Molecular Investigations of the Common Mitochondrial Deletion (MTDNA 49777) Tumoral Tissues as Compared with Adjacent Non-Tumoral Tissues from Gastric Cancer Patients at Baghiyatallah Hospital in Tehran

The human mitochondrial genome has been completely sequenced and each gene has been identified and characterized. The human mtDNA is a supercoiled, double-stranded circular molecule of 16,569 base pairs in size which codes for 13 of the 87 proteins required for oxidative phosphorylation as well as the 12s and 16s rRNAs and 22 tRNAs required for protein synthesis in the mitochondria. One of the common regions and hot spot of the mitochondrial genome so-called the common mitochondrial deletion (AmtDNA4977) was investigated which is at nucleotide number 8470 to 13447. This deletion affects important genes, involved in - OXPHOS (Oxidative Phosphorylation) such as ATPase 6, ATPase 8, Cytochrome Oxidase 111, and NADH subunits ND3, ND4, ND4L, and ND5 which may have a strong metabolic disadvantage. The prevalence of the AmtDNA4977 deletion has been investigated in different cancers. However in this study, we screened the common mitochondrial deletion to determine the prevalence of the common mitochondrial deletion in tumoral tissues as compared with adjacent non-tumoral tissues in gastric cancer by multiplex PCR amplification, polyacrylamide gel electrophoresis and Southern blot. In order to investigate whether a high incidence of mutation exists in mitochondrial DNA of gastric cancer tissues, DNA isolated from these cells was used to amplify hypervariable regions ATPase816, COXIII, ND3, ND4 and ND5 of AmtDNA4977. In 107 cancer patients, AmtDNA4977 was detected in 6 cases (5.60%) of the tumoral tissues and 18 cases (16.82%) of the non-tumoral tissues that were adjacent to the tumors. Levels of AmtDNA4977 deletions were found to be more in non-tumoral tissues than in adjacent tumoral tissues. There was no correlation between the clinical parameters like age, sex, tumor location and tumor size of patients. However there was an obvious relationship with intestinal-type of gastric cancer. The percentage of deleted genome in gastric tumoral tissues ranged from 25% to 74%. The level of the heteroplasmy was quantified by densitometry analysis with a Gel Doc 2000 BIO-RAD instrument (Hercules, CA, USA). The level of heteroplasmy was determined as -25% to 74%. Unknown genetic aspects, ambiguous environmental factors and Reactive Oxygen Species (ROS) can cause the AmtDNA4977 mutation rate to be increased in gastric cancer. The results suggest that percentage level of AmtDNA4977 is less common and intolerable in tumoral tissue, probably because of high metaboIism and ROS generation. We postulate that the cells initially had AmtDNA4977, transformed to tumoral cell and the existence of this deletion confers metabolic disadvantage, thus cells that contain such mtDNA deletion would be overgrown by other cancer cells without this mtDNA deletion. As a result, the presence of AmtDNA4977 will be low in tumoral cell

A Novel Mutation of GDAP1 Associated with Charcot-Marie-Tooth Disease in An Iranian Family

As a result of higher distributed consanguinity in the Mediterranean region and the Middle East, autosomal-recessive forms of Charcot-Marie-Tooth (ARCMT) are more common in these areas. CMT disease caused by mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene is a severe autosomal recessive neuropathy resulting in either demyelinating CMT4A neuropathy or axonal neuropathy with vocal cord paresis. The patient was an 8-year-old boy with AR inheritance that showed some delayed achievement of motor milestones, including walking, also bilateral foot drop, wasting of distal muscles in the legs, pes cavus and marked weakness of the foot dorsiflexors. He had no hoarseness or vocal cord paralysis. Total genomic DNA was extracted from whole peripheral blood of the patient and his family by using standard procedures. PCR- sequencing method were used to analysis the whole coding regions of the GDAP1 gene. A novel homozygote insertion of T nucleotide in codon 34 was detected (c.100_101insT) that probably led to an early stop codon. This mutation may be associated with a common haplotype, suggesting a common ancestor that needs further investigation in the Iranian population

Prevalence of the UGT1A1*6 (c.211G>A) polymorphism and irinotecan toxicity in Iranian populations of different ethnicities

Background: Pharmacogenetic studies on irinotecan treatment in patients with metastatic colorectal cancer have indicated that genetic polymorphisms in UGT1A1*6 can lead to decreased enzyme activity and accumulation of the toxic metabolite SN-38. Here, we compared the prevalence of UGT1A1*6 in an Iranian population of different ethnicities with those of other populations. Materials and Methods: A total of 300 healthy people of different ethnic groups including Persian, Azari, Lure, Kurdish, Arab, Baluch and Caspian in the Iranian population were enrolled. Genotyping of the UGT1A1*6 alleles (G/G, A/G, A/A) was performed by polymerase chain reaction-restriction fragment length polymorphism and direct genomic DNA sequencing. Result: The most predictive genotype among the Iranian ethnic groups, especially Persian, was the G/G genotype (wild-type genotype). The frequency of the A/G genotype among the Persian, Azari, Lure, Kurdish, Arab, Baluch and Caspian ethnicities were 15.69% (n = 27), 11.11% (n = 8), 5.88% (n = 1), 9.09% (n = 1), 10% (n = 1), 20% (n = 1) and 0% (n = 0), respectively. Only one person with Persian ethnicity was homozygous for the mutation in UGT1A1*6 (0.58%). Additionally, the frequency of the A and G alleles in Iranians was 6.83 and 93.16%, respectively. Conclusion: The identification of the UGT1A1*6 alleles is necessary among the different Iranian ethnic groups before irinotecan therapy, suggesting that genotyping would be helpful for clinicians to optimize chemotherapy or identify individuals at risk of adverse drug reactions before clinical trials

Prevalence of the rs7903146C>T polymorphism in TCF7L2 gene for prediction of type 2 diabetes risk among Iranians of different ethnicities

BACKGROUND: Pharmacogenetics is the study of genetic polymorphisms affecting responses to drug therapy. The common rs7903146 (C>T) polymorphism of the TCF7L2 gene has recently been associated with type 2 diabetes (T2D). In this study, prevalence of the rs7903146 (C>T) polymorphism in the TCF7L2 gene for prediction of T2D risk was examined in an Iranian population of different ethnicities. METHODS: The prevalence of rs7903146 (C>T) and the predicted phenotypes, including extensive metabolizers, intermediate metabolizers, and poor metabolizers were investigated in blood samples of 300 unrelated healthy individuals in an Iranian population, including Fars, Turk, Lure, and Kurd, using polymerase chain reaction restriction fragment length polymorphism and direct genomic DNA sequencing. RESULTS: The homozygous wild-type (C/C), heterozygous (C/T), and homozygous (T/T) allelic frequencies of rs7903146 (C>T) in the TCF7L2 gene were 29% (extensive metabolizers), 66.34% (intermediate metabolizers), and 4.66% (poor metabolizers), respectively. The C/C, C/T, and T/T genotypic frequencies of the rs7903146 (C>T) allele were significantly different (P<0.01) among Iranians of different ethnicities. The frequency of the homozygous T/T variant of the rs7903146 (C>T) allele was significantly low in the Lure (P<0.01) and high in the Fars (P<0.001) ethnicities. Additionally, the frequency of the T/T variant of the rs7903146 (C>T) allele in the South of Iran was the highest (P<0.04), while the East of Iran had the lowest frequency (P<0.01). CONCLUSION: The prediction of rs7903146 (C>T) is required in drug research and routine treatment, where the information would be helpful for clinicians to optimize therapy and adverse drug reactions and predict drug response in individuals at risk of T2D

Three Novel Mutations in Iranian Patients with Tay-Sachs Disease

ABSTRACT Background: Tay-Sachs disease (TSD), or GM2 gangliosidosis, is a lethal autosomal recessive neurodegenerative disorder, which is caused by a deficiency of beta-hexosaminidase A (HEXA), resulting in lysosomal accumulation of GM2 ganglioside. The aim of this study was to identify the TSD-causing mutations in an Iranian population. Methods: In this study, we examined 31 patients for TSD-causing mutations using PCR, followed by restriction enzyme digestion. Results: Molecular genetics analysis of DNA from 23 patients of TSD revealed mutations that has been previously reported, including four-base duplications c.1274_1277dupTATC in exon 11 and IVS2+1G&gt;A, deletion TTAGGCAAGGGC in exon 10 as well as a few novel mutations, including C331G, which altered Gln&gt;Glu in HEXB, A&gt;G, T&gt;C, and p.R510X in exon 14, which predicted a termination codon or nonsense mutation. Conclusion: In conclusion, with the discovery of these novel mutations, the genotypic spectrum of Iranian patients with TSD disease has been extended and could facilitate definition of disease-related mutations

Citral induced apoptosis in MDA-MB-231 spheroid cells

Background: Breast cancer remains a leading cause of death in women worldwide. Although breast cancer therapies have greatly advanced in recent years, many patients still develop tumour recurrence and metastasis, and eventually succumb to the disease due to chemoresistance. Citral has been reported to show cytotoxic effect on various cancer cell lines. However, the potential of citral to specifically target the drug resistant breast cancer cells has not yet been tested, which was the focus of our current study. Methods: The cytotoxic activity of citral was first tested on MDA-MB-231 cells in vitro by MTT assay. Subsequently, spheroids of MDA-MB-231 breast cancer cells were developed and treated with citral at different concentrations. Doxorubicin, cisplatin and tamoxifen were used as positive controls to evaluate the drug resistance phenotype of MDA-MB-231 spheroids. In addition, apoptosis study was performed using AnnexinV/7AAD flowcytometry. Aldefluor assay was also carried out to examine whether citral could inhibit the ALDH-positive population, while the potential mechanism of the effect of citral was carried out by using quantitative real time- PCR followed by western blotting analysis. Results: Citral was able to inhibit the growth of the MDA-MB-231 spheroids when compared to a monolayer culture of MDA-MB-231 cells at a lower IC50 value. To confirm the inhibition of spheroid self-renewal capacity, the primary spheroids were then cultured to additional passages in the absence of citral. A significant reduction in the number of secondary spheroids were formed, suggesting the reduction of self-renewal capacity of these aldehyde dehydrogenase positive (ALDH+) drug resistant spheroids. Moreover, the AnnexinV/7AAD results demonstrated that citral induced both early and late apoptotic changes in a dose-dependent manner compared to the vehicle control. Furthermore, citral treated spheroids showed lower cell renewal capacity compared to the vehicle control spheroids in the mammosphere formation assay. Gene expression studies using quantitative real time PCR and Western blotting assays showed that citral was able to suppress the self-renewal capacity of spheroids and downregulate the Wnt/β-catenin pathway. Conclusion: The results suggest that citral could be a potential new agent which can eliminate drug-resistant breast cancer cells in a spheroid model via inducing apoptosis

Boesenbergin A, a chalcone from Boesenbergia rotunda induces apoptosis via mitochondrial dysregulation and cytochrome c release in A549 cells in vitro : involvement of HSP70 and Bcl2/Bax signalling pathways

The anti-cancer effect of Boesenbergin A (BA) isolated from Boesenbergia rotunda, via the induction of apoptosis resulting from mitochondrial dysfunction was assessed in human non-small cell lung cancer (A549) cells. The apoptotic mechanisms of BA induction on cancer cells were studied in the present study for the first time. Nuclear stain, measuring the accumulation of sub-G1 cell population and DNA ladder were done to determine the apoptosis. Further investigations into the depletion of mitochondrial membrane potential and release of cytochrome c determined that BA treatment induced apoptosis via the regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The involvement of both intrinsic and extrinsic caspases (caspase 3/7, 9 and 8) were significantly increased. Moreover the role of free radicals was significantly found to be elevated with concomitant decrease in HSP70. In conclusion the results from the current study indicated BA could be a promising agent for the treatment of lung cancer

Three Novel Mutations in Iranian Patients with Tay-Sachs Disease

ABSTRACT Background: Tay-Sachs disease (TSD), or GM2 gangliosidosis, is a lethal autosomal recessive neurodegenerative disorder, which is caused by a deficiency of beta-hexosaminidase A (HEXA), resulting in lysosomal accumulation of GM2 ganglioside. The aim of this study was to identify the TSD-causing mutations in an Iranian population. Methods: In this study, we examined 31 patients for TSD-causing mutations using PCR, followed by restriction enzyme digestion. Results: Molecular genetics analysis of DNA from 23 patients of TSD revealed mutations that has been previously reported, including four-base duplications c.1274_1277dupTATC in exon 11 and IVS2+1G&gt;A, deletion TTAGGCAAGGGC in exon 10 as well as a few novel mutations, including C331G, which altered Gln&gt;Glu in HEXB, A&gt;G, T&gt;C, and p.R510X in exon 14, which predicted a termination codon or nonsense mutation. Conclusion: In conclusion, with the discovery of these novel mutations, the genotypic spectrum of Iranian patients with TSD disease has been extended and could facilitate definition of disease-related mutations

Pegembangan Model untuk Pembelajaran Keterampilan Menulis di Sekolah Dasar

Pembelajaran Bahasa Indonesia seringkali dianggap mudah oleh sebagian besar peserta didik, hal tersebut menjadikan tujuan pembelajaran dalam suatu kegiatan belajar mengajar tidak dapat dicapai secara optimal, salah satunya keterampilan menulis. Banyaknya model pembelajaran yang ditawarkan dalam dunia pendidikan nyatanya belum mampu mengatasi kekurangan tersebut. Tujuan dari penelitian ini adalah 1) untuk mengetahui pembelajaran keterampilan menulis di SD, 2) untuk mengetahui tingkat keterampilan menulis siswa, 3) untuk mengetahui kebutuhan terhadap model pembelajaran Bahasa Indonesia yang dapat meningkatkan keterampilan menulis siswa. Jenis penelitian yang dilakukan adalah penelitian kualitatif yang dilakukan melalui studi kasus dan studi literature. Subjek penelitiannya meliputi siswa kelas III Sekolah Dasar. Teknik pengumpulan data menggunakan triangulasi data/sumber yaitu data wawancara yang dilakukan dengan guru kelas III di sekolah yang berbeda dan triangulasi metode yaitu data wawancara, dan observasi. Analisis data dilakukan mulai dari pengumpulan data, reduksi data, penyajian data sampai verifikasi. Hasil penelitian pada studi pendahuluan ini adalah 1) kurikulum yang diterapkan di empat sekolah tersebut adalah Kurikulum Tingkat Satuan Pendidikan, 2) model pembelajaran yang digunakan guru sudah mengarah pada student center, 3) media yang digunakan berupa gambar yang sudah tertera pada buku, 4) penilaian dilakukan secara menyeluruh (mencakup empat keterampilan), 5)sebagian siswa belum mampu mencapai KKM. Kesimpulan dari studi pendahuluan ini adalah perlu dikembangkannya model pembelajaran Bahasa Indonesia yang dapat meningkatkan keterampilan menulis siswa

The Implicature in Flouting Maxim of Relation by the Main Character in Iron Man 2 Movie

Language takes an important role in human life. It is a tool for communication. People can get what they want and express their feelings by doing communication. In order to have good communication, the speaker and theThis study uses qualitative approach because the writer analyzes conversations in movie. The result of the study shows that there are 7 reasons why the main character flouts the maxim. This study also shows that there are 20 utterances containing flouting maxim of relation. Furthermore, this study alsoreveals that the using of flouting maxim of relation makes conversation moreinteresting.Hopefully, this study will inspire the next researchers to broaden their pointof view in order to look for the problems of the study. Furthermore, the writer alsosuggests the next researchers to look for other subject to be analyzed and use othertheories to analyze the data. Keywords: Cooperative principle, flouting maxim of relation, implicature, iron man 2 movie.hearer have to obey Cooperative Principle. However, there is a phenomenon when occasionally someone says one thing but he/ she means another. It usually happens with flouting maxim. One kind of flouting maxim is flouting maxim of relation. It occurs when a speaker or hearer responses which is very obviously irrelevant to the topic. The phenomenon is presented in the Iron Man 2 movie. This study aims to reveal the reasons why the main character flouts maxim of relation and what the impact to the conversation is