Effect of growth-promoting 17ß estradiol, 19 nortestosterone and dexamethasone on circulating levels of nine potential biomarker candidates in veal calves

The use of screening methods based on the detection of biological effects of growth promoters is a promising approach to assist residue monitoring. To reveal useful effects on protein metabolism, male and female veal calves at 10 weeks of age were treated thrice with a combination of 25 mg 17ß-estradiol 3-benzoate and 150 mg 19-nortestosterone decanoate with 2 weeks intervals and finally once with 4 mg dexamethasone. Hormone-treated calves showed a significant accelerated growth rate over 6 weeks. Plasma samples of treated and control calves were analysed for immunoreactive inhibin (ir-inhibin), osteocalcin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 2 (IGFBP-2), IGFBP-3, luteinzing hormone (LH), follicle-stimulating hormone (FSH) and prolactin using immunoaffinity assays. Hormone treatment did not affect levels of IGF-1, IGFBP-2, IGFBP-3, LH, FSH and prolactin. The concentration of circulating ir-inhibin decreased, however, significantly (P < 0.05) in bull calves upon administration of the sex steroids, whereas it remained unchanged in the female animals. Dexamethasone treatment decreased significantly (P < 0.05) circulating levels of osteocalcin in both female and male animals. Ir-inhibin and osteocalcin were, therefore, considered as candidates for a protein biomarker-based screening assay for detection of abuse of estrogens, androgens and/or glucocorticoids in cattle fattening, which is being developed in the framework of EU research project BioCop

MicroRNA 203 Expression in Keratinocytes Is Dependent on Regulation of p53 Levels by E6.

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

Meal-induced 24-hour profile of circulating glycated insulin in type 2 diabetic subjects measured by a novel radioimmunoassay

Increasing evidence supports a role for glycated insulin in the insulin-resistant state of type 2 diabetes. We measured 24-hour profiles of plasma glycated insulin, using a novel radioimmunoassay (RIA), to evaluate the effects of meal stimulation and intermittent fasting on circulating concentrations of plasma glycated insulin in type 2 diabetes. Patients (n = 6; hemoglobin A(1c) [HbA(1c)], 7.2% +/- 0.6%; fasting plasma glucose, 7.4 +/- 0.7 mmol/L; body mass index [BMI], 35.7 +/- 3.5 kg/m(2); age, 56.3 +/- 4.4 years) were admitted for 24 hours and received a standardized meal regimen. Half-hourly venous samples were taken for plasma glycated insulin, glucose, insulin, and C-peptide concentrations between 8 Am and midnight and 2-hourly overnight. The mean plasma glycated insulin concentration over 24 hours was 27.8 +/- 1.2 pmol/L with a mean ratio of insulin:glycated insulin of 11:1. Circulating glucose, insulin, C-peptide, and glycated insulin followed a basal and meal-related pattern with most prominent increments following breakfast, lunch, and evening meal, respectively. The mean concentrations of glycated insulin during the morning, afternoon, evening, and night-time periods were 24.4 +/- 2.5, 28.7 +/- 2.3, 31.1 +/- 2.1, and 26.2 +/- 1.5 pmol/L, respectively, giving significantly higher molar ratios of insulin:glycated insulin of 18.0:1, 14.2:1, and 12.7:1 compared with 7.01 at night (P

Ranking of microRNA target prediction scores by Pareto front analysis

Over the past ten years, a variety of microRNA target prediction methods has been developed, and many of the methods are constantly improved and adapted to recent insights into miRNA-mRNA interactions. In a typical scenario, different methods return different rankings of putative targets, even if the ranking is reduced to selected mRNAs that are related to a specific disease or cell type. For the experimental validation it is then difficult to decide in which order to process the predicted miRNA-mRNA bindings, since each validation is a laborious task and therefore only a limited number of mRNAs can be analysed. We propose a new ranking scheme that combines ranked predictions from several methods and - unlike standard thresholding methods - utilises the concept of Pareto fronts as defined in multi-objective optimisation. In the present study, we attempt a proof of concept by applying the new ranking scheme to hsa-miR-21, hsa-miR-125b, and hsa-miR-373 and prediction scores supplied by PITA and RNAhybrid. The scores are interpreted as a two-objective optimisation problem, and the elements of the Pareto front are ranked by the STarMir score with a subsequent re-calculation of the Pareto front after removal of the top-ranked mRNA from the basic set of prediction scores. The method is evaluated on validated targets of the three miRNA, and the ranking is compared to scores from DIANA-microT and TargetScan. We observed that the new ranking method performs well and consistent, and the first validated targets are elements of Pareto fronts at a relatively early stage of the recurrent procedure. which encourages further research towards a higher-dimensional analysis of Pareto fronts. (C) 2010 Elsevier Ltd. All rights reserved.

MicroRNA-130b regulates the tumour suppressor RUNX3 in gastric cancer

Aim: Accumulating evidence indicates that RUNX3 is an important tumour suppressor that is inactivated in many cancer types. This study aimed to assess the role of microRNA (miRNA) in the regulation of RUNX3.

MicroRNA regulation of core apoptosis pathways in cancer

Recent research has demonstrated that microRNAs (miRNAs) are key regulators of many cell processes often deregulated in cancer, including apoptosis. Indeed, it is becoming clear that many miRNAs are anti-apoptotic and mediate this effect by targeting pro-apoptotic mRNAs or positive regulators of pro-apoptotic mRNAs. Conversely, many pro-apoptotic miRNAs target anti-apoptotic mRNAs or their positive regulators. We have reviewed the current knowledge in this area including evidence of miRNA involvement in cancer drug resistance. (C) 2010 Elsevier Ltd. All rights reserved.

MicroRNA-155 Promotes Resolution of Hypoxia-Inducible Factor 1{alpha} Activity

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1a (but not HIF-2a) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1a mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1a in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1a mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1a stabilization and activity. Based on these data and a mathematical model of HIF-1a suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1a activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1a-dependent transcription.

Effect of atorvastatin on circulating hsCRP concentrations: A sub-study of the Achieve Cholesterol Targets Fast with Atorvastatin Stratified Titration (ACTFAST) study

Background: Elevated C-reactive protein (CRP) concentration is a risk factor for cardiovascular events that may add prognostic information. Statin treatment is associated with significant reductions in CRP concentrations, which appear to be unrelated to the magnitude of LDL-cholesterol reduction. We investigated the effect of atorvastatin, across its dose range, on high sensitivity (hs)CRP in subjects at high cardiovascular risk. Methods: ACTFAST was a 12 week, prospective, multicenter, open-label trial in which high-risk subjects were assigned a starting dose of atorvastatin (10, 20, 40 or 80 mg/d) based on LDL-C and status of statin use at screening (1345 statin-free [ SF] and 772 previously statin-treated [ST]). Results: At baseline, ST subjects had significantly lower hsCRP levels than SF subjects (ST group 2.31, 95% CI 2.15, 2.48 mg/L vs. SF group 3.16, 95% CI 2.98, 3.34 mg/L, p

Circulating and synovial antibody profiling of juvenile arthritis patients by nucleic acid programmable protein arrays.

Introduction Juvenile idiopathic arthritis (JIA) is a heterogeneous disease characterized by chronic joint inflammation of unknown cause in children. JIA is an autoimmune disease and small numbers of auto-antibodies have been reported in JIA patients. The identification of antibody markers could improve the existing clinical management of patients. Methods A pilot study was performed on the application of a high-throughput platform, nucleic acid programmable protein arrays (NAPPA), to assess the levels of antibodies present in the systemic circulation and synovial joint of a small cohort of juvenile arthritis patients. Plasma and synovial fluid from ten JIA patients was screened for antibodies against 768 proteins on NAPPA. Results Quantitative reproducibility of NAPPA was demonstrated with >0.95 intra- and inter- array correlations. A strong correlation was also observed for the levels of antibodies between plasma and synovial fluid across the study cohort (r=0.96). Differences in the levels of 18 antibodies were revealed between sample types across all patients. Patients were segregated into two clinical subtypes with distinct antibody signatures by unsupervised hierarchical cluster analysis. Conclusions NAPPA provides a high-throughput quantitatively reproducible platform to screen for disease specific autoantibodies at the proteome level on a microscope slide. The strong correlation between the circulating antibody levels and those of the inflamed joint represents a novel finding and provides confidence to use plasma for discovery of autoantibodies in JIA, thus circumventing the challenges associated with joint aspiration. We expect that autoantibody profiling of JIA patients on NAPPA could yield antibody markers that can act as criteria to stratify patients, predict outcomes and understand disease etiology at the molecular level.