Leishmania (L) are intracellular protozoan parasites that are able to survive and replicate within the harsh and potentially hostile phagolysosomal environment of mammalian mononuclear phagocytes. A ...complex interplay then takes place between the macrophage (MPhi) striving to eliminate the pathogen and the parasite struggling for its own survival. To investigate this host-parasite conflict at the transcriptional level, in the context of monocyte-derived human MPhis (MDM) infection by L. major metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used.
After extracting mRNA from resting human MPhis, Leishmania-infected human MPhis and L. major parasites, three SAGE libraries were constructed and sequenced generating up to 28,173; 57,514 and 33,906 tags respectively (corresponding to 12,946; 23,442 and 9,530 unique tags). Using computational data analysis and direct comparison to 357,888 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, confidently discriminating host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MPhis' and 3,666 tags to L. major parasites transcripts. We focused on these, showing significant changes in their expression that are likely to be relevant to the pathogenesis of parasite infection: (i) human MPhis genes, belonging to key immune response proteins (e.g., IFNgamma pathway, S100 and chemokine families) and (ii) a group of Leishmania genes showing a preferential expression at the parasite's intra-cellular developing stage.
Dual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminating genes of human or parasitic origin in Leishmania-infected human MPhis. The findings presented in this work suggest that the Leishmania parasite modulates key transcripts in human MPhis that may be beneficial for its establishment and survival. Furthermore, these results provide an overview of gene expression at two developmental stages of the parasite, namely metacyclic promastigotes and intracellular amastigotes and indicate a broad difference between their transcriptomic profiles. Finally, our reported set of expressed genes will be useful in future rounds of data mining and gene annotation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We introduce a k-mer-based computational protocol, DE-kupl, for capturing local RNA variation in a set of RNA-seq libraries, independently of a reference genome or transcriptome. DE-kupl extracts all ...k-mers with differential abundance directly from the raw data files. This enables the retrieval of virtually all variation present in an RNA-seq data set. This variation is subsequently assigned to biological events or entities such as differential long non-coding RNAs, splice and polyadenylation variants, introns, repeats, editing or mutation events, and exogenous RNA. Applying DE-kupl to human RNA-seq data sets identified multiple types of novel events, reproducibly across independent RNA-seq experiments.
Human long non-coding RNAs (lncRNAs) are an emerging category of transcripts with increasingly documented functional roles during development. LncRNAs and roles during human early embryo development ...have recently begun to be unravelled.
This review summarizes the most recent knowledge on lncRNAs and focuses on their expression patterns and role during early human embryo development and in pluripotent stem cells (PSCs). Public mRNA sequencing (mRNA-seq) data were used to illustrate these expression signatures.
The PubMed and EMBASE databases were first interrogated using specific terms, such as 'lncRNAs', to get an extensive overview on lncRNAs up to February 2016, and then using 'human lncRNAs' and 'embryo', 'development', or 'PSCs' to focus on lncRNAs involved in human embryo development or in PSC.Recently published RNA-seq data from human oocytes and pre-implantation embryos (including single-cell data), PSC and a panel of normal and malignant adult tissues were used to describe the specific expression patterns of some lncRNAs in early human embryos.
The existence and the crucial role of lncRNAs in many important biological phenomena in each branch of the life tree are now well documented. The number of identified lncRNAs is rapidly increasing and has already outnumbered that of protein-coding genes. Unlike small non-coding RNAs, a variety of mechanisms of action have been proposed for lncRNAs. The functional role of lncRNAs has been demonstrated in many biological and developmental processes, including cell pluripotency induction, X-inactivation or gene imprinting. Analysis of RNA-seq data highlights that lncRNA abundance changes significantly during human early embryonic development. This suggests that lncRNAs could represent candidate biomarkers for developing non-invasive tests for oocyte or embryo quality. Finally, some of these lncRNAs are also expressed in human cancer tissues, suggesting that reactivation of an embryonic lncRNA program may contribute to human malignancies.
LncRNAs are emerging potential key players in gene expression regulation. Analysis of RNA-seq data from human pre-implantation embryos identified lncRNA signatures that are specific to this critical step. We anticipate that further studies will show that these new transcripts are major regulators of embryo development. These findings might also be used to develop new tests/treatments for improving the pregnancy success rate in IVF procedures or for regenerative medicine applications involving PSC.
Genomic integrity of human pluripotent stem cells (hPSCs) is essential for research and clinical applications. However, genetic abnormalities can accumulate during hPSC generation and routine culture ...and following gene editing. Their occurrence should be regularly monitored, but the current assays to assess hPSC genomic integrity are not fully suitable for such regular screening. To address this issue, we first carried out a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications and identified 738 recurrent genetic abnormalities (i.e., overlapping abnormalities found in at least five distinct scientific publications). We then developed a test based on the droplet digital PCR technology that can potentially detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test can be used to routinely screen genomic integrity in hPSCs.
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•A meta-analysis was carried out to list genetic abnormalities detected in hPSCs•Recurrent genetic abnormalities in hPSCs were precisely defined•ddPCR is a robust and sensitive approach to screen these recurrent abnormalities•In culture supernatant digital test can be used to rapidly screen iPSC clones
In this article, De Vos and colleagues performed a comprehensive meta-analysis of genetic abnormalities detected in hPSC lines. They then used this information to devise a ddPCR test targeting the majority of recurrent abnormalities and show that this test can be applied to the cell culture supernatant. Finally, they illustrate the interest of this test for routine hPSC screening.
Progress in assisted reproductive technologies strongly relies on understanding the regulation of the dialogue between oocyte and cumulus cells (CCs). Little is known about the role of long ...non-coding RNAs (lncRNAs) in the human cumulus-oocyte complex (COC). To this aim, publicly available RNA-sequencing data were analyzed to identify lncRNAs that were abundant in metaphase II (MII) oocytes (BCAR4, C3orf56, TUNAR, OOEP-AS1, CASC18, and LINC01118) and CCs (NEAT1, MALAT1, ANXA2P2, MEG3, IL6STP1, and VIM-AS1). These data were validated by RT-qPCR analysis using independent oocytes and CC samples. The functions of the identified lncRNAs were then predicted by constructing lncRNA-mRNA co-expression networks. This analysis suggested that MII oocyte lncRNAs could be involved in chromatin remodeling, cell pluripotency and in driving early embryonic development. CC lncRNAs were co-expressed with genes involved in apoptosis and extracellular matrix-related functions. A bioinformatic analysis of RNA-sequencing data to identify CC lncRNAs that are affected by maternal age showed that lncRNAs with age-related altered expression in CCs are essential for oocyte growth. This comprehensive analysis of lncRNAs expressed in human MII oocytes and CCs could provide biomarkers of oocyte quality for the development of non-invasive tests to identify embryos with high developmental potential.
The development of RNA sequencing (RNAseq) and the corresponding emergence of public datasets have created new avenues of transcriptional marker search. The long non-coding RNAs (lncRNAs) constitute ...an emerging class of transcripts with a potential for high tissue specificity and function. Therefore, we tested the biomarker potential of lncRNAs on Mesenchymal Stem Cells (MSCs), a complex type of adult multipotent stem cells of diverse tissue origins, that is frequently used in clinics but which is lacking extensive characterization.
We developed a dedicated bioinformatics pipeline for the purpose of building a cell-specific catalogue of unannotated lncRNAs. The pipeline performs ab initio transcript identification, pseudoalignment and uses new methodologies such as a specific k-mer approach for naive quantification of expression in numerous RNAseq data. We next applied it on MSCs, and our pipeline was able to highlight novel lncRNAs with high cell specificity. Furthermore, with original and efficient approaches for functional prediction, we demonstrated that each candidate represents one specific state of MSCs biology.
We showed that our approach can be employed to harness lncRNAs as cell markers. More specifically, our results suggest different candidates as potential actors in MSCs biology and propose promising directions for future experimental investigations.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Anaplastic large cell lymphomas associated with ALK translocation have a good outcome after CHOP treatment; however, the 2-year relapse rate remains at 30%. Microarray gene-expression profiling of 48 ...samples obtained at diagnosis was used to identify 47 genes that were differentially expressed between patients with early relapse/progression and no relapse. In the relapsing group, the most significant overrepresented genes were related to the regulation of the immune response and T-cell activation while those in the non-relapsing group were involved in the extracellular matrix. Fluidigm technology gave concordant results for 29 genes, of which FN1, FAM179A, and SLC40A1 had the strongest predictive power after logistic regression and two classification algorithms. In parallel with 39 samples, we used a Kallisto/Sleuth pipeline to analyze RNA sequencing data and identified 20 genes common to the 28 genes validated by Fluidigm technology-notably, the
and
genes. Interestingly, FN1 also belongs to the gene signature predicting longer survival in diffuse large B-cell lymphomas treated with CHOP. Thus, our molecular signatures indicate that the FN1 gene, a matrix key regulator, might also be involved in the prognosis and the therapeutic response in anaplastic lymphomas.
A large number of RNA-sequencing studies set out to predict mutations, splice junctions or fusion RNAs. We propose a method, CRAC, that integrates genomic locations and local coverage to enable such ...predictions to be made directly from RNA-seq read analysis. A k-mer profiling approach detects candidate mutations, indels and splice or chimeric junctions in each single read. CRAC increases precision compared with existing tools, reaching 99:5% for splice junctions, without losing sensitivity. Importantly, CRAC predictions improve with read length. In cancer libraries, CRAC recovered 74% of validated fusion RNAs and predicted novel recurrent chimeric junctions. CRAC is available at http://crac.gforge.inria.fr .
Surgery and cisplatin‐based treatment of hepatoblastoma (HB) currently guarantee the survival of 70%‐80% of patients. However, some important challenges remain in diagnosing high‐risk tumors and ...identifying relevant targetable pathways offering new therapeutic avenues. Previously, two molecular subclasses of HB tumors have been described, C1 and C2, with C2 being the subgroup with the poorest prognosis, a more advanced tumor stage, and the worst overall survival rate. An associated 16‐gene signature to discriminate the two tumoral subgroups was proposed, but it has not been transferred into clinical routine. To address these issues, we performed RNA sequencing of 25 tumors and matched normal liver samples from patients. The transcript profiling separated HB into three distinct subgroups named C1, C2A, and C2B, identifiable by a concise four‐gene signature: hydroxysteroid 17‐beta dehydrogenase 6, integrin alpha 6, topoisomerase 2‐alpha, and vimentin, with topoisomerase 2‐alpha being characteristic for the proliferative C2A tumors. Differential expression of these genes was confirmed by quantitative RT‐PCR on an expanded cohort and by immunohistochemistry. We also revealed significant overexpression of genes involved in the Fanconi anemia (FA) pathway in the C2A subgroup. We then investigated the ability of several described FA inhibitors to block growth of HB cells in vitro and in vivo. We demonstrated that bortezomib, a Food and Drug Administration–approved proteasome inhibitor, strongly impairs the proliferation and survival of HB cell lines in vitro, blocks FA pathway–associated double‐strand DNA repair, and significantly impedes HB growth in vivo. Conclusion: The highly proliferating C2A subtype is characterized by topoisomerase 2‐alpha gene up‐regulation and FA pathway activation, and the HB therapeutic arsenal could include bortezomib for the treatment of patients with the most aggressive tumors. (Hepatology 2018;68:89‐102).
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