Sodium nitroprusside stimulated production of tropane alkaloids and antioxidant enzymes activity in hairy root culture of Hyoscyamus reticulatus L.

Hyoscyamus reticulatus L. is a herbaceous biennial belonging to the solanaceae family. Hyoscyamine and scopolamine as main tropane alkaloids accumulated in henbane are widely used in medicine to treat diseases such as parkinson’s or to calm schizoid patients. Hairy roots media manipulation which uses elicitors to activate defense mechanisms is one of the main strategies for inducing secondary metabolism as well as increasing the production of valuable metabolites. Cotyledon-derived hairy root cultures were transformed by Agrobacterium rhizogenes. Sodium nitroprusside (SNP), a nitric oxide donor), was used in various concentrations (0, 50, 100, 200 and 300 μM) and exposure times (24 and 48 h). Treatment with SNP led to a significant reduction in fresh and dry weight of hairy roots, compared to control cultures. ANOVA results showed that elicitation of hairy root cultures with SNP at different concentrations and exposure times significantly affected the activity of as antioxidant enzymes such as catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). The highest hyoscyamine and scopolamine production (about 1.2-fold and 1.5-fold increases over the control) was observed at 50 and 100 μM SNP at 48 and 24 hours of exposure time, respectively. This is the first report of SNP elicitation effects on the production of tropane alkaloids in hairy root cultures

Ganoderic Acid Production via Aerial Co-cultivation of Ganoderma lucidum with Bacillus subtilis and Aspergillus niger Using Bubble Column Bioreactor

Background and Objective: Ganoderma lucidum, with its medicinal characteristics, is one of the most beneficial fungi in traditional Asian medicine. This fungus low efficiency of ganoderic acid production has limited its use as a valuable secondary metabolite. Environmental stresses and elicitors such as microbial volatile organic compounds in co-cultures can increase ganoderic acid production. To investigate effects of variables of co-culture time and volume on Ganoderma lucidum growth and ganoderic acid production, Bacillus subtilis and Aspergillus niger were aerially co-cultured with Ganoderma lucidum. Material and Methods: To investigate fungus growth and production of ganoderic acid using bubble column bioreactor, effects of independent variables of temperature, initial inoculation, length-to-diameter ratio (L: D) and aeration were investigated using Taguchi method. Then, effects of co-culture of Ganoderma lucidum with Bacillus subtilis and Aspergillus niger under optimum conditions were investigated. Results and Conclusion: Optimizing effects of co-culture time and volume variables led to 2.9-fold increases in production of ganoderic acid, compared to the control sample. Optimization of biomass production in the bioreactor showed that biomass production increased significantly by increasing the initial inoculation percentage and temperature. These two variables significantly affected ganoderic acid production and its optimum production point was 10% of initial inoculation, temperature of 25.6 °C, L: D of 4:8 and aeration rate of 0.64 vvm. Gas holdup investigation for air-water and air-fermentation media systems showed that the presence of suspended solids and aeration rate affected gas holdup. Microbial volatile organic compounds in co-culture of microorganisms can increase ganoderic acid production by Ganoderma lucidum. Conflict of interest: The authors declare no conflict of interest

Effects of Silicon and AgNO3 Elicitors on Biochemical Traits and Antioxidant Enzymes Activity of Henbane (Hyoscyamus reticulatus L.) Hairy Roots

Lattice henbane (Hyoscyamus reticulatus L.) is an herbaceous, biennial plant belonging to Solanaceae family. H. reticulatus hairy roots were established from two-week-old leaves infected by A7 strain of Agrobacterium rhizogenes on solid Murashige and Skoog (MS) medium. In this study, abiotic elicitors including; Sodium silicate (Na2SiO3) with different concentrations (0, 1, 5 and 7 mM) and silver nitrate (AgNO3) concentrations (0, 0.5, 1 and 2 mM) were added to hairy roots culture media. The results showed that, Na2SiO3 and AgNO3 significantly affected hairy roots fresh weight after 24h. Also, the highest hairy root fresh weight was observed in the control, and with broadening elicitor concentrations, fresh weight was decreased in both treated hairy roots with AgNO3 and Na2SiO3 but the effect of exposure duration was not significant. Biochemical analysis showed that total antioxidant activity (TAA), total phenol (TP), catalase (CAT), ascorbate peroxidase (APX) and Guaiacolperoxidase (GPX) activities were enhanced in elicitated hairy roots compared to non elicitated hairy roots. The highest CAT, APX and GPX activities were observed in hairy roots treated with 7mM Na2SiO3 and 2mM AgNO3. Our results suggest that, Na2SiO3 and AgNO3 can stimulate the antioxidant defense systems and protect the plants from subsequent stresses

Increased the specificity and sensitivity of monospecific antibody against host cell protein (HCP) in quality control of hepatitis B recombinant vaccine

     One of the most important aspects in recombinant biologic production, based on GMP rules, is the accuracy of final product quality control, especially assessment of host cell macromolecules contamination rate in final product. The purification requirement can be eliminated when the yeast cell containing the recombinant protein is used as a host cell. It is possibile that the final product contaminated  to the host cell protein during purification stages of HBsAg (HBV vaccine). The protein purification costs depend on the purification  procedures required. Nowadays several companies produce commercial kits for identification and assessment of host cell protein contamination based on ELISA and Western blotting methods. But high prices, difference in sensitivity and lack of easy access to these kits sometimes create problems. So, in this study, two methods of Ammonium sulphate and caprilic acid precipitation technique were used separately for IgG purification. The results showed that IgG purification increased by 97% in caprylic acid method, compared with only a 77% increase in ammonium sulphate method. There were also significant differences in specificity and sensitivity between our standardized ELISA technique and using commercial kit (Cygnus CHO HCP).

High-Frequency in Vitro Direct Shoot Regeneration from Nodal Explants of Hyssop Plant (Hyssopus officinalis L.)

Considering great medicinal value of Hyssopus officinalis L. and possibility of its mass production through in vitro culture, two individual experiments was conducted. Effect of various concentrations (0, 2.2, 4.4 and 11 µmol) of TDZ and BAP in combination with 1 µmol of IAA on direct regeneration from nodal explants were assessed. Significant difference between treatments was observed (P≤ 0.01). In BAP treatments, the maximum shoot-buds induction (9 shoot-buds per explant) and shoot regeneration percentage (96.66%) were observed on MS medium fortified with 2.2 and 4.4 µmol BAP in combination with 1 µmol of IAA. In TDZ treatments, the highest regeneration percentage was achieved in MS medium supplemented with TDZ (2.2 µmol) and IAA (1 µmol), and the maximum shoot-buds induction (19.83 shoot-buds per explant) was observed in medium containing 4.4 µmol of TDZ in combination with 1 µmol of IAA. The highest root production frequency (89.5%) was achieved in medium contained 9.84 µmol of IBA. Rooted plants were acclimatized successfully in greenhouse conditions with 100% survival. The protocol described here could be applicable for mass in vitro production of the valuable medicinal plant Hyssopus officinalis L. for its genetic resource conservation as well as pharmaceutical purpose

Impact of Rifampin Induction on the Fermentation Production of Ganoderic Acids by Medicinal Mushroom Ganoderma lucidum

Backgrounds and Objectives: Ganoderic acids are the most valuable secondary metabolites in Ganoderma lucidum traditional medicinal mushrooms, which have shown antitumor properties in many studies. However, application of ganoderic acids is limited due to low yield production. Recently, it was shown that static liquid culture could be a proven technology for producing ganoderic acids in Ganoderma lucidum, and that applying elicitors could be a potential strategy to improve their production. Materials and Methods: In this work, the effect of rifampin, a cyto-chromes P450 inducer, on production of ganoderic acids was studied, and Response Surface Methodology was applied to optimize the elicitor induction. Then total ganoderic acid in the harvested mycelia was extracted and its absorbency was measured. Results and Conclusion: The results showed an increase in the concen-tration of ganoderic acid in all samples. Moreover, optimum concentration and induction time of rifampin were obtained. The proposed model predicted the maximum ganoderic acid production as 18.6 mg g-1 in which the optimal concentration and time induction obtained were 100 μM and day 9, respectively. This work demonstrated a useful method for the enhanced production of ganoderic acids by Ganoderma lucidum.

The tracheal virome of broiler chickens with respiratory disease complex in Iran: the metagenomics study

Background and Objectives: Avian respiratory disease complex (RDC) is one of the most detrimental economic diseases that affected different parts of the world. Various pathogens cause the disease, but the most significant viral pathogens include avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) are the most prevalent. To detect these pathogens, various methods have been discovered in the last decades. Detection and characterization of viruses by metagenomics methods have improved our knowledge about the role of virome in the avian complex respiratory disease. Materials and Methods: This research investigates the viral pathogen populations that mostly participate in emerging these diseases using the NGS method RNA-sequencing. In surveillance of ten broiler farms from different cities with respiratory symptoms, trachea samples were collected to determine the pathogenic virome causing the disease. Results: In this metagenomics analysis, nine viral families were identified, comprising 72.82% of RNA viruses, 24.32% of RT viruses, and 2.86% of DNA viruses. RNA viruses had the highest contribution to the respiratory disease complex instead of disease, including paramyxoviridae, orthomyxoviridae, coronaviridae, and picornaviridae viruses. Other viruses from the RNA viruses and DNA virus families were also identified in addition to these results. Conclusion: This research suggests that studies of pathogenic viromes in different diseases can help monitor different diseases and predict their future occurrence

Novel approach of vaccination against Brucella abortus 544 based on a combination of fusion proteins, human serum albumin and brucella abortus Lipopolysaccharides.

Lipopolysaccharide (LPS) of Brucella abortus is an essential component for developing the subunit vaccine against brucellosis. B. abortus LPS was extracted by n-butanol, purified by ultracentrifugation and detoxified by alkaline treatment. Pyrogenicity and toxicity of B. abortus LPS and detoxified–LPS (D-LPS) were analyzed and compared with LPS of E. coli. Different groups of mice were immunized intraperitoneally with purified B. abortus LPS, D-LPS, a combination of LPS with human serum albumin (LPS-HSA) and B. abortus S19 bacteria; besides, control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intraperitoneally with virulent B. abortus 544 strain 4 weeks after the second dose of vaccine. Sera and spleens of mice were harvested 4 weeks after challenge. LPS-B. abortus was 10,000-fold less potent in LAL test and 100-fold less potent in eliciting fever in rabbits than in E. coli LPS. And D-LPS was very less potent in LAL test and eliciting fever in rabbits ordinary LPS. The antibody titer of anti-LPS immunoglobulin G (IgG) was higher than D-LPS. However, mice immunized with either LPS, D-LPS or LPS-HSA vaccines showed a significant protection against infection of the spleen (p<0.01). There was no significant difference between mice immunized with LPS and D-LPS in terms of protection (p<0.99). Therefore, it was concluded that D-LPS and LPS-HSA for B. abortus can be used as safer and more potent vaccines than ordinary LPS-B. abortus vaccine