NGS TREX: next generation sequences transcriptome profile explorer

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NGS-Trex : next generation sequencing transcriptome profile explorer

Background: Next-Generation Sequencing (NGS) technology has exceptionally increased the ability to sequence DNA in a massively parallel and cost-effective manner. Nevertheless, NGS data analysis requires bioinformatics skills and computational resources well beyond the possibilities of many "wet biology" laboratories. Moreover, most of projects only require few sequencing cycles and standard tools or workflows to carry out suitable analyses for the identification and annotation of genes, transcripts and splice variants found in the biological samples under investigation. These projects can take benefits from the availability of easy to use systems to automatically analyse sequences and to mine data without the preventive need of strong bioinformatics background and hardware infrastructure.Results: To address this issue we developed an automatic system targeted to the analysis of NGS data obtained from large-scale transcriptome studies. This system, we named NGS-Trex (NGS Transcriptome profile explorer) is available through a simple web interface http://www.ngs-trex.org and allows the user to upload raw sequences and easily obtain an accurate characterization of the transcriptome profile after the setting of few parameters required to tune the analysis procedure. The system is also able to assess differential expression at both gene and transcript level (i.e. splicing isoforms) by comparing the expression profile of different samples.By using simple query forms the user can obtain list of genes, transcripts, splice sites ranked and filtered according to several criteria. Data can be viewed as tables, text files or through a simple genome browser which helps the visual inspection of the data.Conclusions: NGS-Trex is a simple tool for RNA-Seq data analysis mainly targeted to "wet biology" researchers with limited bioinformatics skills. It offers simple data mining tools to explore transcriptome profiles of samples investigated taking advantage of NGS technologies. \ua9 2013 Mignone et al.; licensee BioMed Central Ltd

Identification of novel proteins binding the AU-rich element of \u3b1-prothymosin mRNA through the selection of open reading frames (RIDome)

We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of \u3b1-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome

A Novel Technique for Mitigating Multipactor by Means of Magnetic Surface Roughness

Multipactor phenomena which are closely linked to the SEY (secondary electron yield)can be mitigated by many different methods including groves in the metal surface as well as using electric or magnetic bias fields. However frequently the application of global magnetic or electric bias field is not practicable considering the weight and power limitations on-board satellites. Additionally, surface grooves may degrade the RF performance. Here we present a novel technique which is based on a magnetostatic field pattern on the metallic surface with fast spatial modulation in the order of 30 micron. This field pattern is produced by proper magnetization of an underlying ferromagnetic layer such as nickel. Simulations and preliminary experimental results will be shown and a number of applications, both for particle accelerators and satellite microwave payloads are discussed

On the probabilistic min spanning tree Problem

We study a probabilistic optimization model for min spanning tree, where any vertex vi of the input-graph G(V,E) has some presence probability pi in the final instance G′ ⊂ G that will effectively be optimized. Suppose that when this “real” instance G′ becomes known, a spanning tree T, called anticipatory or a priori spanning tree, has already been computed in G and one can run a quick algorithm (quicker than one that recomputes from scratch), called modification strategy, that modifies the anticipatory tree T in order to fit G ′. The goal is to compute an anticipatory spanning tree of G such that, its modification for any G ′ ⊆ G is optimal for G ′. This is what we call probabilistic min spanning tree problem. In this paper we study complexity and approximation of probabilistic min spanning tree in complete graphs under two distinct modification strategies leading to different complexity results for the problem. For the first of the strategies developed, we also study two natural subproblems of probabilistic min spanning tree, namely, the probabilistic metric min spanning tree and the probabilistic min spanning tree 1,2 that deal with metric complete graphs and complete graphs with edge-weights either 1, or 2, respectively

5′UTR Variants of Ribosomal Protein S19 Transcript Determine Translational Efficiency: Implications for Diamond-Blackfan Anemia and Tissue Variability

Background: Diamond-Blackfan anemia (DBA) is a lineage specific and congenital erythroblastopenia. The disease is associated with mutations in genes encoding ribosomal proteins resulting in perturbed ribosomal subunit biosynthesis. The RPS19 gene is mutated in approximately 25 % of DBA patients and a variety of coding mutations have been described, all presumably leading to haploinsufficiency. A subset of patients carries rare polymorphic sequence variants within the 59untranslated region (59UTR) of RPS19. The functional significance of these variants remains unclear. Methodology/Principal Findings: We analyzed the distribution of transcriptional start sites (TSS) for RPS19 mRNAs in testis and K562 cells. Twenty-nine novel RPS19 transcripts were identified with different 59UTR length. Quantification of expressed w.t. 59UTR variants revealed that a short 59UTR correlates with high levels of RPS19. The total levels of RPS19 transcripts showed a broad variation between tissues. We also expressed three polymorphic RPS19 59UTR variants identified in DBA patients. The sequence variants include two insertions (c.-147_-146insGCCA and c.-147_-146insAGCC) and one deletion (c.-144_-141delTTTC). The three 59UTR polymorphisms are associated with a 20–30 % reduction in RPS19 protein levels when compared to the wild-type (w.t.) 59UTR of corresponding length. Conclusions: The RPS19 gene uses a broad range of TSS and a short 59UTR is associated with increased levels of RPS19. Comparisons between tissues showed a broad variation in the total amount of RPS19 mRNA and in the distribution of TS

Geographical variation in morphology of Chaetosiphella stipae stipae Hille Ris Lambers, 1947 (Hemiptera: Aphididae: Chaitophorinae)

Chaetosiphella stipae stipae is a xerothermophilous aphid, associated with Palaearctic temperate steppe zones or dry mountain valleys, where there are grasses from the genus Stipa. Its geographical distribution shows several populations that are spread from Spain, across Europe and Asia Minor, to Mongolia and China. Geographical variation in chaetotaxy and other morphological features were the basis to consider whether individuals from different populations are still the same species. Moreover, using Ch. stipae stipae and Stipa species occurrences, as well as climatic variables, we predict potential geographical distributions of the aphid and its steppe habitat. Additionally, for Stipa species we projected current climatic conditions under four climate change scenarios for 2050 and 2070. While highly variable, our results of morphometric analysis demonstrates that all Ch. stipae stipae populations are one very variable subspecies. And in view of predicted climate change, we expect reduction of Stipa grasslands. The disappearance of these ecosystems could result in stronger separation of the East-European and Asian steppes as well as European ‘warm-stage’ refuges. Therefore, the geographic morphological variability that we see today in the aphid subspecies Ch. stipae stipae may in the future lead to speciation and creation of separate subspecies or species

Women performing repetitive work: Is there a difference in the prevalence of shoulder pain and pathology in supermarket cashiers compared to the general female population?

Objectives: Shoulder disorders in the occupational environment have been widely studied, but the quality of research and methodology applied vary. Little has been done to ascertain whether shoulder pain in female repetitive workers is due to any verifiable pathology, or to compare findings with the general population. Therefore, we decided to evaluate the prevalence of self-reported shoulder pain in a group of female supermarket cashiers and in the general female population using a standardized questionnaire. Shoulder pain prevalence was then compared to imaging findings in order to assess specific and non-specific pain prevalence. Material and Methods: 196 cashiers and 302 controls filled in a standardized shoulder questionnaire and underwent an imaging examination of a shoulder. Results: The prevalence of shoulder pain was significantly higher in the group of cashiers (46.4%) than in the general population (25.5%) (OR = 1.821; 95% CI: 1.426–2.325). Specific pain prevalence was higher among the controls (19.5%) than among the cashiers (13.2%). Conclusions: The more frequent reports of shoulder pain in the supermarket cashiers are not correlated with a higher prevalence of imaging abnormalities. The causes of these more frequent complaints should be probably sought in the psycho-social and occupational environment

Characterisation of secreted exosomes from the intestinal nematode Heligmosomoides polygyrus

The parasite secretome has been shown to play a key role in both pathogenicity and the regulation of host defence, allowing pathogens, such as helminths, to establish a chronic infection within the host. The recently discovered presence of extracellular vesicles within parasite-derived excretory-secretory products introduces a new mechanism of potential cross-species communication. Extracellular vesicles (EVs), such as exosomes, facilitate cellular communication through the transfer of small RNAs, lipids and proteins between cells and organisms across all three kingdoms of life. In addition to their roles in normal physiology, EVs also transport molecules from pathogens to hosts, presenting parasite antigens and transferring infectious agents. Here, I examine secreted vesicles from the murine gastrointestinal nematode Heligmosomoides polygyrus, and their potential role in the host-helminth interactions. Transmission electron microscopy reveals vesicle-like structures of 50- 100 nM in the ultracentrifuged secretory product, and potential evidence of multi-vesicular bodies in the worm intestine. This, coupled with information from the exoproteome, helped support the hypothesis that exosomes originate from the parasite intestinal tract. I have completed a series of studies looking at the fundamental properties of exosome-cell interactions, providing comparative studies between mammalian and H. polygyrus-derived exosomes. I have determined some of the key factors influencing exosome uptake, including time of incubation, cell type and exosome origin. Through microarray analysis of H. polygyrus exosome-treated small intestinal epithelial cells, we see significant gene expression changes, including those involved in the regulation of signalling and the immune response, such as DUSP1 (dual-specificity phosphatase) and IL1RL1 (the receptor for IL-33). The modest reduction of inflammatory cytokine responses by exosomes in small intestinal cell lines was amplified in immune cells, such as macrophages. Exosomes can significantly reduce expression of classical activation markers, as well as inflammatory cytokine production in the macrophage cell line RAW 264.7, and this is further supported by similar responses in bone marrow-derived macrophages. Owing to their suppressive nature, I demonstrate that immunization of mice with an exosome/alum conjugate generates significant protection from a subsequent H. polygyrus larval challenge, as seen through a reduction in egg counts and worm burden. I have investigated the role of the IL33 receptor (IL-33R); a key molecule associated with parasitic resistance that is suppressed by exosomes in type-2 associated immune responses. Uptake of H. polygyrus-derived exosomes by alternatively activated macrophages caused the suppression of type 2 cytokine/protein release and the reduction of key genes associated with this phenotype. In addition, there was also significant repression of both transcript and surface T1/ST2, a subunit of the IL-33R). Using a model of lung inflammation, in vivo studies demonstrate that, in both prophylactic and co-administration experiments, exosomes modulate the innate cellular response. This is represented by changes in the number of innate lymphoid cells (ILCs), bronchoalveolar lavage eosinophils and type-2 cytokine output. In this system, the expression of T1/ST2 on type 2 ILCs was also significantly reduced. I have extended the investigation on exosome-IL-33R responses by using T1/ST2 knockout mice. Despite generating strong antibody responses, vaccination against exosomes could not protect T1/ST2 knockout mice against a subsequent infection. This work suggests that exosomes secreted by nematodes could mediate the transfer and uptake of parasite products into host cells, establishing cross-species communication to suppress the host ‘danger’ or inflammatory response