Iranian Journal of Biotechnology
Volume:12 Issue: 2, Spring 2014

More than 60 species of the genus Lathyrus are distributed in Southwest Asia. It is the second largest genus of the tribe Fabeae, after Vicia, in the region (and in Iran with 23 species). In the regional Flora (Flora of Turkey, Flora Iranica and flora of Iran), the genus has been divided into 9-10 sections. Here we analyzed the phylogeny of Lathyrus and its relationship with Pisum based on plastid gene matK sequences.. The present study utilized several approaches including maximum parsimony, Bayesian and maximum likelihood methods to evaluate the monophyly and relationship within the genus Lathyrus, both at the sectional level and species level, mainly based on the taxa growing in Iran.. A total of 52 accessions, representing 38 species of Lathyrus, three species of Pisum and four species of Vicia and Lens as out-groups, were analyzed for reconstructing the phylogenetic relationship using chloroplast gene matK sequences. Maximum parsimony, Bayesian and maximum likelihood methods were used to construct phylogenetic trees.. The present study indicated that Pisum was nested among Lathyrus species. Two members of the Lathyrus section, Clymenum (Lathyrus ochrus and L. Clymenum) with Pisum, formed a weakly supported clade as sister to the larger polytomy comprising the remainder of the Lathyrus species. Several sections of Lathyrus including Lathyrostylis, Lathyrus and Clymenum were monophyletic. Lathyrus roseus (of the monotypic section Orobon) were nested among the members of section Lathyrus. The newly taxon described species L. alamutensis, endemic to Iran, were nested among other species of Lathyrostylis. Linearicarpus, Orobus and Pratensis were not monophyletic sections. Pratensis and the monotypic section Aphaca were the closest taxa. In our analysis, L. Pratensis formed a sister group relationship with the Aphaca clade, not its own section.. Shimodaira-Hasegawa (SH) test of the matK dataset showed that all analyzed Lathyrus species formed their own clade and Pisum was sister to them. Furthermore, when we removed the two above-mentioned Lathyrus species, the analysis retrieved Pisum, as a well-supported clade being sister to the Lathyrus calde..

Backgoround: Plant diseases, caused by a wide range of phytopathogenic fungi, could be managed using of Trichoderma sp, as a biocontrol agent. Cell wall degrading enzymes like chitinase from T. harzianum are important means for fungal pathogen inhibition. Overexpression of these chitinase enzymes can improve the antagonistic potential of Trichoderma sp. strains.. This study aimed to produce a new enhanced biocontrol system of Trichoderma harzianum, overexpressing chit42 gene. The improved T. harzianum could be an appropriate biocontrol agent for controlling the stem rot disease of canola caused by Sclerotinia sclerotiorum.. T. harzianum protoplast cotransformation was carried out by pLMRS3-Chit42 and p3SR2 plasmids. The transformants were selected based on their growth on colloidal chitin containing medium. The improvement of transformants was investigated by quantification of mRNA using real-time quantitative polymerase chain reaction (RT-PCR) and measurement of chitinase activity in the medium containing colloidal chitin as the carbon source. Furthermore, the antagonistic activity of transformants against S. sclerotiorum was assessed by dual culture experiments.. The overexpressing transformants of Chit42 displayed higher levels of chitinase activity to inhibit S. sclerotiorum growth compared with the wild type. The results indicated that the value of the chitinase activity (126.42 + 0.07 U.ml -1) of Chit42-9 increased 64.17 fold. Transcriptomic analysis demonstrated that Chit42-9 transformant expressed 5.2 fold of Chit42 transcript as compared with the parent strain. Biocontrol inhibition of this transformant was 4.98-fold more compared with the non-transformant type.. The improved Chit42-9 transformant can be used for biocontrolling S. sclerotiorum, cause of stem rot disease in canola..

Urinary Tract Infections (UTIs) are the most common infectious diseases in childhood. The Uropathogenic Escherichia coli (UPEC) strains account for as much as 80% of UTIs.. From a clinical perspective, it is important to know which virulence factors and antibiotic resistance properties are present in UPEC strains in pediatrics. Therefore, this study was carried out to investigate the molecular characterization and antimicrobial resistance of UPEC strains isolated from hospitalized patients in pediatric ward of Baqiyatallah Hospital in Tehran..Patients and One hundred and twenty-one urine specimens were collected from the patients infected with UTIs (51 boys and 70 girls). The urine samples were cultured immediately, and those with E. coli-positive were analyzed for the presence of antibiotic resistance genes and bacterial virulence factors using Polymerase Chain Reaction (PCR). Also, antimicrobial susceptibility testing was performed using disk diffusion methodology with Mueller–Hinton agar according to the instruction of Clinical Laboratory and Standard Institute.. Nineteen out of 51 (37.25%) urine samples from boys and 47 out of 70 (67.14%) urine samples from girls harbored E. coli. A significant difference was found between the frequency of UPEC strains in boys and girls (P <.05). High resistance levels to tetracycline (69.6%), ampicillin (69.6%) and norfloxacin (63.6%) were also observed. Totally, 1.66% of tested strains were resistant to more than 8 antibiotics. The incidence of genes encoding resistance against gentamicin (aac (3)-IV), sulfonamide (sul1), beta-lactams (blaSHV and CITM), tetracycline (tetA and tetB), trimethoprim (dfrA1), and quinolones (qnr) were 25.7%, 22.7%, 83.2%, 71.1%, 19.6% and 21.2%, respectively. The most commonly detected virulence factors were fim (71.2%), set-1 (66.6%), iha (62.1), papGI (59%), usp (56%) and sen (22.7%).. Resistant strains of uropathogenic E. coli had the lower incidence of uropathogenic virulence factors. We suggested prescription of imipenem and amikacin to treat pediatric patients infected with UTIs..

Recently, applications of nanoparticles in many fields of medicine have developed, due to their specific physical and chemical properties. Therefore, assessment of their toxicity especially in in vivo conditions is necessary.. The aim of this study was to compare the toxicity of Fe3O4 nanoparticles coated with biocompatible compounds and noncoated nanoparticles.. Wetted chemical method was used in order to synthesize Fe3O4 nanoparticles. The synthesized nanoparticles were coated with BSA (Bovine Serum Albumin) and DMSA (Dimercaptosuccinic Acid) and the coating interactions were investigated by FTIR. Magnetic and structure properties of Fe3O4 and coated Fe3O4 nanoparticles were evaluated by AGFM (Alternating Gradient Force Magnetometer), TEM (Transmission Electron Microscope) and XRD (X Ray Diffraction). Toxicity assessment of Fe3O4 and coated Fe3O4 nanoparticles were studied in mice by intra peritoneal injections during a one-month period. Liver enzymes (SGPT, SGOT, ALP, and LDH) were measured 7, 15 and 30 days post injection.. The synthesized nanoparticles have a single phase and spinel structure; their size distribution in the net form is around 5 to 11 nm and in the coated form is 17 to 25 nm. Some mice liver enzymes were changed due to the injection of both uncoated and coated nanoparticles (especially in groups which received concentrations of more than 100 mg per kg of mice weight). Liver enzyme changes were more considerable in groups that received DMSA or DMSA coated nanoparticles in comparison with those that received BSA or BSA coated nanoparticles. Chemical toxicity studies showed that there was no irreversible effect with concentrations less than 200 mg/kg for all control and treated groups.. The results indicated that liver enzymes were changed during seven and 15 days post injection, especially with high doses (200 mg/kg). The results of measurements 30 days post injection showed less change in comparison with the control and this indicates that there was no irreversible effect on the liver. Moreover, DMSA coated nanoparticles were more toxic in comparison with BSA coated nanoparticles..

Over the past century, the areas of genomics, proteomics and lipids have captured the attention of investigators worldwide. Carbohydrates, have recently received increased attention through the expanding field of glycobiology; probably because they are very complex and not encoded in the genome.. The purpose of this study was to express and purify recombinant human galectin 3via the Pichiapastoris expression system.. cDNA of human galectin 3 gene was amplified with specific primers and cloned into a pcDNA3.1 vector with His-tag for easier purification using Ni2andchromatography. Furthermore, galectin 3was purified to homogeneity and confirmed using SDS-PAGE and western blotting.. The protein band corresponding to 29 kDa was excised from the gels, digested with trypsin and processed for mass spectrometric analysis by Matrix Assisted Laser Desorption/Ionization- Time of Flight Mass Spectroscopy (MALDI-TOF MS), using a Reflex III instrument.. Tryptic digest analysis clearly revealed that the purified protein was indeed galectin 3. Similarly, the biological activity of recombinant galectin 3 was confirmed using the hemagglutination inhibition assay..

The dynamic binding capacity (DBC) of a chromatography matrix in protein purification is the amount of the total protein absorbed into the matrix, before occurrence of a significant break in the breakthrough curve. Optimization of the process criteria for maximum DBC avoids extra process scale-up and reduces the processing time, costs and protein loss. Taguchi method is a simple useful tool in experimental design to estimate the optimal condition with minimum experiments.. In this research, linear flow rate, pH and protein concentration of the feed were checked according to an L9 orthogonal Taguchi array, to estimate the best conditions for maximum DBC of Q-sepharose fast flow (QSFF) resin in recombinant human erythropoietin purification process.. A crud sample containing human recombinant erythropoietin was harvested from a cell culture of Chinese hamster ovary (CHO) cell line. Desalted harvests with different total protein concentrations (30, 40 and 50 µg.mL-1) and pH values (5, 6 and 7) were loaded into a packed column of QSFFwith different linear flow rates (60, 120 and 280 cm.h-1) up to 10% of the breakthrough curve. The total protein loading to the column was checked by UV absorbance and Lowry method, and erythropoietin concentration was measured by ELISA. Analysis of variance (ANOVA) was applied to determine the optimum condition.. Finally, total protein concentration of 50 µg.mL-1, pH of 5 and flow rate of 120 cm.h-1, were anticipated as the optimal process conditions with 5.85 mg.mL-1of resin as the dynamic binding capacity.. Experiments with anticipated optimal criteria were performed three times and no significant difference was observed (p = 0.136, and 6.06 mg/mL as the average dynamic binding capacity)..

Epidermal growth factor receptor (EGFR) overexpression is a characteristic of several malignancies and could be considered as an excellent target for designing specific inhibitors such as anti-EGFR monoclonal antibodies for cancer therapy. Drawbacks exerted by large sizes of full-length antibodies have lead to the development of single chain antibodies, which benefit from having smaller sizes and short circulation half-lives..

The aim of this study was cloning, expression and purification of variable regions of anti-EGFR monoclonal antibody in E. coli for production of single chain antibodies..

The RNA, extracted from the C225 hybridoma cells, was reverse transcribed into cDNA and used for PCR amplification of genes encoding light and heavy chains from the variable regions. The PCR products were cloned and expressed in E. coli BL21 for production of a single chain antibody. The expressed protein was analyzed by SDS-PAGE and purified by Ni-NTA affinity chromatography. The reactivity of purified C225-scFv with EGFR-expressing A431 tumor cell line was tested by western blotting and enzyme-linked immunosorbent assays..

The results indicated that C225-scFv was highly expressed in E. coli and appeared as a protein with a mass of 27 kDa in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the induced cell lysate. Reactivity analysis of the purified C225-scFv with A431 tumor cell line by western blotting and enzyme linked immune sorbant assay (ELISA) revealed high binding affinity of the recombinant C225-scFv to the target cells..

The results of this study indicated that C225-scFv is capable of binding to EGFR and could be considered as a useful tool for diagnosis and treatment of EGFR-overexpressing tumor cells..

Host immune system response against blood group antigens is a major problem in blood transfusions, especially for thalassemic patients. Thus, an approach was proposed coating the red blood cell (RBC) surface by polyethylene glycol.. This study aimed to obtain the optimal simultaneous camouflage of the major and minor antigens by activated methoxy polyethylene glycol (mPEG) with succinimidyl valerate (SVA) and succinimidyl carbonate (SC), separately.. The degree of RBC agglutination by antibodies against the major and minor blood groups was used as a surrogate measurement for quantitative assessment of the effectiveness of the surface coating. Also, the RBC morphology was assessed using scanning electron microscope (SEM). In addition, to evaluate the host immune system response, the PEGylated RBCs were transferred between two different mouse strains.. Statistical analysis of the results demonstrated that the optimal reaction conditions for simultaneous coating of the antigens by mPEG-SVA and mPEG-SC are as mPEG20 in the polymer mixture, 91.2 and 90.0%, and polymer concentration, 17.21 and 19.80 mg/mL, respectively. However, according to the SEM results, the maximum polymer concentration of 14.5 mg/mL was suggested as the best condition for mPEG-SVA modified human RBCs.. It is concluded that the membrane PEGylation camouflages the blood group antigens. This effect is observed significantly for non-ABO/Rh(D) antigens. Also, it is found that the mPEG-SVA provide better coverage than mPEG-SC. The results of in vivo analysis showed that the immune reactions against PEGylated RBCs were considerably reduced, so that the levels of the relevant biochemical parameters in serum were similar to those of the normal hosts 24 hours after transfusion..

In the recent decade, the reverse genetics method has been broadly used for rescue of negative-stranded RNA viruses from cDNA or viral minigenomes. This technique has been applied to study different steps in virus replication and virus-host interactions. Reverse genetics could also be implemented for design of new vaccines. The T7 RNA polymerase activity as well as virus (nucleocapsid protein) N, (phosphoprotein) P and (Large) L proteins are necessary to rescue the virus or viral minigenome. Measles virus is a negative-stranded non-segmented RNA virus. There are useful vaccine strains to prevent measles disease.. Here, we describe the construction of a new helper cell line for rescue of measles virus minigenome. The helper cell line stably expresses T7 RNA polymerase as well as measles virus N and P proteins by tricistronic mRNA.. For rescue of measles virus minigenome a stable helper cell line by using tricistronic expression vector was developed which expressed T7 RNA polymerase as well as measles virus N and P proteins. To construct the tricistronic expression vector, T7 RNA polymerase gene was cloned after cytomegalovirus (CMV) promoter and measles virus N and P proteins were under control of IRES (internal ribosome entry site) sequences.. Our results indicated that measles virus minigenome could be rescued in this constructed helper cell line.. Through this system, the measles virus minigenome was rescued. Further studies are necessary to improve the rescue efficiency. This may be possible by replacing the CMV promoter with the T7 promoter..