Evaluation of the expression of Hottip long noncoding RNA in the B16F10 murine melanoma cell line

زمینه و هدف: ملانوما یکی از انواع سرطان پوست است که نسبت به سایر سرطان های پوست خطرناک تر می باشد. یکی از عوامل موثر در ایجاد سرطان ها، گروهی از RNA های غیر کد کننده اند که به نام lncRNA (long noncoding RNA) شناخته می شوند. این مولکول های غیر کد کننده بیش از 200 باز طول دارند و به عنوان یک تنظیم کننده در پیشرفت سرطان عمل می کنند Hottip (HOXA transcript at the distal tip) یک lncRNA ی بین ژنی است که از انتهای '5 لوکوس Hoxa رونویسی می شود و ژن های انتهای '5 این لوکوس را فعال می کند. هدف از این مطالعه، بررسی میزان بیان این مولکول RNA در رده ی سلولی B16F10 ملانومای موشی بود. روش بررسی: در این مطالعه بیان ژن Hottip با انجام تکنیک RT-PCR به صورت کیفی روی رده ی سلولی B16F10 ملانومای موشی بررسی شد. بدین منظور، از رده ی سلولی، RNA کل استخراج و سنتز cDNA انجام گرفت. با استفاده از پرایمرهای اختصاصی طراحی شده، ژن های Hottip و β2m تکثیر گردیدند. یافته ها: نتیجه مطالعه حاضر نشان دهنده ی عدم بیان ژن Hottip موشی در رده ی سلولی B16F10 ملانومای موشی است. نتیجه گیری: در حالی که بر اساس مطالعات صورت گرفته در سرطان های انسانی انتظار می رفت Hottip افزایش بیان داشته باشد، Hottip موشی در رده ی سلولی B16F10 ملانومای موشی بیانی نشان نداد

High prevalence of fluoroquinolone-resistant Escherichia coli strains isolated from urine clinical samples

Background: Fluoroquinolone resistant Escherichia coli isolates have become an important challenge in healthcare settings in Iran. In this study, we have determined Fluoroquinolone resistant E.coli isolates (from both outpatients and inpatients) and evaluated mutations of gyrA and parC within the quinolone resistance-determining regions (QRDR) of these clinical isolates. Materials and Method: A total of 135 E.coli clinical isolates were recovered from urine of 135 patients (91 outpatients and 44 inpatients) admitted at Alzahra hospital, Iran, between September and February 2013. We assessed antimicrobial susceptibility of all isolates and determined mutations in QRDR of gyrA and parC genes from 13 fluoroquinolone-resistant isolates by DNA sequencing. Results: In this study resistance rate of fluoroquinolones (Ciprofloxacin, Norfloxacin and Ofloxacin) were 45.2%. Two E.coli isolates were shown just a single mutation, but other isolates possessed 2,3,4 and 5 mutations in gyrA and parC genes. Mutations in the QRDR regions of gyrA were at positions Ser83 and Asp87 and parC at positions Ser80, Glu84, Gly78. Conclusions: Ciprofloxacin is the most common antimicrobial agent used for treating urinary tract infections (UTIs) in healthcare settings in Iran. Accumulation of different substitutions in the QRDR regions of gyrA and parC confers high-level resistance of fluoroquinolones in clinical isolates

Induction of Apoptosis in Toxoplasma gondii Infected Hela Cells by Cisplatin and Sodium Azide and Isolation of Apoptotic Bodies and Potential Use for Vaccination against Toxoplasma gondii

Background: Toxoplasma gondii can infect a wide range of mammalians, especially humans. It controls several intracellular signals for the inhibition of apoptosis. This study aimed to investigate the apoptogenic effect of cisplatin and sodium azide on T. gondii infected HeLa cells and isolate apoptotic bodies (blebs) as a potent stimulator of the immune system. Methods: The cytotoxic properties of cisplatin and sodium azide (NaN3) on HeLa cells were evaluated by MTT assay. Moreover, the apoptogenic activity of cisplatin and NaN3 was studied using flow cytometry (Annexin V/PI double staining) and scanning electron microscopy (SEM). Finally, apoptotic bodies were separated by centrifugation. Results: MTT assay data showed that the survival rate of cells treated with different concentration of NaN3 was significantly reduced, compared to negative control groups. Concerning cisplatin, only concentration of 20 μM had not a significant impact on the cell viability; however, the other concentration of cisplatin significantly reduced cell viability, compared to negative control groups. The level of early apoptosis in uninfected HeLa cells was higher compared to infected HeLa cells treated with cisplatin and NaN3. Finally, apoptotic bodies were separated from T. gondii infected HeLa cells treated with cisplatin. Conclusion: Apoptosis was induced in both uninfected and infected HeLa cells with T. gondii and apoptotic bodies were isolated from infected cells. Therefore, further studies on apoptotic bodies are required in order to find a proper candidate for vaccine preparation against T. gondii infections

Thyroid Peroxidase Gene Mutation in Patients with Congenital Hypothyroidism in Isfahan, Iran

Background. Thyroid peroxidase gene (TPO) mutations are one of the most common causes of thyroid dyshormonogenesis in patients with congenital hypothyroidism (CH). In this study, the prevalence of TPO gene mutations in patients with thyroid dyshormonogenesis in Isfahan was investigated. Methods. In this cross-sectional study, genomic DNA of 41 patients with permanent CH due to thyroid dyshormonogenesis was extracted using the salting out method. The 17 exonic regions of the TPO gene were amplified. SSCP technique was performed for scanning of the exonic regions of the TPO gene, except exon 8. DNA sequencing was performed for those with different migration patterns in SSCP by chain termination method. Exon 8 was sequenced directly in all patients. In 4 patients, all fragments were also sequenced. Results. One missense mutation c.2669G>A (NM_000547.5) at exon 15 (14th coding exon) in one patient in homozygous form and seven different single nucleotide polymorphisms (SNPs) in exons 1, 7, 8, 11, and 15 of TPO gene. Conclusion. The TPO gene mutations among CH patients with dyshormonogenesis in Isfahan were less frequent in comparison with other similar studies. It may be due to the presence of other unknown gene mutations which could not be detected by SSCP and sequencing methods

Clinical Trial: CYP2D6 Related Dose Escalation of Tamoxifen in Breast Cancer Patients With Iranian Ethnic Background Resulted in Increased Concentrations of Tamoxifen and Its Metabolites

Introduction: The polymorphic enzyme cytochrome P450 2D6 (CYP2D6) catalyzes a major step in the bioactivation of tamoxifen. Genotyping of clinically relevant CYP2D6 alleles and subsequent dose adjustment is a promising approach to individualize breast cancer therapy. The aim of this study was to investigate the relationship between the plasma levels of tamoxifen and its metabolites and different CYP2D6 genotypes under standard (20 mg/day) and dose-adjusted therapy (Registration ID in Iranian Registry of Clinical Trials: IRCT2015082323734N1).Materials and Methods: Using TaqMan® assays common alleles of CYP2D6 (∗1, ∗2, ∗4, ∗5, ∗6, ∗10, ∗17, and ∗41) and gene duplication were identified in 134 breast cancer patients. Based on CYP2D6 genotypes patients with an activity score 1 (n = 15) and 0–0.5 (n = 2) were treated with tamoxifen adjusted dosage of 30 and 40 mg/day, respectively. The concentration of tamoxifen and its metabolites before and after 4 and 8 months of dose adjustment were measured using LC-MS/MS technology.Results: At baseline, (Z)-endoxifen plasma concentrations (33 ± 15.5, 28.1 ± 14, 26.6 ± 23.4, 14.3 ± 8.6, and 10.7 ± 5.5 nmol/l for EM/EM, EM/IM, EM/PM, IM/IM and PM/PM, respectively) and the metabolic ratio (Z)-Endoxifen/N-desmethyltamoxifen (0.0558 ± 0.02, 0.0396 ± 0.0111, 0.0332 ± 0.0222, 0.0149 ± 0.0026, and 0.0169 ± 0.0177 for EM/EM, EM/IM, EM/PM, IM/IM, and PM/PM, respectively) correlated with CYP2D6 genotype (Kruskal–Wallis p = 0.013 and p < 0.0001, respectively). Dose escalation to 30 and 40 mg/day in patients with a CYP2D6 activity score of 1 (n = 15) and 0–0.5 (n = 2) resulted in a significant increase in (Z)-endoxifen plasma levels (22.17 ± 24.42, 34.43 ± 26.54, and 35.77 ± 28.89 nmol/l at baseline, after 4 and 8 months, respectively, Friedman p = 0.0388) along with the plasma concentrations of tamoxifen and its other metabolites. No severe side effects were recorded during dose escalation.Conclusion: For the first time, we show the feasibility of dose escalation of tamoxifen in breast cancer patients with compromised CYP2D6 activity and Iranian ethnic background to increase the plasma concentrations of (Z)-endoxifen

Selection and Characterization of Single-Stranded DNA Aptamers Binding Human B-Cell Surface Protein CD20 by Cell-SELEX

The B-lymphocyte antigen (CD20) is a suitable target for single-stranded (ss) nucleic acid oligomer (aptamers). The aim of study was selection and characterization of a ssDNA aptamer against CD20 using Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX). The cDNA clone of CD20 (pcDNA-CD20) was transfected to human embryonic kidney (HEK293T) cells. Ten rounds of Cell-SELEX was performed on recombinant HEK-CD20 cells. The final eluted ssDNA pool was amplified and ligated in T/A vector for cloning. The plasmids of positive clones were extracted, sequenced and the secondary structures of the aptamers predicted using DNAMAN® software. The sequencing results revealed 10 different types; three of them had the highest thermodynamic stability, named AP-1, AP-2 and AP-3. The AP-1 aptamer was the most thermodynamically stable one (ΔGAP-1 = −10.87 kcal/mol) with the highest binding affinity to CD20 (96.91 ± 4.5 nM). Since, the CD20 is a suitable target for recognition of B-Cell. The selected aptamers could be comparable to antibodies with many advantages. The AP-1, AP-2 and AP-3 could be candidate instead of antibodies for diagnostic and therapeutic applications in immune deficiency, autoimmune diseases, leukemia and lymphoma

Transcription factor 7-like 2 polymorphism and context-specific risk of metabolic syndrome, type 2 diabetes, and dyslipidemia

Background: The transcription factor 7-like 2 gene (TCF7L2) is an element of the Wnt signaling pathway. There is lack of evidence if TCF7L2 has a functional role in lipid metabolism and regulation of the components constitutes the metabolic syndrome (MetSyn). The aims of this study were to evaluate whether the risk allele of TCF7L2 gene polymorphism is associated with dyslipidemia and MetSyn. Materials and Methods: The MetSyn subjects were participated only based on the National Cholesterol Education Program – Third Adult Treatment Panel criteria. In this case–control study, the DNA from MetSyn patients without (n = 90) and with type 2 diabetes (T2D) (n = 94) were genotyped. Results: The results show that the genotype-phenotype for CC, CT/TT of TCF7L2 gene polymorphism correlated with body mass index and waist circumference in MetSyn and MetSyn + T2D subjects (r = −0.949 and r = −0.963, respectively). The subjects that only possess MetSyn but are not diabetics show the 2 h postprandial glucose and fasting blood glucose, glycated hemoglobin significantly lower (P < 0.05) than those subjects have both abnormality. The level of triglyceride in CT/TT carriers in MetSyn was higher than CC carriers (P = 0.025). A comparison with the controls subjects, the frequencies of the T allele in the groups of MetSyn (46.66%) and MetSyn + T2D (47.34%) show significantly different (P < 0.05). The odds ratios for T allele in (MetSyn)/(normal), (MetSyn + T2D)/(normal), and in (MetSyn + T2D)/(MetSyn) were 3.59 (95% confidence interval [CI], 1.33–9.67, P = 0.0093), 3.76 (95% CI, 1.40–10.07, P = 0.0068), and 1.08 (95% CI: 0.55– 2.11, P = 0.834), respectively. Conclusion: The results revealed the important insights essential for the role of TCF7L2 that the T allele of TCF7L2 plays a significant role in the susceptibility to dyslipidemia, MetSyn, and T2D

Monoclonal antibodies production against a 40KDa band of hydatid cyst fluid.

BACKGROUND AND OBJECTIVES Hydatid cyst is the larval stage of the tapeworm Echinococcus granulosus. Hydatid cyst fluid, cyst membrane and Protoscolices, contain a complex mixture of antigens that can induce immune responses in the host. Anti-cancer properties of Protoscolices and hydatid cyst fluid has been shown. In order to identify antigens of hydatid cyst fluid that have anti-cancer effect, in this study production of monoclonal antibodies against one of the hydatid cyst fluid band (40KDa) has been investigated. There are many published patents about applications of monoclonal antibodies. METHOD In this experimental study, 40KDa band of hydatid cyst fluid that has cross reaction with sera of patients with breast cancer was used as antigen. A group of mice were immunized with this antigen, and then their spleen cells were extracted and fused with SP2 cells. Monoclonal antibodies production was checked in wells with signs of cell growth using ELISA and western blotting. The reaction of the produced monoclonal antibodies with breast cancer cells was tested using flow cytometry method. Finally effect of the monoclonal antibodies on growth of breast cancer cells was investigated in vitro. RESULT The results of this study showed that in the first plate antibody against 40KDa was detected in several wells. In the second plate monoclonal antibodies with high titer was detected in one well. The produced monoclonal antibodies reacted with the surface of breast cancer cells. However, they had no significant effect on growth of breast cancer cells in culture medium. CONCLUSION Monoclonal antibodies against hydatid cyst fluid 40KDa band were produced. These antibodies reacted with the surface of breast cancer cells but had no significant effect on growth of these cells

Occurrence of airborne vancomycin- and gentamicin-resistant bacteria in various hospital wards in Isfahan, Iran

Background: Airborne transmission of pathogenic resistant bacteria is well recognized as an important route for the acquisition of a wide range of nosocomial infections in hospitals. The aim of this study was to determine the prevalence of airborne vancomycin and gentamicin (VM and GM) resistant bacteria in different wards of four educational hospitals. Materials and Methods: A total of 64 air samples were collected from operating theater (OT), Intensive Care Unit (ICU), surgery ward, and internal medicine ward of four educational hospitals in Isfahan, Iran. Airborne culturable bacteria were collected using all glass impingers. Samples were analyzed for the detection of VM- and GM-resistant bacteria. Results: The average level of bacteria ranged from 99 to 1079 CFU/m3. The highest level of airborne bacteria was observed in hospital 4 (628 CFU/m3) and the highest average concentration of GM- and VM-resistant airborne bacteria were found in hospital 3 (22 CFU/m3). The mean concentration of airborne bacteria was the lowest in OT wards and GM- and VM-resistant airborne bacteria were not detected in this ward of hospitals. The highest prevalence of antibiotic-resistant airborne bacteria was observed in ICU ward. There was a statistically significant difference for the prevalence of VM-resistant bacteria between hospital wards (P = 0.012). Conclusion: Our finding showed that the relatively high prevalence of VM- and GM-resistant airborne bacteria in ICUs could be a great concern from the point of view of patients' health. These results confirm the necessity of application of effective control measures which significantly decrease the exposure of high-risk patients to potentially airborne nosocomial infections