Journal of Applied Biotechnology Reports
Volume:9 Issue: 1, Winter 2022

The clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) used for genome editing. The usage of CRISPR-Cas9 in gene editing is faced with certain limitations including off-target mutation, decreased homologous recombination (HR) repair, and immune system responses. It seems that if Cas9 expressed in an inducible manner, off-target mutations may decrease. The P53 protein decreases the activity of the HR pathway in the cell cycle, so, the decrease in P53 expression level may increase the activity of this pathway. Based on this topic, for the first time, we designed ''px601-Turbo GFP-TRE-shRNA P53'' as a CRISPR-based vector. The use of this vector can simultaneously induce expression of Cas9 and shutdown transiently P53 under an inducible promoter and an inducing agent. Therefore, shutdown transiently P53 may be leading to reduced off-targets and increased accuracy of genome editing. In the human gastric cancer MKN45 cell line, the P53 gene expresses at a normal level. Moreover, CD44 in this cell line has overexpression and is a gastric cancer stem cell marker. To evaluate this hypothesis, CD44 will be targeted for a specific sequence change (editing) by the px601-Turbo GFP-TRE-shRNA P53 vector. Accordingly, after cloning and virus preparation, MKN45 cell lines will be transduced in the presence of the appropriate doxycycline (DOX) dosage. Ultimately, to evaluate the vector efficiency, DNA extraction and whole-genome sequencing (WGS) will be done and compared with the transduced MKN45 cells without an inducible prompter and DOX as the control group. Furthermore, the Sanger sequencing for the target gene must be done. This temporary inducible expression of P53 may appear to increase the efficiency of the CD44 gene editing and reduce off-targets.

There are limited parasites that can infect both humans and animals; one of these parasites is Toxoplasma. Its primary host is cats but can infect warm-blooded animals like humans, goats, sheep, cattle, dogs, etc. Although the main transmission route in humans is via food, including raw or undercooked meat, the infection route in animals is via mother to child and ingesting sporulated oocysts. Due to the dangerous results of this protozoan, including abortion, stillbirth, different degrees of mental or physical retardation, prevention of such infection has to be seriously considered. Cattle have a natural resistance against Toxoplasma infection. Therefore, its prevalence has more importance in goats and sheep. According to the studies that have measured toxoplasma gondii infection and its prevalence in the world- and countrywide scale, the infection of this protozoan is highly related to the geographic status, susceptible animals, potential hosts, and eating habits. In the present study, we review the prevalence and epidemiology spectrum of T. gondii in sheep and goat meat in Iran. This knowledge could be useful to biologists, public health workers, physicians, and veterinarians.

Double Strand Breaks (DSBs) are the most deleterious DNA lesions among the first signs of cancer in eukaryotic cells. Occurring on exposure to one or more endogenous and/or exogenous factors and leading to fatal consequences like chromosome aberrations and genomic instability. Therefore, programmed and coordinated cellular processes function intra-cellular to stabilize the genomic information and repair damages. These processes are primarily a part of DNA replication and cell-cycle progression. However, various signaling factors also activate specialized DNA Damage Response (DDR) pathways for repairing DSBs. Lately, the phosphorylated histone variant H2AX (γH2AX) has been identified as a biomarker for DNA damage. Studies have shown a correlation between the concentration of cellular γH2AX and the extent of DNA damage. Hence, the quantification of DNA lesions can be done using simple spectroscopic and radiological techniques or immunofluorescent staining. For this reason, γH2AX has especially gained value as a biomarker in translational cancer research. Moreover, this approach may act as a boon in clinical trial studies for understanding the different phases of cancer and studying the pharmacodynamics of prospective drugs. Recently, γH2AX based studies have indicated the indispensable fate of DNA damages occurring during normal neurological development and in disorders like obesity. The current review focuses on the role of γH2AX in DDR pathways, and ways in which the correlation of γH2AX and DNA damage can be applied in monitoring the clinical response of DNA targeted therapies.

The nature and the concentration of the chemical agents responsible for cell flocculation are the bottlenecks for microalgae recovery. The aim of the present study was to evaluate different chemical-based flocculants for Chlorella sorokiniana flocculation. The biomass recovery efficiency was evaluated by comparing self-flocculation and flocculation with the ferric chloride, sodium hydroxide, aluminum sulfate, and zinc sulfate. After identifying the best flocculating agent, its concentration was varied to determine the optimal condition by rapid agitation followed by sedimentation (0.25 to 1 g/L). Zinc sulfate was unsuitable for this strain due to an efficiency lower than 40%. Self-flocculation and sodium hydroxide were fairly efficient (48.65% and 58.06%, respectively). Aluminum sulfate produced moderate results (56.27%), but flocculation took a long time to become efficient. Ferric chloride showed the best potential for flocculation, and in the analysis of different concentrations (0.25 to 1 g/L) showed to be fast and efficient (nearly 80% of biomass recovery in 10 min) at a concentration of 0.75 g/L. All the flocculants tested in this study can be utilized for biomass recovery, except for the zinc sulfate. The procedure was efficient, inexpensive, and contaminant-free for the recovery of C. sorokiniana biomass.

Infectious Laryngotracheitis Virus (ILTV) poses a highly contagious upper respiratory tract illness and is regarded as a major concern for the poultry industry worldwide. Considering the emergence of novel virulent variants of ILTVs, the development of novel vaccines is needed to control the disease. This study was carried out based on immunoinformatics and vaccinology strategies to introduce an effective ILTV vaccine candidate targeting glycoprotein B (gB).

Both T-cell and B-cell epitopes of the gB protein with the potential to induce immune responses were identified. The highly antigenic and immunogenic PH-1 domain was selected as the potential vaccine candidate. The physiochemical properties and tertiary structure model of the domain were predicted, refined, and validated. The resulting high‐quality model was applied for docking analyses with toll‐like receptor 2. The affinity of the vaccine candidate to bind with the immune receptor and generate the appropriate immune responses was calculated based on the free energy level.

The results indicated the PH-1 domain of ILTV gB protein to be immunogenic and was effective in stimulating the T helper, T cytotoxic, and B cells. The domain showed high-affinity binding and stability with the TLR2 immune receptor.

Collectively, this research provides an immunogenic candidate for designing a protein-based vaccine against ILTV. The efficacy of the construct should be examined in lab settings and in animal models.

The prevalence of petroleum oil spills in oceans and seas is on the rise in India, resulting in widespread detrimental effects on the environment. Bioremediation by bacteria is an eco-friendly and safe technique for the removal of these pollutants from seawater. An indigenous bacteria, isolated from garage soil was grown on Bushnell Agar plate with Petroleum Crude Oil (PCO) as a carbon source. It was identified by biochemical characterization and 16S rRNA sequencing. The effect of factors such as concentrations of PCO, inoculum and glucose, agitation speed, pH, and degradation time on the growth of bacteria and PCO degradation in seawater was studied by one factor at a time approach. Screening and optimization were performed by Factorial Design and Central Composite Design respectively. According to findings, isolated bacteria degraded PCO within 48 h and could decolorize 6-dichlorophenol indophenol within 36 h. It was identified as a novel Lysinibacillus sp. SS1, which grew in the pH range of 4.0 to 10.0 and tolerated salinity of 6.0% w/v. Significant factors (concentrations of glucose, inoculum, and pH) were optimized and optimum levels were 11.7% v/v inoculum, 11.36 g/L glucose, and pH 8.6. Maximum degradation of 84 ± 0.13% was achieved when grown in seawater supplemented with 4.0% v/v PCO, at 27 ± 2 °C at 80 RPM in 28 days at optimized conditions. The present study is the first study reporting optimization of degradation of PCO by Lysinibacillus species. Lysinibacillus sp. SS1 could effectively degrade petroleum hydrocarbons in extreme conditions of seawater and can be applied for the treatment of oil spills.

Cannabinoids can be found as the specific secondary metabolites of hemp (Cannabis sativa L.), including Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabichromene (CBC). There are many enzymes, particularly cannabichromene synthase, cannabidiolate synthase, and Δ9-tetrahydrocannabinolate synthase, contributing to the biosynthesis of the cannabinoids. Environmental stress, particularly drought, can induce secondary metabolites. In the present study, we have tried to investigate and understand the key factors such as drouth-induced Transcriptional Factors (TFs) involving in the pathway by employing in vitro and in silico tools. After providing the genes' names and IDs from the National Center for Biotechnology Information (NCBI), Transcription Start Sites (TSS) and TATA-box were predicted by the TSS Plant website, as well as involved transcriptional factors. The expression of the genes was assayed under drought conditions by in silico and in vitro tools, R software, and Real-time PCR, respectively. The findings identified all the genes contributing to biosynthesis cannabinoids in drought conditions. There were actually six TF sites and four TF sites for the gene of olivetolic acid cyclase and AAE1, respectively. Drought stress can induce overexpression of the genes encoding B3 domain-containing proteins, MLP28, MYB binding site, transcriptional repressor OFP7, and WAK1 as TFs respond to biotic and abiotic stresses in Cannabis sativa plants.

DNA cloning plays a crucial role in biotechnology laboratories and biotechnology-related fields, which facilitates the primary yet crucial step for further studies in molecular biology. Many laboratories worldwide still use restriction enzyme-based cloning methods to construct expression vectors owing to financial constraints, time-consuming, and the unavailability of appropriate vectors. In the present study, we introduced a novel method inspired by the restriction enzyme-based cloning method with some modifications named All-In-One (AIO) cloning.

The PCR product and vector for cloning were digested in one 0.2 ml tube with a total volume of 20 µl. Completely digested products were checked and inactivated by heat treatment. Digested genes and vectors were directly used for the ligation step in this 0.2 ml tube, without any purification step required. Finally, ligation products were transformed into competent E. coli DH5α by the heat shock method.

More than eight different clones were generated by using AIO cloning, which all the necessary reactions were performed in one single 0.2 ml tube. This method was efficient in cloning a wide range of DNA fragments, from 200 to 1300 bps.

Collectively, AIO provided an alternative yet sufficient cloning protocol, reducing the loss of DNA components, and saving materials, labor and time, especially where research materials were not abundantly available.

Tannase (tannin acyl hydrolase EC3.1.1.20) is an industrially important enzyme with extensive applications. The current study aimed to optimize the tannase production employing corn leaves as substrate and characterize tannase activity. Tannase producing bacterial strains were isolated from Catla catla fish gut. The highest enzyme-producing bacterial strain was identified as Bacillus subtilis using 16S rDNA sequencing. Fermentation parameters and additional medium components were optimized with the application of one variable at a time and enhanced tannase was obtained with 50% substrate moisture, acetate buffer (pH 4.0) as enzyme extraction medium with 2 ml volume, 45 °C incubation temperature, pH 5, 2% inoculum size, 24 h incubation time, 150 rpm agitation, large-sized substrate particles (4.0 mm), enzyme extraction without centrifugation and medium components (MgSO4, 4% tannic acid and yeast extract). The central composite design was employed to optimize the concentrations of optimal medium components, which were found as 4.0% tannic acid, 0.5% MgSO4 and 1.5% yeast extract for the highest tannase production (211.97±0.08 U/ml). Tannase characterization revealed the maximum tannase activity at pH 8, 30 °C (with 30 min incubation period and 0.35% substrate concentration). The results of the present study revealed the potential of utilization of agricultural resources (corn leaves) as a low-cost substrate to reduce the production cost of tannase.

Bacterial resistance against antibiotics has caused many problems in treating humans and animal infections worldwide. Nowadays, researchers are continuously seeking to develop novel antibacterial to tackle the issue of microbial resistance. Antimicrobial peptides have been introduced as new effective strategies that kill bacteria quickly and cause less antibiotic resistance. In this study, we evaluated the antibacterial and cytotoxic effects of a synthesized peptide (NRWCFAGRR-NH2) on some Gram-positive and –negative bacteria and eukaryotic cells.

Twelve bacterial strains were selected to study the antimicrobial effect of the NRWC peptide. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays were used to study the bacteriostatic and bactericidal activity of these peptides, respectively. The cytotoxic effect of the peptide was evaluated on Hela cell line and human RBC using the MTT assay and hemoglobin release measurement, respectively. The J774 macrophage cell line was used to measure nitric oxide production in response to the peptide.

The results showed that NRWCFAGRR peptide has a bactericidal and inhibitory effect on all 12 bacterial strains' growth in a dosedependent manner. It has also been proven that the toxic effect of the peptide on human cells is evitable at the MIC and MBC concentration. The highest amount of nitric oxide production was induced after 48 hours of treatment.

Considering the research conducted in the field of antimicrobial peptides, our designed peptide has antimicrobial properties that kill some of the pathogenic microorganisms directly and can theoretically kill some organisms indirectly via induction of nitric oxide by macrophages.

The repair of the cartilage continues to be a big challenge. Autologous cartilage is the gold standard, but enough sources are not available. The development of stem cells, biomaterials, and bioactive factors has led to cartilage tissue engineering becoming a promising strategy for the regeneration of cartilage defects. In this study, the effect of Transforming Growth Factor Beta 3 (TGF-β3) and Pomegranate Seed Extract (PSE), as chondrogenesis inducers, were evaluated. Human Adipose-Derived Stem Cells (ADSCs) were seeded in alginate scaffolds and cultivated for two weeks in chondrogenic media. Finally, the MTT assay was used to test the effect of TGF-β3 and PSE on the survival of differentiated cells. The mRNA levels of the cartilage-specific markers such as SRY-box9 (SOX9), Collagen type II (COLII), Collagen type X (COLX), and Aggrecan (ACAN) were determined by RT-PCR. Also, CD markers were evaluated by flow cytometry. Using both natural PSE and chemical TGFβ3 inducers simultaneously, had the best results in chondrogenesis by increasing the expression of the SOX9, COLII, and ACAN genes. Furthermore, the flow cytometry analysis indicated that the expression of CD14 marker of differentiated cells significantly increased, although the expression of CD44 marker decreased two weeks post differentiation. PSE is a suitable inducer and its combined use with TGF-β3 can improve the efficiency of the chondrogenic potential of ADSCs. Our results suggest that PSE may have the potential to be used in tissue engineering as a replacement for TGF-β3.

Breast cancer is the most common cancer among women. Information published by The Iranian Cancer Research Center in 2019 shows that one of every 10 to 15 women is afflicted with this cancer. As one of the active ingredients of turmeric, curcumin has a wide range of biological properties, such as antioxidant and anti-cancer activity. This study aimed to evaluate the anti-cancer effects of curcumin on breast cancer cells in 3-Dimensional (3D) culture conditions. To achieve a 3D environment, we used encapsulation of cells in alginate hydrogel. The anti-cancer effects of curcumin at concentrations of 20, 40, and 80 μM on MCF-7 breast cancer cells in 3D culture were evaluated by MTT, neutral red, comet assay, cytochrome c, Nitric Oxide (NO), catalase, and glutathione assays. The culture medium was used as the negative control and the cell-containing medium was used as the positive control. Data were analyzed by two-way ANOVA using GraphPad InStat software, and the significance was considered at the level of P<0.05. Curcumin reduces the production of cellular NO and increases the production of catalase and glutathione, which confirms the results of the NO test. In addition, the release of cytochrome c from Mitochondria from cells treated with different concentrations of curcumin compared to control cells are significant. The evaluation of the toxicity effect of curcumin at concentrations of 20, 40, and 80 μM using comet assay showed that this substance induces apoptosis in MCF-7 cells in a dose-dependent manner. The findings of this study showed that the anti-cancer effect of curcumin on MCF-7 cells under 3D culture conditions could increase the effectiveness of treatment. The Cell survival rate actually depended on curcumin concentration.

Strongyloidiasis is a neglected disease caused by Strongyloides stercoralis. Fast and robust detection of this parasite can avoid its auto-infection cycle and therefore from the high parasitic load. The culture of stool and stool-based microscopy techniques are the most used methods for parasite diagnosis; however, these methods are not sensitive enough. Immunological methods are more sensitive diagnostic tools and can be employed for the detection of this parasite. Since IgG4 immunoreactive protein (NIE) is a major pathogenicity factor of the parasite, in the present study, an IgM-ELISA method was developed to investigate the efficiency of anti-NIE IgM antibodies in the detection of Strongyloides stercoralis. For this aim, 50 serum samples were gathered from positive patients along with 50 serum patients from healthy people. IgM antibodies were detected by using the ELISA technique and the data were analyzed by statistical analyses. Statistical analyses showed that IgM-ELISA was able to diagnose the disease with the sensitivity and specificity of 65.0%. The developed IgM-ELISA method was relatively but not sufficiently successful at a robust diagnosis of strongyloidiasis. However, the sensitivity and specificity of this method were not good enough to be considered a reliable test for the diagnosis of the disease compared to the IgG- ELISA.

Hyperthermia is rapidly becoming a clinical reality as a tool for the treatment of malignant disease but there are some major problems in the way of using superparamagnetic nanoparticles coated with polymer in medical applications. Modifying the magnetic nanoparticles without using surface coating is more appropriate. In this study, we presented a new physical technique, by surface treatment of nanoparticles with argon gas plasma, to modify the surface of nanoparticles for improving their crystal structure and magnetization.

In this study, Fe3O4 nanoparticles were synthesized using the co-precipitation method. The nanoparticles were then treated with plasma in a vacuum chamber. In this method, the Radio Frequency (RF) generator 13.56 MHz was used as a power supply and the plasma treatment was applied for 10 and 15 min.

Due to the decrease in surface irregularities, the nanoparticle aggregation decreased and their colloidal stability increased. Moreover, with Value Stream Mapping (VSM) analysis the magnetism of the nanoparticles improved along with an increase of plasma power and plasma treatment time due to the reduced crystal defects and crystal growth. By using the AC magnetic field generator with a frequency of 92 kHz and amplitude of 125 Oe, results show that along with an increase of plasma power and plasma treatment time due to the increased magnetization and colloidal stability, heat generation by these nanoparticles increased in a ferrofluids system in the presence of AC magnetic field. In addition, the locking temperature of nanoparticles has also increased.

Our results suggest that the surface modification of Fe3O4 nanoparticles, using plasma treatment, is an appropriate candidate for some medical applications such as magnetic resonance imaging, drug delivery, and especially for magnetic hyperthermia.