A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities

The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage

Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138–Lys139 and Lys142–Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity

Modélisation nD à base d'algèbres géométriques

3ème prix du meilleur article jeunes chercheursDans cet article nous nous intéressons au plongement d'une structure topologique. Pour modéliser un objet, l'approche usuelle en modélisation à base topologique est de séparer la structure de l'objet, sa topologie, de sa forme, son plongement géométrique. Par conséquent, le plus souvent la forme des objets est traitée soit de façon très minimale ; des points d'une certaine dimension sont associés aux sommets de la structure topologique, soit de façon très spécifique ; la forme des objets est définie pour une dimension donnée (généralement un espace à 3 dimensions) et pour un paradigme géométrique donné (continu euclidien, discret). Nous proposons ici une façon de plonger les G-cartes de façon générique ; indépendante de la dimension et du paradigme géométrique. Le résultat est une structure de données qui permet de modéliser et manipuler des objets géométriques facettisés de toutes dimensions

Evaluation of tumor immune contexture among intrinsic molecular subtypes helps to predict outcome in early breast cancer

Background The prognosis of early breast cancer is linked to clinic-pathological stage and the molecular characteristics of intrinsic tumor cells. In some patients, the amount and quality of tumor-infiltrating immune cells appear to affect long term outcome. We aimed to propose a new tool to estimate immune infiltrate, and link these factors to patient prognosis according to breast cancer molecular subtypes.Methods We performed in silico analyses in more than 2800 early breast cancer transcriptomes with corresponding clinical annotations. We first developed a new gene expression deconvolution algorithm that accurately estimates the quantity of immune cell populations (tumor immune contexture, TIC) in tumors. Then, we studied associations between these immune profiles and relapse-free and overall survival among the different intrinsic molecular subtypes of breast cancer defined by PAM50 classification.Results TIC estimates the abundance of 15 immune cell subsets. Both myeloid and lymphoid subpopulations show different spread among intrinsic molecular breast cancer subtypes. A high abundance of myeloid cells was associated with poor outcome, while lymphoid cells were associated with favorable prognosis. Unsupervised clustering describing the 15 immune cell subsets revealed four subgroups of breast tumors associated with distinct patient survival, but independent from PAM50. Adding this information to clinical stage and PAM50 strongly improves the prediction of relapse or death.Conclusions Our findings make it possible to refine the survival stratification of early patients with breast cancer by incorporating TIC in addition to PAM50 and clinical tumor burden in a prognostic model validated in training and validation cohorts

Évaluation intra-tumorale par immunofluorescence multi-paramétrique des potentiels biomarqueurs d’efficacité des nouvelles immunothérapies

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Transverse and longitudinal magneto-optical effects with a functionalized microstructured optical fiber

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Integrated Stacked Parallel Plate Shunt Capacitor for Millimeter-Wave Systems in Low-Cost Highly Integrated CMOS Technologies

International audienceThis letter presents a stacked parallel plate (SPP) shunt capacitor (SC) that benefits from metal stack increment with process nodes advancement. It demonstrated high quality ( Q ) factor and high self-resonance frequency (SRF), promoting the design of analog integrated circuits (ICs) in low-cost highly integrated CMOS technologies at the millimeter-wave (mm-wave) frequency range. As a proof-of-concept, an analytical-equation-based design method is also proposed and three ac-grounded capacitors: 300; 600; and 900-fF, are implemented in STMicroelectronics (STM) 55-nm process. Characterization is performed up to 100 GHz. An effective capacitance density of 0.8 fF/&mu;m2 is obtained. Measurements show Q -values reaching up to 14.7 at 100 GHz and equivalent input series resistances with flat wideband behavior reaching at most an average of 0.55 &Omega; . SRFs of 140 GHz for the 900-fF SPP-SC up to 368 GHz for the 300-fF SPP-SC are also determined from measurements: the highest SRFs for such large capacitances to the authors&rsquo; knowledge.</p

Vibrational measurement of shear modulus and damping of wood: An application of the Vybris-Torsion device

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