International Journal of Medical Laboratory

8

Authors: Behnaz Rahmani, Hossein Zarei Jaliani, Akram Astani, Mohammad Hassan Kheirandish, Ahmad Mosaddegh, Abolghasem Asadi Saghandi

Background and Aims: Enzybiotics are probably the future line of weapons against drug resistant bacteria. They lyse bacteria with the new mechanisms with few likelihood of generating resistance. LasA, which is secreted from Pseudomonas strains degrades Staphylococcus aureus (S. aureus) cell wall and has the potential to use against drug resistant S. aureus infections. Materials and Methods: Codon-optimized gene of the mature form of the LasA protein was ordered. The gene was double digested with NcoI and XhoI restriction enzymes and sub-cloned into pET28a(+) digested with the same enzymes. Recombinant construct was introduced into BL21 DE3 cell. Expression of the gene was induced by 0.2 mM isopropyl β-D-1-thiogalactopyranoside and recombinant protein was affinity purified by Ni-NTA mini-column. The staphylolytic activity of the recombinant LasA protein was evaluated on Methicillin-resistant S. aureus (MRSA) by disk diffusion. Results: Fragment of the LasA gene encoding mature form of the LasA protein was introduced into pET28a(+) expression vector. C-terminal his-tagged recombinant LasA protein was produced in BL21 DE3 E. coli cells. Over 50% purity has been achieved by affinity purification of the LasA protein from the total cell lysate. The yield of purified protein was 5.4 mg.l-1. Growth of MRSA was completely inhibited by dilutions of recombinant his-tagged LasA. Conclusions: The present study shows that the mature form of the LasA can be expressed in E. coli BL21 DE3 cells. C-terminal his-tagged form of the mature LasA protein has staphylolytic activity against MRSA and so it can be a promising therapeutic agent.

9

Authors: Ali Shams , Mohammad Hasan Bargostavan

Background and Aims: Involvement of matrix metalloproteinases 3 (MMP3) in breast cancer tumor progression and metastasis has been revealed. MMP3 -1171 5A/6A and 5A/5A polymorphisms in the gene promoter increase expression of the enzyme. The possible relationship of these polymorphisms and serum levels of major histocompatibility complex class I chain-related protein A and B (MICA/B) in breast cancer patients was goal of the study. Materials and Methods: In this case-control study, 105 breast cancer patients and 100 age-matched healthy women were selected. The MMP3-1171 5A/6A polymorphism was determined by nested and restriction fragment length polymorphism polymerase chain reaction. Concentration of MICB and MICA in serum of breast cancer patients has measured using enzyme-linked immunosorbent assay method. Results: Our results showed that 5A/5A + 6A/5A genotypes in the breast cancer group (77.18%) were more frequent compared to healthy participants (64%). However, the frequency of MMP3 -1171 5A/6A genotypes in the breast cancer patients and healthy donors was similar. Additionally, in the patients with 5A/6A genotypes, the mean of MICB concentration was 51.3±16 pg/ml whereas; in the homozygote 6A/6A genotype, it was 4.7±2.6 pg/ml. Statistical analysis confirmed a significant difference between MMP3 -1171 5A/6A and serum MICB levels in the patients (p=0.02). Based on our results MMP3-1171 5A/6A polymorphism did not alter MICA concentration in the patients (p=0.15) Conclusions: The MMP3 -1171 5A/6A genotype might be associated with shedding of MICB in breast cancer patients.

9

Authors: Behnaz Askarikhah, Mahmood Dehghani Ashkezari , Hamideh Emtiazi , Seyedhossein Hekmatimoghaddam

Background and Aims: Benzopyrones are a group of compounds including coumarins and chromenes. Chromenes have a heterocyclic structure with gamma benzopyran which has anticancer activities. In this investigation we studied the effect of 4 new derivatives of chromene compounds on the human acute lymphoblastic leukemia cell line MOLT4. Materials and Methods: The structure of the new compound was established using spectroscopic method (1H NMR, 13C NMR). MOLT4 cells were cultured in Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum. The cytotoxic effect of different concentrations (0, 50, 250, 500 and 1000 nM) of novel synthetic compounds were evaluated by MTT assay and cell counting after different incubation times (24, 48, 72 h). Expression levels of 3 genes related to apoptosis (p53, Bax, Fas) were measured using real-time quantitative polymerase chain reaction. Results: These compounds all decreased viability of the MOLT4 cells in a time- and dose-dependent manner. Also meaningful difference was found between all concentrations and control groups (p<0.05). The compound C2 had lower IC50 in comparison to other ones. Interestingly, these derivatives showed significantly cell toxicity at nano-molar concentrations, while previously-reported ones had activity at micro-molar concentrations. All three apoptotic genes revealed increased expression. Conclusions: 10,11-dihydrochromeno [4,3-b]chromene-6, 8(7H,9H)-dione derivatives have anti-neoplastic effects on MOLT4 leukemic cell line by induction of apoptosis. These comounds could then be a potential candidate for further investigations.

14

Authors: Mohammad Ali Esmaili, Farhad Razjou, Behzad Nazel Khosroshahi, Ali Amini, Soodeh Namjoo, Peyman Beigi, Mohammad Reza Rezvani

Platelet components (PCs) have widespread applications in clinical cases. Since PCs store in room temperature (between 20-24°C), they are susceptible to bacterial contamination. There are varied approaches for identifying bacterial contamination in PCs. These methods categorized into two groups: Firstly, culture based methods and secondly, non-culture based methods. Both of them have a couple of merits and demerits. BacT/ALERT is a culture-based technique, which has been approved by the food and drug administration. Although sensitivity and specificity of this method could be debatable and is not universal. This method is considered as gold standard contemporary method and it is far more dependable and superb in comparison with the contamination detection methods. It is assumed that, application of rapid methods play an important role in detection of bacterial contamination in the future. Accordingly, this study aimed to represent a summary of each method, which was used for bacterial contamination detection in PCs with detailed assessment of culture-based methods, specifically BacT/ALERT.

4

Authors: Hosein Timori Naghadeh, Zainab Pirmohamadjamaat, Shirin Ferdowsi *

Background and Aims: The quality of platelets (PLT) during storage is influenced by various factors. The purpose of this study was to determine the potential effects of the air bubbles and foam in the bag of platelet and its effects on the quality of the final product. Materials and Methods: In this paired-study, the air bubbles and foam, which develops after the preparation of the platelet units were excluded from the control units (n=10), but not from the test units (n=10). Various in vitro variables after the 5-day storage was evaluated, including measurements of PLT counts, mean PLT volume (MPV), Lactate dehydrogenase (LDH), PH, swirling, aggregation response, and the expression of CD62P. Results: PLT count was lower (p=0.001) and LDH was higher (p=0.006) in the test vs. control units. PH was maintained at >7 (day 5) and swirling remained at the highest level (score=2) for all units throughout storage. No significant difference in MPV and CD62P expression was detected between the groups. Conclusions: It was found that the storage of platelets in bags containing air bubbles and foam after five day storage period increased disintegration of the platelets.

8

Authors: Amin Dehghan Banadkouki, Gilda Eslami, Hengameh Zandi *, Ali Dehghan Banadkouki

Background and Aims: Klebsiella pneumoniae is a gram negative opportunistic pathogen of nosocomial infections that causes pneumonia, urinary tract infection (UTI), meningitis, septicemia and diarrhea. The emergence and spread of carbapenemase producing Klebsiella pneumoniae often cause failure in the treatment of infections. The aim of this study was to evaluate the prevalence of carbapenemase producing Klebsiella pneumoniae strains isolated from clinical urine specimens. Materials and Methods: In this descriptive-sectional study, from December 2013 to August 2014, 130 Klebsiella pneumoniae isolates were collected from hospitalized patients with urinary tract infections and identified by biochemical tests. Antimicrobial susceptibility test was evaluated by the standard disk diffusion method (Kirby-Bauer). E.test method was used for determining of meropenem minimum inhibitory concentration (MIC). Carbapenemase producing was investigated by the Modified Hodge Test (MHT). blaKPC was evaluated by conventional polymerase chain reaction (PCR) method using specific primer pair. Results: The results showed that 61 (46.9%) isolates were non-susceptible to at least one of the carbapenems. 35 (26.9%), 5 (3.8%) and 5 (3.8%) isolates were resistant to imipenem, meropenem and ertapenem, respectively. 45.9% (28 of 61) of isolates were carbapenemase producing and all of them were negative for the presence of blaKPC. Conclusions: With respect to the high prevalence of carbapenemase producing Klebsiella pneumoniae isolates, it is recommended that MHT is performed in routine laboratories for limiting and controlling the spread of the carbapenem resistant isolates.

9

Authors: Anahita Nazari, Mohamad Bagher Khalili *, Akram Astani, Mahmood Vakili, Maryam Sadeh, Mahdiye Mojibiyan, Shahla Rashighi, Fariba Reyhanizadeh

Background and Aims: Group B streptococcus (GBS) may cause neonatal infection during and or after the delivery, and is the leading cause of sepsis, bacteremia, pneumonia and meningitis. The virulence factors are carried by both capsule and surface proteins by which serotypes and genotypes are determined. However, some genotypes are believed to be related in severity of neonatal diseases, therefor this investigation was aimed to determine the surface proteins genotype of detected GBS from both vagina and urine of pregnant women. Materials and Methods: In the present study, a total of 346 vaginal and urine samples were obtained from the same pregnant women. Following culturing the samples on sheep blood agar medium and related tests (CAMP, Hiporate hydrolysis) the suspected colonies were further confirmed by polymerase chain reaction (PCR) technique. The detected GBS species were then genotyped using multiplex PCR assay. Results: Out of 346 specimens, 57 (16.47%), and 33 isolates (9.5%) were identified as vaginal and urine GBS, respectively. Genotype rib was predominant in both vaginal discharge and urine specimens, by frequencies of 56.1% and 54.5%, respectively. Other genotypes of vaginal GBS were as epsilon (36.8%), anlpha-C (3.5%), and alp2/3 (3.5%); while GBS in urine were as epsilon (36.4%), alp2/3 (6.1%), and anlpha-C (3%). Conclusions: The prevalence of GBS in both vaginal and urine samples were reliable in comparison to other societies. These results showed that genotype rib of GBS, which seems to be associated to neonatal diseases, is dominant genotype in both urine and vaginal samples.

11

Authors: Mohammad Hassan Kheirandish, Hossein Zarei Jaliani *, Behnaz Rahmani

Background and Aims: DNA cloning, sub-cloning and site directed mutagenesis are the most common strategies in nearly all projects of recombinant protein production. The classical method of restriction site cloning is unsatisfactory due to the need for supply of restriction enzymes and the inefficiency of the digestion reaction. Many new methods, including recombinatorial cloning and ligation independent cloning need additional enzymes and kits. In this project we insert a full-length streptolysin O gene into an expression plasmid without using any uncommon commercial enzymes. Materials and Methods: Steptolysin O gene was amplified by polymerase chain reaction (PCR) and introduced into the pPSG-IBA35 vector using a quick-change PCR. At the same time the gene was double digested and sub-cloned into pET28a (+). Both constructs were introduced into BL21 DE3 cell. Proteins were purified by Ni-NTA column and hemolytic activity was evaluated by spectrophotometry using human red blood cells. Results: Steptolysin O was subcloned into the pET28a (+) and pPSG-IBA35 vectors and expressed in E. coli. Protein was purified with over 90% purity. The IC50 of C and N terminus his-tagged protein were 0.22 and 0.29 µg/ml, respectively in hemolysis assay. Conclusions: This study showed for the first time that full-length streptolysin O can be expressed in E. coli cytoplasm without any toxicity for the bacteria itself. The only additional amino acids expressed on the protein were his-tag. To study the role of this toxin it would be better to express the protein with the same strategy to have minimal extra amino acids on the protein.

4

Authors: Zakieh Rostamzadeh *, Mohadese Nemati, Mahshid Mohammadian

Background and Aims: Hepatitis B virus (HBV) infection is a serious global health problem affects many people. Recently, because of vaccination, the incidence of HBV infection has been reduced, but in high risk population also health care students (HCS) adequate immunization is serious. The aim of this survey was to evaluate HBV immunization in the population of the paramedical students based on the demographic characteristics in Iran. Materials and Methods: Anti-HBsAg titer was prospectively assayed in all vaccinated of the 95 HCS using Enzyme-linked immunosorbent assay. Results and Conclusions: Anti-HBsAg titer in the studied population was estimated about 92.8±80.5 mIU. Anti-HBsAg titer response was significantly higher in females to males. A low proportion of vaccinated HCS had low titers antibody against HBV infection. Therefore, measuring anti-HBsAg titer may help to drop in HBV incidence in HCS. This reduction can be correlated with the effectiveness of national immunization program.

10

Authors: Shalini N. Maksane*, Rupali Parikh , Lakshmi Vaswani

Background and Aims: Six sigma is the latest version of total quality management. It is quantitative goal for process performance. With increasing demands for improved accuracy and reliability of the results, Six Sigma is gaining increased visibility in the clinical laboratory process outcomes. The aim of study was to evaluate the quality of analytical phase performance in a clinical biochemistry laboratory by calculating sigma metrics. Materials and Methods: The study was conducted in a hospital laboratory of Bhatia Hospital, Mumbai. Mean, coefficient of variation, bias, and sigma values were calculated for 24 biochemistry parameters. The guidelines used for total error allowable (TEa) values were clinical laboratory improvement amendments (CLIA), RILIBAK and college of american pathologists (CAP). Four months’ internal and external quality control data were extracted for the following parameters-albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), amylase, aspartate aminotransferase (AST), bilirubin direct, bilirubin total, Ca, cholesterol, creatine kinase (CK), creatinine, triglycerides (TG), Uric acid (UA), unsaturated iron binding capacity (UIBC), urea, gamma-glutamyl transferase (GGT), glucose, high-density lipoproteins (HDL), iron, lactate dehydrogenase (LDH), lipase, Mg, phosphorus and total protein. Results: Albumin, ALP, AST, ALT, amylase, bilirubin total, Ca, cholesterol, CK, creatinine, TG, uric acid, GGT, glucose, iron, LDH, lipase, Mg, Phosphorus, total protein showed the performance of more than six Sigma for both level of controls. Bilirubin direct, Urea, for level 1; UIBC, urea, HDL for level 2 showed sigma from 3-6. Conclusions: Based upon sigma metrics, laboratory quality control strategy can be planned and reevaluated for continuous monitoring and improvement of test methods.

10

Authors: Arezou Rahimi, Mehrnaz Mesdaghi, Nima Rezaei, Mahboubeh Mansouri, Iraj Mohammadzadeh, Hamid Farajifard, Mehrdad Amirmoini., Delara Babaie, Reza Alimohammadi, Zahra Chavoshzadeh

Background and Aims: Patients with Hyper-IgE syndrome suffer from fungal and bacterial infections, especially Candida albicans and Staphylococcus aureus. Due to the important role of T helper17 (Th17) lymphocytes in defense against fungal infections, the percentage of Th17 lymphocytes was studied in the patients with autosomal recessive hyper-IgE syndrome (AR-HIES). Materials and Methods: In this case-control study, six patients with AR-HIES (with DOCK-8 mutation) and seven healthy age and sex-matched controls were included. Peripheral blood mononuclear cells were isolated from their venous blood and the percentage of Th17 lymphocytes were determined by flow cytometry. Results: There was no statistical difference between the percentage of Th17 lymphocytes (p=0.15) in the case and control groups. Also in comparison to the control subjects, the numbers of eosinophils were dramatically increased (p=0.000). Also, there was a significant negative correlation between serum IgE levels and Th17 lymphocytes’ percentage (r=-0.927, p=0.006) and a significant positive correlation between eosinophils number and Th17 lymphocytes’ percentage (r=0.557, p=0.01). Serum IgE levels showed a significant positive correlation with the numbers of eosinophils in the patients’ peripheral blood with AR-HIES (r=0.961, p=0.003). Conclusions: The numbers of Th17 in the patients with AR-HIES may not show statistical differences between the cases and controls. The numbers of eosinophils significantly increased in the patients AR-HIES compare to the controls.

12

Authors: Khosrow Aghaiypour, Elham Bahreini*, Shiva Jafari

Background and Aims: L-Asparaginase II is a cornerstone of treatment protocols for acute lymphoblastic leukemia. Only asparaginase II obtained from E.coli K12 and Erwinia chrysanthemi have been used in human as therapeutic drug. The therapeutic effects of asparaginase II from E.coli K12 and Erwinia chrysanthemi are accompanied by side effects. It is desirable to search for other asparaginase II sources with novel properties that could be therapeutic and produce an enzyme with less adverse effects. Materials and Methods: Previously, we performed the in vitro studies, including cloning, sequencing and expression of L-asparaginase II genes (ansB) from Citrobacter freundii 1101, Erwinia chrysanthemi DSM 4610, E.coli BL21 and Klebsiella pneumoniae ATCC 10031. In this article, the obtained results were compared bioinformatically. The nucleotide and amino acid sequence alignments were carried out by ClustalW2. Protein localization and signal peptides were predicted by PSORT and SIG-Pred softwares, respectively. Percentages of hydrophobic and hydrophilic residues were calculated by Genscript software. The physicochemical parameters were computed using Expasy’s ProtParam prediction server. The secondary and 3D structures were predicted by SOPMA and the online server Phyre2, respectively. The antigenicity of the asparaginase IIs was predicted using Semi-empirical method. Results: E.coli BL21 and Citrobacter freundii 1101 had the most similarity in physicochemical parameters and antigenicity with E.coli K12. Also, Erwinia chrysanthemi DSM 4610 had the most similarity in physicochemical parameters and antigenicity with Erwinia chrysanthemi. Conclusions: In spite of these similarities with drug types, the potentiality of other low-similar asparaginase IIs should also be determined and compared with drug types.

6

Authors: Salvatore Chirumbolo *

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7

Authors: Hamed Faghihi, Abolghasem Abbasi Sarcheshme, Seyed Hassan Hejazian *

Volume 4, Number 1 (2017) | IJML 2017, 4(1): 56-62 | Back to browse issues page XML Print Download citation: BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks Send citation to: Mendeley Zotero RefWorks Faghihi H, Abbasi Sarcheshme A, Hejazian S H. The Relaxant Effect of Carvacrol on Acetylcholine-induced Contraction in Isolated Rat's Ileum . IJML. 2017; 4 (1) :56-62 URL: http://ijml.ssu.ac.ir/article-1-162-en.html The Relaxant Effect of Carvacrol on Acetylcholine-induced Contraction in Isolated Rat's Ileum Hamed Faghihi, Abolghasem Abbasi Sarcheshme, Seyed Hassan Hejazian * Abstract: (31 Views) Background and Aims: The antioxidant and anti-inflammatory effects of carvacrol were reported. The aim of the current study was an investigation the relaxant effects of carvacrol on acetylcholine-induced contraction in isolated rat's ileum. Materials and Methods: In this study, the tissues separated from ileum were fixed on the organ bath containing Tyrode's solution. The amplitude of contractions has been recorded using isotonic transducer. Before and after subjecting the tissues with saline and different concentrations of carvacrol,the procedure was elicited by cumulative logarithmic concentrations of acetylcholine (Ach). Results: Our results indicated that carvacrol at 10-2, 10-3 and 10-4, but not 10-5 M decreased acetylcholine (10-4 M)-induced contraction (10-2 M at p<0.001; 10-3 and 10-4 at p<0.05 vs. Ach+saline). The antispasmodic effect of carvacrol was examined and showed that in concentrations of 10-5, 10-4 and 10-3 M, but not 10-6 M, the contractive effect of acetylcholine10-3 M was prevented significantly. The concentrations of 1, 2 and 4 µM of verapamil were also tested and indicated that 4 µM of verapamil reduced contraction by 22.1±.5% (p<0.01 vs. saline). In samples treated with combined carvacrol (10-5 M) and verapamil (4 µM) contraction percentage was decreased by 35±14%, which was significantly different compared to the Ach+saline group as well as the single treated groups (p<0.01 and p<0.05, respectively). Conclusions: According to our findings, due to the effect of carvacrol and verapamil, the relaxant mechanism of carvacrol may be mediated by Ca+2 release from the sarcoplasmic reticulum and the response of the contractile system.

8

Authors: Arsalan Salari, Fatemeh Moaddab, Mahsa Pourabdollah Tootkaboni, Maryam Shakiba, Mohadeseh Poursadeghi *

Background and Aims: Bilirubin has been considered an antioxidant that protects against atherosclerosis. The aim of this study was to evaluate the relationship of serum bilirubin level with the severity and complexity of coronary artery disease (CAD) and long-term outcome in the patients undergoing primary percutaneous coronary intervention (PCI). Materials and Methods: This prospective cohort study was performed on 136 patients with STEMI undergoing PCI in Heshmat Hospital, Rasht, Iran during 2 years. The complexity was calculated by using dedicated software of the syntax score and the severity of CAD was determined based on the number of vessel involvement. Also, long-term outcomes were evaluated by major adverse cardiac events (MACE ) after one year. Results: The mean total bilirubin level was 0.95 ± 0.69 mg/dl; 60.3% of the patients were in the low range of syntax score (0-22) and 38.2 % had two vessels involved. In examining the relationship between serum bilirubin level with the severity and complexity of CAD and MACE, There was no statistically significant association (p>0.05); but, the majority of the patients with high bilirubin levels had a lower number of vessels involved and the complexity of the vessels; Also, MACE was observed in the patients with low bilirubin levels. Conclusions: According to the results, attention to serum bilirubin level could be important in CAD and the protective effect of bilirubin can be used for new therapeutic decisions. Further studies on a larger sample of patients have been recommended to confirm and complete these findings.

10

Authors: Yaser Eskandari Torbaghan, Mohammad Kazemi Arababadi, Ali Shams *

Background and Aims: Engagement of the receptor for advanced glycation end products (RAGE) and its ligand “S100A12 protein” induce a cascade of reactions that eventually might lead to develop an inflammatory response dependent on NF-κB. Although involvement of S100A12 and RAGE in some autoimmune disease have proved, in chronic hepatitis B (CHB) infection functions of the proteins are not clear thus far. Determining of expression of S100A12 and RAGE in peripheral blood cells of the CHB patients was the aim of the present study. Materials and Methods: In the case-control study the mRNA levels of S100A12 and RAGE genes of the sixty CHB patients and sixty healthy donors were measured by real-time polymerase chain reaction method. The patients and healthy donors were sex and age-matched. Results: The findings demonstrated expression of S100A12 and RAGE in the CHB patients significantly decreased compared to the healthy donor group (p=0.001). Expression levels of the genes were not altered among HBeAg-positive and HBeAg-negative CHB patients. The HBV-DNA copy number/ml did not affect the expression of S100A12 and RAGE in the patients (p>0.05). Conclusions: The results of the study suggested that down regulation of RAGE and S100A12, because of their role in inducing inflammation, might have a considerable role in chronicity of hepatitis B.