Avicenna Journal of Medical Biotechnology
Volume:13 Issue: 2, Apr-Jun 2021

The coronavirus storm, first reported more than a year ago, has overshadowed all societies' parts and become a challenge to all of the world's health systems 1. Extremely high contagiousness, significant mortality rates, and the lack of a definitive cure have prioritized overcoming this outbreak. In this regard, studies related to coronavirus and especially its clinical studies, became a priority for researchers and decision-makers at the request of governments and the people, as well as by the logic. The superiority of an emergency is not a wrong decision. Still, the problem arose when other research areas were neglected, and their budgets were reduced by decision-makers, resulting in damage to the research and researchers in other fields 2,3. The clinical studies currently being conducted on coronavirus disease 2019 (COVID-19) are due to previous studies in the basic sciences that have provided the background. Obviously, without scientists' tasks over the years and the allocation of research funds to the fields of genetics, biochemistry, immunology, etc., studies and advances would not be possible today 4,5.
Medical biotechnology is a type of applied science that produces or creates products that improve human health, mainly through genetic engineering and tissue culture, using biological systems or living organisms 5,6. However, this area of science can also be harmful through studies' unintended consequences, the production of products without genetic diversity, and deliberate biological manipulation. In medical biotechnology, using basic sciences such as biochemistry, biology, and genetics, and by modifying cells or cell subsets, the prevention of diseases-including the production of vaccines-and the treatment of diseases, especially by creating novel agents, are studied 6-10. It should be noted that the background of these lofty goals has been years of basic science studies, many of which have not reached a positive conclusion or have been rejected by more recent research. Even they more often lead to more questions instead of answers 4.
In other words, the passing of many years and spending on scientific and research projects has enabled human beings today to create advanced products for fighting pathogens and improving society's health. The development of insulin, the production of advanced monoclonal antibodies, and vaccines' production against an RNA virus are some of the notable novel products 6,7.
Since the beginning of the coronavirus outbreak, many ways have been suggested to prevent and treat the disease. Today, after more than a year and the reported deaths of nearly two million people from COVID-19, the highest hopes for overcoming the disorder are with the proposed vaccines 1,11. Science achieves patients' treatment through basic studies 4, so the right decision is a decision that, while meeting the need, does not disrupt the long-established science and research system. We can see that the time and money spent in the past is helping all sections of society today with the production of the coronavirus vaccine, and if those studies had not been done in the past for whatever reason, today we were a few steps behind. Likewise, suppose today, for any reason, even the allocation of all time and budget to the emergency situation, the pace of progress in this area slows down. In that case, it may have detrimental effects on all society in the future.

Out of frame mutations in DMD gene cause Duchenne Muscular Dystrophy (DMD) which is a neuromuscular progressive genetic disorder. In DMD patients, lack of dystrophin causes progressive muscle degeneration, which results in heart and respiratory failure leading to premature death. At present, there is no certain treatment for DMD. DMD gene is the largest gene in human genome by 2.2 mega base pairs and contains 79 exons. In the past few years, gene therapy has been considered a promising DMD treatment, and among various gene-editing technologies, CRISPR/Cas9 system is shown to be more precise and reliable. The aim of this study was to assess the possibility of knocking out exon 48 by using a pair of sgRNAs.

A pair of guide RNAs (gRNAs) was designed to cleave DMD gene and induce deletion of exon 48. gRNAs were transfected to the HEK-293 cell line and then the deletion in genomic DNA was analyzed by PCR and subsequent Sanger sequencing.

Exon 48 was successfully deleted and therefore exon 47 was joined to exon 49.

This result indicated that CRISPR/Cas9 system could be used to edit DMD gene precisely.

Inhibition of angiogenesis using monoclonal antibodies is an effective strategy in cancer therapy. However, they could not penetrate sufficiently into solid tumors. Antibody fragments have solved this issue. However, they suffer from short in vivo half-life. In the current study, a tandem bivalent strategy was used to enhance the pharmacokinetic parameters of an anti-VEGF165 nanobody.

Homology modeling and MD simulation were used to check the stability of protein. The cDNA was cloned into pHEN6C vector and the expression was investigated in WK6 Escherichia coli (E. coli) cells by SDS-PAGE and western blot. After purification, the size distribution of tandem bivalent nanobody was investigated by dynamic light scattering. Moreover, in vitro antiproliferative activity and pharmacokinetic study were studied in HUVECs and Balb/c mice, respectively.

RMSD analysis revealed the tandem bivalent nanobody had good structural stability after 50 ns of simulation. A hinge region of llama IgG2 was used to fuse the domains. The expression was induced by 1 mM IPTG at 25°C for overnight. A 30 kDa band in 12% polyacrylamide gel and nitrocellulose paper has confirmed the expression. The protein was successfully purified using metal affinity chromatography. MTT assay revealed there is no significant difference between the antiproliferative activity of tandem bivalent nanobody and the native protein. The hydrodynamic radius and terminal half-life of tandem bivalent nanobody increased approximately 2-fold by multivalency compared to the native protein.

Our data revealed that the physicochemical as well as in vivo pharmacokinetic parameters of tandem bivalent nanobody was significantly improved.

Oral Squamous Cell Carcinoma (OSCC) is among the ten most common cancers worldwide. Hypermethylation of CpG sites in the promoter region and subsequent down-regulation of a tumor suppressor gene, TGM-3 has been proposed to be linked to different types of human cancers including OSCC. In this study, methylation status of CpG sites in the promoter region of TGM-3 has been evaluated in a cohort of patients with OSCC compared to normal controls.

Forty fresh tissue samples were obtained from newly diagnosed OSCC patients and normal individuals referred to dentistry clinic for tooth extraction. DNA was extracted, bisulfite conversion was performed and it was subjected to PCR using bisulfite-sequencing PCR (BSP) primers. Prepared samples were sequenced on a DNA analyzer with both forward and reverse primers of the region of interest. The peak height values of cytosine and thymine were calculated and methylation levels for each CpG site within the DNA sequence was quantified.

Quantitative DNA methylation analyses in CpG islands revealed that it was significantly higher in OSCC patients compared to controls. DNA methylation at CpG1/CpG3/CpG5 (p=0.004-0.01) and CpG1/CpG3 (p=0.001-0.019) sites was associated with tumor stage and grade, respectively. Male OSCC patients had higher methylation rate at CpG3 (p=0.032), while smoker patients showed higher methylation rate at CpG6 (p=0.045).

These results manifested the contribution of DNA methylation of TGM-3 in OSCC and its potential association with clinico-pathologic parameters in OSCC.

This study investigated the potential effects of Titanium dioxide nanoparticles (Tio2NPs) followed by maternal gavage on fetal development and neural tube formation during pregnancy in mice.

Thirty pregnant mice were randomly divided into five main study groups including the untreated control and 4 experimental groups (n=6 per group). The control group was treated with normal saline and the experimental groups were orally treated with doses of 30, 150, 300, and 500 mg/kg Body Weight (BW) of Tio2NPs during pregnancy. On gestational day 16 and 19 (n=3 per group), pregnant mice were euthanized and then examined for neural tube defects and compared with control. Serial transverse sections were prepared in both cranial region and in lumbar region of spinal cord.

Treatment with Tio2NPs resulted in low fetal weight and short length, dilation of lateral ventricle, thinning of cerebral cortex and spinal cord, spina bifida occulta and an increase in the number of apoptotic neurons in exposed embryos at doses of 300 and 500 mg/kg (p<0.05).

It seems that exposure to nanoparticles of Tio2 during pregnancy induces growth retardation and for the first time, teratogenicity of this nanomaterial in neural tube development and induction of defects such as spinal bifida, reduction in cortical thickness and  dilatation of lateral ventricles were verified which can be related to incidence of apoptosis in central nervous system.

The aim of the present study was to investigate the effect of Sodium Selenite (SS) supplemented media on oocyte maturation, expression of mitochondrial transcription factor A (TFAM) and embryo quality.

Mouse Germinal Vesicle (GV) oocytes were collected after administration of Pregnant Mare Serum Gonadotropin (PMSG); in experimental group 1, oocytes were cultured and then subjected for in vitro maturation in the absence of SS, and in experimental group 2, they were matured in vitro in the presence of 10 ng/ml of SS up to 16 hr. The control group included MII oocytes obtained from the fallopian tubes after ovarian stimulation with PMSG, followed by human chorionic gonadotropin. Then, the expression of TFAM in MII oocytes in all three groups was investigated using real-time RT-PCR. The fertilization and embryo developmental rates were assessed, and finally the quality of the blastocysts was evaluated using propidium iodide staining.

The oocyte maturation rate to MII stage in SS treated group was significantly higher than non-treated oocytes (75.65 vs. 68.17%, p<0.05). Also, the rates of fertilization, embryo development to blastocyst stage as well as the cell number of blastocyst in SS supplemented group were higher than other experimental group (p<0.05). There was a significant decrease in TFAM gene expression in both in vitro groups compared to the group with in vivo obtained oocytes (p<0.05). Moreover, there was a significant increase in TFAM gene expression in oocytes that matured in the presence of SS compared to that of the group without SS (p<0.05).

Supplementation of oocyte maturation culture media with SS improved the development rate of oocytes and embryo and also enhanced TFAM expression in MII oocytes which can affect the mitochondrial biogenesis of oocytes.

Epitope prediction remains a major challenge in malaria due to the unique parasite biology, in addition to rapidly evolving parasite sequence variation in Plasmodium species. Although several models for epitope prediction exist, they are not useful in Plasmodium specific epitope development. Hence, it was proposed to use machine learning based methods to develop a peptide sequence based epitope predictor specific for malaria.

Model datasets were developed and performance was tested using various machine learning algorithms. Machine learning classifiers were trained on epitope data using sequence features and comparison of amino acid physicochemical properties was done to yield a valid prediction model.

The findings from the analysis reveal that the model developed using selected classifiers after preprocessing by Waikato Environment for Knowledge Analysis (WEKA) performed better than other methods. The datasets for benchmarks of performance are deposited in the repository https://github.com/githubramaadiga/epito-pe_dataset.

The study is the first in-silico study on benchmarking Plasmodium cytotoxic T cell epitope datasets using machine learning approach. The peptide based predictors have been used for the first time to classify cytotoxic T cell epitopes in malaria. Algorithms has been evaluated using real datasets from malaria to obtain the model.

Generally, timely diagnosis of micro-organisms is very important to prevent many diseases. Many methods can detect micro-organisms like culture-based methods and molecular methods. The molecular methods are usually preferred because they provide fast and reliable results. In some cases, microbial strains are not accessible, and there is no safety to work with them; therefore, synthetic constructs which are designed according to the available sequences in databases can be used as a positive control for detection of them.

In this study, a synthetic construct was designed for molecular detection of Francisella tularensis (F. tularensis) and the Ebola virus by multiplex real-time PCR reaction. For this, sequences were taken from databases and then multiple alignments were done by software. Also, conventional PCR and two models of real-time PCR (SYBR green and TaqMan) were applied. Finally, multiplex real-time PCR was performed.

The synthetic construct was designed and used for conventional PCR and multiplex PCR. The results of common PCR showed a single band at 148 bp and 167 bp in 1.5% agarose gel stained by ethidium bromide for F. tularensis and Ebola virus, respectively. Also, a dual-band at 148 and 167 bp was observed in multiplex PCR. Results of real-time PCR showed a limit of detection about 0.1 pg of plasmid/µl.

In conclusion, the designed construct can be used as a positive control for an accurate diagnosis of these micro-organisms without any biological danger for laboratory staff. So, this method is useful for diagnosis of these agents in food, water, and blood samples.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen causing a wide range of human infections. The organism is resistant to a wide range of antibiotics. The purpose of this study was to investigate the effect of AgNPs on pyocyanin pigment production of P. aeruginosa bacteria isolated from clinical specimens.

In this study, 15 clinical isolates of P. aeruginosa were collected from different specimens of hospitalized patients. P. aeruginosa was detected by biochemical and molecular (detection of pbo1 gene by colony PCR method) methods and the MIC and MBC of AgNPs were determined by agar dilution method. Inhibition of P. aeruginosa pyocyanin production at AgNPs concentrations of 0, 0.3, 0.5, 1 and 1.5 mg/ml of was studied with OD of 520 nm.

The mean MIC and MBC of AgNPs were 1.229 and 1.687 mg/ml, respectively. Pyocyanin production was investigated for all isolates at different concentrations of nanoparticles, and their comparison showed that with increasing nanoparticle concentration, pyocyanin production significantly decreased (p<0.05).

According to the results of this study, AgNPs had an inhibitory effect on P. aeruginosa and its pigment production and with increasing nanoparticles concentration, pigment production decreased; therefore, it seems that the nanoparticles can be used to treat and prevent diseases caused by P. aeruginosa.