Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

High-Throughput MicroRNA (miRNAs) Arrays Unravel the Prognostic Role of MiR-211 in Pancreatic Cancer

BACKGROUND: Only a subset of radically resected pancreatic ductal adenocarcinoma (PDAC) patients benefit from chemotherapy, and identification of prognostic factors is warranted. Recently miRNAs emerged as diagnostic biomarkers and innovative therapeutic targets, while high-throughput arrays are opening new opportunities to evaluate whether they can predict clinical outcome. The present study evaluated whether comprehensive miRNA expression profiling correlated with overall survival (OS) in resected PDAC patients. METHODOLOGY/PRINCIPAL FINDINGS: High-resolution miRNA profiles were obtained with the Toray's 3D-Gene™-miRNA-chip, detecting more than 1200 human miRNAs. RNA was successfully isolated from paraffin-embedded primary tumors of 19 out of 26 stage-pT3N1 homogeneously treated patients (adjuvant gemcitabine 1000 mg/m(2)/day, days-1/8/15, every 28 days), carefully selected according to their outcome (OS<12 (N = 13) vs. OS>30 months (N = 6), i.e. short/long-OS). Highly stringent statistics included t-test, distance matrix with Spearman-ranked correlation, and iterative approaches. Unsupervised hierarchical analysis revealed that PDACs clustered according to their short/long-OS classification, while the feature selection algorithm RELIEF identified the top 4 discriminating miRNAs between the two groups. These miRNAs target more than 1500 transcripts, including 169 targeted by two or more. MiR-211 emerged as the best discriminating miRNA, with significantly higher expression in long- vs. short-OS patients. The expression of this miRNA was subsequently assessed by quantitative-PCR in an independent cohort of laser-microdissected PDACs from 60 resected patients treated with the same gemcitabine regimen. Patients with low miR-211 expression according to median value had a significantly shorter median OS (14.8, 95%CI = 13.1-16.5, vs. 25.7 months, 95%CI = 16.2-35.1, log-rank-P = 0.004). Multivariate analysis demonstrated that low miR-211 expression was an independent factor of poor prognosis (hazard ratio 2.3, P = 0.03) after adjusting for all the factors influencing outcome. CONCLUSIONS/SIGNIFICANCE: Through comprehensive microarray analysis and PCR validation we identified miR-211 as a prognostic factor in resected PDAC. These results prompt further prospective studies and research on the biological role of miR-211 in PDAC

Factors associated with OS and DFS in the multivariate analysis.

<p>Abbreviations: df, degrees of freedom; DFS, Disease Free Survival; OS, Overall Survival.</p

Heatmap of the clustering of 170 miRs filtered based on t-test p-value<0.05.

<p>In order to perform hierarchical cluster analysis we constructed a distance matrix using the two-tailed Spearman correlation test, due to the non-normal distribution of expression within samples. The cluster analysis shows a good separation between the two groups of samples, based on the significantly different miRNAs. The microRNA expression data were centered by 2 directions (i.e., by miRNA and patients). Red and yellow represent low and high miRNA expression, respectively.</p

Top-10 most discriminative miRNAs, based on RELIEF score.

<p>Top-10 most discriminative miRNAs, based on RELIEF score.</p

Kaplan-Meier curves for OS and DFS according to (A–B) miR-211 above and below median expression (e.g., low vs. high expression), and to (C–D) k-means clustering.

<p>Kaplan-Meier curves for OS and DFS according to (A–B) miR-211 above and below median expression (e.g., low vs. high expression), and to (C–D) k-means clustering.</p

<i>In vitro</i> studies validating the role of miR-211 in gemcitabine chemosensitivity.

<p>(A) MiR-211 expression in 9 PDAC cell lines and 5 primary tumor cultures. Expression was determined by quantitative PCR, using RNU6 as reference, and the values are in a.u. (B) MiR-211 expression in MIA PaCa-2 and LPc028 cells transfected with negative controls, pre-miR-21 or anti-miR-21 oligos. MiR-211 expression was calculated with the 2^−ΔΔCT method with respect to the expression in cells transfected with negative miRNA controls, set as 1, as represented by the dotted line (C) Gemcitabine antiproliferative effects in MIA PaCa-2 and LPc028 cells transfected with miRNA negative controls, pre-miR-21 or anti-miR-21 oligos. *Significantly different from control transfected cells, which were not treated with gemcitabine, set at 100% (P<0.05). (D) RRM2 mRNA expression in MIA PaCa-2 and LPc028 cells transfected with negative controls, pre-miR-21 or anti-miR-21 oligos. RRM2 expression was calculated with the 2^−ΔΔCT method with respect to the expression in cells transfected with miRNA negative controls, set as 1, as represented by the dotted line. Columns, mean values obtained from three independent experiments; bars, SE. *Significantly different from control (P<0.05).</p

Mutations in DDHD2, Encoding an Intracellular Phospholipase A(1), Cause a Recessive Form of Complex Hereditary Spastic Paraplegia

Contains fulltext : 108770.pdf (publisher's version ) (Closed access)We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A(1) (iPLA(1)). The core phenotype of this HSP syndrome consists of very early-onset (<2 years) spastic paraplegia, intellectual disability, and a specific pattern of brain abnormalities on cerebral imaging. An essential role for DDHD2 in the human CNS, and perhaps more specifically in synaptic functioning, is supported by a reduced number of active zones at synaptic terminals in Ddhd-knockdown Drosophila models. All identified mutations affect the protein's DDHD domain, which is vital for its phospholipase activity. In line with the function of DDHD2 in lipid metabolism and its role in the CNS, an abnormal lipid peak indicating accumulation of lipids was detected with cerebral magnetic resonance spectroscopy, which provides an applicable diagnostic biomarker that can distinguish the DDHD2 phenotype from other complex HSP phenotypes. We show that mutations in DDHD2 cause a specific complex HSP subtype (SPG54), thereby linking a member of the PLA(1) family to human neurologic disease

Perspectives on ENCODE

The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community.11Nsciescopu

Expanded encyclopaedias of DNA elements in the human and mouse genomes

AbstractThe human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.11Nsciescopu