A comprehensive review of HVS-I mitochondrial DNA variation of 19 Iranian populations.

Iran is located along the Central Asian corridor, a natural artery that has served as a cross-continental route since the first anatomically modern human populations migrated out of Africa. We compiled and reanalyzed the HVS-I (hypervariable segment-I) of 3840 mitochondrial DNA (mtDNA) sequences from 19 Iranian populations and from 26 groups from adjacent countries to give a comprehensive review of the maternal genetic variation and investigate the impact of historical events and cultural factors on the maternal genetic structure of modern Iranians. We conclude that Iranians have a high level of genetic diversity. Thirty-six haplogroups were observed in Iran's populations, and most of them belong to widespread West-Eurasian haplogroups, such as H, HV, J, N, T, and U. In contrast, the predominant haplogroups observed in most of the adjacent countries studied here are H, M, D, R, U, and C haplogroups. Using principal component analysis, clustering, and genetic distance-based calculations, we estimated moderate genetic relationships between Iranian and other Eurasian groups. Further, analyses of molecular variance and comparing geographic and genetic structures indicate that mtDNA HVS-I sequence diversity does not exhibit any sharp geographic structure in the country. Barring a few from some culturally distinct and naturally separated minorities, most Iranian populations have a homogenous maternal genetic structure.

Differential Expression of miR-21-5p, miR-20a-5p, TGF-ß1, and TGF-ß Receptor 2 in Skin, Serum, and Lung Samples Exposed to Sulfur Mustard.

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-ß1] and TGF-ß 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-ß1, 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-ß1 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-ß1, and TGFR2 expressions could be used as potential biomarkers for discriminating SM-exposed patients from healthy individuals.

Variations of SARS-CoV-2 in the Iranian population and candidate putative drug-like compounds to inhibit the mutated proteins.

The first cases of the novel coronavirus, SARS-CoV-2, were detected in December 2019 in Wuhan, China. Nucleotide substitutions and mutations in the SARS-CoV-2 sequence can result in the evolution of the virus and its rapid spread across the world. Therefore, understanding genetic variants of SARS-CoV-2 and targeting the conserved elements responsible for viral replication have great benefits for detecting its infection sources and diagnosing and treating COVID-19. In this study, we used the SARS-CoV-2 sequence isolated from a 59-year-old man in Ardabil, Iran, in April 2020 and sequenced using Oxford Nanopore technology. A meta-analysis comparing the sequence under study with other sequences from Iran indicated long nucleotide insertions/deletions (indels) that code for NSP15, the NSP14-NSP10 complex, open reading frame ORF9b, and ORF1ab polyproteins. In addition, replicating the NSP8 protein in the study sequence is another topic that can affect viral replication. Then using the DNA structure of NSP8, NSP15, NSP14-NSP10 complex, and ORF1ab as a genetic target can help find drug-like compounds for COVID-19. Potential drug-like compounds reported in this study for their mechanism of action and interactions with SARS-CoV-2 genes using drug repurposing are resveratrol, erythromycin, chloramphenicol, indomethacin, ciclesonide, and PDE4 inhibitor. Ciclesonide appears to show the best results when docked with chosen viral proteins. Therefore, different proteins isolated from nucleotide mutations in the virus sequence can indicate distinct inducers for antibodies and are important in vaccine design.

Overview of advances in CRISPR/deadCas9 technology and its applications in human diseases.

Prokaryotes possess an adaptive immune system using various CRISPR associated (Cas) genes to make an archive of records from invading phages and eliminate them upon re-exposure when specialized Cas proteins cut foreign DNA into small pieces. On the basis of the different types of Cas proteins, CRISPR systems seen in some prokaryotic genomes, are different to each other. It has been proved that CRISPR has a great potential for genome engineering. Studies have also demonstrated that in comparison to the preceding genome engineering tools CRISPR/Cas systems can be harnessed as a flexible tool with easy multiplexing and scaling ability. Recent studies suggest that CRISPR/Cas systems can also be used for non-genome engineering roles. Isolation and identification of new Cas proteins or modification of existing ones are effectively increasing the number of CRISPR applications and helps its development. D10A and H840A mutations at RuvC and HNH endonuclease domains of wild type Streptococcus pyogenes Cas9 (SpCas9) respectively creates a nuclease, dead Cas9 (dCas9) molecule, that does not cut target DNA but still retains its capability for binding to target DNA based on the gRNA targeting sequence. In this article we review the potentials of this enzyme, dCas9, toward development of the applications of CRISPR/dCas9 technology in fields such as; visualization of genomic loci, disease diagnosis and transcriptional repression and activation.

Recent innovations and in-depth aspects of post-genome wide association study (Post-GWAS) to understand the genetic basis of complex phenotypes.

In the past decade, the high throughput and low cost of sequencing/genotyping approaches have led to the accumulation of a large amount of data from genome-wide association studies (GWASs). The first aim of this review is to highlight how post-GWAS analysis can be used make sense of the obtained associations. Novel directions for integrating GWAS results with other resources, such as somatic mutation, metabolite-transcript, and transcriptomic data, are also discussed; these approaches can help us move beyond each individual data point and provide valuable information about complex trait genetics. In addition, cross-phenotype association tests, when the loci detected by GWASs have significant associations with multiple traits, are reviewed to provide biologically informative results for use in real-time applications. This review also discusses the challenges of identifying interactions between genetic mutations (epistasis) and mutations of loci affecting more than one trait (pleiotropy) as underlying causes of cross-phenotype associations; these challenges can be overcome using post-GWAS analysis. Genetic similarities between phenotypes that can be revealed using post-GWAS analysis are also discussed. In summary, different methodologies of post-GWAS analysis are now available, enhancing the value of information obtained from GWAS results, and facilitating application in both humans and nonhuman species. However, precise methods still need to be developed to overcome challenges in the field and uncover the genetic underpinnings of complex traits.

Considering smoking status, coexpression network analysis of non-small cell lung cancer at different cancer stages, exhibits important genes and pathways.

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer among smokers, nonsmokers, women, and young individuals. Tobacco smoking and different stages of the NSCLC have important roles in cancer evolution and require different treatments. Existence of poorly effective therapeutic options for the NSCLC brings special attention to targeted therapies by considering genetic alterations. In this study, we used RNA-Seq data to compare expression levels of RefSeq genes and to find some genes with similar expression levels. We utilized the "Weighted Gene Co-expression Network Analysis" method for three different datasets to create coexpressed genetic modules having relations with the smoking status and different stages of the NSCLC. Our results indicate seven important genetic modules having important associations with the smoking status and cancer stages. Based on investigated genetic modules and their biological explanation, we then identified 13 newly candidate genes and 7 novel transcription factors in association with the NSCLC, the smoking status, and cancer stages. We then examined those results using other datasets and explained our results biologically to illustrate some important genes in relation with the smoking status and metastatic stage of the NSCLC that can bring some crucial information about cancer evolution. Our genetic findings also can be used as some therapeutic targets for different clinical conditions of the NSCLC.

miR-21-5p, miR-141-3p, and miR-205-5p levels in urine-promising biomarkers for the identification of prostate and bladder cancer.

BACKGROUND: Early detection of cancers improves patients' survival and decreases the treatment cost. Unfortunately, the current methods for diagnosis of bladder and prostate cancers, two most common urothelial malignancies, suffer from a low sensitivity and specificity. MicroRNAs, as a group of endogenously produced non-coding RNAs, regulate gene expression and their expression is observed to be altered in many cancers and cancer progression phenomena. The remarkable stability of microRNAs in biofluids and their unique expression pattern in different pathological conditions make them an appealing, noninvasive diagnostic method in cancer diagnosis. Our objective is to identify microRNAs as biomarkers in urine samples of bladder and prostate cancers to improve the existing diagnostic methods in this field. MATERIALS AND METHODS: In this study, urine samples from 110 men with either bladder (n = 45) or prostate (n = 23) cancer, benign prostatic hyperplasia (n = 22) and healthy controls (n = 20) were collected. qPCR was used to evaluate the expression level of miR-21-5p, miR-141-3p, and miR-205-5p in these samples. The sensitivity and specificity of these microRNAs were determined using ROC curve analysis. RESULTS: The analysis of the data revealed that miR-21-5p, miR-141-3p, and miR-205-5p are differentially expressed in urine of bladder and prostate cancer patients. All these three microRNAs were upregulated in these samples and they were also able to differentiate benign prostatic hyperplasia from malignant cases. The statistical analyses revealed a good specificity for each individual microRNA. CONCLUSION: The results show that these three urine-based microRNAs might be a good choice to implement a specific and non-invasive diagnostic tool for bladder and prostate cancer. The expression pattern of all three microRNAs was particularly useful to distinguish benign and invasive tumors in prostate cases. From the patients' perspective the improvement of the diagnostic situation is awaited eagerly.

The functional significance of 14-3-3 proteins in cancer: focus on lung cancer.

The 14-3-3 family proteins are phosphoserine/phosphothreonine binding proteins constituting a conserved class of proteins which are detected in all eukaryotic cells. In mammalians, 14-3-3 proteins have seven distinct isoforms (ß, γ, ε, η, ζ, σ and τ/θ) which are involved in various cellular processes including signal transduction, cell cycle, cell proliferation, apoptosis, differentiation and survival. 14-3-3 proteins do not have a distinct catalytic activity and often regulate the activity, stability, subcellular localization and interactions of other proteins. The 14-3-3 family proteins function through interacting with their client proteins or facilitating the interaction of other proteins likely as adaptor proteins. The versatile functions of these proteins in the regulation of cell growth, cell division, cell death and cell migration make them candidate proteins for which an important role in cancer development could be envisioned. Indeed, analysis of cancer cell lines and tumor-derived tissues have indicated the differential abundance or post-translational modification of some 14-3-3 isoforms. In this review, we aimed to show how deregulation of 14-3-3 proteins contributes to initiation, establishment and progression of cancers with a particular emphasis on lung cancer. The role of these proteins in cancer-relevant processes including cell cycle, cell migration, cell-cell communication and programmed cell death will be discussed in detail.

Aberrant Expression of Breast Development-Related MicroRNAs, miR-22, miR-132, and miR-212, in Breast Tumor Tissues.

PURPOSE: MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that posttranscriptionally regulate the expression of most genes in the human genome. miRNAs are often located in chromosomal fragile sites, which are suscept-ible to amplification or deletion. Chromosomal deletions are frequent events in breast cancer cells. Deletion and loss of heterozygosity at 17p13.3 have been reported in 49% of breast cancers. The aim of the current study was to evaluate potential expression alterations of miR-22, miR-132, and miR-212, which are located on the 17p13.3 locus and are required for mammary gland development. METHODS: A matched case-control study was conducted, which included 36 pairs of tumor and matched nontumor surgical specimens from patients diagnosed with breast invasive ductal carcinoma. Formalin-fixed paraffin-embedded samples from archival collections at the pathology department of Shariati Hospital were prepared for RNA extraction using the xylene-ethanol method before total RNA was isolated with TRIzol Reagent. Specific primers were designed for cDNA synthesis and miRNA amplification. The expression of miRNAs was then evaluated by real-time polymerase chain reaction (RT-PCR). RESULTS: According to our RT-PCR data, the miR-212/miR-132 family was downregulated in breast cancer (0.328-fold, p<0.001), and this reduced expression was the most prominent in high-grade tumors. In contrast, miR-22 exhibited a significant upregulation in breast tumor samples (2.183-fold, p=0.040). CONCLUSION: Consistent with the frequent deletion of the 17p13.3 locus in breast tumor cells, our gene expression data demonstrated a significant downregulation of miR-212 and miR-132 in breast cancer tissues. In contrast, we observed a significant upregulation of miR-22 in breast tumor samples. The latter conflicting result may have been due to the upregulation of miR-22 in stromal/cancer-associated fibroblasts, rather than in the tumor cells.

A systems biology approach for miRNA-mRNA expression patterns analysis in non-small cell lung cancer.

Non-small cell lung cancers (NSCLCs) is a prevalent and heterogeneous subtype of lung cancer accounting for 85 percent of patients. MicroRNAs (miRNAs), a class of small endogenous non-coding RNAs, incorporate into regulation of gene expression post-transcriptionally. Therefore, deregulation of miRNAs' expression has provided further layers of complexity to the molecular etiology and pathogenesis of different diseases and malignancies. Although, until now considerable number of studies has been carried out to illuminate this complexity in NSCLC, they have remained less effective in their goal due to lack of a holistic and integrative systems biology approach which considers all natural elaborations of miRNAs' function. It is able to reliably nominate most affected signaling pathways and therapeutic target genes by deregulated miRNAs during a particular pathological condition. Herein, we utilized a holistic systems biology approach, based on appropriate re-analyses of microarray datasets followed by reliable data filtering, to analyze integrative and combinatorial deregulated miRNA-mRNA interaction network in NSCLC, aiming to ascertain miRNA-dysregulated signaling pathway and potential therapeutic miRNAs and mRNAs which represent a lion' share during various aspects of NSCLC's pathogenesis. Our systems biology approach introduced and nominated 1) important deregulated miRNAs in NSCLCs compared with normal tissue 2) significant and confident deregulated mRNAs which were anti-correlatively targeted by deregulated miRNA in NSCLCs and 3) dysregulated signaling pathways in association with deregulated miRNA-mRNAs interactions in NSCLCs. These results introduce possible mechanism of function of deregulated miRNAs and mRNAs in NSCLC that could be used as potential therapeutic targets.

Decreased expression of miR-20a and miR-92a in the serum from sulfur mustard-exposed patients during the chronic phase of resulting illness.

CONTEXT: Sulfur mustard (SM), with extensive nucleophilic and alkylating properties, was employed during the Iran-Iraq war by Iraqi forces. The most critical complications attributed to SM are related to dangerous pulmonary disorders collectively known as "mustard lung". The symptoms gradually emerge over a long period, becoming chronic, and are dependent on time and the amount of exposed SM. Because of the unknown and complex nature of the disease, no differential diagnostic method or absolute treatment strategy has been formally developed. OBJECTIVE: The aim of our study was to determine the expression pattern of the microRNAs (miRNAs) miR-92a and miR-20a in the serum of patients with mustard lung along with that of normal individuals. miRNAs have been shown to possess stable persistence in biofluids like plasma and serum and are considered non-aggressive biomarkers helpful for diagnosis and treatment of many diseases. MATERIALS AND METHODS: A highly sensitive approach called stem-loop real-time quantitative polymerase chain reaction was employed to study the expression of miRNAs. RESULTS: The expression of miR-92a and miR-20a was significantly down-regulated in the serum of patients with mustard lung compared to the control group. DISCUSSION: Down-regulation of miR-92a and miR-20a may be due to chronic epigenetic alterations after SM exposure, which finally leads to changes in vital cellular processes such as differentiation, proliferation and so forth. CONCLUSION: Our findings may provide a differential diagnostic method that is effective for diagnosing lung diseases caused by SM exposure. Additionally, these miRNAs may be regarded as probable targets for treatment of lung injuries.

Down-regulation of TGF-b1, TGF-b receptor 2, and TGF-b-associated microRNAs, miR-20a and miR-21, in skin lesions of sulfur mustard-exposed Iranian war veterans.

Sulfur mustard (SM) affects divergent cellular pathways including cell cycle, apoptosis, necrosis, and inflammatory responses. SM-induced lesions in skin include late-onset hyper-pigmentation, xerosis, and atrophy. It seems that TGF-b signaling pathway is a major player for SM pathogenesis. Here, we have employed a real-time polymerase chain reaction (PCR) approach to evaluate the expression alterations of all TGF-b variants and their receptors in skin biopsies obtained from 10 Iran-Iraq war veterans. Using specific LNA primers, the expression alteration of a TGF-bR2 regulator, miR-20a, and TGF-b downstream target, miR-21, was also assessed in the same samples Our real-time PCR data revealed a significant down-regulation of TGF-b1 and TGF-bR2, the major mediators of TGF-b signaling pathway, in skin biopsies of SM-exposed patients (p = 0.0015 and p = 0.0115, respectively). Down-regulation of TGF-b signaling pathway seems to contribute in severe inflammation observed in SM-exposed patients' tissues. MiR-20a and miR-21, as two important TGF-b associated microRNAs (miRNAs), were also down-regulated in SM-exposed skin lesions, compared to those of control group (p = 0.0003). Based on our findings, these miRNAs could be directly or indirectly involve in the pathogenesis of SM. Altogether, our data suggest the suitability of TGF-b1, TGF-bR2, as well as miR-20a and miR-21 as potential biomarkers for diagnosis and treatment of SM-exposed patients.

Identification of Reliable Reference Genes for Quantification of MicroRNAs in Serum Samples of Sulfur Mustard-Exposed Veterans.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. CONCLUSION: We demonstrate that non-miRNA reference genes have the least stabil- ity 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.

Selection of DNA aptamers against Human Cardiac Troponin I for colorimetric sensor based dot blot application.

Troponin T and I are ideal markers which are highly sensitive and specific for myocardial injury and have shown better efficacy than earlier markers. Since aptamers are ssDNA or RNA that bind to a wide variety of target molecules, the purpose of this research was to select an aptamer from a 79bp single-stranded DNA (ssDNA) random library that was used to bind the Human Cardiac Troponin I from a synthetic nucleic acids library by systematic evolution of ligands exponential enrichment (Selex) based on several selection and amplification steps. Human Cardiac Troponin I protein was coated onto the surface of streptavidin magnetic beads to extract specific aptamer from a large and diverse random ssDNA initial oligonucleotide library. As a result, several aptamers were selected and further examined for binding affinity and specificity. Finally TnIApt 23 showed beast affinity in nanomolar range (2.69nM) toward the target protein. A simple and rapid colorimetric detection assay for Human Cardiac Troponin I using the novel and specific aptamer-AuNPs conjugates based on dot blot assay was developed. The detection limit for this protein using aptamer-AuNPs-based assay was found to be 5ng/ml.

MiRNA molecular profiles in human medical conditions: connecting lung cancer and lung development phenomena.

MiRNAs are endogenous, single stranded ~22-nucleotide non-coding RNAs (ncRNAs) which are transcribed by RNA polymerase II and mediate negative post-transcriptional gene regulation through binding to 3'untranslated regions (UTR), possibly open reading frames (ORFs) or 5'UTRs of target mRNAs. MiRNAs are involved in the normal physiology of eukaryotic cells, so dysregulation may be associated with diseases like cancer, and neurodegenerative, heart and other disorders. Among all cancers, lung cancer, with high incidence and mortality worldwide, is classified into two main groups: non-small cell lung cancer and small cell lung cancer. Recent promising studies suggest that gene expression profiles and miRNA signatures could be a useful step in a noninvasive, low-cost and repeatable screening process of lung cancer. Similarly, every stage of lung development during fetal life is associated with specific miRNAs. Since lung development and lung cancer phenomena share the same physiological, biological and molecular processes like cell proliferation, development and shared mRNA or expression regulation pathways, and according to data adopted from various studies, they may have partially shared miRNA signature. Thus, focusing on lung cancer in relation to lung development in miRNA studies might provide clues for lung cancer diagnosis and prognosis.

Co-regulated expression of TGF-ß Variants and miR-21 in bladder cancer.

PURPOSE: To investigate a potential alteration in the expression levels of transforming growth factor ß (TGF-ß) and miR-21 in bladder cancer tissues. MATERIAL AND METHODS: Using real-time polymerase chain reaction (PCR) method, we examined a potential correlated expression of miR-21 and TGF-ß variants in 30 bladder tumors and their marginal/non-tumor biopsy specimens obtained from the same patients. RESULTS: Our data revealed a significant down-regulation of TGF-ß 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). CONCLUSION: In accordance to the observed similarity between TGF-ß variants and miR-21 gene expression alterations in bladder tumors, treating 5637 bladder cancer cell line with TGF-ß recombinant protein caused a significant upregulation of miR-21. The later finding further confirmed a correlated expression of TGF-ß and miR-21 in bladder tumors.

Normalization of miRNA qPCR high-throughput data: a comparison of methods.

Low-density quantitative real-time PCR (qPCR) arrays are often used to profile expression patterns of microRNAs in various biological milieus. To achieve accurate analysis of expression of miRNAs, non-biological sources of variation in data should be removed through precise normalization of data. We have systematically compared the performance of 19 normalization methods on different subsets of a real miRNA qPCR dataset that covers 40 human tissues. After robustly modeling the mean squared error (MSE) in normalized data, we demonstrate lower variability between replicates is achieved using various methods not applied to high-throughput miRNA qPCR data yet. Normalization methods that use splines or wavelets smoothing to estimate and remove Cq dependent non-linearity between pairs of samples best reduced the MSE of differences in Cq values of replicate samples. These methods also retained between-group variability in different subsets of the dataset.

Design of a biological method for rapid detection of presence of PCR inhibitors in aged bone DNA.

BACKGROUND: Molecular human identification is one of the most important tests performed in forensic laboratories. Some of these tests are applied for identification of human remains from natural disasters, wars, etc., but problems may occur as a result of DNA degradation and external DNA contamination. We investigated effects of bacterial DNA on identifying the presence or absence of PCR inhibitors in aged bone DNA. METHODS: DNA samples were extracted from blood, bone remains and Escherichia coli. These DNA were amplified using human and bacterial specific primers. RESULTS: Using different blood, aged bone, and bacterial DNA dilutions along with PCR based methods; we checked their positive, negative effects, or detecting presence of inhibitors in aged bone DNA by PCR method. CONCLUSIONS: Our observation indicated that the addition of bacterial DNA could be a valid biological method for testing the quality of bone DNA to enable us to obtain a usable profile for the identification of human remains. This method will help to test the presence of inhibitors, quantity or even quality of DNA which are of importance in profiling archeological remains. Our method will help to determine if PCR failure is due to presence of inhibitors or lack of amplifiable DNA either because of degradation, minute amount or absence of human DNA.

High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli.

The carboxylic domain of the Clostridium botulinum neurotoxin heavy chain (BoNT/A-HC), which has been reported as a vaccine candidate, contains the principle protective antigenic determinants. In this study, the high level expression of the BoNT/A-Hc was achieved by high cell density cultivation of recombinant Escherichia coli in a 2-l batch stirred-tank bioreactor. In order to maximize protein expression, post-induction time and IPTG inducer concentration were optimized by the Taguchi statistical design method. Results showed that the middle of the logarithmic phase and an IPTG concentration of 1 mM presented the optimum conditions for the maximum expression of BoNT/A-HC. High cell density cultivation was subsequently carried out as an effective strategy for the high level expression of recombinant BoNT/A-Hc. Consequently, soluble BoNT/A-Hc was produced at the maximum level of 486 mg l(-1), at 3 h post-induction, which was approximately 9.3 and 7.8 times higher than the levels produced by the shake flask and batch culturing methods, respectively.

Correlation of major histocompatibility complex class I related A (MICA) polymorphism with the risk of developing breast cancer.

In the present study, we examined the association of different alleles of MICA gene with the risk of breast cancer development in Iranian population. Our data showed a significant relationship between longer alleles, alleles with 9- and 6-GCT repeat of MICA gene, and a higher risk of developing breast cancer according to the age of onset. The data indicated a 6-fold increase for developing breast cancer in patients carrying the allele with 6-GCT repeat after age 50 (OR = 5.8333, 95% CI: 1.2976-26/2236, P = 0.0172). In addition, patients carrying longer alleles in their genotype (6/6, 6/9, and 9/9 genotypes) were found significantly at higher risk of developing breast cancer than control individuals (OR = 5.6, P = 0.0038, 95% CI: 1.6578-18.9166). In contrast, alleles with short GCT repeat of 4 and 5.1 showed to play a role in reducing the risk of breast cancer (OR = 0.79, P = 0.3643 and 95% CI: 0.4743-1.3157). Women with allele 4 were found twofold more protected against breast cancer (OR = 0.4597, 95% CI: 0.2164-0.9765, P = 0.0401). The results suggested that women with genotypes with 9- and 6-GCT repeat alleles of MICA gene could be considered more potent to develop breast cancer especially at higher age.