In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination

Homing endonucleases, endonucleases capable of recognizing long DNA sequences, have been shown to be a tool of choice for precise and efficient genome engineering. Consequently, the possibility to engineer novel endonucleases with tailored specificities is under strong investigation. In this report, we present a simple and efficient method to select meganucleases from libraries of variants, based on their cleavage properties. The method has the advantage of directly selecting for the ability to induce double-strand break induced homologous recombination in a eukaryotic environment. Model selections demonstrated high levels of enrichments. Moreover, this method compared favorably with phage display for enrichment of active mutants from a mutant library. This approach makes possible the exploration of large sequence spaces and thereby represents a valuable tool for genome engineering

Mutant library and targets () Localization of the area of the binding interface (bottom view) chosen for randomization (green) and interacting base pairs (−3, orange; −4, pink; −5, magenta)

<p><b>Copyright information:</b></p><p>Taken from " selection of engineered homing endonucleases using double-strand break induced homologous recombination"</p><p>Nucleic Acids Research 2005;33(20):e178-e178.</p><p>Published online 23 Nov 2005</p><p>PMCID:PMC1289081.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Zoom showing residues 44, 68 and 70 chosen for randomization (green), D75 (red) and interacting base pairs (−3, orange; −4, pink; −5, magenta). () Sequences of the target used for selection and screening. C1234, wild-type target C1221; and C4334, palindromic site derived from C1234. H1221 and H4334, palindromic sites related to C1221. Boxes highlight bases not found in sites C1221 or C4334

Le mobilier en verre découvert à Toulouse et dans sa région à travers 15 ans d’archéologie ; évolution du verre, particularités locales et questions de chronologie

International audienceSince the early 2000s, the National Institute of Preventive Archaeological Researches (Inrap) has carried out several archaeological operations in the Toulouse region, revealing an important batch of medieval glass dated between the 13th and the 16th centuries. This glass furniture has been the subject of systematic and exhaustive studies, the results of which are presented in this paper.Depuis le début des années 2000, l‘Institut national de recherches archéologiques préventives (INRAP) a mené, dans la région toulousaine, plusieurs opérations archéologiques ayant permis la découverte d’importants lots de verre d’époque médiévale, issus de contextes datés du XIIIe au XVIe siècle. Ce mobilier a fait l’objet d’études systématiques et exhaustives, dont les résultats sont présentés ici

Effect of Tocilizumab vs Usual Care in Adults Hospitalized With COVID-19 and Moderate or Severe Pneumonia

International audienceImportance Severe pneumonia with hyperinflammation and elevated interleukin-6 is a common presentation of coronavirus disease 2019 (COVID-19).Objective To determine whether tocilizumab (TCZ) improves outcomes of patients hospitalized with moderate-to-severe COVID-19 pneumonia.Design, Setting, and Particpants This cohort-embedded, investigator-initiated, multicenter, open-label, bayesian randomized clinical trial investigating patients with COVID-19 and moderate or severe pneumonia requiring at least 3 L/min of oxygen but without ventilation or admission to the intensive care unit was conducted between March 31, 2020, to April 18, 2020, with follow-up through 28 days. Patients were recruited from 9 university hospitals in France. Analyses were performed on an intention-to-treat basis with no correction for multiplicity for secondary outcomes.Interventions Patients were randomly assigned to receive TCZ, 8 mg/kg, intravenously plus usual care on day 1 and on day 3 if clinically indicated (TCZ group) or to receive usual care alone (UC group). Usual care included antibiotic agents, antiviral agents, corticosteroids, vasopressor support, and anticoagulants.Main Outcomes and Measures Primary outcomes were scores higher than 5 on the World Health Organization 10-point Clinical Progression Scale (WHO-CPS) on day 4 and survival without need of ventilation (including noninvasive ventilation) at day 14. Secondary outcomes were clinical status assessed with the WHO-CPS scores at day 7 and day 14, overall survival, time to discharge, time to oxygen supply independency, biological factors such as C-reactive protein level, and adverse events.Results Of 131 patients, 64 patients were randomly assigned to the TCZ group and 67 to UC group; 1 patient in the TCZ group withdrew consent and was not included in the analysis. Of the 130 patients, 42 were women (32%), and median (interquartile range) age was 64 (57.1-74.3) years. In the TCZ group, 12 patients had a WHO-CPS score greater than 5 at day 4 vs 19 in the UC group (median posterior absolute risk difference [ARD] −9.0%; 90% credible interval [CrI], −21.0 to 3.1), with a posterior probability of negative ARD of 89.0% not achieving the 95% predefined efficacy threshold. At day 14, 12% (95% CI −28% to 4%) fewer patients needed noninvasive ventilation (NIV) or mechanical ventilation (MV) or died in the TCZ group than in the UC group (24% vs 36%, median posterior hazard ratio [HR] 0.58; 90% CrI, 0.33-1.00), with a posterior probability of HR less than 1 of 95.0%, achieving the predefined efficacy threshold. The HR for MV or death was 0.58 (90% CrI, 0.30 to 1.09). At day 28, 7 patients had died in the TCZ group and 8 in the UC group (adjusted HR, 0.92; 95% CI 0.33-2.53). Serious adverse events occurred in 20 (32%) patients in the TCZ group and 29 (43%) in the UC group (P = .21).Conclusions and Relevance In this randomized clinical trial of patients with COVID-19 and pneumonia requiring oxygen support but not admitted to the intensive care unit, TCZ did not reduce WHO-CPS scores lower than 5 at day 4 but might have reduced the risk of NIV, MV, or death by day 14. No difference on day 28 mortality was found. Further studies are necessary for confirming these preliminary results.Trial Registration ClinicalTrials.gov Identifier: NCT0433180

Effect of anakinra versus usual care in adults in hospital with COVID-19 and mild-to-moderate pneumonia (CORIMUNO-ANA-1): a randomised controlled trial

International audienc