Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

Purpose: The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. Methods: To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. Results: The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. Conclusion: It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein

Decontamination of tomato, red cabbage, carrot, fresh parsley and fresh green onion inoculated with Shigella sonnei and Shigella flexneri by some Essential oils (in vitro condition)

Background: Essential oils and their major constituents are useful sources of antimicrobial compounds. There are a few reports on the decontamination and antimicrobial activity of essential oils towards Shigella spp.Materials and Methods: In this study, the antimicrobial and decontamination potentials of essential oils at different concentrations, belonging to plants such as Thymus vulgaris, Saturiea hortensis, Mentha polegium, Cuminum cyminum, Lavandula officinalis and Mentha viridis L. (spearmint), towards Shigella sonnei and Shigella  flexneri were investigated. The disk diffusion method demonstrated the antimicrobial potential of the essential oils.Results: The ability of essential oils to decontaminate vegetables such as, tomato, red cabbage, carrot, fresh parsley and fresh green onion that were previously inoculated with Shigella spp. was determined. Inhibitory effects of essential oils towards Shigella spp. were noted in the disk diffusion method. There was a reduction in Shigella population following inoculation of cultures with 0.5% and 0.1% (v/v) essential oils.Conclusion: This study confirmed that essential oils have the potential to be used for decontamination of vegetables

Optimized Signal Peptide for Secretory Expression of Human Recombinant Somatropin in E. coli

Purpose: The human somatropin is a single-chain polypeptide with a pivotal role in various biological processes. Although Escherichia coli is considered as a preferred host for the production of human somatropin, the high expression of this protein in E. coli results in the accumulation of protein as inclusion bodies. Periplasmic expression using signal peptides could be used to overcome the formation of inclusion bodies; still, the efficiency of each of the signal peptides in periplasmic transportation is varied and often is protein specific. The present study aimed to use in silico analysis to identify an appropriate signal peptide for the periplasmic expression of human somatropin in E. coli. Methods: A library containing 90 prokaryotic and eukaryotic signal peptides were collected from the signal peptide database, and each signal’s characteristics and efficiency in connection with the target protein were analyzed by different software. The prediction of the secretory pathway and the cleavage position was determined by the signalP5 server. Physicochemical properties, including molecular weight, instability index, gravity, and aliphatic index, were investigated by ProtParam software. Results: The results of the present study showed that among all the signal peptides studied, five signal peptides ynfB, sfaS, lolA, glnH, and malE displayed high scores for periplasmic expression of human somatropin in E. coli, respectively. Conclusion: In conclusion, the results indicated that in-silico analysis could be used for the identification of suitable signal peptides for the periplasmic expression of proteins. Further laboratory studies can evaluate the accuracy of the results of in silico analysis

Review of NKG2D function and its related ligands: review article

The natural killer group 2D (NKG2D) is a transmembrane protein and a member of the CD94/NKG2 family of C-type lectin-like receptors. NKG2D is encoded by the KLRK1 gene, which is located in the NK-gene complex (NKC) placed on chromosomes 6 and 12 in mice and humans, respectively. NKG2D forms a homodimer structure and binds through ectodomains with its related ligands. Each of its monomers consists of two β-sheets, two α-helices, and four disulfide bands and also contains a β-strand that distinguishes it from other C-type lectin-like receptors. NKG2D ligands are homologs of major histocompatibility complex (MHC) class I molecules in mice and humans. MHC class I chain-related protein A (MICA) and B (MICB) and human cytomegalovirus UL16-binding proteins (ULBP1-6) are recognized by the human NKG2D. In Natural Killer (NK) cells, NKG2D-mediated cytotoxicity can be elicited via two different systems by signaling from immunoreceptor tyrosine-based activation motifs in DAP12 or via a Syk-independent pathway activated by DAP10. Therefore, NKG2D is an activating immunoreceptor which was first recognized on NK cells but subsequently found on γδT cells, CD8+ αβT cells, and macrophages. NKG2D-ligand diversity may facilitate the detection of the presence of a broad range of viruses and may provide protection against rapidly evolving cancers. NKG2D ligand recognition induces and/or improves immune responses to cancer cells. NK cells recognize a wide range of stressed cells. The activation of NKG2D receptor can lead to the lysis of the target cell and the production of various cytokines and chemokines depending on the nature of the stimulation as a result of NK and myeloid-mediated innate immunity and as well as T γδ and CD8+ mediated-adaptive immune system. However, inappropriate expression of NKG2D ligands could cause autoimmune diseases in healthy cells, including rheumatoid arthritis, colitis, celiac disease, multiple sclerosis, alopecia areata, type 1 diabetes, and chronic obstructive pulmonary disease. Therefore, a precise understanding of the structure and function of NKG2D receptor and its interaction with various ligands may lead to the development of strategies to treat autoimmune diseases. Hence, the purpose of this review is to examine the detailed studies on the function of NKG2D receptor and their related ligands

Prevalence of thrombophilic gene polymorphisms in an Azari population of Iran

There is several evidence suggests that thrombophilic gene polymorphisms may influence susceptibility to thromboembolic events. The prevalence of these polymorphisms is different in various races and ethnics. Accordingly, we studied the prevalence of Factor V (G1691A and A4070G), prothrombin G20210A and PAI-1 4G/5G in healthy northwest population of Iran. In this prospective study, 500 healthy individuals, who had no history of both personal and family history of thromboembolic disorders, were selected as a sample of healthy population in northwestern Iran. Genotyping of these polymorphisms was performed using the amplification refractory mutation system-polymerase chain reaction method. No significant differences were detected between the expected and observed frequencies of FV G1691A and A4070G, prothrombin G20210A polymorphisms (P>0.05), while the expected frequency of 4G allele was significantly more than observed frequency in the studied population (P<0.01). These findings were compared with other reports from various populations. In conclusion, the allele frequency for FV G1691A and PAI-1 4G/5G polymorphisms showed relative consistency compared to those of previous studies, while the incidence pattern of FV A4070G polymorphism in Northwestern population of Iran showed conflicting results regarding other studied population. The prothrombin G20210A polymorphism was observed at a higher frequency than other studied populations

Tumor necrosis factor-alpha and its inhibition strategies: review article

Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine produced by a variety of cells, including hematopoietic and non-hematopoietic cells, malignant cells, macrophages, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, astrocytes, endothelial cells, and smooth muscle cells. TNF-α is a homo-trimeric molecular whose individual subunits are composed of antiparallel beta-sheets, forming a regular triangular prism shape. TNF-α binds to three receptor molecules through its receptor-binding sites, which are at the base of its pyramid structure. Biological responses to TNF-α are mediated through two different receptors: TNFR1 and TNFR2. These receptors are transmembrane glycoproteins with extracellular domains containing multiple cysteine-rich repeats that are structurally and functionally homologous, and the intracellular domains that are discrete and transduce their signals through both overlapping and distinct pathways. However, though TNF-α was initially discovered as an anti-tumor agent, it has been revealed that TNF-α and other ligands of this family are involved in some diseases like cancer, neurological, pulmonary, cardiovascular and autoimmune diseases and metabolic disorders. In general, TNF-α activates the control systems involved in cell proliferation, differentiation, inflammation and cell death, and the regulation of immune system. Although a normal level of TNF-α is very important for the regulation of immune responses, the persistence of the immune response as a result of inappropriate and excessive production of TNF-α can cause some inflammatory or autoimmune diseases. Accordingly, either neutralization TNF-α or blockade of its receptors using TNF-α inhibitors can be an effective therapeutic strategy to prevent or treat such inflammatory diseases. Several methods have been used to inhibit TNF-α, including the production of chimeric or fully human antibodies, soluble TNF-α receptors, or anti-TNF-α small molecules. The two previous agents are mostly capable of inhibiting the binding of TNF-α to its associated receptors, while anti-TNF-α small molecules, in addition to the above, inhibit the biosynthesis of TNF-α by blocking TNF-α mRNA biosynthesis, through the inhibition of its post-translational processing, or by blocking TNF-α receptors. Therefore, in this review article, we discuss the structure and characteristics of TNF-α and its related receptors: TNF-α signaling, TNF-α-mediated inflammatory diseases as well as TNF-α inhibition strategies

Haplotype and linkage disequilibrium of TP53-WRAP53 locus in Iranian-Azeri women with breast cancer.

Among the cancer susceptibility genes, TP53 is one of the crucial genes involved in cell cycle regulations and, therefore, it greatly affects breast cancer initiation and progression. In addition, WRAP53-a natural antisense transcript-regulates TP53 transcription and, as a protein, modulates the normal cell cycle, which results in breast cancer susceptibility. In this study, we aimed to analyze a haplotype comprising four SNPs, including rs1042522, rs17878362, rs2287499, and rs2287498, which are located at 5' regions of the TP53 and WRAP53 genes, in 118 patients and 110 healthy controls of the Iranian-Azeri population. In silico studies were conducted using the SIFT, Polyphen2, Fanthmm, RNAsnp, and SNP&GO online servers. Linkage disequilibrium (LD) and D' for each combination of the markers were calculated via the Haploview program. Our results showed that the GA1CC haplotype was the most frequent in the studied population. Additionally, no significant LD between any pairwise haplotypes was observed. The GA1CC and CA2GC haplotypes were significantly associated with breast cancer susceptibility. Moreover, the in silico analysis revealed the negative effects of rs2287499 and rs1042522 on WRAP53 and P53, respectively. In conclusion, the CA1GC haplotype was strongly identified as a breast cancer risk factor, and the GA1CC haplotype was assumed to be a protective factor against breast cancer risk. Hence, these markers may potentially be used as molecular prognostic and predictive biomarkers for breast cancer

Antibacterial Activity of Pseudonocardia sp. JB05, a Rare Salty Soil Actinomycete against Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacterium that causes many harmful and life-threatening diseases. Some strains of this bacterium are resistant to available antibiotics. This study was designed to evaluate the ability of indigenous actinomycetes to produce antibacterial compounds against S. aureus and characterize the structure of the resultant antibacterial compounds. Therefore, a slightly modified agar well diffusion method was used to determine the antibacterial activity of actinomycete isolates against the test microorganisms. The bacterial extracts with antibacterial activity were fractionated by silica gel and G-25 sephadex column chromatography. Also, the active fractions were analyzed by thin layer chromatography. Finally, the partial structure of the resultant antibacterial compound was characterized by Fourier transform infrared spectroscopy. One of the isolates, which had a broad spectrum and high antibacterial activity, was designated as Pseudonocardia sp. JB05, based on the results of biochemical and 16S rDNA gene sequence analysis. Minimum inhibitory concentration for this bacterium was 40 AU mL −1 against S. aureus. The antibacterial activity of this bacterium was stable after autoclaving, 10% SDS, boiling, and proteinase K. Thin layer chromatography, using anthrone reagent, showed the presence of carbohydrates in the purified antibacterial compound. Finally, FT-IR spectrum of the active compound illustrated hydroxyl groups, hydrocarbon skeleton, and double bond of polygenic compounds in its structure. To the best of our knowledge, this is the first report describing the efficient antibacterial activity by a local strain of Pseudonocardia. The results presented in this work, although at the initial stage in bioactive product characterization, will possibly contribute toward the Pseudonocardia scale-up for the production and identification of the antibacterial compounds

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Not AvailableThe increasing demand for crop production, given worldwide increases in the human population, puts pressure on moving natural resources towards sus-tainable development. This creates a big challenge for the upcoming generation. If improvement is not successful, there exists the unfortunate consequence that global food production may soon become insufficient to feed all of the world’s people. It is therefore essential that agricultural productivity be significantly increased in a more sustainable and environmentally friendly approach. Plant-beneficiary rhizo-bacteria (PBR) naturally activate microorganisms found in the soil. Because they are inexpensive, effective, and environmentally friendly, PBR are gaining impor-tance for use in crop production by restoring the soil’s natural fertility and protect-ing it against drought and soil diseases, thereby stimulating plant growth. PBR decrease the use of chemical fertilisers, pesticides, and artificial growth regulators; the intensive use of these inputs has led to severe health and environmental hazards, such as soil erosion, water contamination, pesticide poisoning, decreased ground-water table, water logging, surface crusting and depletion of biodiversity. The use of PBR has been proven to be an environmentally sound way of increasing crop yields by facilitating plant growth through either a direct or indirect mechanism with the aim of sustaining soil health over the long term. (7) (PDF) Towards Plant-Beneficiary Rhizobacteria and Agricultural Sustainability. Available from: https://www.researchgate.net/publication/325854138_Towards_Plant-Beneficiary_Rhizobacteria_and_Agricultural_Sustainability [accessed Nov 19 2018].Not Availabl