فهرست مطالب mahmood tavallaei

Sulfur mustard (SM) or mustard gas is a blister chemical agent that causes pulmonary damage by triggering inflammation and oxidative injury. Alterations in microRNA (miR) transcript levels are found in pulmonary diseases and even inflammation. Therefore, we evaluated the expression levels of miR-20a-5p, miR-21-5p, and two target transcripts (transforming growth factor-beta [TGF-β1] and TGF-β receptor 2 [TGFR2]) in lung, serum, and skin samples from patients exposed to SM. Total RNA was extracted from lung, serum, and skin samples of patients with moderate (n=10) and high (n=10) SM exposure, as well as 10 healthy subjects. Following the synthesis of complementary deoxyribonucleic acid using real-time polymerase chain reaction, we determined the expression levels of miR-20a-5p, miR-21-5p, TGF-β1, and TGFR2 transcripts. Furthermore, we evaluated the sensitivity and specificity of the chosen miRs by employing receiver operating characteristic (ROC) curves and calculating the area under the ROC curve. The results showed that miR-20a-5p and miR-21-5p expressions in the groups with moderate and high SM exposure were significantly lower than the normal controls. The expression analysis demonstrated that TGFR2 was significantly less expressed in skin samples exposed to SM in both groups of patients compared with healthy controls. Furthermore, the TGF-β1 expression in the skin samples of the group with moderate SM exposure was lower than that of the normal control group. Our findings suggest that miR-20a-5p, miR-21-5p, TGF-β1, and TGFR2 expressions could be used as potential biomarkers for discriminating SM-exposed patients from healthy individuals.

Sulfur mustard or mustard gas is a blistering agent that has been used as a war weapon. Chemical veterans are people who have multiple injuries in different organs, especially in the skin. Studies have shown that the side effects of mustard gas are the result of alkylation with DNA, RNA, protein, and lipids, which leads to metabolic and genetic changes. The respiratory system and the skin are the main targets of mustard gas alkylation. microRNAs are small non-coding and single-stranded RNAs that play a role in the spatial and spatial regulation of protein synthesis and the stability of messenger RNA. The disruption of the expression of any miRNA gene will be equal to the disruption of the expression of several protein-coding genes, each of which can play an essential role in cell biology, so to investigate the changes in the expression of microRNA, miR-20a, in the skin of chemically injured people. In this study, 30 skin biopsy samples were collected, including 10 samples from patients with moderate SM, 10 samples from patients with severe SM, and 10 control samples. RNA extraction and cDNA synthesis were performed. The expression of the miR-20a gene was investigated using the Real-time PCR method. 5s rRNA gene was used as the internal control. GraphPad Prism 6.07 software was used for the statistical analysis of data. Roc curve was used to check the biomarker value of the miR-20a gene. In this study, according to the results of the Roc curve, the miR-20a gene in the skin has a biomarker value, but it needs more study.

Mustard sulfur (SM) is a blister and has destructive effects on the lungs, eyes, and skin. MicroRNAs are non-coding RNA molecules that regulate many important cellular pathways. The clear association of microRNA expression changes in a wide range of diseases and their interception in various organs of the body, including the skin, has made them a suitable biomarker. This study aims at evaluating the changes in miR-21 gene expression in chemical warfare victims infected with SM. In this study, skin biopsy specimens including 10 veterans exposed to SM with moderate complications, 10 veterans exposed to SM with severe complications, and 10 control samples were collected, and then total RNA was extracted. cDNA synthesis was performed. MiR-21 gene expression was assessed using real-time PCR. 5 s rRNA gene was used as the internal control. Graph Pad Prism software version 6.07 was used for the statistical analysis of data. Roc curve was used to evaluate the biomarker value of the miR-21 gene. Increased expression of the miR-21 gene was observed in samples of SM veterans compared with normal samples. There was a statistically significant difference in the expression of the miR-21 gene in the skin samples of veterans exposed to SM with moderate complications and veterans exposed to SM with severe complications compared with the samples of control skin (P-value 0.0001). The expression of the miR-21 gene in the skin samples of veterans exposed to SM with severe complications and veterans exposed to SM with moderate complications in old age was not significantly related to the control sample (P-value = 0.8049, P-value = 0.3802). This study showed that the relative expression of miR-21 could be a potential biomarker in distinguishing SM veterans from healthy individuals but needs further researches..

Colorectal cancer (CRC) is one of the most widespread Cancers across the world. Notwithstanding the diagnoses and treatments developed over thepast four decades, patients' survival rate has improved moderately; and yet, it still has a 5-year survival rate of less than 50%.In this study, hypermethylation of the NDRG4 gene was evaluated as a biomarker in CRC screening.

70samples were examined in this case-control study (45 CRC patients versus 25 healthy controls) totally, and Methylation-Specific PCR (Polymerase Chain Reaction) technique was used to investigate the NDRG4 methylation situation in plasma samples.

The mean age in the control group and CRC patients was 58.4±3.4 years and 64.6±4.4 years, respectively. The male to female ratio in the control group and CRC patients was 1.5:1 and 1.1:1, respectively. Gastrointestinal disease history was positive in 12% and 33% of patients in the control group and CRC patients. In this study, NDRG4 gene hypermethylation was observed in 53.3% of patients, while it was shown in only 23.3% of healthy controls.

The results showed that NDRG4 could be a prognostic biomarker in CRC diagnosis and screen as a noninvasive blood-based biomarker

The role of the lung microbiome in respiratory complications associated with chemicals such as sulfur mustard or chlorine gas has yet to be determined. The aim of this study was to compare the structure and composition of the lung microbiome in chemically injured and healthy individuals in order to understand the relation between the population of the lung microbiota and respiratory complications caused by exposure to these chemicals.

To study lung microbiota, the bronchial alveolar lavage (BAL) fluids were collected from 17 chemically injured and 15 healthy cases during the bronchoscopy procedure. The diversity of lung bacteria present in BAL samples was explored using 16S rRNA gene sequencing.

The lung microbiome dominated by members of phyla Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria and Synergistetes which collectively accounted for > 95% of sequences. At the genus level, members of the genera Prevotella, Leptotrichia, Atopobium, Aggregatibacter, Catonella, and Oribacterium showed more than 2-fold increase in abundance in the lung microbiome of chemically injured patients. Comparing lung bacterial community at the species level, however, revealed an increased prevalence of members of Rothia mucilaginosa (3-fold), Prevotella melaninogenica (2.7-fold), Prevotella pallens (3.5-fold), Actinobacillus parahaemolyticus (2.5-fold), Veillonella parvula (2.5-fold), and Neisseria subflava (1.5-fold) in these patients.

An increased abundance of bacterial species known to associate with airway inflammation suggested their implications in respiratory failure in chemically injured patients. Monitoring and maintaining the homeostasis of the microbial population colonizing lung of chemically injured patients will pave the way to develop a more targeted treatment for these patients.

Oxygen deficiency, referred to as hypoxia, can be divided into two categories of environmental (exogenous) which is sensitized by respiratory cells, and microenvironmental (endogenous) responses by other cells in the body, like cancer cells. Cell responses to oxygen fluctuations are mediated by hypoxia-inducible factors (HIFs). Lung is known as the first organ that encounters the exogenous and environmental oxygen after breathing. However, data are limited for HIF respiratory pathways of environmental oxygen sensing inside the lung cells. Thus, we evaluated the role of both HIF-1 and HIF-2 isoforms in environmental hypoxia sensation by pulmonary vascular and bronchial cells. Then, we introduced the role of HIFs in microenvironmental and endogenous oxygen sensation in tissues and cells other than respiratory cells as well as all of involved molecular mechanisms in those non-lung cells and tissues. Next, a detailed emphasize was put on the strict role of HIF molecule in tumor cells and cancer stem cells.

Chronic obstructive pulmonary disease (COPD) is going to be the third leading cause of death by 2020. Circulating miRNAs are among the most beneficial feasible non-aggressive biomarkers for diagnosis and treatment of many diseases. Among members of the significant miR-17-92 cluster, miR-20a and miR-92a are greatly involved in both inflammation and hypoxia (known as the main reasons for COPD comorbidities). Thus, the expression of these miRNAs was evaluated in the serum of 26 patients and 19 controls using the sensitive stem-loop RT-qPCR approach. The results revealed a significant reduction of these miRNAs in patients relative to controls (P<0.001). Decreased expression of miR-20a in a patient might reflect a progressive stage of the disease. MiR-92a might be used as an early-detection biomarker of COPD. These miRNAs can be used as therapeutic targets specifically in the context of the miR-17-92 cluster to address the various clinicopathological aspects of the disease.

Both Chronic Obstructive Pulmonary Disease (COPD) and mustard lung victims of chemical injury (unlike asthma and allergy) are known respiratory diseases with a substantial genetic background and there are no highly effective medications for either disease. Considering the many similarities between the two diseases, both can be considered simultaneously under one category. Therefore, we assessed the expression of miR-20a and miR-92a as representatives of the miR-17/92 cluster due to the effective interference of these two miRNAs in the signaling pathways of inflammation and hypoxia in the pulmonary serum (consisting of Mustard Lung and COPD, simultaneously).
Stem-Loop Real-Time Quantitative Polymerase Chain Reaction was used to examine the expression of miR-20a and miR-92a simultaneously in patients with COPD (n=25) and mustard lung (n=50).
The results showed that the expression of both miR-20a and miR-92a was significantly downregulated in patients with COPD and chemical injuries when compared to the control group. The expression of each of two miRNAs in the moderate and severe phases of the disease was more downregulated in the mild stage, and miR-92a showed a further downregulation compared to miR-20a (p The downregulation of miR-20a and miR-92a might be due to alterations in 1- genomic and/or 2- environmental and/or 3- ecogenomic (affected by epigenomic) factors which requires intensive research in this field. Our results may shed light on new effective strategies for the treatment of respiratory diseases by regulating the mir17/92 cluster by regulating related factors. The members of the oncogenic miR17/92 cluster are significantly upregulated in lung cancer and have the potential to be used as biomarkers in the non-invasive screening of respiratory diseases. Furthermore, these miRNAs can be used for distinguishing respiratory diseases from lung cancer and cardiovascular diseases (also known as COPD comorbities).

In historical cases, mass disasters, missing person’s identification and ancient DNA investigations, bone and teeth samples are often the best and the only biological material available for DNA typing. This is because of the physical and chemical nature of the protein-mineral matrix of bone which is relatively resistant to the adverse environmental effects and biological attacks. Most bone extraction protocols used in the forensic laboratories involve an incubation period of bone powder in extraction buffer for proteinase digestion, followed by the collection of the supernatant, and the elimination of large quantities of undigested bone powder.
Here we demonstrate an extremely effective protocol for recovery of DNA. This is performed in a method that retains and concentrates all the reagent volume for complete DNA recovery. For our study, we selected challenging bone samples of skeleton remains of the martyred individuals in Iraq’s imposed war on I.R. Iran (1980-1988). The bones that were extracted with our new protocol showed that this new protocol significantly enhances the quantity of DNA that can be used for amplification from degraded skeletal remains. At the same time we tested in parallel the samples by using of QIAamp DNA Investigator Kit and attained the best results by using new protocol. In fact, our new DNA extraction method is based on previous standard methods such as Chelex and salting out. We have used this technique to successfully recover authentic DNA Typing from extremely challenging samples that failed repeatedly using the standard protocols. However, the amount of recovered DNA was very small but it was possible to extract genomic DNA from these challenging bone samples. The results indicated that our procedure for DNA extraction although yielded little amount of genomic DNA; however, it was pure DNA that can be used for further analysis such as PCR amplification and DNA profiling. Since the new procedure is fast and needed less time than previously standard procedures, we have named it FDEB (Fast DNA Extraction of Bone).

The cause of epilepsy in most of cases is unknown but inherited causes can play a role in its incidence. Recently, many researches have been conducted in the field of epilepsy. The aim of this study was to identify gene(s) responsible in an Iranian family with autosomal recessive non-syndromic epilepsy using whole exome sequencing (WES) method.
In this experimental study, genomic DNA was extracted from whole blood belonging to three affected persons and a healthy individual and whole exome sequencing was performed using Illumina Hiseq2000 platform. At first, variants classification were carried out based on mutation types, position of the mutation and their allele frequencies and then, prioritization was performed via two approaches. Sanger sequencing was carried out for RNF216 confirming the variant [NM_207111.3 (RNF216): (g.5780794G > A), (c.854C > T)].
Findings: From 130855 variants, 85% were single nucleotide variants and 15% were indels. One third of them located in intergenic and intronic regions, one third located in 3’and 5’ UTRs and one third located in exonic and splice site regions. 3.8% and 3.4% of variants had allele frequencies below 0.01 in ExAC and 1000 genome project databases, respectively. By using the two prioritization approaches, a variant in RNF216 gene [NM_207111.3 (RNF216): (g.5780794G > A), (c.854C > T)] was selected as a prior candidate. However, this variant did not co-segregate with the disease in all of the members of this family.
Exome sequencing has been considered as a tool for studying genetic causes of Mendelian disorders; but it has some limitations. Mutation in the non-coding regions of the genome, clinical heterogeneity of disease, wrong clinical diagnosis, phenocopy, and technical and analytical limitations can be considered as a reason that we could not find gene(s) responsible for the disease in this family.

In spite of accumulating information about pathological aspects of sulfur mustard (SM), the precise mechanism responsible for its effects is not well understood. Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims. In this case and control experimental study, using quantitative real-time polymerase chain reaction (qRT-PCR), we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war (1980-1988) and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle (Cq) method were employed to find the least variable reference gene. miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. We demonstrate that non-miRNA reference genes have the least stability in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers.

To investigate a potential alteration in the expression levels of transforming growth factor β (TGF-β) and miR-21 in bladder cancer tissues. Using real-time polymerase chain reaction (PCR) method, we examined a potential correlated expression of miR-21 and TGF-β variants in 30 bladder tumors and their marginal/non-tumor biopsy specimens obtained from the same patients. Our data revealed a significant down-regulation of TGF-β variants (P =. 03) along with a non-significant alteration in the expression of miR-21 in tumor vs. non-tumor samples. However, in contrast to low-grade tumors, the expression of miR-21 was upregulated in high-grade ones, and the expression level can efficiently discriminate low-grade tumors from high-grade ones (P =. 03). In accordance to the observed similarity between TGF-β variants and miR-21 gene expression alterations in bladder tumors, treating 5637 bladder cancer cell line with TGF-β recombinant protein caused a significant upregulation of miR-21. The later finding further confirmed a correlated expression of TGF-β and miR-21 in bladder tumors.

MicroRNAs (miRNAs) are short, endogenous non-coding RNAs that function as guide molecules to regulate transcription of their target messenger RNAs. Several methods including low-density qPCR arrays are being increasingly used to profile the expression of these molecules in a variety of different biological conditions. Reliable analysis of expression profiles demands removal of technical variations in data, which is achieved via applying normalization techniques. Most normalization techniques have been developed for mRNA microarrays and new and modified methods should be used or miRNA studies in general and RT-qPCR miRNA arrays in particular, because of low number of miRNAs. Here, we introduce a new method based on Procrustes superimposition of arrays to be normalized on a reference array. To assess the performance of our normalization method, we compared this method to the common miRNA normalization methods. Removal of technical variation was assessed by robust modeling of mean square error (MSE) in different subsets of real miRNA datasets before and after applying normalization. We show that our method outperforms the other normalization methods in concurrent reduction of technical variation and retention of biological variability.

Background and The 50 KDa protein (50 µg) in carboxylic domain of the neurotoxin heavy chain (BoNT/A-Hc) recognizes surface receptors on target neurons and this fragment contains the principle protective antigenic determinants. Recently, this fragment has been used as a recombinant vaccine candidate for botulism. The study aimed to compare the evaluation of BoNT/A-Hc production in fed-batch (high cell density cultivation) and batch cultivation of recombinant Escherichia coli (E. coli.). In this research, growth of recombinant E. coli in batch culture was studied. In order to maximize protein expression, induction time and Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer concentration were optimized by the Taguchi statistical method. Then, fed-batch culture was applied to achieve high cell density cultivation. Finally, the recombinant protein expression level was determined. In optimized conditions, 62 and 486 mg/l of soluble recombinant BoNT/A-Hc were produced in batch and fed-batch cultivation. According to the results, high cell density in fed-batch cultivation is a very effective for improve of recombinant proteins productivity.

MicroRNAs (miRNAs) are short, endogenous non-coding RNAs that function as guide molecules to regulate transcription of their target messenger RNAs. Several methods including low-density qPCR arrays are being increasingly used to profile the expression of these molecules in a variety of different biological conditions. Reliable analysis of expression profiles demands removal of technical variations in data, which is achieved via applying normalization techniques. Most normalization techniques have been developed for mRNA microarrays and new and modified methods should be used or miRNA studies in general and RT-qPCR miRNA arrays in particular, because of low number of miRNAs. Here, we introduce a new method based on Procrustes superimposition of arrays to be normalized on a reference array. To assess the performance of our normalization method, we compared this method to the common miRNA normalization methods. Removal of technical variation was assessed by robust modeling of mean square error (MSE) in different subsets of real miRNA datasets before and after applying normalization. We show that our method outperforms the other normalization methods in concurrent reduction of technical variation and retention of biological variability.

The current methods for bladder cancer diagnosis suffer from low sensitivity and specificity. Therefore, finding a novel tumor markers with high specificity and sensitivity is of great interest. MicroRNAs (miRNA, miR) are small endogenously-produced, non-coding RNAs with an important role in regulating gene expression. Recent studies show that miRNAs expression profiles represent significant tumor-specific changes that are unique for most cancers. The aim of this study was to optimize miRNA containing total RNA extraction from urine and use it as a reliable and repeatable technique for miRNA detection in urine of patients with bladder cancer.

Total RNA was extracted from the urine of patients with bladder cancer and normal individuals using RNX and Trizol solutions with and without modifications of original protocols. Real-time quantitative RT-PCR was then used to detect miRNAs with a potential link to bladder tumorigenesis.

RNX and the modified Trizol are practical methods for RNA extraction from urine samples. The mir-21 amplification of the extracted RNAs using modified Trizol method was more efficient than that of RNX method. It is noteworthy that, the levels of miRNAs expression were much higher in the frozen urines compared to the fresh ones.

We have succeeded to set-up a protocol to easily amplify miRNAs in urine samples. Based on the data, microRNAs seem to be good biomarkers for early detection and screening of bladder cancer.