Investigation of exon 4 mutations of phenylalanine hydroxylase gene in phenylketonuria patients in Guilan Province using PCR-sequencing

Background: Phenylketonuria (PKU) is a heterogeneous and autosomal recessive metabolic disorder that is mainly caused by mutations in the hepatic phenylalanine hydroxylase (PAH) gene. Distribution pattern of mutations in the PAH gene are specific to each population. To date, no reports of phenylketonuria molecular analysis have been found in this population. The aim of this study was to identify PAH mutations within exon 4 in PKU patients in Guilan Province and compare it with the studies in other parts of Iran. Materials and Methods: In this cross-sectional and descriptive study, 25 unrelated PKU patients (age range, 1-21 years) were identified from different regions of Guilan Province during a one-year period. After collecting blood samples and DNA extraction, the DNA fragments containing the exon 4 of the PAH gene and its flanking intronic sequences were amplified and sequenced. Results: In this study, IVS4+5G>T mutation (10) was identified. This mutation was found in two homozygous PKU patients and one heterozygous patient; they had mPKU and cPKU phenotypes, respectively and their parents were third degree relatives. In addition, IVS4+47C>T (28) and IVS3-22C>T (8) polymorphisms were also detected. Conclusion: Investigation of mutations in the PAH gene can be a useful tool for molecular detection of the PKU disease and carrier detection in this population. Moreover, the other 12 remaining exons need to be analyzed to obtain the full spectrum of mutations of this gene among the PKU patients in Guilan Province

First Report of Variable Number of Tandem Repeat Alleles in Phenylalanine Hydroxylase Gene in Patients With Phenylketonuria From Guilan Province, Iran

Aims Mutations in the phenylalanine hydroxylase (PAH) gene are the main reason for phenylketonuria (PKU). The multiplicity of mutations in the PAH gene, which leads to PKU, makes the diagnosis of pathogenic mutations impossible in many cases. In these cases, polymorphic markers in the PAH gene, such as the variable number of tandem repeats (VNTR) are used to determine the carriers in PKU families. This study aims to investigate the allele frequency of this marker in the PKU population of Guilan Province, Iran. Methods & Materials During one year, 25 unrelated PKU patients were identified from different areas of Guilan Province. After extracting DNA from patients’ blood, VNTR containing fragments of the PAH gene were assessed using the polymerase chain reaction (PCR)-sequencing method. Findings PCR products related to PAH VNTR alleles produced 380, 500, and 530 base pairs fragments. They were related to 3, 7, and 8 copies of the repeat units, respectively. In addition, these repetitions had a frequency of 6(12%), 6(12%), and 33(66%), respectively. Also, this study demonstrated that PKU patients in Guilan Province, Iran had VNTR3/VNTR3, VNTR3/VNTR8, ND/VNTR8, VNTR7/VNTR7, VNTR8/VNTR8, and ND/ND genotypes in the PAH gene. Conclusion This study is the first report on the genetic structure of the PKU population using PAH VNTR alleles in Guilan Province, Iran. Given the population diversity in Iran, it is necessary to study the frequency and distribution of VNTR alleles in different parts of the country

Correlation between ERG11 Gene Mutations and Fluconazole Resistance in Candida albicans Strains Isolated from Rasht in Years 2015-2016

Abstract Background: Candida albicans as an opportunistic fungal pathogen in human causes candidiasis. The widespread use of azoles has led to the increase of azole resistance in Candida albicans isolates. Mutation in the ERG11 gene is one of several azole resistance reasons in Candida albicans. The aim of this study was to find ERG11 gene mutations in fluconazole resistant isolates in Rasht. Materials and methods: Candida albicans isolates were identified by standard identification methods such as germ tubes. The fluconazole resistance and susceptibility of the isolates was evaluated by Disc diffusion and MIC methods. For mutation determining, ERG11 gene was amplified by PCR and then sequenced in clinical isolates. Results: From 23 isolates of Candida albicans, 20 isolates were fluconazole resistant. The MIC of fluconazole in these isolates was determined between 128 to 2048µg/ml. Also, sequencing analysis showed that 10 fluconazole resistant isolates had two missense mutations (D116E and E266D) in ERG11 gene. Conclusion: In this study, resistance to high concentration of fluconazole shows that different mechanisms simultaneously implicated in developing azoles resistance in the isolates. Association of ERG11 gene mutation and deregulation of other genes can be led to resistance to high fluconazole concentration in this study

The Effect of Iron Oxide Nanoparticles and Magnetic Field on Angiogenesis and Deregulation of Vegfa Gene After Ischemia Reperfusion in Rat

Background and Aim: Prolonged ischemia in organs with high metabolic rates such as brain and heart is associated with deleterious effects. Therefore, nutritive distribution through angiogenesis after ischemia is necessary for repairing damaged region of tissue. In this study, the effects of iron oxide nanoparticles and magnetic field on angiogenesis after ischemia reperfusion (IR) in rat model have been investigated. Materials and Methods: In this experimental study, fifty male rats aged between 6 -7 weeks at the 220-250gr weight were purchased from Tehran University. Animals were categorized in 5 groups including sham (ischemia reperfusion model), control, iron oxide nanoparticles-treated, magnetic field-exposed, and combination therapy with iron oxide nanoparticles and magnetic field-exposed groups. Angiogenesis was evaluated in hippocampus of 5 groups after 4 days by H&E staining method. The expression of Vegfa gene was studied in 5 groups by Q-RT- PCR. Findings: Iron oxide nanoparticles as well as the magnetic field induced angiogenesis during 4 days in animals after IR (p<0.05), but their combination therapy did not show any significant difference compared to sham group during 4 days. Upregulation of Vegfa gene was observed in iron oxide nanoparticles treated group and the magnetic field exposed group significantly (p<0.05) relative to ischemia reperfusion (IR) model. But overexpression of Vegfa gene in combination therapy group was not significant relative to ischemia reperfusion (IR) group. Conclusion: It seems that iron oxide nanoparticles and magnetic field can separately be two effective methods for angiogenesis after ischemia reperfusion (IR)

Investigation of Mutations of ERG11 Gene in Fluconazole Resistant Strains of Candida Albicans Isolated From Patients With Volvovaginitis in West of Mazandaran

Background and Aims: Candida albicans as an opportunistic fungal pathogen in human is the cause of volvovaginitis candidiasis. Azole resistance is cinsiderable as worldwide problem in treatment of candidiasis. Azole resistance can occur through different mechanisms such as mutation in ERG11 gene. The aim of our study was evaluation of ERG11 gene mutations in fluconazole resistant islotes of C. albicans obtained from patients with volvovaginitis in west of Mazandaran. Materials and Methods: In this study, clinical specimens were obtained from vaginal mucosa of 120 individual. C. albicans isolates were identified by standard methods such as germ tubes and culture in chrome agar media. Susceptibility test to fluconazole in the isolates was evaluated by Disc diffusion and MIC methods. After DNA extraction, ERG11 gene mutations in clinical isolates were determined by PCR- sequencing method. Results: From 45 isolates of C. albicans, 40 isolates were resistant to fluconazole. The MIC of fluconazole in isolates was determined between 2 to 64μg/ml. Also, sequencing analysis showed that 7 fluconazole resistant isolates had three missense mutations (D116E, K128T and A114S) in ERG11 gene.  Conclusion: It apears that high frequency of floconazol resistance is the results of different reasons such as mutations in ERG11 gene in C. albicans isolates

Investigation of TIMP-1 Gene Expression in Patients with Multiple Sclerosis(MS)

Abstract Background: Matrix metalloproteinases (MMPs) comprise a family of proteolytic enzymes.MMPs are capable of disrupting the blood-brain barrier (BBB), mediating the destruction of extracellular matrix and myelin components. Tissue inhibitors of metalloproteinases (TIMPs) are proteins which block the activitiy of MMPs. Matrix metalloproteinase-9 (MMP-9) facilitates T-cell migration into the CNS while the tissue inhibitor matrix metalloproteinase-1 (TIMP-1) inhibits MMP-9 actions. The aim of this study was to evaluate the expression of TIMP-1 gene (in RNA level) in blood cells of relapsing-remitting multiple sclerosis (RRMS) patients treated with IFNb. Materials and Methods: In this study, the expression level of TIMP-1 gene was investigated in blood cells of MS patients compared to healthy subjects by Real-Time PCR. Results: The RRMS patients manifested a lower expression level of TIMP-1 RNA than their normal counterparts, although the result was not significant (p=0.1). Conclusion: The results of this study showed that there was no linear correlation between TIMP-1 expression level and risk of Expanded Disability Status Scale of Kurtzke (EDSS); nor was there any significant correlation between expression status of TIMP-1 and duration of the disease. Further studies are recommended to compare TIMP-1 RNA in patients before and after taking IFN-beta

Molecular analysis of seven Lactobacillus strains isolated from human origin in West of Mazandaran.

Background and Aims: Colorectal cancer is considered to be an important cause of morbidity and mortality worldwide. Anti-cancer properties of probiotic lactic acid bacteria are considerable for treatment of cancers such as colorectal cancer. The aim of this study was to investigate the molecular characterization of probiotic bacterial isolates from collected stool specimens in west of Mazandaran province, in order to confirm the probiotic effect of isolated strains, so to compare their effect on cancer cell lineages, in future studies. Materials and Methods: In this study, out of 60 stool samples collected from healthy children and adult people, 50 bacterial strains were isolated. Antibiotic susceptibility was performed by 15 antibiotics for 7 strains. Then, pH and bile salts tolerance were measured in the strains. Molecular analysis of these strains was carried out by PCR-sequencing and their results were used to draw a phylogenetic tree. Results: Of the 50 isolates, 7 isolates were lactic acid bacteria and two strains were completely probiotic. These strains were resistance to low pH and 0.3% bile salt. Molecular analysis showed that two strains SE and SG were probiotics related to Lactobacillus fermentum (86% homology) and Lactobacillus rhamnosus (61% homology) family, respectively, which were known as new strains. Conclusion: The results suggested that there are 7 strains with good tolerance in the gastrointestinal tract that have probiotic properties and this is the basis of future studies to evaluate their anti-tumor properties and ultimately oral administration

Mutation Spectrum of the Phenylalanine Hydroxylase Gene in Phenylketonuria Patients in Golestan Province, Iran

Abstract: Background. As an autosomal recessive disease, phenylketonuria (PKU) is caused by deficiency in phenylalanine hydroxylase (PAH) gene. The incidence of different mutations in phenylalanine hydroxylase is associated with differences in race and ethnicity. The aim of this study is to investigate the mutation spectrum in hotspot region of PAH gene in PKU patients. Materials and Method. For this purpose, we studied exons 6, 7, 10�11 and 12 and adjacent flanking regions of PAH gene from a total of 26 unrelated hyperphenylalaninemia patients (13 males and 13 females) in Golestan province using PCR-sequencing method. Results. Among 26 analyzed patients, 11 distinct mutations were found in combinations of 18 genotypes. A mutation detection rate of 64 was achieved. The high heterogeneity of PKU was reported in this study. The most prevalent mutations were IVS10-11G>A (19.23), followed by IVS11+1G>C, p.P416HfsX36 and p.R261Q which accounted for 66.67 of mutant alleles. IVS9-1G>T and p.P416HfsX36 are novel mutations. Exonic polymorphisms L385L and Q232Q and intronic polymorphisms IVS10+155T>C, IVS10+156T>G, and IVS10-193G>C were found to have high frequency. Conclusion. A mutation spectrum with high frequency was predicted in Northern region of Iran due to its ethnic heterogeneity. Furthermore, it seems that the presence of some mutations in Golestan province indicates gene flow between Iran and the neighboring countries. © 2020, Pleiades Publishing, Inc

Non-coding RNA-Mediated N6-Methyladenosine (m6A) deposition: A pivotal regulator of cancer, impacting key signaling pathways in carcinogenesis and therapy response

The emergence of RNA modifications has recently been considered as critical post-transcriptional regulations which governed gene expression. N6-methyladenosine (m6A) modification is the most abundant type of RNA modification which is mediated by three distinct classes of proteins called m6A writers, readers, and erasers. Accumulating evidence has been made in understanding the role of m6A modification of non-coding RNAs (ncRNAs) in cancer. Importantly, aberrant expression of ncRNAs and m6A regulators has been elucidated in various cancers. As the key role of ncRNAs in regulation of cancer hallmarks is well accepted now, it could be accepted that m6A modification of ncRNAs could affect cancer progression. The present review intended to discuss the latest knowledge and importance of m6A epigenetic regulation of ncRNAs including mircoRNAs, long non-coding RNAs, and circular RNAs, and their interaction in the context of cancer. Moreover, the current insight into the underlying mechanisms of therapy resistance and also immune response and escape mediated by m6A regulators and ncRNAs are discussed

Progress in targeting PTEN/PI3K/Akt axis in glioblastoma therapy: Revisiting molecular interactions

Glioblastoma (GBM) is one of the most malignant cancers of central nervous system and due to its sensitive location, surgical resection has high risk and therefore, chemotherapy and radiotherapy are utilized for its treatment. However, chemoresistance and radio-resistance are other problems in GBM treatment. Hence, new therapies based on genes are recommended for treatment of GBM. PTEN is a tumor-suppressor operator in cancer that inhibits PI3K/Akt/mTOR axis in diminishing growth, metastasis and drug resistance. In the current review, the function of PTEN/PI3K/Akt axis in GBM progression is evaluated. Mutation or depletion of PTEN leads to increase in GBM progression. Low expression level of PTEN mediates poor prognosis in GBM and by increasing proliferation and invasion, promotes malignancy of tumor cells. Moreover, loss of PTEN signaling can result in therapy resistance in GBM. Activation of PTEN signaling impairs GBM metabolism via glycolysis inhibition. In contrast to PTEN, PI3K/Akt signaling has oncogenic function and during tumor progression, expression level of PI3K/Akt enhances. PI3K/Akt signaling shows positive association with oncogenic pathways and its expression similar to PTEN signaling, is regulated by non-coding RNAs. PTEN upregulation and PI3K/Akt signaling inhibition by anti-cancer agents can be beneficial in interfering GBM progression. This review emphasizes on the signaling networks related to PTEN/PI3K/Akt and provides new insights for targeting this axis in effective GBM treatment