Investigating the simultaneous effect of chitosan and arbuscular mycorrhizal fungi on growth, phenolic compounds, PAL enzyme activity and lipid peroxidation in Salvia nemorosa L.

Considering the importance of Salvia nemorosa L. in the pharmaceutical and food industries, and also beneficial approaches of arbuscular mycorrhizal fungi (AMF) symbiosis and the use of bioelicitors such as chitosan to improve secondary metabolites, the aim of this study was to evaluate the performance of chitosan on the symbiosis of AMF and the effect of both on the biochemical and phytochemical performance of this plant and finally introduced the best treatment. Two factors were considered for the factorial experiment: AMF with four levels (non-inoculated plants, Funneliformis mosseae, Rhizophagus intraradices and the combination of both), and chitosan with six levels (0, 50, 100, 200, 400 mg L-1 and 1% acetic acid). Four months after treatments, the aerial part and root length, the levels of lipid peroxidation, H2O2, phenylalanine ammonia lyase (PAL) activity, total phenol and flavonoid contents and the main secondary metabolites (rosmarinic acid and quercetin) in the leaves and roots were determined. The flowering stage was observed in R. intraradices treatments and the highest percentage of colonization (78.87%) was observed in the treatment of F. mosseae × 400 mg L-1 chitosan. Furthermore, simultaneous application of chitosan and AMF were more effective than their separate application to induce phenolic compounds accumulation, PAL activity and reduce oxidative compounds. The cluster and principal component analysis based on the measured variables indicated that the treatments could be classified into three clusters. It seems that different treatments in different tissues have different effects. However, in an overview, it can be concluded that 400 mg L-1 chitosan and F. mosseae × R. intraradices showed better results in single and simultaneous applications. The results of this research can be considered in the optimization of this medicinal plant under normal conditions and experiments related to abiotic stresses in the future.

Functional characterization of the dimeric form of PDGF-derived fusion peptide fabricated based on theoretical arguments.

A skin wound leads to the loss of skin integrity and the influx of pathogens into the tissue. Platelet-derived growth factors (PDGFs) are cytokines released from alpha granules during wound healing and interact with their cell surface receptors and activate signals involved in chemotaxis, growth, proliferation, and differentiation pathways. Due to the low stability of growth factors (GFs), a new peptide-derived PDGF-BB was designed, expressed in the Shuffle strain of E. coli, and purified by Ni-NTA agarose affinity column chromatography. The effect of fusion peptide was then evaluated on L929 fibroblast cells and animal models with skin lesions. In vitro, studies showed that the peptide led to an increase in the migration of fibroblast cells in the scratch assay. Its positive effect on wound healing was also observed in the skin-injured rats after 3, 7, and 12 days. A significant rise in neutrophils and granular tissue formation, re-epithelialization, angiogenesis, and collagen formation was exhibited on the third day of treatment when compared to the control group. The results showed that, despite reducing PDGF size, the fusion peptide was able to maintain at least some of the known functions attributed to full-length PDGF and showed positive results in wound healing.

Phenolics and terpenoids change in response to yeast extract and chitosan elicitation in Zataria multiflora cell suspension culture.

Zataria multiflora is an important medicinal plant with antioxidant and anticancer properties attributed to its phytochemicals. To develop a method for bulk production of valuable phytochemicals, cell suspension culture of Z. multiflora were grown in liquid B5 medium and then treated in their log growth phase with chitosan (0, 10, 20, and 40 mg L-1) and yeast extract (0, 400, 800, and 1200 mg L-1) for 3 days. The levels of hydrogen peroxide (H2O2), nitric oxide (NO), malondialdehyde (MDA), and the main terpenoids and phenylpropanoids in the cell extracts were determined by HPLC and spectrophotometric techniques. The H2O2 and MDA levels significantly increased in the cells treated with both yeast extract and chitosan, while the NO level increased in those exposed to yeast extract. At their highest concentrations, both elicitors significantly increased PAL and TAL activities, as well as phenolic acids and flavonoids contents. Chitosan only induced the production of caffeic acid (22 µg g-1 DW), benzoic acid (2 µg g-1 DW), 4-hydroxy benzoic acid (6 µg g-1 DW), epicatechin (63 µg g-1 DW), and apigenin (5 µg g-1 DW) in the cells, while yeast extract increased the contents of phenylpropanoids gallic acid (50 µg g-1 DW), vanillin (35 µg g-1 DW), salicylic acid (24 µg g-1 DW), catechin (130 µg g-1 DW) and terpenoids carvacrol (7 µg g-1 DW) and thymol (24 µg g-1 DW). In conclusion, changes in the production of phenolics and terpenoids are a defensive mechanism in Z. multiflora cells treated by yeast extract and chitosan. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03235-x.

Cytokinin and abscisic acid alleviate drought stress through changing organic acids profile, ion immolation, and fatty acid profile to improve yield of wheat (Triticum aestivum L.) cultivars.

There is an increasing interest for plant hormones to modulate the harmful effects of drought on crops. The present study was conducted to assess the effect of foliar-applied cytokin (CK) and abscisic acid (ABA) on yield, organic acids, minerals, and fatty acid profile of wheat (Triticum aestivum L.) cultivars (MV17 and Pishgam) in response to drought stress. The results showed drought significantly decreased grain yield and biomass, but they were enhanced by CK and ABA application. Acetic acid increased under drought stress conditions, and the remarkable increase (~ twofold) in succinic acid content was observed with ABA application under drought stress in MV17 cultivar. In general, drought stress decreased malic acid, pyruvic acid, and citric acid, but CK enhanced them. The leaf accumulations of potassium (K+), calcium (Ca2+), magnesium (Mg2+), iron (Fe2+), and zinc (Zn2+) decreased by drought, where its reduction in MV17 was greater than Pishgam. However, an increased sodium (Na+) content was observed in plants experiencing drought with non-foliar application of ABA and CK. The plant growth hormones especially CK increased K+, Ca2+, Mg2+, Fe2+, and Zn2+, but decreased Na+. Fatty acid profile showed increased polyunsaturated fatty acids and monounsaturated fatty acids upon the drought stress. According to heat map, organic acids represented the maximum variations but fatty acids showed the minimum change during the treatments. The present study recommended foliar-applied CK to alleviate drought stress on wheat yield.

Immunological evaluation of recombinant chimeric construct from Enterotoxigenic E. coli expressed in hairy roots.

Enterotoxigenic Escherichia coli is the most important bacterial agent causing traveller's diarrhea in developing countries. Enterotoxins (LT & ST) and colonization factors (CFs) are two important factors in the ETEC pathogenesis. In the present study, a recombinant four-part fusion protein containing CFAB*ST, CFAE, and LTB (CCL) was expressed in hairy roots of Nicotiana tabacum. The synthetic gene sequence and gene order were designed based on bioinformatics analysis that predicted the best arrangement for antigenicity and stimulation of the immune response. Codon usage was optimized for expression in tobacco plant, under the control of promoter CaMV35S in plasmid pBI121. CCL was efficiently expressed in tobacco hairy roots to yield 1.11% soluble protein as determined by quantitative ELISA and Western blot. In this study, mice were immunized with purified CCL via the oral and subcutaneous route. Humoral immunity especially mucosal immunity with antigen specific IgG and IgA detected in serum and feces. The ability of CCL to elicit neutralizing antibodies was evaluated in the rabbit ileal loop model, using anti-CCL antibodies derived from immunized mice, and co-incubated with ETEC strains. A decrease in fluid accumulation in the intestinal lumen of rabbit ileal loops challenged with ETEC LT and ST positive strains, correlated with the presence of anti-CCL antibodies capable of toxin neutralization. The ability of these antibodies to neutralize toxin confirmed the recognition of epitopes, either linear or conformational, displayed by the recombinant chimeric protein expressed in transgenic tobacco hairy roots. Transgenic plants containing multivalent immunogenic vaccine candidates have the potential to be used for immunization with protection against gastrointestinal pathogens like ETEC.

In Silico designing and immunogenic production of the multimeric CfaB*ST, CfaE, LTB antigen as a peptide vaccine against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most frequent bacterial cause of diarrhea particularly reported in children of developing countries and also travelers. Enterotoxins and colonization factor antigens (CFAs) are two major virulence factors in ETEC pathogenesis. Colonization factor antigen I (CFA/I) includes major pilin subunit CfaB, and a minor adhesive subunit (CfaE), and enterotoxins consisting of heat-labile toxin subunit B (LTB) and heat-stable toxin (ST). Chimeric proteins (CCL) carrying epitopes and adjuvant sequences increase the possibility of eliciting a broad cellular or effective immune response. In the present study, a chimeric candidate vaccine containing CfaB*ST, CfaE, and LTB (CCL) was designed via in silico techniques. This chimeric gene was synthesized by using codon usage of E. coli for increasing the expression of the recombinant protein. After designing the chimeric construct, it showed a high antigenicity index estimated by the vaxiJen server. Linear and conformational B-cell epitopes were identified and indicated suitable immunogenicity of this multimeric recombinant protein. Thermodynamic analyses for mRNA structures revealed the appropriate folding of the RNA representative good stability of this molecule. In silico scanning was done to predict the 3D structure of the protein, and modeling was validated using the Ramachandran plot analysis. The chimeric protein (rCCL) was expressed in a prokaryotic expression system (E. coli), purified, and analyzed for their immunogenic properties. It was revealed that the production of a high titer of antibody produced in immunized mice could neutralize the ETEC using the rabbit ileal loop tests. The results indicated that the protein inferred from the recombinant protein (rCCL) construct could act as a proper vaccine candidate against three critical causative agents of diarrheal bacteria at the same time.

Fast antibody responses by immuno-targeting and nanotechnology strategies versus HBsAg vaccine.

OBJECTIVES: Though immunization with HBsAg has been routine since the 1980s, it has numerous limitations such as low or none humoral immune responses. Today, nanotechnology is used in vaccinology to achieve higher potency. The present study deals with the achievement of fast antibody response of humoral immune responses using immune-targeting through mannosylated nanocarriers of the vaccine. MATERIALS AND METHODS: Mannose sugar and HBsAg were attached to the surface of iron oxide nanoparticles. Mannosylated iron oxide nanoparticles conjugated HBsAg (HBsAg +MLCMNP), iron oxide nanoparticles conjugated HBsAg (HBsAg +LCMNP), hepatitis B vaccine, and mere HBsAg were injected twice to BALB/c mice subcutaneously, while suitable control groups were considered. Specific total IgG antibodies were evaluated on the 7th and 14th days after the final immunization. The avidity maturation of the humoral immune response was assessed with an optimized ELISA. Graph pad prism software was used to analyze statistical data. RESULTS: Results showed that on the seventh day of the final shooting, the mannosylated nano-vaccine caused higher antibody response induction than nano-vaccine without mannose and commercial vaccine groups. After 14 days of the second injection, a significant difference was seen versus the nano-vaccine without mannose but not the commercial vaccine group. In addition, the avidity index in mannosylated nano-vaccine showed a significant increase compared with the nano-vaccine without mannose and mere HBsAg group but not compared with the commercial vaccine. CONCLUSION: It seems that mannosylated nano-vaccine has more potency to achieve fast antibody responses and also higher quality of humoral immune response.

Development of Escherichia coli asparaginase II for the Treatment of Acute Lymphocytic Leukemia: In Silico Reduction of asparaginase II Side Effects by a Novel Mutant (V27F).

Acute lymphoblastic leukemia (ALL) is a common blood disease in children that is accountable for many deaths. Due to major improvements in treatment procedures in the past 50 years, the survivability of this disease has risen dramatically to about 90 percent today. L-asparaginase (ASNase) has been used to treat ALL. The glutaminase (GLNase) activity of this enzyme causes some side effects and is unnecessary for anticancer activity. This study investigated mutagenesis in Escherichia coli ASNase II to find a mutant with lower GLNase activity via molecular dynamics (MD) simulation. Residues with low binding energy to asparagine (Asn) and high binding energy to glutamine (Gln) were chosen for mutagenesis. A mutant with low free binding energy to Gln was then selected for molecular docking and MD studies. The results showed that V27F is a good candidate for reducing GLNase activity and that it has little effect on enzyme ASNase activity. A simulation analysis showed that the V27F mutant was more stable than the WT ASNase and that mutagenesis was quite successful.

Raise up of Scopolamine in Hairy Roots Via Agrobacterium rhizogenes ATCC15834 as Compared with Untransformed Roots in Atropa komarovii.

Atropa komarovii generates tropane alkaloids and three other compounds such as hyoscyamine. Racemate atropine and scopolamine (hyoscine) are the main alkaloids with anticholinergic, antispasmodic, and sedative agents. A proficient convention has been reported for the formation of transgenic Atropa komarovii by the use of Agrobacterium rhizogenes. Root culture, by utilizing leaves explants was contaminated by Agrobacterium rhizogenes ATCC 15834, a strain with the paired vector. The hairy roots after contamination for three weeks were specifically shaped from the cut edges of the leaves. The PCR intensification demonstrated that rol B genes of Ri plasmid of Agrobacterium rhizogenes were coordinated and communicated into the genome of the changed hairy roots. Examination of HPLC revealed that hairy roots can produce scopolamine and hyoscyamine and it was appeared that scopolamine content was essentially expanded in changed roots and hyoscyamine was extremely expanded in non-transgenic roots. According to the results, it was perceived that the scopolamine content in hairy roots was raised significantly compared to the control roots. It was evidenced that hairy roots gather a great number of metabolites that have a commercial significance. Thus, later on we can enhance efficiency for example by building up the biosynthetic route overexpression of gene codifying enzymes in the metabolic route for expanding valuable secondary metabolites in the plant cures.

Aluminium triggers oxidative stress and antioxidant response in the microalgae Scenedesmus sp.

Aluminium (Al) water pollution is an increasing environmental problem and comprehensive analysis of toxic responses of aquatic primary producer organisms is imperative. We characterized the antioxidant response of Scenedesmus sp. microalga to Al-induced oxidative stress. After 72 h of exposure to Al (0, 10, and 100 µM) in a modified Bold Basal Medium (pH 5.0), we observed cell aggregation and alterations in the subcellular structure, strong lipid peroxidation and oxidative stress induction (detected with the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate) in parallel with Al accumulation in cells. At the same time, Al toxicity caused depletion of important macronutrients like Ca, which is important for cell-wall structure. Analysis of antioxidant enzymatic activities in Al-treated Scenedesmus cells revealed that catalase, ascorbate peroxidase, as well as different isoforms of superoxide dismutase were inhibited especially at the highest Al dose (100 µM), cells that accumulated the highest concentration of Al. On the other hand, glutathione reductase activity increased at that Al concentration. Immunodetection after Western-blotting confirmed that only ascorbate peroxidase inhibition was apparently due to a decrease in enzyme levels. However, the inhibition of catalase and activation of glutathione reductase activities seemed related with post-translational modifications in protein function as protein expression decreased or increased, respectively under Al stress. Our results may help to understand toxic mechanisms triggered by Al in freshwater microalgae, which in turn could aid to select suitable biomarkers of Al contamination in aquatic ecosystems.

Recombinant Tandem Repeated Expression of S3 and SΔ3 Antimicrobial Peptides.

BACKGROUND: Antimicrobial peptides (AMPs) are promising candidates for new generations of antibiotics to overcome the threats of multidrug-resistant infections as well as other industrial applications. Recombinant expression of small peptides is challenging due to low expression rates and high sensitivity to proteases. However, recombinant multimeric or fusion expression of AMPs facilitates cost-effective large-scale production of AMPs. In This project, S3 and SΔ3 AMPs were expressed as fusion partners. S3 peptide is a 34 amino acid linear antimicrobial peptide derived from lipopolysaccharide (LPS) binding site of factor C of horseshoe crab hemolymph and SΔ3 is a modified variant of S3 possessing more positive charges. METHODS: Two copy tandem repeat of the fusion protein (named as SΔ3S3-2mer-GS using glycine- serine linker was expressed in E. coli. BL21 (DE3). After cell disruption and solubilization of inclusion bodies, the protein was purified by Ni -NTA affinity chromatography. Antimicrobial activity and cytotoxic properties of purified SΔ3S3-2mer-GS were compared with a previously produced tetramer of S3 with the same glycine- serine linker (S3-4mer-GS) and each of monomeric blocks of S3 and SΔ3. RESULTS: SΔ3S3-2mer-GS was successfully expressed with an expression rate of 26%. The geometric average of minimum inhibitory concentration (MIC GM) of SΔ3S3-2mer-GS was 28%, 34%, and 57% lower than SΔ3, S3-4mer-GS, and S3, respectively. SΔ3S3-2mer-GS had no toxic effect on eukaryotes human embryonic kidney cells at its MIC concentration. CONCLUSION: tandem repeated fusion expression strategy could be employed as an effective technique for recombinant production of AMPs.

HBs antigen and mannose loading on the surface of iron oxide nanoparticles in order to immuno-targeting: fabrication, characterization, cellular and humoral immunoassay.

Mannosylation of nanovaccine is an appropriate strategy for targeting the mannose receptors on DCs. Here, HBsAg and mannose loaded on the surface of iron oxide nanoparticles to increases HBsAg vaccine potency. Nanoparticles are made by co-precipitation method and bonded to the HBsAg and mannose by chemical bonding. The physicochemical properties of nano-vaccines, their toxicity and antigenicity were determined. The synthesized nano-vaccine showed spherical shape with a mean particle size of 60 nm, a zeta potential of -44 mV, an antigen-binding efficiency of around 100% and for mannose 78%. In vitro release of nanoparticles exhibited about 30% at the first day and about 60% until the third day. SDSPAGE analysis confirmed structural integrity of HBsAg loaded on nanoparticles. The HBsAg-loaded LCMNP and MLCMNP nanoparticles had no toxic effects on HEK293 cell line. The quantification of the intracellular Fe by ICP-OES as a criterion of nano-vaccine uptake revealed mannose intensify uptake of MLCMNP. In addition, mannose in the structure of MLCMNP improved IL-6, TNF-α and IFN-γ (>16 fold) cytokines genes expression by macrophage/dendritic cells after exposure in 12 h. Immunization of experimental mice (subcutaneously, two times with 2-week intervals) with 5 µg of HBsAg loaded on MLCMNP nanoparticles increased specific total IgG and IgG2a/IgG1 ratio. In addition, TNF-α, IL-12, IL-2 and IL-4 cytokines in mannosylated nano-vaccine increased versus nano-vaccine group while lymphocyte proliferation and IFN-γ responses in the targeted nano-vaccine group show a tiny increase versus the nano-vaccine group. The results show that mannosylated nano-vaccine promotes higher level of cellular and humoural immune responses against HBsAg nano-vaccine.

Oral immunization with recombinant Lactococcus lactis NZ9000 expressing human papillomavirus type 16 E7 antigen and evaluation of its immune effects in female C57BL/6 mice.

The ORFs of both native and codon-optimized E7 genes were successfully fused to SPusp45 signal peptide and expressed by a nisin-controlled gene expression system in the NZ9000 strains of Lactococcus lactis. Recombinant strains were confirmed by Western blot analysis. To measure immune responses against the E7 antigen, specific-pathogen-free C57BL/6 mice were inoculated with L lactis harboring pNZ8123-rE7 by oral gavage. Then, specific antibodies and cytokines were measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay, respectively. Oral administration of L lactis strains expressing rE7 elicited the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4+ T helper and CD8+ T cell precursors. Our outcomes indicated that the HPV-16 E7 specific IL-2- and IFN-γ-secreting T cells in antigen-stimulated splenocytes and intestinal mucosal lymphocytes were significantly higher than the control groups. Our data also demonstrated that mice vaccinated with recombinant L lactis were able to generate potent protective effects against challenge with the E7-expressing tumor cell line (TC-1). Moreover, L lactis containing pNZ8123-HPV16-optiE7 showed strong therapeutic antitumor effects against established tumors in vivo. These findings demonstrate that recombinant L lactis induce both humoral and cellular immune responses in mice and are therefore recommended for therapeutic treatments in humans after oral administration.

Novel mutant of Escherichia coli asparaginase II to reduction of the glutaminase activity in treatment of acute lymphocytic leukemia by molecular dynamics simulations and QM-MM studies.

L-Asparaginases (ASNase) belong to a family of amidohydrolases, have both asparaginase and glutaminase activity. Acute lymphocytic leukemia (ALL) is an outrageous disease worldwide. Bacterial ASNase has been used for the treatment of ALL. Glutaminase activity of enzyme causes some side effect and it is not essential for anticancer activity. The aim of this study was engineering of Escherichia coli asparaginase II to find a mutant with reduced glutaminase activity by molecular docking, molecular dynamics (MD) and QM-MM (Quantum mechanics molecular dynamics) simulations. Residues with low free energy of binding to Asn and high free binding energy to Gln were chosen for mutagenesis. Then, a mutant with higher glutaminase free binding energy was selected for further studies. Additionally, the MD simulation and QM-MM computation of wild type (WT) were employed and the selected mutated ASNase were analyzed and discussed. Our data showed that V27T is a good candidate to reduction the glutaminase activity, while has no remarkable effect on asparaginase activity of the enzyme. The simulation analysis revealed that V27T mutant is more stable than WT and mutant simulation was successful completely. QM-MM results confirmed the successfulness of our mutagenesis.

Human hemoglobin adsorption onto colloidal cerium oxide nanoparticles: a new model based on zeta potential and spectroscopy measurements.

The nanoparticle (NP)-induced conformational changes of protein and NP agglomeration have gained a remarkable interest in medical and biotechnological fields. Herein, the effect of human hemoglobin (Hb) on the colloidal stability of cerium oxide NP (CNP) was investigated by dynamic light scattering (DLS), zeta potential, and TEM analysis. In addition, the effect of CNP on the heme degradation and structural changes of Hb was studied using fluorescence, circular dichroism (CD), and UV-visible (UV-vis) spectroscopic methods. DLS and TEM analysis showed that the presence of Hb can increase the mean diameter of CNP. Zeta potential measurements revealed that CNP demonstrated a higher charge distribution relative to CNP/Hb complex. Besides, fluorescence studies indicated that two fluorescent heme degradation products are revealed during the interaction of CNP with Hb. Near UV-CD spectroscopy also showed that the microenvironmental changes of heme groups occur after interaction of Hb with CNP. The result of thermal behavior of Hb confirmed the structural changes of protein, which referred to decrease in the Hb stability in the presence of CNP. Indeed, the finding related to structural and functional changes of Hb induced by CNP may be crucial to obtain information regarding the side effects of NPs. Finally, this data reveal much insight into the effects of the interaction on protein structural changes and NP agglomeration, and can correlate the zeta potential of NP-protein complexes with the nature of the principle NP-protein interaction.

Responses of wheat plants to interactions of 24-epibrassinolide and Glomus mosseae in saline condition.

This study was designed to investigate the possible effects of 24-Epibrassinolide (BR), arbuscular mycorrhizal (AM) fungus, Glomus mosseae, singularly and collectively under salt stress in wheat (Triticum aestivum L.) plants. After foliar spraying of mycorrhizal and non-mycorrhizal plants by 5 µM epibrassinolide (24-Epi), they were treated with 0 and 150 mM NaCl for 2 weeks and then harvested. The results showed interactions of G. mosseae and 24-Epi could alleviate the adverse effects of salinity by improving relative water content (RWC) of leaves (62%), relative growth rate (40.74%), shoot fresh weights (39.83%) and shoot phosphorous content (63.93%), stimulating leaf enzymatic antioxidant activities including catalase (2.24 fold) and ascorbate peroxidase (2.18 fold) as well as malondialdehyde (36.17%) and H2O2 concentrations (49.74%) as compared to those of NaCl treatments. Moreover, mycorrhizal dependency of root dry weight (2%) and phosphorus concentration (0.4%) increased with AM infection and 24-Epi application under saline condition. Leaf RWC, also, negatively correlated with membrane electrolyte leakage. Furthermore, the greatest mitigating effects were observed in mycorrhizal plants subjected to NaCl and 24-Epi. This study indicated that 24-Epi application and AM fungi may synergistically mitigate harmful impacts of salinity in wheat plants.

Efficient production and optimization of E7 oncoprotein from Iranian human papillomavirus type 16 in Lactococcus lactis using nisin-controlled gene expression (NICE) system.

The present work was aimed at investigating the expression and optimization of a human papillomavirus (HPV) type 16 gene encoding oncoprotein E7 in Lactococcus lactis. We genetically engineered Lactococcus lactis using nisin-controlled gene expression (NICE) system pNZ8148 to express the native and codon optimized E7 oncogenes isolated from Iranian HPV-16. The results of optimizing fermentation showed, the concentration of produced protein was expressively improved by 10 ng/mL nisin after 3.5, and 4 h induction for NZ9000 harboring the codon-optimized, and native E7 respectively. Furthermore the recombinant NZ9000 strains expressed rE7 by maximum value of 4.7 (Codon-optimized), and 1.82 µg/mL (Native) in static flask experiments at initial glucose concentrations of 50 and 75 g/L respectively. The rE7 yield was further enriched in batch fermenter experiments using controlled pH. Thus, the overall production of rE7 under optimized conditions accumulated in the cytoplasm to nearly 33.25 µg/mL by L. lactis NZ9000 containing codon-optimized E7, which was over ∼2.7-fold higher compared to the NZ9000 having native E7 strain (12.01 µg/mL). Accordingly, the maximum biomass production was calculated 4.87, and 1.51 g/L respectively.

Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363

HPV 16 intratypic sequence variations has been recognized in association with oncogenic potential diverge and geographic distribution. This study aimed to investigate nucleotide modifications and optimization of HPV 16 E7 regions from Iranian infected women. Cervical biopsies from 79/163 HPV 16 positive cancer patients detected in our study were analyzed by PCR in a couple of cloning of a complete ORF of the E7 gene, and sequencing. The most frequently observed variant was C196T in E7 which led to an amino acid change of R66W. In addition, only one common variant T234G was identified from all specimens, but it did not lead to any amino acid change. We also detected nucleotide variations A86G, and C188T in samples. Among 99 codons in E7 gene, 56 codons were improved for Lactococcus lactis subsp. cremoris MG1363 resulting in a reduced G+C content from 43.1% to 34.0%. Also, the AT%, ENC, and CAI values were 66, 20±1.1, and 1.000 instead of 56.90, 60 ±1.1, and 0.406 respectively. Finally we constructed expression vector pNZ8148 encoding optimized E7 oncoprotein of HPV 16. This study declared for the first time, the genetic variations of HPV 16 E7 in IRAN. We conclude that plasmid pNZ8148-HPV 16-opti E7 can be potential vaccine candidates in the future.

Potent Antioxidant Properties of rolB-transformed Catharanthus roseus (L.) G. Don.

Free radicals play an important role in pathological processes such as aging and developing cancer; hence, natural products inhibit free radical production, and can play an important role in preventing diseases. We aimed to evaluate the scavenging activity of free radicals, reducing power, inhibition of lipid peroxidation and malondialdehyde (MDA) formation, and the antioxidant composition of rolB-transformed hairy roots and leaf callus of Catharanthus roseus. Hairy roots of the Catharanthus roseus were induced using Agrobacterium rhizogenes strain ATCC 15834 to transfer the rolB gene. Polymerase chain reaction analysis was used to identify the presence of the gene in the transformed hairy roots. Folin-Ciocalteu reagent, aluminum chloride calorimetry and high performance liquid chromatography were used to determine the content of total phenolic, flavonoid and gallic acid, respectively. To this end, we assayed the free radicals scavenging activity by 1-diphenyl-2-picrylhydrazyl (DPPH), reducing power, inhibition of lipid peroxidation and inhibition of malondialdehyde production. The results showed that phenolic, flavonoid, and gallic acid contents in the ethanol extract of the hairy roots were significantly higher (p ≤ 0.01) than those naturally found in the extracts of root, leaf, and leaf callus of C. roseus. The hairy roots extract showed the lowest IC50 for the inhibition of DPPH(•). Furthermore, the ethanol extract showed the best reducing power and had the highest potential to inhibit lipid peroxidation and to form the MDA. The transformed hairy roots can be considered a rich natural source of antioxidants.

Changes in antioxidant enzymes activities and proline, total phenol and anthocyanine contents in Hyssopus officinalis L. plants under salt stress.

The relationships between salt stress and antioxidant enzymes activities, proline, phenol and anthocyanine contents in Hyssopus officinalis L. plants in growth stage were investigated. The plants were subjected to five levels of saline irrigation water, 0.37 (tap water as control) with 2, 4, 6, 8 and 10 dSm(-1) of saline water. After two months the uniform plants were harvested for experimental analysis. Antioxidant enzymes activities and proline, phenol and anthocyanine contents of the plants were examinated. Enhanced activities of peroxidase, catalase and superoxide dismutase were determined by increasing salinity that plays an important protective role in the ROS-scavenging process. Proline, phenol and anthocyanine contents increased significantly with increasing salinity. These results suggest that salinity tolerance of Hyssopus officinalis plants might be closely related with the increased capacity of antioxidative system to scavenge reactive oxygen species and with the accumulation of osmoprotectant proline, phenol and anthocyanine contents under salinity conditions.