REPORTS OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
Year:2020 | Volume:9 | Issue:2|Papers Count:15

Background: Multiple sclerosis (MS) is a multifactorial condition in which many genetic and environmental factors interfere. The association between genes involved in the immune system and MS was previously reported. The aims of this study were to evaluate 14 SNPs of HLA-DRA, 14 SNPs of IL2RA with severity of MS through Expanded Disability Status Scale (EDSS) and Annualized Relapse Rate (ARR). Methods: 102 patients with MS referred to Sina hospital in Tehran, Iran, were diagnosed and studied based on McDonald’, s guideline, clinical signs, and brain imaging procedures. All patients were included in the study following informed consent. Genotyping study of 14 variants in the HLA-DRA, and 14 variants in IL2RA was conducted by Sanger sequencing. Disease outcomes including EDSS and ARR were registered. Outcome measures between different genotypes of each SNPs were compared separately. Results: Among 14 SNPs in IL 2RA the genotypes of rs12722489 showed a significant association with ARR in two consecutive years. Mean ARR1 was 1. 06±, 1. 12, 0. 20±, 0. 34 and 0. 31±, . 50 for AA, GA, and GG genotypes, respectively (p value= 0. 008). Mean ARR2 was 1. 5±, 1. 08, 0. 28±, 0. 40, and 0. 42±, 0. 55 for AA, GA, and GG, respectively (p value= 0. 001). Regression analysis showed a significant association between rs12722489 with ARR1 and ARR2, removing the potential confounding mediators. No significant association was found between SNPs in HLA-DRA with the attack rate and severity of MS. Conclusions: The rs12722489 of IL-2RA has an association with ARR, but not with EDSS.

Background: Due to extensive damage to the skin, burn victims may acquire life-threatening infections. Though the skin primarily protects against microbial invasions, a large number of bacteria, fungi, and viruses can be isolated from burn patients, specifically Pseudomonas aeruginosa, a gram-negative bacterium with both intrinsic and acquired antibiotic resistance (AR) properties. nalB mutations can be found on the mexR in the P. aeruginosa chromosome. This mutation can induce overexpression of the mexAB-oprMoperon, and affect the MexAB-OprM efflux pump, which removes antimicrobial agents from the bacterial cell. Identifying nalB mutants can be useful for monitoring factors affecting AR. Methods: In this study, 70 P. aeruginosa isolates identified from burn patients and antibacterial sensitivity was evaluated using the Kirby-Bauer method. We also investigated nalB mutations in samples using molecular methods including Polymerase reaction chain (PCR) and Sequencing. Results: We identified nalB mutations in 16 isolates. We also found that the increasing effect of nalB mutants induces hyper production activity of MexAB-OprM resulting in AR. Overall, these findings compliment the findings of previous reports. Conclusions: According to the resistance patterns of the samples, both Amikacin and Ciprofloxacin showed the highest resistance (%). Further, the relationship between Ciprofloxacin resistance and nalB mutations was statistically significant (p= 0. 016). The results confirm that the increasing effect of nalB mutants on hyper production activity of MexAB-OprM leads to AR.

Background: Introduction: Oxytocin (OT) has been proposed to assist in the regulation of bone remodeling and to exert an antiosteoporotic effect. We evaluated the possible protective effect of OT against bone degeneration in ovariectomized (OVX) rats. Methods: The study was performed on three groups of adult female rats,group I was subjected to sham operation, group II was subjected to ovariectomy, and group III was subjected to ovariectomy and intraperitoneal injection with OT for eight successive weeks. At the end of the study, bone mass density (BMD) was measured,then the rats were euthanized and their blood and bone tissues were examined. Results: The group II rats had significantly less BMD and greater serum bone-specific alkaline phosphatase (bALP), osteocalcin (OC), and tartrate-resistant acid phosphatase (TRAP) levels than the group I rats. Furthermore, group II rats had fewer osteocytes and osteoblasts, and less OPG/RANKL mRNA expression than group I rats. The groups I and III and rats showed no significant differences in BMD, bALP, OC, TRAP, OPG/RANKL mRNA expression, or osteocyte and osteoblast numbers. Conclusions: Oxytocin may have an antiosteoporotic effect in OVX rats.

Background: Mycoplasma pneumoniae is one of the widespread causes of community-acquired pneumonia (CAP). Over recent years, the widespread use of macrolides has led to the emergence of macrolide-resistant M. pneumoniae (MRMP) resulted from mutations at specific positions of domain V of the 23S rRNA gene. Methods: We collected 100 samples of throat swabs from patients with respiratory infections. After extraction of DNA from bacterial cell cultured in PPLO broth media using Roche kit (Germany), the PCR was performed on specific samples of M. pneumoniae using specific primers for 23S rRNA gene. Afterwards, for positive samples, minimum inhibitory concentration (MIC) was determined using the broth microdilution with Clarithromycin. Finally, the PCR product was sequenced to detect mutations related to macrolide resistance in domain V of 23S rRNA. Results: According to the analysis of the sequenced PCR product of M. pneumoniae 23S rRNA gene using Clustalw2 online software, one of the samples were shown to have a mutation at A2431G and G2491A positions. The MIC measurement also revealed that all isolates were sensitive to Clarithromycin, and there was no macrolide resistance to Clarithromycin in all isolates. Conclusions: Sequence analysis of the 23S rRNA gene in M. pneumoniae, revealed no macrolide resistance of M. pneumoniae to Clarithromycin. Thus, the use of these antibiotics should be restricted to prevent the development of macrolide-resistant M. pneumoniae in Iran.

Background: The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR). Methods: Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days’,post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8-depleted Hela cells post-3 Gy ionizing radiation (IR) treatment were considered as a control. Results: Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0. 04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of , H2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells. Conclusions: We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage

Background: Over 90% of oral cancers including oral squamous cell carcinoma (OSCC), originate from the oral cavity epithelium. Early detection for this lesion is as important. Evaluating cancer stem cell markers can improve the accuracy of early diagnosis, and be used as an OSCC prognostic indicator. We aimed to evaluate SOX2 and OCT4 gene expression among different grades of OSCC and oral epithelial dysplasia (OED) lesions. Methods: Sixty samples that contains 45 OSCC and 15 OED samples were retrieved from the pathology department archives at the dental school of Mashhad. Demographic and pathological patient data including the tumor stage and tumor grade were assessed. Finally, SOX2 and OCT4 expression was examined using qRT-PCR. Results: There was a significant difference in SOX2 and OCT4 expression between OSCC and OED samples (p< 0. 001). The mean expression of SOX2 and OCT4 in OSCC samples were significantly higher than in the OED group (p< 0. 001). The mean expression of SOX2 and OCT4 was higher in grade II and grade III OSCC compared to grade I. There was no significant relationship between the gene expression of SOX2 or OCT4 to the demographic, site and stage of tumors. The correlation between SOX2 and OCT4 expression (p= 0. 001) was significant in grade III OSCC specimens compared to other grades (p= 0. 005, r= 0. 68). Conclusions: The increased expression of SOX2 and OCT4 in higher grades and the significant correlation of these genes with each other among OSCC specimens could suggest the role of SOX2 or OCT4 in oral mucosal carcinogenesis.

Background: Targeted therapy is an important treatment strategy that is widely used for cancer therapy. Epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of Triple-negative breast cancer (TNBC) patients. Although Cetuximab, which targets EGFR, has shown some inhibitory effects on TNBC cells, Cetuximab resistance cases due to ligand-independent activating mutations in the EGFR gene limit its application. Due to various benefits of single chain antibodies (scFvs), the use of these antibodies in cancer targeted therapy is increasing. In this study, a specific anti-EGFR antibody was isolated and evaluated. Methods: Panning procedure was used against an immunodominant epitope of EGFR in its dimerization arm using a diverse phage library. Polymerase Chain Reaction (PCR) and fingerprinting were applied to identify the specific clones. The MTT tetrazolium assay was performed to evaluate the inhibitory effects of selected anti-EGFR scFv phage antibody on MDA-MB-468, a TNBC cell line. Results: After four round of panning, one dominant pattern was observed in DNA fingerprinting with frequency of 85%. The growth of MDA-MB-468 cells was decreased dose-dependently after treatment with anti-EGFR scFv phage antibody. No significant inhibitory effect of M13KO7 helper phage as negative control on the cell growth of MDA-MB-468 was observed (p> 0. 05). Conclusions: The selected anti-EGFR scFv with high anti proliferative effect on TNBC cells offers an effective alternative for TNBC targeted therapy. The antibody, which binds to the dimerization arm of EGFR and inhibits EGFR dimerization, could also overcome TNBC cases with Cetuximab resistance due to ligandindependent activating mutations.

Background: Janus kinase 2 (JAK2) is a tyrosine kinase located in the cytoplasm that plays a critical role in the signal transduction of cytokines and growth hormones. The conversion of valine to phenylalanine at the polypeptide position 617 results in the JAK2 (V617F) mutation, which often found in patients with myeloproliferative neoplasms (MPNs). As a result of this mutation, JAK2 is constitutively activated leading to uncontrolled cell growth. The present study aimed to investigate the frequency and relationship of the JAK2 (V617F) mutation in a population of patients with MPNs in Iran. Methods: A total of 213 patients with myeloproliferative diseases (MPDs), were included in the study. Real-time PCR was used to detect the presence of the JAK2 (V617F) mutation in the genomic DNA isolated from patient peripheral blood samples. Results: Of the 213 patients with MPDs, approximately 60 (28%) patients were positive for the JAK2 (V617F) mutation. Polycythemia Vera (PV, 42. 11%) was the most common MPD, followed by Essential Thrombocythemia (ET, 29. 82%), Primary Myelofibrosis (MF, 12. 28%), and Chronic Myeloid Leukemia (CML, 10. 5%). A significant relationship between all types of MPDs and the clinical course (p< 0. 05) was observed. The relationship between age and gender among all types of MPD disease was not significant (p> 0. 05). Conclusions: Of the examined cohort in North Eastern Iran, 28% of the patients with MPNs were found to have the JAK2 (V617F) mutation which determining the presence of the JAK2 (V617F) mutation helps to decide the correct form of treatment.

Background: Autosomal dominant polycystic kidney disease (ADPKD), a multisystem disorder, is the most prevalent type of hereditary kidney disease. Here, we aimed to evaluate methylation of the PKD1 gene (PKD1) promoter and its correlation with PKD1 expression in peripheral blood. Methods: In this case-control study methylation of the PKD1 promoter was evaluated using methylation-sensitive high-resolution melt (MS-HRM) analysis. PKD1 expression was assessed by quantitative real-time PCR. The correlation was evaluated using the Pearson correlation test. Results: Twenty subjects from both the patient and control groups (n= 40 for each) were methylated at the PKD1 promoter to various levels (18. 9% in patients and 62. 5% in controls). This difference was statistically significant (p< 0. 0001). PKD1 expression in blood samples was significantly greater in ADPKD patients than in controls (p= 0. 0081). Significant correlation was seen between PKD1 expression and its promoter methylation status in peripheral blood (r case=-0. 5300, p= 0. 0162, and r control =-0. 6265, p= 0. 0031). Conclusions: Methylation of the PKD1 promoter in ADPKD patients was inversely correlated with PKD1 expression.

Background: Multiple sclerosis (MS) is a common demyelinating neurodegenerative disorder with significant heritability. Previous studies have associated genetic variants in human leukocyte antigen (HLA) complex, IL2RA, and HMGB1 genes with the pathophysiology of MS. Methods: In order to investigate the gene association in the Iranian population, we performed a genotyping study of 36 variants in the mentioned genes using Sanger sequencing in 102 MS patients and 113 healthy controls. Results: Our results identified significant associations as well as significant allele frequency differences in some of the studied single-nucleotide polymorphisms including rs4935356, rs3177928, and rs7197 from HLA-DRA gene, and rs12722489 and rs12722490 variants from IL2RA gene (p< 0. 05). Moreover, the strong linkage disequilibrium of two common haplotypes was estimated from the HLA-DRA gene. Conclusions: This association study may suggest the role of these polymorphisms in the genetic susceptibility of MS in the Iranian population and would facilitate the recognition of causative variants in this disease.

Background: Beta thalassemia (,-thalassemia) is a type of inherited blood disorder characterized by the impaired production of beta globin chains. ,-Thalassemia can be categorized into three subtypes according to symptom severity: ,-thalassemia minor, ,-thalassemia intermedia, and ,-thalassemia major. Adipose tissue functions as an endocrine gland by synthesizing and secreting an array of bioactive peptides including leptin, adiponectin, and resistin. Methods: We recruited 30 participants who were transfusion dependent ,-thalassemia patients (major) and 30 participants who were non-transfusion dependent ,-thalassemia patients (minor). The control group consisted of 20 healthy individuals. Analysis of the demographic profile, hematological profile, biochemical parameters, and serum adipokine concentrations (leptin, adiponectin and resistin) were performed for all participants. Results: Our results showed that leptin serum levels were significantly lower in the ,-thalassemia major group compared with the ,-thalassemia minor group or healthy individuals, while serum levels of adiponectin were significantly higher in ,-thalassemic patients compared with healthy controls. Serum levels of resistin were significantly higher in ,-thalassemic patients compared with the healthy control group. A significant negative correlation was noted between adiponectin and BMI in ,-thalassemic patients, whereas leptin was observed to have a significant positive correlation with BMI in the control group. Leptin was observed to have a significant negative correlation with adiponectin and ferritin in the ,-thalassemia major group. Conclusions: The changes we observed in adipokine levels may play a role in the development of the complications related to ,-Thalassemia and disease severity.

Background: Timely identification of Streptococcus pneumoniae infections can lead to a decrease in mortality rates. Differentiation of S. pneumoniae from other similar species using traditional culture-based and molecular methods is problematic. In this study, we assessed the efficacy of identifying the blpA and lytA for the detection of S. pneumoniae from isolates and various clinical samples using molecular methods. Methods: A total of 440 clinical samples were collected from patients with suspected invasive pneumococcal infections during February 2016 to October 2018. Biochemical tests were used to confirm the dubious colonies on 5% sheep blood agar. Fifty-seven confirmed isolates, 57 culture-positive samples, and 57 culture-negative samples were analyzed for the presence of blpA and lytA using both conventional and real-time PCR. Results: All the isolates and culture-positive samples were positive for blpA and lytA by both PCR methods. Of the 57 culture-negative samples, conventional and real-time PCR amplified blpA from six and two samples, and lytA from seven and two samples, respectively. Conclusions: The specificity of real-time PCR assay was significantly higher than that of conventional PCR for the identification of S. pneumoniae. In addition, it is suggested that respiratory secretions are not suitable specimen for direct diagnosis of pneumococcal infections.

Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia. The global increased resistance of M. pneumoniae strains to macrolide (ML) has become a worrisome health problem. The widespread use of these medications has led to increased rate of reported ML-resistant M. pneumoniae (MRMP) throughout the world. This study was aimed to evaluate the resistance of M. pneumoniae against erythromycin due to mutations in the 23S rRNA gene of patients with respiratory infections in Iran. Methods: In this study, 100 samples of throat swab from a patient with respiratory problems were collected. After the cultured of all samples in M. pneumonia-specific PPLO medium, PCR technique was performed with specific primers. Afterwards, the broth micro-dilution MIC assay was employed. Finally, the PCR product of the 23S rRNA gene was sequenced to detect mutations of domain V in 23S rRNA gene of MRMP. Results: It was found that 17 cases (17%) were positive for mycoplasma genus and six cases (6%) positive for M. pneumoniae species. Also, analysis of the sequence of 23S rRNA gene, revealed that one of the samples had mutations at positions A2431G and G2491A. All positive samples M. pneumoniae with 23S rRNA gene were sensitive to erythromycin. Conclusions: These use of these antibiotics should be limited to prevent the emergence of MRMP in Iran.

Background: We aimed to evaluate the effectiveness of Highly Upregulated in Liver Cancer (HULC) and microRNA-372 (miR-372) as biochemical markers in Hepatocellular carcinoma (HCC) and HCV-infected patients. Methods: The present study was conducted on 100 Egyptian individuals divided into 3 groups, 40 patients with HCC and HCV infection, 40 patients only HCV-infected, and 20 individuals as normal controls. They were subject to full history taking, full clinical and laboratory examination, and assessment of HULC and miR-372 levels by real-time PCR. Results: A statistically significant difference was found with p< 0. 05 between HCC and each of HCV and control groups as regards HULC level with high mean among HCC followed by HCV patients. Our results also show a statistically significant difference with p< 0. 05 between each of HCC and HCV compared to control as regards miR-372 level with low mean among HCC patients. Conclusions: HULC could be considered as a potential non-invasive marker for detection and early diagnosis of HCC. Also, it may play an important role in the early prophylaxis and control measures to reduce the incidence of HCC. However, miR-372 cannot be considered as a reliable marker as HULC for early detection of HCC especially in HCV patients.

Background: The objective of this study was to determine the levels of serum ischemia-modified albumin (IMA), fibrinogen (FIB) and high sensitivity C-reactive protein (hs-CRP) in type 2 diabetes mellitus (T2DM) patients with hypertension (HT) (DMT2HTN) and without HT (DMT2). Also, their association with certain biochemical and physical factors were studied to identify possible risk factors that lead to cardiovascular complications. Methods: Fasting blood samples were collected from 35 DMT2 or DMT2HTN patients each to analyze differences in serum and plasma levels of IMA, hs-CRP, FIB, total cholesterol (TC), high and low density lipoproteins (HDL and LDL), triglyceride (TG), hemoglobin A1c (HbA1C), glycated hemoglobin and creatinine. Results: In DMT2 and DMT2HTN patients, IMA, hs-CRP, FIB, TC, TG, HDL, LDL, glycated hemoglobin and creatinine levels, including body mass index (BMI) and waist-to-hip ratio (WHR), were significantly higher relative to healthy controls. In addition, the levels of IMA, hs-CRP and FIB levels showed a strong link to BMI, WHR, TC, TG, LDL and glycated hemoglobin. Lastly, both DMT2 and DMT2HTN patients demonstrated a significant reduction in HDL. Conclusions: DMT2 and DMT2HTN patients have a greater risk of developing cardiovascular related complications. This study suggests that quantifying hs-CRP, IMA and FIB levels can help diagnose the risk of developing complications during the early stages of metabolic and cardiovascular disease. Overall, the specific risk factors may be used for early identification of cardiovascular complications to decrease mortality and morbidity in T2DM patients.