Threshold Cycle Method Research Articles

EFFECT OF PULSED MAGNETIC FIELDS ON THE EXPRESSION LEVELS OF TUMOR SUPPRESSOR GENES IN HUMAN T98G GLYOBLASTOMA CELL LINE

Aim: to study the effect of a pulsed magnetic field (PMF) on the expression of key tumor suppressor genes, such as aPc, MLH, and MGMt in human t98G glioblastoma cell line. material and methods . the PMF with the intensity of 15 and 300 mt was used alone and in combination with ionizing radiation at a single dose of 10 Gy. to perform ionizing radiation, theratron Equinox 60 co unit Best theratronics Ltd., Ottawa, canada) was used. the source of the pulsed magnetic field was Neuro-Ms / D therapeutic advanced device of the Neurosoft company. Live and dead cells were determined in NanoEntekJuliFl cell counter (Korea) using a 0.4 % trypan blue solution to stain dead cells. total RNa was extracted according to the protocol of the manufacturer trizol with changes: the aqueous phase was separated with trizol reagent twice. the quantitative measurement of the isolated RNa was carried out on a Qubit 2.0 fluorimeter using a kit of reagents with the Quant-it RNa assayKit RNa intercalating dye (Life technologies, usa). the expression of MLH, aPc, and MGMt genes was evaluated by Rt-PcR using a cFx96 amplifier (BioRad, usa). Data were analyzed using the cycle threshold (ct) method with normalization for tBP gene expression in each sample. Relative expression of the genetic locus (Exp) was calculated by the 2-Δct method. statistical analysis of the results was carried out using the statictica v10 software package. Results . One day after exposure to PMF, significant differences in the MGMt expression level compared to the control were found (p<0.05). a significant decrease in the transcriptional activity of the MGMt gene in glioblastoma cells was observed with PMF intensity of 15 mt, and correlated with the cell mortality rate. No changes in the mortality rate were observed after radiation exposure combined with 15 mt PMF. However, the mortality rate decreased from 18.7 % to 15 % after radiation exposure combined with 300 mt PMF. Conclusion . the effect of reduction in the transcriptional activity of MGMt in t98G glioblastoma cells and the effect of PMF as a monofactor on their viability characterize the magnetic susceptibility of tumor cell mechanisms. Given the multidirectional nature of the combined interaction of ionizing radiation and PMF, it is necessary to emphasize the importance of choosing and justifying the role of biotropic parameters of PMF in order to exclude a negative effect on the treatment.

18S rRNA, a potential reference gene in the qRT-PCR measurement of bisphenol A contamination in green mussels (Perna viridis) collected from the Gulf of Thailand

Normalization of reference genes through the selection of appropriate housekeeping genes is important for the reliable quantification of mRNA analyzed using real-time polymerase chain reaction (qRT-PCR). The suitability of various housekeeping genes (HKGs), were valuated as internal references to verify the levels of the contaminant bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane) by determining the level of gene expression of cytochrome P450 4 (cyp4) within the digestive gland of green mussels (Perna viridis)—a species used for biomonitoring environmental levels. Additionally, the effect was explored of mussel gender and maturity status (juvenile or mature) on the stability of the expression of the reference genes. Three candidate HKGs were examined, namely 18S rRNA, 28S rRNA and β-actin. The expression stability was calculated using three statistical approaches—NormFinder, BestKeeper and a comparative threshold cycle (Ct) method. The study found that of the three candidate reference genes, 28S rRNA and 18S rRNA were the most stable with no significant differences when maturity, gender and exposure to BPA were considered. It was noted that when these genes (18S rRNA, 28S rRNA, β-actin) were used for normalization using animals exposed to 10 ng BPA and the qRT-PCR method, different levels of cyp4 expression were observed. However, BPA significantly decreased the expression levels of cyp4 compared to the control (animals not exposed to BPA), only when 18S rRNA was used as an internal reference. These results highlighted the importance of selecting appropriate reference genes as the endogenous control in qRT-PCR in biomonitoring programs of BPA.

Molecular Mechanisms of Primary Resistance to Azacitidine in MDS/AML Patients - Data of the Hellenic MDS Study Group

Molecular Mechanisms of Primary Resistance to Azacitidine in MDS/AML Patients - Data of the Hellenic MDS Study Group

Low Expression of MicroRNA335-5p Is Associated with Malignant Behavior of Gallbladder Cancer: A Clinicopathological Study.

Background:MicroRNAs (miRNAs) are non-coding RNAs that regulate multiple cellular processes during cancer progression, identified to be involved in tumorgenesis of several cancers including cancers of digestive system. However its role in gallbladder inflammatory disease (GID) and gallbladder cancer (GBC) has not been well documented. The present study was aimed to investigate the clinical significance of hsa-miRNA-335-5p (miR-335) in GBC and GID. Subjects and Methods:This prospective case control study, conducted from July 1, 2014 to December 1, 2017 in Era’s Lucknow Medical College & Hospital, India, evaluated miR-335 expression by real-time polymerase chain reaction. Hundred tissue samples GID (control; n=50) and GBC (case; n=50) were studied. Relative quantification of target miR-335 expression was examined using the comparative cycle threshold method. Their expression was correlated with different clinicopathological parameters. Fishers’ exact test, Student’s t-test, and Chi-square test were used as appropriate for data analysis. Kaplan-Meier methods were used to calculate overall and disease-free survival rate. Two sided P<0.05 was considered as significant. Results:miR-335 expression was found to be significantly low in GBC lesions when compared with GID lesions (P<0.001). The low expression level of miR-335 was correlated with histological grade (P=0.007), clinical stage (P<0.001), lymph node metastasis (P<0.001) and liver metastasis (P=0.016). Reduced expression of miRNA-335 was associated with a shorter median overall survival (7 months vs. 25 months) in GBC patients (P<0.001). Conclusions:Down regulation of miR-335 is associated with the severity of the disease and thus indicate that miR-335 expression may serve as prognostic marker for GBC.

Serum microRNA 143 as a potential biomarker for the diagnosis of hepatitis C virus-related hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fifth most frequently diagnosed cancer worldwide. Early recognition of the onset of HCC would help to select more effective therapies for patients leading to a better prognosis and life span. So, the development of effective markers for the diagnosis of HCC could have an impact on HCC-related cancer mortality. MicroRNAs (miRNAs) are reported as a group of small noncoding RNAs that can function as endogenous RNA interference to regulate the expression of targeted genes.AimTo study serum miR-143 expression level in patients with hepatitis C virus (HCV)-related HCC.Patients and methodsThe present study was conducted on 60 participants classified into group A: 30 patients with HCV-related cirrhosis with HCC; group B: 15 patients with HCV-related cirrhosis without HCC; and group C: healthy participants as control. Expression of miR-143 in the serum of all participants was obtained in all groups. Total serum RNA was extracted with small RNA enrichment followed by reverse transcription real-time PCR. Expression of miR-143 in the serum of all participants was obtained using the comparative cycle threshold method (2–ΔΔCT) after normalization for the expression of Syn-cel-miR-39 mi script miRNA mimic as control. MiR-143 expression levels were then compared in different groups.ResultsThe mean serum miR-143 levels were significantly higher in cirrhotic patients without and with HCC than in healthy participants (1.69±0.64, 13.0±6.23 vs. 0.56 ±0.29, P< 0.001, respectively) and in patients with HCC than in patients without HCC (P<0.001). The mean serum miR-143 levels were significantly higher in cirrhotic patients with HCC Barcelona Clinic Liver Cancer stage D than stage B (19.91±2.40, 13.41±1.80, P< 0.001, respectively) and in stage B compared with stage A (13.41±1.80, 5.68±1.86, P<0.001, respectively).ConclusionThe significantly higher serum level of miR-143 in cirrhotic patients with HCC compared with those without and to normal participants may suggest its role in hepatocarcinogenesis and may have a value as a potential biomarker for early diagnosis of HCV- related HCC.

Oral Bacterial Signatures in Cerebral Thrombi of Patients With Acute Ischemic Stroke Treated With Thrombectomy.

BackgroundChronic infections have been reported to be risk factors for both coronary heart disease and ischemic stroke. DNA of oral bacteria, mainly from the viridans streptococci group, has been detected in coronary thrombus aspirates of myocardial infarction and cerebral aneurysms. Viridans streptococci are known to cause infective endocarditis and possess thrombogenic properties. We studied the presence of oral bacterial DNA in thrombus aspirates of patients with acute ischemic stroke treated with mechanical thrombectomy.Methods and ResultsThrombus aspirates and arterial blood were taken from 75 patients (69% men; mean age, 67 years) with acute ischemic stroke. The presence of Streptococcus species, mainly the Streptococcus mitis group, belonging to viridans streptococci as well as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in samples were determined using a quantitative polymerase chain reaction with specific primers and probes. The relative amount of bacterial DNA in a sample was determined with the comparative threshold cycle method. Bacterial DNA was detected in 84% (n=63) of aspired thrombi, and 16% (n=12) of samples were considered bacterial DNA negative. DNA of Streptococcus species, mainly the S mitis group, was found in 79% (n=59) of samples. The median relative amount of Streptococcus species DNA was 5.10‐fold higher compared with the control blood samples from the same patients. All thrombi were negative for both P gingivalis and A actinomycetemcomitans.ConclusionsThis is the first study showing the common presence of bacterial DNA from viridans streptococci in aspired thrombi of patients with acute ischemic stroke. Streptococcal bacteria, mostly of oral origin, may contribute to the progression and thrombotic events of cerebrovascular diseases.

MON-178 Vitamin D Treatment of Patients with Type 1 Diabetes Reduces RORy, FoxP3 and CD4 Gene Expression in T Helper Cells

Background: Vitamin D (VD) deficiency and the dysregulation of T helper (Th) cells contribute to the pathogenesis of type 1 diabetes (T1D). Based on the modulatory effects of VD on immune cells, we investigated the gene expression of transcription factors (TFs) regulating the Th cell differentiation before and after VD treatment in a pilot trial. Materials and methods: Nineteen patients with T1D (11 females and 8 males) were randomized and treated for three months with 4000 IU/day cholecalciferol. The gene expression of four Th cell specific TFs (Th1/T-bet, Th2/GATA3, Th1/RORy and Treg/FoxP3) as well as the Th cell surface molecule CD4 was measured before (V1) and after three months (V3) of VD treatment. Th cells were isolated by density gradient centrifugation from venous blood samples and subsequently enriched by magnetic sorting with antibodies. Total RNA from the selected cells was extracted, transcribed and analyzed by RT-qPCR. Transcription levels were calculated using the comparative cycle threshold (CT) method (values presented as 2-[CTtarget -CT18S]= 2-[∆CT]). VD-therapy effects on the gene expression were evaluated by the differences between V1 and V3 using the Wilcoxon matched-pairs test. The median differences are given with Hodges-Lehmann estimator (HLS) and 95% confidence interval (CI). Results: The 25(OH)D3 levels increased to 39.1 ng/ml (HLS: 17.22 ng/ml; 95% CI 9.3 to 23.9, p=8 x10-4) after three months cholecalciferol treatment. In response to this the gene expression of the two TFs FoxP3 (HLS: -12.68; 95% CI -21.47 to -3.36, p=0.01) and RORy (HLS: -1.85; 95% CI -4.68 to 0.02,ptrend=0.07) were downregulated. Additionally, a reduced gene expression of CD4 (HLS: -128; 95% CI -452.36 to -15.46, p=0.009) was detected. Notably, all these gene expression changes occurred in women (FoxP3 HLS: -17.30, p=0.02; RORy HLS: -3.96, p=0.01 and CD4 HLS: -146, p=0.007) but not in men. Furthermore, the downregulation of FoxP3 gene expression after VD supplementation was found in patients who had a 25(OH)D3 level below 20 ng/ml (n=9) at study entry (HLS: -17.36;p=0.03). Changes in the gene expressions of T-bet and GATA3 were not significant. Conclusion: Elevated 25(OH)D3 levels after high dose VD therapy result in downregulated genes (FoxP3, RORy and CD4) which are involved in the differentiation of effector Th cells (Treg and Th17). These VD effects occur predominantly in female patients, highlighting a gender specificity of the Th cell response. The downregulation of FoxP3 gene in VD deficiency patients may reflect other Th cell phenotypes. Our results support a targeted approach for anti-inflammatory VD treatment based on the measurement of peripheral 25(OH)D3 levels and Th cell related TFs. To corroborate our observations, further mechanistic studies and larger groups of patients are necessary.

Identification of Suitable Reference Genes for Quantitative Gene Expression Analysis in Innervated and Denervated Adipose Tissue from Cafeteria Diet-Fed Rats.

Our previous studies show that cafeteria diet increases body adiposity, plasma insulin levels, and sympathetic activity to brown adipose tissue (BAT) and white adipose tissue (WAT) of Wistar rats, leading to rapid and progressive changes in the metabolic profile. The identification of suitable reference genes that are not affected by the experimental conditions is a critical step in accurate normalization of the reverse transcription quantitative real-time PCR (qRT-PCR), a commonly used assay to elucidate changes in the gene expression profile. In the present study, the effects of the cafeteria diet and sympathetic innervation on the gene expression of adrenoceptor beta 3 (Adrb3) from BAT and WAT were assessed using one of the most stable and one of the least stable genes as normalizers. Rats were fed the cafeteria diet and on the 17th day, interscapular BAT or retroperitoneal WAT was denervated and, 7 days after surgery, the contralateral innervated tissue was used as control. Ten reference genes were evaluated (18S, B2m, Actb, CypA, Gapdh, Hprt1, Rpl32, Tbp, Ubc, and Ywhaz) and ranked according to their stability using the following algorithms: geNorm, NormFinder, BestKeeper, and comparative delta threshold cycle (ΔC t ) method. According to the algorithms employed, the normalization of Adrb3 expression by the least stable genes produced opposite results compared with the most stable genes and literature data. In cafeteria and control diet-fed rats, the three most stable genes were Hprt1, Tbp, and Rpl32 for interscapular BAT and Tbp, B2m, and Hprt1 for retroperitoneal WAT, while the least stable genes were 18S, Actb, and Gapdh for both tissues.

Cytokine‐stimulated iNOS Expression Requires Long Chain Fatty Acyl CoA Synthetase (ACSL)

RationaleACSL catalyzes the ligation of fatty acid to coenzyme A, the first and rate limiting step for fatty acid utilization. Triacsin C is a fungal metabolite which inhibits ACSL1, ACSL3, and ACSL4. Earlier, we showed that Triacsin C reduces the infarct volume and inhibits iNOS expression in the MCAO model of stroke. As iNOS is up‐regulated in stroke, we wished to determine whether the Triacsin C effect was linked to iNOS expression.MethodsbEnd.3 cells were stimulated with cytokine mix, (TNFα, 60 ng/mL; Ilβ,2 ng/mL; IFNγ, 100 units/mL), in the presence or absence of 5μM Triacsin C for 24 hours. RNA was extracted following RNeasy Plus Mini Kit(Qiagaen) instruction. The concentration and purity of RNA was determined by Spectrophometer. 20 μl first strand cDNA was synthesized from 1 μg total RNA for each sample using Superscript III First‐Strand(Invitrogen). One μl cDNA was amplified using SYBR Green PCR Master Mix (applied Biosystems) in CFX96 Touch Real‐Time PCR Detection System (BioRad). β‐actin (cat 330001 PPM02945B) and iNOS (cat 330001PPM02928B) primers were purchased from Qiagen. Expression level in fold change was determined by the comparative threshold cycle method (2−ΔΔCt) with β‐actin as the control gene.ResultsCytomix stimulated iNOS expression in the presence of Triacsin C was 52.8 ± 8.33% of control. (p=0.0005; n=5 independent experiments).ConclusionsOur data confirm that iNOS expression is modulated by ACSL activity, and support our hypothesis that the Triacsin C effect on stroke infarct volume is related to suppression of iNOS expression.Support or Funding InformationDepartment of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TXThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Co-expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumors.

The hypoxic tumor microenvironment is the major contributor of chemotherapy resistance in solid tumors. One of the key regulators of hypoxic responses within the cell is the hypoxia inducible factor-1α (HIF-1α) that is involved in transcription of genes promoting cell survival and chemotherapy resistance. Multidrug resistance gene-1 (MDR1) and Lysosome-associated protein transmembrane 4B-35 (LAPTM4B-35) are among those notable players which augment their responses to cellular hypoxia. MDR1 is the hypoxia responsive gene involved in multidrug resistance phenotype while LAPTM4B-35 is involved in chemotherapy resistance by stabilizing HIF-1α and overexpressing MDR1. Overexpression of HIF-1α, MDR1 and LAPTM4B has been associated with poor disease outcome in many cancers when studied individually at tissue level. However, accessibility of the tissues following the course of chemotherapy for ascertaining chemotherapy resistance is difficult and sometimes not clinically feasible. Therefore, indication of hypoxic biomarkers in patient’s blood can significantly alter the clinical outcome. Hence there is a need to identify a blood based marker to understand the disease progression. In the current study the expression of hypoxia associated chemotherapy resistance genes were studied in the peripheral blood lymphocytes of solid tumor patients and any potential correlation with disease progression were explored. The expression of HIF-1α, MDR1 and LAPTM4B was studied in blood of 72 breast, 42 ovarian, 32 colon and 21 prostate cancer patients through real time PCR analysis using delta cycle threshold method. The statistical scrutiny was executed through Fisher’s Exact test and the Spearman correlation method. There was 12–13 fold increased in expression of HIF-1α, two fold increased in MDR1 and 13–14 fold increased in LAPTM4B mRNA level in peripheral blood of breast, ovarian, prostate and colon cancer patients. In the current study there was an association of HIF-1α, MDR1 and LAPTM4B expression with advanced tumor stage, metastasis and chemotherapy treated group in breast, ovarian, prostate and colon cancer patients. The Spearman analysis also revealed a positive linear association among HIF-1α, MDR1 and LAPTM4B in all the studied cancer patients. The elevated expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumor patients can be a predictor of metastasis, disease progression and treatment response in these cancers. However, larger studies are needed to further strengthen their role as a potential biomarker for cancer prognosis.

42 Nrf2 and nuclear factor kappa B cross-talk in bovine granulosa cells under lead challenge

Lead (Pb2+), one of the pervasive and protracted environmental heavy metals, is known to affect the female reproductive system by inducing oxidative stress. The Nrf2 and NF-κB are two key transcriptional factors (TF) known to regulate cellular redox status and response against stress and inflammation, respectively. Evidence showed that these TF undergo functional cross-talk. Although the properties of Pb2+ have been extensively studied, little is known about its effect on bovine granulosa cells with respect to Nrf2/NF-κB interaction. The aim of this study was to investigate the impact of Pb2+ on the response of bovine granulosa cells with regard to the cross-talk between Nrf2 and NF-κB. For this, bovine granulosa cells aspirated from small growing follicles (3-5mm of diameter) from ovaries obtained from a local slaughterhouse were in vitro cultured at 37°C and 5% CO2. Sub-confluent cells were exposed to different doses of lead acetate [Pb (C2H3O2)2] (1, 2, or 3µg mL−1) for 2h. Following this, the accumulation of intracellular reactive oxygen species was measured using the H2DCFDA fluorescent probe (Life Technologies, Darmstadt, Germany). The cell viability was assessed using Cell Counting Kit-8 (CCK-8, Dojindo, Munich, Germany) 24h after treatments. Moreover, cells were harvested and subjected to total RNA isolation using miRNeasy Mini Kit (Qiagen, Hilden, Germany) followed by cDNA synthesis using First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and the gene expression was determined using quantitative PCR. Data were analysed using comparative threshold cycle (ΔΔCT) method and normalized against the expression of GAPDH and β-actin genes. Furthermore, the protein lysate was isolated from cells to assess carbonylated protein using OxyBlot protein oxidation detection kit (abcam, Cambridge, MA, USA). Results showed that exposure of granulosa cells to lead acetate provoked intracellular reactive oxygen species accumulation and protein carbonylation. This was accompanied by cell cycle arrest at G0/G1 phase, reduction in cell viability, and decrease in the expression of cell proliferation marker genes (CCND2 and PCNA). Moreover, lead acetate elicited down-regulation of both Nrf2 and NF-κB and their downstream antioxidant genes (SOD and CAT), whereas no significant difference was shown in the level of other genes involved in both TF pathways (Keap-1, HO-1, Trx, IKK, and TNF-α). In addition, lead acetate challenge increased the expression of endoplasmic reticulum stress marker genes (GRP78 and CHOP) and the pro-apoptotic gene (Caspase-3), whereas it reduced the anti-apoptotic gene (BCL-2). Our findings suggest that Pb+2 driven-oxidative stress deregulates granulosa cell viability and proliferation, enhances endoplasmic reticulum stress, induces cell cycle arrest, and mediates apoptosis probably via disruption of Nrf2/NF-κB cross-talk.

Low tissue levels of miR-125b predict malignancy in solitary fibrous tumors of the pleura

BackgroundSolitary fibrous tumors of the pleura (SFTP) are rare neoplasia of the chest. A subset of SFTP follows a malignant course, sometimes several years after complete resection. Traditional scoring systems based on clinical and histological features are poor predictors of biological behavior. This study aimed to investigate tumor-associated miRNAs expression as novel biomarkers to predict the clinical behavior of SFTP.MethodsFormalin-fixed and paraffin-embedded SFTP tissues blocks from patients surgically resected between 1992 and 2013 at two tertiary care teaching hospitals were included. SFTP tumors were categorized as either malignant or benign variants according to the WHO classification. Following miRNAs levels were measured: let-7a, miR-16b, miR-17, miR-21, miR-31, miR-34a, miR-92a, miR-125a, miR-125b, miR-195-5b, miR-203a, and miR-223. Differential gene expressions which were calculated with the threshold cycle (Ct) method were compared among the two variants.ResultsThirty-eight patients (40% male, mean age 62.2 (±10.9) years) were included. Expression levels of miR-125b showed a significant difference between benign compared to malignant variants (−3.08 ± 0.93 vs. -2.22 ± 1.36, p = 0.0068). Furthermore, lower levels of miR-125b were found to be associated with increased tumor size (p = 0.0414). Thus, downregulation of miR-125b indicates malignant transformation. All other investigated miRNAs were not associated with grading of SFTP.ConclusionsOur data suggest a potential role of miR-125b in the pathogenesis of tumor growth and malignant transformation of SFTP, respectively. Further studies have to address the potential use of miRNA-125b as a biomarker or therapeutic target in SFTP.

117 FIBROBLAST GROWTH FACTOR RECEPTOR-1c, -2c, -3c, and -4 mRNA ABUNDANCE IN GRANULOSA CELLS DURING FOLLICULAR GROWTH IN CATTLE

Fibroblast growth factors (FGF) regulate folliculogenesis of several species, including cattle. The cellular responses to a particular FGF are influenced by the diversity of high affinity fibroblast growth factor receptors (FGFR). There are 4 distinct genes encoding FGFR in vertebrates and the occurrence of mRNA splicing in the immunoglobulin-like domain III generates a diversity of sequences, and results in various isoforms of FGFR1, FGFR2, and FGFR3 (but not of FGFR4). Because FGFR have different ligand specificities, the presence of FGFR in bovine antral follicles is of fundamental importance for the FGF to exert their effects in the ovary. Hence, the objective of this study was to determine if FGFR1c, FGFR2c, FGFR3c, and FGFR4 mRNA abundance in granulosa cells (GC) change according to follicular size, steroidogenic status, and days post-ovulation during growth of first-wave dominant follicles in cattle. Oestrous cycles of non-lactating dairy cattle were synchronized and ovaries were collected on either Day 3–4 (n = 8) or Day 5–6 (n = 8) post-ovulation (as assessed by rectal ultrasonography). Follicular fluid (FFL) was aspirated from small (1–5 mm), medium (5.1–8 mm), or large (8.1–18 mm) follicles for measurement of oestradiol (E2) and progesterone (P4) levels by radioimmunoassay, and GC were collected for mRNA extraction. Relative quantity of target gene mRNA was expressed as 2−ΔΔCt using the comparative threshold cycle (Ct) method. Data were transformed to natural log (x + 1), to correct for heterogeneity of variance, and analysed via factorial ANOVA with the general linear model procedure of SAS and are reported as least squares means ± s.e.M. Follicle group (based on steroidogenic status and size of follicles), but not days post-ovulation or their interaction, significantly affected FGFR1c, FGFR2c, and FGFR3c mRNA abundance, whereas FGFR4 mRNA abundance was not affected by follicle group or days post-ovulation. FGFR1c mRNA abundance was greater (P &lt; 0.01) in large (44.8 ± 11.3; n = 29), medium (63.8 ± 7.6; n = 64), and small (44.6 ± 11.2; n = 29) E2-inactive (FFL E2/P4 ratio &lt; 1) than in large E2-active (FFL E2/P4 ratio &gt; 1) follicles (10.5 ± 15.5; n = 16) and greater (P &lt; 0.05) in medium E2-inactive than in large and small E2-inactive follicles. FGFR2c mRNA abundance was greater (P &lt; 0.01) in large (423.9 ± 131.9), medium (585.8 ± 97.0), and small (273.6 ± 143.2) E2-inactive than in large E2-active (56.2 ± 195.6) follicles. The FGFR3c mRNA abundance was greater (P &lt; 0.05) in large (143.4 ± 40.2) and medium (160.2 ± 29.3) E2-inactive than in large E2-active (43.2 ± 58.6) follicles and tended to be greater (P = 0.06) in small E2-inactive (101.9 ± 42.9) than in large E2-active follicles. Taken together, the findings that FGFR1c, FGFR2c, and FGFR3c mRNA abundance is lower in GC of E2-active follicles during growth of the first dominant follicle support an anti-differentiation role for these FGFR as well as support the idea that some FGF may regulate the selection of dominant follicles in cattle.

176 REGULATORY ROLE OF miR-20a DURING BOVINE OOCYTE MATURATION

The role of microRNA in oocyte maturation is mostly associated with optimal turnover of the accumulated maternal transcripts during their growth to allow maturation. MiR-20a is a member of the miR-17–92 cluster, which has been found to be differentially expressed in bovine granulosa cells derived from preovulatory dominant and subordinate follicles. Our recent study showed that miR-20a is involved in the regulation of granulosa cell proliferation, differentiation, and progesterone synthesis by targeting PTEN and BMPR2 genes. Here, we aimed to investigate the role of miR-20a in the bovine oocyte maturation processes. For this, cumulus-oocyte complexes (COC) were aspirated from small antral follicles (2–8 mm in diameter) and cultured in groups of 50 in 400 µL of maturation media (TCM-199 media supplemented with 12% oestrus cow serum and 10 µg/ml Follitropin®) at 39°C in a humidified atmosphere with 5% (vol/vol) CO2 in the air for 22 h. The cumulus cells and oocytes before (germinal vesicle) and after maturation (metaphase II) were mechanically separated in 0.1% hyaluronidase (in TCM-199 media). To study whether the presence of cumulus cells or oocyte has an impact on the miR-20a expression, we cultured oocytectomized cumulus cells and oocytes with and without their companion cells. Moreover, COC were co-cultured with miR-20a mimic, inhibitor, or corresponding controls to investigate the role of this miRNA in oocyte maturation. The total RNA from cumulus cells and oocytes was extracted using miRNeasy® mini kit (Qiagen GmbH, Hilden, Germany). Total RNA from respective samples was reverse transcribed for mRNA and microRNA expression analysis. Quantitative expression analysis was performed using StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) and subsequent data were analysed using a comparative cycle threshold method. The progesterone released in the spent media was measured using progesterone enzyme-linked immunosorbent assay kit (ENZO Life Sciences GmbH, Loerrach, Germany). Here, we found that miR-20a expression in cumulus cells increased (P &lt; 0.05) during oocyte maturation. Conversely, miR-20a expression in metaphase II stage oocytes was significantly lower (P &lt; 0.001) compared with the germinal vesicle stage. The absence of oocyte cytoplasm resulted in reduced miR-20a expression in cumulus cells. On the other hand, the absent of cumulus cells increased miR-20a expression in oocytes. The miR-20a expression revealed that the microRNA transduction is restricted in the cumulus cells. The overexpression of miR-20a increased oocyte maturation rate (P &lt; 0.05) by 4.8% (as determined by extrusion of the polar body) and the expression of oocyte maturation-related genes (INHBA, MAPK1, PTGS2, PTX3, and EGFR). The progesterone released in spent media of COC co-cultured with miR-20a mimic and inhibitor showed increasing (P = 0.0936) and decreasing (P = 0.0993) trends, respectively. In this study, we also found that miR-20a modulation altered the expression of PTEN and BMPR2 in cumulus cells. In conclusion, the modulation of miR-20a expression in cumulus cells regulates the oocyte maturation and partially involved in the progesterone synthesis by fine-tuning the expression of PTEN and BMPR2 genes.

Analysis of TaqMan Array Cards Data by an Assumption-Free Improvement of the maxRatio Algorithm Is More Accurate than the Cycle-Threshold Method

Quantitative PCR diagnostic platforms are moving towards increased sample throughput, with instruments capable of carrying out thousands of reactions at once already in use. The need for a computational tool to reliably assist in the validation of the results is therefore compelling. In the present study, 328 residual clinical samples provided by the Public Health England at Addenbrooke's Hospital (Cambridge, UK) were processed by TaqMan Array Card assay, generating 15 744 reactions from 54 targets. The amplification data were analysed by the conventional cycle-threshold (CT) method and an improvement of the maxRatio (MR) algorithm developed to filter out the reactions with irregular amplification profiles. The reactions were also independently validated by three raters and a consensus was generated from their classification. The inter-rater agreement by Fleiss' kappa was 0.885; the agreement between either CT or MR with the raters gave Fleiss' kappa 0.884 and 0.902, respectively. Based on the consensus classification, the CT and MR methods achieved an assay accuracy of 0.979 and 0.987, respectively. These results suggested that the assumption-free MR algorithm was more reliable than the CT method, with clear advantages for the diagnostic settings.

BaeR protein acts as an activator of nuclear factor-kappa B and Janus kinase 2 to induce inflammation in murine cell lines.

BaeR, a response regulator protein, takes part in multidrug efflux, bacterial virulence activity, and other biological functions. Recently, BaeR was shown to induce inflammatory responses by activating the mitogen-activated protein kinases (MAPKs). In this study, we investigated additional pathways used by BaeR to induce an inflammatory response. BaeR protein was purified from Salmonella enterica Paratyphi A and subcloned into a pPosKJ expression vector. RAW 264.7 cells were treated with BaeR, and RNA was extracted by TRIzol reagent for RT-PCR. Cytokine gene expression was analyzed by using the comparative cycle threshold method, while western blotting and ELISA were used to assess protein expression. We confirmed that BaeR activates nuclear factor-kappa B (NF-κB), thereby inducing an inflammatory response and increases the production of interleukins (IL-)1β and IL-6. During this process, the Janus kinase 2 (JAK2)-STAT1 signaling pathway was activated, resulting in an increase in the release of interferons I and II. Additionally, COX-2 was activated and its expression increased with time. In conclusion, BaeR induced an inflammatory response through activation of NF-κB in addition to the MAPKs. Furthermore, activation of the JAK2-STAT1 pathway and COX-2 facilitated the cytokine binding activity, suggesting an additional role for BaeR in the modulation of the immune system of the host and the virulence activity of the pathogen.

128 THE ROLE OF ENDOTHELINS IN REGULATING BOVINE GRANULOSA CELLS STEROIDOGENESIS

Endothelins are a group of vasoactive 21 amino acid peptides reported to play roles in steroidogenesis, folliculogenesis, and ovulation (Bridges et al. 2012 Life Sci. 91, 501–506). Nevertheless, the role of endothelins in regulating steroidogenesis in the bovine species requires further investigation. Thus, the objective of this study was to investigate the effects of endothelin 1 (ET-1) and endothelin 2 (ET-2) on bovine granulosa cell (GC) steroidogenesis. Bovine ovaries were obtained from a local abattoir. Follicular fluid was aspirated from small (1–5 mm) follicles and GC were isolated and exposed to various treatments (ET-1, ET-2, or ET-1 plus ET-2 with FSH and with or without insulin-like growth factor-1). In replicated experiments, culture medium was removed and analysed for steroid production via radioimmunoassay. Granulosa cells were either harvested with trypsin and counted using a Coulter Counter or collected with Trizol for RNA extraction and quantification via real-time PCR (18S rRNA was used as a housekeeping gene). Steroid production was expressed as nanograms (in the case of progesterone) and picograms (in the case of oestradiol) per 105 cells per 24 h. Relative quantity of target gene mRNA was expressed as 2–ΔΔCt using the relative comparative threshold cycle (Ct) method. Data were analysed via ANOVA and the general linear models (GLM) procedure of SAS for Windows (SAS Institute Inc., Cary, NC). If a significant main effect was identified, differences among means were determined by Fisher’s protected least significant differences test. The values were reported as least squares means ± standard error of the mean. In the presence of insulin-like growth factor-1, ET-1 significantly inhibited oestradiol production at 300 ng mL–1 (100.30 ± 11.05; P &lt; 0.05), but not at 30 ng mL–1 (114.47 ± 11.05; P &gt; 0.05) in comparison to the control (141.21 ± 11.05), whereas no differences were observed for progesterone production at 300 ng mL–1 (60.11 ± 7.11; P &gt; 0.05) or at 30 ng mL–1 (64.02 ± 7.11; P &gt; 0.05) in comparison to control (76.75 ± 7.11). ET-2 also significantly inhibited oestradiol production at 300 ng mL–1 (91.08 ± 11.87; P &lt; 0.01), but not at 30 ng mL–1 (112.77 ± 11.87; P &gt; 0.05) in comparison to the control in the presence of insulin-like growth factor-1. No significant effect of ET-1 and ET-2 was observed on steroidogenesis of granulosa cells cultured without insulin-like growth factor-1. Consistent with steroids production data, real-time PCR results indicated that, in the presence of IGF-1, ET-1 (5.66 ± 1.05) and ET-2 (5.65 ± 1.05) inhibited (P &lt; 0.05) aromatase gene expression compared to controls (11.33 + 1.05), and ET-1 plus ET-2 (2.42 ± 1.05) reduced (P &lt; 0.05) expression below that observed with either alone. No effect of ET-1 (4.38 ± 0.95; P &gt; 0.05), ET-2 (5.94 ± 0.95; P &gt; 0.05), or ET-1 plus ET-2 (4.57 ± 0.95; P &gt; 0.05) was observed for side-chain cleavage enzyme (CYP11A1) in comparison to controls (4.4 ± 1.07). Altogether, these results indicate that endothelins are involved in the regulation of steroidogenesis of bovine GC.

142 microRNA-17-92 CLUSTER REGULATES BOVINE GRANULOSA CELL FUNCTION BY TARGETING BMPR2 AND PTEN GENES

Normal follicular development, especially from the preantral stage until ovulation, is the critical to ensure the release of a developmentally competent oocyte. We have previously shown that among several clusters of microRNAs, microRNA-17-92 cluster (miR-17-5p, miR-19a, miR-20a, and miR-92a) is differentially expressed between bovine granulosa cells (bGC) derived from preovulatory dominant and subordinate follicles. Here, we aimed to investigate the regulatory role of microRNA-17-92 cluster in bGC function. Among the target genes predicted by the miRWalk database, BMPR2 and PTEN genes were experimentally validated using the pmirGLO Dual Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA). The bGC were aspirated from ovaries obtained from a local slaughterhouse. After determining cell viability and concentration using the trypan blue exclusion method, a total 2.5 × 105 bGC per well were seeded into CytoOne 24-well plate in DMEM/F12-Ham medium (Sigma Aldrich Chemie GmbH, Munich, Germany) supplemented with 10% FBS (Gibco BRL USA, Grand Isalnd, NY, USA) and 1% penicillin/streptomycin (Gibco BRL USA). Then, the bGC were cultured at 37°C with 5% CO2 and O2. To investigate the role of microRNA-17-92 cluster in bGC function, 100 nM of individual and cluster of microRNA-17-92 mimic, inhibitor, and negative controls were transfected into subconfluent-cultured bGC. The bGC were harvested 48 h post-transfection and used for RNA isolation and subsequent cDNA synthesis and expression analysis of candidate genes using real-time qPCR. Data analysis was performed using the comparative cycle threshold (Ct) method. A cell proliferation assay was performed using CCK-8 kit (Dojindo EU GmbH, Munich, Germany). Based on the cell diameter measurement done using ImageJ 1.48v software (National Institutes for Health, Bethesda, MD, USA), those bGC with diameter &gt;14 µm were categorized as differentiated cells, whereas those with diameter = 14 µm were considered as undifferentiated cells. MicroRNA-17-92 cluster overexpression on bGC reduced both mRNA and protein expression of BMPR2 and PTEN genes, whereas inhibition of microRNA-17-92 cluster increased their expression. Bovine GC transfected with microRNA-17-92 cluster mimic showed higher proliferation activity and decreased rate of differentiation. The opposite phenotype was observed in bGC transfected with microRNA-17-92 cluster inhibitor. Similarly, miRNA-17-92 cluster mimic transfection increased the expression of markers of proliferation, CCND2 and PCNA, and resulted in down-regulation of CYP11A1 and STAR genes as markers of differentiation. The opposite expression pattern was observed after transfection of miRNA-17-92 cluster inhibitors. In conclusion, the miRNA-17-92 cluster members coordinately regulate bGC proliferation and differentiation by targeting the expression of BMPR2 and PTEN genes.

232 EFFECT OF SUPEROVULATION PRETREATMENT ON DEVELOPMENTAL CHARACTERISTICS OF IN VITRO-FERTILIZED BOVINE EMBRYOS TRANSFERRED TO THE OVIDUCT-UTERUS ENVIRONMENT

Superovulation is a routine procedure to stimulate growth and ovulation of multiple follicles. However, the hormonal changes in the reproductive tract after superovulation treatment affect the environment and subsequently the early embryo development. The aim of the study was to examine the effect of superovulation pretreatment on embryo development and gene expression of IVM/IVF derived embryos subsequently cultured in vivo. The cumulus‐oocyte complexes derived from slaughterhouse ovaries were in vitro matured and fertilized. The denuded presumptive zygotes were cultured in CR1 medium with 5% oestrous cow serum. A total of 788 cleaved embryos at Day 2 were transferred by transvaginal endoscopy into the oviduct of synchronized and superovulated heifers (superstimulated group, SS) and 784 cleaved embryos were transferred into the ipsilateral oviduct of single ovulated synchronized heifers (single ovulation group, SO). In total, 10 Simmental heifers were used for in vivo culture in a crossover design. The in vivo culture was repeated once at an interval of at least 6 weeks in the same animal. At Day 7, embryos were recovered by combined flushing of the oviducts by endoscopy and the adjacent part of the uterine horns by conventional procedure. The numbers of recovered blastocysts were recorded and the embryos were cultured for the following 48 h to determine the blastocyst rate at Days 8 and 9. Simultaneously, 410 cleaved embryos were cultured in vitro for 9 days (control group, C). Triplicate pools of 10 blastocysts recovered at Day 7 from each treatment group were used for RNA isolation. Real-time PCR using sequence specific primers was performed in StepOnePlus™ real time PCR system (Applied Biosystem, Foster City, CA, USA). A comparative threshold cycle method was used to quantify expression levels of the candidate genes compared to the internal control GAPDH gene. The number of recovered embryos after in vivo culture was significantly lower in the SS group compared with the SO group (66.9 v. 79.5%, respectively; P &lt; 0.05). The blastocyst rates at Days 7, 8, and 9 in the SS, SO, and C groups were not significantly different (31.9, 43.3, and 47.1% v. 35.2, 48.5, and 53.5% v. 37.8, 50, and 56.1%, respectively). Molecular analysis of selected genes playing important roles during pre-implantation development revealed significantly lower expression levels of IL6, IL18, and ABCC2 between both experimental in vivo culture groups and the C-group. The IL18 was also significantly down-regulated in the SS-group compared to the SO-group. The transcription factor NFκB was found to be down-regulated in the SS-group compared to the SO and C groups (P &lt; 0.05). In conclusion, we showed that the superovulation pretreatment did not affect blastocyst yield during the culture period but seemed to influence the expression of developmentally important genes in the resulting embryos.

Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma.

BackgroundDespite its role in increasing the number of offspring during the lifetime of an individual animal, controlled ovarian hyperstimulation (COH) may have detrimental effects on oocyte development, embryo quality and endometrial receptivity. Circulating miRNAs in bio-fluids have been shown to be associated with various pathological conditions including cancers. Here we aimed to investigate the effect of COH on the level of extracellular miRNAs in bovine follicular fluid and blood plasma and elucidate their mode of circulation and potential molecular mechanisms to be affected in the reproductive tract.MethodTwelve simmental heifers were estrous synchronized and six of them were hyperstimulated using FSH. Follicular fluid samples from experimental animals were collected using ovum pick up technique at day 0 of the estrous cycle and blood samples were collected at day 0, 3 and 7 of post ovulation. The expression profile of circulatory miRNAs in follicular fluid and blood plasma were performed using the human miRCURY LNA™ Universal RT miRNA PCR array system. A comparative threshold cycle method was used to determine the relative abundance of the miRNAs.ResultsA total of 504 and 402 miRNAs were detected in both bovine follicular fluid and blood plasma, respectively. Of these 57 and 21 miRNAs were found to be differentially expressed in follicular fluid and blood plasma, respectively derived from hyperstimulated versus unstimulated heifers. Bioinformatics analysis of those circulating miRNAs indicated that their potential target genes are involved in several pathways including TGF-beta signaling pathway, MAPK signaling pathway, pathways in cancer and Oocyte meiosis.Moreover, detail analysis of the mode of circulation of some candidates showed that most of the miRNA were found to be detected in both exosomal and Ago2 protein complex fraction of both follicular fluid and blood plasma.ConclusionOur data provide the consequence of hyperstimulation induced changes of extracellular miRNAs in bovine follicular fluid and blood plasma, which may have a potential role in regulating genes associated not only with bovine ovarian function but also involved in altering various physiological in bovine oocytes, embryos and modulating reproductive tract environment.