MiRNAs related in signaling pathways of women's reproductive diseases: an overview.

BACKGROUND: One of the main health issues that can affect women's health is reproductive diseases, such as polycystic ovary syndrome (PCOS), endometriosis (EMs), uterine leiomyomas (ULs), and ovarian cancer (OC). Although these diseases are very common, we do not have a complete understanding of their underlying cellular and molecular mechanisms. It is important to mention that the majority of patients are diagnosed with these diseases at later stages because of the absence of early diagnostic techniques and dependable molecular indicators. Hence, it is crucial to discover novel and non-invasive biomarkers that have prognostic, diagnostic and therapeutic capabilities. MiRNAs, also known as microRNAs, are small non-coding RNAs that play a crucial role in regulating gene expression at the post-transcriptional level. They are short in length, typically consisting of around 22 nucleotides, and are highly conserved across species. Numerous studies have shown that miRNAs are expressed differently in various diseases and can act as either oncogenes or tumor suppressors. METHODS: The author conducted a comprehensive review of all the pertinent papers available in web of science, PubMed, Google Scholar, and Scopus databases. RESULTS: We achieved three goals: providing readers with better information, enhancing search results, and making peer review easier. CONCLUSIONS: This review focuses on the investigation of miRNAs and their involvement in various reproductive disorders in women, including their molecular targets. Additionally, it explores the role of miRNAs in the development and progression of these disorders.

Significant alteration of IFN stimulated genes expression in MA104 cells infected with bovine rotavirus RF strain.

The pattern recognition receptors (PRRs) trigger signaling cascades, such as nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRFs). Rotavirus (RV) countermeasures against innate responses and understanding of these processes will improve our knowledge regarding immunopathogenesis of RV infection. In this study, we investigated the effect of RV RF strain on the important ISG candidate genes engaging in virus infections for which little information is known in RV RF strain. To this end, MA104 cells were mock/infected with RF followed by incubation in the presence or absence of IFN-α and the expression of MX1, OAS1, STAT1, ISG15, and ISG56 mRNA was analyzed by real-time PCR. All of ISGs' mRNAs showed higher expression levels in IFN I treated cells compared to virus-infected cells except for ISG56. Infecting the cells with RV and treatment with IFN type I led to overexpression of ISG56 compared to cells were either infected with the virus or only treated with IFN I. In conclusion, we showed that the RV RF strain efficiently blocks type I IFN-induced gene expression particularly ISG15, MX1, STAT, and OSA1 as antiviral proteins. Furthermore, viruses may use some ISGs such as ISG 56 to regulate IFN I signaling pathway, negatively.

The role of microRNAs in diseases and related signaling pathways.

MicroRNAs (miRNAs) are epigenetic regulators of the gene expression and act through posttranslational modification. They bind to 3'-UTR of target mRNAs to inhibit translation or increase the degradation mRNA in many tissues. Any alteration in the level of miRNA expression in many human diseases indicates their involvement in the pathogenesis of many diseases. On the other hand, the regulation of the signaling pathways is necessary for the maintenance of natural and physiological characteristics of any cell. It is worth mentioning that dysfunction of the signaling pathways manifests itself as a disorder or disease. The significant evidence report that miRNAs regulate the several signaling pathways in many diseases. Base on previous studies, miRNAs can be used for therapeutic or diagnostic purposes. According to the important role of miRNAs on the cell signaling pathways, this article reviews miRNAs involvement in incidence of diseases by changing signaling pathways.

Increased expression of CD8 marker on T-cells in COVID-19 patients.

BACKGROUND: Cell-mediated immunity including T-cells (T helper and cytotoxic) plays an essential role in efficient antiviral responses against coronavirus disease-2019 (COVID-19). Therefore, in this study, we evaluated the ratio and expression of CD4 and CD8 markers in COVID-19 patients to clarify the immune characterizations of CD4 and CD8 T-cells in COVID-19 patients. METHODS: Peripheral blood samples of 25 COVID-19 patients and 25 normal individuals with similar age and sex as the control group were collected. White blood cells, platelets, and lymphocytes were counted and CD4 and CD8 T lymphocytes were evaluated by flow cytometry. RESULTS: The number of white blood cells, lymphocytes, and platelets were reduced significantly in COVID-19 patients (P < 0.05). The difference in CD4:CD8 ratio, CD4 T-cell frequency, CD8 T-cell frequency, and CD4 mean fluorescence intensity (MFI) was not significant between COVID-19 patients and healthy individuals (P > 0.05); however, the CD8 MFI increased significantly in COVID-19 infected patients (P < 0.05). CONCLUSION: Although, there is no significant difference in the ratio of CD4 to CD8 between two groups, the expression level of CD8 in COVID-19 patients was significantly higher than the normal individuals. This result suggested that the cellular immune responses triggered by COVID-19 infection were developed through overexpression of CD8 and hyperactivation of cytotoxic T lymphocytes.

Assessment of CCL27 and IL-11 in Multiple Sclerosis Patients Treated with Interferon-ß and Glatiramer Acetate.

INTRODUCTION: Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease which involves the central nervous -system. Although the primary cause of MS is obscure, effects of some cytokine and chemokine patterns in both innate and adaptive immune systems have been described. -Objectives: Since limited studies have examined the role of interleukin (IL)-11 and chemokine CCL27 in MS, we aimed to identify changes in IL-11 and CCL27 gene expression and serum levels in relapsing-remitting MS (RRMS) patients, treated with interferon (IFN)-ß and glatiramer acetate (GA). METHODS: The serum level and gene expression of IL-11 and CCL27 were measured and compared between treatment-naïve MS patients and RRMS patients who were treated with high-dose IFN-ß1a, low-dose IFN-ß1a, IFN-ß1b, and GA via enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction. RESULTS: A significant decrease was observed in the serum level of CCL27 in treatment-naïve patients and IFN-ß1b-treated patients compared to the healthy controls. On the other hand, a significant increase was found in the protein level of CCL27 in low-dose and high-dose IFN-ß1a groups compared to the treatment-naïve group. In addition, CCL27 gene expression was higher in patients treated with GA than in the treatment-naïve group. There were no significant changes in the gene expression or protein level of IL-11 in all experimental groups. Additionally, a positive correlation was found between IL-11 and CCL-27. CONCLUSION: Our results suggest the inflammatory role of CCL27 in MS patients, while IFN-ß1a seems to play a compensatory role for this chemokine.

Bone marrow - mesenchymal stem cells impact on the U937 cells in the presence of staphylococcal enterotoxin B (SEB).

The growing resistance against conventional chemotherapy in acute myeloid leukemia (AML) is a noticeable clinical concern. Therefore, many researchers are looking for novel substances to overcome drug resistance in cancer. Staphylococcal enterotoxin B (SEB) is a superantigen (SAg) and a promising compound which has lethal effects on malignant cells. In this unprecedented study, SEB was used against U937 cells in a co-culture system in the presence of human bone marrow-mesenchymal stem cells (hBM-MSCs). The effects of hBM-MSCs on the proliferation and survival of U937 cell line with SEB was assessed using MTT assay and AnnexinV/PI flowcytometry, respectively. Moreover, the expression of IL-6, IL-10, TGF-ß, and inhibitor of nuclear factor kappa-B kinase (IKKb) was evaluated by real-time PCR technique. The same experiments were also carried out using hBM-MSCs-conditioned medium (hBM-MSCs-CM). The results showed that SEB reduced the proliferation and survival of U937 cell line, but hBM-MSCs or hBM-MSCs-CM suppressed the effects of SEB. Furthermore, real-timePCR demonstrated that SEB could decrease the expression of IL-6, IL-10, and TGF-ß in hBM-MSCs (P < .05), while the production of IKKb was increased in comparison with the control group. These findings help us to have a broader understanding ofthe usage of SEB in the treatment of haematological malignancies, especially if it is targeted against hBM-MSCs to disrupt their supportive effects on malignant cells.

Specific detection of common pathogens of acute bacterial meningitis using an internally controlled tetraplex-PCR assay.

Accurate and timely diagnosis of acute bacterial meningitis is critical for antimicrobial treatment of patients. Although PCR-based methods have been widely used for the diagnosis of acute meningitis caused by bacterial pathogens, the main disadvantage of these methods is their high cost. This disadvantage has hampered the widespread use of molecular assays in many developing countries. The application of multiplex assays and "in-house" protocols are two main approaches that can reduce the overall cost of a molecular test. In the present study, an internally controlled tetraplex-PCR was developed and validated for the specific detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in cerebrospinal fluid (CSF) samples. The analysis of a panel of other human pathogens showed no cross-reactivity in the assay. The analytical sensitivity of the in-house assay was 792.3 copies/ml, when all three bacteria were presentin the specimens. This value was calculated as 444.5, 283.7, 127.8 copies/ml when only S. pneumoniae, N. meningitidis and H. influenzae, respectively, were present. To demonstrate the diagnostic performance of the assay, a total of 150 archival CSF samples were tested and compared with a commercial multiplex real-time PCR kit. A diagnostic sensitivity of 92.8% and a specificity of 95.1% were determined for the present tetraplex-PCR assay. The results indicate that the established method is sensitive, specific and cost-effective, and can be used particularly in situations where the high cost of commercial kits prevents the use of molecular methods for the diagnosis of bacterial meningitis.

MicroRNA-124 regulates neuronal differentiation of mesenchymal stem cells by targeting Sp1 mRNA.

MicroRNAs play an important role in neuronal development and function. miR-124 is the most abundantly expressed miRNA in the nervous system. Several different mRNA targets have been proposed for miR-124, but the precise function of endogenous miR-124 and its mRNA targets remain to be further elucidated. Specificity protein 1 (Sp1) is a transcription factor that plays key roles in many cell processes including cell cycle. However, this transcription factor is nearly absent in differentiated neurons and a remarkable suppression of Sp1 expression was shown after neurogenesis. Since miR-124 is expressed abundantly in neurons and because Sp1 levels decrease during neurogenesis, it is possible that miR-124 could regulate the expression of Sp1 during neuronal development. Therefore, the aim of the present study was to evaluate the putative targeting of Sp1 by miR-124. Overexpression of miR-124 using a plasmid coding for pri-miR-124 in HEK293 cells decreased the expression of Sp1 mRNA. The results of dual-luciferase reporter assay demonstrated that miR-124 directly targeted the 3'-untranslated regions of Sp1 mRNA. To evaluate whether Sp1 expression was regulated by miR-124 during the process of neuronal differentiation, Adipose-derived mesenchymal stem cells (A-MSCs) were differentiated into neuron-like cells. The results of qPCR analysis showed that with the gradual increase of miR-124 expression during neurogenesis, the expression of Sp1 mRNA decreased accordingly. In summary, this study demonstrated for the first time that miR-124 is able to suppress Sp1 expression, which in turn affected the neuronal differentiation of mesenchymal stem cells.

Monitoring human cytomegalovirus infection in pediatric hematopoietic stem cell transplant recipients: using an affordable in-house qPCR assay for management of HCMV infection under limited resources.

Quantitative real-time PCR (qPCR) assay is accepted as the method of choice for monitoring human cytomegalovirus (HCMV) infection in hematopoietic stem cell transplant recipients, but the high cost of commercial kits has hampered its use in many developing countries. In this study, an affordable in-house qPCR was used to manage HCMV infection in pediatric patients and the diagnostic value of this method was compared with the conventional pp65 antigenemia assay. A total number of 1179 samples from 82 recipients were used in this study, and the effect of some potential risk factors on HCMV reactivation was evaluated. The qPCR was able to detect HCMV reactivation earlier and with higher sensitivity than antigenemia assay. Forty-six episodes of reactivation were detected in 39 patients, of which all were detected by the qPCR assay, while only 21 episodes were diagnosed by antigenemia. The DNAemia level of 1284 IU/ml plasma was defined as the optimal cutoff value for starting pre-emptive therapy. It was shown that the acute GVHD severity and the relationship of donor and recipient are the most significant risk factors for HCMV reactivation. The data suggest that the antigenemia method for monitoring HCMV reactivation could be substituted by the qPCR assay.

Hsa-miR-15b-5p/miR-195-5p Controls Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells Through Regulating Indian Hedgehog Expression.

Osteoblastogenesis is regulated by several signaling pathways like hedgehog signaling. Of three types of mammalian Hedgehog genes, the Indian Hedgehog (Ihh) plays an important role in the formation of the skeleton. Mesenchymal stem cells (MSCs) isolated from adipose tissue have been considered a good source of osteoblast differentiation. Evidence also suggests that miRNAs play an important role in regulating key stages of osteoblast differentiation. In this study, two miRNAs targeting the Ihh were predicted by using bioinformatics analysis. ASCs were successfully derived, purified, and characterized from human adipose tissue. ASCs were chemically induced into osteoblast cells. Then, differentiation was confirmed by alkaline phosphatase (ALP) activity and Alizarin red staining. The relative expression of Ihh and related miRNAs was evaluated after 0, 7, 14, and 21 from the differentiation duration. The results of bioinformatics data showed that has-miR-195-5p and has-miR-15b-5p target the Ihh gene. The expression of Ihh significantly increased in a time-dependent manner in the differentiation process. In contrast, miR-195-5p and miR-15b-5p were significantly downregulated dependent on time duration (P < 0.01). Overall, the data indicate the antithetical regulation of Ihh versus has-miR-195-5p and has-miR-15b-5p during the differentiation process. These results support the hypothesis that these mi-RNAs could target the Ihh in the pathway of osteoblast differentiation derived from human ASCs.

Cloning, expression and purification of antigenic fragments of the Ureaplasma urealyticum UreD protein and its value in serology.

Background and Objectives: The detection of Ureaplasma urealyticum is usually done through culture. With the change of the smallest effective factor in culture, we face the lack of growth of these bacteria, which is one of the important reasons to find a suitable alternative for the diagnosis of this bacterium. UreD is a protected gene in this bacterium. The aim of this was to evaluate the ability of antigenic regions of UreD protein to bind to patients' serum antibodies. Materials and Methods: Antigenic regions of UreD protein were predicted using IEDB software with five different methods: Emini Surface Accessibility Prediction, Kolaskar and Tongaonkar Antigenicity, Chou and Fasman beta turn prediction, Karplus and Schulz flexibility scale, Ellipro-Epitope prediction based upon structural protrusion. Antigenic regions of UreD gene was clonned, expressed and purified. The antigenicity of this recombinant protein against the antibodies in the serum of people infected with U. urealyticum infections was checked in western blotting. Results: The results showed that the antigenic regions of the UreD protein was producted and its antigenicity was demonstrated in western blotting. Moreover, all sera from patients infected with U. urealyticum reacted to the recombinant antigen. Conclusion: Specimens from people infected with U. urealyticum infection was positive in Western blotting suggesting that UreD protein has antigenic properties. Therefore, it can be used as a suitable candidate for the design of diagnostic kits and U. urealyticum vaccine.

SARS-CoV-2 Viral Shedding and Associated Factors among COVID-19 Inpatients and Outpatients.

Background: According to the contagious ability of the new virus, SARS-CoV-2, characterization of viral shedding duration in the period of infection is highly valuable in terms of providing quarantine guidelines and isolation policies. Therefore, we aimed at viral shedding determination in 58 COVID-19 confirmed Iranian subjects in different stages. Methods: 58 COVID-19 confirmed Iranian subjects including 21 outpatients and 37 inpatients were investigated. The analytical data and clinical properties were documented in the standard questionnaire. The RT-PCR tests were done two and three weeks after the symptoms initiation. Results: Viral eradication occurred in 44.8% two weeks after illness initiation whereas in 71% who achieved a negative PCR test in the third week. Moreover, prolonged viral shedding was observed in hospitalized cases in comparison to outpatients. Almost 30% of patients continued viral shedding three weeks after disease initiation. Conclusion: A longer duration of viral shedding in hospitalized cases rather than outpatients was observed in this study. The results similar to other investigations call into question if the current policies are enough to prevent the viral spread or not. This study should be done on a larger sample to provide an appropriate time in isolation policy.

Diagnostic Value of Plasma miR-145 and miR-185 as Targeting of the APRIL Oncogene in the B-cell Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is one of the most common hematologic malignancy in adults worldwide. This cancer has a poor prognosis at different stages. So, the identification of new biomarkers is important for diagnosis of B-CLL. Considering the oncogenic role of APRIL molecule in this leukemia as well as the regulatory role of miRNAs in different signaling pathways, the present study evaluated the miRNAs targeting APRIL gene in B-CLL. METHODS: The miRNAs were predicted and selected using bioinformatics algorithms. A total of 80 plasma samples were subjected to RNA extraction and synthesis of cDNA. The expressions levels of predicted miRNAs and APRIL gene in plasma of B-CLL patients and healthy individuals were assessed by Real time PCR analysis. ROC analysis was performed to investigate the role predicted miRNAs as novel biomarkers in diagnosis of B-CLL. RESULTS: The results of the prediction showed that miR-145-5p and miR-185-5p target the APRIL gene. The expression level of APRIL gene was strikingly higher in plasma of B-CLL patients than in the healthy individuals (102, P= 0.001). On the other hand, expression levels of miR-145-5p and miR-185-5p were strikingly lower in B-CLL patients than in the healthy individuals (0.07, P= 0.001) (0.29, P= 0.001). Also, ROC curve analyses demonstrated that miR-145-5p and miR-185-5p are specific and sensitive and may serve as new biomarkers for the detection of B-CLL. (AUC; 0.95, sensitivity; %90) (AUC; 0.87, sensitivity; %63). CONCLUSION: These data suggest that miR-145-5p and miR-185-5p target the APRIL gene and might have a role in diagnosis of B-CLL. Therefore, these two miRNAs can be served as a novel and potential biomarker for detection of B-CLL.

Alterations in The Plasma Expression of mir-15b, mir-195 and the Tumor-Suppressor Gene DLEU7 in Patients with B-Cell Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is one of the most prevalent forms of leukemia in adults. Inactivation of the DLEU7 gene is frequently observed in patients with CLL. Furthermore, microRNAs (miRNAs) have been observed to have a critical role in the pathogenesis of several cancers, including leukemia. Considering the tumor-suppressive role of DLEU7, as well as the tumor suppressor or oncogenic role of microRNAs (miRNAs), the aim of the present study was to evaluate the potential miRNAs targeting the DLEU7 gene in B-cells and explore expression changes these genes in the plasma of B-CLL patients. METHODS: The miRNAs interacting with the DLEU7 gene were predicted and selected using bioinformatics tools. A total of 80 plasma samples were collected from 40 patients with B-cells and 40 healthy individuals, then subjected to RNA extraction and cDNA synthesis. The expression profiles of the predicted miRNAs and the DLEU7 gene in the plasma of B-CLL patients and healthy individuals were determined by RT-qPCR analysis. RESULTS: The bioinformatics prediction indicated that miR-15b and miR-195 target the DLEU7 gene. The expression levels of miR-15b and miR-195 were significantly higher in the plasma of patients with B-CLL compared to the healthy individuals (91.6, p= 0.001) (169, p= 0.001). However, the expression level of the DLEU7 gene was found to be significantly lower in the patient group compared to healthy controls (0.304, p= 0.001). CONCLUSION: Both miR-15b and miR-195, have the potential to function as novel and non-invasive biomarkers in the diagnosis and prognosis of patients with B-CLL.

Alteration in Expression of miR-32 and FBXW7 Tumor Suppressor in Plasma Samples of Patients with T-cell Acute Lymphoblastic Leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and malignant neoplasm that arises from the hematopoietic T-cell precursors. Inactivation of FBXW7 gene is frequently observed in T-cell acute lymphoblastic leukemia, suggesting a significant tumor-suppressive role for FBXW7 in the pathobiology of this leukemia. Considering the role of microRNAs in cell proliferation and regulation of apoptosis, the aim of this study was to identify novel oncogenic microRNAs that suppress FBXW7 in patients with T-ALL. PATIENTS AND METHODS: The expression levels of two bioinformatically predicted microRNAs - miR-32 and miR-107 were compared in patients with T-ALL and a control group. A total of 80 plasma samples were subjected to RNA extraction, and the microRNA expression profiles were assessed by the RT-qPCR. The expression level of miR-103 was used as the endogenous reference for normalization of quantitative data. RESULTS: The plasma levels of miR-32 and miR-107 in patients with T-ALL were significantly higher (5.65, P < 0.001) and lower (0.432, P = 0.002), respectively. On the other hand, the expression levels of FBXW7 gene were significantly downregulated by -76.9 fold in T-ALL patients (P < 0.001). The results of the ROC curve analysis indicated that overexpression of miR-32 might be used to distinguish T-ALL patients with reasonable sensitivity and specificity. CONCLUSION: miR-32 is considered as a novel oncomir that targets FBXW7 and might have a role in the etiology or progression of T-ALL. Furthermore, miR-32 can potentially serve as a non-invasive biomarker for detection of T-ALL.

Gene expression of semaphorin-3A, semaphorin-7A, neuropilin-1, plexin-C1, and ß1 integrin in treated-multiple sclerosis patients.

OBJECTIVE: Recently, members of the semaphorin family have received major attention in various medical fields, especially autoimmunity. In this study, we selected semaphorin-3A (Sema3A), semaphorin-7A (Sema7A), and their receptors to determine the possible relationship between these molecules and multiple sclerosis (MS). METHOD: We measured the gene expression of Sema3A, Sema7A, neuropilin-1 (NP-1), plexin-C1, and ß1 integrin in the blood samples of relapsing-remitting multiple sclerosis (RRMS) patients, treated with high-dose interferon-ß1a (IFN-ß1a), low-dose IFN-ß1a, IFN-ß1b, and glatiramer acetate (GA) via quantitative real-time polymerase chain reaction (qRT-PCR) assay, and then, compared the results of treatment-naive patients with the healthy controls. RESULTS: The gene expression of Sema3A (P = 0.02), NP-1 (P < 0.001), and plexin-C1 (P < 0.01) significantly decreased in the treatment-naive group, compared to the healthy controls. Sema3A significantly increased in all treated patients, compared to the treatment-naive patients (P < 0.001). However, expression of NP-1 (P < 0.001), plexin-C1 (P < 0.001), and ß1 integrin (P < 0.05) only increased in patients receiving high-dose IFN-ß1a, IFN-ß1b, and GA. Expression of Sema7A increased in only two groups of patients treated with IFN-ß1b (P < 0.001) and GA (P = 0.018), without any significant decrease in the treatment-naive group, compared to the healthy controls (P > 0.05). CONCLUSION: Our findings confirm that the presence of Sema3A, Sema7A, and their receptors can play critical roles in the treatment of MS patients. Therefore, they can be potential target molecules for MS treatment in the future.

Plasma Level of MicroRNAs, MiR-107, MiR-194 and MiR-210 as Potential Biomarkers for Diagnosis Intestinal-Type Gastric Cancer in Human

Background: Timely and sensitive diagnosis of gastric cancer is crucial for efficient treatment and survival of the patients. microRNAs have been considered as diagnostic biomarkers in different type of cancers including gastric cancer. In the present study, the expression profile of four microRNAs, miR-103, miR-107, miR-194 and miR-210 were evaluated in patients with intestinal-type of gastric cancer (IGC) in order to assess their diagnosis utility as noninvasive biomarkers. Methods: A total number of 100 plasma samples from patients with gastric cancer and healthy controls were obtained and total RNA was extracted using a commercial monophasic solution of phenol and guanidium thiocyanate. Reverse transcription (RT) reactions were performed by specific stem-loop RT primers and M-MuLV RT-enzyme. The expression patterns of microRNAs were assessed using reverse transcription quantitative real-time PCR (RT-qPCR) method and the expression of SNORD47 RNA was used as the reference for normalization. Results: The results indicate that the plasma levels of miR-107, miR-194, and miR-210 were significantly lower in patients. Receiver operating characteristic (ROC) curve analysis showed that the patients could be distinguished from healthy individuals at the cutoff levels of 0.504, 0.266, and 0.394 of miR-107, miR-194, and miR-210, respectively. On the other hand, the expression levels of these miRNAs were not significantly different in different clinicopathological stages of the disease. Conclusion: These findings suggest that the plasma levels of miR-107, miR-194 and miR-210 were downregulated in patients with ICG and propose these molecules as potential non-invasive biomarkers for detection of IGC.

Development of a high resolution melting analysis assay for rapid identification of JAK2 V617F missense mutation and its validation.

BACKGROUND: Myeloproliferative neoplasms (MPN) are heterogeneous diseases that classified by the presence of Philadelphia chromosome into Philadelphia chromosome negative (Ph-neg) and positive (Ph-pos) myeloproliferative neoplasms. In ph-neg group A somatic point mutation (c.1849G>T) in the JAK2 gene, part of the JAK2-STAT signal-transduction pathway, causes substitution of phenylalanine for valine (V617F) in the JAK2 protein and has been identified. This mutation was seen in PV by 65% to 97% and ET (30-57%) and primary myelofibrosis (35-95%). Highly sensitive methods have been used to determine the presence of the JAK2V617F mutation instead of direct sequencing. We aimed to assess JAK2 exon14 mutations by high-resolution melting (HRM) analysis, which allows variation screening in compare to other method for detecting mutation. METHODS: The mutation analysis included 45 individuals who were subjected for diagnosis of ph-neg MPN. Genomic DNA was isolated and different methods are performed. RESULTS: PCR RFLP, ARMS PCR and HRM method has a detection sensitivity comparable with conventional methods (Qiagen) to identify the mutations and sequencing. CONCLUSIONS: For HRM analysis is cost-effective and beside that it is enzyme independence method also this method able to show amount of the mutant allele carried in samples and it's helpful for treatments follow-up and determining MRD for them.

Genotyping of Echinococcus granulosus isolates from livestock based on mitochondrial cox1 gene, in the Markazi province, Iran.

Hydatidosisis a parasitic disease caused by the larval stage of Echinococcus granulosus with different genotypes, and major complications in vital organs such as liver, lungs and, brain. Also, this parasite can infect animals and cause economic damages. Recently, some investigations indicated that the genetic variation of the parasite affects the antigenic, immunogenic and pathogenic features. Therefore, present study conducted to genotyping of the E. granulosus larva based on mitochondrial cox1 gene in livestock in the endemic areas of Markazi province, Iran. In this study, 49 hydatid cysts samples collected from 36 sheep, 11 goats and 2 cattle from different slaughterhouses of Markazi province in central part of Iran, 2017. The mitochondrial cox1 gene was amplified and genotyping were accomplished using sequence analysis. The sequencing analysis indicated that the main genotype G1 (61%) and G3 (37%) were identified. Also, one of the samples shows similarity with the G2 (2%) genotype. The results showed the statistically significant differences between the genotypes in different livestock (P < 0.05). This study indicated that the main genotypes of E. granulosus in Markazi province are G1 and G3 which are related to dog/sheep strain. Therefore, parasite control in dogs and sheep can reduce the risk of transmission of infection to humans.

Viral etiology of acute respiratory infections in children in Southern Iran.

INTRODUCTION: Prevalence of influenza A virus (Flu-A), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) was assessed in children with acute respiratory infections (ARIs). METHODS: Nasopharyngeal aspirates and throat swabs were subjected to real-time polymerase chain reaction (PCR) to detect RSV and Flu-A and to conventional PCR to detect hMPV. RESULTS: Of the 156 children assessed, 93 (59.6%) carried at least one virus, with 35.9% positive for RSV, 14.1% for hMPV, and 9.6% for Flu-A. The prevalence of co-infections was 2.6%. CONCLUSIONS: The high detection rate may reflect increased sensitivity of real-time PCR compared to traditional PCR and viral culture.