Iranian Journal of Biotechnology
Volume:17 Issue: 1, Winter 2019

Magnesium oxide nanoparticles are characterized with a wide variety of applications and are mass-produced throughout the world. However, questions remain regarding their safety. There has been paucity of toxicology research on their side effects, especially under in vivo conditions. The present paper aims at evaluating the toxicity of administering 10-15 nm magnesium oxide nanoparticles to Wistar rat under in vivo conditions. In addition, hematology, biochemistry, and histopathology of the rats are examined at various concentrations (62.5-125-250-500 µg.mL-1) over 28-days period. In this study, 35 male Wistar rats were randomly divided into five groups, comprising one control group and four experimental groups, assigned to various doses of MgO nanoparticles by intraperitoneal injection. Eventually, blood samples were collected, and all animals were sacrificed for liver and kidney tissue investigation. The findings showed that high concentrations of Magnesium oxide nanoparticles (250 and 500 µg.mL-1) significantly increased white blood cells, red blood cells, hemoglobin, and hematocrit compared with the control group (P < 0.05). Moreover, the nanoparticles elevated the levels of aspartate aminotransferase and alkaline phosphatase, whereas no significant difference in levels of alanine aminotransferase, gamma-glutamyl transpeptidase, urea, and creatinine were recorded in comparison with the control group (P < 0.05). Histopathological examinations in the rat's liver showed proliferation of bile ductules, congestion in some regions of the liver sinusoids, and apoptotic cells (probably) in high-dose groups, but no histological changes were found in the kidney functions. The results from the present study showed that the magnesium oxide nanoparticles in concentrations lower than 250 µg.mL-1 are safe for desired applications.

The utilization of methane for production of Poly-β-hydroxybutyrate (PHB) not only cuts the emissions of greenhouse gases but also greatly reduces PHB production cost. The aim of this study was to determine the effects of gas-phase conditions on PHB production by Methylosinus trichosporium OB3b. Bacterial cultivation and PHB production were conducted in a series of sealed serum bottles. Nitrogen-free mineral salts medium was used to induce PHB production in the presence or absence of N2 in the headspace. In the absence of N2, the highest PHB content (i.e., 52.9% of the dry cell weight with a PHB concentration of 814.3 mg.L-1) was obtained at a ratio of CH4:O2=2:1. Further study at different O2 concentrations with a fixed CH4 partial pressure in absence of N2 showed that PHB accumulation by methanotroph could be tolerated high oxygen partial pressure and its respond to the variation of the oxygen concentration depends on the methane partial pressure. In presence of N2, with headspace gas replenished only when oxygen was almost depleted, the degradation of intracellular PHB has appeared. In the regimen of updating headspace gas at the point when the PHB content began to decrease, the highest PHB content (i.e., 55.5% of the dry cell weight with 901.8 mg.L-1 PHB concentration and 12.5 mg.L-1.h-1PHB productivity) was obtained at 0.2 atm O2 and PHB accumulation was depressed with an oxygen concentration greater than 0.3 atm. The methanotroph responses differentially to the increase in the oxygen partial pressure with regard to PHB accumulation either in the presence or in the absence of N2.

Spine gourd (Momordica dioica Roxb. Willd) is one of the important cucurbitaceous crops grown across the world for vegetable and medicinal purposes. Diseases caused by the DNA viruses are becoming the limiting factors for the production of spine gourd reducing its potential yield. For the commercial cultivation of the spine gourd, propagation material used by most of the growers is tuberous roots and stem cuttings, which in turn results in an increased occurrence of the mosaic disease. There is a need for understanding the causal agent; through characterization of which will lead to the designing management strategies for the spine gourd mosaic disease control. Characterization of a begomovirus and its satellites associated with mosaic disease on spine gourd. Total DNA was extracted from spine gourd samples exhibiting symptoms typical to the begomoviruses infection (mosaic mottling, leaf curl) and was tested by PCR using begomovirus specific primers.  Furthermore, the complete genome of begomo viruses (DNA A, DNA B, alpha satellite, and beta satellite) was amplified by rolling circle amplification (RCA) method. The full-length sequences of DNA A, DNA B, alpha satellite, and beta satellite isolated from symptomatic spine gourd were determined. The full length genomes (DNA A and DNA B) of the Tomato leaf curl New Delhi Virus (ToLCNDV) infecting spine gourd were compared with the other begomovirus genomes available in the data base. The sequence analysis has revealed that DNA A and DNA B components of the begomovirus infecting spine gourd share 95.4-96.2 and 86.7-91.2% identical sequence (i.e., nucleotide (nt) identity) with that of ToLCNDV infecting potato and cucurbits in the Indian subcontinent isolates reported earlier (available in GenBank), respectively. Further, alpha satellite and beta satellite were also detected in the begomovirus infected spine gourd samples. The recombination analysis of the DNA A, DNA B, beta satellite, and alpha satellite of the begomovirus infecting spine gourd showed the associated begomovirus and satellite DNAs were driven from the different begomoviruses, leading to emergence as a new variant of the begomovirus infecting spine gourd. The commercial cultivation of the spine gourd by most growers depends on the tuberous roots and stem cutting. The occurrence of begomovirus in spine gourd gives an alarming signal against utilization of such infected plant materials in the crop breeding and improvement programs. Using the clean virus-free vegetative propagation material is considered as one of the most important methods for controlling viral diseases. The study is highly useful for detection of the begomovirus infecting spine gourd in the detection of the virus infection in the clonally propagated planting material.

The tumor necrosis factor alpha (TNFα) is a cytokine that produced principally by monocyte/macrophages and T lymphocytes, respectively. TNFα is recognized as the primary mediator of immunity in inflammation reaction.  One important application of Tumor Necrosis Factor Receptor 2 (TNFR2) is for the treatment of autoimmune diseases like rheumatoid arthritis (RA). The aim of this study is to examine the therapeutic trace of the recombinant humanTNFR2 on collagen-induced arthritis (CIA) in mice. CIA was created in 20 mice by immunization with bovine type II collagen (CII). After the mice were boosted on day 21, they were injected with the recombinant protein in test group (1 mg.kg-1) and assessed edema in paws and knee joints after two weeks. The quantities of inflammatory cytokines such as TNF-α, interleukin-1 beta (IL-β1), interleukin-6 (IL-6), and interleukin-10(IL-10) in serum were evaluated through enzyme-linked immunosorbent assay (ELISA) kit. In addition, the histopathology of joints sections was analyzed. The cytokines TNF-α, IL-1β, and IL-6 values in serum markedly decreased in groups treated with TNFR2 (P < 0.01-0.001). The results showed that treatment with TNFR2 significantly reduced edema in paws and joints (P < 0.01-0.001). Pathological investigations proved that administration of recombinant TNF receptor has blocked or protected joints from progressive damage. This study suggests that the anti-arthritic effectiveness of TNFR2 will repress the symptoms of rheumatoid arthritis. Moreover, it seems that TNFR2 is a strong candidate for the treatment of the RA disease.

According to the epidemiological studies, consuming olive products can decrease the incidence of the different types of cancers mostly due to the high anti-oxidant properties of their polyphenolic compounds. To evaluate the anti-oxidant and anti-proliferative potentials of the olive fruits total polyphenols on the gastric adenocarcinoma MKN45 cells in comparison to the normal Hu02 cells. The total phenolic content of the olive fruits and radical scavenging activity were determined by Folin and 2,2-diphenyl-1-picrylhydrazyl (DPPH) tests respectively. MTT assay was performed for the evaluation of the cell viability. Intracellular reactive oxygen species (ROS) level was measured using DCFH-DA. Statistical analysis was performed using SPSS 16 statistical software. Treatment of the MKN45 cells with the phenolic compounds extracted from olive fruits decreased growth and viability of the cells in a dose- and time-dependent manner. In addition, treatment of the MKN45 cells with a combination of the phenolic compounds extracts and cytarabine further decreased cell compared to monotherapy of the cells with each compound alone. Mechanistically, we showed that the anti-cancer effects of the olive polyphenols in the MKN45 cells are mediated through depletion of ROS. Similarly, polyphenolic extracts were found to decrease ROS level in the normal cells at the concentrations of 500 and 1000 μg.mL-1 and short treatment times (6 h), but the viability of these cells did not significantly change. At high concentrations (2000 μg.mL-1) of the phenolic extracts or at longer times of incubation (12 h), however, both ROS levels and the viability of the cells were significantly decreased in the normal cells. The olive fruits polyphenolic extract modulates ROS levels and selectively targets cancerous cells at low concentrations. Also, the effects of cytarabine could be potentiated by the olive fruits polyphenols. Thus, for a combined protocol of cancer cell therapy, olive fruit polyphenolic compound could be proposed as a proper candidate.

Recombination Activating Genes (RAG) mutated embryonic stem cells are (ES) cells which are unable to perform V (D) J recombination. These cells can be used for generation of immunodeficient mouse. Creating biallelic mutations by CRISPR/Cas9 genome editing has emerged as a powerful technique to generate site-specific mutations in different sequences. The main purposes of this study were to achieve complete knock-out of RAG1 gene by investigating the nature of mutations in mutant mESC and to generate RAG1 knock-out mESCs containing homozygous indels with the aim of creating desired and specific RAG-1 -/- mutant mouse in a shorter period of time. Here, we first utilized CRISPR/Cas9 system to target RAG1/RAG2 genes in NIH3T3 cells to test the activity and efficiency of our CRISPR system. Then we used the system for targeting RAG1 gene in mouse embryonic stem cell (mESCs) to generate knock-out embryonic stem cells. This method combined with highly active single guide RNA (sgRNA) is an efficient way to produce new RAG1-knockout mESCs in the selected regions of early coding DNA sequence, approximately between nucleotide c. 512-c. 513 and nucleotide c. 725-c. 726 of RAG1 coding sequence that had not been targeted previously. CRISPR gene editing resulted in a multitude of engineered homozygous and compound heterozygous mutations, including both in-frame and out-of-frame indels in 92% of mES cell clones. Most of the mutations generated by CRISPR/Cas9 system were out-of-frame, resulting in a complete gene knockout. In addition, 59% of the mutant ES cell clones carried out-of-frame homozygous indel mutations. The RAG1-knockout mESC clones retained normal morphology and pluripotent gene expression. Our study demonstrated that CRISPR/Cas9 system can efficiently create biallelic indels containing both homozygous and compound heterozygous RAG1 mutations in about 92% of the mutant mESC clones. The 59% of mutant ES cell clones carried out-of-frame homozygous indel mutations.

Newcastle disease is a major avian disease that causes enormous economic loss in poultry industry. There have been a number of reports on the suitability of plant-based recombinant vaccine against this disease. Fusion (F) and hemagglutinin-neuraminidase (HN) epitopes of the Newcastle disease virus (NDV) represent the major immunogenic sites for development of recombinant anti-ND vaccines in plant hosts. The main objective of this research was to evaluate the ability of a recombinant anti-ND vaccine in induction of immune responses in animal model. In this study, immunogenicity of recombinant fusion (F) and hemagglutinin-neuraminidase (HN) epitopes of the Newcastle disease virus (NDV) is investigated in an animal model. The corresponding genes encoding amino acids 65-81 of the F protein and 346-353 amino acids of HN were expressed in tobacco seedling using agrobacterium-mediated transformation. Expression of the foreign gene in the tobacco seedlings was investigated by a number of molecular assays including Real-Time PCR and ELISA. Transgenic plant extract was used to induce immunogenic response in animal model. Integration of the foreign gene in plant host genome was confirmed by polymerase chain reaction (PCR). Expression of the foreign recombinant protein was confirmed by Real-Time PCR and ELISA assays. Immunogenicity of the recombinant protein was investigated in rabbit by subcutaneous injection. Results indicated that the transgenic plant extract can induce immune responses in the host as confirmed by presence of specific antibodies in the sera in ELISA assay. Western blot assays showed that the foreign gene was actually expressed in transgenic seedlings. The results obtained in this research provide further evidence on applicability of plant-based recombinant vaccines for protection of poultry against Newcastle disease.

The unique expression of fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL) has been previously reported. Detecting FMOD in CLL patients using specific anti-FMOD mAbs might provide a promising method in detection, monitoring, and prognosis of CLL. In this study, we aimed for producing specific antibodies against FMOD to facilitate further cohort study of CLL, thus addressing FMOD as a potential target of detection. Human FMOD gene (1087 bp) was extracted from genome of the CLL patients, and was cloned into the expression vector of pET-22b (+). The recombinant FMOD protein (rFMOD) was expressed in Escherichia coli. The purified rFMOD protein was used as an immunogen in rabbit and mice. Hybridoma technology was used to develop the monoclonal antibodies (mAbs). Polyclonal antibody (pAb) was purified from the rabbit sera using affinity column. The reactivity of anti-FMOD antibodies was assessed in ELISA, immunocytochemistry (ICC) and Western blot. ICC results showed that the anti-FMOD antibodies specifically detected FMOD in CLL PBMCs and cell lines. The developed anti-FMOD pAb detected FMOD in CLL lysates, compared to healthy PBMCs, in Western blot and ELISA. The developed anti-FMOD mAbs, and pAb specifically detect FMOD in CLL samples and might be used as research tools for further investigations in CLL.

Oat (Avena sativa) with high nutritive value and fiber content is used as the main food grain in many countries for human diet as well as animal feed. Recently, it became difficult to transfer new genes through the conventional breeding due to the lack of desirable traits. The current study aimed at achieving a standardized protocol for Agrobacterium-mediated transformation in oat. For oat transformation, mature seeds were sterilized, germinated, and used for explants generation. Agrobacterium tumefaciens GV3101 with the binary vector pCAMBIA 1305.1, which carries gus as reporter gene, was utilized in the transformation. The co-cultivation treatment assisted with sonication, and vacuum infiltration, and their combination was employed for transformation with different incubation periods of 48, 72, and 96 hours under the dark conditions. Among the different transformation treatments, the vacuum treatment with 72 hours dark incubation had the best results. Vacuum infiltration of the cultures from leaf base produced a maximum of 25% hygromycin-resistant explants. These explants upon GUS assay and PCR analysis revealed 21.85% and 19.04% transformation efficiency, respectively. It could be concluded that vacuum infiltration assisted Agrobacterium-mediated transformation is the most efficient method to conduct the genetic improvement of the oat using transformation protocol.

The UL31 protein of herpes simplex virus 1 (HSV-1) plays an important role in the HSV-1 replication, however, its pinpoint functions in the life cycle of the virus have yet to be adequately elucidated. An antiserum specific for detecting HSV-1 UL31 was prepared as the foundation for future research on the role of UL31 in the course of HSV-1 infection. Recombinant protein of UL31 was expressed in Escherichia coli, which was then purified and employed to raise the level of antiserum in mice. Subsequently, western blot and immunofluorescence assay (IFA) were utilized to detect the specific antiserum. The recombinant UL31 protein consisting of N-terminal 27 aa of UL31 was fused to EYFP and His-tag. It was expressed, purified, and applied to the preparation of the antiserum. Western blot analysis and IFA demonstrated that this antiserum could detect both the recombinant UL31 and the native UL31. Our results manifest that this antiserum could be conducive to further investigations concerning the roles of UL31 in the HSV-1 infection.

Many physical and mechanical phenomena occur during the acupuncture and tuina regime, and pressure is one of the most basic mechanical phenomena. To understand the cellular bio-physical mechanism of basic mechanical stimulation via acupuncture and tuina by investigating the effect of different in vitro pressures on the cell viability and protein expression differences that originate from the facial fibroblasts around the meridians. In vitro culture of the facial fibroblasts around the meridians was conducted using different pressures to perform single and multiple stimulation(s) on the cells. Thus, the changes in the fibroblast cell viability (cell viability rate and diameter) were tested, and changes in the fibroblast protein expression were observed. We found that the pressure stimulation may excite the fascial fibroblast viability at the acupoint and increase cell viability. Two interactive factors are involved: the pressure intensity and the number of pressure stimulations. In addition, we found that all three pressures lead to significant regulation effects on the protein expression of the meridian-related fascial tissue fibroblasts, and clustering analysis revealed that 100 kPa pressure stimulation exhibits the most evident effect on the protein expression which is the pressure inducing the most differentiated protein expression. During the in vitro pressure process, the difference in the cell viability rate and protein expression of the facial fibroblasts around the meridians may (from a cell mechanics’ point-of-view) reveal the cytobiological and therapeutic mechanism of the basic mechanical stimulation via acupuncture and tuina on the facial fibroblasts around the meridians.

While mammalian embryos can adapt to their environments, their sensitivity overshadows their adaptability in suboptimal in vitro conditions. Therefore, the environment in which the gametes are fertilized or to which the embryo is exposed can greatly affect the quality of the embryo and consequently its implantation potential. Since providing an optimal culture condition needs a deep understanding of the environmental effects, and regarding the fact that normal morphology fails to be a reliable indicator of natural embryo development, the current study aimed at comparing in vivo- and in vitro-derived blastocysts at the molecular level. In vivo and in vitro mouse blastocysts were obtained by flushing the uterine horns and in vitro fertilization/culture, respectively. Normal blastocysts of both groups were evaluated in terms of hatching rate and expression of three lineage-differentiation-, apoptosis-, and implantation-related genes. The hatching rate was lower in In vitro fertilization (IVF)-produced blastocysts in comparison with that of the in vivo counterparts. More importantly, the study results indicated significant changes in the expression levels of eight out of ten selected genes, especially Mmp-9 (about -10.7-fold). The expression of Mmp-9 in trophoblast cells is required for successful implantation and trophoblast invasion. The current study, in addition to confirming that the altered gene expression pattern of in vitro-produced embryos resulted in normal morphology, provided a possible reason for lower implantation rate of in vitro-produced blastocysts regarding the Mmp-9 expression.

Root to shoot connection and transfer of information seems to be taken place mostly via the transmissions of signal molecules, secondary metabolites, amino acids, hormones and proteins, through xylem sap. Examination of earlier reports is indicative of relatively high levels of conservation in xylem sap protein compositions. Apparently these protein molecules are being synthesized in roots in response to environmental changes and get transported to aerial plant parts after secretion into xylem sap. In order to comprehend this so-called passive signaling, some questions need to be answered: 1) Do these proteins have the capability to act as signals? 2) How much energy does root spend for the biosynthesis of the secreted proteins? How similar is the amount of energy that root cells spent for the biosynthesis of intra- and extra-cellular proteins? Reported xylem sap proteins curated from Arabidopsis, maize and soybean. Their sequences were put under scrutiny in terms of considering their mobility, and physical and chemical properties. Metabolic energy required for their biosynthesis along with the energy hidden in their peptide bonds were calculated and compared with random non-xylem sap proteins as control. Xylem sap proteins were significantly smaller than the root proteins, while they were bigger in size when compared to the leaf group. Xylem protein pIs were significantly higher than the control proteins in different plants. Similarly, the protein stability was higher for xylem sap proteins in comparison with roots and leaves in all analyzed plants, except for soybean that the stability was indifferent between xylem and root. The data were suggestive a significantly lower energy consumption for the synthesis of xylem sap proteins. Lower energy consumption may suggest an economical route of communication between roots and shoots in plants that mainly rely on symplastic signaling.