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

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated

FCNC Effects in a Minimal Theory of Fermion Masses

As a minimal theory of fermion masses we extend the SM by heavy vectorlike fermions, with flavor-anarchical Yukawa couplings, that mix with chiral fermions such that small SM Yukawa couplings arise from small mixing angles. This model can be regarded as an effective description of the fermionic sector of a large class of existing flavor models and thus might serve as a useful reference frame for a further understanding of flavor hierarchies in the SM. Already such a minimal framework gives rise to FCNC effects through exchange of massive SM bosons whose couplings to the light fermions get modified by the mixing. We derive general formulae for these corrections and discuss the bounds on the heavy fermion masses. Particularly stringent bounds, in a few TeV range, come from the corrections to the Z couplings.Comment: 19 pages, 1 figur

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

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation

Evaluation of the PPAR-γ agonist pioglitazone in mild asthma: a double-blind randomized controlled trial

Background Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a nuclear receptor that modulates inflammation in models of asthma. To determine whether pioglitazone improves measures of asthma control and airway inflammation, we performed a single-center randomized, double-blind, placebo-controlled, parallel-group trial. Methods Sixty-eight participants with mild asthma were randomized to 12 weeks pioglitazone (30 mg for 4 weeks, then 45 mg for 8 weeks) or placebo. The primary outcome was the adjusted mean forced expiratory volume in one second (FEV1) at 12 weeks. The secondary outcomes were mean peak expiratory flow (PEF), scores on the Juniper Asthma Control Questionnaire (ACQ) and Asthma Quality of Life Questionnaire (AQLQ), fractional exhaled nitric oxide (FeNO), bronchial hyperresponsiveness (PD20), induced sputum counts, and sputum supernatant interferon gamma-inducible protein-10 (IP-10), vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and eosinophil cationic protein (ECP) levels. Study recruitment was closed early after considering the European Medicines Agency’s reports of a potential increased risk of bladder cancer with pioglitazone treatment. Fifty-five cases were included in the full analysis (FA) and 52 in the per-protocol (PP) analysis. Results There was no difference in the adjusted FEV1 at 12 weeks (-0.014 L, 95% confidence interval [CI] -0.15 to 0.12, p = 0.84) or in any of the secondary outcomes in the FA. The PP analysis replicated the FA, with the exception of a lower evening PEF in the pioglitazone group (-21 L/min, 95% CI -39 to -4, p = 0.02). Conclusions We found no evidence that treatment with 12 weeks of pioglitazone improved asthma control or airway inflammation in mild asthma

A parsimonious oscillatory model of handwriting

International audienceWe propose an oscillatory model that is theoretically parsimonious, empirically efficient and biologically plausible. Building on Hollerbach’s (Biol Cybern 39:139–156, 1981) model, our Parsimonious Oscillatory Model of Handwriting (POMH) overcomes the latter’s main shortcomings by making it possible to extract its parameters from the trace itself and by reinstating symmetry between the x and y coordinates. The benefit is a capacity to autonomously generate a smooth continuous trace that reproduces the dynamics of the handwriting movements through an extremely sparse model, whose efficiency matches that of other, more computationally expensive optimizing methods. Moreover, the model applies to 2D trajectories, irrespective of their shape, size, orientation and length. It is also independent of the endeffectors mobilized and of the writing direction

Cerebrospinal fluid biomarker candidates associated with human WNV neuroinvasive disease

During the last decade, the epidemiology of WNV in humans has changed in the southern regions of Europe, with high incidence of West Nile fever (WNF) cases, but also of West Nile neuroinvasive disease (WNND). The lack of human vaccine or specific treatment against WNV infection imparts a pressing need to characterize indicators associated with neurological involvement. By its intimacy with central nervous system (CNS) structures, modifications in the cerebrospinal fluid (CSF) composition could accurately reflect CNS pathological process. Until now, few studies investigated the association between imbalance of CSF elements and severity of WNV infection. The aim of the present study was to apply the iTRAQ technology in order to identify the CSF proteins whose abundances are modified in patients with WNND. Forty-seven proteins were found modified in the CSF of WNND patients as compared to control groups, and most of them are reported for the first time in the context of WNND. On the basis of their known biological functions, several of these proteins were associated with inflammatory response. Among them, Defensin-1 alpha (DEFA1), a protein reported with anti-viral effects, presente