132 research outputs found

    Methods to study splicing from high-throughput RNA Sequencing data

    Full text link
    The development of novel high-throughput sequencing (HTS) methods for RNA (RNA-Seq) has provided a very powerful mean to study splicing under multiple conditions at unprecedented depth. However, the complexity of the information to be analyzed has turned this into a challenging task. In the last few years, a plethora of tools have been developed, allowing researchers to process RNA-Seq data to study the expression of isoforms and splicing events, and their relative changes under different conditions. We provide an overview of the methods available to study splicing from short RNA-Seq data. We group the methods according to the different questions they address: 1) Assignment of the sequencing reads to their likely gene of origin. This is addressed by methods that map reads to the genome and/or to the available gene annotations. 2) Recovering the sequence of splicing events and isoforms. This is addressed by transcript reconstruction and de novo assembly methods. 3) Quantification of events and isoforms. Either after reconstructing transcripts or using an annotation, many methods estimate the expression level or the relative usage of isoforms and/or events. 4) Providing an isoform or event view of differential splicing or expression. These include methods that compare relative event/isoform abundance or isoform expression across two or more conditions. 5) Visualizing splicing regulation. Various tools facilitate the visualization of the RNA-Seq data in the context of alternative splicing. In this review, we do not describe the specific mathematical models behind each method. Our aim is rather to provide an overview that could serve as an entry point for users who need to decide on a suitable tool for a specific analysis. We also attempt to propose a classification of the tools according to the operations they do, to facilitate the comparison and choice of methods.Comment: 31 pages, 1 figure, 9 tables. Small corrections adde

    Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

    Get PDF
    The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3-5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h51Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 μM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 μm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction

    Enhanced Functional Recovery in MRL/MpJ Mice after Spinal Cord Dorsal Hemisection

    Get PDF
    Adult MRL/MpJ mice have been shown to possess unique regeneration capabilities. They are able to heal an ear-punched hole or an injured heart with normal tissue architecture and without scar formation. Here we present functional and histological evidence for enhanced recovery following spinal cord injury (SCI) in MRL/MpJ mice. A control group (C57BL/6 mice) and MRL/MpJ mice underwent a dorsal hemisection at T9 (thoracic vertebra 9). Our data show that MRL/MpJ mice recovered motor function significantly faster and more completely. We observed enhanced regeneration of the corticospinal tract (CST). Furthermore, we observed a reduced astrocytic response and fewer micro-cavities at the injury site, which appear to create a more growth-permissive environment for the injured axons. Our data suggest that the reduced astrocytic response is in part due to a lower lesion-induced increase of cell proliferation post-SCI, and a reduced astrocytic differentiation of the proliferating cells. Interestingly, we also found an increased number of proliferating microglia, which could be involved in the MRL/MpJ spinal cord repair mechanisms. Finally, to evaluate the molecular basis of faster spinal cord repair, we examined the difference in gene expression changes in MRL/MpJ and C57BL/6 mice after SCI. Our microarray data support our histological findings and reveal a transcriptional profile associated with a more efficient spinal cord repair in MRL/MpJ mice

    Damage-induced reactive oxygen species enable zebrafish tail regeneration by repositioning of Hedgehog expressing cells.

    Get PDF
    Many aquatic vertebrates have a remarkable ability to regenerate limbs and tails after amputation. Previous studies indicate that reactive oxygen species (ROS) signalling initiates regeneration, but the mechanism by which this takes place is poorly understood. Developmental signalling pathways have been shown to have proregenerative roles in many systems. However, whether these are playing roles that are specific to regeneration, or are simply recapitulating their developmental functions is unclear. Here, we analyse zebrafish larval tail regeneration and find evidence that ROS released upon wounding cause repositioning of notochord cells to the damage site. These cells secrete Hedgehog ligands that are required for regeneration. Hedgehog signalling is not required for normal tail development suggesting that it has a regeneration-specific role. Our results provide a model for how ROS initiate tail regeneration, and indicate that developmental signalling pathways can play regenerative functions that are not directly related to their developmental roles

    The V-region disease hypothesis: evidence from autoimmune encephalomyelitis.

    No full text
    Experimental allergic encephalomyelitis has been shown to have an immunological basis. In fact, the disease can be induced by T cells specific for myelin basic protein, a molecule found in abundance in the central nervous system. In this article, Ellen Heber-Katz and Hans Acha-Orbea discuss the T-cell receptor (TCR) repertoire of the encephalitogenic T-cell response, and show that a limited V gene pool, in fact a single V beta and two V alpha families, are being used by the PL/J and B10.PL mice and by every rat strain examined, even though the antigenic determinants and the major histocompatibility complex (MHC) molecules are different in all cases. This extraordinary finding suggests that the TCR is involved in encephalitogenicity in a way that not only involves the recognition of antigen in association with MHC, but also as an effector molecule that results in encephalitis. If this is true, it implies that TCRs, in general, play more than one role in mammalian physiology
    corecore