TaqMan Universal PCR Master Mix Research Articles

Abstract 6021: Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas

Abstract Background: Uterine leiomyosarcoma (LMS) comprises 60% to 70% of the uterine sarcoma (US). These tumors present high rates of recurrence and metastasis, and the patients’ survival rates are very low (20% in five years). It is known that somatic mutations and modifications in the DNA methylation levels are associated with several types of neoplasms. Previously, our group identified missense and loss of function mutations in KMT2D, CREBBP, ATM, TSC2, and GNAS, using a genetic screening method in LMS samples. Based on these results, we decided to investigate whether a genetic and epigenetic crosstalk could help to understand the biological processes involved in the LMS origin, development, and pathogenesis. The present study aimed to evaluate the KMT2D, CREBBP, ATM, TSC2 and GNAS gene expression profile in LMS cell line, and their methylation levels in formalin-fixed and paraffin-embedded (FFPE) patients´ samples. Methods: We selected 15 LMS - FFPE samples obtained via surgical procedures performed between 2012 and 2019 at the Instituto do Cancer do Estado de São Paulo (ICESP). Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit and we evaluated methylation levels using the Illumina Infinium Methylation EPIC BeadChip system 850k, including samples treated with bisulfite-treated DNA. Leiomyoma (LM) (THESCs CRL-4003) and LMS (SK-UT-1) cell lines were cultivated for gene expression analysis. Cells were growth for 24, 48, 72, and 96 hours, and the total RNA was extracted by TRIzol. High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis and for gene expression evaluation, real-time PCR reactions were performed using TaqMan Universal PCR Master Mix and inventoried TaqMan probes. Results: KMT2D and TSC2 showed increased expression in LMS compared to LM (cut-off ≤ -2 and ≥ 2 for down and upregulation, respectively), with higher expression in 24 hours of KMT2D [Fold Regulation (FR): 2.77] and TSC2 (FR: 2.58). In contrast, CREBBP was downregulated, with lowest expression in 72 hours (FR: -6.57). ATM was upregulated at 24, 48 with higher expression in 72 hours (FR: 2.32), and GNAS was downregulated, with lowest expression in 96 hours (FR: -4.31). Moreover, the methylation analyzes showed an expressive hypomethylation in LMS samples, compared to myometrium, for KMT2D (β value: 0.45; p= 0.004); CREBBP (β value: 0.51 p < 0.0001); ATM (β value: 0.47 p < 0.0001); TSC2 (β value: 0.53 p < 0.0001) and GNAS (β value: 0.41 p < 0.0001). In conclusion, our study showed that potentially pathogenic mutations may be associated with an aberrant hypomethylation profile, as well as increased expression of KMT2D, TSC2, ATM and loss expression of CREBBP and GNAS in LMS. The interconnection of these genetic and epigenetic events is essential for understanding the complex biology of these tumors, in addition to enabling identification of biomarkers. Citation Format: Laura G. dos Anjos, Daniela Bizinelli, Katia C. Carvalho. Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6021.

Association of Polymorphic Variants in Argonaute Genes with Depression Risk in a Polish Population.

Argonaute (AGO) proteins, through their key role in the regulation of gene expression, participate in many biological processes, including cell differentiation, proliferation, death and DNA repair. Accurate regulation of gene expression appears to be important for the proper development of complex neural circuits. Loss of AGO proteins is known to lead to early embryonic mortality in mice with various malformations, including anomalies of the central nervous system. Single-nucleotide polymorphisms (SNPs) of AGO genes can lead to deregulation of the processes in which AGO proteins are involved. The contribution of different SNPs in depression has been extensively studied. However, there are hardly any studies on the contribution of AGO genes. The aim of our research was to assess the relationship between the occurrence of depression and the presence of SNPs in genes AGO1 (rs636882) and AGO2 (rs4961280; rs2292779; rs2977490) in a Polish population. One hundred and one subjects in the study group were diagnosed with recurrent depressive disorder by a psychiatrist. The control group comprised 117 healthy subjects. Study participants performed the HDRS (Hamilton Depression Scale) test to confirm or exclude depression and assess severity. The frequency of polymorphic variants of genes AGO1 (rs636882) and AGO2 (rs4961280; rs2292779; rs2977490) was determined using TaqMan SNP genotyping assays and the TaqMan universal PCR master mix, no AmpErase UNG. The rs4961280/AGO2 polymorphism was associated with a decrease in depression occurrence in the codominant (OR = 0.51, p = 0.034), dominant (OR = 0.49, p = 0.01), and overdominant (OR = 0.58, p = 0.049) models. Based on the obtained results, we found that the studied patients demonstrated a lower risk of depression with the presence of the polymorphic variant of the rs4961280/AGO2 gene—genotype C/A and C/A-A/A.

Ebv-Mir-BART11-3p, Ebv-Mir-BART9 and Ebv-Mir-BART10 Are Present in the Plasma of HIV-Infected Subjects with Lymphoma and Are Potential Lymphoma Biomarkers

Epstein Barr virus (EBV) has been implicated in the lymphomagenesis, particularly of HIV-related lymphomas. EBV-infected cells are able to share miRNAs with other cells via exosomes and certain EBV-miRNAs were found in cells not infected with the virus but from patients with increased levels of EBV copies in plasma such as HIV-infected individuals. Thus, EBV-miRNAs might be detected in the plasma and might be relevant for the diagnosis and follow-up of HIV-related lymphomas.This case-control study included a group of 26 HIV-infected patients with lymphoma that responded to treatment and a control-group of HIV-infected subjetcs without lymphoma matched for age, gender, CD4-lymphocyte counts, plasma HIV viremia and time from HIV infection. We evaluated the presence of 43 EBV-miRNAs in frozen plasma samples from patients with lymphoma at diagnosis (Dx-group, N=26), one year before diagnosis (Pre-group, N=15) and complete response (CR-group, N=26). RNA was extracted from 200µl of plasma using Trizol LS (Ambion, Carlsbad, CA) and cel-miR-39 was used as spike-in. The detection of EBV-miRNAs was assessed by QRT-PCR in a BioMark™ HD System of Fluidigm (San Francisco, CA) using TaqMan® microRNA assays and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA). The following clinical and biological parameters were collected from records: age, gender, ECOG score, extranodal and bulky disease, B symptoms, Ann-Arbor stage, serum lactate dehydrogenase (LDH) and beta2-microglobulin, International Prognostic Index (IPI), HCV and HBV serology, history of opportunistic infection and of AIDS-defining illnesses, onset of combination antiretroviral therapy, CD4-lymphocyte counts, plasma HIV viremia. McNemar's and Wilcoxon tests were used for paired comparisons between groups. P-values of less than .05 were considered statistically significant.Overall, 30 EBV-miRNAs were detected in the Dx-group whereas only 8 EBV-miRNAs where found in the control-group and 19 in the CR-group. EBV-miRNAs were detected more frequently in the Dx-group (17 out of 26) than in the control-group (7 out of 26) (P =.002) and in the CR-group (11 out of 26) (P=.02). The presence of 6 EBV-miRNAs (ebv-miR-BART11-3p, ebv-miR-BART9, ebv-miR-BART10, ebv-miR-BART15, ebv-miR-BART31, and ebv-miR-BART19) could be associated with the lymphoma condition, since they were detected significantly different in the Dx-group and the control-group (Figure 1). Importantly, 3 of those EBV-miRNAs (ebv-miRBART11-3p, ebv-miR-BART9 and ebv-miRBART10) were found to be significantly less frequent in the CR group, reinforcing the idea that they could be derived from lymphoma cells and be used as biomarkers for diagnosis and follow up of HIV-related lymphoma. EBV-miRNAs cannot be used as early biomarkers of lymphoma in HIV-infected patients because none of the 3 EBV-miRNAs associated with lymphoma was significantly found in samples before diagnosis. The detection in plasma of ebv-miR-BART11-3p, ebv-miR-BART10 or ebv-miR-BART9 was not associated with having tumor cells infected with EBV (Figure 1). Only the detection of ebv-miR-BART9 was associated with detectable EBV loads [7/14 (50%) vs. 1/12 (8.3%), P=.036] (Figure 1). The following were the statistically significant associations between the presence of the 3 potential EBV-miRNA lymphoma biomarkers and the clinical-biological features studied. The presence of ebv-miR-BART10 in plasma was associated with IPI>2 [8/14 (57.1%) vs. 1/9 (11.1%), P=.004] and ECOG>2 [8/14 (57.1%) vs. 1/11 (9.1%), P=.011]. Also, more patients with ebv-miR-BART11-3p expression in plasma had ECOG>2 than patients without it [7/13 (53.8%) vs. 1/12 (8.3%), P=.030].Ebv-miR-BART11-3p, ebv-miR-BART9 and ebv-miR-BART10 can be detected in the plasma of patients with HIV-related lymphoma, independently of the presence of EBV in tumor cells or of EBV plasma viremia. They are potential biomarkers of lymphoma to be used in the diagnosis and follow up of HIV-infected subjects with the advantage of being easily determined by QRT-PCR employing noninvasive procedures.This work was supported in part by 2014 SGR225 (GRE) from CERCA Programme/Generalitat de Catalunya, and by funds from Josep Carreras International Foundation, “la Caixa” Foundation, DKMS, and Celgene Spain. [Display omitted] DisclosuresBaptista:DKMS: Research Funding; la Caixa Foundation: Research Funding; Carreras International Foundation: Research Funding; CERCA Programme/Generalitat de Catalunya: Research Funding; Celgene Spain: Research Funding. Sancho:Roche: Honoraria; Laboratorios Servier: Consultancy; Mundipharma: Honoraria; Kern Pharma: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Ribera:Celgene: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Amgen Inc.: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau; ARIAD: Research Funding, Speakers Bureau; Roche: Honoraria. Navarro:Celgene Spain: Research Funding; DKMS: Research Funding; la Caixa Foundation: Research Funding; Josep Carreras International Foundation: Research Funding; CERCA Programme/Generalitat de Catalunya: Research Funding.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Neurokinin-1 Receptor Antagonism Ameliorates Dry Eye Disease by Inhibiting Antigen-Presenting Cell Maturation and T Helper 17 Cell Activation

Neuroinflammation plays an important role in the pathogenesis of ocular surface disease, including dry eye disease (DED), but little is known about the contribution of substance P (SP) to DED. In this study, we investigated the expression of SP at the ocular surface and evaluated its effect on maturation of antigen-presenting cells (APCs), the key cell component involved in the induction of type 17 helper T-cell (Th17) response in DED. The effect of topical blockade of SP signaling was further investigated using neurokinin-1 receptor (NK1R) inhibitors on APC maturation, Th17 cell activation, and disease severity in a mouse model of DED. The results demonstrate that SP is constitutively expressed at the ocular surface, and trigeminal ganglion neurons are the major source of SP in DED. SP derived from trigeminal ganglion enhanced the expression of major histocompatibility complex class II maturation marker by bone marrow-derived dendritic cells, an effect that is abrogated by blockade of SP signaling using NK1R antagonist spantide. Finally, using a well-established murine model of DED, topical treatment of DED mice with NK1R antagonists CP-99,994 and L-733,060 suppressed APC acquisition of major histocompatibility complex class II, reduced Th17 cell activity, and ameliorated DED severity. These findings are of translational value, as they suggest that antagonizing NK1R-mediated SP signaling may be an effective strategy in suppressing Th17-mediated ocular surface disease.

Association of semenogelin (SEMG) gene variants in idiopathic male infertility in Chinese-Han population

ABSTRACTInfertility is known to occur frequently worldwide, and the incidence is continuing to rise in China. It is known that semenogelin (SEMG) protein secreted by human seminal vesicles plays an important role in male reproductive system function. However, an association between alterations in SEMG gene functions and idiopathic male infertility occurrence in Chinese-Han population has not been examined. The aim of this study was thus to investigate the inherent relationship between SEMG gene alterations and idiopathic male infertility using a method of variant genotyping selection and semen quality analysis. A population of 484 males with clinically diagnosed idiopathic male infertility and 246 fertile controls were selected after signing consent forms. Results demonstrated a significantly increased frequency of idiopathic infertility with abnormal semen parameters such as semen volume, sperm concentration, sperm number per ejaculate, and sperm motility in variants carrying the rs2301366 TA genotype. Combined association analysis from target single-nucleotide polymorphisms (SNPs) was selected from the genotype database of unrelated Chinese-Han in Beijing individuals from the Hap Map. SNP array analysis in blood samples in each group was carried out by TaqMan Universal PCR Master Mix and TaqMan SNP Genotyping Assays. In addition, the interaction between SEMG SNPs and binding protein epididymal protease inhibitor (EPPIN) SNPs was determined. Our findings demonstrated that the presence of SEMG SNPs and EPPIN SNPs increased the frequency of idiopathic male infertility in Chinese-Han population. It is proposed that measurement of SEMG SNPs and EPPIN SNPs in carriers may thus be utilized to identify idiopathic male infertility.

Expression changes of microRNAs in menstrual blood samples of different menstrual cycle collection days

Three microRNAs (miR-141-3p, miR-497-5p, miR-143-5p) have been screened out and confirmed to be promising markers to distinguish between menstrual blood and peripheral blood by our previous study. However, studies reported the expression levels of miRNAs might be different in menstrual blood samples of different menstrual cycle collection days. To evaluate the dynamic changes of expression levels of these three miRNAs in menstrual blood samples from different menstrual cycle collection days, 51 menstrual blood (day 1–5 of menstrual cycle) samples were collected and detected. Total RNA was extracted then corresponding cDNA was prepared and three TaqMan Assays were run in triplicate using the TaqMan Universal PCR Master Mix II without UNG (Life Technologies, USA). Bioinformatics analysis was used to search target genes of three miRNAs attempting to explain the results. After comparing with the expression levels of previous peripheral blood samples, we found that miR-141-3p and miR-497-5p were still reliable markers to distinguish menstrual blood from peripheral blood after the sample size was enlarged.

Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

PS 01-18] MECHANISM OF HYPERTENSIVE CARDIAC HYPERTROPHY IN CULTURED NEONATAL RAT VENTRICULAR CARDIAC MYOCYTES

Objective: hypertrophy and impaired cardiac function are commonly observed in some patients with hypertension. Angiotensin II is a critical growth factor to mediate cardiac hypertrophy. MicroRNAs (miRs) have been identified and associated with several cardiovascular diseases, including ischemic cardiomyopathy and cardiac hypertrophy. MiR-21 was reported to be upregulated during adult rat cardiac remodeling. The importance of miR-21* in cardiomyocyte hypertrophy has also been demonstrated. Design and Method: Wistar rats (weight, 120–150 g) were purchased. Ventricular cardiomyocytes were isolated from 1 to 2-day-old neonatal wistar rat as described previously with minor modification. After 24 h of serum starvation, the cardiomyocytes were stimulated with or without 1 umol/L Ang II for 48 h. The hypertrophy cardiomyocytes were identified by semi-quantitative RT-PCR assay. Total miRNAs were extracted using mirVANA miRNA isolation kit. The miRNAs quality and quantity were determined by a NanoDrop 1000 Spectrophotometer. The miRNAs were reverse transcribed by TaqMan MicroRNA Reverse Transcription Kit. qRT-PCR was performed using TaqMan Universal PCR Master Mix II on the ABI 7500 System according to the manufacturer's recommendations. Total proteins isolated from cultured cardiomyocytes, with or without 1 umlo/L Ang II for 48 h, were prepared by M-PER mammalian protein extraction reagent. SDS-polyacrylamide gel electrophoresis was performed using standard procedures. Results: We demonstrated that miR-21* was markedly upregualted in Ang II-induced neonatal rat cardiac myocytes, while miR-21 was reduced. We might speculate that the miRNA expression profile is different between neonatal rat and adult rat cardiac myocytes. Our results further showed that Ang II led to a significant reduction of PTEN expression level and a significant promotion of phosphorylation level of PI3K. In contrast, treatment with prohypertrophic molecule Ang II reduced the phosphorylation level of Akt. Conclusions: We therefore speculate that miR-21* might have two opposite effects on Ang II-induced cardiac hypertrophy through various signaling pathways.

P41. The role of IL-10 polymorphism in pathology of hypertensive disorders in pregnancy

Introduction Hypertensive disorders in pregnancy are serious complications of maternal and/or fetal well being. Hypertension solely (PIH) or preeclampsia (PE), a pregnancy-associated specific disorder characterized by hypertension and excess protein excretion in the maternal urine, are important causes of maternal and fetal morbidity and mortality worldwide. Objectives The etiology of disorder is multi-factorial, including the imbalance between pro- and anti-inflammatory cytokines in impaired immune system. In this study we aimed to analyze the role of IL-10 592 C>A (rs1800872) in healthy pregnant women at term, women with preeclampsia and hypertension, and investigated whether this polymorphism has impact on disease risk and severity. Methods A 100 subjects of whom 36 were preeclamptic patients, 45 healthy pregnant controls and 19 women with PIH were involved in case-control study. Levels of proteinuria for 24 hours were measured and correlated to the IL-10 genotype. The genomic DNA was isolated from peripheral blood leukocytes and genotyping was performed by using TaqMan Universal PCR Mastermix and TaqMan SNP Genotyping Assays for rs1800872on AB7500 Fast Real-Time PCR System. Retrieved results have been correlated to clinico-pathological variables and risk assessment of the disease. For statistical analyses, non-parametric methods were applied. Results There was increased risk for pregnancy induced hypertensive disorders (PE + PIH) in women with aberrant (A) allele in genotype in crude analysis showing the OR = 1.25 (CI95%:0.56–2.76) for subjects carrying genotypes (C/A + A/A). The risk for PE was higher than risk for cases only with hypertension, (OR = 1.31; CI95%:0.54–3.15 vs. OR = 1.16; CI95%:0.39–3.40), respectively. Moreover, there was noted a positive correlation between -592A IL-10 genotype and PE with protein excretion >1 g/24;ħ(OR = 1.35; 95%CI:0.34–5.43 for C/A genotype and OR = 1.84; 95%CI:0.27–12.35 for A/A homozygote). Although insignificant association between IL-10 592C > A and onset of the PE was assessed, a slight trend for higher prevalence of early form ( Conclusion The risk of hypertensive disorders in pregnancy may be elevated among Slovak women with IL-10 592C>A polymorphisms, where the A allele may be associated with severe cases of PE.

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Genetic variation in ERCC1 and XPF genes and breast cancer risk.

Breast cancer is one of the most frequently diagnosed cancer in women worldwide, and we conducted a case-control study by genotyping seven potentially functional SNPs, three in ERCC1 and four in XPF, in a Chinese population of 417 breast cancer cases and 417 cancer-free controls. Three SNPs in ERCC1 and four SNPs in XPF were genotyped by using the Taqman Universal PCR Master Mix in the GeneAmp(®) PCR System 9700 with Dual 384-Well Sample Block Module, and assays were performed on a 384-well plate on the Sequenom MassARRAY platform. We found that elevated breast cancer risk was associated with those who had a family history of breast cancer and history of breast disease, and those who were over 25 years old at first full-term pregnancy. We found that decreased risk of breast cancer was associated with those who had a history of full-term pregnancies. Compared with the ERCC1 rs11615 T/T genotype, a significantly higher risk of breast cancer was found in the C/C genotype in codominant and dominant models after adjusting for potential risk factors. Similarly, we found that ERCC1 rs3212986 C/C genotype was associated with an increased risk of breast cancer in codominant, dominant and recessive models. Our study indicated that the ERCC1 rs11615 and rs2298881 polymorphisms are associated with breast cancer in a Chinese population. Further studies with large sample size are greatly needed to elucidate the SNPs of ERCC1 and XPF genes in the development of breast cancer.

Aurora-A Kinase Expression In Untreated Patients With Chronic Lymphocytic Leukemia

Aurora-A kinase is a cell-cyle regulating kinase required for chromosomal segregation. Overexpression of Aurora-A kinase has been detected some solid tumors and hematological malignancies such as multiple myelomai non-Hodgkin's lymhoma, and acute leukemia. But there are only two studies in chronic lymphocyic leukemia (CLL). This prospective study was approved by Ethical Committee of University. We investigated Aurora-A kinase in bone marrow of 41 untreated patients (22 male and 19 female with mean age of 70±10 years) with CLL and 19 patients (8 male and 11 female with mean age of 54±20 years) with anemia such as megaloblastic, autoimmune hemolytic, and iron deficiency anemia using a quantitative reverse transcriptase-PCR (RT-PCR) method. β-Actin and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA was used as the internal controls. Total RNA was extracted from bone marrow cells using the Trizol method (High Pure Isolation Kit, Roche Diagnostics). cDNA was prepared with the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). Aurora-A cDNA was quantified using TaqMan Universal PCR Mastermix (Applied Biosystems) and the Aurora-A TaqMan Gene Expression Assay. β-Actin was assayed using TaqMan Universal PCR Mastermix with forward (CCCTGGCACCCAGCAC) and reverse (GCCGATCCACACGGAGTAC) primers at 400nM each and probe (fam-ATCAAGATCATTGCTCCTCCTGAGCGC-bhq) at 100nM concentrations. Real-time quantitative RT-PCR was performed in LightCycler 480II (Roche Diagnostics). Relative RNA level was reported via standard delta delta Ct (dd Ct). Immunhistochemical analysis was performed using formalin-fixed, parafin-embedded sections of bone marrow biopsy specimens from patients and controls. Tissue sections were incubated for 60 minutes with Aurora-A (Novus Biologicals Inc. Littleton CO, 1:100 dilution). Aurora-A kinase is establish as positive if exist >10% in the cytoplasms and nuclei of the neoplastic cells in all cases. If this staining is 0-10% of cells, it is accepted as slight positive. If these cells is never (0%) stained, it is negative. For the comparison of values, Mann-Whitney-U, Chi-square, and One-Way ANOVA tests were used by SPSS 15.0 for Windows. With FISH method, 17p and 13q deletions were detected in 10% and 37% of the patients CLL, respectively. There was trisomy 12 in 7% of teh patients. 68% of the patients with CLL were in Binet-A stage. By immunohistochemical analysis, while Aurora-A kinase was positive in 61% of the patients, it was negative in 72% and slight positive 28% of controls. These positivity was statistically significant (p<0.001). By RT-PCR, β-Actin and GAPDH mRNA values were 3.85±2.61 and 3.49±2.32 in the patients with CLL, respectively. These values were 3.80±2.73 and 4.34±2.36 in controls, respectively. There was no difference for both β-Actin and GAPDH mRNA values between two groups (p>0.05). According to Binet classification, there was no difference for Aurora-A kinase expression with RT-PCR and immunohistochemical staining (p>0.05). Moreover there was no difference for both expression of Aurora-A kinase and immunhistochemical staining in between the patients with chromosmal ambnormalities and without (p>0.05). Although overexpression of Aurora-A kinase expression was not detected, significant immunohistochemical staining represented that Aurora-A kinase can be potential therapeutic target in CLL. Disclosures:No relevant conflicts of interest to declare.

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

Type VIII Collagen Mediates Vessel Wall Remodeling after Arterial Injury and Fibrous Cap Formation in Atherosclerosis

Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions invivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8(-/-) mice). After wire injury of the femoral artery, Col8(-/-) mice had decreased vessel wall thickening and outward remodeling when compared with Col8(+/+) mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE(-/-) mice (Col8(-/-);ApoE(-/-)) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture.

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Introduction: Prolonged exposure to hyperoxia leads to the generation of reactive oxygen species (ROS) with subsequent cellular damage and multiple organ dysfunctions in humans. ROS mediate both direct and indirect modulation of signaling mechanisms. The human equivalent of the evolutionarily conserved innate immunity receptors, Toll-like receptors (TLR)-3 and 4, have been implicated in mediating hyperoxia-associated lung injury. Flies lack adaptive immunity and have been useful candidates in studying innate immunity. In fly strains that are highly resistant to high O2 exposure, it was reported that antimicrobial peptides play an important role in tolerance to oxidant stress. Hypothesis: We hypothesized that hyperoxic injury evokes innate immunity pathways in Drosophila. Methods: Flies (Berlin strain) were grown with standard cornmeal media and male flies younger than 7 days were selected for experiments. Hyperoxia chamber was designed and supplied with 1 L/min flow of 100% oxygen with humidification. Four groups (n=30/group) were designated for 1) gene expression assay with hyperoxic injury, 2) survival analysis with hyperoxic injury, 3) gene expression assay with normoxic control and 4) survival analysis control. At the time when 10% of flies of hyperoxic injury were dead, group 1) and 2) were homogenized for gene expression assay. Real-time PCR for Gram(-) pattern recognition receptor of IMD pathway, PGRP-LC (peptidoglycan recognition protein-LC) and end-product of Toll pathway, Def (defensin) were performed using 7300 Real time PCR system with TaqMan universal PCR Master Mix with TaqMan Gene Expression Assay primers. Relative gene expression was calculated after normalization to?-actin 5C. Results: Eleven flies (33.7%) were dead at 5th day after hyperoxic exposure and 28 flies (93.3%) were dead at 8th day. Among 30 flies with normoxia, 5 flies (10.0%) were dead at 8th day (p< 0.05). At 3rd day after hyperoxic exposure, gene expressions for innate immunity, PGRP-LC and Def were increased in flies with hyperoxic injury than in flies with normoxia; PGRP-LC 3.253:1(p<0.05) and Def 2.071:1(p<0.05). Conclusions: Hyperoxic injury caused fatal influences for survival of flies. Hyperoxic injury induces members of Drosophila innate immunity PGRP-LC and Def, suggesting a conserved pattern of adaptive changes against infection and ROS.

Analyses of the immunologically relevant gene transcript by qRT-PCR in primary cutaneous melanoma by the young and the elderly.

e21135 Background: To understand the dynamics of anti tumor immune responses (IR) in the setting of the ageing immune system, we examined immunologically relevant gene transcripts in formalin fixed paraffin embedded (FFPE) primary cutaneous melanoma (PCM) samples derived from five young (age &lt;50 years) and five elderly (age &gt;or=70 years) patients by qRT-PCR. Methods: A total of 10 FFPE tumor blocks were collected between 2004-2007 of patients with PCM at the University of Connecticut Health Center. Total RNA was extracted from FFPE tissue sections and random primed cDNA was generated. Pre amplification using pooled Taqman assays and Universal PCR Master Mix (Applied Biosystems, USA) was performed for 14 cycles to ensure an adequate amount of starting material for qPCR. Using 15 ng total RNA, the quantification of human IR genes (IL-2, INFg, TNFa, TGFb1, IL-10, Foxp3, and IDO-1) and transcripts of succinate dehydrogenase (SDHA) (as endogenous control) was accomplished by qRT-PCR amplification of a cDNA using TaqMan Universal PCR Master Mix and TaqMan gene expressions assay probes. All reaction assays including that of SDHA were performed in triplicate on an ABI 7500 Fast system (Applied Biosystems). Cycle threshold (Ct) values for each mRNA were normalized to SDHA (DCT) and represented as RQ=2-DCT. The average RQ was calculated for all ten samples in both cohorts and Student’s T-test was performed to evaluate the statistical significance of the IR gene expression. Results: While no significant difference of expression of Th1 cytokines (IL-2, IFNg, TNFa) or Th2/3 cytokines (IL-10, TGFb) could be detected within the two groups, FOXp3 expression was significantly decreased in PCM in the elderly. Conclusions: Our results suggest ageing may be associated with less regulatory responses in situ in PCM and that more expanded analyses of the nature of the IR in PCM are likely to provide information with implications on prognosis and immune based treatment options for elderly PCM subjects.

Androgens Affect Myogenesis In Vitro and Increase Local IGF-1 Expression

The mechanism whereby anabolic androgens are associated with hypertrophy of skeletal muscle is incompletely understood but may involve an interaction with locally generated insulin-like growth factor (IGF) 1. The present investigation utilized a cell culture model of human skeletal muscle-derived cell maturation to test the hypothesis that androgens increase differentiation of human muscle precursor cells in vitro and to assess effects of androgen with or without IGF-1 on IGF-1 messenger RNA (mRNA) expression in human muscle precursor cells. Differentiation of muscle-derived cells was induced under standard low-serum conditions. Cultures were then exposed to androgen (testosterone (T)) at 50, 100, and 500 nM or IGF-1 (10-50 ng·mL⁻¹). Immunocytochemistry and real-time polymerase chain reaction (RT-PCR) were used to assess effects of androgens and IGF-1 after 3- (early) or 7-d (late) muscle differentiation, respectively; RT-PCR was used to quantify the effects on androgen receptor expression. Under low-serum conditions, 3-d exposure to androgens or IGF-1 or both resulted in no significant increase in cellular myogenic commitment. After 7-d exposure, however, T and IGF-1 were both found to increase fusion index with no observable synergistic effect. T also increased IGF-1 mRNA generation (P < 0.0001), whereas exogenous IGF-1 (P < 0.001) reduced IGF-1 mRNA transcription relative to control. The T effect was reversible after treatment with flutamide, an androgen receptor antagonist. Both T and IGF-1 increase myogenic commitment after 7-d exposure to a differentiation medium. With T causing a concomitant increase in IGF-1 mRNA underpinning IGF-1 as a central mediator in the cellular pathways associated with muscle hypertrophy, including those affected by androgens. The novel system described has the potential for elucidating the pattern of growth factor effects associated with androgens in skeletal muscle.

Altered expression of microRNA miR-146a correlates with the development of chronic renal inflammation

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that act as post-transcriptional regulators of target mRNA. In this study, we sought to identify the microRNA underlying local inflammation in a murine model of chronic kidney disease (CKD). In microarray analysis of kidneys, the expression of miR-146a/b was elevated in B6.MRLc1 CKD mice that spontaneously develop renal inflammation with age. Primary-microRNA analysis found that elevated miR-146a/b expression in the kidneys of B6.MRLc1 mice was mainly derived from miR-146a rather than miR-146b, and this expression increased with the development of CKD. Histopathological scores for glomerular and interstitial lesions, mRNA expression of inflammatory mediators, and macrophage infiltration were significantly higher in B6.MRLc1 than C57BL/6 mice and were positively correlated with miR-146a expression. In situ hybridization and laser microdissection-RT-PCR showed that miR-146a expression in interstitial lesions containing inflammatory cells was higher than in the glomerulus. The increased expression of the inflammatory-associated genes RELA, IRAK1, IL1B, IL10, and CXCLs was noted in miR-146a/b-silenced human monocytes. The amount of miR-146a was higher in urine sediments of B6.MRLc1 than of C57BL/6 mice. Thus, miR-146a expression in the kidneys and its urinary excretion was specifically associated with the development of interstitial lesions and correlated with inflammatory cell infiltration.

Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 (ENPP1) Protein Regulates Osteoblast Differentiation

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase-1) is an established regulator of tissue mineralization. Previous studies demonstrated that ENPP1 is expressed in differentiated osteoblasts and that ENPP1 influences matrix mineralization by increasing extracellular levels of inorganic pyrophosphate. ENPP1 is also expressed in osteoblastic precursor cells when stimulated with FGF2, but the role of ENPP1 in preosteoblastic and other precursor cells is unknown. Here we investigate the function of ENPP1 in preosteoblasts. We find that ENPP1 expression is critical for osteoblastic differentiation and that this effect is not mediated by changes in extracellular concentration levels of phosphate or pyrophosphate or ENPP1 catalytic activity. MC3T3E1(C4) preosteoblastic cells, in which ENPP1 expression was suppressed by ENPP1-specific shRNA, and calvarial cells isolated from Enpp1 knock-out mice show defective osteoblastic differentiation upon stimulation with ascorbate, as indicated by a lack of cellular morphological change, a lack of osteoblast marker gene expression, and an inability to mineralize matrix. Additionally, MC3T3E1(C4) cells, in which wild type or catalytic inactive ENPP1 expression was increased, exhibited an increased tendency to differentiate, as evidenced by increased osteoblast marker gene expression and increased mineralization. Notably, treatment of cells with inorganic phosphate or pyrophosphate inhibited, as opposed to enhanced, expression of multiple genes that are expressed in association with osteoblast differentiation, matrix deposition, and mineralization. Our results indicate that ENPP1 plays multiple and distinct roles in the development of mineralized tissues and that the influence of ENPP1 on osteoblast differentiation and gene expression may include a mechanism that is independent of its catalytic activity.