Effects of ionizing radiation on the viability and proliferative behavior of the human glioblastoma T98G cell line.

OBJECTIVE: Radiotherapy is the traditional therapy for glioma patients. Glioma has poor response to ionizing radiation (IR). Studying radiation-induced cell death can help in understanding the cellular mechanisms underlying its radioresistance. T98G cell line was irradiated with Co60 source by 2 or 10 Gy. MTT assay was used to calculate the surviving fraction. Cell viability, cell cycle distribution and apoptosis assays were conducted by flow cytometry for irradiated and control cells for the 10 Gy dose. RESULTS: The SF2 value for irradiated cells was 0.8. Cell viability was decreased from 93.29 to 73.61%, while, the Sub G0/G1 phase fraction was significantly increased at 10 Gy after 48 h. On the other hand, there was an increase in the percentage of apoptotic cells which reached 40.16% after 72 h at the same dose, while, it did not exceeds 2% for non-irradiated cells. Our results showed that, the T98G cells is radioresistant to IR up to 10 Gy. Effects of irradiation on the viability of T98G cells were relatively mild, since entering apoptosis was delayed for about 3 days after irradiation.

Mild androgen insensitivity syndrome (MAIS): the identification of c.1783C>T mutation in two unrelated infertile men.

Two unrelated men complaining of primary male infertility presented to Orient Hospital in Damascus city. Physical examination showed moderate hypoandrogenic features. Both men were azoospermic. Hormone profiles revealed an elevation of follicle-stimulating hormone in one patient, but all the other hormones tested were within normal limits for both patients. Further genetic analyses, including karyotype and microdeletions in the AZF region of the Y chromosome, were normal in both patients. Mild androgen insensitivity syndrome was expected in the two patients. Sequencing analysis of the first exon in the androgen receptor (AR) gene have shown c.1783C>T mutation in the two patients with azoospermia. This paper sheds light on the need to screen for mutations in the AR gene, causing male infertility whenever mild hypoandrogenic features are present with unexplained male infertility.

Effects of γ-radiation on cell growth, cell cycle and promoter methylation of 22 cell cycle genes in the 1321NI astrocytoma cell line.

PURPOSE: DNA damage caused by radiation initiates biological responses affecting cell fate. DNA methylation regulates gene expression and modulates DNA damage pathways. Alterations in the methylation profiles of cell cycle regulating genes may control cell response to radiation. In this study we investigated the effect of ionizing radiation on the methylation levels of 22 cell cycle regulating genes in correlation with gene expression in 1321NI astrocytoma cell line. METHODS: 1321NI cells were irradiated with 2, 5 or 10Gy doses then analyzed after 24, 48 and 72h for cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu bromide) assay. Flow cytometry were used to study the effect of 10Gy irradiation on cell cycle. EpiTect Methyl II PCR Array was used to identify differentially methylated genes in irradiated cells. Changes in gene expression was determined by qPCR. Azacytidine treatment was used to determine whether DNA methylation affectes gene expression. RESULTS: Our results showed that irradiation decreased cell viability and caused cell cycle arrest at G2/M. Out of 22 genes tested, only CCNF and RAD9A showed some increase in DNA methylation (3.59% and 3.62%, respectively) after 10Gy irradiation, and this increase coincided with downregulation of both genes (by 4 and 2 fold, respectively). TREATMENT: with azacytidine confirmed that expression of CCNF and RAD9A genes was regulated by methylation. CONCLUSIONS: 1321NI cell line is highly radioresistant and that irradiation of these cells with a 10Gy dose increases DNA methylation of CCNF and RAD9A genes. This dose down-regulates these genes, favoring G2/M arrest.

High prevalence of genetic abnormalities in Middle Eastern patients with idiopathic non-obstructive azoospermia.

INTRODUCTION: Our objective is to detect the frequency and types of major genetic abnormalities of idiopathic non-obstructive azoospermia (NOA) to give appropriate genetic counseling before assisted reproductive techniques (ART) in Middle East and to compare the frequencies with other regions of the world. MATERIAL AND METHODS: A total of 880 Middle Eastern patients with NOA were recruited in this multicenter study for genetic evaluation prior to use of ART. Karyotyping was performed on peripheral blood lymphocytes according to standard G-banding methods, polymerase chain reaction (PCR) was performed to screen the microdeletions in the AZF region of the Y chromosome. RESULTS: The present study shows that the total prevalence of genetic abnormalities is 28.41 %, including 184 patients (20.91 %) with chromosome disorder and 66 patients (7.5 %) with Y chromosome microdeletions. The most prevalent chromosome abnormality is Klinefelter's syndrome, which includes 161 patients (18.3 %), 7 patients had XX reversal male sex (0.8 %), 2 patients had 47XYY (0.23 %) and 2 patients had 45XO/46XY (0.23 %). Structural abnormalities occurred in 12 patients (1.36 %). CONCLUSIONS: The high prevalence of genetic abnormalities (28.41 %) in our study strongly suggests the need for routine genetic testing and counseling prior to assisted reproduction in such population with idiopathic infertility, as a result may help determine the prognosis, as well as the choice of ART. Moreover it allows specific pre-implantation genetic testing to minimize the risk of transmitting genetic defects to offspring.

Characterization of mutations causing rifampicin and isoniazid resistance of Mycobacterium tuberculosis in Syria.

In order to characterize mutations causing rifampicin and isoniazid resistance of M. tuberculosis in Syria, 69 rifampicin resistant (Rif(r)) and 72 isoniazid resistant (Inh(r)) isolates were screened for point mutations in hot spots of the rpoB, katG and inhA genes by DNA sequencing and real time PCR. Of 69 Rif(r) isolates, 62 (90%) had mutations in the rifampin resistance determining region (RRDR) of the rpoB gene, with codons 531 (61%), 526 (13%), and 516 (8.7%) being the most commonly mutated. We found two new mutations (Asp516Thr and Ser531Gly) described for the first time in the rpoB-RRDR in association with rifampicin resistance. Only one mutation (Ile572Phe) was found outside the rpoB-RRDR. Of 72 Inh(r) strains, 30 (41.6%) had a mutation in katGcodon315 (with Ser315Thr being the predominant alteration), and 23 (32%) harbored the inhA(-15C-->T) mutation. While the general pattern of rpoB-RRDR and katG mutations reflected those found worldwide, the prevalence of the inhA(-15C-->T mutation was above the value found in most other countries, emphasizing the great importance of testing the inhA(-15C-->T) mutation for prediction of isoniazid resistance in Syria. Sensitivity of a rapid test using real time PCR and 3'-Minor groove binder (MGB) probes in detecting Rif(r) and Inh(r) isolates was 90% and 69.4%, respectively. This demonstrates that a small set of MGB-probes can be used in real time PCR in order to detect most mutations causing resistance to rifampicin and isoniazid.

Effects of fenofibrate on Semaphorin 6B gene expression in rat skeletal muscle.

Semaphorin 6B is a member of the semaphorin family of signaling proteins, which is implicated in a variety of biological processes, such as axon guidance and muscle regeneration. PPARα is known to be a regulator of semaphorin 6B in human cancer cell lines. In this study, we examined the effect of fenofibrate as a PPARα activator on the expression of the Sema6B gene in rat skeletal muscle. A total of 18 rats were divided into two groups: one fed with standard chow (control group), and the other with fenofibrate chow (treatment group) for 4 weeks by gavage with 30 mg/kg per day. The expression of rSema6B mRNA was analyzed by qPCR. rSema6B protein content in rat skeletal muscle was measured by Western blotting. A significant down-regulation of rSema6B was observed at both the mRNA and protein levels. Using the ChIP assay, a putative PPARα binding region (PPRE) was identified in the rSema6B promoter. We conclude that the expression of Sema6B is down-regulated by fenofibarte in rat skeletal muscle.

Combination of conventional multiplex PCR and quantitative real-time PCR detects large rearrangements in the dystrophin gene in 59% of Syrian DMD/BMD patients.

OBJECTIVES: Adaptation of a low-cost protocol to diagnose large rearrangements of the dystrophin gene in DMD/BMD Syrian patients and to establish the distribution of these mutations in the 2 hotspots. DESIGN AND METHODS: gDNA from 51 unrelated Syrian DMD/BMD male patients was isolated and analyzed by multiplex PCR of 25 hotspot exons in order to detect deletions. Patients who did not show any deletions were further analyzed by quantitative real-time PCR and the DeltaDeltaCt method in order to detect duplications in exons 4, 17, 47 and 52. RESULTS: We found a deletion in 25 (49%) out of 51 patients studied. Quantitative real-time PCR revealed a duplication in 5 (9.8%) out of 51 patients. Combination of traditional multiplex PCR of hotspot exons with real-time PCR quantification of only exons 4, 17, 47 and 52 positively diagnosed 59% of Syrian DMD/BMD patients. CONCLUSION: Our method may be useful as a cost-effective first-line test for the diagnosis of DMD/BMD patients before using exhaustive and expensive methods.