Optimization of nitrogen and phosphorus removal from meat processing wastewaters using microalgal biofilms

Background: The conventional activated sludge processes are not able to completely treat the nitrogen and phosphorus compounds of meat processing wastewaters. Microalgal biofilm, which has the advantages of easiness and inexpensiveness, can be applied as a post-treatment method. Methods: The optimal levels of micronutrients and N/P ratio were aimed in the cultivation of Scenedesmus quadricauda as a biofilm formed on the two types of commercially available bed packing.For this purpose, the Factorial design was run to maximize the removal of ammonium, nitrate and phosphate concentration in the synthetic meat processing wastewater. Results: Experimental data showed better results for the removal of biofilm formed on the sponge filter. In addition, a maximum removal efficiency of 55.6%, 47% and 48.% was respectively achieved for ammonium, nitrate and phosphate at optimal N/P ratio of 10.5 and volumetric percentage of 50% of micronutrients. Conclusion: The results demonstrated that microalgal biofilm of Scenedesmus is able to remove the nutritional components of meat processing wastewaters. The process is needed to be optimized for the effective treatment. Keywords: Meat, Biofilms, Microalgae, Wastewater, Scenedesmu

Qualitative and Quantitative Study of Quercetin and Glycyrrhizin in In Vitro Culture of Liquorice (Glycyrrhiza glabra L.) and Elicitation with AgNO3

Liquorice (Glycyrrhiza glabra L.) is a plant that has been considered for a long time due to its valuable secondary metabolites. This study was conducted to obtain quercetin and glycyrrhizin under controlled conditions and the use of silver nitrate (AgNO3) as an elicitor to increase their production. For this purpose, the seeds were cultured in MS media containing various concentrations of AgNO3 (0, 2, 4, 8 and 10 mg L-1). Quercetin in the aerial parts extract of three-month seedlings prepared with methanol solvent 95% and acetic acid (9:1), and glycyrrhizin in the root extract of four-month seedlings prepared with ethanolic extract (30%) were evaluated qualitatively and quantitatively using HPLC. The results obtained from three replications showed the presence of quercetin and glycyrrhizin in all samples. The amount of quercetin in all samples treated with AgNO3 was significantly higher than control (P≤0.05) and this increase was higher at concentrations of 8 and 10 mg L-1 in comparison with other concentrations. Glycyrrhizin content increased under the influence of different concentrations of AgNO3 as compared to the control; however, this increase was not significant. Our results clearly showed that this method is a practical method to produce and elicit more these compounds with medicinal value

Purification of recombinant C-terminus polyhistidine tagged human calcitonin with cobalt-based and nickel-based affinity chromatography: A comparative study

Human calcitonin (hCT) is a 32 amino acid peptide that participates in the regulation of calcium and phosphorus metabolism in human. It is used in clinics for the treatment of diseases related to bone decalcification, such as Paget disease, osteoporosis imperfecta and parathyroid gland carcinoma. In this research, C-terminus polyhistidine tagged human recombinant calcitonin which was expressed by Pichia pastoris strain KM71H was purified with cobalt-based IMAC resin and Nickel-based IMAC column. The result shows that the yield of purification was higher by cobalt-based IMAC resin in comparison with nickel-based IMAC column.Keywords: Human calcitonin, Pichia pastoris, cobalt-based IMAC resin, nickel-based IMAC colum

Evaluation of Drug Repositioning by Molecular Docking of Pharmaceutical Resources to Identification of Potential <em>SARS-CoV-2</em> Viral Inhibitors

Unfortunately, to date, there is no approved specific antiviral drug treatment against COVID-19. Due to the costly and time-consuming nature of the de novo drug discovery and development process, in recent days, the computational drug repositioning method has been highly regarded for accelerating the drug-discovery process. The selection of drug target molecule(s), preparation of an approved therapeutics agent library, and in silico evaluation of their affinity to the subjected target(s) are the main steps of a molecular docking-based drug repositioning process, which is the most common computational drug re-tasking process. In this chapter, after a review on origin, pathophysiology, molecular biology, and drug development strategies against COVID-19, recent advances, challenges as well as the future perspective of molecular docking-based drug repositioning for COVID-19 are discussed. Furthermore, as a case study, the molecular docking-based drug repurposing process was planned to screen the 3CLpro inhibitor(s) among the nine Food and Drug Administration (FDA)-approved antiviral protease inhibitors. The results demonstrated that Fosamprenavir had the highest binding affinity to 3CLpro and can be considered for more in silico, in vitro, and in vivo evaluations as an effective repurposed anti-COVID-19 drug

Construction of bicistronic cassette for co-expressing hepatitis B surface antigen and mouse granulocyte-macrophage colony stimulating factor as adjuvant in tobacco plant

Context: The co-delivery of adjuvant and antigen has shown to be more effective for targeting the immune response than antigen alone. Therefore, designing an efficient bicistronic system is more assuring for production of both elements in the same tobacco cells as a plant model system. Objective: Comparing the efficient transient co-expression of hepatitis B surface antigen (HBsAg) and mouse granulocyte macrophage colony stimulating factor (mGM-CSF) in tobacco leaves by designing either mono or bicistronic cassettes. Materials and methods: Four expression cassettes containing tobacco etch virus (TEV) leader sequence were constructed with and without above genes in different orders. The cassettes were transferred into tobacco, Nicotiana tabacum L. (Solanaceae), leaves by agroinfiltration technique. The expression levels were compared using ELISA and western blotting and bioactivity of cytokine was assessed by in vitro proliferation of mouse GM-CSF-responsive progenitor cells. Results: Agroinfiltrated leaves contained recombinant HBsAg protein at 20–50 ng/mg and mGM-CSF at 0.2–4 ng/mg in both nonglycosylated and glycosylated forms. The highest expression obtained in HBsAg and mGM-CSF monocistronic co-agroinfiltrated leaves. The expression of mGM-CSF was 1.1 and 0.2 ng/mg in two different orders of bicistronic cassettes. The growth frequency of GM progenitors was approximately 1/187 cells for standard rGM-CSF and 3.2 times less activity for the plant produced. Discussion and conclusions: The recombinant mGM-CSF was produced less in bicistronic cassette than other forms; however, co-presenting of both vaccine candidate and adjuvant is confirmed and could be promising for amelioration of plant expression system as a means for vaccine production

Developing of SARS-CoV-2 fusion protein expressed in E. coli Shuffle T7 for enhanced ELISA detection sensitivity – an integrated experimental and bioinformatic approach

In the recent COVID-19 pandemic, developing effective diagnostic assays is crucial for controlling the spread of the SARS-CoV-2 virus. Multi-domain fusion proteins are a promising approach to detecting SARS-CoV-2 antibodies. In this study, we designed an antigen named CoV2-Pro, containing two RBD domains from SARS-CoV-2 Omicron and Delta variants and one CTD domain of the nucleoprotein in the order of RBD-RBD-N, linked by a super flexible glycine linker. We evaluated the suitability of E. coli Shuffle T7 and BL21 (DE3) strain for expressing CoV2-Pro. Moreover, Bioinformatic studies were conducted first to analyze the tertiary structure of CoV2-Pro. The CoV2-Pro sequences were cloned into a pET-32b (+) vector for expression in E. coli Shuffle T7 and BL21 (DE3). SDS-PAGE and western blot confirmed the protein expression and folding structure. The CoV2-Pro-TRX was purified by Ni-NTA affinity chromatography. Dot blot analysis was performed to evaluate the antigenic characterization of the CoV2-Pro. A molecular docking simulation was conducted to assess the binding affinity of CoV2-Pro with LY-COV555 (Bamlanivimab) monoclonal antibody. A molecular dynamic was performed to analyze the stability of the structure. Bioinformatic and experimental studies revealed a stable conformational 3D structure of the CoV2-Pro. The CoV2-Pro interacted with SARS-CoV-2 antibodies, confirming the correct antigenic structure. We assert with confidence that CoV2-Pro is ideal for developing an ELISA assay for precise diagnosis and rigorous vaccine evaluation during the COVID-19 prevalence. Communicated by Ramaswamy H. Sarma</p

Utility of P19 Gene-Silencing Suppressor for High Level Expression of Recombinant Human Therapeutic Proteins in Plant Cells

Background: The potential of plants, as a safe and eukaryotic system, is considered in the production of recombinant therapeutic human protein today; but the expression level of heterologous proteins is limited by the post-transcriptional gene silencing (PTGS) response in this new technology. The use of viral suppressors of gene silencing can prevent PTGS and improve transient expression level of foreign proteins. In this study, we investigated the effect of p19 silencing suppressor on recombinant human nerve growth factor expression in Nicotiana benthamiana. Materials and Methods: The p19 coding region was inserted in the pCAMBIA using NcoI and BstEII recognition sites. Also, the cloned synthesized recombinant human NGF (rhNGF) fragment was cloned directly into PVX vector by ClaI and SalI restriction enzymes. The co-agroinfiltration of rhNGF with p19 viral suppressor of gene silencing was evaluated by dot-blot and SDS-PAGE. The amount of expressed rhNGF protein was calculated by AlphaEaseFC software. Results: Co-agroinfiltration of hNGF with P19 suppressor showed about forty-fold increase (8% total soluble protein (TSP)) when compared to the absence of P19 suppressor (0.2%TSP). Conclusion: The results presented here confirmed that the use of P19 gene silencing suppressor derived from tomato bushy stunt virus (TBSV) could efficiently increase the transient expression of recombinant proteins in Nicotiana benthamiana manifold

Optimization of nitrogen and phosphorus removal from meat processing wastewaters using microalgal biofilms

Background: The conventional activated sludge processes are not able to completely treat the nitrogen and phosphorus compounds of meat processing wastewaters. Microalgal biofilm, which has the advantages of easiness and inexpensiveness, can be applied as a post-treatment method. Methods: The optimal levels of micronutrients and N/P ratio were aimed in the cultivation of Scenedesmus quadricauda as a biofilm formed on the two types of commercially available bed packing. For this purpose, the Factorial design was run to maximize the removal of ammonium, nitrate and phosphate concentration in the synthetic meat processing wastewater. Results: Experimental data showed better results for the removal of biofilm formed on the sponge filter. In addition, a maximum removal efficiency of 55.6%, 47% and 48.% was respectively achieved for ammonium, nitrate and phosphate at optimal N/P ratio of 10.5 and volumetric percentage of 50% of micronutrients. Conclusion: The results demonstrated that microalgal biofilm of Scenedesmus is able to remove the nutritional components of meat processing wastewaters. The process is needed to be optimized for the effective treatment

Site-specific gene knock-in and bacterial phytase gene expression in Chlamydomonas reinhardtii via Cas9 RNP-mediated HDR

In the present study, we applied the HDR (homology-directed DNA repair) CRISPR-Cas9-mediated knock-in system to accurately insert an optimized foreign bacterial phytase gene at a specific site of the nitrate reductase (NR) gene (exon 2) to achieve homologous recombination with the stability of the transgene and reduce insertion site effects or gene silencing. To this end, we successfully knocked-in the targeted NR gene of Chlamydomonas reinhardtii using the bacterial phytase gene cassette through direct delivery of the CRISPR/Cas9 system as the ribonucleoprotein (RNP) complex consisting of Cas9 protein and the specific single guide RNAs (sgRNAs). The NR insertion site editing was confirmed by PCR and sequencing of the transgene positive clones. Moreover, 24 clones with correct editing were obtained, where the phytase gene cassette was located in exon 2 of the NR gene, and the editing efficiency was determined to be 14.81%. Additionally, site-specific gene expression was analyzed and confirmed using RT-qPCR. Cultivation of the positive knocked-in colonies on the selective media during 10 generations indicated the stability of the correct editing without gene silencing or negative insertion site effects. Our results demonstrated that CRISPR-Cas9-mediated knock-in could be applied for nuclear expression of the heterologous gene of interest, and also confirmed its efficacy as an effective tool for site-specific gene knock-in, avoiding nuclear positional effects and gene silencing in C. reinhardtii. These findings could also provide a new perspective on the advantageous application of RNP-CRISPR/Cas9 gene-editing to accelerate the commercial production of complex recombinant proteins in the food-grade organism “C. reinhardtii”