BackgroundAn adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the underlying epigenetic signatures involved in its regulation.ResultsNOS3 and STAT3 mRNA levels were elevated in HUAEC of patients who suffered from placental insufficiency. 5-hydroxymethylcytosine, H3K9ac and Histone 2A (H2A).Zac at the NOS3 transcription start site directly correlated with NOS3 mRNA levels. Concomitantly, we observed entangled histone acetylation patterns and NOS3 response upon hypoxic conditions in vitro. Knock-down of either NOS3 or STAT3 by RNAi provided evidence for a functional NOS3/STAT3 relationship. Moreover, we recognized massive turnover of Stat3 at a discrete binding site in the NOS3 promoter. Interestingly, induced hyperacetylation resulted in short-termed increase of NOS3 mRNA followed by deferred decrease indicating that NOS3 expression could become self-attenuated by co-expressed intronic 27 nt-ncRNA. Reporter assay results and phylogenetic analyses enabled us to propose a novel model for STAT3-3′-UTR targeting by this 27-nt-ncRNA.ConclusionsAn adverse intrauterine environment leads to adaptive changes of NOS3 expression. Apparently, a rapid NOS3 self-limiting response upon ectopic triggers co-exists with longer termed expression changes in response to placental insufficiency involving differential epigenetic signatures. Their persistence might contribute to impaired vascular endothelial response and consequently increase the risk of cardiovascular disease later in life.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0042-4) contains supplementary material, which is available to authorized users.

BackgroundEpigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore desirable for all epigenetic studies. However, to date little information is available on whether separation protocols might influence epigenetic and/or gene expression signatures and hence might be less beneficial. We investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (IECs) from 6 healthy individuals.Materials and MethodsEpithelial cells were isolated from small bowel (i.e. terminal ileum) biopsies using EDTA/DTT and enzymatic release followed by magnetic bead sorting via EPCAM labeled microbeads. Effects on gene/mRNA expression were analyzed using a real time PCR based expression array. DNA methylation was assessed by pyrosequencing of bisulfite converted DNA and methylated DNA immunoprecipitation (MeDIP).ResultsWhile cell purity was >95% using both cell separation approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable and only revealed subtle differences. Comparison of DNA methylation of the epithelial cell marker EPCAM in unseparated whole biopsy samples with separated epithelium (i.e. EPCAM positive and negative fraction) demonstrated significant differences in DNA methylation between all three tissue fractions indicating cell type specific methylation patterns can be masked in unseparated tissue samples.ConclusionsTaken together, our data highlight the importance of considering the potential effect of cell separation on gene expression as well as DNA methylation signatures. The decision to separate tissue samples will therefore depend on study design and specific separation protocols.

Since the emergence of viral resistance of hepatitis B virus (HBV) during treatment is becoming an important issue even with newer drugs, there is a need for alternative treatment options such as, for example, RNA interference (RNAi) technology. While short-term suppression of HBV replication is easily achieved with small interfering RNA oligonucleotides, this is not the case for long-term suppression due to the lack of an optimal vector system. Based on the nonviral scaffold/matrix attachment region (S/MAR)-based vector system pEPI-1, which is free of common side effects and is stably retained as an episome even in the absence of selection, we designed a short hairpin RNA (shRNA) expression vector called pEPI-RNAi for HBV suppression. HBV-replicating HepG2.2.15 cells were transfected with pEPI-RNAi, and the intracellular status of the plasmid was followed by PCR and Southern analysis. HBV replication was measured on the DNA, RNA, and protein level. HBV RNA expression was reduced by almost 85% 3 months posttransfection with pEPI-RNAi. At 8 months posttransfection in the absence of antibiotic selection pressure, the suppression level was still 70% and the vector was retained as an episome. The reduction of total intracellular HBV DNA at this point was 77%, showing a marked suppression of HBV DNA replication. At a comparable level, secretion of viral antigens, as well as progeny HBV virions, was inhibited. The S/MAR-based vector system pEPI-1 allows long-term suppression of HBV replication by the expression of suitable shRNAs. Due to its unique properties compared to commonly used vectors, it provides an interesting option for the treatment of chronically HBV-infected individuals.

BackgroundIs the patient really suffering from acute appendicitis? Right lower quadrant pain is the most common sign of acute appendicitis. However, many other bowels pathologies might mimic acute appendicitis. Due to fear of the consequences of delayed or missed diagnosis, the indication for emergency appendectomy is liberally made. This has been shown to be associated with high rates of negative appendectomy with risk of potentially serious or lethal complications. Thus there is need for a better preoperative screening of patients with suspected appendicitis.MethodsThis prospective single center single-blinded pilot study was conducted in the Department of surgery at the HELIOS Universitätsklinikum Wuppertal, Germany. Calprotectin was measured in pre-therapeutic stool samples of patients presenting in the emergency department with pain to the right lower quadrant. Fecal calprotectin (FC) values were analyzed using commercially available ELISA kits. Cut-off values for FC were studied using the receiver-operator characteristic (ROC) curve. The Area under the curve (AUC) was reported for each ROC curve.ResultsThe mean FC value was 51.4 ± 118.8 μg/g in patients with AA, 320.9 ± 416.6 μg/g in patients with infectious enteritis and 24.8 ± 27.4 μg/g in the control group. ROC curve showed a close to 80% specificity and sensitivity of FC for AA at a cut-off value of 51 μg/g, AUC = 0.7. The sensitivity of FC at this cut-off value is zero for enteritis with a specificity of 35%.ConclusionFecal calprotectin could be helpful in screening patients with pain to the right lower quadrant for the presence of acute appendicitis or infectious enteritis with the aim of facilitating clinical decision-making and reducing the rate of negative appendectomy.