an accurate pipeline for analysis of ngs data of small non coding rna

Motivations. The discovery of various families of small non-coding RNAs (sncRNAs) in recent years revealed the complexity of the regulation of gene expression at both transcriptional and post-transcriptional level. Of the numerous sncRNAs, microRNAs (miRNAs) constitute a large family of 19-23 nucleotides long RNAs that participate in a variety of processes, such as cell development and differentiation, apoptosis and stress responses to carcinogenesis. Computational analysis indicates that a unique miRNA can regulate hundreds of genes, underlining the potential influence of miRNAs in almost every cellular pathway. Deep sequencing technologies provides a powerful strategy to explore miRNA populations (miRNA-Seq) with high sensitivity and specificity. Different computational approaches have been developed to analyze miRNA-Seq data, allowing to identify known and novel miRNAs, perform differential expression analysis and predict putative miRNAs targets. We combined these algorithms into an analysis pipeline and tested it on data obtained from our experiments in cancer cell lines. Methods. The data obtained from the sequencer were filtered following several criteria. Since the sequence of the adapter is known, a Perl script was used to trim, from the raw data, the adaptors. The sequence reads were then filtered for quality and clustered to unique sequences to remove redundancy, retaining their individual read count information. Unique sequences 18 nucleotides or more in length were mapped, allowing up to one mismatch, on miRNA annotation according to miRBase version 18 using miRanalyzer. This detects the reads which correspond to known miRNAs, giving an estimation of expression level. miRBase repository is used because it offers information about mature (the mature sequence of known miRNAs), mature-star (the sequence which pairs with the mature miRNA in the miRNA secondary structure) and precursor miRNA sequences (sequence of the hairpin). miRNAs have been considered as expressed if they are detected at least 5 reads/sample. After detecting those that correspond to known miRNAs, the remaining reads were mapped to databases of transcribed sequences as mRNA and non-coding RNA (RFam). This step has two goals: (i) the number of matches can be viewed as a sample quality parameter (contamination of the RNA sample with degradation products and poly A tails) and (ii) it might be interesting to see which other known sncRNAs are in the sample. To predict novel miRNAs we used a probabilistic algorithm, miRDeep2, based on miRNA biogenesis model, to score compatibility of the position and frequency of sequenced RNA with the secondary structure of the miRNA precursor. This tool aligns sequencing reads to potential hairpin structures in a manner consistent with Dicer processing and assigns log-odds scores to measure the probability that hairpins are true miRNA precursors. To detect novel miRNAs by miRDeep2, a score cutoff corresponding to a prediction signal-to-noise ratio >10 was used. Identification of differentially expressed miRNAs was performed with the Bioconductor DESeq package. Starting from the expression values, the first step was to minimize the effect of the systematic technical variations, and then a negative binomial distribution model was used to test differential expression in deep sequencing datasets. Only miRNAs with a p-values less or equal to 0.05 and fold-change less or equal to -1.5 and greater or equal to 1.5 were considered as differentially expressed. Given the critical roles of miRNAs in regulating gene expression and cellular functions, we predicted their putative targets, intersecting results obtained from two resources, TargetScan and microRNA.org. TargetScan provide computationally predicted miRNA gene targets by searching for the presence of 8 and 7 mer sites that match the seed region of each miRNA, while microRNA.org target prediction incorporates current knowledge on target rules and on the use of a compendium of mammalian miRNAs. A further step of the analysis was to investigate nucleotide variations relative to the reference genome. To this purpose, preliminary steps were to reduce alignment artifacts and compute a more accurate quality estimation, removing biases due to sequencing cycle and preceding nucleotide. Further evidences were used to identify new miRNA variation sites: (i) Sequencing depth of variation sites should be equal to or larger than 5 reads per site, (ii) frequency of Single Nucleotide Variant occurrence >5% and (iii) variants not found in previous SNP annotation databases, like dbSNP. Results. We developed an accurate pipeline for integral analysis of next generation sequencing data of small RNA molecules. Based on solid statistical methods, this allows both detection of known miRNAs and prediction of new miRNAs, integrating steps for differential analysis, sequence analysis and target prediction. Acknowledgements Research support by: Fondazione con il Sud; Italian Association for Cancer Research; Italian Ministry for Education, University and Research; Regione Campania; University of Salerno; Fondazione Veronesi. Giorgio Giurato is a student of PhD School in Experimental and Clinic Medicine / Doctorate in Experimental Physiopathology and Neuroscience, Second University of Naples. Maria Ravo is supported by a 'Vladimir Ashkenazy' fellowship of Italian Association for Cancer Research. Concita Cantarella and Giovanni Nassa are fellows of Fondazione con il Sud

Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer.

Estrogen effects on mammary epithelial and breast cancer (BC) cells are mediated by the nuclear receptors ERα and ERβ, transcription factors that display functional antagonism with each other, with ERβ acting as oncosuppressor and interfering with the effects of ERα on cell proliferation, tumor promotion and progression. Indeed, hormone-responsive, ERα+ BC cells often lack ERβ, which when present associates with a less aggressive clinical phenotype of the disease. Recent evidences point to a significant role of microRNAs (miRNAs) in BC, where specific miRNA expression profiles associate with distinct clinical and biological phenotypes of the lesion. Considering the possibility that ERβ might influence BC cell behavior via miRNAs, we compared miRNome expression in ERβ+ vs ERβ- hormone-responsive BC cells and found a widespread effect of this ER subtype on the expression pattern of these non-coding RNAs. More importantly, the expression pattern of 67 miRNAs, including 10 regulated by ERβ in BC cells, clearly distinguishes ERβ+, node-negative, from ERβ-, metastatic, mammary tumors. Molecular dissection of miRNA biogenesis revealed multiple mechanisms for direct regulation of this process by ERβ+ in BC cell nuclei. In particular, ERβ downregulates miR-30a by binding to two specific sites proximal to the gene and thereby inhibiting pri-miR synthesis. On the other hand, the receptor promotes miR-23b, -27b and 24-1 accumulation in the cell by binding in close proximity of the corresponding gene cluster and preventing in situ the inhibitory effects of ERα on pri-miR maturation by the p68/DDX5-Drosha microprocessor complex. These results indicate that cell autonomous regulation of miRNA expression is part of the mechanism of action of ERβ in BC cells and could contribute to establishment or maintenance of a less aggressive tumor phenotype mediated by this nuclear receptor

Effects of Oestrogen on MicroRNA Expression in Hormone-Responsive Breast Cancer Cells

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study

Estimating gas accretion in disc galaxies using the Kennicutt-Schmidt law

We show how the existence of a relation between the star formation rate and the gas density, i.e. the Kennicutt-Schmidt law, implies a continuous accretion of fresh gas from the environment into the discs of spiral galaxies. We present a method to derive the gas infall rate in a galaxy disc as a function of time and radius, and we apply it to the disc of the Milky Way and 21 galaxies from the THINGS sample. For the Milky Way, we found that the ratio between the past and current star formation rates is about 2-3, averaged over the disc, but it varies substantially with radius. In the other disc galaxies there is a clear dependency of this ratio with galaxy stellar mass and Hubble type, with more constant star formation histories for small galaxies of later type. The gas accretion rate follows very closely the SFR for every galaxy and it dominates the evolution of these systems. The Milky Way has formed two thirds of its stars after z=1, whilst the mass of cold gas in the disc has remained fairly constant with time. In general, all discs have accreted a significant fraction of their gas after z=1. Accretion moves from the inner regions of the disc to the outer parts, and as a consequence star formation moves inside-out as well. At z=0 the peak of gas accretion in the Galaxy is at about 6-7 kpc from the centre.Comment: 14 pages, 10 figures, accepted for publication in MNRA

Expert consensus document: A 'diamond' approach to personalized treatment of angina.

In clinical guidelines, drugs for symptomatic angina are classified as being first choice (β-blockers, calcium-channel blockers, short-acting nitrates) or second choice (ivabradine, nicorandil, ranolazine, trimetazidine), with the recommendation to reserve second-choice medications for patients who have contraindications to first-choice agents, do not tolerate them, or remain symptomatic. No direct comparisons between first-choice and second-choice treatments have demonstrated the superiority of one group of drugs over the other. Meta-analyses show that all antianginal drugs have similar efficacy in reducing symptoms, but provide no evidence for improvement in survival. The newer, second-choice drugs have more evidence-based clinical data that are more contemporary than is available for traditional first-choice drugs. Considering some drugs, but not others, to be first choice is, therefore, difficult. Moreover, double or triple therapy is often needed to control angina. Patients with angina can have several comorbidities, and symptoms can result from various underlying pathophysiologies. Some agents, in addition to having antianginal effects, have properties that could be useful depending on the comorbidities present and the mechanisms of angina, but the guidelines do not provide recommendations on the optimal combinations of drugs. In this Consensus Statement, we propose an individualized approach to angina treatment, which takes into consideration the patient, their comorbidities, and the underlying mechanism of disease

Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation

Background: Estrogen receptors alpha (ERa) and beta (ERb) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERb being able to modulate the effects of ERa on gene transcription and cell proliferation. ERb is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERb in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. Results: Expression of full-length ERb in hormone-responsive, ERa-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERb and 6024 ERa binding sites in estrogen-stimulated cells, comprising sites occupied by either ERb, ERa or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERb+ vs ERb- cells, 424 showed one or more ERb site within 10 kb. These putative primary ERb target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERb binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. Conclusions: Results indicate that the vast majority of the genomic targets of ERb can bind also ERa, suggesting that the overall action of ERb on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell

Ginkgolic Acid Protects against Aβ-Induced Synaptic Dysfunction in the Hippocampus

Ginkgo leaf is the most used form of supplement for cognitive ailments. The standardized extract formulation EGb 761 is a dietary supplement with proven benefit in several neurological and psychiatric conditions including memory decline in Alzheimer's disease, schizophrenia and dementia. Ginkgolic acid (GA) is a component of this extract which shows pleiotropic effects including antitumoral and anti-HIV action; however, its effect on memory is still unknown. Here, we carried out an electrophysiological analysis to investigate the effects of GA on long term potentiation and synaptic transmission at CA1 hippocampal synapses. We also evaluated the potential rescuing effect of GA on the synaptic dysfunction following in vitro application of Aβ. Data obtained indicate that GA exerts neuroprotective effects against Aβ-induced impairment of neurotransmitter release and synaptic plasticity

A case of shared psychotic disorder (folie à deux) with original aspects associated with cross-cultural elements

Shared psychotic disorder (folie à deux) is a rare condition characterized by the transmission of delusional aspects from a patient (the "dominant partner") to another (the "submissive partner") linked to the first by a close relationship. We report the case of two Moroccan sisters who have experienced a combined delusional episode diagnosed as shared psychotic disorder. In these circumstances, assessment of symptoms from a cross-cultural perspective is a key factor for proper diagnostic evaluation

Distinct Nongenomic Signal Transduction Pathways Controlled by 17b-Estradiol Regulate DNA Synthesis and Cyclin D1 Gene Transcription in HepG2 Cells

Estrogens induce cell proliferation in target tissues by stimulating progression through the G1 phase of the cell cycle. Activation of cyclin D1 gene expression is a critical feature of this hormonal action. The existence of rapid/nongenomic estradiol-regulated protein kinase C (PKC-a) and extracellular signal-regulated kinase (ERK) signal transduction pathways, their cross talk, and role played in DNA synthesis and cyclin D1 gene transcription have been studied herein in human hepatoma HepG2 cells. 17b-Estradiol was found to rapidly activate PKC-a translocation and ERK-2/mitogen-activated protein kinase phosphorylation in this cell line. These actions were independent of each other, preceding the increase of thymidine incorporation into DNA and cyclin D1 expression, and did not involve DNA binding by estrogen receptor. The results obtained with specific inhibitors indicated that PKC-a pathway is necessary to mediate the estradiol-induced Gl-S progression of HepG2 cells, but it does not exert any effect(s) on cyclin D1 gene expression. On the contrary, ERK-2 cascade was strongly involved in both Gl-S progression and cyclin D1, gene transcription. Deletion of its activating protein-1 responsive element motif resulted in attenuation of cyclin D1 promoter responsiveness to estrogen. These results indicate that estrogen-induced cyclin D1 transcription can occuir in HepG2 cells independently of the transcriptional activity of estrogen receptor, sustaining the pivotal role played by nongenomic pathways of estrogen action in hormone-induced proliferation