Modulation of Mouse Coagulation Gene Transcription following Acute In Vivo Delivery of Synthetic Small Interfering RNAs Targeting HNF4α and C/EBPα

Hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4α and C/EBPα in control of genes encoding coagulation factors, a synthetic small interfering (si)RNA approach was used that enabled strong reduction of mouse hepatic HNF4α and C/EBPα under conditions that minimized target-related secondary effects. For both HNF4α and C/EBPα, intravenous injection of specific synthetic siRNAs (siHNF4α and siC/EBPα) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4α, this coincided with marked and significantly reduced transcript levels of the coagulation genes Hrg, Proz, Serpina5, F11, F12, F13b, Serpinf2, F5, and F9 (in order of magnitude of effect) as compared to levels in control siRNA injected animals. Significant decreases in HNF4α target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4α knockdown at this time point. Compared to HNF4α, C/EBPα knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBPα transcript and protein levels resulted in significantly affected transcript levels of the control genes Pck1 and Fasn and a modest downregulation for coagulation genes Fba, Fbg and F5. F5 and F11 were the sole coagulation genes that were significantly affected upon prolonged (5 day) C/EBPα knockdown. We conclude that in the mouse, HNF4α has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBPα is more restricted. In addition, this study demonstrates that synthetic siRNA provides a simple and fast means for determining liver transcription factor involvement in vivo

Virtual pathway explorer (viPEr) and pathway enrichment analysis tool (PEANuT): creating and analyzing focus networks to identify cross-talk between molecules and pathways

BACKGROUND: Interpreting large-scale studies from microarrays or next-generation sequencing for further experimental testing remains one of the major challenges in quantitative biology. Combining expression with physical or genetic interaction data has already been successfully applied to enhance knowledge from all types of high-throughput studies. Yet, toolboxes for navigating and understanding even small gene or protein networks are poorly developed. RESULTS: We introduce two Cytoscape plug-ins, which support the generation and interpretation of experiment-based interaction networks. The virtual pathway explorer viPEr creates so-called focus networks by joining a list of experimentally determined genes with the interactome of a specific organism. viPEr calculates all paths between two or more user-selected nodes, or explores the neighborhood of a single selected node. Numerical values from expression studies assigned to the nodes serve to score identified paths. The pathway enrichment analysis tool PEANuT annotates networks with pathway information from various sources and calculates enriched pathways between a focus and a background network. Using time series expression data of atorvastatin treated primary hepatocytes from six patients, we demonstrate the handling and applicability of viPEr and PEANuT. Based on our investigations using viPEr and PEANuT, we suggest a role of the FoxA1/A2/A3 transcriptional network in the cellular response to atorvastatin treatment. Moreover, we find an enrichment of metabolic and cancer pathways in the Fox transcriptional network and demonstrate a patient-specific reaction to the drug. CONCLUSIONS: The Cytoscape plug-in viPEr integrates –omics data with interactome data. It supports the interpretation and navigation of large-scale datasets by creating focus networks, facilitating mechanistic predictions from –omics studies. PEANuT provides an up-front method to identify underlying biological principles by calculating enriched pathways in focus networks. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-2017-z) contains supplementary material, which is available to authorized users

Performance of the LHCb outer tracker

The LHCb Outer Tracker is a gaseous detector covering an area of 5 × 6 m2 with 12 double layers of straw tubes. The detector with its services are described together with the commissioning and calibration procedures. Based on data of the first LHC running period from 2010 to 2012, the performance of the readout electronics and the single hit resolution and efficiency are presented. The efficiency to detect a hit in the central half of the straw is estimated to be 99.2%, and the position resolution is determined to be approximately 200 μm. The Outer Tracker received a dose in the hottest region corresponding to 0.12 C/cm, and no signs of gain deterioration or other ageing effects are observed

Endpoints in pediatric pain studies

Assessing pain intensity in (preverbal) children is more difficult than in adults. Tools to measure pain are being used as primary endpoints [e.g., pain intensity, time to first (rescue) analgesia, total analgesic consumption, adverse effects, and long-term effects] in studies on the effects of analgesic drugs. Here, we review current and promising new endpoints used in pediatric pain assessment studies

Clinical characteristics of women captured by extending the definition of severe postpartum haemorrhage with 'refractoriness to treatment': a cohort study

Background: The absence of a uniform and clinically relevant definition of severe postpartum haemorrhage hampers comparative studies and optimization of clinical management. The concept of persistent postpartum haemorrhage, based on refractoriness to initial first-line treatment, was proposed as an alternative to common definitions that are either based on estimations of blood loss or transfused units of packed red blood cells (RBC). We compared characteristics and outcomes of women with severe postpartum haemorrhage captured by these three types of definitions. Methods: In this large retrospective cohort study in 61 hospitals in the Netherlands we included 1391 consecutive women with postpartum haemorrhage who received either ≥4 units of RBC or a multicomponent transfusion. Clinical characteristics and outcomes of women with severe postpartum haemorrhage defined as persistent postpartum haemorrhage were compared to definitions based on estimated blood loss or transfused units of RBC within 24 h following birth. Adverse maternal outcome was a composite of maternal mortality, hysterectomy, arterial embolisation and intensive care unit admission. Results: One thousand two hundred sixty out of 1391 women (90.6%) with postpartum haemorrhage fulfilled the definition of persistent postpartum haemorrhage. The majority, 820/1260 (65.1%), fulfilled this definition within 1 h following birth, compared to 819/1391 (58.7%) applying the definition of ≥1 L blood loss and 37/845 (4.4%) applying the definition of ≥4 units of RBC. The definition persistent postpartum haemorrhage captured 430/471 adverse maternal outcomes (91.3%), compared to 471/471 (100%) for ≥1 L blood loss and 383/471 (81.3%) for ≥4 units of RBC. Persistent postpartum haemorrhage did not capture all adverse outcomes because of missing data on timing of initial, first-line treatment. Conclusion: The definition persistent postpartum haemo

Examination of peripheral blood films using automated microscopy; evaluation of Diffmaster Octavia and Cellavision DM96

BACKGROUND: Differential counting of peripheral blood cells is an important diagnostic tool. Yet, this technique requires highly trained staff, is labour intensive and has limited statistical reliability. A recent development in this field was the introduction of automated peripheral blood differential counting systems. These computerised systems provide an automated morphological analysis of peripheral blood films, including a preclassification of both red and white cells (RBCs and WBCs, respectively). AIMS: To investigate the ability of two automated microscopy systems to examine peripheral blood smears. METHODS: Two automated microscopy systems, the Cellavision Diffmaster Octavia (Octavia) and Cellavision DM96 (DM96), were evaluated. RESULTS: The overall preclassification accuracy values for the Octavia and the DM96 systems were 87% and 92%, respectively. Evaluation of accuracy (WBC analysis) showed good correlation for both automated systems when compared with manual differentiation. Total analysis time (including post classification) was 5.4 min/slide for the Octavia and 3.2 min/slide for the DM96 (100 WBC/slide) system. The DM96 required even less time than manual differentiation by an experienced biomedical scientist. CONCLUSIONS: The Octavia and the DM96 are automated cell analysis systems capable of morphological classification of RBCs and WBCs in peripheral blood smears. Classification accuracy depends on the type of pathological changes in the blood sample. Both systems operate most effectively in the analysis of non‐pathological blood samples