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32695275
COVID-19 and Medical Biotechnology
<p>Novel coronavirus disease (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), became a global challenge <sup>1</sup>. The disease which emerged in Wuhan, China, late 2019, has affected more than 6 million individuals in almost all countries and regions, leading to death in more than 350,000 only in a 6-month period (Date: June 1st, 2020). It should be mentioned that SARS-CoV-2 is responsible for the 3rd respiratory syndrome, caused by coronaviruses during last two decades, while SARS-CoV and Middle East Respiratory Syndrome coronavirus (MERS-CoV) were both connected to the emergence of severe respiratory syndromes in 2003 and 2012, respectively <sup>2</sup>. Meanwhile there is no effective treatment or vaccine for the disease.</p>
<p>Although the pathogenesis of SARS-CoV-2 has not been clearly understood yet, it is a large enveloped virus, similar to other coronaviruses, which contains several proteins including M (membrane), S (spike), E (envelope), and N (nucleocapsid), which are good candidates for targeting <sup>3</sup>. Among them, S glycoprotein, with two domains of S1 and S2, has been as of interest of recent studies, while it is responsible for invasion and entry into the host cells; the Receptor Binding Domain (RBD) of S1 interacts with Angiotensin-Converting Enzyme 2 (ACE2) on the cell surface, while the S2 domain is responsible for virus-cell membrane fusion and viral entry with higher affinity <sup>4</sup>.</p>
<p>Considering the fact that the immune system is affected by the SARS-CoV-2, immune-based treatment, including corticosteroids, monoclonal antibodies against pro-inflammatory cytokines, plasma therapy, and intravenous immunoglobulin was practiced in some patients in a few studies <sup>5</sup>. However, the efforts should not be limited to such treatments, while novel therapeutic approaches could be considered, using medical biotechnology.</p>
<p>Such pandemic is complex problem, which needs transdisciplinary studies. The development of medical biotechnology to produce pharmaceutical and diagnostic products is a need, which needs close collaboration with other disciplines <sup>6</sup>. It should be emphasized that it has been clear that coronaviruses know no borders; therefore borderless solution is needed to fight COVID-19 <sup>7</sup><sup>,</sup><sup>8</sup>. It is to be hoped that the lessons we learned from SARS-CoV-2, help us to prevent possible pandemic in the near future.</p>
139
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https://www.ajmb.org/En/Article.aspx?id=30422
https://www.ajmb.org/PDF/En/FullText/30422.pdf
NimaRezaei186
en
32695276
Pasteurella multocida Vaccine Candidates: A Systematic Review
<p><em>Pasteurella multocida (P. multocida)</em> is the highly contagious causative agent of a broad range of diseases in animals as well as an occasional human pathogen. Economically significant infections caused by <em>P. multocida</em> include avian fowl cholera, rabbit snuffles, and hemorrhagic septicemia in cattle, goats and pigs. Chemotherapy of pasteurellosis infections has some limitations, such as high cost of treatment, low efficacy, and the possibility of therapy failure due to antibiotic resistance. Prophylactic immunization offers a safe and effective preventive measure in case of zoonotic diseases. Bacterins, live attenuated and some old traditional vaccines against pasteurellosis remain in use today, beside their limitations. However, the past few years have seen significant progress in research to identify modern, effective vaccine candidates, but there is no new vaccine produced by new strategies. While scientists should struggle with a lot of aspects to design vaccine producing strategies, this review shows how pasteurellosis vaccine evolved and the limitations in its application which need to be overcome. </p>
Pasteurella multocida, Pasteurellosis, Vaccines
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https://www.ajmb.org/En/Article.aspx?id=30423
https://www.ajmb.org/PDF/En/FullText/30423.pdf
SaiedMostaanDepartment of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran41578
AbbasGhasemzadehDepartment of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran41579
SoroushSardari54
Mohammad AliShokrgozarNational Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran202
GholamrezaNikbakht BrujeniDepartment of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran41580
MohsenAbolhassani41581
ParastooEhsaniDepartment of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran514
Mohammad RezaAsadi KaramDepartment of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran41582
en
32695277
Circulating Tumor Cells Detection in Patients with Early Breast Cancer Using MACS Immunomagnetic Flow Cytometry
<p>Background: Circulating Tumor Cells (CTCs) detection in peripheral blood of epithelial cancer patients is an indicator of the presence of primary tumors and metastasis. The CTC phenotype detection uses epithelial markers in defining, detecting, and isolating CTCs. Circulating cell-separation technologies, with the epithelial origin, can be identified by epithelial biomarkers, with different techniques such as flow cytometry. The purpose of this study was to evaluate the expression of molecular Cytokeratins (CKs), CK7, CK8, CK18, CK19 (Pan-CK) and Epithelial Cell Adhesion Molecule (EpCAM) markers for CTC detection.</p>
<p>Methods: The Magnetic Activated Cell Sorting (MACS) was used to identify CTCs in the blood of patients. Specific antibodies to EpCAM and Pan-CK were used and analyzed by flow cytometry. In this study, 35 blood samples of patients with breast cancer were assessed before any treatment and 35 healthy blood samples as the control were evaluated.</p>
<p>Results: Expression of CK markers in the peripheral blood of breast cancer patients was statistically significant with p≤0.05, specifically at stages II-IV, but it was not significant in patients at stage I and healthy controls. Biomarkers expression in the blood of patients and healthy controls was assessed along with the pathologic characteristics of patients.</p>
<p>Conclusion: CTC assessment by flow cytometry in patients with breast cancer could not only be used for detection but also can be considered as a source of specific and subjective evaluation for monitoring the therapy. Besides, the sensitivity and specificity of CTC detection were shown that could be enhanced by specific CK markers.</p>
Circulating tumor cells, Cytokeratins, EpCAM, Epithelial cell adhesion molecule, Flow cytometry
148
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https://www.ajmb.org/En/Article.aspx?id=30424
https://www.ajmb.org/PDF/En/FullText/30424.pdf
NasrinKarimiDepartment of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran41583
ManaOloomi41585
ZahraOrafaDepartment of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran41584
en
32695278
The Role of Dihydropyrimidine Dehydrogenase and Thymidylate Synthase Polymorphisms in Fluoropyrimidine-Based Cancer Chemotherapy in an Iranian Population
<p>Background: The fluoropyrimidine drug 5-Fluorouracil (5-FU) and the prodrug capecitabine have been extensively used for treatment of many types of cancer including colorectal, gastric, head and neck. Approximately, 10 to 25% of patients suffer from severe fluoropyrimidine-induced toxicity. This may lead to dose reduction and treatment discontinuation. Pharmacogenetics research could be useful for the identification of predictive markers in chemotherapy treatment. The aim of the study was to investigate the role of five genetic polymorphisms within two genes (DPYD, TYMS) in toxicity and efficacy of fluoropyrimidine-based chemotherapy.</p>
<p>Methods: Total genomic DNA was extracted from 83 cancer patients treated with fluoropyrimidine-based chemotherapy. In this study, three polymorphisms were genotyped in dihydropyrimidine dehydrogenase gene c.1905+1 G>A (DPYD*2A; rs3918290), c.1679 T>G (I560S; DPYD*13; rs55886062), and c.2846A>T (D949V; rs67376798) and two polymorphisms, besides the Variable Number of Tandem Repeat (VNTR) polymorphism and 6-bp insertion/deletion polymorphism in thymidylate synthase gene. The analysis of polymorphisms for rs3918290, rs55886062, rs67376798 and 6-bp insertion/deletion in TYMS was done by Polymerase Chain Reaction-restriction Fragment Length Polymorphism (PCR-RFLP) TYMS VNTR analysis. 5-FU-related toxicities such as anemia, febrile neutropenia, neurotoxicity, vomiting, nausea, and mucositis were evaluated according to NCI-CTC criteria version 4.0. T-test and chi-square were used and p-values less than 0.05 were considered statistically significant.</p>
<p>Results: DPYD gene polymorphisms were not observed in this study. The frequency of the TYMS +6 bp allele was 40.35% and the -6 bp allele was 59.65% in this study. The frequency of VNTR 2R allele was 48.75% and 3R allele was 51.15%. Toxicity grade II diarrhea, mucositis, nausea, vomiting, and neurotoxicity was 2.2, 24.1, 15.7, 6, and 51.8%, respectively. Thymidylate synthase ins/del polymorphisms were associated with increased grade III neurotoxicity (p=0.02). Furthermore, anemia grade III was significantly associated with 2R/2R genotype (0.009).</p>
<p>Conclusion: Thymidylate synthase gene polymorphisms may play a key role in fluoropyrimidne -based chemotherapy. Although rare DPYD polymorphisms were not observed in our study, according to large population studies, DPYD gene polymorphisms could be used as a predictive biomarker for patient treatments.</p>
5-fluorouracil, Dihydropyrimidine dehydrogenase, Fluoropyrimidines, Pharmacogenetics, Thymidylate synthase
157
164
https://www.ajmb.org/En/Article.aspx?id=30425
https://www.ajmb.org/PDF/En/FullText/30425.pdf
Mohammad HadiAbbasianDepartment of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran41586
NafisehAnsarinejadCancer Pharmacogenetics Research Group (CPGRG), Iran University of Medical Sciences, Tehran, Iran41587
BaharehAbbasiDepartment of Medical Genetic, Medical Biotechnology Ins., National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran41588
MasoudIravaniTehran Gastroenterology and Hepatology Center, Tehran, Iran41589
TayebRamimDepartment of Medicine, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran41590
FahimeHamediDepartment of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran41591
AliM. Ardekani2
en
32695279
Monoclonal Antibody Against ROR1 Induces Apoptosis in Human Bladder Carcinoma Cells
<p><span style="font-size:10pt">Background: Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is one of the promising cell surface antigens for targeting cancer cells. The aim of this study was to evaluate ROR1 cell surface expression in bladder cancer cells using a murine anti-ROR1 monoclonal antibody (mAb) called 5F1-B10 as well as investigate its potential in apoptosis induction.</span></p>
<p><span style="font-size:10pt">Methods: Expression of ROR1 in two human bladder cell lines, 5637 and EJ138, as well as a non-cancerous human cell line, Human Fetal Foreskin Fibroblast (HFFF), was examined by flow cytometry and immunocytochemistry. Immunohistochemical staining of cancer and normal bladder tissues was also performed.</span></p>
<p><span style="font-size:10pt">Results: The flow cytometry results showed that 5F1-B10 mAb could recognize ROR1 molecules in 86.1% and 45.6% of 5637 and EJ138 cells, respectively. The expression level of ROR1 was 5.49% in HFFF cells. The immunocytochemistry and immunohistochemistry staining results also confirmed the presence of ROR1 on the surface of both bladder cancer cells and tissues, respectively. The obtained data from apoptosis assay demonstrated that 5F1-B10 mAb could induce apoptosis in both 5637 and EJ138 cell lines.</span></p>
<p><span style="font-size:10pt">Conclusion: Taken together, our finding indicates the role of ROR1 in bladder cancer cell survival and suggests this receptor might be a promising target for developing novel therapeutic agents against bladder carcinoma.</span></p>
Bladder cancer, Flow cytometry, Monoclonal antibody, ROR1 protein
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https://www.ajmb.org/En/Article.aspx?id=30428
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Ali AhmadBayat Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran835
NiloufarSadeghiMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran41592
RaminaFatemiMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran849
Mohammad RezaNowrooziUro-Oncology Research Center, Tehran University of Medical Sciences, Tehran, Iran41594
SolmazOhadian MoghadamUro-Oncology Research Center, Tehran University of Medical Sciences, Tehran, Iran41595
MohadesehBorzueeUro-Oncology Research Center, Tehran University of Medical Sciences, Tehran, Iran41596
AminRadmaneshLegal Medicine Research Center, Legal Medicine Organization, Tehran, Iran41597
MahmoodKhodadoostFaculty of Traditional Medicine, Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran41598
Ali rezaSarrafzadehDepartment of Pathology, Khatam Al Anbia Hospital, Tehran, Iran840
OmidZareiCellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran190
HodjattallahRabbani24
en
32695280
Effect of Conditioned Medium from IGF1-Induced Human Wharton’s Jelly Mesenchymal Stem Cells (IGF1-hWJMSCs-CM) on Osteoarthritis
<p>Background: Osteoarthritis (OA) is a chronic disease that attacks joints and bones which can be caused by trauma or other joint diseases. Stem cell and Conditioned Medium (CM) of stem cells are developed for OA therapy, which is minimally invasive. It can decrease inflammation and be a replacement for knee surgery. This study aimed to utilize human Wharton’s Jelly-Mesenchymal Stem Cells (hWJMSCs) as an alternative OA therapy.</p>
<p>Methods: CM from hWJMSCs induced by IGF1 was collected. The OA cells model (IL1β-CHON002) culture was treated as follows: 1) with hWJMSCs-CM 15% (v/v); 2) with hWJMSCs-CM 30% (v/v); 3) with IGF1-hWJMSCs (IGF1-hWJMSCs-CM) 15% (v/v); 4) with IGF1-hWJMSCs-CM 30% (v/v). Parameters including inflammatory cytokines (IL10 and TNFα), extracellular matrix degradation (MMP3 expression), and chondrogenic marker (<em>COL2</em> expression) were determined.</p>
<p>Results: The most significant increase in <em>COL2</em> chondrogenic markers was found in IL1β-CHON002 treatment using 15% CM of hWJMSCs induced with IGF1. CM of hWJMSCs can reduce inflammatory cytokines (TNFα and IL10) and matrix degradation mediator MMP3. Better result was gained from IGF1-induced hWJMSCs-CM.</p>
<p>Conclusion: CM of IGF1-hWJMSCs reduce inflammation while repairing injured joint in the human chondrocyte OA model.</p>
Chondrocyte, IGF1, Osteoarthritis, Proinflammatory, Wharton’s jelly
172
178
https://www.ajmb.org/En/Article.aspx?id=30429
https://www.ajmb.org/PDF/En/FullText/30429.pdf
HannaSari Widya KusumaBiomolecular and Biomedical Research Center, Aretha Medika, Utama, Bandung,, West Java, Indonesia41600
WahyuWidowati41601
RimontaFebby GunanegaraFaculty of Medicine, Maranatha Christian University, Bandung, West Java, Indonesia41602
BerryJuliandiDepartment of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, IPB Darmaga Campus, Bogor, West Java, Indonesia41603
NyomanEhrich ListerUniversitas Prima Indonesia, Medan North Sumatera, Indonesia41604
SeilaArumwardanaBiomolecular and Biomedical Research Center, Aretha Medika, Utama, Bandung, West Java, Indonesia41605
DewaniTediana YusepanyBiomolecular and Biomedical Research Center, Aretha Medika, Utama, Bandung, West Java, Indonesia41606
DwiSurya ArtieBiomolecular and Biomedical Research Center, Aretha Medika, Utama, Bandung, West Java, Indonesia41607
EndenDea Nataya Biomolecular and Biomedical Research Center, Aretha Medika, Utama, Bandung, West Java, Indonesia41608
KamilaYashfa GunawanBiomolecular and Biomedical Research Center, Aretha Medika, Utama, Bandung, West Java, Indonesia41609
IkaAdhani SholihahBiomolecular and Biomedical Research Center, Aretha Medika, Utama, Bandung, West Java, Indonesia41610
ErmiGirsangUniversitas Prima Indonesia, Medan North Sumatera, Indonesia41611
ChrismisNovalinda GintingUniversitas Prima Indonesia, Medan North Sumatera, Indonesia41612
IndraBachtiarStem Cell and Cancer Institute, Jakarta, Indonesia41613
HarryMurtiStem Cell and Cancer Institute, Jakarta, Indonesia41614
en
32695281
Conus coronatus and Conus frigidus Venom: A New Source of Conopeptides with Analgesic Activity
<p>Background: Cone snails are a natural source of complex peptides with analgesic properties called conotoxins. These peptides are secreted in a complex venomic mixture and are predominantly smaller than 5 <em>kDa</em>. The present study aimed to document the analgesic activity of two species of <em>Conus coronatus</em> (<em>C.</em> <em>coronatus</em>) and <em>Conus frigidus</em> (<em>C. frigidus</em>) venom collected off the Iranian coast in a mouse behavioral test.</p>
<p>Methods: Conotoxin containing fractions was extracted from the venom ducts and initially purified by column chromatography. The analgesic effect of the fractions was determined on formalin pain model and hot-plate test.</p>
<p>Results: The results led to the identification of four fractions with analgesic activity in <em>C. coronatus</em> and two in <em>C. frigidus</em>. Only one fraction was able to reduce the flinching and licking in both acute pain and chronic pain phases of the formalin test. Moreover, the activity of this fraction remained 30 minutes on the hot-plate test. Purification of the fractions was carried out by RP-HPLC. LC-ESI-MS analysis of the fractions showed that the conotoxins of the analgesic fraction had molecular weights not previously reported.</p>
<p>Conclusion: The findings give insight into the venom of two previously under-investigated <em>Conus</em> species and reveal the therapeutic potential of the containing conopeptides.</p>
Analgesics, Conotoxin, Conus frigidus, Pain, Venoms
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HalimehRajabi Khorramshahr University of Marine Science and Technology , Khorramshahr, Iran41615
HosseinZolgharneinKhorramshahr University of Marine Science and Technology , Khorramshahr, Iran41616
Mohammad TaghiRonagh Khorramshahr University of Marine Science and Technology, Khorramshahr, Iran41617
JamshidAmiri Moghaddam Leibniz Institute for Natural Product Research and Infection Biology- Hans Knöll Institute, Jena, Germany41618
MaxCrüsemann Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany41619
en
32695282
Bioactivity of Bac70 Produced by Bacillus atrophaeus Strain DDBCC70
<p>Background: Recently, using antibacterial peptides has been considered as a strategy to manage the worldwide antibiotic-resistance crisis. Screening of Dasht-Desert Bacterial Culture Collection (DDBCC) for bacteriocin or bacteriocin-like producer was aimed in this study to introduce native antibacterial agent(s).</p>
<p>Methods: In this study, 170 isolates were examined by the cross-streak method against G+ and G- indicators. Isolates with antimicrobial activity were compared using turbidity and well diffusion tests. The candidate isolate, DDBCC70, was molecularly and biochemically characterized. Then, the production of an antibacterial agent was physicochemically optimized. The supernatant was saturated ammonium sulfate. SDS-PAGE and Thin-Layer Chromatography (TLC) analyses, cytotoxicity, and hemagglutination tests were performed.</p>
<p>Results: First, 23 isolates were detected with antimicrobial activity against at least three of the indicator strains. DDBCC70 was distinguished with the broad-spectrum of antibacterial effects of the Cell-Free Supernatants (CFSs). The black pigments on BHI and a 98% similarity in 16S rDNA and similarity in biochemical tests confirmed the strain of DDBCC70 as <em>Bacillus atrophaeus (B. atrophaeus)</em>. The highest amount of the antibacterial agent, Bac70, was obtained from the modified brain heart infusion medium. It was revealed that 70% ammonium sulfate-saturated Bac70 was 3.8 and 1.6 times more effective on <em>Pseudomonas aeuroginosa</em> <em>(P. aeuroginosa)</em> and <em>Klebsiella pneumoniae (K. pneumoniae)</em>. Bac70, a >25 <em>kDa</em> protein and a safe compound for blood cells, neither agglutinated human erythrocyte nor lysed sheep blood. The purified bacteriocin-like molecule destroyed biofilms from <em>P. aeruginosa</em> and <em>Staphylococcus aureus (S. aureus)</em>. Moreover, the fraction of Bac70 from the TLC plate showed higher inhibitory effects against <em>K. pneumoniae</em>.</p>
<p>Conclusion: Based on the above-mentioned features, Bac70 is a potential alternative therapeutic agent in pharmaceutical, food preservative and biotech-related industries.</p>
Antibiofilm, Bacillus atrophaeus, Bacteriocin, Klebsiella pneumoniae, Pseudomonas aeruginosa
186
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https://www.ajmb.org/En/Article.aspx?id=30431
https://www.ajmb.org/PDF/En/FullText/30431.pdf
Mohammad RezaSarjoughianDepartment of Biotechnology, Faculty of Biotechnology, Semnan University, Semnan, Iran41620
ShamsozohaAbolmaali41621
ShakibaDarvish Alipour Astaneh Department of Biotechnology, Faculty of Biotechnology, Semnan University, Semnan, Iran41622
en
32695283
Immunogenic Potency of Formalin and Heat Inactivated E. coli O157:H7 in Mouse Model Administered by Different Routes
<p>Background: Enterohemorrhagic<em> Escherichia coli (E. coli)</em> (EHEC) O157:H7 is a major foodborne pathogen causing severe disease in humans worldwide. Cattle are important reservoirs of <em>E. coli</em> O157:H7 and developing a specific immunity in animals would be invaluable. The administration of Whole Cell Vaccines (WCV) is a well-established method of vaccination against bacterial infections. Route of administration, inactivation and using suitable adjuvant have significant effects on the characteristics and efficacy of WCV.</p>
<p>Methods: In the present study, an attempt was made to evaluate the immunogenic potency of heat and formalin inactivated cells administered orally and subcutaneously in mouse model by ELISA. Mice pretreated with streptomycin were used as a model to evaluate the efficacy of subcutaneous versus oral administration of the vaccine. Following immunization, mice were infected with <em>E. coli</em> O157:H7 and feces were monitored for shedding.</p>
<p>Results: Both forms of inactivated cells induced immune response and hence protection against infectious diseases caused by <em>E. coli</em> O157:H7. However, formalin inactivated cells of <em>E. coli</em> O157:H7 showed superior antigenicity compared to heat inactivated cells. Subcutaneous immunization of mice with both heat and formalin inactivated <em>E. coli</em> O157:H7 induced significant specific levels of IgG antibodies but did not lead to significant antigen-specific IgA rise in feces, whereas oral immunization elicited significant levels of IgG antibodies with some animals developing antigen-specific IgA in feces.</p>
<p>Conclusion: Inactivated <em>E. coli</em> O157:H7 is highly immunogenic and can induce protective immune responses via oral immunization.</p>
Escherichia coli O157:H7, Formaldehyde, Hot temperature, Immunization, Mice, Vaccines
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https://www.ajmb.org/En/Article.aspx?id=30432
https://www.ajmb.org/PDF/En/FullText/30432.pdf
NasimArshadiDepartment of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran41623
Seyed LatifMousavi 41624
JafarAmani Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran41625
ShahramNazarianDepartment of Biology, Imam Hossein University, Tehran, Iran11429
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32695284
CRISPR/Cas as a Potential Diagnosis Technique for COVID-19
<p>Coronaviruses are positive single stranded RNA viruses, and are members of Coronaviridae family. Coronaviruses localize in respiratory tract and are usually known as common cold viruses <sup>1,2</sup>. Seven strains of coronavirus family can infect humans and can cause different signs ranging from cold with major symptoms such as fever and sore throat to upper and lower respiratory tract infections resulting in pneumonia, severe respiratory tract infection and even death. These seven strains include HCoV-229E, HCoV-OC43, SARS-Co- V, human coronavirus NL63, human coronavirus HK-U1, MERS-CoV and SARS-CoV-2, known as 2019-nCoV or "novel corona virus 2019" <sup>3</sup>.</p>
<p>At present, "severe acute respiratory syndrome coronavirus 2" or "coronavirus disease 2019" (COVI D-19) which is closely related to SARS has become a global health problem. The first-ever COVID-19 case was identified in December 2019 in Wuhan, China; however, since then the virus has spread rapidly across the world and has become a worldwide pandemic and an international concern <sup>4</sup>.</p>
<p>To the best of our knowledge until March 2020, COVID-19 has been reported in 161 countries. COVID-19 is typically transmitted by respiratory droplets during sneezing and coughing <sup>5</sup>. There is no evidence of vertical transmission or transmission during pregnancy <sup>6,7</sup>. Incubation period of COVID-19 is estimated between 2-14 days and during this time, infected peoples are considered as asymptomatic carriers. Although infection may be asymptomatic, patients typically have fever, cough and shortness of breath. Occasionally disease progresses acutely and causes severe pneumonia, multiple organ failure and death <sup>8</sup>. Patients with underlying medical conditions such as heart and respiratory diseases, asthma, diabetes and immunodeficiency diseases, in addition to elderly age group are high risk and more susceptible to COVID-19 infection <sup>9</sup>. At present, there is no certain treatment or vaccination for prevention of COVID-19 and infected people are either isolated or, in critical conditions, take nonspecific or supportive care <sup>9</sup>. Diagnosis is made based on the symptoms of the disease, chest CT (Computed tomography scan) scan and qRT-PCR (Quantitative reverse transcription polymerase chain reaction). qRT-PCR technique is the <strong>current </strong>COVID-19 (<em>SARS-CoV-2)</em> <strong>gold </strong>standard molecular <strong>detection method approved by CDC and </strong>World Health Organization (<strong>WHO)</strong> <sup>10,11</sup>. Recently, researchers have proposed a coronavirus rapid detection method based on CRISPR/Cas system <sup>12</sup>. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats) is an adaptive immune system in archaea and bacteria against foreign genetic elements such as phages. Recently, CRISPR/Cas has become a powerful gene editing tool and a promising treatment for genetic diseases and cancers <sup>13,14</sup>. In this technique, a programmable protein attaches to the target site by a guide RNA for cleavage of the target sequence. There are several types of Cas proteins that have different properties. Among them, Cas9 protein has received more attention for gene editing whereas, Cas12a and Cas13a have been more efficient in diagnosis of diseases <sup>15,16</sup>. Cas12a is DNA-specific but Cas13a works with RNA which makes it convenient in detection of <em>SARS-CoV-2</em>. Recently, Zhang <em>et al</em> reported specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) technology which is a CRISPR/Cas13 based nucleic acid detection technique for rapid detection of <em>SARS-CoV-2</em> <sup>17,18</sup>.</p>
<p>They targeted S and ORF1ab protein genes in coronavirus genome. Cas13 identifies and binds to previously determined target sequence which leads to fairly random cleavage of surrounding ssRNA molecules. SHERLOCK technology utilizes a quenched fluorescent ssRNA reporter. The presence of ssRNA coronavirus genome in samples activates Cas13 resulting in the production of quantifiable signals. Amplification of targeted DNA or RNA by Recombinase Polymerase Amplification (RPA) or reverse transcriptase-RPA (RT-RPA) prior to the start of reaction improves the sensitivity of the assay. Subsequently, amplified DNA is converted to RNA by combination of RPA and T7 transcription. Ultimately, detection is made by simultaneous incorporation of the ssRNA reporter (Biotin-RNA-FITC). Viral genome is detected at attomolar concentration using SHERLOCK technology <sup>19</sup>. The test can be carried out starting with RNA purified from patient samples, as used for qRT-PCR assays, and can be read out using a dipstick in less than an hour, without requiring elaborate instrumentation <sup>18</sup>. As a result, application of CRISPR/Cas13 based diagnosis or SHERLOCK for <em>SARS-CoV-2 </em>detection is much faster than detection by qRT-PCR and has high sensitivity. Consequently, SHERLOCK technology could swiftly replace qRT-PCR technique considering the high demand for rapid diagnosis tests in current global pandemic state of COVID-19.</p>
Coronavirus, COVID-19, CRISPR/Cas9, SARS-CoV-2
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https://www.ajmb.org/En/Article.aspx?id=30433
https://www.ajmb.org/PDF/En/FullText/30433.pdf
MahintajDara 41627
MahdiehTalebzadehDepartment of Molecular Medicine, Faculty of Advanced Medical Science and Technology, Shiraz University of Medical Sciences, Shiraz, Iran41628