Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions

A 1-acetamido derivative of 6-epi-valienamine: an inhibitor of a diverse group of β-N-acetylglucosaminidases

The synthesis of an analogue of 6-epi-valienamine bearing an acetamido group and its characterisation as an inhibitor of β-N-acetylglucosaminidases are described. The compound is a good inhibitor of both human O-GlcNAcase and human β-hexosaminidase, as well as two bacterial β-N-acetylglucosaminidases. A 3-D structure of the complex of Bacteroides thetaiotaomicron BtGH84 with the inhibitor shows the unsaturated ring is surprisingly distorted away from its favoured solution phase conformation and reveals potential for improved inhibitor potency

End-users productivity in model-based spreadsheets: an empirical study

Lecture Notes in Computer Science Volume 6654, 2011Spreadsheets are widely used and studies show that most of the existing ones contain non-trivial errors. To improve end-users productivity, recent research proposes the use of a model-driven engineering approach to spreadsheets. In this paper we conduct the first empirical study to assess the effectiveness and efficiency of this approach. A set of spreadsheet end users worked with two different model-based spreadsheets. We present and analyze here the results achieved.(undefined

Visualizing Poiseuille flow of hydrodynamic electrons

Hydrodynamics is a general description for the flow of a fluid, and is expected to hold even for fundamental particles such as electrons when inter-particle interactions dominate. While various aspects of electron hydrodynamics were revealed in recent experiments, the fundamental spatial structure of hydrodynamic electrons, the Poiseuille flow profile, has remained elusive. In this work, we provide the first real-space imaging of Poiseuille flow of an electronic fluid, as well as visualization of its evolution from ballistic flow. Utilizing a scanning nanotube single electron transistor, we image the Hall voltage of electronic flow through channels of high-mobility graphene. We find that the profile of the Hall field across the channel is a key physical quantity for distinguishing ballistic from hydrodynamic flow. We image the transition from flat, ballistic field profiles at low temperature into parabolic field profiles at elevated temperatures, which is the hallmark of Poiseuille flow. The curvature of the imaged profiles is qualitatively reproduced by Boltzmann calculations, which allow us to create a 'phase diagram' that characterizes the electron flow regimes. Our results provide long-sought, direct confirmation of Poiseuille flow in the solid state, and enable a new approach for exploring the rich physics of interacting electrons in real space

Importance of molecular cell biology investigations in human medicine in the story of the Hutchinson-Gilford progeria syndrome

Ranged among laminopathies, Hutchinson–Gilford progeria syndrome is a syndrome that involves premature aging, leading usually to death at the age between 10 to 14 years predominatly due to a myocardial infarction or a stroke. In the lecture I shall overview the importance of molecular cell biology investigations that led to the discovery of the basic mechanism standing behind this rare syndrome. The genetic basis in most cases is a mutation at the nucleotide position 1824 of the lamin A gene. At this position, cytosine is substituted for thymine so that a cryptic splice site within the precursor mRNA for lamin A is generated. This results in a production of abnormal lamin A, termed progerin, its presence in cells having a deleterious dominant effect. Depending on the cell type and tissue, progerin induces a pleiotropy of defects that vary in different tissues. The present endeavour how to challenge this terrible disease will be also mentioned

A Minimal Model of Signaling Network Elucidates Cell-to-Cell Stochastic Variability in Apoptosis

Signaling networks are designed to sense an environmental stimulus and adapt to it. We propose and study a minimal model of signaling network that can sense and respond to external stimuli of varying strength in an adaptive manner. The structure of this minimal network is derived based on some simple assumptions on its differential response to external stimuli. We employ stochastic differential equations and probability distributions obtained from stochastic simulations to characterize differential signaling response in our minimal network model. We show that the proposed minimal signaling network displays two distinct types of response as the strength of the stimulus is decreased. The signaling network has a deterministic part that undergoes rapid activation by a strong stimulus in which case cell-to-cell fluctuations can be ignored. As the strength of the stimulus decreases, the stochastic part of the network begins dominating the signaling response where slow activation is observed with characteristic large cell-to-cell stochastic variability. Interestingly, this proposed stochastic signaling network can capture some of the essential signaling behaviors of a complex apoptotic cell death signaling network that has been studied through experiments and large-scale computer simulations. Thus we claim that the proposed signaling network is an appropriate minimal model of apoptosis signaling. Elucidating the fundamental design principles of complex cellular signaling pathways such as apoptosis signaling remains a challenging task. We demonstrate how our proposed minimal model can help elucidate the effect of a specific apoptotic inhibitor Bcl-2 on apoptotic signaling in a cell-type independent manner. We also discuss the implications of our study in elucidating the adaptive strategy of cell death signaling pathways.Comment: 9 pages, 6 figure

Time-dependent rate of multicomponent dark matter: reproducing the DAMA/LIBRA phase-2 results

The current paradigm for dark matter direct detection is to assume that the dark sector is solely composed of a single particle species. In this short paper, we make the observation that dark matter comprising both a light and a heavy component that modulate out of phase leads to interesting phenomenology in annual modulation experiments. For an illustrative example, we use the recently released DAMA/LIBRA phase-2 results with a lower energy threshold. Immediately after, it was argued that a one-component spin-independent dark matter explanation of the observed annual modulation is strongly disfavored or excluded unless isospin-violating couplings are invoked. We show that a simple two-component extension can reproduce the observed spectrum without the need to invoke fine-tuned couplings. Using the publicly available DAMA/LIBRA data, we perform a fit of the DAMA/LIBRA energy spectrum of the annual modulation amplitude to a scenario with two dark matter components. We also take into account how gravitational focusing affects the phases of the light and a heavy components differently, which leads to nontrivial effects in the total time-dependent rate. Our results show that there exists a unique solution in agreement with the data in the simplest case of isospin-conserving couplings with equal cross sections. The distinctive features found in this work are crucial for a dark matter interpretation of any observed annual modulation.Juan Herrero-Garcia, Andre Scaffidi, Martin White and Anthony G. William

Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von hippel-lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities

E3 ubiquitin ligases are attractive targets in the ubiquitin-proteasome system, however, the development of small-molecule ligands has been rewarded with limited success. The von Hippel-Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degradation. We recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, we report the design and optimization, guided by X-ray crystal structures, of a ligand series with nanomolar binding affinities

Bending modes, elastic constants and mechanical stability of graphitic systems

The thermodynamic and mechanical properties of graphitic systems are strongly dependent on the shear elastic constant C44. Using state-of-the-art density functional calculations, we provide the first complete determination of their elastic constants and exfoliation energies. We show that stacking misorientations lead to a severe lowering of C44 of at least one order of magnitude. The lower exfoliation energy and the lower C44 (more bending modes) suggest that flakes with random stacking should be easier to exfoliate than the ones with perfect or rhombohedral stacking. We also predict ultralow friction behaviour in turbostratic graphitic systems.Comment: 7 pages, 6 figure