Histone Modifications of H3K4me3, H3K9me3 and Lineage Gene Expressions in Chimeric Mouse Embryo.

OBJECTIVE: Chimeric animal exhibits less viability and more fetal and placental abnormalities than normal animal. This study was aimed to determine the impact of mouse embryonic stem cells (mESCs) injection into the mouse embryos on H3K9me3 and H3K4me3 and cell lineage gene expressions in chimeric blastocysts. MATERIALS AND METHODS: In our experiment, at the first step, incorporation of the GFP positive mESCs (GFP-mESCs) 129/Sv into the inner cell mass (ICM) of pre-compacted and compacted morula stage embryos was compared. At the second and third steps, H3K4me3 and H3K9me3 status as well as the expression of Oct4, Nanog, Tead4, and Cdx2 genes were determined in the following groups: i. In vitro blastocyst derived from In vivo morula subjected to mESCs injection (blast/chimeric), ii. In vivo derived blastocyst (blast/In vivo), iii. In vitro blastocyst derived from culture of morula In vivo (blast/morula), and iv. In vitro blastocyst derived from morula In vivo subjected to sham injection (blast/sham). RESULTS: Subzonal injection of GFP-mESCs at the pre-compacted embryos produced more chimeric blastocysts than compacted embryos (P<0.05). The number of trophectoderm (TE), ICM, ICM/TE and total cells in chimeric blastocysts were less than the corresponding numbers in blastocysts derived from other groups (P<0.05). In ICM and TE of chimeric blastocysts, the levels of H3K4me3 and H3K9me3 were respectively decreased and increased compared to the blastocysts of the other groups (P<0.05). Expressions of Oct4, Nanog and Tead4 were decreased in chimeric blastocysts compared to the blastocysts of the other groups (P<0.05), while this was not observed for Cdx2. CONCLUSION: In the present study, embryo compaction significantly reduced the rate of incorporation of injected mESCs into the ICM. Moreover, in chimeric blastocysts, the levels of H3K9me3 and H3K4me3 were altered. In addition, the expressions of pluripotency and cell fate genes were decreased compared to blastocysts of the other groups.

Functional and Molecular Characterization of C91S Mutation in the Second Epidermal Growth Factor-Like Domain of Factor VII.

BACKGROUND: Coagulation Factor VII is a vitamin K-dependent serine protease which has a pivotal role in the initiation of the coagulation cascade. The congenital Factor VII deficiency is a recessive hemorrhagic disorder that occurs due to mutations of F7 gene. In the present study C91S (p.C91S) substitution was detected in a patient with FVII deficiency. This mutation has not been characterized by a functional study. OBJECTIVES: In this study, we aimed to evaluate the impact of C91S substitution on factor VII expression and function. MATERIALS AND METHODS: The F7 complete cDNA was isolated from HepG2 cell line and inserted into the pcDNA3.1 mammalian expression vector. The desired mutation was generated by the site-directed mutagenesis and the wild-type and mutated constructs were transfected into CHO-K1 cells. The protein activity and antigen level (antigen concentration) were validated in the culture medium and cell lysate of the transiently transformed cells. An immunocytochemistry procedure was also performed to evaluate the intracellular localization of the mutated and the wild-type FVII, as well. RESULTS: The present in vitro study has demonstrated that C91S antigen expression was increased in the transfected CHO-K1 cells compared to the wild-type (WT) protein. Despite an increased protein secretion, the factor VII coagulant activity was diminished following C91S substitution when it was assessed by a standard one-stage analysis. In addition, the immunocytochemistry procedure revealed that there was no difference in the intracellular localization of the C91S mutated FVII compared to the WT protein. CONCLUSIONS: Our results present that C91S mutation has an effect on the coagulation activity, secretion, biosynthesis, and probably folding of the FVII leading to the FVII deficiency.

Characterization of a de Novo Constitutional Balanced Translocation t (2;11)(q33.2;q23.2) with Break Point on the Human NBEAL1-GeneHo.

Reciprocal balanced translocations associated with clinical features are very rare. This study reports cytogenetic and molecular cytogenetic findings in a 3-yr-old female patient with mild developmental retardation, slight hypotone with a de novo balanced 46, XX, t(2; 11) (q33; q23) translocation. Her parent attended private office at Tehran, Iran in 2013. G-banded chromosomes and FISH-Analysis were used to examine the patient's karyotype as well as her parents. FISH-probes prepared with specific RP11-BAC clones mapped near 2q33 and 11q23 regions were used to characterize the location of the breakpoints. One of the break points is located within the human NBEAL1-Gene locus on chromosome 2, suggesting a correlation between this gene disruption and the patient's mild developmental retardation.

In vitro expression of mutant factor VII proteins and characterization of their clinical significance.

Factor VII (FVII) serves an essential role in the initiation of blood coagulation. Mutations in conserved residues within its serine protease domain may lead to dysregulated coagulation activity. The objective of the present study was to elucidate the impact of altering two conserved residues, H348R and S282R, on the functional properties of the FVII protein. The mutation­harboring fragments were derived from genomic DNA of a FVII deficient patient. The fragments were integrated into a pcDNA vector containing FVII cDNA of HepG2 cells. The wild-type and mutated FVII constructs were transfected into CHO­K1 cells as a mammalian cell model. The coagulation activity, antigen levels and intracellular localization of the recombinant proteins were studied in association with their pathological importance. Results indicated that FVII activity was not detectable in conditioned media of the cells transfected with the mutated constructs. The H348R mutation reduced the expression of intracellular and secreted forms of the FVII protein. Following S282R transfection, intracellular FVII expression showed no significant variation; however, extracellular protein was reduced. The pattern of intracellular localization of mutated FVII remained unaltered in comparison to the wild-type protein. In conclusion, the present study suggested that missense mutations within the serine protease domain of FVII affect extracellular levels in addition to the coagulation activity of FVII. These results may contribute to further understanding of the molecular pathogenesis of FVII deficiency and the development of pharmaceutical candidates with improved therapeutic properties.

The study of MED12 gene mutations in uterine leiomyomas from Iranian patients.

Uterine leiomyomas are the most common gynecologic benign tumors of the female genital tract that cause a variety of health problems including, abnormal menstrual bleeding, pelvic pain, placenta displacement, premature labor, and miscarriages. Recently, studies showed that recurrent somatic mutations in MED12 exon 2 are the major cause of uterine leiomyomas in different ethnic groups. In order to validate these results in Iranian population, we performed mutational analysis of exon 2 and the flanking intronic regions by using single-strand conformational polymorphism (SSCP) and sequencing analyses in a series of 103 uterine leiomyomas samples. MED12 gene was mutated in 31.07 % of the uterine leiomyomas. Mutations were consisted of 20 missense (62.5 %) and 12 in-frame deletion (37.5 %) mutations and were not detected in normal myometrial tissue. Although this is the lowest mutation frequency reported so far, MED12 mutations are associated with fibroid pathogenesis in the studied population. Understanding the molecular mechanisms responsible for the pathogenesis of uterine leiomyoma will play an important role in designing new therapeutic strategies.

Oxidative stress response in patients infected by diverse hepatitis C virus genotypes.

BACKGROUND: The molecular mechanism of hepatitis C-virus (HCV) genome-specific pathogenesis remains unclear. Oxidative stress is an important pathophysiological mechanism in chronic HCV infection, but its relation to HCV genotypes has not been thoroughly examined. OBJECTIVES: In the present case-control study, the effect of diverse HCV genotypes on oxidative status changes was investigated. PATIENTS AND METHODS: From 310 patients examined by enzyme immunoassay and PCR, 160 patients with positive results for HCV with previously determined genotypes were chosen. For the control group, 160 first time blood donors referred to the Regional Blood Transfusion organization of the West Azerbaijan province, northwestern Iran were selected. Oxidative stress markers such as total antioxidant status (TAS), serum levels of reduced (GSH) and oxidized (GSSG) glutathione, Gamma-glutamyl transferase (GGT) and malondialdehyde (MDA) were evaluated in patients infected with diverse HCV genotypes and those in the control group. RESULTS: In the patient and control groups, the mean ± SE of TAS, GSH, GSSG, GGT and MDA were 1.04 ± 0.35 vs. 2.68 ± 0.77, 1.25 ± 0.37 vs. 3.12 ± 0.58, 0.20 ± 0.05 vs. 0.08 ± 0.04, 26.82 ± 5.62 vs 8.28 ± 2.03 and 2.56 ± 0.60 vs. 0.93 ± 0.34. All markers had statistical difference between the two groups (P <0.05). Obvious differences were found in oxidant/antioxidant balance among diverse HCV genotypes with an ascending trend in antioxidant levels among patients infected with genotypes 1a/b, 4, 2a/c, 2b, 3a and healthy controls and a vice versa trend in measures of oxidative markers except for malondialdehyde with a variable pattern. CONCLUSIONS: More serious disease in HCV genetic subtype 1a/1b might be associated with more severe oxidative stress. Milder damage in subtypes 4, 2a/c, 2b and 3a could be related to lower oxidative response, respectively. A combination of antiviral and antioxidative therapies may enhance the overall response rate of patients with HCV infection, especially with more destructive genotypes.

Diagnostic validity of the chemiluminescent method compared to polymerase chain reaction for hepatitis B virus detection in the routine clinical diagnostic laboratory.

BACKGROUND: Hepatitis B virus (HBV) is the most common significant chronic viral infection world-wide. Hepatitis B surface antigen (HBsAg) has been the principal target for laboratory testing to identify active infection by HBV. We aimed to find out diagnostic validity of the Liaison chemiluminescent method compared to the polymerase chain reaction (PCR) method for HBV detection in the routine clinical diagnostic laboratory. MATERIALS AND METHODS: From 350 patients suspicious of having infection with HBV, serum samples were separated and used for testing HBsAg by two methods of Liaison chemiluminescent immunoassay, with HBsAg confirmatory test and PCR method. RESULTS: According to the PCR results as assumed as gold standard method with 100% sensitivity and specificity, detection rate sensitivity of chemiluminescent with confirmatory test was 96% and its specificity was 100%, and for chemiluminescent without confirmatory test sensitivity and specificity were 100% and 70%, respectively. Also for chemiluminescent with confirmatory test, positive predictive value (PPV) was 100% and its negative predictive value (NPV) was 97%, compared to chemiluminescent without confirmatory test with PPV and NPV equal to 71% and 100%, respectively. CONCLUSIONS: It is possible to conclude that in the majority of the HBV cases, the diagnostic value of chemiluminescent method compared to the PCR method is acceptable, except in low indexes positive cases that need further investigation with the PCR method.

Synthesis and Molecular-cellular Mechanistic Study of Pyridine Derivative of Dacarbazine.

Dacarbazine is an antitumor prodrug which is used for the treatment of malignant metastatic melanoma and Hodgkin's disease. It requires initial activation in liver through an N-demethylationreaction. The active metabolite prevents the progress of disease via alkylation of guanine bases in DNA strands. In order to investigate the importance of imidazole ring and its dynamictautomerization in anticancer activity of dacarbazine, a pyridine analog of this drug was synthesized and the cytotoxic activity and cellular-molecular mechanisms of action for this compound were compared with those of dacarbazine. EC50 values for dacarbazine and the pyridine analog were found to be 56 µM and 33 µM respectively. Both dacarbazine and the pyridine analog resulted in formation of reactive oxygen species (ROS) upon their addition to the isolated rat hepatocytes. They also decreased the mitochondrial membrane potential and causedlysosomal membrane rupture. Cytotoxicity was prevented by ROS scavengers and antioxidants. Cytotoxicity wasalso prevented by CYP450 inhibitors, lysosomalinactivators and MPT (Mitochondrial Permeability Transition Pore) blockers.

Identification of homogeneously staining regions in leukemia patients.

Homogeneously staining regions (HSR) or double minute chromosomes (dmin) are autonomously replicating extra-chromosomal elements that are frequently associated with gene amplification in a variety of cancers. The diagnosis of leukemia patients was based on characterization of the leukemic cells obtained from bone marrow cytogenetics. This study report two cases, one with Acute Myeloblastic Leukemia without maturation (AML-M1), aged 23-year-old female, and the other with chronic myelogenous leukemia (CML)-blast crisis, a 28-year-old female associated with double minute chromosomes. Most cases of acute myeloid leukemia with dmin in the literature (including our cases) have been diagnosed as having acute myeloid leukemia.

shRNA mediated RHOXF1 silencing influences expression of BCL2 but not CASP8 in MCF-7 and MDA-MB-231 cell lines.

RHOXF1 has been shown to be expressed in embryonic stem cells, adult germline stem cells and some cancer lines. It has been proposed as a candidate gene to encode transcription factors regulating downstream genes in the human testis with antiapoptotic effects. Its expression in cancer cell lines has implied a similar role in the process of tumorigenesis. The human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in DMEM medium and transfected with a pGFP-V-RS plasmid bearing an RHOXF1 specific shRNA. Quantitative real- time RT-PCR was performed for RHOXF1, CASP8, BCL2 and HPRT genes. Decreased RHOXF1 expression was confirmed in cells after transfection. shRNA knock down of RHOXF1 resulted in significantly decreased BCL2 expression in both cell lines but no change in CASP8 expression. shRNA targeting RHOXF1 was shown to specifically mediate RHOXF1 gene silencing, so RHOXF1 can mediate transcriptional activation of the BCL2 in cancers and may render tumor cells resistant to apoptotic cell death induced by anticancer therapy. shRNA mediated knock down of RHOXF1 can be effective in induction of apoptotic pathway in cancer cells via BCL2 downregulation, so it can have potential therapeutic utility for human breast cancer.

PCR and Elisa methods (IgG and IgM): their comparison with conventional techniques for diagnosis of Mycobacterium tuberculosis.

In order to establish a rapid and stable method for diagnosis of Mycobacterium tuberculosis infection and minimize the side effects of delayed diagnosis on patients and health system, a cross sectional study was carried out. Since, the infection rate with this bacteria increasing and one of the reasons for this increase is long process of laboratory identification, therefore establishing new diagnosis methods could decrease disease rate. To achieve this aim, collected sputum and blood specimens from 50 patients with clinical suspicion of pulmonary tuberculosis were studied with both traditional, acid-fast stain (AFB) and culture method compare to Enzyme-linked immunosorbent assay (Elisa) (IgG and IgM) and Polymerase Chain Reaction (PCR) methods. The sensitivity and specificity of all methods were determined by using the PCR results as the gold standard. The overall sensitivity, specificity, positive predictive value and negative predictive value of AFB were 17.64, 100, 100 and 70.12%. These values for culture method was 29.41, 100, 100 and 73.33% and for IgG antibody were 66.7, 81.81, 64.7 and 81.81% and IgM antibody were 70.58, 90.9, 80 and 85.71%, respectively. It was concluded that maximum sensitivity and specificity can be achieved by PCR method.