CRISPeering: Bioengineering the Host Cells through CRISPRCas9 Genome Editing System as the Next-generation of Cell Factories.

BACKGROUND: Nowadays, the CRISPR-Cas9 genome editing system has become a popular bioengineering-based tool for various applications. Owing to its high-target specificity, efficiency, versatility, and simplicity, it has gained attention as a robust tool for molecular biology research, which unveils the biological functions of unexplored genes and engineers the metabolic pathways. Chinese hamster ovary (CHO) cells and Escherichia coli are regarded as the most commonly used expression platforms for industrial- scale production of recombinant proteins. The emergence of the CRISPR-Cas9 genome editing system promotes the current status of expression hosts towards controllable and predictable strains. OBJECTIVE: This paper presents the current status of expression hosts for biopharmaceutical production. Some major accomplishments in the utilization of the CRISPR-Cas9 genome editing tool in the different prokaryotic and eukaryotic systems are discussed, and more importantly, the future directions of this newly arrived technology to make the next-generation cell factories with improved or novel properties are suggested. Moreover, the challenges faced in recent patents in this field are also discussed. RESULTS AND CONCLUSION: The CRISPR-Cas9 genome-editing tool has been adopted to be utilized in some major expression platforms. CRISPeering has been successfully employed for genome editing in different prokaryotic and eukaryotic host cells. The emergence of systems metabolic engineering, systems biology, and synthetic biology fortify the current situation of the CRISPR-Cas9 genome editing system.

Drug Delivery Assessment of a Novel Triple Antibiotic-Eluting Injectable Platelet-Rich Fibrin Scaffold: An In Vitro Study.

BACKGROUND: Intracanal disinfection is a critical, yet challenging goal for long-term success in regenerative-based treatments. This in-vitro study aimed to assess the release profile of triple antibiotic- eluting Injectable Platelet-Rich Fibrin (I-PRF) constructs in 28 days. METHODS: I-PRF scaffolds containing triple antibiotic mixture [Metronidazole (MET), Ciprofloxacin (CIP), and Minocycline (MINO)] by immersion (group one), I-PRF scaffolds containing triple antibiotic mixture by integration (group two), and antibiotic-free I-PRF scaffolds (group three) were fabricated. The antibiotic release from the scaffolds was measured using High-Performance Liquid Chromatography (HPLC) (the mobile phase of 0.1% formic acid and methanol (35:65 v/v), a C18 analytical column (150 × 4.6 mm, 5 µm) at a flow rate of 0.7 mL/min, at 25ºC) at days 1, 3, 7, 14, 21, and 28. RESULTS: Retention times for MINO, CIP, and MET were achieved as 2.3, 2.6, and 3.1 min, respectively. The maximum UV absorbance values for CIP, MET, and MINO were 268 nm, 278 nm, and 350 nm, respectively. The results of the first group showed burst release within the first 24 hours followed by sustained maintenance of all three antibiotics up to 14 days. MINO and MET were still detectable in the third week. The second group could not sustainably release the antibiotics. CONCLUSION: The developed method for the simultaneous identification and quantification of each antibiotic in I-PRF was sensitive and quick. Overall, group one could take up the antibiotics in adequate quantities and then subsequently release them over the study period.

In Silico Investigation of Signal Peptide Sequences to Enhance Secretion of CD44 Nanobodies Expressed in Escherichia coli.

BACKGROUND: The selection of a suitable signal peptide that can direct recombinant proteins from the cytoplasm to the extracellular space is an important criterion affecting the production of recombinant proteins in Escherichia coli, a widely used host. Nanobodies are currently attracting the attention of scientists as antibody alternatives due to their specific properties and feasibility of production in E. coli. OBJECTIVE: CD44 nanobodies constitute a potent therapeutic agent that can block CD44/HA interaction in cancer and inflammatory diseases. This molecule may also function as a drug against cancer cells and has been produced previously in E. coli without a signal peptide sequence. The goal of this project was to find a suitable signal peptide to direct CD44 nanobody extracellular secretion in E. coli that will potentially lead to optimization of experimental methods and facilitate downstream steps such as purification. METHODS: We analyzed 40 E. coli derived signal peptides retrieved from the Signal Peptide database and selected the best candidate signal peptides according to relevant criteria including signal peptide probability, stability, and physicochemical features, which were evaluated using signalP software version 4.1 and the ProtParam tool, respectively. RESULTS: In this in silico study, suitable candidate signal peptide(s) for CD44 nanobody secretory expression were identified. CSGA, TRBC, YTFQ, NIKA, and DGAL were selected as appropriate signal peptides with acceptable D-scores, and appropriate physicochemical and structural properties. Following further analysis, TRBC was selected as the best signal peptide to direct CD44 nanobody expression to the extracellular space of E. coli. CONCLUSION: The selected signal peptide, TRBC is the most suitable to promote high-level secretory production of CD44 nanobodies in E. coli and potentially will be useful for scaling up CD44 nanobody production in experimental research as well as in other CD44 nanobody applications. However, experimental work is needed to confirm the data.

Amino Acids Sequence-based Analysis of Arginine Deiminase from Different Prokaryotic Organisms: An In Silico Approach.

BACKGROUND: Arginine deiminase is a bacterial enzyme, which degrades L-arginine. Some human cancers such as hepatocellular carcinoma (HCC) and melanoma are auxotrophic for arginine. Therefore, PEGylated arginine deiminase (ADI-PEG20) is a good anticancer candidate with antitumor effects. It causes local depletion of L-arginine and growth inhibition in arginineauxotrophic tumor cells. The FDA and EMA have granted orphan status to this drug. Some recently published patents have dealt with this enzyme or its PEGylated form. OBJECTIVE: Due to increasing attention to it, we aimed to evaluate and compare 30 arginine deiminase proteins from different bacterial species through in silico analysis. METHODS: The exploited analyses included the investigation of physicochemical properties, multiple sequence alignment (MSA), motif, superfamily, phylogenetic and 3D comparative analyses of arginine deiminase proteins thorough various bioinformatics tools. RESULTS: The most abundant amino acid in the arginine deiminase proteins is leucine (10.13%) while the least amino acid ratio is cysteine (0.98%). Multiple sequence alignment showed 47 conserved patterns between 30 arginine deiminase amino acid sequences. The results of sequence homology among 30 different groups of arginine deiminase enzymes revealed that all the studied sequences located in amidinotransferase superfamily. Based on the phylogenetic analysis, two major clusters were identified. Considering the results of various in silico studies; we selected the five best candidates for further investigations. The 3D structures of the best five arginine deiminase proteins were generated by the I-TASSER server and PyMOL. The RAMPAGE analysis revealed that 81.4%-91.4%, of the selected sequences, were located in the favored region of arginine deiminase proteins. CONCLUSION: The results of this study shed light on the basic physicochemical properties of thirty major arginine deiminase sequences. The obtained data could be employed for further in vivo and clinical studies and also for developing the related therapeutic enzymes.

Effects of Sulfur Starvation on Growth Rates, Biomass and Lipid Contents in the Green Microalga Scenedesmus obliquus.

BACKGROUND: Scenedesmus obliquus, a green unicellular chlorophycean microalga, is well-established as a lipid and biomass production platform. The nutrient starvation strategy is considered as a robust platform for lipid production from different microalgal strains. OBJECTIVE: The study aimed to analyse the influences of sulfur starvation on the growth rates, and also biomass and lipid production and composition in a naturally isolated strain of S. obliquus. METHODS: The BG-11 culture medium was utilized for preservation and microalgal growth. To monitor the cell growth rates, two different methods, including direct cell counting and also dry cell weight measurement were used. The study was conducted in 28 days composed of two distinct growth modes as 10 days of sulfur-rich and 18 days of sulfur starved media. RESULTS: The studied S. obliquus strain displayed higher lipid and carbohydrate production levels (34.68% and 34.02%) in sulfur starved medium compared with the sulfur-rich medium (25.84% and 29.08%). Nevertheless, a noticeable reduction (51.36%) in biomass contents and also in cell growth rates (63.36%) was observed during sulfur starvation. The investigated strain was composed of some important fatty acids with potential applications as food, feed and biodiesel. CONCLUSION: The observed results implied the possibility of the sulfur starvation strategy to increase lipid production in S. obliquus strain. Besides, the available data from recently published patents reveals the promising potential of the identified lipids from S. obliquus in this study for bioenergy production and other biotechnological purposes.

Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine.

BACKGROUND: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. METHODS: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. RESULTS: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund's adjuvant group. CONCLUSION: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

Expanding the Biotherapeutics Realm via miR-34a: "Potent Clever Little" Agent in Breast Cancer Therapy.

BACKGROUND: One of the most prevalent cancers befell to women is considered to be breast cancer (BC). It is also the deadliest among the female population after lung cancer. Additionally, several studies have demonstrated that there is an association between microRNA34-a and breast cancer. METHODS: We searched PubMed, Web of Science, and Google Scholar up to December 2018. Those studies which have been studied miR-34a and its tumor-suppressing capabilities were considered as the most important topics. Moreover, we extracted articles which were solely focused on microRNA-34a in breast cancer therapy. Finally, 80 articles were included. RESULTS: In comparison with the normal tissues, down-regulation of miR-34a expression is shown considerably in tumor cells. Overexpression of miR-34a acts as a tumor suppressor by transcriptional regulating one of the signaling pathways (TP53), NOTCH, and transforming growth factor beta (TGF-ß), Bcl- 2 and SIRT1genes, HDAC1 and HDAC7, Fra-1, TPD52, TLR Via CXCL10. Moreover, drug resistance declines which lead to the apoptosis, cell cycle arrest and senescence. As a result, the proliferation, invasion and metastasis of the tumor are suppressed. The Mrx34 drug contains miR-34a mimic and a lipid vector. MiR-34a as the active ingredient portrays the role of a tumor suppressor. This drug has recently entered the clinical trials studies. CONCLUSION: These findings suggest a robust cause for developing miR-34a as a therapeutic agent to target BC. In that scenario, miR-34a is strongly useful to introduce new therapeutic goals for BC. Moreover, this review aims to confirm the signal pathways, therapeutic and diagnostic values of miR- 34a in BC and beyond.

Maximizing Biomass and Lipid Production in Heterotrophic Culture of Chlorella vulgaris: Techno-Economic Assessment.

BACKGROUND: Nowadays, chlorophycean microalgae have attained a broad-spectrum attention as a potential candidate for biomass and bioenergy production. Despite their appreciated benefits, one of major problems is their low biomass and lipid productivity. Here we investigated the heterotrophic culture in shake flasks and stirred tank bioreactor to improve the lipid and biomass production in a naturally isolated strain of Chlorella vulgaris. METHODS: A naturally isolated C. vulgaris strain was cultivated in BG-11 medium in shake flask and bioreactor. Its biochemical composition and growth kinetic parameters were investigated. RESULTS: The biomass productivity was improved (3.68 fold) under heterotrophic culture compared to basal autotrophic culture condition in shake flask experiment. The total lipid content increased to 44% of total Dry Cell Weight (DCW) during heterotrophic growth after 21 days. Moreover, a great Fatty Acid Methyl Esters (FAME) yield was observed under heterotrophic cultivation. Total biomass and lipid content of microalgae in bioreactor experiment increased to 4.95 and 2.18 g L-1 respectively, during 5 days of the experiment compared to its basic autotrophic culture. CONCLUSION: The techno-economic aspects of exploiting C. vulgaris as a biodiesel feedstock werealso evaluated. The results imply that heterotrophic cultivation could compensate the low biomass productivity in microalgae for green energy production. Ever growing rates of established patents on application of various genetic and bioengineering-based methods have made it possible to achieve higher lipid contents with reduced total costs for microalgal biodiesel production as well.

Extracellular Production of a Potent and Chemically Resistant Nattokinase in Immobilized Escherichia coli Using Response Surface Methodology.

BACKGROUND: Nattokinase is a potent fibrinolytic protease, which is used as a drug for treatment or a supplement for preventing thrombosis besides various industrial applications. The present study aimed to produce a soluble nattokinase in low cost media, which has with high activity and resistance to metal ions, detergents, and organic solvents, and can be easily used in medicines or as detergents. METHODS: Generally, most of the native extracellular proteins, such as nattokinase from Bacillus subtilis, are lysed by secretory proteases. One way for solving this problem is to employ other hosts for nattokinase production. For producing secretory form of nattokinase from Bacillus subtilis, different factors such as a suitable host, optimized media for the maximum enzyme activity and easy purification are important. RESULTS: These factors are studied in this investigation. Escherichia coli BL21 (DE3), as a reliable host was selected for a high yield production of an extracellular recombinant nattokinase. A mature nattokinase gene from Bacillus subtilis 1023, was cloned in the expression vector pET22b by which the host was transformed. The recombinant nattokinase was expressed through induction with IPTG. The expressed protein was confirmed by SDS-PAGE, and its fibrinolytic activity was assayed on the fibrin plates. Afterwards, the enzyme was purified by Ni-NTA native affinity column. Different media components were evaluated for maximum nattokinase production and activity. The highest enzyme activity of 883.107 U/ml was obtained, when a medium approximately consisting of yeast extract (4.38 g/L), tryptone (4 g/L), K2HPO4 (1.61 g/L) and CaCl2 (0.01 g/L) was used. CONCLUSION: Entrapping the transformed host in calcium alginate could lead to more enzyme activity and decrease media cost.

Cell Growth, Lipid Production and Productivity in Photosynthetic Microalga Chlorella vulgaris under Different Nitrogen Concentrations and Culture Media Replacement.

BACKGROUND: The photosynthetic unicellular microalgal strain, Chlorella vulgaris is considered as a robust biomass and lipid producer for human food and feed and also green energy production. The culture media composition plays a crucial role in biomass and lipid production and productivity. METHODS: The photosynthetic green microalga C. vulgaris was studied in three distinct cultivation experiments each for 22 days, using BG11 and f/2 culture media to investigate the effects of different levels of nitrogen and culture media replacement on cell growth, lipid production and productivity. RESULTS: In f/2 culture medium, the total lipid production reached was 0.246 g L-1 (41.55%) which was 147% higher than its counterpart in BG11 medium (0.298 g L-1). On the other hand, the highest biomass (1.492 g L-1) was attained in BG11-3 medium with 6.00 g nitrate L-1 and this was correlated with higher total chlorophyll content (15.189 mg L-1) and cell number (3.812 × 106 cell mL-1). A fatty acid profile, composed of saturated, monounsaturated and polyunsaturated fatty acids was detected and included stearic acid, oleic acid, α-linolenic acid and γ-linolenic acid which have nutritional or combustion importance. CONCLUSION: These data suggest that this C. vulgaris strain may be considered as a robust source for biomass, lipid and green energy production. The newly established patents have shed light on the future directions to achieve higher lipids and fatty acids with improved properties through different bioprocess and genetic engineering approaches.