Iranian Journal of Biotechnology
Volume:18 Issue: 1, Winter 2020

NPs (NPs) with unique chemical and physical properties can be used for therapeutic purposes because of their strong antimicrobial activates. NPs have been used as an antimicrobial agents to inhibit microbial growth. In view of the strong antimicrobial activity of NPs, the biogenic synthesis and leishmanicidal activity of rod-shaped zinc oxide (R-ZnO) NPs was explored using Lilium ledebourii tuber extract. The ensuing NPs are characterized by UV-visible spectroscopy, X-ray diffraction and transmission electron microscopy and their leishmanicidal activity evaluated against the Leishmania major (L. major) by MTT assay. The R-ZnO NPs displayed excellent leishmanicidal activity against the L. major as they significantly inhibited the amastigotes. The IC50 values of R-ZnO NPs being ~ 0.001 mg.mL-1. R-ZnO NPs can inhibit L. major growth in a dose-dependent manner under in vitro conditions. A simple, low-cost feasible and eco-friendly procedure was developed for biosynthesis of R-ZnO NPs using natural bioresource that can inhibit human parasite cells growth in a dose-dependent manner under in vitro conditions.

Shigella is among the most important human pathogenic microorganisms, infecting both humans and nonhuman and causing clinically severe diarrhea. Shigella must be enriched before detection, which is time-consuming. To develop a sensitive, rapid, and specifc method for Shigella detection. Shigella was used as an antigen to generate monoclonal antibodies (mAbs). mAbs were screened via indirect enzyme-linked immunosorbent assay (ELISA) and western blot, and two mAbs were selected. The mAb A3 showed high afnity and specifcity and was used to develop immune magnetic beads (IMBs) for Shigella enrichment. An immunocapture (IC)-PCR primer was designed from the ipaH gene, and IC-PCR was developed based on the IMBs and PCR. This system shortened the Shigella detection time to 70 min. The sensitivity of the IC-PCR was 9 colony-forming units.mL-1 in artifcial milk. The accuracy of the IC-PCR was confrmed using 46 clinical samples collected from monkeys. The IC-PCR results were consistent with the serological and biochemical assays. The IC-PCR described herein accurately detected Shigella from milk samples, monkeys and can thus be used to complement classical detection methods.

Diarrhea caused by Escherichia coli is a major cause of morbidity and mortality in young animals. Few treatment options are available, mainly antibiotic therapy increasingly limited by resistance to commonly used drugs. The aim of this work was to develop immunotherapy based on the use of camel VHH antibody fragments, or nanobodies, to target pathogenic E. coli surface antigens. We immunized a camel with a killed strain we had previously isolated from a diarrheic camel calf and identified as expressing the F17 fimbriae antigen. The immunized animal developed an anti-E.coli immune response including heavy-chain antibodies. Lymphocytes from this animal were purified and RNA isolated to create a VHH library by phage display with a size of about 109 individual transformants. Panning on live E. coli cells resulted in the isolation of VHH fragments specific to the cell surface antigens. The identification of these antigens can lead to the development of new diagnostic and therapeutic tools against diarrhea.

MiR-103a-3p is a small non-coding RNA and has been reported to be involved in osteogenic proliferation and differentiation, but the role of miR-103a-3p in human osteoarthritis (OA) remains unclear. In this study, we aimed to explore its function and molecular target in chondrocytes during OA pathogenesis. Total 12 experimental OA rat models, together with 12 rats without knee OA lesions were established and cartilage samples were collected. Chondrocytes were treated with LPS in vitro. MiR-103a-3p expression was detected in articular cartilage tissues and chondrocytes using quantitative real-time PCR. Knee OA chondrocytes were transfected with miR-103a-3p mimics, and siHMGB1, respectively. Then cellular proliferation, apoptosis, apoptosis related factors and inflammatory cytokines were analyzed by MTT, flow cytometry, western blot, caspase-3 activity and ELISA, respectively. Potential targets of miR-103a-3p were predicted using series of bioinformatics analysis, then confirmed by luciferase reporter assay. We first found miR-103a-3p was significantly down-regulated in the articular cartilage tissues from experimental OA rats, as well as in chondrocytes treated with LPS in vitro. The gain-of-function assay further demonstrated that up-regulation of miR-103a-3p significantly promoted cell proliferation, inhibited apoptosis and inflammation, which was accompanied with elevated expression of PCNA, and reduced expression of caspase-3, PARP, IL-1β, IL-6, IL-10 and TNF-α. Furthermore, high mobility group box 1 (HMGB1), an important inflammatory mediator of OA, was a target of miR-103a-3p. Moreover, knockdown of HMGB1 mimicked the effects of miR-103a-3p on chondrocytes treated with LPS. Taken together, our study suggests that miR-103a-3p inhibits chondrocyte apoptosis and inflammation in OA, which appears to be an attractive approach to OA treatment.

Characterizing the structure and function of superoxide dismutase (SOD), as an antioxidant enzyme providing opportunities for its application in food supplements.
In this study, the features of the Manganese-SOD of Lactococcus lactis (SDLL), subsp. cremoris MG1363, as probiotic bacteria, were determined on the basis of the computational methods.
The protein’s physicochemical properties and the prediction of its secondary structure were determined via the ProtParam server and the GOR program respectively. Moreover, the 3D structures of the proteins were constructed via the MODELLER on the basis of the homology method and the threading algorithm MUSTER. On the other hand, the structural stability of the models was assayed under the quasi-physiological conditions by the GROMACS program via the GROMOS96 43a1 force field in Linux system. Finally, using the Molecular Docking Studies, the functionality features of the models were predicted through their affinity with the corresponding substrates.
The results revealed the physicochemical properties of the SDLL and a 3D model of a chain of the enzyme being similar to the SOD from the Bacillus Subtilis (SDBS). The model of the SDLL was checked for quality control purposes including the Ramachandran plot, the ERRAT and the Verifiy3D. The model was suggestive of the structural stability in quasi-physiological conditions; yet, less than that of the SDBS. Assessing the cause of the instability in the SDLL model was indicative of two unstable regions in the area far from the enzyme’s active position, they were considered suitable for mutagenesis. Accordingly, the loop substitution for the corresponding region of SDBS and the deletion of the loop positioned at the C terminal of SDLL resulted in a mutant of SDLL with more stability and appropriate affinity with the corresponding substrate.
In general, the study provides a new model of SDLL with certain thermostable features, and a new mutant with suitable stability and functionality on the basis of the direct mutagenesis being used in different applications.

Background：Vibrio splendidusVs is an important aquaculture pathogen that can infect a broad host of marine organisms. In our previous study, an antagonistic bacterium Vibrio sp. V33 that possessed inhibitory effects on the growth and virulence of a pathogenic isolate V.splendidusVs was identified.
Here, we further explored the antagonistic substances and antagonistic effects from the viewpoint of iron competition.
The main antagonistic substances in the supernatants from Vibrio sp. V33 were identified using the bioassay-guided method. The response of V. splendidus Vs under the challenge of cell-free supernatant from Vibrio sp. V33 was determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and real-time reverse-transcription PCR.
The main antagonistic substances produced by Vibrio sp. V33 have low molecular weights, are water soluble, and are heat-stable substances. Meanwhile, the iron uptake rate of Vibrio sp. V33 was higher than that of V. splendidus Vs. In the presence of cell-free supernatant from Vibrio sp. V33, expressions of two functional genes, viuB and asbJ related to ferric uptake processes in V. splendidus Vs, were up-regulated, whereas furVs coding the ferric uptake repressor was suppressed below 0.5-fold. One gene coding phosphopyruvate hydratase does not change at mRNA level, but was up-regulated at protein level.
Our results suggested that antagonistic effect of Vibrio sp. V33 on the pathogenic isolate V.splendidusVs was partially due to the stronger ability of Vibrio sp. V33 to seize iron. This cell-free supernatant from Vibrio sp. V33 created an iron-limited milieu for V. splendidusVs, which led to the changed expression profiles of genes that were related to iron uptake in V. splendidusVs.

Clear cell renal cell carcinoma (ccRCC) represents approximately 70% of RCC,as the most frequent histological subtype of RCC. MiR-138-5p, a tumor-related microRNA (miRNA), has been reported to be implicated in the diverse types of human malignancies, but its role in ccRCCremains unclear.
The study was designed to investigate the functional behaviors and regulatory mechanisms of miR-138-5p in ccRCC.
Quantitative real-time PCR and western blotting analyses were performed to determine the expression of miR-138-5p and TMEM40 in ccRCC tissues. Pearson’s correlation coefficient was utilized to evaluate the correlation between miR-138-5p and TMEM40 expression. The function of miR-138-5p and TMEM40 in the cell proliferation, migration and invasion of ccRCC cells (786-O and ACHN) was assessed by CCK-8, colony formation, wound healing and transwell assay, respectively. A luciferase reporter assay was performed to confirm the direct binding of miR-138-5p to the target gene TMEM40.
We found the expression of miR-138-5p was significantly down-regulated, while TMEM40 was remarkably up-regulated in ccRCC tissues. TMEM40 expression was discovered to be inversely correlated with miR-138-5p expression in ccRCC tissues. Functional studies demonstrated that miR-138-5p overexpression or TMEM40 knockdown significantly suppressed ccRCC cell proliferation, migration and invasion in vitro. Notably, we experimentally confirmed that miR-138-5p directly recognizes the 3’-UTR of the TMEM40 transcript and down-regulated its expression in ccRCC cells.
Taken together, our findings provide the first clues regarding the role of miR-138-5p as a tumor suppressor in ccRCC by directly targeting  of TMEM40.

Phenol is an aromatic pollutant in industrial wastes that in combination with salts is highly toxic for all forms of life. Phenol elimination is the foremost challenge to meet the goal of pollutant-free environment.
The present study was carried out to isolate phenol degrading bacteria which can degrade phenol under saline conditions and to identify the isolated strains using 16S rRNA gene sequence analysis.
Sediment samples were collected from Rawal Lake, Islamabad, Pakistan and enriched in mineral salt medium (MSM) containing phenol (150 mg.L-1). Isolated strains were identified on the basis of 16S rRNA gene sequence analysis. Growth of strains were tested at different pH, NaCl concentrations and temperature using Tryptic Soy Agar (TSA). Tolerance to phenol (0-750 mg.L-1) was checked at 5% NaCl and phenol degrading experiment was performed at 4% NaCl, pH 7 and 30 oC. In both, phenol tolerance and degradation study, phenol was used a sole source of carbon and energy.
Thirteen bacterial strains were isolated after enrichment among which, NIGAB-1 was found capable of degrading phenol in saline conditions. This strain was identified as Bacillus sp.NIGAB-1on the basis of 16S rRNA gene sequence analysis and the closest match was Bacillus marisflavi with 99.71% sequence identity. The Bacillus sp.NIGAB-1 exhibited best growth at 30 oC at pH 7 with 10% NaCl. The optimum phenol concentration for growth was recorded as 300 mg.L-1. This strain degraded 300 mg.L-1 of phenol at 4% NaCl in 120 hours with the average degradation rate of 2.63 mg.L-1.h.
These findings suggest that this strain could be efficient in phenol degradation at adverse environmental conditions and helpful in remediation of phenol where the salt concentration is high.

Cysteine proteases of the liver fluke, Fasciola hepatica, participate in catabolism of proteins, migration of the fluke through host tissues and combat host immune system.

In this study, we evaluated proteolytic activity of F. hepatica recombinant cathepsin L1 (rCL1) against gelatin and collagen as common substrates.

The coding sequences of F. hepatica CL1 were cloned and expressed in E. coli, in our previous study. The rCL1 was purified by nickel affinity chromatography with a HisTrap Column. The protein concentrations of the purified fractions were determined by Bradford assay. Rat collagen type-1 was treated with distinct amounts of rCL1 at 37 °C, overnight, and the byproduct was analyzed by SDS-PAGE. Furthermore, we used bovine skin gelatin as zymography substrate to evaluate the gelatinolytic activity of the purified rCL1.

Recombinant CL1 was capable to digest intact type-1 collagen within 24 h and the gelatinlytic activity of rCL1 was visible at approximately 37 kDa region, with optimal activity at acidified conditions (pH 4).

Findings provide a possible mechanism by which a major secretory molecule of F. hepatica could be involved in parasite survival as well as its pathogenesis.

Many problems of combinatorial optimization, which are solvable only in exponential time, are known to be Non-Deterministic Polynomial hard (NP-hard). With the advent of parallel machines, new opportunities have been emerged to develop the effective solutions for NP-hard problems. However, solving these problems in polynomial time needs massive parallel machines and is not applicable up to now.
DNA (Deoxyribonucleic acid) computing provides a fantastic method to solve NP-hard problems in polynomial time. Accordingly, one of the famous NP-hard problems is assignment problem, which is designed to find the best assignment of n jobs to n persons in a way that it could maximize the profit or minimize the cost.
Applying bio molecular operations of Adelman Lipton model, a novel parallel DNA algorithm have been proposed for solving the assignment problem.
The proposed algorithm can solve the problem in  time complexity, and just O(n) initial DNA strand in comparison with  initial sequence, which is used by the other methods.
In this article, using DNA computing, we proposed a parallel DNA algorithm to solve the assignment problem in linear time.

TGF-β isoforms play crucial roles in diverse cellular processes. Therefore, targeting and inhibiting TGF-β signaling pathway provides a potential therapeutic opportunity. TGF-β isoforms bind and bring the receptors (TβRII and TβRI) together to form a signaling complex in an ordered manner.
Herein, an antagonistic variant of TGF-β (AnTβ) has been designed and prepared to inhibit the formation of signaling complex and consequently its signaling pathway. This TGF-β homodimeric variant contains intact TβRII binding sites and blocked TβRI binding sites by substituting three peptide segments. So, AnTβ could only bind to TβRII, but prevent binding and recruitment of TβRI to form a signaling complex.
A reliable model of AnTβ was built and refined using molecular dynamics (MD) simulation, followed by investigating the interactions of AnTβ with the receptors using in silico docking studies. After expression of disulfide-linked AnTβ in a SHuffle strain and purification of the protein using affinity chromatography, its biological activity was evaluated using Mink lung epithelial cells (Mvl Lu).
No meaningful significant changes in AnTβ structure were observed when compared with the native protein. Based on the docking analysis, AnTβ binds to TβRII similar to TGF-β and its binding to TβRI was diminished considerably which was consistent with our design purpose. Cell-based bioassay indicated that AnTβ could modulate TGF-β-induced cell growth inhibition.
Our analysis suggests that the antagonistic potency of AnTβ can be used as an anti-TGFβ signaling factor in the future perspectives.