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Avicenna Journal of Medical Biotechnology - Volume:13 Issue: 4, Oct-Dec 2021

Avicenna Journal of Medical Biotechnology
Volume:13 Issue: 4, Oct-Dec 2021

  • تاریخ انتشار: 1400/07/20
  • تعداد عناوین: 11
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  • Ahmad Shamabadi, Alireza Hasanzadeh, Shahin Akhondzadeh * Pages 172-175

    Besides concerns about the increasing prevalence of psychiatric disorders and the significant burdens and costs, there are concerns about its validity. The dilemma of validity went so far that studies described the diagnoses in psychiatry as scientifically worthless. We suggest integrating psychiatry and medical biotechnology and using biotechnological products in psychiatric aspects help psychiatry become more precise, strengthen its position among other sciences, and increase its scientific credibility by giving examples. For this matter, we need different inputs to choose between the vast outputs. The most common inputs are clinical symptoms, cognitive function, individual and environmental risk factors, molecular markers, genetic markers, neuroimaging signs, and big data. Some molecular markers have been shown to have a relationship with psychiatric disorders such as Interleukin-6 (IL-6) and Tumor Necrosis Factor-  (TNF- ). Genetic studies might evolve the most accurate part of precision psychiatry. Currently, and through the developments in technology, genome-wide association studies have become available. In neuroimaging signs, psychiatric disorders are associated with generalized rather than focal brain network dysfunction, and functional magnetic resonance imaging could be performed to study them. It would exhibit different aberrancies in various psychiatric disorders. In big data, the constitution of predictive models and movement toward precision psychiatry can be led by using artificial intelligence and machine learning.

    Keywords: Behavioral sciences, Cytokines, Integrative medicine, Knowledge, Technology
  • Samira Jafarzadeh, Javad Baharara, Maryam Tehranipour Pages 176-182
    Background

     Ovarian cancer is the leading cause of death caused by genital cancers. One of the most common treatments for this type of cancer is chemotherapy by cisplatin, which induces apoptosis in cancer cells. Apoptosis is a type of physiological cell death. Cisplatin chemotherapy usually has several side effects and cellular resistance to cisplatin is a common incidence. In order to overcome these problems, the use of combination therapies using natural substances has been considered. Fisetin is a flavonoid with anti-cancer activity which induces apoptosis</span>. In this study, the apoptosis induced by cisplatin along with Fisetin in cisplatin-resistant ovarian cancer cell line (A2780) was investigated.

    Methods

      In the present experimental study, the effect of combined use of Fisetin and cisplatin on ovarian cancer cell lines (A2780) was investigated by using MTT assay. Cell death was also determined by DAPI, acridine orange/propidium iodide, and Annexin/PI assay. Apoptotic gene expression of Bax</em>, BCL-2</em>, caspase 3</em>, and caspase 9</em> was also assessed by real time PCR.

    Results

     The results of MTT assay indicated that the combined treatment of Fisetin and cisplatin effectively inhibits proliferation of A2780 cells. The results of DAPI staining showed that fragmentation of chromatin in cells occurred in the combined treatment. Acridine orange-propidium iodide staining and Annexin/PI staining showed an increase in the rate of apoptotic cells in cells under combined treatment. The results of the study regarding changes in gene expression also indicated that Bax</em> pro-apoptotic gene expression and BCL-2</em> anti-apoptotic gene expression increased in cells under treatment; moreover, gene expression of caspases 3</em> and 9</em> significantly increased as well.

    Conclusion

    According to the findings of this study, the combined use of cisplatin and Fisetin increases the induction of apoptosis in cisplatin-resistant ovarian cancer cells (A2780); therefore, the combined use of cisplatin and Fisetin can be considered a promising </span>strategy</span> in the treatment of ovarian cancer.

    Keywords: Apoptosis, Cisplatin, Fisetin, Ovarian neoplasms
  • Fatemeh Sheikhsofla *, Behzad Poopak, Sajjad Firuzyar, Fatemeh Roudbari, Mojtaba Ghadiany Pages 183-191
    Background

    KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods.

    Methods

    This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively.

    Results

    KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method. 

    Conclusion

    These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.

    Keywords: Allele-Specific PCR, BRAF, Colorectal neoplasms, KRAS, PCR-RFLP, Reverse dot blot
  • Nguyen Thi My Trinh, Tran Linh Thuoc, Dang Thi Phuong Thao * Pages 192-200
    Background

    The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimulating Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs.

    Methods

    BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the conjugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography.

    Results

    PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF).

    Conclusion

    These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

    Keywords: Granulocyte colony stimulating factor (G-CSF), Inclusion bodies, Polyethylene glycol
  • Elnaz Faghfuri, Ramak Ajideh, Faranak Shahverdi, Mina Hosseini, Faranak Mavandadnejad, Mohammad Hossein Yazdi, Ahmad Reza Shahverdi * Pages 201-206
    Background

    The inhibitory effect of selenium nanoparticles (SeNPs) on cancer cells has been reported in many studies. In this study,  the purpose was to compare the in vitro effects of SeNPs and calcium sulfate coated selenium nanoparticles (CaSO4@ SeNPs) on breast cancer cells.

    Methods

    CaSO4@SeNPs and SeNPs were chemically synthesized and characterized with Field Emission Scanning Electron Microscope (FESEM) and energy-dispersive X-ray spectroscopy (EDX). By applying MTT assay, the cytotoxicity effect of both nanomaterials on the 4T1 cancer cells was investigated.

    Results

    While LD50 of SeNPs on 4T1 cancer cells was 80 µg, the LD50 of CaSO4@SeNPs was reported to be only 15 µg. The difference between the inhibition rates obtained for SeNPs and CaSO4@SeNPs was statistically significant (p=0.05). In addition, at higher concentrations (50 µg) of CaSO4@SeNPs, the cytotoxicity was 100% more than SeNPs alone. 

    Conclusion

    According to the result of the present work, it can be concluded that decoration of SeNPs with calcium sulfate leads to an increase in potency by decreasing the effective dose. This effect can be attributed to activation of intrinsic apoptosis signaling and/or pH regulatory properties of CaSO4@SeNPs. However, further studies are still needed to determine the exact corresponding mechanisms of this synergistic effect.

    Keywords: Apoptosis, Breast neoplasms, Calcium sulfate, Nanoparticles, Selenium
  • Ige Francis Olaoye, Babatunde Joseph Oso*, Adepeju Aberuagba Pages 207-216
    Background

    A large body of literature suggests that the extracts of Ocimum gratissimum (O. gratissimum) and Thymus vulgaris (T. vulgaris) play protective roles against various inflammatory disorders. However, the possible mechanism of action with reference to the interactions of their respective phytochemical compositions with pro-inflammatory mediators as the indication of their therapeutic effects is less clear. Therefore, the immunomodulatory properties of O. gratissimum and T. vulgaris were investigated in this study.

    Methods

    The in vitro lipoxygenase inhibitory potentials of methanolic extracts of the selected plants were assessed through colorimetric analysis. The pharmacokinetics of some identified compounds in the botanicals were investigated via the Swiss ADME server while the molecular interactions of the compounds with lipoxygenase, IL-1, IL-6, TNF-α, IL-8, and CCL-2 were performed through molecular docking.

    Results

    The assessment of the lipoxygenase inhibition revealed the extracts could possess anti-inflammatory agents. The pharmacokinetic results of some selected compounds identified in the botanicals showed moderate toxic effects compared to indomethacin. The molecular docking study substantiated the report of the in vitro analysis as indicated in the binding score of all the selected compounds compared to indomethacin.

    Conclusion

    The phytochemical components of the extracts of O. gratissimum and T. vulgaris could be effective as anti-inflammatory agents that could be explored in preventing disorders associated with excessive activities of pro-inflammatory mediators.

    Keywords: Anti-inflammatory agents, Lipoxygenase, Ocimum, Phytochemicals, Plant extracts, Thymus plant
  • Farahnaz Zare, Sedigheh Sharifzadeh*, Abbas Behzad-Behbahani, Gholamreza Rafiei Dehbidi, Zahra Yousefi, Reza Ranjbaran, Noorossadat Seyyedi Pages 217-222
    Background

    Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat.

    Methods

    Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity.

    Results

    A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control.

    Conclusion

    MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.

    Keywords: Apoptosis, Gene expression, Gene silencing, Jurkat cells, RNA
  • Zahra Sokhanvar, Ameneh Elikaei, Zohreh Sharifi* Pages 223-225
    Background

    Liver disease is more severe in HDV+HBV co-infected patients than HBV infected patients which seems to be related to differences in the expression of genes and other factors such as MicroRNAs (miRNAs). The aim of this study was to investigate miR-222 expression in HBV infected patients in comparison with HDV+HBV co-infected patients.

    Methods

    First, total RNA was extracted from the serum samples and then, complementary DNA (cDNA) was produced using cDNA synthesis kit. Finally, miR-222 gene expression was measured using U6 as the internal control by quantitative PCR (qPCR).

    Results

    The level of miR-222 expression in HDV+HBV co-infected samples was significantly up regulated. The fold change of the miR-222 expression between two groups was 3.3 (95% CI; 0.011- 17.63) with p<0.001.

    Conclusion

    The expression of miR-222 was higher in HBV+HDV co-infected patients than HBV infected patients. Further studies should be conducted to confirm whether miR-222 can be a biomarker for prognosis of severe liver diseases.

    Keywords: Co-infection with hepatitis B, D, Hepatitis B virus, Hepatitis D virus, MiR-222
  • Mehdi Hassani, Sara Hesami, Nahal Maroofi, Mehdi Banan* Pages 226-229
    Background

    The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously.

    Methods

    In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping.

    Results

    This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids.

    Conclusion

    Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing.

    Keywords: CRISPR-Cas systems, Gene editing, Health services, Plasmids, Restriction mapping
  • Mohammad Dehani, Davood Zare-Abdollahi*, Ata Bushehri, Azadeh Dehghani, Jalil Effati, Seyed Ali Mohammad Miratashi, Hamid Reza Khorram Khorshid Pages 230-233
    Background

    Bardet–Biedl Syndrome (BBS) is a rare pleiotropic autosomal recessive disease related to ciliopathies with approximately 25 causative genes. BBS is a multisystemic disorder with wide spectrum of manifestations including truncal obesity, retinal dystrophy, male hypogenitalism, postaxial polydactyly, learning difficulties, and renal abnormalities.

    Methods

    A consanguineous Iranian family with a 28-year-old daughter affected with BBS, resulting from a first cousin marriage, was examined. After clinical examination, Whole Exome Sequencing (WES) was applied. Following the analysis of exome data, Sanger sequencing was used to confirm as well as to co-segregate the candidate variant with the phenotype.

    Results

    A novel homozygous variant [c. 2035G>A (p.E679K)] in exon 2 of the BBS10 gene was found which was categorized as likely pathogenic based on American College of Medical Genetics and Genomics (ACMG) guidelines and criteria. In this study, the variant was fully co-segregated with the phenotype in the family.

    Conclusion

    Despite overlapping with other ciliopathies in terms of the phenotype, the BBS has high genetic heterogeneity and clinical variability even among affected members of a family. The symptoms observed in patients are largely related to the genes involved and the type of mutations in the BBS. In this study, in addition to phenotype description of the proband harboring a novel disease-causing variant in BBS10 gene, the spectrum of BBS symptoms was expanded. The findings of this study can be useful in genetic counseling, especially for risk estimation and prenatal diagnosis.

    Keywords: Bardet–Biedl syndrome, Mutation, Whole exome sequencing
  • Razieh Dehghan, Mohammad Hasan Soheilifar, Farid Azizi Jalilian, Rezvan Najafi, Razieh Amini* Pages 234-236