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

A-NGR fusion protein induces apoptosis in human cancer cells

The NGR peptide is one of the well-known peptides for targeting tumor cells. It has the ability to target aminopeptidase N (CD13) on tumor cells or the tumor vascular endothelium. In this study, the NGR peptide was used for targeting A subunit of the Shiga toxin to cancer cells. The cytotoxic effect of the A-NGR fusion protein was assessed on HT1080, U937, HT29 cancer cells and MRC-5 normal cells. For this purpose, cells were treated with different concentrations of A-NGR (0.5-40 μg/ml). The evaluation of cell viability was achieved by MTT assay. Apoptosis was determined by annexin-V/PI double staining flow cytometry. Alterations in the mRNA expression of apoptosis - related genes were assessed by real time RT- PCR. The results showed that A-NGR fusion protein effectively inhibited the growth of HT1080 and U937 cancer cells in comparison to negative control (PBS) but for CD13-negative HT-29 cancer cells, only at high concentrations of fusion protein was inhibited growth recorded. On the other hand, A-NGR had little cytotoxic effect on MRC-5 normal cells. The flow cytometry results showed that A-NGR induces apoptosis. Furthermore, the results of real time RT-PCR revealed that A-NGR significantly increases the mRNA expression of caspase 3 and caspase 9. Conclusively, A-NGR fusion protein has the ability of targeting CD13-positive cancer cells, the cytotoxic effect on CD13-positive cancer cells as well as has low cytotoxic effect on normal cells

From classical approaches to new developments in genetic engineering of live attenuated vaccine against cutaneous leishmaniasis: potential and immunization

Despite the development of a vaccine against cutaneous leishmaniasis in preclinical and clinical studies, we still do not have a safe and effective vaccine for human use. Given this situation, the search for a new prophylactic alternative to control leishmaniasis should be a global priority. A first-generation vaccine strategy—leishmanization, in which live Leishmania major parasites are inoculated into the skin to protect against reinfection, is taking advantage of this situation. Live attenuated Leishmania vaccine candidates are promising alternatives due to their robust protective immune responses. Importantly, they do not cause disease and could provide long-term protection following challenges with a virulent strain. In addition to physical and chemical methods, genetic tools, including the Cre-loxP system, have enabled the selection of safer null mutant live attenuated Leishmania parasites obtained by gene disruption. This was followed by the discovery and introduction of CRISPR/Cas-based gene editing tools, which can be easily and precisely used to modify genes. Here, we briefly review the immunopathology of L. major parasites and then present the classical methods and their limitations for the production of live attenuated vaccines. We then discuss the potential of current genetic engineering tools to generate live attenuated vaccine strains by targeting key genes involved in L. major pathogenesis and then discuss their discovery and implications for immune responses to control leishmaniasis

Prospective Prediction of Treatment Response in High-Grade Glioma Patients using Pre-Treatment Tumor ADC Value and miR-222 and miR-205 Expression Levels in Plasma

Background: Treatment response in High-grade Glioma (HGG) patients changes based on their genetic and biological characteristics. MiRNAs, as important regulators of drug and radiation resistance, and the Apparent Diffusion Coefficients (ADC) value of tumor can be used as a prognostic predictor for glioma.Objective: This study aimed to identify some of the pre-treatment individual patient features for predicting the treatment response in HGG patients.Material and Methods: In this prospective study, 18 HGG patients, who were candidated for chemo-radiation treatment, participated after informed consent of the patients. The investigated features were the expression level of miR-222 and miR-205 in plasma, the ADC value of tumor, Body Mass Index (BMI), and age. Treatment response was assessed, and Least Absolute Shrinkage and Selection Operator (LASSO) regression was used to obtain a model to predict the treatment response. Mann-Whitney U test was also applied to select the variables with a significant relationship with patients’ treatment response.Results: The LASSO coefficients for miR-205, miR-222, tumor’s mean ADC value, BMI, and age were 3.611, -1.683, 2.468, -0.184, and -0.024, respectively. Mann-Whitney U test results showed miR-205 and tumor’s mean ADC significantly related to treatment response (P-value˂0.05). Conclusion: The miR-205 expression level of the patient in plasma and tumor’s mean ADC value has the potential for prognostic predictors in HGG

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

Investigating the Exosomes’ Compositions: An Inevitable Necessity in Cancer Treatment

Introduction: Exosomes, small microvesicles of endosomal origin, play a pivotal role in intercellular communication and various physiological and pathological processes. All cell types, especially cancer cells, release exosomes. These vesicles can induce tumor progression, metastasis, and drug resistance by altering the tumor microenvironment and transferring their content to target cells.  Exosomes contain lipids, proteins, and genetic biomolecules such as DNA, mRNA, and miRNA with their contents varying depending on cell type and disease state. Despite their role in cancer progression, exosomes also show potential as therapeutic agents. They are effective carriers for targeted drug delivery due to their high biocompatibility and low immunogenicity. However, more extensive clinical trials are needed to evaluate the efficacy and safety of exosome-based therapies. Oncology researchers are encouraged to investigate exosome contents more precisely and design new therapeutic strategies utilizing these vesicles