Site-Specific mRNA Cleavage for Selective and Quantitative Profiling of Alternative Splicing with Label-Free Optical Biosensors

Alternative splicing of mRNA precursors is a key process in gene regulation, contributing to the diversity of proteomes by the alternative selection of exonic sequences. Alterations in this mechanism are associated with most cancers, enhancing their proliferation and survival, and can be employed as cancer biomarkers. Label-free optical biosensors are ideal tools for the highly sensitive and label-free analysis of nucleic acids. However, their application for alternative splicing analysis has been hampered due to the formation of complex and intricate long-range base-pairing interactions which make the direct detection in mRNA isoforms difficult. To solve this bottleneck, we introduce a methodology for the generation of length-controlled RNA fragments from purified total RNA, which can be easily detected by the biosensor. The methodology seizes RNase H enzyme activity to degrade the upstream and downstream RNA segments flanking the target sequence upon hybridization to specific DNA oligos. It allows the fast and direct monitoring of Fas gene alternative splicing in real time, employing a surface plasmon resonance biosensor. We demonstrate the selective and specific detection of mRNA fragments in the pM-nM concentration range, reducing quantification errors and showing 81% accuracy when compared to RT-qPCR. The site-specific cleavage outperformed random RNA hydrolysis by increasing the detection accuracy by 20%, making this methodology particularly appropriate for label-free quantification of alternative splicing events in complex samples

Transfers from Earth to LEO and LEO to interplanetary space using lasers

New data on some materials at 80ps pulse duration and 1057 nm wavelength give us the option of proportionally combining them to obtain arbitrary values between 35 (aluminum) and 800 N/MW (POM, polyoxymethylene) for momentum coupling coefficient Cm. Laser ablation physics lets us transfer to LEO from Earth, or to interplanetary space using repetitively pulsed lasers and Cm values appropriate for each mission. We discuss practical results for lifting small payloads from Earth to LEO, and space missions such as a cis-Mars orbit with associated laser system parameters

Diversité des agricultures familiales

Dans un contexte d’interrogation sur les modèles agricoles et de profondes transformations des agricultures et des marchés, cet ouvrage s’attache à revisiter la diversité des formes familiales de production et leurs mutations de par le monde. Dès lors, l’analyse intègre les liens aux marchés, aux territoires et à l’Ailleurs — par le fait migratoire — les enjeux d’autonomie et de sécurité alimentaire, les stratégies de survie et d’accumulation ainsi que les formes d’action collective et politique. L’ouvrage est construit autour de dix-huit études de cas, menées dans les cinq continents. Elles ont toutes été conduites avec un cadre méthodologique, identique et original, inspiré du Sustainable Rural Livehoods (cadre d’identification des moyens de subsistance durables en milieu rural). Revisitée pour cet ouvrage, cette méthode d’observations et d’analyses permet aux auteurs de préciser finement « ce qui fait famille », d’analyser les adaptations du travail des actifs familiaux et les mettre en perspective avec le contexte territorial et les politiques publiques de chaque pays. Écrit à plusieurs mains, par un réseau de chercheurs, cet ouvrage contribue autant à l’approfondissement des savoirs scientifiques sur les agricultures familiales dans le monde qu’à la mise à l’épreuve d’un cadre méthodologique d’analyse et d’observations en milieu rural. Il vise un public de chercheurs, d’enseignants et d’étudiants, agronomes, économistes, sociologues et historiens. Les experts du développement agricole et rural y trouveront un grand intérêt. Plus largement, toute personne qui s’intéresse aux agricultures familiales et à leurs évolutions dans divers contextes sociaux trouvera avantage à cette lecture. Les chercheurs qui ont coordonné cet ouvrage sont agronomes, économistes et géographes, au Cirad ou à l’Inra où ils mènent des recherches sur les systèmes agricoles familiaux à des échelles locales, nationales ou internationales. Cet ouvrage est le fruit d’une démarche de recherche collective et partagée permettant à de nombreux scientifiques des pays du Sud d’exprimer la diversité des réalités agraires contemporaines. Certains coordinateurs ou auteurs de cet ouvrage ont également collaboré à Agricultures familiales et mondes à venir, titre paru en 2014 chez Quae. Ce livre est également disponible en anglais sous le titreDiversity of Family Farming Around the Worldauprès des éditions Springer -www.springer.co

Contrôle de l'initiation alternative de la traduction du facteur de croissance fibroblastique 2 humain

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing

Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy.Work in JV’s lab was supported by Fundación Botín, by Banco de Santander through its Santander Universities Global Division and by Consolider RNAREG, MICINN and AGAUR. Work in L.M.L.'s lab was financially supported by Fundación Botín and EPISENS project of the Spanish Ministry of Science and Innovation (TEC2012-3428). The nanoB2A is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (2014 SGR 624). ICN2 acknowledges support of the Spanish MINECO through the Severo Ochoa Centers of Excellence Program under Grant SEV-2013-0295.Peer Reviewe

Insplico: effective computational tool for studying splicing order of adjacent introns genome-wide with short and long RNA-seq reads

Although splicing occurs largely co-transcriptionally, the order by which introns are removed does not necessarily follow the order in which they are transcribed. Whereas several genomic features are known to influence whether or not an intron is spliced before its downstream neighbor, multiple questions related to adjacent introns' splicing order (AISO) remain unanswered. Here, we present Insplico, the first standalone software for quantifying AISO that works with both short and long read sequencing technologies. We first demonstrate its applicability and effectiveness using simulated reads and by recapitulating previously reported AISO patterns, which unveiled overlooked biases associated with long read sequencing. We next show that AISO around individual exons is remarkably constant across cell and tissue types and even upon major spliceosomal disruption, and it is evolutionarily conserved between human and mouse brains. We also establish a set of universal features associated with AISO patterns across various animal and plant species. Finally, we used Insplico to investigate AISO in the context of tissue-specific exons, particularly focusing on SRRM4-dependent microexons. We found that the majority of such microexons have non-canonical AISO, in which the downstream intron is spliced first, and we suggest two potential modes of SRRM4 regulation of microexons related to their AISO and various splicing-related features. Insplico is available on gitlab.com/aghr/insplico.European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [ERCCoG-LS2-101002275 to M.I.]; Spanish Ministry of Economy and Competitiveness [PID2020-115040GB-I00 to M.I.]; ‘Centro de Excelencia Severo Ochoa 2013–2017’ [SEV-2012-0208]. Funding for open access charge: European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [ERCCoG-LS2-101002275 to M.I.]

Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing

Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy