Analysis of microRNAs expression changes in human breast cancer cell lines following exposure to ionizing radiation

Background: the expression alterations of specific miRNAs were analysed in response to gamma-irradiation in two breast cancer cell lines MCF-7 and MDA-MB- 231 with low and high BCSCs content to design probable strategies for sensitizing breast cancer stem-like cells MDA-MB-231 to radiation

Materials and Methods: the expression levels of hsa-miR-34a, hsa-let-7i and hsa-miR-21 were assessed by QRT-PCR in MCF-7 and MDA-MB-231 breast cancer cells at three time points after gamma irradiation using three different doses

Results: the results showed that the over-expression of hsa-miR-34a in MCF-7 cells was much more significant than MDA-MB-231 cells. Furthermore, there was a considerable overexpression of hsa-miR-21 in MDA-MB-231 cells following exposure to ionizing radiation [IR]. The hsa-let-7i expression changes were different, depending on radiation dose, time post irradiation and cell type

Conclusion: according to the results, we may be able to sensitize stem-like MDA-MB-231 breast cancer cells to radiation by increasing miR-34a and decreasing miR-21 expression levels simultaneously

[Cloning and expression of n-teminal domain of pseudomonas aeruginosa flagellin and evaluation of antibodies raised against it on motility inhibition of pseudomonas aeruginosa]

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe and lethal infections in immunocompromised individuals. This bacterium possesses a single polar flagellum. Flagellum and its subunit Flagellin play important roles in the pathogenesis of P. aeruginosa. Flagellin induces immune responses by interaction of its N-terminal domain with TLR-5. Our main aims of this study were cloning and expression of N-terminal domains of flagellin and evaluation of antibodies raised against it on motility inhibition of P. aeruginosa. The DNA sequence coding for the first 161 amino acids of flagellin was PCR amplified and cloned into a pET-28a expression vector. Recombinant protein was over expressed in BL-21[DE3], and purified by Ni-NTA resin. The immune reactivity of recombinant truncated flagellin was evaluated by Western blotting. The recombinant protein was injected into a rabbit and antibodies raised against it were evaluated for the cell motility inhibition of P. aeruginosa 8821M. The N-terminal domain of Flagellin was successfully overexpressed in Escherichia coli BL-21[DE3] host strain. Anti-native and anti-N-terminal flagellin antibodies reacted with the recombinant protein. Motility inhibition assay demonstrated that polyclonal antiserum against N-teminal flagellin is able to inhibit the motility of P. aeruginosa 8821M. The N-terminal domain of flagellin may be used for development of a new recombinant vaccine against P. aeruginosa infections

[Molecular characterization of zoonotic isolates of Enterocytozoon bieneusi in Iran]

Microsporidia infections occur in all invertebrate and vertebrate hosts. The most common microsporidia infecting humans and animals are Enterocytozoon bieneusi. This study aimed to characterize the zoonotic isolates of E. bieneusi using a molecular method among the slaughtered cattle in Tehran. In this descriptive study, 126 fecal samples from slaughtered cattle in Tehran were analyzed for Enterocytozoon bieneusi. A transcribed spacer region [500 bp] for rRNA gene of E. bieneusi was amplified using a nested PCR technique. For genotyping, positive samples were sequenced and the phylogenetic tree was reconstructed to determine the relationship between the isolates from human, animal and zoonotic isolates. Nineteen out of 126 E. bieneusi PCR-positive samples were sequenced. A high degree of genetic polymorphism, represented by four genotypes [IREb4, IREb5, D, M], was found among the E. bieneusi isolated from cattle. In this study, the most common genotypes were D [38.6%], M and IREb4 [26.3%], respectively followed by IREb5 [10.5%] in the next stage. In phylogenetic analysis, 89.5 percent of the isolates [D, IREb4, IREB5] formed a distinct cluster consisting of genotypes from humans and other domestic animals, but one genotype clustered as E. bieneusi genotypes taken from cattle and pig. Only some E. bieneusi isolates taken from cattle may be of public health importance. However, further studies should be conducted on cattle and other hosts to determine the role of animals in the transmission of infection to human

[Prevalence and severity of animal Fasciolosis in six provinces of Iran]

Fasciolosis is one of the most important parasitic disease common among both humans and livestock. Considering the health and economic importance of the disease, an understanding of the epidemiology of Fasciolosis is highly crucial. This study aimed to evaluate the prevalence and severity of Fasciola infection in animals from different geographical regions of Iran during 2009-10. In this descriptive study, 11100 livers taken from slaughtered sheep and cattle were carefully examined for Fasciola parasites at the six industrial slaughterhouses of East Azerbaijan, Khorasan-Razavi, Khuzestan, Fars, Mazandaran and Markazi provinces. All Fasciola parasites isolated from the livers of infected animals were transferred to the laboratory, and then the parasite species were identified and counted. Finally, the frequency distribution and the severity of infection were analyzed. In this study, 1.10% of the total sheep and cattle slaughtered in six industrial slaughterhouses were found positive for Fasciolosis. The severity of Fasciola in sheep and cattle livers was 7.77 +/- 0.42 and 15.24 +/- 1.78, respectively. Khorasan Razavi and Fars provinces had the highest [14.54 +/- 3.16] and lowest [7.75 +/- 0.79] severity of infection, respectively. Rresults of the study show a reduction in the prevalence and severity of Fasciolosis in sheep and cattle. But considering the importance of the disease and its endemicity, the preventive measures should be taken against the animal and human Fasciolosis in Iran

Molecular identification and differentiation of fasciola isolates using PCR-RFLP method based on internal transcribed spacer [ITS1, 5.8S rDNA, ITS2]

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species. The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety tematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp409I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates. The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates

Assessment of piwil2 gene expression pattern upon germ cell development from mouse embryonic stem cell

In order to improve culture conditions for optimal spermatogenesis, quantitative assessment of the male germ cell gene expression profile upon spontaneous ES cell differentiation is necessary. In this study, the quantitative expression profile of Piwil2, germ-line specific marker, during the early stage of embryoid body [EB] formation and differentiation [0-3-day-old EB] was studied. CCE mouse embryonic stem cells [ESCs] were cultured in DMED containing 20% fetal bovine serum [FBS] for 1, 2 and 3 days. The total RNA was isolated from ESCs, 1-3-day-old EBs, and adult testis as positive control. cDNA was prepared and quantitative real-time PCR was done for Oct-4 to study the pluripotency of this cell line. Also, the molecular pattern of PiwiI2 expression in developing EB was investigated. Our quantitative results confirmed the pluripotency of CCE mouse ESC line and showed that PiwiI2 was expressed in undifferentiated CCE mouse ESC line. Our results also showed that expression of Piwil2 increased significantly during the process of EB formation and differentiation up to 2-day-old EB and decreased non- significantly in 3-day-old EB. Our results suggest that EB provide a cellular environment similar to the early embryonic microenvironment and cause the efficient and progressive germ cell lineage differentiation in this system

Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients

Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients Nazemi A1, Sadeghizadeh M2, Forouzandeh Moghaddam M3, Javadi Gh4, Hashemi M5 1 Student of PhD of Molecular Genetics, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 2Associate Professor, Department of Genetics, Tarbiat Modarres University, Tehran, Iran. 3 Associate Professor, Department of Medical Biotechnology, Tarbiat Modarres University, Tehran, Iran. 4 Associate Professor, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 5 Assistant Professor,Department of Molecular Genetics, Islamic Azad University, Tehran Medical Branch, Tehran, Iran. Chronic myeloid leukemia [CML] is characterized by neoplastic overproduction of myelocytes and neutrophils. Affected patients have a Philadelphia chromosome which arises following a translocation between long arms of chromosome 9 and 22 [q34; q11]. This results in abelson murine/breakpoint cluster region [BCR/ABL] fusion. Detection of cells carrying BCR/ABL fusion is extremely important in monitoring response to treatment, remission and relapse in CML patients. In this study, we compared RT-PCR and NASBA techniques to determine quantitatively the number of bcr/abl transcripts. Fusion transcripts were synthesized and RNA was extracted from K562 leukemic cell line. A serial dilution of both fusion transcript and RNA was prepared; then sensitivities of both techniques were determined. RT-PCR and NASBA reaction products were labeled using equal ratios of DIG-11-dUTP and DIG-11-UTP respectively. Following denaturation, hybridization reactions were carried out with specific probes. The products were incubated in streptavidin coated microplates. Then, the plates were washed, anti-DIG conjugated with peroxidase added and using ATBS as substrate, enzymatic activity was determined by absorption at 405 nm. The results showed that specificity of two techniques was equal but RT-PCR-ELISA sensitivity was about 100-fold more than NASBA-ELISA as it could detect 100 pg RNA less than NASBA-ELISA [0.006 versus 0.06 pg RNA]. Furthermore, leukemia cell detection precision by RT-PCR-ELISA and NASBA-ELISA was 4 and 400 cells, respectively. While NASBA technique does not need thermal cycler PCR but has less sensitivity than RT-PCR and is not suitable for quantitative assessment

[ effect of occupational therapy on positive and negative symptoms in schizophrenic patients]

Poor social, self-care, and vocational functioning are criteria for a diagnosis of schizophrenia in most diagnostic systems. Consequently, improving the social behaviors of persons with schizophrenia has been a key target of psychiatric rehabilitation. The occupational therapy is a non organic therapeutic that causes elevated self stem, foppishness and strengthening of occupational behaviour. The aim of this survey is the effect of occupational therapy on the positive and negative symptom's of schizophrenic patients with bear out their symptoms. This survey is an experimental study that, positive and negative symptom's of schizophrenic patients assessed with scale for the assessment of positive and negative symptoms. Then the samples consisted of schizophrenic patients divided randomly into case [30] and control [30] groups. Occupational therapy was performed in case group within a period 20 hours in week for 6 months, then patiants assessed repeatly with SAPS.SANS. Quantative analysis of data was undertaken by using paired and dependent t students tesats and Willcoxon test .Results demonestrated the mean of the total score of negative symptom 72.5 +/- 19.5 and posetive symptom 112 +/- 32.57. Also occupational therapy effected on the posetive and negative symptom's of schizophrenic patiants. In posetive symptom occupational therapy effected on the hallusination and bizzare behaviour [P<0.001], for all noeffected on dellusions and thought. In negative symptom occupational therapy effected on the apathy and involition, attention disorders, anhedonia and thought disorders [P<0.001], for all noeffected on inappropiate affect. The occupational therapy is a non organic therapuitic that causes elevated self steem, foppishness and strengthening of occupational behaviour

Development of covalent reverse dot-blot technique for rapid diagnosis of two common beta-thalassemia mutations: IVS-I-5 and IVS-I-110 in Iran

Beta-thalassemia is an autosomal recessive disorder that most commonly caused by point mutations in the beta-globin gene. IVS-I-5 and IVS-I-110 are the most frequent mutations found among Iranians comprising 12% of all mutations. In this study, we develop the implementation of the reverse Dot- Blot [RDB] hybridization technique as a rapid and simple method for the detection of the two common mutations in the beta-globin gene. Total genomic DNA was extracted and PCR with specific primer [forward and reverse primer] was performed on region of beta-globin gene consisting the two mutations. Labeled dNTPs with DIG-II-dUTP were used in PCR mixture therefore PCR product contained DIG labeling formed. The oligonucleotide probe was immobilized onto a Biodyne C nylon membrane via activation membrane. The strips were hybridized with 10 microliters of denatured PCR product labeled to DIG at 42°C for 60 minutes. After blocking strip with the blocking buffer, the strip exposed to 5 unit anti-DIG antibody conjugates with alkaline phosphatase for 30 minutes at room temperature. The color detection was performed with nitroblue tetrazolium salt [NBT/BCIP] substrate for 120 minutes. The presence of a particular DNA sequence was detected by the appearance of a dot on the membrane. Normal individuals [N/N] showed dots with each wild-type sequence but not with any mutant probe. Heterozygotic individuals showed the appearance of a single mutation dot in addition to all the normal dots and homozygous individuals revealed dots with each mutant probe but not with any wild-type sequence. We described a rapid and simple strategy to detect beta-thalassemia mutations based on PCR followed by reverse Dot-Blot hybridization