AJMB: Avicenna Medical Journal of Biotechnology https://www.AJMB.org/ Avicenna Medical Journal of Biotechnology The Role of Biotechnology in Latest Therapeutic Approaches for Diabetes Mellitus https://www.AJMB.org/En/Article.aspx?ID=60568 Sepideh Hajivalizadeh , Shahin Akhondzadeh Wed, 31 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Poultry Gastrointestinal-derived Lactic Acid Bacteria (pGIT-d-LAB) Inhibit Multiple Antibiotics Resistance Bacterial and Fungal Pathogens <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> To develop a probiotic formulation for poultry feed, a few poultry gastrointestinal derived lactic acid bacteria (pGIT-d-LAB) were isolated from chicken intestinal specimens and <em>in vitro</em> experiment was performed to evaluate their efficacy as potential probiotic candidate.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> A total of 6 strains of LAB: <em>L</em><em>actobacillus brevis</em> (<em>L. brevis</em>)<em>,</em> <em>L</em><em>actobacillus acidophilus </em>(<em>L. acidophilus</em>)<em>, Lactobacillus casei </em>(<em>L. casei</em>)<em>, Pediococci </em>spp.<em>, Lactobacillus fermentum </em>(<em>L. fermentum</em>)<em> </em>and<em> L</em><em>actobacillus plantarum </em>(<em>L. plantarum</em>)<em> </em>were isolated and cultured for collection of Cell Free Supernatant (CFS). CFS collected was tested against pathogenic bacterial isolated from chicken feces as well as prevalent fungal pathogens, utilizing agar-well diffusion techniques. A preliminary investigation into the susceptibility of the pathogens to diverse antibiotics and antifungal drugs was conducted. Bacterial pathogens exhibiting resistance to a minimum of three classes of antibiotics were subsequently identified for pGIT-d-LAB CFS screening. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The observed results revealed that the CFS derived from the isolates exhibited varying degrees of growth inhibition against different pathogens. Among the tested pGIT-d-LAB isolates, <em>L. acidophilus </em>demonstrated the most prominent zone of inhibition, measuring 18 <em>mm</em> against <em>Klebsiella pneumoniae</em> ZTAC 1233. Notably, <em>Citrobacter diversus</em> ZTAC 1255 showed resistance to all tested pGIT-d-LAB. Quantification of the metabolites produced was performed, and peak production levels was determined. <em>L.</em><em> acidophilus </em>produced the highest amount of lactic acid (1.789<em>g/l</em>), <em>Pediococci </em>spp. produced the highest amount of diacetyl and H<sub>2</sub>0<sub>2</sub> (1.918<em>g/l</em>) (0.0025<em>g/l</em>) at 48<em> hr</em> peak values respectively. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> The test isolates are potential probiotic candidates for controlling pathogens in poultry. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60574 Bolanle Adeniyi, Abimbola Adesuyi , Funmilola Ayeni , Temitope Ogunbanwo , Taiwo Agidigbi Wed, 17 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Investigating the Effects of HMGB1 Overexpression on Colorectal Cancer Cell Migration via Oncolytic Herpes simplex Virus Type 1 (oHSV-1) <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Colorectal Cancer (CRC) represents a significant global health challenge, and its progression, resistance to therapy, and metastasis are strongly influenced by the tumor microenvironment, including factors like hypoxia. This study explores the impact of High Mobility Group Box 1 (HMGB1) overexpression on CRC cell migration, while identifying potential genes associated with this process. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> To explore this, we developed oncolytic virotherapy, resulting in HSV-HMGB1, an oncolytic <em>Herpes simplex</em> virus that expresses HMGB1. HMGB1 is known its role in cancer progression, particularly in the context of cancer cell migration. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> Contrary to expectations, our scratch assays indicated that HSV-HMGB1 did not significantly induce migration in CRC cells, suggesting that HMGB1 might not directly contribute to this process. Employing microarray analysis, we investigated gene expression changes linked to CRC cell migration, leading to construction of a Protein-Protein Interaction (PPI) network. This network revealed the presence of hub proteins, including as NDRG1, LGALS1, and ANGPTL4, which are recognized for their roles in cancer cell migration. The differential expression of these genes under hypoxic conditions was further validated using quantitative RT-PCR, aligning with the findings from our microarray data.&nbsp; </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Our findings emphasize the complex regulation of CRC cell migration, and provides valuable insights into potential molecular mechanisms and pathways. These findings have implications for further research into cancer progression and the development of therapeutic strategies.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60575 Sara Shayan, Arash Arashkia, Golnaz Bahramali, Kayhan Azadmanesh Wed, 17 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir CYP21A2 Gene Analysis in Southern Iranian CAH Patients and a Brief Review of the Mutation Spectrum <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><em><span style="font-size:10.0pt"> CYP21A2</span></em><span style="font-size:10.0pt"> gene mutations are responsible for more than 95% of Congenital Adrenal Hyperplasia</span><span style="font-size:10.0pt"> (CAH)</span><span style="font-size:10.0pt"> disorders with</span> <span style="font-size:10.0pt">autosomal recessive inheritance. Most of these pathogenic mutations originate from the <em>CYP21A1P</em>, a neighboring pseudogene with 98% homology, due to unequal crossing over or gene conversion events. Mutation identification of the gene could be beneficial for accurate diagnosis and outcome prediction. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> Twelve unrelated patients with CAH diagnosis</span> <span style="font-size:10.0pt">were recruited for genetic counseling. To ensure distinct amplification of the <em>CYP21A2</em> gene rather than its pseudogene, the complete sequence of the gene was amplified through two overlapping fragments by specific primers. The entire sequences were screened by direct Sanger sequencing using new sequencing primers. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results</span></strong><span style="font-size:10.0pt">: Only two pathogenic point mutations were identified. The c.293-13C&gt;G, also known as In2G, and the c.955C&gt;T mutations were found in 37.5 and 33.3% of alleles, respectively. One patient showed homozygous gene deletion. </span><span style="font-size:10.0pt">We also reviewed recent reports on <em>CYP21A2</em> gene mutations in Iran.</span> </span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Evaluating the ethnicity-specific gene mutation data is significant for populations with diverse ethnic groups including the Iranian population. Although several common mutations have been reported as causative mutations among CAH patients, identifying only two common point mutations in Fars province would help prioritize exon sequencing and reduce the cost and time of genotyping.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60576 Danial Zangene, Ali Moravvej, Homa Ilkhanipoor, Anis Amirhakimi, Zhila Afshar, Mona Entezam Wed, 17 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Factor VIII as a Novel Biomarker for Diagnosis, Prognosis, and Therapy Prediction in Human Cancer and Other Disorders <p><span style="font-size:10.0pt">Coagulation factor VIII (FVIII) is an essential cofactor in the coagulation cascade, encoded by the <em>F8</em> gene on the long arm of chromosome X (Xq28). FVIII is normally circulated in complex with Von Willebrand factor (VWF) and has relevant emerging extracoagulative functions. Dysregulation of FVIII is associated with tumor progression, and could be used as a novel biomarker for tumor screening and monitoring. In breast cancer, bladder cancer, colorectal carcinoma, esophageal carcinoma, hepatocellular carcinoma and lung cancer, <em>F8</em> is regarded as an oncogene. In coronary heart disease, hemophilia A and liver disease, <em>F8</em> dysregulation has been recognized as a potential biomarker for disease diagnosis and prognosis. However, the basis of these differential expression levels remains to be understood. In this review, which is a mixture of literature review and bioinformatics analysis we described the biological functions and characteristics of FVIII, and also its expression level in non-malignant disorders and various cancers.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60569 Sheyda Khalilian , Zahra Mohajer , Soudeh Ghafouri-Fard Mon, 15 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Protective Effect of Crocin on Rat Bone Marrow Mesenchymal Stem Cells Exposed to Aluminum Chloride as an Endocrine Disruptor <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Mesenchymal Stem Cells (MSCs) have the ability to self-renew and proliferate which gives them healing properties in various tissues. Aluminium chloride (AlCl<sub>3</sub>) is a chemical compound with harmful effects on health; oxidative stress caused by Aluminium has been reported previously. Crocin, a major component of <em>Crocus sativus </em>(saffron), has antioxidant properties and has shown therapeutic potential. Researchers have been looking for ways to reduce the harmful effects of AlCl<sub>3</sub>. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> To investigate whether crocin can reduce AlCl<sub>3</sub> cytotoxicity, rat Bone Marrow Mesenchymal Stem Cells (BM-MSCs) were isolated, cultured and divided into four experimental groups. The first group was the control, which was untreated cells. The second and third groups were treated with crocin (</span><span style="font-size:10.0pt">50, 100, 250, 500 </span><em><span style="font-size:10.0pt">&micro;M</span></em><span style="font-size:10.0pt">) and AlCl<sub>3</sub> (20, 25, 30 <em>mM</em>) for 24 <em>hr</em>. The fourth group was pre-treated with crocin (</span><span style="font-size:10.0pt">250, 500 </span><em><span style="font-size:10.0pt">&micro;M</span></em><span style="font-size:10.0pt">) for 24 <em>hr</em> and then treated with AlCl<sub>3</sub> (20 <em>mM</em>) overnight. Cytotoxicity was assessed using the MTT assay. Mineralization was evaluated by alizarin red staining. Sox-2 and E-cadherin expression were measured using real-time PCR.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The results showed that AlCl<sub>3</sub> caused cytotoxicity on BM-MSCs and decreased the mRNA expression of Sox-2 and E-cadherin, which are important for the maintenance of self-renewal and proliferation of BM-MSCs. In contrast, crocin protected the self-renewal characteristic of BM-MSCs by increasing Sox-2 expression and also preserved the proliferative effects on BM-MSCs by upregulating E-cadherin expression (***p&le;0.001). </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Overall, the study suggests that crocin can protect BM-MSCs from AlCl<sub>3</sub>-induced cytotoxicity by upregulate Sox-2 expression and E-cadherin expression. This suggests that crocin may be a potential therapeutic agent for the treatment of AlCl<sub>3</sub>-induced toxicity. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60570 Elaheh Amini, Zahra Baharvand, Azadeh Niknejad, Yasaman Tabari, Sahel Shemshadi Mon, 15 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Protective Effect of N-acetylcysteine against Deltamethrin-Induced Hepatotoxicity in Mice <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Exposure to pesticides is of concern to public health officials worldwide. Deltamethrin is a synthetic <span style="color:black">pyrethroid </span>pesticide which is widely used in agriculture and veterinary medicine. Deltamethrin poisoning is always one of the concerns in medical centers</span><span style="font-size:10.0pt"> due to the </span><span style="font-size:10.0pt">deltamethrin induced </span><span style="font-size:10.0pt">hepatotoxicity.</span><span style="font-size:10.0pt"> This study evaluated the hepatoprotective effects of N-acetylcysteine (NAC) against deltamethrin induced hepatotoxicity in mice</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> A total of 40 BALB/c male mice were randomly divided into four groups; the first group was used as a control (0.5 <em>ml</em> normal saline); Groups 2-4 were treated with NAC [160 <em>mg/kg</em> Body Weight (BW)], deltamethrin (50 <em>mg/kg</em> BW), and NAC plus deltamethrin. At 1 and 24 <em>hr</em> after treatment, the animals were sacrificed and blood and liver samples were obtained for analysis and the liver/body ration, hepatic enzymes as </span><span style="font-size:10.0pt">Aspartate aminotransferase (AST), Alanine Transaminase (ALT), Alkaline phosphatase (ALP), Lactate dehydrogenase (LDH), Glutathione</span><span style="font-size:10.0pt"> (GSH) content and <span style="background-color:white"><span style="color:black">Reactive Oxygen Species</span></span> (ROS) level were measured. For comparison between more than two experimental groups, one-way ANOVA following Tukey test was used by SPSS software.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The deltamethrin significantly increased AST, ALT, ALP, and the level of ROS level at the end of 1 and 24 <em>hr</em> after treatment; while the LDH level and GSH content were decreased. Mice in the deltamethrin treated group had a higher liver/body weight ratio than in other treated groups after 24 <em>hr</em>. On the other hand, NAC in combination with deltamethrin significantly reduced the activities of AST, ALT, ALP, and increased GSH levels.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> This study demonstrated that NAC has a hepatoprotective role against deltamethrin-induced toxicity.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60571 Ali Ameri, Alireza Rahmati, Shadi Soroushfar , Mehdi Lalehzari, Tahereh Dehghani, Hamed Haghi-Aminjan, Jebreil Shamseddin, Mahmoud Omidi Mon, 15 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Simple High Yield Technique for Isolation of Wharton's Jelly-derived Mesenchymal Stem Cell <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> The isolation of Mesenchymal Stem Cells (MSCs) from various tissues is possible, with the umbilical cord emerging as a competitive alternative to bone marrow. In order to fulfill the demands of cell therapy, it is essential to generate stem cells on a clinical scale while minimizing time, cost, and contamination. Here is a simple and effective protocol for isolating MSC from Wharton&#39;s Jelly (WJ-MSC) using the explant method with various supplements. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> Utilizing the explant method, small fragments of Wharton&#39;s jelly from the human umbilical cord were cultured in a flask. The multipotency of the isolated cells, were confirmed by their differentiation ability to osteocyte and adipocyte. Additionally, the immunophenotyping of WJ-MSCs showed positive expression of CD73, CD90, and CD105, while remaining negative for hematopoietic markers CD34 and CD45, meeting the criteria for WJ-MSC identification. Following that, to evaluate cells&#39; proliferative capacity, various supplements, including basic Fibroblast Growth Factor (bFGF), Non-Essential amino acids (NEA), and L-Glutamine (L-Gln) were added to either alpha-Minimal Essential Medium (&alpha;-MEM) or Dulbecco&#39;s Modified Eagle&#39;s Medium-F12 (DMEM-F12), as the basic culture media.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> WJ-MSCs isolated by the explant method were removed from the tissue after seven days and transferred to the culture medium. These cells differentiated into adipocyte and osteocyte lineages, expressing CD73, CD90, and CD105 positively and CD34 and CD45 negatively. The results revealed that addition of bFGF to &alpha;-MEM or DMEM-F12 media significantly increased the proliferation of MSCs when compared to the control group. However, there were no significant differences observed when NEA or L-Gln were added.</span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Although bFGF considerably enhances cell proliferation, our study demonstrates that MSCs can grow and expand when properly prepared Wharton&#39;s jelly tissues of the human umbilical cord.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60572 Bahare Niknam, Arezou Azizsoltani , Neda Heidari, Samaneh Tokhanbigli, Helia Alavifard, Mahsa Haji Valili, Davar Amani, Hamid Asadzadeh Aghdaei, Seyed Mahmoud Hashemi , Kaveh Baghaei Mon, 15 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Simple Determination of Bosentan in Plasma Samples by Reversed-Phase High-Performance Liquid Chromatography <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> In order to measure the plasma levels of Losartan and Bosentan, a sensitive Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) technique was developed.</span></span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> To compare bioavailability, the Area Under the Curve (AUC), peak plasma concentration (Cmax), and time to Cmax (Tmax) were employed. The standard curve (150-2400 <em>ng/ml</em>) was linear (R<sup>2</sup>=0.999), relative errors were between 2.4 to 10.05% and the coefficient of variation (CV%) ranged from 1.52 to 10.88. A single dosage (test and reference) was used for the <em>in vivo</em> investigation, which involved 16 healthy individuals.</span></span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The AUC0-48, AUC0-, Cmax, and Tmax of the test and reference had no statistically significant differences. The C<sub>max</sub> and 95% confidence intervals of the ratio of C<sub>max</sub> of the two formulations were 0.93-0.96 and 97.6-135%, respectively. </span></span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><strong><span style="font-size:10.0pt">Conclusion:</span></strong> <span style="font-size:10.0pt">Therefore, it was established that </span><span style="font-size:10.0pt">generic Bosentan was equivalent to Bosentan from Actelion and that both medications could be regarded as equally effective in clinical settings. The blood level of Bosentan could be measured using this straightforward procedure in all hospital laboratories.</span></span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60573 Zahra Khalighi , Hori Ghaneialvar, Armin Soltani, Ali Khorshidi, Elahe Karimi, Ardeshir Moayeri, Naser Abbasi , Masoumeh Tahmasebi, Ali Aidy Mon, 15 Jan 2024 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir One-step and Rapid Identification of SARS-CoV-2 using Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> SARS-CoV-2 as the cause of novel coronavirus disease (COVID-19) is a member of the family <em>Coronaviridea</em> that has generated an emerging global health concern. Controlling and preventing the spread of the disease requires a simple, portable, and rapid diagnostic method. Today, a standard method for detecting SARS-CoV-2 is </span><span style="font-size:10.0pt">quantitative real-time reverse transcription PCR, which is time-consuming and needs an advanced device. </span><span style="font-size:10.0pt">The aim of this study was to evaluate a faster and more cost-effective field-based testing method at the point of risk. We utilized a one-step RT-LAMP assay and developed, for the first time, a simple and rapid screening detection assay targeting the Envelope (<em>E</em>) gene, using specific primers. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> For this,</span><span style="font-size:10.0pt"> the total </span><span style="font-size:10.0pt">RNA was extracted from respiratory samples of COVID-19</span><span style="font-size:10.0pt"> infected </span><span style="font-size:10.0pt">patients </span><span style="font-size:10.0pt">and </span><span style="font-size:10.0pt">applied to one-step a </span><span style="font-size:10.0pt">RT-LAMP</span><span style="font-size:10.0pt"> reaction.</span> <span style="font-size:10.0pt">The LAMP products were visualized using green fluorescence (SYBR Green I). Sensitivity testing was conducted using different concentrations of the designed recombinant plasmid (TA-E) as positive control constructs. Additionally, selectivity testing was performed using the influenza H1N1 genome. Finally, the results were compared using with conventional real time RT-PCR. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> It was shown that the RT-LAMP assay has a sensitivity of approximately 15 <em>ng</em> for the <em>E</em> gene of SARS-CoV-2 when using extracted total RNA. Additionally, a sensitivity of 112 <em>pg</em> was achieved when using an artificially prepared TA-E plasmid. Accordingly, </span><span style="font-size:10.0pt">for the detection of </span><span style="font-size:10.0pt">SARS-CoV-2 </span><span style="font-size:10.0pt">infection,</span><span style="font-size:10.0pt"> the </span><span style="font-size:10.0pt">RT-LAMP had high sensitivity and specificity and also could be an alternative method for real-time RT-PCR.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Overall, </span><span style="font-size:10.0pt">this method can be used as a portable, rapid, and easy method for detecting </span><span style="font-size:10.0pt">SARS-CoV-2 </span><span style="font-size:10.0pt">in the field and clinical laboratories.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60560 Mohammad Shoushtari, Mehdi Zeinoddini, Javad Fathi, Hani Keshavarz Alikhani, Fatemeh Shiekhi Mon, 04 Dec 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Recombinant Production of TP4-LYC1, A New Chimeric Peptide with Targeted Cytotoxicity to HeLa Cells <p style="text-align:justify"><span style="font-size:11pt"><span style="color:black"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Tilapia Piscidin 4 (TP4) showed potential anti-tumor effects against various cancer cells. Lycosine-1 (LYC1), is another </span><span style="font-size:10.0pt"><span style="background-color:white">Antimicrobial Peptides</span></span><span style="font-size:10.0pt"> (AMP) from spider venom with targeted penetration to cancer cells without any adverse effects on normal cells. The aim of this study was to produce a soluble recombinant fusion peptide in order to diminish the cytotoxicity of TP4 against normal cells. </span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="color:black"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> In order to express of TP4-LYC-1, TP4, and LYC1 in fusion to the inteins1/2 of pTWIN-1 vector, induction condition was optimized to earn soluble peptides. Auto-cleavage induction of inteins1/2 was performed based on IMPACT<sup>&reg;</sup> manual and their effect on cell viability of HeLa and HUVEC cells was surveyed by MTT assay. </span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="color:black"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The best condition for accessing the most soluble peptide in fusion to the inteins was approximately similar for all three peptides (0.1 <em>mM</em> of IPTG, at 22<em>&deg;C</em>). After the induction of self-cleavage of inteins, a band in 3, 3, and 6 <em>kDa</em> was observed on tricine-SDS-PAGE. The IC50 values of TP4-LYC1 and TP4 against HeLa cells were calculated as 0.83, and 2.75 <em>&micro;M</em>, respectively. </span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="color:black"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> In the present study, a novel chimeric peptide, TP4-LYC1, was successfully produced. This fusion protein can act as a safe bio-molecule with potent cytotoxic effects against cancer cells, but the penetration ability and determination of cell death mechanism must be performed in order to have more precise view on the apoptosis induction of this recombinant peptide.&nbsp;</span></span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60561 Hanieh Mohammad Pour, Ali Jahanian-Najafabadi, Fatemeh Shafiee Mon, 04 Dec 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Intra-ovarian Injection of Mesenchymal Stem Cells or its Conditioned Media on Repeated OPU-IVEP Outcomes in Jersey Heifers and Its Relationship with Follicular Fluid Inflammatory Markers <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Repeated Ovum Pick Up (OPU) could have a detrimental effect on ovarian function, reducing <em>In Vitro</em> Embryo Production (IVEP). The present study examined the therapeutic effect of adipose&ndash;derived Mesenchymal Stem Cells (MSCs) or its Conditioned Medium (ConM) on ovarian trauma following repeated OPU. Resolvin E1 (RvE1) and Interleukin-12 (IL-12) were investigated as biomarkers.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> Jersey heifers (n=8) experienced 11 OPU sessions including 5 pre-treatment and 6 treatment sessions. Heifers received intra-ovarian administration of MSCs or ConM (right ovary) and Dulbecco&rsquo;s Modified Phosphate Buffer Saline (DMPBS; left ovary) after OPU in sessions 5 and 8 and 2 weeks after session 11. The concentrations of RvE1 and IL-12 in follicular fluid was evaluated on sessions 1, 5, 6, 9, and 4 weeks after session 11. Following each OPU session, the IVEP parameters were recorded.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> Intra-ovarian administration of MSCs, ConM, and DMPBS did not affect IVEP parameters (p&gt;0.05). The concentration of IL-12 in follicular fluid increased at the last session of pre-treatment (Session 5; p&lt;0.05) and remained elevated throughout the treatment period. There was no correlation between IL-12 and IVEP parameters (p&gt;0.05). However, RvE1 remained relatively high during the pre-treatment and decreased toward the end of treatment period (p&lt;0.05). This in turn was associated with decline in some IVEP parameters (p&lt;0.05).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Intra-ovarian administration of MSCs or ConM during repeated OPU did not enhance IVEP outcomes in Bos taurus heifers. The positive association between RvE1 and some of IVEP parameters could nominate RvE1 as a promising biomarker to predict IVEP parameters following repeated OPU. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60562 Ali Sarvari, Amir Niasari-Naslaji, Abolfazl Shirazi, Banafsheh Heidari, Sara Borjian Boroujeni, Mohammad Hossein Moradi, Mohammad Mehdi Naderi, Bahareh Behzadi, Mohammad-Mahdi Mehrazar, Mohammad Mehdi Dehghan Mon, 04 Dec 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Orexin-1 Receptor Antagonist SB-334867 Enhances Formalin-Induced Nociceptive Behaviors in Adult Male Rats <p style="text-align:justify"><span style="font-size:16pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Orexin (hypocretin) is one of the hypothalamic neuropeptides that plays a critical role in some behaviors including feeding, sleep, arousal, reward processing, and drug addiction. Neurons that produce orexin are scattered mediolaterally within the Dorsomedial Hypothalamus (DMH) and the lateral hypothalamus. In the current research, we assessed the impact of prolonged application of the antagonist of Orexin Receptor 1 (</span><span style="font-size:10.0pt">OXR1) on nociceptive behaviors in adult male rats. </span></span></p> <p style="text-align:justify"><span style="font-size:16pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> Sixteen Wistar rats received subcutaneous (s.c.) injections of the OXR1 antagonist, SB-334867 (20 <em>mg/kg</em>, <em>i.p</em>.), or its vehicle repetitively from Postnatal Day 1 (PND1)-PND30. On the 30<sup>th</sup> day following the final application of the </span><span style="font-size:10.0pt">OXR1</span><span style="font-size:10.0pt"> antagonist formalin-provoked pain was evaluated by injecting formalin. </span></span></p> <p style="text-align:justify"><span style="font-size:16pt"><strong><span style="font-size:10.0pt">Results:</span></strong> <span style="font-size:10.0pt">Administration of the OXR1 antagonist in the long-term augmented the formalin-provoked nociceptive behaviors in interphase and phase II of the formalin-induced pain. </span></span></p> <p style="text-align:justify"><span style="font-size:16pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong> <span style="font-size:10.0pt">Current results showed that the continued inhibiting OXR1 might be implicated in formalin-induced nociceptive behaviors. Therefore, the present study highlighted the effect of orexin on analgesia.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60563 Masoumeh Kourosh-Arami, Alireza Komaki, Masoumeh Gholami Mon, 04 Dec 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Annexin-A5 Overexpression Increases Sensitivity of MCF-7 and MCF-7/ADR Cells to Epirubicin <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Multi-drug resistance is an important challenge in the chemotherapy of cancer. The role of annexin A5 (ANXA5) in the biology of cancer has been the focus of many studies. Breast Cancer (BC) is frequent cancer in women with high morbidity and mortality rate. The present study aimed to investigate the effects of ANXA5 overexpression on the anti-tumor activity of Epirubicin (EPI) in MCF-7 and MCF-7/ADR cells.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> MCF-7 and MCF-7/ADR cells </span><span style="font-size:10.0pt">were transfected with the </span><span style="font-size:10.0pt">pAdenoVator-CMV-ANXA5-IRES-GFP</span><span style="font-size:10.0pt"> plasmid or mock plasmid. The overexpression of ANXA5 was evaluated using qPCR. The effects of ANXA5 overexpression and EPI on the cell viability of </span><span style="font-size:10.0pt">MCF-7 and MCF-7/ADR cells </span><span style="font-size:10.0pt">were measured using an MTT assay. Cell apoptosis was measured by annexin V/7-AAD flow cytometry assay. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong> <span style="font-size:10.0pt">Following the overexpression of ANXA5, the viability of MCF-7 and MCF-7/ADR was significantly decreased. Furthermore, the overexpression of ANXA5 in MCF-7 cells increased the cytotoxic effects of EPI in all doses and reduced the IC50 of EPI from 17.69 <em>&micro;M</em> to 4.07 <em>&micro;M</em>. Similarly, the overexpression of ANXA5 in MCF7-ADR cells reduced the IC50 of EPI from 27.3 <em>&micro;M</em> to 6.69 <em>&micro;M</em>. ANXA5 overexpression alone or combined with EPI treatment increased the apoptosis of MCF7 and MCF7-ADR cells.&nbsp; </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> The results of the present study demonstrate that ANXA5 overexpression increases the sensitivity of MCF-7 and MCF-7/ADR to EPI, suggesting a possible beneficial role of ANXA5 in the therapy of BC. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60564 Mahshad Ghasemi, Niloofar Reiazi , Abbas Behzad-Behbahani, Mohammad Ali Takhshid Mon, 04 Dec 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Extracellular L-Asparaginase Synthesis Bacillus niacin Isolation, Optimization, and Characterization from Marine Saltern Sediment Sources <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Asparagine is an amino acid that can be converted into aspartic acid and ammonia by the enzyme L-asparaginase. Some forms of cancer, such Acute Lymphoblastic Leukaemia (ALL) and Non-Hodgkin Lymphoma (NHL), respond well to this enzyme when employed as a chemotherapeutic drug. The purpose of this research was to find bacteria that can manufacture the enzymes L-asparaginasein marine slattern sediment which can be employed in commercial and industrial scale production. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> All of the strains were identified as <em>Bacillus </em></span><em><span style="font-size:10.0pt">niacini</span></em><em><span style="font-size:10.0pt"> spp</span></em><span style="font-size:10.0pt">. by biochemical and molecular testing. The strain belongs to the <em>Bacillus</em> genus, according to nutritional, biochemical, PCR and 16srRNA sequencing data. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> According to the findings of this research, <em>Bacillus niacin spp.</em> have the potential to create a substance that is helpful in a variety of medical applications. The results of this study hint to the possibility that bacteria have the ability to produce antimicrobial compounds, which have the potential to be successful in a wide variety of environments.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Numerous opportunities may arise for researchers interested in utilizing the medical potential of enzyme-producing bacteria if they are successfully isolated and screened from aquatic and terrestrial habitats. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60565 Mugip Rahaman Abdul Wahab , Thirunavukkarasu Palaniyandi , John Wyson , Asha Sivaji, Swarnakala Thamada Mon, 04 Dec 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Anti-Quorum Sensing and Anti-Biofilm Activity of Ginger (Zingiber officinale) Rhizomes against Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> The aim of this study was to determination of Anti-Quorum Sensing (AQS) and anti-biofilm potential of the methanol extract of ginger (<em>Zingiber officinale</em>) rhizomes against multidrug-resistant clinical isolates of <em><span style="background-color:white"><span style="color:black">Pseudomonas aeruginosa</span></span></em><em> </em>(<em>P. aeruginosa</em>). </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> The AQS activity of ginger was determined against <em>Chromobacterium violaceum</em> (<em>C. violaceum</em>) ATCC 12472 (CV12472), a biosensor strain, in qualitative manner using the agar well diffusion method. The violacein pigment inhibition was assessed to confirm AQS activity of ginger. The AQS potential of sub-minimum Inhibitory Concentrations (sub-MICs) of the ginger extract was determined by targeting different QS regulated virulence factors, including swarming motility (using swarm diameter measurement method), pyocyanin pigment (using chloroform extraction method), Exopolysaccharide (EPS) (using phenol-sulphuric acid method), and biofilm formation (using microtiter plate assay), against clinical isolates (CIs 2, 3, and 4) and standard reference strain of <em>P. aeruginosa </em>(PA01).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The AQS activity of methanol extract of ginger was confirmed against <em>C. violaceum</em> (CV12472) as inhibition of violacein pigment formation without effecting the growth of CIs and PA01 of <em>P. aeruginosa</em>. The ginger extract exhibited concentration-dependent inhibition of virulence factors and biofilm formation. The maximum reduction was found in swarming motility, pyocyanin, EPS and biofilm formation against PA01 (51.38%), CI3 (57.91%), PA01 (63.29%) and CI2 (64.37%), respectively at 1/2 MIC of ginger extract. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> The results of present study revealed the effective AQS and anti-biofilm potential of <em>Zingiber officinale</em> rhizome methanol extract at a reduced dose (sub-MICs). The extract may be explored <span style="color:black">as an agent of antimicrobial compounds having AQS and anti-biofilm activity for controlling microbial infection </span>and also for reducing the chances of emergence of resistance in <em>P. aeruginosa. </em>&nbsp;</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60566 Pankaj Kumar Sagar , Poonam Sharma , Rambir Singh Mon, 04 Dec 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Green Tea Extract Reduced Lipopolysaccharide-Induced Inflammation in L2 Cells as Acute Respiratory Distress Syndrome Model Through Genes and Cytokine Pro-Inflammatory <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Acute Respiratory Distress Syndrome (ARDS) is a severe lung inflammatory condition that has the capacity to impair gas exchange and lead to hypoxemia. This condition is found to have been one of the most prevalent in patients of COVID-19 with a more serious condition. Green tea (<em>Camellia sinensis L.</em>) contains polyphenols that possess many health benefits. The purpose of this study was to assess the anti-inflammatory activities of green tea extract in Lipopolysaccharide (LPS)-induced lung cells as ARDS cells model. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> In this study, rat lung cells (L2) were induced by LPS to mimic the inflammation observed in ARDS and later treated with green tea extract. Pro-inflammatory cytokines such as Interleukin (IL)-12, C-Reactive Protein (CRP) as well as Tumor Necrosis Factor-&alpha; (TNF-&alpha;) were investigated using the ELISA method. Gene expression of NOD-Like Receptor Protein 3 (<em>NLRP-3</em>), Receptor for Advanced Glycation End-product (RAGE), Toll-like Receptor-4 (<em>TLR-4</em>), and Nuclear Factor-kappa B (<em>NF-&kappa;B</em>) were evaluated by qRTPCR. Apoptotic cells were measured using flow cytometry. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The results showed that green tea extract treatment can reduce inflammation by suppressing gene expressions of <em>NF-&kappa;B, NLRP-3, TLR-4</em>, and <em>RAGE</em>, as well as pro-inflammatory cytokines such as IL-12, TNF-&alpha;, and CRP, an acute phase protein. Apoptosis levels of inflamed cells also found to be lowered when green tea extract was administered; thus, also increasing live cells compared to non-treated cells. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> These findings could lead to the future development of supplements from green tea to help alleviate ARDS symptoms, especially during critical moments such as the current pandemic.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60567 Didik Priyandoko , Wahyu Widowati, Lenny Lenny , Sintya Novianti, Revika Revika, Hanna Sari Widya Kusuma, Ika Adhani Sholihah Mon, 04 Dec 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Novel Osteoporosis Therapeutic Targets Derived from Medical Biotechnology <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Osteoporosis is known as the most prevalent metabolic bone disease. Population aging and increasing life span are making osteoporosis one of the most challenging age-related diseases </span><sup><span style="font-size:10.0pt">1</span></sup><span style="font-size:10.0pt">. Based on reports in 2009, over 500 million people are struggling with this disease. Also, 1 in 5 men and 1 in 3 women over 50 years suffer from osteoporotic fractures during their lifetime. 1.5 million osteoporotic fractures are occurred annually in the United States and it cost the healthcare system in this country about $57 billion in 2018. The disease is caused by an imbalance in bone homeostasis, which is induced by bone formation not compensating for bone resorption. This process lowers the Bone Mineral Density (BMD) and makes bone susceptible to fracture </span><sup><span style="font-size:10.0pt">2,3</span></sup><span style="font-size:10.0pt">.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The significant burden of osteoporosis can be decreased since the disease is treatable. Based on the mechanisms resulting in osteoporosis, two main conventional drug categories are used for the treatment of the disease including anti-resorptive agents and anabolic agents </span><sup><span style="font-size:10.0pt">2</span></sup><span style="font-size:10.0pt">. Conventional treatments consist of Selective Estrogen-Receptor Modulators (SERMs), bisphosphonates, parathyroid hormone analogs, and calcitonin. The need for more advanced therapies with fewer adverse effects arises from complications such as increasing risk of cardiovascular diseases, gastrointestinal problems, altering blood calcium levels, potential risks for women in estrogen-related therapies, <em>etc</em>. </span><sup><span style="font-size:10.0pt">4,5</span></sup><span style="font-size:10.0pt">.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">As mentioned previously, the imbalance of bone metabolism causes osteoporosis. In this dynamic complex process of metabolism which is known as bone remodeling, connection or disruption of each specific intra-cellular or inter-cellular link can cause or treat the disease </span><sup><span style="font-size:10.0pt">6</span></sup><span style="font-size:10.0pt">. Hence, this fact represents the remarkable role of medical biotechnology in osteoporosis treatment. Medical biotechnology as an upcoming main pillar of health-related science, has grown so fast in the field of treating disease as well as prevention and diagnosis using a variety of novel approaches </span><sup><span style="font-size:10.0pt">7</span></sup><span style="font-size:10.0pt">. In recent years, biotechnology has introduced some novel therapeutic targets to the medical world to provide more efficient and safe therapies followed by diminishing osteoporosis as a burdensome disease. Monoclonal RANK Ligand (RANKL) antibodies and monoclonal sclerostin antibodies including denosumab, romosozumab, and blosozumab as recently rendered treatments to medicine, have achieved better therapeutic outcomes than conventional treatments </span><sup><span style="font-size:10.0pt">2</span></sup><span style="font-size:10.0pt">.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">One of the main novel therapeutic targets is microRNA (miRNA)-based treatment. Modified nucleoside oligomers are the most common miRNA inhibitors used for new approaches. Since the clinical transformation of miRNA inhibitors is less difficult than lentivirus transfection, using them to affect the progress of osteoporosis is more feasible. Cathepsin K inhibitors which are involved in bone resorption in addition to remodeling, have been studied in many trials and are being investigated to find a promising one </span><sup><span style="font-size:10.0pt">8</span></sup><span style="font-size:10.0pt">. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Another innovation is stem cell therapy which is performed with cell sources from Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs), and Adult Stem Cells (ASCs). Stem cell therapy provides tissue regeneration which in osteoporosis translates to bone formation. Inducing the growth and differentiation of osteoblasts alongside decreasing activity of osteoclasts via cellular crosstalk are the main roles of stem cell therapy </span><sup><span style="font-size:10.0pt">4</span></sup><span style="font-size:10.0pt">. The Wnt signaling pathway components are recently presented as one of the therapeutic targets. So many clinical trials are being conducted for each component to benefit from the bone mass-maintaining ability of these molecules </span><sup><span style="font-size:10.0pt">9</span></sup><span style="font-size:10.0pt">. The sphingosine-1-phos-phate (S1P) signaling pathway and leucine zipper motif (APPL1) extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and the Bone Morphogenetic Protein (BMP) signaling pathway are other targets to point out that play roles in osteogenesis, conducting osteogenic differentiation of stem cells, and promoting angiogenesis </span><sup><span style="font-size:10.0pt">5</span></sup><span style="font-size:10.0pt">. A novel RANKL i-body nominated as ADR3 was introduced with features such as a high affinity for binding to human RANKL (hRANKL) and an ability to tolerate many different physical environments </span><sup><span style="font-size:10.0pt">3</span></sup><span style="font-size:10.0pt">. Also, integrins as cell-adhesion transmembrane molecules have improved postmenopausal women&rsquo;s BMD and made bone loss reversed </span><sup><span style="font-size:10.0pt">6</span></sup><span style="font-size:10.0pt">. Furthermore, in recent years </span><span style="font-size:10.0pt">light has been shed on the bone-related roles of melatonin. Some of its important effects are bone biological rhythm regulatory effects, bone microenvironment modulation, and osteoporosis treatment </span><sup><span style="font-size:10.0pt">10</span></sup><span style="font-size:10.0pt">.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Population aging is greatly increasing osteoporosis prevalence and burden thus promoting osteoporosis to become an immense concern for the healthcare system. Hence, researching more effective therapies with fewer side effects has become urgent. Although studies introduced innovative targets as effective therapies, more investigations such as high-quality clinical trials are necessary to provide more evidence proving their efficiency. Despite all the mentioned conventional treatments, innovative targets, and upcoming therapeutic strategies, studying and developing molecular targets with more accurate and detailed mechanisms is indispensable. </span><span style="font-size:10.0pt">Undoubtedly, cooperation between basic sciences, especially neuroscience and biotechnology, and internal medicine can create a brighter future for the treatment of endocrine diseases <sup>11-14</sup>.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60559 Sepideh Hajivalizadeh , Shahin Akhondzadeh Wed, 15 Nov 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cardiovascular Considerations in Antidepressant Use <div style="text-align:justify"><span style="font-size:12pt"><span style="color:#000000">Depressive disorders are among the most prevalent disorders worldwide. About 1 in every 5 people experience an episode of depression in their lifetime (1,2). Therefore, antidepressants are among the most frequently prescribed medications worldwide. An analysis of a primary care database in the United Kingdom revealed that 23% of patients were ordered to take antidepressants at least once over the course of 17 years (1995-2011) (3). </span><span style="color:black">Antidepressants are categorized into some major groups: 1. Selective Serotonin Reuptake Inhibitors (SSRIs) such as sertraline, fluoxetine, citalopram and escitalopram, 2. Serotonin and Norepinephrine Reuptake Inhibitors (SNRIs) such as duloxetine and venlafaxine, 3. Tricyclic Antidepressants (TCAs) such as amitriptyline and nortriptyline, 4. Monoamine oxidase inhibitors (MAOIs) such as selegiline and 5. Atypical antidepressants such as bupropion and mirtazapine. Despite the production of newer antidepressants, patients taking these medications still experience some cardiovascular adverse effects.</span></span></div> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black">One of the most well-known cardiovascular adverse effects of antidepressants is QT-prolonging. QT-prolonging can lead to fatal ventricular arrhythmias like Torsades de pointes (TdP) and precipitate sudden death. In 2011, the US Food and Drug Administration (FDA) cautioned healthcare professionals about QT-prolongation associated with high doses of citalopram (4). This encouraged various research groups to examine the safety of other antidepressants with respect to QT interval. Studies manifest that while TCAs are not associated with sudden death unless at higher doses; SSRIs are associated with a higher risk of sudden death specifically in adults with cardiovascular comorbidities (5). A meta-analysis revealed that among SSRIs, significant QT prolongations have been demonstrated in using citalopram and escitalopram; whereas fluoxetine, fluvoxamine, and sertraline did not show a clinically significant increase in QT interval at conventional doses in the majority of the studies (6). On the other hand, paroxetine monotherapy showed no QT prolongation in any of the studies (6-8).</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black">While SSRIs are mostly accused of causing QT prolongation; the most common cardiovascular complications caused by high doses of TCA are sinus tachycardia and hypotension (9,10). Sinus tachycardia, seen in 52% of cases with TCA overdose (11), is a result of anticholinergic activity and norepinephrine uptake inhibition by TCAs. Hypotension, on the other hand, happens due to a combination of depressed myocardial contractility and decreased resistance of blood vessels caused by </span><span style="color:black">a</span><span style="color:black">-adrenergic blockage (10). In contrast to TCAs, SNRIs like venlafaxine can cause hypertension. A pooled analysis of controlled studies showed that while venlafaxine has a low risk of causing clinically significant hypertension at doses &lt;200 <em>mg</em>/day; 5.5% of patients using doses &gt;200 <em>mg</em>/day show significant increases in blood pressure (12).</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black">Another issue to be considered is the possibility of cardiovascular birth defects caused by maternal exposure to antidepressants during pregnancy. Maternal exposure to antidepressants 3 months prior to pregnancy or during early pregnancy increases the risk of congenital heart diseases in the newborn (13). A study by Gao <em>et al</em> indicated that newborns with intrauterine exposure to fluoxetine are at a greater risk for cardiovascular defects, especially septal defects(14). Persistent Pulmonary Hypertension of the Newborn (PPHN) is another complication caused by maternal SSRI exposure. research has shown higher rates of PPHN among infants who had intrauterine exposure to SSRIs (15); however, sertraline was proven to be the safest SSRI in this regard (16).</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black">Despite the facts regarding mentioned side effects, there are some evidence showing that antidepressants can be beneficial for the cardiovascular system in some manners. SSRIs are manifested to improve endothelial function, vascular inflammation, arterial stiffening and perhaps delaying atherosclerotic events (17,18). Moreover, antidepressant therapy was associated with lower odds of recurrent Myocardial Infarction (MI) in patients with acute coronary syndrome and concomitant depression (19). With those being said, it is crucial not to deprive depressed patients of their adequate drug therapy and to modify their medications based on cardiovascular safety along with the optimal efficacy of the drug.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60551 Fateme TaghaviZanjani , Saeed Nateghi Sat, 19 Aug 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Radiotherapy Combination: Insight from Tumor Immune Microenvironment (TIME) <p><span style="font-size:10.0pt">The view of Radiotherapy (RT) as a simple inducer of DNA damage resulting in tumor cell death has dramatically changed in recent years, and it is now widely accepted that RT can trigger an immune response which provides a sound basis for combining RT with immunotherapy. Given that, radiation can be delivered with different regimens, its effect on immune responses and Tumor Immune Microenvironment (TIME) may vary with dose and fractionation schedule. This fractional dose dependency may need to be more considered because of recent developments in RT delivery techniques making it possible to deliver precisely higher dosages per fraction (hypofractionation) while reducing exposure to normal tissues. Although combining radiotherapy with immunotherapy could be a promising strategy for synergistic enhancement of treatment efficacy, the selection of the best-matched combination of immunotherapy with each radiotherapy scheme remains to be addressed. Thus, for designing better therapeutic combinations, it is necessary to understand the immunological effects of RT. Here, we review the impact of conventional and different hypofractionation radiation schedules on the TIME. Subsequently, we highlight how knowing about these interactions may have implications </span><span style="font-size:10.0pt">for choosing a rational combination with targeted therapies.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60552 Masoumeh Alimohammadi , Haniyeh Ghaffari-Nazari , Reza Alimohammadi, Mohsen Bakhshandeh, Nima Rezaei, Seyed Amir Jalali Sat, 19 Aug 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Gypenosides Production and Spermatogenesis Recovery Potentials of Extracts from Cell Suspension Cultures of Gynostemma pentaphyllum <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> <em>Gynostemma pentaphyllum (GP)</em>, also called Giao-co-lam, is a traditional Vietnamese herb, also known as the &quot;Herb of Immortality&quot;, that grows throughout Asian countries and is used for the treatment of hematuria, edema in the pharynx and neck, tumors, and trauma.</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> In this study, we investigated the effects of culture conditions on cell growth and total gypenoside accumulation in the GP suspension cells. Cells were cultured on Murashige and Skoog<span style="background-color:white"><span style="color:black">&nbsp;</span></span>(MS) medium supplemented with 2.0 <em>mg/L</em> KIN and 0.5 <em>mg/L</em> IBA</span><span style="font-size:10.0pt">,</span><span style="font-size:10.0pt"> and different inoculum sizes (2-4 <em>g</em>) for 10-24 days. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The results showed that the optimum inoculum size and shaking speed were 3 <em>g</em> of callus and 120 <em>rpm</em>, respectively. The highest cell biomass reached was</span> <span style="font-size:10.0pt">5.833 <em>g </em>of fresh weight, corresponding to 0.136 <em>g</em> of dry weight after 20 days of culture. The</span><span style="font-size:10.0pt"> t</span><span style="font-size:10.0pt">otal gypenoside and Rb1 accumulation was</span><span style="font-size:10.0pt"> the highest</span><span style="font-size:10.0pt"> after 18 days of culture, with 46.498 <em>mg/g</em> and 0.038 <em>mg/g</em> dry weight, respectively. In addition, the crude extract from GP cell suspension cultures remarkably improved pathological changes in mouse testicular tissue after scrotal heat exposure. Blood testosterone levels</span><span style="font-size:10.0pt"> were</span><span style="font-size:10.0pt"> significantly increased in heat-exposed mice treated with the</span> <span style="font-size:10.0pt">GP cell suspension culture extract. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Taken together, these results suggest that GP bio-mass production by cell suspension cultures leads to the accumulation of gypenosides in large amounts</span><span style="font-size:10.0pt">,</span><span style="font-size:10.0pt"> and provides the potential for the</span> <span style="font-size:10.0pt">recovery of</span> <span style="font-size:10.0pt">spermatogenesis following heat stress.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60553 Tung Nguyen-Thanh , Sang Dang-Ngoc , Dung Tran-Quoc, Quang Hoang-Tan Sat, 19 Aug 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Activity of Citrus aurantium and Lavandula angustifolia in Alzheimer’s Disease Symptoms in Male Wistar Rats <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Alzheimer&#39;s Disease (AD) is one of the most prevalent chronic neurodegenerative disorders. The present study aims&nbsp;to better understand the mechanism by which<em> Citrus aurantium</em> (<em>C. aurantium)</em> and <em>Lavandula angustifolia</em> (<em>L. angustifolia)</em> hydro&ndash;alcoholic extracts were used to treat AD and anti&shy;&ndash;oxidant issues in a laboratory model. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> 15 male Wistar rats, weighing 250&plusmn;20 <em>gr</em>, aged 6&ndash;8 weeks, were used. Amyloids in the brain were found and identified using the shuttle box and Congo red test. ELISA testing for norepinephrine and serotonin, Superoxide Dismutase (SOD), Malondialdehyde (MDA), and Real&ndash;time PCR for expression of the <em>APP</em> and <em>GLT1</em> genes were done. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The shuttle box test demonstrated that AD produces behavioral harm, since it significantly reduces passive avoidance learning. The Congo red test revealed that the AD models had much more amyloid beta in their brain tissue than the control. Norepinephrine levels were also decreased by using both extracts in test group. Treatment with both extracts led to a substantial rise in SOD activity and fall in MDA concentration. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> The gene expression data indicated that the relative expression of the <em>APP</em> and <em>GLT1</em> genes was shown to be lower in the groups treated with both extracts. <em>C. aurantium</em> and <em>L. angustifolia</em> may therefore offer a multi&ndash;target treatment strategy for AD, which calls for more research in this area. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60554 Amir Arasteh, Morteza Karimpour, Faezeh Fallah, Sara Kiani, Maedeh Kakavan Sat, 19 Aug 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Transient Co-Expression of Bioactive Murine Interferon-Gamma and HBsAg in Tobacco and Lettuce Leaves <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> The synchronous expression of antigen and adjuvant proteins in plant hosts presents an intriguing potential for vaccine production and the enhancement of appropriate immune responses. In this study, we examined the expression of bioactive murine interferon-gamma (mIFN-&gamma;) along with HBsAg in tobacco and lettuce leaves aimed to further perform the analysis of immune responses in the mouse model. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> Monocistronic and bicistronic cassettes, carrying genes encoding mIFN-&gamma; and HBsAg in various orders, were constructed. These cassettes were placed under the control of the 35S CaMV promoter and included the 5ʹ leader sequence of Tobacco Ech Virus (TEV). Through Agrobacterium infiltration, the cassettes were transferred into plant leaves. The concentration of mIFN-&gamma; in different constructs and HBsAg was tested by ELISA. Murine IFN-&gamma; was characterized through Western blotting, and its bioactivity was evaluated by assessing the up-regulation of MHC class II in macrophages derived from mouse bone marrow.</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> Extracts of agroinfiltrated leaves contained recombinant mIFN-&gamma; and HBsAg proteins at about 14 </span><em><span style="font-size:10.0pt">unit/mg</span></em><span style="font-size:10.0pt"> and 50 </span><em><span style="font-size:10.0pt">ng/mg</span></em><span style="font-size:10.0pt"> of soluble protein, respectively. Subsequently, mIFN-&gamma; was purified from the plant extract and its ability to up-regulate MHC class II in mouse bone marrow-derived macrophages was confirmed by immunofluorescence. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> The co-expression of recombinant HBsAg and mIFN-&gamma; using TEV 5ʹ leader-based cassettes in tobacco and lettuce leaves produced both proteins with active mIFN-&gamma; in different concentrations. The attractive utility and feasibility of using plant transient co-expression systems aimed to co-delivery of vaccine antigen and appropriate cytokine to elicit immune response for different applications.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60555 Sara Mohammadzadeh, Mahshid Amiri, Parastoo Ehsani Sat, 19 Aug 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of Catechol-O-Methyl-Transferase and Estrogen Receptors Polymorphism with Severity of Temporomandibular Disorder in Iranian Patients <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> There are many studies which strongly suggest that the pathophysiology of Temporomandibular joint Disorder (TMD) may also be influenced by genetic conditions. The current study was aimed to evaluate the hypothesis that the polymorphism of estrogen receptor genes, estrogen receptor 1 and 2 (<em>ESR1</em> and <em>ESR2</em>), and the gene Catechol -O-Methyl-Transferase (<em>COMT</em>) could be Predisposing factor for TMD.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods</span></strong><strong><span style="font-size:10.0pt">:</span></strong><span style="font-size:10.0pt"> In this case-control study, blood sample were taken from 100 TMD diagnosed patients based on </span><span style="font-size:10.0pt">Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) </span><span style="font-size:10.0pt">and 103 healthy individuals as the control group. Tetra ARMS-PCR method was used to amplify and identify <em>COMT</em> rs4680,</span> <em><span style="font-size:10.0pt">ESR1</span></em><span style="font-size:10.0pt"> rs1643821, and <em>ESR2 </em>rs1676303 gene polymorphism. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> <em>ESR1</em> genotype AA and GA showed significantly increase probability (OR= 4.80, OR=2.98, respectively) of TMD. <em>ESR2</em> T/T homozygosity was associated with decreased risk for TMD (OR=0.41). The relationship between <em>COMT</em> and TMD was not statistically significant (p&gt;00.05). The relationship between the severity of TMD and <em>ESR1</em> was significant (p=0.003). </span><span style="font-size:10.0pt">According to the inheritance pattern the <em>COMT</em> and <em>ESR1</em> gene, in the dominant pattern can be susceptible to TMD and in <em>ESR2</em> gene, in the recessive pattern can be protective to TMD.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> It seems that SNPs of <em>ESR1</em> rs1643821 has a susceptible role and <em>ESR2</em> rs1676303 has a protective role against TMD. </span><span style="font-size:10.0pt">Also, we add evidences that various genotype of <em>COMT</em> rs4680 were not statistically different between case and control, </span><span style="font-size:10.0pt">but allele A in the dominant inherence pattern can be susceptible to TMD. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60556 Hassan Roudgari, Shamsolmoulouk Najafi, Sheyda Khalilian , Zahra Ghafarzadeh, Aida Hahakzadeh, Sheida Behazin, Nafiseh Sheykhbahaei Sat, 19 Aug 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association between PON1-rs662 Gene Polymorphism and Diabetic Retinopathy in Population of the Qom, Iran <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Diabetic retinopathy is the most severe diabetic microvascular complication that causes changes in the vessel wall. One of the genes involved in this disease is <em>PON1</em>, which encodes paraoxanase1 protein in liver and kidney. It might regulate inflammatory and microvascular responses to the disease. The rs662 T</span><span style="font-size:10.0pt">&gt;</span><span style="font-size:10.0pt">C is one of the single nucleotide polymorphisms of this gene that changes glutamine to arginine at position 192. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> In this study, 300 samples were collected, including 100 healthy and 100 diabetics without retinopathy, and 100 diabetics retinopathies were studied and their age range was from 30 to 80 years. Then 2.5 <em>ml </em>of blood was collected from all relevant individuals in tubes containing EDTA<sub>Na2</sub>. This polymorphism was examined by <em>tetra</em>-ARMS PCR. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> <span style="color:black">Results showed that there is no significant correlation between genotypes and alleles related to PON1 and Diabetes </span>(CC genotype: p=0.609; C allele: p=0.228). On the other hand, an association was observed between PON1 and diabetic retinopathy (CT+CC genotype: p&lt;0.001; CT allele: p&lt;0.001). Considering that the Polyphen database examined the changes caused by replacing the amino acid arginine instead of glutamine at position 129 on the protein, it does not consider these changes dangerous and has introduced this polymorphism as benign. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Based on the findings of this study, the rs662 locus could be considered as one of the molecular markers in future research. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60557 Fateme Sabbaghian Bidgoli, Abasalt Hosseinzadeh Colagar, Roohollah Nakhaei Sistani, Majid Tafrihi Sat, 19 Aug 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of Seroconversion Rate Following SARS COV 2 Vaccination in Health Care Workers at Shahid Beheshti University of Medical Sciences <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Vaccines are the most effective way to prevent Coronavirus 2 severe acute respiratory syndrome (SARS-CoV-2). This</span><span style="font-size:10.0pt"> study examines and compares the efficiency of AstraZeneca, Sinopharm, and Sputnik vaccines and the correlation of antibody response with age, sex, and history of corona disease in employees of Shahid Beheshti University of Medical Sciences. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> 202 participants were included, of which 82 were administered the AstraZeneca, 59 were given the Sinopharm, and 61 were given the Sputnik vaccine. SARS-CoV-2 IgM and IgG antibody levels were checked four weeks after passing the second dose of all three vaccines using the enzyme-linked immunosorbent assay (ELISA) technique.</span><br /> <strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> There was no significant difference between the amount of IgM and IgG antibodies among three vaccines (p=0.056). For all three vaccines, gender and age did not significantly affect the amount of IgM and IgG antibodies. The history of infection with COVID-19 increased the antibody response (p&gt;0.5).</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> The titer of IgM and IgG antibodies were not statistically significantly different. The IgM and IgG antibodies produced by vector-based vaccines are higher than the Sinopharm vaccine. Gender did not affect the produced antibody titer. No significant linear relationship was found between age and antibody titer. In people from this study who received two doses of the AstraZeneca vaccine and had a corona history, the average amount of both IgM and IgG antibodies was measured more than the other participants.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60558 Masoud Alavi, Mohammad Mousavi, Mohammad Jazayeri, Asghar Ebadifar Sat, 19 Aug 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production of Egg Yolk Antibody (IgY) against Vibrio cholerae O1: Protective Effect in Mice <p style="text-align:left"><span style="font-size:12pt"><span style="color:black"><strong>Background:</strong> Cholera is an acute intestinal infection caused by <em>Vibrio cholera </em>(<em>V. cholera</em>). The development of antibodies against specific <em>V. cholerae </em>may have a therapeutic effect. In the present research, we investigated the protective effect of egg yolk Immunoglobulin (IgY), which was produced by immunizing hens with formaldehyde-killed <em>V. cholerae</em> O1 and subsequently the isolated IgY was orally administrated to the <em>V. cholerae</em> O1 infected mice for evaluation of its immunizing capability. </span></span></p> <p style="text-align:left"><span style="font-size:12pt"><span style="color:black"><strong>Methods:</strong> In the current study, hens were immunized three times with<strong> </strong>formaldehyde-killed <em>V. cholerae </em>O1 (1.5&times; 10<sup>7</sup> <em>CFU/mL</em>) and an equal volume of adjuvant. The IgY was isolated from egg yolk by polyethylene glycol method. The validity and activity of isolated IgY were confirmed with SDS-PAGE and ELISA methods, respectively. Subsequently IgY was orally administered to suckling mice following challenge with <em>V. cholerae</em> O1.<strong> </strong>ELISA results showed high antibody titer in the serum and egg yolk. Also, SDS-PAGE analysis showed successful purification of IgY and anti-<em>V.</em> <em>cholerae</em> IgY prevented the death of mice infected with <em>V.</em> <em>cholerae </em>O1. The anti-<em>V.</em> <em>cholera</em> IgY was administered at 2, 4, 6 hours&rsquo; intervals after 3 hours of inoculation of mice with <em>V.</em> <em>cholerae</em> O1. </span></span></p> <p style="text-align:left"><span style="font-size:12pt"><span style="color:black"><strong>Results:</strong> Results showed that the rate of surviving mice (2 <em>mg/mL</em> of IgY) were 60% after 4 hours and 40% after 6 hours and the rate of surviving mice (5 <em>mg/mL</em> of IgY) was 70% after 4 hours and 60% after 6 hours. </span></span></p> <p style="text-align:left"><span style="font-size:12pt"><span style="color:black"><strong>Conclusion:</strong> The findings suggested the egg yolk-driven IgY as a natural antibacterial protein, could be effective in the prevention and treatment of cholera disease.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60550 Mohammad Shoushtari, Ali Barat Shooshtari, Sepideh Asadi, Yousof Karami, Mohsen Honari, Javad Fathi, Mehdi Zeinoddini, Ghorban Ali Alizadeh Wed, 05 Jul 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression of the Hepatitis C Virus core-NS3 Fusion Protein on the Surface of Bacterial Ghosts: Prospects for Vaccine Production <p><span style="font-size:11pt">Background: Antigen presentation using bacterial surface display systems, on one hand, has the benefits of bacterial carriers, including low-cost production and ease of manipulation. On the other hand, the bacteria can help in stimulating the immune system as an adjuvant. For example, using bacterial surface display technology, we developed a hepatitis C virus (HCV) multiple antigens displaying bacteria&#39;s surface and then turned it into a bacterial ghost.</span></p> <p><span style="font-size:11pt">Methods: The HCV core and NS3 proteins&#39; conserved epitopes were cloned into the AIDA gene plasmid as an auto transporter. The recombinant plasmid was then transformed into Escherichia coli (E. coli) Bl21 (DE3). Recombinant bacteria were then turned into a bacterial ghost, an empty cell envelope. Whole-cell ELISA, flow cytometry, and Western blot techniques were used for monitoring the expression of proteins on the surface of bacteria.</span></p> <p><span style="font-size:11pt">Results: A fusion protein of HCV core-NS3-AIDA was successfully expressed on the E. coli Bl21 (DE3) surface and confirmed by western blotting, Enzyme-Linked Immunosorbent Assay (ELISA), and flow cytometry detection techniques.</span></p> <p><span style="font-size:11pt">Conclusion: The presence of HCV antigens on non-pathogen bacteria surfaces holds promise for developing safe and cost-benefit-accessible vaccines with optimal intrinsic adjuvant effects and exposure of heterologous antigens to the immune system.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60545 Minoosadat Tayebinia , Sedigheh Sharifzadeh , Gholamreza Rafiei Dehbidi , Farahnaz Zare, Reza Ranjbaran, Amir Rahimi, Mohammad Reza Miri, Mehdi Mirzakhani , Abbas Behzad-Behbahani Sun, 11 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Glycation Inhibition of Bovine Serum Albumin by Extracts of Momordica charantia L. using Spectroscopic and Computational Methods <p><span style="font-size:11pt">Background: <em>Momordica charantia</em> (<em>M. charantia</em>) has been used in traditional medicine for the management of complications associated with diabetes mellitus. Several phytochemicals with different pharmacological properties have been previously identified from the botanical; however, the mechanisms of actions of this plant vis-&agrave;-vis inhibition of non-enzymatic protein glycation are not known. This study aimed at understanding the putative mechanisms underlying the antiglycation properties of <em>M. charantia</em> extracts experimental and theoretical approaches. </span></p> <p><span style="font-size:11pt">Methods: The antiglycation properties of the plant were evaluated by studying the inhibitory actions of methanol and aqueous extracts on glucose-induced glycation of Bovine Serum Albumin (BSA) and protein aggregation. The mode of binding of identified phenolics of the botanical with BSA, amyloid beta-peptide (1-42) and 3D amyloid beta (1-42) fibrils were also investigated. </span></p> <p><span style="font-size:11pt">Results: The <em>in vitro</em> experimental properties of the extracts showed that the extracts could prevent inductions of protein glycation and protein folding. The molecular docking analyses revealed that phenolics had better binding affinities with chlorogenic acid showing the highest binding score (-7.13&plusmn;0.04 <em>kcal/mol</em>) towards BSA than glucose and their respective interactions with BSA could prevent glucose-induced protein aggregation. </span></p> <p><span style="font-size:11pt">Conclusion: Consequently, the results of this study provide insight into the probable mechanisms of actions of the extracts of <em>M. charantia</em> against the inhibition of advanced glycation end products formation.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60546 Babatunde Oso, Olubukola Agboola , Ige Olaoye Sun, 11 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Anti-proliferative potentials of Aconitum heterophyllum Root Extract in Human Breast cancer (MDA-MB-231) cell lines-Genetic and Antioxidant Enzyme Approach <p><span style="font-size:11pt">Background: One of the most important research activities around the world is the screening of various plant components for novel anticancer medicines. The anticancer activities of Aconitum heterophyllum were studied in human breast cancer MDA-MB- 231 cells in this study. Since tumorigenesis is thought to be the result of a series of pro- gressive gene alterations, including oncogene activation and tumour suppressor gene in- activation, the expression of genes like p53, p21, STAT, and Bcl-2, which are thought to be important in tumorigenesis and cell death, was determined. In the present study there was an upregulation in the level expression of p53and p21 and down regulation in the expression of BCL2 and STAT. However, there is increase and decrease level of gene expression in Aconitum heterophyllum roots loaded Phyto-Niosomes (nEEAH), when compared to ethanolic root extract of Aconitum heterophyllum EEAH extract treated MDA-MB-231 cell lines. </span></p> <p><span style="font-size:11pt">Methods: The enzymatic antioxidants such as CAT, SOD, GR, GST, and GPX as well as non-enzymatic antioxidants such as glutathione, Vitamin E and Vitamin C were esti- mated in the treated MDA-MB-231 cells at the end of incubation. The RT-PCR tech- nique was performed to study the expression patterns of apoptotic genes such as p53 and p21 and anti-apoptotic genes BCL2 and STAT in the drug treated MDA-MB-231 cells</span></p> <p><span style="font-size:11pt">Results: In the present study there was a significant (p&lt;0.05) increase in CAT and glutathione levels and a decrease in Vit C, Vit E and SOD, GR, GST, GPX levels in the untreated MDA-MB-231 cells. Increased apoptotic gene expression and decreased anti-apoptotic gene expression suggest the anti-proliferative nature of the drug extract was comparable to the doxorubicin the positive drug used in the present study. </span></p> <p><span style="font-size:11pt">Conclusion: It can be concluded that the ethanolic extract of Aconitum heterophyllum roots loaded Phyto-Niosomes (nEEAH), when compared to ethanolic root extract of Aconitum heterophyllum EEAH extract treated MDA-MB-231 cell lines exert its anticancer activity by activating the apoptotic genes, suppressing anti-apoptotic genes as well as modulating the antioxidant enzymes.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60547 Sujatha Saravanan, Rajeswary Hari, Karthikeyan Sekar Sun, 11 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Characterization, Cytotoxicity and Anti-oxidant Studies of Phytoniosome Loaded with Ethanolic Leaf Extract of Tinospora Cordifolia <p><span style="font-size:11pt">Background: From time immemorial herbal preparations are been employed for the treatment of several ailments. In recent years due to poor bioavailability the conventional herbal preparations are replaced by phytoniosomes, an advanced novel drug delivery system in which the herbal extracts are incorporated into a non-ionic surfactant to yield higher absorption and remarkable desired pharmacological activity. The present study is aimed to prepare and characterize the ethanolic leaf extract of Tinospora cordifolia (nELETC) loaded phytoniosome and to compare its antioxidant properties with ethanolic leaf extract of Tinospora cordifolia (ELETC). </span></p> <p><span style="font-size:11pt">Methods: The ethanolic leaf extract and ethanolic leaf extract of Tinospora cordifolia loaded phytoniosome (ELETC and nELETC) were prepared. The characterization of the prepared phytoniosomes were performed by UV-Visible spectroscopy, FTIR, XRD, SEM, TEM, DLS and zeta potential. The nontoxic nature of the prepared phytoniosomes was analyzed using MTT assay in vero cell line. The antioxidant potential of ELETC and nELETC were compared by the scavenging activity of DPPH, Hydrogen peroxide and Superoxide radicals.</span></p> <p><span style="font-size:11pt">Results: The formation of ethanolic leaf extract of Tinospora cordifolia loaded phytoniosome (nELETC) was confirmed with UV-Vis spectroscopy. The SEM and TEM images confirmed the spherical shape of the nELETC with average size ranging from 600 to 1800 nm. The zeta potential showed magnitude of -65.55 to -77.83 mV and its crystalline structure was confirmed by XRD analysis. Through the FTIR spectrum presence of alcohols, alkanes, phenols, esters, aliphatic and aromatic compounds as well as alkenes and carbolic acids were identified. MTT assay establishes the non-toxic nature of the synthesized nELETC and excellent antioxidant potential was observed for nELETC than ELETC.</span></p> <p><span style="font-size:11pt">Conclusion: In conclusion, the ethanolic leaf extract of Tinospora cordifolia loaded phytoniosome (nELETC) will serve as a promising drug carrier in scavenging the free radicals and can be used in various biological applications.&nbsp; </span></p> https://www.AJMB.org/En/Article.aspx?ID=60548 Sri Devi Masilamani, Priya Chokkalingam , Rajeswary Hari Sun, 11 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Transmitted Drug Resistance Against Integrase Strand Transfer Inhibitors in Iranian HIV-Infected Naïve Patients <p><span style="font-size:11pt">Background: Human Immunodeficiency Virus (HIV) has claimed the lives of millions of people during the past decades. While several antiretroviral drugs like Integrase Strand Transfer Inhibitors (INSTIs) have been introduced to control HIV, Transmitted Drug Resistance (TDR) in HIV genome caused failure in treatment. This study aimed to investigate TDR and natural occurring mutations (NOPs) in HIV integrase gene in Irani-an HIV patients.</span></p> <p><span style="font-size:11pt">Methods: In this cross-sectional study, blood samples of 30 HIV-positive patients who had never taken integrase inhibitors were considered for CD4 T cell count, RT real-time PCR, and, Nested PCR. The sequencing results were analyzed by CLC sequence viewer software and Stanford University HIV Drug Resistance Database.</span></p> <p><span style="font-size:11pt">Results: In all samples, nine NOPs with a high prevalence were found; however, we did not find any drug resistance mutations, except for a mutation in one sample, which showed a low resistance level. Subtype A1 was dominant in all samples.</span></p> <p><span style="font-size:11pt">Conclusion: Based on the findings and compared to our previous study, all patients were sustainable to main integrase inhibitors, including bictegravir, raltegravir, bicte-gravir, elvitegravir and dolutegravir. It seems the resistant mutation pattern attributed to integrase inhibitors was not diffent among studied patients; hence, the prescription of such inhibitors helps physicians to control HIV infection in Iranian HIV-infected pa-tients.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60549 Ava Hashempour, Zahra Musavi , Javad Moayedi, Zahra Hasanshahi, Behzad Dehghani , Farzaneh Ghasabi, Hassan Joulaei Sun, 11 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of Antidepressant Medication on Brain-derived Neurotrophic Factor Concentration and Neuroplasticity in Depression: A Review of Preclinical and Clinical Studies <p><span style="font-size:10.0pt">Depression is the most prevalent and debilitating disease with great impact on societies. Evidence suggests Brain-Derived Neurotrophic Factor (BDNF) plays an important role in pathophysiology of depression. Depression is associated with altered synaptic plasticity and neurogenesis. BDNF is the main regulatory protein that affects neuronal plasticity in the hippocampus. A wealth of evidence shows decreased levels of BDNF in depressed patients. Important literature demonstrated that BDNF-TrkB signaling plays a key role in therapeutic action of antidepressants. Numerous studies have reported antidepressant effects on serum/ plasma levels of BDNF and neuroplasticity which may be related to improvement of depressive symptoms. Most of the evidence suggested increased levels of BDNF after antidepressant treatment. This review will summarize recent findings on the association between BDNF, neuroplasticity, and antidepressant response in depression. Also, we will review recent studies that evaluate the association between postpartum depression as a subtype of depression and BDNF levels in postpartum women.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60542 Sophia Esalatmanesh , Ladan Kashani, Shahin Akhondzadeh Wed, 07 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Application of Menstrual Blood Derived Stromal (stem) Cells Exert Greater Regenerative Potency Than Fibroblasts/Keratinocytes in Chronic Wounds of Diabetic Mice <p><span style="font-size:11pt">Background: In this study we differentially showed the effects of cell-seeded bilayer scaffold wound dressing in accelerating healing process in diabetic ulcers that still remains as a major clinical challenge. The aim of the study was to compare immunomodulatory and angiogenic activity, and regenerative effect differences between Menstrual blood-derived Stem Cells (MenSCs) and foreskin-derived keratinocytes/fibroblasts.</span></p> <p><span style="font-size:11pt">Methods: The streptozotocin-induced diabetic mice model was developed in male C57/BL6 mice. A bilayer scaffold was fabricated by electrospining silk fibroin nanofibers on human Amniotic Membrane (AM). Dermal fibroblasts and keratinocyte isolated from neonatal foreskin and MenSCs were isolated from the menstrual blood of healthy women. The diabetic mice were randomly divided into three groups including no treatment group, fibroblast/keratinocyte-seeded bilayer scaffold group (bSC+FK), and MenSCs-seeded bilayer scaffold group. The healing of full-thickness excisional wounds evaluations in the diabetic mice model in each group were evaluated at 3, 7, and 14 days after treatment. </span></p> <p><span style="font-size:11pt">Results: The gross and histological data sets significantly showed wound healing promotion via re-epithelialization and wound contraction along with enhanced regeneration in MenSCs-seeded bilayer scaffold group with the most similarity to adjacent intact tissue. Immunofluorescence staining of mouse skin depicted a descending trend of type III collagen along with the higher expression of involucrin as keratinocyte marker in the MenSCs-seeded bilayer nanofibrous scaffold group in comparison with other treatment groups from day 7 to day 14. Moreover, higher levels of CD31 and von Willebrand factor (VWF), and also a higher ratio of M2/M1 macrophages in association with higher levels of the neural marker were observed in the bSC+MenSCs group in comparison with bSC+FK and no treatment groups. </span></p> <p><span style="font-size:11pt">Conclusion: Healing symptoms in wounds dressed with keratinocyte/fibroblast-seeded bilayer scaffold was significantly lower than MenSCs-seeded bilayer scaffold done on impaired diabetic wound chronicity.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60540 Ebrahim Mirzadegan, Hannaneh Golshahi , Zahra Saffarian, Haleh Edalatkhah, Maryam Darzi, Somayeh Khorasani, Kioomars Saliminejad, Somaieh Kazemnejad Wed, 07 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Higher Improvement of Cardiac Function Following Myocardial Infarction using Menstrual Blood Stromal/Stem Cells (MenSCs) Suspended in Conditioned Medium versus Conditioned Medium Alone in Rat Model <p><span style="font-size:11pt">Background: To evaluate the efficiency of Menstrual blood Stromal/Stem Cells (MenSCs) administration in Myocardial Infarction (MI), the effects of MenSCs and their derived conditioned Medium (CM) on cardiac function in MI rat model was assessed. </span></p> <p><span style="font-size:11pt">Methods: Animals were divided into four groups including sham group, MI group, MenSCs derived CM group (CM group), and MenSCs suspended in CM (MenSCs+ CM) group. The injection of different groups was carried out 30 min after ligation of left anterior descending coronary artery into the infarct border zone. </span></p> <p><span style="font-size:11pt">Results: The results showed a significant reduction in scar size after injection of MenSCs+CM compared to MI group. Ejection fraction and fractional shortening of MenSCs+CM group were higher than CM and MI group at day 28. Administration of MenSCs+CM led to much more survival of cardiomyocytes, and prevention of metaplastic development. Moreover, human mitochondrial transfer from MenSCs to cardiomyocytes was seen in group treated by MenSCs+CM. Indeed, MenSCs+CM treatment evoked nuclear factor-&kappa;B (NF-&kappa;B) down-regulation more than other treatments. </span></p> <p><span style="font-size:11pt">Conclusion: MenSCs+CM treatment could significantly ameliorate cardiac function by different mechanisms including inhibition of cartilaginous metaplasia, inhibition of NF-&kappa;B and mitochondrial transfer.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60543 Mahmood Manshori, Somaieh Kazemnejad, Nasim Naderi, Abolfazl Shirazi, Maedeh Arabian, Marzieh Eghtedar Doost, Maryam Darzi, Samaneh Montazeri, Nahid Aboutaleb, Hannaneh Golshahi Wed, 07 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Investigation of Expression Profile of Placenta-specific 1 (PLAC1) in Acute Myeloid and Lymphoid Leukemias <p><span style="font-size:11pt">Background: Placenta-specific 1 (PLAC1) is one of the cancer-testis-placenta antigens that has no expression in normal tissue except placenta trophoblast and testicular germ cells, but is overexpressed in a variety of solid tumors. There is a lack of studies on the expression of PLAC1 in leukemia. We investigated expression of PLAC1 in Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL). </span></p> <p><span style="font-size:11pt">Methods: In this study, we investigated expression pattern of PLAC1 gene in peripheral blood and bone marrow mononuclear cells of newly-diagnosed patients with AML (n=31) and ALL (n=31) using quantitative real-time PCR. Normal subjects (n=17) were considered as control. The PLAC1 protein expression in the samples were also detected using western blotting. </span></p> <p><span style="font-size:11pt">Results: Our data demonstrated that PLAC1 transcripts had 2.7 and 2.9 fold-change increase in AML and ALL, respectively, compared to normal samples. PLAC1 transcript expression was totally negative in all studied normal subjects. Level of PLAC1 mRNA expression in ALL statistically increased compared to normal samples (p=0.038). However, relative mRNA expression of PLAC1 in AML was not significant in comparison to normal subjects (p=0.848). Furthermore, relative mRNA expression of PLAC1 in AML subtypes was not statistically significant (p=0.756). PLAC1 gene expression showed no difference in demographical clinical and para-clinical parameters. Western blotting confirmed expression of PLAC1 in the ALL and AML samples. </span></p> <p><span style="font-size:11pt">Conclusion: Considering PLAC1 expression profile in acute leukemia, PLAC1 could be a potential marker in leukemia which needs complementary studies in the future.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60544 Parastou Gholami , Hossein Asgarian-Omran, Marjan Yaghmaei, Jafar Mahmoudian, Shirin Kianersi, Sina Salari , Ehsan Zaboli, Mahmood Jeddi-Tehrani, Amir-Hassan Zarnani, Mahdi Shabani Wed, 07 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Clinician Scientist Training Program in Iran: Catalyzing Clinical Science Advancements <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt"><span style="background-color:white">The Clinician Scientist Training Program (CSTP) is a transformative educational initiative that combines clinical training with scientific research, producing a unique cohort of physician-scientists. In Iran, the CSTP has emerged as a catalyst for propelling clinical science forward </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">1</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">. By equipping medical professionals with the skills to bridge the gap between research and clinical practice, this program is revolutionizing healthcare, fostering innovation, and contributing to significant advancements in clinical sciences. CSTP represents a paradigm shift in medical education in Iran by emphasizing the integration of clinical practice and scientific inquiry. It recognizes the intrinsic value of research in enhancing patient care, and, thus, cultivates a cohort of physician-scientists capable of seamlessly navigating both domains. By blending clinical training with rigorous research experience, the program nurtures a new generation of medical professionals who can effectively address complex healthcare challenges through a comprehensive understanding of clinical practice and cutting-edge scientific knowledge.</span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt"><span style="background-color:white">Impact of CSTP on clinical science in Iran is remarkable. Although there are no reports on outcomes of CSTP or views from program directors, physician-scientists trained through this program play a pivotal role in advancing medical knowledge, exploring disease mechanisms, and developing innovative therapeutic interventions. The ability to straddle the two worlds of clinical practice and scientific research facilitates uncovering novel insights, contributing to breakthrough discoveries in fields such as molecular biology, genomics, pharmacology, and epidemiology. These advancements translate into tangible benefits for patients, improving diagnosis, treatment options, and overall healthcare outcomes.</span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt"><span style="background-color:white">One of the core strengths of the CSTP lies in its emphasis on translational research. By facilitating collaborations between basic scientists, clinical practitioners, and industry partners, this program accelerates the translation of scientific discoveries into clinical applications Clinician-scientists trained through the CSTP act as crucial mediators, ensuring that research findings are effectively implemented at the bedside. This synergy between research and clinical practice leads to development of innovative diagnostics, therapies, and preventive strategies, ultimately transforming patient care in Iran. Furthermore, CSTP has played a pivotal role in bolstering research infrastructure in Iran. By producing a cohort of highly skilled physician-scientists, the program promotes research excellence, fosters collaborations, and establishes multidisciplinary research teams. This investment in research infrastructure enhances the capacity of research institutions, attracts funding opportunities, and positions Iran as a hub for cutting-edge clinical research. The long-term effects of this strengthened research ecosystem are far-reaching, fueling continuous advancements in clinical science and contributing to the overall development of the healthcare sector.</span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt"><span style="background-color:white">CSTP nurtures a culture of inquiry and scientific curiosity among medical professionals in Iran. By integrating research training into medical education, the program inspires aspiring physicians to embrace evidence-based practice and pursue scientific careers. The exposure to rigorous research methodologies and critical thinking instills a lifelong commitment to advancing medical knowledge and continually improving patient care. This cultural shift fosters a vibrant community of medical professionals dedicated to pushing the boundaries of clinical science, promoting collaboration, and inspiring future generations of clinician-scientists. The Clinician Scientist Training Program in Iran is revolutionizing clinical science by empowering clinician-scientists with a unique blend of clinical expertise and research acumen. This integration is driving advancements in medical knowledge, fostering translational research, strengthening research infrastructure, and cultivating a culture of scientific inquiry. As Iran continues to invest in CSTP, it positions itself at the forefront of clinical science, poised to make significant contributions to global healthcare advancements while addressing the unique healthcare challenges of the nation.</span></span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60541 Hossein Sanjari Moghaddam , Shahin Akhondzadeh Tue, 06 Jun 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Study of DACH1 Expression and its Epigenetic Regulators as Possible Breast Cancer-Related Biomarkers <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Breast carcinogenesis involves both genetic and epigenetic changes. DNA methylation, as well as micro-RNA regulations, are the significant epigenetic phenomena dysregulated in breast cancer<strong>.</strong><strong> </strong>Herein, the expression of&nbsp;<em>DACH1</em>&nbsp;as a tumor suppressor gene and its promoter methylation status was analyzed in breast cancer tumors. Also, the expression of three micro RNAs (miR-217, miR-6807-3p, and miR-552), which had been previously reported to target <em>DACH1</em>, was assessed.&nbsp; </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong> <span style="font-size:10.0pt">The SYBR green-based Real-Time reverse transcription-PCR was used to determine&nbsp;<em>DACH1</em>&nbsp;and micro-RNAs (miR-217, miR-6807-3p, and miR-552) expression in 120 ductal breast cancer tumors compared with standard control. Also, the promoter methylation pattern of&nbsp;<em>DACH1</em>&nbsp;was investigated using the </span><span style="font-size:10.0pt">Methylation-specific PCR<em> </em></span><span style="font-size:10.0pt">technique. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results: </span></strong><em><span style="font-size:10.0pt">DACH1</span></em><span style="font-size:10.0pt">&nbsp;expression was significantly down-regulated in breast tumors (p&lt; 0.05). About 33.5% of tumors showed&nbsp;<em>DACH1</em>&nbsp;promoter hyper-methylation. The studied micro-RNAs, expression was negatively correlated with <em>DACH1</em> expression. The highest expressions of miRNAs and higher&nbsp;<em>DACH1</em>&nbsp;promoter methylation were observed in advanced cancer situations. The Kaplan-Meier survival curves indicated that the overall survival was significantly poor in higher miRNAs and lower <em>DACH1</em> expression in breast cancer patients (p&lt;0.002).</span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><em><span style="font-size:10.0pt"> DACH1</span></em><span style="font-size:10.0pt">&nbsp;down-regulation may be associated with a poor breast cancer prognosis. The&nbsp;<em>DACH1</em> down-regulation may be due to epigenetic regulations such as promoter methylation, especially in triple-negative cases. Other factors, such as micro-RNAs (miR-217, miR-6807-3p, and miR-552), may also have an </span><span style="font-size:10.0pt">impact. The elevated expression of </span><span style="font-size:10.0pt">miR-217, miR-6807-3p, and miR-552, maybe candidates as possible poor prognostic biomarkers in breast cancer management for further consideration.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60537 Mohammad Hossein Nasirpour, Mahdieh Salimi, Faezeh Majidi, Zarrin Minuchehr, Hossein Mozdarani Tue, 28 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cell Surface Vimentin Detection in Cancer Cells by Peptide-Based Monoclonal Antibody <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Vimentin is a prominent Intermediate Filaments (IFs) protein expressed in different mesenchymal origin cell types. Besides a wide range of cellular function roles associated with vimentin expression, its dysregulation and cell surface expression in the induction of malignancy properties have been reported extensively, making it a promising cancer-specific target. Therefore, this study aimed to generate and characterize anti-vimentin monoclonal antibodies. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> A 14-mer synthetic peptide from vimentin was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of Blab/C mice and monoclonal production by conventional hybridoma technology. The monoclonal antibody was purified using affinity chromatography of supernatants from the selected hybridoma cells. ELISA, Immunoprecipitation-Western blotting (IP-WB), Immunocytochemistry (ICC), and flow cytometry were employed to characterize the produced monoclonal antibody in terms of interaction with vimentin immunizing peptide as well as vimentin protein. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> Amid the several obtained producing anti-vimentin antibody hybridomas, the 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, and led to its selection for purification and characterization. The purified antibody could detect vimentin protein in IP-WB, ICC and flow cytometry of the normal and cancerous cells with different origin. No vimentin expression was found in normal healthy <span style="background-color:white">Peripheral Blood Mononuclear Cell</span> (PBMC). </span></span></p> <p><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tool where detection or targeting of vimentin in a wide range of organisms is required.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60532 Niloufar Sadeghi, Ghazaleh Fazli, Ali Ahmad Bayat , Raminasadat Fatemi , Nasim Ebrahimnejhad , Ali Salimi, Omid Zarei, Hodjattallah Rabbani Wed, 22 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of Dental Pulp Stem Cell Preconditioning on Osteogenesis using Conditioned Media of Probiotics Bacteria <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Stem cells are used to treat numerous diseases; however, their lifespan is rather short. Factors such as probiotics affect and improve various cell lineage efficacies. The aim of this study was to investigate the effects of probiotics-conditioned media on dental pulp stem cell potentials in osteogenesis.</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> The experiment was initiated by culturing <em>Lactobacillus casei </em>and <em>Lactobacillus acidophilus </em>probiotics as well as DPS-7 cells. Bacterial supernatants were separated and concentrated as the conditioned media. The DPS-7 cells were treated with various concentrations of the conditioned media. Furthermore, MTT assay and alkaline phosphatase activity were used. The mRNA expression of three genes (bFGF, EGF-&beta; and BMP-2) involved in osteogenesis was analyzed using a real-time polymerase chain reaction.</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The response of dental pulp stem cells to probiotics preconditioning promoted cell proliferation, increased alkaline phosphatase activity and upregulated <em>bFGF</em> and <em>BMP-2</em> gene expression. Increased expression was significant for <em>BMP-2</em> and moderate for <em>bFGF</em>; however, it was non-significant for EGF-&beta;. The use of the two probiotics was the most effective.</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> In general, synergism of the combined probiotics preconditioning induces differentiation of DPS-7 cells into osteoblasts most effectively.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60533 Fatemeh Amini, Mohammad Bagher Rezvani, Ronak Bakhtiari, Elham Tabatabaei Ghomsheh Wed, 22 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Targeted Overexpression of NDRG2 using Survivin Promoter Reduces Viability and Invasiveness of A549 Cell Line <p style="text-align:justify"><span style="font-size:13pt"><strong><span style="font-size:10.0pt">Background: </span></strong><span style="font-size:10.0pt">Anti-tumor effects of N-myc Downstream Regulated Gene2 (NDRG2) have been demonstrated in many tumors. In the present study, NDRG2 was specifically overexpressed in lung cancer cell line using Survivin Promoter (Sur-P). Then, the effects of NDRG2 overexpression on viability, apoptosis, migration, and invasion of A549 cells were evaluated.</span></span></p> <p style="text-align:justify"><span style="font-size:13pt"><strong><span style="font-size:10.0pt">Methods: </span></strong><span style="font-size:10.0pt">Recombinant </span><span style="font-size:10.0pt">pAdenoVator-</span><span style="font-size:10.0pt">Sur-P</span><span style="font-size:10.0pt">-NDRG2-IRES-GFP</span> <span style="font-size:10.0pt">plasmid</span><span style="font-size:10.0pt"> harboring </span><em><span style="font-size:10.0pt">NDRG2</span></em><span style="font-size:10.0pt"> gene under transcriptional control of Sur-P and mock plasmid were constructed. A549 lung tumor cells and LX-2 cells (non-tumor cell line) were transfected with </span><span style="font-size:10.0pt">pAdenoVator-Sur-P-NDRG2-IRES-GFP</span><span style="font-size:10.0pt">, </span><span style="font-size:10.0pt">pAdenoVator-CMV-NDRG2-IRES-GFP</span><span style="font-size:10.0pt">, or mock plasmids. Tumor specificity of </span><span style="font-size:10.0pt">Sur-P </span><span style="font-size:10.0pt">was evaluated using fluorescent microscopy for GFP expression. The effects of <em>NDRG2</em> overexpression on cell viability, apoptosis, and migration of A549 cells were measured using MTT, annexinV/7-AAD flow cytometry, and </span><span style="font-size:10.0pt">transwell migration assay, respectively. </span><em><span style="font-size:10.0pt">NDRG2</span></em><span style="font-size:10.0pt"> and matrix metalloproteinase-2 (<em>MMP-2</em>) expression</span> <span style="font-size:10.0pt">were measured using real time- PCR.</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Results: </span></strong><span style="font-size:10.0pt">pAdenoVator-Sur-P-NDRG2-IRES-GFP</span><span style="font-size:10.0pt"> transfection </span><span style="font-size:10.0pt">resulted in a huge GFP expression in A549 cells, but not in LX-2 cells. The results of real time-PCR analysis also showed that </span><span style="font-size:10.0pt">pAdenoVator-Sur-P-NDRG2-IRES-GFP</span><span style="font-size:10.0pt"> transfection led to an abundant <em>NDRG2</em> expression in A549 cells. <em>NDRG2</em> overexpression decreased A549 cell viability through increasing cell apoptosis. In addition, migration, invasion, and <em>MMP-2</em> expression decreased following <em>NDRG2</em> overexpression in A549 cells. </span></span></p> <p style="text-align:justify"><span style="font-size:13pt"><strong><span style="font-size:10.0pt">Conclusion: </span></strong><span style="font-size:10.0pt">The findings indicate that the targeted overexpression of <em>NDRG2</em> using </span><span style="font-size:10.0pt">Sur-P can reduce the viability and invasiveness of A549 cells, suggesting possible benefits of this approach in lung cancer therapy.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60534 Maryam Fanian, Gholamreza Rafiei , Marzieh Alizadeh Zarei, Mohammad Ali Takhshid Wed, 22 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Is Formulary of Maranta Arundinacea Clarias Gariepinus (F-MaCg) a Potential Immunostimulant? <p><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Background: E</span></strong><span style="font-size:10.0pt">xternal factors have the potential to act as immunostimulants in order to influence the body&#39;s protection from many foreign antigens. We intended to investigate the ethanol extract Formulary of F-MaCg effect as an immunostimulant.&nbsp;</span></span></p> <p><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt">&nbsp;A purely experimental with a completely randomized design was used on twenty-four white male rats. They were divided into four groups:1) G0 [given aquades (5 <em>ml</em>)]; 2) G1 [given F-MaCg-75 <em>mg/gr</em> BW (Body Weight)]; 3) G2 (F-MaCg -150 <em>mg/gr</em> plus Hepatitis B vaccine at the beginning and the end of treatment); and 4) G3 (F-MaCg -300 <em>mg/gr</em> BW plus hepatitis B vaccine at the end of treatment). The rat&#39;s spleen lymphocyte blast transformation was evaluated on the 15th and 37th days. Lymphocytes were examined using microtetrazolium assays. Optical Density (OD) was measured using an ELISA reader [493 <em>n<span style="background-color:white">&mu;</span></em> (nanomicro)]. Observation of lymphocyte viability by a counting chamber using a light microscope and trypan blue 1 % before being cultured with Phytohaemoaglutinin.</span></span></p> <p><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt">&nbsp;Lymphocyte cell viability in the hepatitis B vaccine-induced group on the 15th day showed the highest average value in the G2 (1,484/<em>mcl</em> of blood); on the 37th day, it was in G3 (1,578/<em>mcl</em> of blood). The proliferative activity of spleen lymphocytes indicated by the difference in the OD values of the four treatment groups was 0.467, 0.913, 1.619, and 1.473 <em>n<span style="background-color:white">&mu;</span></em>, respectively. Histological observations of the spleen showed differences at all given formulary dose concentrations.&nbsp;</span></span></p> <p><strong><span style="font-size:10.0pt">Conclusion:&nbsp;</span></strong><span style="font-size:10.0pt">F-MaCg could be an immunostimulant because of its ability to trigger a cellular immune response.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60535 Zulkifli Zulkifli, Darmawi Darmawi, Said Usman, Kurnia Fitri Jamil Wed, 22 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of a High Sensitive Multiplex Lateral Flow Immunoassay (LFIA) System for Rapid Detection of Methicillin-Resistant Staphylococcus Aureus (MRSA) <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background: </span></strong><span style="font-size:10.0pt">Methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) has become a worldwide concern as an epidemic bacterium and a cause of nosocomial and community-acquired infections. One of the major problems in the prevention and treatment of infections caused by MRSA strains is their multi-drug resistant trait, which causes the spread of infections and increases the mortality rate. Therefore, a rapid and accurate method is needed to identify MRSA strains, initiate appropriate antibiotic&nbsp;therapy, and control its infection. The aim of this study was to develop a twin lateral flow immunoassay system to detect methicillin-resistant <em>Staphylococcus aureus </em>(MRSA).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> First, BSA blocked AuNPs-anti<em>-</em>peptidoglycan antibody and AuNPs-anti-BSA antibody were used to detect <em>Staphylococcus aureus </em>(<em>S. aureus</em>). Then, AuNPs</span><span style="font-size:10.0pt">-anti-PBP2a antibody was used to specifically detect </span><span style="font-size:10.0pt">MRSA. Sensitivity, specificity and limit of detection of this twin immunoassay system were assessed using </span><span style="font-size:10.0pt">MRSA, </span><span style="font-size:10.0pt">methicillin susceptible <em>S. aureus </em>and clinical samples. Results </span><span style="font-size:10.0pt">were compared to those of cefoxitin disc diffusion (FOX30) and Polymerase Chain Reaction (PCR) as gold standards.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong> <span style="font-size:10.0pt">The Limit of Detection</span><span style="font-size:10.0pt"> (LOD) of this twin system </span><span style="font-size:10.0pt">were 10<sup>3</sup> and 10<sup>4</sup> CFU/<em>ml</em> for the first and second strips, respectively. Sensitivity and specificity of this innovative assay in detecting MRSA were 92.30 and 97.36%, compared to </span><span style="font-size:10.0pt">FOX30 and PCR, respectively. </span></span></p> <p><span style="font-size:12pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> High rates of sensitivity and specificity of this initiative system show its high potentials for rapid and accurate detection of MRSA.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60536 Masoomeh Amini, Mohammad Reza Pourmand , Reza Faridi-Majidi Wed, 22 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Human T2R38 Bitter Taste Receptor Expression and COVID-19: From Immunity to Prognosis <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt"><span style="color:black">Background: </span></span></strong><span style="font-size:10.0pt"><span style="color:black">Bitter taste-sensing type 2 receptor (T2Rs or TAS2Rs) found on ciliated epithelial cells and solitary chemosensory cells have a role in respiratory tract immunity. T2Rs have shown protection against SARS-CoV-2 by enhancing the innate immune response. The purpose of this review is to outline the current sphere of knowledge regarding this association. </span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt"><span style="color:black">Methods: </span></span></strong><span style="font-size:10.0pt"><span style="color:black">A narrative review of the literature was done by searching (T2R38 OR bitter taste receptor) AND (COVID-19 OR SARS-CoV-2) keywords in PubMed and google scholar.</span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt"><span style="color:black">Results: </span></span></strong><span style="font-size:10.0pt"><span style="color:black">T2R38, an isoform of T2Rs encoded by the <em>TAS2R38 </em>gene, may have a potential association between phenotypic expression of T2R38 and prognosis of COVID-19. Current studies suggest that due to different genotypes and widespread distributions of T2Rs within the respiratory tract and their role in innate immunity, treatment protocols for COVID-19 and other respiratory diseases may change accordingly. Based on the phenotypic expression of T2R38, it varies in innate immunity and host response to respiratory infection, systemic symptoms and hospitalization. </span></span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt"><span style="color:black">Conclusion: </span></span></strong><span style="font-size:10.0pt"><span style="color:black">This review reveals that patients&rsquo; innate immune response to SARS-COV-2 could be influenced by T2R38 receptor allelic variations.</span></span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60538 Lakshmi Deepak Bethineedi , Hediyeh Baghsheikhi , Afsaneh Soltani , Zahedeh Mafi , Noosha Samieefar, Shaikh Sanjid Seraj , Mohammad Amin Khazeei Tabari Wed, 22 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Analysis of SLC26A4 Gene in Individuals with Non Syndromic Hearing Impairment in Relation with GJB2 Associated Mutations <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background: </span></strong><span style="font-size:10.0pt">Hearing Loss (HL) is the most common sensory disorder. HL commonly ranges from mild to severe. Persons with HL face difficulty in hearing conversations or sounds through one ear or both ears, which impacts one&rsquo;s ability to interact with others. Hence it is a communicable disorder that makes people socially isolated, lonely, and frustrated. HL in children severely affects language development. The people who are referred to as &#39;Deaf&#39; with very little or no hearing capabilities, are considered as having profound hearing loss. More than 124 genes are causative for Non-Syndromic HL (NSHL) with varying inheritance, among which the <em>SLC26A4</em> mutations are the second commonest cause of hereditary HL across the globe. </span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> Samples from 70 NSHL patients were analyzed through </span><em><span style="font-size:10.0pt"><span style="background-color:white">Next-Generation<strong> </strong>Sequencing</span></span></em>&nbsp;<span style="font-size:10.0pt">(NGS) and generated five pathogenic variants [N246fs (rs918684449), K564fs (rs746427774), F122fs, V239D (rs111033256), T721M (rs121908363)] each with frequency of 1.42%. Three missense variants [S399P (rs747431002), L597S (rs55638457), and G6V (rs111033423)] were reported under the &quot;uncertain&quot; category. All the collected samples were further genotyped to look for the possibility of having <em>GJB2</em> and HL-associated mutations. </span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> Out of five <em>SLC26A4</em> pathogenic mutations N246fs (rs918684449) and K564fs (rs746427774) were observed in samples which were positive for <em>GJB2</em>-HL associated candidate mutations [W24X (rs104894396), Q124X (rs397516874) and W77X (rs80338944)]. Similarly, pathogenic variants F122fs, V239D (rs111033256) and T721M (rs121908363) were observed in patient samples which were negative for <em>GJB2</em>-HL associated mutations. </span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Our data will expand the list of variants underlying NSHL and encourage further genotype <em>SLC26A4</em> gene concerning the south Indian population with a large sample size.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60539 Krishna Rajalakshmi , Jayakumar Thirunavukkarasu, Meenu Ambika Vikraman , Santosh Maruthy , Charles Sylvester , Rajesh Kundapur Wed, 22 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Interdisciplinary Collaboration between Bench and Bedside in the COVID-19 Pandemic <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt"><span style="background-color:white">The World Health Organization (WHO) announced Coronavirus disease (COVID-19) as a pandemic caused by SARS-CoV-2 on 11 March 2020 </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">1</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">. SARS-CoV-2 primarily affects the human respiratory system cells. Nonetheless, it has been revealed that other systems, such as the gastrointestinal tract, kidney system, liver, pancreas, eyes, and brain are affected by the virus </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">2</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">. The SARS-CoV-2 virus has about 79% and 50% similarity to SARS-CoV and MERS-CoV </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">3</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">. However, it caused millions due to the extremely high transmission rate. The complexity of COVID-19 treatment strategies, as compared to SARS-CoV and MERS-CoV, led to a global crisis. As of February 2023, more than 750 million confirmed cases of COVID-19 and 6.8 million deaths have been reported (https://covid19.who.int/). Extremely high rates of death along with no definitive treatment prompted governments and health institutes around the world to establish strict health and social guidelines and develop and mass-produce COVID-19 vaccines in a very short amount of time, leading to a stark reduction in mortality and morbidity due to COVID-19. </span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The experience from COVID-19 containment and vaccination highlights the important role of </span><span style="font-size:10.0pt">interdisciplinary collaboration between bench (virologist, immunologist, epidemiologist, <em>etc</em>.) and bedside (healthcare providers and clinicians) </span><sup><span style="font-size:10.0pt">4</span></sup><span style="font-size:10.0pt">. Interdisciplinarity is generally defined as when different disciplines investigate a shared topic/object from different perspectives so that each discipline accents a different aspect of that topic. One of the successful interdisciplinary example is the collaboration between psychiatry and neuroscience <sup>5-10</sup>. We, herein, use the example of the COVID-19 pandemic and its containment using vaccination and clinical guidelines to highlight the role of collaboration between basic and clinical sciences. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Various disciplines, such as clinicians, virologists, immunologist, epidemiologists, physicists, and economists have collaborated closely to overcome the COVID-19 pandemic. For example, the diagnostic tests for confirmation of COVID-19 by clinicians were developed and produced by virologists, whereas clinicians deal with how the virus affect the organs of the human host. Clinicians and immunologists investigated new mechanisms of damage to the lungs and blood clotting in patients with COVID-19, and existing interventions were modified to handle the new virus. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">While the clinicians were saving lives in hospitals, preventive measures were implemented by epidemiologists and politicians to impede the most probable routes of transmission. These measures included wearing face masks, washing hands, coughing into elbows, avoiding handshakes, and social distancing. In this regard, physicists investigated what distance would be enough, given the behavior of fluids, to block the transmission of virus. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">On the other hand, biomedical researchers at research institutes and pharmaceutical companies produced the fastest-developed vaccines in history. Multiple vaccines with different mechanisms of action, such as messenger RNA (mRNA) vaccine, vector vaccine, and protein subunit vaccine were generated in a short period of time, mass-produced, and distributed among the population </span><sup><span style="font-size:10.0pt">11</span></sup><span style="font-size:10.0pt">. Vaccination of the population led to a stark decrease in mortality and morbidity. In addition to vaccines, several pharmacological interventions such as remdesivir and convalescent plasma were crafted by biomedical researchers, which significantly helped clinicians in their fight against COVID-19. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">These experiences confirm that close collaboration between different disciplines helped the community to tackle the COVID-19 pandemic with the least possible cost, mortality, and morbidity. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60531 Hossein Sanjari Moghaddam , Shahin Akhondzadeh Tue, 21 Feb 2023 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Inflammation-Schizophrenia: A Bidirectional Causal Association Mediated by Cytokines https://www.AJMB.org/En/Article.aspx?ID=60522 Ahmad Shamabadi, Shahin Akhondzadeh Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Opportunistic Challenges of Computer-aided Drug Discovery of Lipopeptides: New Insights for Large Molecule Therapeutics <p><span style="font-size:10.0pt">Computer-aided drug designing is a promising approach to defeating the dry pipeline of drug discovery. It aims at reduced experimental efforts with cost-effectiveness. Naturally occurring large molecules with molecular weight higher than 500 <em>Dalton</em> such as cationic peptides, cyclic peptides, glycopeptides and lipopeptides are a few examples of large molecules which have successful applications as the broad spectrum antibacterial, anticancer, antiviral, antifungal and antithrombotic drugs. Utilization of microbial metabolites as potential drug candidates incur cost effectiveness through large scale production of such molecules rather than a synthetic approach. Computational studies on such compounds generate tremendous possibilities to develop novel leads with challenges to handle these complex molecules with available computational tools. The opportunities begin with the desired structural modifications in the parent drug molecule. Virtual modifications followed by molecular interaction studies at the target site through molecular modeling simulations and identification of structure-activity relationship models to develop more prominent and potential drug molecules. Lead optimization studies to develop novel compounds with increased specificity and reduced off targeting is a big challenge computationally for large molecules. Prediction of optimized pharmacokinetic properties facilitates development of a compound with lower toxicity as compared to the natural compounds. Generating the library of compounds and studies for target specificity and ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) for large molecules are laborious and incur huge cost and chemical wastage through <em>in-vitro</em> methods. Hence, computational methods need to be explored to develop novel compounds from natural large molecules with higher specificity. This review article is focusing on possible challenges and opportunities in the pathway of computer-aided drug discovery of large molecule therapeutics.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60523 Manisha Yadav, J. Satya Eswari Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An Anti-TAZ Monoclonal Antibody Recognizing Cell Surface Expressed TAZ Protein in Human Tumor Cells <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><strong><span style="font-size:9.5pt">Background:</span></strong><span style="font-size:10.0pt"> WWTR1 or TAZ is a transcriptional co-activator protein expressed in cytoplasm which functions as the main downstream effector of the Hippo signaling pathway. This pathway is an evolutionally conserved signal cascade, which plays a pivotal role in organ size control and tumorigenesis. Ectopic expression of TAZ has already been observed in many malignancies, while the ectopic localization of TAZ is reported for the first time. The aim of this study was to produce a specific monoclonal antibody (mAb) against a synthetic peptide derived from WWTR1 protein to be used as a research tool in human carcinomas. </span></span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><strong><span style="font-size:9.5pt">Methods:</span></strong><strong> </strong><span style="font-size:10.0pt">A 21-mer synthetic peptide (derived from human TAZ protein) was used for immunization of BALB/c mice after conjugation with Keyhole Limpet Haemocyanin (KLH) using hybridoma technology. The generated mAb reacted with the immunizing peptide employing ELISA assay. The reactivity of the antibody with native TAZ protein was assessed through Western blot, immunocytochemistry, and flow cytometry using different cancer cell lines. </span></span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><strong><span style="font-size:9.5pt">Results:</span></strong><span style="font-size:10.0pt"> The produced mAb could recognize the immunizing peptide in ELISA and K<sub>aff</sub> was 0.6&times;10<sup>-9</sup> <em>M</em>. The produced anti-TAZ mAb unlike available commercial anti-TAZ antibody, was capable of specifically recognizing cell surface TAZ in human carcinoma cell lines including MCF-7, Raji, and A431 in Western blot, immunocytochemistry, and flow cytometry assays. As expected, no reactivity was observed using normal<span style="background-color:white"> Peripheral Blood Mononuclear Cell</span> (PBMC) from healthy donors. </span></span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:9.5pt">Conclusion:</span></strong> <span style="font-size:10.0pt">Based on the results, TAZ is ectopically expressed on the surface of tumor cell lines which is not the case in normal cells. The generated mAb has a potential to be used as a research tool in studying the expression of TAZ in human carcinomas in different applications.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60524 Mozhan Haji Ghaffari, Mahsa Mohammadzadeh, Miganoosh Simonian, Mehrdad Hashemi , Niloufar Sadeghi, Babak Negahdari, Mohammadali Mazloomi , Hodjattallah Rabbani Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Proteome Analysis of Adult Acute Lymphoblastic Leukemia by Two-dimensional Blue Native/Sodium Dodecyl Sulfate Gel Electrophoresis <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Despite the significant progress in the treatment of Acute Lymphoblastic Leukemia (ALL) in children, it still remains as one of the most challenging malignancies in adults. Identification of new biomarkers may </span><span style="font-size:10.0pt">improve the management of adult ALL. Proteins expressed on the cell surface can be considered as disease-associated biomarkers with potential for diagnosis and targeted therapies. Thus, membrane proteome studies give essential information about the disease-related biomarkers. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> We applied 2-dimensional blue-native SDS-PAGE technique followed by MALDI-TOF/TOF-mass spectrometry to study the cell membrane proteome of peripheral blood mononuclear cells of adult B-ALL patients in comparison to that of the healthy controls. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> Sixty seven differentially expressed protein spots were detected, among them 52 proteins were found to be up-regulated but the other 15 proteins were down-regulated in B-ALL. Five differentially expressed proteins, involved in energy metabolism pathways, were detected in B-ALL patients compared to the healthy control group. </span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><strong> </strong><span style="font-size:10.0pt">Differentially expressed proteins provide an insight into the molecular biology of B-ALL. Further studies must be done to confirm our data to be </span><span style="font-size:10.0pt">considered as potential targets for detection and treatment of B-ALL.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60526 Servin Bagheralmoosavi , Parastou Gholami , Mahdi Amini , Mahdi Alizadeh , Marjan Yaghmaei, Sahar Tavakkoli, Sina Salari , Mahmood Jeddi-Tehrani, Alireza Ghasempour , Kambiz Gilany, Mahdi Shabani Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Optimization of Degenerate PCR Conditions for Reducing Error Rates in Detection of PKS and NRPS Gene groups in Actinomycetes <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> The screen of Polyketide Synthase (<em>PKS</em>) and Nonribosomal Peptide Synthetase (<em>NRPS</em>) gene groups is a quick way to discover new therapeutic agents. However, errors in laboratory techniques cause a loss of touch with reality. This study aimed to evaluate the presence of <em>PKS</em> and <em>NRPS</em> gene groups in previously isolated strains by optimizing their specialized amplification by degenerate primers and indicating the evolutionary relationships with reference strains.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> <em>PKS-I</em>, <em>II</em>, and <em>NRPS</em> genes PCR amplification was performed using three degenerate primer sets for 22 actinomycete strains with antibacterial activity. Annealing temperature and the amount of template DNA and primers were optimized. PCR products of <em>PKS-I, II</em>, and <em>NRPS</em> from three strains were sequenced after TA cloning. Besides, strains with high antibacterial activity were identified by biochemical features and partial 16S rDNA sequencing and hypothetically classified by a phylogenetic tree.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> High frequencies of <em>PKS-I</em> (86.4%), <em>PKS-II</em> (81.8%), and <em>NRPS</em> (95.4%) genes were found among the strains after optimization. Fourteen strains (64%) contained all of the genes, and 100% of strains had at least two genes. These numbers are pretty distinct in comparison with the previous <span style="background-color:white">researches</span>. All of the sequenced strains were members of <em>Streptomyces</em> genus.</span></span></p> <p><strong><span style="font-size:10.0pt">Conclusion:</span></strong> <span style="font-size:10.0pt">Our research showed that degenerate PCR strongly depends on the variation of annealing temperature and primer concentration, resulting in an unexpected shift in PCR outputs. The sequencing results confirmed the optimized conditions for specialized PCR of <em>PKS-I</em>, <em>PKS-II</em>, and <em>NRPS</em> gene groups.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60528 Sara Ghashghaei , Zahra Etemadifar, Manoochehr Tavassoli, Mohammad Reza Mofid Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Formulation, Characterization, and Evaluation of Wound Healing Potency of a Novel Mattan tailam Nanogel Based on a Famous Traditional Siddha Formula <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> The <em>Mattan tailam</em> mixture has been extensively used to heal ulcerous wounds in traditional Siddha practice. The present study aimed to synthesize a <em>Mattan tailam</em> nanogel and evaluate the enhancement of wound healing potential in an experimental wound model. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> <em>Mattan tailam</em> nanogel was synthesized using the high-energy milling approach, and characterization of nanogel and potency of wound healing was investigated. The novelty of this study was the nanogel preparation of <em>Mattan tailam.</em> </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><strong> </strong><span style="font-size:10.0pt">As expected, a synthesized novel nanogel of <em>Mattan tailam</em> has a distinct, prominent peak with a spherical form, is negatively charged and has an average particle size of 20&ndash;30 <em>nm</em>. <em>Mattan tailam</em> nanogel treated rats showed a remarkable reduction (p&lt;0.001) in the wound area. On the 16<sup>th</sup> day, 10% <em>Mattan tailam</em> nanogel treatment resulted in a higher percentage of wound contraction. The 10% <em>Mattan tailam</em> nanogel group exhibited a faster epithelialization time (14.33 days) and a greater hydroxyproline concentration than the others. The topical application of 10% <em>Mattan tailam</em> nanogel increased tensile strength, signifying a better therapeutic indication. </span></span></p> <p><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion</span></strong><span style="font-size:10.0pt">:</span><span style="font-size:10.0pt"> The present findings prove that polyherbal <em>Mattan tailam</em> nanogel formulation significantly improves collagen production, wound contraction, and tensile strength.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60529 Meenachisundaram Sakthiganapathi , Gnanakumar Prakash Yoganandam , Venkatachalam Gopal Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir MGMT Gene rs1625649 Polymorphism in Iranian Patients with Brain Glioblastoma: A Case Control Study <p><span style="font-size:10.0pt">Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor with poor prognosis and high potential of dispersion to other brain tissues in adult. Effective and modern choices of treatment including chemotherapy with alkylating agents marginally extend survival of GBM. However, alkylating agents can lead to highly harmful mismatch during DNA replication causing apoptosis and cell death. Accordingly, O6-Methylguanine-DNA methyltransferase (MGMT) removes alkyl adducts, thereby causing resistance to alkylating drugs. Single-Nucleotide Polymorphisms (SNPs) in MGMT&nbsp;promoter region may play a role in the regulation of MGMT expression and prediction of glioma development risk. In order to evaluate the clinical significance of rs1625649 SNP in the MGMT promoter region of glioblastoma, genomic DNA from a series of 54 patients with GBM and 50 healthy individuals in Iranian population were collected for tetra ARMS PCR amplification. None of the &quot;A&quot; or &quot;C&quot; alleles were associated with tumor occurrence, the &quot;AA&quot; genotype was more frequent in healthy subjects, and the &quot;AC&quot; genotype was 4.6 times more common in patients with GBM. The longest survival time was observed in the &quot;CC&quot; genotype; however, this difference was not statistically significant. On the other hand, homozygous rs1625649 (AA genotype) was significantly associated with a better survival than the cases with heterozygous rs1625649 (CA genotype) or wild type rs1625649 (CC genotype), predicting better response to temozolomide-based chemotherapy.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60525 Reyhaneh Safaei , Hanieh Mojtahedi , Sara Hanaei , Azadehsadat Razavi , Marzie Esmaeili , Maryam Sadr, Arezou Rezaei, Maryam Edalatfar, Hamidreza Khayat Kashani, Mohsen Sadeghi-Naini, Farzaneh Darbeheshti , Jaber Gharehdaghi , Mehdi Forouzesh , Abdolali Ebrahimi , Nima Rezaei Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Identification of Critical Molecular Factors and Side Effects Underlying the Response to Thalicthuberine in Prostate Cancer: A Systems Biology Approach <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> Uncontrolled mitosis of cancer cells and resistance cells to chemotherapy drugs are the challenges of prostate cancer. Thalicthuberine causes a mitotic arrest and a reduction of the effects of drug resistance, resulting in cell death. In this study, we applied bioinformatics and computational biology methods to identify functional pathways and side effects in response to Thalicthuberine in prostate cancer patients. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> Microarray data were retrieved from <em><span style="background-color:white">Gene Expression Omnibus</span></em>&nbsp;(GEO), and protein-protein interactions and gene regulatory networks were constructed, using the Cytoscape software. The critical genes and molecular mechanisms in response to Thalicthuberine and its side effects were identified, using the Cytoscape software and WebGestalt server, respectively. Finally, GEPIA2 was used to predict the relationship between critical genes and prostate cancer. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The&nbsp;<em>POLQ, EGR1, CDKN1A, FOS, MDM2, CDC20, CCNB1,</em> and <em>CCNB2</em> were identified as critical genes in response to this drug. The functional mechanisms of Thalicthuberine include a response to oxygen levels, toxic substances and immobilization stress, cell cycle regulation, regeneration, the p53 signaling pathway, the action of the parathyroid hormone, and the FoxO signaling pathway. Besides, the drug has side effects including muscle cramping, abdominal pains, paresthesia, and metabolic diseases. </span></span></p> <p><span style="font-size:11pt"><span style="font-size:10.0pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> Our model suggested newly predicted crucial genes, molecular mechanisms, and possible side effects of this drug. However, further studies are required.</span></span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60527 Fatemeh Saberi , Zeinab Dehghan , Effat Noori , Zahra Taheri, Marzieh Sameni, Hakimeh Zali Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Monkeypox Outbreaks in Non-Endemic Countries: Correspondence https://www.AJMB.org/En/Article.aspx?ID=60530 Rujittika Mungmunpuntipantip , Viroj Wiwanitkit Wed, 21 Dec 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir New Biomarkers in Early Diagnosis of Acute Kidney Injury in Children <p><span style="font-size:10.0pt">Acute Kidney Injury (AKI) is a common condition with a high risk of mortality and morbidity, so, early diagnosis and management of AKI is very important in clinical practice. Despite significant progress in the management of AKI, it still carries high morbidity and mortality. BUN and serum creatinine are not very sensitive nor specific for the diagnosis of AKI because they are affected by many renal and non-renal factors that are independent of kidney injury or kidney function and change significantly only after significant kidney injury and with a substantial time delay. Detection of biomarkers of AKI made predominantly by the injured kidney tissue are essential for the early diagnosis of AKI. An ideal biomarker should be one that could be easily measured, with no interference with other biologic variables, and be able to clarify early phases of kidney damage. The most common biomarkers studied are Neutrophil Gelatinase-Associated Lipocalin (NGAL), Interleukin-18 (IL-18), Kidney Injury Molecule-1 (KIM-1), Cystatin-C, L type Fatty Acid-Binding Protein (L-FABP), N-Acetyl- </span><span style="font-size:10.0pt">&beta;</span><span style="font-size:10.0pt">-D Glucosaminidase (NAG), netrin-1, vanin-1, and Monocyte Chemoattractant Protein-1 (MCP-1) and calprotectin. </span></p> https://www.AJMB.org/En/Article.aspx?ID=60513 Behnaz Bazargani , Mastaneh Moghtaderi Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antibodies Produced Toward Recombinant RBD and Nucleocapsid Neutralize SARS-COV-2 <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> The highly contagious SARS-COV-2 virus spread rapidly from China and formed a global pandemic. The virus has infected over 509 million people worldwide and killed about 6.32 million up to date. Up on invasion, the Receptor Binding Domain (RBD) of Spike protein plays a crucial role in the entry of the virus into the host cell. The virus<em> </em>N protein is another protein that has a critical role for genome packaging. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> As bioinformatics approaches, the cassette design, codon adaptation, and protein stability were investigated in this study. Synthetic genes of RBD and N were cloned separately in <em>pET28a </em>+ expression vector. They were transferred into <em>Escherichia coli</em> (<em>E. coli</em>) BL21 DE3 host cell, and expression of recombinant proteins was induced with IPTG. The recombinant proteins were purified by column chromatography and approved by Western blotting. Animal immunization was performed with each of the recombinant proteins individually and in combination of the two. The antibody titer of the blood serum from control and immunized mice groups was determined by ELISA technique. Finally, the anti-spike neutralization test was performed.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The expression and purification of RBD protein were monitored on SDS-PAGE, two bands of about 28 and 45 <em>kDa</em> for RBD and N appeared on gel distinctly, which were further validated by Western blotting. According to ELISA results, related antibodies were traced to a dilution of 1/64000 in immunized sera. The neutralization test exhibited produced antibodies&#39; potency to bind the virus proteins. Using SPSS software, statistical analysis was performed by Duncan&#39;s test and T-test.</span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> According to the present study, recombinant proteins, either RBD alone or in combination with N adequately stimulated the immune response, and the raised antibodies could neutralize the virus in <em>in vitro</em> test.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60514 Amir Rezaei, Shahram Nazarian, Hossein Samiei Abianeh, Emad Kordbacheh, Zahra Alizadeh, Seyed Latif Mousavi Gargari Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Panel of Circulating microRNAs as a Potential Biomarker for the Early Detection of Gastric Cancer <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> The high mortality rate of Gastric Cancer (GC) is a consequence of delayed diagnosis. The early diagnosis of GC could increase the five-year survival rate among patients. We aimed to find a panel of microRNAs (miRNA) for the detection of GC in the early stages. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> In this case-control study, we selected consistently upregulated miRNAs from the results of 12 high-throughput miRNA profiling studies in GC. In the profiling phase, the differential expressions of 13 candidate miRNAs were analyzed by quantitative reverse-transcription PCR (qRT-PCR) in two pooled RNA samples prepared from the plasma of eight GC patients and eight matched controls. In the validation phase, significantly upregulated miRNAs from the profiling phase were further evaluated in the plasma samples of 97 patients with stage I-IV gastric adenocarcinoma and 100 healthy controls.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> In the profiling phase, six miRNAs (miR-18a, 21, 25, 92a, 125b and 221) were significantly upregulated in the GC patients compared to the controls (p&lt;0.05). However, in the validation phase, only significant up-regulation of miR-18a, 21 and 125b was confirmed (p&lt;0.05). A panel of miR-18a/21/125b was able to detect GC patients with stage I-IV from the controls (p&lt;0.001; AUC=0.92, sensitivity=86%; specificity=85%). In addition, the panel could distinguish the early-stage GC (I+II) from the control group with an AUC of 0.83, a sensitivity of 83%, and a specificity of 75%.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> A panel of circulating miR18a/21/125b could be suggested as a potential biomarker for the early detection of GC. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60515 Kioomars Saliminejad, Habibollah Mahmoodzadeh, Shahrzad Soleymani Fard, Marjan Yaghmaei, Hamid Reza Khorram Khorshid, Seyed Asadollah Mousavi, Mohammad Vaezi , Seyed Hamidollah Ghaffari Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Bioactive Materials Derived from Menstrual Blood Stem Cells Enhance the Quality of In Vitro Bovine Embryos <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Backgrounds:</span><span style="font-size:10.0pt"> The aim of this study was to determine whether the addition of bioactive materials derived from Menstrual Blood Stem Cells (MenSCs) to the oocyte maturation medium may improve the quality of bovine embryos <em>in vitro</em>. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> MenSCs were collected from 6 healthy women (between 26 and 36 years old) and after 3 days of culture, their bioactive materials were frozen. The bovine </span><span style="font-size:10.0pt"><span style="background-color:white">Cumulus-Oocyte-Complexes</span> (COCs) were aspirated from ovarian slaughterhouse and the oocytes with more than three layers of cumulus cells were cultured <em>in vitro</em> in media supplemented with (treatment) and without (control) 10% MenSCs&rsquo; bioactive materials. After IVM/IVF, the presumptive zygotes were cultured for 8 days. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The blastocyst rate on day 8 in treatment group was higher than control (40.2&plusmn;1.9 <em>vs.</em> 23&plusmn;4.2.3, p=0.001). The ratio of </span><span style="font-size:10.0pt"><span style="background-color:white">Trophectoderm</span></span><span style="font-size:10.0pt"><span style="background-color:white">&nbsp;(TE) and&nbsp;Inner Cell Mass (ICM)</span> (ICM/TE) cells was also greater in treatment group compared to control (30.3&plusmn;2 <em>vs.</em> 14.9&plusmn;1; p=0.001). The re-expansion of vitrified blastocysts, 24 hours after warming, in treatment group was higher than control (93.3&plusmn;2.5 <em>vs.</em> 66.2&plusmn;8.8; p=0.01). The expression of some genes related to pluripotency and implantation (<em>OCT4, CDX2</em>, and <em>IFNT</em>) were increased in treatment group compared to control (p&lt;0/05). </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> In conclusion, the addition of MenSCs&rsquo; bioactive materials during <em>in vitro</em> maturation of bovine oocytes could improve the quantity and quality of bovine IVP embryos. Also, the expression of some genes associated with pluripotency and implantation in the blastocyst would be increased.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60516 Mohammad Sobhan Amini , Mohammad Mehdi Naderi, Abolfazl Shirazi, Mehdi Aminafshar, Sara Borjian Boroujeni, Mostafa Pournourali , Ali Malekpour Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of PLGA-Encapsulated Recombinant GroEL of S. typhi immune Responses Against Enterohaemorrhagic and Enteropathogenic Escherichia coli <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Heat Shock Proteins (HSPs) elicit humoral and cellular immune responses. Due to their high sequence homology, they can be developed as a new immunogen for cross prophylactic and vaccination effects against infectious agents such as Enteropathogenic and Enterohemorrhagic <em>Escherichia coli</em> (EPEC and EHEC). This study aimed to evaluate the immunogenicity and cross-protective efficacy of rGroEL of <em><span style="background-color:white">Salmonella typhi</span></em> (<em>S. typhi</em>) encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles against EPEC and EHEC.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span> <span style="font-size:10.0pt">Recombinant GroEL was expressed in <em>Escherichia coli</em> (<em>E. coli</em>) and purified using Ni-NTA affinity chromatography. The protein was encapsulated in PLGA by the double emulsion method, and the nanoparticles were characterized physicochemically. BALB/c mice were immunized, and the efficacy of the protein to elicit immune responses was assessed. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt">Over-expression in <em>E. coli</em> led to corresponding 64.5 <em>kDa</em> protein bands in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Non-ag-gregated nanoparticles had a spherical shape with a mean diameter of 194.3&plusmn;3 <em>nm</em> and encapsulation efficiency of 89.5&plusmn;2.5%. Antibody isotyping revealed that GroEL immunization induced both IgG1 and IgG2a antibodies. Moreover, immunization of the mice with recombinant GroEL protein conferred 80 and 60% protection against lethal infections by EPEC and EHEC, respectively. Furthermore, organ burden studies revealed a significant reduction in infection in the immunized mice compared to the non-immunized ones. Passive immunization with anti-GroEL sera also protected 50% of the mice against the lethal doses of EHEC and EPEC strains. </span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The findings indicated that immunization of the mice with recombinant GroEL of <em>S. typhi</em> elicited cross-protection against other bacterial infections. This represented the immense potential of GroEL to be developed as a single vaccine against multiple pathogens</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60517 Milad Parvane , Shahram Nazarian, Emad Kordbacheh, Javad Fathi, Mohamad Ebrahim Minae , Mohammad Reza Ramezani Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparative Antioxidant and Anti-gout Activities of Citrullus colocynthis loaded Fruit Silver nanoparticles with its Ethanolic extract <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> The biological synthesis of silver nanoparticles (AgNPs) using plant materials is a rapidly developing method with several alternative medical applications. This comparative study of ethanolic fruit extract of <em>Citrullus colocynthis (C. colocynthis)</em> (EFECC) and synthesized silver nanoparticles (CC-AgNPs) were carried out for antioxidants and anti-gout arthritic activities. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> The AgNPs were synthesized using <em>C. colocynthis</em> fruit and its characterization was done by UV-visible spectroscopy, TEM, XRD and FT-IR. The 90% ethanol was used for extract preparation. Antioxidant activity was analyzed by DPPH and the Hydrogen Peroxide (H<sub>2</sub>O<sub>2</sub>)<sub> </sub>method. <em>In vitro</em> anti-arthritic activity was tested by xanthine oxidase inhibition, protein denaturation and HRBC membrane stabilization assay. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The synthesized CC-AgNPs were confirmed by UV-vis spectroscopy and TEM images displayed spherical shapes with 10-45 <em>nm</em> size range. Furthermore, the functional groups and crystalline structure of CC-AgNPs were determined by FT-IR and XRD analysis. The biosynthesized CC-AgNPs exhibited an excellent free radical scavenging ability than EFECC. In anti-arthritic activity, the CC-AgNPs showed effective inhibition of xanthine oxidase production, protein denaturation, and damaged RBC membranes compared to EFECC. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The antioxidant activities and <em>in vitro</em> anti-arthritic assays revealed that CC-AgNPs are better anti-gout agents than EFECC. This research suggested that biosynthesized silver nanoparticles from <em>C. colocynthis</em> fruit are an important target in the field of anti-gout drug discovery. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60518 Suganya Karunakaran, Rajeswary Hari Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association between PTCH1 and RAD54B Single-Nucleotide Polymorphisms and Non-syndromic Orofacial Clefts in the Northeast Population of Iran <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background: </span><span style="font-size:10.0pt">Non-Syndromic Cleft Lip with or without cleft Palate (NSCL/P) is a common developmental disorder of the head and neck with a multifactorial etiology. The current study aimed to evaluate the potential association of <em>PTCH1</em> (rs10512248) and <em>RAD54B</em> (rs12681366) polymorphisms with NSCL/P in the Northeast Iranian population.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods: </span><span style="font-size:10.0pt">In the present study, blood samples were taken from 122 subjects with NSCL/P and 161 healthy controls. Polymerase Chain Reaction (PCR) followed by Restriction Fragment Length Polymorphism (RFLP) were used to conduct genotyping of single-nucleotide polymorphisms.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results: </span><span style="font-size:10.0pt">Although differences were observed between cases and controls in rs10512248 and rs12681366, our data did not support a significant association of these polymorphisms with NSCL/P in our population.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion: </span><span style="font-size:10.0pt">Our findings suggest that polymorphisms of rs10512248 and rs12681366 may not be potential risk factors for NSCL/P in the Northeast Iranian population due to the multifactorial and multiethnicity characteristics of some genes.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60519 Reza Morvaridi Farimani , Mohsen Azimi-Nezhad , Hamid Reza Khorram Khorshid, Asghar Ebadifar , Saba Tohidkhah , Zahra Jafarian , Koorosh Kamali, Zeinab Nazari , Reza Ebrahimzadeh-Vesal Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of Poly-N-isopropylacrylamide Microgels Containing Antibiofilm Substances on Pseudomonas aeruginosa Isolated from Chronic Wounds <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Biofilm formation helps <em>Pseudomonas </em></span><em><span style="font-size:10.0pt">&lrm;</span></em><em><span style="font-size:10.0pt">aeruginosa</span></em><span style="font-size:10.0pt"> (<em>P. </em></span><em><span style="font-size:10.0pt">&lrm;</span></em><em><span style="font-size:10.0pt">aeruginosa</span></em><span style="font-size:10.0pt">) survive in various environments. Microgels can be effective in treatment of bacterial infections. The major aim of this study was to investigate effects of poly-N-isopropyl-acrylamide</span> <span style="font-size:10.0pt">microgels (PNIPAM) on <em>P. aeruginosa</em>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> Totally</span><span style="font-size:10.0pt">&lrm;</span><span style="font-size:10.0pt">, </span><span style="font-size:10.0pt">&lrm;</span><span style="font-size:10.0pt">100 <em>P. aeruginosa</em> strains were isolated from chronic wound infections</span><span style="font-size:10.0pt">&lrm;. Quantitative assessments of biofilm formation and antibiotic susceptibility were carried out. Furthermore, <em>alg</em>D, <em>las</em>R, and <em>PA</em>2714<em> </em>genes were amplified to investigate gene frequencies and expression rates.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results: </span><span style="font-size:10.0pt">Significant decreases were seen in <em>las</em>R expression in EDTA-treated samples</span><span style="font-size:10.0pt">&lrm;</span><span style="font-size:10.0pt">. Significant decreases were observed in expression of <em>alg</em>D and <em>las</em>R treated with xylitol. Decreased </span><span style="font-size:10.0pt">&lrm;expression of <em>PA</em>2714 was seen in samples treated with xylitol with no significance.&nbsp;</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The PNIPAM containing xylitol </span><span style="font-size:10.0pt">&lrm;or EDTA could penetrate biofilms of <em>P. aeruginosa</em> and significantly decrease expression of <em>las</em>R and <em>alg</em>D. This can be a novel strategy in the management of chronic wounds.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60520 Akram Etemadinia , Amir Seyfoori , Abbas Rahimi Foroushani , Ramin Mazaheri Nezhad Fard, Ronak Bakhtiari Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Mitochondrial Transfer from Menstrual Blood Stromal/Stem Cells Promotes Survival of Cardiomyocytes Following Myocardial Infarction <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Dear editor,</span></strong></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Recently, Mitochondrial Transfer (MT) from stem cells to injured cells has been proposed as a novel <span style="background-color:white">therapeutic strategy that could restore the bioenergetics requirement of damaged tissues</span> </span><sup><span style="font-size:10.0pt">1</span></sup><span style="font-size:10.0pt">. This organelle is <span style="background-color:white">essential for </span>cellular homeostasis, cell survival, cell growth, cell death induction, <span style="background-color:white">calcium storage,</span> cell signaling, and energy supply, <span style="background-color:white">especially in energy-demanding cells like cardiomyocytes </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">2</span></span></sup><sup><span style="font-size:10.0pt"><span style="background-color:white">,</span></span></sup><sup><span style="font-size:10.0pt"><span style="background-color:white">3</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">.</span></span> <span style="font-size:10.0pt"><span style="background-color:white">Mitochondrial dysfunction is contributed to a majority of pathologic conditions like</span></span><span style="font-size:10.0pt"> <span style="background-color:white">Myocardial Infarction (MI) and cardiomyopathies </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">4</span></span></sup><span style="font-size:10.0pt">. <span style="background-color:white">Mitochondrial impairment results in reduction of Adenosine Triphosphate (ATP) production, induces the generation of intracellular Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), and activates the caspase cleavage pathway </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">5</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">. </span></span><span style="font-size:10.0pt">It has been confirmed that Mesenchymal Stem Cells (MSCs) could transport mitochondria to a range of cells including endothelial cells and cardiomyocytes </span><sup><span style="font-size:10.0pt">6</span></sup><span style="font-size:10.0pt">. <span style="background-color:white">It appears that healthy mitochondrial donation by MSCs is a unique phenomenon that leads to replacement of dysfunctional mitochondria in injured cells</span>. <span style="background-color:white">It has been designated that the transfer of even a small number of Multipotent Mesenchymal Stem Cells (MMSC)-derived mitochondria resulted in enhanced oxidative phosphorylation and promotion of cell proliferation in the recipient damaged cells </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">7</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">.</span></span><span style="font-size:10.0pt"> Although mitochondrial transmission from various sources of stem cells like Bone Marrow Mesenchymal Stem Cell (BM-MSCs), induced Pluripotent Stem Cells (iPSCs), and Dental Pulp Derived Mesenchymal Stem Cell (DP-MSCs) has been stated, there is no report about MT from menstrual blood Stromal/Stem Cells (MenSCs). Recently we have perceived that, transepicardial MenSCs administration could improve cardiac function, prevent metaplastic development, and promote survival of cardiomyocytes following MI conceivably by transfer of their mitochondria to preserved cardiomyocytes and endothelial cells. We tracked the injected MenSCs 28 days&rsquo; post-transplantation by anti-human mitochondrial antibody in MI site in rat model and demonstrated successfully transferred human mitochondria from MenSCs into the targeted cells. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt"><span style="background-color:white">Researchers have showed that mitochondrial dysfunction plays critical role in the loss of cardiomyocytes during MI </span></span><sup><span style="font-size:10.0pt">8</span></sup><span style="font-size:10.0pt">.</span><span style="font-size:10.0pt"> Although exact mechanisms of MT have not been clarified yet, it has indicated that the local microenvironment of injured tissue releases physiological signals that trigger MT </span><sup><span style="font-size:10.0pt">9</span></sup><span style="font-size:10.0pt">. For instance, mitochondrial DNA (mtDNA) released by damaged cells is engulfed by MSCs and that later, prompts the cytoprotective function of MSCs and boosts mitochondrial biogenesis </span><sup><span style="font-size:10.0pt">10</span></sup><span style="font-size:10.0pt">. Furthermore, ROS and RNS can also stimulate mitochondrial donation </span><sup><span style="font-size:10.0pt">11</span></sup><span style="font-size:10.0pt">. It is assumed that, transmission of mitochondria derived from MenSCs may lead to maintenance of cellular homeostasis, preservation of aerobic respiration, reduction the level of ROS, prevention of cell death, <span style="background-color:white">and upregulation of cardio-protective cytokines</span> in the cardiomyocytes </span><sup><span style="font-size:10.0pt">12</span></sup><span style="font-size:10.0pt">. </span><span style="font-size:10.0pt">Mitochondria in MSCs like MenSCs is in dormant state due to lesser energy demands. However upon differentiation, increase in levels of respiratory enzymes, greater oxygen consumption rate, augmented levels of intracellular ATP, increase in mtDNA copy number and mRNA levels may occur </span><sup><span style="font-size:10.0pt">13</span></sup><sup><span style="font-size:10.0pt">,</span></sup><sup><span style="font-size:10.0pt">14</span></sup><span style="font-size:10.0pt">. Interestingly, it has been indicated that the MT phenomenon from MSCs not only results in protection of injured targeted cells, but also, it can lead to more MSCs survival due to decreasing their partially depolarized and dysfunctional mitochondria </span><sup><span style="font-size:10.0pt">15</span></sup><span style="font-size:10.0pt">. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Researchers indicated that </span><span style="font-size:10.0pt">transmission of mitochondria from </span><span style="font-size:10.0pt">BM-MSCs</span><span style="font-size:10.0pt"> to cardiomyocytes can inhibit </span><span style="font-size:10.0pt">apoptosis and reprogrammed differentiated cardiomyocytes to progenitor-like cells </span><sup><span style="font-size:10.0pt">16</span></sup><span style="font-size:10.0pt">. Furthermore, </span><span style="font-size:10.0pt">iPSC-MSCs directly protects cardiomyocytes against <span style="background-color:white">induced cardiomyopathy </span>through bioenergetic preservation by functional MT </span><sup><span style="font-size:10.0pt">17</span></sup><span style="font-size:10.0pt">. </span><span style="font-size:10.0pt">Also </span><span style="font-size:10.0pt">MT from MSCs to endothelial cell rescued the injured endothelial cell by reducing apoptosis and promoting proliferation </span><sup><span style="font-size:10.0pt">18</span></sup><span style="font-size:10.0pt">. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Meanwhile, some resident cells in injured site could transfer mitochondria to injured cells; we believe that only endogenous transfer is transient and cannot inhibit progressive injuries following MI. Our studies have shown that; administration of MenSCs post-MI modifies the metabolism and restores the functionality of heart, and also protect myocardium from further subsequent injuries mainly with donation of their healthy mitochondria to cardiomyocytes and endothelial cells. <span style="background-color:white">It is likely that the donor mitochondria fuse with mitochondria in the recipient cell, and restore bioenergetic requirements; or the recipient cell removes its injured mitochondria and gets the donated healthy mitochondria </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">19</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">. Researchers revealed that human mitochondrial DNA from MSCs could be found in mice 28 days after MSC administration. However, MSC nuclear DNA was not detected 3 days post administration and suggesting that the long-term therapeutic effects of MSCs administration may be related to MT </span></span><sup><span style="font-size:10.0pt"><span style="background-color:white">15</span></span></sup><span style="font-size:10.0pt"><span style="background-color:white">. </span></span><span style="font-size:10.0pt">So, the stem cell-based mitochondria transfer approach from MenSCs can be considered as a newly effective therapeutic strategy to treat cardiomyopathies.<span style="background-color:white"> However, more investigation is needed to explore the exact mechanism of the MenSCs-derived mitochondria communication with the recipient cells, restoration of mitochondrial bioenergetics in these cells, cell-signaling pathways involved to this phenomenon, and understand how this organelle donation would lead to regeneration.</span> </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60521 Mahmood Manshori, Somaieh Kazemnejad, Hannaneh Golshahi Mon, 26 Sep 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Monkeypox Outbreaks in Non-Endemic Countries: What Do We Know and What Do We Need? <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Following the news of the outbreak of the monkeypox virus in non-endemic countries from the media, a new wave of concerns was created. The current susceptibility to epidemics has resulted from successive peaks of the whole-society-inclusive coronavirus and its significant mortality and morbidity. To date, on May 28, 2022, there are 401 confirmed and 85 suspected cases worldwide <sup>1</sup>, almost all of which are from countries with high-income settings. According to Global.health, 3 suspicious and no confirmed cases have been tracked from Iran.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The human monkeypox is a viral zoonosis from the genus <em>Orthopoxvirus</em> in the family Poxviridae that causes flu-like symptoms, including fever, fatigue, and body aches, along with progressive macular-papular, vesicular, pustular, and crusted scab skin lesions that can be itchy <sup>2</sup>. The virus is closely related to the Variola virus, which causes smallpox in humans, but it leads to less severe symptoms and often improves without treatment; however, due to the broad spectrum of the disease severity, it can even be fatal. Previous observations reported fatality rates of 1 to 11%, which appear to be related to the virus clade, patients&#39; age, and concomitant infection with human immunodeficiency virus <sup>2,3</sup>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">This disease is not new, and there is good, but not enough, research and background information about it. The source of the current outbreak is unknown, and scientists are looking for reds to explain its outbreak. Transmission of the disease from animals, humans, including nosocomial and household transmission, and fomites is possible <sup>4</sup>. Like many other viral diseases, there is no definitive cure; however, antiviral drugs, like tecovirimat, vaccines, and immunoglobulins are available in developed countries, which help control complications, transmission, and epidemics <sup>5</sup>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The smallpox vaccine is about 85% effective against the monkeypox virus and reduces the frequency and severity of its signs and symptoms. It should be noted that non-elderly people were born after the cessation of smallpox vaccination <sup>3,6</sup>, and this may justify the storage of vaccines by developed countries. Scientists have previously reported the risks of cessation of smallpox vaccination, including the establishment and propagation of monkeypox, although they have outweighed the benefits due to the complications and costs of vaccination <sup>3,4</sup>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Information on the source and extent of the current cases and outbreaks is insufficient. In addition, although only two strains of the virus have been identified in the past, the possibility of novel strains emerging should be considered. Multiple outbreaks, 2022 outbreaks in non-endemic countries, and the high-consequence nature of the pathogen necessitate significant improvements in the quantity and quality of data collection, basic and clinical science research, providing vaccines, people and medical staff informing and education, and guideline development. These lead to more proper management of disease cases and the promotion of public health. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60503 Ahmad Shamabadi, Shahin Akhondzadeh Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression of Toll-Like Receptors 2, 4 and 5 in Relation to Gut Microbiota in Colon Neoplasm Patients with and without Inflammatory Bowel Disease <h1 style="text-align:justify"><span style="font-size:16pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Toll-Like Receptors (TLRs) are the critical mediators of inflammatory routs in the gut, which play an essential role in regulating the immune responses towards various ligands derived from pathogenic bacteria. Also, TLR signaling has been implicated in the development of Inflammatory Bowel Disease (IBD), Adenomatous Polyp (AP), and Colorectal Cancer (CRC). Here, we aimed to examine the expression of some TLRs concerning certain fecal bacteria in AP and CRC patients with and without IBD.</span></span></h1> <p><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> This case-control study collected fecal and colonic tissue samples from 93 patients versus Normal Controls (NC) <em>via</em> colonoscopy. Fecal samples were used for DNA extraction, and the abundance of selected fecal bacteria was determined by absolute real-time PCR. Also, the gene expression of TLR2, 4, and 5 was analyzed using RT-PCR on the colonic tissues of participants.</span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Compared to NC individuals, in AP and CRC patients, the mRNA expressions of TLR4 and TLR2 were significantly increased while TLR5 was decreased. A meaningful association between TLRs mRNA expression levels and the abundance of some selected fecal bacteria was detected. Also, </span><span style="font-size:10.0pt">there was a significant relationship between participant&rsquo;s food regimes, smoking habit and intestinal TLRs expression.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Our study proposed the important role of TLRs during adenomatous and CRC formation. Alterations in TLRs expression associated with certain gut bacteria may contribute to disease development.</span> </span></p> https://www.AJMB.org/En/Article.aspx?ID=60504 Hamid Asadzadeh Aghdaei, Sama Rezasoltani, Meisam Olfatifar, Ehsan Nazemalhosseini Mojarad, Ghazal Sherkat, Abbas Yadegar, Mohammad Mehdi Feizabadi, Mohammad Reza Zali Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Genome Analysis of an Enterococcal Prophage, Entfac.MY <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Bacteriophages are bacterial parasites. Unlike lytic bacteriophages, lysogenic bacteriophages do not multiply immediately after entering the host cells and may integrate their genomes into the bacterial genomes as prophages. Prophages can include various phenotypic and genotypic effects on the host bacteria. <em>Enterococcus</em> spp. are Gram-positive bacteria that cause infections in humans and animals. In recent decades, these bacteria have become resistant to various antimicrobials, including vancomycin.</span> <span style="font-size:10.0pt">The aim of this study was to analyze genome of an enterococcal prophage.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> In this study, <em>Enterococcus faecium</em> EntfacYE was isolated from biological samples and its genome was analyzed using next-generation sequencing method.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Overall, 254 prophage genes were identified in the bacterial genome. The prophage included 39 housekeeping, 41 replication and regulation, 80 structural and packaging, and 48 lysis genes. Moreover, 46 genes with unknown functions were identified. All genes were annotated in DNA Data Bank of Japan. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> In general, most prophage genes were linked to packaging and structure (31.5%) gene group. However, genes with unknown functions included a high proportion (18.11%), which indicated necessity of further analyses. Genomic analysis of the prophages can be effective in better understanding of their roles in development of bacterial resistance to antibiotics. Moreover, identification and study of prophages can help researchers develop genetic engineering tools and novel infection therapies.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60505 Maryam Yazdanizad, Ramin Mazaheri Nezhad Fard, Golshid Javdani Shahedin, Mohammadreza Salehi, Mahsa Dumanloo, Ali Akbar Saboor Yaraghi Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Isolation, Molecular Identification and Antibacterial Potential of Marine Bacteria from Deep Atlantic Ocean of Morocco <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Antibiotic resistance is an important concern for the public health authorities at global level</span><span style="font-size:10.0pt">. It is detrimental to human and environmental ecosystems, thus, there is a big need for natural bioactive compounds. In this work, we aimed to find out biomolecules derived from marine bacteria that may constitute an alternative to antibiotics. </span></span></p> <p><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> We isolated and identified thirty one marine bacteria collected from deep ocean water in central coast of Safi city, Morocco. Then, we induced biomolecules production in six marine bacterial strains. The extracts were tested for their antibacterial activity against </span><span style="font-size:10.0pt">gram-negative and gram-positive bacteria such as</span><span style="font-size:10.0pt"> <em>Escherichia coli </em></span><span style="font-size:10.0pt">ATCC 25922</span><em><span style="font-size:10.0pt">, Staphylococcus aureus </span></em><span style="font-size:10.0pt">ATCC </span><span style="font-size:10.0pt">33592</span><span style="font-size:10.0pt"> and<em> Listeria monocytogenes </em>ATCC 19117. Furthermore, we partially analyzed the chemical composition of these biomolecules and evaluated their sensibility to different temperatures. </span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The six marine bacteria were able to produce molecules which inhibited the three pathogenic strains with high inhibition zones reaching 27 <em>mm</em>. These molecules were characterized by heat stability from 60 to 121</span><em><span style="font-size:10.0pt">&deg;</span></em><em><span style="font-size:10.0pt">C</span></em><span style="font-size:10.0pt"> relying on each strain. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The produced molecules may offer a great potential to pharmaceutical industries as they may constitute an alternative to antibiotics that are becoming less effective due to the emergence of drugs resistance.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60506 Asmaa Chbel, Jorge Rodriguez-Castro, Javier Quinteiro, Manuel Rey-Méndez, Aurelio Serrano Delgado, Abdelaziz Soukri, Bouchra El Khalfi Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A New Specific DNA Target Sequence for Identification of Staphylococcus epidermidis using Modified Comparative Genomic Analysis <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> <em>Staphylococcus epidermidis (S. epidermidis) </em>is the most frequently isolated pathogen from prostheses infections in the body. </span><span style="font-size:10.0pt">Therefore, improving its diagnostic methods, including rapid Nucleic Acid Amplification Tests (NAAT), seems necessary. </span><span style="font-size:10.0pt">Since the first step in designing a NAAT is to find a specific sequence and all DNA targets that have been introduced so far are not completely specific, we introduced a new 100% specific DNA target sequence to identify <em>S. epidermidis </em>in this study.</span></span></p> <p><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> Modified comparative genomic analysis was used to find the best specific target sequence to detect <em>S. epidermidis</em>. A PCR method was designed for the evaluation of this target. To determine the detection limit and analytical specificity, pure genomic DNA of 18 bacteria include 12 standard strains (one <em>S. epidermidis</em> and 11 non-<em>S. epidermidis</em>) and six clinical isolates (five <em>S. epidermidis</em> and one non-<em>S. epidermidis</em>) were used.</span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The 400 <em>bp</em> sequence of <em>S. epidermidis</em> ATCC 14990 was identified as the most specific sequence (Se400), having a 100% sequence similarity to <em>S. epidermidis</em> genomes but not with other bacteria. The detection limit of Se400-PCR was 10 <em>fg</em>, equal to about 4 copies of <em>S. epidermidis</em> genomic <em>DNA/&mu;l</em>. All pure DNA templates from <em>S. epidermidis</em> generated a detectable amplicon by 264 <em>bp</em> length, but the PCR test was negative for the non-<em>S. epidermidis</em> group.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The Se400 sequence can be considered as a specific target for detecting <em>S. epidermidis, </em>based on our findings.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60507 Reza Khoshbakht, Hosna Zare, Reza Kamali Kakhki, Alireza Neshani, Maryam Arfaatabar Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir GC/MS Analysis and Phyto-synthesis of Silver Nanoparticles Using Amygdalus spinosissima Extract: Antibacterial, Antioxidant Effects, Anticancer and Apoptotic Effects <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> The present study was aimed at phyto-synthesized silver nanoparticles (AgNPs) using <em>Amygdalus spinosissima</em> (<em>A. spinosissima) </em>extract and to investigate the antibacterial, antioxidant effects, anticancer and apoptotic effects of phyto-synthe-sized AgNPs. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> The bio-fabricated AgNPs were characterized using UV-visible spectroscopy (UV-visible), X-ray Diffraction (XRD), Fourier Transform Infrared (FTIR), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and Energy Disper-sive X-ray (EDX). </span></span></p> <p><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The phyto-synthesized AgNPs showed maximum absorption in 438 <em>nm</em>, in the UV-visible spectrum. XRD peaks were observed at 2</span><span style="font-size:10.0pt">&theta;</span><span style="font-size:10.0pt"> values in 38.20</span><span style="font-size:10.0pt">&deg;</span><span style="font-size:10.0pt">, 44.40</span><span style="font-size:10.0pt">&deg;</span><span style="font-size:10.0pt">, 64.60</span><span style="font-size:10.0pt">&deg;</span><span style="font-size:10.0pt">, and 77.50</span><span style="font-size:10.0pt">&deg;</span><span style="font-size:10.0pt"> which are indexed as (111), (200), (220), and (311) bands of Face-Centered Cubic (FCC) structures of silver. FTIR analysis indicated that the AgNPs were capped with <em>A. spinosissima </em>extract. SEM and TEM micrographs revealed that the fabricated AgNPs were spherical and the average size range was 17.89 <em>nm</em>. Also, the EDX results show that the content of Ag was 90%. </span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The phyto-synthesized AgNPs had significant antibacterial activity against Gram-negative bacteria, as well as, the AgNPs exhibited great inhibitory effects on DPPH radicals and their antioxidant properties were favorably comparable to the antioxidant outcomes of ascorbic acid. Moreover, the AgNPs showed anti-cancer activity against the MCF-7 cell line with the IC50=6.1 <em>&micro;g/ml</em>. Moreover, the phyto-synthesized AgNPs could induce apoptosis in the MCF-7 cell line significantly. The GC-MS analysis of the <em>A. spinosissima</em> extract showed that 102 bioactive phyto-chemical compounds, which be of use to the synthesis of AgNPs.&nbsp;&nbsp;&nbsp;&nbsp; </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60508 Azadeh Farmahini Farahani, Seyed Mohammad Mahdi Hamdi, Amir Mirzaee Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Investigation of Durability of SARS-CoV-2-specific IgG and IgM Antibodies in Recovered COVID-19 Patients: A Prospective Study <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Evidence on seroconversion profile of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients is limited. We mainly aimed to evaluate seroconversion and persistence of virus-specific antibodies in patients infected by coronavirus disease 2019 (COVID-19).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> This prospective study was conducted on 118 patients with COVID-19 presentations </span><span style="font-size:10.0pt">admitted to three hospitals in Iran and recovered from the disease, during April and May 2020. Presence of COVID-19 was confirmed by Polymerase Chain Reaction (PCR) testing on nasopharyngeal swabs.</span><span style="font-size:10.0pt"> Serum samples were collected at different time points, including 0-5, 6-15, 16-25, 26-35, and 36-95 days of clinical symptom onset. For measurement of SARS-CoV-2-specific IgG and IgM antibody titers, Iran&#39;s Food and Drug Administration-approved SARS-CoV-2 ELISA kits were used. </span></span></p> <p><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Serologic assay revealed that 37.3% of patients (n=44) were positive for IgM at 0-5 days interval after clinical symptom onset. This rate was 60.2% (n=71) for IgG. There were increasing IgM and IgG seroconversion rates during first 25 days of clinical symptom onset, but seropositivity started to decrease thereafter, which was more evident for IgM (17.9%) than IgG (58.9%) at the 36-95 days post symptoms appearance. In other words, it was found that 83.6% of IgM-positive and 32.9% of IgG-positive patients in the first month of clinical symptom onset became seronegative in the third month of clinical symptom onset. </span></p> <p><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The findings demonstrated that antibody responses to SARS-CoV-2 infection were developed in recovered COVID-19 patients; however, some of them were seronegative three months after onset of relevant symptoms. Furthermore, the stability of anti-SARS-CoV-2 antibodies could also correct our expectations from COVID-19 vaccination responses.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60509 Mohammad Zamani, Ahmad Ghasemi, Morteza Shamshirgaran, Sajjad Ahmadpour, Ahmad Hormati, Javad Khodadadi, Mehran Varnasseri, Fatemeh Amini, Amaneh Shayanrad, Vahid Younesi, Hossein Poustchi, Mahdi Shabani Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Prioritizing Candidate Genes for Type 2 Diabetes Mellitus using Integrated Network and Pathway Analysis <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Type 2 Diabetes Mellitus (T2DM) has emerged as a major threat to global health that fosters life-threatening clinical complications, taking a huge toll on our society. More than 65 million Indians suffer from T2DM, making it one of the leading causes of death. T2DM and associated complications have to be constantly monitored and managed which reduces the overall quality of life and increases socioeconomic burden. Therefore, it is crucial to develop specific treatment and management strategies. In order to achieve this, it is essential to understand the underlying genetic causes and molecular mechanisms.</span></span></p> <p><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> Integrated gene network and ontology analyses facilitate prioritization of plausible candidate genes for T2DM and also aid in understanding their mechanistic pathways. In this study, T2DM-associated genes were subjected to sequential interaction network and gene set enrichment analysis. High ranking network clusters were derived and their interrelation with pathways was assessed. </span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> About 23 significant candidate genes were prioritized from 615 T2DM-associ-ated genes which were overrepresented in pathways related to insulin resistance, type 2 diabetes, signaling cascades such as insulin receptor signaling pathway, PI3K signaling, IGFR signaling pathway, ERBB signaling pathway, MAPK signaling pathway and their regulatory mechanisms. </span></span></p> <p><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Of these, two tyrosine kinase receptor genes-<em>EGFR</em> and <em>IGF1R</em> were identified as common nodes and can be considered to be significant candidate genes in T2DM.</span></p> <div> <div> <div>&nbsp;</div> <div> <div> <div>&nbsp;</div> <div> <p>&nbsp;</p> <p>&nbsp;</p> </div> </div> </div> </div> </div> https://www.AJMB.org/En/Article.aspx?ID=60510 Tejaswini Prakash, Nallur B Ramachandra Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An in silico Design, Expression and Purification of a Chimeric Protein as an Immunogen Candidate Consisting of IpaD, StxB, and TolC Proteins from Shigella spp. <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><em><span style="font-size:10.0pt"> Shigella</span></em><span style="font-size:10.0pt"> spp. is the cause of dysentery and is widespread worldwide. On the other hand, antibiotic resistance is increasing in this bacterium.</span> <span style="font-size:10.0pt">Bioinformatics is a new approach to vaccine and drug design involving the selection of appropriate antigens.</span> <span style="font-size:10.0pt">This study aimed to design a chimeric protein consisting of IpaD, StxB, and TolC proteins from <em>Shigella</em> through a bioinformatics approach as an immunogen candidate.</span></span></p> <p><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> The sequences of <em>ipaD</em>, <em>stxB,</em> and <em>tolC</em> genes were obtained. Additionally, the immunogenic regions of the associated protein, physicochemical characteristics, protein structures, B and T cells epitopes, and molecular docking were determined using <em>in silico </em>servers. Besides,</span> <span style="font-size:10.0pt">the chimeric gene was synthesized following sequence optimization by utilizing the codon usage of <em>Escherichia coli </em>(<em>E. coli)</em>. The expression of the recombinant protein was confirmed <em>via</em> SDS-PAGE and Western blot technique. </span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The residues 41-160 of IpaD, 21-89 of StxB, and 40-335 of TolC were selected. According to half-life, instability, and buried indices, IpaD-StxB-TolC was selected as the best arrangement. The Ramachandran plot showed that 97.077% of the amino acids were in the favored area. Linear and conformational epitopes were also present throughout the chimeric protein sequence. Moreover,</span> <span style="font-size:10.0pt">the C-ImmSim server indicated that IgG and IgM titers could reach desirable values by the third injection</span><span style="font-size:10.0pt">. </span><span style="font-size:10.0pt">Furthermore, the stability of the mRNA-optimized gene was enhanced, increasing the Codon Adaptive Index (CAI) to 0.9. Finally, the chimeric gene was transferred to <em>E. coli</em> BL21, and the expression of the 60.6 <em>kDa</em> recombinant protein was confirmed.&nbsp; </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The results indicated that the recombinant protein could act as a proper immunogen candidate against <em>Shigella</em> spp.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60511 Javad Fathi, Shahram Nazarian, Emad Kordbacheh, Nahal Hadi Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Impact of Single Nucleotide Polymorphism in the ANKRD55 Gene on Occurrence and Clinical Characteristics of Rheumatoid Arthritis <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Rheumatoid Arthritis (RA) has multifactorial etiology and numerous genetic and environmental factors have been related to an increased risk of RA. Recently, Genome-Wide Association Studies (GWAS) suggested a large number of Single Nucleotide Polymorphisms (SNPs) loci affecting the susceptibility to RA. One of these loci is rs6859219 (C&gt;A), a functional polymorphism in the <em>ANKRD55</em> gene which was associated with the expression of <em>ANKRD55</em> and <em>IL6ST</em>. In the current study, we evaluated the possible association between rs6859219 (intronic variant) in the <em>ANKRD55</em> gene with RA risk in the Iranian population.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> A case-control study using 118 RA patients and 115 healthy counterparts was undertaken in order to determine rs6859219 genotypes using real</span><span style="font-size:10.0pt">‑</span><span style="font-size:10.0pt">time polymerase chain reaction High</span><span style="font-size:10.0pt">‑</span><span style="font-size:10.0pt">Resolution Melting (HRM) method. </span></span></p> <p><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> There was a significant difference in the genotype and allele frequencies of rs6859219 between patients and controls (p&lt;0.001). Logistic regression analysis demonstrates that CC genotype and C allele increased the risk of RA (OR <sub>for CC genotype</sub>= 7.12; 95%CI [3.51-15.05]/ OR <sub>for C allele</sub>=4.16; 95%CI [2.78-6.28]). Furthermore, regarding the dominant and recessive model of inheritance, RA patients indicated obvious association of the rs6859219 variant compared to healthy controls (p&lt;0.001). Moreover, in the patient group, there was a significant correlation between <span style="background-color:white">C-Reactive Protein (CRP) </span>concentration with rs6859219 polymorphism (p&lt;0.001).</span></p> <p><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Our findings propose a substantial correlation between rs6859219 polymorphism and RA risk and clinical characteristics of this disease in the Iranian population.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60512 Rasoul Salehi, Mina Motaghi, Amirhossein Salehi, Hadi Karimzadeh, Bahram Pakzad Wed, 13 Jul 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Coronavirus Vaccination and Mortality in the Omicron Outbreak in Iran: Mortality Reduction due to Attenuated Pathogenicity and Booster Vaccine Doses <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">More than two years after the</span><span style="font-size:10.0pt"> first report of the rapidly</span><span style="font-size:10.0pt"> spreading coronavirus disease 2019 (COVID-19), the coronavirus is still unpredictable</span><span style="font-size:10.0pt"> by emerging </span><span style="font-size:10.0pt">highly divergent variants <sup>1</sup></span><span style="font-size:10.0pt">. Through the highly contagious viral sickness the virus causes, it imposes significant morbidity and mortality on global populations, becoming the second cause of death in people aged 25 to 44 years in January 2021 in the United States. </span><span style="font-size:10.0pt">Although mortality rates in disease peaks were significantly reduced as vaccination rates increased, mortality increased again with the outbreak of a variant of concern called Omicron so that the virus became the second leading cause of death </span><span style="font-size:10.0pt">in January 2022 in the United States <sup>1,2</sup></span><span style="font-size:10.0pt">.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The novel variant, first identified in Botswana and named Omicron (B.1.1.529) by World Health Organization on November 26, 2021, exhibited 36 mutations in the spike protein, the target of antibodies, and a total of 59 mutations throughout its genome. More importantly, 15 mutations occurred in the Receptor-Binding Domain (RBD), which can increase infectivity and mediate virus escape from vaccine-induced antibodies <sup>3</sup>.</span><span style="font-size:10.0pt"> In other words, the highly mutated omicron variant can evade immunity in vaccinated individuals and is associated with </span><span style="font-size:10.0pt">vaccine breakthroughs <sup>1,3</sup>.</span><span style="font-size:10.0pt"> The Omicron has a 13-fold increase in viral infectivity, and compared to the Delta variant, it is 2.8 times more contagious and its R0, the indicator of </span><span style="font-size:10.0pt">contagiousness and transmissibility,</span><span style="font-size:10.0pt"> can be as high as 10, while for the Delta, it did not reach 7 <sup>4</sup>. Fortunately, the mortality of this variant is lower than the wild type and Alpha, Beta and Delta variants <sup>5</sup>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">About two years after the first official report of the SARS-CoV-2 outbreak in Iran, like in other countries, the virus is still raging.</span><span style="font-size:10.0pt"> As in other countries, the peak of the Omicron variant in Iran increased infection, hospitalization, and mortality once again. During the Omicron outbreak in Iran, the maximum number of newly identified cases in one day was more than 39,000, so that the maximum number of active cases in one day reached about 375,000 by mid-February 2022. Although in the previous peak, the maximum number of new cases detected in a day and the maximum number of deaths per day were about 50,000 and more than 700, respectively, the number of deaths per day did not reach 250 in this peak <sup>6</sup>. This difference may be due to the lower mortality<sup>5</sup> and progress of the vaccination process, although vaccine efficacy is negatively affected by new variants <sup>3</sup>. Although the percentages of people who have received at least one dose of a vaccine, have been fully vaccinated, and given the booster dose are now about 75%, 65%, and 25%, respectively, </span><span style="font-size:10.0pt">these percentages on August 24, 2021, when the highest number of deaths due to COVID-19 were reported in Iran, were about 20%, 7%, and 0, respectively <sup>7</sup>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The fastest and most accessible ways to protect against the Omicron variant seem to be wearing medical-grade masks and receiving the third dose of vaccine <sup>8</sup>. The protection provided by two shots of vaccines is reduced to less than 40% a few months after the second shot, but the third shot seems to cause about 60 to 70% protection in the two weeks after the injection and protect against severe COVID-19 <sup>9</sup>. It has been shown that vaccine efficacy decreases with increasing time after vaccination <sup>3</sup>. There is not much difference in the treatment of the disease caused by this variant, but of the monoclonal antibodies, only sotrovimab appears to be effective and is authorized <sup>4</sup>. Eventually, updating the current vaccines and pursuing a universal vaccine should be of concern to those in charge <sup>8,10</sup>.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60492 Ahmad Shamabadi, Shahin Akhondzadeh Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cellular Therapy: The Hope for Covid-19 <p><span style="font-size:11.0pt"><span style="background-color:white">Coronaviruses (CoVs) are a group of very diverse viruses that cause a broad spectrum of diseases from mild to severe enteric, respiratory, systemic diseases, and common cold or pneumonia among humans and animals. </span></span><span style="font-size:11.0pt">This virus is associated with Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and lung disease that lead to Acute Respiratory Distress Syndrome (ARDS). In December 2019, researchers <span style="background-color:white">identified </span>a novel coronavirus type, called <span style="background-color:white">Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), </span>which was associated with symptoms of high fever, dry cough, headache, diarrhea, and reduction of White Blood Cells (WBC).</span> <span style="font-size:11.0pt"><span style="background-color:white">Coronavirus-associated acute respiratory disease was named Coronavirus Disease 19 (COVID-19). </span></span><span style="font-size:11.0pt">No proven treatment has been discovered for COVID-19</span> <span style="font-size:11.0pt">so far, but researchers are trying to find the best effective way to treat this disease. Therefore, therapeutic strategies that facilitate the recovery of COVID-19 patients and reduce life-threatening complications are urgently needed now. Today, Mesenchymal Stem Cells (MSCs) and their secretion are utilized as one of the most applied tools to treat various diseases such as inflammation and cancer. MSC-derived vesicles are rich in various growth factors, cytokines, and interleukins that are produced and secreted under different physiological or pathological conditions. These vesicles were considered a suitable and effective tool in regeneration medicine because of their high power in repairing damaged tissues and modulating immune responses. Recently, evidence has shown MSC-derived vesicles through reduced expression of pro-inflammatory cytokines could improve damaged tissues in COVID-19 patients. In addition to MSCs and MSC-derived exosomes, Natural Killer (NK) cells, T cells, and platelet lysates were used against viral infection. In this review, we tried to provide an overview of MSC secretion and immune cells</span> <span style="font-size:11.0pt">for COVID-19 therapy.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60493 Sima Nobari, Motahareh Rezvan, Fariba Dashtestani, Mahdieh Gangi, Hoda Keshmiri Neghab Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Designing a Multi-Epitope Antigen for Serodiagnosis of Strongyloides stercoralis Based on L3Nie.01 and IgG Immunoreactive Epitopes <p><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Serological diagnosis of <em>Strongyloides stercoralis</em>&nbsp; (<em>S. stercoralis</em>) is fre-quently challenging because of cross-reactivity with other parasitic nematodes. There-fore, it is necessary to introduce novel serological tests with high performance to properly diagnose this neglected parasitic infection. The purpose of the current study was to design a multi-epitope construct for the diagnosis of <em>S. stercoralis</em>. </span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> For the purpose of this study, first, highly antigenic segments and potential immunodominant epitopes of <em>S. stercoralis</em> were identified from two antigenic proteins, and then all of the selected parts were linked by an appropriate linker. Next, the physico-chemical features of the designed construct were analyzed. Then, tertiary structures of the construct were built and evaluated to find out the best one. Lastly, the amino acid sequence was reverse-translated and optimized for over-expression in <em>Escherchia coli</em> (<em>E. coli)</em>. </span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The bioinformatic evaluation indicated that the designed protein construct could be hydrophilic, thermostable, and acidic and the estimated half-life was more than 10 <em>hr</em> in <em>E. coli</em>.</span></span></p> <p>Conclusion:<strong> </strong><span style="font-size:10.0pt">According to the results of the study, the designed construct could be used as an efficient antigen in the ELISA system for serological diagnosis of human strong-yloidiasis.</span></p> https://www.AJMB.org/En/Article.aspx?ID=60494 Ahmad Movahedpour, Zohreh Mostafavi-Pour, Bahador Sarkari, Mortaza Taheri-Anganeh, Navid Nezafat , Amir Savardashtaki , Younes Ghasemi Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir CpG-Containing Oligodeoxynucleotides and Freund Adjuvant in Combination with Alum Augment the Production of Monoclonal Antibodies Against Recombinant HBsAg <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Adjuvants are essential to potentiate the immune response to inoculated antigens and play a central role in vaccine development. Alum is generally used as a classic adjuvant, although it does not stimulate proper immunity, and some of the immunized subjects have low or no antibody response. Efforts have been continued to find more efficient adjuvants for better antibody responses. In the present study, the efficacy of three formulations of adjuvants, <em>i.e</em>. Cysteine p Guanine Oligodeoxynucleotide (CpG ODN), alum, and Freund, in the production of monoclonal anti Hepatitis B Surface Antigen (HBsAg) antibodies was investigated.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> To immunize mice, regular hepatitis B vaccine containing recombinant HBsAg and alum was used with CpG ODN or Freund adjuvants, and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by Enzyme-Linked Immunosorbent Assay (ELISA) using HBsAg as coating antigen followed by a limited dilution process.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The results showed that by using all three formulations of adjuvants, monoclonal antibody (mAb) specific to HBsAg was successfully generated. It was also found that the mice immunized with (HBsAg + Alum) + CpG had the highest concentration of antibody production in serum and hybridoma supernatants as well as positive clones. Based on these findings, the addition of CpG ODN also induced a higher antibody response compared with Complete Freund&rsquo;s Adjuvant (CFA).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Results of this study showed that CpG and Freund adjuvants could be efficient partners for alum in the immunization period of the process of monoclonal antibody production.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60495 Mahsa Khayyati Kohnehshahri, Nowruz Delirezh , Leili Aghebati Maleki Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Inhibitory Effect of Thioridazine on adeB Efflux Pump Gene Expression in Multidrug-Resistant Acinetobacter baumannii Isolates Using Real Time PCR <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> The purpose of the present study was to investigate the antimicrobial effects of berberine and thioridazine, as well as their effect on the gene expression of the AdeABC efflux pump system in Multidrug-Resistant (MDR)<em> Acinetobacter baumannii</em> (<em>A. baumannii</em>) isolates.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> This study was carried out in five MDR clinical isolates of <em>A. baumannii</em> and a sample of standard strain (<em>A. baumannii</em> PTCC1797). The effect of Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of berberine, thioridazine, and ciprofloxacin alone and their combination on <em>A. baumannii</em> was evaluated by broth microdilution method. Also, their effect on the expression of <em>adeB</em> efflux pump gene was evaluated using real time PCR method. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The MIC of thioridazine, berberine, ciprofloxacin+thioridazine, ciprofloxacin+ berberine, thioridazine+berberine, and ciprofloxacin+thioridazine+berberine on MDR <em>A. baumannii</em>&nbsp;isolates was 64, 256, 128, 256, 128, and 128<em> &mu;g/ml</em>, respectively. The results showed that treatment of strains with thioridazine alone and in combination with berberine and ciprofloxacin significantly (p&lt;0.05) decreased the expression of <em>adeB</em> efflux pump gene.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Due to the inhibitory effects of thioridazine on bacterial isolates and<em> adeB</em> efflux pump gene, this compound can be used as a potential antimicrobial agent against MDR A. <em>baumannii.</em></span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60496 Fereshteh Ahmadi , Bahman Khalvati , Saba Eslami , Mehdi Mirzaii, Narges Roustaei , Farzad Mazloomirad , Seyed Sajjad Khoramrooz Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Motif-Based Network Analysis of Regulatory Patterns in Doxorubicin Effects on Treating Breast Cancer, a Systems Biology Study <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Breast cancer is the most common malignancy worldwide. Doxorubicin is an anthracycline used to treat breast cancer as the first treatment choice. Never-theless, the molecular mechanisms underlying the response to Doxorubicin and its side effects are not comprehensively understood</span> <span style="font-size:10.0pt">so far. We used systems biology and bio-informatics methods to identify essential genes and molecular mechanisms behind the body response to Doxorubicin and its side effects in breast cancer patients. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> Omics data were extracted and analyzed to construct the protein-protein interaction and gene regulatory networks. Network analysis was performed to iden-tify hubs, bottlenecks, clusters, and regulatory motifs to evaluate crucial genes and molecular mechanisms behind the body response to Doxorubicin and its side effects. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Analyzing the constructed PPI and gene-TF-miRNA regulatory network showed that MCM3, MCM10, and TP53 are key hub-bottlenecks and seed proteins. Enrichment analysis also revealed cell cycle, TP53 signaling, Forkhead box O (FoxO) signaling, and viral carcinogenesis as essential pathways in response to this drug. Besides, SNARE interactions in vesicular transport and neurotrophin signaling were identified as pathways related to the side effects of Doxorubicin. The apoptosis in-duction, DNA repair, invasion inhibition, metastasis, and DNA replication are sug-gested as critical molecular mechanisms underlying Doxorubicin anti-cancer effect. SNARE interactions in vesicular transport and neurotrophin signaling and FoxO signaling pathways in glucose metabolism are probably the mechanisms responsible for side effects of Doxorubicin. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Following our model validation using the existing experimental data, we recommend our other newly predicted biomarkers and pathways as possible mole-cular mechanisms and side effects underlying the response to Doxorubicin in breast cancer requiring further investigations.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60497 Zeinab Dehghan , Seyed Amir Mirmotalebisohi, Marzieh Sameni, Maryam Bazgiri, Hakimeh Zali Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Bioinformatic Investigation of Micro RNA-802 Target Genes, Protein Networks, and Its Potential Prognostic Value in Breast Cancer <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> An increasing number of studies have suggested that unveiling the molecular network of miRNAs may provide novel therapeutic targets or biomarkers. In this study, we investigated the probable molecular functions that are related to microRNA-802 (miR-802) and evaluated its prognostic value in breast cancer utilizing bioinformatics tools.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> PPI network, pathway enrichment and transcription factor analysis were applied to obtain hub genes among overlapping genes of four miRNA target prediction databases. Prognosis value assessments and expression analysis of hub genes using bioinformatics tools, as well as their literature validation were performed.&nbsp; </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Our results showed a significant correlation of the miR-802 overexpression with poor patient survival rate (BC, p=2.7e-5). We determined 247 target genes significant for GO and KEGG terms. Analysis of TFs by TRUST showed that RUNX3, FOXO3, and E2F1 are possible TFs that regulate the miR-802 expression and target genes network. According to our analysis; 21 genes might have an important function in miR-802 molecular processes and regulatory networks. The result shows that among these 21 genes, 8 genes (<em>CASC3, ITGA4, AGO3, TARDBP, MED13L, SF1, SNRPE </em>and<em> CRNKL1</em>) are positively correlated with patient survival. Therefore these genes could be considered and experimentally evaluated as a prognostic biomarker for breast cancer.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The comprehensive bioinformatics study on miR-802 target genes provided insight into miR-802 mediated pathways and processes. Furthermore, representing candidate target genes by prognostic values indicates the potential clinical application of miR-802 in breast cancer.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60498 Maryam Eini, Sepideh Parsi , Mahmoud Barati, Golnaz Bahramali, Marziyeh Alizadeh Zarei , Jafar Kiani, Asaad Azarnezhad, Arshad Hosseini Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of Aberrant Promoter Methylation Changes in the Suppressor of Cytokine Signaling 3 (SOCS3) Gene with Susceptibility to Crohn's Disease <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Growing evidence supports that changes in the methylation state of Inflammatory Bowel Disease (IBD)-associated genes could significantly alter levels of gene expression, potentially contributing to disease onset and progression. We supposed that alterations in DNA methylation status at promoter region within the suppressor of cytokine signaling 3 <em>(SOCS3)</em> gene in intestinal tissues may be involved in the susceptibility to Crohn&#39;s Disease (CD).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> DNA methylation status in the promoter region of the human <em>SOCS3</em> gene of intestinal tissues from 15 patients with CD and 15 age- and sex-matched healthy controls were profiled using the real-time Quantitative Multiplex Methylation Specific PCR (QM-MSP) assay.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Based on methylation assay data profiling, we found that patients with CD showed a higher degree of methylation of the <em>SOCS3</em> gene promoter region than did the healthy controls (unmethylated DNA in CD <em>vs.</em> healthy controls; 0.00048&plusmn;0.0011 <em>vs.</em> 0.07&plusmn;0.142, p&lt;0.000).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The data presented here demonstrate that aberrant methylation of the CpG islands within promoter regions of <em>SOCS3</em> gene in colonic mucosa of CD was associated with mucosal inflammatory status, providing insights into the involvement of methylation could contribute to the initiation of the inflammatory process and development of CD.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60499 Golshid Sanati, Davood Jafari, Mehrdad Noruzinia, Naser Ebrahimi-Daryani, Mohammad Ahmadvand, Shahram Teimourian, Nima Rezaei Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Strong Association of Polymorphism in SPRED2 Gene with Disease Susceptibility and Clinical Characteristics of Rheumatoid Arthritis in the Iranian Population <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> The high heritability of&nbsp;Rheumatoid Arthritis (RA)&nbsp;has been estimated from&nbsp;different studies.&nbsp;Recently, Genome-Wide Association Studies&nbsp;(GWAS) show a large number of Single Nucleotide Polymorphisms (SNPs) loci affecting susceptibility to RA. The rs934734 polymorphism in the <em>SPRED2</em> gene is one of these loci. Studies have shown that the <em>SPRED2</em> gene is involved in the regulation of inflammatory response, leukocyte infiltration, and local chemokine production. In the current study, the possible association between SNP rs934734 (intronic variant) in the <em>SPRED2</em> gene with RA risk in the Iranian population was evaluated.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> One hundred fourteen RA patients and 120 healthy counterparts were recruited in this case-control study to evaluate rs934734 genotypes using the real-time PCR High Resolution Melting method (HRM).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Logistic regression analysis demonstrated that GG and AG genotypes compared with AA genotype increase the risk of RA (GG <em>vs</em>. AA; OR=4.61; 95%CI [2.21-9.35]; p&lt;0.001 and AG <em>vs</em>. AA; OR=2.54; 95%CI [1.36-4.76]; p=0.004). Furthermore, subjects with allele G were more frequently affected with RA than subjects with A allele (OR=2.33; 95%CI [1.61-3.38];<em> </em>p&lt;<span style="background-color:white">0.001</span>). Besides, in the patient group, there was a significant correlation between <span style="background-color:white">Erythrocyte Sedimentation Rate (ESR) and C-reactive protein (CRP) </span>concentration with rs934734 polymorphism (p&lt;0.05).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Our findings suggest that rs934734 in <em>SPRED2</em> strongly underlies RA development and is associated with clinicopathological characteristics of this disease. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60500 Bahram Pakzad, Hamed Moghadammanesh, Mansour Salesi, Rasoul Salehi Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of MTHFR, BMP4, TGFA and IRF6 Polymorphisms with Non-Syndromic Cleft lip and Palate in North Indian Patients <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background: </span><span style="font-size:10.0pt">Non-Syndromic Cleft Lip and Palate (NSCL/P) is a multifactorial birth defect. The world-wide prevalence of NSCL/P is 1 in 1000 live births; it differs with race, ethnicity and gender. The aim of the present study was to find out the status of candidate gene polymorphisms in NSCL/P cases and its association in phenotype of the patients. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods: </span><span style="font-size:10.0pt">We have screened five polymorphisms in four candidate genes <em>MTHFR</em> (rs1801133, rs1801131) <em>BMP4</em> (rs17563), <em>TGFA</em> (rs1146297) and <em>IRF6</em> (rs2235371) by restriction fragment length polymorphism and results were validated by Sanger sequencing. Our dataset consists of 200 NSCL/P cases and 200 healthy controls from the Indian population. Statistical data analysis was performed by SPSS software.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results: </span><em><span style="font-size:10.0pt">MTHFR</span></em><span style="font-size:10.0pt"> (rs1801133), <em>BMP4</em> (rs175563) and <em>TGFA</em> (rs11466297) gene polymorphisms showed significant association with NSCL/P and act as a risk factor in the Indian population (p=&lt;0.05). However, <em>MTHFR</em> (rs1801131), and <em>IRF6</em> (rs2235371) gene polymorphisms did not show significant association with NSCL/P in the Indian population. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion: </span><span style="font-size:10.0pt">The result of the study suggests an association between <em>MTHFR</em> (rs1801133), <em>BMP4</em> (rs175563) and <em>TGFA</em> (rs11466297) polymorphisms with NSCL/P in Indian population. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60501 Kapil Avasthi, Amit Agarwal, Sarita Agarwal Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of rs2013162 and rs2235375 Polymorphisms in IRF6 Gene with Susceptibility to Non-Syndromic Cleft Lip and Palate <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Non-syndromic cleft lip occurs by the interaction of environmental and genetic factors. The purpose of the current study was to analyze the association of<em> </em></span><span style="font-size:10.0pt">Single Nucleotide Polymorphisms (SNPs) in</span><em><span style="font-size:10.0pt"> IRF6 </span></em><span style="font-size:10.0pt">and NS</span><span style="font-size:10.0pt">CL/P in an Iranian population.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span> <span style="font-size:10.0pt">A group of 105 children with NSCL/P and 185 normal controls were included in the current study. Genotyping of <em>IRF6</em> rs2013162 and rs2235375 was performed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt">A substantial association of AA and CA genotypes in rs2013162 with the risk of NSCL/P (AA <em>vs</em>. CC; OR=2.36; 95%CI [1.05-5.29], p=0.004; and CA <em>vs</em>. CC; OR=0.47; 95%CI [0.28-0.79], p=0.018) was found. <span style="background-color:white">However, there were no important associations between A allele and risk of NSCL/P (p=0.980).</span> According to logistic regression analysis results, subjects with GG genotype and G allele in</span><em> </em><span style="font-size:10.0pt">rs2235375</span><span style="font-size:10.0pt"> polymorphism had increased risk of NSCL/P.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The<em> IRF6 </em>polymorphisms are associated with the susceptibility to NS</span><span style="font-size:10.0pt">CL/P</span><span style="font-size:10.0pt"> in Iranian population.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60502 Masoumeh Soleymani, Asghar Ebadifar, Maryam Khosravi, Emran Esmaeilzadeh, Hamid Reza Khorram Khorshid Wed, 23 Mar 2022 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir CRISPR-Cas System: A Promising Diagnostic Tool for Covid-19 <p><span style="font-size:11pt"><span style="font-size:10.0pt">More than a year has passed since the beginning of the 2019 novel coronavirus diseases (COVID-19) pandemic which has created massive problems globally affecting all aspects of people&#39;s life. Due to the emergence of new strains of the SARS-CoV-2, pandemic risk still remains, despite the start of vaccination. Therefore, rapid diagnostic tests are essential to control infection, improve clinical care and stop the spread of the disease. Recently CRISPR-based diagnostic tools have facilitated rapid diagnostic. Here, we review the diagnostic applications of CRISPR-Cas system in COVID-19.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40489 Hashem Khanbabaei, Samaneh Abbasi, Mona Fani, Saber Soltani, Milad Zandi, Zahra Najafimemar, Saeedeh Ebrahimi Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Non-gynaecological Applications of Menstrual-derived Stem Cells: A Systematic Review <p><span style="font-size:11pt"><span style="font-size:10.0pt">Menstrual-derived Stem Cells (MenSC) are a potential novel source of mesenchymal stem cells. There is an increased interest in investigating the therapeutic potential of MenSC due to the various advantages they exhibit</span><span style="font-size:10.0pt">, when </span><span style="font-size:10.0pt">compared to other types of stem cells. MenSC are obtained </span><span style="font-size:10.0pt">non-invasively </span><span style="font-size:10.0pt">from menstrual blood. Thus, collection of MenSC is simple, reproducible and can be </span><span style="font-size:10.0pt">carried out</span><span style="font-size:10.0pt"> periodically</span><span style="font-size:10.0pt">, </span><span style="font-size:10.0pt">with </span><span style="font-size:10.0pt">minimal </span><span style="font-size:10.0pt">complications. MenSC are present in abundance, are highly proliferative, exhibit a low immunogenicity</span> <span style="font-size:10.0pt">and lack ethical issues. MenSC have shown the ability to differentiate into several lineages. </span><span style="font-size:10.0pt">The</span><span style="font-size:10.0pt"> therapeutic potential of MenSC in non-gynaecological applications ha</span><span style="font-size:10.0pt">s</span><span style="font-size:10.0pt"> been investigated in wound healing, neurological, musculo-skeletal,&nbsp; cardiovascular, respiratory</span><span style="font-size:10.0pt">, and </span><span style="font-size:10.0pt">liver disorders, </span><span style="font-size:10.0pt">as well as in </span><span style="font-size:10.0pt">diabetes and cancer. Human clinical trials are limited</span><span style="font-size:10.0pt">. T</span><span style="font-size:10.0pt">o date, therapeutic efficacy and safety have been reported in patients with Avian influenza A subtype H7N9, </span><span style="font-size:10.0pt">COVID-19</span><span style="font-size:10.0pt">, congestive heart failure, multiple sclerosis and Duchene muscular dystrophy. However, further clinical trials in humans should be conducted, to study the long-term therapeutic effects of these stem cells in various diseases and to further explore their mechanism of action. This systematic review focuses on the application of MenSC in non-gynaecological diseases.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40490 Claire Galea, Nicoletta Riva, Jean Calleja-Agius Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir New Generation Vaccines for COVID-19 Based on Peptide, Viral Vector, Artificial Antigen Presenting Cell, DNA or mRNA <p><span style="font-size:11pt"><span style="font-size:10.0pt">At present, effective vaccines have been developed as the most successful approaches for preventing widespread infectious disease. The global efforts are focusing with the aim of eliminating and overcoming the </span><span style="font-size:10.0pt">Coronavirus Disease 2019 (COVID-19)</span><span style="font-size:10.0pt"> and are developing vaccines from the date it was announced as a pandemic disease. In this study, PubMed, Embase, Cochrane Library, Clinicaltrial.gov, WHO reports, Science Direct, Scopus, Google Scholar, and Springer databases were searched for finding the relevant studies</span> <span style="font-size:10.0pt">about the COVID-19 vaccines. This article provides an overview of multiple vaccines that have been manufactured from December 2020 up to April 2021 and also offers a perspective on their efficacy, safety, advantages, and limitations. Currently, there are several categories of COVID-19 vaccines based on Protein Subunit (PS), Inactivated Virus (IV), Virus Like Particle (VLP), Live Attenuated Virus (LAV), Viral Vector (replicating) (VVr) and Viral Vector (non-replicating) (VVnr) in progress or finalized as indicated by the WHO reporting of April 1, 2020.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40491 Marzieh Rezaei, Mahboobeh Nazari Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Monoclonal Antibody Against Sortilin Induces Apoptosis in Human Breast Cancer Cells <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Sortilin has an important role in various malignances and can be used as a promising target to eradicate cancer cells.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> In this study, the expression of sortilin in 4T1 and MDA-MB231 cell lines was evaluated by flow cytometry and immunocytochemistry. Apoptosis assay was also applied to evaluate apoptosis induction in 4T1 and MDA-MB231 cell lines. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Based on cell surface flow cytometry results, anti-sortilin (2D8-E3) mAb could recognize sortilin molecules in 79.2% and 90.3% of 4T1 and MDA-MB231 cell-lines, respectively. The immunocytochemistry staining results confirmed sortilin surface expression. Apoptosis assay indicated that anti-sortilin mAb could induce apoptosis in 4T1 and MDA-MB231 cell lines.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Our study revealed the important role of surface sortilin in breast carcinoma cell survival and its possible application as a therapeutic agent in cancer targeted therapies.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40492 Miganoosh Simonian, Mozhan Haji Ghaffari, Ali Salimi, Ebrahim Mirzadegan, Niloufar Sadeghi, Nasim Ebrahimnezhad, Ghazaleh Fazli, Ramina Fatemi, Ali Ahmad Bayat , Saeideh Milani, Babak Negahdari, Hodjattallah Rabbani Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase<em> </em>(MurNAc-LAA) from a <em>Bacillus subtilis</em>-hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in <em>Escherichia coli (</em><em>E. coli).</em>&nbsp; </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> The sequences of <em>MurNAc-LAA</em> genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of<em> </em>MurNAc-LAA<em> </em>was optimized by GenScript software to generate<em> </em>MurNAc-LAA-MMI (LysM2) for optimal expression in <em>E. coli</em>. Furthermore, the structure and function of<em> </em>LysM2 was evaluated <em>in silico.</em> The optimized gene was synthesized, subcloned in the pET28a, and expressed in <em>E. coli</em> BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> <em>LysM2</em>, on 816 <em>bp</em> gene encoding a 33 <em>kDa</em> protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 <em>hr</em> in <em>E.</em></span><em> </em><em><span style="font-size:10.0pt">coli</span></em><span style="font-size:10.0pt">. <em>In silico</em> analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against <em>Proteus </em>(sp) by turbidity, and an antibacterial activity against <em>Klebsiella pneumoniae</em>, <em>Salmonella typhimurium</em>, and <em>Proteus vulgaris</em> in agar diffusion assays. </span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Taken together, our results confirmed that LysM2 </span><span style="font-size:10.0pt">is an inhibiting agent for gram-negative bacteria.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=50488 Morteza Miri, Sepideh Yazdianpour, Shamsozoha Abolmaali, Shakiba Darvish Alipour Astaneh Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Genome Analysis of the Enterococcus faecium Entfac.YE Prophage <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Bacteriophages are viruses that infect bacteria. Bacteriophages are widely distributed in various environments. The prevalence of bacteriophages in water sources, especially wastewaters, is naturally high. These viruses affect evolution of most bacterial species. Bacteriophages are able to integrate their genomes into the chromosomes of their hosts as prophages and hence transfer resistance genes to the bacterial genomes. Enterococci are commensal bacteria that show high resistance to common antibiotics. For example, prevalence of vancomycin-resistant enterococci has increased within the last decades. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><strong> </strong><span style="font-size:10.0pt">Enterococcal isolates were isolated from clinical samples and morphological, phenotypical, biochemical, and molecular methods were used to identify and confirm their identity. Bacteriophages extracted from water sources were then applied to isolated <em>Enterococcus faecium (E. faecium)</em>. In the next step, the bacterial genome was completely sequenced and the existing prophage genome in the bacterial genome was analyzed.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt">In this study,</span><span style="font-size:10.0pt"> <em>E. faecium</em> </span><span style="font-size:10.0pt">EntfacYE was isolated from a clinical sample. The EntfacYE genome was analyzed and 88 prophage genes were identified. The prophage content included four housekeeping genes, 29 genes in the group of genes related to replication and regulation, 25 genes in the group of genes related to </span><span style="font-size:10.0pt">structure and packaging, and four genes belonging to the group of genes associated with lysis. Moreover, 26 genes were identified with unknown functions. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><strong> </strong><span style="font-size:10.0pt">In conclusion, genome analysis of prophages can lead to a better understanding of their roles in the rapid evolution of bacteria.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=50489 Yara Elahi, Ramin Mazaheri Nezhad Fard, Arash Seifi, Saeideh Mahfouzi, Ali Akbar Saboor Yaraghi Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Optimization of Expression and Purification of Recombinant Mouse plac1 <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><strong> </strong><span style="font-size:10.0pt">Placenta-specific 1 (</span><span style="font-size:10.0pt">PLAC1) is one of the recently-discovered Cancer-Testis-Placenta (CTP) antigen with restricted normal tissue and ectopic expression </span><span style="font-size:10.0pt">in a wide range of cancer cells from </span><span style="font-size:10.0pt">different histological origins. The production of recombinant human PLAC1 has already been optimized; however, no study has been reported so far on the production and purification of mouse plac1. </span><span style="font-size:10.0pt">In this study, mouse </span><span style="font-size:10.0pt">plac1 </span><span style="font-size:10.0pt">expression and purification was optimized in a prokaryotic system and the effects of the generated proteins on inducing humoral responses in mice were investigated. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> A fusion protein containing full extracellular domain of mouse</span><span style="font-size:10.0pt"> plac1</span><span style="font-size:10.0pt">, immunostimulatory peptides, tetanus toxin P2P30 and PADRE and KDEL3 signal (main </span><span style="font-size:10.0pt">plac1</span><span style="font-size:10.0pt">), and the same fragment without immunostimulatory peptides (control </span><span style="font-size:10.0pt">plac1</span><span style="font-size:10.0pt">) was produced. To optimize production and purification steps, different parameters including bacterial strain, cultivation temperature, cultivation time, </span><span style="font-size:10.0pt">IPTG</span><span style="font-size:10.0pt"> concentration</span><span style="font-size:10.0pt">, culture medium, and also different buffers for purification of the recombinant proteins were tested. </span><span style="font-size:10.0pt">After confirming the identity of recombinant </span><span style="font-size:10.0pt">plac1 </span><span style="font-size:10.0pt">proteins with Western Blotting (WB) and ELISA assays, these proteins were </span><span style="font-size:10.0pt">subcutaneously</span><span style="font-size:10.0pt"> injected in mice with Freund&#39;s adjuvant and the anti-</span><span style="font-size:10.0pt">plac1</span><span style="font-size:10.0pt"> antibody response was detected by ELISA. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt">The optimal expression level of </span><span style="font-size:10.0pt">main and control </span><span style="font-size:10.0pt">plac1 </span><span style="font-size:10.0pt">was</span><span style="font-size:10.0pt"> obtained in BL21 (DE3) and TB culture medium in the presence of 0.25 <em>mM</em> IPTG after 24 <em>hr</em> of induction at 15</span><em><span style="font-size:10.0pt">&deg;</span></em><em><span style="font-size:10.0pt">C</span></em><span style="font-size:10.0pt">. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal antibody with both main and control plac1 recombinant proteins in WB and ELISA analysis. Both proteins induced humoral responses in mice; however, anti-plac1 &nbsp;antibody titer was significantly higher in sera of mice immunized with main compared to control plac1. </span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span> <span style="font-size:10.0pt">In this study, an optimized protocol for production and purification of mouse </span><span style="font-size:10.0pt">plac1 </span><span style="font-size:10.0pt">was reported and it was shown that insertion of immunostimulatory peptides in gene construct could efficiently enhance humoral immune responses against mouse</span><span style="font-size:10.0pt"> plac1</span><span style="font-size:10.0pt">, which could potentially augment cellular immune responses against </span><span style="font-size:10.0pt">plac1 </span><span style="font-size:10.0pt">leading to more effective anti-cancer responses.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=50490 Shaghayegh Rahdan, Mahboobeh Nazari, Sorour Shojaeian, Fazel Shokri, Mohammad Mehdi Amiri, Amin Ramezani, Amir-Hassan Zarnani, Seyed Alireza Razavi Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Modern Paradigm Towards Potential Target Identification for Antiviral (SARS-nCoV-2) and Anticancer Lipopeptides: A Pharmacophore-Based Approach <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Lipopeptides are potential microbial metabolites that are abandoned with broad spectrum biopharmaceutical properties ranging from antimicrobial, antiviral and anticancer, <em>etc</em>. Clinical studies are not much explored beyond the experimental methods to understand drug mechanisms on target proteins at the molecular level for large molecules. Due to the less available studies on potential target proteins of lipopeptide based drugs, their potential inhibitory role for more obvious treatment on disease have not been explored in the direction of lead optimization. However, Computational approaches need to be utilized to explore drug discovery aspects on lipopeptide based drugs, which are time saving and cost-effective techniques. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> Here a ligand-based drug discovery approach is coupled with reverse pharmacophore-mapping for the prediction of potential targets for antiviral (</span><span style="font-size:10.0pt">SARS-nCoV-2</span><span style="font-size:10.0pt">) and anticancer lipopeptides.&nbsp;Web-based servers PharmMapper and SwissTargetPrediction are used for the identification of target proteins for lipopeptides surfactin and iturin produced by&nbsp;<em>Bacillus subtilis</em>. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The studies have given the insight to treat the diseases with next-generation large molecule therapeutics. Results also indicate the affinity for Angiotensin-Converting Enzymes (ACE) and proteases as the potential viral targets for these categories of peptide therapeutics. A target protein for the Human Papilloma Virus (HPV) has also been mapped. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The work will further help in exploring computer-aided drug designing of novel compounds with greater efficiency where the structure of the target proteins and lead compounds are known.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=50492 Manisha Yadav, J. Satya Eswari Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Fluorescent Detection of Methicillin Resistant Staphylococcus aureus by Loop-mediated Isothermal Amplification Assisted with Streptavidin-coated Quantum Dots <p><span style="font-size:11pt">Background:<span style="font-size:11.5pt"> Methicillin Resistance<em> Staphylococcus aureus</em> (<em>MRSA</em>) could be considered as a major concern in medicine can cause nosocomial infection and bacteremia, especially in patients using catheter and household medical devices. </span></span></p> <p><span style="font-size:11pt">Methods: <span style="font-size:11.5pt">Using molecular diagnostic methods are important for identification of<em> MRSA </em>from the Methicillin Sensitive<em> Staphylococcus aureus</em> (<em>MSSA</em>). Here we described a fluorescent assay using biotin-labelling Loop-mediated isothermal amplification (LAMP) method assisted with streptavidin-coated Quantum Dots (QDs) for detection of <em>MRSA</em>. For comparison, another fluorescent assay using LAMP assisted with Green Viewer (GV; a fluorescent dye) was applied for detection of <em>MRSA</em>. The <em>mecA</em> gene was selected as the target for amplification by LAMP and for biotin-labeling of the LAMP amplicons, biotin-11-dUTP was mixed with the dNTPs (deoxy Nucleotide Phosphates) in LAMP reaction. For determining the clinical performance of the developed assay, 30 blood samples with <em>MRSA</em> positive results were tested with QD-LAMP</span><span style="font-size:11.5pt">, the conventional LAMP, GV-LAMP, and </span><span style="font-size:11.5pt">Polymerase Chain Reaction</span><span style="font-size:11.5pt"> (PCR). </span></span></p> <p><span style="font-size:11pt">Results: <span style="font-size:11.5pt">Obtained results indicated that % sensitivity of QD-LAMP was 86.66% for detection of <em>mecA</em> positive <em>MRSA</em> samples; however, the Limit of Detection (LoD) of QD-LAMP was </span><span style="font-size:11.5pt">1.5&times;10<sup>4</sup></span> <span style="font-size:11.5pt">Colony Forming Unit</span><span style="font-size:11.5pt"> (CFU). </span></span></p> <p><span style="font-size:11pt">Conclusion: <span style="font-size:11.5pt">The results suggested that </span><span style="font-size:11.5pt">the QD-LAMP </span><span style="font-size:11.5pt">assay was easy to operate and could be used for detection of <em>MRSA</em> in parallel to the blood culture with less sensitivity for detection of bacteremia and pediatric septicemia with low counts of <em>MRSA</em>.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=60491 Aily Aliasgharian, Pooria Gill, Mohammad Ahanjan, Adele Rafati Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antitumor Activities of Green Tea by Up-regulation of miR -181a Expression in LNCaP Cells Using 3D Cell Culture Model <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Prostate Cancer (PCa) is the major reason for the high mortality rates among men worldwide. In fact, current therapeutic approaches are not successful. It appears that discovering more effective methods considering several parameters such as availability, low cost, and no toxicity to normal cells is one of the biggest challenges for interested researchers. Green tea (extracted from the plant <em>Camellia sinensis</em>) with high level of polyphenolic compounds and as the most globally consumed beverage has attracted considerable interest. MicroRNAs (or miRNAs) were considered as novel tools in cancer therapy which modulate various biological events in cell by regulation of gene expression. The aim of the current study was to evaluate the antitumor activity of green tea in LNCaP cells through up-regulation of miR-181a expression.</span></span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> First, </span><span style="font-size:10.0pt">LNCaP cells were cultured and by using quantitative real time PCR (qRT-PCR) and western blot methods, the expression levels of Bax and BCL2 were analyzed. Next, a 3D cell culture model was applied to evaluate the expression of miRNA-181a </span><span style="font-size:10.0pt">in </span><span style="font-size:10.0pt">LNCaP cells. </span></span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt">It was shown that green tea induced cellular apoptosis. The high number of apoptotic nuclei was also shown by using DAPI staining. The inhibition of tumor growth was revealed by analyzing the size and number of spheroids. Also, up-regulation of miR-181a expression </span><span style="font-size:10.0pt">in </span><span style="font-size:10.0pt">LNCaP cells was revealed after treatment with green tea. </span></span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="color:black"><span style="font-size:9.5pt">Conclusion: </span><span style="font-size:9.5pt">Our results are helpful to design antitumor regimens based on consumption of green</span><span style="font-size:10.0pt"> tea through up-regulation of miRNA-181a expression and induction of apoptosis.</span></span></span></p> https://www.AJMB.org/En/Article.aspx?ID=50494 Fatemeh Safari, Narjes Rayat Azad, Ali Alizadeh Ezdiny , Safoora Pakizehkar, Zeinab Khazaei Koohpar, Najmeh Ranji Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Pulse Voltage Electrical Stimulation for Bacterial Inactivation and Wound Healing in Mice with Diabetes <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Treatment of wounds in diabetes often gets less than perfect healing. One of the reasons for the difficulty in treating wounds in diabetes is the growth of aerobic and anaerobic bacteria. This study aims to determine the pulse voltage and treatment time that can optimally inactivate bacteria, and their effect on wound healing in mice suffering from diabetes.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> The study used electrical stimulation with a direct voltage of 10 <em>volts</em> given a pulse voltage of 50-80 <em>volts</em>, a width of 50 <em>&micro;s</em>, and the number of pulses of 65 per second. The research samples were <em>Staphylococcus aureus</em> (<em>S. aureus</em>) and <em>Pseudomonas aeruginosa</em> (<em>P. aeruginosa</em>) bacteria that grew on beef and mice (<em>Mus musculus</em>) with diabetes. The treatment for <em>S. aureus</em> and <em>P. aeruginosa</em> bacteria was carried out using a pulse voltage of 50-80 <em>volts</em> for 5-15 <em>min/day</em> and repeated for 3 days. Meanwhile, treatment of mice wounds was carried out with a pulse voltage of 80 <em>volts</em> for 15 <em>min/day</em> and repeated for 7 days.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The results showed that treatment with a pulse voltage of 50-80 <em>volts</em> and a treatment time of 5-15 <em>min</em> significantly reduced the number of <em>S. aureus</em> and <em>P. aeruginosa</em> bacteria in beef (p</span><span style="font-size:10.0pt">&pound;</span><span style="font-size:10.0pt">0.05). Treatment with a pulse voltage of 80 <em>volts</em> for 15 <em>min</em> made beef free from bacteria. Meanwhile, treatment with a pulse voltage of 80 <em>volts</em> for 15 <em>min</em> per day for seven days resulted in the wound state of three mice in the maturation phase and two mice in the proliferation phase on day 8 with an average wound area of 0.108 <em>cm <sup>2</sup></em>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The treatment with a pulse voltage of 80 <em>volts</em> for 15 <em>min</em> made the beef sterile, the mice wounds healed quickly, and the mice not stressed. The higher the blood glucose level, the slower the wound healing process.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=50495 Mokhamad Tirono, Farid Samsu Hananto, Ahmad Abtokhi Fri, 31 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Protection against the Omicron and Subsequent Coronavirus Variants: Medical-grade Masking, Third Dose Vaccination, Updating Vaccines, and Pursuing Universal Vaccine https://www.AJMB.org/En/Article.aspx?ID=60489 Ahmad Shamabadi, Shahin Akhondzadeh Tue, 28 Dec 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Integrating Psychiatry and Medical Biotechnology as a Way to Achieve Scientific, Precision, and Personalized Psychiatry <p><span style="font-size:10.0pt">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-</span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt"> (TNF-</span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">). 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.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40485 Ahmad Shamabadi, Alireza Hasanzadeh, Shahin Akhondzadeh Mon, 20 Sep 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Cross-Sectional Study for Evaluation of KRAS and BRAF Mutations by Reverse Dot Blot, PCR-RFLP, and Allele-Specific PCR Methods Among Patients with Colorectal Cancer <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> <em>KRAS</em> and <em>BRAF</em> 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 <em>KRAS</em> and <em>BRAF</em> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, <em>KRAS</em> and <em>BRAF</em> gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for <em>KRAS</em> and <em>BRAF</em> mutations, respectively.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><em><span style="font-size:10.0pt"> KRAS</span></em><span style="font-size:10.0pt"> 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 <em>KRAS</em> were related to G12D and 10% of patients had <em>BRAF</em> mutated genes. </span><span style="font-size:10.0pt">The type of <em>KRAS</em> gene mutations could be evaluated by reverse dot blot method. </span><span style="font-size:10.0pt">In</span><span style="font-size:10.0pt"> general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method.&nbsp; </span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in <em>KRAS</em> and <em>BRAF</em> 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 <em>KRAS</em> point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40475 Fatemeh Sheikhsofla, Behzad Poopak, Sajjad Firuzyar, Fatemeh Roudbari , Mojtaba Ghadiany Mon, 20 Sep 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Apoptosis Induction with Combined Use of Cisplatin and Fisetin in Cisplatin-resistance A2780 Ovarian Cancer Cells <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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><span style="font-size:10.0pt">. In this study, the apoptosis induced by cisplatin along with Fisetin in cisplatin-resistant ovarian cancer cell line (A2780) was investigated.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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 <em>Bax</em>, <em>BCL-2</em>, <em>caspase 3</em>, and <em>caspase 9</em> was also assessed by real time PCR.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> 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 <em>Bax</em> pro-apoptotic gene expression and <em>BCL-2</em> anti-apoptotic gene expression increased in cells under treatment; moreover, gene expression of <em>caspases 3</em> and <em>9</em> significantly increased as well.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> 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><span style="font-size:10.0pt">strategy</span><span style="font-size:10.0pt"> in the treatment of ovarian cancer.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40474 Samira Jafarzadeh, Javad Baharara, Maryam Tehranipour Sat, 18 Sep 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production of PEGylated G-CSF from non-classical inclusion bodies expressed in Escherichia coli <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span> <span style="font-size:10.0pt">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</span> <span style="font-size:10.0pt"><span style="background-color:white">Granulocyte Colony Stimulating Factor</span></span><span style="font-size:10.0pt"> (</span><span style="font-size:10.0pt">GCSF</span><span style="font-size:10.0pt">)</span><span style="font-size:10.0pt"> could be expressed as non-classical </span><span style="font-size:10.0pt">I</span><span style="font-size:10.0pt">nclusion </span><span style="font-size:10.0pt">B</span><span style="font-size:10.0pt">odies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, i</span><span style="font-size:10.0pt">n this study, a simple process was developed to produce PEGylated GCSF</span><span style="font-size:10.0pt"> from ncIBs.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span> <span style="font-size:10.0pt">BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 <em>L</em> bioreactor and</span><span style="font-size:10.0pt"> the expression of GCSF was induced by adding 0.5 <em>mM</em></span><span style="font-size:10.0pt"> IPTG. </span><span style="font-size:10.0pt">After 24 <em>hr</em> of fermentation, c</span><span style="font-size:10.0pt">ells were collected, resuspended, and disrupted. The insoluble fraction</span><span style="font-size:10.0pt"> was obtained from cell lysates and </span><span style="font-size:10.0pt">dissolved in 0.1% N-lauroylsarcosine solution</span><span style="font-size:10.0pt">. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used </span><span style="font-size:10.0pt">for the conjugation with 5 <em>kDa</em> PEG<span style="background-color:white">. The PEGylated GCSF was purified using two purification</span></span> <span style="font-size:10.0pt"><span style="background-color:white">steps, including anion exchange chromatography and gel filtration chromatography.</span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt"><span style="background-color:white">PEGylated GCSF was obtained with high purity (</span></span><span style="font-size:10.0pt"><span style="background-color:white">~</span></span><span style="font-size:10.0pt"><span style="background-color:white">97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 <em>kDa</em> PEG molecule (monoPEG-GCSF).</span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span> <span style="font-size:10.0pt">These results clearly indicate that the process developed in this study might</span><span style="font-size:10.0pt"> be</span><span style="font-size:10.0pt"> a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in </span><em><span style="font-size:10.0pt">Escherichia coli</span></em><em><span style="font-size:10.0pt"> (E. coli).</span></em></span></p> https://www.AJMB.org/En/Article.aspx?ID=40477 Nguyen Thi My Trinh, Tran Linh Thuoc, Dang Thi Phuong Thao Sat, 11 Sep 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Molecular Mechanisms of Anti-inflammatory Activities of the Extracts of Ocimum gratissimum and Thymus vulgaris <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> A large body of literature suggests that the extracts of <em>Ocimum gratissimum (</em><em>O</em>. <em>gratissimum) </em>and <em>Thymus vulgaris</em> (<em>T</em>. <em>vulgaris</em>) 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 <em>O</em>. <em>gratissimum</em> and <em>T</em>. <em>vulgaris </em>were investigated in this study. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> The <em>in vitro</em> 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-</span><span style="font-size:10.0pt">&alpha;, IL-8, and CCL-2 were performed through molecular docking. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The assessment<em> </em>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 <em>in vitro</em> analysis as indicated in the binding score of all the selected compounds compared to indomethacin. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The phytochemical components of the extracts of <em>O. gratissimum </em>and <em>T. vulgaris</em> could be effective as anti-inflammatory agents that could be explored in preventing disorders associated with excessive activities of pro-inflammatory mediators.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40479 Ige Francis Olaoye, Babatunde Joseph Oso, Adepeju Aberuagba Sat, 11 Sep 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Fabrication of Calcium Sulfate Coated Selenium Nanoparticles and Corresponding In-Vitro Cytotoxicity Effects Against 4T1 Breast Cancer Cell Line <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> The inhibitory effect of selenium nanoparticles (SeNPs) on cancer cells has been reported in many studies. In this study,&nbsp; the purpose was to compare the <em>in vitro</em> effects of SeNPs and calcium sulfate coated selenium nanoparticles (CaSO<sub>4</sub>@ SeNPs) on breast cancer cells. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> CaSO<sub>4</sub>@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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt">While LD<sub>50</sub> of SeNPs on 4T1 cancer cells was 80</span> <em><span style="font-size:10.0pt">&micro;g</span></em><span style="font-size:10.0pt">, the LD<sub>50</sub> of </span><span style="font-size:10.0pt">CaSO<sub>4</sub>@SeNPs was </span><span style="font-size:10.0pt">reported to be only 15</span> <em><span style="font-size:10.0pt">&micro;g</span></em><span style="font-size:10.0pt">. </span><span style="font-size:10.0pt">The difference between the inhibition rates obtained for SeNPs and CaSO<sub>4</sub>@SeNPs was statistically significant (p=0.05). </span><span style="font-size:10.0pt">In addition, at higher concentrations (50 <em>&micro;g</em>) of CaSO<sub>4</sub>@SeNPs, the cytotoxicity was 100% more than SeNPs alone.&nbsp;</span></span></p> <p style="text-align:justify"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> 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 </span><span style="font-size:10.0pt">CaSO<sub>4</sub>@SeNPs.</span><span style="font-size:10.0pt"> However, </span><span style="font-size:10.0pt">further studies are still needed to determine the exact corresponding mechanisms of this synergistic effect.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40478 Elnaz Faghfuri , Ramak Ajideh, Faranak Shahverdi , Mina Hosseini, Faranak Mavandadnejad, Mohammad Hossein Yazdi, Ahmad Reza Shahverdi Thu, 19 Aug 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes&rsquo; 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40480 Farahnaz Zare, Sedigheh Sharifzadeh , Abbas Behzad-Behbahani, Gholamreza Rafiei Dehbidi, Zahra Yousefi, Reza Ranjbaran, Noorossadat Seyyedi Thu, 19 Aug 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of miR-222 Expression in HBV Infected Patients in Comparison with HDV and HBV Co-infected Patients <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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). </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> 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)</span><span style="font-size:10.0pt"> with p&lt;0.001.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> 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.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40481 Zahra Sokhanvar, Ameneh Elikaei , Zohreh Sharifi Thu, 19 Aug 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span> <span style="font-size:10.0pt">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.</span> <span style="font-size:10.0pt">Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span> <span style="font-size:10.0pt">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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt">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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span> <span style="font-size:10.0pt">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. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40483 Mehdi Hassani, Sara Hesami, Nahal Maroofi, Mehdi Banan Thu, 19 Aug 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Identification of a Novel Homozygous Mutation in BBS10 Gene in an Iranian Family with Bardet-Biedl Syndrome <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Bardet&ndash;Biedl Syndrome (BBS) is a rare pleiotropic autosomal recessive disease related to ciliopathies with </span><span style="font-size:10.0pt">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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods: </span><span style="font-size:10.0pt">A consanguineous Iranian family with a 28-year-old daughter affected with BBS, resulting from a first cousin marriage, was examined. After clinical examination, </span><span style="font-size:10.0pt">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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> A </span><span style="font-size:10.0pt">novel</span> <span style="font-size:10.0pt">homozygous variant [</span><span style="font-size:10.0pt">c. 2035G&gt;A (p.E679K)</span><span style="font-size:10.0pt">] in exon 2 of the <em>BBS10</em> gene was found which was </span><span style="font-size:10.0pt">categorized as likely pathogenic</span><span style="font-size:10.0pt"> based on American College of Medical Genetics and Genomics (</span><span style="font-size:10.0pt">ACMG) guidelines and criteria.</span><span style="font-size:10.0pt"> In this study, </span><span style="font-size:10.0pt">the variant was fully co-segregated with the phenotype in the family.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> 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 <em>BBS10</em> 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.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40482 Mohammad Dehani, Davood Zare-Abdollahi, Ata Bushehri , Azadeh Dehghani , Jalil Effati, Seyed Ali Mohammad Miratashi , Hamid Reza Khorram Khorshid Thu, 19 Aug 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Potential Anti-inflammatory Effects of Zerumbone in COVID-19 Patients https://www.AJMB.org/En/Article.aspx?ID=40484 Razieh Dehghan, Mohammad Hasan Soheilifar, Farid Azizi Jalilian, Rezvan Najafi, Razieh Amini Thu, 19 Aug 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Medical biotechnology in the service of coronavirus vaccine discovery and production https://www.AJMB.org/En/Article.aspx?ID=40472 Ahmad Shamabadi, Shahin Akhondzadeh Wed, 16 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Designing a Strategy for pH Control to Improve CHO Cell Productivity in Bioreactor <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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 CO<sub>2</sub> 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 <em>L</em> bioreactor to assess the efficiency of an alternative strategy in controlling culture pH.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> Factors inducing partial pressure of CO<sub>2</sub> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The results indicated that the major factor affecting pH is non-efficient CO<sub>2</sub> removal. CO<sub>2</sub> 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 <em>L</em> scale bioreactor culture, indicating the scalability of the approach.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> The obtained results showed that pH fluctuations could be effectively controlled by optimizing CO<sub>2</sub> stripping. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40464 Zohreh Ahleboot, Mahdi Khorshidtalab, Paria Motahari, Rasoul Mahboudi, Razieh Arjmand, Aram Mokarizadeh, Shayan Maleknia Wed, 16 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Determining the Specific Activity of Anti-Rabies Sera and Immunoglobulin Using Atomic Force Microscopy of Cell Cultures <p>Background: Mouse neutralization test is widely used to determine the level of anti-rabies 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 anti-rabies sera and immunoglobulin using AFM of cell cultures.&nbsp;</p> <p>Methods: 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.</p> <p>Results: 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.</p> <p>Conclusion: 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.</p> https://www.AJMB.org/En/Article.aspx?ID=40466 Sergey V Generalov, Pavel S Erokhin, Oleg S Kuznetsov, Elena G Abramova, Ivan M Zhulidov, Natalya A Osina Wed, 16 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Detection of Aneuploidies in Products of Conception and Neonatal Deaths in Iranian Patients Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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&nbsp; 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> 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%). </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> 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.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40467 Sara Khorami Sarvestani, Maryam Rafati, Haleh Soltanghoraee, Azadeh Hoseini, Azadeh Soltani, Koosha Jalilian, Saeed Reza Ghaffari Wed, 16 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An Integrated Bioinformatics Analysis of the Potential Regulatory Effects of miR-21 on T-cell Related Target Genes in Multiple Sclerosis <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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 IFN</span><span style="font-size:10.0pt">&gamma;</span><span style="font-size:10.0pt"> and TNF-</span><span style="font-size:10.0pt">&alpha; following stimulation of T-cells <em>via</em> 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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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 T-cells 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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> It found that seven genes (NRIP1, ARNT, KDM7A, S100A10, AK2, TGF</span><span style="font-size:10.0pt">&beta;</span><span style="font-size:10.0pt">R2, 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.&nbsp; </span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Our findings suggest that miR-21 and MS-related drugs can act synergistically to regulate several genes</span><span style="font-size:10.0pt"> in the existing datasets, and miR-21 inhibitors have the potential to be used in MS treatment.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40468 Mostafa Manian, Ehsan Sohrabi, Nahid Eskandari, Mohammad-Ali Assarehzadegan, Gordon A. Ferns, Mitra Nourbakhsh, Mir Hadi Jazayeri, Reza Nedaeinia Wed, 16 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of a Functional Single Nucleotide Polymorphism (rs874040) in the RBPJ Gene with Susceptibility to Rheumatoid Arthritis in Iranian Population <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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&gt;C) near the recombination signal-binding protein for the immunoglobulin kappa J region (<em>RBPJ</em>) gene. <em>RBPJ</em> 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&gt;C) upstream of the <em>RBPJ </em>gene with RA risk was assessed in Iranian population.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> A case-control study including 60 RA patients and 44 control subjects was conducted to estimate rs874040 genotypes using real</span><span style="font-size:10.0pt">‑</span><span style="font-size:10.0pt">time polymerase chain reaction High</span> <span style="font-size:10.0pt">Resolution Melting (HRM) method.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Logistic regression analysis indicated that homozygous CC and heterozygous GC genotypes increase the risk of RA compared with GG genotype (CC <em>vs</em>. GG; OR=11.36; 95% CI [3.93-33.33] and CG <em>vs</em>. 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<span style="background-color:white"> C-reactive protein </span>concentrations and rs874040 polymorphism (p&lt;0.05).</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Our findings propose a substantial correlation between rs874040 polymorphism and RA risk in Iranian population.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40469 Mansour Salesi, Mahdieh Oboodiyat, Rasoul Salehi, Bahram Pakzad Wed, 16 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Novel Drug Discovery to Combat COVID-19 by Repressing Important Virus Proteins Involved in Pathogenesis Using Medicinal Herbal Compounds <p style="text-align:justify"><span style="font-size:12pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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 SARS-CoV-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. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The outcomes of the study revealed that Linarin, Amentoflavone, (-)-Catechin Gallate and Hypericin from <em>Chrysanthemum morifolium</em>, <em>Hypericum perforatum</em>, <em>Humulus lupulus</em>, and <em>Hibiscus sabdariffa</em> had the highest affinity for these basic proteins and in some cases, their affinity was much higher than antiviral medicines. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> 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.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40462 Samira Mahmoudi, Negar Balmeh, Niloofar Mohammadi, Tahereh Sadeghian-Rizi Sat, 05 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Impact of Nrf2 Silencing on Nrf2-PD-L1 Axis to Overcome Oxaliplatin Resistance as well as Migration in Colon Cancer <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods</span><span style="font-size:10.0pt">:</span> <span style="font-size:10.0pt">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 </span><span style="font-size:10.0pt">resistance </span><span style="font-size:10.0pt">was investigated.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> 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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> 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. </span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40463 Zahra Payandeh, Abbas Pirpour Tazehkand, Behzad Mansoori, Vahid Khaze, Milad Asadi, Behzad Baradaran, Nasser Samadi Sat, 05 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression and Characterization of Two DNA Constructs Derived from HIV-1-vif in Escherichia coli and Mammalian Cells <p style="text-align:justify"><span style="font-size:12pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Acquired immunodeficiency syndrome&nbsp;(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. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> In this study, vif sequence was amplified from pLN4-3 plasmid including HIV-1 <em>vif</em> 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. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> vif protein was expressed in BL21</span><span style="font-size:10.0pt">)DE3) strain and identified as a23 <em>kDa</em> band in SDS-PAGE and confirmed by anti-His antibody in western blotting. The purified protein concentration was 173.3 <em>&mu;g/ml</em> using Bradford assay. HEK-293T cells were successfully transfected by recombinant pEGFP plasmids and flow cytometry confirmed the cell transfection. </span></span></p> <p style="text-align:justify"><span style="font-size:12pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> vif protein can be expressed in mammalian cells and may be a proper protein subunit vaccine candidate against HIV-1.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40465 Fatemeh Zamani, Azam Bolhassani, Sepideh Shahbazi , Ahmad Faghih, Seyed Mehdi Sadat Sat, 05 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir ZHER2 Affibody as a Good Candidate for Detection of Metastatic Prostate Cancer <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Prostate cancer (PCa) is the second leading cause of cancer death in men. About one in 39 will die of prostate cancer and about one man out of seven is diagnosed with the problem. The expression of the Epidermal Growth Factor Receptor (EGFR) is shown in the progression of androgen independent PCa. EGFR has emerged as a promising therapeutic target for patients with castration‑resistant PCa. There is an urgent need for detection of EGFR expression and monitoring the treatment in prostate cancer. Affibodies&nbsp;are small engineered proteins with a high affinity to a large number of target proteins or peptides. Affibodies are three-helix bundles of 58 amino acids based on Z domain of staphylococcal protein A; ZHER2 is one of them with the high-affinity to Human Epidermal growth factor Receptor 2 (HER2). Positron Emission Tomography (PET) imaging with 18F-Labeled ZHER2: 2891 affibody can be a good candidate for prostate cancer detection.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40470 Mahboobeh Nazari, Ramin Radmanesh Sat, 05 Jun 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Requisiteness for not Sacrificing Medical Biotechnology in the Coronavirus Era <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">The coronavirus storm, first reported more than a year ago, has overshadowed all societies&#39; parts and become a challenge to all of the world&#39;s health systems <sup>1</sup>. 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 <sup>2,3</sup>. 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&#39; tasks over the years and the allocation of research funds to the fields of genetics, biochemistry, immunology, <em>etc</em>., studies and advances would not be possible today <sup>4,5</sup>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">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 <sup>5,6</sup>. However, this area of science can also be harmful through studies&#39; 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 <sup>6-10</sup>. 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 <sup>4</sup>. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">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&#39;s health. The development of insulin, the production of advanced monoclonal antibodies, and vaccines&#39; production against an RNA virus are some of the notable novel products <sup>6,7</sup>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">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 <sup>1,11</sup>. Science achieves patients&#39; treatment through basic studies <sup>4</sup>, 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.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40441 Shahin Akhondzadeh, Ahmad Shamabadi Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Using CRISPR/Cas9 System to Knock out Exon 48 in DMD Gene <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> Out of frame mutations in <em>DMD</em> 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. <em>DMD</em> 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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> A pair of guide RNAs (gRNAs) was designed to cleave <em>DMD</em> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Exon 48 was successfully deleted and therefore exon 47 was joined to exon 49.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> This result indicated that CRISPR/Cas9 system could be used to edit <em>DMD</em> gene precisely.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40450 Mahintaj Dara , Vahid Razban, Mahdieh Talebzadeh, Sepideh Moradi, Mehdi Dianatpour Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Designing and Development of a Tandem Bivalent Nanobody against VEGF165 <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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 <em>Escherichia coli</em> (<em>E. coli)</em> cells by SDS-PAGE and western blot. After purification, the size distribution of tandem bivalent nanobody was investigated by dynamic light scattering. Moreover, <em>in vitro</em> antiproliferative activity and pharmacokinetic study were studied in HUVECs and Balb/c mice, respectively.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> RMSD analysis revealed the tandem bivalent nanobody had good structural stability after 50 <em>ns</em> of simulation. A hinge region of llama IgG2 was used to fuse the domains. The expression was induced by 1 <em>mM</em> IPTG at 25<em>&deg;C</em> for overnight. A 30 <em>kDa</em> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Our data revealed that the physicochemical as well as <em>in vivo</em> pharmacokinetic parameters of tandem bivalent nanobody was significantly improved.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40451 Farnaz Khodabakhsh, Morteza Salimian, Pardis Ziaee, Fatemeh Kazemi-Lomedasht, Mahdi Behdani, Reza Ahangari Cohan Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Methylation of TGM-3 Promoter and Its Association with Oral Squamous Cell Carcinoma (OSCC) <p style="text-align:justify"><span style="font-size:10pt"><span style="font-size:9.5pt">Background: </span>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. </span></p> <p style="text-align:justify"><span style="font-size:10pt"><span style="font-size:9.5pt">Methods:</span> 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. </span></p> <p style="text-align:justify"><span style="font-size:10pt"><span style="font-size:9.5pt">Results:</span> 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). </span></p> <p style="text-align:justify"><span style="font-size:10pt"><span style="font-size:9.5pt">Conclusion:</span> These results manifested the contribution of DNA methylation of TGM-3 in OSCC and its potential association with clinico-pathologic parameters in OSCC. </span></p> https://www.AJMB.org/En/Article.aspx?ID=40452 Sorour Shojaeian, Abdolkarim Moazeni-Roodi, Abdolamir Allameh, Ata Garajei, Anoshirvan Kazemnejad, Kourosh Kabir, Amir-Hassan Zarnani Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Developmental Toxicity of the Neural Tube Induced by Titanium Dioxide Nanoparticles in Mouse Embryos <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> This study investigated the potential effects of Titanium dioxide nanoparticles (Tio<sub>2</sub>NPs) followed by maternal gavage on fetal development and neural tube formation during pregnancy in mice.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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 <em>mg/kg</em> Body Weight (BW) of Tio<sub>2</sub>NPs 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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Treatment with Tio<sub>2</sub>NPs 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 <em>mg/kg</em> (p&lt;0.05). </span></span></p> <p style="text-align:justify"><span style="font-size:10pt"><span style="font-size:9.5pt">Conclusion:</span> It seems that exposure to nanoparticles of Tio<sub>2</sub> 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&nbsp; dilatation of lateral ventricles were verified which can be related to incidence of apoptosis in central nervous system.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40453 Nahid Mohamadzadeh, Masoumeh Zirak Javanmard, Mojtaba Karimipour, Gholamhosain Farjah Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of Sodium Selenite on Expression of Mitochondrial Transcription Factor A during In Vitro Maturation of Mouse Oocyte <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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 <em>in vitro</em> maturation in the absence of SS, and in experimental group 2, they were matured <em>in vitro</em> in the presence of 10 <em>ng/ml</em> of SS up to 16 <em>hr</em>. 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 <em>TFAM</em> 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. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The oocyte maturation rate to MII stage in SS treated group was significantly higher than non-treated oocytes (75.65 <em>vs</em>. 68.17%, p&lt;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&lt;0.05).</span> <span style="font-size:10.0pt">There was a significant decrease in <em>TFAM</em> gene expression in both <em>in vitro</em> groups compared to the group with <em>in vivo</em> obtained oocytes (p&lt;0.05). Moreover, there was a significant increase in <em>TFAM</em> gene expression in oocytes that matured in the presence of SS compared to that of the group without SS (p&lt;0.05). </span></span></p> <p><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Supplementation of oocyte maturation culture media with SS improved the development rate of oocytes and embryo and also enhanced<em> TFAM</em> expression in MII oocytes which can affect the mitochondrial biogenesis of oocytes.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40454 Tina Moshaashaee, Saeed Zavareh, Shahram Pourbeiranvand, Mojdeh Salehnia Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Benchmarking Datasets from Malaria Cytotoxic T-cell Epitopes Using Machine Learning Approach <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion: </span><span style="font-size:10.0pt">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.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40455 Rama Adiga Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Designing Two Synthetic Constructs for Real Time PCR Detection of Francisella tularensis and Ebola Virus <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span> <span style="font-size:10.0pt">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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods: </span><span style="font-size:10.0pt">In this study, a synthetic construct was designed for molecular detection of <em>Francisella tularensis</em> (<em>F. tularensis</em>) and the <em>Ebola</em> 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.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results: </span><span style="font-size:10.0pt">The synthetic construct was designed and used for conventional PCR and multiplex PCR. The results of common PCR showed a single band at 148 <em>bp</em> and 167 <em>bp</em> in 1.5% agarose gel stained by ethidium bromide for <em>F. tularensis</em> and <em>Ebola</em> virus, respectively. Also, a dual-band at 148 and 167 <em>bp</em> was observed in multiplex PCR. Results of real-time PCR showed a limit of detection about 0.1 <em>pg</em> of plasmid/<em>&micro;l</em>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion: </span><span style="font-size:10.0pt">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.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40456 Mohammadjavad Dehghan Esmat Abadi, Hesam Motalebzadeh, Mahmoud Barati, Mohammadali Yaghobi Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of Silver Nanoparticles on Pyocyanin Production of Pseudomonas aeruginosa Isolated From Clinical Specimens <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> <em>Pseudomonas aeruginosa (P. aeruginosa)</em> 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 <em>P. aeruginosa</em> bacteria isolated from clinical specimens.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> In this study, 15 clinical isolates of <em>P. aeruginosa</em> were collected from different specimens of hospitalized patients. <em>P. aeruginosa</em> 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 <em>P. aeruginosa</em> pyocyanin production at AgNPs concentrations of 0, 0.3, 0.5, 1 and 1.5 <em>mg/ml</em> of was studied with OD of 520 <em>nm</em>.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> The mean MIC and MBC of AgNPs were 1.229 and 1.687 <em>mg/ml</em>, 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&lt;0.05).</span></span></p> <p style="text-align:justify"><span style="font-size:9.5pt">Conclusion:</span> <span style="font-size:10.0pt">According to the results of this study, AgNPs had an inhibitory effect on <em>P. aeruginosa</em> 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 <em>P. aeruginosa</em>.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40457 Mahboobeh Najafi, Mahboobeh Nakhaei Moghaddam, Ehsan Yousefi Mon, 01 Mar 2021 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An Evaluation of Transmission Dynamics of Cryptosporidium Using Molecular Methods <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Dear Editor-in-Chief, </span></strong></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The members of genus <em>Cryptosporidium</em> are intracellular parasites that infect mammals, poultry, reptiles and amphibians. From the total of 30 valid species mentioned currently, 14 have been determined to infect the human being. Two species,<span style="background-color:#dddddd"><em> </em></span><em><span style="background-color:white">Cryptosporidium hominis</span></em> (<em>C. hominis)</em> (Anthroponotic species) and<em> <em><span style="background-color:white">Cryptosporidium parvum</span></em> (C. parvum)</em> bovine genotype (Zoonotic species), are considered of major public health importance </span><sup><span style="font-size:10.0pt">1</span></sup><span style="font-size:10.0pt">. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Commonly, the ubiquitous oocysts of <em>Cryptosporidium</em> are transmitted <em>via</em> direct contact with infected hosts or indirectly <em>via</em> contaminated food and water </span><sup><span style="font-size:10.0pt">2</span></sup><span style="font-size:10.0pt">. The low infectious dose and its resistance against common water disinfectants make it a challenge for the drinking water plants </span><sup><span style="font-size:10.0pt">3</span></sup><span style="font-size:10.0pt">. Common laboratory techniques which are being used for diagnosis of <em>Cryptosporidium</em> cannot discriminate it at species and genotype level. However, the genetic tools allow species determination of the parasite as well as tracing its transmission routes </span><sup><span style="font-size:10.0pt">2</span></sup><span style="font-size:10.0pt">.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">In this study, a total of 55 drinking water samples were collected from 11 different areas of Tabriz, the largest city in North West of Iran.</span><span style="font-size:10.0pt"> Each sample contained 30 <em>L</em> of water. To collect the suspended particles, samples were filtered through a membrane filter with 1.2 <em>&mu;m</em> pore size (Sartorius, Germany). The pellets trapped on the filter were collected </span><sup><span style="font-size:10.0pt">4</span></sup><span style="font-size:10.0pt">. All water pellets were subjected to DNA extraction by a method described previously </span><sup><span style="font-size:10.0pt">5</span></sup><span style="font-size:10.0pt">. Then, the amplification of small ribosomal subunit RNA (SSU-rRNA; 18S rRNA) gene was done using a two step nested PCR method </span><sup><span style="font-size:10.0pt">6</span></sup><span style="font-size:10.0pt">. A sample of <em>C. parvum </em>DNA that was extracted by the extraction method was included in each round of PCR as a positive control. The multi copy nature of 18S rRNA gene and nested format of the PCR make it a very sensitive method for detection of <em>Cryptosporidium</em> oocysts in water samples <sup>7</sup>. However, the expected amplicon (826-864-<em>bp</em>) was not detected in any of the water samples.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Various</span><span style="font-size:10.0pt"> reports from many areas of the world provide strong evidence that contaminated water is an important risk factor for cryptosporidiosis <sup>8</sup>. </span><span style="font-size:10.0pt">Several factors, including</span><span style="font-size:10.0pt"> water source, location of sampling, number and volumes of samples, type of ecosystem, climate and detecting procedures are effective for detection of <em>Cryptosporidium</em> oocysts in the water samples <sup>9</sup>. </span><span style="font-size:10.0pt">The drinking water supplied from surface water sources is more susceptible to contamination. </span><span style="font-size:10.0pt">Thus, underground</span><span style="font-size:10.0pt"> water is a more protected source</span><span style="font-size:10.0pt"> <sup>10</sup>. In our study area, the main </span><span style="font-size:10.0pt">part of the drinking water supplies comes from rivers.</span> </span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The role of rainfall as a determining risk factor for the waterborne transmission of <em>Cryptosporidium</em> can be&nbsp;</span></span><span style="font-size:11pt"><span style="font-size:10.0pt">significant</span><span style="font-size:10.0pt">. The rela</span><span style="font-size:10.0pt">tionship between increased rainfall and an increase in the concentration of <em>Cryptosporidium</em> oocysts in nearby river waters has been reported <sup>10</sup>. The North West part of Iran is an area with lower than average rainfall, </span><span style="font-size:10.0pt">which could be the most important reason for the lack of parasites in the water.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">There are not many reports about the prevalence of <em>Cryptosporidium</em> in water samples in Iran. Meanwhile, the prevalence of</span><span style="font-size:10.0pt"> <em>Cryptosporidium</em> on the surface and recreational water was 36 and 20%, respectively </span><sup><span style="font-size:10.0pt">4,9</span></sup><span style="font-size:10.0pt">. The two researches conducted in </span><span style="font-size:10.0pt">Ardabil and </span><span style="font-size:10.0pt">Chaharmahal va Bakhtiyari provinces as well as our study are limited studies with small sample size. Thus, more comprehensive studies with large samples from different sources and in different seasons are required to assess the real risk of waterborne cryptosporidiosis in Iran.&nbsp; </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Previous studies in Tabriz showed that 1.76% of diarrheic children and 3.8% of cattle have been infected with <em>C. parvum</em> <sup>2,6</sup> (Table 1). The presence of <em>C. parvumm</em> in children, as a sensitive group and in cattle, as a major source for zoonotic disease may be associated with zoonotic transmission of the parasite in the study area. Lack of parasite in drinking water may indicate that this cannot be an important route for transmission; instead, it can be a reason for the low prevalence of the infection in children. Lack of <em>C. hominis</em> (</span><span style="font-size:10.0pt">Anthroponotic species</span><span style="font-size:10.0pt">) in children and the prevalence of <em>C. parvum</em>, potentially zoonotic species, in cattle and its presence in </span><span style="font-size:10.0pt">diarrheic rural children</span><span style="font-size:10.0pt"> would raise the possibility that zoonotic transmission originally occurs through direct contact with farm animals</span> <span style="font-size:10.0pt">in this region.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Therefore, cattle and other domestic animals should be considered as important sources of infection in the North-western part of Iran.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=40448 Behroz Mahdavi Poor, Jalil Rashedi, Mohammad Asgharzadeh, Esmaeel Fallah Mon, 28 Dec 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antibiotic Resistance Pattern in Pseudomonas aeruginosa Isolated from Clinical Samples Other than Burn Samples in Iran <p><span style="font-size:11pt">Background: The purpose of this study was to systematically review the prevalence of class 1 integrons, antibiotic resistance pattern in Pseudomonas aeruginosa (P. aeruginosa) isolated from clinical samples other than burn samples.</span></p> <p><span style="font-size:11pt">Methods: The Web of Science, PubMed, Scopus, and Science Direct databases were searched using keywords based on the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. The cross-sectional studies published from 1st January 2000 until 1st January 2019 were included which addressed the prevalence of class 1 integrons and antibiotic-resistance in P. aeruginosa isolated from clinical samples other than burn samples. Meta-analysis was conducted using Comprehensive Meta-Analysis (CMA) software. The random-effects model, Cochran&rsquo;s Q and I2 tests were applied for statistical analyses.</span></p> <p><span style="font-size:11pt">Results: Eight articles met the eligibility standards for including in the present meta-analysis. The combined prevalence of class 1 integrons in P. aeruginosa isolated from clinical samples other than burn samples was reported by 40% (95% CI:26.1-55.8%). The pooled prevalence of Multi-Drug Resistant (MDR) P. aeruginosa isolates was 70.1%. The highest prevalence of combined antibiotic resistance was related to carbenicillin with a resistance rate of 79.9%. In general, 6 (75%) out of the 8 included studies showed the correlation between the presence of class 1 integrons and antibiotic resistance.</span></p> <p><span style="font-size:11pt">Conclusion: Regarding the correlation between the presence of integrons and the high antibiotic resistance reported by studies included in the present review, there is the need for preventive measures to prevent the spread of resistance by integrons and transferring to other micro-organisms.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40445 Ebrahim Karimi, Fatemeh Ghalibafan, Akram Esfandani, Niusha Manoochehri Arash, Sassan Mohammadi, Azad Khaledi, Hakimeh Akbari, Maria Khurshid Wed, 28 Oct 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir β-sitosterol Mediated Silver Nanoparticles Induce Cytotoxicity in Human Colon Cancer HT-29 Cells <p><span style="font-size:11pt">Background: Silver nanoparticles (AgNP) are commonly used metallic nanoparticles in health care systems. Colon cancer incidence is increasing worldwide. In this study, AgNP was synthesized using &beta;-sitosterol and its cytotoxic potential was evaluated in human colon cancer (HT-29) cells. </span></p> <p><span style="font-size:11pt">Methods: Characterization of AgNP was analyzed by TEM and spectrophotometry analysis. HT-29 cells were treated with different concentrations (2, 4, 6, 8 and 10 ng/ml) of AgNPs and cytotoxicity was evaluated by MTT assay. The apoptosis was analyzed by the flow cytometry. The expression of p53 protein was analyzed by western blotting. </span></p> <p><span style="font-size:11pt">Results: &beta;-sitosterol mediated AgNP are spherical in shape and induced concentration-dependent cytotoxicity in HT-29 cells. AgNP caused apoptosis related morphological changes as evidenced by annexin positive staining. AgNP treatments also induced the p53 expression in HT-29 cells. </span></p> <p><span style="font-size:11pt">Conclusion: Our present result suggests that &beta;-sitosterol mediated AgNP induce apoptosis in colon cancer cells and this finding may pave the way for further experimental analysis <em>in vivo</em>.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40446 Palaniappan Chithambara Shathviha, Devaraj Ezhilarasan, Shanmugam Rajeshkumar, Jayaraman Selvaraj Wed, 28 Oct 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparison of Antifungal Properties of Gold, Silver, and Selenium Nanoparticles Against Amphotericin B-Resistant Candida glabrata Clinical Isolates <p><span style="font-size:11pt">Background: The present study aimed to investigate the antifungal activity of Nanoparticles (NPs) against amphotericin B-resistant <em>Candida glabrata</em> (<em>C. glabrata</em>) strains. </span></p> <p><span style="font-size:11pt">Methods: Twelve resistant (<em>C. glabrata</em>) strains were isolated from archived clinical isolates. Antifungal activity was conducted according to Clinical and Laboratory Standards Institute&rsquo;s (CLSI) guidelines, document M27-A3/S4. The Scanning Electron Microscope (SEM) was used to observe the morphological changes of strains exposed to each nanoparticle. </span></p> <p><span style="font-size:11pt">Results: Minimum Inhibitory Concentration (MIC) of nanoparticles of all strains was in the concentration range of 0.125 to 0.5 <em>&micro;g/Ml</em>. The synthesized Ag-NPs showed superior antifungal activity against (<em>C. glabrata</em>) compared to Se-NPs and Au-NPs. The scanning electron microscope images revealed the difference in the fungal morphology between the untreated and treated fungi with nanoparticles. </span></p> <p><span style="font-size:11pt">Conclusion: The Ag-NPs, followed by Se-NPs synthesized, revealed significant antifungal activity against resistance regardless of their antifungal-resistant mechanisms.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40447 Ensieh Lotfali, Hossein Toreyhi, Kamyab Makhdoomi Sharabiani, Azam Fattahi, Amirali Soheili, Reza Ghasemi, Mahyar Keymaram, Yasaman Rezaee, Sayna Iranpanah Wed, 28 Oct 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Basic Science and Clinical Studies on Non-COVID-19 Topics, of Coronavirus Victims <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Despite advancements of societies in terms of health, epidemics do happen; the latest is the outbreak of coronavirus. Re-assortment of viruses is a biological phenomenon that creates new subtypes of viruses, indicating that pandemics are unavoidable </span><sup><span style="font-size:10.0pt">1</span></sup><span style="font-size:10.0pt">.</span> <span style="font-size:10.0pt">The SARS-CoV-2 crisis has so far affected all countries of the world, which has been more pronounced in aspects related to health and economy.</span> <span style="font-size:10.0pt">One area in which COVID-19 has had a significant impact is research </span><sup><span style="font-size:10.0pt">2</span></sup><span style="font-size:10.0pt">.</span><span style="font-size:10.0pt"> The fact that </span><span style="font-size:10.0pt">a pandemic of a virus has caused and continues to cause deaths worldwide and dire consequences for the economies of nations are of main reasons for the focus of research in this field.</span> <span style="font-size:10.0pt">Currently,</span><span style="font-size:10.0pt"> people and the media are paying the most attention to the disease among other health-related issues, and governments are asking researchers to find solutions to the problem has negatively affected all of society. As a result, </span><span style="font-size:10.0pt">research centers&#39; budgets are prioritized with issues related to COVID-19, and the majority of researchers have focused on the disease </span><sup><span style="font-size:10.0pt">2-4</span></sup><span style="font-size:10.0pt">.</span> <span style="font-size:10.0pt">In this situation, are basic science research and diseases other than COVID-19 given </span><span style="font-size:10.0pt">necessitous</span><span style="font-size:10.0pt"> attention <sup>5-7</sup>?</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">The large number of researchers focusing on the field and the rapidity of the publication of research, despite its many advantages, </span><span style="font-size:10.0pt">has several significant disadvantages that affect both COVID-19 related and other research.</span> <span style="font-size:10.0pt">Rapid and immature conclusions from some trials and their use in the clinic, despite the low quality of several studies and small size of study samples-compared to the COVID-19 spectrum-along with disruption of the peer review process even in reputable journals are of the significant drawbacks affecting COVID-19-related research </span><sup><span style="font-size:10.0pt">3,8</span></sup><span style="font-size:10.0pt">.</span> <span style="font-size:10.0pt">The negative impact of this pandemic on clinical trials on topics other than COVID-19 is worrying.</span> <span style="font-size:10.0pt">Due to the transmission problem required to perform many trials, performing such studies is problematic in terms of implementation and practice, but the main problem goes back to before this stage.</span> <span style="font-size:10.0pt">Governments want to resolve the coronavirus crisis, people and the media are following the issue of COVID-19, COVID-19 is a priority for research center budgets, and journals publish research on the field faster; as a result, individual interests such as advancement in academic status and financial profits in this field are better secured </span><sup><span style="font-size:10.0pt">2-8</span></sup><span style="font-size:10.0pt">.</span> <span style="font-size:10.0pt">Delays in progress in basic science and clinical research other than COVID-19 due to budget cuts and reduced researcher focus will have long-term adverse effects </span><sup><span style="font-size:10.0pt">9</span></sup><span style="font-size:10.0pt">.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:10.0pt">Doing basic science and free studies-that is, studies for which there are no questions or requests-often leads to other questions and sometimes to answers to which we have never asked a question.</span> <span style="font-size:10.0pt">The results of these studies are knowledge and understanding and are necessary for further studies.</span> <span style="font-size:10.0pt">These studies are the entrance to a bridge built from the laboratory to the clinic to the community </span><sup><span style="font-size:10.0pt">9</span></sup><span style="font-size:10.0pt">. Science works this way, from free studies to patient treatment, and the result of a reduction in funding and a reduction in basic science and clinical studies other than COVID-19 will be nothing but a delay in the advancement of science in general.</span></span></p> https://www.AJMB.org/En/Article.aspx?ID=30441 Shahin Akhondzadeh, Ahmad Shamabadi Sat, 24 Oct 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Silencing SALL-4 Gene by Transfecting Small Interfering RNA with Targeted Aminoglycoside-Carboxyalkyl Polyethylenimine Nano-Polyplexes Reduced Migration of MCF-7 Breast Cancer Cells <p><span style="font-size:11pt">Background: The application of non-viral systems for delivering genes to cells is becoming a very interesting issue, especially in the treatment of neoplasms such as Breast Cancer (BC). Polymer-based non-viral systems are safe and feasible gene carriers to be used in targeted cancer therapy. SALL4 gene encodes a transcription factor and is overexpressed in some cancers.</span></p> <p><span style="font-size:11pt">Methods: In this study, carboxyalkylated-PEI25 (25 kDa) was used to deliver plasmids expressing SALL4-siRNA into MCF-7 cells. DLS and AFM were applied to determine the size of nanoparticles. The MTT method was used to assess cytotoxicity, and the efficiency of transfection was confirmed both qualitatively and quantitatively. Finally, the effect of silencing SALL4 was investigated on the migration of MCF7 cells using the scratch test.</span></p> <p><span style="font-size:11pt">Results: The results showed that transferring the SALL4-siRNA using PEI25G10C50 reduced the expression of the corresponding transcription factor by 14 folds which attenuated the migration of MCF-7 cells by 58%. </span></p> <p><span style="font-size:11pt">Conclusion: In conclusion, PEI25G10C50 can serve as an effective gene delivery system for treating BC by targeting SALL-4.</span></p> https://www.AJMB.org/En/Article.aspx?ID=30437 Somaye Noruzi, Mehran Vatanchian, Amir Azimian, Arash Niroomand, Reza Salarinia, Fatemeh Oroojalian Sat, 24 Oct 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of Metastasis Suppressor Genes Expression and In Vitro Anti-Cancer Effects of Zinc Oxide Nanoparticles in Human Breast Cancer Cell Lines MCF-7 and T47D <p><span style="font-size:11pt">Background: Metallic nanoparticles are useful materials to be applied in biomedical research. In this study, the possible apoptotic and anti-metastatic activity of Zinc Oxide Nanoparticles (ZnONPs) was assessed in breast cancer cells. </span></p> <p><span style="font-size:11pt">Methods: First, in vitro cell viability was investigated by MTT assay in two human breast cancer cells (MCF-7 and T47D) and normal Human Embryonic Kidney (HEK293) cells at 37&deg;C overnight. Apoptosis induced by ZnONPs was evaluated by annexin V/PI staining, cell cycle analysis and caspase assay in cancerous cells. Moreover, quantitative real-time PCR was employed for the detection of two metastasis suppressor genes (KAI-1 and NM23) expression in cancerous cells. </span></p> <p><span style="font-size:11pt">Results: Data demonstrated that ZnONPs exert a dose-dependent inhibitory effect on the viability of T47D and MCF-7 cells, while no cytotoxic effect was observed on normal HEK293 cells. The mRNA expression levels of KAI-1 and non-metastatic protein (NM23) genes were up-regulated in ZnONP-exposed cancerous cells. ZnONPs were also found to enhance the apoptosis properties of cells by annexin V/PI staining, and caspase assay in cancerous cells. Furthermore, ZnONPs can increase sub-G1 population as compared to negative control. </span></p> <p><span style="font-size:11pt">Conclusion: Our findings showed that ZnONPs induce apoptotic activity and can modulate metastasis by up-regulating of KAI-1 and NM23 gene expression in two breast cancer (MCF-7 and T47D) cells.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40442 Seyed Ataollah Sadat Shandiz, Faryad Sharifian, Sorayya Behboodi, Fatemeh Ghodratpour, Fahimeh Baghbani-Arani Sat, 24 Oct 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Integrated Network and Gene Ontology Analysis Identifies Key Genes and Pathways for Coronary Artery Diseases <p><span style="font-size:11pt">Background: The prevalence of Coronary Artery Disease (CAD) in developing countries is on the rise, owing to rapidly changing lifestyle. Therefore, it is imperative that the underlying genetic and molecular mechanisms be understood to develop specific treatment strategies. Comprehensive disease network and Gene Ontology (GO) studies aid in prioritizing potential candidate genes for CAD and also give insights into gene function by establishing gene and disease pathway relationships. </span></p> <p><span style="font-size:11pt">Methods: In the present study, CAD-associated genes were collated from different data sources and protein-protein interaction network was constructed using STRING. Highly interconnected network clusters were inferred and GO analysis was performed. </span></p> <p><span style="font-size:11pt">Results: Interrelation between genes and pathways were analyzed on ClueGO and 38 candidates were identified from 1475 CAD-associated genes, which were significantly enriched in CAD-related pathways such as metabolism and regulation of lipid molecules, platelet activation, macrophage derived foam cell differentiation, and blood coagulation and fibrin clot formation.</span></p> <p><span style="font-size:11pt">Discussion: Integrated network and ontology analysis enables biomarker prioritization for common complex diseases such as CAD. Experimental validation and future studies on the prioritized genes may reveal valuable insights into CAD development mechanism and targeted treatment strategies.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40443 Tejaswini Prakash, Nallur B Ramachandra Sat, 24 Oct 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Role of Phosphorylation and Hyperphosphorylation of Tau in Its Interaction with βα Dimeric Tubulin Studied from a Bioinformatics Perspective <p><span style="font-size:11pt">Background: Tau is a disordered Microtubule Associated Protein (MAP) which prefers to bind and stabilize microtubules. Phosphorylation of tau in particular enhances tau-tubulin interaction which otherwise detaches from tubulin during hyperphosphorylation. The reason behind their destabilization, detachment and the role of &beta; subunit (from microtubule) and the projection domain (Tau) in microtubule stability remains elusive till date. Thus, a complete 3D structural investigation of tau protein is much needed to address these queries as the existing crystal structures are in fragments and quite limited. </span></p> <p><span style="font-size:11pt">Methods: In this study, the modelled human tau protein was subjected to phosphorylation and hyperphosphorylation which were later considered for docking with microtubules (&beta;&alpha; subunits-inter dimer) and vinblastine.</span></p> <p><span style="font-size:11pt">Results: Phosphorylated tau protein interacts with both &alpha;- and &beta; subunits. But stronger bonding was with &alpha;- compared to &beta; subunits. Regarding &beta; subunit, proline rich loop and projection domain actively participated in tau binding. Interestingly, hyperphosphorylation of tau increases MAP domain flexibility which ultimately results in tau detachment, the main reason behind tangle formation in Alzheimer&rsquo;s disease. </span></p> <p><span style="font-size:11pt">Conclusion: This study being the first of its kind emphasizes the role of projection domain and proline rich region of &beta;-subunit in stabilizing the tau-tubulin interaction and also the effect of hyperphosphorylation in protein-protein and protein-drug binding.</span></p> https://www.AJMB.org/En/Article.aspx?ID=40444 Hrushikesh Dixit , Selvaa Kumar C, Ruchi Chaudhary, Divya Thaker, Nikhil Gadewal, Debjani Dasgupta Sat, 24 Oct 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Removal of Cefixime from Water Using Rice Starch by Response Surface Methodology <p>Background: Remaining pharmaceutical compounds cause environmental pollution. Therefore, refining these compounds has become a major challenge. In this study, the function of eliminating Cefixime (CFX)&nbsp;using rice starch was evaluated under controlled conditions.</p> <p>Methods: Response Surface Methodology (RSM) was used to design, analyze, and optimize experiments, and the interaction between four variables including pH (3-9), rice starch dose (0&ndash;300 <em>mg/L</em>), CFX initial concentration (0&ndash;16 <em>mg/L</em>) and time (20&ndash;120 <em>min</em>) was investigated on CFX removal.</p> <p>Results: The optimum pH, starch dose, initial concentration and time were 4.5, 225 <em>mg/L</em>, 7.9 <em>mg/L</em> and 95 <em>min</em>, respectively. The maximum efficiency of CFX removal was 70.22%. According to RSM, this study follows a quadratic model (R2=0.954).</p> <p>Conclusion: Rice starch has been successful in removing CFX from the aqueous solution. Therefore, it is recommended to utilize this process to remove CFX from aqueous solutions.</p> https://www.AJMB.org/En/Article.aspx?ID=30434 Fatemeh Sadat Tabatabaei, Mahdi Asadi-Ghalhari , Rahim Aali, Fatemeh Mohammadi, Roqiyeh Mostafaloo, Rezvaneh Esmaeili, Zohreh Davarparast, Zahra Safari Wed, 19 Aug 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Pharmacokinetic Effect of MDR Gene Polymorphism rs2032582 on the Therapeutic Response in Iraqi Patients with Acute Myeloid Leukemia <p>Background: The main problem in treatment of leukemia patients is the chemotherapy resistance which is a main concern in recent years. The cause of chemotherapy drug resistance is related to <em>MDR</em> gene which is located on chromosome 7 (7q21-31) and it is mainly connected with energy-dependent efflux (P-glycoprotein). This study was conducted to assess the correlation between MDR polymorphism and chemotherapy efficiency with Vincristine in a sample of Iraqi Acute Myeloid Leukemia (AML) patients.</p> <p>Methods: The blood sample of 200 AML patients and 200 controls were collected and the frequency of rs2032582 was calculated through sequencing and then the role of different genetic patterns was evaluated on cancer cells by MTT assay.</p> <p>Results: The results indicate that GG and TT genotypes (20 and 20.5% from total patients count) are more frequent in Iraqi AML patients than other genetic patterns in <em>MDR</em> gene and also the genotype TA is more sensitive to Vincristine chemotherapy than other genotypes.</p> <p>Conclusion: It seems that genetic pattern is the main factor in determination of chemotherapy of AML patients, and patients should not undergo chemotherapy with such drugs, especially Vincristine.</p> https://www.AJMB.org/En/Article.aspx?ID=30436 Rafid A Abdulkareem, Tamadher Abbas Rafaa, Hamsa Ahmed Jasim, Ahmed Abdul Jabbar Suleiman Wed, 19 Aug 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effective Anti-SARS-CoV-2 RNA Dependent RNA Polymerase Drugs Based on Docking Methods : The Case of Milbemycin, Ivermectin, and Baloxavir Marboxil <p>Background: Severe Acute Respiratory Syndrome-coronavirus 2 (SARS-CoV-2) is a new virus with a global pandemic. Yet, no vaccine or efficient treatments are found against the disease. The viral RNA dependent RNA Polymerase (RdRP) is a suitable target for developing antiviral agents. SARS-CoV-2 RdRP was employed to test its binding activity with some drugs.</p> <p>Methods: Using some docking methods, RdRP was targeted by Milbemycins (MMs), Ivermectin (IMT), Baloxavir Marboxil (BM), and Tadalafil (TF), a phosphodiesterase type 5 inhibitor.</p> <p>Results: MM-A3 5-oxime (MMA35O), MM-A3 (MMA3), MM-A4 5-oxime (MMA45O), IMT, BM, and TF showed the highest binding affinity to RdRp.</p> <p>Conclusion: The drugs used in the present computational investigation are effective against the SARS-CoV-2 RdRP with high affinity values especially, milbemycins, ivermectin, and Baloxavir marboxil, which could further be studied in laboratory and clinical trials for saving millions of lives around the world.</p> https://www.AJMB.org/En/Article.aspx?ID=30438 Ali Hassan Daghir Janabi Sat, 15 Aug 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Application of Electrospray in Preparing Solid Lipid Nanoparticles and Optimization of Nanoparticles Using Artificial Neural Networks <p>Background: Electrospray (Electrohydrodynamic atomization) has been introduced as a novel approach to prepare nanoparticles. This work aimed to prepare SLNs through electrospray and evaluate factors affecting particle size of prepared Solid Lipid Nanoparticles (SLNs).</p> <p>Methods: SLNs were prepared by electrospray method. To study the factors affecting particle size of SLNs, Artificial Neural Networks (ANNs) were employed. Four input variables, namely, Tween 80 concentration, lipid concentration, flow rate, and polymer to lipid ratio were analyzed through ANNs and particle size was the output.</p> <p>Results: The analyzed model presented concentration of Tween 80 (surfactant) and lipid as effective parameters on particle size. By increasing surfactant and decreasing lipid concentration, minimum size could be obtained, while flow rate and polymer to lipid ratio appeared not to be effective.</p> <p>Conclusion: Concentration of surfactant/lipid plays the most important role in determining the size.</p> https://www.AJMB.org/En/Article.aspx?ID=30439 Elaheh Shanaghi, Mahdi Aghajani, Fariba Esmaeli, Mohammad Ali Faramarzi, Hoda Jahandar, Amir Amani Sat, 15 Aug 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Nanoformulation-Based Antiviral Combination Therapy for Treatment of COVID-19 https://www.AJMB.org/En/Article.aspx?ID=30440 Hoda Keshmiri Neghab, Seyedeh Sara Azadeh, Mohammad Hasan Soheilifar, Fariba Dashtestani Sat, 15 Aug 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Screening PAX9, MSX1 and WNT10A Mutations in 4 Iranian Families with Non-Syndromic Tooth Agenesis <p>Background: Tooth agenesis is one of the most common developmental anomalies in human and the main reasons for its occurrence are still unknown. Mutations of several genes such as <em>PAX9</em>, <em>MSX1</em>, <em>AXIN2</em>, <em>KDF1</em> and <em>WNT10A</em> have been reported which are associated with non-syndromic tooth agenesis. However, <em>PAX9</em>, <em>MSX1</em> and <em>WNT10A</em> are commonly reported in the literature. Hence, the aim of this study was to investigate the mutations of these genes in 4 Iranian families with non-syndromic tooth agenesis.</p> <p>Methods: DNA extractions from peripheral blood cells of patients with non-syndromic tooth agenesis from 4 unrelated Iranian families were performed by salting out method, and the candidate genes were amplified then followed by Sanger sequencing method.</p> <p>Results: One missense variant (rs4904210) and 4 Single Nucleotide Polymorphisms (SNPs) (rs2236007, rs12883298, rs12882923 and rs12883049) were found in <em>PAX9</em> gene. Five variants (rs149370601, rs8670, rs186861426 and rs774949973) including a missense variant (rs36059701) were detected in <em>MSX1</em> gene and no variants were found in <em>WNT10A</em> gene.</p> <p>Conclusion: All variants were analyzed based on bioinformatics websites and Iranian gene databases, and as a result, it was revealed that variants of <em>PAX9</em>, <em>MSX1</em> and <em>WNT10A</em> may not play a role in non-syndromic tooth agenesis among Iranian cases.</p> https://www.AJMB.org/En/Article.aspx?ID=30435 Shiva Safari, Asghar Ebadifar, Hossien Najmabadi, Koorosh Kamali, Seyedeh Sedigheh Abedini Mon, 29 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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> https://www.AJMB.org/En/Article.aspx?ID=30422 Nima Rezaei Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Pasteurella multocida Vaccine Candidates: A Systematic Review <p><em>Pasteurella multocida (P.&nbsp;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.&nbsp;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.&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=30423 Saied Mostaan, Abbas Ghasemzadeh, Soroush Sardari, Mohammad Ali Shokrgozar, Gholamreza Nikbakht Brujeni, Mohsen Abolhassani, Parastoo Ehsani, Mohammad Reza Asadi Karam Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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&le;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> https://www.AJMB.org/En/Article.aspx?ID=30424 Nasrin Karimi, Mana Oloomi, Zahra Orafa Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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&gt;A (DPYD*2A;&nbsp; rs3918290), c.1679 T&gt;G (I560S; DPYD*13; rs55886062), and c.2846A&gt;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> https://www.AJMB.org/En/Article.aspx?ID=30425 Mohammad Hadi Abbasian, Nafiseh Ansarinejad, Bahareh Abbasi, Masoud Iravani, Tayeb Ramim, Fahime Hamedi, Ali M. Ardekani Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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> https://www.AJMB.org/En/Article.aspx?ID=30428 Ali Ahmad Bayat , Niloufar Sadeghi, Ramina Fatemi, Mohammad Reza Nowroozi, Solmaz Ohadian Moghadam, Mohadeseh Borzuee, Amin Radmanesh, Mahmood Khodadoost, Ali reza Sarrafzadeh, Omid Zarei, Hodjattallah Rabbani Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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.&nbsp; This study aimed to utilize human Wharton&rsquo;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 (IL1&beta;-CHON002) culture was treated as follows: 1) with hWJMSCs-CM 15% (v/v); 2) with hWJMSCs-CM&nbsp; 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 TNF&alpha;), 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 IL1&beta;-CHON002 treatment using 15% CM of hWJMSCs induced with IGF1. CM of hWJMSCs can reduce inflammatory cytokines (TNF&alpha; 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> https://www.AJMB.org/En/Article.aspx?ID=30429 Hanna Sari Widya Kusuma, Wahyu Widowati, Rimonta Febby Gunanegara, Berry Juliandi, Nyoman Ehrich Lister, Seila Arumwardana, Dewani Tediana Yusepany, Dwi Surya Artie, Enden Dea Nataya , Kamila Yashfa Gunawan, Ika Adhani Sholihah, Ermi Girsang, Chrismis Novalinda Ginting, Indra Bachtiar, Harry Murti Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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 &gt;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> https://www.AJMB.org/En/Article.aspx?ID=30431 Mohammad Reza Sarjoughian, Shamsozoha Abolmaali, Shakiba Darvish Alipour Astaneh Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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> https://www.AJMB.org/En/Article.aspx?ID=30432 Nasim Arshadi, Seyed Latif Mousavi , Jafar Amani , Shahram Nazarian Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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&nbsp; pneumonia, severe respiratory tract infection and even death.&nbsp; 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 &quot;novel corona virus 2019&quot; <sup>3</sup>.</p> <p>At present, &quot;severe acute respiratory syndrome coronavirus 2&quot; &nbsp;or &quot;coronavirus disease 2019&quot; (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>.&nbsp; 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&nbsp; 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&nbsp; 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&nbsp; 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> https://www.AJMB.org/En/Article.aspx?ID=30433 Mahintaj Dara , Mahdieh Talebzadeh Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Appropriate Scaffold Selection for CNS Tissue Engineering <p>Cellular transplantation, due to the low regenerative capacity of the Central Nervous System (CNS), is one of the promising strategies in the treatment of neurodegenerative diseases. The design and application of scaffolds mimicking the CNS extracellular matrix features (biochemical, bioelectrical, and biomechanical), which affect the cellular fate, are important to achieve proper efficiency in cell survival, proliferation, and differentiation as well as integration with the surrounding tissue. Different studies on natural materials demonstrated that hydrogels made from natural materials mimic the extracellular matrix and supply microenvironment for cell adhesion and proliferation. The design and development of cellular microstructures suitable for neural tissue engineering purposes require a comprehensive knowledge of neuroscience, cell biology, nanotechnology, polymers, mechanobiology, and biochemistry. In this review, an attempt was made to investigate this multidisciplinary field and its multifactorial effects on the CNS microenvironment. Many strategies have been used to simulate extrinsic cues, which can improve cellular behavior toward neural lineage. In this study, parallel and align, soft and injectable, conductive, and bioprinting scaffolds were reviewed which have indicated some successes in the field. Among different systems, three-Dimensional (3D) bioprinting is a powerful, highly modifiable, and highly precise strategy, which has a high architectural similarity to tissue structure and is able to construct controllable tissue models. 3D bioprinting scaffolds induce cell attachment, proliferation, and differentiation and promote the diffusion of nutrients. This method provides exceptional versatility in cell positioning that is very suitable for the complex Extracellular Matrix (ECM) of the nervous system.</p> https://www.AJMB.org/En/Article.aspx?ID=30426 Akram Shafiee, Hanie Ahmadi, Behnaz Taheri, Simzar Hosseinzadeh, Yousef Fatahi, Masoud Soleimani, Fatemeh Atyabi, Rassoul Dinarvand Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antibiofilm Activity of Kefir Probiotic Lactobacilli Against Uropathogenic Escherichia coli (UPEC) <p>Background: Inhibition of biofilm formation is essential for the prevention and treatment of urinary tract infection. This study was aimed to identify the probiotic potential of Lactobacillus strains isolated from kefir and evaluate their antimicrobial and antibiofilm activities against Uropathogenic&nbsp;<em>Escherichia coli</em>&nbsp;(UPEC).&nbsp;</p> <p>Methods: Twelve Lactobacillus strains were evaluated. Antimicrobial and antibiofilm activities of Cell Free Supernatant (CFS) of the Lactobacillus strains against UPEC isolates were evaluated by agar well diffusion method and crystal violet assay, respectively. Probiotic potential of selected isolates was assessed by analyzing their tolerance to acidic pH and bile salts, auto-aggregation ability, co-aggregation with&nbsp;<em>Escherichia coli (E. coli)</em>&nbsp;and hemolytic activity. The isolates were identified by phenotypic and 16S rRNA gene sequencing.</p> <p>Results:&nbsp; The CFS of all lactobacilli strains was able to inhibit UPEC isolates even after neutralization. Four out of 12 isolates inhibited the biofilm formation by UPEC in the range 62-75%. The viability under acidic condition varied among the isolates ranging from 6-89.8%. All the isolates could tolerate the 0.3% bile and eight isolates showed the adaptation time of less than 1&nbsp;<em>hr</em>. All the strains exhibited co-aggregation with&nbsp;<em>E. coli</em>. Auto-aggregation was highly correlated with co-aggregation of all lactobacilli strains with&nbsp;<em>E. coli</em>&nbsp;(r=0.889, p&lt;0.001). The isolates with satisfactory probiotic potential and higher ability of biofilm inhibition and antibacterial activity belonged to the species&nbsp;<em>Lactobacillus</em>&nbsp;<em>rhamnosus</em>&nbsp;and&nbsp;<em>Lactobacillus&nbsp;paracasei</em>.</p> <p>Conclusion: All four selected probiotic strains exhibited antimicrobial and antibiofilm activities, which suggest potential applications for controlling or preventing infections caused by UPEC.</p> https://www.AJMB.org/En/Article.aspx?ID=30427 Maryam Ghane, Laleh Babaeekhou, Seyedeh Sepideh Ketabi Sat, 20 Jun 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Bone Regeneration Using Bio-Nanocomposite Tissue Reinforced with Bioactive Nanoparticles for Femoral Defect Applications in Medicine <p>Background: In recent years, the method of constructing and evaluating the properties of polymer nanocomposite and bioactive ceramics in tissue engineering such as biocompatible scaffolds was studied by some researchers.&nbsp;</p> <p>Methods: In this study, the bio-nanocomposite scaffolds of Chitosan (CS)&ndash;Hydroxya-patite (HA)&ndash;Wllastonite (WS), incorporated with 0, 10, 20 and 30 wt% of zirconium were produced using a freeze-drying method. Also, the phase structure and morphology of scaffolds were investigated using X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS). By analyzing the SEM images, the porosity of the scaffolds was observed in the normal bone area of ​​the body. In the next step, bioactivity and biodegradability tests of the scaffolds were carried out. Due to the presence of hydrophilic components and the high-water absorption capacity of these materials, the bio-nanocomposite scaffolds were able to absorb water properly. After that, the mechanical properties of the scaffolds were studied.</p> <p>Results: The mechanical test results showed that the preparation of reinforced bio-nanocomposites containing 10 wt% of zirconium presented better properties compared to incorporated bio-nanocomposites with different loadings of zirconium.</p> <p>Conclusion: According to MTT assay results, the prepared scaffolds did not have cytotoxicity at different concentrations of scaffold extracts. Consequently, the investigated scaffold can be beneficial in bone tissue engineering applications because of its similarity to natural bone structure and its proper porosity.</p> https://www.AJMB.org/En/Article.aspx?ID=20406 Mohammad Ali Maghsoudlou, Ehsan Nassireslami, Saeed Saber-Samandari, Amirsalar Khandan Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Polyaniline Based Electrochemical Sensor for the Detection of Dengue Virus Infection <p>Background: Dengue burden is increasing day-by-day globally. A rapid, sensitive, cost-effective early diagnosis kit is the need of the hour. In this study, a label-free electrochemical immunosensor was proposed for dengue virus detection. A modified Polyaniline (PANI) coated Glassy Carbon (GC) electrode, immobilized with DENV NS1 antibody was used to detect the circulating DENV NS1 antigen in both spiked and infected sample.</p> <p>Methods: Cloning, purification and expression of DENV NS1 protein in <em>Escherichia coli</em> (<em>E. coli)</em> was performed and sensor design, PANI modification on GC electrode surface by electrochemical polymerization and immobilization of NS1 antibody on the modified electrode surface was done and finally the analytical performance of the electrochemical immunosensor was done using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS).</p> <p>Results: CV and EIS were used to study and quantitate the circulating DENV antigen. The calibration curve showed wide linearity, good sensitivity (Slope=13.8% IpR/<em>ml.ng</em><sup>-1</sup>) and distribution of data with a correlation coefficient (R) of 0.997. A lower Limit of Detection (LOD) was found to be 0.33 <em>ng.ml</em><sup>-1</sup> which encourages the applicability of the sensor.</p> <p>Conclusion: Thus, a PANI based new electrochemical immunosensor has been developed which has the potential to be further modified for the development of cost effective, point of care dengue diagnostic kit.</p> https://www.AJMB.org/En/Article.aspx?ID=10373 Reshmi Dutta, K Thangapandi, Sumantra Mondal, Amalesh Nanda, Shreyosi Bose, Shairee Sanyal, Saikat Kumar Jana, Suvankar Ghorai Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cysteine/Histidine-Dependent Amidohydrolase/Peptidase (CHAP)-Displayed Nano Phages: Antimicrobial Function against Methicillin-Resistant Staphylococcus aureus (MRSA) <p>Background: Emergence and prevalence of multi drug resistance strains such as Methicillin-Resistant <em>Staphylococcus aureus</em> (MRSA) call for new antibacterial option. Endolysins as a new option is suggested. The phage display technique is suggested for production of recombinant endolysins. The recombinant endolysins displayed nano phages specifically lysis bacteria, which penetrate to the depth of tissue and the effective dose is reduced.</p> <p>Methods: <em>CHAPK</em> gene was ligated in T7Select vector arms in T7Select10-3b cloning kit. To produce recombinant nano phages, ligation reaction was added directly to the packaging extract. Recombinant nano phages were amplified by Double Layer Agar assay (DLA). The recombinant nano phages were characterized using TEM. Size of recombinant nano phages was determined using DLS. The spot test was performed to confirm CHAPk -displayed on the surface of nano phages. The turbidimetry was used to investigate lytic activity of recombinant nano phages against MRSA ATCC No. 33591.</p> <p>Results: The results showed recombinant nano phages belonged to order Caudovirales and family Podoviridae with titer 2&times;10<sup>7</sup> <em>PFU/ml</em>. According to the results of DLS, size of recombinant nano phages was 71 <em>nm</em>. Formation inhibition zone&nbsp;confirmed the presence of CHAPk on the surface of nano phage phenotypically. The turbidimetry showed lytic activity recombinant nano phages against MRSA after 5 <em>min</em>.</p> <p>Conclusion: This study suggests that CHAPk -displayed nano phages can be effective in MRSA infections.</p> https://www.AJMB.org/En/Article.aspx?ID=20404 Golnar Rahimzadeh, Pooria Gill, Mohammad Sadegh Rezai Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Design of Anti-Angiogenic Peptidomimetics and Evaluation their Biological Activity by In Vitro Assays <p>Background: One of the important&nbsp;therapeutic approaches in cancer field is development of compounds which can block the initial tumor growth and the progression of tumor metastasis with no side effects. Thus, the recent study was carried out to design anti-VEGFR2-peptidomimetics as the most significant factor of angiogenesis process- and evaluate their biological activity by <em>in vitro</em> assays.</p> <p>Methods: We designed anti-VEGFR2 peptidomimetics with anti-angiogenic activity, including compound P (lactam derivative) and compound T (indole derivative) by using in silico methods. Then, the inhibitory activity on angiogenesis was evaluated by using angiogenesis specific assays such as Human Umbilical Vein Endothelial Cell (HUVEC) proliferation, tube formation in Matrigel, MTT and Real-Time PCR. IC50 values of the compounds were also determined by cytotoxicity plot in MTT assay.</p> <p>Results: Compounds P and T inhibited HUVEC cell proliferation and viability in a dose-dependent manner. The IC50 for compound T and compound P in HUVEC cell line were 113 and 115 <em>&mu;g/ml</em>, respectively. Tube formation assay revealed that both compounds can inhibit angiogenesis effectively. The results of Real-Time PCR also showed these compounds are able to inhibit the expression of <em>CD31</em> gene in HUVEC cell line.</p> <p>Conclusion: Our study suggested that compounds P and T may act as therapeutic molecules, or lead compounds for development of angiogenesis inhibitors in VEGF-related diseases.</p> https://www.AJMB.org/En/Article.aspx?ID=20409 Mona Ghadam, Soroush Sardari, Mohammad Ali Shokrgozar, Mahdiyeh Sadat Mahdavi Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Methylation Analysis of P16, RASSF1A, RPRM, and RUNX3 in Circulating Cell-Free DNA for Detection of Gastric Cancer: A Validation Study <p>Background: Most of Gastric Cancer (GC) patients are diagnosed at an advanced stage with poor prognosis. Hypermethylations of several tumor suppressor genes in cell-free DNA of GC patients have been previously reported. In this study, an attempt was made to investigate the methylation status of <em>P16</em>, <em>RASSF1A</em>, <em>RPRM</em>, and <em>RUNX3 </em>and their potentials for early diagnosis of GC.</p> <p>Methods: Methylation status of the four tumor suppressor genes in 96 plasma samples from histopathologically confirmed gastric adenocarcinoma patients (Stage I-IV) and 88 healthy controls was determined using methylation-specific PCR method. Receiver operating characteristic curve analysis was performed and Area Under the Curve (AUC) was calculated. Two tailed p&lt;0.05 were considered statistically significant.</p> <p>Results: Methylated <em>P16</em>, <em>RASSF1A</em>, <em>RPRM</em>, and <em>RUNX3</em> were significantly higher in the GC patients (41.7, 33.3, 66.7, and 58.3%) compared to the controls (15.9, 0.0, 6.8, and 4.5%), respectively (p&lt;0.001). Stratification of patients showed that <em>RPRM</em> (AUC: 0.70, Sensitivity: 0.47, Specificity: 0.93, and p&lt;0.001) and <em>RUNX3 </em>(AUC: 0.77, Sensitivity: 0.59, Specificity: 0.95, and p&lt;0.001) had the highest performances in detection of early-stage (I+II) GC. The combined methylation of <em>RPRM </em>and <em>RUNX3 </em>in detection of early-stage GC had a higher AUC of 0.88 (SE=0.042; 95% CI:0.793&ndash;0.957; p&lt;0.001), higher sensitivity of 0.82 and reduced specificity of 0.89.</p> <p>Conclusion: Methylation analysis of <em>RPRM </em>and <em>RUNX3 </em>in circulating cell free-DNA of plasma could be suggested as a potential biomarker for detection of GC in early-stages.</p> https://www.AJMB.org/En/Article.aspx?ID=10370 Kioomars Saliminejad, Shahrzad Soleymani Fard, Hamid Reza Khorram Khorshid, Marjan Yaghmaei, Habibollah Mahmoodzadeh, Seyed Asadollah Mousavi, Seyed Hamidollah Ghaffari Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Analysis of Glioblastoma Multiforme Tumor Metabolites Using Multivoxel Magnetic Resonance Spectroscopy <p>Background: Glioblastoma Multiforme (GBM) is the most common and deadly type of primary brain tumor in adults. Magnetic Resonance Spectroscopy (MRS) is a non-invasive imaging technique used to study metabolic changes in the brain tumors. Some metabolites such as Phosphocholine, Creatine, NAA/Cr, and Pcho/Cr have been proven to show a diagnostic role in GBM. The present study was conducted to analyze important metabolites using MRS multivoxel in GBM tumor.</p> <p>Methods: In this study, information was collected from 8 individuals diagnosed with GBM using Siemens multivoxel MRS with a magnetic field strength of 3 T. Data were obtained by Point-Resolved Spectroscopy (PRESS) protocol with TE=135 <em>ms</em> and TR=1570 <em>ms</em>. NAA, Pcho, Cr, Ala, Gln, Gly, Glu, Lac, NAAG, and Tau metabolites were extracted and evaluated statistically.</p> <p>Results: Given total number of normal voxels and total number of all voxels, levels of Cr, Glu, NAA, NAAG, and Gly/Tau ratio in healthy voxels were significantly higher than tumoral voxels (p=0.005, p=0.03, p&lt;0.001, p&lt;0.001 and p=0.041, respectively). In contrast, levels of Gly, Gln, Tau, Lac/Cr, Pcho/Cr, Pcho/NAA, Lac/NAA, and Gln/Glu ratios in tumoral voxels were significantly more than healthy voxels (p=0.001, p=0.037, p&lt;0.001, p=0.010, p&lt;0.001, p&lt;0.001, and p=0.024, respectively). However, levels of Lac and Pcho had no significant difference in the two types of voxels.</p> <p>Conclusion: In summary, compared to patients with glioblastoma with <sup>1</sup>H-MRS, the Pcho/Cr and Pcho/NAA ratios, and NAAG are the most important parameters to differentiate between tumoral and normal voxels.</p> https://www.AJMB.org/En/Article.aspx?ID=20407 Meysam Siyah Mansoory, Ayob Faramarzi, Karim Khoshgard, Hadi Mozafari Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Optimization of Fermentation Conditions to Enhance Cytotoxic Metabolites Production by Bacillus velezensis Strain RP137 from the Persian Gulf <p>Background: Isolation, introduction and producing bioactive compounds from bacteria, especially marine bacteria, is an attractive research area. One of the main challenges of using these metabolites as drug and their industrialization is the optimization of production conditions.</p> <p>Methods: In the present study, the response surface methodology was applied to optimize the production of a cytotoxic extract (C-137-R) by <em>Bacillus velezensis</em>&nbsp;(<em>B.</em>&nbsp;<em>velezensis)&nbsp;</em>strain RP137. Initially, among the three carbon and three nitrogen sources, rice starch and potassium nitrate were selected as the best, with cell toxicity equal to IC50=54.4 and 45.1 <em>&mu;g/ml</em> in human lung and liver cancer cell lines, respectively (A549 and HepG2). In the next step, fractional factorial design was performed to survey effect of seven physical and chemical factors on the amount of production, and the most important factors including carbon and nitrogen sources with the positive effect and the sea salt with negative effect were determined. Finally, using the central composite design with 20 experiments, the best concentrations of rice starch and potassium nitrate (1.5%) and sea salt (1%) were obtained.</p> <p>Results: The average amount of dried extract produced in the optimum conditions was 131.1 <em>mg/L</em> and the best response was 71.45%, which is more than 28-fold better than the pre-optimized conditions.</p> <p>Conclusion: In general, it can be suggested that the use of modern statistical methods to optimize environmental conditions affecting the growth and metabolism of bacteria can be a highly valuable tool in industrializing the production of bioactive compounds.</p> https://www.AJMB.org/En/Article.aspx?ID=20405 Roya Pournejati, Hamid Reza Karbalaei-Heidari Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Investigation of Integron-Associated Resistance Gene Cassettes in Urinary Isolates of Klebsiella pneumoniae in Yasuj, Southwestern Iran during 2015-2016 <p>Background: Growing antibiotic resistance among urinary opportunistic pathogens such as <em>Klebsiella pneumoniae (K. pneumonia)</em> has created a worrisome condition in the treatment of the Urinary Tract Infections (UTIs) in recent years. Integrons play a significant role in the dissemination of antibiotic resistance genes. The present study was conducted to investigate class 1-3 integrons and the corresponding resistance gene cassettes in urinary <em>K. pneumoniae</em> isolates.</p> <p>Methods: In this study, from December 2015 to September 2016, a total of 196 <em>K. pneumoniae</em> isolates were collected from the patients with UTI referred to medical diagnostic laboratories in Yasouj, Southwestern Iran. Antibiotic susceptibility patterns of isolates were determined using 12 antibiotics by the disc diffusion method. Polymerase Chain Reaction (PCR) was used for detection of integron genes (<em>intI1</em>, <em>intI2</em>, and <em>intI3</em>). The variable regions of integrons were amplified by PCR and sequenced to identify the corresponding gene cassettes.</p> <p>Results: Thirty-nine different antibiotic resistance profiles were observed among <em>K. pneumoniae</em> isolates. Only 12.2% of <em>K. pneumoniae</em> isolates were found to harbor the <em>intI1</em> gene. While 17 (60.7%) out of 28 Multidrug Resistance (MDR) <em>K. pneumoniae </em>isolates carried the <em>intI1</em> gene, only 4.2% of non-MDR isolates harbored <em>intI1</em> gene. Totally 7 different gene cassette arrays were found in the <em>intI1</em> gene of <em>K. pneumoniae</em> isolates. The <em>aadA1 </em>was the most prominent gene cassette. Also, high frequency of <em>dfrA</em> containing gene cassettes was observed.</p> <p>Conclusion: Continuous monitoring and characterization of integrons and their associated gene cassettes could be helpful in controlling the rising rate of antibiotic resistance.</p> https://www.AJMB.org/En/Article.aspx?ID=20408 Fariba Jahanbin, Masoud Marashifard, Sanaz Jamshidi, Maryam Zamanzadeh, Masumeh Dehshiri, Seyed Ali Asghar Malek Hosseini, Seyed Sajjad Khoramrooz Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Gene Expression and Levels of TGF-B in PBMC is Associated with Severity of Symptoms in Chronic Heart Failure <p>Background: TGF-&beta;1 is known to promote cardiac remodeling and fibrosis during Congestive Heart Failure (CHF). In this study, an attempt was made to investigate expression of Transforming Growth Factor beta1 (TGF-&beta;1) and relative expansion or contraction of regulatory T-cell (Tregs) population in peripheral blood of patients with Chronic Heart Failure (CHF).</p> <p>Methods: Real-time PCR assay was used to investigate expression and post-stimulation levels of TGF-&beta;1 in cell culture supernatant of Peripheral Blood Mononuclear Cells (PBMC) of 42 patients with CHF and 42 controls. Flow cytometry was used to identify relative counts of CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> Tregs.</p> <p>Results: PBMCs in patients with CHF expressed higher levels of TGF-&beta;1 compared to controls. Post-stimulation levels of TGF-&beta;1 expression were significantly higher in New York Heart Association (NYHA) functional class IV patients compared to stage I patients. Tregs were significantly expanded in PBMC in CHF, while the CD4<sup>+&shy;</sup> helper T-cells were unchanged. Treg expansion was more significant in NYHA functional class I patients compared to class IV patients.</p> <p>Conclusion: Expansion of Treg population in CHF provides an extrinsic source for TGF-&beta;1 production to induce reactive fibrosis and cardiac remodeling. Relative decrease in Treg population at advanced stages of CHF is indicative of a loss of regulatory characteristics in these cells and unopposed proinflammatory milieu.&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=20410 Samaneh Saadati, Vajiheh Eskandari, Farzaneh Rahmani, Mohammad Jafar Mahmoudi, Zahra Rahnemoon, Zahra Rahmati, Fatemeh Gorzin, Mona Hedayat, Ali Akbar Amirzargar, Nima Rezaei Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Construction, Cloning, and Expression of CagA Recombinant Protein of Helicobacter pylori <p>Background: This study aimed to assess construction and expression of CagA recombinant protein of <em>Helicobacter pylori (H. pylori)</em> in <em>Escherichia coli</em> <em>(E. coli)</em> BL21.</p> <p>Methods: Bioinformatics was used in designing the desired gene by Gene Runner. Next, the construct was subcloned to pET21b vector and this process was confirmed by Polymerase Chain Reaction (PCR), enzyme digestion and sequencing techniques. Then, it was cloned in the Escherichia coli BL21 as an expression host. Expression of protein was verified using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting technique. For purification of the protein, the Ni-NTA column was used. Protein concentration was determined by the Bicinchoninic Acid Protein Assay Kit (Parstoos). Finally, Western blotting was performed using CagA antibodies and normal human serum for determining immunogenicity feature with human antiserum.</p> <p>Results: According to the results of the present study, CagA construct was cloned into the pET21b vector and after confirmation and cloning in host expression, recombinant protein with the size of 38 <em>kDa</em> was successfully expressed and purified. The recombinant CagA protein showed immunogenicity characteristics with human antiserum.</p> <p>Conclusion: In conclusion, only 5&prime;-end of recombinant protein CagA with high immunogenicity effects was successfully constructed, cloned and expressed.&nbsp; Also, CagA recombinant protein showed good immunogenicity activity with human antiserum.</p> https://www.AJMB.org/En/Article.aspx?ID=10375 Abbas Shapouri Moghaddam, Shamseddin Mansouri, Alireza Neshani, Farzaneh Firoozeh, Azade Matinpur, Azad Khaledi, Mehran Ghazalibina Sat, 14 Mar 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Optimization of In vitro Expansion and Activation of Human Natural Killer Cells against a Breast Cancer Cell Line <p>Background: Regarding to the increase of cancer deaths in recent years and disability of common therapies to eradicate cancers, as well as expansion of Natural Killer (NK) cell therapy, it seems so vital to find new useful therapies against cancers. Breast cancer is the second main cause of cancer death among women. As it is impossible for a majority of patients to receive NK cell therapy, an attempt was made to establish a low-cost and efficient method for expanding and activating NK cells against breast cancer cell line (MCF7).</p> <p>Methods: NK cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) applying either MACS based NK cell enrichment kit or antibodies and complement as cytotoxic method. Then, the NK cells were cultured in Stem Cell Growth Medium (SCGM) with feeder layer (irradiated PBMCs) along with PHA or OKT3. IL-2, IL-15 and IL-21 were used to expand NK cells and finally their cytotoxic activity was investigated by flow cytometry.</p> <p>Results: Highly pure NK cells were obtained and no significant difference between the two isolation methods was found. Using IL-2 plus IL-15, the number of NK cells increased up to100 fold after 16 days. No significant effect was observed after IL-21 treatment.</p> <p>Conclusion: Our data indicated that cytotoxicity method can be considered a low-cost alternative for NK cell isolation kits. It seems that culturing NK cells for 14 days in either PHA or OKT3 supplemented SCGM medium would be more effective than culturing for 16 days in the presence of IL-21.</p> https://www.AJMB.org/En/Article.aspx?ID=10400 Farzaneh Peighambarzadeh, Anahita Najafalizadeh, Nafiseh Esmaeil, Abbas Rezaei, Farzaneh Ashrafi, Mazdak Ganjalikhani-Hakemi Wed, 01 Jan 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression of Human Placenta-specific 1 (PLAC1) in CHO-K1 Cells <p>Background: Placenta-specific 1 (PLAC1), as a new Cancer/Testis Antigen (CTA), is frequently expressed in a variety of cancers and localized to cytoplasm and plasma membrane. Surface expression of cancer target antigens is of great importance that enables antibody-mediated cancer immunotherapy. The aim of the current study was to express the intact human PLAC1 protein on plasma membrane of a eukaryotic cell as a model for future anti-PLAC1-based cancer immunotherapy.</p> <p>Methods: In the first approach, entire human PLAC1 gene including its own Signal Peptide (SP) was cloned into pIRES2-EGFP and LeGO-iG2 vectors and expressed in CHO-K1 cells. In the second approach, cytosolic and Signal-Anchor (SA) sequence of Transferrin Receptor Protein 1 (TFR1) were fused to extracellular portion of PLAC1 and expressed as above. Expression of PLAC1 was then assessed using Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western Blot (WB), Immunocytochemistry (ICC), Immunofluorescence (IF) and Flow Cytometry (FC).</p> <p>Results: The first approach resulted in the expression of PLAC1 in submembranous but not in the surface of transfected CHO-K1 cells. Using the chimeric human PLAC1 construct, the same intracellular expression pattern was observed.</p> <p>Conclusion: These results indicated that there are some yet unknown PLAC1 localization signals employed by cancer cells for surface expression of PLAC1.</p> https://www.AJMB.org/En/Article.aspx?ID=10402 Jafar Mahmoudian, Mahboobeh Nazari, Roya Ghods, Mahmood Jeddi-Tehrani, Seyed Naser Ostad, Mohammad Hossein Ghahremani, Sedigheh Vafaei, Mohammad Mehdi Amiri, Amir-Hassan Zarnani Wed, 01 Jan 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Poly-Histidine Tag Position toward Inhibition Activity of Secretory Leukocyte Protease Inhibitor as Candidate for Material Wound Healing <p style="text-align:justify"><span style="font-size:9.5pt">Background:</span> The Secretory Leukocyte Protease Inhibitors (SLPI) has many biological functions including anti-bacterial, anti-fungal, anti-viral, anti-inflammatory, and immuno-modulatory. Previous studies have shown that gene-encoding human SLPI have successfully been expressed in <em>Escherichia coli (E. coli)</em> with a C-terminal polyhistidine tag (His-tag). The aim of this research was to investigate the inhibition activity of N-terminal His-tag position (NSLPI) and C-terminal His-tag position (CSLPI). We hypothesized that a His-tag close to an active site SLPI domain may interfere with the inhibition activity of SLPIs.<span style="font-size:2.0pt">&nbsp;</span></p> <p style="text-align:justify"><span style="font-size:9.5pt">Methods:</span> A NSLPI and CSLPI were constructed with polymerase chain reaction (PCR) amplification. The PCR products were then ligated into pET-30a plasmid and transformed into <em>E. coli </em>TOP10. Recombinant plasmids were verified by using restriction analysis and nucleotide sequence analysis. pET-NSLPI and pET-CSLPI were then subcloned in <em>E. coli</em> BL21(DE3) for its expression. The SLPI protein was expressed using Isopropyl &beta;-D-1-thiogalactopyranoside induction (IPTG). The inhibition effect of both SLPI against Porcine Pancreatic Elastase (PPE) enzyme was tested using the N-succinyil-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-4-nitroanalide (NPN) substrate.<span style="font-size:2.0pt">&nbsp;</span></p> <p style="text-align:justify"><span style="font-size:9.5pt">Results:</span> The SLPI gene was successfully cloned and expressed in <em>E. coli</em> BL21. Fusion proteins of NSLPI and CSLPI were generated with His-tag in the N-terminal and C-terminal position, respectively. The inhibition effect of NSLPI and CSLPI on PPE indicated that both SLPI were active. The inhibition activity of NSLPI was 66.7%, higher than CSLPI by 44.4%.<span style="font-size:2.0pt">&nbsp;</span></p> <p><span style="font-size:9.5pt">Conclusion:</span> The His-tag position on the C-terminal of SLPI reduced the inhibition activity of SLPI.</p> https://www.AJMB.org/En/Article.aspx?ID=20413 Elly Munadziroh, Evi Ulfa, Amaliah Labiqah, One Asmarani, Ni Nyoman Puspaningsih Wed, 01 Jan 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments <p>Background: Despite the ease of conventional splicing by overlap-extension (SOEing) PCR technique in theory, when splicing more than two fragments, and especially if one of the complementary sequences is A-T rich, the attachment of the fragments would be challenging. A new rapid and highly efficient SOEing PCR assay was developed for simultaneous splicing of multiple DNA fragments and induction of site-directed mutagenesis in a single tube.</p> <p>Methods: The method was adapted for splicing human beta-globin UTRs to OCT4, SOX2, KLF4, C-MYC, LIN28A, and destabilized GFP for the construction of chimeric DNA fragments for <em>in vitro</em> transcription. In addition, the native Kozak sequence of beta-globin (K1) was replaced by the strongest Kozak sequence (K2) using site-directed mutagenesis to enhance the expression of target genes.</p> <p>Results: ChimericGFPd2/K1, GFPd2/K2, OCT4, and KLF4 were created by the optimized conventional SOEing PCR. The single tube method was able to create the chimeric SOX2, C-MYC, and LIN28A in high quality and quantity in comparison with the conventional SOEing PCR. Moreover, using single tube SOEing PCR, the reaction time and materials that are required in the conventional SOEing PCR were significantly reduced. Fluorescent microscopy and flow cytometry examinations indicated highly efficient translation of K2 sequence in comparison with the K1sequence.</p> <p>Conclusion: Single tube SOEing PCR is a valuable method to construct more multiple fragments with high yield. The method can successfully be applied for construction of various kinds of complex chimeric genes.</p> https://www.AJMB.org/En/Article.aspx?ID=20415 Farzaneh Zarghampoor, Abbas Behzad-Behbahani, Negar Azarpira, Saeed Reza Khatami, Maryam Fanian, Mahdokht Hossein-Aghdaie, Gholam Reza Rafiei Dehbidi Wed, 01 Jan 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Investigation of the Correlation between Androgen Receptor and ZEB1 and its Value in Progression of Gastric Cancer <p><strong>Background:</strong> Zinc-finger Enhancer Binding protein (ZEB1) acts as a transcription factor to promote cancer progression through regulating Epithelial to Mesenchymal Transition (EMT). It is well-known that ZEB1 mRNA expression is directly induced by both Estrogen Receptor (ER) and Progesterone Receptor (PR). Moreover, Androgen Receptor (AR) and PR could bind to the same regulatory element. Since it has been shown that AR overexpresses in Gastric Cancer (GC) as a male-predominant tumor, the goal of this study was to evaluate whether AR could regulate ZEB1 expression in GC.</p> <p><strong>Methods:</strong> The expression profile of ZEB1 in 60 fresh GC and adjacent non-tumor tissues and 50 normal gastric specimens was assessed by qRT-PCR, and the association of ZEB1 expression with clinicopathological features was investigated. Furthermore, possible correlation between ZEB1 and AR was evaluated to elucidate a novel prognostic marker using Kaplan-Meier method and Cox regression model. Finally, molecular interaction of ZEB1 and AR was assessed using a potent AR antagonist in GC cells.</p> <p><strong>Results:</strong> Among GC patients, 70.2% (40/57) overexpressed ZEB1 and 64.91% (37/57) overexpressed AR relative to normal gastric tissues. ZEB1 overexpression was significantly correlated with the AR overexpression in GC patients. Moreover, ZEB1 overexpression was remarkably associated with lower overall survival; however, it was not an independent prognostic factor. Evidence shows that simultaneous evaluation of ZEB1 and AR expression could independently predict survival of GC patients (HR=2.193, p=0.047).</p> <p><strong>Conclusion:</strong> These findings have clinical importance suggesting simultaneous evaluation of ZEB1 and AR expression as a potential prognostic marker. Moreover, AR may regulate ZEB1 expression in GC cells proposing a possible promising targeted therapy for GC patients.</p> https://www.AJMB.org/En/Article.aspx?ID=20419 Shahrzad Soleymani Fard, Masoud Sotoudeh, Kioomars Saliminejad, Mansour Yazdanbod, Habibollah Mahmoodzadeh, Shaghayegh Kouchaki, Marjan Yaghmaei, Seyed Asadollah Mousavi, Reza Malekzadeh, Kamran Alimoghaddam, Seyed Hamidollah Ghaffari Wed, 01 Jan 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of the Bioactive Potential of Secondary Metabolites Produced by a New Marine Micrococcus Species Isolated from the Persian Gulf <p><strong>Background</strong><strong>:</strong> In the present work, a newly isolated marine bacterium, <em>Micrococcus</em> sp. MP76, from coastal area of Persian Gulf around Bushehr province, Iran, was identified with the ability to produce bioactive compounds.</p> <p><strong>Methods:</strong> The pigment production was optimized by changing carbon and nitrogen sources in bacterial growth media at 28<em>&deg;</em><em>C</em> and 220 <em>rpm</em> for 5 days. Partial purification of the pigment was carried out using suitable solvents.</p> <p><strong>Results:</strong> Maximum pigment extract was achieved (1.4 <em>g/l</em>) when cultured in the medi<em>um</em> containing 0.5% (v/v) molasses, 0.5% (w/v) peptone, 1% (w/v) sea salt, 0.01% (w/v) potassium phosphate, and 0.05% (w/v) yeast extract, pH=7.0. Antibacterial effect assessment of the extract against pathogenic bacteria revealed the MIC values in the range of 4.2-7.5 <em>mg/ml</em> depending on different pathogens. The pigment extracted from medium supplemented by molasses and ammonium sulfate had 81% radical scavenging activity, and its IC<sub>50</sub> value was 0.28 <em>mg/ml</em>.</p> <p><strong>Conclusion:</strong> The newly isolated strain of <em>Micrococcus</em> genus from the Persian Gulf revealed a valuable source to access worth medicinal ingredients when cultured under optimized conditions.</p> https://www.AJMB.org/En/Article.aspx?ID=20420 Hamid Reza Karbalaei-Heidari, Mozhdeh Partovifar, Mina Memarpoor-Yazdi Wed, 01 Jan 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Importance of Argan Oil in Human Health According to the Dosage of Antioxidants in the Algerian Argan Fruits (Argania spinosa) https://www.AJMB.org/En/Article.aspx?ID=20421 Zohra Benaouf, Imen Benbahi, Oussama Djorf, Zahira Souidi, Reda Kechairi Wed, 01 Jan 2020 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 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> https://www.AJMB.org/En/Article.aspx?ID=30430 Halimeh Rajabi , Hossein Zolgharnein, Mohammad Taghi Ronagh , Jamshid Amiri Moghaddam , Max Crüsemann Mon, 30 Dec 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir AS-LAMP: A New and Alternative Method for Genotyping <p>In recent decades, different methods have been introduced for the genotyping of Single Nucleotide Polymorphisms (SNPs) and mutations in nucleic acid sequences. These methods have several applications ranging from agriculture to medicine. The Loop-mediated isothermal amplification (LAMP) method was first introduced by Notomi et al. Since then, different methods derived from LAMP have been extensively applied in detecting pathogens. The LAMP method is an isothermal technique that amplifies the target DNA segment using four different primers that have been uniquely designed for recognizing six distinct zones on the objective gene; the process of reaction continues at a constant temperature via a strand displacement reaction. Amplifying and detecting the targeted zone can be accomplished in one stage. Although the LAMP method is mostly used for pathogen detection, several studies have used this method for genotyping. The present article reviewed various studies that used the LAMP method for SNP detection. The outcomes indicated that the LAMP technique could be a reliable and alternative technique for genotyping. Further studies are recommended to use this approach for genotyping.</p> https://www.AJMB.org/En/Article.aspx?ID=20412 Pooria Gill, Arash Hadian-Amree Sat, 28 Dec 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of Immune Response and Protection Induced by V-ATPase Subunit F as DNA Vaccine Against Leishmania tropica (LCED Syrian 01) After Detection and Sequencing <p>Background: Leishmaniasis is one of the major emerging health problems worldwide and <em>Leishmania tropica</em> (<em>L. tropica</em>) is most prevalent in the Middle East due to conflict and environmental factors, and there is no effective prevention strategy available until now. An effective vaccine has not been developed to date. DNA vaccines are considered a promising approach to protect against this infection. In this study, since vacuolar (H+)-ATPase (V-ATPase) enzyme has an essential role in the life cycle of eukaryotes, V-ATPase subunit F gene has been chosen to design DNA vaccine and evaluate its immunogenicity in BALB\c mice.&nbsp;</p> <p>Methods: Genomic DNA was isolated from promastigote culture, synthesized complementary DNA (cDNA) after standardization of Polymerase Chain Reaction (PCR) conditions. The V-ATPase subunit F gene was placed into plasmid PCI. Then, recombinant plasmids were transformed into competent cells. Cloning was confirmed by PCR, restriction enzyme assays, and finally, DNA sequence analysis, after making miniprep from positive colonies and finally the gene was sequenced. BALB/c mice were immunized subcutaneously three times at an interval of two weeks with designed vaccine. BALB\c mice were challenged with 106 promastigotes of <em>L. tropica </em>7 days post-immunization. IL-12, IFN-&gamma; and IL-4 were quantified by RT-qPCR.&nbsp;</p> <p>Results: The present study proved the existence of subunit F gene in Syrian strain of <em>L. tropica </em>(LCED Syrian 01) promastigotes genome. Its expression was also proved in these parasites and the gene length was 414 <em>bp</em>.&nbsp;</p> <p>Conclusion: This study showed that vaccination of BALB\c mice with this gene induced partial protection against Leishmania by reduction of lesion size by 41.9% and parasite burden reduction by 3-log in the dLNs when compared with control group. IFN-&gamma;\IL-4 was 1.6 after challenge test, so the immune response consisted of both Th1 and Th2.</p> https://www.AJMB.org/En/Article.aspx?ID=10397 Amira Orabi, Mohammad Maarouf, Mustafa Alammori Sat, 28 Dec 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Protein Subcellular Mislocalization in Human Cancers <p>Several sets of proteins exist in eukaryotic cells wherein accurately localized in appropriate cellular locations containing plasma membrane, cytoplasm, nucleus and different membrane-enclosed organelles. Each protein is involved in distinct biochemical processes within the normal cell; therefore, the correct subcellular localization of protein is vital; as it provides the physiological context for protein function. However, protein mislocalization described as changing the appropriate subcellular localization of protein, was reported as a key feature of many proteins in a variety of human cancers <sup>1</sup>. Mislocalization has important implications in alteration of activation condition, biological function and interaction network of a protein.&nbsp;Particularly,&nbsp; the aberrant localization of tumor-suppressor proteins and proto-oncoproteins can alter their functions, respectively in either suppressing or supporting the cancer initiation in normal cells whereby cancer development, metastasis and drug resistance are increased <sup>2</sup>.&nbsp;</p> <p>The mechanisms by which subcellular mislocalization is arising in cancer are various and deeply reviewed by Wang and Li <sup>2</sup>. They can be described as modification of signals directing proteins into a particular location, dysregulation of sorter and transporter machinery, Endoplasmic Reticulum (ER) retention of misfolded proteins, aberrant endocytosis and vesicular trafficking, dysregulation of signal transduction and protein post-translational modification and so on <sup>2</sup>.</p> <p>The aberrant subcellular position of proteins in cancer tissues has been investigated broadly by antibody-based strategies including Immunohistochemistry (IHC). The major localization of sortilin on cell surface of ovarian carcinoma tissues rather than the main resident in ER-Golgi compartment of normal tissues was detected using IHC technique <sup>3</sup>. The new method of Dissociable Antibody Microarray (DAMA) combining the power of IHC staining with protein microarray, provides the facility of high-throughput detection of protein subcellular localization <sup>4</sup>.</p> <p>The property of malignant cells in differently subcellular localization of proteins might be recruited as an intelligent strategy for detection of malignant but not normal cells in clinical diagnostic and prognostic applications <sup>5</sup>.&nbsp; For instance, fibromodulin, a type of proteoglycan resides in extracellular matrix, has been irregularly located on cell surface of Chronic Lymphocytic Leukemia (CLL) cells but not in normal samples by which a new CLL diagnostic biomarker appropriate for cell surface flow cytometric detection&nbsp; might be suggested <sup>6</sup>. Moreover, directly targeting of locations in where proteins are accumulated in cancer cells plus selecting the structural dissimilarities of proteins by biologic weapons help us to specifically eradicate malignant but not normal cells.</p> <p>In conclusion, the subscellular mislocalization of proteins is a frequent event in cancers defined as an accessory approach for proliferation, survival and invasion of tumors. However, targeting and trapping proteins in specific cellular compartments has been conceptualized as a promising approach for diagnostic, prognostic and therapeutic clinical use.</p> https://www.AJMB.org/En/Article.aspx?ID=20411 Fatemeh Ghaemimanesh Mon, 23 Dec 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir HR9: An Important Cell Penetrating Peptide for Delivery of HCV NS3 DNA into HEK-293T Cells <p>Background: The delivery of exogenous genes into cells for functional expression is required for development of DNA vaccine and gene therapy in medicine and pharmacology. Cell Penetrating Peptides (CPPs) were considered to mediate gene and drug delivery into living cells. In this study, an attempt was made to evaluate the efficiency of an arginine-rich CPP, HR9, in HCV NS3 gene delivery compared to TurboFect cationic polymer and supercharged +36 GFP into HEK-293T cells.</p> <p>Methods: The recombinant pEGFP-NS3 was constructed and their accuracy was confirmed by digestion and sequencing. Then, the recombinant plasmid was transfected into HEK-293T cells by TurboFect, +36 GFP and HR9 gene delivery systems. The expression of NS3 protein was assessed by fluorescent microscopy, flow cytometry and western blotting.</p> <p>Results: Our data indicated that HR9 peptide was able to form stable complexes with plasmid DNA and increased its delivery into HEK-293T cells in a non-covalent manner. Furthermore, treatment of cells with HR9 and HR9/DNA complexes resulted in a viability of 90-95% indicating this CPP was not cytotoxic. The analysis of zeta potential and size showed the importance of interactions between positively-charged HR9/pEGFP-NS3 complexes and negatively-charged plasma membranes.&nbsp;&nbsp;</p> <p>Conclusion: The non-toxic HR9 CPP can be considered an effective carrier for delivering plasmid DNA harboring <em>Hepatitis C </em>virus (HCV) gene in therapeutic vaccine design.</p> https://www.AJMB.org/En/Article.aspx?ID=20416 Sina Alizadeh, Shiva Irani, Azam Bolhassani, Seyed Mehdi Sadat Mon, 23 Dec 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Repositioning Drugs for Psychiatry <p>Drug development can be time-consuming and expensive. Recent estimates suggest that, on average, it takes 10 years and at least $1 billion to bring a drug to market. Since last decade, 30-40% of drugs or biologics that were approved or launched for the first time in the US were either drugs repositioned for new indications, reformulations or new combinations of existing drugs. This is the lifecycle business with repositioning as a major contributor, and it is rarely given much attention outside of its practitioners <sup>1,2</sup>.</p> <p>In general, drug repurposing or drug repositioning alludes to the development of existing drugs or pro-drugs for new indications, not necessarily related to the original disease focus. These drugs have probably failed in late-stage clinical trials by lacking in efficacy or safety, or have problems associated with commercial strategies, patent expiration or geographic expansion. Repositioning existing drug substances for the treatment of different indications can significantly reduce the cost and time required for the development of new medicines. Therefore, drug repurposing brings forth the benefit of quickening patient access to innovative and effective treatment at lower risk and development cost for the industry <sup>1,2</sup>.</p> <p>There are a number of different definitions of drug repurposing. All of&nbsp;them contain two&nbsp;key elements:</p> <p>Taking&nbsp;existing scientific or medical knowledge and technology that is &quot;approved&quot; for human use in&nbsp;one disease or condition; and</p> <p>Applying this knowledge and technology to another disease or condition.</p> <p>Aspirin, a drug that&rsquo;s been in use in some form or other for many hundreds of years was originally employed, and indeed still is, as a mild pain-relieving analgesic. But it&rsquo;s probably more commonly used today as an antiplatelet agent helping to prevent blood clotting that can occur in thromboembolic disease.</p> <p>Nervous system diseases represent a major health concern worldwide. Although important financial and professional investment, their etiology and pathophysiology still remain mostly elusive. Moreover, the clinical need of disease-modifying therapies is still unmet. In medicine in general and in psychiatry in particular, repositioning was the result of serendipitous but astute clinical observation of an unexpected benefit or expected or unexpected adverse effects <sup>3</sup>.&nbsp;A number of old drugs have been reintroduced for psychiatric indications such as celecoxib for schizophrenia, tamoxifen for mania and scopolamine for depression <sup>4-7</sup>. Drug repurposing has become a new business segment for the life science services industry. In conclusion, drug repurposing emerges as a new value proposition for the industry, patients and payers.</p> https://www.AJMB.org/En/Article.aspx?ID=20422 Shahin Akhondzadeh Mon, 23 Dec 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Microbiome and Schizophrenia <p>Schizophrenia is a debilitating psychiatric disorder that contributes to a large cascade of emotional, occupational, and cognitive impairments. Treatment involves combination of psychosocial rehabilitation and pharmacotherapy. In most cases, chronic antipsychotic therapy is required to treat symptoms, avoid relapse and attenuate episode recurrence <sup>1-3</sup>. Despite the growing number of pharmacologic agents for the treatment of schizophrenia, many patients do not adequately benefit from or tolerate currently available antipsychotics 1-3. Existing typical and atypical antipsychotic medications are relatively equally effective in treating what are known as the positive symptoms of schizophrenia. What has been prominently lacking, however, is an agent that also treats the negative symptoms as well as substantial cognitive impairment 1-3. Despite growing numbers of antipsychotic drugs for the treatment of schizophrenia, the management of this disorder remains to be a major challenge. Therefore, there is a need to find new strategies to improve treatment plans for schizophrenia patients. New studies have found that people with schizophrenia have differences in their gut biomes compared to people without the mental disorder <sup>4,5</sup>. The researchers found a smaller subset of bacteria that were clearly different between schizophrenia patients and those without the disorder. They report that when they introduced samples of the subset from the schizophrenia patients into the biomes of healthy mice, the mice displayed behavior changes<sup> 6,7</sup>. The researchers claim that their results show that people with schizophrenia have differences in their gut biomes and that those differences may be associated with schizophrenia symptoms. They suggest that certain bacteria in the biome may be associated with schizophrenia-related symptoms due to interactions with microbiota gut-brain amino acids, and possibly lipid metabolic pathways. In conclusion, researchers have started to find interesting links between the naturally occurring bacteria that live in our guts, and things we&rsquo;ve traditionally attributed to the brain. Things like our mood, feelings, and even thoughts <sup>8</sup>.&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=20402 Shahin Akhondzadeh Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Sortilin as a Novel Diagnostic and Therapeutic Biomarker in Chronic Lymphocytic Leukemia <p>Background: The overexpression of sortilin/neurotensin receptor 3 has previously been reported in various human solid tumors but not in hematological malignancies. Here, we report the overexpression of sortilin in leukemic cells from patients with Chronic Lymphocytic Leukemia (CLL).<br /> Methods: Flow cytometry was used to compare the expression of sortilin in CLL pa-tients (n=52) and healthy individuals (n=26). Also, in vitro apoptosis induction was assessed in CLL Peripheral Blood Mononuclear Cell (PBMCs) following directly targeting of sortilin.<br /> Results: The results showed a significant expression of sortilin on the surface of CLL PBMCs (range from 2.2 to 71.5%) in comparison to healthy individuals (range from 0.03 to 7.4%) (p&le;0.0001). The optimal cut-off value of sortilin expression was deter-mined at 7.2% with high sensitivity and specificity. Treatment of leukemic cells with anti-sortilin antibody could induce apoptosis without any effect on normal cells.<br /> Conclusion: Apoptosis induction in CLL cells together with a significant correlation between the expression of sortilin and CD23 represent a possible functional role of sortilin in leukemogenesis of CLL cells. Therefore, sortilin might be considered as a promising novel biomarker in diagnosis, monitoring, and therapy of patients with CLL.</p> https://www.AJMB.org/En/Article.aspx?ID=10387 Lia Farahi, Fatemeh Ghaemimanesh, Saeideh Milani, Seyed Mohsen Razavi, Mohammad Mehdi Akhondi, Hodjattallah Rabbani Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Design and Fabrication of a Novel Microfluidic System for Enrichment of Circulating Tumor Cells with the Assistance of Computer Simulations <p>Background: Cancer is the first cause of death in developed countries. The heteroge-neous nature of cancer requires patient-specified treatment plans. One reliable ap-proach is collecting Circulating Tumour Cells (CTCs) and using them for prognosis and drug response assessment purposes. CTCs are rare and their separation from normal cell requires high-accuracy methods.<br /> Methods: A microfluidic cell capture device to separate CTCs from peripheral blood is presented in this study. The CTC separation device applies hydrodynamic forces to categorize cells according to their sizes. The proposed device is designed and evaluated by numerical simulations and validated experimentally. The simulation modified design was fabricated by soft lithography which allows prototyping the device in a few hours. For experimental setup two solutions: 1) fixed cells spiked in Phosphate Buffered Saline (PBS), and 2) fixed cells in blood were used. The CTC separation device was validated by tracking the flow and separation of cancer cell lines in the solutions.<br /> Results: It is demonstrated that the setup is capable of CTC enrichment up to 50 times.<br /> Conclusion: The presented CTC enrichment method reduces costs by eliminating the use of antibodies. The high-throughput method has the potential to be used in pre-clinical studies of cancer.</p> https://www.AJMB.org/En/Article.aspx?ID=10396 Dina Dorrigiv, Manouchehr Vossoughi, Iran Alemzadeh Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Anti-Influenza Virus Activity and Phenolic Content of Pomegranate (Punica granatum L.) Peel Extract and Fractions <p>Background: Influenza virus, associated with high level of morbidity and mortality, has been recently considered a public health concern while the choices for the control and treatment of the disease are limited. The present study was conducted to evaluate activity of pomegranate peel extract and its fractions against <em>Influenza A</em> virus <em>in vitro</em>.&nbsp;<br /> Methods: In this research, ethyl alcohol extract of pomegranate peel was prepared and subjected to fractionation with different polarities. The potential <em>in vitro</em> anti-influenza A virus activity of the extract and fractions was assessed using Cytopathic Effect (CPE) reduction assay, Hemagglutinin Assay (HA), and 50% Tissue Culture In-fectious Doses (TCID<sub>50</sub>) method in Madin-Darby Canine Kidney (MDCK) cells.<br /> Results: The crude pomegranate peel extract and its n-butanol and ethyl acetate fractions had the highest inhibitory effect against influenza A virus with IC<sub>50</sub> value of 6.45, 6.07 and 5.6 <em>&mu;g/ml</em> in MDCK cells, respectively. Our results also showed that, the production of virus was significantly reduced upon treatment with crude extract, n-butanol and ethyl acetate fractions in a dose-dependent manner (p&lt;0.05).<br /> Conclusion: Based on our results, the ethyl alcohol extract and its polar fractions of pomegranate peel can inhibit influenza A virus replication<em> in vitro</em>. Therefore, further characterization of its active ingredients and the mechanism of action should be carried out.&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=10395 Mohammad-Taghi Moradi, Ali Karimi, Mehrdad Shahrani, Leila Hashemi, Mohammad-saleh Ghaffari-goosheh Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir G2 Dendrimer as a Carrier Can Enhance Immune Responses Against HCV-NS3 Protein in BALB/c Mice <p>Background: Hepatitis C virus (HCV) infection is a major issue of public health. It seems of paramount importance to find an effective vaccine against HCV infection. The best vaccine candidate should induce robust cellular responses. The aim of the current study was to evaluate immunogenicity effects of novel conjugated dendrimer G2 with the recombinant NS3 antigen as a vaccine candidate for eliciting Th1-oriented cellular responses.<br /> Methods: Female BALB/c mice were immunized with different regimes especially with NS3 conjugated with G2 dendrimer. The humoral responses (Total IgG and IgG isotyping) and cellular responses (<em>Ex vivo</em> IFN-&gamma; and IL-4 ELISpot assays, <em>in vitro </em>CTL assay and proliferation) were evaluated and compared in immunized mice.<br /> Results: The results indicated that induced specific total IgG in all mice groups im-munized with rNS3 formulated with different adjuvants and IgG2a subclass was the predominant isotype in rNS3-G2 (p&le;0.05). For preliminary evaluation of cellular re-sponse, <em>ex vivo</em> ELISpot assay has shown that the higher frequency of IFN-&gamma; producing cells was in groups immunized with rNS3+M720 and rNS3-G2 (p= 0.0012) than control groups. Finally, the rNS3-specific CTLs activity showed the highest percentage of specific lysis (LDH release) of the target cells in rNS3-G2 and rNS3+M720 groups.&nbsp;<br /> Conclusion: In the present study, as our knowledge, this is first time that the immunogenicity of nanodendrimer G2 as a biocompatible adjuvant with the HCV-NS3 antigen was evaluated. The results showed high capability of the regimen to induce strong Th1-orinted cellular response in mice model, indicating the dendrimer G2 as a novel adjuvant candidate for HCV vaccine studies.&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=10398 Foozieh Javadi, Pooneh Rahimi, Mohammad Hossien Modarresi, Azam Bolhassani, Mehdi Shafiee-Ardestani, Seyed Mehdi Sadat Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Metabolomic Study to Identify Potential Tissue Biomarkers for Indomethacin-Induced Gastric Ulcer in Rats <p>Background: Gastric Ulcer (GU) is the most prevalent gastrointestinal disorder in-duced by various factors and Non-Steroid Anti-Inflammatory Drugs (NSAIDs) as one of the most common reasons. Due to the absence of appropriate molecular markers for GU, the aim of this study was to utilize a metabolomics approach in order to find potential metabolite markers for the disease.&nbsp;<br /> Methods: Stomach tissue samples from indomethacin-treated rats and normal con-trols were used to perform a 1H-NMR metabolomics study. The altered metabolites were identified using random forest multivariate analysis.<br /> Results: ROC curves showed that the random forest model had a good predictive performance with AUC of 1 for the test and 0.708 for the training sets. Seventeen differentially expressed metabolites were found between GU and normal tissue sample. These metabolites included trimethylamine, betaine, carnitine, methionine, acetylcho line, choline, N,N-Dimethylglycine, cis-aconitate, tryptophan, spermidine, acetylcar-nitine, creatinine, pantothenate, taurine, isoleucine, glucose and kynurenine.<br /> Conclusion: The results of the study demonstrated that metabolomics approach could serve as a viable method to find potential markers for GU. Surely, further studies are needed for the validation of the results.</p> https://www.AJMB.org/En/Article.aspx?ID=10390 Reyhaneh Farrokhi-Yekta, Nasrin Amiri-Dashatan, Mehdi Koushki, Masoomeh Dadpay, Fatemeh Goshadrou Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Menstrual Blood Stem Cell Transplantation in Mice Model of Acute Liver Failure: Does Gender of Recipient Affect the Outcome? <p>Background: There exists a dramatic rise in liver failure and numerous patients un-dergo liver transplant for life-saving reasons annually. Introducing alternatives to allograft transplantation is necessary due to present limitations. Recently, a noninvasive stem cell population from Menstrual blood-derived Stem Cells (MenSCs) has been identified. There is an increasing interest in the application of MenSCs in tissue engineering; however, the fact that these gender-specific stem cells are safe for use in male sex is still not well defined.<br /> Methods: In this research, a model of acute liver failure was created in male and fe-male immunocompetent Balb-C mice through intraperitoneal injection of Carbon tetrachloride (CCl4) and MenSCs were transplanted intravenously 48 hrs after induc-tion of liver injury to evaluate their therapeutic potential. All mice were sacrificed on days 1, 7, and 30 post-transplantation to examine biochemical and molecular markers and pathological appearances.<br /> Results: Results showed the liver engraftment of MenSCs by immunofluorescence staining using anti-human mitochondrial antibody in both male and female treated groups. The restoration of serum markers of liver injury, aspartate aminotransferase and alanine aminotransferase, as well as expression levels of liver-specific genes, tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase, were more significant in the female treated group compared with the male treated group on day 7 (p&lt;0.05); however, after 30 days, there were no significant differences. Furthermore, hematoxylin and eosin and periodic acid-Schiff staining of liver sections demonstrated the considerable liver regeneration post cell therapy in both groups. Notably, data has shown that MenSCs could engraft into injured liver tissues and result in the same effect in the regeneration of liver function in both genders.&nbsp;<br /> Conclusion: Results of this study introduce MenSCs therapy as an attractive alterna-tive approach for liver repairing and regeneration which has no gender constraints.</p> https://www.AJMB.org/En/Article.aspx?ID=10391 Mina Fathi-Kazerooni, Gholamreza Tavoosidana Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Sodium Butyrate on <I>LHX1</I> mRNA Expression as a Transcription Factor of HDAC8 in Human Colorectal Cancer Cell Lines <p>Background: LHX1 is an important transcription factor for the <em>HDAC8</em> gene. The aim of this study was to investigate the effect of Sodium Butyrate (SB), as a histone deacetylase inhibitor, on the expression of <em>LHX1</em> gene in colorectal cancer cell lines.&nbsp;<br /> Methods: HT-29 and HCT-116 cell lines were treated with 6.25 to 200 <em>mM</em> concentrations of SB at 24, 48, and 72 <em>hr</em>. The cytotoxicity effect on cell viability was evaluated by MTT assay. The 50% Inhibiting Concentration (IC<sub>50</sub>) was determined graphically. Quantitative real-time PCR was performed to investigate the LHX1 mRNA expression level.&nbsp;<br /> Results: Our study revealed that SB inhibited the proliferation of these cell lines in a concentration and time-dependent manner. The IC<sub>50</sub> values for HT-29 cell line were 65, 18.6, and 9.2 <em>mM</em> after 24, 48, and 72<em> hr</em> of treatment, respectively. The IC<sub>50</sub> values for HCT-116 cell line were 35.5, 9.6, and 10 <em>mM </em>after 24, 48, and 72 <em>hr </em>of treatment, respectively. Furthermore, real-time PCR findings demonstrated that the LHX1 mRNA expression in treated HT-29 cell line significantly increased in comparison with untreated cells (p&lt;0.05). However, in treated HCT-116 cell line, SB led to a significant decrease in the level of LHX1 mRNA (p&lt;0.05), as compared to untreated cells.&nbsp;<br /> Conclusion: In this study, different effects of SB on LHX1 mRNA expression level were revealed in two distinct human colorectal cancer cell lines.</p> https://www.AJMB.org/En/Article.aspx?ID=10389 Mahsa Ghiaghi, Flora Forouzesh, Hamzeh Rahimi Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Interleukin 10 and Transforming Growth Factor Beta Polymorphisms as Risk Factors for Kawasaki Disease: A Case-Control Study and Meta-Analysis <p>Background: Alteration in serum expression of Transforming Growth Factor-beta (TGF-&beta;) and IL-10 have been suggested to play a role in the pathogenesis of Kawa-saki Disease (KD). Inconsistent reports exist on the association of IL-10 polymorphisms with KD susceptibility and Coronary Artery Aneurysms (CAA).&nbsp;<br /> Methods: A number of 110 paediatric patients with KD and 140 healthy individuals were recruited to investigate the frequency of Single Nucleotide Polymorphisms (SNPs) of TGF-&beta; C/T at codon 10 (rs1982073), C/G at codon 25 (rs1800471) and IL-10 A/G at -1082 (rs1800896), C/T at -819 (rs1800871) and A/C at -592 (rs1800872) and their respective genotype and haplotypes. A comprehensive search was performed in MEDLINE and SCOPUS using the keywords of interleukin 10, transforming growth factor beta, and Kawasaki disease. Moreover, previous studies investigating the TGF-&beta; and IL-10 polymorphisms in KD were evaluated. Review Manager Version 5.1 Software was used to perform meta-analysis.<br /> Results: There was no significant association between allelic or genotypic variants in the mentioned polymorphisms in TGF-&beta; or IL-10 with KD or CAA. The only significant haplotypic variant was TC variant at codon 10, and 25 of TGF-&beta; polymorphisms were associated with higher risk of KD. Meta-analysis of a total number of 770 patients vs. 1471 healthy controls showed no difference in the frequency of any of the IL-10 genetic variants in KD patients, regardless of the presence of CAA.<br /> Conclusion: Polymorphisms of TGF-&beta; or IL-10 are not associated with additional risk for KD in Iranian population. IL-10 polymorphisms at -1082, -819 and -592 positions are not associated with KD, nor do they predict coronary artery aneurysm formation.</p> https://www.AJMB.org/En/Article.aspx?ID=10392 Farzaneh Rahmani, Vahid Ziaee, Raheleh Assari, Maryam Sadr, Arezou Rezaei, Zeinab Sadr, Seyed Reza Raeeskarami, Mohammad Hassan Moradinejad, Yahya Aghighi, Nima Rezaei Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Integrity and Quantity Evaluation of Plasma Cell-Free DNA in Triple Negative Breast Cancer <p>Background: Triple-Negative Breast Cancer (TNBC) is a subtype of breast cancer that lacks expression of the estrogen and progesterone receptor and does not overexpress human epidermal growth factor 2 receptor protein. TNBC is associated with special characteristics, including aggressiveness, poor prognosis, and treatment response. Non-invasive blood-based molecular markers such as cell-free DNA (cfDNA) variables have been shown to be putative markers in breast cancer prognosis.&nbsp;<br /> Methods: The cfDNA quantity and integrity were assessed in a case-control study of 96 breast cancer patients including 46 triple negative and 50 non-triple negative compared with 50 unaffected controls. A quantitative real-time PCR approach based on the quantification of two amplicons of the &beta;-actin gene with different lengths (99 and 394 bp) was used to evaluate the integrity index 394/99.&nbsp;<br /> Results: Both cfDNA integrity index and quality were significantly elevated in breast cancer patients but integrity index can be considered as the more reliable diagnostic marker. The statistically significant increase of cfDNA quantity and integrity was ob-served in TNBC patients, somehow associated with nodal metastasis (p&lt;0.001).&nbsp;<br /> Conclusion:&nbsp;Elevated cfDNA concentration and integrity index in breast cancer pa-tients compared with normal control and significant difference observed between TNBC and non-TNBC may be considered as a possible effective non-invasive diagnostic and prognostic molecular marker in breast cancer.</p> https://www.AJMB.org/En/Article.aspx?ID=10394 Mahdieh Salimi, Somayeh Sedaghati-Burkhani Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Importance of Fluctuating Amino Acid Residues in Folding and Binding of Proteins <p>Background: Conformational flexibility of proteins remains as one of the major events in protein-protein/DNA/ligand/small molecule binding to achieve its biological function in the cell. The availability of high-resolution structures of protein complexes is a valuable resource for researchers to understand the mechanisms behind such interactions and it is found that the flexibility of amino acid residues at binding sites is crucial for many important functions in the cell.<br /> Methods: In this article, our statistical method (PreFRP) developed based on fluctuating amino acid residues and various amino acid indices related to flexibility/rigidity were used to study the importance of fluctuating amino acid residues in thermonuc-leases from pathogenic bacteria, cell penetrating peptides and intrinsically disordered proteins responsible for many neural disorders.&nbsp;<br /> Results: The results from our analysis reveal the importance of fluctuating amino acid residues in folding and binding of proteins. The role of moderate and high fluctuating residues in themonucleases, cell penetrating peptide and disordered regions are discussed in detail.<br /> Conclusion: Therefore, our analysis will help in understanding the importance of fluc-tuating amino acid residues in proteins which undergo a conformation change phenomenon.</p> https://www.AJMB.org/En/Article.aspx?ID=10393 Renganathan Senthil, Singaravelu Usha, Konda Saravanan Wed, 25 Sep 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Depression and Inflammation: Is There any Role for Biomarkers? <p>Despite the advent of several antidepressant medications, treatment of Major Depressive Disorder (MDD) is still far from optimal <sup>1-3</sup>.&nbsp; A large proportion of patients with MDD do not respond to their first medication. To achieve favorable response, these patients are generally treated with either switching to another treatment or augmentation therapy. In the recent decade, several augmentative strategies for treatment of MDD have been developed. Some of these treatment modalities focus on recently developed hypotheses of pathophysiological processes in patients with MDD <sup>1-3</sup>. These mainly include immune system dysfunction, hypothalamic-pituitary-adrenal (HPA) axis and metabolic derangements, impaired neuroprotection, or neuroinflammation <sup>1-3</sup>.<br /> Growing body of evidence suggests that inflammation is implicated in the pathophysiology of MDD 4-6. Sickness be-havior which is a result of inflammatory activation, shares many clinical features such as anhedonia, anorexia, irritability, and mild cognitive problems with MDD <sup>4-6</sup>. Several studies have shown an elevation of proinflammatory cytokines [particularly IL-6 and Tumor Necrosis Factor (TNF-&alpha;)] in patients with MDD <sup>7</sup>. A large body of research now suggests that depression is associated with a low-grade, chronic inflammatory response and is accompanied by increased oxidative stress.&nbsp;<br /> &bull; depression frequently is comorbid with many inflammatory illnesses&nbsp;<br /> &bull; increased inflammatory biomarkers are associated with major depressive disorder (MDD)&nbsp;<br /> &bull; exposure to immunomodulating agents may increase the risk of developing depression&nbsp;<br /> &bull; stress can activate proinflammatory pathways&nbsp;<br /> &bull; antidepressants can decrease inflammatory response&nbsp;<br /> &bull; inhibition of inflammatory pathways can improve mood <sup>4-7</sup>.<br /> IL-6 is one of the most widely studied cytokines in patients with MDD <sup>8,9</sup>. In addition to elevation of this cytokine in patients with MDD, relation of IL-6 concentration to severity of depression, a shift in circadian rhythm <sup>8,9</sup>, and a reduction in its concentration in response to antidepressants have been shown in several studies.&nbsp;&nbsp;<br /> Previous studies have already shown that elevated levels of inflammation are associated with poor response to antidepressants. The scientists found that they could pinpoint a threshold and precisely predict which patients would respond to conventional antidepressants. None of the patients with MIF and IL-1&beta; levels above the threshold responded to the antidepressants most often prescribed. Those with inflammation levels below the threshold would likely respond. One reason for the lack of predictive biomarkers in MDD is that little is known with absolute certainty about how antidepressants improve mood. All currently approved medications for depression act in a similar way, increasing the availability of monoamine neurotransmitters like serotonin in the brain. Psychiatrists continue to search for biomarkers to help guide therapy and, potentially, improve chances of discovering new drugs <sup>9</sup>.&nbsp;<br /> In conclusion, the link between depression and the body&#39;s inflammatory response continues getting stronger, with more research showing an ever-tighter correlation.</p> https://www.AJMB.org/En/Article.aspx?ID=20401 Shahin Akhondzadeh Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The p53 Modulated Cytotoxicity of <i>Ophiocoma scolopendrina</i> Polysaccharide Against Resistance Ovarian Cancer Cells <p>Background: Marine environment is a valuable source of bioactive compounds with variable medicinal properties. Previously, it was shown that <em>Ophiocoma erinaceus</em> extracted polysaccharide has prominent cytotoxic effect on HeLa human cervical cancer cells. In the present study, the anti-cancer properties of polysaccharide extracted from <em>Ophiocoma scolopendrina (O. scolopendrina) </em>were examined in comparison with paclitaxel as a conventional drug against resistant ovarian cancer; also, its related mechanism against A2780cp ovarian cancer cells was investigated.&nbsp;<br /> Methods: The A2780cp cancer cells and NIH3T3 normal cells were cultured and treated with different concentrations of polysaccharide extracted from <em>O. scolopendrina</em> for 24 hr and 48 hr. Then, cell toxicity was studied by MTT assay, morphology of cells was observed under inverted microscopy and the type of induced cancer cell death was assessed by annexin V-FITC, propodium iodide and acridine orange staining. Finally, the apoptosis pathway was determined by measurement of caspase-3 and caspase-9 activity and assessment of p53 and Bcl-2. The statistical analysis was performed by SPSS software, one way ANOVA and p&lt;0.05 was considered significant.&nbsp;<br /> Results: Our observations from MTT assay and morphological assessment exhibited that <em>O. scolopendrina</em> isolated polysaccharide inhibited proliferation of ovarian cancer cells with IC<sub>50</sub> of 35 <em>&micro;g/ml</em>, while paclitaxel suppressed tumor cell growth with IC<sub>50</sub>=10 <em>&micro;g/ml</em>. In contrast, MTT observations revealed low cytotoxicity of these chemotherapeutic agents against NIH3T3 normal cells. Also, the analysis correlated with induced cell death elucidated that concurrent treatment of polysaccharide plus paclitaxel had a further anti-cancer effect against A2780cp cells mainly through restoration of p53 and mitochondrial apoptosis cell death induction.&nbsp;<br /> Conclusion: Taken together, our research supports the finding that application of polysaccharide extracted from <em>O. scolopendrina</em> can be considered a promising marine chemotherapeutic approach for advancing efficacy of paclitaxel in treatment of resistant ovarian cancer. Additional <em>in vivo</em> experiments are required to elucidate the role of brittle star polysaccharides in animal and clinical trials.</p> https://www.AJMB.org/En/Article.aspx?ID=10376 Elaheh Amini, Javad Baharara, Mahbube Afzali, Najme Nikdel Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir MIG1 Glucose Repression in Metabolic Processes of <i>Saccharomyces cerevisiae</i>: Genetics to Metabolic Engineering <p>Background: Although <em>Saccharomyces cerevisiae</em> has several industrial applications, there are still fundamental problems associated with sequential use of carbon sources. As such, glucose repression effect can direct metabolism of yeast to preferably anaerobic conditions. This leads to higher ethanol production and less efficient production of recombinant products. The general glucose repression system is constituted by <em>MIG1</em>, TUP1 and SSN6 factors. The role of <em>MIG1</em> is known in glucose repression but the evaluation of effects on aerobic/anaerobic metabolism by deletion of <em>MIG1 </em>and constructing an optimal strain brand remains unclear and an objective to be explored.&nbsp;<br /> Methods: To find the impact of <em>MIG1</em> in induction of glucose-repression, the Mig1 disruptant strain (∆<em>MIG1</em>) was produced for comparing with its congenic wild-type strain (2805). The analysis approached for changes in the rate of glucose consumption, biomass yield, cell protein contents, ethanol and intermediate metabolites production. The <em>MIG1</em> disruptant strain exhibited 25% glucose utilization, 12% biomass growth rate and 22% protein content over the wild type. The shift to respiratory pathway has been demonstrated by 122.86 and 40% increase of glycerol and pyruvate production, respectively as oxidative metabolites, while the reduction of fermentative metabolites such as acetate 35.48 and ethanol 24%.&nbsp;<br /> Results: Results suggest that ∆<em>MIG1</em> compared to the wild-type strain can significantly present less effects of glucose repression.&nbsp;<br /> Conclusion: The constructed strain has more efficient growth in aerobic cultivations and it can be a potential host for biotechnological recombinant yields and industrial interests.</p> https://www.AJMB.org/En/Article.aspx?ID=10377 Iraj Alipourfard, Nelly Datukishvili, Salar Bakhtiyari, Karimeh Haghani, Laura Di Renzo, Renata de Miranda, David Mikeladze Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Phylogenetic Analysis of <i>Hepatitis B</i> Virus among Household Members with HBV Chronic Infection <p>Background: Intrafamilial spread of <em>Hepatitis B virus </em>(HBV) infection in Iran has only been investigated with serological testing without using molecular studies as the most informative and definitive type of analysis.&nbsp;<br /> Methods: In the present study, intrafamilial transmission of HBV among family members of Iranian index HBsAg carriers was investigated using phylogenetic analysis of the S region of the viral genome. Nested polymerase chain reaction was used for detection of HBV DNA in serum samples from 22 index and 43 contact patients with chronic HBV infection. HBV DNA was detected in 37 samples (14 indexes, 23 contacts). The S gene region of the DNA isolates was subjected to direct sequencing and phylogenetic analysis by Bioedit, Mega and Phylip programs.&nbsp;<br /> Results: All isolates (from 26 patients) were clustered with genotype D, of which 24 strains were of subgenotype D1, subtype<em> ayw2</em>, while 2 additional strains were of subgenotype D2, subtype <em>ayw3</em>. Evidence of intrafamilial transmission of the virus was found in 8 families studied phylogenetically. Overall, 60 changes were detected in the amino acid sequences of the surface antigen protein in 23 patients. Four premature stop codons occurred in 3 isolates at residues 69 and 182. Seven out of 8 families displayed 25&minus;100% common amino acid substitutions among their members.&nbsp;<br /> Conclusion: Our data corroborated intrafamilial transmission of HBV, as evidenced by concordant HBV genotype among household members, viral sequence homology and close genetic relatedness of the strains on the phylogenetic tree, and horizontal transmission of S gene mutations among family members.</p> https://www.AJMB.org/En/Article.aspx?ID=10378 Shahnaz Sali, Shirin Azarmmanesh, Hediyeh Ghalikhani, Maryam Vaezjalali Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Recombinant Helicobacter Outer Membrane Protein H (HopH) on Nitric Oxide Production by Peripheral Macrophage in BALB/c Mice <p>Background: Some products of bacteria are reported as an immunomodulator. The <em>Helicobacter pylori (H. pylori) </em>outer membrane proteins play an important role in stimulation of immune system. The present study was performed to determine the in vitro effect of recombinant HopH of <em>H. pylori</em> on Nitric Oxide (NO) production and viability of mouse peritoneal macrophages.<br /> Methods: <em>H. pylori </em>recombinant HopH was produced in this study. Mice peritoneal macrophages were purified and cultured. Different concentrations of recombinant HopH were used for stimulation of macrophages in order to evaluate NO production. The cell viability was detected by MTT assay. NO amounts released in to the supernatants of cultured macrophages and LPS-stimulated macrophages (10 <em>&mu;g/ml</em>) were detected by Griess reagent.<br /> Results: Results demonstrated that the suppressive effect of high concentrations of recombinant HopH on NO release and the stimulation effect of protein was shown in 15 <em>&micro;g/ml</em>, compared to the control group. NO stimulation was significant in all the concentrations of LPS stimulated with HopH groups.<br /> Conclusion: According to our findings, recombinant HopH has a toxic effect in high concentration on cell. So it can be an anticancer candidate.</p> https://www.AJMB.org/En/Article.aspx?ID=10379 Masoumeh Navidinia, Neda Soleimani, Narges Bodagh abadi Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Hydroalcoholic Ginger Extract on Brain HMG-CoA Reductase and CYP46A1 Levels in Streptozotocin-induced Diabetic Rats <p>Background: Patients with diabetes present with lipid disorders, including hypercholesterolemia, which can be a high-risk factor for atherosclerosis. Recently, increasing interest has been focused on anti-lipidemic function of herbal medicines, especially <em>Zingiber officinale</em> (known as ginger), in diabetes. However, the mechanism underlying the effect of ginger on some players involved in cholesterol homeostasis of Central Nervous System (CNS) among diabetic patients remains unclear. To our knowledge, this is the first study to investigate the effect of ginger on brain regulation of Hydroxymethylglutaryl-CoA Reductase (HMG-CoA reductase) and Cholesterol 24-hydroxylase (CYP46A1), which provides a rational model for understanding brain dyslipidemia mechanisms associated with diabetes.&nbsp;<br /> Methods: Brains of rats were isolated from four groups: control, non-treated diabetic, and treated diabetic groups receiving 200 or 400 <em>mg/kg</em> of hydroalcoholic extracts of ginger for eight weeks. HMG-CoA reductase and CYP46A1 levels in brain homogenates were determined by western-blot technique.&nbsp;<br /> Results: Ginger root extract caused a significant decrease in HMG-CoA reductase and an increase in CYP46A1 levels in treated diabetic groups compared to diabetic control. In comparison to diabetic group, these effects were more remarkable with 400 <em>mg/kg </em>concentration of ginger extract.&nbsp;<br /> Conclusion: The findings showed that ginger extract has a regulatory effect on proteins involved in cholesterol homeostasis in CNS by a significant down- and up-regulation of HMG-CoA reductase and CYP46A1 levels, respectively. It can be suggested that adding ginger to daily diet of diabetic patients has useful effects and may ameliorate diabetes complications.</p> https://www.AJMB.org/En/Article.aspx?ID=10380 Shirin Azizidoost, Zahra Nazeri, Asma Mohammadi, Ghorban Mohammadzadeh, Maryam Cheraghzadeh, Alireza Jafari, Alireza Kheirollah Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Investigation of the Association between 5-Hydroxytryptamine Transporter Gene-Linked Polymorphic Region with Type 2 Diabetes Mellitus, Obesity and Biochemical Profiles of Serum in Iranian Population <p>Background: Type 2 Diabetes Mellitus (T2DM) is a serious problem in the world. 5-Hydroxytryptamine (5-HT, serotonin) plays an important role in obesity, glucose control and insulin resistance. The polymorphism of the serotonin transporter gene linked promoter region (5-HTTLPR) might influence 5-HTT expression and serotonin uptake. The polymorphism results in two alleles of L (Long) and S (Short). The aim of the present study was to evaluate the association between 5-HTTLPR genotypes in type 2 diabetes mellitus (T2DM), obesity as well as serum biochemical profiles in Iranian population from 2012 until 2015.<br /> Methods: 180 patients with T2DM and 180 controls were selected and the frequency of S and L alleles was determined by PCR. Then, the relationship between genotypes, body mass index (BMI) and serum biochemical variables was investigated.<br /> Results: The frequency of S and L alleles in experimental and control groups was the same [for the L allele p=0.754, OR (95%CI)=1.103 (0.597 to 2.041) and for the S allele p=0.906, OR (95%CI)=(0.490 to 1.676)]. However, the mean triglyceride, cholesterol, LDL-C, systolic and diastolic blood pressure levels in the diabetic subjects with LL genotype were significantly higher than LS and SS genotypes (p&lt;0.001) in this population.<br /> Conclusion: The L allele of 5-HTTLPR was related to the increased serum lipids and blood pressure in the diabetic patients. However, there was no relationship between the polymorphism of 5-HTTLPR L/S and T2DM in Iranian population.&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=10381 Azizeh Asadzadeh, Hooria Seyedhosseini Ghaheh, Fatemeh Sholehvar, Mohammadali Takhshid, Mohammad Mehdi Naghizadeh Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Sericin Ameliorates the Capacitation State and Chromatin Integrity of Frozen-Thawed Stallion Spermatozoa by Reducing Oxidative Stress <p>Background: In the process of sperm cryopreservation, apart from cryoinjury, the production of Reactive Oxygen Species (ROS) can adversely affect the integrity of chromatin and cellular membranes. Addition of natural antioxidants to freezing me-dium is an approach to reduce the destructive effects of ROS on sperm.&nbsp;<br /> Methods: In this study, during 60 <em>min</em> of cooling process, the ejaculates of five stallions were diluted in the following media: INRA 82 medium as Control (C), INRA 82 medium supplemented with 0.25% Sericin (S), INRA 82 medium supplemented with 1.5 <em>mM</em> Glutathione (G), and INRA 82 medium supplemented with 0.25% Sericin+1.5 <em>mM</em> Glutathione (S+G).<br /> Results: In the frozen/thawed sericin supplemented group, while the integrity of DNA and the activity of catalase and Glutathione Peroxidase (GPx) were increased, the lipid peroxidation and midpieceab normality decreased, compared with other groups (p&lt;0.05). The proportions of sperms with abnormal head in group S and the sperm with distal droplet in G and S+G groups decreased, compared with group C (p&lt;0.05). In CTC assay, the percentage of capacitated spermatozoa in treatment groups was lower than control (p&lt;0.01).&nbsp;<br /> Conclusion: In conclusion, the presence of sericin in freezing medium of stallion semen could improve sperm DNA integrity and its resistance to ROS and lipid peroxidation.</p> https://www.AJMB.org/En/Article.aspx?ID=10382 Mahboobeh Heidari Nasirabadi, Abolfazl Shirazi, Ali Kadivar, Naser Shams-Esfandabadi, Abdolnaser Mohebbi, Ebrahim Ahmadi Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Whole Exome Sequencing of an X-linked Thrombocytopenia Patient with Normal Sized Platelets <p>Wiskott-Aldrich Syndrome (<em>WAS</em>) is a rare X-linked recessive Primary Immunodeficiency (PID) caused by mutations in <em>WAS</em> gene which encodes a protein known as WASp. WASp plays important roles in cytoskeletal functions that compromise multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion and directed migration. WASp defect particularly causes platelets abnormality which is presented in forms of decrease of Mean Platelet Volume (MPV) and thrombocytopenia in most <em>WAS</em> conditions; nevertheless, some studies reported WAS patients with a normal or large size of platelets in recent years. This phenomenon is unique and the exact mechanism of thrombocytopenia with a normal or large size of platelets is still unknown. In this study, Next Generation Sequencing (NGS) was utilized to discover the causing mutation in WAS gene; furthermore, an attempt was made to evaluate the possibility of other mutations or genes especially WASp interacting proteins and inherited platelet disorder genes in patient clinical symptoms for the purpose of understanding the origin of such unique symptom and to perform further analysis if it is required. Therefore, clinical manifestations and immunologic functions of the patient were checked and Whole Exome Sequencing (WES) was performed to analyze all exonic variations which can be associated with patient phenotypes. Finally, a novel de novo mutation in <em>WAS</em> gene which truncates WASp to half of its normal size was determined as the only cause of clinical manifestation.</p> https://www.AJMB.org/En/Article.aspx?ID=10383 Majid Fathi, Hojat Shahraki, Edris Sharif Rahmani, Hamzeh Rahimi, Pouria omidi, Saeedeh Darvishi, Mohammad Foad Abazari, Arshad Hosseini Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>In Vitro</i> Pre-validation of Gene Editing by CRISPR/Cas9 Ribonucleoprotein <p>Background: The CRISPR/Cas9 genome editing system is a powerful and simple gene editing method. The format of the CRISPR components is one of the important factors in targeting efficiency. Compared to plasmid or mRNA (IVTs) format, using the CRISPR/Cas9 system as Cas9&ndash;crRNA&ndash;tracrRNA RNP format is more efficient and rapid, especially in minimizing some of the pitfalls of CRISPR-mediated gene editing. In addition to efficient <em>in vivo</em> applications of the CRISPR RNP format in a variety of cell types and organisms, another advantage of this approach is usability for <em>in vitro</em> applications in which the crRNAs in the tracrRNA&ndash;crRNA structure guides the Mg2+-dependent RNAdirected DNA endonuclease to introduce double-strand breaks at specific sites in DNA.<br /> Methods: Here, Cas9&ndash;crRNA&ndash;tracrRNA RNP system was used to test the designed crRNAs for <em>in vitro</em> DNA cleavage by Cas9 protein in RAG1, RAG2 and IL2RG genes.&nbsp;<br /> Results: The results of cleavage reveal theCas9&ndash;crRNA&ndash;tracrRNA RNP system is a rapid and efficient way to pre-validate the efficiency of CRISPR cleavage with crRNAs designed for RAG1, RAG2 and IL2RG genes.<br /> Conclusion: one step <em>in vitro</em> cleavage of DNA by CRISPR/Cas9 ribonucleoprotein complex can be used to pre-validate the functionality and relative efficiency of CRISPR system for targeting genes.</p> https://www.AJMB.org/En/Article.aspx?ID=10384 Maryam Mehravar, Abolfazl Shirazi, Mohammad Mehdi Mehrazar, Mahboobeh Nazari Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning and Expression of <i>B. mellitensis bp26</i> Gene in <i>Lactococcus lactis</i> as a Food Grade Vaccine <p>Background: Brucellosis is still an important health problem in under developing countries and researches for finding efficient vaccine are going on. <em>Brucella melitensis (B. mellitensis) bp26</em> gene is a good candidate for brucellosis vaccine and investigations showed that<em> Lactococcus lactis (L. lactis)</em> with several positive characteristic are attractive for protein expression as a live delivery vectors. These fast growing bacteria need no aeration, are easy to handle, have no exotoxin, endotoxin and protease, so the cost of culturing is inexpensive.<br /> Methods: <em>B. mellitensis</em> <em>bp26</em> gene was cloned in food grade pNZ 8149 vector and expressed in <em>L. lactis</em> NZ 3900.<br /> Results: Results showed that we can produce a food-grade recombinant <em>L. lactis </em>producing the <em>B. melitensis </em>BP26 protein.&nbsp;<br /> Conclusion: In this study, for Future evaluation about ability of L. lactis as a live delivery vector, a food-grade recombinant <em>L. lactis</em> producing the <em>B. melitensis</em> BP26 protein was produced.&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=10385 Maryam Azizpour Maghvan, Parvaneh Jafari, Seyyed Davood Hosseini, Ali mohammad Behrozikhah Mon, 03 Jun 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Microbiota and Autism spectrum disorder <p>Autism spectrum disorder (ASD) represents a neurodevelopmental condition characterized by two main deficits: impaired social communication and interaction; restricted and repetitive patterns of interests, behaviors or activities <sup>1</sup> with prevalence ranges from 2 to 20 per 1,000, worldwide <sup>1,2</sup>. Presently, core symptoms of autism have no approved treatment. Autistic disorder management emphasis is on behavioral and educational modalities that target the core symptoms <sup>3</sup>. Psychopharmacologic interventions are introduced to improve daily function and treat associated behavioral problems including hyperactivity, irritability, and aggression; leading to support the implementation of behavioral approaches through reducing the interfering symptoms <sup>3</sup>.<br /> In recent years, a growing number of studies have found evidence implicating dysregulation of immune responses and neuroinflammatory mechanisms in patients with ASD <sup>3</sup>. Dysregulation of T helper cells <sup>4</sup>, increased plasma levels of proinflammatory cytokines such as interleukin 1, 6 and 8 <sup>1-3</sup>, increased proliferation and activation of B cells and natural killer cells <sup>4</sup>, decreased serum levels of immunoglobulin G and M <sup>5</sup> in presence of immunoglobulin G autoantibodies against neuron-axon filament and glial fibrillary acidic proteins <sup>6</sup>, and increased microglial and astrocytic density and activation <sup>5</sup> have been documented.<br /> Risperidone, as a serotonin 5-HT (2A) receptor antagonist that can attenuate dopamine release as well <sup>3</sup>, is the only drug approved by the Food and Drug Administration (FDA) for the treatment of irritability in children with ASD. In recent years, research correlates risperidone use with significant weight gain <sup>3</sup> and with high rate of relapse after discontinuation of the medication in children with ASD <sup>4</sup>. Despite this, due to lack of insight into the exact pathogenesis of ASD, no new medication could be approved as an adjuvant or standalone treatment for patients with ASD.&nbsp;<br /> The fact that diet has a huge influence on our health should be common knowledge by now. But what research has been showing us in recent years is just how fundamental the influence of diet on our health can be. Surprising links between diet and a number of previously unsuspected diseases are being continuously established. But food does not affect us only after we are born, it actually starts to shape our health during pre-natal development. Many of these associations between diet and disease, including neurological diseases, are now known to be modulated by the gut microbiota. Research on the gut-brain axis is a blooming field where new and important findings keep streaming. Individuals with ASD often also have gastrointestinal problems and dysbiosis of the gut microbiota, being unclear whether an altered gut flora is a cause of a comorbidity of ASD. It has been suggested that changes in the gut microbiota may indeed play a role in the development of the behavioral symptoms associated with ASD, but the possible mechanisms of this link remain unknown <sup>6</sup>.<br /> As for autism, this link may come down to a particular molecule called interleukin-17a (or IL-17a), which is produced by the immune system. The molecule has already been associated with conditions like rheumatoid arthritis, multiple sclerosis, and psoriasis, and has been shown to serve an important role in preventing infections, notably those of the fungal kind. Importantly, it can also influence the way the brain develops in the womb.<br /> The effects of the microbiome on the development of MIA-induced autism could be prevented either by modifying the pregnant mother&rsquo;s microbiome, or by directly blocking IL-17a signaling.<br /> Although ASD primarily impacts the brain, over recent years, links with other systems have become clear, in particular, gastrointestinal (issues seem to occur more often in individuals with ASD than in the rest of the population <sup>6</sup>.</p> https://www.AJMB.org/En/Article.aspx?ID=10348 Shahin Akhondzadeh Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Review of Different Sequence Motif Finding Algorithms <p>The DNA motif discovery is a primary step in many systems for studying gene function.&nbsp; Motif discovery plays a vital role in identification of Transcription Factor Binding Sites (TFBSs) that help in learning the mechanisms for regulation of gene expression. Over the past decades, different algorithms were used to design fast and accurate motif discovery tools. These algorithms are generally classified into consensus or probabilistic approaches that many of them are time-consuming and easily trapped in a local optimum. Nature-inspired algorithms and many of combinatorial algorithms are recently proposed to overcome these problems. This paper presents a general classification of motif discovery algorithms with new sub-categories that facilitate building a successful motif discovery algorithm. It also presents a summary of comparison between them.</p> https://www.AJMB.org/En/Article.aspx?ID=10368 Fatma Hashim, Mai Mabrouk, Walid Al-Atabany Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir CRISPR/Cas9 System for Efficient Genome Editing and Targeting in the Mouse NIH/3T3 Cells <p>Background: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for targeting <em>Rag</em> genes to produce mutant mouse NIH/3T3 cell line. The <em>Rag1</em> and <em>Rag2</em> genes are essential for generation of mature B and T lymphocytes. Disruption of <em>Rag</em> genes causes disease like Severe Combined Immunodeficiency syndrome (SCID). Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line.<br /> Methods: Four single guide RNAs were designed to target sequences in the coding region of the <em>Rag1</em> and <em>Rag2</em> genes. Four sgRNA-CAS9 plasmids were tested to target <em>Rag1</em> and <em>Rag2</em>.&nbsp;<br /> Results: Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in <em>Rag1</em> resulted in deletion of the intervening DNA fragment. The expression of sgRNAs with Cas9 targeting two sites in <em>Rag2 </em>gene resulted in indel mutations at both sites. In this report, fragment deletion in <em>Rag1</em>&nbsp;gene was detected in about 50% of transfected cells.<br /> Conclusion: Therefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engineering of cells and model animals.</p> https://www.AJMB.org/En/Article.aspx?ID=10369 Maryam Mehravar, Abolfazl Shirazi, Mohammad Mehdi Mehrazar, Mahboobeh Nazari, Mehdi Banan Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression Patterns for <i>TETs</i>, <i>LGR5</i> and <i>BMI1</i> in Cancer Stem-like Cells Isolated from Human Colon Cancer <p>Background: Colon tumor is generated and maintained by a small subset of chemo-resistant cancer cells known as Cancer Stem-like Cells (CSCs) that are able to self-renew and differentiate into various cell types within the cancer milieu. CSCs are identified through expression of CD133 that is the most important surface marker of these cells. Epithelial Cell Adhesion Molecule (EpCAM) is another colon CSCs marker. Other markers that are probably involved in colon tumorigenesis are Leucine-rich repeat-containing G-protein-coupled Receptor 5 (LGR5), B cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) and Ten-Eleven Translocations (TETs).&nbsp;<br /> Methods: Here, mRNA expression rates of <em>LGR5</em>, <em>BMI1</em> and <em>TETs</em> were surveyed by real-time PCR. After collection and digestion, colon samples were used to isolate CD133 and EpCAM positive CSCs through evaluation of AC133 EpCAM by Magnetic Activated Cell Sorting (MACS) and flow cytometry. Real-time PCR was carried out for assessing expressions of LGR5, BMI1 and TETs.&nbsp;<br /> Results: High expressions for <em>LGR5</em>, <em>BMI1</em>, <em>TET1</em> and <em>TET2</em> in the CD133 and EpCAM positive CSCs (p&le;0.05 <em>vs.</em> non-CSCs) were found. <em>TET3</em>, however, showed no significant changes for mRNA expression in the CSCs.&nbsp;<br /> Conclusion: In conclusion, high mRNA expressions for <em>LGR5</em>, <em>BMI1</em>, <em>TET1</em> and <em>TET2</em> in the CD133 and EpCAM positive CSCs may be a useful criterion for better identification of the cells involved in colon cancer in order to specify therapeutic targets against this type of cancer.</p> https://www.AJMB.org/En/Article.aspx?ID=10361 Nader Atlasy, Fardin Amidi, Keywan Mortezaee, Mohammad Sadegh Fazeli, Seyed Javad Mowla, Fatemeh Malek Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Optimization of Fermentation Conditions for Reteplase Expression by Escherichia coli Using Response Surface Methodology <p>Background: Expression of heterologous proteins at large scale is often a challenging job due to plasmid instability, accumulation of acetate and oxidative damage in bioreactors. Therefore, it is necessary to optimize parameters influencing cell growth and expression of recombinant protein.&nbsp;&nbsp;<br /> Methods: In the present study, the optimal culture conditions for expression of reteplase by <em>Escherichia coli (E. coli)</em> BL21 (DE3) in a bench-top bioreactor was determined. Response Surface Methodology (RSM) based on Box-Behnken design was used to evaluate the effect of three variables (<em>i.e</em>., temperature, shaking speed and pH) and their interactions with cellular growth and protein production. The obtained data were analyzed by Design Expert software.<br /> Results: Based on results of 15 experiments, a response surface quadratic model was developed which was used to explain the relation between production of reteplase and three investigated variables. The high value of &quot;R-Squared&quot; (0.9894) and F-value of 51.99 confirmed the accuracy of this model. According to the developed model, the optimum fermentation conditions for reteplase expression were temperature of 32&deg;<em>C</em>, shaking speed of 210 <em>rpm</em>, and pH of 8.4. This predicted condition was applied for the production of reteplase in the bioreactor leading to a protein yield of 188 <em>mg/l</em>.<br /> Conclusion: Our results indicate the significant role of culture conditions (<em>e.g</em>., pH, temperature and oxygen supply) in protein expression at large scale and confirm the need for optimization. The proposed strategy here can also be applied to experimental set-up of optimization for fermentation of other proteins.</p> https://www.AJMB.org/En/Article.aspx?ID=10371 Hamze Zare, Hamid Mir Mohammad Sadeghi, Vajihe Akbari Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of Sensitive and Rapid RNA Transcription-based Isothermal Amplification Method for Detection of <i>Mycobacterium tuberculosis</i> <p>Background: The accurate and early diagnosis of tuberculosis is important for its effective management. During the last decade, several molecular methods for detection of Tuberculosis (TB) have been developed. Since RNA especially mRNA has a generally much shorter half-life than DNA, its detection may be useful for the assessment of viability of bacteria. This research is a Nucleic Acid Sequence Based Amplification-Enzyme Linked Immunosorbent Assay (NASBA-ELISA) which was designed and developed for rapid detection of viable <em>Mycobacterium tuberculosis (M. tuberculosis)</em>.&nbsp;<br /> Methods: Oligonucleotide primers targeting <em>tuf</em> gene encoding viability marker EF-Tu mRNAs were selected and used for the amplification of mycobacterial RNA by the isothermal NASBA Digoxigenin (DIG) labeling process and incorporated with DIG-UTP, reverse transcriptase and T7 RNA polymerase.&nbsp;<br /> Results: Using the NASBA-ELISA system, as little as 17.5 <em>pg</em> of RNA of <em>M. tuberculosis</em> was detected within 4 <em>hr</em> and no interference was encountered in the amplification and detection of viable <em>M. tuberculosis</em> in the presence of non-target RNA or DNA. Results obtained from the clinical specimens showed 97 and 75% of sensitivity and specificity, respectively.<br /> Conclusion: The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity for detection of <em>M. tuberculosis</em>. Furthermore, due to its simplicity and high sensitivity feature, it could be used in limited access laboratories in a cost-effective manner.</p> https://www.AJMB.org/En/Article.aspx?ID=10372 Reihaneh Ramezani, Mahdi Forouzandeh Moghadam, Mohammad Javad Rasaee Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Bacteriostatic Potency of Fe2O3 Against <i>Enterococcus faecalis</i> in Synergy with Antibiotics by DDST Method <p>Background: In this study, bacteriostatic potency of the Iron oxide nanoparticles against <em>Enterococcus faecalis (E. faecalis) </em>(a clinical sample and the ATCC11700 strain) was investigated.<br /> Methods: Nanoparticles&rsquo; bacteriostatic concentration was determined and used to appraise the characteristics of the Iron Oxide (Fe<sub>2</sub>O<sub>3</sub>) against the isolates. Antimicrobial examinations with 10<sup>8</sup><em>cfu.ml</em><sup>-1</sup> were performed at the baseline. Due to evaluation level of potency, after performing Minimum Inhibitory Concentration (MIC), the assessment of death kinetic and susceptibility constant of nanoparticles was done by suspension at two MICs in 0 to 360 <em>min</em> treatment time.<br /> Results: Fe<sub>2</sub>O<sub>3</sub> nanoparticles in size range of 10-50 <em>nm</em> demonstrated the most effective susceptibility reaction against <em>E. faecalis</em> and ATCC11700 strain in Z=78.125 <em>ml/&mu;g</em><sup>-1</sup> and 39.0625 <em>ml/&mu;g</em><sup>-1</sup>, respectively. The kinetic reaction of E. faecalis against Fe<sub>2</sub>O<sub>3</sub> suspension was supposed to be decreased through the elapse of treatment time, whereas increased concentration was along with bacteria growth after a certain time. So, the efficient concentration of nanoparticles was applied with semi-sensitive and antibiotic resistant for both strains. However, synergism of Fe<sub>2</sub>O<sub>3</sub> nanoparticles with Ceftazidime and Clindamycin revealed a higher susceptibility compared with Fe2O3 nanoparticles alone against <em>E. faecalis</em>.<br /> Conclusion: The experimental results reveal that Fe<sub>2</sub>O<sub>3 </sub>has a strong antimicrobial effect at a certain concentration over the time so could potentially be used for bacterial inhibition and this feature will be strengthened in combination with antibiotics.</p> https://www.AJMB.org/En/Article.aspx?ID=10366 Erfan Shahbazi, Firouzeh Moreshedzadeh, Davood Zaeifi Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Immunogenicity of Cork and Loop Domains of Recombinant Baumannii acinetobactin Utilization Protein in Murine Model <p>Background: <em>Acinetobacter baumannii (A. baumannii) </em>is a bothersome fatal patho-gen, particularly in healthcare system. Persistence and successful invasion of <em>A. baumannii </em>in vertebrate host cells largely depends on iron acquisition methods. Sidero-phore molecules and Iron-Regulated Outer Membrane Proteins (IROMPs) are the two essential members of iron acquisition system. Siderophores are secreted by bacteria to bind peripheral ferric iron and the IROMPs are expressed at the bacterial outer membrane as the receptor of ferric-siderophore complex. BauA is the corresponding siderophore receptor of <em>A. baumannii</em>. In this study, an attempt was made to assess the immunogenicity of antigenic domains of BauA which could be effective in iron uptake restriction and protection against bacterial invasion of the host cells.&nbsp;<br /> Methods: The antigenic domains of <em>bauA</em> were amplified from <em>A. baumannii </em>ATCC-19606. The PCR products were ligated into pET32a and expressed in<em> Escherichia coli (E. coli)</em> BL21 (DE3). Purification of recombinant domains was done by Nickel-Nitri-lotriacetic Acid (Ni-NTA) affinity chromatography. The recombinant domains were injected into BALB/C mice separately and in combination. Sero-reactivities of the recombinant proteins and mouse challenge tests were carried out.&nbsp;<br /> Results: The antibodies raised in mice could successfully recognize and bind antigenic domains. Passive immunization studies accomplished by immune rabbit serum inhibited the establishment of infection in mice.<br /> Conclusion: The results adapted from the present study disclose the protective role of functional domains of BauA, especially the cork domain, suggesting a novel recombinant immunogen candidate.</p> https://www.AJMB.org/En/Article.aspx?ID=10367 Hamid Esmaeilkhani, Iraj Rasooli, Masoomeh Hashemi, Shahram Nazarian, Fatemeh Sefid Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Single Nucleotide Polymorphism of <i>TYK2</i> Gene and Susceptibility to Rheumatoid Arthritis in Iranian Population <p>Background: Rheumatoid Arthritis (RA) is a debilitating disorder in which the immune system mainly targets the synovial tissue. Janus kinase family including tyrosine kinase 2 (TYK2) is one of the crucial mediators of the downstream signaling pathway of inflammatory cytokines that further contributes to RA pathogenesis. In this study, the association of <em>TYK2</em> gene rs34536443 polymorphism, which may affect the function of TYK protein and, hence, the inflammatory settings, with RA susceptibility was investigated. Moreover, its correlation with demographic and serological features of the patients was assessed.&nbsp;<br /> Methods: In the present study, 700 RA patients and 700 sex, age and ethnicity-matched healthy individuals as the control group were included. MGB TaqMan real-time allelic discrimination method was used to determine the rs34536443 polymorphism. Rheumatoid factor, anti-cyclic citrullinated peptide antibody, erythrocyte sedimentation rate and C-reactive protein were also measured.<br /> Results: The frequency of rs34536443 minor allele (C allele) was not different between patients and control group [1.7 <em>vs</em>. 2.61 percent, OR (95% CI)=1.35 (0.78-2.33);p=0.27]. There was not a statistically significant association between rs34536443 genotypes and RA susceptibility. Genotypes of rs34536443 polymorphism were associated nor with demographic neither with serological features of RA patients.&nbsp;<br /> Conclusion: In the present study, there was not any association between <em>TYK2</em> gene rs34536443 polymorphism with either disease susceptibility, demographic and serological features of Iranian RA patients. These findings are not compatible with previous works from other ethnicities, further supporting the role of genetics in disease susceptibility.</p> https://www.AJMB.org/En/Article.aspx?ID=10374 Azadeh Mohamadhosseini, Reza Mansouri, Ali Javinani, Amir Ashraf-Ganjouei, Massoumeh Akhlaghi, Saeed Aslani, Elham Hamzeh, Ahmadreza Jamshidi, Nooshin Ahmadzadeh, Mahdi Mahmoudi Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antibiotic Susceptibility Patterns and Prevalence of Some Extended Spectrum Beta-Lactamases Genes in Gram-Negative Bacteria Isolated from Patients Infected with Urinary Tract Infections in Al-Najaf City, Iraq <p>Background: Urinary Tract Infection (UTI) in patients with Chronic Kidney Disease (CKD) caused by multi-drug resistance and Extended Spectrum Beta Lactamase (ESBL)-producing gram-negative bacteria has been increased in different countries. The aim of the present study was to detect the antibiotic susceptibility patterns and the distribution of <em>Bla-TEM, Bla-SHV </em>and<em> Bla-CTX-M</em> genes in gram-negative bacteria isolated from outpatients infected with UTI, with and without CKD in Al-Najaf city, Iraq.&nbsp;<br /> Methods: A total of 120 non-duplicate urine samples were collected from outpatients (37 male and 83 female) infected with UTI in Al-Najaf city, Iraq; 60 samples from patients Without Kidney Disease (WKD) and 60 samples from patients with CKD. The antibiotic susceptibility testing was done according to Kirby-Bauer method. PCR technique was performed to investigate the prevalence of <em>Bla-TEM</em>, <em>Bla-SHV</em> and<em> Bla-CTX-M </em>genes.<br /> Results: A total of 126 different gram-negative bacterial strains were isolated. <em>Escherichia coli (E. coli)</em> was the most prevalent bacterium (49 isolates) followed by <em>Idebsiella pneumonia (K. pneumonia) </em>(35 isolates), <em>Pseudomonas aeruginosa (P. aeruginosa)</em> (18 isolates), <em>Citrobacter freundii (C. freundii) </em>(12 isolates), <em>Enterobocter aerogenes (E. aerogenes)</em> (8 isolates) and <em>Proteus mirabilis (P. mirabilis)</em> (4 isolates). All bacterial isolates from UTI patients with CKD were resistant to antibiotics and carried <em>Bla-TEM</em>, <em>Bla-SHV </em>and <em>Bla-CTX-M</em> genes more than isolates from UTI patients with WKD.<br /> Conclusion: This study demonstrated that all bacterial isolates from UTI patients with CKD were more virulent than isolates from UTI patients with WKD.</p> https://www.AJMB.org/En/Article.aspx?ID=10364 Heba Takleef Majeed, Ahmed Abduljabbar Jaloob Aljanaby Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of the Effect of Morphine and Imiquimodon Expression of TLR2 and TLR4 from Lesion RNA Extracted from BALB/c Mice Infected with Leishmania major <p>Background: Toll-Like Receptors (TLRs) are the cause of phagocytosis activation and destruction of the infection agents. In addition, new evidences support the idea that TLRs play a vital role in starting the acquired immunity reactions.<br /> Methods: In this study, it has been attempted to infect the BALB/c mice with <em>Leishmania major (L. major)</em> and treat them using morphine and imiquimod; then the expressions of TLR2,4 from treated lesion were studied by using Real-Time PCR method. Treatment with morphine 1 <em>mg/kg</em>, imiquimod 5% and nalmefene 1<em> mg/kg</em> began four weeks after the challenge. After treatment period, half of the mice of each group were killed and their lesions were isolated for RNA extraction and making cDNA. For the rest of mice, lesion size was measured weekly.<br /> Results: The results showed increase of expression of <em>TLR2</em> gene among all treated groups relative to the control, and the difference was significant (p&lt;0.05). The expression of<em> TLR4</em> gene only was reduced in groups under treatment with morphine and morphine plus nalmefene relative to the control group and in the other groups increased. The highest expression of <em>TLR2</em> was seen in the group treated by glucantime (p&lt;0.0001).&nbsp;<br /> Conclusion: However, in this study it was found that despite decreasing the size of lesion in all treated groups, expression of <em>TLR4</em>&nbsp; in the morphine, nalmefene, morphine plus nalmefene treated groups compared to the control group was decreased. Therefore, morphine may have a different function mechanism in treatment of the Leishmaniasis with the <em>L. major</em>.</p> https://www.AJMB.org/En/Article.aspx?ID=10401 Parisa Ebrahimisadr, Fatemeh Ghaffarifar, John Horton, Abdolhossein Dalimi, Zohreh Sharifi Mon, 18 Mar 2019 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Monoclonal Antibody a Promising Treatment for Depression <p>Major depressive disorder (MDD) is one of the prominent causes of disability in the world, affecting 15-20% of people over the period of a lifetime. Patients commonly experience continuous remaining symptoms, functional impairment, and diminished health <sup>1</sup>.<br /> Treatment of MDD is still far from optimal and drug resistance in MDD is still considered a serious clinical challenge. Only about one-third of patients completely respond to their first antidepressant medication, first with an approximate lag time of 2 months. This notable lag time for onset of therapeutic efficacy is associated with significant morbidity and suicidal risk. As a result, there is a widely accepted need for fast acting antidepressants. Another downside of the currently available treatments is their side effects which are documented in large proportion of patients <sup>1,2</sup>.<br /> Patients who do not respond to their first-line medication are generally treated by either switching to another treatment or with augmentation therapy to achieve favorable response. Combination therapy from start of treatment has been suggested recently to gain quicker and better response and remission rates, however, not all studies have supported this opinion. Most MDD pathophysiology etiological theories used to focus on brain modulatory monoamine systems (dopamine, serotonin and norepinephrine) <sup>3,4</sup>. A more recent line of evidence points to glutamate, the brain&rsquo;s principal excitatory neurotransmitter, as playing a role in MDD&rsquo;s pathophysiology. Additionally, glutamate dysregulation is known to cause impairments in structural plasticity and cellular resilience, which seems to be implicated in mood disorders as well. It is therefore reasonable to hypothesize that medications which reduce glutamatergic tone may be able to play a role in treatment of depression <sup>5</sup>.<br /> Depression is demonstrated to be accompanied with parallel increases in the immuno-inflammatory biomarkers including significantly higher levels of pro-inflammatory cytokines interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-&alpha;, and C-reactive protein (CRP) in depressed patients compared to normal individuals. The dynamic interaction between pro-inflammatory cytokines, prostaglandin (PG)-E2 synthesis and depression has led to suggestions that anti-inflammatory agents could be useful for treatment of depression <sup>6,7</sup>. Celecoxib, a nonsteroidal anti-inflammatory drug that acts <em>via</em> the selective inhibition of cyclooxygenase (COX)-2, has shown promising outcomes in several psychiatric diorders, including autistic disorder, schizophrenia, and depression. Antidepressive effects of celecoxib is suggested to be largely attributed to its inhibitory effect on the levels of pro-inflammatory cytokines. In line with this idea, baseline levels of serum IL-6 is demonstrated to be significantly correlated with the Hamilton Depression Rating Scale (HDRS) score. In the same article, adjunctive therapy with celecoxib, as an antidepressant, resulted in significant decreases in both serum IL-6 levels and HDRS scores. Although most of the previous research has focused on the antidepressant effects of celecoxib as an add-on treatment, few reports have investigated the safety and efficacy of celecoxib as a monotherapy <sup>8-10</sup>.<br /> In clinical trials, two new classes of anti-inflammatory drugs-anti-cytokine monoclonal antibodies and cytokine inhibitors have been shown to reduce inflammation in a range of autoimmune diseases, and these drugs have already started to be administered to patients who do not respond to standard treatments <sup>11,12</sup>.</p> https://www.AJMB.org/En/Article.aspx?ID=10336 Shahin Akhondzadeh Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antibody-Drug Conjugates: Possibilities and Challenges <p>The design of Antibody Drug Conjugates (ADCs) as efficient targeting agents for tumor cell is still in its infancy for clinical applications. This approach incorporates the antibody specificity and cell killing activity of chemically conjugated cytotoxic agents. Antibody in ADC structure acts as a targeting agent and a nanoscale carrier to deliver a therapeutic dose of cytotoxic cargo into desired tumor cells. Early ADCs encountered major obstacles including, low blood residency time, low penetration capacity to tumor microenvironment, low payload potency, immunogenicity, unusual off-target toxicity, drug resistance, and the lack of stable linkage in blood circulation. Although extensive studies have been conducted to overcome these issues, the ADCs based therapies are still far from having high-efficient clinical outcomes. This review outlines the key characteristics of ADCs including tumor marker, antibody, cytotoxic payload, and linkage strategy with a focus on technical improvement and some future trends in the pipeline.</p> https://www.AJMB.org/En/Article.aspx?ID=10350 Mohammad Reza Nejadmoghaddam, Arash Minai-Tehrani, Ramin Ghahremanzadeh, Morteza Mahmoudi, Rassoul Dinarvand, Amir-Hassan Zarnani Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Recovery of MicroRNA from Stored Bone Marrow Aspirate Slides <p>Background: Archived bone marrow aspirate slides are almost infinite, readily available resource of biospecimens that enable retrospective molecular investigations of diseases. RNAs obtained from slides has limitations in utility because of their low quality and highly fragmented nature. MicroRNAs are small (&lt;22 nt) noncoding RNAs with various cellular regulatory roles. Due to their small size, microRNAs are less prone to degradation and modification, therefore, can be preserved well in archived tissues.<br /> Methods: The current study investigated the efficacy of archived bone marrow aspirate slides for miRNA expression analysis in pediatric leukemia. Total RNA was isolated from air-dried unstained archived slides using High pure miRNA isolation Kit with some modifications and from fresh samples using TRizol. After cDNA synthesis, RT-qPCR was then carried out using specific hsa-miR-326 LNA primers. Finally, statistical analyses were conducted using GraphPad Prism 6 software.&nbsp;<br /> Results: The difference observed in miRNA expression due to disease state was far greater than the differences between archived slides and their matching fresh bone marrow specimens. In fact, the expression of archival slide smears for the miR-326 closely mimicked that of fresh-frozen tissues (0.035&plusmn;0.04 <em>vs.</em> 0.03&plusmn;0.04) (Mean&plusmn;SD, p&gt;0.05). Differential expression of hsa-miR-326 was detected between leukemic and non-leukemic samples from archived slides or fresh frozen bone marrows.<br /> Conclusion: The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role that miRNAs play in the development and long term outcome of hematologic, as well as non-hematologic diseases.</p> https://www.AJMB.org/En/Article.aspx?ID=10356 Elaheh Sadat Ghodousi, Soheila Rahgozar Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of Differential Gene Expression during Transdifferentiation of Bone Marrow Stromal Cells to Glial phenotype in the Presence of Cerebrospinal Fluid <p>Background: The present study assessed the alteration of gene expression during transdifferentiation of Bone Marrow Stromal Cells (BMSCs) into oligodendrocyte in the presence of Cerebrospinal Fluid (CSF).<br /> Methods: BMSCs were collected from female Sprague-Dawley rats and were cultured in DMEM/F12 medium supplemented with Retinoic Acid (RA), basic Fibroblast Growth Factor (bFGF), and Epidermal Growth Factor (EGF). CSF was added daily to the culture media. The oligoprogenitor and oligodendrocyte generation was assessed by immunocytochemistry for Oligo 2, A2B5, CNP and MBP markers.&nbsp;<br /> Results: The mean percentages of immunopositive cells for Olig2 and A2B5 were 52.1&plusmn;1.74 and 56.34&plusmn;2.55%, respectively. The number of immunopositive cells for glial markers CNP and MBP were 48.8&plusmn;3.12 and 40.5&plusmn;8.92%, respectively. Alteration of gene expression of Oct4, Olig 2, PDGFR-&alpha; and PLP were examined by real time PCR during transdifferentiation of BMSC to oligodendrocyte. Immunocytochemical results indicate that oligoprogenitor cells were immunopositive for Oligo2 and A2B5 markers. Also, oligodendrocytes expressed the mature glial markers of CNP and MBP indicating successful differentiation.&nbsp;<br /> Conclusion: In conclusion, CSF promotes the transdifferentiation of BMSC into mature oligodendrocyte via providing an appropriate niche for glial maturation.&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=10345 Hatef Ghasemi Hamidabadi, Maryam Nazm Bojnordi, Nourollah Rezaei, Sara Soleimani Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Preconditioning with SDF-1 Improves Therapeutic Outcomes of Bone marrow-derived Mesenchymal Stromal Cells in a Mouse Model of STZ-induced Diabetes <p>Background: Nowadays, transplantation of bone marrow-derived Mesenchymal Stromal Cells (BMSCs) is currently an important alternative therapy for patient&rsquo;s type 1 diabetes mellitus. But a number of critical obstacles lie ahead of this new strategy including reducing stem cell homing to the damaged tissue due to oxidative stress. The purpose of the present study was to investigate whether preconditioning of BMSCs with SDF-1 could enhance their homing to the pancreas and promote regeneration of the pancreatic &beta; cells after being intravenously injected.<br /> Methods: Mice BMSCs were isolated and expanded. Cell proliferation was assayed by MTT Assay. Preconditioning was performed with 10 <em>ng/ml</em> SDF-1&alpha; for 24 <em>hr</em>. Male NMRI mice were injected with high-dose STZ (150 <em>mg/kg</em>). The preconditioned or unpreconditioned BMSCs at a dose of 1&times;10<sup>6 </sup>cells were infused via the tail vein. Blood and pancreatic tissue samples were taken from all mice for flow cytometry, biochemical and histological studies.<br /> Results: Proliferation and homing of BMSCs to the pancreas were significantly increased in the BMSCs with SDF-1&alpha; preconditioning. Differentiation of transplanted BMSCs, were significantly increased in preconditioning group. Although BMSCs without SDF-1 preconditioning exhibited remarkable recovery of pancreatic islets structure but this recovery were significantly increased in the BMSCs with SDF-1&alpha; preconditioning.<br /> Conclusion: Our results showed the effectiveness of SDF-1&alpha;preconditioning in BMSCs transplantation of STZ induced diabetes mice which might be achieved through improvement of BMSCs homing into the injured pancreas.</p> https://www.AJMB.org/En/Article.aspx?ID=10337 Mohammad Sadegh Gholami Farashah, Parichehr Pasbakhsh, Ameneh Omidi, Saied Nekoonam, Roya Aryanpour, Iraj Regardi Kashani Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Anti-cancer Effects of <i>Capparis spinosa</i> Hydroalcoholic Extract <p>Background: Recently, due to the steep increase in cancer lethality statistics, pharmaceutical societies seek approaches for designing drugs with higher efficiency and lower expenses. Plant-based drugs have therefore gained much attention, due to their abundance and ease of accessibility, and their higher effectiveness.&nbsp;<br /> Methods: Wild-grown caper [<em>Capparis spinosa (C. spinosa)</em>] was collected from northern Iran and next 100 <em>g</em> of the powder was added to 300<em> ml</em> of a solvent (Ethanol 80), the solution was mixed for 72 <em>hr</em> and later filtered <em>via</em> Whatman filter papers. The solvent was taken out under vacuum conditions and extracts were then collected and stored in glass vials. The High Pressure Liquid Chromatography (HPLC) method was used to assay quercetin which consisted of the following specifications: C18 column, UV detector wavelength of 260 <em>nm</em>, mobile phase acetonitrile and water and flow rate of 1 <em>ml/min</em>. In this study, the anti-cancer effects of <em>C. spinosa</em> extract on HeLa, MCF7, Saos and Fibroblast cancer cell lines have been investigated.<br /> Results: The amount of quercetin was assessed by HPLC. The anti-tumor activity and the antioxidant level of hydroalcoholic extract of <em>C. spinosa</em> have been evaluated with MTT assay and FRAP technique, respectively. HPLC data showed quercetin form the major component of <em>C. spinosa</em> extract. In addition, FRAP data indicated that <em>C. spinosa</em> extract had high antioxidant activity and MTT assay indicated that <em>C. spinosa</em> extract effectively decreased the cancer cell lines.<br /> Conclusion: The quercetin in <em>C. spinosa </em>extract had significant anti-tumor effects and may be regarded as an ideal natural drug for cancer therapy.</p> https://www.AJMB.org/En/Article.aspx?ID=10352 Yasaman Moghadamnia, Seydeh Narges Mousavi Kani, Maryam Ghasemi-Kasman, Mohamad Taghi Kazemi Kani, Sohrab Kazemi Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Multidisciplinary Study to Evaluate the Anti-quorum Sensing Ability of Phyto-compounds in <i>Ruellia patula</i> Jacq <p>Background: <em>Staphylococcus aureus (S. aureus)</em> causing numerous diseases in humans, have become resistant to antibiotics, hence, urging the need for alternative medicines.&nbsp;<br /> Methods: In this study, the Indian medicinal weed, <em>Ruellia patula (R. patula)</em> extracted and fractioned through column chromatography was subjected to antibacterial and anti-quorum sensing activity against <em>S. aureus</em> and Methicillin Resistant Staphylococcus aureus MRSA.<br /> Results: The obtained results confirmed fraction F44 to have significant effect as antimicrobial and anti-biofilm agent against both the micro-organism. Therefore, few of such highly active fractions were chemical finger printed using GC-MS and the compounds identified were further docked with DNA binding (LytTR) domain of agrA, which revealed that compounds identified from fraction were interactive to the protein.<br /> Conclusion: <em>R. patula</em> is promising antimicrobial and anti-biofilm agent against S. aureus and MRSA.</p> https://www.AJMB.org/En/Article.aspx?ID=10338 P Chemmugil, PTV Lakshmi, A Annamalai Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Statistical Optimization of Process Parameters by Central Composite Design (CCD) for an Enhanced Production of L-asparaginase by Myroides gitamensis BSH-3, a Novel Species <p>Background: The present study focused on the production of L-asparaginase using Solid State Fermentation (SSF) by <em>Myroides gitamensis</em>.&nbsp;<br /> Methods: Initially, five significant parameters (Carbon source; Nitrogen source, temperature, pH and incubation period) were identified that affect the production process of L-asparaginase using Classical One Factor at a Time (OFAT) optimization. An optimized L-asparaginase specific activity obtained by OFAT was recorded as 85.7 <em>IU</em>. Central Composite Design (CCD) was also employed successively to optimize the multiple parameters at a time and their results were compared.&nbsp;<br /> Results: Maximum L-asparaginase enzyme specific activity obtained by CCD method was 295.6 <em>IU</em> under the hold values of carbon source (wheat bran) 12 <em>g/L</em>, nitrogen source (yeast extract) 7 <em>g/L</em>, temperature 37<sup><span style="font-size:10.8333px">o</span></sup><em>C</em>, pH=7.5 and incubation period 47 <em>hr</em>. Upon validation, the obtained results proved that there was a good relation existing between the experimental and the predicted model (p&lt;0.05). L-asparaginase activity was enhanced in statistical method up to 3.4 folds compared to that of classical method.&nbsp;<br /> Conclusion: Utilization of wheat bran as a low cost carbon source in SSF for the production of L-asparaginase enzyme makes the process economical and in turn reduces the environmental pollution by biotransformation to commercially useful bio product.</p> https://www.AJMB.org/En/Article.aspx?ID=10355 VSSL Talluri, Sri Lanka, SV Rajagopal Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of <i>TAX-1</i> Gene of Human T-cell Leukemia Virus Type -1 on the Expression of CCR5 in K562 Cell Line <p>Background: Tax-1 protein of Human T-cell Leukemia Virus type 1(HTLV-1) serves as a key transcriptional regulatory gene product and has a crucial role in transactivating genes of infected cells by employing their transcriptional factors. This modulation includes induction of genes which encode CC-chemokines and their receptors. In this study, a recombinant vector containing Tax-1 gene was made and tested for its ability to induce CCR5 (CC chemokine receptor 5) expression in K562 cell line.<br /> Methods: In order to perform this research, two blood samples of HTLV-1 positive were obtained from Urmia blood transfusion center. After DNA extraction, a complete sequence of <em>Tax-1</em> gene was amplified by specific primers. Recombinant vectors carrying Tax-1 gene were synthesized and transformed into <em>Escherichia coli (E. coli)</em>. After bacteria transformation, bacteria containing recombinant plasmid were selected and purified. Then, the recombinant shuttle vectors, <em>pCDNA3.1-TAX</em>, were transfected into the cell culture (K562 cell line). Expression of <em>CCR5</em> was measured after 72 <em>hr</em> by Syber Green Real-Time PCR method compared to control cell culture. Normalization was done with GAPDH as a standard gene.<br /> Results: Cloning of <em>Tax-1</em> gene in the vector, <em>pCDNA3</em>.1 was confirmed by colony PCR, restriction digestion, and sequencing methods. Expression of <em>Tax-1</em> and <em>CCR5</em> genes were confirmed by real time PCR and also, expression of <em>CCR5</em> gene showed an 8-fold increase in comparison to mock-treated controls (p&lt;0.05).<br /> Conclusion: Our data suggested that recombinant <em>Tax-1</em> may have the enhancing effect on CCR5 expression rate at mRNA levels in K562 cell line. Further studies are necessary to evaluate the effect of<em> pCDNA3.1-TAX</em> on cell surface CCR5 expression.</p> https://www.AJMB.org/En/Article.aspx?ID=10354 Nasrin Haghnazari Sadaghiani, Lila Pirayeshfard, Afsaneh Aghaie, Zohreh Sharifi Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Characterization of Monoclonal and Polyclonal Antibodies Recognizing Prostate Specific Antigen: Implication for Design of a Sandwich ELISA <p>Background: Prostate cancer is the second most common cancer in men. Prostate-Specific Antigen (PSA) is a tumor-associated glycoprotein with enzymatic activity which is secreted by the prostate gland. Following entry to the blood, 70-90% of PSA forms complexes with protease inhibitors and its enzymatic activity is inhibited. The serum level of PSA is increased and the rate of free PSA (fPSA) to total PSA is decreased in prostate cancer patients. Therefore, measurement of PSA and fPSA in serum is very valuable for diagnosis and prognosis of prostate cancer.<br /> Methods: In the present study, five anti PSA monoclonal Antibodies (mAb) were characterized by Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting. For designing a sandwich ELISA, epitope specificity of these antibodies was studied by a competition ELISA. Free PSA was purified by electroelution technique from seminal plasma and used to produce polyclonal anti-fPSA antibody in rabbit. Purified polyclonal antibody (pAb) and mAbs were conjugated with HRP enzyme and Biotin (Bio) to set up the sandwich ELISA.<br /> Results: Three of the mAbs were found to recognize PSA similarly. One of these mAbs (2G3) was paired with anti-fPSA pAb to detect fPSA in serum. Eventually, serum fPSA concentration of 356 subjects was measured and compared by our designed ELISA and a commercial ELISA kit. Our results demonstrated a significant correlation (r=0.68; p&lt;0.001) between the two assays. Sensitivity and specificity of our designed ELISA was 72.4 and 82.8%, respectively.<br /> Conclusion: These results imply suitability of our designed ELISA for detection of fPSA in patients with prostate cancer.</p> https://www.AJMB.org/En/Article.aspx?ID=10351 Sahar Raoofi Mohseni, Forough Golsaz-Shirazi, Mostafa Hosseini, Jalal Khoshnoodi, Tannaz Bahadori, Mohammad Ali Judaki, Mahmood Jeddi-Tehrani, Fazel Shokri Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development and Comparison of In-house Line Probe Assay (LiPA) and SYBR Green Real-time PCR Regarding the Detection of Periodontal Pathogens <p>Background: Periodontal disease, which can become a chronic condition, is an inflammatory disease that upsets the soft and hard structures supporting the teeth. The aim of the present study was to design and develop an in-house Line Probe Assay (LiPA), to detect putative periodontitis-related bacterial pathogens, and compare it with SYBR Green Real-time PCR.<br /> Methods: The LiPA method was launched using biotinylated 16s rRNA universal primers and specific probes for each of the five bacteria including <em>Aggregatibacter actinomycetemcomitans</em>, <em>Prevotella intermedia</em>, <em>Tannerella forsythia</em>, <em>Porphyromonas gingivalis</em> and <em>Treponema denticola</em>. For this, optimized quantities of the primers and specific probes were dotted onto nylon membrane stripes in a defined pattern. Hybridization was performed between the probes and the single-stranded biotinylated PCR products. The stripes were developed <em>via</em> biotin-streptavidin reaction. Ultimately, the analytical and diagnostic sensitivity and specificity of the in-house LiPA was evaluated and compared with SYBR Green Real-time PCR.<br /> Results: The detection limit of the LiPA was 2760 copies of targeted genes. In testing analytical specificity, only signals corresponding to the specific biotinylated products were produced. The calculated diagnostic sensitivity of the LiPA for the five bacterial targets ranged from 96.4 to 100%, whereas the diagnostic specificity was between 90.9 and 100%. Comparing the results, no noticeable difference (p=0.4795) was observed between the two methods.<br /> Conclusion: To screen periodontal pathogens, a simple, inexpensive and accurate method is desirable. The in-house LiPA, having advantages such as high specificity and sensitivity, and the ability to detect five major periodontal pathogens, offers the option of evaluating samples without the need for a post-PCR platform.</p> https://www.AJMB.org/En/Article.aspx?ID=10353 Mohammad Soleimani, Mohammad Reza Zolfaghari Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression and Activity of Platelet Endothelial Nitric Oxide Synthase are Decreased in Patients with Coronary Thrombosis and Stenosis <p>Background: Nitric Oxide (NO) which is synthesized by endothelial Nitric Oxide Synthase (eNOS) in both vascular tissues and platelets plays an important role as a protective mediator in the cardiovascular system. It modulates blood pressure, vasodilation and thrombosis. In this regard, eNOS activity and gene expression in platelets and NO levels in patients&rsquo; plasmas with Coronary Thrombosis (CT) and stenosis diseases were determined.&nbsp;&nbsp;<br /> Methods: Blood samples were collected from 60 subjects that where divided into three equal groups [without coronary artery disease, with CT and less than 70% Coronary Stenosis (CS)]. NO concentration in plasma was measured by the Griess reagent system. The eNOS activity was assessed based on a fluorimetric detection system in platelets and the gene expression was quantified by the real time-reverse transcription-polymerase chain reaction.&nbsp;<br /> Results: There was a significant decrease in the amount of NO concentration in the plasma of subjects with CT (0.53&plusmn;0.09 <em>&micro;M</em>, p&lt;0.01) and CS (1.31&plusmn;0.11 <em>&micro;M</em>, p&lt;0.01) compared to the control group (2.6&plusmn;0.10 <em>&micro;M</em>). The activity levels of eNOS enzyme were significantly lower in patients&rsquo; platelets with CT (0.68&plusmn;0.013 <em>UF/mn</em>, p&lt;0.01) and CS (0.85&plusmn;0.017 <em>UF/mn</em>, p&lt;0.01) than the control cases (1.29&plusmn;0.019 <em>UF/mn</em>). These data were consistent with the reduction of the expression levels of eNOS in patients with CT (75 folds) and CS (4 folds) lower than the control cases.<br /> Conclusion: Patients with CT and CS possessed reduced eNOS activity and gene expression in their platelets. Decreased plasma concentration of NO in these patients confirmed the potential significance of the diagnostic and prognostic value of NO in the subjects&rsquo; plasma with vascular disease risk.</p> https://www.AJMB.org/En/Article.aspx?ID=10349 Zahra Emami, Alireza Mesbah Namin, Javad Kojuri, Farideh Mashayekhi Jalali, Mozhgan Rasti Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Identification of Potential Lead Molecules for Zika Envelope Protein from in Silico Perspective <p>Background: Zika virus is the family member of flavivirus with no reported clinically approved drugs or vaccines in the market till date. This virus is spread by Aedes mosquitoes, and can also be transmitted through sexual contact or blood transfusions. There are reported medical conditions like microcephaly among new-borns delivered by infected pregnant women. The envelope protein of Zika virus is associated with virulence, tropism, mediation of receptor binding and membrane fusion. ED1-EDII domain (K1 loop pocket) is an integral part of the envelope protein and a potential drug target. In the present study, the purpose was to identify the potential lead molecules to dock against K1 loop which could be later considered as flavivirus entry inhibitors.<br /> Methods: Multiple sequence alignment method was considered for the analysis of indels in envelope protein. Phylogenetic tree was constructed based on the alignment. Aliphatic index, GRAVY scores and hydropathy plot of the envelope proteins were calculated for the flavivirus family members. Zika envelope protein was homology modeled and considered for protein-ligand docking analysis with chemical compounds of known functions.<br /> Results: As per in silico based analysis, the envelope protein of Zika virus is highly hydrophilic with the least number of amino acid deletions compared to rest of the family members. During docking studies, it was observed that compounds like NITD, compound 6, P02, Doxytetracycline and Rolitetracycline show better binding affinity with Zika envelope protein compared to dengue virus.<br /> Conclusion: These better binding compounds could be the promising lead molecules for Zika envelope protein which could better block the viral entry.</p> https://www.AJMB.org/En/Article.aspx?ID=10340 Selvaa Kumar C, Shine Devarajan Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Prediction of RNA- and DNA-Binding Proteins Using Various Machine Learning Classifiers <p>Background: Nucleic acid-binding proteins play major roles in different biological processes, such as transcription, splicing and translation. Therefore, the nucleic acid-binding function prediction of proteins is a step toward full functional annotation of proteins. The aim of our research was the improvement of nucleic-acid binding function prediction.<br /> Methods: In the current study, nine machine-learning algorithms were used to predict RNA- and DNA-binding proteins and also to discriminate between RNA-binding proteins and DNA-binding proteins. The electrostatic features were utilized for prediction of each function in corresponding adapted protein datasets. The leave-one-out cross-validation process was used to measure the performance of employed classifiers.<br /> Results: Radial basis function classifier gave the best results in predicting RNA- and DNA-binding proteins in comparison with other classifiers applied. In discriminating between RNA- and DNA-binding proteins, multilayer perceptron classifier was the best one.<br /> Conclusion: Our findings show that the prediction of nucleic acid-binding function based on these simple electrostatic features can be improved by applied classifiers. Moreover, a reasonable progress to distinguish between RNA- and DNA-binding proteins has been achieved.</p> https://www.AJMB.org/En/Article.aspx?ID=10341 Mehdi Poursheikhali Asghari, Parviz Abdolmaleki Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Enhancing Stability of Destabilized Green Fluorescent Protein Using Chimeric mRNA Containing Human Beta-Globin 5′ and 3′ Untranslated Regions <p>Background: In spite of recent progress in mRNA technologies and their potential applications for treatment of human diseases, problems such as the transient nature of mRNA limit the stability of gene up-regulation and, thus, potentially reduce mRNA efficiency for gene therapy. Using human &beta;-globin 5&prime; and 3&prime; untranslated regions (UTRs), this study aimed to develop the different chimeric constructs of mRNAs to increase the stability of destabilized green fluorescent protein (EGFPd2) in HEK 293 cells.&nbsp;<br /> Methods: Purified human &beta;-globin (HBG) 5&prime;-3&prime;UTRs, and the coding sequence of destabilized green fluorescent protein (EGFPd2) were amplified separately and ligated to each other using SOEing PCR method in a different format. As controls, the original construct of EGFPd2 under the control of T7 promoter was used. Following <em>in vitro </em>transcription, HEK 293 cells were then transfected with several constructs and incubated at 37<sup>o</sup><em>C</em> in a CO<sub>2</sub> incubator. They were monitored under a fluorescence microscope every four hours for the first 24 <em>hr</em>, then every 12<em> hr</em> afterwards. The resulting fluorescence was measured as a surrogate for translation efficiency and duration.&nbsp;<br /> Results: By monitoring the HEK cells over 48 <em>hr</em>, cells transfected with mRNA with various HBG UTRs showed significantly different fluorescence intensity and stability in comparison with the pEGFPd2 prototype (control transcript) overtime. Overall, the images show that replacement of the 3&prime; UTR end of the prototype vector pGFPd2 with the 3&prime; end of &beta;- globin mRNA increases the half-life of the chimeric mRNA for more than 32 <em>hr</em>.&nbsp;<br /> Conclusion: This result indicates that &beta;-globin 3&prime; UTR would definitely increase the half-life of mRNA and may help to decrease the mRNA therapeutic dosage in the treatment of diseases associated with mRNA therapy.</p> https://www.AJMB.org/En/Article.aspx?ID=335 Setare Adibzadeh, Majid Fardaei, Mohammad Ali Takhshid, Mohammad Reza Miri, Gholam Reza Rafiei Dehbidi, Ali Farhadi, Reza Ranjbaran, Parnian Alavi, Negin Nikouyan, Noorossadat Seyyedi, Samaneh Naderi, Alireaz Eskandari, Abbas Behzad-Behbahani Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of One-Step Tetra-primer ARMS-PCR for Simultaneous Detection of the <i>Angiotensin Converting Enzyme (ACE)</i> I/D and <i>rs4343</i> Gene Polymorphisms and the Correlation with CAD Patients <p>Background: The <em>Angiotensin Converting Enzyme (ACE)&nbsp;</em>Insertion/Deletion and <em>rs4343</em> gene polymorphisms could be associated with pathogenesis of essential hypertension and cardiovascular disorders and Coronary Artery Disease (CAD). In the present study, a fast and novel approach of multiplex Tetra-Primer Amplification Refractory Mutation System-PCR (T-ARMS-PCR) was developed for simultaneous detection of two SNPs including <em>ACE</em> I/D (rs4340) and 2350A&gt;G (rs4343) of <em>Angiotensin Converting Enzyme (ACE)</em> gene.&nbsp;<br /> Methods: The present research was performed using 148 blood samples taken from patients with CAD and 135 healthy individuals. One set of inner primers (for rs4343) and one set of outer primer pairs were designed for genotyping of Insertion/Deletion and rs4343 polymorphisms in single tube T-ARMS-PCR.&nbsp;<br /> Results: Our results manifested that genotypes and alleles frequency of the <em>ACE </em>polymorphisms showed no statistically significant association between CAD patients and the control group. In addition, complete concordance was seen between sensitive Tetra-ARMS-PCR and sequencing method.&nbsp;<br /> Conclusion: The technique is the first work for simultaneous detection of Insertion/Deletion polymorphism and rs4343 SNPs in <em>ACE</em> gene and the results were entirely according to those from an independent procedure.</p> https://www.AJMB.org/En/Article.aspx?ID=10365 Mohammad Mehdi Heidari, Mehdi Hadadzadeh, Hossein Fallahzadeh Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Triploidy and Routine Combined First Trimester Pregnancy Screening <p>Background: This report is about a pregnancy with a triploid fetus and underscores the potential of first trimester combined screening to detect this devastating chromosomal aberration earlier in pregnancy. This report is about a pregnancy with a triploid fetus identified from the first trimester combined screening and confirmed by amniocentesis.&nbsp;<br /> Methods: A 28 year old, G5P2AB2 woman was referred to our clinic at 15 weeks of gestation due to a remarkable decrease of her first trimester double biochemical markers and therefore in the high-risk range for trisomy 13 and 18. The woman underwent amniocentesis which revealed a karyotype of 69,XXX. The parents opted for termination and in post mortem physical examination, a hydrocephalus fetus with marked Intra-Uterine Growth Retardation (IUGR) in addition to syndactyly of third and fourth digits, low set malformed ears, micrognathia and club foot, was seen.&nbsp;<br /> Results: Our results and previous reports highlight the need to consider a somewhat consistent pattern of the first trimester combined screening in a pregnancy with triploidy and underscore the potential of this screening strategy to detect this chromosomal aberration earlier in pregnancy.&nbsp;<br /> Conclusion: Early prenatal diagnosis of this syndrome would provide women an opportunity to terminate an affected pregnancy earlier. This is also important in preventing the risks of associated later induced abortion or obstetric complications.</p> https://www.AJMB.org/En/Article.aspx?ID=10362 Mitra Eftekhariyazdi, Ali Khaligh, Behnaz Suizi, Maryam Naghibi Nasab, Davood Zare-Abdollahi Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Retraction: The Association of PON1 192 Q/R Polymorphism with the Risk of Idiopathic Male Infertility in Northern Iran <p>The Editorial Board of <em>Avicenna Journal of Medical Biotechnology (AJMB)</em> has decided to retract the original article entitled &quot;The Association of PON1 192 Q/R Polymorphism with the Risk of Idiopathic Male Infertility in Northern Iran&quot; published in the October-December 2018 issue due to a fact which is contrary to the scientific rules and regulation of AJMB.</p> https://www.AJMB.org/En/Article.aspx?ID=10388 Setareh Behrouzi, Farhad Mashayekhi, Mohammad Hadi Bahadori Sat, 22 Dec 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An Overview of 3D Bioprinting as a Novel Strategy in the Field of Tissue Engineering <p>Tissue engineering and regenerative medicine have typically matured from benchtop ideas to commercially applicable products in the clinic <sup>1</sup>. However, despite of typical advances in tissue engineering field, some limitations such as no reproducibility, no control of structure geometry including pore size and pore distribution and no integrity of cell distribution and migration in the construct have impelled the scientists into bioprinting technology. The most advantage of 3D bioprinting sounds to be precise fabrication of 3D deposition with controlled geometric structure and cells distribution <sup>2</sup>. Over the past decade, lots of researches in bioprinting of different tissues and organs has been carried out using different bioprinting modalities particularly inkjet based printing for skin tissue engineering and extrusion based printing for 3D depositions like bone, cartilage, heart, liver and heart valve. The key factor in extrusion-based bioprinting is bioink preparation, cell encapsulation in the bioink and bioprinting procedure. Indeed, preparation of bioink with appropriate gelation rate, suitable mechanical strength and elasticity which preserve cell viability and proliferation is the most challenge of bioprinting technology. So far, different strategies such as dual bioink cross-linkers, multi-step polymerization and using of core-shell nozzle have been reported to improve viability, quality and functionality of the printed product <sup>3</sup>. However, some issues including creation of constructs supporting <em>in vivo</em> vascularization, scaling up tissue constructs and in situ bioprinting have been remained to resolve. A few bioprinting products have been commercialized especially in orthopedic and skin tissue engineering fields and given the fast development of this industry over the past years; it supposed that the bioprinting products will eventually take a big proportion of the medical market to help patients suffering from a wide range of diseases in the future.</p> https://www.AJMB.org/En/Article.aspx?ID=285 Somaieh Kazemnejad Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Inclusion Body Expression and Refolding of Recombinant Bone Morphogenetic Protein-2 <p>Background: Bone Morphogenetic Protein-2 (BMP-2) is a cysteine rich growth factor expressed in homodimeric form and has a pivotal role in osteochondral development and fracture healing. Recent studies have benefited more from recombinant BMP-2 in osteochondral tissue engineering. Cost-effective and easy production at large scale makes <em>Escherichia coli (E. coli)</em> the first choice for recombinant protein expression programs. However, inclusion body aggregation and refolding process limits production and purification of recombinant BMP-2 in bacterial systems.<br /> Methods: BMP-2 encoded gene was optimized for expression in bacterial expression system and synthesized with proper restriction sites. The optimized sequence was then cloned in a pET28a expression vector and expressed in Origami<sup>TM</sup> <em>E. coli </em>strain. The aggregated and monomeric BMP-2 was refolded and purified comparing two oxidoreductase systems and refolding methods as well as different purification techniques. The biological activity of recombinant protein was investigated by increasing alkaline phosphatase activity (ALK) of ATDC-5 cell line.<br /> Results: No difference was observed between oxidoreductase systems in improving the efficiency of protein refolding. However, comparisons between two refolding methods showed that pooling monomeric BMP-2 that was refolded under mild condition with equal volume of it refolded under severe oxidoreductase condition resulted in production of more active dimeric protein.<br /> Conclusion: A new method for production of biologically active dimeric form of BMP-2 in <em>E. coli </em>expression system was established in this study.</p> https://www.AJMB.org/En/Article.aspx?ID=326 Davood Nasrabadi, Siamak Rezaeiani, Ali Sayadmanesh, Mohammadreza Baghaban Eslaminejad, Aliakbar Shabani Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Improvement Efficacy of Influenza Nanovaccine in Combination with Hemokinin-1 Molecular Adjuvant <p>Background: H9N2 avian influenza viruses have the potential to become the next human pandemic threat and next generation vaccine technologies are needed. Current studies introduce nanoparticles as a proper vaccine delivery vehicle for induction of protective immunity. In this study, the efficacy of chitosan nanoparticle-based H9N2 influenza vaccine with and without hemokinin-1 (HK-1) as a molecular adjuvant to induce protective immunity against the virus was examined.<br /> Methods: The H9N2 antigen was prepared in MDCK cells and inactivated with formalin. The inactivated antigen alone and in combination with HK-1 was encapsulated into chitosan nanoparticles. Groups of BALB/c mice received chitosan nanoparticle-based H9N2 antigen alone or in combination with HK-1 in a prime/boost platform via eye drop method. To evaluate the efficacy of the adjuvanted-nanovaccine candidate, systemic antibody responses were compared among the groups of animals.<br /> Results: Serological analysis indicated that mice receiving the HK-1/H9N2 nanoparticles formulation induced higher antibody titers that were sustained until the end of experiment. However, in the immunized mice, influenza specific antibody titers were comparable to that in the animals which were immunized either with inactivated antigen alone or the H9N2 nanoparticles without HK-1 adjuvant.<br /> Conclusion: The data demonstrate the synergy between HK-1 as an adjuvant and chitosan nanoparticles as a delivery antigen/adjuvant carrier in the improvement of influenza immune responses.</p> https://www.AJMB.org/En/Article.aspx?ID=327 Atefeh Dehghan, Shahla Shahsavandi, Leila Jabalameli Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of Dietary Polyunsaturated Fatty Acids on DNA Methylation and the Expression of <i>DNMT3b</i> and <i>PPARα</i> Genes in Rats <p>Background: Previous studies have suggested a protective role for Polyunsaturated Fatty Acids (PUFA) against cancer, cardiovascular, and other diseases. To provide new insights into the<em> in vivo</em> effects of PUFA on gene expression, the effects of dietary PUFA on <em>DNMT3b</em> and <em>PPAR&alpha;</em> gene expression and global DNA methylation were investigated in selected rat tissues.&nbsp;<br /> Methods: Thirty sprague-dawley rats were allotted into 3 dietary groups of ten animals each, received experimental diets containing PUFAs every day by gavages for 12 weeks as follows: control group fed a normal diet and water; n-3 PUFAs group received 300 <em>mg/kg/day</em> n-3 PUFAs supplementation; mixed-PUFAs group received 300 <em>mg/kg/day</em> of a mixture of n-3, -6, -9 PUFAs supplementations. The expressions of <em>DNMT3b</em> and <em>PPAR&alpha;</em> genes were quantitated using real-time RT-PCR. The genome-wide 5-methylcytosine contents in rat tissues were determined by ELISA method.&nbsp;<br /> Results: The average expression of the <em>DNMT3b </em>mRNA was 50% lower in the colon and liver of rats fed the n-3- or mixed-PUFAs supplemented diet than control group (p=0.00). However, <em>PPAR&alpha;</em> expression was significantly upregulated both in the colon and liver of PUFAs-supplemented rats (p&lt;0.001). No significant difference was observed in the blood, colon, and liver DNA methylation levels between PUFAs-supplemented and control animals.<br /> Conclusion: The results indicate that dietary PUFAs could modulate the expressions of <em>PPAR&alpha;</em> and <em>DNMT3b</em> genes in various rat tissues. The findings of this study provide additional insights into the in vivo mechanism of PUFA-mediated regulation of gene expression and could provide an opportunity to develop personalized diets for related disease control.</p> https://www.AJMB.org/En/Article.aspx?ID=325 Ehsan Maktoobian Baharanchi, Mostafa Moradi Sarabi, Fakhraddin Naghibalhossaini Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of <i>Anethum graveolens L.</i> on <i>In Vitro</i> Matured Mouse Oocytes and Granulosa Cells <p>Background: According to previous studies, <em>Anethum graveolens L.</em> (dill) aqueous extracts decreased the fertility of female rats. Therefore, the present study aimed to examine the effects of this herb on cultured granulosa cells and immature oocytes.&nbsp;<br /> Methods: The cells were obtained from 27-29 day immature superovulated mice. The oocytes were cultured in a petri dish consisting of 30 <em>&mu;l </em>drops of MEM-&alpha; and granulosa cells in a 24-well plate consisting of DMEM/F12 and different concentrations of 0, 10, 50, 100, 500, 1000, 10000 <em>&mu;g/ml </em>of dill seed aqueous extract (DSAE) in 37<sup>o</sup><em>C</em> and 5% CO<sub>2</sub>. Then, the <em>in vitro</em> maturation of oocytes, including Germinal Vesicle (GV), Germinal Vesicle Breakdown (GVBD), and meiosis ІІ (MІІ) and oocyte bioviability were determined. Granulosa cells were then extracted and their bioviability, apoptosis, chromatin condensation, and lipid synthesis were examined. Estrogen and progesterone concentrations and Alkaline Phosphatase (ALP) activity were measured by RIA and spectrophotometry respectively from the supernatant of granulosa cell culture.<br /> Results: The results revealed that concentration of 10000 <em>&mu;g/ml </em>of DSAE were toxic and damaged granulosa cell growth and oocytes maturation. Lower concentrations were the same in the control group and did not have any side effects on cell growth. The number of lipid droplets, estrogen and progesterone concentrations, and ALP activity increased with higher doses of DSAE compared to those in the control culture. Additionally, apoptosis and chromatin condensation increased in higher concentrations of DSAE-(500 and 1000 <em>&mu;g/ml</em>) treated cells. This herb extract decreased the oocytes maturation in dose-dependent manner.&nbsp;<br /> Conclusion: It was concluded that DSAE increased granulosa cells activity but damaged oocytes maturation, therefore it might be introduced as infertility agent.</p> https://www.AJMB.org/En/Article.aspx?ID=328 Malihezaman Monsefi, Bahareh Khalifeh, Samaneh Nikeghbal Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir HER-3 Knocking Down Induces G2/M Arrest in Gastric Cancer Cells <p>Background: The Human Epidermal growth factor Receptor-3 (HER-3) is a member of ErbB receptor family and has deficient kinase activity. HER-3 should heterodimerize with other members of ErbB receptor family, especially with HER-2, to transduce downstream signaling pathways. HER-3 co-expresses with other ErbB receptors in different cancers and overexpresses while the oncogenic signaling pathways such as Jak/Stat, MAPK, and PI3K/Akt are activated and promoted. Here, the expression level of HER-3 was evaluated in Iranian gastric adenocarcinoma&#39;s patients and the effects of HER-3 knocking down was investigated on cell cycle and cell viability of human gastric adenocarcinoma cell line of MKN45.<br /> Methods: In this study, 38 paraffin-embedded surgical adenocarcinoma specimens and their marginal non-tumor tissue samples were collected. Total RNAs were extracted and cDNAs were synthesized. Finally, the expression level of HER-3 was evaluated by real time PCR approach. Moreover, the human adenocarcinoma cell line of MKN45 was transfected with siRNA against HER-3 and the effects of its down-regulation were evaluated using MTT assay and cell-cycle analysis.<br /> Results: The data obtained from this study revealed HER-3 is significantly overexpressed in gastric tumors rather than non-tumor marginal tissues. Also, it was found that the expression level of HER-3 is elevated with tumor depth of invasion. Moreover, HER-3 knocking down promotes cell accumulation in G2/M phase of cell cycle and decreases cell viability in MKN45 cells which suggests a potential role for HER-3 in gastric adenocarcinoma tumorigenesis.<br /> Conclusion: Taken together, these results emphasize the importance of HER-3 receptor in diagnosis and prognosis of gastric adenocarcinoma.</p> https://www.AJMB.org/En/Article.aspx?ID=329 Ehsan Mokhtari, Hesamodin Mokhtari, Elham Moslemi Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Exploring Potential Biomarkers Underlying Pathogenesis of Alzheimer’s Disease by Differential Co-expression Analysis <p>Background: Alzheimer&#39;s Disease (AD) is the most common form of dementia in the elderly. Due to the facts that biological causes of AD are complex in addition to increasing rates of AD worldwide, a deeper understanding of AD etiology is required for AD treatment and diagnosis.&nbsp;<br /> Methods: To identify molecular pathological alterations in AD brains, GSE36980 series containing microarray data samples from temporal cortex, frontal cortex and hippocampus were downloaded from Gene Expression Omnibus (GEO) database and valid gene symbols were subjected to building a gene co-expression network by a bioinformatics tool known as differential regulation from differential co-expression (DCGL) software package. Then, a network-driven integrative analysis was performed to find significant genes and underlying biological terms.&nbsp;<br /> Results: A total of 17088 unique genes were parsed into three independent differential co-expression networks. As a result, a small number of differentially co-regulated genes mostly in frontal and hippocampus lobs were detected as potential biomarkers related to AD brains. Ultimately differentially co-regulated genes were enriched in biological terms including response to lipid and fatty acid and pathways mainly signaling pathway such as G-protein signaling pathway and glutamate receptor groups II and III. By conducting co-expression analysis, our study identified multiple genes that may play an important role in the pathogenesis of AD.&nbsp;<br /> Conclusion: The study aimed to provide a systematic understanding of the potential relationships among these genes and it is hoped that it could aid in AD biomarker discovery.</p> https://www.AJMB.org/En/Article.aspx?ID=330 Fereshteh Izadi, Mohammad Hasan Soheilifar Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of G/C (rs638405) Polymorphism in <i>β-secretase</i> Gene with Alzheimer’s Disease <p>Background: Alzheimer&#39;s Disease (AD) is a neurodegenerative disorder, which is the most common cause of dementia in the elderly. Accumulation of &beta;-amyloid plaques outside neurons is the most important pathological hallmark of AD, which is produced by cleavage of amyloid precursor protein by the Alzheimer&#39;s &beta;-secretase (<em>BACE1</em>). Since&nbsp;<em>BACE1 </em>is a key enzyme in the formation of &beta;-amyloid peptides, the purpose of this study was to assess the association between polymorphisms of G/C (rs638405) <em>BACE1</em> gene with sporadic AD in Khuzestan, Isfahan and Fars provinces in Iran.<br /> Methods: Genotypes were determined by the PCR&ndash;Restriction Fragment Length Polymorphism (PCR&ndash;RFLP) technique in two groups including 89 sporadic AD patients and 73 healthy subjects.<br /> Results: The findings of the <em>BACE1</em> G/C (rs638405) polymorphism revealed that there was no significant difference between AD patients and controls in men group; however, there was a weak difference in the frequency of CC genotype between patients and controls in women group (<em>&chi;</em><sup>2</sup>=3.333, df=1, p=0.068).<br /> Conclusion: The results of this study suggest that the G/C (rs638405) polymorphism of BACE1 gene might not be related with sporadic AD in Khuzestan, Isfahan and Fars provinces in Iran. However, our results do not support a genetic risk factor of this polymorphism for developing AD in male group of this study.</p> https://www.AJMB.org/En/Article.aspx?ID=331 Mostafa Chashmpoosh, Hossein Babaahmadi, Rouhollah Mousavidehmordi, Bita Shalbafan, Asma Mohammadi, Alireza Kheirollah Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Interaction Effect of RsaI and BamHI Polymorphisms of TGFα, BMP2 and BMP4 on the Occurrence of Non-Syndromic Cleft Lip and Palate in Iranian Patients <p>Background: Orofacial cleft is the most common congenital defect of the maxillofacial region. Its non-syndromic type is multi-factorial, and several genes are involved in its occurrence. This study aimed to assess the interaction effect of Rsal and BamHI polymorphisms of <em>Transforming Growth Factor-alpha (TGF&alpha;)</em> gene and Bone Morphogenetic Protein-2 (BMP2) and BMP4 variants on the occurrence of Non-Syndromic Cleft Lip and Palate (NSCLP) in the Iranian population.<br /> Methods: This case-control study was conducted on 120 children with NSCLP and 215 healthy children. Genotyping of the TGFA/BamHI (rs11466297), TGFA/RsaI (rs3732248), BMP4 (rs17563) and BMP2 (rs235768) was performed by Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) methods. Logistic regression was applied to determine the effective factors and the interaction effect of different variants on the occurrence of NSCLP.&nbsp;<br /> Results: Gender of patients had no significant association with the occurrence of NSCLP (p=0.335). Multiple logistic regression showed that the interaction effect of the&nbsp;aforementioned polymorphisms on the occurrence of NSCLP was not statistically significant (p=1.000).<br /> Conclusion: Although the individual effect of each of the BMP4, BMP2, RsaI and BamHI variants on the occurrence of NSCLP in the Iranian population has been previously confirmed, their interaction does not play a role in this respect.</p> https://www.AJMB.org/En/Article.aspx?ID=332 Saba Samadi, Asghar Ebadifar, Hamid Reza Khorram Khorshid, Koorosh Kamali, Mohammadreza Badiee Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Retraction: The Association of PON1 192 Q/R Polymorphism with the Risk of Idiopathic Male Infertility in Northern Iran <p>Background: Infertility is defined as the inability to achieve pregnancy after 12 months of regular unprotected sexual intercourse. Environmental and genetic factors are involved in male infertility. The polymorphism studies have a crucial role in disease recognition. Paraoxonase (PON) is an oxidant enzyme which is associated with inflammation, oxidative stress and lipid metabolism. The present study aimed to evaluate the relationship between <em>PON1 192 Q/R</em> polymorphism and the susceptibility to idiopathic male infertility.&nbsp;<br /> Methods: Samples were collected from 220 patients diagnosed with male infertility and 230 controls genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).&nbsp;<br /> Results: A significant difference in genotype distributions of <em>PON1 192 Q/R</em> polymorphism was observed between patients and controls (p=0.001). Our findings revealed that individuals with the variant QR had a significant decreased risk of idiopathic male infertility (OR=0.49, 95%CI=0.33&ndash;0.73, p=0.0004). Moreover, analyses showed that R allele may have a protective effect on susceptibility of idiopathic male infertility (OR=0.31, 95%CI=0.21-0.47, p=0.0001).&nbsp;<br /> Conclusion: The data from this study indicates that the <em>PON1 192 Q/R</em> polymorphism is associated with decreased risk of idiopathic male infertility. However, more studies should be considered with larger number of patients and control subjects to confirm our results.</p> https://www.AJMB.org/En/Article.aspx?ID=10358 Setareh Behrouzi, Farhad Mashayekhi, Mohammad Hadi Bahadori Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Pattern Analysis of Short Tandem Repeats Allele Frequencies among the Population of Khuzestan Province, South of Iran <p>Background: The basis of genetic fingerprinting and DNA profiling in forensic laboratories is the use of Short Tandem Repeats (STRs) according to local and ethnical genetics characteristics.<br /> Methods: Forensic parameters and allele frequencies for 15 autosomal STRs in 100 unrelated individuals from Khuzestan province, south Iran were determined. PCR was carried out for amplification of STRs and GeneMapper ID software was used for genotyping and allelic analyzing.&nbsp;<br /> Results: The Power of Exclusion (PE) varied between 0.332 (TPOX) and 0.768 (FGA). With exception of the THO1 (0.020), TPOX (0.014) and D18S51 (0.003), other STRs showed no deviation from the Hardy-Weinberg equilibrium (p&gt;0.05).<br /> Conclusion: Out of 15 STRs, 12 repeats seemed to be more useful and more powerful tools in identity and paternity determination for our studied population. Variation in our data analysis revealed that effective use of these 15 STR loci in forensic cases needed to be localized by collection and analysis of population data from the general population.</p> https://www.AJMB.org/En/Article.aspx?ID=10344 Seyed Farzad Hosseini, Mahdi Bijanzadeh, Elham Modheji Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A study on Association Between <i>CCRΔ32</i> Mutation and HCV Infection in Iranian Patients <p>Background: Mutations in the coding region of the Chemokine Receptor 5 (<em>CCR5</em>) genes reduce or eliminate <em>CCR5</em> expression in immune cells and progression of HCV infection. This study aimed to investigate the role of this mutation in HCV infection in Iranian patients in comparison with healthy individuals.<br /> Methods: 100 HCV infected patients and 100 healthy individuals were randomly selected. The <em>CCR5&Delta;32</em> genotypes were determined using specific primers and PCR method.<br /> Results: The agarose gel electrophoresis showed a189-bp fragment from wild type for both alleles of <em>CCR5 </em>gene. The <em>CCR5-&Delta;32</em> allele was not found in any HCV infected and healthy subjects.<br /> Conclusion: The mutation in <em>CCR5</em> gene was not detected in any of the two groups; therefore, the role of<em> CCR5</em> gene expression in immune cells and progression of HCV infection needs to be studied in larger samples in our country.</p> https://www.AJMB.org/En/Article.aspx?ID=10334 Farahnaz Bineshian, Asieh Hosseini , Zohreh Sharifi, Afsaneh Aghaie Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Study of Recombinant Factor IX in Drosophila Insect S2 Cell Lines Through Transient Gene Expression Technology <p>Background: Since the mass production of recombinant proteins requires the development of stable cell lines which is a time-consuming complex process, the use of transient expression on a large scale can be a comparatively useful alternative. Although various cell lines have been used for the expression of recombinant proteins, only a limited number of cells enjoy a high transfection characteristic and the ability to adapt to serum-free suspension culture easily. In the present study, the S2 cells from Drosophila insect with the ability to grow in suspension and serum-free cultures were used for the expression of factor IX (FIX) using Transient Gene Expression (TGE) technique.<br /> Methods: <em>Drosophila Schneider</em> (S2) cells were seeded in special roller bottles, and then, the cells were transfected with pMT-hFIX plasmid employing the calcium phosphate co-precipitation method. The stable S2-hFIX cells were also seeded in special roller bottles, separately. After the induction, recombinant FIX was quantified in conditioned media employing an ELISA. Moreover, its functional activity was examined using an aPTT assay.<br /> Results: The results showed that the expression of FIX through TGE technology was 1.6 times as high as that obtained through S2-FIX stable cells. Furthermore, the comparison of the FIX expression in S2 cells through TGE techniques with that obtained in previous studies in HEK cells or CHO cells revealed that S2 cells were more efficient in terms of FIX expression.<br /> Conclusion: The S2 cells with the capability to grow in suspension and serum-free cultures are a suitable alternative for transient expression for the large scale production of proteins.</p> https://www.AJMB.org/En/Article.aspx?ID=321 Jafar Vatandoost, Kambiz Kafi Sani Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Rapid and Simple Detection of <i>Escherichia coli</i> by Loop-Mediated Isothermal Amplification Assay in Urine Specimens <p>Background: To improve urinary tract infection detection, we evaluated the specificity and sensitivity of Loop-mediated isothermal Amplification Method (LAMP) for detection of the <em>Eschericia coli (E. coli)</em> in urine samples, for the first time.&nbsp;<br /> Methods: Primers were designed to target the malB gene of <em>Escherichia coli.</em> LAMP assay was performed on urine specimens collected from patients with urinary tract infection symptoms.<br /> Results: As expected, LAMP was more specific and sensitive than direct microscopic tests. LAMP assay showed the best detection limit of DNA copies with 1.02 copies.<br /> Conclusion: LAMP method offers several advantages in terms of sensitivity, rapidness and simplicity for detection of <em>E. coli</em> infection in urine samples. The LAMP method would be highly suitable for the early detection of the UTIs and also comfort quick diagnosis of UTI in clinical laboratories with limited equipment.</p> https://www.AJMB.org/En/Article.aspx?ID=333 Reihaneh Ramezani, Zahra Kardoost Parizi, Nassim Ghorbanmehr, Hamideh Mirshafiee Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>KIF21A</i> Gene c.2860C>T Mutation in CFEOM1A: The First Report from Iran <p>Congenital Fibrosis of the Extra Ocular Muscles1 (CFEOM1) is an autosomal dominant condition, caused by mutation in the <em>KIF21A</em> and <em>TUBB3</em>. It is characterized by congenital non-progressive restrictive ophthalmoplegia and ptosis. Mutational analysis of the known genes in such rare diseases by Sanger sequencing not only prevents wasting the time and expenses but also speeds diagnosis process, genetic counseling, and the possibility of prenatal diagnosis. Here, for the first time, association of pathogenic variant c.2860C&gt;T in <em>KIF21A</em> gene in an Iranian family with positive history of CFEOM1A was reported.</p> https://www.AJMB.org/En/Article.aspx?ID=10357 Masoomeh Ramahi, Abolfazl Rad, Ebrahim Shirzadeh, Maryam Najafi Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Letter to: Arylamine N-acetyltransferase 2 Polymorphisms and the Risk of Endometriosis https://www.AJMB.org/En/Article.aspx?ID=10386 Fabio Barra, Lorenzo Ferro Desideri, Carolina Scala, Simone Ferrero Mon, 01 Oct 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Monoclonal Antibody for Reducing Memory and Learning Problems in Schizophrenia <p>Schizophrenia is a chronic debilitating psychiatric illness that accounts for a significant portion of the burden caused by mental illnesses worldwide. Primary negative symptoms of schizophrenia are not secondary to extrapyramidal, depressive or positive symptoms <sup>1,2</sup>. Negative symptoms are the core features of the illness which are associated with long-term functional disability and poor outcome <sup>1,3</sup>. These symptoms include deficits in social and emotional functioning, blunted affect and lack of spontaneity. There is a growing body of evidence for the role of inflammation and immune system dysregulation in psychiatric disorders <sup>4</sup>. Although the precise pathophysiology of schizophrenia is not completely known, a number of recent studies support the probable pathologic role of immunologic dysfunction in this disorder. Assessing serum cytokine levels such as interleukin 1 (IL-1), IL-2, IL-6, and chemokine CCL11 in schizophrenic patients demonstrates profound alterations compared to healthy matched controls <sup>4</sup>. Furthermore, increased cyclooxygenase-2 (COX-2) expression as well as prostaglandin E2 production in schizophrenia, are among other postulated etiologies supported by recent studies <sup>4</sup>. On the other hand, it has been shown that immune response imbalance is associated with decreased activity of indoleamine 2, 3-dioxygenase enzyme which subsequently leads to accumulation of kynurenic acid, an endogenous antagonist of glutamate N-methyl-D-aspartate (NMDA) receptor. Compared with anit-inflammatory agents like celecoxib and NAC, monoclonal antibodies also have more potent anti-inflammatory properties. Indeed, COX-2 inhibitors and N-acetylcysteine have moderate efficacy in treatment of schizophrenia and autism <sup>1,2,5</sup>. British scientists have begun testing a radically new approach to treating schizophrenia based on emerging evidence that it could be a disease of the immune system. Evidence for prenatal and premorbid immune risk factors for the development of schizophrenia in the offspring is highlighted <sup>6,7</sup>. Then key evidence for immune dysfunction in patients with schizophrenia is considered. A collaboration between the Medical Research Council (MRC) and King&rsquo;s College London, is based on emerging evidence that schizophrenia may be an immune disease. The drug, natalizumab, works by targeting microglia, a type of immune cell residing in the brain which are thought to be overactive in people at risk of developing schizophrenia <sup>6,7</sup>.</p> https://www.AJMB.org/En/Article.aspx?ID=315 Shahin Akhondzadeh Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Apoptosis of Adipose-Derived Stem Cells Induced by Liposomal Soybean Phosphatidylcholine Extract <p>Background: Recently, Phosphatidylcholine (PC) has been used as an off-label treatment for lipolysis injection, which is associated with inflammatory reaction due to sodium deoxycholate, an emulsifier, so that inflammation as side effect occurs in those patients. Liposome formulation from <em>soybean</em> lipid was thought to be a better and safer alternative. This study aimed to analyze the mechanism of Liposomal Soybean Phosphatidylcholine (LSPC) extract from Indonesian soybeans (containing 26% PC) to induce Adipose-derived Stem Cells (ASCs) death <em>in vitro</em>.&nbsp;<br /> Methods: Liposomes were prepared using thin film hydration method followed by a stepwise extrusion process to produce a small amount of 41.0-71.3 <em>nm</em>. Liposomal soybean phosphatidylcholine extract (LSPCE), liposomal purified PC (LPCC), and solution of PC+SD were used for comparison. Annexin V fluorescein Isothiocyanate/ Propidium Iodide (FITC/PI) double staining by flow cytometry and also measurement of caspase-3 activity using ELISA were used to quantify the rate of apoptosis. ASCs viability was measured using MTT assay after induction with liposomes. Morphological changes were shown using a phase-contrast, inverted microscope and Transmission-Electron Microscope (TEM).&nbsp;<br /> Results: The flow cytometry results showed that cells treated with both LSPCE and LPCC showed increase in early apoptosis beginning at 6 <em>hr </em>after incubation, which was confirmed by caspase 3 measurement. MTT assay showed that both LSPCE and LPCC could decrease viability of cells. Cells treated with LSPCE and LPCC showed some rounded cells, which was an early sign of cell death. Cells treated with SD showed extensive membrane damage with necrosis features using TEM.&nbsp;<br /> Conclusion: The results above demonstrated that LSPCE induced apoptosis of ASCs.</p> https://www.AJMB.org/En/Article.aspx?ID=316 Reza Purwoko, Iis Rosliana, Siti Sobariah, Nabila Hermana, Silvani Permatasari, Dewi Wulandari, Puji Sari, Ernie Purwaningsih, L Chaidir, Hans-Joachim Freisleben, Jeanne Pawitan, Kusmarinah Bramono Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>LACK</i> Gene’s Immune Response Induced by Cocktail DNA Vaccine with <i>IL-12</i> Gene Against Cutaneous Leishmaniasis in BALB/c Mice <p>Background: Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which is an obligate intracellular parasite in the infected host. Individuals who have been recovered from clinical leishmaniasis develop strong immunity against reinfection. DNA vaccines are the new type of vaccines that induce expression of protein eukaryotic cells. DNA vaccines can be stimulated by the cellular and humoral immune responses using one or several genes.&nbsp;<br /> Methods: A DNA vaccine containing plasmids encoding the <em>pcLACK+pcTSA</em> genes of <em>Leishmania major (L. major) </em>(MHRO/IR/75/ER) in the vicinity of <em>IL-12</em> gene expression was made and then its protective efficacy in comparison with single-gene of LACK was evaluated. Also, BALB/c mice were immunized intramuscularly three times. The humoral and cellular immune responses were evaluated after immunization with pcLACK, pcLACK+pcTSA+pCAGGS-IL12, and then challenged with <em>L. major</em>.&nbsp;&nbsp;<br /> Results: Humoral response and IFN-&gamma; values were significantly higher than control groups after immunization with pcLACK, pcLACK+pcTSA+pCAGGS-IL12 and challenge with<em> L. major</em> (p&le;0.05). IL-4 values were increased in the control groups in such a way that they were remarkably higher than the pcLACK, pcLACK+pcTSA+ pCAGGS-IL12 groups (p&le;0.05) after immunization and challenge with <em>L. major</em>.<br /> Conclusion: The survival time of the immunized mice with pcLACK, pcLACK+pcTSA+ pCAGGS-IL12 groups was higher than the control groups. Then, DNA vaccine of pcLACK appeared to be likely able to induce more protection against infection with L. major in mice. Therefore, cocktail DNA is effective to enhance specific immunity.</p> https://www.AJMB.org/En/Article.aspx?ID=317 Oghlniaz Jorjani, Fatemeh Ghaffarifar, Zohreh Sharifi, Abdolhossein Dalimi, Hajar Ziaei-Hezarjaribi, Benyamin Talebi Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Soluble Expression of Humanized Anti-CD20 Single Chain Antibody in <i>Escherichia coli</i> by Cytoplasmic Chaperones Co-expression <p>Background: CD20 is an important cell surface receptor that is used for target therapy of B cell lymphoma and some related blood diseases due to vital function of CD20. In previous studies, a Rituximab based humanized single chain variable fragment (scFv) antibody showed good reactivity against B cell related cancer cells. But this recombinant protein produced Inclusion Bodies (IBs) in <em>Escherichia coli (E. coli)</em> cytoplasm. The aim of this study was to investigate the effect of coexpression with cytoplasmic chaperones on expression and solubility of humanized anti-CD20 scFv in <em>E. coli</em>.<br /> Methods: For this purpose, the fragment coding for anti-CD20 huscFv subcloned into the pET22b (+) and transformed into the <em>E. coli</em> BL21 (DE3) was evaluated. In order to inhibit the production of IBs, the effects of co-expression with cytoplasmic chaperones GroEL, DnaK, GroES, Tig, DnaJ and GrpE were investigated.&nbsp;<br /> Result: Coexpression with cytoplasmic chaperones led to increased soluble expression of anti-CD20 recombinant protein. Among investigated chaperones, pKJE7 chaperone plasmid containing DnaJ, GrpE, DnaK chaperone genes had significant effects with an expression yield of 325 <em>&micro;g/ml</em> soluble anti-CD20 scFv.&nbsp;<br /> Conclusion: The result of this study demonstrated remarkable effect of pKJE7 chaperone on enhancement of soluble expression of anti-CD20 huscFv antibody in <em>E. coli</em>.</p> https://www.AJMB.org/En/Article.aspx?ID=318 Mohammadreza Yousefi, Safar Farajnia, Ahad Mokhtarzadeh, Bahman Akbari, Shiva Ahdi Khosroshahi, Mina Mamipour, Hassan Dariushnejad, Vahideh Ahmadzadeh Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Feasibility Study to Evaluate <i>Bacillus subtilis</i> as a Host for Producing Recombinant Human Parathyroid Hormone <p>Background: Biosynthetic teriparatide (1-34) (TPD) is a N-terminally truncated version of human parathyroid hormone (hPTH). The recombinant form of this polypeptide has been expressed in <em>Escherichia coli (E. coli)</em> and approved as the first anabolic treatment of osteoporosis in the EU and the USA. Feasibility of expression and secretion of a tag- fused form of TPD into <em>Bacillus subtilis (B. subtilis)</em> was examined due to several advantages of <em>B. subtilis </em>over <em>E. coli </em>in production of recombinant proteins with pharmacological activities.<br /> Methods: A codon optimized gene containing TPD open reading frame carrying enterokinase site in its upstream was fully synthesized. According to our cloning scheme, this synthetic polynucleotide was used as a template for PCR amplification using engineered primers in such a way that a polyhistidin tag was added in frame to the upstream of the amplicon as well as two restriction sites at its ends. The resulted amplicon, a cassette containing His-tag, enterokinase site and TPD, from 5&rsquo; to 3&rsquo;, was cloned into pTZ57R/T vector and subjected to sequencing.The cassette was then subcloned into pHT43 shuttle vector and transformed into <em>B. subtilis</em>. Expression of target protein was analyzed by SDS-PAGE and western blotting upon induction by IPTG.<br /> Results: The accuracy of construction of pHT43-TPD was confirmed by sequencing and restriction map analyses. SDS-PAGE and western blotting results showed that the recombinant fusion form of hPTH was successfully expressed and secreted into cytoplasm and extracellular medium.<br /> Conclusion: TPD may be successfully expressed and secreted in <em>B. subtilis</em>; however, optimization of expression conditions is required for enhancing target protein yield.</p> https://www.AJMB.org/En/Article.aspx?ID=319 Mahdi Karimi, Farida Behzadian, Hamideh Rouhaninejad, Sanaz Yari Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Distinct Role of Small Heat Shock Protein 20 on HCV NS3 Expression in HEK-293T Cell Line <p>Background: Hepatitis C (HCV) is known as a serious blood-borne disease that infects millions of people globally. NS3 is a conserved non-structural sequence of hepatitis C virus which has a major role in activating specific CTL responses. As known, there is no effective vaccine against HCV infection, thus it is required to design a specific regimen of vaccination. Recently, the strong immunological properties of Heat shock proteins (Hsps) led to their use as immunomodulators and an antigen carrier for subunit vaccine candidates. In the current study, the role of Hsp20 was evaluated as a HCV NS3 gene carrier in mammalian cell line.<br /> Methods: At first, the recombinant plasmids of pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3 were constructed and their accuracy was confirmed by digestion and sequencing. Then, all recombinant plasmids were transfected into HEK293T cells by Lipofectamine and TurboFect gene delivery systems. Finally, the expression of proteins was assessed by fluorescent microscopy, western blotting, and flow cytometry.&nbsp;<br /> Results: In western blotting, the 47, 59, and 79 kDa bands were detected for pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3, respectively. The percentage of NS3-Hsp20-GFP protein expression was ~67% by TurboFect and ~50% by Lipofectamine indicating high potency of TurboFect delivery system. Furthermore, the expression of Hsp20 (~83%) was higher than NS3 (~58%) in the cells transfected by TurboFect using flow cytometry analysis. This result was confirmed in the expression of Hsp20-NS3 fusion (~67%) in which Hsp20 increased the delivery of HCV NS3 <em>in vitro</em>. The same data were obtained by Lipofectamine transfection reagent.&nbsp;<br /> Conclusion: Briefly, our data confirmed the role of Hsp20 as a suitable antigen carrier for DNA vaccine design.</p> https://www.AJMB.org/En/Article.aspx?ID=320 Marzieh Basirnejad, Azam Bolhassani, Seyed Mehdi Sadat Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>In silico</i> Biological Activity of Steroids from the Marine Gastropods <i>Telescopium telescopium</i> Collected from South West Coast of India <p>Background: The purpose of this study was to investigate the sterol profiling and predict the pharmacological potential of marine gastropod <em>Telescopium telescopium (T. telescopium)</em>, collected from mangrove ecosystem in the South west coast of India.<br /> Methods: Sterol fractions were separated from the crude lipids using 15% ethyl acetate. Ethyl acetate fractions were dried under ultrahigh purity N<sub>2</sub> and analyzed using GC-MS. The biological activity was predicted using the software CLC-Pred; <em>In silico</em> predictions of cytotoxicity for tumor and non-tumor cell lines and PASS.&nbsp;<br /> Results: This study proved the existence of four sterols, of which cholesterol was abundant. It was found that most of the steroids profiled from <em>T. telescopium</em> displayed activity against reproductive system as well as skin related diseases.&nbsp;<br /> Conclusion: The predicted anti infertility and skin related activity of the steroids identified from the marine gastropod <em>T. telescopium</em> is useful to attract industrial interest towards this species which will be helpful in rising new combinations with added therapeutic and nutritional worth.</p> https://www.AJMB.org/En/Article.aspx?ID=322 A Ragi, P Leena, K Prashob Peter, S Nair Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Arylamine N-acetyltransferase 2 Polymorphisms and the Risk of Endometriosis <p>Background: Human arylamine N-acetyltransferase 2 (<em>NAT2</em>) gene has a key role in xenobiotic metabolism through the conjugation of acetyl group to xenobiotic substances. <em>NAT2</em> has been suggested as a susceptibility factor in endometriosis; however, the results of studies have been controversial. In this study, the association of <em>NAT2</em> polymorphisms with susceptibility to endometriosis was evaluated in an Iranian population.&nbsp;<br /> Methods: This is an association study and totally 141 women with diagnosis of endometriosis and 158 healthy women as control group were analyzed for <em>NAT2</em> gene polymorphisms (C481T, A803G, G857A and G590A) by PCR-RFLP methods.<br /> Results: The 590 GA genotype was significantly lower (p=0.001; OR=0.42, 95% CI: 0.25-0.71) in the patients (38.3%) than the control group (55.1%). The 590A allele was significantly lower (p=0.033; OR=0.69, 95% CI: 0.49-0.79) in the patients (31.2%) compared with the controls (39.6%). Analysis of haplotypes showed that NAT2 481C, 803A, 590A, 587A combination was significantly different between the case and control women (p= 0.029; OR=3.11, 95% CI: 1.13-8.52).<br /> Conclusion: The <em>NAT2</em> G590A SNP may be associated with susceptibility to endometriosis and the 590A allele may have a protective role in development of endometriosis. The <em>NAT2</em> 481C, 803A, 590A, 587A haplotype was associated with a higher risk of endometriosis in Iranian population.</p> https://www.AJMB.org/En/Article.aspx?ID=10342 Diman Fayez, Kioomars Saliminejad, Shiva Irani, Koorosh Kamali, Toktam Memariani, Hamid Reza Khorram Khorshid Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of <i>WNT3</i> Variations and Risk of Non-Syndromic Cleft Lip and Palate in a Population of Iranian Infants <p>Background: Nonsyndromic cleft lip and/or palate (NSCL/P) is the most common orofacial birth defect, often attributed to ethnic and environmental differences. Up to now, linkage analyses and genome-wide association studies have identified several genomic susceptibility regions for NSCL/P. The <em>WNT</em> genes including <em>WNT3</em> are strong candidates for NSCL/P, since they are involved in regulating mid-face development and upper lip fusion. This study tested association of the WNT3 polymorphisms, rs3809857 G/T and rs9890413 G/A, with the risk of NSCL/P in a population of Iranian infants.<br /> Methods: The allelic and genotypic frequencies for each participant were determined in 113 unrelated Iranian subjects with NSCL/P and 220 control subjects using PCR and restriction fragment length polymorphism (RFLP) methods. A p-value of 0.05 was considered statistically significant.&nbsp;<br /> Results: The <em>WNT3</em> rs3809857 GT genotype was significantly lower (p=0.039, OR=0.55, 95% CI=0.30-0.97) in the NSCL/P (21.2%) than the control group (30.42%). For the WNT3 rs9890413 G/A polymorphism, neither genotype nor allele frequencies were significantly different between the case and control groups.<br /> Conclusion: Our results indicated that the <em>WNT3</em> rs3809857 GT genotype may have a protective effect against NSCL/P in Iranian population.</p> https://www.AJMB.org/En/Article.aspx?ID=323 Homa Farrokhi Karibozorg, Nahid Masoudian, Kioomars Saliminejad, Asghar Ebadifar, Koorosh Kamali, Hamid Reza Khorram Khorshid Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>Interleukin-2</i> and <i>Interferon-Gamma</i> Single Nucleotide Polymorphisms in Iranian Patients with Chronic Heart Failure <p>Background: Inflammatory cytokines have been known to be associated with Chronic Heart Failure (CHF). Given the importance of cytokines in the context of the failing heart, the prevalence of Interleukin-2 (IL-2) and Interferon-gamma (IFN-&gamma;) polymorphisms was studied in patients with CHF due to ischemic heart disease in a case-control study.<br /> Methods: Fifty-six Iranian patients with CHF were enrolled in this study as the case group and compared with 139 healthy subjects, using polymerase chain reaction with sequence-specific primers method, so as to determine the frequency of alleles, genotypes and haplotypes of <em>IFN-&gamma;</em> (+874 A/T) and <em>IL-2</em> (-330 G/T, +166 G/T) SNPs.&nbsp;<br /> Results: The GG genotype at <em>IL-2</em> -330 in patients with CHF was significantly overrepresented in comparison with the control group (p=0.013). Such a positive genotypic association was also observed for <em>IL-2</em> +166/TT (p=0.022). Meanwhile, the GT genotype frequency at <em>IL-2</em> -330/GT in the patient group was significantly lower than the one in healthy controls (p=0.049). No significant association was detected between the <em>IFN-&gamma;</em> gene polymorphisms and individuals&rsquo; susceptibility to CHF.<br /> Conclusion: Certain genotypes in <em>IL-2</em> gene were overrepresented in patients with CHF, which could render individuals more vulnerable to this disease.</p> https://www.AJMB.org/En/Article.aspx?ID=10343 Mohammad Jafar Mahmoudi, Sara Harsini, Elham Farhadi, Mona Hedayat, Mohammad Taghvaei, Maryam Mahmoudi, Maryam Sadr, Nilufar Esfahanian, Ebrahim Nematipour, Keramat Nourijelyani, Ali Akbar Amirzargar, Nima Rezaei Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association Between rs6759298 and Ankylosing Spondylitis in Iranian Population <p>Background: Ankylosing Spondylitis (AS) is a chronic autoinflammatory Spondyloar-thropathy (SpA) which is characterized by sacroiliitis, which progresses to the axial skeleton. It seems that non-Human Leukocyte Antigen (HLA) and also HLA-B27 are associated with the susceptibility and pathogenesis of the disease. The recent Genome-Wide Association Studies (GWASs) have reported intergenic rs6759298 to be associated with AS etiology. The aim of this study was investigation of the rs6759298 polymorphism in Iranian AS patients. In addition, probable correlations with clinical indices and manifestations were considered.<br /> Methods: This study included 403 patients with AS. The control group consisted of 506 healthy individuals who were matched for sex, age, and ethnicity with AS group. Genotyping of rs6759298 was determined using the Amplification-Refractory Muta-tion System-Polymerase Chain Reaction (ARMS-PCR).<br /> Results: The GG genotype and G allele were found to be significantly more prevalent in the patient group in comparison to the control group [(p=2&times;10<sup>-6</sup> and 7.44&times;10<sup>-9</sup>; OR (95% CI) =2.16 (1.56-2.98) and 1.73 (1.43-2.08)], respectively.<br /> Conclusion: No associations were found between patients with three genotypes and any disease manifestations or clinical indices. This investigation confirmed a highly significant association of rs6759298 with disease susceptibility, with no effect on disease progress or clinical presentations. Since rs6759298 belongs to the 2p15 gene desert, further studies would elucidate the exact role of this polymorphism in the pathogenesis of AS.</p> https://www.AJMB.org/En/Article.aspx?ID=324 Mahdi Mahmoudi, Masoud Garshasbi, Amir Ashraf-Ganjouei, Ali Javinani, Mahdi Vojdanian, Masoumeh Saafi, Nooshin Ahmadzadeh, Ahmadreza Jamshidi Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Menstrual Blood-derived Stromal Stem Cells Augment CD4+ T Cells Proliferation <p>Background: It is more than sixty years that the concept of the fetal allograft and immunological paradox of pregnancy was proposed and in this context, several regulatory networks and mechanisms have been introduced so far. It is now generally recognized that mesenchymal stem cells exert potent immunoregulatory activity. In this study, for the first time, the potential impact of Menstrual blood Stem Cells (MenSCs), as surrogate for endometrial stem cells, on proliferative capacity of CD4+ T cells was tested.<br /> Methods: MenSCs and Bone marrow Mesenchymal Stem Cells (BMSCs) were isolated and assessed for their immunophenotypic features and multi-lineage differentiation capability. BMSCs and MenSCs with or without IFN&gamma; pre-stimulation were co-cultured with purified anti-CD3/CD28-activated CD4+ T cells and the extent of T cell proliferation at different MenSCs: T cell ratios were investigated by CSFE flow cytometry. IDO activity of both cell types was measured after stimulation with IFN&gamma; by a colorimetric assay.<br /> Results: MenSCs exhibited dual mesenchymal and embryonic markers and multi-lineage differentiation capacity. MenSCs significantly increased proliferation of CD4+ cells at ratios 1:2, 1:4 and 1:8. IFN&gamma; pre-treated BMSCs but not MenSCs significantly suppressed CD4+ T cells proliferation. Such proliferation promoting capacity of MenSCs was not correlated with IDO activity as these cells showed the high IDO activity following IFN&gamma; treatment.<br /> Conclusion: Although augmentation of T cell proliferation by MenSCs can be a basis for maintenance of endometrial homeostasis to cope with ascending infections, this may not fulfill the requirement for immunological tolerance to a semi-allogeneic fetus. However, more investigation is needed to examine whether or not the immunomodulatory properties of these cells are affected by endometrial microenvironment during pregnancy.</p> https://www.AJMB.org/En/Article.aspx?ID=10363 Mehdi Aleahmad, Alireza Ghanavatinejad, Mahmood Bozorgmehr, Mohammad-Reza Shokri, Shohreh Nikoo, Maryam Tavakoli, Somaieh Kazemnejad, Fazel Shokri, Amir-Hassan Zarnani Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>TNFRSF13B/TACI</i> Alterations in Turkish Patients with Common Variable Immunodeficiency and IgA Deficiency <p>Background: The Transmembrane Activator and Calcium modulator ligand Interactor (TACI), encoded by <em>TNFRSF13B/TACI</em> gene, is mutated in some patients with Common Variable Immunodeficiency (CVID) and IgA Deficiency (IgAD). The purpose of the study was to investigate for the first time in Turkish patients the prevalence of <em>TNFRSF13B</em> alterations in CVID, selective and partial IgAD patients.&nbsp;<br /> Methods: Forty two CVID, 36 selective IgAD, 34 partial IgAD and 25 healthy controls were included. All patients were examined for <em>TNFRSF13B</em> gene mutations by PCR.&nbsp;<br /> Results: The percentages of <em>TNFRSF13B</em> mutations in CVID, selective and partial IgAD patients were 7.1, 2.7 and 2.9%, respectively. No disease causing <em>TNFRSF13B</em> mutation in healthy controls was found. Patients with TACI mutations had recurrent respiratory tract infections. None of them experienced autoimmunity, bronchiectasis or granulomatous disease. In conclusion, <em>TNFRSF13B </em>mutations were present not only in CVID patients, but also in IgAD cases.<br /> Conclusion: Modifier genes as well as their combination with other genetic or environmental factors may play an important role in the development of the immunodeficiency phenotype.</p> https://www.AJMB.org/En/Article.aspx?ID=10346 Neslihan Karaca, Ezgi Severcan, Burcu Guven, Elif Azarsiz, Guzide Aksu, Necil Kutukculer Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Polymorphisms in the Cholinergic Receptors Muscarinic (<i>CHRM2 and CHRM3</i>) Genes and Alzheimer’s Disease <p>Background: Disruption of the cholinergic neurotransmitter pathway which is important for cognition, memory and learning abilities has been reported in Alzheimer&rsquo;s Disease (AD) patients. The receptors involved include the Cholinergic Receptors Muscarinic (<em>CHRM</em>). <em>CHRM2</em> gene has been associated with intelligence, personality traits, substance dependence and depression. <em>CHRM3</em> has been found to heterodimerize with <em>CHRM2</em>.<br /> Methods: DNA samples from 240 AD patients with SNPs rs6962027 of <em>CHRM2</em> gene and rs7511970 of <em>CHRM3</em> gene were amplified using PCR and genotyped using Restriction Fragment Length Polymorphism (RFLP). Chi-squared test was done to check if the genes are in Hardy-Weinberg equilibrium.&nbsp;<br /> Results and Conclusion: Although the results did not show significant associations, these data denote plausible interaction between TT in SNP rs6962027 in <em>CHRM2 </em>gene and TT in SNP rs7511970 in CHRM3 gene affecting AD risk. SNP rs7511970 of <em>CHRM3</em> gene may also exert an influence on late-onset AD.</p> https://www.AJMB.org/En/Article.aspx?ID=10347 Lim Ya Chee, Alaistair Cumming Tue, 19 Jun 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Therapeutic Monoclonal Antibodies and Emergence of Their Biosimilars <p>Antibodies are proteins of the immune system that are produced by B-lymphocytes. These proteins exert their effects by recognizing and binding to their targets (antigens). A monoclonal antibody (mAb) is originally produced by a single B-cell. Production of mAbs was first introduced in 1975 using cell fusion technique and hybridoma cell production. A Hybridoma is formed by fusion of an antibody producing B-lymphocyte and a myeloma cell line. These cells have two main characteristics, production of uniform, monospecific antibodies (mAbs) that originate from the B-cell and immortality that comes from the myeloma cell line. A hybridoma cell line is thus acting like a biological factory that produces and secretes mAbs into the cell culture medium. Most of the mAbs have been produced in mice and are thus proteins of murine origin. mAbs were later found to be able to bind biological targets like tumor antigens, molecules involved in autoimmune and infectious disease-related molecules, etc. This led to emergence of therapeutic monoclonal antibodies. However, since the first mAbs were murine proteins (OKT3 was injected to kidney transplant patients to prevent graft rejection), their repeated administration raised Human Anti Mouse Antibody (HAMA) in the patients that resulted in neutralization of the injected mAb (OKT3). The solution was to genetically change the murince antibodies to human antibodies. In this regard chimeric (%80 -%90 human), humanized (&ge; %90 human) and fully human (%100 human) therapeutic mAbs were produced. At present more than 50 therapeutic mAbs are on the market with more than 120 billion USD global market share. These mAbs have shown very good therapeutic effect in treatment of cancers, autoimmune and infectious diseases. However, these drugs are very expensive and thus their patient accessibility is limited. Although, they are protected by patents, some patents have already expired and some are close to expiration dates. Here, biosimilar versions of these drugs have started to immerge. Biosimilars are defined as biological drugs that are highly similar but not identical to the biological reference (original or originator) drug. The approval of biosimilars to enter the biopharmaceutical market is governed by the regulatory bodies in different countries. This happens under strict and comprehensive comparability exercise. The biosimilarity determination procedure is planned to ensure that the difference between the originator and the biosimilar drug is not clinically significant. The assessment includes immunochemical and physicochemical properties, biological activity, structural similarity, purity, contamination with impurities like host cell protein and DNA. In addition, in vivo pharmacology studies like pharmacokinetic and pharmaco-dynamic characteristics as well as, efficacy, safety and tolerability must be determined and approved. Moreover, after market analyses like pharmaco-epidemiological studies should also be done.<br /> These procedures are also necessary to be performed to assure the prescribing physicians to suggest them to their patients. Thus, the biosimilars, due to their lower production costs, are expected to introduce huge amounts of cost savings to healthcare systems and more importantly, increase affordability/accessibility of biological treatment.</p> https://www.AJMB.org/En/Article.aspx?ID=295 Mahmood Jeddi-Tehrani Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of Hypoxia on Biology of Human Leukemia T-cell Line (MOLT-4 cells) Co-cultured with Bone Marrow Mesenchymal Stem Cells <p>Background: One of the most significant problems in the treatment of leukemia is the expansion of resistance to chemotherapeutic agents. Therefore, assessing the drug resistance and especially the drug resistance genes of leukemic cells is important in any treatment. The impact of Mesenchymal Stem Cells (MSCs) and hypoxic condition have been observed in the biological performance of majority of leukemic cells.<br /> Methods: MOLT-4 cells were co-cultured with MSCs in the hypoxic condition induced by Cobalt Chloride (CoCl<sub>2</sub>) for 6 and 24 <em>hr</em>. Then, apoptosis of cells was analyzed using annexin-V/PI staining and expression of the drug resistance genes including MDR1, MRP, and BCRP along with apoptotic and anti-apoptotic genes, including BAX and BCL2, was evaluated by real-time PCR.<br /> Results: The hypoxic condition for MOLT-4 cells co-cultured with MSCs could significantly increase the expression of MDR1 and BCRP genes (p&lt;0.05) which are involved in drug resistance. Also, the results indicated that this condition significantly increases the expression of BCL2 (p&lt;0.05) and reduces the apoptosis in MOLT- 4 cells co-cultured with MSCs in the hypoxic condition.<br /> Conclusions: These effects can demonstrate the important role of hypoxia and MSCs on the biological behavior of Acute Lymphoblastic Leukemia (ALL) cells that may lead to particular treatment outcomes.</p> https://www.AJMB.org/En/Article.aspx?ID=312 Sina Baharaghdam, Mehdi Yousefi, Ali Akbar Movasaghpour, Saeed Solali, Mehdi Talebi, Milad Ahani-Nahayati, Hamid Lotfimehr, Karim Shamsasanjan Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Evaluation of Astaxanthin Effects on Differentiation of Human Adipose Derived Stem Cells into Oligodendrocyte Precursor Cells <p>Background: Multiple Sclerosis (MS) has been explained as an autoimmune mediated disorder in central nerve system. Since conventional therapies for MS are not able to stop or reverse the destruction of nerve tissue, stem cell-based therapy has been proposed for the treatment of MS. Astaxanthin (AST) is a red fat-soluble xanthophyll with neuroprotection activity. The aim of this study was evaluation of pre-inducer function of AST on differentiation of human Adipose- Derived Stem Cells (hADSCs) into oligodendrocyte precursor cells.<br /> Methods: After stem cell isolation, culture and characterization by flow cytometry, hanging drop technique was done for embryoid body formation. In the following, hADSCs were differentiated into oligodendrocyte cells in the presence of AST at various concentrations (1, 5, and 10 <em>ng/ml</em>). Finally, immunocytochemistry and real-time PCR techniques were used for assessment of oligodendrocyte differentiation.&nbsp;<br /> Results: Flow cytometry results indicated that hADSCs were CD44, CD49-positive, but were negative for CD14, CD45 markers. In addition, immunocytochemistry results revealed that, in AST treated groups, the mean percentage of Olig 2 and A2B5 positive cells increased especially in 5 <em>ng/ml</em> AST treated group compared to control group (p&lt;0.001). Moreover, real-time PCR analysis confirmed the results of immunocytochemistry.<br /> Conclusion: Since hADSCs have the potential to differentiate into multi lineage cells and due to important functions of AST in regulating various cellular processes, it seems that AST can be used as a promoter for oligodendrocyte differentiation of hADSCs for being used in cell transplantation in multiple sclerosis.</p> https://www.AJMB.org/En/Article.aspx?ID=307 Nazem Ghasemi Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Ectopic Expression of Human <i>DPPA2</i> Gene in ESCC Cell Line Using Retroviral System <p>Background: Cancer/Testis Antigens (CTAs) are a subgroup of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2(DPPA2) with unknown biological function. Considering the importance of <em>DPPA2</em> in developmental events and cancer, preparing a suitable platform to analyze DPPA2 roles in the cells seems to be necessary.<br /> Methods: In this study, the coding sequence of <em>DPPA2</em> gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYSE-30 cells) and the stable transducted cells were confirmed for ectopic expression of <em>DPPA2</em> gene by real-time PCR.<br /> Results: According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression of <em>DPPA2</em> gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression.<br /> Conclusion: Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of DPPA2 ectopic expression in target cells. Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cells <em>in vivo</em> leads to an understanding of the biological importance of DPPA2 in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of DPPA2 existence in any biological fluid through ELISA system.</p> https://www.AJMB.org/En/Article.aspx?ID=305 Maryam Khaleghizadeh, Mohammad Mahdi Forghanifard, Abolfazl Rad, Moein Farshchian, Zahra Hejazi, Mehran Gholamin, Bahram Memar, Mohammad Reza Abbaszadegan Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>Lavandula angustifolia</i> Effects on Rat Models of Alzheimer’s Disease Through the Investigation of Serum Metabolic Features Using NMR Metabolomics <p>Background: Alzheimer&rsquo;s Disease (AD) is the most prevalent cause of memory impairment in the elderly population, but the diagnosis and treatment of the disease is still challenging. Lavender aqueous extract has recently been shown to have the potential in clearing Amyloid-beta plaques from AD rat hippocampus. To elucidate the therapeutic mechanisms of lavender, serum metabolic fingerprint of A&beta;-induced rat Alzheimer&rsquo;s models was investigated through nuclear magnetic resonance spectrometry.<br /> Methods: For the establishment of rat Alzheimer&rsquo;s models, 10 <em>&mu;g</em> of Amyloid beta 1-42 was injected to male Wistar rats. The lavender aqueous extract was injected 20 days after the establishment of the models, once daily for 20 days. Serum samples were collected and metabolite fingerprints were obtained using 500 <em>MHz</em> 1H-NMR spectrometry, following multivariate statistical analyses. The resulted metabolites were then subjected to pathway analysis tools to reveal metabolic pathways affected by the lavender extract treatment.<br /> Results: Levels of 10 metabolite markers including alanine, glutamine, serine, isoleucine, valine, carnitine, isobutyrate, pantothenate, glucose and asparagine were reversed nearly to control values after treatment with lavender extract. The results revealed that the most significantly affected pathways during treatment with lavender extract belonged to carbohydrate and amino acid metabolism, including pantothenate and CoA metabolism, glyoxilate and dicarboxylate metabolism, alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism.<br /> Conclusion: As lavender extract reversed the direction of changes of some metabolites involved in AD pathogenesis, it was concluded that the extract might play a role in the disease improvement and serve as a potential therapeutic option for the treatment of AD. Moreover, the metabolites which were found in AD rats could serve as a potential marker panel for the disease; however, much further investigation and validation of the results is needed.</p> https://www.AJMB.org/En/Article.aspx?ID=306 Afsaneh Arefi Oskouie, Reyhaneh Farrokhi Yekta, Mostafa Rezaie Tavirany, Masoud Soheili Kashani, Fatemeh Goshadrou Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Isolation and Proliferation of Spermatogonial Cells from Ghezel Sheep <p>Background: Sheep industry has taken steps toward transforming itself into a more efficient and competitive field. There are many varieties of sheep breeds in the world that each of them serves a useful purpose in the economies of different civilizations. Ghezel sheep is one of the Iranian important breeds that are raised for meat, milk and wool. Field of spermatogonial cell technologies provides tools for genetic improvement of sheep herd and multiple opportunities for research. Spermatogonial cells are the only stem cells capable of transmitting genetic information to future generations.&nbsp;<br /> Methods: This study was designed to extend the technique of isolation and in vitro proliferation of spermatogonial cells in Ghezel sheep.&nbsp;<br /> Results: Isolated cells were characterized further by using specific markers for type A spermatogonia, including PLZF. Also, sertoli cells were characterized by vimentin which is a specific marker for sertoli cells. After 10 days of co-culture, viability rates of the cells was above 94.7%, but after the freezing process the viability rates were 74 percent.<br /> Conclusion: In this study, a standard method for isolation and in vitro proliferation of spermatogonial stem cells in Ghezel sheep was developed.</p> https://www.AJMB.org/En/Article.aspx?ID=308 Babak Qasemi-Panahi, Mansoureh Movahedin, Gholamali Moghaddam, Parviz Tajik, Mortaza Koruji, Javad Ashrafi-Helan, Seyed Abbas Rafat Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Clinically Significant Dysregulation of <i>hsa-miR-30d-5p</i> and <i>hsa-let-7b</i> Expression in Patients with Surgically Resected Non-Small Cell Lung Cancer <p>Background: The cyclin E2 (CYCE2) is an important regulator in the progression and development of NSCLC, and its ectopic expression promoted the proliferation, invasion, and migration in several tumors, including Non-Small Cell Lung Cancer (NSCLC). However, the upregulation of CYCE2 in NSCLC cells suggested that it has a key role in tumorigenicity. In addition, the RAS family proteins as oncoproteins were activated in many major tumor types and its suitability as the therapeutic target in NSCLC was proposed. Considering the crucial role of microRNAs, it was hypothesized that altered expression of <em>hsa-miR-30d-5p</em>&nbsp;and <em>hsa-let-7b</em> might provide a reliable diagnostic tumor marker for diagnosis of NSCLC.<br /> Method: Real-time RT-PCR approach could evaluate the expression alteration of <em>hsa-miR-30d-5p </em>and <em>hsa-let-7b</em> and it was related to the surgically resected tissue of 24 lung cancer patients and 10 non-cancerous patients. The miRNAs expression was associated with clinicopathological features of the patients.<br /> Results: <em>Hsa-miR-30d</em> showed a significant downregulation (p=0.0382) in resected tissue of NSCLC patients compared with control group. Its expression level could differentiate different stages of malignancies from each other. The ROC curve analysis gave it an AUC=0.73 (p=0.037) which was a good score as a reliable biomarker. In contrast, <em>hsa-let-7b</em> was significantly overexpressed in tumor samples (p=0.03). Interestingly, our findings revealed a significant association of <em>hsa-let-7b</em> in adenocarcinoma tumors, compared to Squamous Cell Carcinomas (SCC) (p&lt;0.05). Also, analysis of ROC curve of <em>hsa-let-7b</em> (AUC=0.74, p-value=0.042) suggests that it could be as a suitable biomarker for NSCLC.<br /> Conclusion: Together, these results suggest a possible tumor suppressor role for <em>hsa-miR-30d</em> in lung tumor progression and initiation. Moreover, upregulation of <em>hsa-let-7b</em> was associated with the tumor type.</p> https://www.AJMB.org/En/Article.aspx?ID=311 Sayed Mostafa Hosseini, Bahram Mohammad Soltani, Mahmoud Tavallaei, Seyed Javad Mowla, Elham Tafsiri, Abouzar Bagheri, Hamid Reza Khorram Khorshid Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Tumor Necrosis Factor-Alpha and Interleukin-6 Gene Polymorphisms in Iranian Patients with Ischemic Heart Failure <p>Background: Proinflammatory cytokines have been known to be elevated in patients with Chronic Heart Failure (CHF). Given the importance of proinflammatory cytokines in the context of the failing heart, the prevalence of Tumor Necrosis Factor-&alpha; (TNF-&alpha;), Interleukin (IL)-6 polymorphisms in patients with CHF was studied due to ischemic heart disease.<br /> Methods: Forty three patients with ischemic heart failure were enrolled in this study and compared with 140 healthy individuals. The allele and genotype frequency of four Single Nucleotide Polymorphisms (SNPs) within the IL-6 (-174, nt565) and TNF-&alpha; (-308, -238) genes were determined, using Polymerase Chain Reaction with Sequence-Specific Primers (PCR-SSP) assay.<br /> Results: The frequency of the TNF-&alpha; (-238) A/A genotype was significantly higher in patients comparing to controls (p=0.043), while TNF-&alpha; G/A genotype at the same position decreased significantly, in comparison with controls (p=0.018). The most frequent haplotype for TNF-&alpha; was A/A in the patient group in comparison with controls (p=0.003). There was no significant difference in allele and genotype frequencies of IL-6 at positions -174 and nt565, and TNF-&alpha; at position -308.<br /> Conclusion: Certain alleles, genotypes, and haplotypes in TNF-&alpha;, but not IL-6, gene were overrepresented in patients with ischemic heart failure, which may, in turn, predispose individuals to this disease.</p> https://www.AJMB.org/En/Article.aspx?ID=309 Mona Hedayat, Mohammad Jafar Mahmoudi, Mohammad Taghvaei, Ebrahim Nematipour, Elham Farhadi, Nilufar Esfahanian, Maryam Mahmoudi, Maryam Sadr, Keramat Nourijelyani, Ali Akbar Amirzargar, Nima Rezaei Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of <i>AIRE</i> Polymorphism and the Susceptibility to Multiple Sclerosis in Iranian Population <p>Background: Multiple Sclerosis (MS) is the most common cause of neurologic disability in young adults. Recently, the <em>AIRE</em> gene was identified as a genetic risk factor for several autoimmune diseases in genome wide association studies. The aim of this study was to further investigate the possible role of the <em>AIRE</em> gene in susceptibility to MS in Iranian population.<br /> Methods: A total of 112 MS patients and 94 ethnically matched controls were included in the study. The Single-Nucleotide Polymorphism (SNP) (rs1800520, C&gt;G) with a global MAF=0.2282/1143 was selected and genotyped using HRM real-time PCR method.<br /> Results: Results showed that <em>AIRE</em> SNP rs1800520 was significantly less common in the MS patients than in healthy controls (17.8 <em>vs.</em> 28.7%, pc=0.032, OR=0.54,95% CI 0.279,1.042). Also, the frequency of allele G was significantly higher among the control group than in the case group (37.77 <em>vs.</em> 25%, pc=0.014). Interestingly, mRNA transcribed on the rs1800520 SNP showed decreased free energy than the wild type suggesting that its increased stability may be responsible for the different activities of the polymorphic AIRE molecule.&nbsp;<br /> Conclusions: This is the first study investigating the relationship between <em>AIRE</em> gene and the susceptibility to MS. These results indicated that the rs1800520 SNP is not a susceptibility gene variant for the development of MS in Iranian population.</p> https://www.AJMB.org/En/Article.aspx?ID=310 Tahereh Sadeghian-Rizi, Fereshteh Alsahebfosoul, Mohammad Kazemi, Hossein Khanahmad, Ali Jahanian Najafabadi Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluating the Frequency of <i>aac(6')-IIa, ant(2'')-I, intl1,</i> and <i>intl2</i> Genes in Aminoglycosides Resistant <i>Klebsiella pneumoniae</i> Isolates Obtained from Hospitalized Patients in Yazd, Iran <p>Background: <em>Klebsiella pneumoniae (K. </em>pneumoniae<em>)</em> is an opportunistic pathogen that could be resistant to many antimicrobial agents. Resistance genes can be carried among gram-negative bacteria by integrons. Enzymatic inactivation is the most important mechanism of resistance to aminoglycosides. In this study, the frequencies of two important resistance gene <em>aac(6&#39;)-IIa</em> and <em>ant(2&#39;&#39;)-I</em>, and genes coding integrase I and II, in <em>K. pneumoniae</em>&nbsp;isolates resistant to aminoglycosides were evaluated.<br /> Methods: In this cross-sectional study, an attempt was made to assess the antibiotic susceptibility of 130 <em>K. pneumoniae</em> isolates obtained from different samples of patients hospitalized in training hospitals of Yazd evaluated by disk diffusion method. The frequencies of <em>aac(6&#39;)-IIa, ant(2&#39;&#39;)-I, intl1,</em> and intl2 genes were determined by PCR method. Data were analyzed by chi-square method using SPSS software (Ver. 16).<br /> Results: our results showed that resistance to gentamicin, tobramycin, kanamycin, and amikacin were 34.6, 33.8, 43.8, and 14.6%, respectively. The frequencies of <em>aac(6&#39;)-IIa, ant(2&#39;&#39;)-I, intl1</em>, and <em>intl2</em> genes were 44.6, 27.7, 90, and 0%, respectively.<br /> Conclusion: This study showed there are high frequencies of genes coding aminoglycosides resistance in <em>K. pneumoniae</em> isolates. Hence, it is very important to monitor and inhibit the spread of antibiotic resistance genes.</p> https://www.AJMB.org/En/Article.aspx?ID=313 Hesam Mokhtari, Gilda Eslami, Hengameh Zandi, Amin Dehghan-Banadkouki, Mahmood Vakili Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Assessment of <i>EGFR</i> Gene Expression Following Vitrification of 2-cell and Blastocyst Mouse Embryos <p>Background: Exact mechanisms of fetal harm following vitrification are still unknown. This study was conducted to evaluate the cryopreservation impact on the expression of Epidermal <em>Growth Factor Receptor</em> (<em>EGFR</em>) gene in mouse 2-cell and blastocysts.<br /> Methods: To stimulate ovulation in mice, hCG was injected, followed by collecting 2-cells and blastocysts after 44-46 and 88-89 <em>hr</em>, respectively. These embryos were divided into two case and control groups. The fresh case group was cryopreserved using cryotop and warmed after 4 mounts. Normal 2-cells were selected based on their morphology and their RNA was extracted. Quantitative expression of <em>EGFR</em> gene in both groups was investigated by applying real time-PCR.<br /> Results: The statistical real-time (RT)-PCR analyses performed using SPSS revealed that the expression level of EGFR gene was diminished in the case group compared to the control group.&nbsp;<br /> Conclusion: The current study indicated the negative effect of cryopreservation on expression amount of <em>EGFR</em> gene in 2-cell and blastocyst mouse embryos.</p> https://www.AJMB.org/En/Article.aspx?ID=334 Rouhollah Gazor, Mozhgan Eskandari, Alireza Sharafshah, Mohammad Hadi Bahadori, Mohammad Ghasem Golmohammadi, Parvaneh Keshavarz Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Retraction: The Frequency and Importance of Common α-globin Gene Deletions Among β-Thalassemia Carriers in an Iranian Population <p>The Editorial Board of Avicenna Journal of Medical Biotechnology (AJMB) has decided to retract the original article entitled &ldquo;The Frequency and Importance of Common &alpha;-globin Gene Deletions Among &beta;-Thalassemia Carriers in an Iranian Population&rdquo; published in the October-December 2017 issue due to a fact which is contrary to the scientific rules and regulation of AJMB.</p> https://www.AJMB.org/En/Article.aspx?ID=10360 Azam Moosavi, Ali M. Ardekani Sun, 11 Mar 2018 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir New Hopes for Treatment of Alzheimer’s Disease <p>Alzheimer&rsquo;s Disease (AD), the leading cause of dementia worldwide, is an irreversible progressive neurodegenerative disorder characterized by cognitive impairment and functional disability <sup>1,2</sup>. Devastating nature of AD leads to serious social and economic impacts on the healthcare systems which implies the necessity of its proper management. It has been demonstrated that patients&rsquo; quality of life and their overall prognosis has a significant negative correlation with the severity of AD. Patients with severe AD need full-time care and assistance with some basic activities of daily living such as feeding and dressing in addition to severe deterioration in various domains of their cognitive functioning. Moreover, behavioral aberrancy and neuropsychiatric symptoms such as depression, apathy, psychosis, agitation, and aggression are observed more frequently in moderate to severe AD. Despite such an enormous burden, most practical guidelines focus on mild to moderate stages of the illness and there is still a serious lack of evidence regarding the management of severe AD. Among currently FDA-approved drugs, very few medications have shown to be effective in attenuating some of the AD-related symptoms in severe stages. Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, and donepezil, an acetylcholinesterase inhibitor (ACEI) are the most widely accepted agents in this regard. Unfavorable side effects of these agents along with lack of optimal efficacy have led to many researches trying to find novel pharmacologic strategies for AD based on its underlying pathophysiological defects <sup>3</sup>.<br /> More than 400 clinical trials are currently looking at new treatments for AD and many of them are actively recruiting. Many of these studies are based on decreasing the harmful effects of a toxic protein called amyloid-beta in the brain, but others reflect a broadening range of possible treatment approaches based on other theories about AD.<br /> For example, the importance of inflammation in exacerbation of amyloid-beta&rsquo;s neuron-destroying effects has led to trials of medications with anti-inflammatory properties <sup>4</sup>.<br /> Serotonin neurotransmission failure is a demonstrated aspect of AD, and several experimental medications attempt to correct that problem. RVT-101 and LuAE58054 are two examples of medications that are in clinical trials. Altering the brain&rsquo;s serotonin activity seems to help cognitive difficulties in schizophrenia, and may also prove helpful in cognitive difficulties associated with AD <sup>5</sup>. In conclusion, a number of biotechnologists are working on effective drugs for treatment of AD and in particular severe type.</p> https://www.AJMB.org/En/Article.aspx?ID=296 Shahin Akhondzadeh Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of Immune Responses Induced by <i>GRA7</i> and <i>ROP2</i> Genes by DNA Vaccine Cocktails Against Acute Toxoplasmosis in BALB/c Mice <p>Background: The severe damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with plasmid DNA is a promising vaccination technique. Therefore, <em>GRA7</em> plasmid was prepared to be used as a vaccine. The purpose of this study was evaluation of immunization with cocktail DNA vaccine including plasmids encoding <em>Toxoplasma gondii</em> <em>ROP2</em> and <em>GRA7</em> in BALB/c mice.<br /> Methods: In this study, 733 <em>bp</em> of <em>GRA7</em> gene was cloned in pCDNA3.1 plasmid as an expression vector. The plasmids containing <em>GRA7</em> and <em>ROP2</em> genes were administered via <em>IM </em>according to immunized mice three times with a 3 week interval. For lymphocyte proliferation and cytokine assay, splenocytes of immunized mice were cultured for proliferation and cytokine assay. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis.<br /> Results: The cytokine assay results and lymphocyte proliferation response in cocktail DNA vaccines showed that IFN-&gamma; levels were significantly higher than controls (p&lt;0.05), whereas IL-4 expression level in BALB/c mice immunized with cocktail was lower than that in control groups and these results are confirmed by MTT test. Predominance of the levels of IgG2a over IgG1 was observed in sera of the immunized mice. Furthermore, IgG2a values in cocktail DNA vaccines pcGRA7 were significantly higher than control group (p&lt;0.01). In contrast, IgG1 antibodies were similar between the two groups (p&gt;0.5). So, survival time in the immune groups was significantly prolonged in comparison to control ones (p&lt;0.01).<br /> Conclusion: The immunized mice by DNA vaccine produce higher titration of IFN&gamma;, indicated with Th1 response which is confirmed by high level of IgG2a. These data demonstrate that the cocktail <em>GRA7/ROP2</em> is a potential vaccine candidate against toxoplasmosis.</p> https://www.AJMB.org/En/Article.aspx?ID=297 Hossein Vazini, Fatemeh Ghafarifar, Zohreh Sharifi, Abdolhossein Dalimi Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Novel Recombinant Traceable c-Met Antagonist-Avimer Antibody Mimetic Obtained by Bacterial Expression Analysis <p>Background: Avimers are originally types of artificial proteins with multiple binding sites for specific binding to certain antigens. Various radioisotopes and nanoparticles link these molecules, which are widely used in early detection in tissue imaging, treatment and study on carcinogenesis. Among these, c-Met antagonist avimer (C426 avimer), with ability to bind the c-Met receptor of tyrosine kinase (RTK) is an attractive candidate for targeted cancer therapy. In this study, a novel traceable C426 avimer gene was designed and introduced by adding the 12nt tracer binding site encoded four specific amino acid residues at the C-terminal region of C426 avimer coding sequence.<br /> Methods: The 282 <em>bp</em> DNA sequence encoded 94aa avimer protein was synthesized and sub-cloned into prokaryotic pET26b expression vector. The expression of the mature peptide encoding the traceable avimer molecule was carried out in <em>Escherichia coli</em> strain BL21 using IPTG (Isopropyl &beta;-D-1-thiogalactopyranoside) induction process. The expression level of the 11&nbsp;<em>kDa</em> traceable avimer was&nbsp; studied by SDS-PAGE, western blot and ELISA analysis.<br /> Results: Docking analysis of C426 avimer protein and its ligand c-Met showed that the traceability related changes happened at the best conformation and optimal energy. The SDS-PAGE, western blotting and ELISA analysis results demonstrated that the expression of the 11 <em>kDa</em> C426 avimer molecule was detectable without any degradation compared with the control group.<br /> Conclusion: Concerning the consequences of this work, this new approach can be widely used in the medical field and provide an opportunity to evaluate the affinity and traceability features.</p> https://www.AJMB.org/En/Article.aspx?ID=298 Bahram Baghban Kohnehrouz, Afsaneh Talischian, Alireza Dehnad, Shahnoush Nayeri Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir RNA Loading on Nano-Structured Hyperbranched β-Cyclodextrin <p>Background: &beta;-Cyclodextrin functionalized hyper-branched polyglycerol (HBCD: &beta;-CD-g-PG), a biocompatible polymer, has recently been proposed for delivery of poorly water soluble compounds.<br /> Methods: The present study examines the interaction of HBCD with RNA, utilizing a constant concentration of RNA and different HBCD/RNA ratios of 1/16 to 1/1, at physiological condition in an aqueous solution. Circular Dichroism (CD), UV-visible, FTIR spectroscopic methods, zeta potential and Dynamic Light Scattering (DLS) were used to analyze the particle formation, particle charge, particle size, aggregation, RNA conformation, binding constant and mode, and the effect of polymer complexation on RNA stability.<br /> Results: The results indicate that the interaction of RNA with HBCD leads to the formation of a linear dendritic supramolecule biopolymer with an overall binding constant of KHBCD/RNA= 1.25 &times; 10<sup>3</sup>.&nbsp;<br /> Conclusion: The small sized synthesized polymer can be considered as an appropriate system for preventing RNA aggregation and protecting the gene by host-guest interaction.</p> https://www.AJMB.org/En/Article.aspx?ID=301 Sorina Hirbod, Shohreh Nafisi, Howard I Maibach Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Microwave-Assisted Hydro-Distillation of Essential Oil from Rosemary: Comparison with Traditional Distillation <p>Background: Hydro-distillation (HD) method is a traditional technique which is used in most industrial companies. Microwave-assisted Hydro-distillation (MAHD) is an advanced HD technique utilizing a microwave oven in the extraction process.&nbsp;<br /> Methods: In this research, MAHD of essential oils from the aerial parts (leaves) of rosemary (<em>Rosmarinus officinalis L.</em>) was studied and the results were compared with those of the conventional HD in terms of extraction time, extraction efficiency, chemical composition, quality of the essential oils and cost of the operation.&nbsp;<br /> Results: Microwave hydro-distillation was superior in terms of saving energy and extraction time (30 <em>min</em>, compared to 90 <em>min</em> in HD). Chromatography was used for quantity analysis of the essential oils composition. Quality of essential oil improved in MAHD method due to an increase of 17% in oxygenated compounds.&nbsp;<br /> Conclusion: Consequently, microwave hydro-distillation can be used as a substitute of traditional hydro-distillation.</p> https://www.AJMB.org/En/Article.aspx?ID=300 Sara Moradi, Alireza Fazlalia, Hamid Hamedi Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Relative Expression of PBMC MicroRNA-133a Analysis in Patients Receiving Warfarin After Mechanical Heart Valve Replacement <p>Background: MicroRNAs (miRNAs) are implicated in various biological processes including anticoagulation. However, the modulation of miRNA by pharmacological intervention such as warfarin treatment in patients receiving warfarin has not been disclosed yet. The aim of this study work was to assess the effect of warfarin drug on expression level of mir-133a-3p in patients with mechanical heart valve replacement.<br /> Methods: In this research, the expression level of miRNA-133a-3p was analyzed in Peripheral Blood Mononuclear cells (PBMCs) from mechanical valve replacement patients who had received warfarin for at least 3 months continuously. Quantitative RT-PCR method was used for this assay.<br /> Results: Our findings indicated a significant diffrence between the rate of miR-133a-3p expression in individuals receiving warfarin and the control group (p&lt;0.01). There was also a statistically significant difference in miR-133a-3p expression in patients with different ages (p&lt;0.05) suggesting that the rate of miR-133a-3p expression in persons receiving warfarin is related to age. However, other variables like warfarin dose, International Normalized Ratio (INR), gender, and Body Mass Index (BMI) were not significantly effective on the miR-133a-3p experssion rate in individuals receving warfarin.&nbsp;<br /> Conclusion: Based on our results, it can be concluded that miR-133a-3p is involved in the coagulation pathway. The recent result indicates that warfarin affects the expression of miR-133a. This expression may be potentially important for treatment by anticoagulants. Awareness of the time course of miRNA expression profile can improve efficiency of response to warfarin.</p> https://www.AJMB.org/En/Article.aspx?ID=299 Hamid Kabiri Rad, Mahta Mazaheri, Ali Dehghani Firozabadi Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Molecular Characterization and Functional Analysis of the PilQ380-705: a Novel Secretin Domain in <i>Pseudomonas aeruginosa</i> <p>Background: Type 4 pili (T4P) is an important virulence factor of<em> Pseudomonas aeruginosa (P. aeruginosa)</em>. T4P pass the outer membrane through a large oligomeric channel made of a single PilQ protein that is most highly conserved at their C-termini. To develop a functional vaccine that can be used in clinical application, the secretin domain of the PilQ (PilQ<sub>380-706</sub>) was produced as a recombinant protein.<br /> Methods: A 981 <em>bp</em> fragment of C-terminal of the <em>pilQ</em> secretin (<em>pilQ</em><sub>1138-2118</sub>) from was designed into the prokaryotic expression vector pET28a. The presence of the <em>pilQ</em><sub>1138-2118</sub> gene in the recombinant construct (pET28a/<em>pilQ</em>) was assessed by double digestion and PCR. After transformation, expression of the recombinant <em>PilQ</em> was induced by addition of IPTG. The expressed recombinant protein was purified by a modified method using a HisTrap affinity column and finally confirmed by SDS-PAGE. The functional activities of the produced <em>PilQ</em><sub>380-706</sub> confirmed by Western blot analysis and twitching inhibition assay.&nbsp;<br /> Results: The PCR and enzymatic digestion results showed the presence of the <em>pilQ</em><sub>1138-2118</sub> gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant <em>PilQ</em><sub>380-706</sub> is approximately 37 <em>kDa</em>. The Western blot analysis confirmed the specificity of specific IgG against the <em>PilQ</em><sub>380-706</sub> protein. The<em> PilQ</em><sub>380-706</sub> protein showed high biological activity in all of these standard assays.<br /> Conclusion: Since, the <em>PilQ</em><sub>380-706</sub> protein plays an important role in the biogenesis of pili; and thus, the primary establishment of <em>P. aeruginosa</em>; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies.</p> https://www.AJMB.org/En/Article.aspx?ID=302 Sobhan Faezi, Iraj Nikokar, Ali Elmi, Yusuf Ghasemi, Mojtaba Farahbakhsh, Alireza Salimi Chirani, Mehdi Mahdavi Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Male Pronuclear Formation and Embryo Development Following Intracytoplasmic Injection of Ovine Pretreated Sperm <p>Background: Failure of Male Pronucleus (MPN) formation is a major concern in the success of Intracytoplasmic Sperm Injection (ICSI) in some species. Despite the conducted unsuccessful efforts to improve ICSI efficiency in ovine, the present study was aimed to improve MPN formation and embryo development in ovine ICSI procedure through accompaniment of sperm pretreatment with co-injection of some compounds.&nbsp;<br /> Methods: In experiment 1, sperm were treated with either 2-mercaptoethanol (2ME), glutathione (GSH), or DTT (dithiothreitol) in combination with Heparin (Hep). Following DNA integrity and fragmentation assessments, the best sperm pretreatment approach in induction of sperm head decondensation was applied for the second and third experiments. In experiment 2, <em>in vitro </em>matured oocytes were subjected to ICSI using pretreated sperm with or without GSH and Sperm Extract (SE) co-injection. In experiment 3, the procedure was followed as experiment 2 with acrosome reacted sperm.&nbsp;<br /> Results: The highest percentages of oocyte activation were observed in Hep+GSH and Hep+2ME groups. The greatest MPN formations were also observed in the same groups when ICSI procedure was accompanied with GSH co-injection. Despite the higher percentage of MPN formation and oocyte activation in Hep+GSH and Hep+2ME groups, none of the employed strategies could increase the cleavage and blastocyst rates compared to the control.&nbsp;<br /> Conclusion: In our study condition, despite the lack of significant increase in embryo development in treated groups, the significant increase in MPN formation in Hep+GSH+co.GSH and Hep+2ME+co.GSH groups indicates the lower chance of parthenote formation that means a higher chance of normal fertilization compared with control.</p> https://www.AJMB.org/En/Article.aspx?ID=304 Abolfazl Shirazi, Arefeh Golestanfar, Masomeh Bashiri, Ebrahim Ahmadi, Naser Shams-Esfandabadi Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Optimization of Effective Minerals on Riboflavin Production by <i>Bacillus subtilis subsp. subtilis</i> ATCC 6051 Using Statistical Designs <p>Background: Riboflavin (vitamin B<sub>2</sub>) is an essential component of the basic metabo-lism, and an important nutritional and growth factor in humans, animals, plants and micro-organisms. It has been widely used in the fields of pharmaceuticals, feed and food additives. The industrial production of riboflavin mostly relies on the microbial fermentation. Designing an appropriate fermentation medium is of crucial importance to improve the riboflavin production.&nbsp;<br /> Methods: In this study, sequential methodology combining a screening test of minerals by Plackett-Burman (PB) and an optimization test by Central Composite Design (CCD) was applied to enhance riboflavin production by <em>Bacillus subtilis</em> ATCC 6051 in shake flasks.<br /> Results: Initially, one-factor-at-a-time approach was applied to evaluate the effect of different carbon sources. The results showed that fructose was significantly most effective on biomass and riboflavin production. After that, 13 minerals [CaCl<sub>2</sub>, CuCl, FeCl<sub>3</sub>, FeSO<sub>4</sub>, AlCl<sub>3</sub>, Na<sub>3</sub>MoO<sub>4</sub>, Co(NO<sub>3</sub>)<sub>2</sub>, NaCl, KH<sub>2</sub>PO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, MgSO<sub>4</sub>, ZnSO<sub>4</sub>, and MnSO<sub>4</sub>] were studied with the screening test. The results revealed that concentration of MgSO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, and FeSO<sub>4</sub> had greater influence on riboflavin production (p&lt;0.05). A CCD with five factors (concentration of fructose, MgSO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, FeSO<sub>4</sub>, and yeast extract) at five levels was then used to determine the maximum riboflavin concentration. The optimal concentrations (<em>g/l</em>) of these variables determined by Response Surface Methodology (RSM) were fructose, 38.10; MgSO<sub>4</sub>, 0.85; K<sub>2</sub>HPO<sub>4</sub>, 2.27; FeSO<sub>4</sub>, 0.02; and yeast extract, 4.37.<br /> Conclusion: Statistical experimental design offers a practicable approach to the implementation of medium optimization. From an industrial view point, our optimum medium, besides fructose and a small amount of yeast extract, is mainly composed of common and cheap inorganic salts, which are available to the industrial riboflavin production.</p> https://www.AJMB.org/En/Article.aspx?ID=10335 Marjan Oraei, Seyed Hadi Razavi, Faramarz Khodaiyan Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of Serotonin Transporter Gene Polymorphism with Recurrent Aphthous Stomatitis <p>Background: Recurrent Aphthous Stomatitis (RAS) is one of the most common diseases of the oral cavity all over the world (5-66%). RAS has a multifactorial etiology, while psychological factors such as stress and anger play a role in its manifestation. The serotonergic mechanisms particularly the serotonin-transporter gene (<em>5-HTT</em>) may affect the risk of psychological alterations and stress response. The aim of the present study was to evaluate the polymorphism of the promoter region of <em>5-HTT (5-HTTLPR)</em> in the patients with RAS, compared to that in the control subjects.<br /> Methods: In this case-control study, 100 patients with RAS and 100 healthy subjects were enrolled. PCR was performed on DNA of the samples, using a pair of primers capable of distinguishing S/L alleles and replicating <em>5-HTTLPR</em>.<br /> Results: No statistically significant difference existed between LL and LS genotype frequencies in the case and control groups. However, SS genotype frequency was significantly higher in the case group, as compared to the control group (p=0.001).<br /> Conclusion: The conclusion of the present study demonstrated that S allele could approximately double the risk of RAS.</p> https://www.AJMB.org/En/Article.aspx?ID=303 Shamsolmoulouk Najafi, Mahsa Mohammadzadeh, Amirabbas Zahedi, Mansour Heidari, Nima Rezaei Sat, 09 Dec 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>Lactococcus lactis</i>: A New Strategy for Vaccination <p>Needle free vaccines have a several advantages and very attractive way for vaccination. In a body, mucosal surfaces provide a universal entry portal for all the known and emerging infectious pathogenic microbes. Therefore, it seems, vaccination strategies need to be reorganized for vaccines that are hindering the entry capability of pathogenic microbes through mucosal surfaces. Lactic acid Bacteria (LAB) are widely used in the food industry and at the present, used as delivery vehicles for biological investigations. In this review, we summarized the Results of several studies which <em>Lac-tococcus lactis (L. lactis)</em> used as a live vector for vaccines. These bacteria are considered as promising candidates for heterologous expression of proteins and biotechnological usage. LAB are considered as promising candidates for heterologous expression of proteins and biotechnological usage. The results showed that these bacteria have an ability to deliver antigen to immune system. Therefore, developing mucosal live vaccines using lactic acid bacterium,<em> L. lactis</em>, as an antigen delivery vector, is an attractive alternative choice and a safer vaccination strategy against pathogens.</p> https://www.AJMB.org/En/Article.aspx?ID=289 Maryam Azizpour, Seyyed Davood Hosseini, Parvaneh Jafari, Neda Akbary Tue, 19 Sep 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning, Expression and Purification of <i>Pseudomonas putida ATCC12633</i> Creatinase <p>Background: <em>Pseudomonas putida (P. putida)</em> ATCC12633 can produce creatinase. It is a microbial enzyme which degrades creatinine in bacteria and provides source of carbon and nitrogen. Also, this enzyme is used in the enzymatic measurement of creatinine concentration for diagnosis of renal and muscles functions and diseases. Our purpose was recombinant production of creatinase for using in clinical measurement of serum or urine creatinine.<br /> Methods: A 1209bp of open reading frame of creatinase was amplified by PCR from P. putida ATCC12633 genome and cloned into pET28a expression vector which was digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony PCR, double digestion analysis and sequencing. Recombinant pET28a vector was transformed to <em>Escherichia coli (E. coli)</em> <em>BL21 (DE3)</em>. Creatinase expression was induced in <em>E.coli BL21 (DE3)</em> using IPTG and confirmed by SDS-PAGE and western blotting. Purification of creatinase was performed using Ni-NTA column. The specific activity of this enzyme was also investigated.<br /> Results: The creatinase gene cloning was confirmed by DNA sequencing. Successful expression of creatinase was performed in <em>E. coli</em> (57.4% of total protein). SDS-PAGE and western blot analysis showed a 45 kDa creatinase protein. Purification of creati-nase was done with high purity. The specific activity of recombinant enzyme is 26.54 unit/mg that is much higher than other creatinase used in the commercial kits (9 <em>unit/mg</em>).<br /> Conclusion: The P. putida ATCC12633 recombinant creatinase was expressed effi-ciently in <em>E. coli </em>BL21 and 57% of total protein was the recombinant creatinase. Also, expressed creatinase has high solubility and also the enzyme has good activity com-pared to enzymes used in commercial kits, so a new source of creatinase was produced for creatinine assay kit in this study.</p> https://www.AJMB.org/En/Article.aspx?ID=286 Elnaz Afshari, Zahra Amini-bayat, Saman Hosseinkhani, Nahid Bakhtiari Tue, 19 Sep 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Lithium Chloride can Induce Differentiation of Human Immortalized RenVm Cells into Dopaminergic Neurons <p>Background: Stem cell-based therapy is a novel strategy for the treatment of neuro-degenerative diseases. The transplantation of fully differentiated cells instead of stem cells in order to decrease serious adverse complications of stem cell therapy is a new idea. In this study, the effect of lithium chloride on dopaminergic differentiation of human immortalized RenVm cells was investigated in order to access a population of fully differentiated cells for transplantation in Parkinson disease.<br /> Methods: The immortalized RenVm cells were induced to dopaminergic differentiation using a neurobasal medium supplemented with N2 and different concentrations (1, 3, 6 <em>mM</em>) of Lithium Chloride (LiCl) for 4, 8 and 12 days. The efficiency of dopaminergic differentiation was evaluated using immunocytochemistry and western blot techniques for tyrosine hydroxylase and &beta;-catenin marker expression.<br /> Results: Our results indicated that LiCl can promote dopaminergic differentiation of RenVm cells in a dose-dependent manner.<br /> Conclusion: It can be concluded that LiCl is able to facilitate dopaminergic differenti-ation of cultured cells by affecting Wnt-frizzled signaling pathway.</p> https://www.AJMB.org/En/Article.aspx?ID=287 Mitra Soleimani, Nazem Ghasemi Tue, 19 Sep 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Magnetic Iron Oxide Nanoparticles as T2 MR Imaging Contrast Agent for Detection of Breast Cancer (MCF-7) Cell <p>Background: Advances of nanotechnology have led to the development of nano-materials with both potential diagnostic and therapeutic applications. Among them, Super Paramagnetic Iron Oxide Nanoparticles (SPIONs) have received particular at-tention. Modified EDC coupling fraction was used to fabricate the SPION-C595 as an MR imaging contrast agent for breast cancer detection in early stages.<br /> Methods: Nanoprobe characterization was confirmed using Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy with Energy Dispersive X-Ray Spectroscopy (SEM-EDAX), and Photon Correlation Spectroscopy (PCS). Protein and iron concentration of nanoprobe was examined by standard method. MTT assay was performed to evaluate the cytotoxicity of the nanoprobe in breast cancer cell line (MCF-7). T<sub>2</sub>-weighted MR imaging was performed to evaluate the signal enhancement on T<sub>2</sub> relaxation time of nanoprobe using spin-echo pulse sequence.<br /> Results: As results showed, SPIONs-C595 provided active targeting of breast cancer cell (MCF-7) at a final concentration of 600 <em>&micro;gFe/ml</em>. The final concentration of protein was calculated to be at 0.78 <em>&micro;gprotein/ml</em>. The hydrodynamic size of the nano-probe was 87.4&plusmn;0.7 <em>nm</em>. The MR imaging results showed a good reduction of T<sub>2</sub> relaxation rates for the highest dose of SPIONs-C595.<br /> Discussion: Based on the results, SPIONs-C595 nanoprobe has a potential in T<sub>2</sub>-weighted MR imaging contrast agent for breast cancer cell (MCF-7) detection.</p> https://www.AJMB.org/En/Article.aspx?ID=288 Pegah Moradi Khaniabadi, Daryoush Shahbazi-Gahrouei, Mohammad Suhaimi Jaafar, Amin Malik Shah Abdul Majid, Bita Moradi Khaniabadi, Saghar Shahbazi-Gahrouei Tue, 19 Sep 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Altered miR-223 Expression in Sputum for Diagnosis of Non-Small Cell Lung Cancer <p>Background: Diagnosis of Non-small Cell Lung Cancer (NSCLC) at an early stage is a daunting challenge due to the deficiency of specific noninvasive markers. MicroRNAs (miRNAs) play important roles in the initiation and progression of NSCLC. Measuring miRNA expression levels could provide a potential approach for the diagnosis of NSCLC. Our goals were to examine miR-223, miR-212, miR-192, miR-3074, SNORD33 and SNORD37 expression levels in tissue and sputum of NSCLC patients and cancer free subjects for molecular diagnosis of NSCLC.<br /> Methods: Relative expressions of miR-223, miR-212, miR-192, miR-3074, SNORD33 and SNORD37 were examined with quantitative real-time RT-PCR assay in tissue and sputum obtained from 17 NSCLC patients and 17 controls.<br /> Results: miR-3074 was upregulated in tissue samples of NSCLC patients compared with control group. miR-223 was upregulated, miR-212 and SNORD37 were downer-gulated in sputum samples of patients compared with controls. miR-223 quantification produced 82% sensitivity and 95% specificity with areas under the ROC curve at 0.90 in detection of NSCLC.<br /> Conclusion: miR-223 clearly discriminated cancer patients from cancer-free subjects and our results suggest that miR-223 could be a diagnostic useful biomarker. The measurement of altered miRNA expression in sputum samples manifested the potential noninvasive approach for detection of lung cancer.</p> https://www.AJMB.org/En/Article.aspx?ID=290 Abouzar Bagheri, Hamid Reza Khorram Khorshid, Seyed Javad Mowla, Hassan Ali Mohebbi, Azam Mohammadian, Mehdi Yaseri, Masoud Solaymani-Dodaran, Masih Sherafatian, Mahmood Tavallaie Tue, 19 Sep 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Retraction: The Frequency and Importance of Common α-globin Gene Deletions Among β-Thalassemia Carriers in an Iranian Population <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span><span style="font-size:10.0pt"> &beta;</span><span style="font-size:10.0pt">-thalassemia&nbsp;is the most common monogenic disorder in Iran, and one of the challenges in the screening of the carriers is the coinheritance of </span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">-thalassemia mutations. In the view of high prevalence of </span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">-thalassemia mutations in many parts of the country, the aim of this study was to determine the carrier frequency&nbsp;of common alpha deletions, as a secondary modifier in clinical manifestations of beta thalassemia, in known beta-thalassemia carriers and some hematology parameter changes. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span><span style="font-size:10.0pt"> The study included families referred from different primary health care centers with microcytic hypochromic anemia [MCV&lt;80fl; MCH&lt;27 <em>pg</em>]&nbsp;and&nbsp;A2&gt;3.4%]. Genomic DNA was extracted from peripheral blood leukocytes by salting out method. For common </span><span style="font-size:10.0pt">&beta;</span><span style="font-size:10.0pt">-globin gene mutation analysis, amplification refractory mutation system- polymerase chain reaction (ARMS-PCR) and for rare </span><span style="font-size:10.0pt">&beta;</span><span style="font-size:10.0pt">-thal alleles, DNA sequencing were used. Also, for investigation of common </span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">-globin gene cluster deletions (-</span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">3.7, -</span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">4.2, --MED and -</span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">20.5), multiplex Gap-PCR was performed.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span><span style="font-size:10.0pt"> Among 227 </span><span style="font-size:10.0pt">&beta;</span><span style="font-size:10.0pt">-thalassemia minor individuals studied, </span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">-globin gene deletions were found in 43 cases:&nbsp;37 heterozygote&nbsp;-</span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">3.7 (16.3%), 5 homo -</span><span style="font-size:10.0pt">&alpha;</span><span style="font-size:10.0pt">3.7 (2.2%) and 1 --MED (0.44%). Also, </span><span style="font-size:10.0pt">the co-inheritance of </span><span style="font-size:10.0pt">&alpha;-globin gene deletion and triplication was not found in the studied individuals.</span></span></p> <p>&nbsp;</p> <p><span style="font-size:9.5pt">Conclusion:</span><span style="font-size:10.0pt"> Although it is highly recommended that physicians and genetic counselors involved in the screening program of beta-thal major in the country consider this phenomenon because of high prevalence of this coinheritance, hematologic indices changes are very slight.</span></p> https://www.AJMB.org/En/Article.aspx?ID=291 Azam Moosavi, Ali M. Ardekani Tue, 19 Sep 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Alternative Splicing Generates Different 5' UTRs in OCT4B Variants <p>Background: The human <em>OCT4</em> gene, responsible for pluripotency and self-renewal of Embryonic Stem (ES) and Embryonic Carcinoma (EC) cells, can generate several tran-scripts (OCT4A, OCT4B-variant 2, OCT4B-variant 3, OCT4B-variant 5, OCT4B1, OCT4 B2 and OCT4B3) by alternative splicing and alternative promoters. OCT4A that is responsible for ES and EC cell stemness properties is transcribed from a promoter upstream of Exon1a in those cells. The OCT4B group variants (OCT4B-variant2, OCT4B-variant3, OCT4B-variant5, OCT4B1, OCT4B2 and OCT4B3) are transcribed from a different promoter located in intron 1 and some of them respond to the cell stresses, but cannot sustain the ES/EC cell self-renewal. However, the exact function of OCT4B group variants is still unclear.<br /> Methods: In the present study, we employed RT-PCR and sequencing approaches to explore different forms of <em>OCT4</em> transcripts.<br /> Results: Our data revealed that the OCT4B group variants (OCT4B-variant2, OCT4 B-variant3, OCT4B1, OCT4B2 and OCT4B3) have longer 5&#39; UTR in the human bladder carcinoma cell line of 5637.<br /> Conclusion: These <em>OCT4</em> variants undergo alternative splicing in their 5&#39; UTR which might exert regulatory roles in transcription and translation mechanisms.</p> https://www.AJMB.org/En/Article.aspx?ID=314 Ensieh M. Poursani, Majid Mehravar, Alireza Shahryari, Seyed Javad Mowla, Bahram Mohammad Soltani Tue, 19 Sep 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Novel Mutation in <i>SNX10</i> Gene Causes Malignant Infantile Osteopetrosis <p>Background: Osteopetrosis is a group of genetically heterogonous diseases and the main feature of that is increased bone density due to osteoclast&rsquo;s abnormality. It has three clinical forms based on inheritance pattern, severity and age of onset: the dominant benign form (ADO), the intermediate form (IRO) and the recessive severe form (ARO). One of the recently discovered genes for ARO form is <em>SNX10</em> that accounts for 4% of affected persons by this type.<br /> Methods: In this paper, a 15 years old girl affected by osteopetrosis has been analyzed for detecting causal mutation in known osteopetrosis genes. To get it done, amplified exons of the genes were sequenced and then were analyzed.<br /> Results: Direct sequencing of <em>SNX10</em> gene showed a homozygous c.43delG variant in the patient. Both healthy parents were heterozygous for this variant. In silico analysis revealed that this novel variant can be considered as the cause of disease in the patient.<br /> Conclusion: In this paper, a girl affected by osteopetrosis with a novel deletion in <em>SNX10</em> gene was reported.</p> https://www.AJMB.org/En/Article.aspx?ID=292 Akbar Amirfiroozy, Amir A. Hamidieh, Zahra Golchehre, Azim Rezamand, Mahin Yahyaei, Fatemeh Beiranvandi, Soheyla Amirfiroozy, Mohammad Keramatipour Tue, 19 Sep 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Innovation and Technology in Iran <p>Scientific production of Iran&rsquo;s scientists holds the needed potential and wealth to be the world&rsquo;s reference in science and knowledge. Based on this and by following guidelines of the supreme leader of the Islamic revolution, preservation, per-sistence, and reinforcement of the scientific production debate has unavoidable urgency and priority in academic and scientific circles and gatherings. We must support innovative and technological institutes more than before.<br /> Even though the country has achieved regional first rank in number of published articles and enjoys noticeable ranking in the world either in basic or in clinical medicine <sup>1,2</sup>, it seems the process of commercializing articles and other research products and transforming research into product and innovation production is still in need of more attention. It is such that Iran&rsquo;s rank in international innovation indicators, or Global Innovation Index, is still not noticeable. Despite holding first rank in the region for article publication and scientific production, in regards to global innovation indicators, Iran holds 11<sup>th</sup> rank in the region, ranking which places Iran after countries like United Arab Emirates and Kuwait. In this ranking system, unfortunately, Iran holds rank of 78 among the 143 countries of the world. In the same ranking system, Iran&rsquo;s rank in the world in regards to infrastructure for innovation is 91, in regards to creative output is 75, and in regards to knowledge and technology output is 65. It seems, in this field, more drive and effort should be put forth to shape a system for transforming science into innovation, commercializing research and pivoting research on production so Iran can be transformed into an innovative country with an economy pivoted on science and also in the innovation arena can acquire needed authority.</p> https://www.AJMB.org/En/Article.aspx?ID=276 Shahin Akhondzadeh Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Embryonic Stem Cell Conditioned Medium Supports In Vitro Maturation of Mouse Oocytes <p>Background: This study aimed to investigate the maturation and fertilization rates of immature mouse oocytes using Embryonic Stem Cell Conditioned Medium (ESCM).<br /> Methods: Germinal Vesicle (GV) stage oocytes were observed in 120 NMRI mice, aged 4-6 weeks. GV oocytes with or without cumulus cells were subjected to IVM in either ESCM, Embryonic Stem Cell Growth Medium (ESGM), or &alpha;-minimum essential medium (&alpha;-MEM). After recording the Metaphase II (MII) oocyte maturation rate, the oocytes were fertilized<em> in vitro</em>. The fertilization success rate was recorded after 24 <em>hr. </em>The embryos were maintained in potassium Simplex Optimization Medium (KSOM) for 96 <em>hr</em> and allowed to grow until the blastocyst stage. After recording developmental competence, they were transferred into the uteri of pseudopregnant mice and their birth rates were recorded.<br /> Results: No significant difference existed between the maturation rates in &alpha;-MEM (68.18%) and ESCM (64.67%; p&gt;0.05), whereas this rate was significantly higher for both &alpha;-MEM and ESCM compared to ESGM (32.22%; p&lt;0.05). A significant difference in IVF success rate existed for oocytes grown in &alpha;-MEM (69.44%), ESCM (61.53%), and ESGM (0%). A significantly higher developmental competence was observed at the blastocyst stage for oocytes grown in &alpha;-MEM (51.2%) compared to ESCM (35%; p&lt;0.05). 17 days after embryo transfer into the uteri of pseudopregnant mice, there was a nonsignficant (p&gt;0.05), similar birth rate between &alpha;-MEM and ESCM (47 <em>vs</em>. 40%).<br /> Conclusion: ESCM is an effective medium for preantral follicle growth, oocyte maturation, and subsequent embryo development.</p> https://www.AJMB.org/En/Article.aspx?ID=277 Saber Miraki, Aram Mokarizadeh, Omid Banafshi, Vahideh Assadollahi, Mahdad Abdi, Daem Roshani, Fardin Fathi Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Biosynthesis of Silver Nanoparticles using <i>Chlorella vulgaris</i> and Evaluation of the Antibacterial Efficacy Against <i>Staphylococcus aureus</i> <p>Background: It is well documented that Silver Nanoparticles (SNPs) are potent antimicrobial agents. However, little is known about antimicrobial effects of biologically synthesized SNPs at molecular level. In the present study, efficacy of the green microalgae <em>Chlorella vulgaris</em> in biosynthesis of silver nanoparticles and inhibitory effect of the biosynthesized SNPs on growth and virulence of <em>Staphylococcus aureus (S. aureus)</em> were investigated.<br /> Methods: Algal suspension was incubated in the presence of silver nitrate to induce formation of nanoparticles. The experiment was conducted under a pH range to evaluate pH effect on the shape and properties of nanoparticles. Characterization was performed by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy Dispersive Spectrometry (EDS) and X-ray diffraction analysis (XRD). Moreover, concentration of biosynthesized SNPs was measured by high resolution ICP-OES spectrometer. Antibacterial effect of SNPs on growth of <em>S. aureus</em> was evaluated by broth micro-dilution method. Inhibitory effect of SNPs on alpha hemolysin, a well-known virulence factor of <em>S. aureus</em> was investigated through real time PCR assay.<br /> Results: Spherical SNPs were produced with characteristic monodispersity at low and neutral pHs; however, in alkaline condition, nanorod structures were formed. SNPs inhibited growth of <em>S. aureus</em> at concentration of 50 <em>&mu;g/ml</em>. Alpha hemolysin expression was also effectively inhibited by SNPs treatment.<br /> Conclusion: In general, results revealed formation of spherical silver nanoparticles with inhibitory effects on bacterial growth and antagonist activity on the expression of alpha hemolysin. Moreover, increase in pH to basic condition resulted in aggregation of nanoparticles and formation of rod-like nanostructures.</p> https://www.AJMB.org/En/Article.aspx?ID=278 Mohammad Soleimani, Maziar Habibi-Pirkoohi Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of Factors Affecting Size and Size Distribution of Chitosan-Electrosprayed Nanoparticles <p>Background: Size and size distribution of polymeric nanoparticles have important effect on their properties for pharmaceutical application. In this study, Chitosan nanoparticles were prepared by electrospray method (electrohydrodynamic atomization) and parameters that simultaneously affect size and/or size distribution of chitosan nanoparticles were optimized.<br /> Methods: Effect of formulation/processing three independent formulation/processing parameters, namely concentration, flow rate and applied voltage was investigated on particle size and size distribution of generated nanoparticles using a Box&ndash;Behnken experimental design.<br /> Results: All the studied factors showed important effects on average size and size dis-tribution of nanoparticles. A decrease in size and size distribution was obtainable with decreasing flow rate and concentration and increasing applied voltage. Eventually, a sample with minimum size and polydispersity was obtained with polymer concentration, flow rate and applied voltage values of 0.5 %w/v, 0.05 <em>ml/hr</em> and 15 <em>kV</em>, respectively. The experimentally prepared nanoparticles, expected having lowest size and size distribution values had a size of 105 <em>nm</em>, size distribution of 36 and Zeta potential of 59.3 <em>mV</em>.<br /> Conclusion: Results showed that optimum condition for production of chitosan nano-particles with the minimum size and narrow size distribution was a minimum value for flow rate and highest value for applied voltage along with an optimum chitosan concentration.</p> https://www.AJMB.org/En/Article.aspx?ID=279 Morteza Abyadeh, Ali Akbar Karimi Zarchi, Mohammad Ali Faramarzi, Amir Amani Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effects of <i>Melilotus officinalis</i> Extract on Expression of <i>Daxx</i>, <i>Nfkb</i> and <i>Vegf</i> Genes in the Streptozotocin-Induced Rat Model of Sporadic Alzheimer's Disease <p>Background: Possible mechanisms of Alzheimer Disease (AD) such as inflammation and oxidative stresses in the brain led us to investigate potential AD therapeutics of <em>Melilotus officinalis</em>, an herbal extract, with possible role as an anti-inflammatory and anti-oxidant agent. Among different genes which had important role in Sporadic AD (SAD), three genes including<em> DAXX</em>, <em>NFkB</em> and <em>VEGF</em> have shown significant statistical diversity in the brains of Alzheimer patients.<br /> Methods: These genes were chosen to be investigated for neuroprotective effects of the extract by comparing the expression level in the hippocampus of Sporadic AD (SAD) rat model using quantitative polymerase chain reaction (qPCR) in the treated and untreated groups. In addition, therapeutic effects at the behavioral, learning and memory level by Morris Water Maze (MWM) test were investigated.<br /> Results: The results represented significant decreased expression in <em>Daxx</em>, <em>Nfkb</em> and <em>Vegf</em> genes in the SAD rat&rsquo;s model treated with the herbal extract compared to the Streptozotocin-induced (STZ-induced) rats. Furthermore, no significant changes were seen in swimming distance and time for finding the hidden platform in the herbal-treated compared to the STZ-induced group. In memory level, no significant changes were observed among treated and untreated groups.<br /> Conclusion: It seems that the herbal extract may have significant effect on Alzheimer-related gene expression changes but not on clinical levels.</p> https://www.AJMB.org/En/Article.aspx?ID=280 Niloofar Bazazzadegan, Marzieh Dehghan Shasaltaneh, Kioomars Saliminejad, Koorosh Kamali, Mehdi Banan, Hamid Reza Khorram Khorshid Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Distribution of Class I Integron and <i>smqnr</i> Resistance Gene Among <i>Stenotrophomonas maltophilia</i> Isolated from Clinical Samples in Iran <p>Background: <em>Stenotrophomonas maltophilia (S. maltophilia)</em> is a multiple-antibiotic-resistant opportunistic pathogen that is being isolated with increasing frequency from patients with health-care-associated infections. <em>S. maltophilia</em> is inherently resistant to most of the available antimicrobial agents. Spread of resistant strains has been attributed, in part, to class I integrons. <em>In vitro</em> susceptibility studies have shown trimethoprim-sulfamethoxazole and new floroquinolones as two important agents with activity against these organisms.<br /> Methods: 150 isolates of <em>S. maltophilia </em>were isolated from clinical samples such as respiratory discharges, sputum, and catheter and hospital environments. These isolates were also subjected to susceptibility testing and polymerase chain reaction for four groups of genes including <em>int </em>encoding integron elements, <em>sulI</em> and <em>sulII</em> encoding trimethoprim-sulfamethoxazole resistance and <em>smqnr</em> encoding quinolone resistance.<br /> Results: The rate of resistance to trimethoprim-sulfamethoxazole was up to 27 (18%) and the highest resistance to quinolone family belonged to ofloxacin (20%) and the lowest rate was for gatifloxacin (16%). The results showed that 14% of isolates contained integron elements concomitantly with <em>sulI</em> and <em>sulII</em> genes.<br /> Conclusion: Resistance rate of <em>S. maltophilia</em> to co-trimoxazole and fluoroquinolones and detection of integron elements between isolates in this study showed that this rate corresponded to other data obtained from other studies.</p> https://www.AJMB.org/En/Article.aspx?ID=281 Mohammadali Malekan, Bahman Tabaraie, Ladan Akhoundtabar, Parviz Afrough, Ava Behrouzi Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Novel Variant of <i>OCT4</i> Entitled OCT4B3 is Expressed in Human Bladder Cancer and Astrocytoma Cell Lines <p>Background: Alternative splicing is an important mechanism that regulates gene expression and function in human cells. <em>OCT4</em>, a crucial pluripotency marker in embryonic stem/carcinoma cells generates several spliced variants in different cell types and cancers. The expression of <em>OCT4</em> in cancers has been challenged in many studies. The existence of several <em>OCT4</em> spliced variants and absence of specific discriminating primers is the main reason of this controversy. Therefore, using specific primers and discriminating <em>OCT4</em> variants from each other might help to reduce these discrepancies&nbsp; in carcinogenesis and stem cell researches.<br /> Methods: 17 various human cancer, pluripotent and normal cells were cultured and their RNAs were extracted. Related cDNAs were synthesized and the expression pattern of <em>OCT4</em> variants was investigated by RT-PCR assay. PCR products were cloned into pTZ57R/T vector and their authenticity was confirmed by DNA sequencing.<br /> Results: Expression pattern of <em>OCT4</em> variants (OCT4A, OCT4B and OCT4B1) was analyzed by RT-PCR assay and the authenticity of PCR products was confirmed by DNA sequencing. A novel spliced variant of OCT4 was discovered and named as OCT4B3. This variant was very similar to OCT4B2 transcript except that 207-nt of exon 1b is lost. Moreover, the expression pattern of OCT4B3 variant was investigated in 17 human cell types, where its expression was only found in astrocytoma and bladder cancer cell types 1321N1 and 5637, respectively.<br /> Conclusion: <em>OCT4</em> variants are differentially expressed in various human cancer cell lines. Moreover, a novel variant of <em>OCT4</em>, OCT4B3, was detected in two human cancer cell lines of bladder carcinoma (5637) and brain astrocytoma (1321N1) for the first time.</p> https://www.AJMB.org/En/Article.aspx?ID=282 Ensieh M. Poursani, Majid Mehravar, Bahram Mohammad Soltani, Seyed Javad Mowla, James E. Trosko Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Novel Variant in the <i>PAH</i> Gene Causing Phenylketonuria in an Iranian Pedigree <p>Background: <em>Phenylalanine hydroxylase (PAH) </em>gene is the well-known causative gene for classic Phenylketonuria (PKU) (OMIM#261600) disease, with more than 500 reported mutations. Through this study, a novel mutation in the <em>PAH </em>gene in an Iranian pedigree with phenylketonuria was introduced.<br /> Methods: A consanguineous family with a 10-year old affected girl was referred for genetic analysis. Mutation screening of all exons and exon-intron boundaries was performed by Sanger sequencing, and mini haplotype analysis was carried out by genotyping of Short Tandem Repeat (STR) and Variable Number Tandem Repeat (VNTR) alleles.<br /> Results: Mutation analysis revealed a novel homozygous insertion of a single adenine nucleotide at position 335 in exon 3 of the PAH gene. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the change is interpreted as a pathogenic mutation which produces a premature termination signal (TAA) at codon 113 according to in silico assessments. The mini haplotype analysis showed that this mutation was linked to STR (15) &ndash;VNTR (3).<br /> Conclusion: In this study, a novel mutation was reported in a patient who had PKU symptoms without any previously reported mutations in the <em>PAH</em> gene (NM_000277.1: p.Asp112Glufs*2) that can be responsible for the classical PKU phenotype in the Iranian population. Detection of novel mutations indicates notable allelic heterogeneity of the PAH locus among this population.</p> https://www.AJMB.org/En/Article.aspx?ID=283 Elaheh Alavinejad, Seyede Zahra Sajedi, Masoumeh Razipour, Mona Entezam, Neda Mohajer, Aria Setoodeh, Saeid Talebi, Mohammad Keramatipour Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Polymorphisms in the Estrogen Receptor Beta Gene and the Risk of Unexplained Recurrent Spontaneous Abortion <p>Background: Recurrent Spontaneous Abortion (RSA) is caused by multiple genetic and non-genetic factors. Around 50% of the RSA cases have no known etiology and are considered as Unexplained RSA (URSA). Estrogens, <em>via</em> binding to their receptors, play an important role in female reproduction. This study aimed to investigate whether single nucleotide polymorphisms (SNPs; +1082G/A, +1730G/A and rs1256030C/T) in the estrogen receptor beta (<em>ESR2</em>) gene are associated with susceptibility to URSA in a population of Iranian women.<br /> Methods: In this case-control study, the study groups consisted of 240 subjects with a history of URSA and 102 fertile women as controls. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were measured on day 2-3 of menstrual cycle. Two functional SNPs, +1082G/A (a silent mutation in exon 5) and +1730G/A (3&#39; untranslated region of the exon 8),and one intron,rs1256030C/T, in the ESR2 gene were genotyped, using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis.<br /> Results: Serum levels of LH were significantly increased in URSA women. No significant differences in distribution of +1082G/A, +1730G/A and rs1256030C/T between URSA and control groups were observed.<br /> Conclusion: Our findings suggest that the studied SNPs on <em>ESR2</em> gene may not be associated with URSA.</p> https://www.AJMB.org/En/Article.aspx?ID=284 Marzieh Mahdavipour, Saeed Zarei, Ramina Fatemi, Haleh Edalatkhah, Hamed Heidari-Vala, Mahmood Jeddi-Tehrani, Farah Idali Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Major Components of Metabolic Parameters and Nutritional Intakes in Different Genotypes of Adiponectin +276 G>T Gene Polymorphism in Non-Diabetes and Non-Alcoholic Iranian Fatty Liver Patients <p>Background: Genetic and environmental factors are both involved in the etiology of Non-Alcoholic Fatty Liver Disease (NAFLD). Among the genetic factors, certain polymorphisms of adiponectin gene are associated with NAFLD. In the current study, we investigated the association between metabolic parameters with different genotypes of adiponectin +276 G&gt;T polymorphism among the Iranian NAFLD patients, and the effect of nutritional intake with development of NAFLD.<br /> Methods: In this study, 75 patients with NAFLD and 76 healthy individuals were enrolled. Dietary intakes were assessed using a semi- quantitative Food-Frequency Questionnaire (FFQ). Body Mass Index (BMI) and Waist to Hip Ratio (WHR) were calculated. Biochemical assays including FSG (Fasting Serum Glucose), liver enzymes, lipid profiles, Malondialdehyde, insulin resistance and Total Antioxidant Capacity (TAC) were measured after 12 <em>hr</em> fasting. Gene polymorphism study was done by using of sequencing method.<br /> Results: Although, T allele frequency was more prevalent in patients with NAFLD than control, adiponectin +276 G&gt;T polymorphism was not associated with risk of NAFLD. Among the metabolic parameters, TAC in TT genotype was significantly lower 1.44(0.69 to 2.81) p&gt;0.05, AST in GT, GG genotypes, and ALT in all three genotypes were higher in NAFLD patients in compared to healthy subjects (p&lt;0.05). Patients with GT genotype have significantly lower fat consumption and vitamin E intake as compared to control group with the same genotype (p&lt;0.05).<br /> Conclusion: In this study, we showed the association of different genotypes of +276 G&gt;T polymorphism in adiponectin gene with some metabolic parameters.</p> https://www.AJMB.org/En/Article.aspx?ID=294 Fatemeh Mohseni, Sahar Moghbelinejad, Reza Najafipour Wed, 07 Jun 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Research in Iran: An Overview <p>Islamic Iran, on the Horizon of the 2025 Outlook Document, should obtain first regional rank in economic, social, scientific, and cultural domains. Country&rsquo;s noticeable growth in scientific production and dynamic movement of Iranian scientists and specialists in scientific frontiers resulted in the country earning first rank in regional scientific production, leaving behind Turkey, in the year 2011, 14 years ahead of the Horizontal Outlook prediction. In the year 2016, Iran, with publication of 51187 articles in the Scopus database, acquired 16th world-wide ranking. In the same year, citation to the Iranian articles was 28965, has achieved 18th international ranking and 1st regional ranking. Overall, Iran, in the year 2016, has been responsible for an equivalent of 1.7% of the world&rsquo;s total scientific production. Certainly, preserving Iran&rsquo;s scientific growth trend is an important and fundamental subject and should consistently be brought to attention and consideration of government officials and policy makers in the research arena. There is no doubt; this support should include both basic and clinical studies <sup>1-3</sup>.<br /> Presence of national universities in international ranking institutions is considered one of the credible indicators for international scientific production for universities. Multiple international ranking institutions exist and university ranks in these institutions is considered to represent their qualitative and quantitative scientific production standing. One of the in-ternational ranking institutions, Essential Science Indicators (ESI), evaluates and ranks universities based on institution&rsquo;s citation standing for the past ten years in the ISI database. From the sixty present national universities, currently thirteen national medical science universities are among world&rsquo;s top universities in ESI. Before, this number was less and limited to a few universities. Among the top 500 universities in the world, just Tehran University of Medical Sciences is present.<br /> Scientific production of Islamic Iran&rsquo;s scientists holds the needed potential and wealth to be the world&rsquo;s reference in sci-ence and knowledge.&nbsp; Based on this and by following guidelines of the supreme leader of the Islamic revolution, preservation, persistence, and reinforcement of the scientific production debate has unavoidable urgency and priority in academic and scientific circles and gatherings. Therefore, we must support innovative and technological institutes more than before.</p> https://www.AJMB.org/En/Article.aspx?ID=267 Shahin Akhondzadeh Wed, 08 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Current State of Cartilage Tissue Engineering using Nanofibrous Scaffolds and Stem Cells <p>Cartilage is an avascular, aneural, and alymphatic connective tissue with a limited capacity caused by low mitotic activity of its resident cells, chondrocytes. Natural repair of full thickness cartilage defects usually leads to the formation of fibrocartilage with lower function and mechanical force compared with the original hyaline cartilage and further deterioration can occur. Tissue engineering and regenerative medicine is a promising strategy to repair bone and articular cartilage defects and rehabilitate joint functions by focusing on the optimal combination of cells, material scaffolds, and signaling molecules. The unique physical and topographical properties of nanofibrous structures allow them to mimic the extracellular matrix of native cartilage, making an appropriate resemblance to induce cartilage tissue regeneration and reconstruction. To improve simulation of native cartilage, the incorporation of nanofibrous scaffolds with suitable corresponsive cells could be effective. In this review article, an attempt was made to present the current state of cartilage tissue engineering using nanofibrous scaffolds and stem cells as high proliferative immune privilege cells with chondrogenic differentiation ability. The comprehensive information was retrieved by search of relevant subject headings in Medline/Pubmed and Elsevier databases.</p> https://www.AJMB.org/En/Article.aspx?ID=268 Somaieh Kazemnejad, Manijeh Khanmohammadi, Nafiseh Baheiraei, Shaghayegh Arasteh Wed, 08 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Gestational Exposure to Silymarin Increases Susceptibility of BALB/c Mice Fetuses to Apoptosis <p>Background: Silymarin is a flavonolignan that has been the subject of research to evaluate the beneficial properties for decades. Silymarin has been known for its potent cytoprotective, hepatoprotective and antioxidant activities. The goal of the present study was to gain a deeper understanding of possible molecular mechanisms of apoptosis of the injuries induced by silymarin on BALB/c mice fetuses.<br /> Methods: The present experimental study was carried out in virgin female BALB/c mice. The animals were divided randomly into 4 groups. Three test groups were injected intraperitoneally with silymarin at doses of 50, 100 and 200 <em>mg/kg/day</em> during gestational days 6-15. The control group received the solvent by the same route at equivalent volume. Western blot analysis was conducted to determine the levels of caspase-3 and caspase-8 in fetal heart, kidney, lungs and brain tissue.<br /> Results: The results of this study showed that silymarin administration during organogenesis at doses of 50, 100 and 200 <em>mg/kg</em> can significantly increase the protein levels of caspase-3 and 8 in heart, kidneys and brain tissues of mice fetuses compared with control group (p&lt;0.001). Silymarin exposure could not change the level of apoptotic markers in fetal lung tissue.<br /> Conclusion: According to the results, programmed cell death, especially via the intrinsic pathway, plays a pivotal role in the pathogenesis of silymarin-induced malformations in some tissue including heart, kidneys and brain. More studies are needed to determine other molecular mechanisms underlying silymarin- induced embryo toxicity.</p> https://www.AJMB.org/En/Article.aspx?ID=269 Mahbobe Gholami, Seyed Adel Moallem, Mohammad Afshar, Leila Etemad, Gholamreza Karimi Wed, 08 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction <p>Background: Autophagy as a cellular pathway facilitates several immune responses against infection. It also eliminates invading pathogens through transferring content between the cytosol and the lysosomal vesicles and contributes to the cross-presentation of exogenous antigens to T lymphocytes via MHC class I pathway. Autophagy induction is one of the main targets for new drugs and future vaccine formulations. Nanoparticles are one of the candidates for autophagy induction. Cysteine Peptidase A (CPA) and Cysteine Peptidase B (CPB) are two members of papain family (Clan CA, family C1) enzyme that have been considered as a virulence factor of Leishmania (L.) major, making them suitable vaccine candidates. In this research, Leishmania major cysteine peptidase A and B (CPA and CPB) conjugation to alpha alumina nanoparticle was the main focus and their entrance efficacy to macrophages was assessed.<br /> Methods: For this purpose, CPA and CPB genes were cloned in expression vectors. Related proteins were extracted from transformed <em>E. coli</em> and purified using Ni affinity column. Alpha alumina nanoparticles were conjugated to CPA/CPB proteins using Aldehyde/Hydrazine Reaction. Autophagy induction in macrophages was assessed using acridine orange staining.<br /> Results: CPA/CPB protein loading to nanoparticles was confirmed by Fourier Transform Infrared Spectroscopy. &alpha;-alumina conjugated CPA/CPB antigen uptake by macrophages at different concentrations was confirmed using fluorescence microscope and flowcytometry. Highly efficient CPA/CPB protein loading to &alpha;-alumina nanoparticles and rapid internalization to macrophages introduced these nanocarriers as a delivery tool. Acridine orange staining demonstrated higher autophagy induction in CPA/CPB protein conjugated with &alpha;-alumina nanoparticles.<br /> Conclusion: &alpha;-alumina nanoparticles may be a promising adjuvant in the development of therapeutic leishmania vaccines through antigen delivery to intracellular compartments, induction of autophagy and cross presentation to CD<sub>8</sub> lymphocytes.</p> https://www.AJMB.org/En/Article.aspx?ID=270 Fatemeh Beyzay, Ahmad Zavaran Hosseini, Sara Soudi Wed, 08 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Efficient Media for High Lipase Production: One Variable at a Time Approach <p>Background: Lipase enzymes have applications in a wide range of industries. A crucial determining factor of industrial prices of these enzymes is the culture media composition that is constantly under review by researchers. In this work, for maximum lipase production by <em>Bacillus sp. ZR-5</em>, culture media compositions were optimized using &quot;one variable at a time&quot; strategy.<br /> Methods: For this purpose, the culture medium parameters such as low and high cost carbon and nitrogen sources, substrates and incubation times were evaluated.<br /> Results: Maximum lipase activity was achieved after 24<em> hr</em> of incubation with 1.5% of glucose syrup (1600&plusmn;69.1 <em>u/mg</em>), 1% of fish powder (1238&plusmn;36.7 <em>u/mg</em>) and olive oil (1407&plusmn;2.1 <em>u/mg</em>) as low cost carbon and nitrogen sources and substrate, respectively.<br /> Conclusion: Our results show a significant increase in lipase activity with usage of low cost sources; this could help in reducing the media prices for industrial application of lipase enzyme.</p> https://www.AJMB.org/En/Article.aspx?ID=271 Safoura Soleymani, Houri Alizadeh, Hossein Mohammadian, Emad Rabbani, Fatemeh Moazen, Hamid MirMohammad Sadeghi, Ziaedin Samsam Shariat, Zahra Etemadifar, Mohammed Rabbani Wed, 08 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of IgY Antibody as a Polyspecific Coombs-Reagent <p>Background: During the last twenty years, the extraction of specific egg yolk (IgY) antibodies from the immunized chickens has been accepted as a useful alternative to the immunization of mammals. The aim of the present study was immunizing the chickens with Human Umbilical Cord Serum (HUCS) and the extraction of specific anti-human globulins (IgG, C3b, and C3d) antibodies from egg yolk in order to obtain polyspecific Coombs reagent.<br /> Methods: The novelty of this work was the achievement of a polyclonal reagent through a very cheap alternative method in accordance with all ethical regulations required for obtaining it. Three Leghorn hens (21 weeks old) were immunized four times for a period of 66 days with 20uL of HUCS mixed with PBS/FCA or FIA each time. The extraction of IgY antibodies was performed according to the method of lipid precipitation of yolk and using water soluble fraction as the reagent material. The resulting IgY antibody&nbsp; was characterized by SDS-PAGE and immunoelectrophoresis and tested for the presence of hetero-agglutinins by means of direct agglutination using human erythrocytes of all blood groups treated with 0.1% papain and for indirect Coombs-test to evaluate its specificity to fractions (C3b, C3d, C4d) of human complement and human IgG, respectively.<br /> Results: Our findings show, that, the reagent obtained contains IgY and other 3 proteins (SDS-PAGE), and reacts specifically with plasma proteins, that migrate in &beta; and &upsih; regions. In immunoelectrophoresis, in addition, there is the presence of low hetero-agglutinins levels in IgY-preparation (3 lots), and the possibility to produce high amount (more than 500 <em>ml/egg</em>) of polyspecific Coombs-reagent in chickens is also discussed.<br /> Conclusion: IgY-preparation (3 lots), and the possibility to produce high amount (more than 500 ml/egg) of polyspecific Coombs-reagent in chickens with the originality to achieve a polyclonal reagent through a very cheap alternative method in accordance with all ethical regulations required for obtaining it, was also discussed.</p> https://www.AJMB.org/En/Article.aspx?ID=272 Gutiérrez Calzado Esteban Justo, Toledano Heredia Marlene, Fernadez Duharte Jeorge, Amir-Hassan Zarnani Wed, 08 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of Interactions of Single Nucleotide Polymorphisms of APOA1/APOC3 with Food Group Intakes on the Risk of Metabolic Syndrome <p>Background: The aim of this study was to examine the interaction of dietary food groups and genetic variants of APOA1/APOC3, relative to Metabolic Syndrome (MetS) risk in adults.<br /> Methods: In this matched nested case-control study, 414 MetS subjects and 414 controls were selected from among participants of Tehran Lipid and Glucose Study. Dietary intake was assessed with the use of a valid and reliable semi-quantitative food frequency questionnaire. Single Nucleotide Polymorphisms (SNPs), APOA1 (rs670, -75G&gt;A and rs5069, +83C&gt;T/APOC3 rs5128 C3238&gt;G) were genotyped by the conventional polymerase chain reaction and restriction fragment length polymorphism.<br /> Results: The mean (SD) of age was 40.7 (13) and 41.2 (13) years in male cases and controls versus 44.0 (11) and 44.0 (12) years in female case and controls. A significant interaction between intake quartiles of the sugar group and APOA1 combined group (GA+AA/CT+TT) SNPs was found; The ORs for these genotype carriers were (1, 0.44, 0.36, 0.23; P trend&lt;0.001) in quartiles of intake, relative to other combined genotypes (P interaction=0.02). MetS risk appeared to be increased significantly in higher quartiles of sweet beverages and fish intakes in the GA+AA/CT+TT/CC genotypes of APOA1/APOC3 SNPs, compared to other genotypes (P interaction=0.01). The combined effect of genotypes of APOC3/APOA1 showed further decrease in MetS risk in higher quartiles of sugar group intakes (OR: 1, 0.24, 0.26, 0.14, P trend=0.001) relative to other combinations (P interaction=0.008).<br /> Conclusion: Results obtained demonstrate that some dietary food groups (sugar, fish, and sweet beverages) modulate the effect of APOA1/APOC3 SNPs in relation to MetS risk.</p> https://www.AJMB.org/En/Article.aspx?ID=273 Firoozeh Hosseini-Esfahani, Parvin Mirmiran, Maryam S. Daneshpour, Azadeh Mottaghi, Fereidoun Azizi Wed, 08 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of Flow Cytometry-Fluorescent In Situ Hybridization (Flow-FISH) Method for Detection of PML/RARa Chromosomal Translocation in Acute Promyelocytic Leukemia Cell Line <p>Background: Acute Promyelocytic Leukemia (APL) is a subclass of acute myeloid leukemia. The chromosomal aberration in 95% of APL cases is t(15; 17) (q22; q21), which prevents cell differentiation. Characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. The goal of this study was to develop a new and powerful Flow- FISH technique to detect the long isoform (L) of PML-RARa fusion transcript in NB4 cell line.<br /> Methods: To achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% Triton X-100 and 0.2% saponin were used for the permeabilization step .In hybridization, a wide range of times and temperatures were used and probe was designed in FRET system. Results were confirmed by fluorescent microscope assay and reverse transcription PCR.<br /> Results: In the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of PML-RARa transcript. Using standard fixation and permeabilization protocol of 2% PFA and 0.2% saponin gave the best fluorescent results in flow cytometry. Also, results indicated that the optimum time and temperature for hybridization was 2 <em>hr</em> at 42<sup>o</sup><em>C</em>. The results of reverse transcription PCR and fluorescent microscopy confirmed the presence of PML-RARa transcript.<br /> Conclusion: The concordance between the results of Flow-FISH and those of two other techniques including reverse transcription PCR and FISH indicated that this method would be applicable as a diagnostic test for APL in clinical samples and MRD monitoring.</p> https://www.AJMB.org/En/Article.aspx?ID=274 Fatemeh Zahedipour, Reza Ranjbaran, Abbas Behzad-Behbahani, Khalil Tavakol Afshari, Mohammad Ali Okhovat, Gholamhossein Tamadon, Sedigheh Sharifzadeh Wed, 08 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Can Aptameric Ligands Specific to Plasma Coagulation Factor VII Bind the Recombinant Form with High Affinity: Affinity Measurement by Fluorescence Method <p>Background: Among diverse protein purification systems, affinity chromatography is the most attractive one in the purification process of coagulation factors. Coagulation factor VII is a plasma serine protease that has a significant role in natural human hemostasis and its recombinant form such as AryoSeven<sup>TM</sup>, has been applied in clinical treatment of bleeding disorders. Immunoaffinity chromatography is the purification method of choice that is currently applied in the development of coagulation factor VIIa products. Aptamers as nucleic acid based affinity ligands are more promising than monoclonal antibodies. In addition, DNA aptamers are more acceptable than RNA ones in this regard.<br /> Methods: In this study, two of the aptameric DNA oligonucleotides that showed acceptable affinities for purification of coagulation factor VIIa from plasma, were selected to evaluate their affinity against Aryoseven. A serial dilution of fluorescence labeled aptamers was incubated against the concentration of 1 <em>nM</em> from Aryoseven. Then, a fluorescence index was calculated according to the fluorescence intensity data measured from test and control samples. The dissociation constant of aptamers was calculated using according to the fluorescence index Prism5 software.<br /> Results: Results showed that the binding affinity of the 44 nucleotide aptamer was more than 81 nucleotide aptamer sequence. As a result, this aptamer could be optimized in order to develop aptamer based affinity chromatography process for this form of recombinant coagulation factor VIIa.<br /> Discussion: Aptamers with shorter length of sequence could show higher affinity in target binding, as they could adapt more easily to suitable conformation according to target interaction. However, it should be considered that the selectivity of affinity ligands is also important for target purification and analytical applications.</p> https://www.AJMB.org/En/Article.aspx?ID=275 Maryam Tabarzad, Marzieh Jafari, Nastaran Nafissi-varcheh Wed, 01 Mar 2017 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Research in Iran: Hopes and Disappointments <p>In the September 15, 2016 issue of Science, Dr. Richard Stone wrote a report regarding selling theses and research articles in Iran. There are noteworthy points in this report which truly disturb the Iranian scientific community, but the present report is not the whole truth about the Iranian scientific community. Iran&rsquo;s scientific infrastructure was destroyed in the eight-year imposed war by Saddam Hossein in a way that production of Iranian scientific articles in the Web of Sciences, which was twice Turkey&rsquo;s scientific articles before the Islamic Revolution, decreased to one-tenth of Turkey&rsquo;s scientific articles after the imposed war. Luckily, with creation of scientific infrastructure in the country&rsquo;s universities in a 25-year period after the war, Iranian scientific production increased noticeably in such a way that in 2011 Iran had the greatest impetus in scientific production and also in industry, for example in production of drugs, we succeeded in producing 90% of country&rsquo;s needed drugs <sup>1, 2</sup>. We succeeded in training enough physicians, dentists, and specialized physicians so that even small Iranian cities have neurosurgeons and orthopedic surgeons. Unfortunately, two problems arose in continuation of this policy: 1. Many private universities and on-line public universities began training students without logical policies especially postgraduate students without appropriate scientific infrastructure, qualified professors, and qualified students; 2. Western economic sanctions which began in 2011, although the economy was the primary focus, also targeted the Iranian scientific infrastructure. For example, university and researcher access to ISI journals via the digital library was terminated or purchase of research material such as laboratory kits for research and educational objectives encountered serious problems. Researchers and the society of Iranian scientists believe this act has not been scientific and fair. We must accept that the above mentioned two factors delivered injuries to the body of higher education. On the other hand, one must confess that at international levels, credible Iranian scientific universities move forward in education and research with power as evidenced by acceptance of Iranian medical graduates with high scores on the USMLE.<br /> Many weak scientific journals which daily appear like mushrooms in many Southeast Asian countries and even European countries have disrupted the correct international scientific environment. The Iranian scientific society is hopeful. The Joint Comprehensive Plan of Action (JCPOA) and the vision of Dr. Rouhani&rsquo;s cabinet will increase the Iranian scientific society&rsquo;s endurance and will put a stop to present worries. Along the same lines, vision of the Iranian Ministry of Health and Medical Education in research evaluation of universities, in the past three years, has returned from quantity to quality. Many, in place of number of articles, put emphasis on article quality. Also, for improvement of medical university research infrastructures, a national granting body at the Health Ministry, titled NIMAD, was created which financially supports large national projects. In addition, ten large comprehensive research laboratories opened in the past three years. More than ten large cohort studies and thirty national disease registries have begun operating across the country. We are hopeful that in the next few years, exit of these large studies will result in research quality improvement in the country&rsquo;s medical universities.</p> https://www.AJMB.org/En/Article.aspx?ID=250 Shahin Akhondzadeh Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effects of Berberine and Palmatine on Efflux Pumps Inhibition with Different Gene Patterns in <i>Pseudomonas aeruginosa</i> Isolated from Burn Infections <p>Background: Related Multidrug Resistance (MDR) to efflux pumps is a significant problem in treating infections caused by <em>Pseudomonas aeruginosa (P. aeruginosa)</em>. Plant compounds have been identified as Pump Inhibitors (EPIs). In the current study, the potential effect of Berberine and Palmatine as EPIs were investigated on efflux pump inhibition through focusing on different gene patterns in <em>P. aeruginosa</em> isolated from burn infections.<br /> Methods: All isolates were collected and identified using the standard biochemical tests. Antimicrobial sensitivity was performed based on disk agar diffusion method for 12 antibiotics. MIC-MBC tests were also performed based on the broth microdilution method to detect synergistic relationship between ciprofloxacin, Berberine and Palmatine. Detection of <em>mexA</em>, <em>mexB</em>, <em>mexC</em>, <em>mexD</em>, <em>mexE</em>, <em>mexF</em> and <em>mexX </em>was conducted by PCR assay. Fisher&#39;s Exact test and Logistic Regression were used as statistical tools.<br /> Results: A total of 60 <em>P. aeruginosa</em> isolates were collected. The highest and lowest levels of resistance were found to be respectively against clindamycin and tigecycline. Comparing the MIC with MBC distribution, it was found that Berberine and Palmatine lower the MIC-MBC level of ciprofloxacin. The PCR results indicated that the highest frequency is about MexAB-OprM operon. The statistical analysis among different gene patterns of efflux pumps showed that there were no significant relationships between the effectiveness of Berberine and Palmatine (p&gt;0.05).<br /> Conclusion: It can be speculated that Berberine and Palmatine both act as EPIs and can be used as auxiliary treatments with the purpose of increasing the effect of available antibiotics as well as decreasing the emergence of MDR bacteria. The efficiency of these combinations should be studied further under<em> in vivo</em> conditions to have a more comprehensive conclusion regarding this issue.</p> https://www.AJMB.org/En/Article.aspx?ID=259 Seyed Sadjjad Aghayan, Hamidreza Kalalian Mogadam, Mozhgan Fazli, Davood Darban-Sarokhalil, Seyed Sajjad Khoramrooz, Fereshteh Jabalameli, Somayeh Yaslianifard, Mehdi Mirzaii Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Role of M2000 as an Anti-inflammatory Agent in Toll-Like Receptor 2/microRNA-155 Pathway <p>Background: M2000 is a newly designed and safe Non-Steroidal Anti-Inflammatory Drug (NSAID). The aim of this study was to assess the effects of M2000 on expression levels of Suppressor of Cytokine Signaling-1 (SOCS-1) and Src Homology-2 domain-containing inositol-5&rsquo;-phosphatase 1 (SHIP1) proteins <em>via</em> Toll-Like Receptor (TLR) 2/microRNA-155 pathway.<br /> Methods: HEK293 TLR2 cell line and Peripheral Blood Mononuclear Cells (PBMCs) were treated by different concentrations of M2000 in MTT assay. RNA was extracted by miRNeasy Mini kit. Then, cDNA was synthesized and the expression levels of SOCS1, SHIP1 and miRNA155 were evaluated by Quantitative Real time PCR.<br /> Results: Our results showed that M2000 significantly increased the expression levels of SOCS1 and SHIP-1 in Lipopolysachride (LPS)-treated and non-treated cells. Moreover, M2000 decreased expression level of miR-155 in LPS treated PBMCs.<br /> Conclusion: M2000 can be used as NSAID in LPS induced inflammation and decrease inflammatory cytokines production by targeting SOCS1, SHIP1 and miR-155 in autoimmune and inflammatory diseases.</p> https://www.AJMB.org/En/Article.aspx?ID=260 Fatemeh Pourgholi, Mahsa Hajivalili, Rasoul Razavi, Shadi Esmaeili, Behzad Baradaran, Ali Akbar Movasaghpour, Sanam Sadreddini, Hamidreza Goodarzynejad, Abbas Mirshafiey, Mehdi Yousefi Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Induction of Epigenetic Alteration by CPUK02, An Ent- kaurenoid Derivative of Stevioside <p>Background: Dietary polyphenols, such as those found in green tea and red wine, are linked to antitumor activity. They are known to influence many signaling pathways epigenetically within the human body. In this regard, CPUK02 (15-Oxosteviol benzyl ester) is a new ent-kaurenoid derivative of stevioside and exhibits strong anti-cancer activity <em>in vitro</em> and <em>in vivo</em>. Nowadays, the role of epigenetics in cancer has been the subject of intensive study and DNA methylation targeting represents a relevant strategy for cancer treatment. There are no reports regarding the effects of CPUK02 on epigenetic alterations in colorectal cancer cell line. This study was an attempt to compare CPUK02 with 5-AZA as DNMT inhibitor agent and evaluate whether it can induce its anti-cancer effects via altering the level of DNMT3b mRNA, MGMT and SFRP2 methylation pattern in HCT 116 cell line.<br /> Methods: To evaluate DNMT3b expression, DNMT3B mRNA levels in HCT116 CRC cell line were quantified by real-time reverse-transcriptase Polymerase Chain Reaction (PCR) assay after 24 <em>hr</em> of incubation time with CPUK02 and 5-AZA. In addition, the methylation patterns of 2 CpG islands in this cell line were examined by methylation-specific PCR methods.<br /> Results: CPUK02 surprisingly, decreased the DNMT3b mRNA level. The average expression levels of DNMT3b in HCT116 treated with CPUK02 and 5-AZA relative to the GAPDH expression level in control were 0.16 and 0.5%, respectively. Furthermore, CPUK02 could decrease the methylated allele of MGMT and SFRP2 genes in HCT 116 after 24 <em>hr</em>.<br /> Conclusion: In this study, positive correlation was found between mRNA expression of DNMT3b and gene promoter hypermethylation after treatment with CPUK02 and 5-AZA. Our data confirmed that CPUK02 like 5-AZA exhibits demethylating properties.</p> https://www.AJMB.org/En/Article.aspx?ID=261 Pooneh Mokarram, Zeinab Mohammadi, Saeid Khazayel, Zhang Dayong Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Overexpression of Recombinant Human Teriparatide, rhPTH (1-34) in <i>Escherichia coli</i>: An Innovative Gene Fusion Approach <p>Background: Parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. Its physiological role is maintenance of normal serum calcium level and bone remodeling. Biological activity of this hormone is related to N-terminal 1-34 amino acids. The recombinant form of hormone (1-34) has been approved for treatment of osteoporosis from 2002. In this study, a novel fusion partner has been developed for preparation of high yield recombinant 1-34 amino acids of hPTH.<br /> Methods: Novel nucleotide cassette designed encoding a chimeric fusion protein comprising of a fusion partner consisting of a His-tag in N-terminal, 53 amino acids belong to <em>Escherichia coli (E. coli) </em>&beta;-galactosidase (LacZ) gene, a linker sequence for increasing of expression and protection of target peptide structure from fusion tag effect, an Enteropeptidase cleavage site, rhPTH (1-34) gene fragment. Optimized fusion gene was synthesized and ligated into pET-28a vector under control of T7 promoter, and then transformed in <em>E. coli</em> (DH5&alpha;) cells. Positive clones containing this gene were double digested with NcoI and-BamHI and also approved by sequencing. Gene overexpression was observed in SDS-PAGE after induction with 0.2 <em>mM</em> IPTG. Confirmation of gene expression was performed by western blotting using anti-His-tag antibody conjugated with peroxidase.<br /> Results: By this fusion gene design approach, we achieved a high level expression of the rhPTH, where it represented at least 43.7% of the total protein as determined by SDS-PAGE and confirmed by western blotting.<br /> Conclusion: In addition to high level expression of the designed gene in this work, specific amino acid sequence of bacterial &beta;-galactosidase was selected as major part of carrier tag for protection of this hormone as important step of recombinant rhPTH with relevant isoelectronic point (pI). This innovation resulted in recombinant production of hPTH very well and the gene construct could be applied as a pattern for similar recombinant peptides where recombinant protein degradation is a critical issue.</p> https://www.AJMB.org/En/Article.aspx?ID=262 Nahid Bakhtiari, Zahra Amini Bayat, Sepideh Sagharidouz, Mohsen Vaez Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Anti-Quorum Sensing Activity of Substances Isolated from Wild Berry Associated Bacteria <p>Background: Quorum Sensing (QS) is a mechanism used by bacteria to determine their physiological activities and coordinate gene expression based on cell to cell signaling. Many bacterial physiological functions are under the regulation of quorum sensing such as virulence, luminescence, motility, sporulation and biofilm formation. The aim of the present study was to isolate and characterize Quorum Sensing Inhibitory (QSI) substances from epiphytic bacteria residing on wild berries surfaces.<br /> Methods: Fifty nine bacterial isolates out of 600 screened bacteria were successfully isolated. These bacteria were obtained from berry surfaces of different plants in the wild forests of Ajloun-Jordan. Screening for QSI activity using <em>Chromobacterium violaceum</em> ATCC 12472 monitor strain, resulted in isolating 6 isolates exhibiting QSI activity only, 11 isolates with QSI and antibacterial activity, and 42 isolates with antibacterial activity only. Three potential isolates S 130, S 153, and S 664, were gram positive rods and spore formers, catalase positive and oxidase negative. These were chosen for further testing and characterization.<br /> Results: Different solvent extraction of the QSI substances based on polarity indicated that the activity of S 130 was in the butanol extract, S 153 activity in both chloroform and butanol; and for S 664, the activity was detected in the hexane extract. The chloroform extract of S 153 and hexane extract of S 664 were proteinaceous in nature while QSI substances of the butanol extract of S 130 and S 153 were non-proteinaceous. All the tested QSI substances showed a marked thermal stability when subjected at several time intervals to 70<sup>o</sup><em>C</em>, with the highest stability observed for the butanol extract of S 153. Assessing the QSI substances using violacein quantification assay revealed varying degrees of activity depending upon the extracting solvent, type of the producer bacteria and the concentration of the substances.<br /> Conclusion: This study highlighted the potential of untapped reservoirs in nature to be used as a source of unique metabolite that may be further developed for therapy. The potential QSI substances included in this study are just one aspect to be further analyzed for use as biopharmaceutical agents.</p> https://www.AJMB.org/En/Article.aspx?ID=263 Suha M. Abudoleh, Adel M. Mahasneh Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Isolation and Identification of Phyllospheric Bacteria Possessing Antimicrobial Activity from <i>Astragalus obtusifolius</i>, <i>Prosopis juliflora</i>, <i>Xanthium strumarium</i> and <i>Hippocrepis unisiliqousa</i> <p>Background: The widespread utilization of antimicrobial compounds has caused emergence of resistant microorganisms in the world. Hence, the research to probe the products with antimicrobial features has led to finding natural habitats and discovering new pharmaceutical products.<br /> Methods: In this study, an attempt was made to explore the niche of novel habitat to isolate pyllospheric bacteria from the above ground parts (stems and leaves) of <em>Astragalus obtusifolius, Prosopis juliflora, Xanthium strumarium,</em> and <em>Hippocrepis unisiliqousa</em> to evaluate their antimicrobial features. The inhibitory effects of these strains on the growth of two fungi <em>(Aspergillus niger, Aspergillus fumigatus)</em>, two yeasts <em>(Saccharomyces cerevisiae, Candida albicans)</em> and six bacteria <em>(Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella typhi, Streptococcus pyogenes)</em> were tested.<br /> Results: In total, 113 bacterial strains were isolated. Twenty five bacterial strains (B-1 to B-25) indicated promising antimicrobial (antibacterial and antifungal) activities against aforementioned pathogens. The identification of the bacterial strains was ascertained by morphological, physiological, biochemical tests and two strains with the strongest antimicrobial activities were further characterized based on 16s rRNA sequencing. These two strains were identified as <em>Bacillus amyloliquefaciens</em>.<br /> Conclusion: Our results provide evidence that phyllospheric microorganisms are capable of producing some compounds with antimicrobial properties.</p> https://www.AJMB.org/En/Article.aspx?ID=264 Zohreh Mazinani, Marzieh Zamani, Soroush Sardari Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of ATP-Binding Cassette Transporter A1 (<i>ABCA1</i>)-565 C/T Gene Polymorphism with Hypoalphalipoproteinemia and Serum Lipids, IL-6 and CRP Levels <p>Background: ATP-binding cassette transporter A1 (<em>ABCA1</em>) is a membrane integral protein which plays a vital role in High Density Lipoprotein (HDL) metabolism and exerts a protective effect against Hypoalphalipoproteinemia (HA) by mediation of rate-limiting step in HDL biogenesis. In addition, this protein possesses anti-inflammatory effects by inhibiting the production of some inflammatory cytokines in macrophages. This study investigated the association of <em>ABCA1</em>-565 C/T gene polymorphism with HA and serum lipids, IL-6 and CRP levels.<br /> Methods: A population which consisted of 101 HA and 95 normal subjects were genotyped for <em>ABCA1</em>-565C/T polymorphism by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The serum concentrations of lipids, IL-6 and high sensitive-CRP (hs-CRP) were measured by the relevant methods.<br /> Results: The frequency of T allele was significantly higher in the HA group than the controls (31.7 <em>vs</em>. 19.5%, p=0.002). Thus, carriers of the T allele (CT and TT genotypes) had a higher risk for HA (p=0.016, OR=2.04, 95% CI=1.14-3.63). T allele carriers demonstrated decreased HDL-C and increased triglyceride, IL-6 and CRP levels than those with the CC genotype. &nbsp;<br /> Conclusion: This study suggests that the-565 C/T polymorphism of <em>ABCA1</em> gene is associated with an increased risk of HA, decreased HDL-C and increased TG, IL-6 and CRP.</p> https://www.AJMB.org/En/Article.aspx?ID=265 Mohammad Mahdi Babashamsi, Sohrab Halalkhor, Hamid Moradi Firouzjah, Hadi Parsian, Seyed Farzad Jalali, Mohammad Babashamsi Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir <i>In Vitro</i> Activity of Linezolid in Combination with Photodynamic Inactivation Against <i>Staphylococcus aureus</i> Biofilms <p>Background: Biofilm infections are a major challenge in medical practice. Bacteria that live in a biofilm phenotype are more resistant to both antimicrobial therapy and host immune responses compared to their planktonic counterparts. So, there is need for new therapeutic strategies to combat these infections. A promising approach [known as Photodynamic Inactivation (PDI)] to kill bacteria growing as biofilms uses light in combination with a photosensitizer to induce a phototoxic reaction which produces reactive oxygen species that can destroy lipids and proteins causing cell death. PDI does not always guarantee full success, so, combination of PDI with antibiotics may give increased efficiency. This study aimed to determine if PDI was effective in the eradication of <em>Staphylococcus aureus (S. aureus)</em> biofilms in combination with linezolid.<br /> Methods: The susceptibility of biofilm cultures of three <em>S. aureus</em> strains to Methylene Blue (MB) and Toluidine Blue O (TBO)-mediated PDI was determined alone and in combination with linezolid.<br /> Results: Bactericidal activity (&ge;3 log<sub>10</sub> reduction in viable cell count) was not achieved with MB/TBO-PDI or antibiotic treatment alone. When antibiotic treatment was combined with TBO-PDI, a greater reduction in viable count than antibiotic alone was observed for two strains.<br /> Conclusion: This study showed that although TBO-PDI did not have good bactericidal activity against <em>S. aureus</em> biofilms; it increased the antimicrobial activity of linezolid against these bacteria.</p> https://www.AJMB.org/En/Article.aspx?ID=266 Nasim Kashef, Mahboobeh Akbarizare, Mohammad Reza Razzaghi Tue, 06 Dec 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Importance of Clinical Trials in Drug Development <p>While preclinical research answers basic questions about a drug&rsquo;s safety, it is not a substitute for studies of ways the drug will interact with the human body. &ldquo;Clinical research&rdquo; refers to studies, or trials, that are done in people <sup>1,2</sup>. As the developers design the clinical study, they will consider what they want to accomplish for each of the different Clinical Research Phases and begin the Investigational New Drug Process, a process they must go through before clinical research begins. The ultimate goal of drug development is to bring a new compound with proven therapeutic effect to the market. In this context, the transition from preclinical research to clinical stages marks a critical turning point, as it nears the new medicinal product to the market <sup>3,4</sup>. With the promise of marketing authorization, though far ahead in the road, hanging on the horizon, the approval of a clinical trial usually attracts investors and leads to a respectable rise of the company shares. However, everything comes at a price. Clinical trials are not without risks, and while the perspective of success is encouraging, the crude reality is that most compounds fail before reaching the market. As explained in previous entries, despite higher R&amp;D expenditures, attrition rates are high and, what is worse, on the rise. Data collected between 1990 and 2004 show that the number of unsuccessful clinical trials has been steadily increasing during the last years: from 30% to 50% at Phase 1, from 40% to 70% at Phase 2 and from 20% to nearly 50% at Phase 3 <sup>3,4</sup>. As a result, less than 10% of the drugs that enter clinical trials end up being approved by regulatory agencies. Clinical trials are only a small part of the research that goes into developing a new treatment. Drugs of the future, for example, first have to be discovered or created, purified, described, and tested in labs (in cell and animal studies) before ever reaching human clinical trials. Of all the substances that are tested in these early stages, very few are promising enough to be tested in humans. Drug development is the process of bringing a new pharmaceutical drug to the market once a lead compound has been identified through the process of drug discovery. It includes pre-clinical research on microorganisms and animals, filing for regulatory status, such as via the United States Food and Drug Administration for an investigational new drug to initiate clinical trials on humans, and may include the step of obtaining regulatory approval with a new drug application to market the drug. Indeed, of every 5,000 cancer molecules identified in the laboratory, about 250 will enter pre-clinical testing. Of this 250, fewer than 10 are tested in clinical trials and on average only one will be approved by regulatory authorities. The process of bringing a new treatment from the research stage (laboratory) to clinic is estimated to take between 10-13 years<sup> 5</sup>.</p> https://www.AJMB.org/En/Article.aspx?ID=249 Shahin Akhondzadeh Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Paroxetine Can Enhance Neurogenesis During Neurogenic Differentiation of Human Adipose-derived Stem Cells <p>Background: Some antidepressant drugs can promote neuronal cell proliferation <em>in vitro</em> as well as hippocampal neurogenesis in human and animal models. Furthermore, adipose tissue is an available source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human Adipose-Derived Stem Cells (hADSCs) may be a suitable source for regenerative medical applications. Since there is no evidence for the effect of Paroxetine as the most commonly prescribed antidepressant drug for neurogenic potential of hADSCs, an attempt was made to determine the effect of Paroxetine on proliferation and neural differentiation of hADSCs.<br /> Methods: ADSCs were isolated from human abdominal fat. These cells differentiated to neuron-like cells and were treated with Paroxetine. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and immunofluorescence technique were used for assessment of cell proliferation and neurogenic differentiation potential of induced cells, respectively.<br /> Results: MTT assay analysis showed that Paroxetine significantly increased the proliferation rate of induced hADSCs (p&lt;0.05), while immunofluorescent staining indicated that Paroxetine treatment during neurogenic differentiation could enhance the mean percentage of Nestin and MAP2 (Microtubule-associated protein-2) positive cells but the mean percentage of GFAP (Glial acidic fibrillary protein) positive cells significantly decreased relative to control group (p&lt;0.05).<br /> Conclusion: Our results provide evidence that Paroxetine can promote proliferation and differentiation rate during neurogenic differentiation of hADSCs. Moreover, Paroxetine can reduce gliogenesis of induced hADSCs during neurogenic differentiation.</p> https://www.AJMB.org/En/Article.aspx?ID=251 Maliheh Jahromi, Shahnaz Razavi, Nushin Amirpour, Zahra Khosravizadeh Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Exogenous Secreted Frizzled-Related Protein-4 Modulates Steroidogenesis of Rat ‎Granulosa Cells Through Wnt/Bcatenin and PI3K/AKT Signaling Pathways‎ <p>Background: It has been reported that secreted frizzled-related protein-4 known as an antagonist of Wnt signaling pathway plays a role in luteinization process of rodent granulosa cells. The purpose of this study was twofold: 1) to determine whether recombinant human secreted frizzled-related protein-4 (rhSFRP-4) could directly induce terminal differentiation of rat Granulosa Cells (GCs) and 2) to understand how the modulation of &beta;-catenin and Protein Kinase B (PKB)/AKT activity by exogenous SFRP-4 could be involved in steroidogenesis.<br /> Methods: GCs were firstly stimulated with Follicle-Stimulating Hormone (FSH) named as FSH-primed cells then were treated with luteinizing hormone (LH). Then estradiol (E<sub>2</sub>) and progesterone (P<sub>4</sub>) production levels were assessed in the absence or presence of rhSFRP-4 treatment. The expression levels of activated &beta;-catenin, pAKTser<sup>473</sup>, pGSK3&beta;ser<sup>9</sup> were assessed by western blot or immuno-fluoresence.<br /> Results: In the presence of rhSFRP-4, there was 38% decreased E<sub>2</sub> levels compared to untreated FSH-primed cells (p&lt;0.05), and P<sub>4</sub> production subsequently decreased. However, in GCs pre-treated with rhSFRP-4 prior to addition of FSH, P<sub>4</sub> levels increased 2-fold compared with untreated cells (p&lt;0.05). Unexpectedly, treatment with rhSFRP-4 prior to LH stimulation inhibited LH-induced P<sub>4</sub> secretion. Treatment with low (0.5 <em>ng/ml</em>) but not high (50 <em>ng/ml</em>) concentrations of rhSFRP-4 led to significantly increased levels of pGSK3&beta;ser<sup>9</sup> (1.6-fold) and nuclear active &beta;-catenin (2.8-fold) in GCs compared with untreated cells. Interestingly, pre-treating GCs with rhsFPR4 prior to LH stimulation resulted in a 38% decrease in pAKTser<sup>473</sup> levels compared with those in LH-treated cells (p&lt;0.05).<br /> Conclusion: Taken together, our results showed that rhSFRP-4 could directly induce terminal differentiation in GCs via the modulation of &beta;-catenin and PKB/AKT pathways and that it does so in a dose-dependent manner.</p> https://www.AJMB.org/En/Article.aspx?ID=252 Ghamartaj Hossein, Manijeh Khanmohammadi, Parissa Sahranavard Fard, Yasaman Heidarian, Somaieh Kazemnejad, Mohammad Mehdi Akhondi Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Novel Combinations of Synthesized ZnO NPs and Ceftazidime: Evaluation of their Activity Against Standards and New Clinically Isolated Pseudomonas aeruginosa <p>Background: Antibiotic resistant bacteria can be considered as a main problem in infection management. Zinc oxide nanoparticles (ZnO NPs), individually or in combination with antibiotics, can be considered as good candidates for struggling against drug resistant bacteria.<br /> Methods: In this study, Zinc oxide nanoparticles were synthesized using sol-gel method in low temperature as a cost effective procedure and characterized by X-ray diffraction and Scanning Electron Microscopy. Antibacterial activity of 9 new combinations of Zinc oxide nanoparticles and ceftazidime was assessed against standards and new clinically isolated multi drug resistant <em>Pseudomonas aeruginosa (P. aeruginosa)</em>, in order to evaluate enhancement effect of synthesized Zinc oxide nanoparticles on antibacterial activity of ceftazidime.<br /> Results: The results indicated that desirable effects can be seen at 6 and 7 <em>mM</em> of Zinc oxide nanoparticles (60 to 100% inhibition). Moreover, after evaluation of 9 new combinations with various concentrations of both components, it was demonstrated that Zinc oxide nanoparticles can enhance the antibacterial activity of ceftazidime, against some bacterial strains of <em>P. aeruginosa</em>. The highest activity was observed with the concentration of 20 <em>&mu;g/ml</em> ceftazidime in the presence of 5, 6 or 7 <em>mM</em> of Zinc oxide nanoparticles.<br /> Conclusion: Zinc oxide nanoparticles in appropriate concentrations can be proposed as new and promising candidates for overcoming bacterial resistance.</p> https://www.AJMB.org/En/Article.aspx?ID=253 Elham Isaei, Shahla Mansouri, Fereshteh Mohammadi, Sadegh Taheritarigh, Zohreh Mohammadi Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein <p>Background: As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in <em>E. coli</em> and investigation of its immunoreactivity.<br /> Methods: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in <em>Escherichia coli (E. coli)</em> BL21. Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA.<br /> Results: Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100<em> &mu;g/ml</em>. Immunogenicity of rPR was confirmed by Western blotting and ELISA.<br /> Conclusion: Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests.</p> https://www.AJMB.org/En/Article.aspx?ID=254 Asaad Azarnezhad, Zohreh Sharifi, Rahmatollah Seyedabadi, Arshad Hosseini, Behrooz Johari, Mahsa Sobhani Fard Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of Recombinant Human Growth Hormone Secretion in E. coli using the L-asparaginase II signal Peptide <p>Background: In the recent years, there has been an increasing interest in secretory production of recombinant proteins, due to its various advantages compared with intracellular expression. Signal peptides play a critical role in prosperous secretion of recombinant proteins. Accordingly, different signal peptides have been assessed for their ability to produce secretory proteins by trial-and-error experiments. The aim of this study was to evaluate the effect of L-asparaginase II signal peptide on the recombinant human Growth Hormone (hGH) protein secretion in the <em>Escherichia coli (E. coli)</em> host.<br /> Methods: Cloning and expression of a synthetic hGH gene, containing L-asparaginase II signal sequence was performed in <em>E. coli</em> BL21 (DE3) using 0.1 <em>mM</em> IPTG as an inducer at 23<sup>o</sup><em>C</em> overnight. Periplasmic protein extraction was performed using three methods, including osmotic shock, osmotic shock in the presence of glycine and combined Lysozyme/EDTA osmotic shock. Afterwards, the hGH expression was determined by SDS-PAGE.<br /> Results: Based on experimentally obtained results, hGH protein is expressed as inclusion body even in the presence of L-asparaginase II signal peptide.<br /> Conclusion: Therefore, this signal peptide is not effective for secretory production of the recombinant hGH.</p> https://www.AJMB.org/En/Article.aspx?ID=255 Mozhdeh Zamani, Navid Nezafat, Younes Ghasemi Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Preliminary Study on Cost-effective L-Tryptophan Production from Indole and L-Serine by E. coli Cells <p>Background: L-tryptophan is used widespread in the pharmaceutical industry. The majority of L-Trp production depends on microbial processes that produce L-tryptophan from indole and L-serine. These processes are very costly due to the costs of precursors, especially L-serine. Use of inexpensive substitutions as the L-serine source of L-tryptophan production enables us to reach a cost-effective process. In this paper, effect of Triton X-100 on L-Trp production and the ability to use Iranian cane molasses as inexpensive L-serine source was investigated.<br /> Methods: <em>Escherichia coli (E. coli)</em> <em>ATCC 11303</em> cells were grown in 10-L fermenter containing minimal medium supplemented with beet molasses as an inexpensive carbon source and indole as tryptophan synthase inducer. Whole cells of stationary phase were used as biocatalyst for L-Trp production. Triton X-100 addition to the production medium as indole reservoir was investigated. Then, cane molasses was used as L-Ser source in L-Trp production medium. Amount of L-Tryptophan and theoretical yield of L-Trp production was determined by HPLC and by a colorimetrically method on the basis of remaining indole assay, respectively.<br /> Results: As a result, triton X-100 increased L-Trp production three times.&nbsp; Also, the result showed that 0.68 <em>mM</em> L-Tryptophan was produced in the presence of cane molasses at 37<sup>o</sup><em>C</em> for 8 <em>hr</em>.<br /> Conclusion: This result showed that cane molasses of Qazvin sugar factory includes significant amounts of L-Ser that makes it a suitable substitution for L-Ser in L-Trp production. Therefore, it has the potential to be used for cost-effective L-Trp production in industrial scale.</p> https://www.AJMB.org/En/Article.aspx?ID=256 Tahereh Sadeghiyan-Rizi, Jamshid Fooladi, Sima Sadrai Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of a High-Resolution Melting Analysis Method for CYP2C19*17 Genotyping in Healthy Volunteers <p>Background: Genetic polymorphisms of drug metabolisms by cytochrome P450 (P450s) could affect drug response, attracting particular interest in the pharm-acogenetics. Due to the importance of CYP2C19* 17 allele and its capability of super- fast metabolism and also lack of information about distribution of the alleles in Iranian population, this research aimed to use High Resolution Melting (HRM) method compared to PCR-RFLP for genotyping healthy Iranian population.<br /> Methods: Blood samples were collected from 100 healthy Iranian volunteers. DNA was extracted by salting out method. Real-time PCR was used for amplification of the CYP2C19 gene and the alleles were identified by HRM. Sequencing was used to confirm the amplified DNA fragments and data were analyzed using SPSS software ver.18.<br /> Results: The frequency of alleles CYP2C19*1/*1, CYP2C19*1/*17 and CYP2C19*17/*17 were estimated as 58.33, 29.1 and 11.1%, respectively. Specificity and sensitivity of HRM method were 90% and 100%, with respect to PCR-RFLP. Also, HRM analysis has been evaluated as a faster and more effective approach.<br /> Conclusion: Comparison of our results based on HRM analysis with PCR-RFLP showed that our developed method is rapid, accurate, fast and economic to study the CYP2C19*17 allele and it is appropriate for other similar population genetic studies.</p> https://www.AJMB.org/En/Article.aspx?ID=257 Zahra Ghasemi, Mehrdad Hashemi, Mahsa Ejabati, Seyyed Meisam Ebrahimi, Hamidreza Kheiri Manjili, Ali Sharafi, Ali Ramazani Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Lack of Association between Interleukin-12 Gene Polymorphisms and Recurrent Aphthous Stomatitis https://www.AJMB.org/En/Article.aspx?ID=258 Isaac Firouze Moqadam, Shamsolmoulouk Najafi, Mahsa Mohammadzadeh, Alireza Zare Bidoki, Hila Yousefi, Elham Farhadi, Arghavan Tonekaboni, Ghasem Meighani, Mohsen Mohammadzadeh, Ali Akbar Amirzargar, Nima Rezaei Mon, 26 Sep 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Future directions for translation of tissue engineering products into clinic <p>At present, Iran has been known one of the up-warding countries in the world in regenerative medicine using stem cells therapy. In fact, the outcomes of some clinical trials on stem cell therapy of myocardial infarction, vitiligo, decompensated cirrhosis, and osteoarthritis narrate the feasibility of stem cell-based therapy for treatment of human diseases <sup>1,2</sup>. However, in a similar manner with global configuration, the commercialization and translation of tissue engineering products into clinical phase has been restricted. It might be due to weak collaboration of different specialties for technology transfer of the multidisciplinary projects of tissue engineering field into clinical phase. Basic tissue engineers mostly prefer elegant studies, whereas physicians have tendency to solve medical problems with products indicating efficiency, easy to use, and cost benefit. Actually, a surgeon encountered with a dilemma between a partially effective tissue-engineered product that is both expensive and difficult to apply and a more traditional approach may choose the latter option. Therefore, a coherent teamwork between basic sciences and medicine as well as acquisition of competent knowledge about target tissue is necessary to conduct tissue engineering in the clinic. Moreover, it should be considered that in developing countries including Iran the high cost of high-tech biomedical research necessitates government investment <sup>3</sup>. Currently, the policy makers have established some action plans to support of science-based companies financially. This is a suitable opportunity to ligature basic research and market for commercialization of tissue engineering products. However, because private investors beyond academic laboratories should provide financing of tissue engineering products, incentive of private companies for investment should not be neglected. It is notable that tissue-engineered products will fulfill small market size unless they could indicate much superior results than competitive alternatives.<br /> It is noticeable that tissue engineers should determine the requirements of community and develop strategies to penetrate the products into clinic. Indeed, the communication between scientists and policy makers should be increased to better definition of national research priorities. On the other hand, considering local necessities and natural resources should be rather than subjective experts&rsquo; notions or international superiorities. Finally, ethical and legal regulations should be actually defined that indubitably make great profits to the society.</p> https://www.AJMB.org/En/Article.aspx?ID=240 Somaieh Kazemnejad Wed, 29 Jun 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of a Single Stranded DNA Aptamer as a Molecular Probe for LNCap Cells Using Cell-SELEX <p>Background: Nowadays, highly specific aptamers generated by cell SELEX technology (systematic evolution of ligands by exponential enrichment) are being applied for early detection of cancer cells. Prostate Specific Membrane Antigen (PSMA), over expressed in prostate cancer, is a highly specific marker and therefore can be used for diagnosis of the prostate cancer cells. The aim of the present study was to select single-stranded DNA aptamers against LNCap cells highly expressing PSMA, using cell&ndash;SELEX method which can be used as a diagnostic tool for the detection of prostate cancer cells.<br /> Methods: After 10 rounds of cell-SELEX, DNA aptamers were isolated against PSMA using LNCaP cells as a target and PC-3 cell lines for counter SELEX. Five DNA aptamers with more than 70% affinity were selected up on flow cytometry analysis of positive clones.<br /> Results: Dissociation constants of two selected sequences (A12-B1) were estimated in the range of 33.78&plusmn;3.77 and 57.49&plusmn;2.214 <em>pmol</em>, respectively. Conserved secondary structures of A12 and B1 sequences suggest the necessity of these structures for binding with high affinity to native PSMA. Comparison of the secondary structures of our isolated aptamers and aptamer A10 obtained by protein SELEX showed similar stem-loop structures which could be responsible for the recognition of PSMA on LNCap cell surface.<br /> Conclusion: Our results indicated that selected aptamers may turn out to be ideal candidates for the development of a detection tool and also can be used in targeted drug delivery for future smart drugs.</p> https://www.AJMB.org/En/Article.aspx?ID=242 Faezeh Almasi, Seyed Latif Mousavi Gargari, Fatemeh Bitaraf, Samaneh Rasoulinejad Wed, 29 Jun 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Cytotoxicity of Dacarbazine Potentiated by Sea Cucumber Saponin in Resistant B16F10 Melanoma Cells through Apoptosis Induction <p>Background: Malignant melanoma is a highly aggressive malignant melanocytic neoplasm which resists against the most conventional therapies. Sea cucumber as one of marine organisms contains bioactive compounds such as polysaccharide, terpenoid and other metabolites which have anti-cancer, anti-tumor, anti-inflammatory and antioxidant properties. The present study was designed to investigate the anticancer potential of saponin extracted from sea cucumber <em>Holothuria leucospilata</em> alone and in combination with dacarbazine on B16F10 melanoma cell line.<br /> Methods: The B16F10 cell line was treated with different concentrations of saponin (0, 4, 8, 12, 16, 20 <em>&micro;g/ml</em>), dacarbazine (0, 1200, 1400, 1600, 1800, 2000<em> &micro;g/ml</em>) and co-administration of saponin-dacarbazine (1200 da+8 sp, 1200 da+4 sp) for 24 and 48 <em>hr</em> and the cytotoxic effect was examined by MTT, DAPI, acridine orange/propodium iodide, flow cytometry and caspase colorimetric assay.<br /> Results: The results exhibited that sea cucumber saponin, dacarbazine, and co-administration of saponin-dacarbazine inhibited the proliferation of melanoma cells in a dose and time dependent manner with IC<sub>50</sub> values of 10, 1400 and 4+1200 <em>&micro;g/ml</em>, respectively. Morphological observation of DAPI and acridine orange/propodium iodide staining documented typical characteristics of apoptotic cell death. Flow cytometry assay indicated accumulation of IC<sub>50</sub> treated cells in sub-G1 peak. Additionally, saponin extracted induced intrinsic apoptosis <em>via</em> up-regulation of caspase-3 and caspase-9.<br /> Conclusion: These results revealed that the saponin extracted from sea cucumber as a natural anti-cancer compound may be a new treatment modality for metastatic melanoma and the application of sea cucumber saponin in combination with dacarbazine demonstrated the strongest anti-cancer activity as compared with the drug alone.</p> https://www.AJMB.org/En/Article.aspx?ID=243 Javad Baharara, Elaheh Amini, Najme Nikdel, Farzaneh Salek-Abdollahi Wed, 29 Jun 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Neuroprotective Effects of Herbal Extract (Rosa canina, Tanacetum vulgare and Urtica dioica) on Rat Model of Sporadic Alzheimer’s Disease <p>Background: Sporadic Alzheimer&rsquo;s Disease (SAD) is caused by genetic risk factors, aging and oxidative stresses. The herbal extract of <em>Rosa canina (R. canina)</em>, <em>Tanacetum vulgare (T. vulgare)</em> and <em>Urtica dioica (U. dioica)</em> has a beneficial role in aging, as an anti-inflammatory and anti-oxidative agent. In this study, the neuroprotective effects of this herbal extract in the rat model of SAD was investigated.<br /> Methods: The rats were divided into control, sham, model, herbal extract -treated and ethanol-treated groups. Drug interventions were started on the 21<sup>st</sup> day after modeling and each treatment group was given the drugs by intraperitoneal (I.P.) route for 21 days. The expression levels of the five important genes for pathogenesis of SAD including <em>Syp, Psen1, Mapk3, Map2</em> and <em>Tnf-&alpha;</em> were measured by qPCR between the hippocampi of SAD model which were treated by this herbal extract and control groups. The Morris Water Maze was adapted to test spatial learning and memory ability of the rats.<br /> Results: Treatment of the rat model of SAD with herbal extract induced a significant change in expression of <em>Syp</em> (p=0.001) and <em>Psen1</em> (p=0.029). In Morris Water Maze, significant changes in spatial learning seen in the rat model group were improved in herbal-treated group.<br /> Conclusion: This herbal extract could have anti-dementia properties and improve spatial learning and memory in SAD rat model.</p> https://www.AJMB.org/En/Article.aspx?ID=244 Parvaneh Daneshmand, Kioomars Saliminejad, Marzieh Dehghan Shasaltaneh, Koorosh Kamali, Gholam Hossein Riazi, Reza Nazari, Pedram Azimzadeh, Hamid Reza Khorram Khorshid Wed, 29 Jun 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Inhibitory Effects of Some Carbohydrates on Nano-Globular Aggregation of both Normal and Glycated Albumin <p>Background: Protein aggregation is one of the important, common and troubling problems in biotechnology, pharmaceutical industries and amyloid-related disorders.<br /> Methods: In the present study, the inhibitory effects of some carbohydrates (alginate, &beta;-cyclodextrin and trehalose) on the formation of nano-globular aggregates from normal (HSA) and glycated (GHSA) human serum albumin were studied; when the formation of aggregates was induced by the simultaneous heating and addition of dithiotheritol. For the investigations, the biophysical methods of UV-vis spectrophotometry, circular dichroism spectroscopy, transmission electron microscopy and tensiometry were employed.<br /> Results: The effect of inhibitory mechanism of these inhibitors on the aggregation of HSA and GHSA was expressed and compared together.<br /> Conclusion: The results showed that the nucleus formation step of the aggregation process of HSA and GHSA was different in the presence of alginate (compared to &beta;-cyclodextrin and trehalose). The inhibition efficiencies of the carbohydrates on the aggregate formation of HSA and GHSA were different, arising from the differences in the hydrophobicities of HSA and GHSA, and also, the differences between HSA- and GHSA-carbohydrate interactions.</p> https://www.AJMB.org/En/Article.aspx?ID=245 Ali Akbar Moosavi-Movahedi, Naghmeh Sattarahmady, Esmaeil Sharifi, Hossein Heli Wed, 29 Jun 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Removal of Spermatogonial Stem Cells (SSCs) from In Vitro Culture on Gene Expression of Niche Factors in Bovine <p>Background: Niche cells, regulating Spermatogonial Stem Cells (SSCs) fate are believed to have a reciprocal communication with SSCs. The present study was conducted to evaluate the effect of SSC elimination on the gene expression of Glial cell line-Derived Neurotrophic Factor (GDNF), Fibroblast Growth Factor 2 (FGF2) and Kit Ligand (KITLG), which are the main growth factors regulating SSCs development and secreted by niche cells, primarily Sertoli cells.<br /> Methods: Following isolation, bovine testicular cells were cultured for 12 days on extracellular matrix-coated plates. In the germ cell-removed group, the SSCs were removed from the<em> in vitro</em> culture using differential plating; however, in the control group, no intervention in the culture was performed. Colony formation of SSCs was evaluated using an inverted microscope. The gene expression of growth factors and spermatogonia markers were assessed using quantitative real time PCR.<br /> Results: SSCs colonies were developed in the control group but they were rarely observed in the germ cell-removed group; moreover, the expression of spermatogonia markers was detected in the control group while it was not observed in the germ cell-removed group, substantiating the success of SSCs removal. The expression of Gdnf and Fgf2 was greater in the germ cell-removed than control group (p&lt;0.05), whereas the expression of Kitlg was lower in the germ cell-removed than control group (p&lt;0.05).<br /> Conclusion: In conclusion, the results revealed that niche cells respond to SSCs removal by upregulation of GDNF and FGF2, and downregulation of KITLG in order to stimulate self-renewal and arrest differentiation.</p> https://www.AJMB.org/En/Article.aspx?ID=246 Vahid Akbarinejad, Parviz Tajik, Mansooreh Movahedian, Reza Youssefi Wed, 29 Jun 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of Media Supplementation with Angiotensin on Developmental Competence of Ovine Embryos Derived from Vitrified-warmed Oocytes <p>Background: This study was aimed to assess the effects of angiotensin II (Ang II) supplementation to the In Vitro Maturation (IVM) and In Vitro Culture (IVC) media of vitrified-warmed ovine oocytes on their developmental competence and expression of Na<sup>+</sup>/K<sup>+</sup>/ATPase in resulting embryos.<br /> Methods: The slaughterhouse-derived immature oocytes (n=1069) were randomly distributed into four experimental groups: groups I and II) IVM/IVF and IVC of fresh and vitrified oocytes without angiotensin supplementation (Control-Fresh and Control-Vit groups, respectively); group III) IVM of vitrified oocytes in the presence of Ang II followed by IVF/IVC (Vit-IVM group); and group IV) IVM/IVF of vitrified oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC (Vit-D4 group). The embryos were immunostained with primary antibodies against Na<sup>+</sup>/K<sup>+</sup>/ATPase &alpha;<sub>1</sub> and &beta;<sub>1</sub> subunits.<br /> Results: In Vit-IVM and Vit-D4 groups, the rates of expanded and total blastocysts on day 7 as well as the proportion of blastocysts on day 8 were increased. The expression of Na<sup>+</sup>/K<sup>+</sup>/ATPase &alpha;1 and &beta;<sub>1</sub> subunits were positively influenced by the addition of Ang II on day 4 (Vit-D4 group).<br /> Conclusion: The addition of Ang II to the IVM and IVC media could improve blastocysts formation in vitrified sheep oocytes. This improvement might be related to the greater expression of Na<sup>+</sup>/K<sup>+</sup>/ATPase &alpha;<sub>1</sub> and &beta;<sub>1</sub> subunits when Ang II was added during IVC.</p> https://www.AJMB.org/En/Article.aspx?ID=247 Mohammad Mehdi Naderi, Sara Borjian Boroujeni, Ali Sarvari, Banafsheh Heidari, Mohammad Mehdi Akhondi, Amir-Hassan Zarnani, Abolfazl Shirazi Wed, 29 Jun 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Leptin Receptor Gene Polymorphism may Affect Subclinical Atherosclerosis in Patients with Acromegaly <p>Background: Acromegaly is associated with increased morbidity and mortality related to cardiovascular diseases. Leptin (LEP) and Leptin Receptor (LEPR) gene polymorphisms can increase cardiovascular risks. The aim of this study was to investigate association between the frequencies of LEP and LEPR gene polymorphisms and subclinical atherosclerosis in acromegalic patients.<br /> Methods: Forty-four acromegalic patients and 30 controls were admitted to study. The polymorphisms were identified by using polymerase chain reaction from peripheral blood samples. The levels of systolic and diastolic blood pressure, BMI, fasting plasma glucose, fasting insulin, IGF-I, GH, IGFBP3, leptin, triglyceride, carotid Intima Media Thickness (cIMT) and HDL and LDL cholesterol concentrations were evaluated.<br /> Results: There was statistically significant difference between the LEPR genotypes of acromegalic patients (GG 11.4%, GA 52.3%, and AA 36.4%) and controls (GG 33.3%, GA 50%, and AA 16.7%) although their LEP genotype distribution was similar. In addition, the prevalence of the LEPR gene G and A alleles was significantly different between patients and controls. No significant difference was found among the G(-2548)A leptin genotypes of groups in terms of the clinical parameters. cIMT significantly increased homozygote LEPR GG genotype group compared to AA subjects in patients. But the other parameters were not different between LEPR genotypes groups of patients and controls.<br /> Conclusion: It can be said that the LEPR gene polymorphism may affect cIMT in patients. The reason is that LEPR GG genotype carriers may have more risk than other genotypes in the development of subclinical atherosclerosis in acromegaly.</p> https://www.AJMB.org/En/Article.aspx?ID=248 Sebahat Turgut, Senay Topsakal, Melek Tunç Ata, Duygu Herek, Fulya Akın, Şeyma Özkan, Günfer Turgut Wed, 29 Jun 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Medical Biotechnology and Alzheimer's Disease: New Hopes <p>Alzheimer&rsquo;s Disease (AD), the leading cause of dementia worldwide, is an irreversible progressive neurodegenerative disorder characterized by cognitive impairment and functional disability <sup>1-3</sup>. Devastating nature of AD leads to serious social and economic impacts on the healthcare systems which implies the necessity of its proper management <sup>1-3</sup>. It has been demonstrated that patients&rsquo; quality of life and their overall prognosis has a significant negative correlation with the severity of AD. Patients with severe AD need full-time care and assistance with some basic activities of daily living such as feeding and dressing in addition to severe deterioration in various domains of their cognitive functioning. Progress to a cure for AD has been hampered by the lack of information about the biology of the disease. The therapies currently approved for Alzheimer&rsquo;s disease work by treating the patients&rsquo; symptoms, improving their cognitive and overall functions <sup>4-8</sup>. Increasingly, however, experts are intent on slowing or halting the disease process, before it has ravaged patients&rsquo; brains. A lot of data is being generated on changes in imaging biomarkers before patients really become clinically significantly impaired. For example, there has been a lot of great work done in identifying patients early based on these biomarkers. The current therapeutic market is valued at $3 to $4 billion, shared among drugs that temporarily delay disease progression or address the symptoms but do not alter the underlying disease. Currently, medical biotechnology has brought new hopes in the treatment of Alzheimer&#39;s disease. For example one of the ongoing trials is related to bapineuzumab. Bapineuzumab is a monoclonal antibody (mAb) to target and clear &szlig;-amyloid. This vaccine is the first new drug aimed at slowing or even halting AD progression.</p> https://www.AJMB.org/En/Article.aspx?ID=233 Shahin Akhondzadeh Tue, 01 Mar 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Role of Superoxide Dismutase 2 Gene Ala16Val Polymorphism and Total Antioxidant Capacity in Diabetes and its Complications <p>Diabetes Mellitus (DM) is a chronic heterogeneous disorder and oxidative stress is a key participant in the development and progression of it and its complications. Anti-oxidant status can affect vulnerability to oxidative damage, onset and progression of diabetes and diabetes complications. Superoxide dismutase 2 (SOD2) is one of the major antioxidant defense systems against free radicals. SOD2 is encoded by the nuclear SOD2 gene located on the human chromosome 6q25 and the Ala16Val polymorphism has been identified in exon 2 of the human SOD2 gene. Ala16Val (rs4880) is the most commonly studied SOD2 single nucleotide polymorphism (SNP) in SOD2 gene. This SNP changes the amino acid at position 16 from valine (Val) to alanine (Ala), which has been shown to cause a conformational change in the target sequence of manganese superoxide dismutase (MnSOD) and also affects MnSOD activity in mitochondria. Ala16Val SNP and changes in the activity of the SOD2 antioxidant enzyme have been associated with altered progression and risk of different diseases. Association of this SNP with diabetes and some of its complications have been studied in numerous studies. This review evaluated how rs4880, oxidative stress and antioxidant status are associated with diabetes and its complications although some aspects of this line still remain unclear.</p> https://www.AJMB.org/En/Article.aspx?ID=234 Katayoun Pourvali, Mehrnaz Abbasi, Azadeh Mottaghi Tue, 01 Mar 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Construction and Production of Foxp3- Fc (IgG) DNA Vaccine/Fusion Protein <p>Background: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell (DC) based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc(IgG) can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I (cross priming).<br /> Methods: DNA construct containing fragment C (Fc) portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, <em>Escherichia coli (E. coli)</em> strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining.<br /> Results: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells.&nbsp; Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification.<br /> Conclusion: Due to successful expression of Foxp3-Fc (IgG), it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means.</p> https://www.AJMB.org/En/Article.aspx?ID=235 Neda Mousavi Niri, Arash Memarnejadian, Jamshid Hadjati, Mohammad Reza Aghasadeghi, Mehdi Shokri, Yones Pilehvar-soltanahmadi, Abolfazl Akbarzadeh, Nosratollah Zarghami Tue, 01 Mar 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Hypoglycemic Effect of Aquatic Extract of Stevia in Pancreas of Diabetic Rats: PPARγ -dependent Regulation or Antioxidant Potential <p>Background: Traditional medicines with anti-diabetic effects are considered suitable supplements to treat diabetes. Among medicinal herbs, <em>Stevia Rebaudiana Bertoni</em> is famous for its sweet taste and beneficial effect in regulation of glucose. However, little is known about the exact mechanism of stevia in pancreatic tissue. Therefore, this study investigated the possible effects of stevia on pancreas in managing hyperglycemia seen in streptozotocin-induced Sprague-Dawley rats.<br /> Methods: Sprague-Dawley rats were divided into four groups including normoglycemic, diabetic and two more diabetic groups in which, one was treated with aquatic extract of stevia (400 <em>mg/kg</em>) and the other with pioglitazone (10 <em>mg/kg</em>) for the period of 28 days. After completion of the experimental duration, rats were dissected; blood samples and pancreas were further used for detecting biochemical and histopathological changes. FBS, TG, cholestrol, HDL, LDL, ALT and AST levels were measured in sera. Moreover, MDA (malondialdehyde) level, catalase activity, levels of insulin and <em>PPAR&gamma; </em>mRNA expression were also measured in pancreatic tissue.<br /> Results: Aquatic extract of stevia significantly reduced the FBS, triglycerides, MDA, ALT, AST levels and normalized catalase activity in treated rats compared with diabetic rats (p&lt;0.05). In addition to this, stevia surprisingly, increased <em>PPAR&gamma;</em> and insulin mRNA levels in treated rats (p&lt;0.05). Furthermore, stevia compensated for the histopathological damage in diabetic rats.<br /> Conclusion: It is concluded that stevia acts on pancreatic tissue to elevate the insulin level and exerts beneficial anti-hyperglycemic effects through the PPAR&gamma;-dependent mechanism and stevia&rsquo;s antioxidant properties.</p> https://www.AJMB.org/En/Article.aspx?ID=236 Raheleh Assaei, Pooneh Mokarram, Sanaz Dastghaib, Sara Darbandi, Mahsa Darbandi, Fatemeh Zal, Masoumeh Akmali, Gholam Hossein Ranjbar Omrani Tue, 01 Mar 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells <p>Background: Gold Nanoparticles (GNPs) are used in imaging and molecular diagnostic applications. As the development of a novel approach in the green synthesis of metal nanoparticles is of great importance and a necessity, a simple and safe method for the synthesis of GNPs using plant extracts of <em>Zataria multiflora</em> leaves was applied in this study and the results on GNPs&rsquo; anticancer activity against HeLa cells were reported.<br /> Methods: The GNPs were characterized by UV-visible spectroscopy, FTIR, TEM, DLS and Zeta-potential measurements. In addition, the cellular up-take of nanoparticles was investigated using Dark Field Microscopy (DFM). Induction of apoptosis by high dose of GNPs in HeLa cells was assessed by MTT assay, Acridin orange, DAPI staining, Annexin V/PI double-labeling flow cytometry and caspase activity assay.<br /> Results: UV-visible spectroscopy results showed a surface plasmon resonance band for GNPs at 530 <em>nm</em>. FTIR results demonstrated an interaction between plant extract and nanoparticles. TEM images revealed different shapes for GNPs and DLS results indicated that the GNPs range in size from 10 to 42 <em>nm</em>. The Zeta potential values of the synthesized GNPs were between 30 to 50 <em>Mev</em>, indicating the formation of stable particles. As evidenced by MTT assay, GNPs inhibit proliferation of HeLa cells in dose- dependent GNPs and cytotoxicity of GNPs in Bone Marrow Mesenchymal Stem Cell (BMSCs) was lower than cancerous cells. At nontoxic concentrations, the cellular up-take of the nanoparticles took place. Acridin orange and DAPI staining showed morphological changes in the cell&rsquo;s nucleus due to apoptosis. Finally, caspase activity assay demonstrated HeLa cell&rsquo;s apoptosis through caspase activation.<br /> Conclusion: The results showed that GNPs have the ability to induce apoptosis in HeLa cells.</p> https://www.AJMB.org/En/Article.aspx?ID=237 Javad Baharara, Tayebe Ramezani, Adeleh Divsalar, Marzieh Mousavi, Arefeh Seyedarabi Tue, 01 Mar 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR <p>Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time.<br /> Methods: A DNA fragment of 167 <em>bp</em> chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 <em>bp </em>EDC fragment in final concentration of 1.1 <em>pg</em>/ 500 <em>&mu;l</em> was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples.<br /> Results: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them.<br /> Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis.</p> https://www.AJMB.org/En/Article.aspx?ID=238 Maryam Eini, Abbas Behzad-Behbahani, Mohammad Ali Takhshid, Amin Ramezani, Gholam Reza Rafiei Dehbidi, Mohammad Ali Okhovat, Ali Farhadi, Parniyan Alavi Tue, 01 Mar 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir LC-MS Method for Studying the Pharmacokinetics and Bioequivalence of Clonidine Hydrochloride in Healthy Male Volunteers <p>Background: A simple and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been evaluated for the assignment of clonidine hydrochloride in human plasma.<br /> Methods: The mobile phase composed of acetonitrile-water 60:40 (<em>v/v</em>) and 0.2% formic acid 20<em> &micro;l</em> of sample was chromatographically analyzed using a repacked ZORBAX-XDB-ODS C<sub>18</sub> column (2.1 <em>mm</em>x30 <em>mm</em>, 3.5 <em>&mu;</em>). Detection of analytes was achieved by tandem mass spectrometry with Electrospray Ionization (ESI) interface in positive ion mode operated under the multiple-reaction monitoring mode (m/z 230.0 &rarr;213). Sample pretreatment consisted of a one-step Protein Precipitation (PPT) with methanol and perchloric acid (HClO<sub>4</sub>) of 0.10 <em>ml</em> plasma.<br /> Results: Standard curve was linear (r=0.998) over the concentration range of 0.01-10.0 <em>ng/ml</em> and showed suitable accuracy and precision. The Limit of Quantification (LOQ) was 0.01 <em>ng/ml</em>. The mean (SD) Cmax, Tmax, AUC<sub>0&ndash;t </sub>and AUC<sub>0&ndash;&infin;</sub> values after administration of the test and reference formulations, respectively, were in this manner: 6.16 (0.32) versus 6.21 (0.07) <em>ng/ml</em>, 30.12 (0.86) versus 30.13 (0.73) <em>hr</em>, 290.37 (1.13) versus 293.39 (1.22) <em>ng/ml/hr</em>, and 350.17 (1.98) versus 352.96 (1.67) <em>ng/ml/hr</em>. The mean (SD) t1/2 was 120.12 (1.90) <em>hr</em> for the test formulation and 120.96 (1.54) hr for the reference formulation. No statistical differences were showed for Cmax and the area under the plasma concentration-time curve for test and reference tablets.<br /> Conclusion: The method is rapid, simple, very steady and precise for the separation, assignment, pharmacokinetic and bioavailability evaluation of clonidine in healthy Iranian adult male volunteers.</p> https://www.AJMB.org/En/Article.aspx?ID=239 Hossein Danafar, Mehrdad Hamidi Tue, 01 Mar 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Distribution and Diversity of <i>hmw1A</i> Among Invasive Nontypeable <i>Haemophilus influenzae</i> Isolates in Iran <p>Background: The pathogenesis of nontypeable <em>Haemophilus influenzae</em> (NTHi) begins with adhesion to the rhinopharyngeal mucosa. Almost 38-80% of NTHi clinical isolates produce proteins that belong to the High Molecular Weight (HMW) family of adhesins, which are believed to facilitate colonization.<br /> Methods: In the present study, the prevalence of <em>hmwA</em>, which encodes the HMW adhesin, was determined for a collection of 32 NTHi isolates. Restriction Fragment Length Polymorphism (RFLP) was performed to advance our understanding of <em>hmwA</em> binding sequence diversity.<br /> Results: The results demonstrated that <em>hmwA</em> was detected in 61% of NTHi isolates. According to RFLP, isolates were divided into three groups.<br /> Conclusion: Based on these observations, it is hypothesized that some strains of nontypeable <em>Haemophilus influenzae</em> infect some specific areas more than other parts.</p> https://www.AJMB.org/En/Article.aspx?ID=241 Milad Shahini Shams abadi, Seyed Davar Siadat, Farzam Vaziri, Mehdi Davari, Abolfazl Fateh, Shahin Pourazar, Farid Abdolrahimi, Morteza Ghazanfari Tue, 01 Mar 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Economic Aspects of Medical Biotechnology <p>The biotechnology industry has grown rapidly in recent years, doubling in size between 2004 and 2014. Much attention is given to the potential of the biotechnology industry, from drugs and environmental products currently in the pipeline. These products have the potential to generate tremendous opportunities for society, by improving the quality of health care and producing a cleaner environment. Over the past few decades biotechnology sometimes described as the oldest profession in the world has evolved into a modern technology without which medical progress would be scarcely imaginable. Modern biotechnology plays a crucial role both in the elucidation of the molecular causes of disease and in the development of new diagnostic methods and better targeted drugs. Diagnosis and treatment are thus becoming increasingly intertwined. When a disease, rather than being diagnosed on the basis of more or less vague signs and symptoms, can be detected on the basis of molecular information, the possibility of successful treatment depends largely on what diagnostic techniques are available. Biotechnology is an important component of the worldwide economy, and could take on an increasingly significant role as the industry continues to develop. The economic impact of biotechnology as a distinct industry is currently difficult to evaluate because of the manner in which data is collected; however, it is possible to calculate the combined impact of the biotech and pharmaceutical industries <sup>1,2</sup>.</p> https://www.AJMB.org/En/Article.aspx?ID=225 Shahin Akhondzadeh Fri, 01 Jan 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Differentiation of Definitive Endoderm from Human Induced Pluripotent Stem Cells on hMSCs Feeder in a Defined Medium <p>Background:<strong> </strong>The Definitive Endoderm (DE) differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells (hiPSCs) on an appropriate feeder in a more defined medium.<br /> Methods: Human Induced Pluripotent Stem Cells (hiPSCs) were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3-ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts (MEFs) and in the fifth method were human adult bone marrow Mesenchymal Stem Cells (hMSCs). DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry.<br /> Results: QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences.<br /> Conclusion: Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine.</p> https://www.AJMB.org/En/Article.aspx?ID=226 Zahra Jaafarpour, Masoud Soleimani, Saman Hosseinkhani, Mohammad Hossein Karimi, Marjan Yaghmaei, Naser Mobarra, Bita Geramizadeh Fri, 01 Jan 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of Angiotensin on the Quality of <i>In Vitro</i> Produced (IVP) Sheep Embryos and Expression of Na<sup>+</sup>/K<sup>+</sup>/ATPase <p>Background: The presence of rennin-angiotensin components in mammalian ovaries and their involvement in ovarian physiology have been established. In the present study, effects of angiotensin II (Ang II) on sodium-potassium adenosine triphosphatase (Na<sup>+</sup>/K<sup>+</sup>/ATPase) expression and development of sheep embryos was evaluated.<br /> Methods: The abattoir-derived Cumulus Oocyte Complexes (COC) were randomly allocated into three experimental groups; group I) <em>in vitro</em> Maturation (IVM) of oocytes in the presence of Ang II followed by <em>in vitro</em> fertilization (IVF)/<em>in vitro</em> Culture (IVC) (IVM group), group II) IVM/IVF of oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC (D4 group), and&nbsp; group III) IVM/IVF and IVC of oocytes without any angiotensin (Control). The blastocyst and hatching rates were recorded on days 6 to 8. Day 8 embryos were immunostained with primary and secondary antibodies against Na<sup>+</sup>/K<sup>+</sup>/ATPase &alpha;<sub>1</sub> and &beta;<sub>1</sub> subunits.<br /> Results: Addition of Ang II during IVM and IVC significantly increased the hatching rate of blastocysts on day 8 compared to the control. The trophectoderm and total blastocyst cells&rsquo; numbers were significantly increased by addition of Ang II to the IVM and IVC media, though the expression of Na<sup>+</sup>/K<sup>+</sup>/ATPase &alpha;<sub>1</sub> and &beta;<sub>1</sub> subunits were positively influenced by the addition of Ang II on day 4 (D4 group).<br /> Conclusion: In conclusion, it seems Ang II through positive effects on embryos, expressed as the greater hatching rate and blastocyst cell number, could increase the sheep embryo developmental rate. These improvements might be partly related to the greater expression of Na<sup>+</sup>/K<sup>+</sup>/ATPase &alpha;<sub>1</sub> and &beta;<sub>1</sub> subunits when Ang II was added during IVC.</p> https://www.AJMB.org/En/Article.aspx?ID=227 Mohammad Mehdi Naderi, Sara Borjian Boroujeni, Ali Sarvari, Banafsheh Heidari, Mohammad Mehdi Akhondi, Amir-Hassan Zarnani, Abolfazl Shirazi Fri, 01 Jan 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparison of Three <i>Escherichia coli</i> Strains in Recombinant Production of Reteplase <p>Background: <em>Escherichia coli (E. coli)</em> is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. Reteplase as a highly disulfide-bonded recombinant protein is an example of difficult to express protein in <em>E. coli</em>.<br /> Methods: In this study, a codon optimized reteplase gene was synthetically prepared and cloned under the control of an IPTG inducible T7 promoter. The vector was simultaneously transformed and expressed in three different <em>E. coli</em> strains. The ability of strains for expression of this recombinant pharmaceutical was compared. Also, an attempt was made to increase the soluble production of reteplase in SHuffle T7 <em>E. coli</em> with alterations of expression condition like temperature, inducer concentration and oxygen supply.<br /> Results: High amounts of reteplase were expressed as inclusion bodies in all three strains. BL21 (DE3) showed the highest level of expression in inclusion bodies followed by Rosetta-gami (DE3) and Shuffle T7. Changes of expression conditions were insufficient for soluble expression of reteplase in SHuffle T7 as a genetically engineered host for production of disulfide bonded proteins.<br /> Conclusion: The oxidizing cytoplasm of Rosetta-gami and Shuffle T7 in addition to alterations of cultivation parameters could not result in soluble production of reteplase, although the inclusion bodies produced in these two strains might increase the rate of refolding procedure likely due to formation of folding intermediates.</p> https://www.AJMB.org/En/Article.aspx?ID=228 Mehrnoosh Fathi-Roudsari, Asal Akhavian-Tehrani, Nader Maghsoudi Fri, 01 Jan 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning and Optimization of Soluble Vascular Endothelial Growth Factor165 Expression in <i>Escherichia coli</i> <p>Background: Vascular Endothelial Growth Factor (VEGF) is a coordinate regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. There are several types of VEGF, including VEGF<sub>165</sub>. VEGFs stimulate endothelial cell growth, angiogenesis, and capillary permeability. Low induction temperature is a major factor for expression of the recombinant VEGF<sub>165</sub> in soluble form. The purpose of this study was cloning and optimization of soluble vascular endothelial growth factor165 expression in <em>Escherichia coli (E. coli)</em>.<br /> Methods: In this study, total RNA of HeLa cell [cervix epithelium] was extracted. The VEGF<sub>165</sub> gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), and then VEGF<sub>165</sub> was subcloned into prokaryotic expression vectors pET-32a(+) and transformed into BL21 (DE3) <em>E. coli</em> strain. VEGF<sub>165</sub> expression was optimized by fine adjustments such as induction time and incubation temperature. VEGF<sub>165</sub> was analyzed by DNA sequencing prior to expression and the protein was further characterized by SDS-PAGE and immunoblotting using His&bull;tag specific polyclonal antibody.<br /> Results: Our results demonstrated that VEGF<sub>165</sub> was successfully cloned and expressed in pET-32a(+) vector. Optimization of the expression procedure showed that, induction by 1 <em>mM</em> IPTG at OD600=0.7 and overnight incubation at 22<sup>o</sup><em>C</em> resulted in the highest expression levels of soluble VEGF<sub>165</sub>.<br /> Conclusion: In this study, the expression of VEGF<sub>165</sub> in a high soluble level was successfully cloned and optimized.</p> https://www.AJMB.org/En/Article.aspx?ID=229 Ali Salimi, Mohammad Babashamsi Fri, 01 Jan 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of the Anti-proliferative Effects of <i>Ophiocoma erinaceus</i> Methanol Extract Against Human Cervical Cancer Cells <p>Background: Marine organisms provide appreciable source of novel bioactive compounds with pharmacological potential. There is little information in correlation with anti-cancer activities of brittle star. In the present study, anti-neoplastic efficacy of <em>Ophiocoma erinaceus</em> methanol extract against human cervical cancer cells was investigated.<br /> Methods: The HeLa cells were cultured and exposed to brittle star methanol extract for 24 and 48 <em>hr</em>. The anti-proliferative properties were examined by MTT assay and the type of cell death induced was evaluated through morphological changes, flow cytometry, Annexin kit and caspase assay. To assess the anti-metastatic activity, wound healing assay was conducted and photographs were taken from the scratched areas. Further, to understand molecular mechanism of cell apoptosis, the expression of Bax was evaluated.<br /> Results: The morphological analysis and MTT assay exhibited that the brittle star methanol extract can exert dose dependent inhibitory effect on cells viability (IC<sub>50</sub>, 50 <em>&mu;g/ml</em>). Flow cytometry and fluorescence microscopy demonstrated increment of sub-G1 peak, early and late apoptosis in HeLa treated cells. Wound healing migration assay showed that brittle star extract has anti-neoplastic efficacy by inhibiting cell migration. Caspase assay and RT-PCR analysis revealed that brittle star methanol extract induced caspase dependent apoptosis in HeLa cells through up-regulation of caspase-3 followed by up-regulation of Bax gene which is a hallmark of intrinsic pathway recruitment.<br /> Conclusion: These results represented further insights into the chemopreventive potential of brittle star as a valuable source of unknown therapeutic agents against human cervical cancer.</p> https://www.AJMB.org/En/Article.aspx?ID=230 Javad Baharara, Elaheh Amini, Farideh Namvar Fri, 01 Jan 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Computational Detection of piRNA in Human Using Support Vector Machine <p>Background: Piwi-interacting RNAs (piRNAs) are small non-coding RNAs (ncRNAs), with a length of about 24-32 nucleotides, which have been discovered recently. These ncRNAs play an important role in germline development, transposon silencing, epigenetic regulation, protecting the genome from invasive transposable elements, and the pathophysiology of diseases such as cancer. piRNA identification is challenging due to the lack of conserved piRNA sequences and structural elements.<br /> Methods: To detect piRNAs, an appropriate feature set, including 8 diverse feature groups to encode each RNA was applied. In addition, a Support Vector Machine (SVM) classifier was used with optimized parameters for RNA classification. According to the obtained results, the classification performance using the optimized feature subsets was much higher than the one in previously published studies.<br /> Results: Our results revealed 98% accuracy, Mathew&rsquo; correlation coefficient of 98% and 99% specificity in discriminating piRNAs from the other RNAs. Also, the obtained results show that the proposed method outperforms its competitors.<br /> Conclusion: In this paper, a prediction method was proposed to identify piRNA in human. Also, 48 heterogeneous features (sequence and structural features) were used to encode RNAs. To assess the performance of the method, a benchmark dataset containing 515 piRNAs and 1206 types of other RNAs was constructed. Our method reached the accuracy of 99% on the benchmark dataset. Also, our analysis revealed that the structural features are the most contributing features in piRNA prediction.</p> https://www.AJMB.org/En/Article.aspx?ID=231 Atefeh Seyeddokht, Ali Asghar Aslaminejad, Ali Masoudi-Nejad, Mohammadreza Nassiri, Javad Zahiri, Balal Sadeghi Fri, 01 Jan 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Outer Membrane Protein C (<i>ompC</i>) Gene as the Target for Diagnosis of <i>Salmonella</i> Species Isolated from Human and Animal Sources <p>Background: The use of selective and differential plating media is a simple method for the isolation of <em>Salmonella spp</em>. Recently, there has been a general move toward molecular methods of <em>Salmonella</em> detection and typing.<br /> Methods: A total of 1200 different specimens collected from human and animal sources were involved in his study. 600 stool specimens from patients suffering from diarrhea and 600 specimens from gall bladder (bile) of cattle from Al-Diwaniya slaughter house, Iraq were used. <em>Salmonella spp</em>. were isolated and identified using bacterial culturing on selective media and colonies were tested by API 20Eand then serotyping through polyvalent antisera and conformation by Polymerase Chain Reaction (PCR). PCR was used to detect <em>ompC</em> gene encoding biosynthesis of outer membrane protein C of <em>Salmonella</em> genus.<br /> Results: The results revealed that the rate of <em>Salmonella</em> isolates was 0.5% (3/600) from human and 1% (6/600) from animals. The PCR technique revealed that 9 isolates of <em>Salmonella spp</em>. harbored <em>ompC</em> gene. The results of this study revealed that the PCR technique had a high specificity in detection of <em>Salmonella spp</em>., in comparison to culture and biochemical test, Mini API 20 E and serological tests. The present study found no significant differences between human and animal isolates.<br /> Conclusion: Detection of <em>ompC</em> gene is a good method for detection of <em>Salmonella</em> species isolated from clinical specimens. It has a high specificity in comparison with other tests, with its advantages of greater speed and effectiveness than conventional detection methods.</p> https://www.AJMB.org/En/Article.aspx?ID=232 Alaa Abdel-Kadhim Jawad, Alaa H. Al-Charrakh Fri, 01 Jan 2016 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evidence Based Medicine and Medical Biotechnology <p>Evidence Based Medicine (EBM) can be found as far back as the 1940s. However, it was in 1972 that the concept first came into play, originated by Professor Archie Cochrane, in his book, Effectiveness &amp; Efficiency: Random Reflections on Health Services. This was the foundation for evidence based research, and in 1992 a facility was funded by the UK government, with the aim of performing randomly controlled tests on health services.<br /> This is no coincidence since evidence-based medicine suggests a personal responsibility for clinicians to keep abreast of research that would be difficult without the information access that the web provides. Evidence-based medicine is now generally perceived to be the dominant operating system in conventional medicine. The term &ldquo;evidence-based medicine&rdquo; first appears in 1991, in a piece by Gordon Guyatt (1). But EBM came to the attention of a wider audience in 1992 with an article by the Evidence-Based Medicine Working Group (2) that boldly proclaimed EBM as a &ldquo;new paradigm&rdquo; in medicine. The National Institutes of Health defines &ldquo;clinical research&rdquo; as research conducted with human subjects (or on material of human origin such as tissues, specimens and cognitive phenomena) for which an investigator (or colleague) directly interacts with human subjects. Excluded from this definition are in vitro studies that utilize human tissues that cannot be linked to a living individual. Indeed, Clinical trials are important component of evidence based medicine. A clinical trial is a research study that finds new ways to prevent, diagnose or treat disease (3-6). For example, cancer clinical trials test new treatments in people with cancer. These treatments investigate promising new drugs, drug combinations, new approaches to surgery or radiation therapy, and advances in new areas such as gene therapy. Clinical trials are the final step in a long process. There is no doubt that EBM plays an important role in the future of medical biotechnology.</p> https://www.AJMB.org/En/Article.aspx?ID=217 Shahin Akhondzadeh Mon, 17 Aug 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Comprehensive Review of Clinical Trials on EGFR Inhibitors Such as Cetuximab and Panitumumab as Monotherapy and in Combination for Treatment of Metastatic Colorectal Cancer <p>Metastatic colorectal cancer is the fourth most common cause of death due to cancer after those of lung, stomach, and liver. Anti epidermal growth factor receptor drugs as a targeting therapy seem to be good candidates for curing metastatic colorectal cancer. Two available anti epidermal growth factor receptor monoclonal antibodies are cetuximab and panitumumab which have been approved for metastatic colorectal cancer treatment. Through the available literature on NCBI and clinical trials, 31 clinical trials in which cetuximab or panitumumab as monotherapy or in combination with chemotherapy were used for the treatment of metastatic colorectal cancer patients in different line settings and 12 clinical trials in which bevacizumab was used for being compared with anti epidermal growth factor receptor monoclonal antibodies or chemotherapy were chosen for reviewing and comparing the results of overall survival, progression free survival and adverse effects. Cetuximab and panitumumab are well accepted for the treatment of mCRC patients at all stages in different line settings. Although cetuximab administration in metastatic colorectal cancer patients is mostly associated with better overall survival and panitumumab results in better progression free survival, to confirm the superiority of each of them in the treatment protocol of epidermal growth factor receptor monoclonal antibodies, more clinical trials with larger sample size are needed. Through current available data from clinical studies, it can be concluded that the best treatment outcome is achieved by a combination of anti epidermal growth factor receptor monoclonal antibodies with conventional chemotherapy.</p> https://www.AJMB.org/En/Article.aspx?ID=218 Mohammad Hossein Yazdi, Mohammad Ali Faramarzi, Shekoufeh Nikfar, Mohammad Abdollahi Mon, 17 Aug 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies <p>Background: Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes.<br /> Methods: To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37&deg;C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%.<br /> Results: Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose.<br /> Conclusion: Nowadays, application of appropriate additives is important for increasing the stability of antibodies.&nbsp; It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage.</p> https://www.AJMB.org/En/Article.aspx?ID=219 Yaghoub Yazdani, Saeed Mohammadi, Mehdi Yousefi, Fazel Shokri Mon, 17 Aug 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Potential of Brittle Star Extracted Polysaccharide in Promoting Apoptosis via Intrinsic Signaling Pathway <p>Background: Anti-cancer potential of marine natural products such as polysaccharides represented therapeutic potential in oncological researches. In this study, total polysaccharide from brittle star [<em>Ophiocoma erinaceus (O. erinaceus)</em>] was extracted and chemopreventive efficacy of Persian Gulf brittle star polysaccharide was investigated in HeLa human cervical cancer cells. &nbsp;<br /> Methods: To extract polysaccharide, dried brittle stars were ground and extracted mechanically. Then, detection of polysaccharide was performed by phenol sulfuric acid, Ultra Violet (UV)-sulfuric acid method and FTIR. The anti proliferative activity of isolated polysaccharide was examined by MTT assay and evaluation of cell death was done through morphological cell changes; Propodium Iodide staining, fluorescence microscopy and caspase-3, -9 enzymatic measurements. To assess its underlying mechanism, expression of Bax, Bcl-2 was evaluated.<br /> Results: The polysaccharide detection methods demonstrated isolation of crude polysaccharide from Persian Gulf brittle star. The results revealed that <em>O. erinaceus</em> polysaccharide suppressed the proliferation of HeLa cells in a dose and time dependent manner. Morphological observation of DAPI and Acridine Orange/Propodium Iodide staining was documented by typical characteristics of apoptotic cell death. Flow cytometry analyses exhibited the accumulation of treated cells in sub-G1 region. Additionally, polysaccharide extracted induced intrinsic apoptosis via up-regulation of caspase-3, caspase-9 and Bax along with down-regulation of Bcl-2 in HeLa cells.<br /> Conclusion: Taken together, the apoptosis inducing effect of brittle star polysaccharide via intrinsic pathway confirmed the anti tumor potential of marine polysaccharide. Therefore, these findings proposed new insight into anti cancer properties of brittle star polysaccharide as a promising agent in cervical cancer treatment.</p> https://www.AJMB.org/En/Article.aspx?ID=220 Javad Baharara, Elaheh Amini Mon, 17 Aug 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Interferences in the Optimization of the MTT Assay for Viability Estimation of Proteus mirabilis <p>Background: The chromogenic assay based on MTT bioreduction was adapted to <em>Proteus mirabilis viability</em> estimations. We primarily intended to use the assay for the evaluation of novel antimicrobial compounds, including structures with possible permeabilizing activity. Therefore, the influence of basic permeabilizing agents like Triton X-100 and EDTA upon the MTT assay was studied.<br /> Methods: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was used as a substrate for the whole-cell dehydrogenase activity estimations. The amount of formazan product was evaluated in the end-point reactions terminated with acidic isopropanol or in the continuous reactions run in the presence of low detergent concentrations.<br /> Results: The generally established procedure of the end product dissolution with acidic isopropanol caused absorbance instability which strongly affected the results accuracy. The disadvantage was especially pronounced when the assay was conducted in Mueller-Hinton Broth. PBS with 0.01% Triton X-100 used as the reaction medium allowed to omit the formazan dissolution step and follow the microbial MTT reduction in a continuous mode. It was observed that in Proteus mirabilis with a compromised outer membrane the assay score was artificially increased above the untreated control.<br /> Conclusion: The dependence of the assay results on the cell integrity might be a major drawback of the MTT assay application for the evaluation of novel antimicrobials against Gram-negative microorganisms. On the other hand, the MTT reduction could be conveniently used to assay the permeabilization degree in biotechnological protocols.</p> https://www.AJMB.org/En/Article.aspx?ID=221 Ewa Grela, Adam Ząbek, Agnieszka Grabowiecka Mon, 17 Aug 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of Transforming Growth Factor Alpha Polymorphisms with Nonsyndromic Cleft Lip and Palate in Iranian Population <p>Background: Cleft lip with or without cleft palate (CL/P) is one of the most common congenital anomalies and the etiology of orofacial clefts is multifactorial. <em>Transforming growth factor alpha (TGFA)</em> is expressed at the medial edge epithelium of fusing palatal shelves during craniofacial development. In this study, the association of two important <em>TGFA </em>gene polymorphisms, BamHI (rs11466297) and RsaI (rs3732248), with CL/P was evaluated in an Iranian population.<br /> Methods: The frequencies of BamHI and RsaI variations were determined in 105 unrelated Iranian subjects with nonsyndromic CL/P and 218 control subjects using PCR and RFLP methods, and the results were compared with healthy controls. A p-value of &lt;0.05 was considered statistically significant.<br /> Results: The BamHI AC genotype was significantly higher (p=0.016) in the patients (12.4%) than the control group (5.0%). The BamHI C allele was significantly higher (p=0.001; OR=3.4, 95% CI: 1.6-7.4) in the cases (8.0%) compared with the control group (2.5%).<br /> Conclusion: Our study showed that there was an association between the TGFA BamHI variation and nonsyndromic CL/P in Iranian population.</p> https://www.AJMB.org/En/Article.aspx?ID=222 Asghar Ebadifar, Roya Hamedi, Hamid Reza Khorram Khorshid, Kioomars Saliminejad, Koorosh Kamali, Fatemeh Aghakhani Moghadam, Nazanin Esmaeili Anvar, Nazila Ameli Mon, 17 Aug 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association between Serum Paraoxonase 1 Activities (PONase/AREase) and L55M Polymorphism in Risk of Female Infertility <p>Background: The risk of developing female infertility has been associated with gene polymorphisms that decrease the activity of enzymes involved in systemic Oxidative Stress (OS). In this study, <em>PON1 L55M</em> polymorphism for association with susceptibility to infertility was investigated among Iranian female population.<br /> Methods: Samples from 120 Iranian females [20 endometriosis; 30 Polycystic Ovary Syndrome (PCO); 70 controls] were analyzed and PCR-RFLP assay was used to determine the PON1 rs854560 (L55M) frequencies. The paraoxonase (PONase) and arilesterase (AREase) activities of PON1 enzyme were also assessed in order to investigate the association between serum PON1 activities, female infertility, and <em>PON1 L55M</em> polymorphism.<br /> Results: The women with a MM genotype (p=0.021; OR=2.55) showed more possibilities of experiencing infertility than those with a LM genotype (p=0.039; OR=1.91). According to LSD test, endometriosis subjects had significantly lower paraoxonase enzyme activity compared to control group (p=0.0024; CI=95%). No significant difference was found in women with PCOS for both PONase and AREase activity in comparison with control group (p=0.469; CI=95%). Furthermore, PON1 activities were the highest in LL genotype followed by LM and then MM genotype (MM&lt;LM&lt;LL) in both patients and controls.<br /> Conclusion: <em>PON1 L55M</em> polymorphism may be associated with serum PON1 activity and the risk of developing female infertility.</p> https://www.AJMB.org/En/Article.aspx?ID=223 Majid Motovali-Bashi, Saeid Sedaghat, Fariba Dehghanian Mon, 17 Aug 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Construction of CTLA-4-Ig Fusion Gene in pBudCE4.1 Expression Vector <p>Background: CTLA-4 inhibitory signals prevent cell cycle progression and IL-2 production, leading to a halt on an ongoing immune response. CTLA4-Ig fusion proteins contain the extracellular domain of CTLA-4 and Fc fragment of human IgG antibody. In this study we aimed to fuse the <em>ctla-4</em> gene encoding the extracellular domain of CTLA-4 molecule with <em>igg1</em> gene encoding Fc region of human IgG.<br /> Methods: After primer design, PCR reaction was performed using pfu polymerase enzyme and specific primers. PCR amplified fragment was ligated into the vector containing the human <em>igg1</em> gene. The resulting fusion fragment of <em>ctla-4</em> and human <em>igg1</em> genes was ligated to pBudCE4.1 expression vector.<br /> Results: Extracellular domain of <em>ctla-4</em> gene was ligated to <em>igg1</em> gene and then <em>ctla4-ig</em> fragment was cloned into pBudCE4.1 vector. Construction of the expression vector was confirmed by restriction pattern analysis and sequencing.<br /> Conclusion: By confirming the construct, in the next step, the recombinant DNA will be used to produce CTLA4-Ig recombinant protein for the clinical uses.</p> https://www.AJMB.org/En/Article.aspx?ID=224 Mahsa Yazdanpanah-Samani, Elham Mahmoudi Maymand, Tayebeh Jahangeerfam, Abbas Ghaderi Mon, 17 Aug 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Importance of Ethics in Medical Biotechnology <p>The rapid progress of modern biotechnology has presented a number of new and unique ethical and social challenges within the context of human medical science. Research in &nbsp;medical biotechnology has led to increased knowledge of disease, acceleration of the treatment process, improved pharmacotherapy for infectious diseases and hope for the struggle against incurable diseases such as ALS, MS and Alzheimer&rsquo;s <sup>1</sup>. Medical biotechnology promises major advances in human health and therefore, any limitations on the right to freedom of scientific research should be for significant reasons only, and as least restrictive as possible, so as not to impede scientific wisdom and prevent damage to the scientific undertaking. At the same time a duty exists to ensure that research in this area of biotechnology is conducted in ethically acceptable ways. A balance needs to be struck between recognizing the potential benefits which biotechnology research offers to individuals and the community as a whole, and the duty to ensure that research in this area is conducted ethically <sup>2</sup>.</p> <p>Researchers in the biotechnology industry face challenges unlike researchers in other sectors. Unlike most other industries, advances and research in the biotechnology industry are often front page news and has to face intense scrutiny by press, academics, government and the public. As biotechnology is a newly emerging field, a further challenge facing the industry is the lack of historical precedence in the sector to provide guidance for the safe and ethical development of the technology. In biotechnology research, the usual ethical principles applicable to health research involving animals and human participants must be observed and such research must be scientifically sound.</p> https://www.AJMB.org/En/Article.aspx?ID=209 Shahin Akhondzadeh Wed, 24 Jun 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Immunosuppressive Activity of Amniotic Membrane Mesenchymal Stem Cells on T Lymphocytes <p>Background: Mesenchymal Stem Cells (MSCs) are isolated from different sources like placenta. The placenta and its membranes like Amniotic Membrane (AM) are readily available and easy to work with. There is only limited knowledge on the immunomodulatory properties of human Amniotic Membrane-derived Mesenchymal Stem Cells (hAM-MSCs). The aim of this study was to survey the suppressive activity of hAM-MSCs on T lymphocytes in vitro.<br /> Methods: Human AMs were obtained after caesarean section births from healthy women. After enzymatic digestion, cells were cultured and hAM-MSCs were obtained. In addition, human T lymphocytes were isolated and co-cultured with hAM-MSCs for 72 hr in the presence or absence of phytohemagglutinin (PHA). Subsequently, proliferation of T cells was analyzed using BrdU and subsequently flow cytometry technique. Besides, the production of IL-4 and IFN-&gamma; was examined by ELISA method. Additionally, the expression of activation markers (CD38, HLA-DR) was studied on T lymphocytes by flow cytometry technique.<br /> Results: It was revealed that hAM-MSCs could significantly suppress the proliferation of T lymphocytes (p&le;0.01) and significantly decrease the production of IFN-&gamma; by T cells (p&lt;0.05). hAM-MSCs also down regulated the expression of activation markers on the surface of T lymphocytes, CD38 and HLA-DR. The difference was significant between the case and control samples (p&lt;0.05). All the comparisons were carried out between the case (Tcell+PHA+hAM-MSCs) and control (Tcell+PHA) groups.<br /> Conclusion: In conclusion, hAM-MSCs could inhibit the (mitogen-activated) T cells even in the absence of blood monocytes. Besides, hAM-MSCs-mediated inhibition of T lymphocytes was combined with down regulation of activation markers.</p> https://www.AJMB.org/En/Article.aspx?ID=210 Fatemeh Alikarami, Fatemeh Yari, Naser Amirizadeh, Mahin Nikougoftar, Mohammad Ali Jalili Wed, 24 Jun 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir High Prevalence of Y Chromosome Partial Microdeletions in Overweight Men <p>Background: Microdeletions of the Y chromosome are one of the most frequent genetic causes of spermatogenic failure in infertile men. But their role in gaining weight is unclear. The present study investigated the possible association of these partial microdeletions and obesity.<br /> Methods: In a case-control study, 180 males were selected. The prevalence of microdeletions was assessed using PCR in AZFc area of Y chromosome and statistical analysis was done using the Fisher exact test and Pearson correlation.<br /> Results: In our study, inverse relationship was observed between body mass index and testosterone level (p-value: 0.005). Fisher exact tests showed that there was a significant association between gr/gr mutation and BMI (p-value: 0.044).<br /> Conclusion: Our study revealed that Y chromosome microdeletions are more common in obese men. Furthermore, microdeletions such as gr/gr, which were observed in normal men, could cause decreased testosterone level. So, they may contribute to gaining weight.</p> https://www.AJMB.org/En/Article.aspx?ID=211 Atefeh Biabangard zak, Masoud Golalipour, Gholamreza Hadadchi Wed, 24 Jun 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression of Recombinant Human Insulin-like Growth Factor Type 1 (rhIGF-1) in Escherichia coli <p>Background: Human insulin-like growth factor type 1 (hIGF-1) is a protein consisting of 70 amino acids (MW=7.6 kDa) and mainly synthesized by liver. Mecasermin (Trade name INCRELEX) is the synthetic form of the protein which is used as an effective treatment for particular disorders such as short stature, type 1 and 2 diabetes, and wound healing. Current study was aimed to investigate the expression of human insulin-like growth factor type1 in Escherichia coli (E. coli) BL21 (DE3) expression system in order to produce an active recombinant form of the protein.<br /> Methods: For the purpose of the study, firstly codon optimization was done for hIGF-1 gene, using bioinformatics databases. Then, the gene was synthesized and inserted in pET-24a vector by a cutting strategy included NdeI and BamHI-HF enzymes. In the next step, gene was run in agarose gel and purified. The constructed expression cassette was transformed into E. coli BL21 (DE3) cells through CaCl2 heat shock method. Identification and confirmation of the transformed colonies were performed using screening PCR method. Synthesis of hIGF-1 was induced by IPTG. The expression in induced strains was analyzed by SDS-PAGE and western blotting techniques. Confirmation of cloning and IGF-1 expression cassette was carried out through genetic engineering procedures.<br /> Results: Analysis of transformed E. coli strain with SDS-PAGE and western blotting techniques confirmed that gene was expressed in host cells. Molecular weight of the expressed protein was estimated to be 7.6 kDa.<br /> Conclusion: hIGF-1 expression cassette for cloning and expression in E. coli was designed and the protein of interest was successfully induced and identified. In addition, E. coli BL21 (DE3) can be used as a suitable host for production of recombinant hIGF-1 and this technology has a potential to be localized.</p> https://www.AJMB.org/En/Article.aspx?ID=212 Hamidreza Iranpoor, Eskandar Omidinia, Venus Vatankhah, Vahid Gharanjik, Majid Shahbazi Wed, 24 Jun 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Retinoic Acid as the Stimulating Factor for Differentiation of Wharton’s Jelly- Mesenchymal Stem Cells into Hepatocyte-like Cells <p>Background: Wharton&rsquo;s Jelly-Mesenchymal Stem Cells (WJ-MSCs) are pluripotent cells with differentiation capability into most cell lineages. The aim of the current work was to examine the role of Retinoic Acid (RA) in differentiation process of these cells into hepatocyte-like cells and determine the morphological and functional patterns.<br /> Methods: Human WJ-MSCs were extracted, cultured and expanded; after approximately 95% of confluence, the cells were treated with hepatogenic media containing RA. The cells were subsequently analyzed for morphological changes, glycogen storage, albumin production, and specific gene expression.<br /> Results: WJ-MSCs expressed high levels of CD90 (93.6%) and CD105 (90.7%), but low levels of CD34 (0.3%) and CD45 (0.8%). Albumin production had significant difference in the two groups (p&le;0.05). The data showed specific characteristics in favor of considering the differentiated cells as hepatocyte-like cells such as obtaining morphologic, functional, and &alpha;FP and HNF1-&alpha; expression patterns which in turn were higher in cells exposed to RA.<br /> Conclusion: Based on the data of present study, RA is an effective molecule in inducing differentiation of WJ-MSCs into hepatocyte-like cells; therefore, it may be considered as a promising factor for targeting therapy of liver disorders.</p> https://www.AJMB.org/En/Article.aspx?ID=213 Keywan Mortezaee, Bagher Minaii, Fatemeh Sabbaghziarani, Iraj Ragerdi Kashani, Gholamreza Hassanzadeh, Parichehr Pasbakhsh, Mohammad Barbarestani Wed, 24 Jun 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate <p>Background: Coenzyme Q10 (CoQ10) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ10-producing Escherichia coli (E. coli).<br /> Methods: Two CoQ10-producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or PBAD promoters.<br /> Results: Over-expression of ispA under the control of PBAD promoter led to a relative increase in CoQ10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629&plusmn;40 to 30&plusmn;13 and 564&plusmn;28 to 80&plusmn;14 &micro;g/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other.<br /> Conclusion: The reduction in CoQ10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ10.</p> https://www.AJMB.org/En/Article.aspx?ID=214 Mojtaba Samoudi, Negar Omid Yeganeh, Hossein Shahbani Zahiri, Parvin Shariati, Reza Hajhosseini Wed, 24 Jun 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir In Silico Evaluation of Nonsynonymous Single Nucleotide Polymorphisms in the ADIPOQ Gene Associated with Diabetes, Obesity, and Inflammation <p>Background: The human ADIPOQ gene encodes adiponectin protein hormone, which is involved in regulating glucose levels as well as fatty acid breakdown. It is exclusively produced by adipose tissue and abundantly present in the circulation, with concentration of around 0.01% of total serum proteins, with important effect on metabolism.<br /> Methods: Most deleterious nonsynonymous single nucleotide polymorphisms in the coding region of the ADIPOQ gene were investigated using SNP databases, and detected nonsynonymous variants were analyzed in silico from the standpoint of relevant protein function and stability by using SIFT, PolyPhen-2, PROVEAN and MUpro, I-Mutant2.0 tools, respectively.<br /> Result: A total of 58 nonsynonymous SNPs consisting of 55 missense variations, 3 nonsense variations were found in the ADIPOQ gene. Next, 14 of the 55 missense variants were predicted to be damaging or deleterious by three different software programs (PolyPhen-2, SIFT, and PROVEAN), and 38 of them were predicted to be less stable (I-Mutant 2.0 and MUpro software). Totally, 10 variants out of 55 missense variants were predicted to be both deleterious and reduce protein stability. Additionally, 3 nonsense variants were predicted to produce a truncated ADIPOQ protein. RMSD and total energy were calculated for 4 nsSNPs out of 10 nsSNPs which were both deleterious and showed a decrease in protein stability.<br /> Conclusion: rs144526209 has high root-mean-square deviation (RMSD) and lower total energy value compared to the native modeled structure. It was concluded that this nsSNP, potentially functional and polymorphic in the ADIPOQ gene, might be associated with diabetes, obesity, and inflammation.</p> https://www.AJMB.org/En/Article.aspx?ID=215 Narayana Swamy A, Harika Valasala, Sreenivasulu Kamma Wed, 24 Jun 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An Association Study on IL16 Gene Polymorphisms with the Risk of Sporadic Alzheimer’s Disease <p>Background: Interleukin-16 (IL-16) is an important regulator of T cell activation and was reported to act as a chemoattractant agent. There are evidences that IL16 can control the neuroinflammatory processes in Alzheimer&rsquo;s Disease (AD). This study was performed to investigate the role or association of IL16 polymorphisms, rs11556218 and rs4778889 with the risk of late-onset Alzheimer&rsquo;s disease (LOAD) in Iranian population.<br /> Methods: Totally, 148 AD patients and 137 nondemented and age-matched subjects were recruited in this study. Genotyping of rs11556218 T/G and rs4778889 T/C polymorphisms was performed by PCR-RFLP method using the NdeI and AhdI restriction enzymes, respectively.<br /> Results: Statistical analysis of rs11556218 genotypes showed a protective effect against AD in the heterozygote genotype (p=0.001, OR=0.16) as well as rs4778889 (p=0.001, OR=0.23). Frequency of rs11556218 allele T was higher in controls than patients (p=0.001, OR=0.32). However, there was no significant difference in the frequencies of rs4778889 alleles between the AD patients and controls. &nbsp;<br /> Conclusion: Our results indicate that the rs11556218 and rs4778889 polymorphisms have a protective role in the development of sporadic AD in Iranian population.</p> https://www.AJMB.org/En/Article.aspx?ID=216 Tayyebeh Khoshbakht, Mohsen Soosanabadi, Maryam Neishaboury, Koorosh Kamali, Masoud Karimlou, Niloofar Bazazzadegan, Hamid Reza Khorram Khorshid Wed, 24 Jun 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir IVF Technology <p>The Parliament of Great Britain approved three-parent IVF project with 382 votes for and 128 votes against it on 3rd February, 2015. In case of the project approval in House of Lords, Great Britain would be the first country which legalizes the action. The purpose of this procedure is prevention of genetic diseases with mitochondrial defects in which the defective mitochondria in the cytoplasm of the mother&rsquo;s egg is transferred to the embryo. The consequence is the death of the child, muscle weakness, blindness and heart diseases. If the healthy mitochondria of another person are used in this procedure, the defects would disappear. This is done in two ways:</p> <p>1. Nucleus transfer from the mother&#39;s egg with mitochondrial defects or egg cytoplasm to the donated egg in which the nucleus has been removed,</p> <p>2. Transfer of parents&rsquo; nuclei from the early embryo (zygote) containing the cytoplasm to a donated early embryo (zygote) in which the parents&rsquo; nuclei have been removed.</p> <p>In each of these cases, the mitochondria in the egg or the donated zygote replace defective mitochondria and protect the child from related diseases.</p> <p>The advancement of IVF technology brought up the possibility in replacing the egg of patients with mitochondrial diseases with healthy donated eggs. In this case, the child produced has no genetic relationship with the patient, while in the current practice, 23 pairs of parental chromosomes construct the genetic essence of the child. In this practice, 37 mitochondrial genes, which comprise 1.10% of the total genetic essence of the child and play key roles in production of energy in cytoplasm of the egg, can be replaced with donated mitochondria in case of any defects, and thereby culminate in preventing mitochondrial defects in 1200 cases of children in Great Britain.</p> <p>The support of majority of reproduction scientists from the action and the concern and opposition of the Catholic Church together with some law and ethics specialists for authorizing the birth of engineered children denote the contrary position of the parties in applying this preventive and therapeutic action. The advent and development of reproductive technologies have always been intermingled with theological, ethical, legal and social issues and consequences. While great emphasis has been placed on ethical, legal and social dimensions and implications of cutting edge advances in reproduction, it seems that under supervision and surveillance of law makers and regulations, the possibility for amending inappropriate decisions is always provided. This is the focal point that the Parliament of Great Britain adhered to. However, there is no doubt that if necessary practical measures are not taken for discussing problems and compiling rules and regulations in treatment of reproductive diseases with higher incidence (such as application of IVF in indications for surrogacy in Iran), more theoretical and practical vexing issues will emerge which are complicated enough to be handled optimally. Although further studies are required to evaluate the action approved by the Parliament of Great Britain, the legal aspect of the action is worthy of attention since timely evaluation of it has been done. Also, as a prominent advancement in the field of biotechnology, the potential for its modification and amendment is permitted by the law.</p> https://www.AJMB.org/En/Article.aspx?ID=201 Mohammad Mehdi Akhondi Wed, 11 Mar 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparison between Platinum-Azidothymidine and Azidothymidine Effects on Bcl-2 and Telomerase Gene Expression in Rats with Hepatocellular Carcinoma <p>Background: High expression of telomerase and Bcl-2 are reported in hepatocellular carcinoma. Some anticancer drugs show their effects through reduction of these factors. In this study, it was aimed to investigate the effects of a new synthetic compound, platinum azidothymidine, on inhibition of telomerase and Bcl-2 expression in hepatocellular carcinoma compared to azidothymidine.<br /> Methods: To study the effects of Pt-AZT on hepatocellular carcinoma and compare its effects with AZT in inhibition of telomerase and Bcl-2 gene expression, pathogen-free male Wistar rats (n=100) were used. They were randomly divided to 4 groups (n=25). Group A as the control group contained 25 healthy rats; in the rest of animals, preneoplastic lesions were induced in their livers (groups B, C, and D) using Solt-Farber resistant hepatocyte protocol. Cancer development was approved by a pathology laboratory. Group B was negative control (untreated), groups C and D were treated by intraperitoneal injection (IP) of Pt-AZT (0.9 <em>mg/kg/day</em>) and AZT (0.3<em> mg/kg/day</em>), respectively for 14 days. At the end of the protocol, all rats were sacrificed and Bcl-2 and telomerase gene expression was determined using real -time PCR.<br /> Results: No tumor in the livers was found in group A at any point of the study, but it was present in livers of all animals in B, C and D groups. Results showed that telomerase and Bcl-2 expression was significantly lower in group C compared with group B (0.473&plusmn;0.231 <em>vs</em>. 5.137&plusmn;5.08, p&lt;0.001, for telomerase expression, and 0.41&plusmn;0.276 <em>vs</em>. 7.25&plusmn;11.6, p&lt;0.001, for Bcl-2 expression) and also compared with group D (0.473&plusmn;0.231 <em>vs</em>. 3.48&plusmn;4.02, p&lt;0.001, for telomerase expression, and 0.41&plusmn;0.276 <em>vs</em>. 4.93&plusmn;18, p&lt;0.001, for Bcl-2 expression).<br /> Conclusion: For the first time, it was demonstrated that Pt-AZT has more inhibitory effect on telomerase and Bcl-2 expression than AZT. It effectively inhibits the growth of liver tumor in rats by extending apoptosis.</p> https://www.AJMB.org/En/Article.aspx?ID=202 Abdolreza Sabokrouh, Asad Vaisi-Raygani, Mohammad Taghi Goodarzi, Shohreh Khatami, Masoud Taghizadeh-Jahed, Nahid Shahabadi, Niknam Lakpour, Yadollah Shakiba Wed, 11 Mar 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effects of Ultraviolet Light and Riboflavin on Inactivation of Viruses and the Quality of Platelet Concentrates at Laboratory Scale <p>Background: This study investigated the effects of Riboflavin (RB) combined with different doses of UV on Platelet Concentrate (PC) which was infected by three models of virus. Platelet quality after treatment was also assessed.<br /> Methods: Three models of virus used in this study were Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV), and Polio virus, which were added to PC. After photochemical treatment with RB and UV light, residual viral infectivity was titrated using 50% Tissue Culture Infective Dose (TCID<sub>50</sub>)/<em>ml</em>. This treatment was done with concentration of 50 <em>&micro;M</em> of RB and different doses of UV light (0.24, 0.48, 0.97, 1.29 <em>J/cm<sup>2</sup></em>). Platelet quality was assessed by measuring pH, Lactate Dehydrogenase (LDH), MTT assay and cell count after treatments and during 4 days of storage against control groups.<br /> Results: Concentration of 50 <em>&micro;M</em> RB with combination of 1.29 <em>J/cm<sup>2</sup></em> dose of UV resulted in the highest titer reduction of VSV (4 log <sub>10</sub>) and HSV (4.26 log<sub>10</sub>) and lowest titer reduction of Polio virus (2.6 log<sub>10</sub>). No significant difference was observed between different doses in comparison with control groups. In all treatment groups, the storage stability of platelets in PC was in the acceptable range in comparison with control group.<br /> Conclusion: This study indicated that RB/UV treatment was a promising pathogen reduction technique in PC and had limited effects on platelet quality. However, further optimization of this method is necessary to deal with blood-borne viruses like non-enveloped viruses.</p> https://www.AJMB.org/En/Article.aspx?ID=203 Hamideh Mirshafiee, Zohreh Sharifi, Seyed Masoud Hosseini, Fatemeh Yari, Hamed Nikbakht, Hamid Latifi Wed, 11 Mar 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cobalt Zinc Ferrite Nanoparticles as a Potential Magnetic Resonance Imaging Agent: An In Vitro Study <p>Background: Magnetic Nanoparticles (MNP) have been used for contrast enhancement in Magnetic Resonance Imaging (MRI). In recent years, research on the use of ferrite nanoparticles in T<sub>2</sub> contrast agents has shown a great potential application in MR imaging. In this work, Co<sub>0.5</sub>Zn<sub>0.5</sub>Fe<sub>2</sub>O<sub>4</sub> and Co<sub>0.5</sub>Zn<sub>0.5</sub>Fe<sub>2</sub>O<sub>4</sub>-DMSA magnetic nanoparticles, CZF-MNPs and CZF-MNPs-DMSA, were investigated as MR imaging contrast agents.<br /> Methods: Cobalt zinc ferrite nanoparticles and their suitable coating, DMSA, were investigated under <em>in vitro</em> condition. Human prostate cancer cell lines (DU145 and PC3) with bare (uncoated) and coated magnetic nanoparticles were investigated as nano-contrast MR imaging agents.<br /> Results: Using T<sub>2</sub>-weighted MR images identified that signal intensity of bare and coated MNPs was enhanced with increasing concentration of MNPs in water. The values of 1/T<sub>2</sub> relaxivity (r<sub>2</sub>) for bare and coated MNPs were found to be 88.46 and 28.80 (<em>mM</em><sup>&ndash;1</sup> <em>s</em><sup>&ndash;1</sup>), respectively.<br /> Conclusion: The results show that bare and coated MNPs are suitable as T<sub>2</sub>-weighted MR imaging contrast agents. Also, the obtained r<sub>2</sub>/r<sub>1</sub> values (59.3 and 50) for bare and coated MNPs were in agreement with the results of other previous relevant works.</p> https://www.AJMB.org/En/Article.aspx?ID=204 Zeinab Ghasemian, Daryoush Shahbazi-Gahrouei, Sohrab Manouchehri Wed, 11 Mar 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Release Studies on Ciprofloxacin Loaded Non-ionic Surfactant Vesicles <p>Background: Development of new drug carriers would be an interesting approach if it allowed increased efficacy of antibiotics and a reduction in doses, thus reducing the risk of developing resistance. As with most drug carriers, niosomes have been used to improve the selective delivery and the therapeutic index of antimicrobial agents.<br /> Methods: In this study, different formulation of niosomes containing ciprofloxacin (CPFX), Span (20, 60 or 80), Tween (20, 60 or 80) and cholesterol were prepared by film hydration method. The release of the drug from different formulations was studied by using Franz diffusion cell. The niosomes were further characterized by optical microscopy and particle size analysis, and their antimicrobial activity was evaluated.<br /> Results: Size of the niosomes was significantly dependent on the amount of cholesterol and surfactant type and varied from 8.56 to 61.3 <em>&mu;m</em>. The entrapment efficiency of CPFX niosomes prepared by remote loading was more than 74%. Niosomes composed of Span/Tween 60 provided a higher CPFX release rate than other formulations. The obtained results indicated a diffusion-based mechanism for drug leakage through bilayers. All formulations presented more antibacterial activity as compared to free CPFX solution.<br /> Conclusion: Niosomal CPFX appears to be a promising approach in the management of bacterial infections, especially ophthalmic ones, and should be further evaluated by<em> in vivo</em> experiments.</p> https://www.AJMB.org/En/Article.aspx?ID=205 Vajihe Akbari, Daryoush Abedi, Abbas Pardakhty, Hojjat Sadeghi-Aliabadi Wed, 11 Mar 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir TP53 Binding to BRCA1 and RAD51 in MCF7 and MDA-MB-468 Breast Cancer Cell Lines In vivo and In vitro <p>Background: Tumour suppressor genes such as TP53, BRCA1 and RAD51 are involved in DNA repair and their malfunctions result in genomic instability and cancer. Wild type (WT) TP53 binds to BRCA1and RAD51 <em>in vivo</em> and<em> in vitro</em>. However, mutated TP53 in tumours can interfere with WT TP53 function. We studied how mutation of TP53 in MDA-MB-468 cell line could affect its binding capacity and interfere with WT TP53 interaction with these DNA repair proteins.<br /> Methods: Binding capacity of mutated TP53 in MDA-MB-468 breast cancer cell line to BRCA1 and RAD51 proteins in comparison to WT TP53 in MCF7 cell line was studied by Immunoprecipitation. <em>In vitro</em> studies were performed by GST-WT p53 pull-down assays in these cell lines to assess the interaction of GST-WT p53 with BRCA1 and RAD51 proteins.<br /> Results: The results showed that mutated TP53 in MDA-MB-468 cells interacted with BRCA1 protein<em> in vivo</em> and did not effect WT TP53 binding to this protein <em>in vitro</em>. The Immunoprecipitation assays revealed that the mutated TP53 did not bind to RAD51 in comparison to WT TP53. However, this mutated protein could not interfere with binding of RAD51 to GST-WT p53 in MDA-MB-468 cell line by in vitro experiment.<br /> Conclusion: It was found that WT TP53 interactions with BRCA1 and RAD51 did not interfere with mutated TP53 in MDA-MB-468 cell line. In addition, RAD51 did not bind to TP53 with R273C mutation<em> in vivo</em>.</p> https://www.AJMB.org/En/Article.aspx?ID=206 Mozhgan Rasti, Tayebeh Azimi Wed, 11 Mar 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Maternal Supplementary Folate Intake, Methylenetetrahydrofolate Reductase (MTHFR) C677T and A1298C Polymorphisms and the Risk of Orofacial Cleft in Iranian Children <p>Background: The purpose of this study was to describe the association of MTHFR gene single nucleotide polymorphisms (C677T and A1298C) and maternal supplementary folate intake with orofacial clefts in the Iranian population.<br /> Methods: In this case-control study, peripheral venous blood was taken from 65 patients with orofacial clefts and 215 unaffected controls for DNA extraction and kept in EDTA for further analysis. The genotyping was carried out using Polymerase Chain Reaction (PCR) followed by Restriction Fragment Length Polymorphism (RFLP) and gel electrophoresis. Data were analyzed using Chi square test and logistic regression tests.<br /> Results: Genotype frequencies of 677TT were reported to be 13.5 and 36.1% in controls and CL/P patients, respectively, which showed a significant difference compared to CC as reference (OR=4.118; 95% CI=1.997-8.492; p=0.001). Conversely, 1298CC with frequencies of 10.8 and 12.7% in controls and patients, respectively, showed no significant difference compared to AA (OR=2.359; 95% CI=0.792-7.023; p=0.123). Comparing patients whose mothers did not report the folate supplement intake during pregnancy, to controls, it was observed that lack of folate intake was a predisposing factor for having a child with oral clefts (OR=5/718, p=0.000).<br /> Conclusion: Children carrying the 677TT variant of the MTHFR gene may have an increased risk of CL/P. In addition, the finding that the risk associated with this allele was obviously higher when the mothers didn&#39;t use folic acid, supports the hypothesis that folic acid may play a role in the etiology of CL/P.</p> https://www.AJMB.org/En/Article.aspx?ID=207 Asghar Ebadifar, Hamid Reza Khorram Khorshid, Koorosh Kamali, Mehdi Salehi Zeinabadi, Tayyebeh Khoshbakht, Nazila Ameli Wed, 11 Mar 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum <p>Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification.<br /> Methods: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women&#39;s plasma was collected, then real-time PCR for <em>RHD</em> exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results.<br /> Results: The results indicated significant differences between two extraction methods (p=0.001). The mean&plusmn;SD of Ct-value using THP protocol was 33.8&plusmn;1.6 and 36.1&plusmn;2.47 using QIAamp DNA Blood mini Kit.<br /> Conclusion: our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results.</p> https://www.AJMB.org/En/Article.aspx?ID=208 Zeinab Keshavarz, Leili Moezzi, Reza Ranjbaran, Farzaneh Aboualizadeh, Abbas Behzad-Behbahani, Masooma Abdullahi, Sedigheh Sharifzadeh Wed, 11 Mar 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Autistic Disorders and Medical Biotechnology <p>Autistic Disorders (ADs) are neuro-developmental disorders in the category of pervasive developmental disorders chiefly described by three main deficits: 1) deviant communication, 2) impaired reciprocal social interaction, and 3) limited, repetitive and stereotypic patterns of behaviors or interests <sup>1</sup>. The world-wide prevalence of ADs is estimated to be 62/10,000 <sup>1</sup>. Although various treatment regimens have been proposed for improving overall function of autistic patients, a definite efficient treatment which can target both core and associated symptoms in ADs has not been established so far. For example, current approved drugs by the Food and Drug Administration (FDA) such as risperidone and aripiprazole have not been proven to pose significant effect on the core features of this disorder <sup>2-4</sup>.</p> <p>While the absolute pathophysiologic mechanism of ADs is still debated, several genetic, environmental and neurobiological factors such as immune dysfunction, oxidative stress and imbalance of the inhibitory-excitatory systems are implicated in the pathogenesis of these disorders <sup>5-7</sup>. Neurobiological models have become research areas of interest for development of novel therapeutic agents in this regard <sup>1</sup>. Increased neuronal excitation in various central pathways has been proposed as one of the main neurobiological dysregulations in autistic patients <sup>1</sup>. Indeed, biotechnology and in particular gene therapies plays an important role in the future of research in autism <sup>1</sup>.</p> <p><em><strong>Neurexin 1:</strong></em> Part of family of genes that play a role with the neurotransmitter glutamate which is linked to autism. Gene chip technology was used to scan for genetic similarities in people with autism. DNA was scanned to search for copy number variations (CNVs), or insertions and deletions of genetic material linked to autism <sup>1</sup>.</p> <p><em><strong>Adult Form of Fragile X Syndrome:</strong></em> Expression of toxic RNA leads to Fragile X Tremor Ataxia Syndrome is modifiable by gene therapy <sup>1</sup>.</p> <p><strong>Fragile X Syndrome:</strong> Caused by loss of a gene called <strong>FMPR</strong> which acts as a break on a protein synthesis in specific area of the brain. This allows another protein, mGluR5 <strong>Metabotropic Glutamate Receptor</strong>, to function unchecked resulting in over activity in the brain. Reducing <strong>mGluR5</strong> reduces symptoms associated with fragile x syndrome <sup>1</sup>.</p> <p><strong>MECP2</strong> Activation of the gene reversed Rett syndrome. The MECP2 gene mutation is present in 95 percent of individuals with the disease <sup>1</sup>.</p> https://www.AJMB.org/En/Article.aspx?ID=193 Shahin Akhondzadeh Sat, 03 Jan 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production and Characterization of Monoclonal Antibodies against Human Prostate Specific Antigen <p>Background: Prostate Specific Antigen (PSA) is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies (mAbs) against PSA have been presented.</p> <p>&nbsp;Methods: Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer (PCa), Benign Prostatic Hyperplasia (BPH) and brain cancer tissues by Immunohistochemistry (IHC).</p> <p>Results: Five anti-PSA mAbs (clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/К) and clones (2C8-E9, 2G3-E2, IgG2a/К) were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 <em>kDa</em> in human seminal plasma in western blot.</p> <p>Conclusion: These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids.</p> https://www.AJMB.org/En/Article.aspx?ID=194 Ali Ahmad Bayat , Roya Ghods, Mahdi Shabani, Ahmad Reza Mahmoudi, Omid Yeganeh, Hadi Hassannia, Ali Sadeghitabar, Leila Balay-Goli, Farzaneh Noutash-Haghighat, Ali reza Sarrafzadeh, Mahmood Jeddi-Tehrani Sat, 03 Jan 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparative Analysis of Prostate Cancer Gene Regulatory Networks via Hub Type Variation <p>Background: Prostate cancer is one of the most widespread cancers in men and is fundamentally a genetic disease. Identifying regulators in cancer using novel systems biology approaches will potentially lead to new insight into this disease. It was sought to address this by inferring gene regulatory networks (GRNs). Moreover, dynamical analysis of GRNs can explain how regulators change among different conditions, such as cancer subtypes.</p> <p>Methods: In our approach, independent gene regulatory networks from each prostate state were reconstructed using one of the current state-of-art reverse engineering approaches. Next, crucial genes involved in this cancer were highlighted by analyzing each network individually and also in comparison with each other.</p> <p>Results: In this paper, a novel network-based approach was introduced to find critical transcription factors involved in prostate cancer. The results led to detection of 38 essential transcription factors based on hub type variation. Additionally, experimental evidence was found for 29 of them as well as 9 new transcription factors.</p> <p>Conclusion: The results showed that dynamical analysis of biological networks may provide useful information to gain better understanding of the cell.</p> https://www.AJMB.org/En/Article.aspx?ID=195 Pegah Khosravi, Vahid H. Gazestani, Mohammad Akbarzadeh, Samira Mirkhalaf , Mehdi Sadeghi, Bahram Goliaei Sat, 03 Jan 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of Combined Soy Isoflavone Extract and Docetaxel Treatment on Murine 4T1 Breast Tumor Model <p>Background: Emergence of drug resistance has brought major problems in chemotherapy. Using nutrients in combination with chemotherapy could be beneficial for improvement of sensitivity of tumors to drug resistance. Soybean-derived isoflavones have been suggested as chemopreventive agents for certain types of cancer, particularly breast cancer. In this study, the synergistic effects of soy isoflavone extract in combination with docetaxel in murine 4T1 breast tumor model were investigated.</p> <p>Methods: In this study, mice were divided into 4 groups (15 mice per group) of control, the dietary Soy Isoflavone Extract (SIE, 100 <em>mg/kg</em> diet), the Docetaxel (DOCE, 10 <em>mg/kg</em>) injection and the combination of dietary soy isoflavone extract and intravenous docetaxel injection (DOCE+SIE). After 3 injections of docetaxel (once a week), 7 mice were sacrificed to analyze MKI67 gene and protein expressions and the rest were monitored for diet consumption, tumor growth and survival rates.</p> <p>Results: In DOCE+SIE group, diet consumption was significantly higher than DOCE group. While lifespan showed a trend towards improvement in DOCE+SIE group, no significant difference was observed among the 4 studied groups. Tumor volume was not significantly affected in treated groups. A lower but not significant MKI67 protein expression was detected in western blot in DOCE+SIE group. The mRNA expression was not significantly different among groups.</p> <p>Conclusion: The results suggest that the combination of soy isoflavone as an adjunct to docetaxel chemotherapy can be effective in improving diet consumption in breast cancer.</p> https://www.AJMB.org/En/Article.aspx?ID=196 Ehsan Hejazi, Javad Nasrollahzadeh , Ramina Fatemi, Leila Barzegar-Yar MohamadI, Kioomars Saliminejad, Zohre Amiri, Masoud Kimiagar, Mohammad Houshyari, Maryam Tavakoli, Farah Idali Sat, 03 Jan 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Intravenous Transplantation of Very Small Embryonic Like Stem Cells in Treatment of Diabetes Mellitus <p>Background: Diabetes Mellitus (DM), simply known as diabetes, refers to a group of metabolic diseases in which there are high&nbsp;blood sugar&nbsp;levels over a prolonged period. In this study, the feasibility and safety of intravenous transplantation of Very Small Embryonic Like stem cells (VSELs) were investigated for diabetes repair, and finally the migration and distribution of these cells in hosts were observed.</p> <p>Methods: Mouse bone marrow VSELs were isolated by Fluorescent Activating Cell Sorting (FACS) method by using fluorescent antibodies against CD45, CXCR4 and Sca1 markers. Sorted cells were analyzed for expression of oct4 and SSEA1 markers with immunocytochemistry staining method. To determine multilineage differentiation, sorted cells were differentiated to Schwann, osteocyte and beta cells. Ten days after the establishment of a mouse model of pancreas necrosis, DiI-labeled VSELs were injected into these mice <em>via</em> tail vein. Pancreases were harvested 4 weeks after transplantation and the sections of these tissues were observed under fluorescent microscope.</p> <p>Results: It was proved that CD45-, CXCR4+, and Sca1+ sorted cells express oct4 and SSEA1. Our results revealed that intravenously implanted VSELs could migrate into the pancreas of hosts and survive in the diabetic pancreas. In treated groups, blood glucose decreased significantly for at least two month and the weights of mice increased gradually.&nbsp;&nbsp;</p> <div> <p>Conclusion: This study provides a strategy for using VSELs for curing diabetes and other regenerative diseases, and the strategy is considered an alternative for other stem cell types.</p> </div> https://www.AJMB.org/En/Article.aspx?ID=197 Morteaz Abouzaripour, Iraj Ragerdi Kashani, Parichehr Pasbakhsh, Nader Atlasy Sat, 03 Jan 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir ADSCs on PLLA/PCL Hybrid Nanoscaffold and Gelatin Modification: Cytocompatibility and Mechanical Properties <p>Background: Development of tissue engineering and regenerative medicine has led to designing scaffolds and their modification to provide a better microenvironment which mimics the natural niche of the cells. Gelatin surface modification was applied to improve scaffold flexibility and cytocompatibility.</p> <p>Methods: PLLA/PCL aligned fibrous scaffold was fabricated using electrospinning method. ADSCs were seeded after O2 plasma treatment and gelatin coating of the scaffolds. The morphological and mechanical properties of blends were assessed by Scanning Electron Microscopy (SEM), tensile test and ATR-FTIR. The cells proliferation was evaluated by MTT assay.&nbsp;</p> <p>Results: Based on the results, it is supposed that gelatin coating is a brilliant method of surface modification which significantly increases the mechanical properties of scaffold without any changes on the construction or on the direction of nanofibers which conducts cell&rsquo;s elongation. MTT analysis exhibited that ADSCs attachment, viability and proliferation significantly (p&lt;0.05) increased after gelatin treatment.</p> <p>Conclusion: Gelatin surface modification is a highly beneficial method to improve cytocompatibility, flexibility and mechanical features of the scaffolds which doesn&rsquo;t affect the nanofibers construction. Proliferation of Adipose Derived Stem Cells (ADSCs) as a remarkable source of stem cells was investigated for the first time on PLLA/PCL hybrid scaffold.</p> https://www.AJMB.org/En/Article.aspx?ID=198 Maedeh Mashhadikhan , Masoud Soleimani, Kazem Parivar , Marjan Yaghmaei Sat, 03 Jan 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line <p>Background: CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study.</p> <p>Methods: Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5&alpha; competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic.</p> <p>Results: Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively).</p> <p>Conclusion: Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera.</p> https://www.AJMB.org/En/Article.aspx?ID=199 Hajar Abbasi-Kenarsari, Farzaneh Shafaghat, Behzad Baradaran, Ali Akbar Movassaghpour, Dariush Shanehbandi, Tohid Kazemi Sat, 03 Jan 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Use of Raman Spectroscopy to Decrease Time for Identifying the Species of Candida Growth in Cultures <p>Background: The objective of this study is to establish Raman signatures from pure cultures of different Candida species using Raman Spectroscopy (RS) and use these signatures for rapid identification of unknown Candida species.</p> <p>Methods: Pure cultures of five Candida species were evaluated using RS to build a limited signature library. &lsquo;Raman Processing&rsquo; (RP) software was used for Principal Component Analysis (PCA) and Differential Functional Analysis (DFA).</p> <p>Results: Eleven principal components described at least 95% variance in the spectra. Raman signatures from these known Candida species were able to identify the species of unknown Candida cultures with 100% accuracy.</p> <p>Conclusion: Raman spectroscopy can improve early identification of Candida species and may facilitate early optimal antifungal therapy.</p> <p style="text-align:justify">&nbsp;</p> https://www.AJMB.org/En/Article.aspx?ID=200 Nitin S. Chouthai, Anuj A. Shah , Hossein Salimnia, Olena Palyvoda , Suneetha Devpura , Michael Klein , Basim Asmar Sat, 03 Jan 2015 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Personalized Medicine: A Tailor Made Medicine <p>In the past, disease diagnosis was based on symptoms that might be indicative of several diseases. Nowadays, diagnosis of some diseases has become more accurate because we are able to test for genes known to be associated with the disease. This method not only clearly identifies the presence of a particular disease; it can also precisely determine the subtype of the disease. Throughout history, the practice of medicine has largely been reactive. Even now days, we have to wait until the onset of diseases and then try to treat or cure them. As we don&rsquo;t fully understand the genetic and environmental factors that cause major diseases such as cancer, Alzheimer&rsquo;s and diabetes, our efforts to treat them are often imprecise, unpredictable and ineffective. In addition, the drugs and treatments we devise are tested on broad populations and are prescribed using statistical averages. For example, on average, any given prescription drug now on the market only works for half of those who take it. Anti-depressants are effective in only about 60 percent of those who take them 1-6. Personalized medicine is beginning to transform the practice of medicine. Personalized medicine is the tailoring of medical treatment to the individual characteristics of each patient. The approach relies on scientific breakthroughs in our understanding of how a person&rsquo;s unique molecular and genetic profile makes them susceptible to certain diseases. This same research is increasing our ability to predict which medical treatments will be safe and effective for each patient, and which ones will not be. Personalized medicine may be considered an extension of traditional approaches to understanding and treating disease. Personalized medicine has the potential to change the way we think about, identify and manage health problems. It is already having an exciting impact on both clinical research and patient care, and this impact will grow as our understanding and technologies improve.</p> https://www.AJMB.org/En/Article.aspx?ID=185 Shahin Akhondzadeh Mon, 29 Sep 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir New Insights into VEGF-A Alternative Splicing: Key Regulatory Switching in the Pathological Process <p>Vascular endothelial growth factor (VEGF-A) is one of the most important regulatory factors in pathological and physiological angiogenesis. Alternative splicing is a complicated molecular process in VEGF-A gene expression which adds complexity to VEGF-A biology. Among all VEGF-A exons, alternative splicing of exon 8 is the key determinant of isoform switching from pro-angiogenic VEGF-xxx to anti-angiogenic VEGF-xxxb. This is known as a key molecular switching in many pathological situations. In fact, the balance between VEGF-xxx and VEGF-xxxb isoforms is a critical controlling switch in both conditions of health and disease. Here, the properties of VEGF-xxx and VEGF-xxxb isoforms were discussed and their regulatory mechanism and their roles in certain pathological processes were evaluated. In summary, it was suggested that C-terminal VEGF-A alternative splicing can provide a new treatment opportunity in angiogenic diseases.</p> https://www.AJMB.org/En/Article.aspx?ID=186 Fariba Dehghanian, Zohreh Hojati, Maryam Kay Mon, 29 Sep 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of Treatment with Platinum Azidothymidine and Azidothymidine on Telomerase Activity and Bcl-2 Concentration in Hepatocellular Carcinoma- Induced Rats <p>Background: Telomerase activity increases in cancer cells. Bcl-2 is an antiapoptotic factor that its concentration grows in many cancer cells including hepatocellular carcinoma cells. In this study, an attempt was made to investigate the effects of a new synthetic compound, platinum azidothymidine (Pt-AZT) on treatment of rats with Hepatocellular Carcinoma (HCC) and to compare its effects with azidothymidine (AZT) in alteration of telomerase activity and Bcl-2 concentration in HCC. Methods: Healthy adult male Wistar rats (n=100) were randomly divided into 4 groups (A, B, C, and D). Group A contained 25 healthy rats and was considered as the control group. Liver preneoplastic lesions were induced in remaining animals (n=75) using Solt-Farber resistant hepatocyte protocol. These animals were randomly allocated in groups B, C and D. Group B was negative control (untreated), groups C and D were treated by intraperitoneal injection (IP) of Pt-AZT (0.9 mg/kg/day) and AZT (0.3 mg/kg/day), respectively for 14 days. After the treatment period, telomerase activity and Bcl-2 concentration were determined in the rats&rsquo; liver. Results: No HCC was developed in group A, but tumors were present in all other groups. Telomerase activity and Bcl-2 concentration were significantly lower in group C compared to groups B (0.1590.06 vs. 0.5770.116 IU/L, p&lt;0.001, respectively and 0.9310.388 vs. 3.940.74 ng/ml, p&lt;0.001, respectively). Similar results were observed in comparison with group D (0.3310.06 vs. 0.5770.116 IU/L, p&lt;0.001, respectively and 0.9310.388 vs. 2.940.594 ng/ml, respectively). There was a significant negative correlation between telomerase activity and Bcl-2 concentration only in untreated cancer group (p=0.034). Conclusion: In this study, higher anticancer activity of Pt-AZT in comparison to AZT was demonstrated. It effectively inhibits the growth of liver tumor in rats through extending apoptosis.</p> https://www.AJMB.org/En/Article.aspx?ID=187 Abdolreza Sabokrouh, Mohammad Taghi Goodarzi, Asad Vaisi-Raygani, Shohreh Khatami, Masoud Taghizadeh-Jahed Mon, 29 Sep 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Mitochondrial Distribution and ATP Content of Vitrified, In vitro Matured Mouse Oocytes <p>Background: The objective of this study was to investigate the effect of vitrification and in vitro maturation on the mitochondrial distribution and ATP content of oocytes. Methods: The oocytes at Germinal Vesicle (GV) and Metaphase II (MII) stages were recovered from 6-8 week old NMRI strain female mice. The oocytes were divided into vitrified and non-vitrified groups. Vitrification was done by the cryotop method using ethylene glycol, dimethylsulfoxide and sucrose as cryoprotectants. The GV oocytes were cultured in maturation medium for 24 hrs. The collected in vitro matured oocytes (IVM-MII) and ovulated metaphase II (OV-MII) oocytes were inseminated with capacitated sperm. The ATP content of the oocytes was measured by luciferin-luciferase reaction. Distribution of oocyte mitochondria was studied using Mito Tracker Green staining under fluorescent microscope. Results: The survival rates of vitrified oocytes at GV and MII stages were 87.39 and 89.5%, respectively. There was no significant difference in the developmental and hatching rates of vitrified and non-vitrified oocytes. The ATP content of GV and MII oocytes derived from in vivo and in vitro condition was not significantly different in vitrified and non-vitrified samples. The pattern of mitochondrial distribution in vitrified and non-vitrified GV and MII oocytes was similar but it was different between MII oocytes collected from fallopian tube and in vitro matured MII oocytes. However, the florescent intensity of mitochondrial staining was different in all the groups in the study. Conclusion: Vitrification did not affect mouse oocyte developmental competence, ATP content at different developmental stages but some alteration was seen in mitochondria distribution of in vitro matured oocytes in comparison to their controls.</p> https://www.AJMB.org/En/Article.aspx?ID=188 Zohreh Nazmara, Mojdeh Salehnia, Saman Hosseinkhani Mon, 29 Sep 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Differentiation of Human Umbilical Cord Matrix-Derived Mesenchymal Stem Cells into Germ-Like Cells <p>Background: Mesenchymal Stem Cells (MSCs) are multipotent cells that can be collected from different sources. Under specific conditions, MSCs can be differentiated to tissue specific cells in vitro. Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) can easily be harvested and cultured in in vitro conditions. Production of germ cells from mesenchymal stem cells is a very interesting and promising area in the field of reproductive medicine. In the present study, the possible trans-differentiation of hUCMSCs into Primordial like Germ Cell (PGC) was performed in vitro under specific condition. Methods: Human umbilical cord mesenchymal stem cells were cultured and expanded in DMEM medium containing 10% FBS. The cultured cells were studied for differentiation ability to adipocytes and osteocytes. Furthermore, MSCs related markers were identified by flow cytometry method. For PGC differentiation, hUCMS cells were cultured in differentiation medium containing Bone Morphogenetic Protein 4 (BMP4) and it was followed by retinoic acid (RA). Real time PCR and immunocytochemistry analysis were performed to evaluate the expression of PGC specific genes and proteins, respectively. Results: Our results showed that hUCMSCs cultured in the presence of BMP4 and RA are able to transdifferentiate in to PGC like cells in vitro. Real time PCR and immunocytochemistry results showed that differentiated cells expressed PGC specific markers after 14 days of culture. Conclusion: Based on these results, it was concluded that hUCMSC may be considered as a promising alternative cell source in reproductive medicine. More studies including laboratory and also animal models are needed to evaluate the functionality of differentiated PGCs before introducing them to clinical applications.</p> https://www.AJMB.org/En/Article.aspx?ID=189 Mostafa Latifpour, Yadollah Shakiba, Fardin Amidi, Zohreh Mazaheri, Aligholi Sobhani Mon, 29 Sep 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Quantitative Analysis of ErbB1 and ErbB2 Genes Amplification by a High Performance Liquid Chromatography <p>Background: Genes for human epidermal growth factor receptors B1 (ErbB1) and B2 (ErbB2) were amplified in breast and ovarian cancers. Both of them were associated with aggressive disease and worse prognosis. The ErbB1 or ErbB2 status of a tumor may provide an indication of the response to ErbB1 and ErbB2 -targeted therapies. For accurate and rapid assessment of amplification of ErbB1 and ErbB2 oncogenes, a High Performance Liquid Chromatography (HPLC) method was developed in this study. Methods: DNA was extracted from 30 primary breast tumors and 20 blood samples of healthy donors. ErbB1 and ErbB2 genes along with a reference gene were co-amplificated by Polymerase Chain Reaction (PCR). The PCR products were separated and quantified using an anion- exchange column within 30 min and in a single step. Optimum resolution was obtained when a sodium chloride gradient and a column temperature of 35˚C were used. The results of HPLC analysis of ErbB1 and ErbB2 PCR products were compared with real time PCR method as a gold standard test for 7 tumor samples. Results: The proposed HPLC method was confirmed by real time PCR method. Twenty two and ten of the specimens in our breast cancer cohort showed more than a two-fold amplification of ErbB2 and ErbB1 oncogenes, respectively. Conclusion: Our results were confirmed by real time PCR and showed that HPLC method is a specific, cheap and clinically applicable analytical approach for assessment of ErbB1 and ErbB2 statuses in breast tumors.</p> https://www.AJMB.org/En/Article.aspx?ID=190 Mozhgan Rasti, Zohreh Honardar, Mohsen Nikseresht, Aliakbar Owji Mon, 29 Sep 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Induction of Strong and Specific Humoral and T-helper 1 Cellular Responses by HBsAg Entrapped in the Methanobrevibacter smithii Archaeosomes <p>Background: Application of adjuvants with microbial origins is a recently highlighted approach in the vaccinology trials. Archaeosomes are among these microbial compounds with both adjuvant and liposomal activities and features. Methods: In the present study, recombinant HBsAg encapsulated into Methanobrevibacter smithii (M. smithii) archaeosomes. Balb/c mice immunized with this compound and humoral and cytokine secretion pattern of immunized models analyzed. Results: Frequency of IFN-&gamma; secreting cells in the HBsAg-containing archaeosomes group was significantly higher than HBsAg and HBsAg+C/IFA groups (p&le;0.05). IgG2a titer in the sera of HBsAg-containing archaeosomes group was also significantly higher than this subclass titer in the other groups (p&le;0.05). Conclusion: Analysis of induced responses revealed the Immunopotentiating characteristics of M. smithii archaeosomes in the induction of T-helper 1 responses according to the dominance of IgG2a subtype and IFN-&gamma; secreting splenocytes of immunized mice.</p> https://www.AJMB.org/En/Article.aspx?ID=191 Mohammad Reza Aghasadeghi, Seyed Ali Delbaz, Seyed Mehdi Sadat, Seyed Davar Siadat, Mehdi Shafiee Ardestani, Pooneh Rahimi, Azam Bolhassani, Rouhollah Vahabpour Roudsari, Golnaz Bahramali, Fateme Motevalli, Mehdi Davari, Habib Vakily, Ali Sharifat Salmani, Maryam Borhan Nobari Mon, 29 Sep 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Variable Number Tandem Repeat (VNTR) Genotyping of Hydatidiform Mole in Iranian Patients <p>Background: Classification of molar gestation into Complete Hydatidiform Mole (CHM) and Partial Hydatidiform Mole (PHM) is done according to clinical, ultrasonographic, histologic and genetic criteria. However, making a distinction between CHM and PHM using histologic criteria alone may be difficult and several studies have shown that misclassifications are frequent, even for experienced pathologists. CHM is the most common precursor to choriocarcinoma and heterozygous moles carry an increased predisposition to transformation. Methods: Formalin-fixed, paraffin-embedded tissue sections of patients as well as peripheral blood of patients and their partners&rsquo; were collected in EDTA tubes. Tissue samples were obtained by curettage. Histological evaluation was performed on routine section stained with Hematoxylin and Eosin. Variable Number Tandem Repeats (VNTRs) genotyping was performed for 30 cases in two groups of CHM (n=21) and PHM (n=9), with Polymerase Chain Reaction (PCR) amplification of 2 different polymorphic loci, namely the Col2A1 and D1S80. Results: The results of DNA analysis by VNTR genotyping showed that in 16 cases of CHM, amplification of the VNTR polymorphic loci showed androgenetic mono-spermic moles (homozygote) and in 5 cases of CHM androgenetic dispermic moles (heterozygote) in molar tissue. In cases of PHM, 6 samples were triploid dispermic and 3 samples were diploid biparental. Conclusion: This study confirmed that VNTR genotyping can identify the parental source of polymorphic alleles in hydatidiform mole. Compared to STR genotyping, VNTR genotyping was performed by PCR amplification of several minisatellite markers of DNA. This method significantly requires less time and is cost-effective.</p> https://www.AJMB.org/En/Article.aspx?ID=192 Zahra Pakzad, Hossein Mozdarani, Narges Izadi-Mood, Shirin Niromanesh Mon, 29 Sep 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Ethical Issues in Medical Biotechnology The extraordinary revolution in biotechnology has created new possibilities for curing disease and manipulating our genetic heritage. It is easy to see how biotechnology can be used for medicinal purposes. Knowledge of the genetic makeup of our species, the genetic basis of heritable diseases, and the invention of technology to manipulate and fix mutant genes provides methods to treat the disease 1. But it has also created numerous ethical problems that need close philosophical attention. In another words, since biotechnology involves modifying living things for human purposes, there is great potential for ethical concerns. The recent advances in biotechnology present both benefits and risks. They have revolutionized the process of drug manufacture, diagnosis and treatment and the production of animal models for human diseases. There is a tremendous potential for creating new drugs and treatment. This technology raises important ethical issues in the social structures including families, preventive medicine, employment, health insurance etc. We must interact with the general public, to educate them, and prepare them better for the impact of biotechnology. The scientific and medical communities and the public, in general, have to use these powerful tools responsibly, for the maximum benefit of mankind 2. https://www.AJMB.org/En/Article.aspx?ID=176 Shahin Akhondzadeh Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cytogenetic Alterations in Preimplantation Mice Embryos Following Male Mouse Gonadal Gamma-irradiation: Comparison of Two Methods for Reproductive Toxicity Screening Background: Genome instability is a main cause of chromosomal alterations in both somatic and germ cells when exposed to environmental, physical and chemical genotoxicants. Germ cells especially spermatozoa are more vulnerable to suffering from DNA damaging agents during spermatogenesis and also more potent in transmitting genome instability to next generation. Methods: To investigate the effects of γ-rays on inducing abnormalities manifested as numerical Chromosome Aberrations (CA) and Micronucleus (MN) in preimplantation embryos, adult male NMRI mice were irradiated with 4 Gy of γ-rays. They were then mated at weekly intervals with superovulated, non-irradiated female mice in 6 successive weeks. About 68 hr post coitous, four to eight cell embryos were retrieved and fixed on slides using standard methods in order to screen for CA and MN. Results: In embryos generated from irradiated mice, the frequency of aneuploidy and MN increased dramatically at all post-irradiation sampling times as compared to the control (p<0.01). The frequency of embryos expressed MN was much higher than chromosomally abnormal embryos, although the trend of MN formation was similar to chromosomal abnormalities seen in corresponding sampling times. Conclusion: Irradiation of sperms at any stages of spermatogenesis may lead to stable chromosomal abnormalities affecting pairing and disjunction of chromosomes in successive preimplantation embryos that are expressed as MN. Although chromosome analysis of embryos showed various types of chromosomal abnormalities, MN assay provide a simpler and faster technique for investigating the genotoxicity of agents affecting embryos at preimplantation stages. https://www.AJMB.org/En/Article.aspx?ID=177 Mahdieh Salimi, Hossein Mozdarani, Elmina Nazari Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Male Pronuclear Formation using Dog Sperm Derived from Ectopic Testicular Xenografts, Testis, and Epididymis Background: Testis tissue xenografting and the resultant sperm in a xenograft may provide a unique approach to rescue the genetic material of males that die prematurely and is a model for the study of human spermatogenesis and can represent an alternative approach for fertility preservation in cancer patients. This study was aimed to evaluate the xenogenic dog sperm in formation of male pronucleus following injection into the sheep oocytes. Methods: The in vitro matured slaughterhouse derived sheep oocytes were subjected to Intracytoplasmic Sperm Injection (ICSI) with epididymal, testicular, and xenogenic dog sperm. The ICSI was performed after scoring of the sperm midpiece using an IX71-Olympus inverted microscope with Nomarsky optics. Within 1 hr after injection, the injected oocytes in activated group were exposed to 5 µM ionomycin for 5 min. The data were analyzed by Chi-square and ANOVA using SigmaStat, version 3.5, and p<0.05 was considered significant. Results: The formation of female pronucleus after ICSI of xenogenic sperm was higher than epididymal and testicular sperm in non-activated oocytes. The corresponding rate in activated oocytes was higher or comparable with testicular and epididymal sperm. The rate of male pronucleus formation after ICSI of xenogenic sperm was comparable with injection of two other sperm sources. Oocyte activation had an inductive role in female and male pronuclear formation. Conclusion: Dog xenogenic sperm was capable to induce oocyte activation and proportion of male pronucleous formation was comparable to the testicular and epididymal sperm. https://www.AJMB.org/En/Article.aspx?ID=178 Abolfazl Shirazi, Asma Khadivi, Naser Shams-Esfandabadi Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Peptone Feeding on Transient Gene Expression Process in CHO DG44 Background: Transient Gene Expression (TGE) gained popularity over the last decade as a rapid method for the production of milligram to gram quantities of recombinant proteins for preclinical studies in biophama industry. Thereby, the optimization of the TGE technique for Chinese hamster ovary (CHO) as the dominant host for the production of biotherapeutics is of great interest to reach the values for Human Embryo Kidney-293 (HEK-293) cells in terms of transfection efficiencies and production titers. TGE efficiencies are cell line and vector dependant. Methods: In transfection efficiency optimization experiments, different starting cell densities, different amounts of plasmid DNA and PEI transfection reagent were investigated to achieve the best conditions leading to maximum transfection efficiencies. Furthermore, in order to investigate the effect of peptone feeding on transfection efficiency, three different sources of peptones with the greatest effect in the CD DG44 basal media were selected; Casein Tryptone N1, Soy petone A2SC and Soy peptone E110. Results: The transfection strategy performed here was able to make an outstanding increase in transfection efficiency of CHO DG44 cell line transfected with pTracer-SV40-mutated t-PA plasmid from 3.6% in our starting non-optimized condition to 66.93% in finally optimized situation. Moreover, peptone feeding strategy used here was successful to increase volumetric productivities up to 37%. In addition, the amounts of both PEI and plasmid DNA were reduced up to 66% and 25% respectively compared to our previous protocol. Conclusion: Here we described an optimization process for TGE in suspension-adapted CHO cells based on Polyethylenimine (PEI)/DNA concentration, DNA: PEI ratio, starting cell densities and peptone feeding strategy. https://www.AJMB.org/En/Article.aspx?ID=179 Fatemeh Davami, Farnaz Eghbalpour, Farzaneh Barkhordari, Fereidoun Mahboudi Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An Investigation on Mitochondrial DNA Deletions and Telomere Shortening during Multiple Passages of Adult Stem Cells Background: Limited resources for adult stem cells necessitate their in vitro culture prior to clinical use. Investigating mitochondrial DNA (mtDNA) and telomere shortening has proved to be important indications of stem cell validity. This study was designed to investigate these indicators in multiple passages of three adult stem cell lines which were produced in our stem cell laboratory. Methods: In this study, Dental Pulp Stem Cells (DPSCs), Periapical Follicle Stem Cells (PAFSCs) and Human Foreskin Fibroblast (HFF) cell lines were expanded for 20 passages. After 1, 5, 10, 15 and 20 passages, expanded cells were harvested and DNA was extracted for further studies. Common mtDNA mutation was detected by multiplex PCR and telomere shortening was tested by Southern blot analysis. Results: The common deletion was not detected in any of the stem cells or cell lines after several passages. In addition, Southern blot analysis indicated that the mean difference of telomere length between first and last passage was 0.25 kb in DPSC, 0.1 kb in PAFSC and 0.32 kb in HFF which indicates that the mean telomere length in various passages of the samples showed insignificant changes. Conclusion: Absence of mtDNA mutations in adult stem cell lines indicates good mitochondrial function even after 20 passages. In addition, absence of telomere shortening indicates stem cells validity after multiple passages. It is hoped this information could pave the way for using in vitro expansion of adult stem cells for future clinical applications. https://www.AJMB.org/En/Article.aspx?ID=180 Farzaneh Fesahat, Mohammad Hasan Sheikhha, Azam Rasti, Fatemeh Sadeghian Nodoshan, Hadi Zare-Zardini, Ali Reza Navabazam Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production of L-glutamic Acid with Corynebacterium glutamicum (NCIM 2168) and Pseudomonas reptilivora (NCIM 2598): A Study on Immobilization and Reusability Background: L-glutamic acid is one of the major amino acids that is present in a wide variety of foods. It is mainly used as a food additive and flavor enhancer in the form of sodium salt. Corynebacterium glutamicum (C. glutamicum) is one of the major organisms widely used for glutamic acid production. Methods: The study was dealing with immobilization of C. glutamicum and mixed culture of C. glutamicum and Pseudomonas reptilivora (P. reptilivora) for L-glutamic acid production using submerged fermentation. 2, 3 and 5% sodium alginate concentrations were used for production and reusability of immobilized cells for 5 more trials. Results: The results revealed that 2% sodium alginate concentration produced the highest yield (13.026±0.247 g/l by C. glutamicum and 16.026±0.475 g/l by mixed immobilized culture). Moreover, reusability of immobilized cells was evaluated in 2% concentration with 5 more trials. However, when the number of cycles increased, the production of L-glutamic acid decreased. Conclusion: Production of glutamic acid using optimized medium minimizes the time needed for designing the medium composition. It also minimizes external contamination. Glutamic acid production gradually decreased due to multiple uses of beads and consequently it reduces the shelf life. https://www.AJMB.org/En/Article.aspx?ID=181 Rajaram Shyamkumar, Innasi Muthu Ganesh Moorthy, Karuppiah Ponmurugan, Rajoo Baskar Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of Sortilin Silencing on Ovarian Carcinoma Cells Background: Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin (NTR3), the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Methods: Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Results: Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis (27.5±0.48%) in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation (40.1%) in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart (p<0.05). Conclusion: As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma. https://www.AJMB.org/En/Article.aspx?ID=182 Fatemeh Ghaemimanesh, Gholamreza Ahmadian, Saeid Talebi, Amir-Hassan Zarnani, Mehrdad Behmanesh, Shayda Hemmati, Reza Hadavi, Mahmood Jeddi-Tehrani, Maryam Farzi, Mohammad Mehdi Akhondi, Hodjattallah Rabbani Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Genetic Profile Variation in Vaccine Strains and Clinical Isolates of Bordetella pertussis Recovered from Iranian Patients Background: Re-emergence of pertussis has been reported in Iran despite a high rate of vaccination coverage. Low efficacy of the vaccine might be due to the genetic divergence between clinical versus vaccine strains. In the current study, the genetic profiles of clinical isolates and vaccine strains of Bordetella pertussis (B. pertussis) were assessed by using Pulsed Field Gel Electrophoresis (PFGE). Methods: Following phenotypic and molecular identification of isolates, XbaI-digested genomic DNA of 5 clinical isolates, 2 vaccine strains and a Tohama I strain were analyzed by PFGE along with B. parapertussis as a control. Results: Seven distinct PFGE profiles were found among all examined isolates/strains. In 5 clinical isolates, 4 profiles were identified whereas the vaccine strains displayed 2 distinct profiles. The reference strain, Tohama I had a distinct profile. Vaccine and clinical profiles had low similarity, with relatedness of approximately 40%. Conclusion: The genetic profiles of B. pertussis were different between circulating isolates and vaccine strains used in the national vaccination programs. Since new genetic profiles of B. pertussis can be disseminated periodically, the profiles of isolates circulating in the population should be monitored over the course of the re-emergence. https://www.AJMB.org/En/Article.aspx?ID=183 Faezeh Haghighi, Fereshteh Shahcheraghi, Ebrahim Abbasi, Seyed Saeed Eshraghi, Hojjat Zeraati, Seyed Ali Javad Mousavi, Hossein Asgarian-Omran, Masoumeh Douraghi, Fazel Shokri Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Inactivation of aprE Gene in Bacillus subtilis 168 by Homologus Recombination Background: One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis (B. Subtilis). One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. Methods: The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate (FITC)–labeled casein-substrate. Results: The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. Conclusion: It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase. https://www.AJMB.org/En/Article.aspx?ID=184 Mohammad Rabbani, Safoura Soleymani, Hamid Mir Mohammad Sadeghi, Narjes Soleimani, Fatemeh Moazen Wed, 25 Jun 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Research Institutions without Walls A network can be loosely defined as a structure linking together individual and organizational actors with shared goals or values, though often not a shared geography. A large body of literature highlights the important interaction between knowledge and networks. Interest in the impact of networking on knowledge translation and exchange, diffusion of innovations, knowledge management, and organizational outcomes is also increasing 1. There has been a growing interest in research networks and its implications on the creation of new knowledge. For example, there seems to be a consensus that those "scientists who collaborate with each other are more productive, oftentimes producing 'better' science, than are individual investigators. An open science platform can empowers researchers in their daily work and where everybody has equal opportunity to seek, share and generate knowledge. A value network can be defined as a network of relationships, which creates both tangible and intangible value through a complicated dynamic exchange between individuals, groups and organisations 2. The partnership for research and innovation in the health system funding opportunity recognizes the need to create networks of health researchers and clinical practitioners that can generate solutions to improve sustainable quality and value for money in the health system. The partnership for health research and innovation in the health system will support research and innovation. It seems that the concept of research networking in developing countries with several limitations such as research budgets should be engraved in the minds rather than papers. https://www.AJMB.org/En/Article.aspx?ID=153 Shahin Akhondzadeh Sat, 05 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Computational Survey of FHIT, A Putative Human Tumor Suppressor, Truncates Structure Background: Fragile Histidine Triad protein (FHIT), as a known tumor suppressor protein, has been proposed to play crucial role in inhibiting p53 degradation by MDM2. Studies have confirmed FHIT interaction with p53 or MDM2, although functional interacting domains of FHIT with MDM2 and/or p53 are not completely defined. Thus, through determining the significant structural interacting domains of FHIT, information with regard to MDM2 and p53 would be provided. As there were no previous studies evaluating the interaction of optimized important parts of target molecules, docking study was employed. Methods: Truncated structures of FHIT were screened to reveal critical sections engaging in FHIT interaction. HEX program was used in order to study the interaction of target structures. Results: Given the total energy, FHIT structures (β5-7, α1) and (α1) of FHIT were showed to be better candidates in comparison with other structures in interaction with optimized MDM2 part. Furthermore, FHIT structures (β4-7, α1) and (β5-7, α1) were considered to be better than other structures in interaction with optimized p53 part. FHIT truncates which interact with MDM2 optimized part exhibited lower energy levels than FHIT truncates which interact with p53 optimized part. Conclusion: Our results can be useful for designing new inhibitors of this protein complex interaction which would result in tumor repression. https://www.AJMB.org/En/Article.aspx?ID=144 Ameneh Eslamparast, Mohammad Hossein Ghahremani, Soroush Sardari Tue, 01 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of a High-resolution Melting Analysis Method Based on SYBR Green-I for rs7216389 Locus Genotyping in Asthmatic Child Patients Background: Asthma is caused by the combination of different factors. Current concepts of asthma pathogenesis emphasize on gene-environment interactions. Mega-genome scanning projects revealed that different Single Nucleotide Polymorphisms (SNPs) are related to asthma susceptibility. rs7216389-T is one of them that is related to childhood asthma and its effect on childhood asthma severity has been proved in different nations, however no study has been performed in Eastern Mediterranean and Middle East countries yet. Methods: To perform population genetic studies, a rapid and high-throughput screening method is necessary. High-resolution melting analysis is a rapid, powerful and accurate method, which is suitable for this type of studies. Therefore, it has been decided to develop a high-resolution melting method for rs7216389 locus genotyping in Iranian asthmatic children. In the current study, a high-resolution melting analysis method based on SYBR Green-I was developed to check the frequency of rs7216389-T mutation in Iranian asthmatic children for the first time. Results: Second and third classes of intercalating dyes are commonly used for high-resolution melting method. However, in this study, SYBR Green-I was used for rs7216389 locus genotyping for the first time. Our results for 60 samples showed that SYBR Green-I has good efficacy for rs7216389 locus genotyping through high-resolution melting method in comparison with PCR-RFLP and sequencing. Conclusion: Comparison of our results based on HRM analysis with PCR-RFLP showed that our developed method is rapid, accurate, high-throughput and economic to study the rs7216389 locus in asthmatic children and it is applicable for other similar population genetic studies. https://www.AJMB.org/En/Article.aspx?ID=145 Zahra Vali, Abbasali Raz, Hanieh Bokharaei, Mohammad Nabavi, Mohammad Hassan Bemanian, Mina Sharifi Yazdi, Navid Dinparast Djadid Tue, 01 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An In silico Based Comparison of Drug Interactions in Wild and Mutant Human β-tubulin through Docking Studies Background: Tubulin protein being the fundamental unit of microtubules is actively involved in cell division thus making them a potential anti-cancer drug target. In spite of many reported drugs against tubulin, few of them have started developing resistance in human β-tubulin due to amino acid substitutions. Methods: In this study we generated three mutants (F270V, A364T and Q292E) using Modeller9v10 which were targeted with compounds from higher and lower plants along with marine isolates using iGEMDOCK2.0 to identify their residual interactions. Results: The mutant F270V does not bring in any increase in the binding affinity in comparison with the taxol-wild type due to their conservative substitutions. However, it increases the volume of the active site. A364T mutant brings a better binding among few of the marine and higher plants isolates due to the substitution of the non-reactive methyl group with the polar residue. But this leads to reduced active site volume. Finally the mutant Q292E from epothilone binding site brings a remarkable change in drug binding in the mutants in comparison with the wild type due to the substitution of uncharged residue with the charged one. But as such there was no change in the volume of the active site observed in them. Conclusion: Lower plants extracts were reported to exhibit better interactions with the taxol and epothilone binding sites. Whereas marine and higher plants isolates shows significant interactions only in the wild type instead of the mutants. In addition to this, the residual substitutions were also found to alter the conformations of the active sites in mutants. https://www.AJMB.org/En/Article.aspx?ID=146 Selvaakumar Chellasamy, Sudheer M. M. Mohammed Tue, 01 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Enrichment of Undifferentiated Type A Spermatogonia from Goat Testis Using Discontinuous Percoll Density Gradient and Differential Plating Background: The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Methods: Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). Results: The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p≤0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p<0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p<0.001). Conclusion: Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes. https://www.AJMB.org/En/Article.aspx?ID=147 Banafsheh Heidari, Minoo Gifani, Abolfazl Shirazi, Amir-Hassan Zarnani, Behzad Baradaran, Mohammad Mehdi Naderi, Bahareh Behzadi, Sara Borjian-Boroujeni, Ali Sarvari, Niknam Lakpour, Mohammad Mehdi Akhondi Tue, 01 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparative Proteomics Study of Streptozotocin-induced Diabetic Nephropathy in Rats’ Kidneys Transfected with Adenovirus-mediated Fibromodulin Gene Background: Transforming Growth Factor-beta (TGF-β) activation appears to be crucial for tissue injury in Diabetic Nephropathy (DN). Fibromodulin, the small leucine-rich proteoglycan, has been proposed to be the potent TGF-β modulator. In this study, the therapeutic effects of fibromodulin in the kidneys of streptozotocin (STZ)-induced diabetic rats were investigated. Methods: Diabetic rats received intraperitoneal (IP) injections of recombinant adenovirus expression vectors (RAd5) containing fibromodulin (RAd-FMOD) and were killed after 10 weeks. Proteins were isolated from the rat kidney and separated using two-dimensional gel electrophoresis. The differentially expressed proteins were analyzed using Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Results: Ten spots were identified using MALDI-TOF-MS. The identified proteins were primarily responsible for cell metabolism, cytoskeleton formation, and oxidative stress. RAd-FMOD treatment markedly attenuated the albuminuria in diabetic rats. Conclusion: Taken together, these results provide a valuable clue in exploring the mechanism underlying the therapeutic effects of fibromodulin in diabetic nephropathy suggesting that it can be a potential agent in the treatment of this disease. https://www.AJMB.org/En/Article.aspx?ID=148 Akram Maleki, Ali Ramazani, Maryam Foroutan, Alireza Biglari, Parisa Ranjzad, Ali Awsat Mellati Tue, 01 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir In vitro Therapeutic Effects of Low Level Laser at mRNA Level on the Release of Skin Growth Factors from Fibroblasts in Diabetic Mice Background: Numerous in vitro reports suggest that Low Level Laser Therapy (LLLT) affects cellular processes by biostimulation, however most of them emphasize on using visible light lasers which have low penetration. The aim of this study was to determine the effect of infrared laser light (which is more useful in clinic because of its higher penetration) on secretion of Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF), as important growth factors in wound healing. Methods: Fibroblasts were extracted from the skin of 7 diabetic and 7 nondiabetic mice and cultured. Cell cultures of experimental group were irradiated with single dose of LLLT (energy density of 1 J/cm2) using an 810 nm continuous wave laser and the control group was not irradiated. Secretion of growth factors by skin fibroblasts were quantified through real time polymerase chain reaction. Results: Diabetic irradiated group showed significant increase in FGF (p=0.017) expression, although PDGF increased and VEGF decreased in both diabetic and nondiabetic irradiated groups, but these variations were not statistically significant. Conclusion: These results suggest that LLLT may play an important role in wound healing by stimulating the fibroblasts. https://www.AJMB.org/En/Article.aspx?ID=149 Nooshafarin Kazemi khoo, Mohammad Ali Shokrgozar, Iraj Ragerdi Kashani, Amir Amanzadeh, Ehsan Mostafavi, Hassan Sanati, Laleh Habibi, Saeid Talebi, Morteaz Abouzaripour, Seyed Mohammad Akrami Tue, 01 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An In vitro Study on Chick Somite Ability to Express Cerberus, Chordin, FGF8,Follistatin, and Noggin Transcripts Background: In vitro simulation of developmental processes is an invaluable tool to shed light on the intrinsic mechanism of developmental biosystems such as central nervous system in mammals. Chick somites have been used to simulate the neural differentiation of human neural progenitor cells. In the present study, we aimed to indicate whether somites have the ability to express required neural differentiation factors at mRNA level. Methods: Chick embryos were isolated from the yolk surface of the fertilized eggs and somites were subsequently isolated from embryos under a dissecting microscope. Total RNA of the somites was extracted and RT-PCR carried out with specific primers of cerberus, chordin, FGF8, follistatin and noggin. Results: Data showed that five aforementioned factors were co-expressed after 7 days in vitro by somites. Conclusion: We concluded that neural induction property of somites appeared by production of required neural differentiation factors including cerberus, chordin, FGF8, follistatin and noggin. https://www.AJMB.org/En/Article.aspx?ID=150 Samaneh Sadat Hosseini Farahabadi, Khadijeh Karbalaie, Hossein Salehi, Farzaneh Rabiee, Kamran Ghaedi, Mohammad-Hossein Nasr-Esfahani Tue, 01 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Synergic Effects of Crocus Sativus L. and Low Frequency Electromagnetic Field on VEGFR2 Gene Expression in Human Breast Cancer Cells Background: Angiogenesis, which is required for embryonic development and many physiological events, plays crucial role in many pathological conditions such as tumor growth and metastasis. Recent studies indicate anticancer and antitumor properties of saffron against human cancers. Many processes are affected by Electromagnetic Field (EMF) and its effect on proliferation and gene expression were examined. In this experimental study, the synergic effects of saffron and EMF on VEGFR2 gene expression in MCF7 cells were investigated. Methods: Saffron was extracted using freeze dryer. MCF7 cells were grown in RPMI1640 medium supplemented with 10% FBS and incubated at 37C with 5% CO2. After 24 hr cells were treated with saffron extract at concentrations of 100, 200, 400 and 800 μg/ml. Forty eight hr after treatment all flasks were exposed with EMF (50 Hz, 0.004 T). Then total RNA was extracted and cDNA was synthetized using specific primer. Synthetized products were analyzed by Real Time PCR to determine expression level of VEGFR2. Data were analyzed by SPSS (ANOVA & Tukey). Results: Critical inhibitory effect on VEGFR2 gene expression was 20% at 400 μg/ml. Synergic use of EMF and saffron extract showed most reduction (38%) at 100 μg/ml. On the other hand synergic use of 200, 400 and 800 μg/ml saffron aqua extract and EMF decline noticeably the VEGFR2 level of gene expression to 29, 35 and 36%, respectively. EMF itself also reduced VEGFR2 up to 25% in comparison with control group which is remarkable at p<0.001. Conclusion: Results indicate a decrease in the expression of vascular endothelial growth factor receptor in the treated samples with saffron extract compared to control. This reduction in VEGFR2 level induced by synergic treatment of saffron and EMF which reveals induction of inhibitory effects of saffron on angiogenesis and could be also considered as a promising chemotherapeutic agent in breast cancer treatment. https://www.AJMB.org/En/Article.aspx?ID=151 Marzieh Mousavi, Elaheh Amini, Khadijeh Shahrokhabadi Tue, 01 Apr 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Avicenna and Evidence Based Medicine Evidence Based Medicine (EBM) is now generally perceived to be the dominant operating system in con-ventional medicine. It is unsurprising then that some have counseled complementary and alternative medicine practitioners to resist EBM 1,2. The efficacy of medicinal herbs does need to be established and toxicity, contraindications and side effects also need to be investigated, and this is best done with clinical research and trials that at this time are being conducted almost exclusively on efficacy and are limited in number most probably because of funding. Very little to no attention is being given to the more traditional fresh herbal extracts 3,4. Many herbal medicines are now being supported by scientific evidence and have been shown to exert sig-nificant effects in the body, relieve symptoms, treat disease and improve everyday function. Any "expert" who still states there’s no scientific evidence to support the use of herbal medicines hasn’t done their homework. One of interesting example is saffron (Crocus sativus) for Alzheimer’s disease and depression that has been mentioned by Avicenna in his famous book. Avivenna’s famous works is the Canon of Medicine, which was a standard medical text at many medieval universities. The Canon of Medicine was used as a text-book in the universities of Montpellier and Leuven as late as 1650. Avicenna Canon of Medicine provides a complete system of medicine according to EBM. Saffron is the world’s most expensive spice, derived from the flower of Crocus sativus. Each saffron crocus grows to 20–30 cm and bears up to four flowers, each with three vivid crimson stigmas 3,4. Indeed, it is a Persian herb with a history as long as the Persian Empire itself. Iran, the world's largest producer of saffron has been investing in research into saffron's potential medicinal uses 3,4. To date, five published randomized controlled trials have been published about effects of saffron on depression. The first evidence-based study on this subject was published in 2004 showing that saffron was as efficacious as imipramine in the short-term treatment of mild to moderate depression in adults 5. Importantly, saffron was more tolerable than imipramine (which often causes anticholinergic side effects). Subsequently, saffron was compared to placebo in a six-week randomized controlled trial of 40 adult patients with mild to moderate depression. Saffron resulted in about 12-point reduction on Hamilton Depression Rating Scale (HDRS) compared with only five points seen with the placebo. Tolerability profile of saffron was similar to the placebo 5. Later, several studies provided evidence for antidepressant effects of different Crocus sativus L. constituents compared with both placebo and fluoxetine. Both petal and stigma of Crocus sativus L. have shown beneficial effects for treatment of depression 6,7. Crocus sativus L. is increasingly being studied as a memory enhancer. Saffron can attenuate the deleterious effect of ethanol on memory registration and retrieval, and prevent ethanol-induced inhibition of hippocampal long-term potentiation 3,4. Crocin seems to be involved in spatial memory and recognition and blocked scopolamine-induced performance deficits in the step-through passive avoidance and radial water maze tests 3,4. Saffron showed similar protective effects on recognition and spatial memory in chronic stress and hypoperfusion models of memory impairment 3,4. In an animal model of Alzheimer’s Disease (AD) induced by intraventricular injection of streptozocin, Khalili et al showed that administration of crocin resulted in significantly better results in passive avoidance test 3,4. In a 16-week placebo-controlled study, 46 patients with mild to moderate AD were assigned to saffron 15 mg twice daily or placebo. At the end of the trial, saffron was associated with a significantly better outcome on cognitive function than placebo. Importantly, tolerability of saffron was similar to placebo 8. In a 22-week donepezil-controlled study, saffron 15 mg twice daily was compared to donepezil 5 mg twice daily. Saffron was as efficacious as donepezil, but was associated with lower frequency of side effects than donepezil 9. Now if we read again the monograph regarding saffron in the Avicenna’s book we will find an evidence based medicine approach. https://www.AJMB.org/En/Article.aspx?ID=173 Shahin Akhondzadeh Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Hepatitis B Virus X Gene on the Expression Level of p53 Gene using Hep G2 Cell Line Background: The HBV-X (HBX) protein is believed to contribute to the development of HCC. However, the molecular mechanisms involved in HBX-mediated hepatocarcinogenesis remain obscure. In this study, the effect of hepatitis B virus X gene and its protein product HBxAg on expression of p53 gene in Hep G2 cell line was investigated. Methods: Viral DNA extracted from HBV-positive serum and HBX gene region was amplified using polymerase chain reaction (PCR). Then, PCR product was cloned into the pcDNA3 vector. After confirmation of cloning, the recombinant plasmid pcDNA3-X was transfected into HepG2 cell line using lipid-mediated DNA-transfection procedure. SDS-PAGE and western blotting methods were used to identify expression of HBX protein. Relative quantification was used to analyze the p53gene expression using the 2-ΔΔ Ct method. Results: Recombinant plasmid pcDNA3–HBX was confirmed by restriction endonucleases digestion and colony-PCR. The results of SDS-PAGE and western blot assays showed that HBX gene could be expressed in Hep G2 cell line. There was no significant difference between the expression levels of p53 compared with GAPDH gene as housekeeping gene (p<0.05). Conclusion: There was no significant difference in the protein levels between the transfected cells with X gene containing HBX130 and HBX131 double mutations and p53 gene. It is necessary to do more studies on Hepatitis B virus to understand the role of HBX on the development of liver cancer and its function on p53 tumor suppressor protein. https://www.AJMB.org/En/Article.aspx?ID=136 Roghyeh Kordestani, Hamideh Mirshafiee, Seyed Masoud Hosseini, Zohreh Sharifi Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Isolation and Partial Characterization of Human Amniotic Epithelial Cells: The Effect of Trypsin Background: Despite the extensive information available in the literature, cell surface marker signature of human Amniotic Epithelial Cells (hAECs) remains controversial. The aim of the present study was to characterize immuno-phenotypic features, proliferative capacity and immunogenicity of hAECs. We also tested whether expression of some cell surface markers is influenced by the type of trypsin used for tissue digestion. Methods: Single cell suspensions of amniotic membranes from four human placentas were isolated by enzymatic digestion and expression of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, CD133, HLA-I, HLA-DR, HLA-G, SSEA-4, STRO-1 and OCT-4 was then evaluated by flow cytometry. The differential impact of four trypsin types on the yield and expression of CD105 and HLA-I was also determined. The proliferative capacity of cultured hAECs was assessed and compared in the presence and absence of Epidermal Growth Factor (EGF). To test their immunogenicity, hAECs were injected into Balb/c mice and the reactivity of hyperimmunized sera was examined by immunofluorescence staining. Results: Nearly all purified cells expressed mesenchymal markers, CD9, CD10, CD29, and CD73 and the embryonic marker, SSEA-4. A large proportion of the cells also expressed STRO-1 and OCT-4. The purified cells also expressed HLA-G and HLA-I. A very small proportion of hAECs expressed CD34, CD38, CD44, CD133 and HLA-DR. The type of trypsin used for enzymatic digestion affected both the percentage and expression of HLA-I and CD105. hAECs revealed substantial proliferative capacity only when cultured in the medium supplemented with EGF. These cells were shown to be capable of inducing high amounts of anti-donor antibodies. Conclusion: Here we provided evidence that hAECs are immunogenic cells with high level of HLA-I expression. Furthermore, this work highlighted the impact of isolation procedure on the immunophenotype of hAEC. https://www.AJMB.org/En/Article.aspx?ID=137 Meraj Tabatabaei, Nariman Mosaffa, Shohreh Nikoo, Mahmood Bozorgmehr, Roya Ghods, Somaieh Kazemnejad, Simin Rezania, Bahareh Keshavarzi, Soheila Arefi, Fahimeh Ramezani-Tehrani, Ebrahim Mirzadegan, Amir-Hassan Zarnani Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Messenger RNA Expression Patterns of Neurotrophins during Transdifferentiation of Stem Cells from Human-Exfoliated Deciduous Teeth into Neural-like Cells Background: Stem cells from Human Exfoliated Deciduous teeth (SHED) have the capability to differentiate into neural cells. Neurotrophins including Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) have neurogenesis, neurotrophic, or neuroprotective effects and are expressed in developing teeth. The aim of this study was to measure quantitative changes in mRNA expression levels of neurotrophins in neural-like cells differentiated from dental pulp stem cells. Methods: Isolated total RNA from SHED, dental pulp and neural-like cells (n=3) were transcribed into cDNA. Then real time PCR was done. Expression levels of mRNA for NGF, BDNF, NT-3, and NT-4 genes were compared in these three cells. Results: In neural like cells, BDNF mRNA increased (372.1113.5) significantly (p<0.01) after differentiation. NGF mRNA increased to more than 266 times the dental pulp level after differentiation. A similar pattern was seen for the expression of NT3 after differentiation. NT4 mRNA enhancement was 1344630.8 and 30.77.9 fold in neural like cells and SHED cells, respectively. Results show alterations with different degrees and direction in neurotrophins mRNA expression levels in these cells. Conclusion: Our results suggest that neurotrophins dental pulp cells, SHED cells and neural like cells derived from SHED cells produce neurotrophic factors. Since the large amounts of neurotrophins are expressed in SHED and neural like cells they may have important role in survival and differentiation of dental pulp stem cells and obtained information may lead to a novel method for tooth regeneration. https://www.AJMB.org/En/Article.aspx?ID=138 Abolghasem Esmaeili, Sedigheh Alifarja, Nosrat Nourbakhsh, Ardeshir Talebi Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Downregulation of MMP2 and Bcl-2 in Adipose Derived Stem Cells (ASCs) following Transfection with IP-10 Gene <p>Background: Mesenchymal Stem Cells (MSCs) are recently introduced as novel immunological gene carriers for treatment of cancer. It is believed that balance between the expression of angiogenic and anti-angiogenic factors, such as SDF-1 and IP-10, may regulate neovascularization within the tumor. Methods: In this study, we compared the expression of important tumor promoting mediators in IP-10-transfected Adipose Derived Stem Cells (ASCs) to those transfected with SDF-1. ASCs were isolated from adipose tissue of a normal subject undergoing cosmetic mamoplasty surgery using collagenase. ASCs were transfected with IP-10 or SDF-1 propagated plasmids by electroporation method and Lipofectamin 2000. Expressions of SDF-1, CXCR4, IP-10, Bcl-2, MMP2, IL-10, IGF-1, and VEGF were detected in transfected ASCs using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Results: Results showed that the expressions of SDF-1, CXCR4, Bcl-2, MMP2, IL-10, IGF-1, and VEGF were upregulated in SDF-1-transfected ASCs. In contrast, Bcl-2 and MMP2 transcripts showed 45&times;103 and 10 fold lower expression in ASCs transfected with IP-10 compared to non-transfected cells. Conclusion: Anti-angiogenic chemokines such as IP-10 may modulate tumor promoting properties of ASCs and would be introduced as novel candidates for tumor immunotherapy; however, further studies are needed to be conducted.</p> https://www.AJMB.org/En/Article.aspx?ID=139 Mahboobeh Razmkhah, Mansooreh Jaberipour, Abbas Ghaderi Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Assessment of Different Permeabilization Methods of Minimizing Damage to the Adherent Cells for Detection of Intracellular RNA by Flow Cytometry Background: Various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells. Methods: HeLa cells were fixed in 2% paraformaldehyde. A variety of detergents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets. Results: In comparison with other methods, maximum cell frequency in percentage and fluorescent intensity (M1=2.1%, M2=97.9%) were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min (p=0.001). Conclusion: Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized. https://www.AJMB.org/En/Article.aspx?ID=140 Zahra Amidzadeh, Abbas Behzad-Behbahani, Nasrollah Erfani, Sedigheh Sharifzadeh, Reza Ranjbaran, Leili Moezzi, Farzaneh Aboualizadeh, Mohammad Ali Okhovat, Parniyan Alavi, Negar Azarpira Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Fourier Transform Infrared Spectroscopy: A Potential Technique for Noninvasive Detection of Spermatogenesis Background: The seminal plasma is an excellent source for noninvasive detection of spermatogenesis. The seminal plasma of normospermic and azoospermic men has been analyzed for detection of spermatogenesis. Methods: Optical spectroscopy (Attenuated Total Reflectance-Infrared spectroscopy (ATR-IR) and Fourier Transform infrared spectroscopy (FT-IR) has been used to analyze the seminal plasma and the metabolome of seminal plasma for detection of spermatogenesis. Results The seminal plasma of normospermic and azoospermic men has been analyzed by ATR-IR. The results show that there is a pattern variation in the azoospermic men compared to normospermic men. However, the seminal plasma is too complex to show significant pattern variation. Therefore, the metabolome which is a subcomponent of the seminal plasma was analyzed. The seminal plasma metabolome of normospermic and azoospermic men has been analyzed by FT-IR. A significant pattern change was observed. The data combined with chemometrics analysis showed that significant changes are observed at metabolome level. Conclusion: We suggest that FT-IR has the potential as a diagnostic tool instead of testicular biopsy. https://www.AJMB.org/En/Article.aspx?ID=141 Kambiz Gilany, Roudabeh Sadat Moazeni Pouraci, Mohammad Reza Sadeghi Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Evaluation of the Effect of miR-26b Up-Regulation on Hb-F Expression in Erythroleukemic K-562 Cell Line Background: The major hemoglobin in the fetus is hemoglobin F (𝛼2𝛾2), whereas in adult humans, hemoglobin A (𝛼2𝛽2) is predominately expressed. Several studies have indicated that expression of the HbF subunit 𝛾-globin might be regulated post-transcriptionally. This could be done by small non-coding RNAs called microRNAs which target mRNAs in a sequence-specific manner and lead to translational repression or mRNA decay. The aim of this study is to evaluate the effect of miR-26b up-regulation on 𝛾-globin gene expression in K-562 cell line. Methods: These cells were grown in RPMI 1640 and pre miR-26b and were transfected within K-562 cell line using lentiviral vector. After RNA extraction and cDNA synthesis in selected days, miRNA up-regulation was confirmed by miRNA real time PCR and then 𝛾and 𝛽chain and GATA-1 expression were investigated by RT and QRT-PCR. Results: The viability of cells before transfection was 90%. Three and 7 days after transfection, through the use of relative Q-PCR, the 𝛾 chain expression increased 3.7, 6.8 and 3.8 folds and GATA-1 expression increased 2.1, 6.0 and 8.0 in comparison with untransfected cells. Conclusion: The data suggest that miR-26b can be involved in the increase of 𝛾-globin gene expression in K-562 cell line. We suggest that miR-26b may be a significant therapeutic target for increasing HbF levels in patients with sickle cell disease and 𝛽-thalassemia. https://www.AJMB.org/En/Article.aspx?ID=142 Sadegh Alijani, Shaban Alizadeh, Ahmad Kazemi, Zahra Kashani Khatib, Masoud Soleimani, Mohamadreza Rezvani, Neda Minayi, Farshid Karami, Behnoosh Tayebi Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Fine Structures of the Oocyte in Relation to Serum, Follicular Fluid Steroid Hormones and IGF-I in the Ovulatory-Sized Follicles in One-Humped Camel (Camelus dromedarius) Background: The following study was carried out to determine the ultrastructural features of the oocyte of the ovulatory-sized follicles in relation to concentrations of steroids and IGF-I in the follicular fluid and serum in the dromedary camel. Methods: Camel follicles with a clear and healthy appearance were categorized into three classes: follicles 10 to 13.9, 14-17.9 and 18-30 mm diameter. The Follicular Fluid (FF) and serum samples were assayed for estradiol-17β, progesterone and IGF-I. Recovered Cumulus-Oocyte Complexes (COCs) were prepared for transmission electron microscopy. Results: The mean (±SD) FF concentrations of progesterone and IGF-I was significantly (p<0.05) higher in follicles 18 to 30 mm diameter compared to other groups of follicles. There was no difference in the mean (±SD) serum estradiol-17β, progesterone and IGF-I concentrations between camels with different ovulatory-sized follicles (p>0.05). Oocytes from follicles 18 to 30 mm diameter (group 3) showed more advanced signs of maturation including the disappearance of the nuclear envelope, increased number of microvilli in erect position, the increase in number and size of vesicles and more even distribution of the mitochondria throughout the ooplasm. Conclusion: The final stages of oocyte maturation in dromedary camel is associated with increasing progesterone and IGF-I concentrations and constant high estradiol concentration in the follicular fluid which are paralleled with well-defined ultrastructural changes in oocytes. https://www.AJMB.org/En/Article.aspx?ID=143 Mojtaba Kafi, Seyed Fakhroddin Mesbah, Najmeh Davoodian, Ali Kadivar Thu, 02 Jan 2014 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus : A Study on Immunoreactivity in Balb/C Mouse Background: Staphylococcus aureus (S. aureus) is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of S. aureus comprises modification in binding proteins to penicillin. Vaccine strategy may be useful in controlling the infections induced by this organism. This study aimed at developing and producing the recombinant protein PBP2a as a vaccine candidate and evaluating the related humoral immune response in a murine model. Methods: A 242 bp fragment of mecA gene was amplified by PCR from S. aureus COL strain and then cloned into prokaryotic expression vector pET-24a. For expression of recombinant protein, pET24a-mec plasmid was transformed into competent E. coli BL21 (DE3) cells. Recombinant protein was over expressed with 1 mM isopropythio-β-D-galctoside (IPTG) and purified using Ni-NTA agarose. SDS-PAGE and western blotting were carried out to confirm protein expression. For immunization of experimental groups, Balb/c mice were injected subcutaneously with 20 µg of recombinant PBP2a three times with three weeks intervals. The sera of experimental groups were collected three weeks after the last immunization and then specific antibodies were evaluated by ELISA method. Results: Successful cloning of mecA was confirmed by colony-PCR, enzymatic digestion, and sequencing. SDS-PAGE and western blot analysis showed that recombinant protein with molecular weight of 13 kDa is over expressed. In addition, high titer of specific antibody against PBP2a in vaccinated mice was developed as compared with the control group and confirmed the immunogenicity of the vaccine candidate. Conclusion: Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies. https://www.AJMB.org/En/Article.aspx?ID=126 Setareh Haghighat, Seyed Davar Siadat, Seyed Mehdi Rezayat Sorkhabadi, Abbas Akhavan Sepahi, Mehdi Mahdavi Mon, 30 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production and Characterization of a Murine Monoclonal Antibody Against Human Ferritin Background: Ferritin is an iron storage protein, which plays a key role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody (mAb) against human ferritin was reported. Methods: Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma (clone: 2F9-C9) was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Results: Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity (2.34×109 M-1) and the isotype was determined to be IgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. Conclusion: This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic kit if other requirements of the kit are met. https://www.AJMB.org/En/Article.aspx?ID=127 Ali Ahmad Bayat, Omid Yeganeh, Roya Ghods, Amir-Hassan Zarnani, Reza Bahjati Ardekani, Ahmad Reza Mahmoudi, Jafar Mahmoudian, Farzaneh Haghighat-Noutash, Mahmood Jeddi-Tehrani Mon, 30 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production and Characterization of Recombinant Light Chain and Carboxyterminal Heavy Chain Fragments of Tetanus Toxin Background: Light chain (LC) and heavy chain carboxyterminal subdomain (HCC) fragments are the most important parts of tetanus neurotoxin (TeNT) which play key roles in toxicity and binding of TeNT, respectively. In the present study, these two fragments were cloned and expressed in a prokaryotic system and their identity was confirmed using anti-TeNT specific polyclonal and monoclonal antibodies. Methods: LC and HCC gene segments were amplified from Clostridium tetani genomic DNA by PCR, cloned into pET28b(+) cloning vector and transformed in Escherichia coli (E. coli) BL21(DE3) expression host. Recombinant proteins were then purified through His-tag using Nickel-based chromatography and characterized by SDS-PAGE, Western blotting and ELISA techniques. Results: Recombinant light chain and HCC fragments were successfully cloned and expressed in (E. coli) BL21 (DE3). Optimization of the induction protocol resulted in production of high levels of HCC (~35% of total bacterial protein) and to lesser extends of LC (~5%). Reactivity of the His-tag purified proteins with specific polyclonal and monoclonal antibodies confirmed their renatured structure and identity. Conclusion: Our results indicate successful cloning and production of recombinant LC and HCC fragments of TeNT. These two recombinant proteins are potentially useful tools for screening and monitoring of anti-TeNT antibody response and vaccine production. https://www.AJMB.org/En/Article.aspx?ID=128 Mehdi Yousefi, Roya Khosravi-Eghbal, Azam Hemmati, Fazel Shokri Mon, 30 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression and Purification of Recombinant ROP1 of Toxoplasma gondii in Bacteria Background: Toxoplasmosis is a worldwide-distributed infection which is mostly asymptomatic but can cause serious health problems in congenitally-infected newborns and immunecompromised individuals. Research is undergoing both to improve Toxoplasma serological tests, which play the main role in laboratory diagnosis of the infection, and develop an effective vaccine to prevent the infection. Some studies showed usefulness of rhoptry protein 1 (ROP1) antigen of Toxoplasma gondii (T. gondii) in serodiagnosis of the infection and induction of protective immunity. The purpose of this study was to produce recombinant ROP1 and evaluate its antigenicity against human infected sera. Methods: DNA encoding ROP1, amino acids 171 to 574, was obtained from T. gondii RH strain by polymerase chain reaction amplification and cloned in prokaryotic expression plasmid pET-15b. rROP1 was expressed in Escherichia coli (E. coli) and purified in a single step by immobilized metal ion affinity chromatography. Results: DNA sequencing showed 99% similarity between the cloned sequence and the corresponding sequence in Gene bank. Results indicated the proper antigenicity of rROP1. Sera from Toxoplasma infected individuals specifically recognized rROP1 in Western blotting. Conclusion: rROP1 is antigenic toward human infected sera and can be used in studies for development of both a Toxoplasma serological test and a protective vaccine. https://www.AJMB.org/En/Article.aspx?ID=129 Reyhaneh Mohabati, Jalal Babaie, Samira Amiri, Mohammad Talebzadeh, Pezhman Fard-Esfahani, Mojtaba Darbouy, Majid Golkar Mon, 30 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Micropatterning of ECM Proteins on Glass Substrates to Regulate Cell Attachment and Proliferation Background: Micropatterning is becoming a powerful tool for studying cells in vitro. This method not only uses very small amount of material but also mimic the microenvironment structure present in living tissues better than flask culturing techniques. In previous studies using micropatterning of extracellular matrix proteins on glass surfaces, the rate of protein detachment from the surface was so high that the proteins and the cultivated cells detached after 3 three days of cell seeding. Methods: Here we optimized the glass surface modification method to fulfill the requirement of most in vitro studies. Results: in our study we showed that the optimum time for glass surface modification reaction is 1.5 hr, and the cells could be tracked in vitro for over 15 days after cell seeding which is enough for the most in vitro studies. As a model, we cultivated HEK 293T and HepG2 cells on the collagen micropatterns and showed that they have normal growth and morphology on these micropatterns. The HEK cells also transfected with pmaxGFP plasmid vector to show that the cells on collagen micropatterns could also used in transfection studies. Conclusion: Taking these together, this novel method is promising for efficient cell culture studies on micropatterened surfaces in the future. https://www.AJMB.org/En/Article.aspx?ID=130 Omid G. Alamdari, Ehsan Seyedjafari, Masoud Soleimani, Nasser Ghaemi Mon, 30 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of Polymer-coated Glass Slides as Optical Oligonucleotide Microarrays Background: The microarray technology is in needed of cost-effective, low background noise and stable substrates for successful hybridization and analysis. Methods: In this research, we developed a three-dimentional stable and mechanically reliable microarray substrates by coating of two polymeric layers on standard microscope glass slides. For fabrication of these substrates, a thin film of oxidized agarose was prepared on the Poly-L-Lysine (PLL) coated glass slides. Unmodified oligonucleotide probes were spotted and immobilized on these double layered thin films by adsorption on the porous structure of the agarose film. Some of the aldehyde groups of the activated agarose linked covalently to PLL amine groups; on the other side, they bound to amino groups of adsorbed tail of biomolecules. These linkages were fixed by UV irradiation at 254 nm using a CL-1000 UV. These prepared substrates were compared to only agarose-coated and PLL-coated slides. Results: Atomic Force Microscope (AFM) results demonstrated that agarose provided three-dimensional surface which had higher loading and bindig capacity for biomolecules than PLL-coated surface which had two-dimensional surface. The nano-indentation tests demonstrated the prepared double coating was more reliable and flexible for mechanical robotic spotting. In addition, the repeated indentation on different substrates showed uniformity of coatings. The stability of novel coating was sufficient for hybridization process. The signal-to-noise ratio in hybridization reactions performed on the agarose-PLL coated substrates increased two fold and four fold compared to agarose and PLL coated substrates, respectively. Conclusion: Finally, the agarose-PLL microarrays had the highest signal (2920) and lowest background signal (205) in hybridization, suggesting that the prepared slides are suitable in analyzing wide concentration range of analytes. https://www.AJMB.org/En/Article.aspx?ID=131 Atefeh Pourjahed, Mohammad Rabiee, Mohammadreza Tahriri Mon, 30 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Mutation Analysis of SLC20A2 and SPP2 as Candidate Genes for Familial Idiopathic Basal Ganglia Calcification Background: Familial Idiopathic Basal Ganglia Calcification (IBGC) is a rare neurodegenerative disorder which is usually transmitted as an autosomal dominant trait. IBGC is genetically heterogeneous and SLC20A2, on chromosome 8p21.1–8q11.23, is the first gene found in IBGC-affected patients with varied ancestry. On the other hand, several candidate genes for IBGC on chromosome 2q37, including the SPP2 gene, may play a role in inhibiting calcification. Methods: Totally, 22 members of a three generational Iranian family affected by IBGC, with an autosomal dominant pattern of inheritance were included in this study. DNA was extracted from the whole blood using standard salting out method. To find a mutation responsible for IBGC, we sequenced the coding region of SLC20A2 as well as promoter and coding region of SPP2 in the index subject of IBGC-affected family. Results: Pathogenic mutation was found neither in SLC20A2 nor in SPP2. Conclusion: Our results strengthen genetic heterogeneity of this condition. Additional mutation studies are necessary to find a gene or genes responsible for IBGC in this affected family. https://www.AJMB.org/En/Article.aspx?ID=132 Fereshteh Ashtari, Kioomars Saliminejad, Ali Ahani, Koorosh Kamali, Zhamak Pahlevanzadeh, Hamid Reza Khorram Khorshid Mon, 30 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Retraction: Genotyping Analysis of Circulating Fetal Cells Reveals High Frequency of Vanishing Twin Following Transfer of Multiple Embryos <p>The author has requested that the original article entitled &quot;Genotyping Analysis of Circulating Fetal Cells Reveals High Frequency of Vanishing Twin Following Transfer of Multiple Embryos&quot; that was published in the April-June 2013 issue of <em>Avicenna Journal of Medical Biotechnology (AJMB)</em> be withdrawn because the results were published without the co-workers being aware of this publication. Therefore this paper is retracted, considering the fact that it is contrary to the scientific rules and unrespectful of the contribution of the other authors.</p> https://www.AJMB.org/En/Article.aspx?ID=133 Hussein Mouawia Mon, 30 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir US Editors and Reviewers can no Longer Handle Submissions by Authors Employed by the Government of Iran: Is it Fair and Logical? We have recently become aware that the Office of Foreign Assets Control (OFAC) of the US Department of the Treasury has imposed new sanctions against Iran. Regulation 560.538 provides that US persons should not handle publishing services for written publications (whether journals or books) if any of the authors of the manuscript are employed by the Government of Iran. The Government of Iran is defined in Regulation 560.304. The regulation does not include authors at academic and research institutions or authors based in clinical settings such as hospitals, and should therefore only affect a very small number of submissions. Never-theless, some editors reject the manuscripts from Iran even from academic settings with abovementioned sanction. In practice the result of these sanctions will mean that: • Submissions where any author is based in Iran, and is not at an academic and research institution, cannot be handled by US-based editors, US Elsevier staff, US reviewers, or any US citizens based outside of the US. • If an Iranian author has dual affiliations (Eg. University and government), their submission cannot be handled by US-based editors, US Elsevier staff, US reviewers, or any US citizens based outside of the US. • Affiliations of Iranian authors should therefore be checked, and any manuscripts which fall under this OFAC regulation delegated to a non-US editor, before handling. • When assigning reviewers, affiliations of Iranian authors should also be checked, and any papers which fall under this OFAC regulation should only be sent to non-US reviewers. (As an editor you should do what is reasonable to determine the nationalities of a reviewer e.g. check their email address. This check does not extend to emailing reviewers directly to confirm their nationality or location). Unless there is specific knowledge that a non-US-based reviewer is a US citizen, editors can send such submissions to reviewers based outside the US. Iranian researchers and academicians are disappointed that some publishers have created the impression that work from Iran should be discriminated against. This attitude is contrary to the spirit and values of global science. Over the last two decades everyone has spoken regarding global village and this action is against that. As an editor from Iran I ask my counterparts over the world that do you think this behavior and action is fair and logical. https://www.AJMB.org/En/Article.aspx?ID=172 Shahin Akhondzadeh Sat, 07 Sep 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Role of Different Supplements in Expression Level of Monoclonal Antibody against Human CD20 Background: Recombinant monoclonal antibodies have been marketed in last three decades as the major therapeutic proteins against different cancers. However choosing a proper medium and supplements to reach the high expression is a challenging step. Despite of commercial serum free and chemically defined media, there are still numerous researches seeking the optimum media to gain higher expression titer. Selecting the best basal media followed by proper supplementation to increase the cell density and expression titer needs proper and accurate investigation. Methods: In this study, we have determined the expression titer of monoclonal antibody against human CD20 using soy extract, Essential Amino Acid, Non- Essential Amino Acid, Panexin NTS, Peptone, Yeast extract, Insulin-transferrin selenite, Human Serum Albumin, Bovine Serum Albumin, Lipid, and two commercially available supplements, Power and Xtreme feed. In each experiment, the expression level was compared with a well defined media, ProCHO5, RPMI 1640 and DMEM-F12. Results: It has been shown that supplementing the ProCHO5 basal medium with 10% power feed or combination of 5% PanexinNTS,1.5 g/L yeast and 1.5 g/L peptone results in the best production levels with 450 and 425 mg/L of anti CD20 mAb expression level, respectively. Conclusion: Panexin NTS, yeast and peptone cane be proper supplement for fed-batch cell culture instead of commercial Power feed supplement which is a cost effective way to increase expression level. And thereby ProCHO5 may be replaced with common media such as RPMI 1640 and DMEM-F12. https://www.AJMB.org/En/Article.aspx?ID=119 Fereidoun Mahboudi, Mohammad Reza Abolhassan, Armita Azarpanah, Hamideh Aghajani-Lazarjani, Mohammad Amin Sadeghi-Haskoo, Shaian Maleknia, Behrouz Vaziri Sat, 06 Jul 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Predictions of Protein-Protein Interfaces within Membrane Protein Complexes Background: Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods: In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results: The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion: Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. https://www.AJMB.org/En/Article.aspx?ID=120 Ebrahim Barzegari Asadabadi, Parviz Abdolmaleki Sat, 06 Jul 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning and Expression of Functional Reteplase in Escherichia coli TOP10 <p>Background: Production of tissue Plasminogen Activator protein (t-PA) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. Methods: In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli (E. coli) bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method. Results: Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification. Conclusion: We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein.</p> https://www.AJMB.org/En/Article.aspx?ID=122 Fatemeh Khodabakhsh, Zohreh Dehghani, Mohammad Farid Zia, Mohammad Rabbani, Hamid Mir Mohammad Sadeghi Sat, 06 Jul 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Overexpression and Purification of C-terminal Fragment of the Passenger Domain of Hap Protein from Nontypeable Haemophilus influenzae in a Highly Optimized Escherichia coli Expression System Background: Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae (H. influenzae) Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called “HapS” and a 45 kDa C-terminal translocator domain called “Hapβ”. Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain (CBD) which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. Methods: To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24a-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography (IMAC) using Ni-NTA columns. Results: The highest expression level of target protein was achieved when CBD expressing E. coli BL21 (DE3) cells were grown at 37C in 2xTY medium with 1.0 mM IPTG at mid-log phase (OD600 nm equal to 0.6) for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than %97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Conclusion: Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process. https://www.AJMB.org/En/Article.aspx?ID=123 Akram Tabatabaee, Seyed Davar Siadat, Seyed Fazllolah Mousavi, Mohammad Reza Aghasadeghi, Arash Memarnejadian, Mohammad Hassan Pouriayevali, Neda Yavari Sat, 06 Jul 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production of Cloned Mice by Nuclear Transfer of Cumulus Cells Background: Over the past several years, mammals have been successfully cloned by either the splitting of an early stage embryo or nuclear transfer of adult somatic cells (NT) into oocytes. Although it has been 15 years since the generation of the first cloned mammals from somatic cells by NT, the success rate for producing live offspring by this technique is low regardless of the cell type and animal species used. However, these techniques have the potential to be important tools for future research in basic biology. In the present study, we described our experiences in producing successfully cloned mouse using NT method and piezo-actuated micromanipulator. Methods: B6D2F1 mice, 8-12 weeks old, were superovulated with injections of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin administered 48 hr apart. Enucleation and donor nuclei cumulus cell injection were performed with a piezo-actuated micromanipulator after which activation and trichostatin A treatment were used for reconstructed oocytes. Two-cell stage cloned embryos that developed in the mWM medium were transferred into the oviducts of pseudopregnant NMRI mice. Results: Of 367 oocytes collected, 131 (69%) developed into 2-cell stage embryos. Of these, 5 (1%) live pups were successfully delivered. We used NMRI foster mother to raise the pups by lactation. One adult cloned mouse was mated, after which she delivered and raised normal offspring. Conclusion: For mouse cloning, the present study also successfully tested the capability of somatic cell nuclear transfer SCNT using a piezo unit. https://www.AJMB.org/En/Article.aspx?ID=124 Soleiman Kurd, Mohammad Ali Zarei, Fardin Fathi, Tayyeb Ghadimi, Mohammad Saeed Hakhamaneshi, Ali Jalili Sat, 06 Jul 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Novel Approach for High Level Expression of Soluble Recombinant Human Parathyroid Hormone (rhPTH 1-34) in Escherichia coli Background: Parathyroid hormone (PTH) secreted by parathyroid glands regulates the metabolism of calcium and phosphorus in bone and kidney. Thereby, it can stimulate bone formation, and is a promising agent in the treatment of osteoporosis. Mature form of PTH consists of 84 amino acids; however, the first 34 residues of PTH cover the majority of hormonal action. Methods: In this study, the fusion form of highly soluble rhPTH was expressed at high level in Escherichia coli (E. coli). His6-thioredoxin as an extension for rhPTH improves the solubility of inclusion body. His6-thioredoxin-hPTH (1-34) was ligated into pET32a expression vector. The insertion of 5 amino acids (Asp-Asp-Asp-Asp-Lys) in the N-terminal of PTH made this protein to be digestable specifically by enterokinase enzyme. The fusion form of rhPTH was harvested and purified by immobilized affinity chromatography followed by digestion with enterokinase. Digested rhPTH was purified by applying on size exclusion and ion exchange chromatography to get the highest purity. Results: The mass spectroscopy analysis shows rhPTH molecular weight was 4117.5 Da. The purity was measured by HPLC column which showed more than 97%. Bioassay analysis of rhPTH was performed on rat sarcoma cell UMR-106 in parallel with commercially available rhPTH, Forteo. The result was measured through immunofluorescence detection kit. The data showed that the potency of rhPTH was comparable with commercially available medicine. Conclusion: Thioredoxin was applied as a fusion partner for production of highly soluble rhPTH. This specific fusion partner increased protein solubility and decreased protease reactivity. Purification process was optimized for high recovery and for purity more than 99%. As its biological activity is comparable with marketed drug, this protein is qualified for biopharmaceutical usage. https://www.AJMB.org/En/Article.aspx?ID=125 Haleh Hamedifar, Firoozeh Salamat, Mohammad Saffarion, Mohammad Ghiasi, Alireza Hosseini, Hadi Lahiji, Zomorrod Nouri, Hamed Arfae, Fereidoun Mahboudi Sat, 06 Jul 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effect of Oral Supplementation of Biogenic Selenium Nanoparticles on White Blood Cell Profile of BALB/c Mice and Mice Exposed to X-ray Radiation Background: Radiation therapy is an effective method used for treatment of many types of cancers. However, this method can cause unwanted side effects such as bone marrow suppression. In this study, the effect of oral administration of biogenic selenium nanoparticles (SeNPs) on total and differentiated white cells profile of BALB/c mice exposed to X-ray radiation was investigated and compared with non-irradiated mice. Methods: Sixty female BALB/c mice between six to eight weeks olds were divided into 4 test and control groups in two categories of normal and irradiated mice. In normal mice SeNPs administration was started from the day 0 and followed for a month. Irradiated mice were divided into three groups and were exposed to doses of 2, 4 and 8 Gy. After 72 hr of irradiation, the SeNPs treatment was started and continued for a month. Total and differentiated blood cells counts of both irradiated and non-irradiated groups were monitored during 30 days and the obtained results were compared. Also, the deposition of Se in different tissues and blood serum of normal mice was determined in normal mice after 30 days period of supplementation. Results: In normal mice an increase in the count of neutrophils was observed after 30 days of supplementation. In irradiated mice, SeNPs supplementation led to increase in both lymphocytes and neutrophils counts especially in mice exposed to 2 and 4 Gys radiation. Conclusion: Radiotherapy is categorized as an invasive method which can cause tissue damage and suppress the host immune defense. A restore of lymphocytes which was observed after SeNPs supplementation in irradiated mice can be highly interesting and provide cellular immunity against malignant diseases or other bacterial or fungal infections after radiotherapy. https://www.AJMB.org/En/Article.aspx?ID=121 Mohammad Hossein Yazdi, Maryam Masoudifar, Bardia Varastehmoradi, Ehsan Mohammadi, Erfan Kheradmand, Somayeh Homayouni, Ahmad Reza Shahverdi Fri, 05 Jul 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Iranian science shows world's fastest growth: ranks 17th in science production in 2012 The Islamic Republic of Iran ranked 17th in terms of science production in the world in 2012, according to the latest statistics released by the Scopus database. According to the statistics, Iran produced 34,155 articles in 2012, which gained the country the world’s 17th rank in science production and fixed its top position in the region, above Turkey. Scientific progress over the past few years was the result of the country’s recent policies and programs to develop knowledge and facilitate researchers' access to the world’s top academic resources. Iran has the world's fastest-growing scientific output, measured by the number of peer-reviewed papers published in international journals. In addition, Iran ranked first in scientific growth in the world in 2011. In 2000, the Islamic Republic of Iran ranked 53rd in the world in terms of highly cited medical articles, but improved to the 23rd rank in 2011. According to the Institute for Scientific Information (ISI), Iranian researchers and scientists published a total of 60,979 scientific articles in major international journals from 1999 to 2008. It is being said that scientific growth in Iran has been fastest in the world, even more than China. Whatever the reason, it is commendable that the country has achieved this even at the worst possible sanctions from the most powerful and most influential country in the world. Iran with a science and technology yearly growth rate of 25% is doubling its total output every three years and at this rate will reach the level of Canadian annual output in 2017. "If Iran keeps moving with the present momentum, it can ascend to the 4th place from the current 17th (in the world ranking of scientific growth and scientific production) in the next 6 years. How about quality and impact? The USA still has a clear lead; taking as a measure of world impact the share of the most highly cited papers (taken as the top 1% in each discipline) puts the USA in the lead with 61%, while the UK outperforms its volume share with 13% of highly cited papers. Iran still underperforms on this measure but the gap is closing, and is likely to close further as citation counts are a lagging indicator – it takes some years for spending on science to translate, first into publication outputs, and only later into citations of those papers by other scientists. Under Iran's 'comprehensive plan for science', the country plans to be spending four per cent of GDP on research and development by 2030. Unfortunately, heavy sanctions against Iran and financial crisis over the last two years slowed down this scientific growth. Now Iranian scientific society hopes to next president to fasten this growth and again back to future. https://www.AJMB.org/En/Article.aspx?ID=171 Shahin Akhondzadeh Sat, 08 Jun 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line Background: Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods: In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results: Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion: This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. https://www.AJMB.org/En/Article.aspx?ID=111 Loghman Salimzadeh, Mansooreh Jaberipour, Ahmad Hosseini, Abbas Ghaderi Sat, 16 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Design of Small Molecules with HIV Fusion Inhibitory Property Based on Gp41 Interaction Assay Background: Gp41 of HIV (Human Immunodeficiency Virus) is a protein that mediates fusion between viral and cellular membranes. The agent, T-20, which has been approved for HIV inhibition, can restrain Gp41 function in the fusion process; nevertheless, it has disadvantages like instability, high cost of production and injection form to be delivered twice a day. Methods: Several molecules like NB-2 and NB-64 have been discovered that can inhibit HIV infection. These molecules were used as template compounds to design and develop more effective small molecules functioning as HIV-1 fusion inhibitors targeting Gp41. The process included in silico docking protocols using HEX and ArgusLab applications. A multisource database was created, after choosing the best molecules; they were tested in vitro for inhibitory activity by HIV-1 single-cycle model, transfected in HEK cells (293T). Results: Computational analysis and experimental data were combined to explore molecular properties and the most potent ones were found, with the best suitable criteria for interaction with Gp41. Several examples (DAA-6, DAA-9 and DAA-12) could inhibit infection in vitro as effective as NB-2, NB-64. Conclusion: Since disadvantages of available fusion inhibitor (T-20), it seems necessary to find similar molecules to be approved and have small size providing suitable bioactivity profile. The molecules explored in this study can be good candidates for further investigations to be used as oral HIV fusion inhibitors in the future. https://www.AJMB.org/En/Article.aspx?ID=112 Soroush Sardari, Kayhan Azadmanesh, Fereidoun Mahboudi, Asghar Davood, Ruhollah Vahabpour, Rezvan Zabihollahi, Hosna Gomari Sat, 16 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression Enhancement in Trastuzumab Therapeutic Monoclonal Antibody Production using Genomic Amplification with Methotrexate Background: Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. Methods: According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method. Results: Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l/day. According to the results, the produced mAb had similar affinity to HER2+ tumor cells to that of Herceptin. Conclusion: High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase (DHFR). It is usually accepted that DHFR gene can be amplified in DHFR− CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR. https://www.AJMB.org/En/Article.aspx?ID=113 Soudabeh Akbarzadeh-Sharbaf, Bagher Yakhchali, Zarrin Minuchehr, Mohammad Ali Shokrgozar, Sirous Zeinali Sat, 16 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Preparation and Cytotoxic Evaluation of Magnetite (Fe3O4) Nanoparticles on Breast Cancer Cells and its Combinatory Effects with Doxorubicin used in Hyperthermia Background: Magnetic nanoparticles in a variable magnetic field are able to produce heat. This heat (42-45°C) has more selective effect on fast dividing cancer cells than normal tissues. Methods: In this work magnetite nanoparticles have been prepared via co-precipitation and phase identification was performed by powder x-ray diffraction (XRD). Magnetic parameters of the prepared nanoparticles were measured by a Vibrating Sample Magnetometer (VSM). A sensitive thermometer has been used to measure the increase of temperature in the presence of an alternating magnetic field. To evaluate the cytotoxicity of nanoparticles, the suspended magnetite nanoparticles in liquid paraffin, doxorubicin and a mixture of both were added to the MDA-MB-468 cells in separate 15 ml tubes and left either in the RT or in the magnetic field for 30 min. Cell survival was measured by trypan blue exclusion assay and flow cytometer. Particle size distribution of the nanoparticles was homogeneous with a mean particles size of 10 nm. A 15°C temperature increase was achieved in presence of an AC magnetic field after 15 min irradiation. Results: Biological results showed that magnetite nanoparticles alone were not cytotoxic at RT, while in the alternative magnetic filed more than 50% of cells were dead. Doxorubicin alone was not cytotoxic during 30 min, but in combination with magnetite more than 80% of the cells were killed. Conclusion: It could be concluded that doxorubicin and magnetite nanoparticles in an AC magnetic field had combinatory effects against cells. https://www.AJMB.org/En/Article.aspx?ID=114 Hojjat Sadeghi-Aliabadi, Morteza Mozaffari, Behshid Behdadfar, Maryam Raesizadeh, Hamid Zarkesh-Esfahani Sat, 16 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparison of Proliferative and Multilineage Differentiation Potential of Sheep Mesenchymal Stem Cells Derived from Bone Marrow, Liver, and Adipose Tissue Background: Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow Mesenchymal Stem Cells (MSCs) has remained to be challenged. In this study successful isolation, multilineage differentiation, and proliferation potentials of sheep MSCs derived from bone marrow, adipose tissue, and liver were widely investigated. Methods: The primary cell cultures were prepared form tissue samples obtained from sheep 30-35 day fetus. Passage-3 cells were plated either at varying cell densities or different serum concentrations for a week. The Population Doubling Time (PDT), growth curves, and Colony Forming Unit (CFU) of MSCs was determined. The stemness and trilineage differentiation potential of MSCs were analyzed by using molecullar and cytochemical staining approaches. The data was analyzed through one way ANOVA using SigmaStat (ver.2). Results: The highest PDT and lowest CFU were observed in adipose tissue group compared with other groups (p<0.001). Comparing different serum concentrations (5, 10, 15, and 20%), irrespective of cell sources, the highest proliferation rate was achieved in the presence of 20% serum (p<0.001). Additionally, there was an inverse relation between cell seeding density at culture initiation and proliferation rate, except for L-MSC at 300 cell seeding density. Conclusion: All three sources of fetal sheep MSCs had the identical trilineage differentiation potential. The proliferative capacity of liver and bone marrow derived MSCs were similar at different cell seeding densities except for the higher fold increase in B-MSCs at 2700 cells/cm2 density. Moreover, the adipose tissue derived MSCs had the lowest proliferative indices. https://www.AJMB.org/En/Article.aspx?ID=115 Banafsheh Heidari, Abolfazl Shirazi, Mohammad Mehdi Akhondi, Hossein Hassanpour, Bahareh Behzadi, Mohammad Mehdi Naderi, Ali Sarvari, Sara Borjian Sat, 16 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression of Shigella flexneri ipaB Gene in Tobacco Background: Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotype-specific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System (TTSS) of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli (E. coli), the feasibility of the expression of this protein in tobacco has been investigated. Methods: The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens (A. tumefaciens) strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum (N. tobaccum) leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. Results: The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. Conclusion: This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting. https://www.AJMB.org/En/Article.aspx?ID=116 Mandana Ohadi, Rahimeh Rasouli, Elham Darzi-Eslam, Anis Jafari, Parastoo Ehsani Sat, 16 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Retraction: Genotyping Analysis of Circulating Fetal Cells Reveals High Frequency of Vanishing Twin Following Transfer of Multiple Embryos Background: Detection of Circulating Fetal Trophoblastic Cells (CFTC) by single cell genotyping not only allows to identify fetal cells from maternal blood, but also to characterize their bi-parental genome. Methods: We have tested intact fetal trophoblastes recovered at 4th to 10th weeks of gestation (WG) from blood (10 ml per mother) of 13 women after In Vitro Fertilization (IVF) and transfer of one or several embryos. Large cells isolated from blood were individually microdissected and studied by genetic fingerprinting with a mean number of 3 Short Tandem Repeats (STR) markers, known to be informative by testing paternal and maternal blood DNA. Results: CFTC were found in all mothers starting from the 5th WG. A mean number of 2.5 CFTC per ml of blood was found in all the analyzed samples collected at the different terms of pregnancy. All mothers who received the transfer of two or three embryos, including one who delivered twins and one with vanishing twin (identified by ultrasounds), were found to have CFTC with two or three different bi-parental genotypes, belonging to different embryos derived from the same parents. Conclusion: CFTC circulation is detectable starting from the 5th WG. A "vanishing twin" phenomenon frequently develops after IVF and transfer of multiple embryos, being undetectable by ultrasounds and revealed by genetic CFTC fingerprinting. https://www.AJMB.org/En/Article.aspx?ID=117 Hussein Mouawia Sat, 16 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Culture of Ovine IVM/IVF Zygotes in Isolated Mouse Oviduct: Effect of Basal Medium Background: The basal medium that supports Isolated Mouse Oviduct (IMO) is important for supporting embryo development and quality. Methods: The culture of ovine IVM/IVF zygotes was done in IMO using SOFaaciBSA and SOFaaBSA as basal medium of IMO and in SOFaaBSA alone as control. For preparation of IMO mature inbred strain C57BL/6 female mice were synchronized and mated with vasectomized males. The females with vaginal plug were sacrificed and the zygotes were transferred in to the isolated oviduct at 20 hpi. The oviducts were cultured with SOFaaciBSA and SOFaaBSA for 6 days. Another group of zygotes were cultured in SOFaaBSA alone as control. Results: Culture of zygotes in the IMO with SOFaaciBSA and SOFaaBSA, did not significantly affect the development and quality of embryos (p>0.05). The hatching rate, total and trophectoderm cells number in IMO groups’ blastocysts were significantly higher than SOFaaBSA alone. The morphological appearance of IMO blastocysts was superior to SOFaaBSA alone. When the quality of oocytes was poor, IMO could better support ovine embryo development either with SOFaaBSA or SOFaaciBSA than SOFaaBSA alone and there was a significant difference in blastocyst formation at day 6 with SOFaaBSA alone. Conclusion: The culture of ovine IVM/IVF zygotes in IMO using two highly efficient ruminant embryo culture media not only could support development of ovine embryos similar to the level in non IMO culture system (SOFaaBSA alone) but also could improve the quality of resulting embryos. Additionally, IMO could better support the development of ovine embryos derived from poor quality oocytes compared to the SOFaaBSA alone. https://www.AJMB.org/En/Article.aspx?ID=118 Abbas Farahavar, Abolfazl Shirazi, Hamid Khoram, Ahmad Zareh Shahneh, Ali Sarvari, Mohammad Mehdi Naderi, Sara Borjian Boroujeni, Mahdi Zhandi Sat, 16 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Chairman’s statement In recent decades, increasing advancement in biological sciences brings the need for reliable updated re-sources. Scientific research journals facilitate accessibility to comprehensive resources and lay a ground for dis-seminating the latest findings of research projects to achieve constructive experiences and satisfy the scientific community. Accordingly, Avicenna Research Institute has published Avicenna Journal of Medical Biotechnology (AJMB) since four years ago as a mission to promote infrastructure in biotech research. Now, we are very delighted to being indexed in PubMed after four years of working in a skilled group of staff and enjoying the cooperation of a competent editorial board, executive manager and specifically the editorial assistance of Dr. Nima Rezaei. It is my great pleasure to appreciate the invaluable efforts of Dr. Ali Motevalizadeh Ardakani, the former editor in chief of AJMB, in promoting the quality of the journal and an-nounce that Professor Shahin Akhondzadeh, the Iranian prominent scientist and neuropsychopharmacologist, has been appointed to the position for the next three years. I am hopeful that taking advantage of his inval-uable experiences in other journals would be a precious asset to AJMB’s promotion in line with modern ad-vances in biological and medical sciences. Hereby, I like to express my gratitude for the precious efforts of the previous editorial board members, Dr. Montazeri, Dr. Dinarvand, Dr. Bahram, Dr. Rafi, Dr. Vasei, and Dr. Zakeri and warmly send my greetings to the new board members. Finally, I would like to ask all experts, scientists, and intellectuals to join hands to support AJMB and improve its quality by their productive and brilliant ideas. https://www.AJMB.org/En/Article.aspx?ID=170 Mohammad Mehdi Akhondi Sat, 09 Mar 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial Iran has taken significant steps in the fields of animal cloning, stem cell therapy and recombinant protein drugs in the past decade. The cloning of animals, stem cell therapies and transplantation by the Iranian stem cell transplantation research centers and production of a few recombinant proteins by the Iranian biotech companies and academic institutes is clearly positive for the development of advanced technologies in the field of medical biotechnology. According to the most recent reports in the media, recombinant proteins such as blood coagulation factors VII and VIII are in the process of being manufactured. These are certainly quite significant steps in the development process of recombinant protein drugs. As these recombinant protein drugs come to the Iranian market in the future, it is reasonable to expect prospective studies are designed in order to evaluate the effectiveness and side effects of these drugs in large populations. It is hoped to see a sustained support and a stable environment for the biotech industry in Iran for the continued production of recombinant protein drugs to not only meet the needs of domestic market but also find its way into the international markets in the near future. https://www.AJMB.org/En/Article.aspx?ID=169 Ali M. Ardekani Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Constructing a Mouse Oct4 Promoter/EGFP Vector, as a Whole-Cellular Reporter to Monitor the Pluripotent State of Cells Background: The transcription factor Oct-4, is an important marker of undifferentiating level and a key regulating factor for maintenance of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter system is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. Methods: In the present study, we report construction of a recombinant vector, pDB2 Oct4 promoter/EGFP, in which expression of Enhanced Green Fluorescent Protein (EGFP) was controlled by the mouse Oct-4 promoter. Results: In transfected mouse embryonic stem cells with this vector, EGFP was predicted to be specifically expressed in pluripotency state. After transfection, high-level expression of EGFP under the control of Oct-4 promoter was observed in manipulated embryonic stem cells. Conclusion: Thus, our new cellular reporter showed that both the properties of embryonic cells and expression the EGFP could be of great help in studying the differentiating and reprogramming mechanisms of mESCs. https://www.AJMB.org/En/Article.aspx?ID=103 Reza Ghorbani, Abdolrahman Emamzadeh, Yahya Khazaie, Kianoush Dormiani, Kamran Ghaedi, Mohammad Rabbani, Mahboubeh Foruzanfar, Khadijeh Karbalaie, Fereshteh Karamali, Liana Lachinani, Abbas Kiani-Esfahani, Marzieh Nematollahi, Mohammad-Hossein Nasr-Esfahani Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir RORC2 Gene Silencing in Human Th17 Cells by siRNA: Design and Evaluation of Highly Efficient siRNA Background: RNA interference-based gene silencing has recently been applied as an efficient tool for functional gene analysis. RORC2 is the key transcription factor orchestrating Th17 cells differentiation, the cells that are known as the pathogenic elements in various autoimmune diseases. The aim of this study was to design efficient siRNAs specific for RORC2 and to evaluate different criteria affecting their functionality. Methods: Three siRNA duplexes specific for RORC2 mRNA were designed. Th17 cells were produced from IL-6 and IL-1 treated cord blood CD4+ T cells. The T cells were transfected with three different designed siRNAs against RORC2 and the expression of RORC2 gene was measured using quantitative real time PCR. Results: Different levels of RORC2 down regulation were observed in the presence of each of the designed siRNAs. Efficient siRNA with 91.1% silencing activity met the majority of the established bioinformatics criteria while the one with 46.6% silencing activity had more deviations from these criteria. Conclusion: The more bioinformatics criteria are considered, the more functionality were observed for silencing RORC2. However, the importance of the type of criteria per se should not be neglected. Although all recommended criteria are important for designing siRNA but their value is not the same. https://www.AJMB.org/En/Article.aspx?ID=104 Mazdak Ganjalikhani-Hakemi, Kamran Ghaedi, Alireza Andalib, Vida Homayouni, Mohsen Hosseini, Abbas Rezaei Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression, Purification and Characterization of Three Overlapping Immunodominant Recombinant Fragments from Bordetella pertussis Filamentous Hemagglutinin Background: Filamentous hemagglutinin (FHA) is one of the most important immunoprotective antigens of Bordetella pertussis (B.pertussis) and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated. Methods: Three overlapping coding sequences of FHA antigen were amplified from B.pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli (E. coli) BL21(DE3) strain and purified through His-tag using Nickel-based chromatography. Purified fragments were characterized by SDS-PAGE and Western blotting techniques. In vitro peripheral blood mononuclear cells (PBMC) proliferation and IFN-γ production were assessed in a limited number of healthy adults vaccinated with a commercial acellular pertussis vaccine in response to all purified FHA fragments by H3-Thymidine incorporation and ELISA, respectively. Results: Recombinant FHA segments were successfully cloned and produced at high levels in E. coli BL21(DE3). SDS-PAGE and Western blot analyses confirmed their purity and reactivity. All three recombinant fragments together with a commercial native FHA were able to induce in vitro PBMC proliferation and IFN-γ production. Conclusion: Our preliminary results suggest that these overlapping recombinant FHA fragments are immunogenic and may prove to be immuno-protective. https://www.AJMB.org/En/Article.aspx?ID=105 Hossein Asgarian-Omran, Ali Akbar Amirzargar, Mohammad Arjmand, Mohammadreza Eshraghian, Behrooz Nikbin, Saeid Eshraghi, Marzieh Mahdavi, Jalal Khoshnoodi, Mahmood Jeddi-Tehrani, Hodjattallah Rabbani, Fazel Shokri Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Generation of a Uracil Auxotroph Strain of the Probiotic Yeast Saccharomyces boulardii as a Host for the Recombinant Protein Production Background: Saccharomyces boulardii (S. boulardii) is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. Methods: Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA (5-Fluoroorotic acid), uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3 , the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants Results: Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. Conclusion: A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmacuetics into the intestinal lumen. https://www.AJMB.org/En/Article.aspx?ID=106 Hassan Hamedi, Ali Misaghi, Mohammad Hossein Modarressi, Taghi Zahraei Salehi, Dorsa Khorasanizadeh, Vahid Khalaj Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning and Expression of Gumboro VP2 Antigen in Aspergillus niger Background: Infectious Bursal Disease Virus (IBDV) causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger (A. niger). Methods: Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG - protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein. Results: A number of pyrG+ positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken. Conclusion: In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry. https://www.AJMB.org/En/Article.aspx?ID=107 Mohammad Azizi, Bagher Yakhchali, Abdolreza Ghamarian, Somayeh Enayati, Mahvash Khodabandeh, Vahid Khalaj Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antifungal Indole and Pyrrolidine-2,4-Dione Derivative Peptidomimetic Lead Design Based on In Silico Study of Bioactive Peptide Families Background: The rise of opportunistic fungal infections highlights the need for development of new antimicrobial agents. Antimicrobial Peptides (AMPs) and Antifungal Peptides (AFPs) are among the agents with minimal resistance being developed against them, therefore they can be used as structural templates for design of new antimicrobial agents. Methods: In the present study four antifungal peptidomimetic structures named C1 to C4 were designed based on plant defensin of Pisum sativum. Minimum inhibitory concentrations (MICs) for these structures were deter-mined against Aspergillus niger N402, Candida albicans ATCC 10231, and Saccharomyces cerevisiae PTCC 5052. Results: C1 and C2 showed more potent antifungal activity against these fun-gal strains compared to C3 and C4. The structure C2 demonstrated a potent antifungal activity among them and could be used as a template for future study on antifungal peptidomemetics design. Sequences alignments led to identifying antifungal decapeptide (KTCENLADTY) named KTC-Y, which its MIC was determined on fungal protoplast showing 25 (μg/ml) against Asper-gillus fumigatus Af293. Conclusion: The present approach to reach the antifungal molecules seems to be a powerful approach in design of bioactive agents based on AMP mimetic identification. https://www.AJMB.org/En/Article.aspx?ID=108 Shoeib Moradi, Parisa Azerang, Vahid Khalaj, Soroush Sardari Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Isolation and Culture of Human Spermatogonial Stem cells Derived from Testis Biopsy Background: In cancer patients, chemo and radiotherapy can cause infertility by damaging spermatogenesis process. This process is based on self-renewal and differentiation of a rare population of the testicular cells called Spermatogonial Stem Cells (SSCs). Scientists have tried to isolate, enrich and culture Human spermatogonial stem cells, hoping to resolve infertility problems in cancer recovered patients in the future. Methods: Spermatogonial stem cells were isolated and purified from human testicular biopsies sample consisting of at least 500,000 and at most 2,000,000 cells. Two enzymatic digestion steps were performed. Enriching methods, differential plating, and specific culture in serum-free medium with added growth factors: human GDNF, bFGF, EGF and LIF was performed on coated dishes. Results: Human spermatogonial stem cell clusters were observed after 7 to 10 days in specific culture, then after several passages and successful expanding duration of 52 days, the cells were evaluated by three layer immunocytochemistry test (LSAB) to stain GPR125 protein as a surface marker in human spermatogonial stem cells. Conclusion: In current study human spermatogonial stem cell were isolated and expanded with the least manipulations in comparison with the other usual isolation methods like florescent or magnetic activated cell sorting. In contrast to the other SSCs isolation and culture methods, this system is based on the testicular biopsies against large samples, thus suggested method in this study is closer to clinical usage in the future. https://www.AJMB.org/En/Article.aspx?ID=109 Leila Goharbakhsh, Arash Mohazzab, Sheida Salehkhou, Mahnaz Heidari, Amir-Hassan Zarnani, Kazem Parivar, Mohammad Mehdi Akhondi Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Technique for Facile and Precise Transfer of Mouse Embryos Background: Successful Embryo Transfer (ET) technique is a fateful step of all efforts to achieve live births from in vitro produced embryos in assisted reproductive techniques or in knockout, transgenic or cloned animal projects. Small reproductive tract of mice and limitation of current techniques may not well satisfy the requirements for mass production of genetically modified mice. Genetic abnormalities of embryos, receptivity and uterine contractions, expulsion of embryos, blood, mucus or bacterial contamination on the transfer pipette tip, technical problems and even animal strain may affect embryo transfer outcome. Methods: In this study, two techniques of embryo transfer in mice were compared. In conventional technique the oviduct wall was punctured with a 30-gauge needle and the loaded Pasteur pipette with embryos and medium was inserted into the hole. In new technique, embryos that were loaded in modified micropipette with minimal medium were transferred directly to the oviduct by manual piston micro-pump easily. Embryo viability was evaluated considering the percentage of live healthy newborns. Results: Results of the two techniques were compared by t-test within the NPAR1WAY procedure of SAS software (ver. 9.2). The average live birth rates in the novel methods was significantly higher (42.4%) than the conventional method (21.7%, p<0.05). Conclusion: In conclusion, using new embryo transfer technique improved birth rate by preventing embryos expulsion from the oviduct, saving time and easy transfer of embryos with minimum volume of medium. https://www.AJMB.org/En/Article.aspx?ID=110 Ali Sarvari, Mohammad Mehdi Naderi, Mohammad Reza Sadeghi, Mohammad Mehdi Akhondi Thu, 03 Jan 2013 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Characterization and Functional Assessment of Mouse PPARγ1 Promoter Background: Peroxisome Proliferator Activated Receptor gamma (PPARγ), a member of nuclear receptor superfamily, comprises two isoforms in mouse. These two isoforms are encoded by different mRNAs, which are arisen by alternative promoter usage. There are two promoter regions upstream of PPARγ gene. A 3 kb fragment, containing several transcription factor binding sites, acts as PPARγ1 promoter region. Thus, expression pattern of PPARγ1 isoform is due to the potential transcription factors that could influence its promoter activity. PPARγ, Retinoid X Receptor (RXR) and Vitamin D Receptor (VDR), as nuclear receptors could influence PPARγ gene expression pattern during several differentiation processes. During neural differentiation, PPARγ1 isoform expression reaches to maximal level at neural precursor cell formation. Methods: A vast computational analysis was carried out to reveal the PPARγ1 promoter region. The putative promoter region was then subcloned upstream of an EGFP reporter gene. Then the functionality of PPARγ1 promoter was assessed in different cell lines. Results: Results indicated that Rosiglitazone increased PPARγ1 promoter regulated EGFP expression of neural precursor cells during Embryoid Body (EB) formation. Furthermore vitamin D reduced PPARγ1 promoter regulated EGFP expression of neural precursor cells during EB formation through binding to its receptor. Conclusion: This study suggests that there are potential response elements for PPAR/RXR and VDR/RXR heterodimers in PPARγ1 isoform promoter. Also VDR/RXR heterodimers may decrease PPARγ expression through binding to its promoter. https://www.AJMB.org/En/Article.aspx?ID=95 Liana Lachinani, Kamran Ghaedi, Somayeh Tanhaei, Ahmad Salamian, Fereshteh Karamali, Abbas Kiani-Esfahani, Farzaneh Rabiee, Marjan Yaghmaei, Hossein Baharvand, Mohammad Hossein Nasr-Esfahani Fri, 28 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production and Characterization of Mouse Monoclonal Antibodies Recognizing Human Pan-IgG Specific Conformational or Linear Epitopes Background: Pan-IgG specific monoclonal antibodies (MAbs) are essential tools for assessment of humoral immunity, immune deficiency and gammopathy. In this study, four hybridoma clones producing MAbs with different specificities for human IgG isotypes were established. Methods: Splenocytes from Balb/c mice immunized with Fc fractions of human IgG were fused with SP2/0 myeloma cells. Hybridoma cells were selected in HAT selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed using a panel of purified myeloma IgG proteins by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. Results: Immunoblotting studies revealed recognition of either linear or conformational epitopes by these MAbs. The most abundant cross-reactivity (100%) was observed with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen sera. Two of the MAbs cross reacted with either dog or horse sera. The affinity constant of two MAbs was measured by ELISA and found to be 0.74×108 M-1 and 0.96×107 M-1. Conclusion: Our results indicate that these pan-IgG specific MAbs recognize restricted linear or conformational epitopes located on all human IgG subclasses with no cross-reactivity to IgG from most species studied. These MAbs are potentially useful tools for quantification of total or antigen-specific IgG levels. https://www.AJMB.org/En/Article.aspx?ID=96 Fatemeh Hajighasemi, Jalal Khoshnoodi, Fazel Shokri Fri, 28 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Periplasmic Expression of a Novel Human Bone Morphogenetic Protein-7 Mutant in Escherichia coli Background: Bone Morphogenetic Proteins (BMPs) belong to the transforming growth factor-β (TGF-β) superfamily, and play an important role in bone metabolism. Recombinant forms of BMP-2 and BMP-7 are the only BMPs used clinically. In this study the mature part of human bone morphogenetic protein-7 (BMP-7) was engineered through substitution of the BMP-7 N-terminal sequence by heparin-binding site of BMP-2. This targeted substitution was made to enhance the binding affinity of the novel protein to the extracellular matrix components such as heparin and heparan sulfate proteoglycans (HSPGs). Methods: The engineered protein was expressed in Escherichia coli (E.coli). The PelB signal sequence was used to translocate soluble pro¬teins into the periplasmic space of E.coli. The protein was purified from periplasmic extract using Ni-NTA chromatography and the SDS-PAGE and western blot analysis confirmed the successful expression of the novel protein. Results: The novel hBMP-7 mutant was produced as approximately 16 kDa monomer. It was found that the heparin binding of this protein was approximately 50% more than that of the wild-type at a protein concentration of 500 ng/ml. Conclusion: The findings showed that the periplasmic expression may be suitable to produce complex proteins like BMPs. https://www.AJMB.org/En/Article.aspx?ID=97 Leila Nematollahi, Vahid Khalaj, Seyedeh Maliheh Babazadeh, Azam Rahimpour, Hoda Jahandar, Fatemeh Davami, Fereidoun Mahboudi Fri, 28 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning and Expression of Leishmania infantum LPG3 Gene by the Lizard Leishmania Expression System Background: Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum). Methods: The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Results: Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. Conclusion: These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis. https://www.AJMB.org/En/Article.aspx?ID=98 Leila Pirdel, Ahmad Zavaran Hosseini, Bahram Kazemi, Manoochehr Rasouli, Mojgan Bandehpour, Sara Soudi Fri, 28 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Genotyping of Polymorphic Microsatellite Markers Linked to RB1 Locus in Iranian Population Background: Retinoblastoma is the most common intraocular tumor in childhood and mutation in the RB1 gene will trigger the tumorigenesis. So far, a wide range of the mutations along the length of RB1 gene have been reported. However, some could not be detected by common detection methods. In such condition, linkage analysis using microsatellite markers is suggested to trace unknown RB1 mutations in the affected family. The aim of the present study was to evaluate the heterozygosity rates and genotyping of three microsatellite markers near or inside of the RB1 gene. Methods: Totally, 120 unrelated healthy people from Fardis, Karaj, Iran were recruited and from each participant genomic DNA was extracted from 5 ml of peripheral blood. Three microsatellite markers D13S153, D13S156 and D13S128 located within or adjacent to the RB1 gene were selected for linkage analysis. The reliability of microsatellite markers and linkage analysis were investigated in 10 members of 2 families with familial retinoblastoma. Results: Our results showed that heterozygosity rates for the three markers D13S153, D13S156 and D13S128 were 74, 70 and 78%, respectively. On the other hand, 2 and 36 out of 120 people were homozygote and heterozygous for all loci, respectively. Conclusion: Given the heterozygosity rates, it may be concluded that all microsatellite markers D13S153, D13S156 and D13S128 are informative and have a high rate of heterozygosity and sensitivity. Therefore, tracing the unknown RB1 mutated alleles using linkage analysis in Iranian family with familial retinoblastoma could be recommended by means of these three microsatellite markers. https://www.AJMB.org/En/Article.aspx?ID=99 Saman Mohamad Zahery, Kioomars Saliminejad, Hamid Reza Khorram Khorshid, Ali Ahani Fri, 28 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Medical Biotechnology Trends and Achievements in Iran A healthcare system has been the most important priority for all governments worldwide. Biotechnology products have affected the promotion of health care over the last thirty years. During the last several decades, Iran has achieved significant success in extending healthcare to the rural areas and in reducing the rates of infant mortality and increasing population growth. Biomedical technology as a converging technology is considered a helpful tool to fulfill the Iranian healthcare missions. The number of biotechnology products has reached 148 in 2012. The total sales have increased to 98 billion USD without considering vaccines and plasma derived proteins in 2012. Iran is one of the leading countries in the Middle East and North Africa in the area of Medical biotechnology. The number of biotechnology medicines launched in Iran is 13 products until 2012. More than 15 products are in pipelines now. Manufacturers are expecting to receive the market release for more than 8 products by the end of 2012. Considering this information, Iran will lead the biotechnology products especially in area of biosimilars in Asia after India in next three years. The present review will discuss leading policy, decision makers’ role, human resource developing system and industry development in medical biotechnology. https://www.AJMB.org/En/Article.aspx?ID=100 Fereidoun Mahboudi, Haleh Hamedifar, Hamideh Aghajani Fri, 28 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Amplification of GC-rich Putative Mouse PeP Promoter using Betaine and DMSO in Ammonium Sulfate Polymerase Chain Reaction Buffer Background: Recently, we have shown that peroxisomal protein expression was induced upon retinoic acid treatment in mouse embryonic stem cells during the process of neurogenesis. Thus, characterization of the respective promoter could elucidate the molecular aspects of transcriptional regulation of this gene. Methods: Using the conventional software programs for promoter prediction, a putative promoter region was identified approximately 561 bp upstream of the peroxisomal protein coding sequence. In order to clone this region with a GC-content of 71.01%, a cocktail of ammonium sulfate buffer supplied with two additive components, betaine and dimethyl sulfoxide, and a high concentration of MgCl2 was used. Results: The modulated polymerase chain reaction composition significantly improved the amplification of GC-rich DNA target sequences. Improved amplification of this region was due to reduction in the formation of secondary structures by the GC-rich region. Conclusion: Therefore, this polymerase chain reaction composition could be generally used to facilitate the amplification of other GC-rich DNA sequences as verified by amplification of different GC rich regions. https://www.AJMB.org/En/Article.aspx?ID=101 Tahere Seifi, Kamran Ghaedi, Ahmad Salamian, Somayeh Tanhaei, Forouzan Safari, Zohreh Hojati, Manuchehr Tavassoli, Hossein Baharvand, Mohammad Hossein Nasr-Esfahani Fri, 28 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Host-Microbiota Interaction is MyD88-Independent in the Intestinal Tract under Physiologic Condition The role of microbiota in health and disease is the subject of rigorous investigation. Several studies have demonstrated that microbiota and the pattern-recognition receptors contribute to intestinal tumourigenesis; the exact mechanism of which is still obscure. MyD88 is the downstream effector of all Toll-like receptors (TLRs) except TLR3. However, the alternative MyD88-independent pathway is functional downstream of not only TLR3, but also TLR1/2, 2/6, 4, and 5. TLR4 stimulation with intraperitoneal lipopolysaccharide exerts distinct functional effect on the intestinal motility via MyD88-dependent and-independent pathways. https://www.AJMB.org/En/Article.aspx?ID=102 Shirin Moossavi, Nima Rezaei Fri, 28 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial Virtual Institute of Medical Biotechnology (VIMB) (http://vimb.ir) is a website designed and developed in Iran to dedicate itself to providing the latest world news related to medical biotechnology. This web site also has accumulated a wealth of information on: e-books, conferences, references, articles, dissertations, films and animations, journals and biotech companies in Iran. The website fills one of the major gaps in the area of medical biotechnology in the country and is now serving those interested in the subject, free of charge. Although over the past 20 years many institutes and organizations have established biotechnology laboratories and initiated hundreds of projects, unfortunately many were not aware of the activities or sometimes existence of each other in the country. With the help of Internet and dedicated staff at VIMB, all of us working in any organizations, societies, universities, and biotech companies in Iran can now visit this website and get many of the information needed instantly. Staff at the VIMB are truly doing a great job and I recommend all individuals working in the field of biotechnology/medical biotechnology to spend a little bit of time to discover this informative and helpful web site. Another exciting news in the field of Biotechnology in Iran has been the recent announcement of the first international biotechnology exposition “IranBiotech 2013” which is to be held in Tehran-Iran from 14-18 February 2013. The “IranBiotech 2013” has declared, for the first time, to bring all the major international and Iranian biotech companies, universities, financial organizations, governmental and private institutes in the field of biotechnology together. This is great news for all of us who are involved in research, education and business aspects of biotechnology in general and medical biotechnology in particular. For more detailed information, please see the website www.irbiotech.com. https://www.AJMB.org/En/Article.aspx?ID=168 Ali M. Ardekani Sat, 08 Sep 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Regulations and Ethical Considerations in Animal Experiments: International Laws and Islamic Perspectives Growing usage of animals in the research projects has drawn more attention to their welfare and ethics surrounding this practice. Dissemination of information about the existing ethical consideration and alternatives in animal experiments has two important functions; first, it increases the researcher's awareness of the possible methods of using animals in the experiment, and second, to ensure that potential users are aware of the established alternatives. For example, legislations enacted in many countries during the 1980s state that laboratory animal applications should be reduced, refined and replaced wherever possible according to principles of the 3Rs. Thus, scientists around the world tried to apply the 3Rs in their biomedical researches regarding welfare of the laboratory animals. However, the Qur’an, the holy book of Muslims, and also Hadiths contain the obligatory ways to keep and treat animals since their revelations. According to Islamic viewpoint, animals represent Allah's ability and wisdom, and humans must pay attention to their health and living conditions. Several Islamic manuscripts state that animals have their own position in the creation hierarchy and humans are responsible for supplying minimal facilities and their welfare. This paper has tried to review ethical consideration in animal experiments and regarding Islamic resources in this case to encourage providing comprehensive ethical regulations in animal experiments which its establishment could be beneficial for animal ethics committees or research institutes. https://www.AJMB.org/En/Article.aspx?ID=89 Mohammad Mehdi Naderi, Ali Sarvari, Alireza Milanifar, Sara Borjian Boroujeni, Mohammad Mehdi Akhondi Sun, 22 Jul 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Thioredoxin System: A Model for Determining Novel Lead Molecules for Breast Cancer Chemotherapy Background: Thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP) also known as thioredoxin binding protein 2 or vitamin D3-upregulated protein 1 are key players in oxidative stress control. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes. Computational analyses of TXNRD1, TXNIP and TRX expressions have not been analyzed in relation to prognosis of breast cancer. High expression of TXNRD1 and low expression of TXNIP are associated with worst prognosis in breast cancer. Methods: Using bioinformatics applications we studied sequence analysis, molecular modeling, template and fold recognition, docking and scoring of thioredoxin as a target. Results: The resultant model obtained was validated based on the templates from I-TASSER server and binding site residues were predicted. The predicted model was used for Threading and Fold recognition and was optimized using GROMACS. The generated model was validated using programs such as Procheck, Ramachandran plot, verify-3d and Errat value from Saves server, and the results show that the model is reliable. Next we obtained small molecules from pubchem and chembank which are databases for selecting suitable ligands for our modeled target. These molecules were screened for docking, using GOLD and scoring was obtained using Chemscore as a scoring function. Conclusion: This study predicted the ligand interaction of four molecules with the minimized protein modeled structure and the best ligand with top scores from about 500 molecules screened. These were 3-hydroxy-2,3-diphenylbutanoic acid, 4-amino-3-pentadecylphenol, 3-(hydroxyimino)-2,4-diphenylbutanenitrile and 2-ethyl-1,2-diphenylbutyl carbamate, which are proposed as possible hit molecules for the drug discovery and development process. https://www.AJMB.org/En/Article.aspx?ID=90 Kaiser Jamil, Sabeena Muhammed Mustafa Sun, 22 Jul 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir 3’-RACE Amplification of Aminopeptidase N gene From Anopheles stephensi Applicable in Transmission Blocking Vaccines Background: Because of the lack of an effective and economical control strategy against malaria (the most devastating infectious disease in developing countries) Transmission-Blocking Vaccines (TBVs) concept has been raised in recent years, promising a more efficient way to malaria control. TBVs aim at interfering and/or blocking pathogen development within the vector, halting transmission to non-infected vertebrate host. Aminopeptidase N (APN) is one of the most potent proteins in parasite development in Anopheles malaria vectors, which is strongly co-localized with human malaria parasites in the mosquito midgut epithelium. Therefore, Aminopeptidase N is one of the best choices for a new TBV. Methods: In this study for the first time we used 3'-RACE to amplify APN gene in Anopheles stephensi (An.stephensi), a major malaria vector in Iran, Indian subcontinent up to China by using different sets of primers including exon junction, conserved and specific region primers. Results: Full length of APN was sequenced stepwise, which could be applied in designing a new regional TBV and act as an essential component of malaria elimination program in An. stephensi distribution areas. Conclusion: Primers design and method modification should be set up exactly in approach based amplifications. From results we came to this conclusion that that 3'-RACE could be applied to amplified key regions which are be-yond reach. https://www.AJMB.org/En/Article.aspx?ID=91 Hanieh Bokharaei, Abbasali Raz, Sedigheh Zakeri, Navid Dinparast Djadid Sun, 22 Jul 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of an Immunoaffinity Method for Purification of Streptokinase Background: Streptokinase is a potent activator of plasminogen to plasmin, the enzyme that can solubilize the fibrin network in blood clots. Streptokinase is currently used in clinical medicine as a thrombolytic agent. It is naturally secreted by β-hemolytic streptococci. Methods: To reach an efficient method of purification, an immunoaffinity chromatography method was developed that could purify the streptokinase in a single step with high yield. At the first stage, a CNBr-Ac-tivated sepharose 4B-Lysine column was made to purify the human blood plasminogen. The purified plasminogen was utilized to construct a column that could purify the streptokinase. The rabbit was immunized with the purified streptokinase and the anti-streptokinase (IgG) purified on another streptokinase substituted sepharose-4B column. The immunoaffinity column was developed by coupling the purified anti-Streptokinase (IgG) to sepharose 6MB–Protein A. The Escherichia coli (E.coli) BL21 (DE3) pLysS strain was transformed by the recombinant construct (cloned streptokinase gene in pGEX-4T-2 vector) and gene expression was induced by IPTG. The expressed protein was purified by immunoaffinity chromatography in a single step. Results: The immunoaffinity column could purify the recombinant fusion GST-SK to homogeneity. The purity of streptokinase was confirmed by SDS-PAGE as a single band of about 71 kD and its biological activity determined in a specific streptokinase assay. The yield of the purification was about 94%. Conclusion: This method of streptokinase purification is superior to the previous conventional methods. https://www.AJMB.org/En/Article.aspx?ID=92 Zohreh Karimi, Mohammad Babashamsi, Ezat Asgarani, Ali Salimi Sun, 22 Jul 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Reduction of Sodium Arsenite-Mediated Adverse Effects in Mice using Dietary Supplementation of Water Hyacinth (Eichornia crassipes) Root Powder Background: In this study, we evaluated the protective effects of water Hyacinth Root Powder (HRP) on arsenic-mediated toxic effects in mice. Methods: Swiss albino mice, used in this study, were divided into four different groups (for each group n=5). The control group was supplied with normal feed and water, Arsenic group (As-group) was supplied with normal feed plus arsenic (sodium arsenite)-containing water, and arsenic+hyacinth group (As+Hy group) was supplied with feed supplemented with HRP plus arsenic water. The remaining Hy-group was supplied with feed supplemented with HRP plus normal water. Results: Oral administration of arsenic reduced the normal growth of the mice as evidenced by weight loss. Interestingly, tip of the tails of these mice developed wound that caused gradual reduction of the tail length. Supplementation of HRP in feed significantly prevented mice growth retardation and tail wounding in As+Hy group mice. However, the growth pattern in Hygroup mice was observed to be almost similar to that of the control group indicating that HRP itself has no toxic or negative effect in mice. Ingested arsenic also distorted the shape of the blood cells and elevated the serum enzymes such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and serum glutamic pyruvic transaminase (SGPT). Importantly, elevation of these enzymes and distortion of blood cell shape were partially reduced in mice belong to As+Hy group, indicating HRP-mediated reduction of arsenic toxicity. Conclusion: Therefore, the preventive effect of hyacinth root on arsenic-poisoned mice suggested the future application of hyacinth to reduce arsenic toxicity in animal and human. https://www.AJMB.org/En/Article.aspx?ID=93 Rim Sabrina Jahan Sarker, Nazmul Ahsan, Khaled Hossain, Paritosh Kumar Ghosh, Chowdhury Rafiqul Ahsan, Anwarul Azim Akhand Sun, 22 Jul 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Anticonvulsant and Neuroprotective Effects of Walnuts on the Neurons of Rat Brain Cortex Background: Epilepsy is a chief communal health problem. Antiepileptic drugs only provide symptomatic treatment. Walnut Kernels (WK) have high concentrations of phenolic compounds, which have beneficial effects on human health because of their antioxidant and anti-atherogenic properties. The present study was designed to evaluate the efficacy of WK supplementation for the prevention of experimental epilepsy in male rats. Methods: Wistar adult male rats were divided into three groups: a control group (PTZ injection, fed with ordinary food), experimental group (PTZ injection, fed with WK) and a sham group (no PTZ injection, only for histological studies). Pentylenetetrazole (PTZ) was administered after the prescribed time. Results: WKs displayed anti-epileptogenic properties, and WK supplementation was associated with increased seizure threshold and reduced mortality in the experimental group versus controls. Conclusion: Use of WK may be helpful in prevention of PTZ-induced seizure and its further neurodegeneration in male rats. https://www.AJMB.org/En/Article.aspx?ID=94 Majid Asadi-Shekaari, Taj Pari Kalantaripour, Fatemeh Arab Nejad, Elaheh Namazian, Azam Eslami Sun, 22 Jul 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial Genetically modified organisms (GMOs) are now used in the production of pharmaceutical drugs, gene therapy, and golden rice (containing provitamin A). Although options of buying GMOs in the developing countries are limited now, their availability is expected to increase in these countries in the near future and along with it debates will definitely pursue. The creation and use of GMOs have increased continuously in the past few decades. Since creation of the first recombinant bacteria (E.coli) was reported in 1973, expressing an exogenic Salmonella gene (1), Genentech (now a member of the Roche Group) was the first company to announce the creation of an E.coli strain producing the human protein insulin in 1978 (2). More recently (2009), transgenic animals (forms of GMOs) were approved by the U.S. Food and Drug Administration for the production of the first human biological drug (an anticoagulant) to reduce the probability of blood clots during surgery or childbirth (3). Genetically modified bacteria are now used to produce clotting factors to treat haemophilia (4) and human growth hormone to treat different forms of dwarfism (5). In 2012, genetically modified male mosquitoes containing a lethal gene have been developed and released to the environment to fight Dengue fever which is responsible for the death of thousands of people worldwide (6). Considering the anxiety of public over the safety of these organisms, international organizations such as WHO and FAO have been issuing guidances on assessing the safety of genetically engineered organisms during the past two decades (7). Other societies including International Society for Biosafety Research –ISBR– have also been established to promote scientifically sound biosafety research worldwide. ISBR is organizing an International Symposium on the Biosafety of Genetically Modified Organisms (ISBGMO) which is to be held in St Louis, Missouri, USA, 16-20 September 2012. This symposium is expected to be attended by scientists, regulators and developers from all over the world. Main topics in this symposium will include: Genetically modified animals, biosafety policy and practice, RNAi applications and considerations, genetically modified biofuels, current regulatory challenges, defining environmental harm, concepts and applications for environmental risk assessment and regulatory decision-making, biotechnology and crop improvement in developing countries, and new applications of modern biotechnology in agriculture and future implications. It is very appropriate for the leaders of biotech industry and government regulatory agencies in Iran to attend such conferences for learning, exchanging ideas and participation in the discussions on the safety of GMOs. It looks as certain that the products from GMOs will enter the Iranian animal and food market(s) in the coming years. Therefore, the appropriate regulatory agencies in the Ministries of Health and Agriculture should prepare themselves to scientifically explain to the public the importance of GMOs and why they are safe to use either as food or pharmaceutical drugs. https://www.AJMB.org/En/Article.aspx?ID=167 Ali M. Ardekani Sat, 16 Jun 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Generation of In-vitro Spermatogonial Stem Cells following Genetic Manipulation of Primordial Germ-like Cells Research about potential use of stem cells for the development of germ line cells in vitro had been challenged. In the present study, we reported a novel protocol consisting of cocktail growth factor addition for germ cell differentiation followed by transfection. The cells were purificated based on the expression on the cell surface of a protein. This protein is not present in normal cells of mice and does not interfere with cellular function. This cell surface marker is efficiently recognized by monoclonal antibodies. Bone marrow mesenchymal stem cells derived primordial germ like cells were differentiated to spermatogonial stem like cells by inducer cocktail including Retinoic acid (RA)+Leukemia inhibitory factor (LIF)+Basic fibroblast growth factor (bFgF). Co-culture system was used as a feeder under differentiated cells. A 400 bp fragment of spermatogonia-specific Stra-8 locus was enough to direct gene expression to the germ line stem cells. Stra8-CD4HAglo construct was used for purification of premeiotic differentiated cells. Expression of pluripotency (Pou5F1, Nanog, c-Myc) and specific germ cell )Mvh, Piwil2, Stra-8) genes in each stage were analyzed. The purified cells expressed the known molecular markers of PGC-like cells such as Mvh, Piwil2 & Stra-8. The outcomes of qPCR showed that ratio pluripotency of genes expression in selective group significantly decreased (p≤0.05) in the initial differentiation process. This results showed that ratio of Pou5F1, Nanog, c-Myc, Mvh, Piwil2 & Stra-8 expression to purified PGC-like cells were 0.41, 0.204, 1.1, 0.003, 0.184 and 2.276, respectively. Treatment of cells with RA affected up regulation of Stra-8. Although, c-Myc gene as an oncogenic gene had significantly increased (p≤0.05) at the end of differentiation stage compared to initial phase of study, this level of expression could not be tumorgenic. qPCR results of the differentiation stage showed higher expression of Stra-8 in co-culture+cocktail and co-culture groups, Also, there was a significant difference (p≤0.05) in the expression of Pou5F1 & Nanog. Our results suggest that selection and purification of PGC-like cells based on Stra-8 as a pre-meiotic marker is a useful tool for getting in vitro spermatogonial stem cell. This method facilitates identification of safely differentiated germ cells in vitro. https://www.AJMB.org/En/Article.aspx?ID=82 Zohreh Mazaheri, Mansoureh Movahedin, Fatemeh Rahbarizadeh, Saied Amanpour Wed, 16 May 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Design and Fabrication of Anatomical Bioreactor Systems Containing Alginate Scaffolds for Cartilage Tissue Engineering The aim of the present study was to develop a tissue-engineering approach through alginate gel molding to mimic cartilage tissue in a three-dimensional culture system. The perfusion biomimetic bioreactor was designed to mimic natural joint. The shear stresses exerting on the bioreactor chamber were calculated by Computational Fluid Dynamic (CFD). Several alginate/bovine chondrocyte constructs were prepared, and were cultured in the bioreactor. Histochemical and immunohistochemical staining methods for the presence of glycosaminoglycan(GAG), overall matrix production and type II collagen protein were performed, respectively. The dynamic mechanical device applied a linear mechanical displacement of 2 mm to 10 mm. The CFD modeling indicated peak velocity and maximum wall shear stress were 1.706×10-3 m/s and 0.02407 dyne/cm2, respectively. Histochemical and immunohistochemical analysis revealed evidence of cartilage-like tissue with lacunas similar to those of natural cartilage and the production of sulfated GAG of matrix by the chondrons, metachromatic territorial matrix-surrounded cells and accumulation of type II collagen around the cells. The present study indicated that when chondrocytes were seeded in alginate hydrogel and cultured in biomimetic cell culture system, cells survived well and secreted newly synthesized matrix led to improvement of chondrogenesis. https://www.AJMB.org/En/Article.aspx?ID=83 Anneh Mohammad Gharravi, Mahmoud Orazizadeh, Karim Ansari-Asl, Salem Banoni, Sina Izadi, Mahmoud Hashemitabar Wed, 16 May 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Combined Treatment of Androgen-Independent Prostate Cancer Cell Line DU145 with Chemotherapeutic Agents and Lithium Chloride: Effect on Growth Arrest and/or Apoptosis Hormone-independent prostate cancer cell lines are resistant to antineoplastic drugs, this study sought to determine the usefulness of lithium chloride as an inhibitor of glycogen synthase kinase-3β to increase the cytotoxic effect of doxorubicin, etoposide or vinblastine antineoplastic drugs on DU145 cells. Combination effect was assessed by using low and IC50 doses of drugs + lithium chloride. Subsequently, cell cycle analysis and p53 levels and its subcellular localization as a key regulator of cell cycle were assessed. Lithium chloride showed cytotoxic effect in a dose and time dependent manner (p<0.001). Both drugs doxorubicin and etoposide in combination with lithium chloride showed higher percent of cells in SubG1 compared to control (p<0.001). Combination of IC50 dose of doxorubicin and lithium chloride led to S phase arrest (p<0.001, compared to control, lithium chloride or doxorubicin alone). Moreover, G2/M arrest was significantly increased when low dose of doxorubicin and vinblastine were combined with lithium chloride (p<0.001, compared to control and lithium chloride alone). DU145 cells were highly sensitive to vinblastine and no significant changes were observed when combined with lithium chloride. The IC50 doses of all three drugs combined with lithium chloride demonstrated decreased cell percent in G1 phase compared to control or lithium chloride alone (p<0.001). Moreover, in the presence of lithium chloride there were increased levels of p53 in cytoplasm and nucleus (p<0.05). Our results suggest that combination of lithium chloride with chemotherapeutic agents may increases their cytotoxic effect on hormone non-responsive human prostate cancer cells. https://www.AJMB.org/En/Article.aspx?ID=84 Ghamartaj Hossein, Vajihe Azimian Zavareh, Parissa Sahranavard Fard Wed, 16 May 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production of Pentameric Cholera Toxin B Subunit in Escherichia coli Cholera toxin B subunit (CTB) has been extensively studied as an immunogen, adjuvant, and inducer of oral tolerance in many investigations. Production of CTB has been carried out in the bacterial, plant, insect and yeast expression systems. In this study the expression of the CTB containing a 6XHis-tagged was performed by Escherichia coli (E.coli) M15. The yield of purified pentameric recombinant CTB was about 1 mg/l. Western blot analysis demonstrated that the recombinant CTB was antigenically active. In addition, GM1-ganglioside ELISA showed that recombinant CTB binds to GM1-gangelioside receptor, confirming disulfide bond formation and proper folding of the recombinant protein in E.coli. Overall, in regard to the vast applications of CTB in medicine, this bacterial expression system will be a fast, cost-effective and simple system for production of pentameric CTB and CTB conjugated proteins. https://www.AJMB.org/En/Article.aspx?ID=85 Farida Dakterzada, Ashraf Mohabati Mobarez, Mehryar Habibi Roudkenar, Mehdi Forouzandeh Wed, 16 May 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Variation of ATM Gene Expression in Peripheral Blood Cells of Sporadic Breast Carcinomas in Iranian Patients The ataxia telangiectasia mutated gene (ATM), candidate for breast cancer susceptibility gene, encode a 350-kDa protein belongs to the core components of DNA-damage response machinery. Female AT carriers have at least 5-fold increase risk for breast cancer. Reduction in ATM expression is shown in multiple studies in breast tissues. We aimed to perform a research to measure the ATM mRNA expression in peripheral blood cells in breast cancer patients. Peripheral blood sample from 40 newly diagnosed, histologically confirmed female breast cancer patients was collected before surgery. Total RNA was isolated from blood cells using the RNX-Plus solution and reverse transcribed into cDNA. Real-time PCR was performed using the 2-∆∆CT method to calculate relative changes in gene expression by REST software. The Relative Quantitation (RQ) mean was 1.27 with the min. and max. equal to 0.20 and 3.34, respectively. Calculation of patient frequencies in different groups revealed that 17.5% had reduced expression lower than two fold decreases and 15% high expression more than two fold increases, but according to REST software there was no up-regulation or down-regulation compared to normal females. The findings of multiple studies consistent with this study indicate that the ATM gene may play an important role in breast cancer development and progression, and ATM expression is down-regulated in breast cancer tissues. Although, some of the results do not support a suppressor role for ATM in the development of sporadic breast cancer, 17.5% of our patients had under expression of ATM mRNA less than two fold relative to control. https://www.AJMB.org/En/Article.aspx?ID=86 Mohsen Foroughizadeh, Hossein Mozdarani, Keyvan Majidzadeh-A, Ahmad Kaviani Wed, 16 May 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of HLA-DRB1, DQA1 and DQB1 Alleles and Haplotypes with Common Variable Immunodeficiency in Iranian Patients Common Variable Immunodeficiency (CVID) is an antibody deficiency syndrome that often co-occurs in families with selective IgA deficiency (IgAD). This study was designed to investigate the frequency of DR and DQ loci of HLA class II region in common variable immunodeficiency (CVID) patients. Fifteen Iranian patients with CVID or IgAD (mean age 14.6±5.4, range 4-25 years; 9 male and 6 female) and 63 healthy controls were studied. Establishment of B-lymphoblastoid cell lines was performed using Epstein-Barr-virus (EBV) immortalization technique and HLA alleles were typed using polymerase chain reaction based on sequence specific primers (PCR-SSP). DRB1 alleles including DRB1 *04 (p=0.03) and DRB1 *11 (p=0.01) significantly showed higher frequency in the studied subjects. In contrast, DRB1 *301 (p=0.04) and DRB1 *07 (p=0.02) alleles were negatively associated with CVID. For DQB1 and DQA1 loci, DQB1 *0302 (p=0.047) and DQA1 *03011 (p=0.001) demonstrated high frequency in cases, while DQB1 *0201 (p=0.02) and DQA1 *0201 (p=0.01) were detected to be low when compared to controls. Haplotype analysis indicated that frequency of DRB1*04-DQB1*03011-DQA1 *03011 (p=0.02), DRB1 *11-DQB1 *03011-DQA1 *0505 (p=0.047), DRB1 *11-DQA1 *0505 (p=0.04) and DRB1*04-DQA1*03011 (p=0.02) haplotypes were significantly higher in patient group, while only the frequency of the DRB1 *07-DQA1 *0201 haplotype gene was statistically lower in control group (p=0.02). According to the results, it could be deduced that the HLA-DR and DQ loci may contribute to the pathogenesis of CVID or they might be considered as suitable markers for the possibility of the occurrence of this genetic defect. https://www.AJMB.org/En/Article.aspx?ID=87 Amir Amanzadeh, Ali Akbar Amirzargar, Nilufar Mohseni, Zohreh Arjang, Asghar Aghamohammadi, Mohammad Ali Shokrgozar, Fazel Shokri Wed, 16 May 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Non-Invasive Detection of Esophageal Cancer using Genetic Changes in Circulating Cell-Free DNA Cell free DNA (cfDNA) is a genetic biomarker that is present in serum or plasma in high concentration in many types of cancer. Identification of circu-lating cancer related DNA molecules in serum or plasma is a non-invasive tool for early diagnosis and prognosis in many cancer patients. For this review, study selection and data extraction were performed by the authors. Detection of point mutations, microsatellite alterations, DNA hypermethylations and losses of heterozygosity in circulating cell free DNA have been characterized in esophagus cancer. Application of circulating cell free DNA as a biomarker, provide the best opportunity for constructing non-invasive tests for early de-tection, prognosis and management of cancer patients, after therapy in many types of cancer. https://www.AJMB.org/En/Article.aspx?ID=76 Saeid Ghorbian, Ali M. Ardekani Sat, 24 Mar 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Ligation Independent Cloning of Polycistronic, Genetically Modified, HuMAb4D5-8 F (ab') 2, in Bacterial Plasmid In recent years, recombinant monoclonal antibodies and their derivatives have emerged as important targeted therapy agents. Monoclonal antibodies are ex-tremely difficult to produce. So, the cost of production is very high and many people cannot afford these drugs. In this regard, choosing inexpensive and easy ways to manipulate production systems such as bacterial hosts to reduce the cost of manufacturing these critical components are considered as vital step for developmental issues in recombinant expression systems. We, therefore, at-tempted to generate a polycistronic construct of anti HER-2 F(ab')2 fragment antibody for insertion in an expression bacterial plasmid. Also some modifica-tions were made in the hinge region to express antibody F(ab')2 fragment in its authentic form preventing from multiple varieties of disulfide bond formation. Finally, synthesized construct was cloned in pET-32 Ek/LIC vector without using restriction enzyme digestion or ligation reactions. The results of this study show-ed that modified F(ab')2 fragment was simply and successfully inserted in Escherichia coli (E.coli) using the Ligation Independent Cloning technology. https://www.AJMB.org/En/Article.aspx?ID=77 Leila Farahmand, Keivan Majidzadeh, Zargham Sepehrizadeh, Mohammad Reza Mofid, Rezvan Esmaeili, Mojtaba Tabatabaei Yazdi Sat, 24 Mar 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir In silico Evaluation of Crosslinking Effects on Denaturant meq values and ΔCp upon Protein Unfolding Important thermodynamic parameters including denaturant equilibrium m values (meq) and heat capacity changes (ΔCp) can be predicted based on changes in Solvent Accessible Surface Area (SASA) upon unfolding. Crosslinks such as disulfide bonds influence the stability of the proteins by decreasing the entropy gain as well as reduction of SASA of unfolded state. The aim of the study was to develop mathematical models to predict the effect of crosslinks on ΔSASA and ultimately on meq and ΔCp based on in silico methods. Changes of SASA upon computationally simulated unfolding were calculated for a set of 45 proteins with known meq and ΔCp values and the effect of crosslinks on ΔSASA of unfolding was investigated. The results were used to predict the meq of denaturation for guanidine hydrochloride and urea, as well as ΔCp for the studied proteins with overall error of 20%, 31% and 17%, re-spectively. The results of the current study were in close agreement with those obtained from the previous studies. https://www.AJMB.org/En/Article.aspx?ID=78 Maryam Hamzeh-Mivehroud, Ali Akbar Alizade, Monire Ahmadifar, Siavoush Dastmalchi Sat, 24 Mar 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Toxicity Study of Silver Nanoparticles Synthesized from Suaeda monoica on Hep-2 Cell Line Recently there has been fabulous excitement in the nano-biotechnological area for the study of nanoparticles synthesis using some natural biological system, which has led the growth advanced nanomaterials. This intention made us to assess the biologically synthesized silver nanoparticles from the leaf of Suaeda monoica (S.monoica) using 1 mM silver nitrate. The leaf extract of S.monoica incubated with 1 mM silver nitrate solution and characterized by UV- spectrometer and AFM. The effect of synthesized silver nanoparticles on Human Epidermoid Larynx Carcinoma cell line was evaluated by the MTT colorimetric technique. As a result we observed gradual change in the colour of extract from greenish to brown. The synthesized silver nanoparticles con-firmed by UV at 430 nm and spherical shape identified in the range of 31 nm under AFM. The effect of silver nanoparticles on Human Epidermoid Larynx Carcinoma cell line exhibits a dose-dependent toxicity for the cell tested and the viability of Hep-2 cells decreased to 50% (IC50) at the concentration of 500 nM. Further findings will be determined the exact mechanisms of this cost effective Nano-treatments. https://www.AJMB.org/En/Article.aspx?ID=79 Kaliyamurthi Satyavani, Selvaraj Gurudeeban, Thiruganasambandam Ramanathan, Thangavel Balasubramanian Sat, 24 Mar 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Construction and Stable Expression of a Truncated Human Receptor Tyrosine Kinase Ror1 (Ror1-ECD) Expression of receptor tyrosine kinase Ror1 in a wide variety of cancers has emerged as a new era focusing on targeting this receptor in cancer therapy. Our preliminary re-sults indicate the presence of a truncated transcript of Ror1 in tumor cells. The trun-cated Ror1 encompasses extracellular and transmembrane domains, lacking catalytic kinase domain (Ror1-ECD). As enzyme activity is highly dependent on the catalytic domain, we were wondering how this transcript and its encoded protein could play a possible role in tumorigenesis. To understand the function of this truncated transcript and whether or not the encoded protein translocates to the cell surface, we construct-ed a mammalian expression vector containing exon 1 to exon 8 of human Ror1 gene as a model system. The encoded protein by this construct covers the entire extracellular and transmembrane domains of Ror1. The Chinese Hamster Ovary Cell line (CHO) was used for transfection. Our results showed that this construct could express Ror1-ECD at protein level and also the protein could effectively translocate to the surface of transfected cells. Such model may suggest that a proportion of Ror1 molecules ex-pressed by tumor cells are not full-length Ror1. This notion may be considered when applying flow cytometry using antibodies against Ror1 for screening of tumor cells in order to avoid any miscalculation in the number of Ror1 molecules expressed by tumor cells. Furthermore, such expression may bring about assumptions on functional roles of Ror1-ECD in tumorigenesis, which requires extensive functional studies. https://www.AJMB.org/En/Article.aspx?ID=80 Flora Forouzesh, Samira Shakeri Tabarian, Shaghayegh Emami, Mahmood Jeddi-Tehrani, Reza Hadavi, Hodjattallah Rabbani Sat, 24 Mar 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Optimization of the Expression of Genes Encoding Poly (3-hydroxyalkanoate) Synthase from Pseudomonas aeruginosa PTCC 1310 in Escherichia coli Over the years, the use of plastics has complicated the problem of disposal of solid wastes. One strategy to reduce plastic waste is the use of biodegradable plastics. A group of these plastics are polyhydroxyalkanoates (PHAs). To date more than 250 different microorganisms are known to synthesize and accumulate PHA. Most Pseu-domonas strains are able to accumulate mcl-PHA. In previous studies, the phaC1 and phaC2 genes were identified in Pseudomonas aeruginosa (P.aeruginosa) PTCC 1310 and were cloned. The aim of this study was to express these genes and optimize the conditions for their expression. The inserts obtained from vectors pTZPHAC1 and pTZPHAC2 were subcloned into pET15b expression vector. After transformation of competent Escherichia coli (E.coli) BL21 (DE3) cells with recombinant plasmids, expres-sion was induced using IPTG. By changing expression conditions such as IPTG concen-tration, time and temperature of incubation with IPTG, the expression conditions for these enzymes were optimized, and the obtained results were compared using proper statistical analysis. The PHA synthase genes were induced with IPTG and the expres-sed 62 kDa protein was observed and purified. By changing expression conditions, 1 mM IPTG, 37 °C and a 2 hr incubation provided the highest level of protein pro-duction in E.coli cells. These results suggest that induction condition of PhaC genes can influence expression of PHA synthase enzymes. https://www.AJMB.org/En/Article.aspx?ID=81 Daryoush Abedi, Maryam Beheshti, Ali Jahanian Najafabadi, Hamid Mir Mohammad Sadeghi, Vajihe Akbari Sat, 24 Mar 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial In recent years, due to increase in infertility rates, Assisted Reproductive technologies (ART) have been very much in use worldwide. The first phase of the study of national infertility amongst the Iranian women, be-tween the ages of 20-40 was completed recently by the Academic Center for Education, Culture and Re-search (ACECR). This study, which was widely reported in the Iranian media, indicates that the infertility rate in this group of women is around 20.2%. The 20.2% infertility rate appears to be above the average reported by the World Health Organization (WHO), which is around 15%. This study was conducted by scientists from Tehran University and Avicenna Research Institute which is also affiliate of the ACECR. This is the first scientific study of the infertility rate in the Iranian women population, and raise awareness of a medical condition that must be addressed by the medical community and health officials in Iran. The use of ART is anticipated to grow rapidly in Iran in the coming years, and this growth will bring about some very important bioethical issues to consider. These include: creation, selection and disposal of unwanted embryos, access, insurance coverage and resource allocation, the rights and responsibilities of individuals who agree to collaborate with each other in donating or receiving reproductive materials. As the use of ART increases in Iran, appropriate ethical laws must be written and passed in the parliament to regulate this rapidly growing industry in order to safeguard the rights of individuals involved in receiving and donating reproductive materials. Also, it would be appropriate for the Iranian Ministry of Health to ap-point a special task force to make sure that all the fertility and infertility centers in Iran abide by the bio-ethical guidelines that are specifically designed and written to safeguard the rights of patients and individuals involved in the use of ART. https://www.AJMB.org/En/Article.aspx?ID=166 Ali M. Ardekani Sat, 10 Mar 2012 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Stress-Induced Proteomic Changes in the Hippocampus of Pregnant Wistar Rats Stress is a threatening factor that all living organisms encounter throughout life. Depending on the type of stress, there are several mechanisms for keeping body homeostasis to minimize stress effects. Brain is an organ which shows high sensitivity to stress conditions. Although many studies have shown induced-stress effects on rat embryos, little is known about the mechanisms involved in coping with stress by female rats during pregnancy. In the present study, restraint stress method was applied because this technique has been widely used in animal models to induce both psychological and physical stress. Restraint stress was applied in regular sessions (1 and 3 hrs) in two groups of 6 pregnant Wistar rats and similar number of animals was used as control group receiving no stress. ACTH and corticosterone levels in plasma samples were shown to increase in response to stress treatments. On the last day of pregnancy, rat hippocampus from the brain of each animal in all three groups was removed and analyzed using 2 Dimensional Gel Electrophoresis (2DE) technique. Using Image Master Software, approximately 2000 proteins were detected in the 2D gels analyzed, among which 34 proteins exhibited differential expression. These results indicate that the proteome patterns from the hippocampus of pregnant rats subjected to 1 and 3 hr of stress differs significantly from the control (unstressed) group. Future mass spectrometry identification of the 34 protein spots discovered in this study should allow a more precise understanding of molecules and cellular pathways involved in stress-induced responses during pregnancy. https://www.AJMB.org/En/Article.aspx?ID=70 Ali M. Ardekani, Nader Maghsudi, Anna Meyfour, Rasool Ghasemi, Niknam Lakpour, Elahe Nooshinfar, Zahra Ghaempanah Sat, 24 Dec 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparison of Epothilone and Taxol Binding in Yeast Tubulin using Molecular Modeling Microtubules are unique cytoskeletal structures that have structural subunits of αβ tubulin. Taxol is a typical microtubule stabilizing drug. The epothilones are other natural products with similar mechanism of action totaxol. Despite the highly conserved nature of β-tubulin, some organism like Saccharomyces cerevesia is resistance to taxol, but sensitive to epothilones. In order to find differences in sensitivity of yeast tubulin to these molecules, we investigated binding mode of the taxol and epothilone A to yeast tubulin using molecular modeling. The multiple sequence alignment of β-tubulin of different species was performed using ClustalW2. Protein structure of yeast β-tubulin was constructed with Swiss Model 8.05 by using 1TVK. Modeled tubulin was superimposed with PyMol on1JFF for comparison of three-dimensional structure of two proteins. Our results showed that one of the most interesting differences in binding mode of these molecules is residue 227. The His227 in bovine makes a hydrogen bond by means of its δ-nitrogen with epothilone A and by means of its ε-nitrogen with taxol. The Asn227 of yeast can play role of the δ-nitrogen of imidazole ring of H227, but not of ε-nitrogen of it. So yeast tubulin in contrast to taxol can interact with epothilone A. Due to conservation of essential residues for binding (T274, R282 and Q292), epothilone A in comparison with taxol can tolerate the interchange in the binding pocket (R276I). Our findings may be of a great aid in the rational design of anti-tumor agents that bind to the taxol binding region of tubulin. https://www.AJMB.org/En/Article.aspx?ID=73 Vajihe Akbari, Sharareh Moghim, Mohammad Reza Mofid Sat, 24 Dec 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Bryostatin-1, Fenretinide and 1α,25 (OH)2D3 Induce Growth Inhibition, Apoptosis and Differentiation in T and B Cell-Derived Acute Lymphoblastic Leukemia Cell Lines (CCRF-CEM and Nalm-6) In many acute leukemias, normal differentiation does not occur. However, in many cell lines derived from hematologic malignancies, differentiation or apoptosis can be induced by variety of agents. Despite advances in the treatment of Acute Lymphoblastic Leukemia (ALL), in most patients long-term survival rates remain unsatisfactory, especially in T-cell derived ALL. Thus we studied the anti-cancer effects of fenretinide, 1α,25(OH)2D3, and bryostatin-1 in CCRF-CEM (T-cell derived) and Nalm-6 (B-cell derived) ALL cell lines. Using MTT assays, both cell lines were shown to exhibit increased inhibition of proliferation at micro (fenretinide) and nanomolar (1α,25(OH)2D3, bryostatin-1) concentrations. These anti-cancer agents were shown to induce apoptosis and activate caspase-3 pathway in both ALL cell lines. Furthermore, for the first time we are reporting consistent anti-proliferative and apoptotic effects of Bryostatin-1 in ALL T-cell derived cell line with the lowest ED50 (ranging 4.6 nM - 7.4 nM). To evaluate the differentiation induction by fenretinide, 1α,25(OH)2D3, and bryostatin-1 in ALL cell lines, we assayed for the expressions of CD19, CD38 markers on Nalm-6 and CD7 marker on CCRF-CEM cell line. The flow cytometric analysis showed a significant increase in expression of CD markers in response to anticancer drug treatments. To assay the effects of anti-cancer drugs on cell cycle distribution, cell cycle analysis using flow cytometry was employed. These anti-cancer drugs appear to affect the CCRF-CEM and Nalm-6 cell cycles differently (G0/G1 and G2/M, respectively). Overall results demonstrate that the anticancer agents used in this study are strong inhibitors of ALL cell proliferation and inducers of apoptosis and differentiation in vitro. These findings may be quite helpful if these drugs are to be used for differentiation therapy of ALL patients in clinics in the future. Further studies are warranted to establish the in vivo effect of these drugs particularly in patients with T-cell derived ALL. https://www.AJMB.org/En/Article.aspx?ID=71 Ali M. Ardekani, Shahrzad Soleymani Fard, Mahmood Jeddi-Tehrani, Ramin Ghahremanzade Sat, 24 Dec 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An Enhancing Effect of Gold Nanoparticles on the Lethal Action of 2450 MHz Electromagnetic Radiation in Microwave Oven Today, there is an increasing interest in the use of metal nanoparticles in health sciences. Amongst all nanoparticles, the gold nanoparticles have been known to kill the cancer cells under hyperthermic condition by near-infrared frequency electromagnetic waves. On the other hand, although there are different physiochemical methods for disinfection of microbial pollution, however applications of irradiated gold nanoparticles against microorganisms have not yet been investigated. In this study, gold nanoparticles were prepared using D-glucose and characterized (particle size <26 nm). In the next step, the enhancing effect of the non toxic level of gold nanoparticles (50 µg/mL) on the antimicrobial activity of 2450 MHz electromagnetic radiation generated at a microwave oven operated at low power (100 W), was investigated by time-kill course assay against Staphylococcus aureus (S. aureus) ATCC 29737. The results showed that application of gold nanoparticles can enhance the lethal effect of low power microwave in a very short exposure time (5 s). https://www.AJMB.org/En/Article.aspx?ID=75 Kamyar Mollazadeh-Moghaddam, Bardia Varasteh Moradi, Reza Dolatabadi-Bazaz, Mojtaba Shakibae, Ahmad Reza Shahverdi Sat, 24 Dec 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Fetal Sex Determination using Non-Invasive Method of Cell-free Fetal DNA in Maternal Plasma of Pregnant Women During 6th– 10th Weeks of Gestation In previous years, identification of fetal cells in maternal blood circulation has caused a new revolution in non-invasive method of prenatal diagnosis. Low number of fetal cells in maternal blood and long-term survival after pregnancy limited the use of fetal cells in diagnostic and clinical applications. With the discovery of cell-free fetal DNA (cffDNA) in plasma of pregnant women, access to genetic material of the fetus had become possible to determine early gender of a fetus in pregnancies at the risk of X-linked genetic conditions instead of applying invasive methods. Therefore in this study, the probability of detecting sequences on the Y chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women at 6th to 10th weeks of gestation and then the fetal DNA was extracted from the plasma. Nested PCR was applied to detect the sequences of single copy SRY gene and multi copy DYS14 & DAZ genes on the Y chromosome of the male fetuses. At the end, all the obtained results were compared with the actual gender of the newborns. In 40 out of 42 born baby boys, the relevant gene sequences were identified and 95.2% sensitivity was obtained. Conclusion: Non-invasive early determination of fetal gender using cffDNA could be employed as a pre-test in the shortest possible time and with a high reliability to avoid applying invasive methods in cases where a fetus is at the risk of genetic diseases. https://www.AJMB.org/En/Article.aspx?ID=72 Maryam Zargari, Mohammad Reza Sadeghi, Mohammad Hassan Shahhosseiny, Koorosh Kamali, Kyomars Saliminejad, Ali Esmaeilzadeh, Hamid Reza Khorram Khorshid Sat, 24 Dec 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Construction and Evaluation of an Expression Vector Containing Mtb32C (Rv0125) of Mycobacterium tuberculosis Expressions of recombinant proteins for different applications are important objectives in molecular biotechnology; however, expression of some recombinant proteins is difficult. Several methods have been designed for expression of these proteins. The aim of this study was to construct a vector containing Mtb32C fragment of Mycobacterium tuberculosis (M.tuberculosis) as a fusion partner in order to improve the expression of fused recombinant proteins. Mtb32C was amplified by polymerase chain reaction (PCR). The amplified fragment was ligated into pET21b+ vector. Colony-PCR, enzyme digestion and DNA sequencing methods were used to confirm the recombinant vector. Colony-PCR showed a 420 bp fragment in size corresponding to the correct size of our fragment. In addition the recombinant plasmids sequencing showed the accuracy of the cloned fragment. For confirming the expression, reverse transcriptase (RT)-PCR analysis was performed showing a 420 bp fragment in agarose gel electrophoresis using specific primers. The construction of a vector containing Mtb32C fragment is promising as a fusion partner for future studies as it affected the expression of the fused proteins and increased immune responses against the partner. https://www.AJMB.org/En/Article.aspx?ID=74 Maryam Sadat Nabavinia, Mahboobeh Naderi Nasab, Zahra Meshkat, Mohammad Derakhshan, Mehrangiz Khaje-Karamadini Sat, 24 Dec 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial Recently I was invited to attend the bi-annual Steering committee meeting of the EMGEN network, for-merly known as Eastern Mediterranean Health Genomics and Biotechnology Network (EMHGBN). This was held at the Pasteur Institute of Iran in Tehran on January 29-30, 2012. Attending this meeting as the chief editor of Avicenna Journal of Medical Biotechnology (AJMB) was very informative and I became familiar with the goals of this international organization. The EMGEN network was established in 2004 on the recom-mendation and support of WHO/EMRO and participation of representatives from selected centers of excel-lence in Molecular biology, Biotechnology, and genomics from 22 countries in the Eastern Mediterranean Re-gion including Iran. The names of these countries are listed on the website for the network (www.emgen.net). It also has useful information on the goals, membership, training programs, job opportunities, and newsletters of the organization. I recommend it to those interested in the role of biotechnology and genomics in health to visit the website and participate in the activities of the organization. As I understand, one of the major goals of EMGEN network is to increase awareness on the potentials and capacities of member countries in the fields of Biotechnology and genomics. More importantly, they aim to facilitate scientific collaborations among the scientists in the Eastern Mediterranean Region. After listening to the representatives from member countries attending the meeting, I was convinced that many more scientists from the fields of biotechnology and genomics in EMGEN network member countries ought to interact and collaborate on scientific projects before resources in member countries can be ef-fectively shared in joint projects. For my part, I encourage eminent biotech and genomic scientists from the member countries to participate in AJMB's scientific activities through submission of original articles, opinions, reviews in fields such as Medical Biotechnology, Genomics, Nanobiotechnology, Bioinformatics, and Molecular Biology. Furthermore, the chief scientists from the member countries can participate in the AJMB's scientific activities in the capacity of associate editors and reviewers in their fields of expertise. As the chief editor of AJMB, I would like to invite all the scientists in the EMGEN network countries working in the fields of Medical Biotechnology and genomics to actively participate in this network's activities. I would also like to inform those interested in obtaining research grants that funds have been made available by some member countries in the form of 50/50 contribution. https://www.AJMB.org/En/Article.aspx?ID=165 Ali M. Ardekani Sat, 10 Dec 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial The seventh National Biotechnology Congress in Iran was held in Tehran from 12-14 September 2011. Since many of our readers most likely did not find the opportunity to attend this conference, I take this opportunity to provide a summary of activities in this three day congress. In the seventh National Biotech-nology Congress in Iran, dozens of talks and several hundred posters were presented. The topics of biotech-nology-related subjects presented in this congress were wide spread and included: Transgenic animals, plant biotechnology, systems biology, next generation DNA sequencing, biotech market, biotech manage-ment, environment, pharmaceuticals, food industry, medical biotechnology, marine biotechnology, bio-informatics and ethics. This congress was attended by hundreds of students, researchers, government officials and industry leaders from all corners of Iran. This congress was sponsored and/or supported by more than 25 national research centers, private institutes and government organizations including the presidential office. Holding regular National biotechnology meetings in the field of biotechnology in Iran is hoped to deepen the discussions of biotech-related issues in both academic and non-academic centers and encourage inter-actions between the interested parties. An interesting and novel aspect of this congress was an attempt to initiate a dialogue between the biotech leaders and religious authorities. The reality is that application of biotechnologies in different disciplines including genetic manipulation of plant and animal cells, food, … has caused anxieties and social concerns in recent years. Therefore, any attempt to encourage dialogues between the biotech and religious leaders should be helpful in presenting biotechnology as a helpful technology to solve societal problems. Today biotechnology is one of the fastest growing technologies worldwide as was predicted a couple of decades ago. The market value of biotech-related technologies is estimated to be over $ 1000 billion worldwide and is dominated by the developed countries mainly due to the massive investments they did in biotech industry a couple of decades earlier. The question is whether the developing countries can develop the biotech industry segments of their economies soon enough to capture a portion of this huge market worldwide. Hopefully holding the seventh National biotechnology meeting in Iran would focus the attention of the scientific, economic and political leaders on this important issue. https://www.AJMB.org/En/Article.aspx?ID=164 Ali M. Ardekani Sat, 10 Sep 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Genetically Modified Foods and Social Concerns Biotechnology is providing us with a wide range of options for how we can use agricultural and commercial forestry lands. The cultivation of genetically modified (GM) crops on millions of hectares of lands and their injection into our food chain is a huge global genetic experiment involving all living beings. Considering the fast pace of new advances in production of genetically modified crops, consumers, farmers and policymakers worldwide are challenged to reach a consensus on a clear vision for the future of world food supply. The current food biotechnology debate illustrates the serious conflict between two groups: 1) Agri-biotech investors and their affiliated scientists who consider agricultural biotechnology as a solution to food shortage, the scarcity of environmental resources and weeds and pests infestations; and 2) independent scientists, environmentalists, farmers and consumers who warn that genetically modified food introduces new risks to food security, the environment and human health such as loss of biodiversity; the emergence of superweeds and superpests; the increase of antibiotic resistance, food allergies and other unintended effects. This article reviews major viewpoints which are currently debated in the food biotechnology sector in the world. It also lays the groundwork for deep debate on benefits and risks of Biotech-crops for human health, ecosystems and biodiversity. In this context, although some regulations exist, there is a need for continuous vigilance for all countries involved in producing genetically engineered food to follow the international scientific biosafety testing guidelines containing reliable pre-release experiments and post-release track of transgenic plants to protect public health and avoid future environmental harm. https://www.AJMB.org/En/Article.aspx?ID=64 Behrokh Mohajer Maghari, Ali M. Ardekani Sun, 04 Sep 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Comparative Expression Profile of Orphan Receptor Tyrosine Kinase ROR1 in Iranian Patients with Lymphoid and Myeloid Leukemias It has recently been shown that ROR1, a member of the receptor tyrosine kinase family, is overexpressed in leukemic B cells of Chronic Lymphocytic Leukemia (CLL) and a subset of Acute Lymphoblastic Leukemia (ALL). In this comparative study the expression profile of ROR1 mRNA was investigated in Iranian patients with CLL and Acute Myelogenous Leukemia (AML) and the results were compared with those previously reported in our Iranian ALL patients. RT-PCR was performed on bone marrow and/or peripheral blood samples of 84 CLL and 12 AML patients. CLL samples were classified into immunoglobulin heavy chain variable region (IGHV) gene mutated (n=55) and unmutated (n=29) and also indolent (n=42) and progressive (n=39) subtypes. ROR1 expression was identified in 94% of our CLL patients, but none of the AML patients expressed ROR1. No significant differences were observed between different CLL subtypes for ROR1 expression. Taken together the present data and our previous results on ROR1 expression in ALL, our findings propose ROR1 as a tumor-associated antigen overexpressed in a large proportion of lymphoid (CLL and ALL), but not myeloid (AML) leukemias. Expression of ROR1 seems to be associated to lineage and differentiation stages of leukemic cells with a potential implication for immunotherapy. https://www.AJMB.org/En/Article.aspx?ID=65 Mahdi Shabani, Hossein Asgarian-Omran, Mohammad Hojjat-Farsangi, Parvaneh Vossough, Ramazan A.Sharifian, Gholam Reza Toughe, Seyed Mohsen Razavi, Jalal Khoshnoodi, Mahmood Jeddi-Tehrani, Hodjattallah Rabbani, Fazel Shokri Sun, 04 Sep 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression and Purification of Functionally Active Recombinant Human Alpha 1-Antitrypsin in Methylotrophic Yeast Pichia Pastoris Human alpha 1-antitrypsin (AAT) cDNA was obtained from HepG2 cell lines. After PCR and construction of expression vector pPICZα-AAT, human AAT was expressed in the yeast Pichia pastoris (P.pastoris) in a secretary manner and under the control of inducible alcohol oxidase 1 (AOX1) promoter. The amount of AAT protein in medium was measured as 60 mg/l 72 hr after induction with methanol. Results indicated the presence of protease inhibitory function of the protein against elastase. Purification was done using His-tag affinity chromatography. Due to the different patterns of glycosylation in yeast and human, the recombinant AAT showed different SDS-PAGE patterns compared to that of serum-derived AAT while pI shifted from 4.9 in native AAT compared to 5.2 in recombinant AAT constructed in this study. https://www.AJMB.org/En/Article.aspx?ID=66 Sareh Arjmand, Elham Bidram, Abbas Sahebghadam Lotfi, Mehdi Shamsara, Seyed Javad Mowla Sun, 04 Sep 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Construction and In vitro Expression Analyses of a DNA Plasmid Encoding Dense Granule GRA5 Antigen of Toxoplasma gondii Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii (T.gondii) throughout the world. Although usually asymptomatic, the infection can cause serious medical problems in immunocompromised individuals and fetuses. Toxoplasmosis also causes considerable economic loss because of abortion in livestock. DNA vaccination is a promising approach against intracellular parasites such as T.gondii. The goal of this study was to construct and evaluate functionality of a mammalian plasmid expressing GRA5 anti-gen of T.gondii as a possible DNA vaccine. GRA5 gene fragment, devoid of the signal sequence, was amplified from genomic DNA of T.gondii RH strain, and cloned into pcDNA3.1 plasmid. The pcDNA3.1-GRA5 (pGRA5) was analyzed by restriction enzyme digestion followed by sequence determination. The pGRA5 was transfected into HEK 239-T human kidney cells, and expression of GRA5 antigen was investigated by Western blotting and immunofluorescence staining. The sequence encoding GRA5 was cloned into pcDNA3.1 plasmid. Restriction digestion of pGRA5 with Pst I enzyme showed correct insertion of GRA5 DNA into the plasmid. Sequence analysis revealed 100% homology with the published sequence of gra5. Immunofluorescence and Western blotting analyses of HEK 293-T cells transfected with pGRA5 showed specific expression of GRA5. Immunogenicity of pGRA5 will be evaluated in mice. https://www.AJMB.org/En/Article.aspx?ID=67 Jalal Babaie, Ghazaleh Sadeghiani, Majid Golkar Sun, 04 Sep 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Biogenic Silver Nanoparticles by Gelidiella acerosa Extract and their Antifungal Effects The synthesis, characterization and application of biologically synthesized nanomaterials are an important aspect in nanotechnology. The present study deals with the synthesis of silver nanoparticles (Ag-NPs) using the aqueous extract of red seaweed Gelidiella acerosa as the reducing agent to study the antifungal activity. The formation of Ag-NPs was confirmed by UV-Visible Spectroscopy, X-Ray Diffraction (XRD) pattern, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The synthesized Ag-NPs was predominately spherical in shape and polydispersed. Fourier Transform Infra-Red (FT-IR) spectroscopy analysis showed that the synthesized nano-Ag was capped with bimolecular compounds which are responsible for reduction of silver ions. The antifungal effects of these nanoparticles were studied against Humicola insolens (MTCC 4520), Fusarium dimerum (MTCC 6583), Mucor indicus (MTCC 3318) and Trichoderma reesei (MTCC 3929). The present study indicates that Ag-NPs have considerable antifungal activity in comparison with standard antifungal drug, and hence further investigation for clinical applications is necessary. https://www.AJMB.org/En/Article.aspx?ID=68 Marimuthu Vivek, Palanisamy Senthil Kumar, Sesurajan Steffi, Sellappa Sudha Sun, 04 Sep 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Differentiation of Bovine Spermatogonial Stem Cells into Osteoblasts Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining. https://www.AJMB.org/En/Article.aspx?ID=69 Babak Qasemi-Panahi, Parviz Tajik, Mansooreh Movahedian, Gholamali Moghaddam, Younes Barzgar, Hamed Heidari-Vala Sun, 04 Sep 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir How Does Iranian's Legal System Protect Human Vulnerability and Personal Integrity in Medical Research? The astonishing advance of medical science in recent decades has had endless advantages for humans, including improved level of health, prevention of disease and advances in treatment. These advances depend to a great extent on conducting continuous research. However, besides its enormous advantages, the sole interest of medical science undermines the principles of respect for human vulnerability and personal integrity, in both positive and negative approaches. The positive approach refers to the people who participate in research and practice, while the negative approach refers to people who are deprived of research and practice. The authors of this work, based on legal or moral grounds try to analyse the tension between the principle of respect for human vulnerability and personal integrity and the interest of medical science. Undoubtedly, in applying scientific knowledge and medical practice human vulnerability should be taken into account. In this regard, especially vulnerable individuals and groups should be protected and the personal integrity of such individuals respected. In the light of the merits of Islamic law, this paper is designed to examine the significance of the principles of human vulnerability and personal integrity in medical research by studying the international documents as formalised by UNESCO in order to explore the place of these principles in the Iranian legal system. https://www.AJMB.org/En/Article.aspx?ID=60 Mohammad Taghi Karoubi, Mohammad Mehdi Akhondi Sat, 02 Jul 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir miR-155 Down Regulation by LNA Inhibitor can Reduce Cell Growth and Proliferation in PC12 Cell Line MicroRNAs (miRNAs) are a class of small non coding regulatory RNAs that have key functions in multiple cell processes. Deregulation of these tiny miRNAs are involved in various human diseases. MiR-155 is one of the multifunctional miRNA that its over-expression has been found to be associated with different kinds of cancer such as leukemia, breast and colon cancers. It is thought that deregulation and over-expression of this microRNA may be associated with PC12 cell proliferation. So, the aim of this study was to investigate the role of miR-155 expression on PC12 cell growth. For this reason, PC12 cells were cultured and transfected by 3 different concentration (25, 50 and 75 nmol) of either LNA anti-miR-155 or scramble antisense in 24-well plate. Then, total RNA was extracted from transfected cells. miRNA cDNAs were synthesized from isolated total RNA. In the second step, miR-155 expression level was analyzed using the quantitative real-time polymerase chain reaction (QRT-PCR). MTT test was performed to evaluate cell viability. In the next step, apoptosis assay was assessed to investigate anti miR-155 effect on PC12 cells death. Obtained results were analyzed with t-test. MTT test revealed that cell viability of transfected cells with 75 nM of anti-miR- 155 to be reduced by half of the control and scramble groups (0.5 vs. 0.97 and 0.94). Our data suggest that miR-155 over-expression is associated with PC12 cell growth. So, miR-155 down regulation by anti-miR-155 could open up new ways to restrain brain tumor growth, as anti-miR-155 causes PC12 cells to repress. https://www.AJMB.org/En/Article.aspx?ID=61 Fatemeh Kouhkan, Shaban Alizadeh, Saeid Kaviani, Masoud Soleimani, Ali Akbar Pourfathollah, Naser Amirizadeh, Saeid Abroun, Mehrdad Noruzinia, Shahin Mohamadi Sat, 02 Jul 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Expression and Single-step Purification of GRA8 Antigen of Toxoplasma gondii in Escherichia coli Diagnosis of Toxoplasma gondii (T.gondii) infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several tToxoplasma antigens, including dense granule antigens (GRAs) has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 (TGRA8GRA8), excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli. (E.coli). TGRA8GRA8 was purified using an optimized single-step Iimmobilized Mmetal ion Aaffinity Cchromatography (IMAC). The purity and yield of TGRA8GRA8 was highest at pH= 9.25. At this pH, 13.6 mg of TGRA8GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with TGRA8GRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute tToxoplasma infection in pregnant women, an indirect immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed using TGRA8GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic TGRA8GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. TGRA8GRA8-IgM-ELISA was useful for detection of acute tToxoplasma infection. https://www.AJMB.org/En/Article.aspx?ID=58 Jalal Babaie, Mandana Miri, Ghazaleh Sadeghiani, Mehrak Zare, Ghader Khalili, Majid Golkar Sat, 02 Jul 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Peptide-based Polyclonal Antibody Production against P110 Protein of Mycoplasma genitalium Mycoplasma genitalium (M.genitalium) is a sexually transmitted pathogen. Detection of this microorganism in clinical specimens by culture is rather difficult and time consuming. The aim of this study was to produce polyclonal antibody against a synthetic peptide from P110 protein of M.genitalium in order to develop a diagnostic tool for detection of this microorganism in clinical specimens. A synthetic peptide from P110 protein was conjugated to Keyhole Limpet Hemocyanin )KLH( and used for immunization of a white New Zealand rabbit. The produced antibody was purified by affinity chromatography and its specific interaction with immunizing peptide was determined by ELISA. Immunoreactivity of the antibody was also tested by Western blotting in bacterial cell lysate prepared from M.genitalium G-37. To confirm its application as a diagnostic tool, indirect immunofluorescent staining method was performed on M.genitalium-infected PBMC using anti-P110 as the primary antibody. The results showed that produced antibody has excellent reactivity with immunizing peptide and also detected a single band of 110 kDa corresponding to P110 protein. M.genitalium-infected PBMC showed a bright fluorescent signal in IF staining. This antibody might be used as a tool in diagnostic applications. https://www.AJMB.org/En/Article.aspx?ID=63 Omid Zarei, Gholam Reza Irajian, Amir-Hassan Zarnani, Leili Chamani-Tabriz, Shaghayegh Emami, Mahmood Jeddi-Tehrani, Hodjattallah Rabbani Sat, 02 Jul 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Nephroprotective and Nitric oxide Scavenging Activity of Tubers of Momordica tuberosa in Rats Hydroalcoholic extract (70% ethanol extract) of tubers of Momordica tuberosa Cogn. (Cucurbitaceae) was subjected to preliminary phytochemical screening by qualitative tests. Nitric oxide scavenging activity was performed by Griess reagent method. And nephroprotective activity was assessed in gentamicin, cisplatin and paracetamol induced renal damage in wistar rats (150-200 g) by standard methods. The protective property of 70% ethanol extract was assessed by measuring the levels of body weight, blood urea, serum creatinine, tissue glutathione and lipid peroxidation in administered doses. The extract exhibited free radical scavenging activity in dose dependant manner. And 100 g/ml dose produced significantly higher scavenging activity than standard sodium metabisulphate at 25 g/ml. Also, it significantly reduced the renal damage caused by cisplatin, gentamicin and paracetamol at a dose of 40 mg/kg. https://www.AJMB.org/En/Article.aspx?ID=62 Pramod Kumar, G. Devala Rao, - Lakshmayya, Ramachandra Setty Sat, 02 Jul 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antidiarrheal, Antioxidant and Antimicrobial Activities of the Musa Sapientum Seed Musa sapientum (M.sapientum) commonly known as ‘banana’ is widely used in Bangladeshi folk medicine for the treatment of various ailments including diarrhea. Hence, the present study was designed to investigate antidiarrheal, antioxidant and antibacterial potential of the methanolic extract of M.sapientum seed (MMSS). The extract was studied for antidiarrheal property using castor oil and magnesium sulfate induced diarrheal model and charcoal induced gastrointestinal motility test in mice. Total phenolic and flavonoids content, total antioxidant activity, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, as well as nitric oxide (NO) and assessment of reducing power were used to evaluate antioxidant potential of MMSS. In addition, disc diffusion methods were used for antibacterial assay using various diarrheal induced bacterial strains. At the doses of 100 and 200 mg/kg body weight, the extract reduced the frequency and severity of diarrhea in test animals throughout the study period. At the same doses, the extracts significantly (p0.001) delayed the intestinal transit of charcoal meal in test animals as compared to the control. In DPPH and NO scavenging method, MMSS showed good antioxidant potentiality in a dose dependent manner with the IC50 value of 12.32±0.33 g/ml and 18.96±1.01 g/ml, respectively with a significant (p0.001) good reducing power. The extract also displayed strong antibacterial effect against when tested against Escherichia coli, Shigella dysenteriae, and Pseudomonas aeruginosa. Altogether, these results suggest that the MMSS could be used as a potential antidiarrheal agent along with its antioxidant and antibacterial potentiality. https://www.AJMB.org/En/Article.aspx?ID=59 Md. Sarowar Hossain, Md. Badrul Alam, Md. Asadujjaman, Ronok Zahan, M. Monirul Islam, Mohammed Ehsanul Hoque Mazumder, Md. Ekramul Haque Sat, 02 Jul 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial Genetically modified organisms (GMOs) have received a lot of attention in recent years, initially for their great potential in aiding the food shortage for a growing world population and later for the lower price, durability (resistance to insects, herbicides, viruses) and nutritional value of GM plants. In this issue of AJMB, a review article has addressed some of the major issues and concerns related to uses of GMOs. Due to recent progress in production of several GMOs by the agricultural biotechnology institutes in Iran, it is appropriate that issues such as potential health and environmental risks of GMOs are discussed in a biotechnology Journal such as AJMB. Since there are many issues that need to be addressed on the production and uses of GMOs, I have summarized some of the issues of concern for our readers and encourage submission of articles on this topic to AJMB. One of the major issues in GM foods is potential risks to human health. Safety assessment is an important method to address direct health effects (toxicity), allergenicity, nutritional or toxic properties, stability of the inserted gene, nutritional effects associated with genetic modification. Gene transfer and outcrossing are the two major concerns in the area of food safety and food security. If gene transfer (particularly antibiotic resistance genes) from GM foods to cells or bacteria in the gastrointestinal tract occurs, it would cause concern if the transferred genetic material adversely affects human health. The outcrossing can also occur if the movement of genes from GM plants into conventional crops or related species in the wild occurs. This problem has occurred when traces of a maize type only approved for feed use appeared in maize products for human consumption in the United States of America. Another major issue of concern is how GMO affects the environment? It is known that GMOs are capable to escape and potentially introduce the newly engineered genes into wild populations which then can potentially lead to loss of biodiversity on earth. Intellectual property rights and monopolization are also issues that are likely to become very significant as competition increases among nations and companies in applying biotechnology in improving food and medicine. The release of GMOs into the environment and the marketing of GM foods have in recent years resulted in public debates in many parts of the world, especially in the European Union. This debate is predicted to continue and intensify even more in the coming years mainly due to uses of biotechnology in medicine and its implications for human societies. It is appropriate that the leaders of biotechnology institutes and appropriate governmental bodies supervising the production and release of GMOs in Iran, actively participate in a national dialogue on the use and safety of GMOs. Furthermore, they should provide the right information to the public on matters of safety and security of GM foods produced domestically or imported to Iran from GM food producing countries. https://www.AJMB.org/En/Article.aspx?ID=175 Ali M. Ardekani Sat, 11 Jun 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Protective Effects of Capparis zeylanica Linn. Leaf Extract on Gastric Lesions in Experimental Animals The aim of the study was to study the anti-ulcer activity of the methanolic extract of the leaves of Capparis zeylanica Linn on experimental animal models. The methanol extract of Capparis zeylanica Linn. leaves was investigated for anti-ulcer activity against aspirin plus pylorus ligation induced gastric ulcer in rats. HCl-Ethanol induced ulcer in mice and Indomethacin induced ulcer in rats at 200 mg/kg body weight p.o. A significant (p<0.01, p<0.001) anti-ulcer activity was observed in all the models. Pylorus ligation showed significant (p<0.01) reduction in gastric volume, free acidity and ulcer index as compared to control. It also showed 88.5% ulcer inhibition in HCl-ethanol induced ulcer and 83.78% inhibition in Indomethacin induced ulcer. https://www.AJMB.org/En/Article.aspx?ID=56 Karanayil R.Sini, Barij N.Sinha, Aiyolu Rajasekaran Sun, 08 May 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Design and Construction of Two Yeast Shuttle Vectors Containing Human Procollagen Genes Expression Cassette for Expression in Yeast Collagens are the most abundant proteins in the human body. Their main function is to provide structural and mechanical support for the tissues, but they are also involved in a number of other biological functions including cell attachment, migration and differentiation. Collagens and gelatins are widely used in pharmaceutical and medical applications. Every year, more than 50,000 tons of collagen and gelatin are used in medical applications. These materials may have some viral and prion impurity and/or stimulate allergic responses in human body. Therefore, scientists have produced human collagen in recombinant systems. In this study we have constructed two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast. Total RNA was extracted from human skin fibroblast cell line, and cDNA synthesis was done by oligo dt. Then gene fragments were amplified from the cDNA with the necessary changes by Polymerase Chain Reaction (PCR). Finally they were cloned in yeast vector pPICZαA containing regulatory sequences for expressing and secreting the polypeptide product. Two yeast shuttle vectors containing human COL1A1 and COL1A2 expression cassettes were created. Final constructs were confirmed by enzymatic digestion, PCR of desired fragment and sequencing. The yeast shuttle vectors containing human COL1A1 and COL1A2 can be transferred into the yeast in the later stages to determine the scale of expression. https://www.AJMB.org/En/Article.aspx?ID=57 Baharak Abdemami, Mohammad Ali Shokrgozar, Hossein Khanahmad, Mehdi Ghavami Sun, 08 May 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Extraction, Purification and Characterization of Lipopolysaccharide from Escherichia coli and Salmonella typhi Lipopolysaccharide (LPS) is an important structural component of the outer cell membrane complex of gram-negative microorganisms. Its causative role in gram-negative bacteria-induced diseases and broad applications in different kinds of cell stimulation experiments provided a conceptual basis for studies directed at the isolation, purification, and detailed chemical characterization of LPS. The main problem with LPS purification protocols is the contamination of the end product with nucleic acids and proteins in variable proportions which could potentially interfere with downstream applications. In this study, a simple procedure for purification of LPS from Escherichia coli (E. coli) and Salmonella typhi (S. typhi) with high purity and very low contaminating nucleic acids and proteins based on the hot phenol-water extraction protocol has been introduced. The purity of extracted LPS was evaluated by silver and coomassie blue staining of SDS-PAGE gels and HPLC analysis. Limulus Amebocyte Lysate (LAL) coagulation activity and rabbit pyrogen assay were exploited to monitor the functionality of purified LPS. The results showed that DNase and RNase treatment of the sample is essential after the sonication step to eliminate nucleic acid contamination in the LPS fraction. Silver staining demonstrated ladder pattern which is characteristic of LPS. No contaminating protein was found as assessed by Coomassie blue staining. HPLC fractionation revealed high degree of purity comparable with commercial LPS. Parenteral administration of purified LPS resulted in substantial increase of rabbits’ body temperature (mean: 1.450C). LAL coagulation assay confirmed the functional activity of the purified LPS. In conclusion, the protocol presented here could be employed for isolation of LPS with high purity and functional activity. https://www.AJMB.org/En/Article.aspx?ID=53 Simin Rezania, Noor Amirmozaffari, Bahman Tabarraei, Mahmood Jeddi-Tehrani, Omid Zarei, Reza Alizadeh, Faramarz Masjedian, Amir-Hassan Zarnani Sun, 08 May 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Nonlinear Pattern Recognition of Pandemic H1N1 Using a State Space Based Methods Genomic Signal Processing is a relatively new field in bioinformatics, in which signal processing algorithms and methods are used to study functional structures in the DNA. An appropriate mapping of the DNA sequence into one or more numerical sequences enables the use of many digital signal processing tools in the analysis of different genomic sequences. Also, a novel Influenza A (H1N1) virus of swine origin emerged in the spring of 2009 and spread very rapidly among people. The severity of the disease and the number of deaths caused by a pandemic virus varies greatly and can change over time. Throughout this work, Pandemic H1N1 genomic sequences were characterized according to nonlinear dynamical features such as moment invariants and largest Lyapunov exponents and then compared to those features that extracted from classical H1N1 genomic sequences. The proposed methods were applied to a number of sequences encoded into a time series using a coding measure scheme employing Electron-Ion Interaction Pseudopotential (EIIP). The aim of this work is to extract genomic features that can distinguish the new swine flu from the classical H1N1 existed before using sequences from segment 8 of the influenza genome that consists of 8 RNA segments which encodes two important proteins for immune system attack (NS1 and NS2). According to the obtained results it is evident that variability is present based on a significance test in both groups; pandemic and classical H1N1 sequences. https://www.AJMB.org/En/Article.aspx?ID=54 Mai S.Mabrouk Sun, 08 May 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial We all know that cancer is a major medical condition that millions of individuals are afflicted by it throughout the world. No cure is found for it yet and the diagnostic tools currently available to detect cancer at an early stage are not considered to be as sensitive and specific to meet the requirement in the clinics. However, for some cancers there are treatments available in major industrial countries but not available in the developing countries with low to medium income range. Indeed why the developing countries are not investing more in prevention and early diagnosis methods? Obviously if health officials and the public are educated to focus on prevention of cancer and early detection of cancer, the burden of cancer would be much lower for any population in the near and long term. In fact for some countries (including Iran) it is predicted cancer incidence to be on the rise which could reach its climax by year 2025. The gap between the rich and poor countries is a well established fact and international organizations such as IMF, world bank,… have been established to help the economies of such developing countries which may take many decades depending on a given country. However, what is urgently needed for many of these low- to medium income level countries is an assistance in education and research programs, enabling these countries to defend their populations against cancer. In the most recent issue of Science (1), some numbers on the gap between the rich and poor countries are given that are shocking, for example: "A child suffering from leukemia in Western Europe has an 85% chance of survival; in the 25 poorest countries in the world, it's just over 10%. For a man with testicular cancer, the numbers are about 95% and just over 40%. Estimates suggest that less than 5% of the world's cancer resources are spent in the developing world". These data suggest that the developing world should pay more attention to the problem of cancer, especially in the areas of prevention and diagnosis and establish organizations that can effectively work with the developed countries in order to transfer their experiences in fighting cancer. Hopefully the Iranian ministry of health would spend more resources for fighting cancer in the coming years simply because all data suggest that cancer will be the number one killing disease in Iran in the near future if mechanisms are not found to stop its spread due to both life-style changes and environmental pollutants that are considered among the major contributors to cause cancer. https://www.AJMB.org/En/Article.aspx?ID=161 Ali M. Ardekani Sun, 08 May 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cloning and Expression of S1 Subunit of Pertussis Toxin in Escherichia coli Bordetella pertussis is a gram negative bacterium that causes respiratory tract infection in human (whooping cough). Pertussis toxin (PT) is the main component of current acellular pertussis vaccine and the S1 (subunit1) is the main immunogenic part of it. Thus, S1 has been the target of many studies as a potent candidate of acellular vaccine against Bordetella pertussis, lacking the side effects of whole cell based ones. S1 gene was amplified and inserted in three expression vectors including pET-14, pET-22b(+) and pAED4. The possibility and level of expression of these constructs were investigated in BL21 (DE3) strain of Escherichia coli (E.coli) as expression host. The highest expression was in pET-22b(+)-S1. Best expression achieved 6 hr post induction with 0.2 mM IPTG in LB broth containing ampicillin, at 30°C with shaking (250 rpm). Recombinant S1 protein was observed in two distinct separated proteins with 28 and 31 kDa estimated molecular weight. In spite of toxicity of PT and S1 in the E.coli, considerable amount of S1 was expressed in E.coli. Two rS1 bands were detected on SDS-PAGE. Both were confirmed as S1 in western blot with specific monoclonal and polyclonal antibodies against pertussis toxin. Appearance of two distinct bands could be the result of leader peptidase activity or nonspecific peptidase from E.coli on recombinant S1. As the recombinant S1 is a suitable antigen for studies as a candidate acellular vaccine or development of ELISA for detection of Bordetella pertussis, further studies are underway. https://www.AJMB.org/En/Article.aspx?ID=51 Abolfazl Khafri, Khosrow Aghaiypour, Shahin Najar Peerayeh, Reihaneh Ghorbani Sun, 08 May 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antidiabetic Activity of Aqueous Leaves Extract of Sesbania sesban (L) Merr. in Streptozotocin Induced Diabetic Rats The aqueous leaves extract of Sesbania sesban (L) Merr. (Family: Fabaceae) was evaluated for its antidiabetic potential on normal and streptozotocin (STZ)-induced diabetic rats. In the chronic model, the aqueous extract was administered to normal and STZ- induced diabetic rats at the doses of 250 and 500 mg/kg body weight (b.w.) p.o. per day for 30 days. The fasting Blood Glucose Levels (BGL), serum insulin level and biochemical data such as glycosylated hemoglobin, Total Cholesterol (TC), Triglycerides (TG), High Density Lipoproteins( HDL) and Low Density Lipoproteins (LDL) were evaluated and all were compared to that of the known anti-diabetic drug glibenclamide (0.25 mg/kg b.w.). The statistical data indicated significant increase in the body weight, liver glycogen, serum insulin and HDL levels and decrease in blood glucose, glycosylated hemoglobin, total cholesterol and serum triglycerides when compared with glibenclamide. Thus the aqueous leaves extract of Sesbania sesban had beneficial effects in reducing the elevated blood glucose level and lipid profile of STZ-induced diabetic rats. https://www.AJMB.org/En/Article.aspx?ID=55 Ramdas B.Pandhare, B. Sangameswaran, Popat B.Mohite, Shantaram G.Khanage Sun, 08 May 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Lack of Association between Interleukin 12 C(-1188)A Polymorphism and Irritable Bowel Syndrome Irritable Bowel Syndrome (IBS) is a functional gastrointestinal disorder, characterized by recurrent abdominal pain and altered bowel habits. This study was performed to investigate the important role of interleukin-12 (IL-12) in intestinal inflammation. For this study seventy one patients with IBS and 140 controls were investigated. The allele and genotype frequencies of IL-12 C(-1188)A were determined using polymerase chain reaction with sequence-specific primers. The allele A was more common that the allele C in both groups of patients and controls. There was not any significant difference on IL-12 alleles and genotypes between patients and controls. The AA genotype was the most common genotypes, which was seen in 57.4% of the patients and 51.4% of the controls (p=0.53). Although frequency of the CC genotype in the control group was lower than the patient group, this difference was not significant (5.7% vs. 11.5%, respectively, p=0.16). Considering the lack of association between IL-12 C(-1188)A polymorphism and IBS, this cytokine gene polymorphism may not have significant role in the pathophysiology of disease. https://www.AJMB.org/En/Article.aspx?ID=52 Elham Barkhordari, Ali Akbar Amirzargar, Naser Ebrahimi-Daryani, Mahdi Mahmoudi, Bita Ansaripour, Maryam Alighardashi, Hamid Reza Ahmadi-Ashtiani, Mohammad Bashashati, Nima Rezaei Sun, 08 May 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial Humans have used animals for nourishments, body parts to help survival, in sports, in fields, in experiments and also have domesticated them as companions for a long time. Although the animal rights groups have been raising the issue of animal cruelty for the past few decades, however, animals are still exploited in the industrial processing. Billions of chickens, turkeys, sheep, cows, pigs, geese, and other animals are maltreated everyday around the world in order to prepare them to feed humans. A vast literature exists on the issue of animal suffering and the cruelties humans exert on them, and its ethical implications have been discussed. However, there are conflicting viewpoints on animal experimentations and particularly genetic manipulations of animals in societies where biotechnology sector has reached a level of potentiality to produce such animals. For example, is it ethical to engineer hundreds of transgenic pigs and keep them in standby positions to provide heart valves and whole heart transplants? Or even engineer a pig to grow a heart using certain genes from the intended heart patients? Is it ethical to use animals entirely as a means to save a human? How is it that the animal can be eaten entirely for food purposes, but may be questioned ethically when the animal is to be helping a human to survive? In recent years, the biotechnology community has found the capacity to make any kind of transgenic animals they wish including: animals which glow green, become diabetic or oncogenic in a given span of time, serve as bioreactors or bodies that can produce drug molecules. The question is where to draw the line with regard to ethics in using biotechnology to manipulate animals in serving the needs of humans? Obviously, the lines are not quite clear and the standards may differ in different countries and cultures. But, what is clear is that as more societies find the capacity to create animals whose bodies are pharmaceutical manufacturing plants, whose organs provide transplants, and whose bodies are genetically altered to provide experimental platforms; the ethical bodies in the government and non-government should scrutinize the motives and actions where biotechnologies are applied. As some argue, the best way to measure our humanity is not only in respecting the individual human beings but also in how we treat animals in our societies. https://www.AJMB.org/En/Article.aspx?ID=162 Ali M. Ardekani Sat, 12 Mar 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial In the previous editorial I made mention of the two important conferences on ethical aspects of genetic technologies that had been planned to be held in Iran (November 2010 and February 2011). In the November conference which was held by the Avicenna Research Institute, the ethical and social aspects of using genetic technologies were presented to the academic and non-academic audiences. This conference was widely covered by the national media and the subject of genetics with its interdisciplinary nature initiated discussion between experts in both fields of natural and social sciences. Since some of our colleagues were not able to attend the conference, I thought it would be appropriate to provide a brief summary of the theme of presentations in the conference. The two day conference was organized in four panels and the topics presented in each panel were discussed from ethical, legal and social points of view. The four panels were as follows: 1) Genetics and the Emergence of Life 2) Genetics in Diagnosis and Treatment of Diseases 3) Plant and Animal Genetics 4) New Findings on the Science of Genetics In the first panel 10 lectures were presented on topics of eugenics, cloning, genetic diagnostics and abortion. In the second panel 7 lectures were presented on topics of stem cells, Pre-implantation Genetic Diagnosis (PGD), pharmacogenomics and gene therapy. In the third panel on the second day, 8 lectures were presented on topics of genetically modified organisms (GMO), transgenic plants, bio-safety issues and its implementations at the national level, agricultural genetic engineering, Intellectual Property, issues of patent and bio-safety and the rights of consumers with regards to transgenic products. In the fourth panel on the second day, 9 lectures were presented on topics of mental health and human genome, genetic enhancements, completion of human genome and social implications, genetic predisposition to crime and legal responsibility, DNA tests and proof of identity. The topics listed above clearly demonstrate that genetic engineering has had a profound impact on human life in societies worldwide at multilevel. Therefore, it is important for leaders of societies in the modern world to pay attention to the advances in genetic technologies and prepare themselves and institutions under their control to face the challenges which these new technologies induce in the areas of ethics, law and social policies. https://www.AJMB.org/En/Article.aspx?ID=160 Ali M. Ardekani Tue, 11 Jan 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Role of MicroRNAs in Human Diseases MicroRNAs (miRNAs) are short RNA molecules which bind to target mRNAs, resulting in translational repression and gene silencing and are found in all eukaryotic cells. Approximately 2200 miRNA genes have been reported to exist in the mammalian genome, from which over 1000 belong to the human genome. Many major cellular functions such as development, differentiation, growth, and metabolism are known to be regulated by miRNAs. Proximity to other genes in the genome and their locations in introns of coding genes, noncoding genes and exons have been reported to have a major influence on the level of gene expressions in eukaryotic cells. miRNAs are well conserved in eukaryotic system and are believed to be an essential and evolutionary ancient component of gene regulatory networks. Therefore, in recent years miRNAs have been studied as a likely candidate for involvement in most biologic processes and have been implicated in many human diseases. https://www.AJMB.org/En/Article.aspx?ID=44 Ali M. Ardekani, Mozhgan Moslemi Naeini Tue, 11 Jan 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Regulation Studies of Telomerase Gene in Cancer Cells by Lentinan Lentinan a polysaccharide from medicinal mushroom i.e Lentinus, has been known to have anticancer properties. Telomerase activity is not observed in normal healthy cells, whereas in cancerous cells telomerase expression is high. Telomerase represents a promising cancer therapeutic target. We investigated the inhibitory effect of lentinan on telomerase reverse transcriptase gene (hTERT) which is essential for telomerase activity. To assess the transcriptional effect, DLD -1 cancer cells were cultured in the presence of various concentrations of lentinan. TRAP assay, RT-PCR analysis were performed to find telomerase activity and hTERT gene expression respectively. Since C-myc is known to regulate hTERT, expression of C-myc was also determined. Culturing cells with lentinan resulted in down regulation of hTERT and C-myc expression.These results indicate that lentinan inhibits telomerase activity by down regulating hTERT expression via suppression of C-myc in cancer cells. https://www.AJMB.org/En/Article.aspx?ID=45 Kamma Sreenivasulu, Muvva Vijayalakshmi, Krothapalli RS. Sambasivarao Tue, 11 Jan 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Cytotoxic Activities of Silver Nanoparticles and Silver Ions in Parent and Tamoxifen-Resistant T47D Human Breast Cancer Cells and Their Combination Effects with Tamoxifen against Resistant Cells Studies on biomedical applications of nanoparticles are growing with a rapid pace. In medicine, nanoparticles may be the solution for multi-drug-resistance which is still a major drawback in chemotherapy of cancer. In the present study, we investigated the potential cytotoxic effect of silver nanoparticles (Ag NPs) and silver ions (Ag+) in both parent and tamoxifen-resistant T47D cells in presence and absence of tamoxifen. Ag NPs were synthesized (< 28 nm) and MTT assay was carried out. The associated IC50 values were found to be: 6.31 ?g/ml for Ag NPs/parent cells, 37.06 ?g/ml for Ag NPs/tamoxifen-resistant cells, 33.06 ?g/ml for Ag+/parent cells and 10.10 ?g/ml for Ag+/resistant cells. As a separate experiment, the effect of subinhibitory concentrations of Ag NPs and Ag+ on the proliferation of tamoxifen resistant cells was evaluated at non-toxic concentrations of tamoxifen. Our results suggested that in non-cytotoxic concentrations of silver nanomaterials and tamoxifen, the combinations of Ag+-tamoxifen and Ag NPs-tamoxifen are still cytotoxic. This finding may be of great potential benefit in chemotherapy of breast cancer; since much lower doses of tamoxifen may be needed to produce the same cytotoxic effect and side effects will be reduced. https://www.AJMB.org/En/Article.aspx?ID=46 Seyed Naser Ostad, Shahrzad Dehnad, Zeinab Esmail Nazari, Shohreh Tavajohi Fini, Narges Mokhtari, Mojtaba Shakibaie, Ahmad Reza Shahverdi Tue, 11 Jan 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir High Expression of Methylotrophic Yeast-Derived Recombinant Human Erythropoietin in a pH-Controlled Batch System To accomplish the worldwide demand for recombinant human erythropoietin (rHuEpo) as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris (P. pastoris) rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZaA vector under control of AOX1 promoter, downstream of the secretion-a-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin (1.0 mg/ml), followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDS- PAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems. https://www.AJMB.org/En/Article.aspx?ID=47 Ahmad Maleki, Farzin Roohvand, Hosnieh Tajerzadeh, Hossein Khanahmad, Maryam B. Nobari, Ahmad Beiruti, Abdolhossein Rouholamini Najafabadi Tue, 11 Jan 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of a Sensitive Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis B Surface Antigen Using Novel Monoclonal Antibodies Hepatitis B virus (HBV) infection is the 10th leading cause of death worldwide. The most important diagnostic and screening marker for HBV infection is Hepatitis B surface antigen (HBsAg), and the most widely used HBsAg screening test is Enzyme-linked Immunosorbent Assay (ELISA). In this study, an ELISA assay has been developed for detection of HBsAg using two novel monoclonal antibodies (mAb) as capture layer and a polyclonal biotinylated Ab as detector phase. We evaluated the sensitivity, specificity, detection limit, seroconversion time, positive and negative predictive values and reproducibility of our assay with standard panels and different serum samples. The results were compared with a well established commercial kit. Both assays showed similar detection limit values of 0.5 to 0.7 ng/ml and the same seroconversion periods of 42 and 65 days for “ad” and “ay” serotypes of HBsAg, respectively. Sensitivity and specificity of the assay were 98.98% and 99.6%, respectively. The positive and negative predictive values of our assay were also calculated as 99.49% and 99.2%, respectively. Analysis of reproducibility of the present assay demonstrated 3.96% and 5.85% intra-and inter-assay coefficient of variations, respectively, which were less than those obtained by the commercial kit. There was a highly significant correlation between our designed assay and the commercial ELISA kit (p < 0.0001, r = 0.957). Altogether, our results indicate that the designed assay is comparable to the commercial kit in terms of sensitivity, specificity, positive and negative predictive values and reproducibility and could be employed for diagnosis of HBV infection in blood samples. https://www.AJMB.org/En/Article.aspx?ID=48 Yaghoub Yazdani, Azam Roohi, Jalal Khoshnoodi, Fazel Shokri Tue, 11 Jan 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Hypolipidemic Activity of Chloroform Extract of Mimosa pudica Leaves Mimosa pudica Lin., known as chue Mue, is a stout straggling prostrate shrubby plant, with spinous stipules and globose pinkish flower heads, and grows as weed in almost all parts of the country. It is traditionally used for its various properties and hence in the present study, chloroform extract of Mimosa pudica leaves has been screened for its hypolipidemic activity. Hypolipidemic activity is screened by inducing hyperlipidemia with the help of atherogenic diet in wistar albino rats and serum levels of various biochemical parameters such as total cholesterol, triglycerides, LDL, VLDL and HDL cholesterol were determined. Atherogenic index shows the measure of the atherogenic potential of the drugs. Chloroform extract showed significant (p < 0.05) hypolipidemic effect by lowering the serum levels of biochemical parameters such as significant reduction in the level of serum cholesterol, triglyceride, LDL, VLDL and increase in HDL level which was similar to the standard drug Atorvastatin. Chloroform extract exhibited significant atherogenic index and percentage protection against hyperlipidemia. These biochemical observations were in turn confirmed by histopathological examinations of aorta, liver and kidney sections and are comparable with the standard hypolipidemic drug Atorvastatin. Preliminary phytochemical analysis revealed the presence of phytoconstituents such as steroids, flavonoids, glycosides, alkaloids, phenolic compounds which is further confirmed by the thin layer chromatography, High Performance Thin Layer Chromatography (HPTLC). The overall experimental results suggests that the biologically active phytoconstituents such as flavonoids, glycosides alkaloids present in the chloroform extract of Mimosa pudica, may be responsible for the significant hypolipidemic activity and the results justify the use of Mimosa pudica as a significant hypolipidemic agent. https://www.AJMB.org/En/Article.aspx?ID=49 Rekha Rajendran, Ekambaram Krishnakumar Tue, 11 Jan 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir A Brief View on Molecular Diagnosis and Surveillance of West Nile Virus <p>&nbsp;West Nile Virus (WNV) is an important zoo-notic agent having a wide host range. Due to its emergence with increased virulence in a wide geographical range, its monitoring becomes im-perative. Development of more rapid and sensitive molecular techniques for instance Reverse Trans-criptase-Polymerase Chain Reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Nucleic Acid Se-quence Based Amplification (NASBA) assays are vital for detection of the virus. Various surveil-lance techniques according to epidemiological, climatic and geographical conditions in the ex-posed area have also been developed. The surveil-lance can be set up at different levels of the WNV transmission cycle using birds, horses and mos-quitoes as sentinels.</p> https://www.AJMB.org/En/Article.aspx?ID=50 Pranay Kumar, Shanker K.Singh, Yogranjan R.Singh, Mayurdhvaj K.Jhala Tue, 11 Jan 2011 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Synchronous Comparison of Mycobacterium tuberculosis Epidemiology Strains by "MIRU-VNTR" and "MIRU-VNTR and Spoligotyping" Technique Molecular epidemiology analyses are frequently used in determining epidemiology of tuberculosis. Recently, Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) and Spoligotyping has become an important method, as it allows high-through put, discriminatory and reproducible analysis of clinical isolate. The purpose of this study is to compare techniques of “MIRU-VNTR” versus “MIRU-VNTR and Spoligotyping” together for study of genetic pattern of Mycobacterium tuberculosis (M. tuberculosis) strains. Sixty M. tuberculosis (MTB) isolates were selected (30 susceptible, 30 multi-drug resistant) for this study. Thereafter, the "MIRU-VNTR and spoligotyping" were performed to identify their genetic patterns. The frequency of unknown genetic pattern of MTB was compared using technique of “MIRU-VNTR” alone versus “MIRU-VNTR and Spoligotyping” together. The MIRU-VNTR allelic diversity at each of the loci was calculated by Hunter – Gaston Discriminatory Index (HGDI). Based on differentiation index of all strains 10, 16, 26, 31 and 40 loci were identified as the most distinctive (HGI = 0.6) and 2, 4, 20 and 24 as the weakest distinctive locus (HGI = 0.3). By using MIRU-VNTR technique 38% (n= 23) of isolates could not be typed, whereas by applying "MIRU-VNTR and Spoligotyping" together only 15% (n= 9) of isolates remained unknown (p = 0.004). For sensitive strains, the difference was significant (67% vs. 90%, p = 0.028), but only marginally significant for drug resistant strains (57% vs. 80%, p = 0.052). The discrimination power of 12-locus MIRU-VNTR and Spoligotyping was equal to that of MIRU-VNTR analysis. If appropriate loci are added to the standard MIRU analysis, MIRU-VNTR genotyping could be a valuable tool for strain typing and epidemiological research of M. tuberculosis. With this approach a more clear understanding about genetic pattern of MTB can be achieved. https://www.AJMB.org/En/Article.aspx?ID=42 Mehdi Jafarian, Muayed Aghali-Merza, Parissa Farnia, Mojtaba Ahmadi, Mohammad Reza Masjedi, Ali Akbar Velayati Wed, 06 Oct 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Teratogen Screening: State of the Art Due to the number of new substances coming into use every year and the increasing amounts of chemicals, which are introduced into the environment, there is a high demand for a rapid, reliable and cost-effective method for detection of developmental toxicity. To meet this challenge various in vitro techniques have been established additional to in vivo animal testing. This review introduces the techniques in existence at the moment. Requirements on an ideal in vitro teratogenicity test system are stated, and the advantages and disadvantages of the present methods are discussed. https://www.AJMB.org/En/Article.aspx?ID=38 Julia Schumann Tue, 28 Sep 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. https://www.AJMB.org/En/Article.aspx?ID=39 Mahdi Shabani, Shayda Hemmati, Reza Hadavi, Zahra Amirghofran, Mahmood Jeddi-Tehrani, Hodjattallah Rabbani, Fazel Shokri Tue, 28 Sep 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm. https://www.AJMB.org/En/Article.aspx?ID=40 Keivan Majidzadeh, Fereidoun Mahboudi, Mahdi Hemayatkar, Fatemeh Davami, Farzaneh Barkhordary, Ahmad Adeli, Mohammad Soleimani, Noushin Davoudi, Vahid Khalaj Tue, 28 Sep 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir In Silico Design and Selection of Anti-fungal AmB-polyene-analog Lead Molecules by Virtual Screening Method A major group of drugs that have been approved for the therapy of systemic fungal infections are polyene antibiotics. Amphotericin B (AmB), one of the polyene antibiotics, has been used to treat serious systemic fungal infections by binding to sterols such as ergosterol in fungal cells membrane, and is believed to form pores in the membrane and create a transmembrane ion-channel. Since all eukaryotic cells contain sterols, using AmB can cause toxicity in mammalian cells; this is the most serious unwanted side effect. Therefore, there is still a need to develop suitable antifungal compounds to be entered in the drug development pipeline. In this study, we report the screening of various compounds from the Enhanced NCI database against ergosterol and cholesterol as receptors. The strategy employed is divided into two categories, screening and docking, respectively. Screening was performed using structure search based on AmB and molecular constraints to filter compounds with physico-chemical properties similar to the polyene macrolid antibiotics. The selected compounds were docked and scored to identify structurally novel ligands that make similar interactions to AmB. Our screening approach identified several molecules with high ranking criteria mentioned above. Among these compounds, two molecules, NSC 89270 and NSC 62792 were tested for their bioactivity against three fungal strains using broth microdilution assay that presented to have moderate antifungal activity against tested fungi. Thus, they could be possible lead compounds that grant further research on them to improve their potency and compare their mechanism of action in comparison to AmB. https://www.AJMB.org/En/Article.aspx?ID=41 Marziyeh Ferdosiyan, Soroush Sardari Tue, 28 Sep 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Association of CALHM1 Gene Polymorphism with Late Onset Alzheimer’s Disease in Iranian Population Alzheimer's disease (AD) is a genetically heterogeneous neurodegenerative disease and Late-Onset type (LOAD) is the most common form of dementia affecting people over 65 years old. CALHM1 (P86L) encodes a transmembrane glycoprotein that controls cytosolic Ca2+ concentrations and Aß levels and P86L polymorphism in this gene is significantly associated with LOAD in independent case controls in a number of studies. This study was performed to determine whether this polymorphism contributes to the risk for LOAD in Iranian population. One hundred and forty one AD patients and 141 healthy controls were recruited in this study. After extraction of genomic DNA, the genotype and allele frequencies were determined in case and control subjects using PCR/RFLP method. The statistical analysis showed a significant difference in the heterozygote genotype frequency in case and control groups and polymorphic allele had a protective role between two groups. Also after stratifying the subjects by their APOE-e4 status, no significant association was observed. Our study suggests that P86L polymorphism could be a protective factor for late-onset Alzheimer's disease (LOAD) in Iranian population. However, to confirm these results, further study with a bigger sample size may be required. https://www.AJMB.org/En/Article.aspx?ID=43 Meysam Jafari Aqdam, Koorosh Kamali, Mehdi Rahgozar, Mina Ohadi, Mehdi Manoochehri, Ali Tahami, Leila Bostanshirin, Hamid Reza Khorram Khorshid Tue, 28 Sep 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Angiotensin II Differentially Induces Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 Production and Disturbs MMP/TIMP Balance Angiotensin II, the main component of the renin-angiotensin system, is associated with cardiovascular diseases such as hypertension, vascular remodeling and inflammation. Remodeling process results from dysregulation of Matrix Metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). MMPs are considered as important target genes for angiotensin II. The aim of this study was to determine the effects of angiotensin II on MMP-9 and TIMP-1 production and MMP/TIMP balance in a monocytic cell type. Human monocytic U-937 cells were cultured and treated with 100 nM angiotensin II. Supernatants were analyzed for MMP-9 and TIMP-1 using ELISA and zymography methods. Real-time PCR was utilized to evaluate relative MMP-9 and TIMP-1 genes expression following treatments. Cytotoxicity potentials of treatments were determined by assaying lactate dehydrogenase leakage from the cells. Stimulation of the monocytic cells with angiotensin II significantly increased MMP-9 and TIMP-1 secretion as measured by ELISA (p<0.05). It also augmented gelatinolytic activity of MMP-9 in the conditioned media as much as 49% (p<0.05). Incubation of the cells with angiotensin II for 12 hr increased MMP-9 and TIMP-1 gene expression 2.7 and 1.8 folds, respectively (p<0.05). Angiotensin II treatments did not establish significant cytotoxic effects. In summary, our data provide further evidences that monocytic MMP-9 is a major effector of angiotensin II. It is induced more efficiently than TIMP-1 by angiotensin II that leads to MMP/TIMP imbalance. Our data also reveal the pivotal participation of these cells in pathological cardiovascular remodeling mediated by angiotensin II. https://www.AJMB.org/En/Article.aspx?ID=33 Hamid Yaghooti, Mohsen Firoozrai, Soudabeh Fallah, Mohammad Reza Khorramizadeh Mon, 19 Jul 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Conjugation of R-Phycoerythrin to a Polyclonal Antibody and F (ab')2 Fragment of a Polyclonal Antibody by Two Different Methods R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab')2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity. https://www.AJMB.org/En/Article.aspx?ID=34 Jafar Mahmoudian, Mahmood Jeddi-Tehrani, Hodjattallah Rabbani, Ahmad Reza Mahmoudi, Mohammad Mehdi Akhondi, Amir-Hassan Zarnani, Leila Balaei Goli, Mahdokht Babaei, Roya Ghods Mon, 19 Jul 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir An Investigation into the Antifungal Property of Fabaceae using Bioinformatics Tools Chemodiversity in plants provides sources of great value which might be helpful for finding new leads in drug discovery programs. Fabaceae as the third largest family of flowering plants was chosen to investigate its possible antifungal activity. In order to increase the effectiveness of the result, molecular similarity methods and chemical data were used. Twelve plants were selected from Fabaceae and collected from the North and South of Iran. Percolation method with 80% ethanol was used for extraction of collected plants. Antifungal activities of these extracts were determined using broth microdilution method against Candida albicans (C. albicans) ATCC 10231, Aspergillus fumigatus (A. fumigatus) AF 293 and Asperigillus niger (A. niger) ATCC 16404. Extracts with promising activity were screened for toxicity with larvae of Artemia salina (brine shrimp). Dalbergia sissoo, Lathyrus pratensis, Oreophysa microphyalla, Astragalus stepporum, Ebenus stellata, Sophora alopecuroides, Ammodendron persicum and Taverniera cuneifolia showed activity against at least one of the microorganisms used in this study. According to the results of our experiment, the extracts of these plants can be used for further investigation in therapeutic research. https://www.AJMB.org/En/Article.aspx?ID=35 Zahra Arabi, Soroush Sardari Mon, 19 Jul 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Anti-Arthritic Activity of Premna serratifolia Linn., Wood against Adjuvant Induced Arthritis Adjuvant induced arthritis is a chronic crippling, skeleton-muscular disorder having nearest approximation to human rheumatoid arthritis for which there is currently no medicine available effecting a permanent cure. Even modern drugs used for the amelioration of the symptoms, offer only temporary relief and also produce severe side effects. In the indigenous system of medicine, wood of Premna serratifolia Linn., is reported to be useful in the treatment of arthritis. It is a large shrub, distributed throughout Asia, used against a wide variety of diseases. However, no systematic study has been reported regarding its anti-arthritic activity. This work was aimed at the scientific validation of the ethno-pharmacological claim about its anti-arthritic property. In the present study, anti-arthritic activity of ethanol extract of Premna serratifolia Linn., wood is done by Freund's adjuvant induced arthritis model. Loss in body weight during arthritis condition was corrected on treatment with ethanol extract and standard drug, indomethacin. Biochemical parameters such as hemoglobin content, total WBC, RBC, erythrocyte and sedimentation rate were also estimated. The ethanol extract at the dose of 300 mg/kg body weight inhibited the rat paw edema by 68.32% which is comparable with standard drug indomethacin 74.87% inhibition of rat paw edema after 21 days. The results of the current investigation concluded, ethanol extract of Premna serratifolia Linn., wood possess a significant anti-arthritic activity against adjuvant induced arthritis and justifying its therapeutic role in arthritic condition. The observed anti-arthritic activity may be due to the presence of phytoconstituents such as irridiod glycosides, alkaloids, phenolic compounds and flavonoids. https://www.AJMB.org/En/Article.aspx?ID=36 Rekha Rajendran, Ekambaram Krishnakumar Mon, 19 Jul 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of Biopsy During Precompacted Morula Stage on Post Vitrification Development of Blastocyst Derived Bovine Embryos Improvements on embryo micromanipulation techniques led to the use of embryo biopsy in commercial embryo transfer programs for genetic analysis of preimplantation bovine embryos. The aim of this study was to evaluate the quality of bovine blastocyst derived from embryos biopsied at different precompacted morulae stages by assessment of cryosurvivability of the resulting blastocysts. The in vitro produced bovine embryos were subjected to biopsy at days 2, 3, and 4 post-insemination with different cell numbers (4 to 16-cells). Embryo cell biopsy was carried out in a 100 ?l drop of H-SOF following pronase drilling by aspiration of one blastomere. The biopsied embryos were then cultured in SOFaaBSA co-cultured with oviduct cells-monolayer until blastocyst formation. The blastocysts were cryopreserved at room tempera-ture after exposure of equilibration (glycerol 1.4 M for 5 min and then glycerol 1.4 M and ethylene glycol 3.6 M for 5 min) and vitrification solutions (3.4 M glycerol and 4.6 M ethylene glycol). The blastocysts were loaded into the center of 0.25 ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. There was no significant difference in cryosurvivability of vitrified-warmed blastocysts derived form biopsied embryos at different pre-compacted morula stages. The quality of biopsy derived blastocysts was identical to that of non-biopsy derived ones in terms of post vitrifcation survival and hatching rates. In conclusion there was no preference between different times of embryo biopsy at precompacted morula stages in term of cryosurvivability of biopsy derived bovine blastocysts. https://www.AJMB.org/En/Article.aspx?ID=37 Abolfazl Shirazi, Sara Borjian, Ebrahim Ahmadi, Hassan Nazari, Banafsheh Heidari Mon, 19 Jul 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production of Monoclonal Antibody against Human Nestin We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. https://www.AJMB.org/En/Article.aspx?ID=32 Reza Hadavi, Amir-Hassan Zarnani, Negah Ahmadvand, Ahmad Reza Mahmoudi, Ali Ahmad Bayat, Jafar Mahmoudian, Mohammad Reza Sadeghi, Haleh Soltanghoraee, Mohammad Mehdi Akhondi, Majid Tarahomi, Mahmood Jeddi-Tehrani, Hodjattallah Rabbani Sun, 18 Jul 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial In the past few decades, as new medical technologies have been incorporated into the common medical practice around the world, new ethical challenges have been introduced in the realms of philosophy and ethics. These issues include: the consent of cell donors and subject of property rights in the field of tissue engineering and enhancement of human capabilities for longevity; cloning of animals and humans (potentially) in the future; genetic manipulation for diagnosis and treatment of diseases; use of stem cells and gene therapy for treatment of human diseases; genetic manipulation of plants to produce therapeutic drugs and food for people and many other related issues. The bioethical aspects of many of these new medical biotechnologies have been fervently debated for many years in private bioethics centers, universities and governments’ sponsored studies in many developed countries. However, little attention has been paid to these issues in the developing countries, perhaps due to limited application of such technologies in a given country. Fortunately, in the past few years, some national and international conferences on ethical aspects of using these new technologies have been held in Iran. Incidentally, later this year, in the month of November, two conferences are to be held in Iran which will focus on bioethics and new technologies. The international conference on this issue will be held by the National Research Center for Genetic Engineering and Biotechnology of Iran and the national conference will be held by the Avicenna Research Institute. I must mention that the conference held by the Avicenna Research Institute will focus on genetics as a central theme and will consider the legal, ethical and psychological aspects as they relate to genetics and human health. These two conferences will provide an excellent opportunity and should be very beneficial to those who are interested in bioethics, ethics, law, philosophy, sociology, psychology as they relate to human health. I encourage researchers, students, leaders of research institutions and government offices currently involved with issues related to medical biotechnology research, genetics and bioethics to participate in these conferences. https://www.AJMB.org/En/Article.aspx?ID=159 Ali M. Ardekani Sat, 12 Jun 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development in Immunoprophylaxis against Rabies for Animals and Humans Rabies is a fatal neurological disease and a persistent global problem. It is spread primarily by domestic dogs but other canid, viverrid (skunks and raccoons) and chiropteran species are considered as the most efficient vectors of the disease. Since dogs are the main perpetuator of rabies, special attention has to be given to bring all the dogs including unauthorized stray dogs under immunization umbrella in order to control rabies. Vaccination is the only way to combat the disease before and after exposure or infection as there is no treatment available once the symptoms have appeared. After the first crude nerve tissue vaccine developed by Pasteur in 1885, a number of rabies vaccines for animal and human use have been developed with varying degree of safety and efficacy over the years. Presently, cell culture based inactivated rabies vaccines are largely used in most of the parts of the world. However, these vaccines are too expensive and unaffordable for vaccination of people and animals in developing countries. The comparatively cheaper inactivated nerve tissues vaccines can cause serious side-effects such as autoimmune encephalomyelitis in inoculated animals and production has been discontinued in several countries. Although attenuated live vaccines can efficiently elicit a protective immune response with a smaller amount of virus, they sometimes can cause rabies in the inoculated animals by its residual virulence or pathogenic mutation during viral propagation in the body. New-generation rabies vaccines generated by gene manipulation although in experimental stage may be a suitable alternative to overcome the disadvantages of the live attenuated vaccines. So, awareness must be created in general public about the disease and the cell culture based vaccines available in the market should be recommended for wide scale use to prevent and control this emerging and reemerging infectious disease in foreseeable future. https://www.AJMB.org/En/Article.aspx?ID=29 Sukdeb Nandi, Manoj Kumar Sat, 17 Apr 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Intermittent Feeding Attenuates Clinical Course of Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice Multiple Sclerosis (MS) is an autoimmune inflammatory, demyelinating disease of human central nervous system. Experimental Autoimmune Encephalomyelitis (EAE) is the commonly used animal model of MS. Calorie restriction has been found to reduce inflammation and autoimmune responses and promote neuroprotection. In this study we evaluated the effects of intermittent feeding protocol of the calorie restriction in a mouse model of EAE. Fifty four female mice (C57BL/6) were used in this study. The animals were divided into two dietary groups: ad libitum (AL) (n=29) with free access to food and water and intermittent feeding (IF) (n=25) with access to food on alternate days. After 8 weeks, EAE was induced in animals by immunization with MOG antigen (Hooke labs, Lawrence, MA, USA) subcutaneously. AL and IF groups were then further divided into two groups each: AA (ad libitum until the end of study) (n=16) and AI (subjected to intermittent feeding regimen after immunization day) (n=13). The IF group was divided into II (continued intermittent feeding regimen until the end of study) (n=13) and IA (changed to AL regimen after immunization day) (n=12). All the animals were behaviorally monitored for 35 days after immunization and observed daily for the signs and severity of disease with EAE scoring scale [0-5] and cumulative disease index (CDI) score. Intermittent feeding significantly reduced the incidence of EAE in IF groups (AI 0%, II 18.5%, IA 22.2%, p<0.05). In addition, intermittent feeding significantly delayed the onset of EAE in AI group (p<0.05) and also, intermittent feeding significantly reduced the severity of disease in II and IA groups (AA vs. II, p<0.05 & AA vs. IA p<0.05) groups. The CDI was also significantly reduced in intermittent feeding fed groups [AI, II and IA compared to AA group (P<0.05, <0.01, <0.05 respectively)]. Intermittent feeding regimen protocol of the calorie restriction significantly suppressed EAE incidence, induction, and severity. The results of this study suggest possible role of intermittent feeding in the treatment of Multiple Sclerosis patients. https://www.AJMB.org/En/Article.aspx?ID=30 Laya Kafami, Mohsin Raza, Alireza Razavi, Abbas Mirshafiey, Mansooreh Movahedian, Mohammad Reza Khorramizadeh Sat, 17 Apr 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial New advances in Genetic Technologies are changing the lives of millions of people around the world in important ways. These advances have also raised difficult ethical and legal questions for policy makers in many countries. Therefore, there is a great interest and need by experts in both governmental and non-governmental institutions to have a deeper understanding of the issues involved for establishing the necessary societal rules to regulate the use of genetic technologies in the fields of Medicine, Veterinary Science and Agriculture. To address these issues and provide a forum for a scientific discussion at a national level, the Avicenna Research Institute is planning to hold a conference in November 2010, entitled 'Genetics: Law, Ethics and Psychology'. The conference will particularly focus on the use of new genetic technologies and its impacts on the society from the legal and ethical point of view. In view of the fact that better understanding of the genetic basis of human behavior and physiology is imperative to comprehend the more complex topics of the conference, the genetics of human behavior will also be discussed in the convention. The conference will aim to address the specific areas of concern in the use of genetic technologies in human health as follows: • Assisted Reproduction Techniques (e.g. in-vitro fertilization)- ART are used to help fertility problems. Ethical issues are around the creation, selection, and disposal of embryos. These technologies can also require the use of sperm, eggs, or wombs from other women who are unrelated to the real parents and are not expected to play a role in raising the child. • Pre-implantation Genetic Diagnosis – In this technique an embryo at 6-10 cell stage can be tested and selected for or against a specific sex, disease and physical condition. Although, this method can be used for treatment purposes, however the ethical issues are around the use of this technology by parents to select a specific baby based on personal desires. • Cloning and Stem cells - Cloning is an essential tool of modern biology which has led to important drugs and new therapies. Cloning also has helped the understanding of genetic basis of human development and disease. Cloning has a potential to be used in producing a lifetime supply of therapeutic stem cells that are genetically matched to a patient. The ethical issues around cloning concern the production and destruction of a two-to-four-day-old embryo to make a line of embryonic stem cells. Another, concern is assuring that women donating eggs for research give proper informed consent. Some fear that a cloned embryo could be implanted into a woman resulting in a baby; a cloned human being. • Animal and Plant Cloning – Cloning animals and plants for specific purposes are becoming possible with new genetic technologies. Specific recombinant proteins to be used as therapeutic drugs are being produced in some animals and specific plants with certain characteristics are now possible to be made and some are currently available in the market for human use. The ethical issues around the animal and plant cloning are the consequence of releasing such new species in the environment and its impact on the society and human health. • Biobank – Biospecimens are being stored in public and private repositories and contain genetic material to identify gene variations associated with human diseases and lead to diagnostic tests and targeted treatments for specific diseases. Ethical issues are around the methods used in obtaining informed consent, protect privacy and disclose of research results, ownership of biospecimens as intellectual property and the ethical use of the biospecimens. • New frontiers in genetics research–There have been many new genetic technologies developed and there are plans to be applied in human health. • The research areas such as Human Behavior, Epigenetics, Nutrigenomics are some of the topics that are planned to be addressed in the forum. I look forward to your active participation in the conference and receive articles examining the issues related to ethical and legal aspects of Medical Biotechnology. https://www.AJMB.org/En/Article.aspx?ID=157 Ali M. Ardekani Sat, 17 Apr 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Development of a Latex Agglutination Test as a Simple and Rapid Method for Diagnosis of Trichomonas vaginalis Infection Trichomoniasis is a worldwide infection and due to its complications rapid and accurate diagnosis of infection especially in pregnant women is very important. In this study, development of a latex agglutination test using native antigens for rapid diagnosis of trichomoniasis is investigated. Trichomonas vaginalis was harvested from TYIS33 culture medium and anti Trichomonas vaginalis antiserum was raised in rabbits. Salt precipitation method was used for antibody purification. Polyesteren latex particles coated with purified antibody and used for detection of Trichomonas vaginalis. Clinical samples of vaginal discharge were collected from 500 women and examined for Trichomonas vaginalis by using wet mount, culture and latex agglutination tests. Sensitivity and specificity of latex test was determined considering culture as golden standard. Sensitivity and specificity of latex agglutination test was 100% and 81% and those of wet mount were 33.3% and 100%, respectively. Positive and negative predictive values of latex agglutination test were 6% and 100%, respectively. Due to inconvenient sensitivity and specificity of the latex agglutination test developed in this study, further work is recommended to improve the test. https://www.AJMB.org/En/Article.aspx?ID=31 Hossein Yousofi Darani, Firuzeh Ahmadi, Nozhat Zabardast, Hossein Ali Yousefi, Hedayat Shirzad Sat, 17 Apr 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production and Characterization of Mouse Monoclonal Antibodies Recognizing Multiple Subclasses of Human IgG Different IgG subclass profiles are produced in response to different antigenic stimuli in a variety of diseases. IgG subclass levels may reflect disease severity. Quantification of IgG subclasses depends on the availability of specific Monoclonal antibodies (MAbs). In the present study seven hybridoma clones producing MAbs reactive with multiple subclasses of human IgG were established. Splenocytes from Balb/c mice immunized with Fc fractions of human IgG1 or IgG2 myeloma proteins were fused with mouse myeloma cells. Fused cells were selected and cloned by limiting dilution assay. Antibody secreting cells were screened by Enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified human myeloma paraproteins of different IgG subclasses by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. The MAbs were found to react with triple IgG subclasses, including IgG1,2,4 (n=4) and IgG1,2,3 (n=3). Immunoblotting studies revealed recognition of linear (n=4) or conformational (n=3) epitopes by these MAbs. The most abundant cross-reactivity (71.4%) was observed with monkey Ig while no cross-reactivity was detected with hen and cat sera. The MAbs mostly displayed a restricted pattern of cross-reactivity and one of them did not bind to any of the animal sera tested. The affinity constant of 3 MAbs was measured by ELISA. Based on the data obtained from this study, mouse MAbs reactive with multiple human IgG subclasses are directed to a variety of immunogenic epitopes, mostly shared with IgG of other species. These MAbs are valuable tools for purification of non-reactive IgG subclasses through negative affinity chromatography. These MAbs could also provide an opportunity for epitope mapping of the Fc region of IgG, as well as serological phylogenetic studies. https://www.AJMB.org/En/Article.aspx?ID=28 Fatemeh Hajighasemi, Fazel Shokri Thu, 15 Apr 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of Human Chorionic Gonadotropin Treatment on Recipient Mouse Germ Cell Proliferation Following Spermatogonial Stem Cell Transplantation of Neonatal Donor Mice Spermatogonia are the male germ line stem cells whose life long expansion is needed for permanent production of spermatozoa. The present study was designed to examine the effect of hCG treatment on germ cell proliferation following stem cell transplantation in mice. Spermatogonial stem cells were isolated from neonatal mice testes and characterized by alkaline phosphatase, immunoreactivity and morphological analysis. hCG was injected into normal and cell transplanted mice. We then evaluated the testosterone levels and cell number in normal mice. After that, cyclin B1 gene expression was investigated in transplanted mice. Different doses of busulfan were injected to investigate the effects of chemotherapy on morphological criteria and preparation of recipient mice for transplantation. In this report we show proliferative potential of spermatogonial stem cells after cytotoxic treatment, transplantation efficiency by semi-quantitative RT-PCR, and hCG effect on stem cell regeneration in normal mice and following cell transplantation. The results indicate that spermatogonial stem cells can proliferate after transplantation, and the efficiency of their transplantation depends on hormonal treatment. Therefore, hormonal treatment after stem cell transplantation will be a powerful avenue for increasing the efficiency of transplantation and fertility restoration. https://www.AJMB.org/En/Article.aspx?ID=27 Mohammad Mehdi Akhondi, Reza Akbarzadeh Najar, Mahmood Jeddi-Tehrani, Mohammad Reza Sadeghi, Amir-Hassan Zarnani, Hodjattallah Rabbani, Sheida Salehkhou, Leila Eini, Fatemeh Hoseinzadeh, Mahnaz Heidari Wed, 14 Apr 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Flow Cytometric Analysis of 4-HPR-induced Apoptosis and Cell Cycle Arrest in Acute Myelocytic Leukemia Cell Line (NB-4) In many acute leukemias, normal differentiation does not occur. However, in many cell lines derived from hematologic malignancies, differentiation or programmed cell death (apoptosis) can be induced by variety of agents including: Vitamin analogs, demethylating agents, cyclic AMP analogs and anti-proliferative agents. To the best of our knowledge there has been not any study specifically to analyze apoptotic and anti-proliferative effects of 4-HPR (a vitamin analog) in NB-4 cell line. To test whether this drug has activity in acute myeloid leukemia (AML), we first analyzed the anti-proliferative effect of 4-HPR in one AML cell line (NB-4) using MTT Assay. Next we tested whether this drug induced apoptotic cell death. The ability of this compound to induce apoptosis of cancer cells was examined by Annexin V-FITC Assay using Flow cytometry. We also analyzed the cell cycle progression by PI staining using flow cytometry. Using MTT assay, NB-4 cells exhibited increased inhibition of proliferation at micromolar concentrations of 4-HPR at 24, 48 and 72 hrs post treatment. Flow cytometry analysis indicates that 4-HPR is a potent inducer of in vitro apoptotic cell death, and cell cycle analysis revealed an increase in S phase population. In total, the results indicate that 4-HPR is a strong inhibitor of AML cell proliferation and a potent inducer of in vitro apoptotic cell death. Further studies are required to evaluate the in vitro effects of 4-HPR in AML blasts derived from AML patients. https://www.AJMB.org/En/Article.aspx?ID=26 Shahrzad Soleymani Fard, Mahmood Jeddi-Tehrani, Mohammad Mehdi Akhondi, Mehrdad Hashemi, Ali M. Ardekani Wed, 14 Apr 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial I have recently sent out a letter to many of our colleagues and friends in the Iranian scientific community informing them of the recent addition of AJMB to two of the world’s largest databases, namely: Scopus and Embase. In case the spectrum of the coverage by these databases are not known to the AJMB readers in Iran, I have decided to provide you with some information about these two databases obtained from the official company website. Scopus (launched in November 2004) is a database of abstracts and citations and covers nearly 18,000 titles from more than 5,000 international publishers, including coverage of 16,500 peer-reviewed journals in the scientific, technical, medical and social sciences as well as fields in arts and humanities. It is owned by Elsevier and searches in Scopus incorporate searches of scientific web pages (435 Million), patent databases (23 Million) from 5 patent offices (US Patent and Trademark Office, European Patent Office, Japan Patent Office, World Intellectual Property Organization and UK Intellectual Property Office), article-in-press from over 3000 journals and full coverage of Medline titles. Embase or the Excerpta Medica Database is a biomedical and pharmacological database produced also by Elsevier and contains over 11 million records from 1947 to the present date. Each record is fully indexed and covers over 5,000 biomedical journals from 70 countries and is available online through a number of database vendors. Embase has a holding of more than 2,000 biomedical titles that are not offered by the Medline. Embase delivers comprehensive, authoritative, and reliable coverage of the most relevant biomedical literature. Now that AJMB has been recognized as a journal deserving to be included in these databases, it is up to the Iranian scientists to support the AJMB’s mission of publishing high quality articles in the field of Medical Biotechnology from Iran and worldwide. I continue to look forward to receiving your high quality articles and would appreciate if you could inform and encourage your colleagues to submit their articles to AJMB and enjoy its international exposure among thousands of other medical scientific journals in the world. https://www.AJMB.org/En/Article.aspx?ID=158 Ali M. Ardekani Mon, 15 Mar 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial As our valued readers you may well be aware that, the Avicenna Journal of Medical Biotechnology (AJMB) has recently been approved by the Commission for the Accreditation and Improvement of Iranian Medical Journals (CAIMJ) in the Ministry of Health and Medical Education. The AJMB is now included in the final list of accredited scientific and research journals in Iran and articles published in the AJMB will receive the appropriate credits from all the research/teaching institutes and Universities throughout the country. This is a great news for all our readers, contributors and particularly members of the editorial board and associates whose efforts in the past two years have brought this recognition from the Ministry of Health and Medical Education. I hope such acknowledgement will further facilitate the submission of high quality articles from the researchers in Iran who are working in the field of Medical Biotechnology. I must also inform our dear readers that the AJMB in the past year has been abstracted/ indexed in the databases as follows: Google Scholar, Genamics JournalSeek, Index Copernicus, EBSCO, Academic Search Complete, Serials Solution, Ulrichs Periodical Directory, Open J-Gate, Electronic Journals Library EZB, Scientific Information Database (SID), EMRmedex, IranMedex and Magiran. The AJMB will be added to further databases gradually as the number of published issues increases in the near future. Inclusion in the databases that index so many international journals in the field of medicine and medical biotechnology would certainly bring wider readership worldwide for the articles published in the AJMB. Therefore, I would like to invite all scientists worldwide who work in the field of Medical Biotechnology to submit their works for publication in the AJMB. We would like to consider this journal as an international journal rather than a journal of a particular geographical region. https://www.AJMB.org/En/Article.aspx?ID=154 Ali M. Ardekani Fri, 01 Jan 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Hepatic Tissue Engineering Using Scaffolds: State of the Art <p>Severe hepatic failure accounts for many deaths and raises medical costs each year worldwide. Currently, liver transplantation is the most common therapeutic option for patients with end-stage chronic liver disease. Due to decrease in the number of organ donors, many in need of transplantation continue to remain on the waiting list. Hepatic Tissue Engineering is a step toward alleviating the need for organ donors. Regenerative medicine and tissue engineering require two complementary key ingredients as follows: 1) biologically compatible scaffolds that can be readily adopted by the body system without harm, and 2) suitable cells including various stem cells or primary cells that effectively replace the damaged tissues without adverse consequences. Yet many challenges must be overcome such as scaffold choice, cell source and immunological barriers. Today, hepatogenic differentiation of stem cells has created trust and promise for use of these cells in hepatic tissue engineering and liver replacement. However, using suitable scaffolds is an important key to achieving the necessary functions required for hepatic replacement. In recent years, different scaffolds have been used for liver tissue engineering. In this review, we have presented different concepts in using cell /scaffold constructs to guide hepatic tissue engineering.</p> https://www.AJMB.org/En/Article.aspx?ID=19 Somaieh Kazemnejad Fri, 01 Jan 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Sperm Chromatin Integrity: Etiologies and Mechanisms of Abnormality, Assays, Clinical Importance, Preventing and Repairing Damage <p>The standard semen analysis is the first line and the most popular laboratory test in the diagnosis of male fertility. It evaluates sperm concentration, motility, morphology and their vitality. However, it is well-known that normal results of semen analysis can not exclude men from the causes of couples? infertility. One of the most important parameters of sperm in its fertilizing potential is &quot;Sperm chromatin integrity&quot; that has direct positive correlation with Assisted Reproductive Techniques (ART) outcomes including; fertilization rate, embryo quality, pregnancy and successful delivery rate. It seems that sperm DNA chromatin integrity provides better diagnostic and prognostic approaches than standard semen parameters. For these reasons under-standing the sperm chromatin structure, etiology of sperm chromatin abnor-mality, identification factors that disturbs sperm chromatin integrity and the mechanism of their action can help in recognizing the causes of couples? infertility. Various methods of its evaluation, its importance in male fertility, clinical relevance in the outcomes of ART and application of laboratory and medical protocols to improve this integrity have valuable position in diagnosis and treatment of male infertility. There has recently been interest in the subject and its application in the field of andrology. Therefore, with regard to the above mentioned importance of sperm chromatin integrity, this review article describes details of the useful information pertaining to sperm DNA damage including the origins, assessments, etiologies, clinical aspects, and prevention of it.</p> https://www.AJMB.org/En/Article.aspx?ID=20 Azita Hekmatdoost, Niknam Lakpour, Mohammad Reza Sadeghi Fri, 01 Jan 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir TSGA10 is Specifically Expressed in Astrocyte and Over-expressed in Brain Tumors <p>In this study TSGA10 has been demonstrated as a testis-specific human gene that encodes a protein localized in sperm-tail and conserved in ciliary structure. Further investigations showed TSGA10 signalling and expression during embryogenesis, brain development and some malignancies including brain tumors. Given the role of this protein in neuronal development and in certain tumors, it could potentially serve as a diagnostic marker and therapeutic target in brain tumors. Therefore, using immunohistochemistry, we evaluated the localization of TSGA10 in different regions of brain, and its pattern/level of expression in tissue microarray (Cybrdi) containing human brain tumors and normal brain. In rat specimens, TSGA10 was mainly expressed in subventricular zone, hippocampus and granular layer of cerebellum of the brain. The antibody also stained the diverse and different types of human brain cancers. The TSGA10 was strongly over-expressed in glioblastoma and astrocytoma when compared to normal human brain. The expression of TSGA10 was also confirmed in astrocyte derived from a human astroctyoma cell line by immunocytochemistry. This study indicates that TSGA10 can be used as an immunohistochemical marker for human neuroglia and astrocyte cells and is over-expressed in brain tumors.</p> https://www.AJMB.org/En/Article.aspx?ID=21 Babak Behnam, Ali Chahlavi, Jogi Pattisapu, Jonathan Wolfe Fri, 01 Jan 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Antinociceptive and Antipyretic Activities of Amaranthus Viridis Linn in Different Experimental Models Methanolic extract of whole plant of Amaranthus viridis L (MEAV), was screened for antinociceptive activity using acetic acid induced writhing test, hot plate test and tail immersion test in mice. In a similar way a screening exercise was carried out to determine the antipyretic potential of the extract using yeast induced pyrexia method in rats. Administration of the extracts was applied to both laboratory animals at the doses of 200 and 400 mg/kg body weight, respectively. The results of the statistical analysis showed that MEAV had significant (p<0.01) dose dependent antinociceptive and antipyretic properties at 200 and 400 mg/kg. Hence present investigation reveals the antinociceptive and antipyretic activities of methanolic extract of Amaranthus viridis. https://www.AJMB.org/En/Article.aspx?ID=22 Bagepalli Srinivas Ashok Kumar, Kuruba Lakshman, Korala Konta Narsimha Jayaveera, Devangam Sheshadri Shekar, Chinna SwamyVel Muragan, Bachappa Manoj Fri, 01 Jan 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Effects of Sperm Chromatin Integrity on Fertilization Rate and Embryo Quality Following Intracytoplasmic Sperm Injection Sperm chromatin integrity has been being recognized as an important factor in male fertility. During normal fertilization, high quality sperm with intact chromatin are selected through natural selection in journey from vagina to fallopian tube. However, using Assisted Reproductive Techniques, particularly ICSI, the natural selection is bypassed. Therefore sperm with DNA breakage have the opportunity to fertilize the egg which may lead to decreased embryo quality and implantation rate. The aim of this study was to evaluate the effects of sperm chromatin integrity on ICSI outcomes. A total of 200 semen samples were collected from couples undergoing ICSI and were analyzed according to WHO criteria. Each sample was evaluated for sperm chromatin integrity using four cytochemical assays and semen processing by swim up method. The ICSI was carried out according to a long-term pituitary down-regulation protocol. The correlation between sperm parameters, sperm chromatin integrity and ICSI outcomes (fertilization rate and embryo quality) was examined. The mean number of oocyte, fertilization rate and cleavage embryos per cycles was 7.5±5.0, 74.06%±25 and 5.4±3.6, respectively. There was not significant correlation between the results of chromatin assays (AO, AB, TB, and CMA3) and fertilization outcomes following ICSI. The fertilization rate was significantly higher for a group with less than 10% chromatin abnormality (p<0.05). Sperm chromatin integrity is essential for successful fertilization, embryo development and normal pregnancy. A protamine deficiency appeared to affect fertilization rate and embryo quality. However, the presence of confounding factors such as selection of spermatozoa according to normal morphology may influence the effect of sperm chromatin status on ICSI outcomes. https://www.AJMB.org/En/Article.aspx?ID=23 Mohammad Reza Sadeghi, Mahshid Hodjat, Niknam Lakpour, Soheila Arefi, Naser Amirjannati, Tahereh Modarresi, Hossain Hossaini Jadda, Mohammad Mehdi Akhondi Fri, 01 Jan 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Effect of the Duration of In Vitro Maturation (IVM) on Parthenogenetic Development of Ovine Oocytes The aim of this study was to compare the effect of time of parthenogenetic activation (22 hr versus 27 hr after In Vitro Maturation-IVM) on in vitro development of ovine oocytes using either single (Ionomycin 5 ?M for 5 min or Ethanol 7% for 7 min) or combined (ionomycin and ethanol with 6-DMAP 2 mM for 3 hr) activation treatments. The abattoir-derived in vitro matured activated oocytes were cultured in modified synthetic oviductal fluid and assessed for the cleavage, blastocyst, and hatching rates. The single-activated oocytes had a reduction in cleavage, blastocyst and hatching rates compared to the combined-activated oocytes (except for the cleavage at 27 hr). In single-treated groups the rates of cleavage and blastocyst were increased as the maturation time was extended from 22 hr to 27 hr. The numbers of total cells and Inner Cell Mass (ICM), though insignificant, were greater in combined-treated groups compared to the single treatment. The number of ICM in Eth+6-DMAP group activated at 27 hr was lower than 22 hr. Nonetheless, irrespective of the activation protocol, development to the blastocyst stage, the numbers of total cell, ICM, and cell allocation (ICM/total cells) were significantly lower in parthenogenetic than fertilized embryos. In conclusion, though the cleavage and blastocyst rates in single-treated groups were positively influenced by the extension of duration of IVM (27 hr), there was a trend of decreased numbers of total cells and ICM in slightly aged oocytes. Moreover, developmental potential of ovine parthenotes, especially in young oocytes, was improved by the addition of 6-DMAP to the activation regimen. https://www.AJMB.org/En/Article.aspx?ID=24 Abolfazl Shirazi, Amin Bahiraee, Ebrahim Ahmadi, Hassan Nazari, Banafsheh Heidari, Sara Borjian Fri, 01 Jan 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Lack of Association Between Tumor Necrosis Factor-alpha -308 G/A Polymorphism and Risk of Developing Late-Onset Alzheimer's Disease in an Iranian Population Late-onset Alzheimer's Disease (LOAD) is a neurodegenerative disorder and the most common form of dementia affecting people over 65 years old. Alzheimer’s disease is a complex disease with multi-factorial etiology. Inflammation has been approved to have an important role in the pathogenesis of Alzheimer’s disease (AD). TNF-a is a main pro-inflammatory cytokine that plays an essential role in initiation and regulation of inflammatory responses. Several studies have shown the probable association of polymorphism at TNF-a gene’s promoter with AD pathogenesis. This study was performed to determine whether this polymorphism contributes to the risk for late-onset Alzheimer's disease (LOAD) in Iranian population. One hundred and forty AD patients and 158 healthy controls were recruited in the study. Following extraction of genomic DNA, using PCR/RFLP methods the genotype and allele frequencies were determined in case and control subjects. The statistical analysis showed no significant difference in the allele and genotype frequencies due to this polymorphism between the two groups. Also after stratifying the subjects by their APOE-e4 status, no significant association was observed. Our results suggest that Tumor necrosis factor-alpha (TNF-?) -308 G/A is not a risk or protective factor for late-onset Alzheimer’s disease in Iranian population. However, to confirm these results further study with a bigger sample size may be required. https://www.AJMB.org/En/Article.aspx?ID=25 Mehdi Manoochehri, Koorosh Kamali, Mehdi Rahgozar, Mina Ohadi, Homa Farrokhi Karibozorg, Hamid Reza Khorram Khorshid Fri, 01 Jan 2010 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Noncoding RNAs and Cancer The eukaryotic complexity involves the expression and regulation of genes via RNA-DNA, RNA-RNA, DNA-protein and RNA-protein interactions. Recently, the role of RNA molecules in the regulation of genes in higher organisms has become more evident, especially with the discovery that about 97% of the transcriptional output in higher organisms are represented as noncoding RNAs: rRNA, snoRNAs, tRNA, transposable elements, 5' and 3' untranslated regions, introns, intergenic regions and microRNAs. MicroRNAs function by negatively regulating gene expression via degradation or translational inhibition of their target mRNAs and thus participate in a wide variety of physiological and pathological cellular processes including: development, cell proliferation, differentiation, and apoptosis pathways. MicroRNA expression profiles in many types of cancers have been identified. Recent reports have revealed that the expression profiles of microRNAs change in various human cancers and appear to function as oncogenes or tumor suppressors. Abnormal microRNA expression has increasingly become a common feature of human cancers. In this review, we summarize the latest progress on the involvement of microRNAs in different types of cancer and their potential use as potential diagnostic and prognostic tumor biomarkers in the future. https://www.AJMB.org/En/Article.aspx?ID=9 Mozhgan Moslemi Naeini, Ali M. Ardekani Tue, 01 Sep 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir DNA Immunization as an Efficient Strategy for Vaccination The field of vaccinology provides excellent promises to control different infectious and non-infectious diseases. Genetic immunization as a new tool in this area by using naked DNA has been shown to induce humoral as well as cellular immune responses with high efficiency. This demonstrates the enormous potential of this strategy for vaccination purposes. DNA vaccines have been widely used to develop vaccines against various pathogens as well as cancer, autoimmune diseases and allergy. However, despite their successful application in many pre-clinical disease models, their potency in human clinical trials has been insufficient to provide protective immunity. Several strategies have been applied to increase the potency of DNA vaccine. Among these strategies, the linkage of antigens to Heat Shock Proteins (HSPs) and the utilization of different delivery systems have been demonstrated as efficient approaches for increasing the potency of DNA vaccines. The uptake of DNA plasmids by cells upon injection is inefficient. Two basic delivery approaches including physical delivery to achieve higher levels of antigen production and formulation with microparticles to target Antigen-Presenting Cells (APCs) are effective in animal models. Alternatively, different regimens called prime-boost vaccination are also effective. In this regimen, naked DNA is utilized to prime the immune system and either recombinant viral vector or purified recombinant protein with proper adjuvant is used for boosting. In this review, we discuss recent advances in upgrading the efficiency of DNA vaccination in animal models. https://www.AJMB.org/En/Article.aspx?ID=10 Azam Bolhassani, Sima Rafati Yazdi Tue, 01 Sep 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Biotechnology-Related Intellectual Property Law of Iran In this study, an attempt has been made to expound the Iranian law of intellectual property in relation to biotechnology. The most important themes studied are patents, industrial designs and trade marks. The latest relevant piece of legislation concerning the subject matters was passed in March 2008. However, the history of laws and regulations in this field goes back to early twentieth century (i.e. 1925). In this review, on the basis of the latest law passed in 2008, the topics explored are the responsible authority, patentable items and criteria, excluded items, registration procedure, rights conferred and sanctions. At the end, an attempt is made to put forward a few points as an analysis of the above Law from a critical point of view. https://www.AJMB.org/En/Article.aspx?ID=11 Mohammad Rasekh Tue, 01 Sep 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Forward Modeling of the Coumarin Antifungals; SPR/SAR Based Perspective Although, coumarins are a group of compounds which are naturally found in some plants, they can be synthetically produced as well. Because of their diverse derivatives, origin and properties most of them can be used for medicinal purposes. For example, they can be used against fungal diseases or in studying structure and biological properties of antifungal agents to discover new compounds with the similar activity. A Structure Property/Activity Relationship (SAR) can be utilized in prediction of biological activity of desired molecules. https://www.AJMB.org/En/Article.aspx?ID=12 Saeed Soltani, Shima Dianat, Soroush Sardari Tue, 01 Sep 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Anticancer Activity Compared Between Triptorelin and a New Gonadotropin Releasing Hormone Analogue Gonadotropin releasing hormone (GnRH) plays a key role in reproduction. This decapeptide is synthesized and released by hypothalamus and induces the pituitary gonadotrop cells to release pituitary gonadotropin hormones. In some extrapituitary compartments GnRH and its receptor act as part of the autocrine regulatory system of cell proliferation. The anticancer activity of GnRH and its analogues has been observed by many researchers. In this study the anticancer activity of a new analogue of GnRH and triptorelin was investigated by cell proliferation assay. Results indicate that proliferation of human breast and ovarian cancer cell lines are dose-dependently inhibited. The inhibitory efficiency of the new analogue is proved to be higher than the original triptorelin. In addition to its antimitogenic activity, evidence was found for the involvement of the apoptotic mechanism in the action of the new analogue and triptorelin. In conclusion, the new analogue can be considered as a good pharmaceutical candidate. https://www.AJMB.org/En/Article.aspx?ID=13 Mohammad Mirzaei Saleh-Abady, Abdolali Alizadeh, Fereshteh Shamsipour, Hossein Naderi-Manesh Tue, 01 Sep 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Green Synthesis of Small Silver Nanoparticles Using Geraniol and Its Cytotoxicity against Fibrosarcoma-Wehi 164 Many reports have been published about the biogenesis of silver nanoparticles using several plant extracts such as Pelargonium graveolens (P.graveolens- geranium) and Azadirachta indica (neem) but the capacity of their natural reducing constituents to form silver nanoparticles has not yet been studied. In this research the synthesis of silver nanoparticles using geraniol has been investigated. We successfully synthesized uniformly dispersed silver nanoparticles with a uniform size and shape in the range of 1 to 10 nm with an average size of 6 nm. Also the cytotoxicity of the prepared silver nanoparticles was investigated using a cancer cell line (Fibrosarcoma-Wehi 164). The cytotoxicity analysis of the sample shows a direct dose-response relationship; cytotoxicity increased at higher concentrations. At concentration of 1 µg/ml, silver nanoparticles was able to inhibit the cell line’s growth by less than 30%. Conversly, the presence of 5 µg/ml of silver nanoparticlse significantly inhibited the cell line’s growth (> 60%). The concentration necessary to produce 50% cell death was 2.6 µg/ml for this silver nanoparticles preapared with geraniol. https://www.AJMB.org/En/Article.aspx?ID=14 Mona Safaepour, Ahmad Reza Shahverdi, Hamid Reza Shahverdi, Mohammad Reza Khorramizadeh, Ahmad Reza Gohari Tue, 01 Sep 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir New Variations in the Promoter Regions of Human DOCK4 and RAP1A Genes, and Coding Regions of RAP1A in Sporadic Breast Tumors Breast cancer is the most common cancer among women in developed countries. The prevalence of the disease is increasing in the world. Its annual incidence among Iranian women is about 7000 cases. RAP1A, a tumor suppressor gene, is located at 1p13.3 and plays an important role in the cellular adhesion pathway and is involved in the pathogenesis of breast cancer. The DOCK4 gene, which is located at 7q31.1, specifically activates RAP1A gene. In the present study, DNA samples from 64 cases of sporadic breast tumors (referred to Mehrad Hospital in Tehran) were screened using PCR-SSCP method and the number of observed variations compared with the control group (100 normal women). Mutation detection for coding exons of RAP1A gene and the 500 bp upstream of transcription initiation site as promoters of both DOCK4 and RAP1A were carried out and compared with the control group. The promoter region of DOCK4 showed a heterozygous mutation with G>A transition at nucleotide -303 in a fibroadenoma case. With regard to RAP1A we found a heterozygous mutation, G>A transition in an adenoid cystic carcinoma case, and another heterozygous mutation, G>T transversion in an intraductal papilloma case both at nucleotide +45. A homozygous variation, T>A transversion was also found at nucleotide +29 of a fibroadenoma case. The differences in the frequency of variations mentioned above were not statistically significant. However Fisher’s exact showed significant difference for T>A transversion. Although, the higher frequency of these mutations and variations may be related to the disease, a larger sample size is needed for the confirmation of our findings. https://www.AJMB.org/En/Article.aspx?ID=15 Akram Jalali, Hassan Ebrahimi, Mina Ohadi, Masood Karimloo, Atena Irani Shemirani, Behrokh Mohajer Maghari, Hamid Reza Khorram Khorshid Tue, 01 Sep 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Ectopic Expression of Sortilin 1 (NTR-3) in Patients with Ovarian Carcinoma Gene expression profiling of ovarian carcinoma tissues has shown an increase of four-fold expression of SORT1 gene. Sortilin 1 (NTR-3) is a 95-100 kDa protein normally expressed in heart, brain, placenta, skeletal muscle, spinal cord, thyroid, and testis. However, its expression has never been reported in normal ovary. Here, we report expression of sortilin 1 in ovarian carcinoma tissues both at gene and protein levels. Sortilin 1 was expressed in all ovarian carcinoma patients (n=15) as well as ovarian carcinoma cell lines (n=5) regardless of their phenotypic characteristics. Non-malignant ovaries (n=6) did not express sortilin 1. The molecular basis for this ectopic expression is not yet clear. Our results showed a major cell surface expression of sortilin 1 rather than ER-Golgi compartment where it is mainly expressed. This finding may introduce sortilin 1 as a novel tumor marker for diagnosis of ovarian carcinoma and may signify its therapeutic value in targeted therapy. https://www.AJMB.org/En/Article.aspx?ID=16 Shayda Hemmati, Amir-Hassan Zarnani, Ahmad Reza Mahmoudi, Mohammad Reza Sadeghi, Haleh Soltanghoraee, Mohammad Mehdi Akhondi, Majid Tarahomi, Mahmood Jeddi-Tehrani, Hodjattallah Rabbani Tue, 01 Sep 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial Welcome to the second issue of the AJMB. I hope you have read and enjoyed the first edition. In this issue, like the first one, there is a mix of articles covering a wide range of topics including: Promoter analysis, DNA immunization, synthesis of nanoparticles, non-coding RNAs and Intellectual property (IP) issues. I believe the article entitled "Biotechnology-Related IP Law of Iran" is the first article of its kind published in Iran in which a comprehensive review of Iranian laws pertaining to biotechnology is addressed. Therefore, I encourage those who are interested in patenting a discovery and/or product in the field of biotechnology in Iran to read this article. On different note, the sixth National Biotechnology Congress took place on 13-15 August, 2009 at the Milad Tower Convention Center in Tehran. This three day congress appeared to be one of the most successful congresses held in the field of biotechnology in Iran. It was understood that about 1000 abstracts were accepted and the majority of them were in the disciplines of agriculture, medicine and marine biology. It was interesting to learn that (according to a research carried out by the organizers of the congress) from a good number of articles published in the area of biotechnology in 2009 (and indexed by ISI), ninety nine articles belonged to the authors and research centers in Iran. As the editor, I would like to take the opportunity to invite all researchers in the field of medical biotechnology to send in their original research works, review articles, short articles, etc. for publication in the AJMB. The AJMB is now indexed in several databases worldwide and it is anticipated to be included in further lists in the near future. https://www.AJMB.org/En/Article.aspx?ID=155 Ali M. Ardekani Sat, 15 Aug 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir The Place of Avicenna in the History of Medicine Avicenna, a Muslim scientist of the tenth and eleventh centuries has an important place in the history of medicine in Iran and the world. Furthermore, the modern medicine is laid upon the infrastructure of his medicine. In this article, the position of Avicenna in the medical history and the scientific influence of his medical works in particular Al-Canon in the development of medical literature and medical educational programs have been studied in a historical approach. In reviewing the position of Avicenna in the history of medicine in the Islamic world and the Europe, it was concluded that during 11th to 17th centuries, the scientific and educational activities of medicine in the world were moving on the pivot of Avicenna medicine or was under its intensive influence. https://www.AJMB.org/En/Article.aspx?ID=1 Jamal Moosavi Mon, 01 Jun 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Nutrigenomics and Cancer Cancer incidence is projected to increase in the future and an effectual preventive strategy is required to face this challenge. Alteration of dietary habits is potentially an effective approach for reducing cancer risk. Assessment of biological effects of a specific food or bioactive component that is linked to cancer and prediction of individual susceptibility as a function of nutrient-nutrient interactions and genetics is an essential element to evaluate the beneficiaries of dietary interventions. In general, the use of biomarkers to evaluate individuals susceptibilities to cancer must be easily accessible and reliable. However, the response of individuals to bioactive food components depends not only on the effective concentration of the bioactive food components, but also on the target tissues. This fact makes the response of individuals to food components vary from one individual to another. Nutrigenomics focuses on the understanding of interactions between genes and diet in an individual and how the response to bioactive food components is influenced by an individual’s genes. Nutrients have shown to affect gene expression and to induce changes in DNA and protein molecules. Nutrigenomic approaches provide an opportunity to study how gene expression is regulated by nutrients and how nutrition affects gene variations and epigenetic events. Finding the components involved in interactions between genes and diet in an individual can potentially help identify target molecules important in preventing and/or reducing the symptoms of cancer. https://www.AJMB.org/En/Article.aspx?ID=2 Ali M. Ardekani, Sepideh Jabbari Mon, 01 Jun 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Generation and Characterization of Mouse Hybridomas Secreting Monoclonal Antibodies Specific for Human IgG3 Mammalians express several subclasses of the IgG molecule. In human being there are four homologous IgG subclasses, each of which is structurally unique and has different functions. Quantification of IgG subclasses is fundamental to clinical assessment and diagnosis of many diseases as such assessments depends on the availability of subclassspecific antibodies (Abs), particularly monoclonal antibodies (MAbs). In the present study, we produced and characterized two murine MAbs specific for human IgG3 molecule. These MAbs were obtained by the fusion of myeloma cells with splenocytes from Balb/c mice immunized with heavy chain of a human IgG3 myeloma protein. Fused cells were selected in hypoxanthine, aminopterine and thymidine (HAT) medium and cloned by limiting dilution assay. Ab-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. Two stable hybridomas designated 1F18G7 and 1F18A11 were obtained secreting MAbs specific for Fc fragment of human IgG3. None of these MAbs showed cross-reactivity with other immunoglobulin isotypes derived from human and nine other animals, except 1F18A11 which displayed a weak cross-reactivity with only dog serum. Immunoblotting results indicate that these MAbs react with linear epitope(s) located in the heavy chain of human IgG3 molecules. The affinity constant of 1F18G7 and 1F18A11 MAbs was found to be 0.81×109 Mol –1 and 0.71×109 Mol –1, respectively, as measured by ELISA. These two MAbs with relatively high affinity can be useful tools for quantification of IgG3 subclass levels in human serum. https://www.AJMB.org/En/Article.aspx?ID=3 Fatemeh Hajighasemi, Fazel Shokri Mon, 01 Jun 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Conjugation of Monoclonal Antibodies to Super Paramagnetic Iron Oxide Nanoparticles for Detection of her2/neu Antigen on Breast Cancer Cell Lines Conjugation of monoclonal antibodies to super paramagnetic nanoparticles is an effective method for cancer diagnosis and treatment. In this study the humanized anti her2/neu monoclonal antibody- Herceptin- was conjugated to super paramagnetic iron oxide (SPIO) nanoparticles using EDC method. The concentration of the conjugated antibodies was measured by Bradford assay. The antibody-nanoparticle conjugates were incubated with SKBR-3 and T47D human breast carcinoma cell lines and the presence of the conjugates on cell surface was confirmed by Prussian blue iron staining method. Conjugation of Herceptin to SPIO resulted in a precipitate-free conjugate containing 20µg antibody/mg SPIO. Prussian blue iron-staining of cells showed successful binding of the conjugates to the cell surfaces. Conjugation of monoclonal antibodies to SPIO may be a useful method for detection of tumor cells, especially by MRI techniques. https://www.AJMB.org/En/Article.aspx?ID=4 Fereshteh Shamsipour, Amir-Hassan Zarnani, Roya Ghods, Mahmood Chamankhah, Flora Forouzesh, Sedigheh Vafaei, Ali Ahmad Bayat, Mohammad Mehdi Akhondi, Mohammad Ali Oghabian, Mahmood Jeddi-Tehrani Mon, 01 Jun 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Producing a Mammalian GFP Expression Vector Containing Neomycin Resistance Gene The green fluorescent protein (GFP) was originally isolated from the Jellyfish Aequorea Victoria that fluoresces green when exposed to blue light. GFP protein is composed of 238 amino acids with the molecular mass of 26.9 kD. The GFP gene is frequently used in cellular and molecular biology as a reporter gene. To date, many bacterial, yeast , fungal, plants, fly and mammalian cells, including human, have been created which express GFP. Martin Chalfie, Osamu Shimomura, and Roger Tsien were awarded the 2008 noble prize in chemistry for their discovery and development of GFP. In many studies on mammalian cells, GFP gene is introduced into cells using vector-based systems or a recombinant virus to track the location of a target protein or to study the expression level of the gene of interest, but in these studies there is no selection marker to normalize transfection. According to the importance of neomycin gene as a selection marker in mammalian cells, we aimed to produce a GFP expression vector that contains neomycin gene. GFP gene was separated from pEGFP-N1 vector and was inserted in the back-bone of pCDNA3.1/His/LacZ vector that contained the neomycin gene. The resulted vector contained GFP beside neomycin gene. https://www.AJMB.org/En/Article.aspx?ID=5 Manizheh Izadi, Maryam Abiri, Mohammad Keramatipour Mon, 01 Jun 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Construction of a High Efficiency PCR Products Cloning T Vector Using pGEM-5zf (+) A highly efficient cloning vector was constructed for cloning PCR products by inserting an 80 bp DNA fragment into pGEM-5zf (+) vector. The Xcm I digestion of this vector gave rise to a 3’ overhanging deoxythymidine offering the possibility of cloning PCR products with 3' adenosine overhang created by Taq DNA polymerase. Furthermore, two EcoR I sites were added to the construct for identification of recombinant plasmids using a single restriction enzyme. Taken together, the more efficient cloning performance and the lower cost of this vector as compared to the commercial T vector, suggests that it may be one of the best T vectors for cloning of PCR products. https://www.AJMB.org/En/Article.aspx?ID=6 Yaofeng Zhao, Zhancai Liu, Shuyang Yu, Sicheng Wen, Lennart Hammarstrom, Hodjattallah Rabbani Mon, 01 Jun 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Assessment of Thyroglobulin Expression in Reproductive Organs at Different Stages of Mouse Estrous Cycle Prevalence of abortion is higher in women with autoimmune thyroid disease. In the majority of cases, however, no abnormality of thyroid function is detected despite the high levels of antithyroid antibodies. The direct influence of such harmful autoantibodies in female reproductive organs may serve a role in pregnancy loss. In this study, expression of thyroglobulin in the reproductive tissues of cycling mice has been evaluated. Stages of estrous cycle were determined by cellular morphology and ratio of epithelial cells to leukocytes in vaginal smear of Balb/C mice. At each phase, the mice were sacrificed and their uterus, ovary and fallopian tubes were removed. Expression of thyroglobulin-specific transcript in endometrium was investigated by two sets of primers using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, expression of thyroglobulin in reproductive tissues was assessed by immunohistochemistry and dot blot analysis. The results showed that thyroglobulin mRNA is not expressed in endometrial tissue of Balb/C mice at any stage of estrous cycle. Immunohistochemical analysis also confirmed that thyroglobulin or its cross reactive-antigens are not expressed at the protein level in the female reproductive organs. The results showed that thyroglobulin was not expressed in the reproductive organs of female mice. It is plausible that antithyroglobulin antibodies could interact with newly-generated antigens during placentation and pregnancy. https://www.AJMB.org/En/Article.aspx?ID=7 Ali Moravvej, Mahmood Jeddi-Tehrani, Ali Reza Salek Moghaddam, Pouneh Dokouhaki, Mahdi Shekarabi, Roya Ghods, Mahdi Shahbazi, Jamileh Ghasemi, Parivash Danesh, Ahmad Reza Mahmoudi, Amir-Hassan Zarnani Mon, 01 Jun 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Production and Purification of Streptokinase by Protected Affinity Chromatography Streptokinase is an extracellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy. The rate of bacterial growth and streptokinase production was studied in condition of excess glucose addition to culture media and its pH maintenance. The streptokinase product of the bacterial culture was preliminary extracted by salt precipitation and then purified by affinity chromatography on plasminogen substituted sepharose-4B in a condition that the plasminogen active site was protected from streptokinase-induced activation. The purity of streptokinase was confirmed by SDS-PAGE and its biological activity determined in a specific streptokinase assay. The results showed that in the fed –batch culture, the rate of streptokinase production increased over two times as compared with the batch culture while at the same time, shortening the streptokinase purification to a single step increased the yield over 95 % at the chromatography stage. https://www.AJMB.org/En/Article.aspx?ID=8 Mohammad Babashamsi, Mohammad Hossein Razavian, Mohammad Reza Nejadmoghaddam Mon, 01 Jun 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir Editorial In this first editorial for the “first issue” of Avicenna Journal of Medical Biotechnology (AJMB), I thought I should provide our dear readers a little background as well as the reasons used in making decision to publish an English journal in the field of Medical Biotechnology in Iran. Although the field of Medical Biotechnology is still at its infancy in many ways, it has travelled a long journey since its inception in early 1980s, a period parallel to the emergence of DNA recombinant technology and genetic engineering. At that time, the impact of genetic engineering in medicine was predicted to be great and this proved to be correct since the science of Medical Biotechnology expanded later on to include a variety of fields such as molecular medicine, cellular and molecular therapies, applied immunology, nanomedicine, genomics and proteomics, bioinformatics, tissue engineering, biosensors and pharmaceuticals. As a consequence of the great impacts that Medical Biotechnology has had in commercial, legal, ethical and social areas, AJMB is intending to provide a venue for articles discussing these issues in specific areas pertaining to the application of technologies in medicine in general. Since the establishment of biotechnology research centers in Iran, including National Center for Biotechnology and Genetic Engineering more than 15 years ago as well as Avicenna Research Institute, several English language journals have been published in the field of Biotechnology in Iran. AJMB is another English journal in this field but what makes it distinguishable from other journals is its scope of coverage and the comprehensiveness of the fields in medical biotechnology. Regarding the journal’s name, after many hours of deliberations, the scientific staff at Avicenna Research Institute voted for the selection of the name “Avicenna” because of three reasons: 1. Avicenna simply best represents the Iranian-Islamic scientific culture and medicine in history 2. Avicenna is a name that is well recognized and respected in science and medicine throughout the world. 3. Avicenna is a name that has been chosen for a research institute that is focused on the application of biotechnologies in the areas of reproductive biology and cancer. Therefore, it is most appropriate to have Avicenna as the name for a Journal that publishes articles in Medical Biotechnology. As we all know, Avicenna is a Latinized name of Ibn-e-Sina and his contributions to almost all fields of medical sciences and philosophy in the world is well known. Recently, Avicenna’s contributions to science and medicine have been recognized by major international organizations. For example, the United Nations Educational, Scientific and Cultural Organization (UNESCO) has established a virtual campus dedicated to open distance learning in eleven Mediterranean countries under the name of The Avicenna Virtual Campus Project. Also another project with the name of Avicenna Directories of Educational Institutions for the Health Professionals has been initiated under the auspices of the World Health Organization (WHO). I hope the name of “Avicenna” at both Avicenna Research Institute and Avicenna Journal of Medical Biotechnology provides the inspiration for conducting good science and become a place where high quality articles are published. I look forward to receiving your articles in the future and hope the readers of AJMB make this journal, the journal of their choice to publish the results of their research work in the field of Medical Biotechnology. https://www.AJMB.org/En/Article.aspx?ID=156 Ali M. Ardekani Sun, 15 Mar 2009 00:00:00 GMT en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir https://www.AJMB.org/En/Article.aspx?ID=60577 en eslamifar@gmail.com (Mohsen Eslamifar) ajmb@avicenna.ac.ir