Production of nanocalcite crystal by a urease producing halophilic strain of <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis> and analysis of its properties by XRD and SEM

Cementation of salt-containing soils can be achieved by salt-tolerant or halophilic calcite precipitation bacteria. Therefore, the isolation of calcite-producing bacteria in the presence of salt is the first step in the microbial cementation of saline soils. Urease producing bacteria can cause calcite nano-crystals to precipitate by producing urease in the presence of urea and calcium. The purpose of this study was to isolate urease producing halophilic bacteria in order to make calcite precipitate in saline soil. The calcite and the properties of the strains were further analyzed by X-ray diffraction (XRD) and scanning electron microscope equipped with an energy dispersive X-ray detector. In this study, a total of 110 halophilic strains were isolated, from which 58 isolates proved to have the ability of urease production. Four strains were identified to produce nano-calcite using urease activity in the precipitation medium. The XRD studies showed that the size of these particles was in the range of 40–60 nm. Strain H3 revealed that calcite is mostly produced in the precipitation medium containing 5% salt in comparison with other strains. This strain also produced calcite precipitates in the precipitation medium containing 15% salt. Phylogenetic analysis indicated that these isolates are about 99–100% similar to Staphylococcus saprophyticus.     

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (R_p)α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.     

Reference Genes Selection for Quantitative Real-Time PCR Using RankAggreg Method in Different Tissues of Capra hircus

Identification of reference genes with stable levels of gene expression is an important prerequisite for obtaining reliable results in analysis of gene expression data using quantitative real time PCR (RT-qPCR). Since the underlying assumption of reference genes is that expressed at the exact same level in all sample types, in this study, we evaluated the expression stability of nine most commonly used endogenous controls (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, and B2M) in four different tissues of the domestic goat, Capra hircus, including liver, visceral, subcutaneous fat and longissimus muscles, across different experimental treatments (a standard diet prepared using the NRC computer software as control and the same diet plus one mg chromium/day). We used six different software programs for ranking of reference genes and found that individual rankings of the genes differed among them. Additionally, there was a significant difference in ranking patterns of the studied genes among different tissues. A rank aggregation method was applied to combine the ranking lists of the six programs to a consensus ranking. Our results revealed that HSP-90 was nearly always among the two most stable genes in all studied tissues. Therefore, it is recommended for accurate normalization of RT-qPCR data in goats, while GAPDH, ACTB, and RPS18 showed the most varied expressions and should be avoided as reference genes.     

The interaction of beta-lactoglobulin with ciprofloxacin and kanamycin; a spectroscopic and molecular modeling approach

A vast research has been conducted to find suitable and safe carriers for vital and pH-sensitive drugs including antibiotics. This article reports the use of easily accessible and abundant purified beta-lactoglobulin (β-LG) protein as the potential carrier of widely used Kanamycin (Kana) and Ciprofloxacin (Cip) antibiotics. Spectroscopic techniques (Fluorescence, UV–vis, Circular Dichroism) combined with molecular docking were used to determine the binding mechanism of these drugs. Fluorescence studies showed moderate binding affinity with the calculated binding constants K_Cip = 60.1 (±0.2)?×?10³ M^?1 and K_kana = 2.5 (±0.6)?×?10³ M^?1 with the order of Cip > Kana. Results of UV–vis were consistent with fluorescence measurements and demonstrated a stronger complexation for Cip rather than Kana. The secondary structure of β-LG was preserved upon interaction with Kana; however, a reduction in β-sheet content from 39.1 to 31.9% was convoyed with an increase in α-helix from 12.8 to 20.5% due to complexation of Cip. Molecular docking studies demonstrated that preferred binding sites of these drugs are not the same and several amino acids are involved in stabilizing the interaction. Based on the achieved results, Kana and Cip can spontaneously bind to β-LG and this protein may serve as their transport vehicle.     

The role of anti-CCD antibodies in grape allergy diagnosis

Background:

Allergens are mostly composed of glycoprotein structures. It is believed that glycan-specific antibodies may lead to false-positive reactions in immunoassays. In this study we investigated the glycosylation state of grape allergens as well as the presence of antibodies to cross-reactive carbohydrate determinants (anti-CCDs) in sera from grape-sensitive individuals.

Methods:

Grape extract proteins were electrotransferred onto PVDF membranes and their glycosylation states were analyzed by blotting methods. To assess the presence of anti-CCDs, natural and mildly deglycosylated proteins were immunoblotted with grape-allergic subjects'' sera. We also measured the IgE reactivity of each subject’s sera with other fruit extracts via an indirect ELISA.

Results:

Immunoblotting studies showed that mildly deglycosylated grape proteins had lower IgE-binding capacity than their intact natural counterparts, which could be due to the presence of anti-CCDs. Biotinylation studies confirmed that the glycosylation levels of the 24, 32, and 60 kDa IgE-reactive proteins were higher than those of the 38 and 45 kDa ones. Lectin blotting showed that the 24 and 60 kDa bands were highly mannosylated, with the highest level of mannosylation on the 24 kDa allergen.

Conclusion:

This study showed that some grape allergens are glycosylated and that anti-CCD antibodies may cause weakly false-positive results during assessment of IgE reactivity to grape allergens.Key Words: Allergy, Grape, Cross-reactive carbohydrate determinants, Antibody     

Hyperinsulinemia: effect on cardiac mass/function, angiotensin II receptor expression, and insulin signaling pathways

To investigate the association between hyperinsulinemia and cardiac hypertrophy, we treated rats with insulin for 7 wk and assessed effects on myocardial growth, vascularization, and fibrosis in relation to the expression of angiotensin II receptors (AT-R). We also characterized insulin signaling pathways believed to promote myocyte growth and interact with proliferative responses mediated by G protein-coupled receptors, and we assessed myocardial insulin receptor substrate-1 (IRS-1) and p110 alpha catalytic and p85 regulatory subunits of phospatidylinositol 3 kinase (PI3K), Akt, MEK, ERK1/2, and S6 kinase-1 (S6K1). Left ventricular (LV) geometry and performance were evaluated echocardiographically. Insulin decreased AT1a-R mRNA expression but increased protein levels and increased AT2-R mRNA and protein levels and phosphorylation of IRS-1 (Ser374/Tyr989), MEK1/2 (Ser218/Ser222), ERK1/2 (Thr202/Tyr204), S6K1 (Thr421/Ser424/Thr389), Akt (Thr308/Thr308), and PI3K p110 alpha but not of p85 (Tyr508). Insulin increased LV mass and relative wall thickness and reduced stroke volume and cardiac output. Histochemical examination demonstrated myocyte hypertrophy and increases in interstitial fibrosis. Metoprolol plus insulin prevented the increase in relative wall thickness, decreased fibrosis, increased LV mass, and improved function seen with insulin alone. Thus our data demonstrate that chronic hyperinsulinemia decreases AT1a-to-AT2 ratio and increases MEK-ERK1/2 and S6K1 pathway activity related to hypertrophy. These changes might be crucial for increased cardiovascular growth and fibrosis and signs of impaired LV function.     

Structural and Electrochemical Study of Al2O3 and TiO2 Coated Li1.2Ni0.13Mn0.54Co0.13O2 Cathode Material Using ALD

Nanolayers of Al₂O₃ and TiO₂ coatings were applied to lithium‐ and manganese‐rich cathode powder Li_1.2Ni_0.13Mn_0.54Co_0.13O₂ using an atomic layer deposition (ALD) method. The ALD coatings exhibited different surface morphologies; the Al₂O₃ surface film appeared to be uniform and conformal, while the TiO₂ layers appeared as particulates across the material surface. In a Li‐cell, the Al₂O₃ surface film was stable during repeated charge and discharge, and this improved the cell cycling stability, despite a high surface impedance. The TiO₂ layer was found to be more reactive with Li and formed a Li_xTiO₂ interface, which led to a slight increase in cell capacity. However, the repetitive insertion/extraction process for the Li⁺ ions caused erosion of the surface protective TiO₂ film, which led to degradation in cell performance, particularly at high temperature. For cells comprised of the coated Li_1.2Ni_0.13Mn_0.54Co_0.13O₂ and an anode of meso‐carbon‐micro‐beads (MCMB), the cycling stability introduced by ALD was not enough to overcome the electrochemical instability of MCMB graphite. Therefore, protection of the cathode materials by ALD Al₂O₃ or TiO₂ can address some of the capacity fading issues related to the Li‐rich cathode at room temperature.     

Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples

In diagnostic research challenges, quantitative real-time PCR (QPCR) has been widely utilized in gene expression analysis because of its sensitivity, accuracy, reproducibility, and most importantly, quantitativeness. Real-time PCR base kits are wildly applicable in cancer signaling pathways, especially in cancer investigations. T-cell acute lymphoblastic leukemia (T-ALL) is a type of leukemia that is more common in older children and teenagers. Deregulation of the Notch signaling pathway promotes proliferation and inhibits apoptosis of the lymphoblastic T cells. The aim of this study was to investigate the effect of Notch signaling activation on the expression of target genes using real-time QPCR and further use this method in clinical examination after validation. Two T-ALL cell lines, Jurkat and Molt-4, were used as models for activation of the Notch signaling via over-expression of the Notch1 intracellular domain. Expression analysis was performed for six downstream target genes (NCSTN, APH1, PSEN1, ADAM17, NOTCH1 and C-MYC) which play critical roles in the Notch signaling pathway. The results showed significant difference in the expression of target genes in the deregulated Notch signaling pathway. These results were also verified in 12 clinical samples bearing over-expression of the Notch signaling pathway. Identification of such downstream Notch target genes, which have not been studied inclusively, provides insights into the mechanisms of the Notch function in T cell leukemia, and may help identify novel diagnoses and therapeutic targets in acute lymphoblastic leukemia.     

Inhibition of mushroom tyrosinase by a newly synthesized ligand: inhibition kinetics and computational simulations

Alterations in the synthesis of melanin contribute to a number of diseases; therefore, the design of new tyrosinase inhibitors is very important. Mushroom tyrosinase (MT) is a metalloenzyme, which plays an important role in melanin biosynthesis. In this study, the inhibitory effect of a novel designed compound, i.e. 2-((1Z)-(2-(2,4-dinitrophenyl)hydrazin-1-ylidene)methyl)phenol, as a specific ligand which can bind to the copper ion of MT, has been assessed. The ligand was found to competitively inhibit both the cresolase and catecholase activities of MT, with small inhibition constants of 2.8 and 2.6?μM, respectively. Intrinsic fluorescence studies were performed to gain more information on the binding constants. Docking results indicated that the ligand binds to copper ions in the active site of MT via the OH group of the ligand. The ligand makes four hydrogen bonds with aspartic acid and one hydrogen bond with the histidine residue in the active site. Molecular dynamics results show that ligand binds to the MT via both electrostatic and hydrophobic interactions with its different parts.