Avicenna Journal of Medical Biotechnology
Volume:13 Issue: 3, Jul-Sep 2021

The cause of COVID-19 global pandemic is SARS-CoV-2. Given the outbreak of this disease, it is so important to find a treatment. One strategy to cope with COVID-19 is to use the active ingredients of medicinal plants. In this study, the effect of active substances was surveyed in inhibiting four important druggable targets, including S protein, 3CLpro, RdRp, and N protein. RdRp controls the replication of SARSCoV-2 and is crucial for its life cycle. 3CLpro is the main protease of the virus and could be another therapeutic target. Moreover, N protein and S protein are responsible for SARS-CoV-2 assembly and attaching, respectively.

The 3D structures of herbal active ingredients were prepared and docked with the mentioned SARS-CoV-2 proteins to obtain their affinity. Then, available antiviral drugs introduced in other investigations were docked using similar tools and compared with the results of this study. Finally, other properties of natural compounds were uncovered for drug designing.

The outcomes of the study revealed that Linarin, Amentoflavone, (-)- Catechin Gallate and Hypericin from Chrysanthemum morifolium, Hypericum perforatum, Humulus lupulus, and Hibiscus sabdariffa had the highest affinity for these basic proteins and in some cases, their affinity was much higher than antiviral medicines.

In addition to having high affinity, these herb active ingredients have antioxidant, vasoprotective, anticarcinogenic, and antiviral properties. Therefore, they can be used as extremely safe therapeutic compounds in drug design studies to control COVID-19.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a key role in promoting chemoresistance in various cancers. PD-L1 is one of the downstream targets of the Nrf2 signaling pathway. This molecule has some beneficial impacts on tumors growth by inhibition of the immune system. This study aimed to investigate the potential role of the Nrf2-PD-L1 axis in the promotion of oxaliplatin resistance in colon cancer cells.

We examined Nrf2, PD- L1, and CD80 expression in the tumor and margin tissue samples from CRC patients. After that role of the Nrf2-PD-L1 axis in promotion of Oxaliplatin resistance was investigated.

Our data revealed that Nrf2 and PD-L1 mRNA expressions were markedly higher in tumor tissues compared to margin tissues. The PD-L1 mRNA expression level was also increased in the resistant cells. However, Nrf2 expression was decreased in SW480/Res cells and increased in LS174T/Res cells. The inhibition of Nrf2 through siRNA treatment in SW480/Res and LS174T/Res cells has decreased the IC50 values of oxaliplatin. Inhibition of the Nrf2 has significantly increased the oxaliplatin-induced apoptosis, and reduced the migration in SW480/Res cells.

It is suggested that effective inhibition of Nrf2-PD-L1 signaling pathways can be considered as a novel approach to improve oxaliplatin efficacy in colon cancer patients.

Drastic pH drop is a common consequence of scaling up a mammalian cell culture process, where it may affect the final performance of cell culture. Although CO2 sparging and base addition are used as common approaches for pH control, these strategies are not necessarily successful in large scale bioreactors due to their effect on osmolality and cell viability. Accordingly, a series of experiments were conducted using an IgG1 producing Chinese Hamster Ovary (CHO-S) cell culture in 30 L bioreactor to assess the efficiency of an alternative strategy in controlling culture pH.

Factors inducing partial pressure of CO2 and lactate accumulation (as the main factors altering culture pH) were assessed by Plackett-Burman design to identify the significant ones. As culture pH directly influences process productivity, protein titer was measured as the response variable. Subsequently, Central Composite Design (CCD) was employed to obtain a model for product titer prediction as a function of individual and interaction effects of significant variables.

The results indicated that the major factor affecting pH is non-efficient CO2 removal. CO2 accumulation was found to be affected by an interaction between agitation speed and overlay air flow rate. Accordingly, after increasing the agitation speed and headspace aeration, the culture pH was successfully maintained in the range of 6.95-7.1, resulting in 51% increase in final product titer. Similar results were obtained during 250 L scale bioreactor culture, indicating the scalability of the approach.

The obtained results showed that pH fluctuations could be effectively controlled by optimizing CO2 stripping

Acquired immunodeficiency syndrome (HIV/AIDS) is still a major global concern and no effective therapeutic vaccine has been produced to prevent the problem. Among HIV-1 proteins, vif as a basic cytoplasmic protein of HIV-1 is involved in late stages of viral generation and plays important role in HIV-1 virion replication. It also increases the stability of virion cores, which probably inhibits early degradation of viral entry. Therefore, it seems rational to apply this protein as a vaccine based on its impact on HIV-1 life cycle. This study aimed at cloning, expression and production of vif protein as an HIV-1 vaccine candidate.

In this study, vif sequence was amplified from pLN4-3 plasmid including HIV-1 vif gene and then cloned in pET23a to generate the recombinant plasmids of pET23a/vif with hexahistidine tags. BL21 competent cells were transformed to obtain the protein of interest. Ni-NTA column was used to purify the protein of interest and western blotting confirmed vif protein using anti-His tag antibody. In order to express the gene of interest in eukaryotic cells, vif was sub-cloned into pEGFP plasmids and HEK 293-T cells were transfected. Flow cytometry was then applied to evaluate GFP expression.

vif protein was expressed in BL21)DE3) strain and identified as a23 kDa band in SDS-PAGE and confirmed by anti-His antibody in western blotting. The purified protein concentration was 173.3 μg/ml using Bradford assay. HEK-293T cells were successfully transfected by recombinant pEGFP plasmids and flow cytometry confirmed the cell transfection.

vif protein can be expressed in mammalian cells and may be a proper protein subunit vaccine candidate against HIV-1.

Mouse neutralization test is widely used to determine the level of antirabies antibodies, but it is labor-intensive and time consuming. Alternative methods for determining the neutralizing activity of anti-rabies sera and immunoglobulin in cell cultures are also known. Methods such as FAVN and RFFIT involve the use of fluorescent diagnostics. Determination of Cytopathic Effect (CPE) is often complicated due to features of rabies virus replication in cells. Atomic Force Microscopy (AFM) is able to detect the interaction of the virus with the cell at an early stage. Therefore, in this study, a method has been developed for determining the specific activity of antirabies sera and immunoglobulin using AFM of cell cultures.

The method is based on the preliminary interaction of rabies virus with samples of rabies sera or immunoglobulin drug, adding the specified reaction mixture to cell culture (Vero or BHK-21), and then measuring the surface roughness of the cells using AFM. AFM was carried out in the intermittent contact mode by the mismatch method in the semi-contact mode. The results were compared with the values obtained in the mouse neutralization test. The consistency of the results obtained by both methods was evaluated by Bland-Altman method.

The increment in the surface roughness of the cells is a consequence of the damaging effect of the virus, which is weakened as a result of its neutralization by rabies antibodies. A dilution allowing 50% suppression of the increase in the surface roughness of cells was selected as the titer of rabies sera or immunoglobulin. In this case, the recommended range for determining the antibody titer is from 1:100 to 1:3000.

For the first time, a new methodological approach in virology and pharmaceutical research is presented in this study. The use of the proposed methodological technique will reduce the time from 21 to 2 days to obtain results in comparison with the mouse neutralization test; also, fewer laboratory animals are required in this approach which is in agreement with 3 R Principle.

Around 70% of all pregnancies (Including 15% of clinically-recognized ones) are lost due to various fetal or maternal disorders. Chromosomal aneuploidies are among the most common causes of pregnancy loss. Standard chromosome analysis using G-banding technique (Karyotype) is the technique of choice in studying such abnormalities; however, this technique is time-consuming and sensitive, and limited by vulnerabilities such as cell culture failure. The use of molecular cytogenetic techniques, including array-based techniques and Multiplex Ligation-Dependent Probe Amplification (MLPA), has been proposed to overcome the limitations of this method to study the products of conception. This study has been designed to investigate the feasibility of using MLPA technique as a standalone genetic testing, with histopathologic examinations and genetic counseling to detect aneuploidies in products of conception and neonatal deaths.

Forty-two verified fetal and neonatal samples were studies and genetic counseling was scheduled for all parents. Histopathologic examinations were carried out on the products of conception, and appropriate fetal tissues were separated for genetic studies. Following DNA extraction and purification, MLPA was carried out to investigate chromosomal aneuploidies.

Nine samples (21.42%) were diagnosed to be affected with aneuploidy. Detected aneuploidies were trisomy 22 (n=3), trisomy 21(n=1), trisomy 18 (n=2), trisomy 16 (n=1), trisomy 13 (n=1), and monosomy of chromosome X (n=1). The MLPA analysis results were conclusive for all of the fetal samples (Success rate: 100%).

These results suggest that MLPA, as a standalone genetic testing, is an accurate, rapid, and reliable method in overcoming the limitations of standard cytogenetic techniques in genetic investigation of products of conception.

Overexpression of miR-21 is a characteristic feature of patients with Multiple Sclerosis (MS) and is involved in gene regulation and the expression enhancement of pro-inflammatory factors including IFNγ and TNF-α following stimulation of T-cells via the T Cell Receptor (TCR). In this study, a novel integrated bioinformatics analysis was used to obtain a better understanding of the involvement of miR-21 in the development of MS, its protein biomarker signatures, RNA levels, and drug interactions through existing microarray and RNA-seq datasets of MS.

In order to obtain data on the Differentially Expressed Genes (DEGs) in patients with MS and normal controls, the GEO2R web tool was used to analyze the Gene Expression Omnibus (GEO) datasets, and then Protein-Protein Interaction (PPI) networks of co-expressed DEGs were designed using STRING. A molecular network of miRNA-genes and drugs based on differentially expressed genes was created for Tcells of MS patients to identify the targets of miR-21, that may act as important regulators and potential biomarkers for early diagnosis, prognosis and, potential therapeutic targets for MS.

It found that seven genes (NRIP1, ARNT, KDM7A, S100A10, AK2, TGFβR2, and IL-6R) are regulated by drugs used in MS and miR-21. Finally, three overlapping genes (S100A10, NRIP1, KDM7A) were identified between miRNA-gene-drug network and nineteen genes as hub genes which can reflect the pathophysiology of MS.

Our findings suggest that miR-21 and MS-related drugs can act synergistically to regulate several genes in the existing datasets, and miR-21 inhibitors have the potential to be used in MS treatment.

Rheumatoid Arthritis (RA) is a progressive, heterogeneous, and common multifactorial autoimmune disease. Several Genome-Wide Association Studies (GWASs) have revealed more than 100 risk loci for RA. One of these loci is a functional single nucleotide polymorphism (rs874040; G>C) near the recombination signalbinding protein for the immunoglobulin kappa J region (RBPJ) gene. RBPJ can convert into a transcriptional activator upon activation of the canonical Notch pathway. Notch signaling has recently emerged as an important regulator of immune responses in inflammation and autoimmune diseases. In the present study, the possible association between SNP rs874040 (G>C) upstream of the RBPJ gene with RA risk was assessed in Iranian population.

A case-control study including 60 RA patients and 44 control subjects was conducted to estimate rs874040 genotypes using real‑time polymerase chain reaction High Resolution Melting (HRM) method.

Logistic regression analysis indicated that homozygous CC and heterozygous GC genotypes increase the risk of RA compared with GG genotype (CC vs. GG; OR=11.36; 95% CI [3.93-33.33] and CG vs. GG; OR=3.78; 95% CI [1.30-10. 98]). Besides, subjects with C allele were more frequently affected with RA than subjects with G allele (OR=10.42; 95% CI [5.21-20.83]). Furthermore, in the patient group, a significant correlation was found between C-reactive protein concentrations and rs874040 polymorphism (p<0.05).

Our findings propose a substantial correlation between rs874040 polymorphism and RA risk in Iranian population.