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Larrouy-Maumus G.,CNRS Institute of Pharmacology and Structural Biology | Larrouy-Maumus G.,Universitcrossed gn Of Toulouse | Larrouy-Maumus G.,Imperial College London | Gilleron M.,CNRS Institute of Pharmacology and Structural Biology | And 10 more authors.
Glycobiology | Year: 2015

Mycobacterium tuberculosis lipoarabinomannan (LAM) and biosynthetically related lipoglycans and glycans play an important role in host-pathogen interactions. Therefore, the elucidation of the complete biosynthetic pathways of these important molecules is expected to afford novel therapeutic targets. The characterization of biosynthetic enzymes and transporters involved in the formation and localization of these complex macromolecules in the bacterial cell envelope largely relies on genetic manipulation of mycobacteria and subsequent analyses of lipoglycan structural alterations. However, lipoglycans are present in relatively low amounts. Their purification to homogeneity remains tedious and time-consuming. To overcome these issues and to reduce the biomass and time required for lipoglycan purification, we report here the development of a methodology to efficiently purify lipoglycans by sodium deoxycholate-polyacrylamide gel electrophoresis. This faster purification method can be applied on a small amount of mycobacterial cells biomass (10-50 mg), resulting in tens of micrograms of purified lipoglycans. This amount of purified products was found to be sufficient to undertake structural analyses of lipoglycans and glycans carbohydrate domains by a combination of highly sensitive analytical procedures, involving cryoprobe NMR analysis of intact macromolecules and chemical degradations monitored by gas chromatography and capillary electrophoresis. This glycomic approach was successfully applied to the purification and structural characterization of a newly identified polysaccharide, structurally related to LAM, in the model fast-growing species Mycobacterium smegmatis. © 2015 The Author.

Laurent G.S.,St Laurent Institute | Laurent G.S.,Brown University | Vyatkin Y.,St Laurent Institute | Vyatkin Y.,Academy Lavrentjev ave | And 22 more authors.
Nucleic Acids Research | Year: 2016

Functionality of the non-coding transcripts encoded by the human genome is the coveted goal of the modern genomics research. While commonly relied on the classical methods of forward genetics, integration of different genomics datasets in a global Systems Biology fashion presents a more productive avenue of achieving this very complex aim. Here we report application of a Systems Biology-based approach to dissect functionality of a newly identified vast class of very long intergenic non-coding (vlinc) RNAs. Using highly quantitative FANTOM5 CAGE dataset, we show that these RNAs could be grouped into 1542 novel human genes based on analysis of insulators that we show here indeed function as genomic barrier elements. We show that vlincRNAs genes likely function in cis to activate nearby genes. This effect while most pronounced in closely spaced vlincRNA-gene pairs can be detected over relatively large genomic distances. Furthermore, we identified 101 vlincRNA genes likely involved in early embryogenesis based on patterns of their expression and regulation. We also found another 109 such genes potentially involved in cellular functions also happening at early stages of development such as proliferation, migration and apoptosis. Overall, we show that Systems Biology-based methods have great promise for functional annotation of non-coding RNAs. © 2016 The Author(s).

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