect (production of transgenic plant with Agrobacterium tumefaciens and then polluting it with non-transgenic A. rhizogenes). To compare the 2 methods, GUS protein expression was used as a reporter and chimeric protein LSC (consisting of ltB-stxB-ctxB) as an antigenic protein. Transformation of tobacco hairy root was confirmed by genomic Polymerase chain reaction (PCR). Gene expression comparison was investigated by semi-quantitative ELISA and enzymatic activity. The results show that the activity of GUS reporter enzyme in the indirect method is seven times more than that of the direct method. Likewise, the expression of LSC recombinant protein in the indirect transformation was 1.5 times more than in direct method. Comparing of these two methods indicated that the hairy roots in the indirect method yield higher protein content than in the direct method.

  Breast cancer, as a multifactorial disease is the most frequent cancer among women and second most commonly diagnosed cancer in worldwide. Breast cancer is associated with mutations in several genes such as PIK3CA. Phosphoinositide 3 kinase (PI3K) is an important group of lipid kinases that regulate the vital cellular functions such as survival, proliferation, cell growth, motility, differentiation, and intracellular trafficking. The aim of this study is to evaluate the association of rs2230461 of PIK3CA gene with the incidence of breast cancer. A total of 198 healthy donors and 205 breast cancer patients were recruited. Genomic DNA was extracted from peripheral blood leukocytes by Triton X100 technique. Genotyping was performed using RFLP-PCR protocol. Chi-square test, odds ratios (ORs) and 95% CIs were used to determine associations. There were no significant differences observed regarding the PIK3CA genotype frequencies at codon 391 between patient and control groups (P = 0.17). However, by comparing stage III breast cancer patients and control groups, there was a significantly higher frequency of the GG genotype among stage III cases compared to control (P = 0.01). Although the PIK3CA I391M polymorphism has been located in the C2 domain and doesn’t involve in the binding site, it can affect the protein function. Since even those mutations that are far from the binding site can affect the protein function and change its dynamic behavior through allosteric impacts and lead to tumorigenesis at last. Since PIK3CA mutations mainly appear late in tumorigenesis, exactly before or coincident with invasion, and may be involved in tumor formation, it is suggested that this polymorphism may be involved in breast cancer invasion.

  Chronic kidney disease is a worldwide health problem and Glomerular filtration rate (GFR) is the most frequently used criteria in the assessment of rental function. Cystatin C as a member of type 2 cystatin superfamily and cysteine-protease inhibitor is found in high concentrations in all biological fluids. This study is aimed to study cystatin C as a potent biomarker for clinical measurement of renal disorders and other diseases. In this study, the human cystatin C construct was analyzed by bioinformatics software. It was cloned and expressed to produce an appropriate antigen for anti-cystatin C (anti-Cys C) obtained from mice Balb/C as a crucial point in the improvement of an enzyme-linked immunosorbent assay (ELISA) method. Serum samples were given from 32 hospitalized patients with renal failure and cardiovascular disease and non-hospitalized patients were tested by ELISA method using anti-Cys C obtained from mice Balb/C. Our findings indicated 0.36-2.4 mg/L as the best conclusion for antigen cystatin C in patients’ sera and 1/50 dilution for anti-Cys C obtained from mice Balb/C and showed a relationship between patients with high creatinine and high concentration of cystatin C. In case of five cardiovascular disease patients with normal upper limit of Creatinine we obtained cystatin C lower than kidney failure and raising of cystatin C in 6 patients with increased TSH were seen. Polyclonal anti-Cys C antibodies were obtained through the immunization of Balb/C mice can be employed as an anti-Cys C in ELISA for diagnosis of some renal dysfunction.

  Amylases are used in various industries, mainly, starch processing that hydrolyze polysaccharides. Insoluble and solid supports are noteworthy in immobilization of enzymes for industry because of increasing enzyme stability. In this study, immobilization of alpha amylase in electrospun polyethersulfone (PES) nanofibers was studied. Covalent immobilization of the enzyme was done through the carboxyl groups made by oxygen plasma treatment and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a carboxyl group activator agent. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) and contact angle analysis proved enzyme immobilization. The optimum conditions determined and the catalytic parameters of immobilized enzyme were calculated. The results of this investigation showed that the optimum pH of immobilized enzyme was displaced toward acidic region by 1 unit. Comparison of the optimum temperature for immobilized and free amylase revealed 10°C increasing for the immobilized enzyme. Furthermore, the kinetic parameters, Vmax and Km for the immobilized enzyme were the same and higher than those of free ones, respectively. Storage stability of the immobilized amylase was obviously improved. The results illustrated that nanofibrous supported alpha amylase is a new and suitable matrix for industrial applications.

  This study represents efficient ring opening of epoxides to the corresponding vicinal diacetates in the presence of acetic anhydride. Different catalyst including molecular sieves, zirconyl triflate, LiClO4, zeolite, LiAlH4 and some other catalyst were reported so far as efficient catalyst for epoxide ring opening and subsequent diacetylation. In the present study, catalytic amount of commercially available TiO2 (anatase) nanoparticles (NPs) were used to promote reactions. According to X-ray diffraction (XRD) patterns, anatase is the main phase of the TiO2 and scanning electron microscope (SEM) images revealed almost all the particles are under 100 nm in size. Existence of just oxygen and titanium peaks in EDS spectrum confirms high purity of catalyst. It was found that catalyst revealed good reusability and could be used in 3 cycles without marked loss of efficiency for synthesis of vic-diacetates from epoxides. Reactions of structurally diverse epoxides in the presence of acetic anhydride and catalytic amount of titanium dioxide were set at 100°C, progress of the reactions were monitored by thin layer chromatography (TLC) and gas chromatography (GC) which resulted in good to excellent yields. In brief, the current work represents an efficient procedure for one-pot conversion of structurally different epoxides to the corresponding vic-diacetates. Comparison of cyclohexene oxide acetylation in the presence of titanium dioxide NPs and other catalyst confirmed acceptable efficiency of anatase titanium dioxide as a clean, safe and low cost catalyst for reaction promotion.

  Pomegranate (Punica granatum L.) is an ancient fruit with numerous phytochemical bioactive compounds. In this study, the antibacterial activity of ethanolic extracts of pomegranate peels and seeds were investigated against Pseudomonas aeruginosa and Staphylococcus aureus clinical isolates from Tabriz health centers (2017). The ethanolic extracts of pomegranate seed and peel were prepared and GC-MS chromatogram analyzed using Agilent 7890B gas chromatography. The antibacterial activities of extracts were evaluated by agar diffusion and microbroth dilution methods against clinical isolates of P. aeruginosa (n = 10), S. aureus (n = 10) and standard strains. The ethanolic extracts of pomegranate seed and peel showed inhibitory effects on clinical isolates of P. aeruginosa and S. aureus. The minimum inhibitory concentrations (MICs) of pomegranate peel and seed extracts were 12.5 and 25.0 mg/mL, respectively. In addition, the minimum bactericidal concentrations (MBCs) of pomegranate peel and seed extracts were found to be 25.0 and 50 mg/mL, respectively. In all of the studied bacterial isolates, the MICs and MBCs values for pomegranate seed extract were significantly higher than those for pomegranate peel extract (P < 0.05). The mean inhibition zones and MICs values indicated that the antibacterial activity of pomegranate peel extract had more potent effect on studied bacterial isolates compared with pomegranate seed extract. According to the findings, the pomegranate peel and seed extracts showed antibacterial activities against bacterial isolates in this study; however, the peel extract had a stronger antibacterial effect than the seed extract. Therefore, further studies including cell toxicity assay and in vivo investigations are recommended

  Many study results have suggested that infection by the Epstein-Barr virus is a possible agent of human breast cancer. But the role of Epstein-Barr virus in breast cancer is still controversial. Paraffin embedded formalin fixed specimens were prepared from 40 breast and 40 healthy tissues in Khuzestan province of Iran. After DNA extraction, the purity of all DNA samples was evaluated by amplification of constitutive beta-actin gene. Then the presence of EBV gene in DNA extraction with appropriate purity was assessed by polymerase chain reaction (PCR) using virus specific primers. only 2 out 39 cases of tumor (5.12%) and none of the 37 healthy samples were positive for the presence of Epstein-Barr virus. Statistically, Cramer’s index for EBV infection was 0.160 in cancer samples. Our results indicated that there is no significant relationship between breast cancer and Epstein-Barr virus. Further investigation on more patients needed to determine the exact relationship between EBV and breast cancer in this province.