Comparison of three methods for mitochondria isolation from the human liver cell line [HepG2]

Aim: The aim of this study was to evaluate and compare three available methods for mitochondrial isolation from a human cell line to predict the best method for each probable application

Background: Organelle isolation is gaining importance in experimental laboratory settings. Mitochondrial dysfunction may affect tumorgenesis process. There are some evidences that transplantation of healthy, intact and active mitochondria into cells containing defective mitochondria may reduce the proliferation. Therefore, isolated mitochondria could be considered as an effective tool for assessment and management of mitochondrial related disorders

Patients and methods: Mitochondrial isolation from the human liver cell line [HepG2] was performed using two commercially available kits, including Qproteome [Qiagen] and MITOISO2 [Sigma-Aldrich], as well as a manual method. Integrity of inner membrane of mitochondria was assessed by JC-1 staining. Activity of isolated mitochondria was evaluated by DCFH-DA staining, and total yield of isolated mitochondria determined by micro-Lowry method. Finally, relative quantification using Real-time PCR was conducted to compare the mtDNA copy number of mitochondria isolated by three different methods

Results: Compared to other methods, manual kit resulted in higher yields of total amount of mitochondrial protein and mtDNA copy numbers. Isolated mitochondria by Qproteome kit, showed a higher activity. Finally, the integrity of inner-membrane of isolated mitochondria was significantly higher in Qproteome when compared with the other two methods

Conclusion: Due to differences in quality, quantity and activity of isolated mitochondria using three techniques discussed here, the method in which best-suited to each research project should be selected according to the distinct features of isolated mitochondria

Association study of glutathione S-transferase polymorphisms and risk of endometriosis in an Iranian population

Background: Endometriosis influenced by both genetic and environmental factors. Associations of glutathione S-transferases [GSTs] genes polymorphisms in endometriosis have been investigated by various researchers; however, the results are not consistent

Objective: We examined the associations of GSTM1 and GSTT1 null genotypes and GSTP1 313 A/G polymorphisms with endometriosis in an Iranian population

Materials and Methods: In this case-control study, 151 women with diagnosis of endometriosis and 156 normal healthy women as control group were included. The genotyping was determined using multiplex PCR and PCR- RFLP methods

Results: The GSTM1 null genotype was significantly higher [p=0.027] in the cases [7.3%] than the control group [1.3%]. There was no significant difference between the frequency of GSTT1 genotypes between the cases and controls. The GSTP1 313 AG genotype was significantly lower [p=0.048] in the case [33.1%] than the control group [44.4%]

Conclusion: Our results showed that GSTM1 and GSTP1 polymorphisms may be associated with susceptibility of endometriosis in Iranian women

Neuroprotective effects of herbal extract [rosa canina, tanacetum vulgare and urtica dioica] on rat model of sporadic alzheimer's disease

Background: Sporadic Alzheimer's Disease[SAD] is caused by genetic risk factors, agingand oxidative stresses. The herbal extract of Rosa canina [R. canina], Tanacetumvulgare [T. vulgare]and Urtica dioica [U. dioica]has a beneficial role in aging, as ananti-inflammatory and anti-oxidative agent. In this study, the neuroprotective effectsof this herbal extract in the rat model of SAD was investigated

Methods: The rats were divided into control, sham, model, herbal extract -treated andethanol-treated groups. Drug interventions were started on the 21st day after modelingand each treatment group was given the drugs by intraperitoneal [I.P.] route for21 days. The expression levels of the five important genes for pathogenesis of SAD includingSyp, Psen1, Mapk3, Map2 and Tnf-alphawere measured by qPCR between thehippocampi of SAD model which were treated by this herbal extract and controlgroups. The Morris Water Maze was adapted to test spatial learning and memoryability of the rats

Results: Treatment of the rat model of SAD with herbal extract induced a significantchange in expression of Syp [p=0.001] and Psen1 [p=0.029]. In Morris Water Maze,significant changes in spatial learning seen in the rat model group were improved inherbal-treated group

Conclusion: This herbal extract could have anti-dementia properties and improve spatiallearning and memory in SAD rat model

Association study of the TREM2 gene and identification of a novel variant in Exon 2 in Iranian patients with Late-Onset Alzheimer's disease

Objective: To analyze the association between TREM2 exon 2 variants and late-onset [sporadic] Alzheimer's disease [AD] in an elderly Iranian population

Materials and Methods: Exon 2 of TREM2 in a total of 131 AD patients and 157 controls was genotyped using polymerase chain reaction and Sanger sequencing. Fisher's exact test was used to compare the allele and genotype frequency between the 2 study groups

Results: One homozygous and 2 heterozygous carriers of rs75932628-T in the AD patients and 1 heterozygous carrier in the control group were identified. One novel damaging variant, G55R, was also detected in the AD patient group. The frequency of rs75932628-T as well as the amount of rare variants were higher in the AD patients than in the controls, but this did not reach a statistically significant association with AD [odds ratio: 4.8; 95% confidence interval: 0.54 to 43.6; p = 0.270]

Conclusion: The rs75932628-T allele frequency in the elderly Iranian population [0.86%] was high

association study on IL16 gene polymorphisms with the risk of sporadic alzheimer's disease

Interleukin-16 [IL-16] is an important regulator of T cell activation and was reported to act as a chemoattractant agent. There are evidences that IL16 can control the neuroinflammatory processes in Alzheimer's Disease [AD]. This study was performed to investigate the role or association of IL16 polymorphisms, rs11556218 and rs4778889 with the risk of late-onset Alzheimer's disease [LOAD] in Iranian population. Totally, 148 AD patients and 137 nondemented and age-matched subjects were recruited in this study. Genotyping of rs11556218 T/G and rs4778889 T/C polymorphisms was performed by PCR-RFLP method using the NdeI and AhdI restriction enzymes, respectively. Statistical analysis of rs11556218 genotypes showed a protective effect against AD in the heterozygote genotype [p=0.001, OR=0.16] as well as rs4778889 [p=0.001, OR=0.23]. Frequency of rs11556218 allele T was higher in controls than patients [p= 0.001, OR=0.32]. However, there was no significant difference in the frequencies of rs4778889 alleles between the AD patients and controls. Our results indicate that the rs11556218 and rs4778889 polymorphisms have a protective role in the development of sporadic AD in Iranian population

Association of transforming growth factor alpha polymorphisms with nonsyndromic cleft lip and palate in Iranian population

Cleft lip with or without cleft palate [CL/P] is one of the most common congenital anomalies and the etiology of orofacial clefts is multifactorial. Transforming growth factor alpha [TGFA] is expressed at the medial edge epithelium of fusing palatal shelves during craniofacial development. In this study, the association of two important TGFA gene polymorphisms, BamHI [rs11466297] and RsaI [rs3732248], with CL/P was evaluated in an Iranian population. The frequencies of BamHI and RsaI variations were determined in 105 unrelated Iranian subjects with nonsyndromic CL/P and 218 control subjects using PCR and RFLP methods, and the results were compared with healthy controls. A p-value of <0.05 was considered statistically significant. The BamHI AC genotype was significantly higher [p=0.016] in the patients [12.4%] than the control group [5.0%]. The BamHI C allele was significantly higher [p=0.001; OR=3.4, 95% CI: 1.6-7.4] in the cases [8.0%] compared with the control group [2.5%]. Our study showed that there was an association between the TGFA BamHI variation and nonsyndromic CL/P in Iranian population

Early fetal gender determination using real-time PCR analysis of cell-free fetal DNA during 6[th]-10[th] weeks of gestation

Nowadays, new advances in the use of cell free fetal DNA [cffDNA] in maternal plasma of pregnant women has provided the possibility of applying cffDNA in prenatal diagnosis as a non-invasive method. In contrary to the risks of invasive methods that affect both mother and fetus, applying cffDNA is proven to be highly effective with lower risk. One of the applications of prenatal diagnosis is fetal gender determination, which is important in fetuses at risk of sex-linked genetic diseases. In such cases by obtaining the basic information of the gender, necessary time management can be taken in therapeutic to significantly reduce the necessity of applying the invasive methods. Therefore in this study, the probability of detecting sequences on the human Y-chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women with gestational age between 6[th] to 10[th] weeks and the fetal DNA was extracted from the plasma. Identification of SRY, DYS14 and DAZ sequences, which are not present in the maternal genome, was performed using Real-Time PCR. All the obtained results were compared with the actual gender of the newborns to calculate the test accuracy. Considerable 97.3% sensitivity and 97.3% specificity were obtained in fetal gender determination which is significant in the first trimester of pregnancy. Only in one case, false positive result was obtained. Using non-invasive method of cffDNAs in the shortest time possible, as well as avoiding invasive tests for early determination of fetal gender, provides the opportunity of deciding and employing early treatment for fetuses at risk of genetic diseases

Prevalence of intratubular germ cell neoplasia in cryptorchid testes of infertile men

Cryptorchidism is a common malformation in neonates; surgery or medical treatments are applied during childhood. Untreated cryptorchid testes are in the risk of intratubular germ cell neoplasia [IGCN] and consequently invasive testicular tumors which could be shown by immunohistochemistry staining for placental like acid phosphatase [PLAP] marker. We designed this study to know the prevalence of IGCN in untreated cryptorchid testes of infertile men, in our infertility center as a refferal center. In this cross-sectional study we assessed H and E slides of testicular samples of 13 adult infertile patients with impalpable intra-abdominal testes seeking infertility treatment; then we stained them by PLAP marker. Three [23.08%] samples were positive for PLAP marker means presence of IGCN in testis. One of them showed seminoma besides IGCN. According to the results of this study and the fact that there are adult untreated cryptorchid patients in our country yet, it is suggested to pay more attention in clinical examination, assessment and follow up of such patients for malignancy screening

Mutation analysis of slc2oa2and spp2as candidate genes for familial idiopathic basal ganglia calcification

Familial Idiopathic Basal Ganglia Calcification [IBGC] is a rare neurodegenerative disorder which is usually transmitted as an autosomal dominant trait. IBGC is genetically heterogeneous and SLC2OA2, on chromosome 8p21.1-8q11.23, is the first gene found in IBGC-affected patients with varied ancestry. On the other hand, several candidate genes for IBGC on chromosome 2q37, including the SPP2 gene, may play a role in inhibiting calcification. Totally, 22 members of a three generational Iranian family affected by IBGC, with an autosomal dominant pattern of inheritance were included in this study. DMA was extracted from the whole blood using standard salting out method. To find a mutation responsible for IBGC, we sequenced the coding region of SLC2OA2 as well as promoter and coding region of SPP2 in the index subject of IBGC-affected family. Pathogenic mutation was found neither in SLC2OA2 nor in SPPZ Our results strengthen genetic heterogeneity of this condition. Additional mutation studies are necessary to find a gene or genes responsible for IBGC in this affected family

Genotyping of polymorphic microsatellite markers linked to RB1 locus in Iranian population

Retinoblastoma is the most common intraocular tumor in childhood and mutation in the RB1 gene will trigger the tumorigenesis. So far, a wide range of the mutations along the length of RB1 gene have been reported. However, some could not be detected by common detection methods. In such condition, linkage analysis using microsatellite markers is suggested to trace unknown RB1 mutations in the affected family. The aim of the present study was to evaluate the heterozygosity rates and genotyping of three microsatellite markers near or inside of the RB1 gene. Totally, 120 unrelated healthy people from Fardis, Karaj, Iran were recruited and from each participant genomic DNA was extracted from 5 ml of peripheral blood. Three microsatellite markers D13S153, D13S156 and D13S128 located within or adjacent to the RB1 gene were selected for linkage analysis. The reliability of microsatellite markers and linkage analysis were investigated in 10 members of 2 families with familial retinoblastoma. Our results showed that heterozygosity rates for the three markers D13S153, D13S156 and D13S128 were 74, 70 and 78%, respectively. On the other hand, 2 and 36 out of 120 people were homozygote and heterozygous for all loci, respectively. Given the heterozygosity rates, it may be concluded that all microsatellite markers D13S153, D13S156 and D13S128 are informative and have a high rate of heterozygosity and sensitivity. Therefore, tracing the unknown RB1 mutated alleles using linkage analysis in Iranian family with familial retinoblastoma could be recommended by means of these three microsatellite markers

Fetal sex determination using non-invasive method of cell-free fetal DNA in maternal plasma of pregnant women during 6[th]- 10[th] weeks of gestation

In previous years, identification of fetal cells in maternal blood circulation has caused a new revolution in non-invasive method of prenatal diagnosis. Low number of fetal cells in maternal blood and long-term survival after pregnancy limited the use of fetal cells in diagnostic and clinical applications. With the discovery of cell-free fetal DNA [cffDNA] in plasma of pregnant women, access to genetic material of the fetus had become possible to determine early gender of a fetus in pregnancies at the risk of X-linked genetic conditions instead of applying invasive methods. Therefore in this study, the probability of detecting sequences on the Y chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women at 6th to 10th weeks of gestation and then the fetal DNA was extracted from the plasma. Nested PCR was applied to detect the sequences of single copy SRY gene and multi copy DYS14 and DAZ genes on the Y chromosome of the male fetuses. At the end, all the obtained results were compared with the actual gender of the newborns. In 40 out of 42 born baby boys, the relevant gene sequences were identified and 95.2% sensitivity was obtained. Conclusion: Non-invasive early determination of fetal gender using cffDNA could be employed as a pre-test in the shortest possible time and with a high reliability to avoid applying invasive methods in cases where a fetus is at the risk of genetic diseases

Characterization of immune responses induced by combined clade-A HIV-1 recombinant adenovectors in mice

Numerous evidences indicate that in some HIV-1 positive patients, the humoral and cellular immune responses are induced against HIV-1 proteins and this is inversely related to the progress of infection. The aim of this study was the evaluation of the Adenovectors containing HIV genes in induction of immune responses in mice. The HIV-1 genes including gag p24, rev, nef and exon-1 of tat were amplified from HIV-1 RNA [clade-A]. The cDNA of each gene was cloned into a transfer vector. The transfer vector was then co-transformed into E. coli strain BJ5183 together with pAdenovector ?E1/E3. The recombinant adenoviral construct was transfected into QBI-293A cells. Recombinant viruses were purified and titrated on 293 cell plates. Expression of transgenes was evaluated using western blotting. Then 1012 viral particles were injected into 15 groups of 5 mice and all patterns of combination of these 4 HIV-1 genes were evaluated. After 2 weeks, humoral and cellular immune responses were evaluated using ELISA, cell proliferation and ELISpot [IL-2, IL-4 and IFN- gamma] assays, consecutively. It was demonstrated that each gene was expressed. The response targets were mostly toward Th1, though several Th2 responses were also observed. Single injection in our study induced a good cellular response but the humoral responses were not as strong as the cellular ones. Considering and comparing all results and evaluating the various possible interactions revealed that simultaneous injection of tat and gag has enhanced the humoral and cellular responses

Association of CALHM1 gene polymorphism with late onset Alzheimer's disease in Iranian population

Alzheimer's disease [AD] is a genetically heterogeneous neurodegenerative disease and Late-Onset type [LOAD] is the most common form of dementia affecting people over 65 years old. CALHM1 [P86L] encodes a transmembrane glycoprotein that controls cytosolic Ca[2+] concentrations and A beta levels and P86L polymorphism in this gene is significantly associated with LOAD in independent case controls in a number of studies. This study was performed to determine whether this polymorphism contributes to the risk for LOAD in Iranian population. One hundred and forty one AD patients and 141 healthy controls were recruited in this study. After extraction of genomic DNA, the genotype and allele frequencies were determined in case and control subjects using PCR/RFLP method. The statistical analysis showed a significant difference in the heterozygote genotype frequency in case and control groups and polymorphic allele had a protective role between two groups. Also after stratifying the subjects by their APOE-epsilon status, no significant association was observed. Our study suggests that P86L polymorphism could be a protective factor for late-onset Alzheimer's disease [LOAD] in Iranian population. However, to confirm these results, further study with a bigger sample size may be required

Association of vascular endothelial growth factor [VEGF] +405 G>C polymorphism with endometriosis in an Iranian population

Angiogenesis, growth of new blood vessels from pre-existing vessels, is a crucial physiological process for tissue regeneration. This state is also seen in pathological processes such as malignancies and endometriosis. Vascular endothelial growth factor [VEGF] is a major mediator of angiogenesis and vascular permeability which is known to play an important role in the development of endometriosis. The aim of this study was to investigate the relationship between +405 G>C VEGF polymorphism and endometriosis in an Iranian population. The study population was comprised of 105 women with and 150 women without laparoscopic evidence of endometriosis. Genomic DNA from blood cells was extracted using salting out method. Genotype and allele frequency of +405 G>C polymorphism was compared between women with endometriosis and the controls using PCR-RFLP. Statistical analysis was performed using SPSS 13.0 software. Chi-squared test and odds ratio plus 95% confidence interval were determined. A p-value less than 0.05 was considered statistically significant. While the +405 VEGF genotype frequencies in the case group were 41.3% G/G, 46.2% C/G and%12.5 C/C, they were 32% GG,%53.3 GC and 14.7% CC in the control group. The distribution of three genotypes and allele frequencies of+405 G>C VEGF polymorphism between the case and control groups did not demonstrate any significant difference. In contrast to previous studies, no significant correlation was found between +405 G>C VEGF polymorphism and endometriosis. Since this was the first study in an Iranian population, further investigation with bigger sample sizes may be indicated to be able to generalize the findings

Lack of association between tumor necrosis factor-alpha -308 C/A polymorphism and risk of developing late-onset alzheimer's disease in an Iranian population

Late-onset Alzheimer's Disease [LOAD] is a neurodegenerative disorder and the most common form of dementia affecting people over 65 years old. Alzheimer's disease is a complex disease with multi-factorial etiology. Inflammation has been approved to have an important role in the pathogenesis of Alzheimer's disease [AD]. TNF-alpha is a main pro-inflammatory cytokine that plays an essential role in initiation and regulation of inflammatory responses. Several studies have shown the probable association of polymorphism at TNF-alpha gene's promoter with AD pathogenesis. This study was performed to determine whether this polymorphism contributes to the risk for late-onset Alzheimer's disease [LOAD] in Iranian population. One hundred and forty AD patients and 158 healthy controls were recruited in the study. Following extraction of genomic DMA, using PCR/RFLP methods the genotype and allele frequencies were determined in case and control subjects. The statistical analysis showed no significant difference in the allele and genotype frequencies due to this polymorphism between the two groups. Also after stratifying the subjects by their APOE-epsilon 4 status, no significant association was observed. Our results suggest that Tumor necrosis factor-alpha [TNF-alpha] -308 C/A is not a risk or protective factor for late-onset Alzheimer's disease in Iranian population. However, to confirm these results further study with a bigger sample size may be required