Mapping SNP-anchored genes using high-resolution melting analysis in almond

Peach and almond have been considered as model species for the family Rosaceae and other woody plants. Consequently, mapping and characterisation of genes in these species has important implications. High-resolution melting (HRM) analysis is a recent development in the detection of SNPs and other markers, and proved to be an efficient and cost-effective approach. In this study, we aimed to map genes corresponding to known proteins in other species using the HRM approach. Prunus unigenes were searched and compared with known proteins in the public databases. We developed single-nucleotide polymorphism (SNP) markers, polymorphic in a mapping population produced from a cross between the cloned cultivars Nonpareil and Lauranne. A total of 12 SNP-anchored putative genes were genotyped in the population using HRM, and mapped to an existing linkage map. These genes were mapped on six linkage groups, and the predicted proteins were compared to putative orthologs in other species. Amongst those genes, four were abiotic stress-responsive genes, which can provide a starting point for construction of an abiotic resistance map. Two allergy and detoxification related genes, respectively, were also mapped and analysed. Most of the investigated genes had high similarities to sequences from closely related species such as apricot, apple and other eudicots, and these are putatively orthologous. In addition, it was shown that HRM can be an effective means of genotyping populations for the purpose of constructing a linkage map. Our work provides basic genomic information for the 12 genes, which can be used for further genetic and functional studies.     

Agrobacterium-mediated transformation of alfalfa (Medicago sativa) using a synthetic cry3a gene to enhance resistance against alfalfa weevil

To introduce genetic resistance against alfalfa weevil (Hypera postica), leaves and petiole explants of three commercial alfalfa genotypes, including Km-27, Kk-14 and Syn-18 were transformed with Agrobacterium tumefaciens strains GV101, LBA4404 and AGL01. All the Agrobacterium strains used harbored the recombinant binary vector pBI121 containing a synthetic cry3a gene under the control of CaMV35S promoter as well as the nptII gene as selectable marker. Transformed explants were cultured on callus-induction medium, and the germinated somatic embryos were then transferred to the regeneration medium. The primary transformants were evaluated by PCR and Southern blot analysis. The results indicated successful integration of the target gene into the genomes of primary transgenic lines. Moreover, the expression of Cry3a protein in the transgenic plants was confirmed by ELISA method. Three transgenic lines, including TL6, TL8 and TL11 showed significantly higher levels of insect resistance against H. postica larvae (mortality rate of 73–90 % after infestation), in comparison with the control plants during the two-year bioassays. All transgenic plants were fertile and no irregular behavior in terms of growth and the morphological traits were observed. Transgenic plants developed during the course of this study are currently being grown in greenhouse and will be crossed with each other for seed production.     

A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum

Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile-phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.     

Distribution and diversity of Dipteran-specific cry and cyt genes in native Bacillus thuringiensis strains obtained from different ecosystems of Iran

One hundred and twenty-eight Bacillus thuringiensis isolates from fields of different ecological regions of Iran were collected to study the distribution and diversity of Dipteran-specific cry and cyt genes. The percentage of samples with Bt showed significant differences between different regions and also between different fields. The most Bt frequency was observed in the soil samples collected from Caspianic zone (7%) and soils of cotton (17%). Characterization of isolates was based on morphological characteristics of crystals, plasmid profiles and protein band patterns as well as PCR analysis using general and specific primers for 22 different cry and cyt genes encoding proteins active against mosquitoes. Thirty-eight different cry gene profiles were detected in this collection. Several of them were found to be different from all previously published profiles and none of the previous researches reported these numbers of profiles. Strains containing cry2-type genes were the most abundant and represent 57.1% of the isolates. Strains harboring cry24 and cry10 genes were also highly abundant (38.7 and 32.8%, respectively). cry11, cry4, cry17, cry19, cry21, cry29, cyt1, and cry9 genes were less abundant, found in 25.7, 14.3, 11.4, 1.4, 4.3, 1.4, and 10% of the strains, respectively. Among the cry2 gene containing isolates, 37.5% strains harbored cry2Aa, 55% cry2Ab, 2.5% cry2Ac, and 5% other or novel cry2-type genes. Among the cry4 gene containing isolates, 0% strains harbored cry4A, 60% cry4B, and 40% cry4C, cry4D or novel cry4 type genes. Finally, based on crystal morphology, protein patterns and PCR, 21 strains were selected as potentially high Dipteran-active for bioassays. Also our results showed that some of the isolates may harbor minimum a putative novel cry gene.     

Leishmania major: Reactive oxygen species and interferon gamma induction by soluble lipophosphoglycan of stationary phase promastigotes

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is membrane glycoconjugate lipophosphoglycan (mLPG). In addition, it has been shown that mLPG could be used as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we attempted to compare the immunological properties of culture supernatant and membrane LPG prepared from stationary phase promastigotes of Leishmania major. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by thin layer chromatography. The effect of sLPG and mLPG on the production of reactive oxygen species (ROS) was studied using PBMCs isolated from healthy individuals. In addition, induction of IL-12, IFN-gamma and IL-10 secretion in the presence of sLPG and mLPG was investigated. Reactive oxygen species in addition to IL-10 and IL-12 were induced by both sLPG and mLPG. However, IFN-gamma production was promoted only in response to sLPG suggesting its ability to promote Th1 response and implication in vaccine design.     

On the parasitoid complex (Hymenoptera: Chalcidoidea) of Anophococcus abaii (Hemiptera: Eriococcidae), with description of Metaphycus anophococcusi Lotfalizadeh,n. sp.

The parasitoid community of Anophococcus abaii (Danzig, 1990) – one of the main pests of Haloxylon in Iran – was studied. Four species of Chalcidoidea, belonging to the families Aphelinidae, Encyrtidae, Pteromalidae, and Signiphoridae, were found. Metaphycus anophococcusi Lotfalizadeh, n. sp. is described in the zebratus species group and compared with closely allied species of Metaphycus.     

High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

Intestinal Parasitic Infections among Inhabitants of Karaj City, Tehran Province, Iran in 2006-2008

Karaj is an area with large influx of refugee people in Iran. To increase knowledge about parasitic infections, we carried out this research during 2006-2008. We recorded the stool examination results and some of their personal characteristics. A total of 13,915 human stools were examined, and 649 (4.7%) were positive for intestinal parasites. Among them, 13 (0.09%) had worm and 636 (4.6%) had protozoan infections. Maximum infections belonged to Giardia intestinalis, and 534 (3.8%) samples had this infection. Other parasitic infections included Entamoeba coli (0.39%), Entamoeba histolytica (0.021%), Blastocystis hominis (0.08%), Trichomonas hominis (0.1%), Iodamoeba butschlii (0.06%), Chilomastix mesnili (0.007%), Endolimax nana (0.05%), Enterobius spp. eggs (0.028%), Taenia proglottids (0.028%), and Strongyloides stercoralis larvae (0.03%). The maximum numbers of referred people to laboratories were in July and the maximum percentage of infections was in August. There is a point that all 5 Strongyloides stercoralis infections were pertained to 2008. With attention to the rate of parasitic infections (4.7%), it seems that we should take additional educational information to wide spectrum of people living in this city.     

Investigating the Parameters Affecting the Stability of Superparamagnetic Iron Oxide-Loaded Nanoemulsion Using Artificial Neural Networks

Nanoemulsions are increasingly being investigated for their fascinating capability of loading both hydrophobic and hydrophilic molecules while their stability is still an issue, being affected by various factors. In this study, to evaluate the dominant factors affecting the stability of nanoemulsions, artificial neural networks (ANNs) were implemented. Nanoemulsions of almond oil in water containing oleic acid-coated superparamagnetic iron oxide nanoparticles were prepared using a mixture of Tween 80 and Span 80 as surfactant system and ethanol as a co-surfactant. The ratio of transparency of the samples at 30 min and 7 days after preparation was taken as an indication of the stability of samples. Four independent variables, namely, concentration of nanoparticle, surfactant, oil, and alcohol were investigated to find their relations with the dependent variable (i.e., transparency ratio). Using ANNs modeling, it was concluded that the stability is affected by all variables, with all variables showing reverse effect on the stability beyond an optimum amount.Key words: artificial neural networks, nanoemulsion, optimization, stability, superparamagnetic iron oxide