Iranian Journal of Biotechnology
Volume:17 Issue: 4, Autumn 2019

Programmed cell death protein-1 (PD-1)/PD-L1 pathway is one of the immune checkpoint pathways involved in the regulation of the immune responses and the suppression of anti-tumor defense. PD-1/PD-L1 blocking antibodies improve immune responses such as cytotoxic activity of CD8+/CD4+T cells and increase mortality of tumor cells as well; however, their use is accompanied by adverse side effects.
We aimed to produce a native blocker of human PD-1/PD-L1, for developing T cells cytotoxicity and tumor cells apoptosis.
We designed and cloned soluble human PD-1-GFP-pcDNA3.1/hygro construct in Escherichia coli strain TOP10 cells and then transfected this construct into the HEK cells. The concentration of the secreted shPD-1 in the supernatant was measured and the supernatant was used for blocking PD-L1 on the MDA-MB-231 cells. The cytotoxicity of CD8+/CD4+T cells and the apoptosis of MDA-MB-231 cells, under the influence of shPD-1 in the co-culture of T cells with the MDA-MB-231 cells, were evaluated using flow cytometry technique.
The GFP expression in the transfected cells illustrated the successful designing, transfection, and production of shPD-1. Soluble human PD-1 concentration in the supernatant of the transfected HEK cells was significantly higher than the untransfected cells. In addition, shPD-1 significantly blocked PD-L1 on the MDA- MB-231 cells, improved the cytotoxicity of CD4+T cells, and increased the apoptosis of MDA-MB-231 cells.
Overall, increased CD4+T cell cytotoxicity and tumor cells apoptosis under the influence of shPD-
1, confirmed the effectiveness of shPD-1 as a natural blocker of PD-L1and as an augmenter of the anti-tumor
immune responses.

Probiotics are food supplements that benefit the host by improving its intestinal microbial balance. Probiotics are used as diet supplements to prevent diarrhoea and improve lactose tolerance. The present study deals with the isolation of a potent probiotic strain capable of inducing healing properties in rat model. Probiotic VITSAMJ1 was isolated from goat milk using MRS media. The antimicrobial assay was carried out against S. aureus (MTCC 3160) and the wound healing properties were assessed on female Wistar rats. A 1.5 cm2 subcutaneous wound was induced in the rats, and a probiotic gel formulation was tropically applied onto the wounds. Tissue biopsy was carried out after days 1, 3, 5, 7, 9, and 11. Total leucocyte count and Histopathological analysis were performed after each interval. VITSAMJ1 can be effectively used for wound healing. VITSAMJ1 can be effectively used for wound healing.

Today, the use of maggot therapy has become widespread due to the increase in chronic ulcers in the world. The recombinant production of secreted enzymes from these larvae is a novel non-invasive method for the treatment of chronic ulcers. Lucilia sericata (L. sericata) collagenase (MMP-1) has been expressed in insect cells. Collagenase is an enzyme that is widely used in clinical therapy and industry. It has been indicated that collagenase is expressed and secreted in salivary glands of L. sericata while using for maggot debridement therapy.
In the present study we decided to produce the recombinant form of collagenase enzyme in Spodoptera frugiperda (SF9) insect cells using the baculovirus expression system (Bac-to-Bac).
cloned the coding sequences (residues 494-1705) of L. sericata collagenase into the pFastBacHTA as donor plasmid. After transposition in the bacmid of DH10Bac host, the bacmid was transfected into the Sf9 cell line, then the expressed recombinant collagenase (MMP-1) was purified using the Ni-NTA agarose.
The recombinant protein was verified by Western blotting. Furthermore, the biological activity of purified protein was measured in the presence of its specific substrate and its inhibitor, which was 67 IU.mL-1 based on our results, it was revealed that the characterized gene in our previous study codes L. sericata collagenesa enzyme.
Considering to the broad applications of collagenase in medical sciences, for the first time, we cloned the L. sericata collagenase (MMP-1) gene into the insect cell line to establish a method for the expression and purification of L. sericata collagenase (MMP-1). The result help for preparing and designing a safe and versatile recombinant drug in future.

Heterogeneous breast cancer is the most common cause of cancer-related mortality. Obesity defined by BMI is a known major risk factor for breast cancer.
The purpose of this study was to explore the role of obesity related-polymorphisms rs9939609 Fat Mass and Obesity-associated (FTO) and rs17782313 MC4R in breast cancer development.
Matched peripheral blood serum was obtained from 64 breast cancer patients and 83 normal controls. Height and weight were measured to calculate BMI. All were genotyped for the SNPs rs9939609 and rs17782313 using a Tetra-primer ARMS-PCR method. For statistical analysis, the chi-square test and SPSS software were used.
In subgroup analyses defined by BMI, FTO rs9939609 genotypes (TT/AA/AT) were significantly associated with the risk of breast cancer only in non-obese subjects (p < 0.005). TT genotypes of MC4R rs17782313 in non-obese and genotypes TT/CC in the overweight group were also statistically associated with breast cancer (p < 0.005). No significant associations between any variants and breast cancer risk were seen in obese subjects.
Based on the absence of an association between obesity-related SNPs and breast cancer in obese subjects, it is proposed that weight gain in Iranian women will help prevent breast cancer risk. The result help for preparing and designing a safe and versatile recombinant drug in future.

Non-Iranian Primary Tritipyrum (2n=6x=42, AABBEbEb) set seed after Triticale (2n=6x=42, AABBRR) and Tritordeum (2n=6x=42, AABBHcHc) but, due to a few undesirable agronomic traits, it cannot fulfil the commercial expectations of farming.  To remove these deficiencies, six hexaploid Tritipyrum lines were crossed with four Iranian bread wheat cultivars which led to the production of 107 (F1), 479 (F2), 768 (F3), and 1539 (F4) Iranian Secondary Tritipyrum Genotypes (ISTG) seeds. This study was carried out for selecting the plants potentially carry the 5Eb chromosome/s and are good candidates for salt tolerant by GISH and RFLP markers. The procedure involved extracting the total DNA content of 209 plants, including non-Iranian primary Tritipyrum lines, Iranian wheat cultivars, Chinese Spring addition, and substitution lines for 5Eb and Iranian secondary Tritipyrum genotypes (ISTG: F1, F2, F3, F4). Genomic in situ Hybridization (GISH) on mitotic spreads of fertile new Iranian secondary Tritipyrum genotypes (ISTG) was carried out to demonstrate the feasibility of single Eb chromosomes. There were three trials of 18 Fragment Length Polymorphism (AFLP) EcoRI/MseI primers to identify the presence of the 5Eb chromosome in 105 ISTG plants, along with four wheat addition lines and substitution lines for the 5Eb chromosome.  GISH on mitotic spreads demonstrated the feasibility of producing 75 plants out of 105 fertile new Iranian secondary Tritipyrum genotypes (ISTG) with 0-14 single Eb chromosomes. Among the mentioned markers, only the E36/M59 marker showed 43, 50, 30 and 47 identical bands, respectively, in contrast to 53 expected bands in all plants with the 5Eb chromosome which indicated 21, 33, 9 and 6 out of 75 ISTG plants, respectively, with the 5Eb chromosome.  This study indicated that 69 ISTG Tritipyrum plants were potentially carry the 5Eb chromosome/s and are good candidates for salt tolerant tests in comparison with Iranian modern bread wheat cultivars.

Quality of bread baking is affected by gluten genes and balance between their expressions. Hence, it is necessary for a comprehensive research to study and compare all gluten genes and their regulating elements simultaneously.
The aim of this study was to evaluate the molecular mechanism of bread quality at the level of coding genes and regulating elements via comparative transcriptome analysis of two extreme wheat cultivars.
RNAs were extracted from the grain of two wheat cultivars with high (Pishtaz) and low (Navid) bread making qualities, collected during endosperm development at five stages. mRNAs were sequenced and gluten transcripts were assessed to find differentially expressed genes. Then, transcription factors interacting with gluten genes were detected and evaluated for expression.
Results showed that Ɣ-gliadin and LMW-GS genes had a higher expression in Pishtaz and Navid, respectively. Most identified transcription factors were active at the early stage of growth and it seemed that NAC and ERF transcription factors had significant roles in regulating genes with different expressions. There was no significant difference in the expression level of NACs between two cultivars. It is proposed that the ERF transcription factor which classified as BREB2C transcription factor could control the expression of LMW-GS genes in two cultivars and functionally act as a repressor for their target genes.
The priority of Pishtaz wheat cultivar in bread quality originated from high expression levels of Ɣ-gliadin gene and ERF transcription factor.

The microbial genome sequences provide solid in silico framework for interpretation their drug-like chemical scaffolds biosynthetic potential. The Pseudomonas fluorescens species is metabolically versatile and producing therapeutically important natural products. The main objective of the present study was to mine the publically available data of P. fluorescens strains genomes for putative drug-like metabolites identification. We implemented the computational biology resources of AntiSMASH and BAGEL3 for the secondary metabolites prediction from the P. fluorescens strains genome sequences. The predicted secondary metabolites were evaluated using drug discovery chemoinformatics resources, like Drugbank database search and molecular docking inspection. The analyses unveiled a wide array of chemical scaffolds biosynthesis in different P. fluorescens strains. Subsequently, the drug-like potential evaluation of these metabolites identified few strains, including P. fluorescens PT14, P. fluorescens A5O6, and P. fluorescens FW300-N2E3 that harbor the biosynthetic gene clusters for salicylic acid like metabolite biosynthesis. The molecular docking inspection of this metabolite against human cyclooxygenase and aldo-keto reductase targets revealed its feasible inhibitory potentials like other salicylates compounds. The computational biology and drug discovery analyses identified different gene clusters in P. fluorescens genomes coding for salicylic acid like chemotypes biosynthesis. These gene clusters may worthy to target through metabolic engineering for the massive production of salicylates-like chemical scaffolds from microbial resources.

Probiotics have attracted a great attention aiming to develop natural non-toxic antioxidants, because of their role in decreasing the risk of reactive oxygen species (ROS) accumulation.
The purpose of this study was to assess the antioxidant activity of a probiotic Streptococcus salivarius ssp thermophillus (St.sa) and to evaluate its protective effect against the oxidative stress induced by a toxic dose of paracetamol in Wistar rats.
Several assays were used to investigate the in vitro antioxidant capacity of the strain. To evaluate the protective effect against oxidative stress induced by paracetamol in liver, hepatic marker enzymes, the antioxidant enzyme activities, malondialdehyde (MDA) and glutathione (GSH) content in liver tissues were investigated.
The strain has shown a considerable ability to scavenge DPPH free radical (89.43%),a good resistance to hydroxyl radicals (47%), a considerable ability to chelate iron ions (33.21%) and a good inhibitory effect against plasma lipid peroxidation (54.36%). Significant changes in liver function tests, antioxidant enzyme activities, MDA and GSH levels in paracetamol treated group were obtained compared to control group. Pretreatment with probiotic removed significantly the inhibition of antioxidant enzymes and suppressed MDA increase and GSH depletion. The analysis of the level of mRNA expression of antioxidant enzymes showed no significant differences in the expression of the enzymes in treated or non-treated groups.
This finding emphasizes the protective role of probiotics against ROS generated during the treatment with paracetamol.

Etanercept is prescribed for the rapid and effective treatment of chronic immune-mediated inflammatory disorders. Due to the expiration of etanercept patents and worldwide demand for comparable and more affordable substitutes, several biosimilars of etanercept have been approved in different countries and new ones are in the process of approval.
In the present study, Altebrel™ as an etanercept proposed biosimilar was investigated in a side by side comparison using various orthogonal analytical methods.
Three batches of the Altebrel™ and Enbrel® samples were used for the study. Several physicochemical properties of samples were compared according to international guidelines, incliding; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion high performance liquid chromatography (SE-HPLC), hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and its biological activity was evaluated using surface plasmon resonance affinity analysis and tumor necrosis factor alpha (TNFα) neutralization biological assay. Amino acid analysis was applied to check the primary sequence and far-UV circular dichroism (CD) spectroscopy investigated the secondary structure.
The obtained results indicated a high degree of similarity between Altebrel™ and Enbrel®. Results of SDS-PAGE, CE-SDS, HIC-HPLC and SE-HPLC implied a comparable pattern of size variants for all samples. Based on the data achieved via in vitro bioactivity assays and SPR analysis, the functional property of Altebrel™ was proved comparable to that of the reference product. Moreover, amino acid analysis indicated similar primary structure and circular dichroism study implied a similar secondary structure for Altebrel™ and Enbrel®.
Overall, our data provide analytical evidence for structural and in vitro functional similarity between Altebrel™ and Enbrel®.

The bovine AGPAT6 gene is one of the potential candidate genes governing milk fat synthesis.

Identification of single nucleotide polymorphisms (SNP) in the targeted region of AGPAT6 gene and their effect on expected breeding values (EBV) of first lactation milk production traits viz. fat %, fat yield and 305 days milk yield in Karan Fries (KF) breeding bulls were sought.

A tetra-primer ARMS PCR technique was adapted to genotype an SNP, g.36,175,805C>T located on 5’ flanking region of AGPAT6 gene. The relationship between EBV of milk production traits and polymorphic locus of AGPAT6 gene was assessed.

Three kinds of genotype (CC, CT, and TT) with respect to g.36,175,805C>T SNP locus were observed. The identified SNP had significant (P<0.05) influence on EBV of fat % (EBV-FP). The KF bulls with CC and CT genotype had comparatively higher EBV-FP than the bulls with the TT genotype. The substitution of “C” allele by “T” allele led to a decrease of 0.0045 % in the EBV-FP.

The identified SNP was significantly associated with EBV-FP, thus it may be utilized as a molecular marker for developing marker-assisted selection strategy to enhance the milk fat content in KF cattle population.