Journal of Sciences, Islamic Republic of Iran
Volume:30 Issue: 3, Summer 2019

CRISPR/Cas9 system is a powerful gene editing tool in vivo and in vitro. Currently, CRISPR/Cas9 delivery cells or tissue with different vehicles are available, and Adeno- associated virus (AAV) in one of them. Due to AAV packaging size limitation, AAV base vectors that carry CRISPR/Cas9 system do not have florescent tag like GFP for simple detection and navigation of cells, containing AAV. The aim of this study was to modify and synthesis AAV base vector for CRISPR/cas9 system containing sgRNA and GFP.Px602 plasmid was double digested with NcoI and HindIII restriction enzyme. Gfp gene was amplified from px458 plasmid. Linear digested px602 and amplified Gfp gene were ligated together. After transformation and colony PCR on white colonies, plasmid was extracted and transfected to HEK-293 cell line. Gfp expression was monitored by florescent microscopy. After transfection of modified plasmid, florescent microscopy of HEK-293 cells showed shining green florescent cells, which indicate that Gfp gene, was replaced in the correct place according to our design.We modified an AAV base vector carrying CRISPR/Cas9 system, and synthesized a new vector carrying Gfp gene and sgRNA that can be packaged as reporter AAV for navigation and detection of cells, containing AAV.

This study concerns to evaluate the morphological and flavonoid variations, and chemotaxonomy among seven Scutellaria species. The limits of Scutellaria species were disturbed by different factors including hybridization and polymorphism. For this purpose, 39 Scutellaria accessions were collected from different natural habitats of Zagros region, Iran. A total of 15 quantitative and 20 qualitative morphological characters were studied. Leaf flavonoids were extracted using MeOH solution. The flavonoid classes were investigated using thin layer chromatography, column chromatography, UV-spect and LC-MS/MS (liquid chromatography mass spectrometry). To detect the taxonomic status of Scutellaria species, statistical analyses such as cluster, dissimilarity tree, and ordination methods were applied. The results of this research showed five flavonoid classes in different Scutellaria species including isoflavone, flavone, flavanone, flavonol and chalcone. Based on the cluster analysis of flavonoid and morphological data, the members of Scutellaria section Scutellaria were accurately separated from those of Scutellaria section Lupulinaria. Our study revealed a relationship between Scutellaria patonii and Scutellaria multicaulis. Moreover, the trichomes such as strigose, lanate, tomentose, pannous in leaf and stem, petiole, calyx, the form of leaf apex, and inflorescence length were found as diagnostic characters. Based on our results, the flavonoid and morphological markers display the taxonomic status of inter and intra-specific levels in Scutellaria.

Evaluation of gene expression in urinary sediment has been considered as a promising non-invasive approach for biomarker identification of kidney diseases. Nonetheless, there are several challenges in extraction of RNA from this valuable source of biomarkers, mostly because of the factors that have influence on quality of isolated RNA such as low cellular content. Accordingly, we compared the quality of RNA from urine sediment samples that was isolated by four different methods. TRIzol reagent with basic protocol (method 1), modified procedure of TRIzol (method 2), a column-based protocol (method 3) and combination of method 1 and 3 (method 4) were applied for isolation of RNA from identical aliquots of five healthy urine samples. The quality and yield of isolated RNA were evaluated based on concentration and purity. Expression levels of GAPDH and miR-21 were studied by quantitative RT-PCR. Methods 1 and 2 showed the highest RNA yield while no difference in purity of RNA in different methods was noticed. Quantitative RT-PCR findings indicated that Ct values in samples of method 1 had the lowest level. Although higher concentrations of RNA were isolated by method 2, the declined Ct values in this method might indicate degradation of isolated RNA. Column based protocols (method 3 and 4) were failed to show significant recovery of RNA. It seems that isolation procedure using TRIzol, as a phenol based method, is the most efficient, robust and reliable procedure for RNA isolation from urinary sediment cells.

A recently constructed polyglycine modified carbon paste electrode (PGMCPE) was employed to evaluate the electrochemical behavior of methyl orange (MO). The developed electrode was characterized by FESEM to understand the morphology of the modified electrode and bare carbon paste electrode (BCPE). The modified electrode exhibits an excellent electrochemical activity towards the oxidation of MO. Through the cyclic voltammetry (CV) measurement it was found that MO oxidized at the potential of 651 mV. The peak current obtained for the electrode which was modified with glycine was 4.2 times higher than that exhibited for BCPE. The influence of the experimental parameter on electrochemical oxidation of MO like pH, scan rate, and concentration was studied. Simultaneous analysis (with indigo carmine) was also studied by Differential pulse voltammetry (DPV) technique. Under optimized condition, the linear calibration curve obtained for MO is in the concentration range 1.5×10-5 to 1.8×10-4 M, leading to the detection limit of 7.4×10-6 M.

This study focuses on RR, SS and RS medetomidine (MM) and inclusion of several achiral acids to distinguish which acid can help conglomerate formation instead of crystallizating racemic mixtures by defining the low-lying energy of their structures. Favorable orientation of acids was determined in interaction with the MM enantiomers after optimization. The most noticeable interactions include hydrogen transfer from acids to nitrogen (N) atom of the MM enantiomers, which was confirmed through quantum theory of atom in molecule (QTAIM) and natural bond orbital (NBO) analysis. In addition, nature and source of change of these bonds were investigated by determination of significant donor-acceptor interactions. The results revealed for obtaining RR and SS conglomerates; oxalic acid solvent provides the optimum stability. Furthermore, application of propanoic acid solvent should be neglected since MM crystallization is nonspontaneous with this solvent. Therefore, oxalic acid is the acid of choice for preferential conglomerate formation.

The present article pays to some Alpine-Himalayan ophiolites of Late Cretaceous age from Darepahn area in the southern part of Nain-Baft Ophiolite belt. Whole rock processed data clearly shows that the metaperidotite rocks are silica-poor with low CaO, Al2O3, and TiO2 that reflect the predominance of olivine over calcic pyroxene in the protolithes. The considerable content of Co (85.6–124.6 ppm) and very low contents of Sc (4.3–15.3 ppm) and Sr (0.7-14 ppm) provided more evidence for olivine enrichment in the rock parentages. Calculating numbers of cation per formula unites based on EMPA results for distinguished Serpentine polymorphs (classified structurally via Raman spectroscopy) display that Tschermak substitution of trivalent cations for lizardite is tetrahedrally, and for chrysotile and antigorite, it is octahedrally. These chemical differentiations, lead to identifying chemical field plots in separator diagrams between SiO2, MgO, H2O, and Al2O3. Lizardite polymorphs can consider as primarily metasomatism results thru an iron-rich fluid occurrence. It has a considerable occupation of trivalent cations (especially Fe3+) and simultaneous less than 4 Si atoms in formula structure that reflected tetrahedral Tschermak substitutions. Antigorites have some deformed recrystallized textures along with high Σoct (Total Octahedral Cations) and low H2O content that could be considered as thermal originated polymorph. Chrysotiles have bimodal MgO and FeO contents that return to magnetite formations in groundmass and octahedral Tschermak substitutions on late-stage veins. The results exemplify compositional variations of Darepahn Serpentine polymorphs as a function of textural behaviors, structural position and thermodynamic condition of the formation process.

In the linear regression models with AR (1) error structure when collinearity exists, stochastic linear restrictions or modifications of biased estimators (including Liu estimators) can be used to reduce the estimated variance of the regression coefficients estimates. In this paper, the combination of the biased Liu estimator and stochastic linear restrictions estimator is considered to overcome the effect of collinearity on the estimated coefficients. In addition, the deletion formulas for the detection of influential observations are presented for the proposed estimator. Finally, a simulation study and numerical example have been conducted to show the superiority of the proposed procedures.