Iranian Journal of Biotechnology
Volume:15 Issue: 1, Winter 2017

Transforming growth factor (TGF)-β is over-expressed in a wide variety of cancers such as lung adenocarcinoma. TGF-β plays a major role in cancer progression through regulating cancer cell proliferation and remodeling of the tumor micro-environment. However, it is still a great challenge to explain the phenotypic effects caused by TGF-β stimulation and the effect of TGF-β stimulation on tumor micro-environment.
To address this issue, in the present study we used two time-course microarray data in human lung adenocarcinoma cells and applied bioinformatics methods to explore the gene regulation network responding to TGF-β stimulation in lung adenocarcinoma cells.
The time-dependent reverse-engineering method, protein-protein interaction network analyses, and calculation of the similarity measures between the links were used to construct gene regulatory network and to extract gene clusters.
Utilizing the constructed gene regulation network, we predicted NEFL and LUC7A show the opposite and the same change with C21orf90 if HAND2 is knocked-out after treatment with TGF-β1 for 4 hours and for 12 hours respectively. FGG and HSPC009 are predicted to display the opposite change with NEFL if CSMD1 is knocked out after treatment with TGF-β1 for 12 hours. Additionally, by integrating two datasets, we specially identified several nested clusters which included those genes regulated by TGF-β stimulation in lung adenocarcinoma cells.
Our analysis can help a better understanding regarding how TGF-β stimulation causes the expression change of a number of the genes and provide a novel insight into TGF-β stimulation effect on lung adenocarcinoma cells.

Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS). The main cause of the MS is yet to be revealed, but the most probable theory is based on the molecular mimicry that concludes some infections in the activation of T cells against brain auto-antigens that initiate the disease cascade.
The Purpose of this research is the prediction of the auto-antigen potency of the myelin proteolipid protein (PLP) in multiple sclerosis.
As there wasn’t any tertiary structure of PLP available in the Protein Data Bank (PDB) and in order to characterize the structural properties of the protein, we modeled this protein using prediction servers. Meta prediction method, as a new perspective in silico, was performed to fi nd PLPs epitopes. For this purpose, several T cell epitope prediction web servers were used to predict PLPs epitopes against Human Leukocyte Antigens (HLA). The overlap regions, as were predicted by most web servers were selected as immunogenic epitopes and were subjected to the BLASTP against microorganisms.
Three common regions, AA58-74, AA161-177, and AA238-254 were detected as immunodominant regions through meta-prediction. Investigating peptides with more than 50% similarity to that of candidate epitope AA58-74 in bacteria showed a similar peptide in bacteria (mainly consistent with that of clostridium and mycobacterium) and spike protein of Alphacoronavirus 1, Canine coronavirus, and Feline coronavirus. These results suggest that cross reaction of the immune system to PLP may have originated from a bacteria or viral infection, and therefore molecular mimicry might have an important role in the progression of MS.
Through reliable and accurate prediction of the consensus epitopes, it is not necessary to synthesize all PLP fragments and examine their immunogenicity experimentally (in vitro). In this study, the best encephalitogenic antigens were predicted based on bioinformatics tools that may provide reliable results for researches in a shorter time and at a lower cost.

Micro RNAs (miRNAs) are a pivotal part of non-protein-coding endogenous small RNA molecules that regulate the genes involved in plant growth and development, and respond to biotic and abiotic environmental stresses posttranscriptionally.
In the present study, we report the results of a systemic search for identifi cation of new miRNAs in B. rapa using homology-based ESTs (Expressed Sequence Tags) analysis and considering a series of fi ltration criteria.
Plant mature miRNA sequences were searched in non-protein coding ESTs registered in NCBI EST database. Zuker RNA folding algorithm was used to generate the secondary structures of the ESTs. Potential sequences were candidate as miRNA genes and characterized evolutionarily only and if only they fi t some described criteria. Also, the web tool psRNATarget was applied to predict candidate B. rapa miRNA targets.
In this study, 10 novel miRNAs from B. rapa belonging to 6 miRNA families were identifi ed using EST-based homology analysis by considering a series of fi ltration criteria. All potent miRNAs appropriate fold back structure. Several potential targets with known/unknown functions for these novel miRNAs were identifi ed. The target genes mainly encode transcription factors, enzymes, DNA binding proteins, disease resistance proteins, carrier proteins and other biological processes.
MicroRNA having diverse functions in plant species growth, development and evolution by post transcriptionally regulating the levels of specifi c transcriptome so by eff ecting on their translation products. Research in miRNA led to the identifi cation of many miRNAs and their regulating genes from diverse plant species.

TRR14 protein is a small member of a multi-gene family in Arabidopsis and is the first ones found during screening of seedlings for their resistant to the trehalose sugar.
Characterization ofTRR14 over-expressed plants with respect to morphological changes, growth and photosynthesis related parameters.
TRR14gene was isolated from Arabidopsis and cloned intopBin-35S vector. Recombinant vector was transferred to Arabidopsis (Col-0) via Agrobacterium tumefaciens by Floral Dipping method. Seeds from TRR14over-expressed (TRR14) and Col-0 wild type (WT) plant were sown on soil under long day conditions. Several measurements was then performed such as determination of fresh and dry weights, leaf area, Chl a and Chl b content, Chl a/b ratio, total chlorophyll and carotenoids content, soluble and insoluble sugars content, total and soluble protein content, the Hill reaction rate, chlorophyll fluorescence and photorespiration rate. Meanwhile, the chloroplastic proteins were investigated by SDS-PAGE analysis.
TRR14 plants showed significant increase in both fresh and dry weights, leaf area, total and soluble protein content and significant decrease in insoluble sugar contents, compared to WT plants. Chl a, Chl b, total chlorophyll content, Chl a/b ratio, carotenoids content, Hill reaction rate, chlorophyll fluorescence had not showed significant differences between TRR14 and WT plants. The SDS-PAGE gel electrophoresis of chloroplastic proteins showed one ticker band with a molecular mass of 25kDain TRR14-over expressed plants, compared to WT plants. Remarkably, Photorespiration rate was decreased in TRR14 plants compared to WT plants.
The increased biomass of TRR14 transformed plants may be due to its ability in reducing photorespiration through concentrating CO 2 in the leaf intercellular spaces.

Plants have various defense mechanisms such as production of antimicrobial peptides, particularly pathogenesis related proteins (PR proteins). PR10 family is an essential member of this group, with antifungal, antibacterial and antiviral activities.
The goal of this study is to assess the antifungal activity of maize PR10 against some of fungal phytopathogens.
Zea mays PR10 gene (TN-05-147) was cloned from genomic DNA and cDNA and overexpressed in Escherichia coli. The existence of a 77- bp intron and two exons in PR10 was confi rmed by comparing the genomic and cDNA sequences. The PR10 cDNA was cloned in pET26b () expression vector and transformed into E. coli strain Rosetta DE3 in order to express PR10 recombinant protein. Expression of the recombinant protein was checked by western analysis.
Recombinant PR10 appeared as insoluble inclusion bodies and thus solubilized and refolded. PR10 was isolated using Ni-NTA column. The activity of the refolded protein was confi rmed by DNA degradation test. The antifungal activity of PR10 was assessed using radial diff usion, disc diff usion and spore germination. The hemolytic assay was performed to investigate the biosafety of recombinant PR10.
Recombinant maize PR10 exerted broad spectrum antifungal activity against Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Verticillium dahlia and Alternaria solani. Hemolysis biosafety test indicated that the protein is not poisonous to mammalian cells.
Maize PR10 has the potential to be used as the antifungal agent against different fungal phytopathogens. Therefore, this protein can be used in order to produce antifungal agents and fungi resistance transgenic plants.

Erythropoietin, as a principal hormone promotes red blood cell production in bone marrow. Varieties of erythropoietin biosimilar are being produced by recombinant DNA technology in cell cultures. The detection or quantification of these molecules are being performed by diff erent methods which some of theme such as Western blot and enzymelinked immunosorbent assay (ELISA) require specifi c antibodies. High cost, inappropriate shipping (cold chain failures), reduced sensitivity and thus poor detection performance are common pitfalls of using commercial kits for performing immunological tests.
To produce in-house polyclonal antibody against active pharmaceutical ingredient (API) of recombinant human erythropoietin (rh-EPO) was the aim of this study.
Two healthy female albino rabbits were injected four times in 14 days interval using rh-EPO API as antigen. The produced antibody was separated from plasma via either caprylic acid or saturated ammonium sulfate precipitation and the results were compared from each purifi cation methodologies. The antibody was further purified by ion exchange chromatography. Acceptable purity and good immunogenicity were detected respectively by SDS-PAGE and western blot analysis. The purifi ed antibody was compared with a commercial kit to determine rh-EPO concentration in different steps of production batches via ELISA.
The purity of antibodies after ion exchange chromatography, obtained from caprylic acid and ammonium sulfate precipitation were 97 and 80%, respectively.
As producing in house kits is one of the important challenges of bio-pharmaceutical manufacturers, a simple, cost- and time-eff ective, and easy to scale up strategy for making in-house polyclonal antibody was set up. Caprylic acid precipitation resulted higher purity than ammonium sulfate and finally purified antibody (97% purity) used as a capture antibody in sandwich ELISA test was able to detect erythropoietin antigen as sensitive (100%) and specific (100%) as commercial kits.

A few reports confirm the ability of Helicobacter pylori to form biofilm. However, conclusive data do not exist concerning the factors that favor this ability.
Evaluation of the factors associated with the biofilm formation ability of H. pylori including bacterial, physical and chemical, and environmental factors was the research’s aim.
H. pylori isolates from gastric biopsy specimens of patients infected chronically were screened for biofi lm formation ability. Association of bacterial properties such as motility, auto-aggregation, cell hydrophobicity, and extracellular polymeric substances (EPS) with in vitro biofilm formation ability of H. pylori was evaluated. The effects of environmental factors such as growth-medium, temperature, oxygen-tension, pH, β-cyclodextrin, gastric secreted mucins, and sub-inhibitory concentration of amoxicillin were also evaluated.
Ability of clinical H. pylori isolates to form biofi lm in was quantitatively compared. The coccoid shape H. pylori cells were observed by scanning electron microscopy, the images were illustrative of the attachment of cells to form microcolony. The levels of hydrophobicity, motility and auto aggregation of two isolates with highest and lowest biofilm formation ability were the same. However, the signifi cant role of mucins (P Mucins have a significant role in elevating the biofilm formation, also pH, atmosphere and sub-MIC of antibiotics influence biofilm formation.

Benzaldehyde dehydrogenase (BZDH) is encoded by the xylC that catalyzes the conversion of benzaldehyde into benzoate in many pathways such as toluene degradation.
In this study, the xylC gene from Rhodococcus ruber UKMP-5M was expressed in Escherichia coli, purified, and characterized.
The xylC was amplified and cloned in E. coli. The recombinant plasmid pGEMT-xylC was digested by NdeI and HindIII to construct plasmid pET28b-xylC and transformed in E. coli BL21 (DE3). Expression of the recombinant protein was induced by 1 mM isopropyl β-D-thiogalactoside (IPTG) at 37 °C. The BZDH was purified by ion exchange chromatography, in which the product was an NAD-dependent enzyme using benzaldehyde as a substrate for enzyme characterization. The end metabolite was identified via gas chromatography mass spectrometry (GC-MS).
The recombinant BZDH is 27 kDa, purified by ion exchange chromatography. The activity of BZDH was 9.4 U/ml. The optimum pH and temperature were 8.5 and 25 ºC, respectively. The Michaelis constant (Km) and maximum velocity (Vmax) were 4.2 mM and 19.7 U/ml, respectively. The metabolite of BZDH was benzene carboxylic acid as determined by GC-MS analysis.
BZDH has the ability to degrade benzaldehyde to less toxic compounds. The BZDH is a critical enzyme for the degradation of aromatic hydrocarbons in Rhodococcus sp. The BZDH from R. ruber UKMP-5M is showed similar function with other aldehyde dehydrogenases.

Olive pomace, as the main by-product of the olive oil industry, is recently recycled as fermentation substrate for enzyme production.
Actinobacteria isolates were separated from an Algerian soil under olive pomace cultivation and were evaluated for their lignocellulolytic enzymes production.
Isolates of Actinobacteria were separated from soils around oil mills using four isolation media, among them three were enriched by olive pomace. The isolates were screened for their cellulolytic, xylanolytic and ligninolytic activities. Isolates with potential of producing lignocellulose-degrading enzymes were selected under submerged fermentation based olive pomace.
Ninety isolates of Actinobacteria were separated from soil samples. M3 medium (raw pomace autoclaved alone) was the best isolation medium (68 strains), whereas, the soil from oil mill with continuous system (S1) led to separation of 52 strains. Among the 90 isolates, 82 were shown promising enzyme activity, 19 isolates were presented the largest zone diameter ( Olive pomace with medium low cost and high titers of enzymes can be valorized by culture of Actinobacteria to produce lignocellulolytic enzymes for industrial applications.