Type distribution of human papillomaviruses in ThinPrep cytology samples and HPV16/18 E6 gene variations in FFPE cervical cancer specimens in Fars province, Iran.

BACKGROUND: There exists strong evidence that human papillomavirus (HPV) is associated with cervical cancer (CC). HPV E6 is a major oncogene whose sequence variations may be associated with the development of CC. There is not sufficient data on the distribution of HPV types in ThinPrep cytology specimens and HPV 16/18 E6 gene variations among CC patients in the southwest of Iran. This study was conducted to contribute to HPV screening and vaccination in Iran. METHODS: A total of 648 women screened for cervicitis, intraepithelial neoplasia or CC were included in the study. All participants underwent ThinPrep cytology testing, single-step HPV DNA detection and allele-specific reverse hybridization assays. Moreover, a total of 96 specimens previously tested positive for single infection with HPV16 or 18 were included for variant analysis. HPV16/18 lineages and sublineages were determined by PCR assays followed by sequencing the E6 gene and the construction of neighbor-joining phylogenetic trees. RESULTS: Overall, HPV DNA was detected in 62.19% of all the screened subjects. The detection rates of HPV DNA among individuals with normal, ASC-US, ASC-H, LSIL, and HSIL cervical cytology were 48.9%, 93.6%, 100%, 100%, and 100%, respectively. Low-risk HPVs were detected more frequently (46.9%) than high-risk (38.9%) and possible high-risk types (11.1%). Of 403 HPV-positive subjects, 172 (42.7%) had single HPV infections while the remaining 231 (57.3%) were infected with multiple types of HPV. Our results indicated a remarkable growth of high-risk HPV66 and 68 and low-risk HPV81 which have rarely been reported in Iran and HPV90 and 87 that are reported for the first time in the country. In addition, 3 lineages (A, D, and C) and 6 sublineages (A1, A2, A4, C1, D1, and D2) of HPV16, and one lineage and 4 sublineages (A1, A3, A4, and A5) of HPV18 were identified. The studied HPV16 and 18 variants mainly belonged to the D1 and A4 sublineages, respectively. CONCLUSION: The present study suggests that the prevalence of HPV infection in women of all age groups with or without premalignant lesions in the southwestern Iran is high and the predominant HPV types in the southwest of Iran may differ from those detected in other parts of the country. This study also highlights the necessity of not only initiating HPV vaccination for the general population but also developing new vaccines that confer immunity against the prevalent HPV types in the area and national cervical screening programs using a combination of thinPrep cytology test and HPV detection assays in order to improve the accuracy of the screening.

A Small Molecule Targeting the Intracellular Tyrosine Kinase Domain of ROR1 (KAN0441571C) Induced Significant Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

A ROR1 Small Molecule Inhibitor (KAN0441571C) Induced Significant Apoptosis of Mantle Cell Lymphoma (MCL) Cells.

The receptor tyrosine kinase orphan receptor 1 (ROR1) is absent in most normal adult tissues but overexpressed in various malignancies and is of importance for tumor cell survival, proliferation, and metastasis. In this study, we evaluated the apoptotic effects of a novel small molecule inhibitor of ROR1 (KAN0441571C) as well as venetoclax (BCL-2 inhibitor), bendamustine, idelalisib (PI3Kδ inhibitor), everolimus (mTOR inhibitor), and ibrutinib (BTK inhibitor) alone or in combination in human MCL primary cells and cell lines. ROR1 expression was evaluated by flow cytometry and Western blot (WB). Cytotoxicity was analyzed by MTT and apoptosis by Annexin V/PI staining as well as signaling and apoptotic proteins (WB). ROR1 was expressed both in patient-derived MCL cells and human MCL cell lines. KAN0441571C alone induced significant time- and dose-dependent apoptosis of MCL cells. Apoptosis was accompanied by decreased expression of MCL-1 and BCL-2 and cleavage of PARP and caspase 3. ROR1 was dephosphorylated as well as ROR1-associated signaling pathway molecules, including the non-canonical WNT signaling pathway (PI3Kδ/AKT/mTOR). The combination of KAN0441571C and ibrutinib, venetoclax, idelalisib, everolimus, or bendamustine had a synergistic apoptotic effect and significantly prevented phosphorylation of ROR1-associated signaling molecules as compared to KAN0441571C alone. Our results suggest that targeting ROR1 by a small molecule inhibitor, KAN0441571C, should be further evaluated particularly in combination with other targeting drugs as a new therapeutic approach for MCL.

The effects of high fructose fruits and honey on the serum level of metabolic factors and nonalcoholic fatty liver disease.

INTRODUCTION: The effect of the natural sources of fructose such as high fructose fruits and honey on the risk of fatty liver is still challenging. This study aimed to compare the effect of fructose, high fructose fruits, and honey on the metabolic factors and non-alcoholic fatty liver disease (NAFLD). METHODS: Forty-four rats were divided into four groups including normal diet group, high fructose group (HF), high fructose fruits group (HFF), and honey group (HO). After 120 days of intervention, the levels of insulin resistance, hepatic enzyme, and lipid profile were measured. Also, the expression levels of the acetyl-coA carboxylase (ACC), sterol regulatory element-binding protein 1c (SREBP-1c), Interleukin 6 (IL-6), and transforming growth factor-beta (TGF-ß) genes were assessed. In addition, a histopathologic assessment was performed on liver tissues. RESULTS: Insulin resistance (IR) increased significantly in the HF, HFF, and HO groups (All P < 0.05). The levels of liver enzymes was significantly increased only in the group receiving the HF regimen (P < 0.01). A significant decrease in total cholesterol and HDL-C (high density lipoprotein cholesterol) levels was found in HO group compared to the control group (P < 0.05). The expression levels of ACC and SREBP-1c genes in HF, HFF, and HO groups were significantly higher than the control group (All P < 0.05). The HF group had a greater increase in the level of gene expression of IL-6 and TGF-ß (All P < 0.05). Histopathological assessment did not find any changes in fatty liver formation and inflammatory damage. CONCLUSION: Consumption of fructose-rich honey and fruits improved the status of inflammatory markers and liver enzymes compared with the industrial fructose-rich products.

A ROR1 small molecule inhibitor (KAN0441571C) induced significant apoptosis of ibrutinib-resistant ROR1+ CLL cells.

ROR1 - a receptor tyrosine kinase - is overexpressed in CLL. Ibrutinib, a Bruton's tyrosine kinase inhibitor, is clinically effective in CLL but patients may develop resistance. We evaluated the effect of an ROR1 inhibitor, KAN0441571C, in CLL cells from six patients obtained before and after developing resistance to ibrutinib. The ROR1 inhibitor induced apoptosis in ibrutinib-resistant CLL cells to the same degree as in ibrutinib-sensitive cells and dephosphorylated ROR1. This was also noted in one patient who became resistant to both ibrutinib and the Bcl-2 inhibitor venetoclax. The combination of ROR1 inhibitor and venetoclax had a synergistic apoptotic effect on ibrutinib-resistant cells.

A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens.

Early diagnosis and genotyping of high-risk human papillomavirus (HR-HPV) in cervical tissue specimens is significant for cervical cancer prevention. A sensitive microplate fluorometric hybridization assay (MFHA) was designed for the detection of HPV DNA 16 and 18 in cervical tissue. Following optimization and validation of the method, 60 formalin-fixed and paraffin-embedded cervical samples representing different cervical intraepithelial neoplasia grades of HPV-associated lesions were tested to determine the sensitivity and specificity of the assay. Using consensus GP5+/6+ biotin-labeled primers to amplify a conserved region within the L1 gene, the amplicons were added to the microplate wells coated with specific probes for the hybridization of HPV 16 and 18 individually. Final detection was performed with streptavidin-AlexaFluor488 conjugated. The results were then compared with type-specific nested polymerase chain reaction (PCR) and colorimetric microplate assay. While the agreement between the results obtained by the type-specific nested PCR and fluorometric assay for the detection of both HR-HPV types was 100%, this agreement for the detection of HPV type 16 and 18 using microplate colorimetric assay was 94.2% and 85% respectively. Overall, the results of the fluorometric and colorimetric assays are promising for detecting both HR-HPV types in a large number of cervical tissue samples with the higher MFHA assay sensitivity.

Expression of transforming growth factor-beta and interferon gamma biomarkers after whole body gamma irradiation.

CONTEXT: There are plenty of evidence that suggest that the potential high doses of radiation result in severe health effects to exposed individuals, although there is no consensus about the health impact of low dose of ionizing radiation (IR). AIMS: This study aimed to discuss the effect a range of IR doses on the changes of gene expression and serum protein levels of two immune factors transforming growth factor-ß (TGF-ß) and interferon gamma (IFN-γ) in rats. Findings from this study can be useful to develop a suitable biomarker for biological dosimetry applications. SUBJECTS AND METHODS: After 24 h of irradiation of rats with the doses of 1000, 500, 100, 50, and 20 mGy, the gene expression of TGF-ß and IFN-γ in lymphocytes was assessed using quantitative polymerase chain reaction. Besides, the protein level of these two factors in blood plasma was determined by enzyme-linked immunosorbent assay (ELISA) kits. STATISTICAL ANALYSIS USED: One-way analysis of variance Tukey-Kramer Multiple Comparisons Test was used. P <0.05 was considered statistically significant. RESULTS: Significant increases in the expression levels of TGF-ß and IFN-γ genes were observed by increasing the dose from 100 to 500 mGy and then 1000 mGy compared to the control (P < 0.05). The ELISA tests showed significant differences in the serum level of TGF-ß cytokine in the dose of 1000 mGy, while the serum level of IFN-γ cytokine showed significant differences in doses of 20 mGy and 1000 mGy compared to the control (P < 0.05). CONCLUSIONS: The results of this study showed the changes in the expression of TGF-ß and IFN-γ genes after irradiation more than 100 mGy in lymphocytes compared to the control group; the changes in the serum levels of these cytokines only occurred in the specific doses compared to the control group.

Evaluation of the effect of hesperidin on vascular endothelial growth factor gene expression in rat skin animal models following cobalt-60 gamma irradiation.

INTRODUCTION: Skin is highly prone to radiation damage. Radiation burn is defined as damage to the skin or other biological tissues induced by radiofrequency or ionizing radiation. Vascular endothelial growth factor (VEGF) is a heparin-binded pro-angiogenic factor. Flavonoids belong to a family of polyphenol chemical compounds that are frequently present in fruits and vegetables. Hesperidin is an agent belonging to the flavonoid family. The aim of this study is to investigate whether hesperidin can affect the VEGF gene expression in rat skin following gamma irradiation or not. MATERIALS AND METHODS: A total number of 36 male Sprague-Dawley rats were divided into three groups. First group: radiation group (n = 12), second group: radiation + hesperidin-treated group (n = 12), and third group: untreated control group (n = 12). The hesperidin administration dose was 100 mg/kg body weight. The rats received a 22 Gy single dose at a dose rate of about 0.3 Gy/min using a cobalt-60 external beam radiotherapy unit. The animals were euthanized 24 h postirradiation. VEGF gene expression data were analyzed using the equation 2-ΔΔCT, where ΔΔCT = (Threshold cycle [CT], of target gene - CT of housekeeping gene)treated group- (CT of target gene - CT of housekeeping gene)untreated control group. Glyceraldehyde-3-phosphate dehydrogenase gene was used as a housekeeping gene. RESULTS: VEGF gene in the radiation + hesperidin group overexpressed 25-fold relative to the control group. In addition, VEGF gene in the radiation group underexpressed 0.15-fold relative to the control group. When the three groups were compared relative to each other using the Kruskal-Wallis test, P < 0.001 was obtained. Based on the Mann-Whitney U-test, when all groups were compared to each in a binary model, P = 0.001 was achieved. These tests all showed statistically significant changes in VEGF gene expression. CONCLUSIONS: We can conclude that hesperidin is a potent angiogenic factor. Hesperidin as a radioprotector can initiate angiogenesis by VEGF gene induction. It may stimulate epithelialization, collagen deposition, and enhanced cellular proliferation. These changes can together accelerate wound healing, in particular, radiation-induced skin damage.

The Synergistic Effects of Celecoxib and Sodium Valproate on Apoptosis and Invasiveness Behavior of Papillary Thyroid Cancer Cell Line In-vitro.

Metastasis to lymph nodes and distant organs is the main challenge in the treatment of papillary thyroid cancer. In the current investigation, we aimed to evaluate the synergistic effects of celecoxib (CX) and sodium valproate (VPA) against cell survival, invasiveness properties, and expression of metalloproteinase-2 and -9 (MMP-2 and MMP-9) in papillary thyroid cancer cell line, B-CPAP cells. The effect of CX and VPA on B-CPAP cells viability and apoptosis were investigated using MTT assay and annexin V/7-AAD flowcytometry, respectively. The effects of the drugs on invasiveness properties of B-CPAP cells and expression of MMP-2 and MMP-9 were evaluated using transwell assay and real time PCR, respectively. MTT assay showed that CX and VPA decreased viability of B-CPAP cells dose dependently (IC50 32.4µM and 6.8 mM, respectively). Combination of CX (5 µM) and VPA (2.5 and 5 mM) increased apoptosis, and reduced cell migration and invasion of B-CPAP cell, synergistically. Real time PCR results showed that both CX (5 µM) and VPA (2.5 and 5 mM) reduced MMP-2 expression (P < 0.05) but had no significant effects on the expression of MMP-9. Our findings suggest that CX and VPA synergistically increase apoptosis and suppress migration and invasion of B-CPAP cells through inhibition of MMP-2 expression.

The effect of histone deacetylase inhibitors on AHSP expression.

Alpha-hemoglobin stabilizing protein (AHSP) is a molecular chaperone that can reduce the damage caused by excess free α-globin to erythroid cells in patients with impaired ß-globin chain synthesis. We assessed the effect of sodium phenylbutyrate and sodium valproate, two histone deacetylase inhibitors (HDIs) that are being studied for the treatment of hemoglobinopathies, on the expression of AHSP, BCL11A (all isoforms), γ-globin genes (HBG1/2), and some related transcription factors including GATA1, NFE2, EKLF, KLF4, and STAT3. For this purpose, the K562 cell line was cultured for 2, 4, and 6 days in the presence and absence of sodium phenylbutyrate and sodium valproate. Relative real-time qRT-PCR analysis of mRNA levels was performed to determine the effects of the two compounds on gene expression. Expression of all target mRNAs increased significantly (p < 0.05), except for the expression of BCL11A, which was down-regulated (p < 0.05) in the cells treated with both compounds relative to the levels measured for untreated cells. The findings indicated that sodium valproate had a more considerable effect than sodium phenylbutyrate (p < 0.0005) on BCL11A repression and the up-regulation of other studied genes. γ-Globin and AHSP gene expression continuously increased during the culture period in the treated cells, with the highest gene expression observed for 1 mM sodium valproate after 6 days. Both compounds repressed the expression of BCL11A (-XL, -L, -S) and up-regulated GATA1, NFE2, EKLF, KLF4, STAT3, AHSP, and γ-globin genes expression. Moreover, sodium valproate showed a stronger effect on repressing BCL11A and escalating the expression of other target genes. The findings of this in vitro experiment could be considered in selecting drugs for clinical use in patients with ß-hemoglobinopathies.

The effects of specific expression of apoptin under the control of PSES and PSA promoter on cell death and apoptosis of LNCaP cells.

OBJECTIVES: Apoptotic effect of apoptin has been demonstrated in numerous studies. However, its tumor specificity has been questioned by some reports. The aim of this study was to confine the expression of apoptin in the prostate tumor cells by inducing its gene expression under the control of a chimeric enhancer composing of prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) regulatory elements (PSES). Furthermore, we investigated the effects of apoptin expression on LNCaP prostate carcinoma cell survival and apoptosis using MTT assay and annexin V/7-AAD flow cytometry assay. MATERIALS AND METHODS: Recombinant plasmids containing apoptin gene under the control of PSES/PSA promoter or Cytomegalovirus (CMV) promoter were constructed. Tumor cell lines including LNCaP cells and HeLa cells, and LX-2 cells (as a normal control) were transfected with the plasmids and the expression of apoptin was evaluated by real time-PCR and western blot analyses. The effects of apoptin expression on cell survival and apoptosis were then investigated using MTT and annexinV/7-AAD flow cytometry assay, respectively. RESULTS: Western blot and real time PCR analyses confirmed the specific expression of apoptin under the control of PSES/PSA regulatory element in the LNCaP cells, while CMV promoter caused apoptin expression in both tumor and normal cell lines. Apoptin expression significantly increased cell death and apoptosis in tumor cells when compared with the normal cells (P<0.001). CONCLUSION: These results suggest that PSES/PSA regulatory element may be considered as an efficient approach for specific expression of apoptin gene in prostate tumor cells and treatment of prostate cancer.

Melatonin Ameliorates The Production of COX-2, iNOS, and The Formation of 8-OHdG in Non-Targeted Lung Tissue after Pelvic Irradiation.

In this study, we evaluated the bystander effect of radiation on the regulation of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and 8-hydroxydeoxyguanosine (8-OHdG) in lung tissues of Sprague-Dawley rats with and without pre-administration of melatonin. A 2×2 cm2 area of the pelvis of male Sprague-Dawley rats with and without pre-administration of melatonin (100 mg/kg) by oral and intraperitoneal injection was irradiated with a 3 Gy dose of 1.25 MeV γ-rays. Alterations in the levels of COX-2, iNOS, and 8-OHdG in the out-of-field lung areas of the animals were detected by enzyme immunoassay. The bystander effect significantly increased COX-2, iNOS, and 8-OHdG levels in non-targeted lung tissues (P<0.05). Melatonin ameliorated the bystander effect of radiation and significantly reduced the level of all examined biomarkers (P<0.05). The results indicated that the ameliorating effect of a pre-intraperitoneal (IP) injection of melatonin was noticeably greater compared to oral pre-administration. Our findings revealed that the bystander effect of radiation could induce oxidative DNA damage and increase the levels of imperative COX-2 and iNOS in non-targeted lung tissues. Interestingly, melatonin could modulate the indirect destructive effect of radiation and reduce DNA damage in non-targeted cells.

Development of Flow Cytometry-Fluorescent In Situ Hybridization (Flow-FISH) Method for Detection of PML/RARa Chromosomal Translocation in Acute Promyelocytic Leukemia Cell Line.

BACKGROUND: Acute Promyelocytic Leukemia (APL) is a subclass of acute myeloid leukemia. The chromosomal aberration in 95% of APL cases is t(15; 17) (q22; q21), which prevents cell differentiation. Characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. The goal of this study was to develop a new and powerful Flow- FISH technique to detect the long isoform (L) of PML-RARa fusion transcript in NB4 cell line. METHODS: To achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% Triton X-100 and 0.2% saponin were used for the permeabilization step .In hybridization, a wide range of times and temperatures were used and probe was designed in FRET system. Results were confirmed by fluorescent microscope assay and reverse transcription PCR. RESULTS: In the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of PML-RARa transcript. Using standard fixation and permeabilization protocol of 2% PFA and 0.2% saponin gave the best fluorescent results in flow cytometry. Also, results indicated that the optimum time and temperature for hybridization was 2 hr at 42°C. The results of reverse transcription PCR and fluorescent microscopy confirmed the presence of PML-RARa transcript. CONCLUSION: The concordance between the results of Flow-FISH and those of two other techniques including reverse transcription PCR and FISH indicated that this method would be applicable as a diagnostic test for APL in clinical samples and MRD monitoring.

Fetal RHD Genotyping from Circulating Cell-Free Fetal DNA in Plasma of Rh Negative Pregnant Women in Iran.

The prenatal determination of the fetal Rh genotype could lead to a substantial reduction in the use of anti-D immunoglobulin and prevention of unnecessary exposure of pregnant women carrying RhD negative fetus. The aim of this study was fetal RHD genotyping through the analysis of cffDNA in plasma samples of RhD negative pregnant women by real-time PCR technique. In this experiment, 30 plasma samples were collected from RhD negative pregnant women. DNA were extracted and real-time PCR reactions were done by specific primers for RHD, SRY and beta-globin (GLO) genes. The Rh phenotypes of mothers and their babies were determined by agglutination method and specific anti-serums. From the 30 maternal plasma samples considered for SRY genotyping, 16 samples revealed the presence of the SRY gene. Regarding the fetal RHD genotyping, 26 samples were positive for RhD and 4 samples were negative. In all cases, the predicted RhD and SRY genotypes were in concordance with the serologically determined phenotypes. The sensitivity, specificity and precision of the fetal RHD and SRY genotyping test were calculated 100 % (p value <0.0005; K = 100 %). The present study confirms the precision of fetal RHD and SRY genotyping in maternal plasma by real-time PCR technique. This method helps RhD negative pregnant women about the appropriate use of anti-D immunoglobulin and also on the management and prevention of HDFN. However, superior and confirmatory studies are recommended before fetal RHD genotyping by real-time PCR is introduced as a non-invasive prenatal screening test.

Detection of Hepatitis E Virus Genotype 1 Among Blood Donors From Southwest of Iran.

BACKGROUND: Infection with hepatitis E virus (HEV) is endemic in developing countries and reveals significant regional differences. Several studies have reported virus transmission via blood transfusion. To date, however, no cases of HEV RNA detection in blood donors have been reported from Iran. OBJECTIVES: The aim of this study was to determine the presence of HEV RNA in plasma samples of blood donors referred to a blood transfusion center in Shiraz in the southwest of Iran. The HEV genotypes were also investigated using nucleotide sequencing. PATIENTS AND METHODS: Blood samples were collected from 700 blood donors who were referred to Fars blood transfusion organization from January to March 2014. Plasma samples were screened for the presence of HEV IgG and IgM antibodies by standard enzyme immunoassay. Samples seroreactive to anti-HEV were further tested for the presence of HEV RNA using nested polymerase chain reaction (PCR) with universal primers for detection of all four HEV genotypes. Positive PCR samples were then subjected to DNA sequencing for further analysis. RESULTS: Fifty (50, 7.1%) out of 700 plasma samples tested positive for anti-HEV antibodies. HEV RNA was detected in 7/50 (12%) of the antibody-positive samples, the majority of which were IgM positive. Sequence analysis of seven isolates of the HEV RNA ORF 2 gene region revealed > 80% similarity with genotype 1. CONCLUSIONS: The analysis indicates that the HEV isolated from blood donors in the southwest of Iran belongs to genotype 1. However, more samples from other geographic regions of Iran are needed to confirm these findings. Because transmission of HEV by administration of blood or blood components is likely to occur, it may be sensible to screen donor blood for HEV to eliminate transfusion-transmitted HEV infection when the recipient is immunocompromised.

Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR.

BACKGROUND: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time. METHODS: A0 DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/500 µl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples. RESULTS: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them. CONCLUSIONS: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis.

Development of an In-House TaqMan Real Time RT-PCR Assay to Quantify Hepatitis C Virus RNA in Serum and Peripheral Blood Mononuclear Cells in Patients With Chronic Hepatitis C Virus Infection.

BACKGROUND: Viral load measurements are commonly used to monitor HCV infection in patients with chronic diseases or determining the number of HCV-genomes in serum samples of patients after sustained virological response. However, in some patients, HCV viral load in serum samples is too low to be detected by PCR, especially after treatment. OBJECTIVES: The aim of this study was to develop a highly specific, sensitive, and reproducible in-house quantitative PCR using specific primers and probe cited in highly conservative region of HCV genome that allows simultaneous detection of HCV genotypes 1 - 4. MATERIALS AND METHODS: In this study, three sets of primer pairs and a TaqMan probe for amplification and detection of selected region within 5'-non-coding (5'NCR) of four HCV genotypes were used. Using plasmid containing 5'NCR region of HCV, standard curve, threshold, and threshold cycle (CT) values were determined. Real-time and nested PCR were performed on HCV genotypes 1 - 4 extracted from plasma and peripheral blood mononuclear cells (PBMCs) samples collected from patients with chronic HCV infection. RESULTS: The lower limit detection of this in-house HCV real-time RT-PCR was determined as 100 RNA copies/mL. Inter- and intra-assay coefficient of variation (CV) of this in-house HCV real-time RT-PCR ranged from 0.9% to 1.8% and 1.76% to 3.94%, respectively. The viral load of the genotyped samples ranged from 2.0 × 10(6) ± 0.31 to 2.7 × 10(5) ± 0.46 copies/mL in serum samples and 5 × 10(2) ± 0.36 to 4.0 × 10(3) ± 0.51 copies/10(6) cells/mL of PBMCs. CONCLUSIONS: The quite sensitive in-house TaqMan real time RT-PCR assay was able to detect and quantify all four main HCV genotypes prevailing around all geographical regions of Iran.

The effect of a new impregnated gauze containing bentonite and halloysite minerals on blood coagulation and wound healing.

In recent years, a wide variety of research has been carried out in the field of novel technologies to stop severe bleeding. In several studies, coagulation properties of minerals such as zeolite, bentonite and halloysite have been proven. In this study, the effect of a new impregnated sterile gauze containing bentonite and halloysite minerals was studied on blood coagulation and wound healing rate in male Wistar rats. Initially, impregnated sterile gauze was prepared from the mixture of bentonite and halloysite minerals and petroleum jelly (Vaseline). Then, the effect of gauze was studied on the blood coagulation time and wound healing process in 40 Wistar rats. SPSS software was used for data analysis and P values less than 0.05 were considered significant. The coagulation time of 81.10 ± 2.532 s in the control group and 33.00 ± 1.214 s in the study group (bentonite-halloysite treated) were reported (P < 0.0005). Time for complete wound healing in the group, which is treated with impregnated sterile pads, was calculated approximately from 10 to 12 days. However, in the control group, there was no complete wound healing (P < 0.0005). According to the results of the present study, topical application of the bentonite-halloysite impregnated sterile gauze significantly decreases the clotting time and increase the wound healing rate.

Relationship between AHSP gene expression, ß/α globin mRNA ratio, and clinical severity of the ß-thalassemia patients.

BACKGROUND: Alpha hemoglobin stabilizing protein (AHSP) is a chaperone-like molecule specialized for erythroid series which binds to free α-globin chain. According to this characteristic, AHSP can be considered an important factor which reduces beta thalassemia symptoms. MATERIALS AND METHODS: Reticulocytes RNA extraction and a subsequent cDNA synthesis were performed, followed by Relative qRT-PCR for AHSP, alpha, and beta globin chain genes. The beta actin gene was used as an endogenous reference as well. The relationship between AHSP gene expression, disease severity, and the ß/α globin mRNA ratio was studied among different homozygote ß-thalassemia patients (mild, moderate and severe) and compared with minor thalassemia and the normal population. RESULTS: Analysis of the ß-globin/α-globin mRNA ratio has shown that disease severity enhanced with a decrease in this proportion. Evaluation of the correlation between AHSP gene expression and the average of the ß-globin/α-globin expression ratio indicated a significant but indirect relationship in considered groups. Our results demonstrated that the AHSP gene expression increases in accordance with augmentation of clinical symptoms. CONCLUSIONS: Although one of the main reasons for reduced clinical severity in homozygote ß-thalassemia can be the high level of AHSP gene expression as a chaperon molecule, our findings indicated that AHSP gene expression decreased in a mild category as compared to that in severe and moderate groups.

Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry.

BACKGROUND: Various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells. METHODS: HeLa cells were fixed in 2% paraformaldehyde. A variety of detergents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets. RESULTS: In comparison with other methods, maximum cell frequency in percentage and fluorescent intensity (M1 = 2.1%, M2 = 97.9%) were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min (p = 0.001). CONCLUSION: Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized.