فهرست مطالب abbas ali raz

Programmable nucleases are powerful genomic tools for precise genome editing. These tools precisely recognize, remove, or change DNA at a defined site, thereby, stimulating cellular DNA repair pathways that can cause mutations or accurate replacement or deletion/insertion of a sequence. CRISPR-Cas9 system is the most potent and useful genome editing technique adapted from the defense immune system of certain bacteria and archaea against viruses and phages. In the past decade, this technology made notable progress, and at present, it has largely been used in genome manipulation to make precise gene editing in plants, animals, and human cells. In this review, we aim to explain the basic principle, mechanisms of action, and applications of this system in different areas of medicine, with emphasizing on the detection and treatment of parasitic diseases.

Although the study on the bacteria residing in the mid-gut, salivary gland, and reproductive organs of insect vectors have drawn appeal to the host-pathogen interactions, we know comparatively less about microbiota that naturally exist in different mosquito organs within Iran.

In the current investigation, PCR assay by using 16S rRNA gene amplification and DNA sequenc- ing, in addition to the traditional culture-based approach utilized for the detection of cultivable bacterial assemblages in mid-gut and reproductive tracts of Culex quinquefasciatus.

The identified bacteria isolated from different tissues of 45 individuals were consisted of Achromobacter, Aero- monas, Arthrobacter, Asaia, Enterobacter, Gluconobacter, Klebsiella, Lysinibacillus, Micrococcus, Psuedomonas and Serra- tia. The results showed that Proteobacteria was the most prevalent phylum in both genders' mid-gut and reproductive tracts, and Asaia was the most common bacteria that originated in adult females and males’ tissues.

These outcomes recommend that the discovered microbiome may span through Cx. quinquefasciatus popula- tions. This data can be utilized to interfere with the transmission of pathogens and design new strategies for the control of mosquito-borne diseases.

A given amino acid sequence can be encoded by a huge number of different nucleic acid sequences. These sequences, however, have proved not to be equally useful. The choice of sequence can significantly impact the expression of an encoded protein. Given the importance of protein-coding sequence and promising industrial and medicinal applications of Clostridium histolyticum collagenase, this study examined the codon optimization of the Col H gene so as to enhance collagenase expression in Escherichia coli (E. coli).
This is an experimental study in which the CDS of Col H gene was optimized according to the codon usage of E. coli, using Gene Designer software (DNA 2.0).
The results revealed that relative frequency of codon usage in Col H gene was adapted to the most preferred triplets in E. coli in such a way that codon usage bias in E. coli was enhanced after codon optimization. The higher level of collagenase expression was more likely the result of substituting rare codons with optimal codons.
The findings of this study suggest that codon optimization provides a theoretical improvement in Col H gene expression in E. coli. Nevertheless, experimental research is needed to confirm the improvement.

Today, the use of maggot therapy has become widespread due to the increase in chronic ulcers in the world. The recombinant production of secreted enzymes from these larvae is a novel non-invasive method for the treatment of chronic ulcers. Lucilia sericata (L. sericata) collagenase (MMP-1) has been expressed in insect cells. Collagenase is an enzyme that is widely used in clinical therapy and industry. It has been indicated that collagenase is expressed and secreted in salivary glands of L. sericata while using for maggot debridement therapy.
In the present study we decided to produce the recombinant form of collagenase enzyme in Spodoptera frugiperda (SF9) insect cells using the baculovirus expression system (Bac-to-Bac).
cloned the coding sequences (residues 494-1705) of L. sericata collagenase into the pFastBacHTA as donor plasmid. After transposition in the bacmid of DH10Bac host, the bacmid was transfected into the Sf9 cell line, then the expressed recombinant collagenase (MMP-1) was purified using the Ni-NTA agarose.
The recombinant protein was verified by Western blotting. Furthermore, the biological activity of purified protein was measured in the presence of its specific substrate and its inhibitor, which was 67 IU.mL-1 based on our results, it was revealed that the characterized gene in our previous study codes L. sericata collagenesa enzyme.
Considering to the broad applications of collagenase in medical sciences, for the first time, we cloned the L. sericata collagenase (MMP-1) gene into the insect cell line to establish a method for the expression and purification of L. sericata collagenase (MMP-1). The result help for preparing and designing a safe and versatile recombinant drug in future.

Some mosquito species which belong to the Culex. pipiens complex are primary vectors for West Nile virus, Sindbis, Dirofilaria immitis, and many arboviruses. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene of Cx. pipiens that is inherited, is one of the important threats for the efficacy of pyrethroids insecticides. Knockdown resistance (kdr) mutation, L1014F, is a well-defined mechanism of resistance to pyrethroids and DDT in many insect species. The aim of study was to determine the mechanisms of Insecticide resistance in this species Specimens of Cx. pipiens, the major vector of West Nile virus, were obtained in Tehran, Iran by collecting larvae from polluted wastewater in Qarchak of Tehran. In 2016 Insecticide susceptibility tests were performed according to WHO methods with deltamethrin 0.05%. We focused on determination of this point mutation in the VGSC gene of Cx. pipiens by Real-time PCR. Our results revealed high levels of resistance to deltamethrin 0.05%. The lethal times i.e. LT50 and LT90 for deltamethrin were 2.1530 and 8.5117 h respectively. The result of Real-time PCR confirmed the presence of resistant genotype in all the members of tested population. This study is the first report on kdr genotyping of Cx. pipiens from Tehran and our results on the VGSC gene in position L1014F confirmed the TTA to TTT nucleotide change. This finding will provide a clue for management of insecticide resistance in mosquito which are vectors of arboviruses and decision for replacement of novel approach for vector control.

Dirofilaria immitis is a cosmopolitan zoonotic, vector-borne parasite of carnivorous animals causing dirofilariasis in human beings. Common commercial serodiagnostic tests for canine dirofilariasis usually lead to different results in their sensitivity and specificity. The present study reports development of recombinant DgK (rDgK) antigen of D. immitis for accurate immunodiagnosis of D. Immitis-infected dogs using indirect ELISA test.
The rDgK coding sequence was successfully sequenced, codon optimized and cloned into pET-24a() expression vector and then expressed in Escherichia coli. The recombinant DgK was affinity purified using Ni²+charged HiTrap chelating column, followed by testing in Western blotting and enzyme-linked immunosorbent assays (ELISA) with dog sera from a dirofilariasis endemic area. The performance of rDgK ELISA was evaluated using 60 sera collected from suspected dogs, while molecular technique was used as a reference test.
Sera from positive control D. immitis infection produced a strong IgG antibody response to rDgK both in ELISA and Western blotting tests. The sensitivity and specificity related to diagnostic potential of rDgK for ELISA were 92.5% and 87.5%, respectively. The results of rDgK ELISA showed a high agreement (0.764) with molecular identification.
The findings revealed that the developed new rDgK antigen is sensitive and specific for immunodiagnosis of canine dirofilariasis using ELISA test.

Type 2 diabetes is the most common type of diabetes, and a complex endocrine metabolic disorder that one of its main complications is diabetic foot ulcers. Matrix metalloproteinases (MMPs) are one of the key enzymes in the reconstruction of extracellular matrix which have proteolytic activity. The aim of this research is based on evaluating relationship between -1562 C>T allele at MMP-9 gene promoter with diabetic foot ulcer in type II diabetic patients. If such correlation proves, it can be used as a prognostic biomarker in patients with high-risk.
This is a case-control study. The single nucleotide polymorphism of -1562C>T allele of MMP9 gene promoter was genotyped by hit Tetra ARMS PCR technique in 100 diabetic patients with foot ulcer grade 1 or 2 as the case group according Wagner classification and in 100 diabetic patients without foot ulcer as the control group.
The Chi-square test revealed significant difference in genotype frequency of CC, CT and TT alleles of -1562C>T allele of MMP9 gene promoter between case and control groups (P=0.000).
According to this study, there is a relationship between -1562C>T allele of MMP9 gene promoter with diabetic foot ulcer in type2 diabetes patients. Thus we can introduce this biomarker for evaluation of risk and prognosis of diabetic foot ulcers.

Malarious areas in Iran are close to Afghanistan and Pakistan that urge the researchers to extend their knowledge on malaria epidemiology to the neighboring countries as well. Vectorial capacity differs at species or even at population level, the first essential step is accurate identification of vectors. This study aimed to identify Anopheles species composition in selected malarious areas of Afghanistan and Iran, providing further applied data for other research in two countries.
Adults Anopheles spp. were collected from four provinces in Afghanistan (Badakhshan, Herat, Kunduz, Nangarhar) by pyrethrum spray catch, hand collection methods through WHO/EMRO coordination and from Chaba­har County in Iran by pyrethrum spray catch method. Identification was performed using reliable identification key.
Totally, 800 female Anopheles mosquitos, 400 from each country were identified at species level. Anophe­les composition in Afghanistan was An. superpictus, An. stephensi and An. hyrcanus. Most prevalent species in Ba­dakhshan and Kunduz were An. superpictus, whereas An. stephensi and An. hyrcanus were respectively found in Nangarhar and Heart. Anopheles species in Chabahar County of Iran were An. stephensi, An. fluviatilis, An. culicifa­cies and An. sergentii. The most prevalent species was An. stephensi.
Current study provides a basis for future research such as detection of Plasmodium infection in col­lected samples which is on process by the authors, also for effective implementation of evidence-based malaria vec­tor intervention strategies.

Type 2 diabetes is a metabolic disorder characterized by high blood sugar levels that can damage nerves. Many organs are affected ¡especially the foot that leading to loss of sensation. These factors make favorable conditions for the development of diabetic foot ulcers. Polymorphisms (Thr399Ile) of Toll Like Receptor4 (TLR4) gene due to malfunction of TLR4 protein which plays an important role in immunity. The purpose of this study was to determine the parameters which are affecting the imbalance resulting in chronic inflammation and wound healing. By showing the relationship between single nucleotide polymorphism (Thr399Ile) of TLR4 gene with diabetic foot ulcer we can identify a biomarker for prediction of diabetic foot ulcer.
This is a case-control study. Single nucleotide polymorphisms of TLR4 gene were genotyped by hit Tetra ARMS PCR technique. In this study, 100 and 120 diabetic patients with and without foot ulcer were selected as the cases and controls, respectively.
The Chi-square test revealed significant difference in frequency of TT, CC and TC alleles of (Thr399Ile) between case and control groups (P=0.021).
According to this study, there is a relationship between single nucleotide polymorphisms (Thr399Ile) of TLR4 gene with diabetic foot ulcer in type 2 diabetes patients

Insulin resistance and progressive β-cells failure are the key factors in type 2 diabetes mellitus (T2DM) pathogenesis. Many studies support a primary role of RBP4 in insulin resistance and suggest that genetic variations which alter the expression level of RBP4 might influence the risk of T2DM and its complications. Diabetic foot is one of the main complications of diabetes leading to disability and hospitalization. In addition, it reduces quality of life and imposes great cost to patients. The purpose of this study was to evaluate the correlation between two single nucleotide polymorphisms (rs10882273 and rs10882283) of RBP4 genes with diabetic foot ulcer in order to identify a biomarker for prediction of diabetic foot ulcer.
This is a case-control study. Two single nucleotide polymorphisms of RBP4 genes were genotyped by hit Tetra ARMS PCR technique. In this study, 100 and 133 diabetic patients with and without foot ulcer were selected as the cases and controls, respectively.
The Chi-square test revealed no significant difference in frequency of TT, CC and TC alleles of rsl0882273 between case and control groups (P=0.414). Also, Comparison of AA, CC and AC alleles of rsl0882283 in both groups did not show significant difference (P=0.85).
According to this study, there is no relationship between two single nucleotide polymorphisms of RBP4 genes (rs10882273 and rs10882283) with diabetic foot ulcer in type2 diabetes patients.

HBV and HTLV-I are life threatening infectious agents in patients who receive blood and blood products. Although serological methods have been proved to be useful, detection of these viruses has remained a challengingissue due to the many obstacles. By the advent of Nucleic Acid Testing methods, especially in multiplex format, more precise detection is possible.The objective of this study was to develop a reliable, rapid and cost- effective method tosimultaneously detect HBV and HTLV-I.
We have developed a multiplex Real time-PCR assay for simultaneous detection of HBV and HTLV-I. Primer sets were designed for highly conserved regions of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex Real-time PCRs were performed.
Analytical sensitivity was considered to be 1000 and 100 copies/ml for HBV and HTLV-I, respectively. High concentration of one virus had no adverse effect on detection of t low concentrations of the other one. By analyzing 30 samples, clinical sensitivity of the assay was determined to be 87% and 96% for HBV and HTLV-I, respectively. Using different viral and human genome samples, the specificity of the assay was verified to be 100%.
We have developed a reliable, rapid and cost effective method tosimultaneously detect HBV and HTLV-I.Our results indicatedthe high capability of this simple and rapid method for detecting these viruses in clinical samples.

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. 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. 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. 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.

Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 (human immunodeficiency virus) and HCV (hepatitis C virus), it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument.. The current research recruits a multiplex Nucleic Acid Sequence Base Amplification (NASBA) in order to simultaneously detect HIV-1 and HCV genomes in patients’ plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples.. A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5’-NCR of HCV genome were used. A total of 40 samples of HIV-1 (20 samples) and HCV (20 samples) were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated.. Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively.. By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients’ plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner..

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. 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. 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. 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.

HIV-1 and HCV infections are life threatening problems in patients who receive blood products. Serological methods have proven useful in detecting these infections, but there are setbacks that make it challenging to detect these infectious agents. By the advent of Nucleic Acid Testing (NAT) methods, especially in multiplex format, more precise detection is possible. We have developed a multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV. Primers were designed for highly conserved region of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex RT-PCR were performed. Analytical sensitivity was considered to be 100 and 200 copies/ml for HIV-1 and HCV, respectively. High concentration of one virus had no significant effect on the detection of the other one with low concentration. By analysis of 40 samples, clinical sensitivity of the assay was determined to be 97.5%. Using different viral and human genome samples, the specificity of the assay was evaluated to be 100%. The aim of this study was to develop a reliable, rapid and cost effective method to detect HIV-1 and HCV simultaneously. Results showed that this simple and rapid method is perfectly capable of detecting two viruses in clinical samples.