Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Genomics of Divergence along a Continuum of Parapatric Population Differentiation

MM received funding from the Max Planck innovation funds for this project. PGDF was supported by a Marie Curie European Reintegration Grant (proposal nr 270891). CE was supported by German Science Foundation grants (DFG, EI 841/4-1 and EI 841/6-1)

The Sertindole Safety Survey: A retrospective analysis under a named patient use programme in Europe

<p>Abstract</p> <p>Background</p> <p>After sertindole's suspension, health authorities established a specific named-patient use (NPU) programme in order to supply sertindole to patients who did not respond to or did not tolerate alternative treatments. This programme provided the possibility of prospectively following an exhaustive cohort of patients treated with sertindole after its suspension. A survey was performed to assess sertindole's modalities of prescription, assess and document any serious adverse events (SAEs), and assess the mortality rate within the NPU cohort.</p> <p>Methods</p> <p>The study comprised a survey of sertindole-treated patients in eleven European countries. All patients treated with sertindole within the NPU programme were eligible for the study.</p> <p>Results</p> <p>1,432 patients were included in the study. The reason for sertindole prescription was lack of efficacy (approximately 50%) or adverse events (approximately 20%) of other antipsychotic treatments. The mean sertindole dose was 13.4 mg daily. Lack of efficacy and adverse events were reported as reasons for sertindole discontinuation.</p> <p>A total of 97 SAEs were recorded, including ten fatal outcomes, which occurred during the study period or within thirty days after sertindole discontinuation. The all-cause mortality rate was 0.51 per 100 Person-Years of Exposure (95% Poisson confidence interval: 0.23–0.97). QTc prolongation was reported in 15 patients (1.05% of total patients), being a rate of 0.85 per 100 Person-Years of Exposure [95% CI: 0.48–1.41].</p> <p>Conclusion</p> <p>Although prescribing and supplying sertindole were subject to administrative constraints, a significant number of patients were treated with sertindole, thus supporting the need for sertindole in specific cases.</p> <p>Trial registration number</p> <p>Not applicable.</p

The utility of existing passerine microsatellite markers for genetic studies in endangered species: as demonstrated for a critically endangered forest bird endemic to Réunion Island, the Réunion cuckooshrike (Coracina newtoni)

Genetic data are increasingly recognized for their utility in conservation programs. However, many endangered species belong to families that have been understudied. Due to the urgency of their conservation status it is important to quickly identify polymorphic microsatellite loci from available resources. We show for the Re´union Cuckoo shrike Coracina newtoni, that this strategy can be very useful. Using 110 passerine microsatellite primer sets we identified eighteen polymorphic loci and tested them in 25 C. newtoni individuals. Following a Bonferroni correction one pair of loci displayed linkage disequilibriu

Switching of magnetization by non-linear resonance studied in single nanoparticles

Magnetization reversal in magnetic particles is one of the fundamental issues in magnetic data storage. Technological improvements require the understanding of dynamical magnetization reversal processes at nanosecond time scales. New strategies are needed to overcome current limitations. For example, the problem of thermal stability of the magnetization state (superparamagnetic limit) can be pushed down to smaller particle sizes by increasing the magnetic anisotropy. High fields are then needed to reverse the magnetization that are difficult to achieve in current devices. Here we propose a new method to overcome this limitation. A constant applied field, well below the switching field, combined with a radio-frequency (RF) field pulse can reverse the magnetization of a nanoparticle. The efficiency of this method is demonstrated on a 20 nm cobalt particle by using the micro-SQUID technique. Other applications of this method might be nucleation or depinning of domain walls.Comment: 11 pages, 5 figure

Transcriptomic Analysis of Host Immune and Cell Death Responses Associated with the Influenza A Virus PB1-F2 Protein

Airway inflammation plays a major role in the pathogenesis of influenza viruses and can lead to a fatal outcome. One of the challenging objectives in the field of influenza research is the identification of the molecular bases associated to the immunopathological disorders developed during infection. While its precise function in the virus cycle is still unclear, the viral protein PB1-F2 is proposed to exert a deleterious activity within the infected host. Using an engineered recombinant virus unable to express PB1-F2 and its wild-type homolog, we analyzed and compared the pathogenicity and host response developed by the two viruses in a mouse model. We confirmed that the deletion of PB1-F2 renders the virus less virulent. The global transcriptomic analyses of the infected lungs revealed a potent impact of PB1-F2 on the response developed by the host. Thus, after two days post-infection, PB1-F2 invalidation severely decreased the number of genes activated by the host. PB1-F2 expression induced an increase in the number and level of expression of activated genes linked to cell death, inflammatory response and neutrophil chemotaxis. When generating interactive gene networks specific to PB1-F2, we identified IFN-γ as a central regulator of PB1-F2-regulated genes. The enhanced cell death of airway-recruited leukocytes was evidenced using an apoptosis assay, confirming the pro-apoptotic properties of PB1-F2. Using a NF-kB luciferase adenoviral vector, we were able to quantify in vivo the implication of NF-kB in the inflammation mediated by the influenza virus infection; we found that PB1-F2 expression intensifies the NF-kB activity. Finally, we quantified the neutrophil recruitment within the airways, and showed that this type of leukocyte is more abundant during the infection of the wild-type virus. Collectively, these data demonstrate that PB1-F2 strongly influences the early host response during IAV infection and provides new insights into the mechanisms by which PB1-F2 mediates virulence

The Pioneer Anomaly

Radio-metric Doppler tracking data received from the Pioneer 10 and 11 spacecraft from heliocentric distances of 20-70 AU has consistently indicated the presence of a small, anomalous, blue-shifted frequency drift uniformly changing with a rate of ~6 x 10^{-9} Hz/s. Ultimately, the drift was interpreted as a constant sunward deceleration of each particular spacecraft at the level of a_P = (8.74 +/- 1.33) x 10^{-10} m/s^2. This apparent violation of the Newton's gravitational inverse-square law has become known as the Pioneer anomaly; the nature of this anomaly remains unexplained. In this review, we summarize the current knowledge of the physical properties of the anomaly and the conditions that led to its detection and characterization. We review various mechanisms proposed to explain the anomaly and discuss the current state of efforts to determine its nature. A comprehensive new investigation of the anomalous behavior of the two Pioneers has begun recently. The new efforts rely on the much-extended set of radio-metric Doppler data for both spacecraft in conjunction with the newly available complete record of their telemetry files and a large archive of original project documentation. As the new study is yet to report its findings, this review provides the necessary background for the new results to appear in the near future. In particular, we provide a significant amount of information on the design, operations and behavior of the two Pioneers during their entire missions, including descriptions of various data formats and techniques used for their navigation and radio-science data analysis. As most of this information was recovered relatively recently, it was not used in the previous studies of the Pioneer anomaly, but it is critical for the new investigation.Comment: 165 pages, 40 figures, 16 tables; accepted for publication in Living Reviews in Relativit

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio