Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.

INTRODUCTION: Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1. OBJECTIVES: In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli. METHODS: The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process. RESULTS: Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity. CONCLUSION: It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

Important structural features of antimicrobial peptides towards specific activity: Trends in the development of efficient therapeutics.

Proteins and peptides, as polypeptide chains, have usually got unique conformational structures for effective biological activity. Antimicrobial peptides (AMPs) are a group of bioactive peptides, which have been increasingly studied during recent years for their promising antibacterial, antifungal, antiviral and anti-inflammatory activity, as well as, other esteemed bioactivities. Numerous AMPs have been separated from a wide range of natural resources, or produced in vitro through chemical synthesis and recombinant protein expression. Natural AMPs have had limited clinical application due to several drawbacks, such as their short half-life due to protease degradation, lack of activity at physiological salt concentrations, toxicity to mammalian cells, and the absence of suitable methods of delivery for the AMPs that are targeted and sustained. The creation of synthetic analogs of AMPs would both avoid the drawbacks of the natural analogs and maintain or even increase the antimicrobial effectiveness. The structure-activity relationship of discovered AMPs or their derivatives facilitates the development of synthetic AMPs. This review discovered that the relationship between the activity of AMPs and their positive net charge, hydrophobicity, and amino acid sequence and the relationship between AMPs' function and other features like their topology, glycosylation, and halogenation.

Heterologous Production of Antimicrobial Peptides: Notes to Consider.

Heavy and irresponsible use of antibiotics in the last century has put selection pressure on the microbes to evolve even faster and develop more resilient strains. In the confrontation with such sometimes called "superbugs", the search for new sources of biochemical antibiotics seems to have reached the limit. In the last two decades, bioactive antimicrobial peptides (AMPs), which are polypeptide chains with less than 100 amino acids, have attracted the attention of many in the control of microbial pathogens, more than the other types of antibiotics. AMPs are groups of components involved in the immune response of many living organisms, and have come to light as new frontiers in fighting with microbes. AMPs are generally produced in minute amounts within organisms; therefore, to address the market, they have to be either produced on a large scale through recombinant DNA technology or to be synthesized via chemical methods. Here, heterologous expression of AMPs within bacterial, fungal, yeast, plants, and insect cells, and points that need to be considered towards their industrialization will be reviewed.

Ultrasensitive detection of vital biomolecules based on a multi-purpose BioMEMS for Point of care testing: digoxin measurement as a case study.

Rapid and label-free detection of very low concentrations of biomarkers in disease diagnosis or therapeutic drug monitoring is essential to prevent disease progression in Point of Care Testing. For this purpose, we propose a multi-purpose optical Bio-Micro-Electro-Mechanical-System (BioMEMS) sensing platform which can precisely measure very small changes of biomolecules concentrations in plasma-like buffer samples. This is realized by the development of an interferometric detection method on highly sensitive MEMS transducers (cantilevers). This approach facilitates the precise analysis of the obtained results to determine the analyte type and its concentrations. Furthermore, the proposed multi-purpose platform can be used for a wide range of biological assessments in various concentration levels by the use of an appropriate bioreceptor and the control of its coating density on the cantilever surface. In this study, the present system is prepared for the identification of digoxin medication in its therapeutic window for therapeutic drug monitoring as a case study. The experimental results represent the repeatability and stability of the proposed device as well as its capability to detect the analytes in less than eight minutes for all samples. In addition, according to the experiments carried out for very low concentrations of digoxin in plasma-like buffer, the detection limit of LOD = 300 fM and the maximum sensitivity of S = 5.5 × 1012 AU/M are achieved for the implemented biosensor. The present ultrasensitive multi-purpose BioMEMS sensor can be a fully-integrated, cost-effective device to precisely analyze various biomarker concentrations for various biomedical applications.

The Analgesic and Anti-inflammatory Effects of Partially Purified Polysaccharide Fractions of Cell-free Medium and Biomass of Spirulina platensis PCST5.

Background: Spirulina is a cyanobacteria species containing various bioactive compounds. Spirulina is a known source of nutrients in some traditional diets. Different activities have been reported for various extracts of S. platensis. Objectives: In this study, the polysaccharide content of culture media and biomass extract of one species of Spirulina was partially purified, and its analgesic and anti-inflammatory effects were evaluated. Methods: Spirulina platensis PCST5 was cultured in a sterile Zarouk medium at 27°C and 16/8h of light/ dark exposure cycle for 25 days. Then, the polysaccharide content of biomass and cell-free culture medium samples (BPSs and CFPSs, respectively) was partially purified. The analgesic and anti-inflammatory effects were evaluated using animal models. Results: 16S rRNA gene analysis confirmed that the organism was genetically similar to Spirulina platensis. The CFPSs (30 and 100 mg/kg) and BPSs (30 mg/kg) significantly reduced pain-related behaviors in rats. Similarly, all samples could significantly reduce carrageenan-induced paw inflammation volume compared with the control group. Our results suggest Spirulina's polysaccharide fractions (CFPSs and BPSs) had significant analgesic and anti-inflammatory effects. Conclusions: Since Spirulina is a readily available source of bioactive compounds, finding such potent anti-inflammatory and anti-nociceptive compounds can provide promising leads for novel drug development.

Culture Optimization to Produce High Yields of Mycosporine-Like Amino Acids by Fischerella sp. F5.

Fischerella sp. is a valuable source of active metabolites, including UV-protecting compounds, among which mycosporin-like amino acids (MAAs) can be mentioned. Mycosporine-like amino acids are attractive secondary metabolites of a wide range of microorganisms, including microalgae and cyanobacteria. Enhanced production of MAAs has been studied in different sources. This study aimed to optimize the phosphate and nitrate concentrations of the culture medium on BG11 to maximize MAAs production from Fischerella sp. F5, using response surface methodology. The extraction process from the cultures, grown in adjusted conditions, was also optimized. The results confirmed that increasing both, nitrate and phosphate concentration, in the culture medium had a positive effect on the MAAs production by Fischerella sp. F5. While, optimization of the extraction process was not led to a highly accurate predictive model; temperature, sonication time, methanol ratio, and solvent/biomass ratio exhibited significant effects on the final MAAs' concentration in partially purified extracts. In general, more optimization cultures studies need to complete these findings in reference to MAAs production and extraction from Fischerella sp. F5, for commercial-scale applications.

Antimicrobial peptides with anticancer activity: Today status, trends and their computational design.

Some antimicrobial peptides have been shown to be able to inhibit the proliferation of cancer cell lines. Various strategies for treating cancers with active peptides have been pursued. According to the reports, anticancer peptides are important therapeutic peptides, which can act through two distinct pathways: they either just create pores in the cell membrane, or they have a vital intracellular target. In this review, publications up to Sep. 2021 had extracted form Scopus and PubMed using "antimicrobial peptide" and "anticancer peptide" as keywords. In second step, "computational design" related publications extracted. Among publications, those have similar scopes were classified and selected based on mechanisms of action and application. In this review, the most recent advances in the field of antimicrobial peptides with anti-cancer activities have been summarized. Freely available webservers such as AntiCP, ACPP, iACP, iACP-GAEnsC, ACPred are discussed here. In conclusion, despite some limitations of ACPs such as production cost and challenges, short half-life and toxicity on normal cells, the beneficial properties of AMPs make some of them good therapeutic agents for cancer therapy. Towards designing novel ACPs, the computational methods have substantial position and have been used progressively, today.

Design and Evaluation of a Novel Anti-microbial Peptide from Cathelicidin-2: Selectively Active Against Acinetobacter baumannii.

Background: Infections caused by pathogenic microorganisms have increased the need for hospital care and have thus represented a public health problem and a significant financial burden. Classical treatments consisting of traditional antibiotics face several challenges today. Anti-microbial peptides (AMPs) are a conserved characteristic of the innate immune response among different animal species to defend against pathogenic microorganisms. Objectives: In this study, a new peptide sequence (mCHTL131-140) was designed using the in silico approach. Methods: Cathelicidin-2 (UniprotID: Q2IAL7) was used as a potential antimicrobial protein, and a novel 10 - 12 amino acids sequence AMP was designed using bioinformatics tools and the AMP databases. Then, the anti-bacterial, anti-biofilm, and anti-fungal properties of the peptide, as well as its hemolytic activity and cytotoxicity towards human fibroblast (HDF) cells, were investigated in vitro. Results: Online bioinformatics tools indicated that the peptide sequence could have anti-bacterial, anti-viral, anti-fungal, and anti-biofilm properties with little hemolytic properties. The experimental tests confirmed that mCHTL131-140 exhibited the best anti-bacterial properties against Acinetobacter baumannii and had fair anti-fungal properties. Besides, it did not cause red blood cell lysis and showed no cytotoxicity towards HDF cells. Conclusions: In general, the designed peptide can be considered a promising AMP to control hospital-acquired infections by A. baumannii.

A Study on the Effect of Nitrate and Phosphate Concentrations on the Production of Mycosporine-Like Amino Acids by Chlorella Vulgaris.

Background: Cyanobacteria can produce compounds absorbing ultraviolet irradiation. Mycosporine like amino acids (MAAs) are some of these important metabolites, which can be potentially considered as a sunscreen agent in the pharmaceutical and cosmetic industry. Different factors have been reported that can affect the biosynthesis of MAA. Objective: In this study, the influence of different concentrations of phosphate and nitrate under different environmental conditions on MAA production by Chlorella vulgaris was investigated using an experimental design method, in order to enhance MAAs production in this specious. Materials and Methods: A 23 full factorial design (FFD) using Design-Expert v7.0.0 software was used to optimize simultaneously all the three factors of nitrate and phosphate concentration and condition of incubation environment on the MAA production by this species of C. vulgaris. Two milliliter of organism stock were grown in 200 mL BG11 medium and after 21 days, the biomasses of all samples were separated. Then, the MAA was extracted from dried biomass using methanol extraction. The extracts were analyzed by reverse-phase high performance liquid chromatography (RP-HPLC). After complete analysis, four samples were then cultured at the optimized conditions and analyzed by liquid chromatohraphy coupled to mass spectrometry (LC/MS). Results: The results showed that this microalga could produce compounds with λmax of 330 nm and a retention time of about 2 min. According to the central composite analysis, phosphate at 0.51 g.L-1 and nitrate at 2.5 g.L-1 can be considered as the optimum concentrations, resulting to the preferable conditions concerning the culture in germinator. Based on LC/MSS analysis, the major compound had a m/z of 332 at the optimum condition. Conclusion: Thus, this species is expected to have the capability of MAA production (maybe Shinorine) or one of its glycosylated derivatives.

Anti-Microbial Peptides: Strategies of Design and Development and Their Promising Wound-Healing Activities.

BACKGROUND: Current approaches used to overcome microbial infections are becoming inefficient due to the overuse or misuse of antibiotics. Antimicrobial peptides (AMPs) are one of the most promising substitutional candidates for commercial antibiotics. METHODS AND RESULTS: The publications in the field of in silico design of AMPs with focus on the wound-healing AMPs were searched though SCOPUS and PubMed. Through publications, it was reported that a number of AMPs approved for clinical use have also showed efficient wound-healing activity. Todays, the design and production of synthetic types of AMPs have attracted attention in order to expand their applications and to cope with their limitations and adverse effects. In this paper, the currently published researches in the field of AMPs and their wound-healing features were summarized and the strategies used in AMPs design and development have been reviewed. Moreover, different databases and online servers used in this regard were summarized. CONCLUSION: Design and development of active AMPs, especially those with wound-healing activity, can be performed using bioinformatics servers and software, regarding AMPs structure-activity relationships.

Isolation of the Anticoagulant and Procoagulant Fractions of the Venom of Iranian Endemic Echis carinatus.

Background: The venom of Echis carinatus contains both procoagulant and anticoagulant components that can either promote or block the blood coagulation cascade, and some of these components affect platelet function in different ways. Objectives: The present study focuses on setting up a procedure for the purification of crude venom and designing appropriate clotting tests in order to characterize the procoagulant and anticoagulant fractions of E. carinatus venom. Methods: Chromatographic methods, including gel filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC), were applied for purifying these fractions. Coagulant activity testing, prothrombin time (PT), and activated partial thromboplastin time (APTT) were used to determine procoagulant and anticoagulant properties. For measuring molecular weight, 15% SDS-PAGE electrophoresis with a molecular weight standard ranging from 6.5 to 200 kDa was used. Results: We obtained five fractions named F1, F2, F3, F4, and F5. The F1 and F2 fractions showed procoagulant activity, and the F5 fraction had anticoagulant activity. The molecular weight of F2.4.2 from fraction F2 and F5.1 from fraction F5 were analyzed by SDS-PAGE electrophoresis under the reducing condition. These factors were identified as a single protein band at the end of purification. The molecular weights of these purified fractions were estimated to be 7.5 kDa and 38 kDa for F5.1(b) and F2.4.2(b), respectively. Conclusions: Our findings suggest an efficient and suitable procedure for the identification and purification of the procoagulant and anticoagulant factors of the venom of Iranian E. carinatus using the PT and APTT assays.

In silico design of novel aptamers utilizing a hybrid method of machine learning and genetic algorithm.

Aptamers can be regarded as efficient substitutes for monoclonal antibodies in many diagnostic and therapeutic applications. Due to the tedious and prohibitive nature of SELEX (systematic evolution of ligands by exponential enrichment), the in silico methods have been developed to improve the enrichment processes rate. However, the majority of these methods did not show any effort in designing novel aptamers. Moreover, some target proteins may have not any binding RNA candidates in nature and a reductive mechanism is needed to generate novel aptamer pools among enormous possible combinations of nucleotide acids to be examined in vitro. We have applied a genetic algorithm (GA) with an embedded binding predictor fitness function to in silico design of RNA aptamers. As a case study of this research, all steps were accomplished to generate an aptamer pool against aminopeptidase N (CD13) biomarker. First, the model was developed based on sequential and structural features of known RNA-protein complexes. Then, utilizing RNA sequences involved in complexes with positive prediction results, as the first-generation, novel aptamers were designed and top-ranked sequences were selected. A 76-mer aptamer was identified with the highest fitness value with a 3 to 6 time higher score than parent oligonucleotides. The reliability of obtained sequences was confirmed utilizing docking and molecular dynamic simulation. The proposed method provides an important simplified contribution to the oligonucleotide-aptamer design process. Also, it can be an underlying ground to design novel aptamers against a wide range of biomarkers.

Primary Assessment of Mycosporine-Like Amino Acids Production by Two Species of Fischerella sp.

Mycosporin-like amino acids (MAAs) are a group of UV-absorbing compounds, which can be produced by various organisms such as algae and cyanobacteria, particularly if they survive in highly irradiated environments. In this study, the production of MAAs by two species of Fischerlla sp. (F5 and F14), isolated from the North of Iran, was investigated. Both species, which had previously been morphologically detected as Fisherella sp., were confirmed molecularly by sequencing the PCR amplicon of the 16S rRNA gene. The species were cultured in sterilized BG.11 medium for 21 days, then biomasses were separated, and their MAAs content was extracted by methanol and partially purified using chloroform liquid-liquid extraction. The extract was analyzed using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). In both species, the compounds with MAAs characteristics were observed. They had maximum absorbance (λmax) in the range of 300-400 nm, which was confirmed by the LC-MS analysis. In F5 species, the peaks with m/z 340 and 391 and in another one (F14), a peak with m/z 333.2 were recorded, that the latter might be Shinorine. In general, further analysis should be performed to elucidate the exact structural aspects of these compounds. In conclusion, both Fischerella sp. studied here were capable of producing MAAs and can be evaluated for use in sunscreen pharmaceutical and cosmetic products.

Anti-inflammatory Activity of Bioactive Compounds from Microalgae and Cyanobacteria by Focusing on the Mechanisms of Action.

Microalgae and cyanobacteria are the potentially valuable source of bioactive compounds applied in the various industries and human usage in different fields of pharmaceutical, nutraceutical, and cosmetic disciplines. One of the interesting aspects is their application as the anti-inflammatory agents for treatment of inflammation related mal-conditions. Natural compounds are of great importance in the treatment of inflammations to reduce the reaction of immune system against pathogens, toxic compounds and damaged cells. A wide range of different metabolites with various chemical structures, including small molecules and peptides and proteins, polysaccharides, fatty acids and their derivatives have been found in microalgae and cyanobacteria which have anti-inflammatory activity. In this review, we summarized different metabolites with anti-inflammatory activity that were extracted from these microorganisms and their mechanisms. The bioactive compounds from microalgae and cyanobacteria have exhibited anti-inflammatory activity through different mechanisms acting intra- or extra- cellularly. So, they could be considered as promising anti-inflammatory agents in treatment of related diseases.

RPINBASE: An online toolbox to extract features for predicting RNA-protein interactions.

Feature extraction is one of the most important preprocessing steps in predicting the interactions between RNAs and proteins by applying machine learning approaches. Despite many efforts in this area, still, no suitable structural feature extraction tool has been designed. Therefore, an online toolbox, named RPINBASE which can be applied to different scopes of biological applications, is introduced in this paper. This toolbox employs efficient nested queries that enhance the speed of the requests and produces desired features in the form of positive and negative samples. To show the capabilities of the proposed toolbox, the developed toolbox was investigated in the aptamer design problem, and the obtained results are discussed. RPINBASE is an online toolbox and is accessible at http://rpinbase.com.

Therapeutic applications of nucleic acid aptamers in microbial infections.

Today, the treatment of bacterial infections is a major challenge, due to growing rate of multidrug-resistant bacteria, complication of treatment and increased healthcare costs. Moreover, new treatments for bacterial infections are limited. Oligonucleotide aptamers are single stranded DNAs or RNAs with target-selective high-affinity feature, which considered as nucleic acid-based affinity ligands, replacing monoclonal antibodies. The aptamer-based systems have been found to be talented tools in the treatment of microbial infections, regarding their promising anti-biofilm and antimicrobial activities; they can reduce or inhibit the effects of bacterial toxins, and inhibit pathogen invasion to immune cell, as well as they can be used in drug delivery systems. The focus of this review is on the therapeutic applications of aptamers in infections. In this regard, an introduction of infections and related challenges were presented, first. Then, aptamer definition and selection, with a brief history of aptamers development against various pathogens and toxins were reviewed. Diverse strategies of aptamer application in drug delivery, as well as, the effect of aptamers on the immune system, as the main natural agents of human defense against pathogens, were also discussed. Finally, the future trends in clinical applications of this technology were discussed.

Biomedical and Pharmaceutical-Related Applications of Laccases.

The oxidation of a vast range of phenolic and non-phenolic substrates has been catalyzed by laccases. Given a wide range of substrates, laccases can be applied in different biotechnological applications. The present review was conducted to provide a broad context in pharmaceutical- and biomedical- related applications of laccases for academic and industrial researchers. First, an overview of biological roles of laccases was presented. Furthermore, laccase-mediated strategies for imparting antimicrobial and antioxidant properties to different surfaces were discussed. In this review, laccase-mediated mechanisms for endowing antimicrobial properties were divided into laccase-mediated bio-grafting of phenolic compounds on lignocellulosic fiber, chitosan and catheters, and laccase-catalyzed iodination. Accordingly, a special emphasis was placed on laccase-mediated functionalization for creating antimicrobials, particularly chitosan-based wound dressings. Additionally, oxidative bio-grafting and oxidative polymerization were described as the two main laccase-catalyzed reactions for imparting antioxidant properties. Recent laccase-related studies were also summarized regarding the synthesis of antibacterial and antiproliferative agents and the degradation of pharmaceuticals and personal care products.

Silymarin antiproliferative and apoptotic effects: Insights into its clinical impact in various types of cancer.

Silymarin is a complex extract isolated from the plant Silybum marianum, widely known for its prominent antioxidant and hepatoprotective effects, although increasing evidences have reported extraordinary antiproliferative and apoptotic abilities. As a result, several signaling pathways involved in cell cycle control, cell proliferation, and cell death have been deconvoluted as critical mechanisms. In this regard, cyclin and cyclin-dependent pathways have been the most studied ones. Following that, apoptotic pathways, such as p53, Akt, STAT-3, Ras, and caspases pathways, have been extensively studied, although other mechanisms involved in inflammation and angiogenesis have also been highlighted as silymarin-likely targets in cancer therapy. Therefore, the main challenge of this review is to discuss the diverse molecular mechanisms for silymarin antiproliferative and apoptotic effects; most of them largely studied in various types of cancers so far. Clinical trials and combination therapies related to silymarin application in cancer prevention and treatment are presented as well.

Peptide dendrimers as valuable biomaterials in medical sciences.

Peptides are oligomers of amino acids, which have been used in a wide range of applications, particularly in medical and pharmaceutical sciences. Linear peptides have been extensively developed in various fields of medicine as therapeutics or targeting agents. The branched structure of peptide dendrimers with peptide (commonly, poly l­Lysine) or non-peptide (commonly poly­amidoamine) core, often exhibits valuable novel features, improves stability and enhances the functionality of peptide in comparison with small linear peptides. The potential applications of Branched and hyper-branched peptidic structures which are known as peptide dendrimers in biomedical sciences have been approved vastly. A peptide dendrimer contains three distinct parts including core, building blocks and branching units or surface functional groups. These structures provide a lot of opportunities in the pharmaceutical field, particularly for novel drug development. In this review, a brief summary of different biomedical applications of peptide dendrimers is presented, and peptide dendrimers as active pharmaceutical ingredients and drug delivery carriers are discussed. Applications of peptide dendrimers in vaccines and diagnostic tools are also presented, in brief. Generally, peptide dendrimers are promising biomaterials with high evolution rate for clinical and non-clinical applications in medicine.

Construction of a Mammalian IRES-based Expression Vector to Amplify a Bispecific Antibody; Blinatumomab.

Blinatumomab, the bispecific T cell engager antibody (BsAb), has been demonstrated as the most successful BsAb to date. Throughout the past decade, vector design has great importance for the expression of monoclonal antibody in Chinese hamster ovary (CHO) cells. It has been indicated that expression vectors based on the elongation factor-1 alpha (EF-1 alpha) gene and DHFR selection marker can be highly effective to produce populations of stably transfected cells in the selection medium. Moreover, the phiC31 integrase system is considered as an attractive and safe protein expression system in mammalian cells and it could integrate a donor plasmid of any size, as a single copy, in to the host genome with no cofactors. In this study, phiC31 integrase technology in combination with DHFR amplification system was used to have an expression vector for future expression of blinatumomab in CHO cells. The gene of interest (BsAb gene) could be joined to DHFR selection marker with the insertion of an internal ribosome entry site (IRES). By positioning the DHFR downstream of BsAb gene and IRES, the transcription of the selection marker can depend on the successful transcription of the BsAb gene, which was located upstream in the expression construct. In this study, FC550A-1 vector was used as the backbone and DHFR selection marker was successfully combined with phiC31 integrase technology to generate a high-expressing construct for BsAb expression in CHO-DG44 cells in future studies.