TRIM28 and β-Actin Identified via Nanobody-Based Reverse Proteomics Approach as Possible Human Glioblastoma Biomarkers

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.     

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     

Contrasting soil microbial responses to fertilization and tillage systems in canola rhizosphere

Information regarding the simultaneous evaluation of tillage and fertilization on the soil biological traits in canola production is not available. Therefore, field experiments were conducted in 2007–2010 in a split plot based on randomized complete block design with three replications. Main plots consisted of conventional tillage (CT); minimum tillage (MT) and no tillage (NT). Six strategies of fertilization including (N₁): farmyard manure (cattle manure); (N₂): compost; (N₃): chemical fertilizers; (N₄): farmyard manure + compost; (N₅): farmyard manure + compost + chemical fertilizers and (N₆): control, were arranged in sub plots. Results showed that the addition of organic manure increased the soil microbial biomass. No tillage system increased microbial biomass compared to other tillage systems. The activities of all enzymes were generally higher in the N₄ treatment. The activity of phosphatase and urease tended to be higher in the no tillage treatment compared to the CT and MT treatments.     

The dengue virus M protein localises to the endoplasmic reticulum and forms oligomers

The dengue virus membrane (M) protein is a key component of the mature virion. Here, we characterised the cellular behaviour of M using a recombinant protein construct to understand its inherent properties. Using confocal microscopy, we showed that M and its intracellular precursor, prM, localised to the endoplasmic reticulum. M protein was also detected on the cell surface and secreted, suggesting that M can enter the secretory pathway. In addition, cross-linking studies showed that M can form dimers and tetramers. These findings suggest that M behaves as a secretory protein analogous to the major envelope protein E.     

Acetylcholinesterase/butyrylcholinesterase inhibition activity of some new carbacylamidophosphate derivatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl2 with R = pCl-C6H4 1a, pBr-C6H4 2a, C6H5 3a, and pMe-C6H4 4a and RC(O)NHP(O)(NC4H8O)2 R = pCl-C6H4 1b, pBr-C6H4 2b, C6H5 3b, pMe-C6H4 4b, were selected to compare the inhibition kinetic parameters, IC50, Ki, kp and KD, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.     

Biological denitrification by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> immobilized on microbial cellulose

This study demonstrated the feasibility of a biological denitrification process using immobilized Pseudomonas stutzeri. The microbial cellulose (MC) from Acetobacter xylinum was used as the support material for immobilization of the bacterium. Nitrate removal took place mainly in the anoxic system. The effects of various operating conditions such as the initial nitrate concentration, pH, and carbon source on biological denitrification were demonstrated experimentally. The system demonstrated a high capacity for reducing nitrate concentrations under optimum conditions. The denitrification rate increased up to a maximal value of 1.6 kg NO₃-N m⁻³ day⁻¹ with increasing nitrate loading rate. Because of its porosity and purity, MC may be considered as appropriate supports for adsorbed immobilized cells. The simplicity of immobilization and high efficiency in operation are the main advantages of such systems. To date, the immobilization of microorganisms onto MC has not been carried out. The results of this research shows that a pilot bioreactor containing P. stutzeri immobilized on MC exhibited efficient denitrification with a relatively low retention time.     

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.     

Heterologous Expression,Purification and Characterization of a Peroxidase Isolated from <Emphasis Type="Italic">Lepidium draba</Emphasis>

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.     

Bacillus thuringiensis: a successful insecticide with new environmental features and tidings

Bacillus thuringiensis (Bt) is known as the most successful microbial insecticide against different orders of insect pests in agriculture and medicine. Moreover, Bt toxin genes also have been efficiently used to enhance resistance to insect pests in genetically modified crops. In light of the scientific advantages of new molecular biology technologies, recently, some other new potentials of Bt have been explored. These new environmental features include the toxicity against nematodes, mites, and ticks, antagonistic effects against plant and animal pathogenic bacteria and fungi, plant growth-promoting activities (PGPR), bioremediation of different heavy metals and other pollutants, biosynthesis of metal nanoparticles, production of polyhydroxyalkanoate biopolymer, and anticancer activities (due to parasporins). This review comprehensively describes recent advances in the Bt whole-genome studies, the last updated known Bt toxins and their functions, and application of cry genes in plant genetic engineering. Moreover, the review thoroughly describes the new features of Bt which make it a suitable cell factory that might be used for production of different novel valuable bioproducts.