16 research outputs found

    Long non-coding RNAs in the epigenetic regulation of oligodendrocyte differentiation

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    Long non-coding RNAs (lncRNAs) constitute a heterogeneous class of RNAs with limited coding potential, united by an arbitrarily placed cut off of >200 ntd. The past decade has seen the emergence of lncRNAs as versatile regulators of gene expression, amidst skepticism regarding the biological usefulness of pervasive genomic transcription and its non-coding RNA products prevalent in most eukaryotes. A significant portion of lncRNAs operate in the development and functioning of the mammalian CNS. Oligodendrocytes (OLs) are the myelinating cells of the CNS that are essential for efficient saltatory conduction and axonal survival. They are derived from OL precursors (OPCs) and progress into transcriptomically heterogeneous OL sub-populations along the differentiation pathway to produce mature OLs, capable of myelination. These epigenetic transitions between different OL subpopulations are carefully regulated, spatially and temporally, by a network of transcription factors, chromatin modulators and lncRNAs. In demyelinating diseases like multiple sclerosis (MS), patients suffer immune mediated attacks against myelin. Eventually, remyelination strategies fail due to deficits in OPC migration and OL differentiation at the site of lesions. Thus, understanding molecular mechanisms governing OL differentiation and myelination is crucial not only for understanding OL function in health but also in disease, in order to develop suitable therapeutic interventions. The investigations presented in this thesis explore the role of lncRNAs and RNA-binding proteins in neurodevelopment, particularly in embryonic stem cells (ESCs) and cells of the OL lineage. Article 1 provides a resource for the protein interactome of a key pioneering transcription factor, Sox2, in different nuclear fractions of mouse ESCs. We found Sox2 to be a multifaceted regulator forming interactions with HP1 family of proteins, whose members perform as both activators and repressors in a context dependent manner. In addition to interacting with RBPs involved in post-transcriptional processes, Sox2 also interacted with Rn7sk, a well-known ncRNA involved in the regulation of transcriptional elongation at promoters and enhancers. Although they did not influence each other‘s recruitment to the chromatin, this interaction opens up the possibility for ncRNA mediated modulation of ES transcriptional programs dependent on Sox2. Article 2 draws important insights regarding lncRNAs from a broad transcriptomic resource established from single cell- as well as bulk RNA- sequencing of OL lineage cells from different developmental stages. From a subset of lncRNAs which were found to be specific for certain OL subpopulations, we investigated the role of 2610035D17Rik in modulating the expression of its neighboring gene, Sox9, a transcription factor essential for OPC specification. We decoupled the role of lncRNA transcript from its genomic locus using various loss-of-function strategies and found that the regulation of Sox9 was dependent on the regulatory elements and/or ongoing transcription at the 2610035D17Rik locus, rather than the transcript itself. In Article 4, we investigated a hitherto unexplored RNA-binding function of myelin gene expression factor 2 (Myef2), a known transcriptional repressor of myelin basic protein (MBP). To this end, we uncovered the RNA interactome of Myef2 in a mouse oligodendroglial cell line with individual nucleotide resolution CLIP (iCLIP) followed by sequencing. We show that Myef2 interacts with CUG motifs located within introns and 3‘UTRs of protein-coding genes, a finding which implicates Myef2 in post-transcriptional processes like splicing and RNA stability. Finally, in Article 3 we have identified disease specific transcriptomic profiles of OL lineage cells through single-cell RNA sequencing of OPCs and OLs derived from experimental autoimmune encephalomyelitis (EAE) mice, a model that recapitulates several aspects of MS. EAE specific OPC and OL clusters were enriched for genes involved in antigen processing and presentation (MHC class I/II). We could demonstrate that OPCs can phagocytose myelin debris and MHC-II-expressing OPCs can activate memory and effector CD4-positive T cells. These findings show OL lineage cells as active participants in MS pathology than passive targets. Further, the findings of Article 2 implicate 2610035D17Rik as a regulator of immunomodulatory properties of oligodendroglia, as 2610035D17Rik KO cells showed reduced expression of IFNγ responsive genes and elevated expression of those involved in antigen presentation, compared to the controls, following IFNγ stimulation

    In search of the right literature search engine(s)

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    *Background*
Collecting scientific publications related to a specific topic is crucial for different phases of research, health care and ‘effective text mining’. Available bio-literature search engines vary in their ability to scan different sections of articles, for the user-provided search terms and/or phrases. Since a thorough scientific analysis of all major bibliographic tools has not been done, their selection has often remained subjective. We have considered most of the existing bio-literature search engines (http://www.shodhaka.com/startbioinfo/LitSearch.html) and performed an extensive analysis of 18 literature search engines, over a period of about 3 years. Eight different topics were taken and about 50 searches were performed using the selected search engines. The relevance of retrieved citations was carefully assessed after every search, to estimate the citation retrieval efficiency. Different other features of the search tools were also compared using a semi-quantitative method.
*Results*
The study provides the first tangible comparative account of relative retrieval efficiency, input and output features, resource coverage and a few other utilities of the bio-literature search tools. The results show that using a single search tool can lead to loss of up to 75% relevant citations in some cases. Hence, use of multiple search tools is recommended. But, it would also not be practical to use all or too many search engines. The detailed observations made in the study can assist researchers and health professionals in making a more objective selection among the search engines. A corollary study revealed relative advantages and disadvantages of the full-text scanning tools.
*Conclusion*
While many studies have attempted to compare literature search engines, important questions remained unanswered till date. Following are some of those questions, along with answers provided by the current study:
a)	Which tools should be used to get the maximum number of relevant citations with a reasonable effort? ANSWER: _Using PubMed, Scopus, Google Scholar and HighWire Press individually, and then compiling the hits into a union list is the best option. Citation-Compiler (http://www.shodhaka.com/compiler) can help to compile the results from each of the recommended tool._
b)	What is the approximate percentage of relevant citations expected to be lost if only one search engine is used? ANSWER: _About 39% of the total relevant citations were lost in searches across 4 topics; 49% hits were lost while using PubMed or HighWire Press, while 37% and 20% loss was noticed while using Google Scholar and Scopus, respectively._ 
c)	Which full text search engines can be recommended in general? ANSWER: _HighWire Press and Google Scholar._
d)	Among the mostly used search engines, which one can be recommended for best precision? ANSWER: _EBIMed._
e)	Among the mostly used search engines, which one can be recommended for best recall? ANSWER: _Depending on the type of query used, best recall could be obtained by HighWire Press or Scopus.

    RADICL-seq identifies general and cell type–specific principles of genome-wide RNA-chromatin interactions

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    Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure

    Disease-specific oligodendrocyte lineage cells arise in multiple sclerosis

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    Multiple sclerosis (MS) is characterized by an immune system attack targeting myelin, which is produced by oligodendrocytes (OLs). We performed single-cell transcriptomic analysis of OL lineage cells from the spinal cord of mice induced with experimental autoimmune encephalomyelitis (EAE), which mimics several aspects of MS. We found unique OLs and OL precursor cells (OPCs) in EAE and uncovered several genes specifically alternatively spliced in these cells. Surprisingly, EAE-specific OL lineage populations expressed genes involved in antigen processing and presentation via major histocompatibility complex class I and II (MHC-I and -II), and in immunoprotection, suggesting alternative functions of these cells in a disease context. Importantly, we found that disease-specific oligodendroglia are also present in human MS brains and that a substantial number of genes known to be susceptibility genes for MS, so far mainly associated with immune cells, are expressed in the OL lineage cells. Finally, we demonstrate that OPCs can phagocytose and that MHC-II-expressing OPCs can activate memory and effector CD4-positive T cells. Our results suggest that OLs and OPCs are not passive targets but instead active immunomodulators in MS. The disease-specific OL lineage cells, for which we identify several biomarkers, may represent novel direct targets for immunomodulatory therapeutic approaches in MS

    Synthesis and Cytotoxicity of Novel Analogues of Podophyllotoxin

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    We have synthesized four novel derivatives of podophyllotoxin, the chalcone derivatives namely 2-benzylidene-6,7-dimethoxy-4-phenyl-3,4-dihydro-2H-naphthalen-1-one (4a), 2-benzylidene-5,7-dichloro-6-hydroxy-4-phenyl-3,4-dihydro-2H-naphthalen-1-one(4b),andthe amino-thiazolyl derivatives namely7,8-dimethoxy-5-phenyl-4,5-dihydro-naphtha[1,2-d] thiazol-2-yl amine (5a)and 2-amino-6,8-dichloro-5-phenyl-4,5-dihydro-naphtho[1,2-d] thiazol-7-ol (5b). The aryl tetralin ring system in these compounds were attached to a fused aminothiazolyl ring or a chalcone ring. Synthesis of the tetralin ring in these podophyllotoxin derivatives was accomplished by the cyclization of the γ-hydroxyketones. These derivatives were evaluated for the induction of cytotoxicity in mouse mammary carcinoma cells in vitro. All the four compounds exhibited time dependent cytotoxicity at a concentration of 100μM when assessed by Trypan blue dye exclusion assay on Ehrlich Ascites Tumor (EAT) cells in vitr

    De novo design and synthesis of a 30-cistron translation-factor module

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    Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0
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