Avicenna Journal of Medical Biotechnology
Volume:14 Issue: 1, Jan-Mar 2022

More than a year has passed since the beginning of the 2019 novel coronavirus diseases (COVID-19) pandemic which has created massive problems globally affecting all aspects of people's life. Due to the emergence of new strains of the SARS-CoV-2, pandemic risk still remains, despite the start of vaccination. Therefore, rapid diagnostic tests are essential to control infection, improve clinical care and stop the spread of the disease. Recently CRISPR-based diagnostic tools have facilitated rapid diagnostic. Here, we review the diagnostic applications of CRISPR-Cas system in COVID-19.

Menstrual-derived Stem Cells (MenSC) are a potential novel source of mesenchymal stem cells. There is an increased interest in investigating the therapeutic potential of MenSC due to the various advantages they exhibit, when compared to other types of stem cells. MenSC are obtained non-invasively from menstrual blood. Thus, collection of MenSC is simple, reproducible and can be carried out periodically, with minimal complications. MenSC are present in abundance, are highly proliferative, exhibit a low immunogenicity and lack ethical issues. MenSC have shown the ability to differentiate into several lineages. The therapeutic potential of MenSC in non-gynaecological applications has been investigated in wound healing, neurological, musculo-skeletal, cardiovascular, respiratory, and liver disorders, as well as in diabetes and cancer. Human clinical trials are limited. To date, therapeutic efficacy and safety have been reported in patients with Avian influenza A subtype H7N9, COVID-19, congestive heart failure, multiple sclerosis and Duchene muscular dystrophy. However, further clinical trials in humans should be conducted, to study the long-term therapeutic effects of these stem cells in various diseases and to further explore their mechanism of action. This systematic review focuses on the application of MenSC in non-gynaecological diseases.

At present, effective vaccines have been developed as the most successful approaches for preventing widespread infectious disease. The global efforts are focusing with the aim of eliminating and overcoming the Coronavirus Disease 2019 (COVID-19) and are developing vaccines from the date it was announced as a pandemic disease. In this study, PubMed, Embase, Cochrane Library, Clinicaltrial.gov, WHO reports, Science Direct, Scopus, Google Scholar, and Springer databases were searched for finding the relevant studies about the COVID-19 vaccines. This article provides an overview of multiple vaccines that have been manufactured from December 2020 up to April 2021 and also offers a perspective on their efficacy, safety, advantages, and limitations. Currently, there are several categories of COVID-19 vaccines based on Protein Subunit (PS), Inactivated Virus (IV), Virus Like Particle (VLP), Live Attenuated Virus (LAV), Viral Vector (replicating) (VVr) and Viral Vector (non-replicating) (VVnr) in progress or finalized as indicated by the WHO reporting of April 1, 2020.

 Sortilin has an important role in various malignances and can be used as a promising target to eradicate cancer cells.

 In this study, the expression of sortilin in 4T1 and MDA-MB231 cell lines was evaluated by flow cytometry and immunocytochemistry. Apoptosis assay was also applied to evaluate apoptosis induction in 4T1 and MDA-MB231 cell lines.

 Based on cell surface flow cytometry results, anti-sortilin (2D8-E3) mAb could recognize sortilin molecules in 79.2% and 90.3% of 4T1 and MDA-MB231 cell-lines, respectively. The immunocytochemistry staining results confirmed sortilin surface expression. Apoptosis assay indicated that anti-sortilin mAb could induce apoptosis in 4T1 and MDA-MB231 cell lines.

 Our study revealed the important role of surface sortilin in breast carcinoma cell survival and its possible application as a therapeutic agent in cancer targeted therapies.

 To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase (MurNAc-LAA) from a Bacillus subtilis-hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in Escherichia coli (E. coli). 

 The sequences of MurNAc-LAA genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of MurNAc-LAA was optimized by GenScript software to generate MurNAc-LAA-MMI (LysM2) for optimal expression in E. coli. Furthermore, the structure and function of LysM2 was evaluated in silico. The optimized gene was synthesized, subcloned in the pET28a, and expressed in E. coli BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied.

 LysM2, on 816 bp gene encoding a 33 kDa protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 hr in E. coli. In silico analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against Proteus (sp) by turbidity, and an antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium, and Proteus vulgaris in agar diffusion assays.

 Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria.

 Bacteriophages are viruses that infect bacteria. Bacteriophages are widely distributed in various environments. The prevalence of bacteriophages in water sources, especially wastewaters, is naturally high. These viruses affect evolution of most bacterial species. Bacteriophages are able to integrate their genomes into the chromosomes of their hosts as prophages and hence transfer resistance genes to the bacterial genomes. Enterococci are commensal bacteria that show high resistance to common antibiotics. For example, prevalence of vancomycin-resistant enterococci has increased within the last decades.

 Enterococcal isolates were isolated from clinical samples and morphological, phenotypical, biochemical, and molecular methods were used to identify and confirm their identity. Bacteriophages extracted from water sources were then applied to isolated Enterococcus faecium (E. faecium). In the next step, the bacterial genome was completely sequenced and the existing prophage genome in the bacterial genome was analyzed.

 In this study, E. faecium EntfacYE was isolated from a clinical sample. The EntfacYE genome was analyzed and 88 prophage genes were identified. The prophage content included four housekeeping genes, 29 genes in the group of genes related to replication and regulation, 25 genes in the group of genes related to structure and packaging, and four genes belonging to the group of genes associated with lysis. Moreover, 26 genes were identified with unknown functions.

 In conclusion, genome analysis of prophages can lead to a better understanding of their roles in the rapid evolution of bacteria.

 Placenta-specific 1 (PLAC1) is one of the recently-discovered Cancer-Testis-Placenta (CTP) antigen with restricted normal tissue and ectopic expression in a wide range of cancer cells from different histological origins. The production of recombinant human PLAC1 has already been optimized; however, no study has been reported so far on the production and purification of mouse plac1. In this study, mouse plac1 expression and purification was optimized in a prokaryotic system and the effects of the generated proteins on inducing humoral responses in mice were investigated.

 A fusion protein containing full extracellular domain of mouse plac1, immunostimulatory peptides, tetanus toxin P2P30 and PADRE and KDEL3 signal (main plac1), and the same fragment without immunostimulatory peptides (control plac1) was produced. To optimize production and purification steps, different parameters including bacterial strain, cultivation temperature, cultivation time, IPTG concentration, culture medium, and also different buffers for purification of the recombinant proteins were tested. After confirming the identity of recombinant plac1 proteins with Western Blotting (WB) and ELISA assays, these proteins were subcutaneously injected in mice with Freund's adjuvant and the anti-plac1 antibody response was detected by ELISA.

 The optimal expression level of main and control plac1 was obtained in BL21 (DE3) and TB culture medium in the presence of 0.25 mM IPTG after 24 hr of induction at 15°C. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal antibody with both main and control plac1 recombinant proteins in WB and ELISA analysis. Both proteins induced humoral responses in mice; however, anti-plac1  antibody titer was significantly higher in sera of mice immunized with main compared to control plac1.

 In this study, an optimized protocol for production and purification of mouse plac1 was reported and it was shown that insertion of immunostimulatory peptides in gene construct could efficiently enhance humoral immune responses against mouse plac1, which could potentially augment cellular immune responses against plac1 leading to more effective anti-cancer responses.

 Lipopeptides are potential microbial metabolites that are abandoned with broad spectrum biopharmaceutical properties ranging from antimicrobial, antiviral and anticancer, etc. Clinical studies are not much explored beyond the experimental methods to understand drug mechanisms on target proteins at the molecular level for large molecules. Due to the less available studies on potential target proteins of lipopeptide based drugs, their potential inhibitory role for more obvious treatment on disease have not been explored in the direction of lead optimization. However, Computational approaches need to be utilized to explore drug discovery aspects on lipopeptide based drugs, which are time saving and cost-effective techniques.

 Here a ligand-based drug discovery approach is coupled with reverse pharmacophore-mapping for the prediction of potential targets for antiviral (SARS-nCoV-2) and anticancer lipopeptides. Web-based servers PharmMapper and SwissTargetPrediction are used for the identification of target proteins for lipopeptides surfactin and iturin produced by Bacillus subtilis.

 The studies have given the insight to treat the diseases with next-generation large molecule therapeutics. Results also indicate the affinity for Angiotensin-Converting Enzymes (ACE) and proteases as the potential viral targets for these categories of peptide therapeutics. A target protein for the Human Papilloma Virus (HPV) has also been mapped.

 The work will further help in exploring computer-aided drug designing of novel compounds with greater efficiency where the structure of the target proteins and lead compounds are known.

 The present study was aimed at phyto-synthesized silver nanoparticles (AgNPs) using Amygdalus spinosissima (A. spinosissima) extract and to investigate the antibacterial, antioxidant effects, anticancer and apoptotic effects of phyto-synthe-sized AgNPs.

 The bio-fabricated AgNPs were characterized using UV-visible spectroscopy (UV-visible), X-ray Diffraction (XRD), Fourier Transform Infrared (FTIR), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX).

 The phyto-synthesized AgNPs showed maximum absorption in 438 nm, in the UV-visible spectrum. XRD peaks were observed at 2θ values in 38.20°, 44.40°, 64.60°, and 77.50° which are indexed as (111), (200), (220), and (311) bands of Face-Centered Cubic (FCC) structures of silver. FTIR analysis indicated that the AgNPs were capped with A. spinosissima extract. SEM and TEM micrographs revealed that the fabricated AgNPs were spherical and the average size range was 17.89 nm. Also, the EDX results show that the content of Ag was 90%.

 The phyto-synthesized AgNPs had significant antibacterial activity against Gram-negative bacteria, as well as, the AgNPs exhibited great inhibitory effects on DPPH radicals and their antioxidant properties were favorably comparable to the antioxidant outcomes of ascorbic acid. Moreover, the AgNPs showed anticancer activity against the MCF-7 cell line with the IC50=6.1 µg/ml. Moreover, the phyto-synthesized AgNPs could induce apoptosis in the MCF-7 cell line significantly. The GC-MS analysis of the A. spinosissima extract showed that 102 bioactive phytochemical compounds, which be of use to the synthesis of AgNPs.

 Prostate Cancer (PCa) is the major reason for the high mortality rates among men worldwide. In fact, current therapeutic approaches are not successful. It appears that discovering more effective methods considering several parameters such as availability, low cost, and no toxicity to normal cells is one of the biggest challenges for interested researchers. Green tea (extracted from the plant Camellia sinensis) with high level of polyphenolic compounds and as the most globally consumed beverage has attracted considerable interest. MicroRNAs (or miRNAs) were considered as novel tools in cancer therapy which modulate various biological events in cell by regulation of gene expression. The aim of the current study was to evaluate the antitumor activity of green tea in LNCaP cells through up-regulation of miR-181a expression.

 First, LNCaP cells were cultured and by using quantitative real time PCR (qRT-PCR) and western blot methods, the expression levels of Bax and BCL2 were analyzed. Next, a 3D cell culture model was applied to evaluate the expression of miRNA-181a in LNCaP cells.

 It was shown that green tea induced cellular apoptosis. The high number of apoptotic nuclei was also shown by using DAPI staining. The inhibition of tumor growth was revealed by analyzing the size and number of spheroids. Also, up-regulation of miR-181a expression in LNCaP cells was revealed after treatment with green tea.

 Our results are helpful to design antitumor regimens based on consumption of green tea through up-regulation of miRNA-181a expression and induction of apoptosis.

 Treatment of wounds in diabetes often gets less than perfect healing. One of the reasons for the difficulty in treating wounds in diabetes is the growth of aerobic and anaerobic bacteria. This study aims to determine the pulse voltage and treatment time that can optimally inactivate bacteria, and their effect on wound healing in mice suffering from diabetes.

 The study used electrical stimulation with a direct voltage of 10 volts given a pulse voltage of 50-80 volts, a width of 50 µs, and the number of pulses of 65 per second. The research samples were Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) bacteria that grew on beef and mice (Mus musculus) with diabetes. The treatment for S. aureus and P. aeruginosa bacteria was carried out using a pulse voltage of 50-80 volts for 5-15 min/day and repeated for 3 days. Meanwhile, treatment of mice wounds was carried out with a pulse voltage of 80 volts for 15 min/day and repeated for 7 days.

 The results showed that treatment with a pulse voltage of 50-80 volts and a treatment time of 5-15 min significantly reduced the number of S. aureus and P. aeruginosa bacteria in beef (p£0.05). Treatment with a pulse voltage of 80 volts for 15 min made beef free from bacteria. Meanwhile, treatment with a pulse voltage of 80 volts for 15 min per day for seven days resulted in the wound state of three mice in the maturation phase and two mice in the proliferation phase on day 8 with an average wound area of 0.108 cm 2.

 The treatment with a pulse voltage of 80 volts for 15 min made the beef sterile, the mice wounds healed quickly, and the mice not stressed. The higher the blood glucose level, the slower the wound healing process.