NEW CELLULAR & MOLECULAR BIOTECHNOLOGY JOURNAL
Year:2014 | Volume:4 | Issue:16|Papers Count:15

Aim and Background: Extended spectrum b-lactamases (ESBLs) have emerged as a major threat worldwide with limited treatment options. This study aimed to determine the antibiotic susceptibility pattern and the evaluation of TEM genes producing in Escherichia coli and Klebsiella pneumonia isolates collected from clinical samples.Materials and methods: Bacteria were isolated and identified from the different samples in patients sent to laboratory of shahid Beheshti University in Tehran in 2011. Isolates were then tested for antimicrobial susceptibility by disc diffusion and examined for TEM genes production by polymerase chain reaction using specific primer.Results: Out of 100 studied nosocomial infection specimens, 50 isolates were klebsiella pneumonia of which 34 percent were ESBL producer and all were positive for TEM gene, resistance of Cefotaxime was 90 percent which is the highest degrees of resistance and lowest degrees of resistance to Imipenem was 4 percent. Among the 50 isolates of Escherichia coli of which 14 percent were ESBL producer and all were positive for TEM gene, resistance of Cefexime was 90 percent which is the highest degrees of resistance and lowest degrees of resistance to Meropenem was 6 percent.Conclusion: Due to relatively high prevalence of ESBL-producing bacteria in the studied population, antibiogram test are advised for appropriate treatment.

Aim and Background: The cancer stem cell model postulates that a small subset of cancer cells with stem like properties drive tumor initiation, progression and drug resistance. Since Three-dimensional (3D) in vitro models provide a well-defined environment for cancer research in contrast to the complex host environment of an in vivo model, our aim in this study is to compare cancer stem cell properties in 3D and 2D culture.Materials and methods: MDA-MB468 Cells cultured in 2D and 3D culture were compared for their colony formation and spheroid formation efficiency, stemness markers expression (CD133, CD44) by flowcytometry and stemness gene expression (Oct4,Sox2,Nanog,Aldh, Cmyc) by Real time PCR.Results: Cells cultured in 3D showed no significant difference in colonogenic and spheroid forming capacity in comparison with those cultured in 2D. While Cells in 2D showed higher proportion of CD133+ phenotype than those in 3D (98.55±0.34 vs. 52.69±5.84), no significant difference was observed in CD44+ expression. Additionally, stemness genes including Aldh, Cmyc, Oct4, Nanog, and Sox2 were strikingly down regulated in 3D culture.Conclusion: We demonstrated that 3D culture which could mimic the tumor architecture and in vivo conditions decrease cancer stem cell properties of MDA-MB468 cell line.

Aim and Background: Understanding phylogenic relationships and genetic diversity in Citrus is considered to be important in clarifying their genetic relationships, germplasm characterization and the registration of new varieties. There are some Citrus biotypes in the country Citrus collections which have been classified merely based on their morphological traits and many genetic researches were not done on them. Molecular markers would help to infer their relations with known cultivars.Materials and Methods: In order to investigate about phylogenic relationships among 45 Citrus genotypes including 33 unknown local genotypes and 12 commercially important Citrus varieties at the collection in Agricultural Research Institute of Kotra, Ten SSR primer pairs were used. DNA was extracted from young leaf tissues according to the procedure of Murry and Thompson (1980). One SSR primer was chosen from each corn chromosome and totally ten primers were assayed using the sample of 45 genotypes. The amplified products were separated using 6% polyacrylamide gel with 7 M urea under denaturing conditions and visualized by staining with silver nitrate. Gels were then scored based on either the presence or absence of bands. Polymorphism information content (PIC) for each SSR marker was determined with 1-  Sfi 2- S2fi2 fj2 where fi is the frequency of the ith allele and fj is frequency of the ith allele + 1. Estimation of genetic similarities was done among these lines using the NTSYS program. The 45 genotypes were clustered based on the matrix of Jaccard genetic similarity using the complete clustering algorithm.Results: Ten primers revealed 0.685 polymorphism rate. The number of alleles per locus ranged from 3 to 13. Among all, 72 alleles were identified. Cluster analysis was resulted in designating the 45 samples into six major groups. Orange, mandarin and 24 unknown genotypes into group A, grapefruit, Pummelo, sour orange and six unknown genotypes into group B, three unknown genotypes into group C, two unknown genotypes and Yuzo into groups D and E, citron, Eureka lemon, sweet lime, Mexican lime and two unknown genotypes into group F. Conclusion: SSR markers revealed different levels of diversity which can be useful in germplasm and genetic resources management. Genetic diversity and phylogenetic analysis in Citrus provide useful information for further breeding programs, selection, and registration of new varieties.

Aim and Background: Olive tree (Olea europaea L.) has a high economic value and many countries such as Iran and Mediterranean countries use its oil and canned fruits. Conventional propagation programs are time consuming. Tissue culture technique allows producing high quality and rapid growing plants. The objective of the present study was to evaluate the effect of chelated iron and nano-chelated iron on the growth rate of in vitro microshoots.Material and Methods: Uninodal explants of young shoots from a mature olive Oleaeuropaea L ((cv. Dezful), were cultured in DKW medium supplemented with 2ip (4m[g.L]^(-1)). Sterile microshoots were subcultured in DKW medium supplemented with different concentrations of nano-Fe and Fe chelate (60, 120,180, 240 mM). Forty-five days after subculture, growth parameters were investigated. Results: Results indicated that the growth parameters (number of nodes, microshoots, leaves and stem length) significantly decreased in the presence of nano Fe. The maximum level of leaves number and stem length was achieved in the high concentration of Fe (180 mM). Upon increasing the Fe or nano Fe content in the medium, the chlorophyll b and carotenoid levels were significantly increased. Fe chelate was more effective than nano Fe.Conclusion: Microshoots couldn't survive in the highest concentration of nano Fe and Fe (240 mM). The best results were obtained in the presence of Fe (120, 180 mM).

Aim and Background: The aim of this study to compare the coding area of the exon 17 DGAT1 gene polymorphism in Mehraban sheep.Materials and Methods: DNA was extracted using Kit method, 120 samples of Mehraban sheep were obtained, and also polymerase chain reaction was performed to amplify 309 bp of exon 17 DGAT1 gene using a specific primer. In RFLP method, PCR products using AluI enzyme digestion were digested and the three genotypes TT, TC and CC were found.Conclusion: Mehraban sheep in two alleles T and C respectively, with frequency of 0.37 and 0.63 were observed. Also the population was not found to follow Hardy-Weinberg equilibrium.

Aim and Background: The infection outbreak and formation of urinary tract stones is one of the basic and general problems in humans. The purpose of this research was to study on stones and identify bacteria in patient’s urinary tract (pelvic) attending Tehran Labafi-Nejad hospital.Material and Methods: Specimens of the pelvic and urine of 100 patients from 2-day infants to 75-years old persons) with urinary tract stones were collected during PCNL operation and bacteria were identified by microbiological standard methods. The small piece of patient’s urinary tract stones was put (in sterile condition) in two tube containing BHI medium for culture and identification of bacteria, then, another part of stone was retained for chemical analysis using stones diagnosis kits.Result: In this study, out of 100 stone culture samples, the bacteria grew in 31 cases (31%) and there weren’t any microorganisms in the remaining 69 cases (69%). Two bacteria were grown together in some cases. The isolated microorganisms from tube’s culture, containing stone included: E. coli (29%, 3), Staphylococcus aureus (9%,6), Proteus (6%,4). Isolated bacteria from pelvic urine cultures were the same as stone’s culture. The percentage of obtained stones include: calcium oxalate stones (62%), calcium phosphate (18%), magnesium ammonium phosphate (struvite stones) (11%), uric acid (7%) and cystein stones (2%). In 69% of pelvic sample, no bacteria were grown. It might be due to other bacteria such as (Mycoplasma urealyticum, Nanobacteria, etc.), which couldn’t grow in general laboratory media. Therefore, it’s recommended to use specific media instead of general cultures, for the identification of bacteria. Also PCR technique can be used for the detection of urinary tract infection. Determination of compound of urinary tract stones is essential and inevitable for the treatment and prevention of recurrent stone formation. It’s necessary for stone formers to follow food diet and use special drug treatment, in order to prevent the recurrent stone formation.

Aim and Background: Nowadays demands for clean power source is highly enhanced. Bio-fuel cell (BFC) can convert chemical energy to electrical energy. The enzyme-based bio fuelcells (BFC) is a special fuel cell using enzyme as catalyst and can directly convert chemical energy to electrical energy. Bio-fuel cells are energy conversion devices based on bio- electrocatalysis leveraging on enzymes or microorganisms.Materials and Methods: In the present paper, Sol-gel is used to laccase Encapsulation and immobilization on the electrode. Mediator is immobilized into porous silicate-carbon heterogeneous structure of carbon ceramic electrode (CCE). In the next step, laccase is immobilized in hydrophilic silicate thin film deposited on the ABTS modified CCE (ABTS-CCE) surface. ABTS polymer is located in sol-gel function as the mediator for the electron transfer.Results: Cyclic voltammetric results indicate low electron transfer rate because of weak contact between enzyme and electrode surface while maximum redox pick achived in 10 micro amper in a solution containing 15 micro molar o-dianisidin as substrate.Conclusion: By using of carbon nanotubes connection were improved and maximum redox pick achived in 14 micro amper in the same concentration.

Aim and Background: Tissue engineering is a promising area in biomedical engineering to repair or replace the function of defective or damaged tissues or organs. Recently, tissue engineering has provided a new medical therapy as an alternative to conventional transplantation methods by using polymeric biomaterials with living cells. The scaffold provides the microenvironment (synthetic temporary extracellular matrix) for regenerative cells, supporting cell attachment, proliferation, differentiation, and neo tissue genesis due to their suitable chemical, physical and biological. In this research, chitosan/laminin nanocomposite was exploited as scaffold for suitable cell proliferation.Materials and Methods: Freeze-drying technique was used to fabricate chitosan/laminin-nanocomposites for L929 fibroblast cellsseeding, proliferation and attachment. The physico-chemical properties of the scaffold were fully characterized by using scanning electron microscopy (SEM). Consequently, the biocompatibility of scaffold was evaluated by biocompatibility test.Results: The microstructure observation with SEM suggests the formation of cylinder-shaped porous structure and interconnected porosity.Conclusion: Our results demonstrated that the nano-sized chitosan/laminin scaffolds are nontoxic and biocompatible which can promote proliferation and attachment of L929 fibroblast cells. And their appropriate adhesion to nanocomposite for improved tissue engineering applications.

Aim and Background: Symbiodinium is a unicellular biflagellate alga that performs symbiosis with certain invertebrates. Different clades of Symbiodinium show variety of physiological features that could have uneven affect on the hermatypic corals stability and survival in various environmental conditions and geographical locations. In this study, Clades of symbiotic zooxanthellae of hermatypic corals of eastern Kish Island were compared to samples taken in 2007.Materials and Methods: Samples were taken at depth of 2 to 5 meters from 5 coral species including Acropora arabiensis, Platygyra daedalea, Porites compressa, Acropora downingi and Psammacora contigua. Coral colonies were air brushed in order to separate Symbiodinium cells. DNA was extracted using CTAB-Chloroform method. LSU (Large Subunit Ribosomal DNA) region was used as target for amplification. Finally PCR products were sequenced.Results: Results did not show any difference between Corals’ symbionts and the study in the area during 2007. None of the studied coral species have switched or shuffled their symbionts populations.Conclusion: Taking into account inhospitable condition and shallowness of eastern parts of the Kish Island, presence of clade D might be an adaptive or acclimatized response due to dominant regional condition.

Aim and Background: L-asparaginaseΙΙ (E.C. 3.5.1.1) has effective usage for treatment of acute lymphoblastic leukemia. This enzyme is isolated from bacterial sources and it is commercially available as an anti-cancer drug. This enzyme catalyzes the hydrolysis of L-asparagine to ammonium and aspartate. Unlike Normal cells, the cancer cells strongly require L-Asparagine leading to destruction of these cells. The propose of this project is cloning of L-asparaginaseΙΙ gene from E. coli in B. subtilis to produce the mass of this enzyme.Materials and methods: L-asparaginaseΙΙ gene is isolated from E. coli by PCR approach. The amplified fragment and expression shuttle vector pMR12 are digested by HindΙΙΙ and BamHΙ enzymes. The ligation between DNA fragment and the cloning shuttle vector is done by standard method. In the next step recombinant vector is transferred to E. coli JM101 by cold calcium chloride treatment and finally introduced into B. subtilis by a chemical method.Results: in this experiment, ansB gene is isolated from E. coli by PCR and cloned into the expression shuttle vector pMR12. Existence of gene with 1047 bp lenght is confirmed by enzymatic analysis and PCR reaction. Then recombinant vector cloned in E. coli at first and then in B. subtilis. Finally the plasmid extracted and compared with the band of expersion shuttle vector containing ansB gene to confirm.Conclusions: In this study, we tried to clone the ansB gene in B. subtilis using expression shuttle vector pMR12. This is the first report of ansB gene cloning in B. subtilis.

Aim and Background: Charybdis hellerii is a well-known species in the Indian Ocean. There are many molecular researches on this species all over the word. Molecular researches on Iranian water crabs are very scarce; however there are several comprehensive morphological studies. In this paper, for the first time, phylogeny of Charybdis hellerii from Iranian waters are studied.Materials and Methods: Specimens were collected in 2013 from sandy sediments of shallow-waters in Chabahar Bay at full tide. Collections were transported to the laboratory and preserved in -20 degrees. The gill tissue was used for DNA extraction. The Hexadecyltrimethylammonium bromide (CTAB) method was used to extract DNA. Polymerase Chain Reaction (PCR) was then carried out. The sequencing alignments were conducted and phylogenic tree was obtained.Results: The crab sample was identified as Charybdis hellerii by morphological analysis confirmed by molecular analysis. Nucleotides sequence of the cytochrome oxidase subunit 1 (CO1) gene was analyzed. The phylogenetic analysis was conducted and found that the Iranian species with other Charybdis hellerii are in a clade and support with 94% bootstrap.Conclusion: The maximum Likelihood phylogenetic analysis showed that the Iranian species is in the Eubrachyura clad and with 94% bootstrap support is a sister group Charybdis hellerii. The molecular results were also confirmed by morphological features.

Aim and Background: P53 tumor suppressor gene, also known as “genome guardian” is mutated in more than half of all kinds of cancers. P53 protein acts as a tetramer and is a transcription factor, controlling the transcription of genes that are necessary for apoptosis or cell proliferation arrest. Also it inhibits the transcription of genes that are responsible for cell growth. Experimental researches revealed that acidic pH raised the rate of cancer and mutation in 248CGG codon of P53 gene. This may be due to protonation of this three-nucleotide codon. Mutation in this codon changes the encoding amino acid and subsequently produces a protein, which has oncogenic features instead of tumor suppressor characteristics of original p53 protein. In current study, we perform an investigation on the impact of protonation on stability of codon 248CGG in this gene.Materials and Methods: Molecular mechanic methods used in this study to calculate energy levels. HYPERCHEM software used in AMBER, BIO+, MM+ and OPLS.Results: Obtained results from different force fields all suggest that acidic condition can increase molecular instability in given sequence in comparison with physiological pH.Conclusion: Our results suggested a reliable answer about the effect of protonation on mentioned codon and its stability. From theoretical point of view, acidity can increase the instability of this specific codon. Along with the experimental investigations, our results can, to some extent, elaborate the mutagenesis of acidic pH.

Aim and Background: The genus Micromonospora is a prolific source of various bioactive metabolites, such as antibiotics and enzyme inhibitors. Members of Micromonospora are widely distributed in a variety of habitats, notably soil rich. The aim of current study was to isolation, identification of the isolates to genus level by 16S rRNA gene amplification and determines the isolates antimicrobial activity.Material and Methods: Sixty soil samples collected from different parts of Iran. Each soil sample treated by 1.5% phenol and was cultured on different suitable media to isolation of Micromonospora. The isolates were subjected to genus identification by means of 16S rRNA amplification with genus specific primers. Extract of the isolates belongs to Micromonospora, were tested agianst pathogenic bacteria to determine their antibacterial activity.Results: From 60 soil samples, 200 actinomycets were isolated which 15 isolates were confirmed as Micromonospora by means of 16S rRNA amplification with genus specific primers. Extract of the 5 isolates had antimicrobial activity against Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 and Bacillus cereus ATCC 1399.Conclusion: Isolated Micromonospora spp during this study, were able to produce the antimicrobial components against MRSA, one of the most challenge in the clinical setting.

Aim and Background: Screening of actinomycest for the production of novel antibiotics has been intensively evaluated for many years by scientists.Material and Methods: From Lout desert, Sistan and Balouchestan and Khuzestan deserts, 30 soil samples were collected. The actinomycets from each sample were isolated and their antibiotics acivities agains pathogenic bacteria were investigated.Results: A total of 300 actinomycetes isolates from soil samples were isolated. Out of 300 isolates, 24 isolates shown antimicrobial activity agains S. aureus, E. faecium, K. pneumonia and A. baumannii.Conclusion: The study indicated that desert soil had diverse group of actinomycetes which some of the isolates had broader spectrum antibacterial activity which showed potential as a source of antibiotics for pharmaceutical interest.